V CONGRESS OF THE SERBIAN GENETIC SOCIETY

BOOK OF PAPERS

KLADOVO - BELGRADE, Serbia, 28 September-02 October 2014

Publisher

Serbian Genetic Society, Belgrade

www.dgsgenetika.org.rs

Editors

Snežana Mladenović Drinić

Violeta Anđelković

ISBN 978-86-87109-11-7

Honorary Committee

Dragoslav Marinković Marko Anđelković Kosana Konstantinov Draga Simić Vukosava Diklić Ljubiša Topisirović Vasilije Isajev Marija Kraljević Balalić Nada Barjaktarević Vučinić

Scientific Committee

Branka Vasiljević (Chair) Marina Stamenković-Radak Jelena Milašin Branka Vuković-Gačić Janoš Berenji Dragana Obreht Jelena Knežević-Vukčević Dragana Miladinović Violeta Anđelković Guiseppe Damante Olivera Francetić Malgorzata Dobrzynska Vittorio Venturi Jeffrey D. Jensen Domagoj Šimić Cino Pertoldi George Patrinos Segey Zotchev Borut Peterlin Vera Garaj-Vrhovac Metka Filipić Goradana Nikčević Branka Zukić Mirjana Šijačić Nikolić Dragica Radojković Jelena Blagojević Dragana Cvetković Gordana Joksić Ninosalv Đelić Vesna Milankov

Sofija Pavković-Lučić Marija Guč-Šćekić Ivana Novaković Đorđe Fira

Organizing Committee

Snežana Mladenović Drinić (Chair) Zorana Kurbalija Novičić Ivana Strahinić Vladan Ivetić Slaviša Stanković Bojana Banović Novo Pržulj Ana Nikolić Aleksandar Lučić Jasmina Ludoški Mihailo Jelić Tanja Adnadjević Tatjana Savić Katarina Zeljić Biljana Nikolić Milan Stevanović Branka Popović Biljana Jekić

Secretariat

Aleksandra Patenković Marija Tanasković Vesna Kandić Nađa Nikolić Boško Toljić Vladan Popović Jovan Pavlov Marko Đurakić Goran Vukotić

Content Dragana Antonić, Borut Bohanec, Anna De Carlo, Carla Benelli, Maurizio Lambardi, Angelina Subotić, Slađana Jevremović: Ploidy level stability of Iris

reichenbachii plants regenerated after cryopreservation

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Violeta Anđelković, Jelena Vančetović, Dragana Ignjatović-Micić, Natalija Kravić, Ana Obradović and Slavica Stanković: Tolerance to ear rot in maize inbred lines within drought tolerant mini-core collection

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Milka Brdar-Jokanović, Milan Zdravković, Mirjana Vasić, Aleksandra Savić, Zdenka Girek, Jasmina Zdravković: Yield-related traits in a collection of ‘trešnjevac’ beans (Phaseolus vulgaris L. forma versicolor)

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Zoran Čamdžija, Milomir Filipović, Slaven Prodanović, Sofija Božinović, Milan Stevanović, Jovan Pavlov and Nikola Grčić: Mode of inheritance of kernel row number of ZP maize material

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Nebojša Deletić, Slaviša Stojković, Slaviša Gudžić, Miroljub Aksić, Miodrag Jelić: The effect of some agronomic traits on grain yield of small grain crops

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Jovana Devetaković, Vladan Ivetić, Mirjana Šijačić-Nikolić, Vladan Popović, Boris Ivanović: Breeding of european white elm in Serbia – selection and progeny test

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Vesna Dragičević, Milomir Filipović, Natalija Kravić, Milovan Stojiljković, Snežana Mladenović Drinić. Selection of maze inbred lines for improved ratio between phytic acid and Mg, Fe, Mn and Zn content in grain

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Vesna Dragičević, Natalija Kravić, Milovan Stojiljković, Violeta Anđelković, Jelena Vančetović : Increased availability of Mg, Fe, Mn and Zn in drought tolerant maize inbred lines

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Zoran Dumanović, Vojka Babić, Života Jovanović, Milomir Filipović, Violeta Anđelković: Evaluation of selection indices for drought tolerance in maize (Zea mays L.) hybrids

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Snežana Đorđević, Dragana Stanojević, Dušan Kovačević, Željko Dolijanović, Zvonko Radan:Wheat genotype response on bacterial auxin treatment

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Jelena Filipović, Andreja Leskovac, Ana Valenta Šobot, Sandra Petrović, Dragana Vujić, Gordana Joksić:High incidence of radial chromosomes in circulation lymphocytes predicts bone marrow failure in Fanconi anemia patient – a case report

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Dragana Ignjatovic-Micic, Ana Nikolic, Sofija Bozinovic, Jelena Vancetovic: SSR

analysis of genetic diversity and relationships among maize drought tolerant and susceptible inbred lines

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Dragan Kovačević, Vesna Perić, Mirjana Srebrić, Aleksandra Sudarić, Snežana Mladenović Drinić:Determination of KTI in soybean genotypes by molecular markers

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Natalija Kravic, Violeta Andjelkovic, Jelena Vancetovic, Ana Nikolic, Danijela Ristic, Dragana Ignjatovic-Micic:Assessment of genetic diversity among drought tolerant maize local landraces using SSR markers

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Natalija Kravić, Vesna Dragičević, Milovan Stojiljković, Mile Sečanski, Jelena Vančetović, Violeta Anđelković:Variation of availability in mineral elements content from grain in maize landraces

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Dragana Lalević, Milan Biberdžić:Response of winter triticale genotypes on nitrogen fertilization

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Ana Milošević-Đerić, Boško Ristanović, Milena Aćimović, Gordana Šošic, Tanja Novaković:Prenatally detected trisomy 20 mosaicism- a single case report

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Ana Nikolic, Dragana Ignjatovic-Micic, Sofija Bozinovic, Violeta Andjelkovic, Jelena Vancetovic:SSR allele frequency differences among drought tolerant and susceptible maize inbred lines

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Biljana Nikolić, Dragana Mitić-Ćulafić, Branka VukoviĆ-Gačić, Jelena Knežević-Vukčević:Genotoxic and cytotoxic effect of camphor, eucalyptol and thujone in repair proficient and mismatch repair deficient eukaryotic cells

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Biljana Nikolić, Dragana Mitić-Ćulafić, Branka Vuković-Gačić, Jelena Knežević-Vukčević: Opposite effect of camphor, eucalyptol and thujone on DNA repair and mutagenesis: a question of dose

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Jovan Pavlov , Nenad Delić, Nikola Grčić, Milan Stevanović, Zoran Čamdžija, Miloš Crevar and Sofija Božinović: Correlation between genetic distance, specific combining ability and heterosis in maize

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Jasna Paradiž, Jože Bavcon, Danijel Vrhovšek: Pollen grain deformation assay on Galanthus for variability assessment and environmental biomonitoring

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Vesna Peric, Ana Nikolic, Mirjana Srebric, Snezana Mladenovic Drinic: Genetic relationship between soybean cultivars from different breeding institutions

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Danijela Ristic, Marija Kostadinovic, Dragana Ignjatovic-Micic, Jelena Vancetovic: Utilization of maize genetic resources in grain quality improvement

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Mirjana Srebric, Vesna Peric, Gordana Surlan Momirovic: Genetic gain from selection in progeny of soybean sister crosses

153

Hadi Waisi, Bogdan Nikolić, Vesna Dragičević, Danijela Pavlović, Milorad Vujičić, Sanja Đurović, Lana Đukanović: Influence of brassinosteroid based fertilizer on the germination of two maize hybrids

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Milica Zivkovic, Ewelina Stefanovic, Marija Miljkovic, Jovanka Lukic, Maja Tolinacki, Jelena Begovic, Natasa Golic, Milan Kojic, Ivana Strahinic: Probiotic potential of lactobacilli of different orgin

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1

PLOIDY LEVEL STABILITY OF Iris reichenbachii PLANTS REGENERATED AFTER CRYOPRESERVATION

Dragana ANTONIĆ1, Borut BOHANEC2, Anna DE CARLO3, Carla BENELLI3, Maurizio LAMBARDI3, Angelina SUBOTIĆ1, Slađana JEVREMOVIĆ1

1Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar despota

Stefana 142, 11060 Belgrade, Serbia (sladja@ibiss.bg.ac.rs) 2Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia

3CNR, National Research Council of Italy, IVALSA (Istituto per la Valorizzazione del Legno e Specie Arboree), 50019 Sesto Fiorentino, Firenze, Italy

Abstract The main aim of this work was to investigate clonal fidelity of Iris reichenbachii plants regenerated after cryopreservation procedure, plants regenerated by tissue culture and plants collected from natural habitat. I. reichenbachii plants were successfully regenerated after cryopreservation of shoot tips using PVS2-vitrification based procedure (droplet vitrification). Fully developed plantlets were acclimatized in greenhouse conditions. Flow cytometric analyses revealed that all tested plants regenerated after cryopreservation procedure had the same ploidy level and genome size as plants that were regenerated by tissue culture or collected from natural habitat. Key words: flow cytometry, plant tissue culture, cryopreservation Introduction

Dwarf iris, Iris reichenbachii Heuff. (2n = 24, 48; Syn: I. balkana Janka, I. bosniaca (Beck) Dörfl., I. serbica Pancic, I. skorpilii Velen.) is an endemic iris species of Balkan Peninsula. I. reichenbachii belongs to a group of bearded irises which, in addition to the ornamental value have pharmacologically important substances like xanthones (Wiliams et al., 1997; Šavikin-Fodulović et al., 2000). An efficient protocol for in vitro propagation was developed for I. reichenbachii by using somatic embryogenesis and organogenesis techniques (Jevremović et al., 2006).

Apart from ex situ collections, in vitro slow growth storage and cryopreservation represent additional safe methods for long term storage of different plant genetic resources (Kulis and Zalewska, 2014). There are two reports about methods for cryopreservation of Iris sp. genetic resources. Firstly, Shibli (2000) reported about cryopreservation of encapsulated somatic embryos of black iris where survival of somatic embryos was 90% and only 10% of somatic

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embryos showed secondary somatic embryogenesis. Recently, Jevremović et al. (2011) reported that vitrification protocol with Plant Vitrification Solution 2 (PVS2, Sakai et al., 1990) could be successfully used for cryopreservation of I. pumila shoot tips. Furthermore, in assessment of clonal fidelity of I. pumila plants regenerated after cryopreservation showed the same genome size as plants regenerated by organogenesis in culture in vitro (Jevremović et al., 2011). Some alterations in genome size of I. pumila plants regenerated by somatic embryogenesis were reported earlier (Jevremović et al., 2010).

The potential impact of in vitro culture and cryopreservation on the size of the genome was analyzed through comparison with plants originated from natural habitat. Material and methods

Plant material

The material for analysis were leaves of I. reichenbachii plants, germinated from seeds collected from natural habitat (Mountain Divčibare, Serbia), shoot cultures grown on medium for shoot multiplication before cryopreservation experiments and plants regenerated after cryopreservation and acclimatized in greenhouse conditions. Plant tissue culture

Shoot cultures of I. reichenbachii were established according to procedure described in Jevremović et al. (2006). Culture media were supplemented with MS medium (Murashige and Skoog, 1962), α-naphthalene acetic acid (NAA, 0.1 mg/l) and 6-benzyladenine (BA, 1.0 mg/l). Cryopreservation techniques employed

For cryopreservation PVS2-based vitrification procedure with two weeks of cold hardening and two days of excised shoot tips preculture at 4°C were applied (Jevremović et al. 2009). In brief: loading was 30 min; PVS2 treatment at 0°C for 20 min at aluminium foil (droplet vitrification); immersion in liquid nitrogen; re-warming at 40°C for 1 min, unloading 1.2 M sucrose for 20 min. Shoot development after cryopreservation was achieved on the same medium for shoot multiplication supplemented with NAA and BAP (0.1; 1.0 mg/l, respectively). Shoot rooting was attained on MS medium without plant growth regulators. Fully developed plantlets were potted in mix of peat and perlite (3:1) and acclimatized in greenhouse conditions. Flow cytometry analysis The level of ploidy of I. reichenbachii plants was determined by flow cytometry analysis according to the method reported in Doležel et al. (1992). Nuclei were isolated from 1 cm2 portion of leaves with 0.4 ml of nuclei extraction buffer (0.1 M Tris, 2 mM MgCl2, 0.1 M NaCl, 0.05 % Triton X-100, pH 7.0). After filtration through a 30 µm nylon sieve, nuclei were stained with 2.0 ml DAPI stock solution. Flow cytometric analysis was performed with PAS flow-cytometer (Partec Münster, Germany) with argon laser (488 nm, 20 mW) and Vicia faba (nuclear DNA content = 26.90 pg) as the internal standard. The positions of G0/G1 peak and coefficient of variations (CV) were evaluated with softwear FLOMAX (Partec Münster, Germany).

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Results and discussion Cryopreservation represents a good method for long term conservation of different

genetic resources. Shoot tip cryopreservation is shown as the most reliable, a cost-effective and efficient option for ex situ conservation of many plant species (Panis and Lambardi, 2005; Kulus and Zalewska, 2014) as well as iris species (Jevremović et al. 2011). I. reichenbachii plants can be successfully regenerated three months after cryopreservation procedure using shoot tips and following droplet-vitrification procedure. Whole plantlets could be regenerated after three months of thawing of shoot tip samples. In our study the ploidy level was estimated by flow cytometry of small pieces of I. reichenbachii leaves sampled from shoots of different origin. In Fig. 1 representative histograms of control plants collected in natural habitat (Fig. 1A), shoot cultures multiplied in tissue culture before cryopreservation (Fig. 1B) and plants regenerated after cryopreservation (Fig. 1C) were shown. The channel position (around 200) of G0/G1 peak and total number of peaks (2) corresponded to control plants taken from natural habitat as well as multiplied in culture in vitro. Linear FL2-DAPI histograms of relative nuclear content in leaf tissue of all types of material showed single distinct G0/G1 peaks and coefficient of variation (CV) was low. Also, there was no significant difference in DNA content in samples of in vitro grown plants, plants regenerated after cryopreservation and plants derived from seeds collected in natural habitat (Table 1). Table 1. DNA content in leaf samples in Iris reichenbachii of different origin.

Origin of plant material DNA content (pg/2C)*

Coefficients of variation (CV %)

Plants collected in natural habitat 11.08 3.43 In vitro multiplied shoots 10.85 3.79 Ex vitro grown plants after cryopreservation 10.84 1.89 * DNA content was calculated according to Doležel et al. (2007). Vicia faba was used as the internal standard (nuclear DNA content = 26.90 pg).

Somaclonal variation is a well-studied phenomenon in regenerated plants derived from

organ cultures, calli, protoplasts and somatic embryos (Wang and Wang, 2009). Clonal stability of plants regenerated after cryopreservation using shoot tips was not investigated so far. Cells in culture in vitro divide mitotically during dedifferentiation and growth but callus formation is a main source of ploidy instability in tissue culture (Shao and Wang, 2010). Results of DNA amount measurement performed in this study suggested that changes of ploidy level were not occurred in I. reichenbachii plants after cryopreservation procedure. The results of flow cytometric analysis revealed that relative DNA content of plants regenerated after cryopreservation procedure as well as in vitro grown shoots remained unaffected compared with plants collected in natural habitat. The CV value of DNA peaks is the measure of precision of flow cytometry analysis and CVs below 5% are considered as acceptable (Doležel and Bartoš, 2005). One of the explanations for confirmed ploidy level stability in our study lies in the way of shoot regeneration of I. reichenbachii plants in tissue culture. During shoot initiation and multiplication the callus phase was very short and shoots multiplication was mainly achieved by axillary shoot development. Conclusion

Flow cytometric analyses showed that cryopreservation had no effect on the ploidy level stability of I. reichenbachii plants. Analysis revealed that all tested I. reichenbachii plants

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regenerated after cryopreservation procedure had the same genome size as plants regenerated by tissue culture or collected in natural habitat.

Fig. 1 Representative flow cytometric histograms of DAPI-stained nuclei isolated from leaf tissue of Iris

reichenbachii plants collected from natural habitat (A), regenerated by tissue culture (B) and regenerated after cryopreservation by droplet-vitrification procedure (C). Left peak = G0/G1-phases of cell cycle of sample; right pick = G0/G1 peak of an internal standard (Vicia faba).

Acknowledgements This research was sponsored by the Ministry for Education, Science and Technological Development, Serbia (Project TR31019).

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References Doležal, J., Sgorbati, S., Lucretti, S. (1992): Comparasion of three DNA fluoro-chromes for flow

cytometric estimation of nuclear DNA content in plants. Physiol. Plant., 85:625-631. Doležel, J., Bartoš, J. (2005): Plant DNA flow cytometry and estimation of nuclear genome size.

Ann. Bot., 95:99-100. Doležel, J., Greilhuber, J., Suda J. (2007): Estimation of nuclear DNA content in plants using

flow cytometry. Nat. Protoc., 2:2233-2244. Jevremović, S., Subotić, A., Radojević, Lj. (2006): In vitro morphogenesis of dwarf irises. In: J.T

da Silva (ed.). Floriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues Vol 2. Global Science Books, Japan, p.551-557. Jevremović, S., Subotić, A., de Carlo, A., Benelli, C., Lambardi, M. (2009): Development of

cryopreservation procedures for dwarf irises (Iris spp). 1st International Symposium on Cryopreservation in Horticultural Species, Leuven, Belgium, Book of abstracts, p.45.

Jevremović, S., Ćalić, D., Subotić, A., Trifunović, M., Petrić, M., Bohanec, B. (2010): Ploidy variation and flowering of dwarf iris derived from tissue culture. Acta Hort., 855:153-158.

Jevremović, S., Subotić, A., Benelli, C., de Carlo, A., Lambardi, M. (2011): Cryopreservation of Iris pumila shoot tips by vitrification. Acta Hort., 908:355-360.

Kulus D., Zalewska, M. (2014): Cryopreservation as a tool used in long term storage of ornamental species – A review. Sci. Hort., 168:88-107.

Murashige T., Skoog, F. (1962): A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 15:473-497.

Panis, B., Lambardi, M. (2005): Status of cryopreservation technologies in plants (crops and forest trees). In: FAO (ed.) Proc. Int. Workshop 'The Role of Biotechnology for the Characterization and conservation of Crop, Forestry, Animal and Fishery genetic Resources', Turin (Italy), pp.43-54.

Sakai, A., Kobayashi, S., Oiyama, I. (1990): Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. Var. brasiliensis) by vitrification. Plant Cell Rep., 9:30-33.

Shao, L., Wang, Z.C. (2010): Review of genetic stability research for plant germplasm cryopreservation. Plant Physiol. Comm., 46 (11):1109-1113.

Shibli, R.A. (2000): Cryopreservation of black iris (Iris nigricans) somatic embryos by encapsulation-dehydration. Cryoletters, 21:39-46.

Šavikin-Fodulović, K., Stojanović, D., Menković, N. (2000): Fitohemijska i anatomska analiza vrste Iris reichenbachii Heuff. Lekovite sirovine, 20:21-26.

Wang, Q.M., Wang L. (2012): An evolutionary view of plant tissue culture: Somaclonal variation and selecton. Plant Cell Rep., 31 (9):1535-1547.

Williams, C.A., Harborne, J.B., Colasante, M. (1997): Flavonoid and xanthone patterns in bearded iris species and the pathway of chemical evolution in the genus. Biochem. Systematics and Ecology, 25 (4):309−325.

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TOLERANCE TO EAR ROT IN MAIZE INBRED LINES WITHIN DROUGHT TOLERANT MINI-CORE COLLECTION

Violeta ANĐELKOVIĆ*, Jelena VANČETOVIĆ, Dragana IGNJATOVIĆ-MICIĆ,

Natalija KRAVIĆ, Ana OBRADOVIĆ and Slavica STANKOVIĆ

Maize Research Institute Zemun Polje, Slobodana Bajića 1, 11185 Belgrade, Serbia *e-mail: avioleta@mrizp.rs

Abstract Adverse abiotic or biotic factors seriously reduce maize yields and grain quality. In 2012, as implication of drought, maize yields reduction in Serbia was 48%. Besides, ear rot caused by Fusarium species is one of the most common worldwide diseases of maize, causing yield reduction and contamination of grain by mycotoxins. Drought and various insects are key factors affecting disease severity. Development of core collections within genebank could be an efficient tool to evaluate and characterize large collections, and to increase usage of genetic diversity. About 6000 accessions of Maize Research Institute Zemun Polje gene bank were screened for drought tolerance in Egypt and in temperate climate, and the mini-core collection of 41 accessions (15 inbred lines, 13 introduced population and 13 local landraces) was established. Ears of chosen inbreds and drought susceptible lines (B73 and Mo17) were inoculated with suspension of F. verticillioides. Considering the importance of genotype effect and low specificity of disease resistance to the environment, the lines L1, L3, L4, L5 and L15 were chosen as potential sources of genes for stable resistance to ear rot in drought conditions. Key words: Disease resistance, Drought stress, Fusarium verticillioides, Stress indices, Zea

mays L. Introduction

The need to breed robust and high-productivity crops is more important than ever, due to increasingly adverse environmental conditions and scarce natural resources. A broad range of stress factors divided into two major categories, namely abiotic stresses encompassing a variety of unfavorable environmental conditions, such as drought, submergence, salinity, heavy metal contamination or nutrient deficiency, and biotic stresses caused by infectious living organisms, such as bacteria, viruses, fungi, or nematodes, negatively affect the productivity and survival of plants (Atkinson and Urwin 2012).

Under field conditions, plants are often subjected to multiple stresses simultaneously, requiring efficient molecular mechanisms to perceive a multitude of signals and to elicit a

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tailored response (Mittler 2006). However, research programmes aimed at developing tolerance to a particular stress do not necessarily test susceptibility to other biotic or abiotic stresses. This can have unforeseen consequences, as improved varieties may respond unpredictably when grown in field conditions (Collins et al., 2008).

Fusarium graminearum was the most important pathogen of maize and small grains in Serbia. However, during the last several years due to extremely high temperatures (>35˚C) and drought in the period from pollination to harvest of maize, increasing occurrence of F.

verticillioides has been recorded. Fusarium ear rot (FER), caused by this species is one of the most common worldwide diseases of maize, causing yield and quality reductions as well as contamination of grain by mycotoxigenic metabolites, fumonisins and other mycotoxins, harmful to human and animal health (Proctor et al. 2006). Fumonisin B1 is the most commonly found, not only in corn (maize) and corn-based foods, but also in wheat and other cereals in Serbia (Stanković et al., 2012). The accumulation of fumonisins in maize kernels is highly correlated with fungal biomass in the kernels (Waalwijk et al., 2008) and its biosyntesis is regulated by environmental conditions that favor the growth of the pathogen (Miller 2001). Seed infection by F. verticillioides can reduce emergence and stand quality in the subsequent crop generation, and often leaves grain contaminated with mycotoxins. Symptoms usually develop initially on ear tips, but may also manifest as scattered areas of infection over the ear surface (Payne 1999). Severely infected ears are covered with white to pink fungal mycelia, which colonize kernels, sap from ruptured kernel tissue, and husk leaves. When infection is extremely severe, husk leaves are sometimes fused to the ear by the fungal mycelium. Fungal metabolism within the kernels results in a net decrease in grain dry matter content, kernel density, and total grain yield (Presello et al., 2008). In addition to symptomatic kernels, F. verticillioides can be isolated from a large proportion of symptomless kernels, as well as host root, stalk, and leaf tissues (Munkvold and Carlton 1997).

Miller (2001) suggested five factors of prime importance in determining risk of Fusarium ear rot and fumonisin contamination: temperature, water deficit, insect damage, other fungal diseases, and maize genotype. Since dry conditions typically accompany excessive heat, it can be difficult to separate the two factors and determine the individual effects of each. Shelby (1994) reported a strong inverse correlation between precipitation near pollination and fumonisin accumulation in maize grain.

Recent breeding and heritability studies suggest that selection against visible Fusarium ear rot symptoms should be an effective method to reducing susceptibility to fumonisin contamination (Robertson et al., 2006). Thus, the objective of this study was to conduct preliminary screening on inbred lines differing in drought tolerance, in order to determine the direct effect of F. verticillioides ear rot resistance by evaluating disease severity and measuring yield of each genotype under both inoculated and non-inoculated conditions. Material and methods Plant material

The subset of fifteen maize inbred lines from Maize Research Institute Zemun Polje (MRIZP) gene bank drought tolerant mini-core collection (L1 - L15) and two drought susceptible public inbred lines (B73 and Mo17) were the objectives of the present study.

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Screening for Fusarium ear rot resistance The experiment was carried out in 2013 in Zemun Polje, Serbia (44˚52´N, 20˚19´E, 81 m

asl). The inbreds were tested in field experiment for resistance to ear rot by application of Silk Channel Inoculation Assay (SCIA) on adult plants. The inoculum for F. verticillioides was produced from the isolate MRIZP 2920, obtained from naturally infected seed at MRIZP Phytopathology experimental station. For SCIA protocol, isolate of F. verticillioides was grown on potato dextrose agar (PDA) plates at 25 ˚C, with a 12-h day-night NUV light cycle for seven days. After three weeks of growth in a liquid medium, the cultures were filtered through cheesecloth to remove mycelium and conidial concentration was adjusted to 1 × 105 conidia ml−1 with sterile water. Suspensions were stored at 4 ˚C for a maximum of three days prior to inoculation.

For the field experiments, a randomized complete block design (RCBD) with two replications was used. Each inbred line was presented with 20 plants within a row, while hills, i.e. rows were 0.4 m, i.e. 0.7 m apart, respectively (2 plants per hill). Plants were hand-pollinated and SCIA was applied at 3 days after silking (treatment). For inoculation, 2 ml of conidial suspension was injected into the silk channel of each primary ear (Reid et al., 1996). For control plants, sterile water was used as inoculum.

Ears were manually harvested and hand de-husked. The severity of ear rot symptoms was evaluated using rating scales based on the percentage of kernels with visible symptoms of infection as follows:

��������� ������ �%� � No. of rotten kernels ear��Total No. kernels ear�� x 100

The visual rating scale consists of 7 classes, based on percentage of visibly infected

kernels (Disease Severity Rating - DSR: 1 = 0% - no infection; 2 = 1–3%; 3 = 4–10%; 4 = 11–25%; 5 = 26–50%, 6 = 51–75%; 7 =76–100%). Results and discussion

Several lines of evidence indicated that drought stress was associated with elevated levels of F. verticillioides infection and fumonisin accumulation in kernels (Miller 2001). The optimum temperature for F. verticillioides has been reported as about 30 ˚C (Reid et al. 1999). Since Fusarium ear rot is generally favored by warm, dry weather during the grain-filling period (Marín et al. 1999), year 2013 could be considered as sufficiently relevant for screening for ear rot resistance. Namely, high air temperatures characterized flowering (June-July) and grain filing (July-August), e.g. eight days in June and 14 days in July were with the maximal temperatures above 35 ˚C, i.e. above 37 ˚C, respectively, while 16 days in August were with the maximal temperatures above 37 ˚C (Fig 1).

Since disease severity after natural infection varies strongly from year to year, from location to location and with the duration of the vegetation period, artificial inoculation method was used in the present study. Its advantages reflect in much higher disease severity and results in more uniform ratings that make genotypic comparisons easier and reproducible especially when phenotyping for QTLs-(Quantitative Trait Loci, Oberforster and Felder 2010). However, the genotypes selected under artificial infection pressure must demonstrate their superior resistance under natural infection pressures.

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Fig 1. The average monthly temperatures (˚C) and total precipitations (mm) during vegetative period in 2013 at Zemun Polje, Serbia (URL: http://www.accuweather.com)

For obtaining a sufficient level of infection to define genotypic differences in resistance, but not so severe that are hard to observe, choise of an inoculation technique is important. Lemmens (2010) observed a close correlation between the results of the silk channel method and natural infection (r = 0.75-0.96). These findings support the view that artificial and natural infection data tend to be closely correlated, indicating that some form of complex resistance to different Fusarium spp. exists.

Conidia have been shown to be as good infection materials as mycelium and in fact, in natural infections, conidia are often the source of infection. The amount of inoculum used for silk or silk channel resistance can vary; in this study was used 2 ml, as in studies of Reid et al. (1996) and Presello et al. (2008). The conidium concentration for silk channel inoculation, which appears to be more important for the entrance of F. verticillioides, also varies: Presello et al. (2008) and Löffler et al. (2010) used 1 x 106 conidia ml-1 for FER, whereas in our study was used lower concentration of conidial suspension.

For understanding disease severity and consequently resistance differences among chosen inbred lines differing in drought tolerance, the optimum timing of inoculation is of great importance. In numerous studies has been shown that silk inoculation performed at earlier ear developmental stage (2-3 days after mid-silking, as was the case in our study), led to very high infection severities, while later inoculations (10 days or more) resulted in little or no infection (Reid et al., 2002). Bush et al. (2004) indicated that FER symptoms reach a maximum 7 weeks after pollination and that toxin levels reach a maximum at 9 weeks. The later is therefore an optimum harvest time for ear rot evaluations.

In this study, symptoms of FER from F. verticillioides infection occured mainly on individual kernels or on limited areas of the ear. Also, in some ears, many independent infection sites were developed. Infected kernels developed a cottony growth and white streaks on the pericarp and fungal growth on the cob.

Considerable debate exists whether or not toxin evaluations should also be used to evaluate disease resistance. In many cases, the extent of toxin contamination is proportional to the visual severity of infection; however, asymptomatic kernels may also be infected and may contain toxins, usually in trace amounts (Reid et al., 2009). Although for F. verticillioides significant differences in amount of toxins can be detected at the same visual severity rating

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(Butron et al., 2006), artificial inoculation followed by selection on the basis of visual disease severity rating is sufficient during the inbred selection process.

Considering environmental conditions, F. verticillioides was found to have different impact on the yield. Thus, results obtained from the evaluation of visual severity of ear rot induced by F. verticillioides among the tested inbred lines, indicated high influence of drought tolerance in maize inbreds from MRIZP gene bank drought tolerant mini-core collection (Table 1). Table 1. Effect of Fusarium verticillioides ear rot on grain yield of investigated maize inbred lines

differing in drought tolerance and disease severity rating (DSR)

Inbred line Yield (g plant-1) Tot. no. kern. ear-1

No. infect. kern. ear-1 DSR

Control Treatment IL 1 85.8 83.0 429.1 14.2 2.6 IL 2 79.3 71.1 317.2 32.8 3.5 IL 3 93.5 90.3 593.9 20.8 2.7 IL 4 63.0 61.6 340.5 7.5 2.0 IL 5 49.4 47.5 290.4 10.7 2.8 IL 6 53.0 47.4 243.6 25.5 3.6 IL 7 70.7 63.2 344.8 36.3 3.4 IL 8 56.8 51.0 291.2 29.7 3.2 IL 9 67.0 64.0 319.2 14.4 3.0

IL 10 52.8 50.3 242.8 11.3 3.1 IL 11 80.4 75.2 335.0 21.6 3.3 IL 12 52.7 46.9 334.5 36.6 3.6 IL 13 52.4 49.8 322.2 15.6 3.1 IL 14 65.8 58.7 333.2 36.2 3.6 IL 15 53.1 51.1 354.2 13.8 2.9 B73 38.1 10.7 171.4 123.5 6.0

Mo17 74.5 36.7 323.7 164.1 5.8 For drought tolerant inbreds, grain yield reduction ranged from 2.0% to 10.95%, whereas

for drought suceptible ones (B73 and Mo17), this reduction was 52.65% and 40.03%, respectively. Although Eller et al. (2008) did not find a relation between ear rot resistance and yield performance, which would support the hypothesis that high resistance and high yield are not mutually exclusive for FER, highly significant and positive correlation (P ≤ 0.001) was found between yield obtained under treatment and visual rate of disease severity (Table 1). Conclusion

In this study, silk channel resistance to F. verticillioides ear rot was evaluated on seventeen maize inbred lines differing in drought tolerance. Results obtained from visual scoring of disease severity had shown medium level of infection in all drought tolerant inbred lines compared to drought susceptible inbreds (B73 and Mo17). However, the lines L1, L3, L4, L5 and L15 displayed the lowest rate of infection, ranging from 2.0 to 2.6. Although further investigations concerning level of toxin contamination and toxin analyses are necessary to be

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conducted, these genotypes could be considered as potential sources of favorable genes for stable resistance to F. verticillioides ear rot under drought conditions. Acknowledgments

This work was supported by Project TR31028 “Exploitation of maize diversity to improve grain quality and drought tolerance” from the Ministry of Education, Science and Technological Development, Republic of Serbia. References Atkinson N.J., Urwin P.E. (2012). The interaction of plant biotic and abiotic stresses: from genes

to the field. J Exp Bot 63: 3523-3543. Bush B.J., Carson L.M., Cubeta M.A., Hagler W.M., Payne G.A. (2004). Infection and

fumonisin production by Fusarium verticillioides in developing maize kernels. Phytopathology 94: 88-93.

Butron A., Santiago R., Mansilla P., Pintos-Varela C., Ordas A., Malvar R.A. (2006). Maize (Zea mays L.) genetic factors for preventing fumonisin contamination. J Agric Food Chem 54: 6113-6117.

Collins N.C., Tardieu F., Tuberosa R. (2008). Quantitative trait loci and crop performance under abiotic stress: where do we stand? Plant Physiol 147: 469-486.

Eller M.S., Robertson-Hoyt L.A., Payne G.A., Holland J.B. (2008). Grain yield and Fusarium ear rot of maize hybrids developed from lines with varying levels of resistance. Maydica 53: 231-237.

Lemmens M. (2010). Fusarium ear rot resistance testing in maize: natural infection versus artificial inoculation. In: Á. Mesterházy (ed.), Workshop for Variety Registration in Cereals for Fusarium Resistance, 18-20. March 2010, Szeged, Hungary. Pp. 23-24.

Löffler M., Kessel B., Ouzunova M., Miedaner T. (2010). Population parameters for resistance to Fusarium graminearum and Fusarium verticillioides ear rot among large sets of early, mid-late and late maturing European maize (Zea mays L.) inbred lines. Theor Appl Genet 120: 1053-1062.

Marín S., Magan N., Belli N., Ramos A.J., Canela R., Sanchis V. (1999). Two-dimensional profiles of fumonisin B1 production by Fusarium moniliforme and Fusarium

proliferatum in relation to environmental factors and potential for modelling toxin formation in maize grain. Int J Food Microbiol 51: 159-167.

Miller J.D. (2001). Factors That Affect the Occurrence of Fumonisin. Environ Health Persp 109: 321-324.

Mittler R. (2006). Abiotic stress, the field environment and stress combination. Trends Plant Sci 11: 15-19.

Munkvold G.P., Carlton, W.M. (1997). Influence of inoculation method on systemic Fusarium

moniliforme infection of maize plants grown from infected seeds. Plant Dis 81: 211-216. Oberforster M., Felder H. (2010). Systems for evaluating the susceptibility of cereal and maize

cultivars to Fusarium in Austria. In: Á. Mesterházy (ed.), Workshop for Variety Registration in Cereals for Fusarium Resistance, March 23-24 2010, Szeged, Hungary. Pp. 31-33.

Payne G.A. (1999). Ear and kernel rots. Pp 44-47. In: White D.G. (Ed.). Compendium of Corn Diseases. 3rd ed. St. Paul, MN: APS Press.

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Presello D.A., Botta G., Iglesias J., Eyhérabide G.H. (2007). Effect of disease severity on yield and grain fumonisin concentration of maize hybrids inoculated with Fusarium

verticillioides. Crop Prot 27: 572-576. Proctor R.H., Plattner R.D., Desjarins A.E., Busman M., Butchko A.E. (2006). Fumonisin

production in the maize pathogen Fusarium verticillioides: Genetic basis of naturally occurring chemical variation. J Agric Food Chem 54: 2424-2430.

Reid L.M., Hamilton R.E., Mather D.E. (1996). Screening maize for resistance to gibberella ear rot. Agriculture and Agri-Food Canada, Ottawa, ON, Pp. 62. Tech Bull Publ 1996-5E.

Reid L.M., Nicol R.W., Ouellet T., Savard M., Miller J.D., Young J.C., Stewart D.W., Schaafsma AW (1999). Interaction of Fusarium graminearum and F. moniliforme in maize ears: Disease progress, fungal biomass, and mycotoxin accumulation. Phytopathology 89: 1028-1037.

Reid L.M., Woldemariam T., Zhu X., Stewart D.W., Schaafsma A.W. (2002). Effect of inoculation time and point of entry on disease severity in Fusarium graminearum, Fusarium verticillioides, or Fusarium subglutinans inoculated maize ears. Can J Plant Pathol 24: 162-167.

Robertson L.A., Kleinschmidt C.E., White D.G., Payne G.A., Maragos C.M., Holland J.B. (2006). Heritabilities and correlations of Fusarium ear rot resistance and fumonisin contamination resistance in two maize populations. Crop Sci 46: 353-361.

Stanković S., Lević J., Ivanović D., Krnjaja V., Stanković G., Tančić S. (2012). Fumonisin B1 and its co-occurrence with other fusariotoxins in naturally-contaminated wheat grain. Food Control 23: 384-388.

Waalwijk C., Koch S. H., Ncube E., Allwood J., Flett B., de Vries I., Kema G.H.J. (2008). Quantitative detection of Fusarium spp. and its correlation with fumonisin content in maize from South African subsistence farmers. World Mycotoxin J 1: 39-47.

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15

YIELD-RELATED TRAITS IN A COLLECTION OF ‘TREŠNJEVAC’ BEANS (Phaseolus vulgaris L. forma versicolor)

Milka BRDAR-JOKANOVIĆ1*, Milan ZDRAVKOVIĆ2, Mirjana VASIĆ1, Aleksandra

SAVIĆ1, Zdenka GIREK2, Jasmina ZDRAVKOVIĆ2

1Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000 Novi Sad, Serbia 2Institute for Vegetable Crops, Karađorđeva 71, 11420 Smederevska Palanka, Serbia

*Corresponding author’s E-mail: milka.brdar@nsseme.com Abstract

In order to facilitate the selection of high-yielding ’trešnjevac’ bean variety adapted to Serbian agro ecological conditions, local populations and a foreign (control) variety have been examined in terms of several traits of agronomic importance. Significant variability was found among the tested accessions and between the two seasons of experiment in terms of all investigated traits. Yield per plant correlated positively to pod weight, seed weight, number of seeds per plant, number of seeds per pod and number of pods per plant. Out of 13 examined populations, six over-yielded check variety in both seasons. The population with high and the most stable yield (P-36, 22.2 g/plant) would be a valuable starting material for breeding ’trešnjevac’ bean variety. Key words: local populations, ’trešnjevac’ bean, yield Introduction

As an important source of nutrients in human diet, dry bean (Phaseolus vulgaris L.) is grown on 25-30 million hectares worldwide, with annual production of 21-23 million tons. It is the third most important food legume crop in the world, following only soybean and peanut. In Serbia dry beans occupy approximately 20 thousand hectares. The average annual production for both pure and interplanted crops is 43 thousand tons, which does not fully meet the needs of the local population.

Although more than twenty high-yielding and high-quality dry bean varieties have been developed at Institute of Field and Vegetable Crops from Novi Sad and Institute for Vegetable Crops from Smederevska Palanka, a significant part of Serbian dry bean crops are still domestic, local populations. This is especially true for beans intended for producer’s own consumption or for selling in local markets. The fact that the producers frequently choose local populations over commercial varieties imply the specific requirements of consumers, but also the possibility that at least a part of this material perform better than commercial varieties in terms of important traits; primarily quality, tolerance to abiotic and biotic stress, and yield. In addition, Serbian

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commercial varieties are mostly white-seeded and seeds of certain types, such as two-colored ’trešnjevac’, are not available on the market. Although seed color and shape do not affect dry bean nutritional composition, they represent basic market characteristic. Therefore, the collected local populations of ’trešnjevac’ should be evaluated in field trials in order to distinguish the superior ones that could be included in breeding programs (Vasić, 2004; Gonzáles et al., 2006; Vasić et al., 2007; Stat. Yearb. Serb., 2012; Kahraman and Önder, 2013; FAOSTAT, 2014).

The purpose of this study was to investigate the collection of ’trešnjevac’ dry bean local populations in terms of yield and several traits of agronomic importance. The results should facilitate the selection of high-yielding variety adapted to Serbian agro ecological conditions. Material and methods

This study comprised 13 ’trešnjevac’ type dry bean local populations (P-2, P-11, P-18, P-19, P-20, P-29, P-34, P-36, P-62, P-69 and P-92 collected in Vojvodina, P-86 from south Serbia and P-93 from Bosnia) and one variety (P-74, Butmirski trešnjo, old cultivar from Bosnia and Herzegovina). The accessions are part of the joint dry bean collection founded by Institute for Vegetable Crops, Smederevska Palanka and Institute of Field and Vegetable Crops, Novi Sad.

The trial was conducted during two growing seasons (2010, 2011) at the experimental field of the Institute for Vegetable Crops, Smederevska Palanka, Serbia (44o 22’ N, 20o 57’ E, elevation 121 m). The soil type is vertisol. The trial was set in complete randomized blocks, with three replications. The main plot consisted of three 1 m long rows; with intra and interspacing of 5 and 70 cm, respectively. Sowing and harvest were performed at regular dates. The official weather data (Republic Hidrometeorological Service of Serbia) covering the two seasons are given in Table 1. Table 1. Weather data for dry bean growing seasons (April – July) of 2010, 2011 and the corresponding

long-term averages (Smederevska Palanka, Serbia)

Parameter 2010 2011 2004-2013 Average daily temperature (oC) 18.1 18.2 18.4 Minimum temperature (oC) 1.0 0.1 -3.4 Maximum temperature (oC) 34.2 38.7 44.9 Sum of temperatures (oC) 2208.6 2223.3 2245.3 Sum of precipitation (mm) 373.6 197.6 251.8

The following traits were analyzed: yield per plant (g), plant height (cm), the first pod height (cm), weight of vegetative plant parts (g), pod weight (without seeds, g), 1000 seed weight (g), number of seeds per plant, number of seeds per pod and number of pods per plant.

Data were processed by analysis of variance. Basic statistic parameters (mean, minimum, maximum values and coefficients of variation), as well as Pearson’s correlation coefficients were calculated. The calculations were performed using Statistica 12 software package (StatSoft, Tulsa, OK, USA). Results and discussion

Statistically significant differences in terms of all the investigated traits were found among the tested ’trešnjevac’ dry bean accessions (Table 2). The highest variation was noted for pod weight and yield per plant (54.2 and 48.6%, respectively), while the first pod height, number of seeds per pod, plant height and 1000 seed weight varied in significantly lower range (19.9, 23.4, 23.9 and 26.8%, respectively). Comparatively low coefficients of variation were reported for the

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same traits investigated in a three-year field trial comprising collection of 129 dry bean accessions of different types (Vasić, 2004). Table 2. Basic statistic parameters and LSD test for the investigated traits in fourteen ’trešnjevac’ dry

bean accessions, two-year averages. CV – coefficient of variation

Trait Mean Min Max CV (%) LSD0.05 LSD0.01 Yield per plant (g) 15.3 7.3 23.0 48.6 5.3 7.4 Plant height (cm) 49.0 38.0 71.1 23.9 6.9 9.6 The first pod height (cm) 22.4 18.3 26.2 19.9 5.4 7.6 Weight of vegetative plant parts (g) 8.1 4.7 14.3 44.3 4.3 5.9 Pod weight (g) 5.2 3.1 8.8 54.2 3.9 5.4 1000 seed weight (g) 404.0 302.8 503.8 26.8 82.9 115.0 Number of seeds per plant 37.2 19.5 49.2 33.8 8.3 11.4 Number of seeds per pod 3.6 2.0 4.3 23.4 1.8 2.5 Number of pods per plant 10.3 8.5 13.1 31.3 3.7 5.2 Table 3. Pearson’s coefficients of correlation for the investigated ’trešnjevac’ dry bean traits

Traits (1) (2) (3) (4) (5) (6) (7) (8) (9) Yield per plant (1) 1.00 0.32 -0.29 0.16 0.86** 0.77** 0.88** 0.66** 0.84** Plant height (2) 1.00 0.24 0.51** 0.30 0.09 0.22 -0.03 0.47* First pod height (3) 1.00 0.31 -0.22 -0.43* -0.36 -0.41* -0.32 Weight of veg. parts (4) 1.00 0.38* 0.04 -0.08 -0.25 0.25 Pod weight (5) 1.00 0.72** 0.64** 0.50** 0.82** 1000 seed weight (6) 1.00 0.52** 0.48** 0.61** No of seeds per plant (7) 1.00 0.68** 0.80** No of seeds per pod (8) 1.00 0.52** No of pods per plant (9) 1.00

As for the mean values of yield per plant and traits directly related to yield (1000 seed weight, number of seeds per plant, number of pods per plant), the analyzed accessions performed better than the mentioned collection, probably due to the fact that the latter consisted of a large number of accessions differing in seed coat color, shape and chemical composition, growth habit etc. Plant height was in the range of most widely grown Serbian cultivars (Vasić et al., 1999), while the first pod height mean value of 22.4 cm meets the requirements of mechanized harvesting (Zdravković et al., 2005). Number of seeds per pod (3.6) was not significantly different from the value of 3.2 reported by Vasić (2004), implying high stability of the trait.

In addition, Pearson’s correlation coefficients were calculated among the investigated traits in order to analyze their interrelationships and to identify those with the strongest effects on yield (Table 3). Positive correlations of yield per plant and number of seeds per plant, number of seeds per pod and number and weight of pods per plant are in accordance to the results of Vasić (2004). However, the yields of ’trešnjevac’ beans analyzed in this study, as well as the yield of commercial Serbian varieties in general (Vasić et al., 1997) correlate positively to 1000 seed weight, whereas this correlation was negative in the study comprising large number of accessions differing in morphology and origin. According to Vasić (2004), the discrepancy is due to the requirements of the local market which demands high-yielding varieties with large seeds. In addition, the interrelationships among the mentioned traits were generally positive. Yield per plant was not significantly related to plant and the first pod height, implying the possibility for

breeding high-yielding ’trešnjevac’ varieties of different growth types. However, the first pod height correlated negatively to the majority of the traits directly related to yield, which should be taken into account in further research. In anegatively affected by both first pod and plant height.

Figure 1. The average effect of the season on the investigated ’trešnjevac’ dry bean traits

The average effect of the seasons in which the experiment has been conducted on the investigated traits is depicted in Figure 1. The analyzed ’trešnjevac’ beans performed significantly better in 2010 than in 2011 growing season in terms of all investigatedthe first pod height which was slightly increased in the unfavorable season. Since the average daily temperature and sum of temperatures in both 2010 and 2011 fell in the range of longaverages (Table 1), the poor performance in 2011 iprecipitation. In addition, maximum daily temperatures were 4.5 than in the favorable season. The highest seasonal effect was noted for pod weight, yield per plant and number of pods per plant, other traits was also reported by Shenkut and Brick (2003).

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yielding ’trešnjevac’ varieties of different growth types. However, the first pod height correlated negatively to the majority of the traits directly related to yield, which should be taken into account in further research. In a study performed by Önder et al. (2013) yield was negatively affected by both first pod and plant height.

Figure 1. The average effect of the season on the investigated ’trešnjevac’ dry bean traits

The average effect of the seasons in which the experiment has been conducted on the investigated traits is depicted in Figure 1. The analyzed ’trešnjevac’ beans performed significantly better in 2010 than in 2011 growing season in terms of all investigatedthe first pod height which was slightly increased in the unfavorable season. Since the average daily temperature and sum of temperatures in both 2010 and 2011 fell in the range of longaverages (Table 1), the poor performance in 2011 is probably due to the low sum of precipitation. In addition, maximum daily temperatures were 4.5 oC higher in the unfavorable than in the favorable season. The highest seasonal effect was noted for pod weight, yield per plant and number of pods per plant, respectively. Seasonal variation in dry bean yield and several other traits was also reported by Shenkut and Brick (2003).

yielding ’trešnjevac’ varieties of different growth types. However, the first pod height correlated negatively to the majority of the traits directly related to yield, which should be

. (2013) yield was

Figure 1. The average effect of the season on the investigated ’trešnjevac’ dry bean traits

The average effect of the seasons in which the experiment has been conducted on the investigated traits is depicted in Figure 1. The analyzed ’trešnjevac’ beans performed significantly better in 2010 than in 2011 growing season in terms of all investigated traits, except the first pod height which was slightly increased in the unfavorable season. Since the average daily temperature and sum of temperatures in both 2010 and 2011 fell in the range of long-term

s probably due to the low sum of C higher in the unfavorable

than in the favorable season. The highest seasonal effect was noted for pod weight, yield per respectively. Seasonal variation in dry bean yield and several

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Figure 2. Yield per plant (g) in the analyzed ’trešnjevac’ dry bean accessions

Yield per plant of the particular accessions grown in the two seasons of the experiment is shown in Figure 2. Out of thirteen examined populations, six (P-11, P-18, P-19, P-29, P-36 and P-69) over-yielded check variety (P-74, Butmirski trešnjo), implying wide possibilities for developing new ’trešnjevac’ cultivar adapted to local environment. The highest average yields are noted for accessions P-29, P-19 and P-36 (23.0, 22.3 and 22.2 g/plant, respectively). However, the yield of the agricultural plants should be both high and stable across environments, therefore only P-36 is recommended for further research. Since the two seasons of the investigation differed in terms of precipitation, yield stability of the particular accessions might be used as a starting point for breeding for drought tolerance. Conclusions

Significant variability was found among the tested fourteen ’trešnjevac’ dry bean accessions and between the two seasons of experiment in terms of all investigated traits. Yield per plant correlated positively to pod weight, seed weight, number of seeds per plant, number of seeds per pod and number of pods per plant. Six populations over-yielded check variety, implying the possibility for developing new ’trešnjevac’ dry bean cultivar. The population with high and the most stable yield (P-36, 22.2 g/plant) would be a valuable starting material for further research. Yield stability of the analyzed accessions might be considered in breeding for drought tolerance. Acknowledgement

This study was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Research Grant: TR-31059). References FAOSTAT (2014). http://faostat.fao.org González, A.M., Monteagudo, A.B., Casquero, P.A., De Ron, A.M., Santalla, M. (2006). Genetic

variation and environmental effects on agronomical and commercial quality traits in the main European market classes of dry bean. Field Crops Research, 95 (2-3), 336-347.

Kahraman, A., Önder, M. (2013). Correlations between seed color and nutritional composition of dry bean. Ratarstvo i povrtarstvo, 50 (2), 8-13.

Önder, M., Kahraman, A., Ceyhan, E. (2013). Correlation and path analysis for yield and yield components in common bean genotypes (Phaseolus vulgaris L.). Ratarstvo i povrtarstvo, 50 (2), 14-19.

Shenkut, A.A., Brick, M.A. (2003). Traits associated with dry edible bean (Phaseolus vulgaris L.) productivity under diverse soil moisture environments. Euphytica, 133 (3), 339-347.

Statistical office of the Republic of Serbia (2012). Statistical yearbook of the Republic of Serbia. Statistical office of the Republic of Serbia, Belgrade.

Vasić, M., Gvozdanović-Varga, J., Červenski, J. (1997). The interdependence of morphological characters in Yugoslavian bean varieties (Phaseolus vulgaris L.). Acta Horticulturae, 462, 235-239.

Vasić, M., Gvozdanović-Varga, J., Červenski, J. (1999). Varijabilnost i heritabilnost komponenti visine biljke kod domaćih sorti pasulja (Phaseolus vulgaris L.). Zbornik apstrakata II Kongresa genetičara Srbije, Sokobanja, Društvo genetičara Srbije, 129-130.

Vasić, M. (2004). Genetička divergentnost pasulja. Zadužbina Andrejević, Beograd.

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Vasić, M., Milić, S., Pejić, B., Gvozdanović-Varga, J., Maksimović, L., Bošnjak, D. (2007). Mogućnost postrne proizvodnje pasulja (Phaseolus vulgaris L.) u agroekološkim uslovima Vojvodine. Zbornik radova Instituta za ratarstvo i povrtarstvo, 43 (1), 283-291.

Zdravković, M., Zdravković, J., Stanković, Lj., Pavlović, N. (2005). Combining abilities of inheriting first pod height of some french bean lines (Phaseolus vulgaris L.). Genetika, 37 (1), 65-70.

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MODE OF INHERITANCE OF KERNEL ROW NUMBER OF ZP MAIZE MATERIAL

Zoran ČAMDŽIJA1*, Milomir FILIPOVIĆ1, Slaven PRODANOVIĆ2, Sofija BOŽINOVIĆ1, Milan STEVANOVIĆ1, Jovan PAVLOV1 and Nikola GRČIĆ1

1 Maize Research Institute, “Zemun Polje”, Slobodana Bajića 1, 11185 Belgrade, Serbia

*e-mail: zcamdzija@mrizp.rs 2 University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade-Zemun, Serbia

Abstract

Seventeen inbreds (fifteen mothers and two testers) and their 30 hybrids were used in this study in order to evaluate mode of inheritance of kernel row number (KRN). Mothers derive from three sources A, B and C (five inbreds per source), for which is known to be opposite heterotic group to two Lancaster Sure Crop testers (Z1 and Z2). Average KRN of parents ranged from 11.90 to 18.99, and hybrids from 13.93 to 18.97. LSD0.05 distiguished five inbreds with a highest value of KRN: B1 (18.27), B2 (18.70), B3 (18.80), B4 (18.99) and C4 (18.58). Exept from C4, all other inbreds menaged to create hybrids with a highest value of KRN, but only with Z1 tester, B1 x Z1 (18.97), B2 x Z1(18.0), B3 x Z1 (18.97) and B4 x Z1 (18.15). Concerning the mode of inheritance, (+) dominance was most frequent way (10 cases), folowed by intermediar (9 cases) and (+) superdominance (7 cases). Mothers crossed to Z1 tester predominately had (+) dominance and (+) superdominance as mode of inheritance, while crossed to tester Z2 intermediar way. Superdominance which resulted in high KRN, as most desired case, happened only once with C3 x Z1 (18.65), while other superdominance cases had significantly lower KRN.

Key words: kernel row number, heterosis, mode of inheritance

Introduction

Grain yield represents ultimate goal of maize breeder, and is achieved by creating high yielding hybrids. Since F1 maize hybrids represent crosses, made by cross-fertilisation of two or rarely three inbred lines, emphasis is put on parent components and their good performance per se, as well as their good combining abilities. Considering the fact that grain yield can be defined as multidimensional phenomena which comprises several different characteristics and which is influenced by number of factors (Prodanović et al., 1996), breeding program is complexed work.

Mode of inheritance and heterosis of grain yield and yield components has been for long time a main subject of maize research. Alongside plant height, upper ear height, ear lenght number of grains per row and others, kernel row number (KRN) is one of the important grain yield components. KRN as yield component icreased throught time, from Teosinte ssp. which had two, modern varietes have 8-20 KRN (Bommert et al. 2013).

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Earlier maize hybrids had lower KRN in comparing to new ones. Smaller plants, medium-sized ears, greater kernel depth and greater number of KRN (14-20) allowed higher planting densities which all resulted in increasment of grain yield (Troyer, 1999). Kernel-row number is determined in relatively early stages of plant ontogeny and is less affected by environmental conditions at the time of flowering and from flowering to maturity, and this trait has high average heritability estimate (57.0%) (Hallauer et al. 2010).

In order to obtain high heterosis of grain yield and quantitative traits, a maize breeder should follow heterotic pattern. A heterotic pattern is the cross between known genotypes that expresses a high level of heterosis. For example, it is known that inbred lines derived from Iowa Stiff Stalk Synthetic cross best with inbred lines derived from Lancaster Sure Crop in order to obtain high heterosis (Carena and Hallauer, 2001). Mode of inheritance of KRN is usually intermediate, ie. additive variance has greater influence on inheritance of this trait. Such results were found by Živanović et al. (2007), Sečanski et al. (2005) and Srdić et al. (2011). Boćanski et al., (2001 and 2002), on the other hand, found mode of inheritance for KRN to be superdomination, intermediate and one hybrid combination showed same KRN as parents.

The aim of this study was to determinate mode of inheritance of KRN of fiveteen latest generation inbred lines originating from maize breeding program in Maize Research Institute „Zemun Polje“.

Material and methods

Seventeen inbred lines (fifteen mothers and two testers) and their 30 hybrids were used in this study in order to evaluate mode of inheritance of KRN. Fifteen mothers derive from three sources A, B and C, for which is known to be opposite heterotic group of Lancaster Sure Crop. Inbreds from source A (A1, A2, A3, A4 and A5) derive from self-pollination of hybrid, and are representatives of maturity group FAO 600. Inbreds from source B (B1, B2, B3, B4 and B5) derive from F2 population, created by crossing two public inbred lines B 84 and B 14, and are representatives of maturity group FAO 500. Inbreds from source C (C1, C2, C3, C4 and C5) derive from crossing public inbred line B 14 and maize germplazm from Zambia, and are representatives of maturity group FAO 400. The trial was set in year 2010 on three locations Zemun Polje, Školsko dobro and Srbobran in two repetitions in randomised block design. Hybrids were sawn near to the parents in order to share same agroecological conditions. Kernel row number was calculated as average value of 10 randomly selected plants per repetiotion.

Calculations were done in MSTAT-C software (MSTAT, 1989), using average values of all three locations. Determination of mode of inheritance (MOI) was done using statistics presented by Šurlan et al. (2007). MOI was defined as intermediar (im), partial domination of better parent ((+) pd), domination of better parent ((+) d) and superdomination ((+) sd). In case that parents value of KRN was not statisticaly different, it was unable to determine mode of inheritance and sign (-) was used. Heterosis for KRN was calculated over better parent (BPH) for 2010th year according to the formula:

H � F1 – BPBP ) 100

where: F1 – average value of KRN of F1 hybrid BP – average value of KRN of better parent

23

The significance of calculated heterosis values was tested by t-test according to formula given by Wynne et al. (1970):

t � F1 * BP�+* √MSe

where: MSe - Error mean square Results and disscusion

KRN values of parent components are shown in Table 1. Average KRN for all components was 15.70, KRN of mothers ranged from 12.23 (C2) to 18.99 (B4), while fathers had 14.15 (Z1) and 11.90 (Z2) respectivelly. Based on the LSD0.05 value, inbreds B1, B2, B3, B4 and C4 proved to be the highest KRN inbreds with average values just over 18. On the other hand A3 and C2 had average KRN value just over 12. B source proved to be the source with highest KRN with values above 18, except inbred B5 (15.70). Table. 1 Values of KRN

1 of parental components

Genotype KRN Genotype KRN Genotype KRN Genotype KRN A1 15.70 cd B1 18.27 a C1 13.63 ef Z1 14.15 e A2 16.52 bc B2 18.70 a C2 12.23 gh Z2 11.90 h A3 12.93 fg B3 18.80 a C3 17.05 b / / A4 15.17 d B4 18.99 a C4 18.58 a / / A5 15.17 d B5 15.70 cd C5 13.52 ef / / 1 KRN – kernel row number; Average KRN =15.70; LSD 0.05 = 0.85; CV=4.65%

Results of KRN of hybrids, BPH and MOI are presented in Table 2. KRN of all hybrids

was 15.90. Based on the LSD0.05, hybrids B1 × Z1 (18.97), B2 × Z1 (18.00), B3 × Z1 (18.97), B4 × Z1 (18.15), C3 × Z1 (18.65) had the highest average KRN values. All mentioned hybrids were made by crossing with Z1 tester. Except C5 × Z1 with KRN 14.97, the rest of the hybrids with the lowest average of 14 KRN were made by crossing mother with Z2 tester: A1 × Z2, A3 × Z2, A4 × Z2, A5 × Z2, B5 × Z2, C1 × Z2, C2 × Z2 and C5 × Z2. B source inbred lines, which per se had the highest average KRN, also created hybrids with highest value of KRN, proving to be the best source for desired trait.

Better parent heterosis showed values from -20,53** (B3 x Z2) to 18,38** (C2 × Z2). Besides C2 × Z2, five more significant and highly significant values of heterosis were obtained: A3 × Z1 (16,12**), C1 × Z1 (9,24*), C2 × Z1 (13,52**), C3 × Z1 (10,27**) and A3 × Z2 (11,01*), showing high heterosis for observed trait. On the contrary, nine combinations showed significant and highly significant negative BPH. Sush results are in contrast to Aghaei et al. (2012), who reported predominately negative values for BPH for KRN. High heterosis, that was obtained within six hybrid combinations, represents predominant role of non-additive genes in control of inheritance of certain trait, which is in accordance with results of Sečanski et al., (2005). Nine hybrids had significant and highly significant negative heterosis in comparing to parent with higher KRN.

24

Table 2. KRN1 of hybrids, BPH (%)

2 and MOI

3

Hybrid KRN BPH MOI Hybrid KRN BPH MOI A1 × Z1 16.27 cdefg 3,80 (+) d A1 × Z2 13.97 n -12,56** im A2 × Z1 16.93 c 2,67 (+) d A2 × Z2 15.23 hijkl -9,08* (+) pd A3 × Z1 16.33 cdef 16,12** (+) sd A3 × Z2 14.3 lmn 11,01* - A4 × Z1 16.30 cdef 7,73 (+) sd A4 × Z2 14.83 jklmn -2,46 (+) d A5 × Z1 15.89 defghi 4,91 (+) d A5 × Z2 13.93 n -9,14* im B1 × Z1 18.97 a 4,32 (+) d B1 × Z2 16.10 cdefgh -14,36** im B2 × Z1 18.00 b -4,26 (+) d B2 × Z2 15.70 efghij -19,61** im B3 × Z1 18.97 a 1,01 (+) d B3 × Z2 15.65 efghij -20,53** im B4 × Z1 18.15 ab -5,07 (+) pd B4 × Z2 15.33 ghijk -23,67** im B5 × Z1 16.73 cd 6,91 (+) sd B5 × Z2 14.23 mn -10,63* im C1 × Z1 15.43 fghij 9,24* (+) sd C1 × Z2 13.93 n 2,35 (+) d C2 × Z1 15.93 defgh 13,52** (+) sd C2 × Z2 14.45 klmn 18,38** (+) sd C3 × Z1 18.65 ab 10,27** (+) sd C3 × Z2 16.3 cdef -5,17 (+) d C4 × Z1 16.58 cde -12,22** im C4 × Z2 15.67 efghij -19,09** im C5 × Z1 14.97 ijklm 5,90 - C5 × Z2 14.03 mn 4,03 (+) d 1 KRN-Kernel row number; 2 BPH-Better parent heterosis; 3 MOI- Mode of inheritance; Average KRN =15.90; LSD 0.05 = 0.95; Average BPH=-1.52; CV=5.18%

Concerning the mode of inheritance, (+) dominance was most frequent way (10 cases),

folowed by intermediar (9 cases) and (+) superdominance (7 cases). Mothers crossed to Z1 tester predominately had (+) dominance and (+) superdominance as mode of inheritance, while crossed with tester Z2 intermediar way. Superdominance which resulted in high KRN, as most desired case, happened only once with C3 x Z1 (18.65), while other superdominance cases had significantly lower KRN. Boćanski et al. (2001) in their resarch, also found intermediar, dominance and superdominance to be the mode of inheritance of KRN, but obtained results were contradictory with Živanović et al. (2007), Sečanski et al. (2005) and Srdić et al. (2011), who found intermediar way to be the most frequent mode of inheritance.

Conclusion

Increasing number of grains per area unit represents leading cause of increasing the grain yield itself. Therefore, superdominance as mode of inheritance of KRN is most desired case, since the value of KRN in that case would overcome values of KRN of parents from which hybrid was created. In examined material, such mode happened only once, reaching value just over 18. Dominance, on the other hand was the most frequent mode of inheritance of KRN in examined material. Acknowledgments

This research was supported by the Ministry of education, science and technological development of Republic of Serbia through the Project TR31068. References Aghaei Sh., Aharizad S., Shiri M. R., Mohammadi S. A. (2012): Average heterosis of maize

hybrids under terminal water stress at Moghan region. Annals of Biological Research, 3 (12): 5462-5465

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Boćanski, J., Petrović Z., Milić D. (2001): Međusobna povezanost i nasleđivanje broja redova, mase 100 zrna i prinosa zrna kukuruza (Zea mays L.). Zbornik radova Instituta za ratarstvo i povrtarstvo, (35), 113-120.

Boćanski, J., Petrović Z., Vasić N., Šeremešić S. (2002): Kvantitativno ispitivanje načina nasleđivanja nekih komponenti prinosa kukuruza - Zea mays L.. Zbornik radova Instituta za ratarstvo i povrtarstvo, (36), 327-335.

Bommert P., Nagasawa N. S., Jackson D. (2013): Quantitative variation in maize kernel row number is controlled by the FASCIATED EAR2 locus. Nature Genetics 45, 334–337.

Carena, M. J., Hallauer A. R. (2001): Expression of heterosis in Leaming and Midland Corn Belt dent populations. J. Iowa Acad. Sci. 108: 73–8.

Hallauer A. R., Carena M. J., Miranda Filho J. B. (2010): Quantitative genetics in maize breeding. Springer New York, 1-655 .

MSTAT Development Team (1989): MSTAT-C: A microcomputer program for the design, management and analysis of agronomic research experiments, Michigan State University, East Lansing.

Prodanović S., Sabljarević V., Surlan-Momirović G., Zorić D., Perović D., Živanović T. (1996): Genetic values of yield components and protein content in F1 generation of maize (Zea mays L.) hybrids. Abstracts, EUCARPIA, XVIIth Conference on genetics, biotechnology and breeding of maize and sorghum. Thessaloniki, Greece, 20-25.10. p.126.

Sečanski M., Živanović T., Todorović G. (2005): Komponente genetičke varijabilnosti i heritabilnost broja redova zrna silažnog kukuruza. Biotechnology in Animal Husbandry 21(1-2): 109-121.

Srdić J., Pajić Z., Filipović M., Babić M., Sečanski M. (2011). Nasleđivanje prinosa klipa i njegovih komponenti kod kukuruza šećerca (Zea mays L. saccharat). Genetika 43(2): 341-348.

Šurčan-Momirović G., Rakonjac V., Prodanović S., Živanović T. (2007): Genetika i oplemenjivanje biljaka-Praktikum. Poljoprivredni fakultet Beograd-Zemun.

Troyer, A.F.(1999): Background of U.S. hybrid corn. Crop Sci., 39: 621-626. Wynne J. C., Emery D. A., Rice P. H. (1970): Combining ability estimation in Arachis hypogaea

L. II. Field performance of F1 hybrids. Crop Sci., 10: 713-715. Živanović T., Sečanski M., Filipović M. (2007): Kombinacione sposobnosti za broj redova zrna

silažnog kukuruza. Selekcija i semenarstvo 13(3-4): 13-1

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27

THE EFFECT OF SOME AGRONOMIC TRAITS ON GRAIN YIELD OF SMALL GRAIN CROPS

Nebojša DELETIĆ, Slaviša STOJKOVIĆ, Slaviša GUDŽIĆ, Miroljub AKSIĆ, Miodrag JELIĆ

Faculty of Agriculture Kosovska Mitrovica – Lešak, Kopaonička street bb, 28888 Lešak, Serbia,

nebojsa.deletic@pr.ac.rs Abstract

This paper presents the results of a study dealing with individual and joint effect of plant height, spike length and 1000 grain weight, on grain yield of representative Serbian cultivars of winter wheat, winter barley, triticale and rye. The trials have been set at two locations (Kraljevo and Zaječar, Serbia), where four cultivars of winter wheat, four of winter barley, two of triticale, as well as one cultivar of rye, were grown in five fertilization and liming variants. Trials were set in split-plot design. Multiple regression analysis of individual and joint effect of the studied parameters on wheat grain yield showed that plant height (β=0.639***) and 1000 grain weight (β=0.322**) had significant effect on grain yield, while the effect of spike length was not significant. Grain yield of barley was under significant influence of the all three studied traits at the level of significance P<0.01 (β=0.407**, β=0.448** and β=0.477**, respectively). In triticale, significant effect was shown by plant height (β=1.146**) and 1000 grain weight (β=0.446**). Grain yield of rye was significantly affected only by 1000 grain weight (β=0.768**). Key words: wheat, barley, triticale, rye, grain yield, multiple regression. Introduction

Grain yield is a complex trait of outstanding economic significance, dependent upon a number of hereditarily determined traits and environmental conditions in which plant is developing (Madić et al., 2005). Therefore, contribution of various plant traits to grain yield is a permanent subject of studies in plant breeding.

Plant height is an important agronomic trait for morphogenesis and grain yield formation in wheat. An appropriate plant height is a prerequisite for attaining the desired yield in wheat breeding programs. The introduction of dwarfing genes into plants has achieved tremendous increase in wheat grain yield during the “Green Revolution” (Peter, 2003). The same can also be applied to the other small grains. Therefore, it is essential to elucidate the genetic basis of plant height in order to gain further increase of grain yield (Cui et al., 2011). Deletić et al. (2013)

28

found significant individual effect of this trait on grain yield of barley and triticale, as well as the joint effect with other yield components.

Spike length is one of the important yield components, not only because longer spike offers more room for spikelets, but also because it is the source of assimilates closest to grains. Spike structure is more effective in utilizing illumination than the other parts of the plant, and it also will stay green and functional for a longer time. Because of these features, it contributes up to 20-30% of the dry matter accumulated in grains (Sharma et al., 2003).

Grain weight is also one of the crucial yield components. In fact grain yield is the product of productive tiller number per square unit, number of grains per spike and 1000 grain weight. Saleem et al. (2006) found strong, significant genotypic and phenotypic correlation between grain yield and 1000 grain weight in several small grains.

This paper presents the results of a study dealing with individual and joint effect of plant height, spike length and 1000 grain weight on grain yield of representative Serbian cultivars of winter wheat, winter barley, triticale and rye. Material and methods

The trials have been set at two locations (Kraljevo and Zaječar, Serbia), where four cultivars of winter wheat (Pobeda, Planeta, ZA-75, Nora) and winter barley (Jagodinac, Rekord, Premium, Kristal), two cultivars of triticale (KG-20, Tango), as well as one cultivar of rye (Raša), were grown in five fertilization and liming variants (1. unfertilized control; 2. N120P80K53; 3. N120P160K53; 4. N120P80K53 + 5 t/ha CaCO3; 5. N120P80K53 + 5 t/ha CaCO3 + 20 t/ha of manure). Trials were set in split-plot design, with three replications. Individual and joint effect of those three traits on grain yield was studied by multiple regression analysis (Hill and Lewicki, 2007). The input data were for the all cultivars and fertilization variants from both locations, in order to get a better estimate of the part of grain yield variation explained by the investigated agronomic traits. Results and discussion

Multiple regression analysis of individual and joint effect of the studied parameters on wheat grain yield (tab. 1) showed that plant height (β=0.639***) and 1000 grain weight (β=0.322**) had significant effect on grain yield, while the effect of spike length was not significant. Adjusted R2 value (0.829) showed that 82.9% of the observed variation in wheat grain yield was explained by the studied three traits. F test for goodness of fit was significant at the level of P<0.001. Significant effect of 1000 grain weight (at P<0.001) on wheat grain yield was previously reported by Deletić et al. (2012), based on the investigation of twenty wheat cultivars. Table 1. Effects of the observed traits on wheat genotypes’ grain yield calculated by multiple regression

analysis. β SE (β) B SE (B) t (d.f. 36) P value

Intercept -- -- -9457.77 1279.250 -7.39321 0.000000

Plant height 0.639024 0.099731 120.61 18.824 6.40750 0.000000 Spike length 0.042695 0.069783 75.62 123.601 0.61183 0.544498 1000 grain weight 0.321563 0.102351 102.56 32.643 3.14175 0.003352

R=0.918; R2=0.842; adjusted R2=0.829; goodness of fit: F(3, 36)=64.174; P<0.00000

29

Table 2. Effects of the observed traits on barley genotypes’ grain yield calculated by multiple regression

analysis.

β SE (β) B SE (B) t (d.f. 36) P value

Intercept -- -- -3311.94 1346.552 -2.45957 0.018846 Plant height 0.407320 0.111756 57.84 15.870 3.64472 0.000838 Spike length 0.448350 0.095903 891.07 190.602 4.67504 0.000040 1000 grain weight 0.476790 0.107708 86.45 19.528 4.42670 0.000085

R=0.913; R2=0.833; adjusted R2=0.820; goodness of fit: F(3, 36)=60.030; P<0.00000 Grain yield of barley (tab. 2) was under significant influence of the all three studied traits

at the level of significance P<0.01 (β=0.407**, β=0.448** and β=0.477**, respectively). Adjusted R2 value was 0.820 and F test for goodness of fit was significant at the level of P<0.001. In another trial, Deletić et al. (2013) did not found significant influence of plant height and spike length on barley grain yield. Table 3. Effects of the observed traits on triticale genotypes’ grain yield calculated by multiple regression

analysis.

β SE (β) B SE (B) t (d.f. 16) P value

Intercept -- -- -2703.81 834.5438 -3.23987 0.005129 Plant height 1.146198 0.217208 130.29 24.6906 5.27695 0.000075 Spike length -0.12629 0.233213 -112.41 207.5739 -0.54153 0.595605 1000 grain weight 0.446176 0.081286 75.20 13.7004 5.48896 0.000050

R=0.965; R2=0.932; adjusted R2=0.919; goodness of fit: F(3, 16)=72.884; P<0.00000 Table 4. Effects of the observed traits on rye genotype’s grain yield calculated by multiple

regression analysis.

β SE (β) B SE (B) t (d.f. 6) P value

Intercept -- -- -3421.49 4191.693 -0.81625 0.445544

Plant height 0.287749 0.213824 49.85 37.044 1.34573 0.227012

Spike length 0.439498 0.221911 1061.04 535.740 1.98052 0.094956

1000 grain weight 0.767590 0.158424 360.99 74.505 4.84517 0.002866

R=0.935; R2=0.875; adjusted R2=0.812; goodness of fit: F(3, 6)=13.958; P<0.00410 In triticale (Tab. 3), significant effect was shown by plant height (β=1.146**) and 1000

grain weight (β=0.446**). Adjusted R2 value was 0.919 and F test for goodness of fit was significant at the level of P<0.001. Deletić et al. (2013) also found significant influence of plant height (P<0.001) on triticale grain yield, but not one of spike length.

Grain yield of rye (tab. 4) was significantly affected only by 1000 grain weight (β=0.768**). Adjusted R2 value was 0.812 and F test for goodness of fit was significant at the level of P<0.01.

30

Conclusion

Multiple regression analysis of individual and joint effect of the studied parameters on wheat grain yield showed that plant height (P<0.001) and 1000 grain weight (P<0.01) had significant effect on grain yield, while the effect of spike length was not significant.

Grain yield of barley was under significant influence of the all three studied traits at the level of significance P<0.01.

In triticale, significant effect was shown by plant height (P<0.01) and 1000 grain weight (P<0.01).

Grain yield of rye was significantly affected only by 1000 grain weight (P<0.01). Acknowledgement

The investigation published in this paper is a part of the project “The development of new technologies of small grains cultivation on acid soils using contemporary biotechnology” financed by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant No TR-31054. References Cui, F., Li, J., Ding, A., Zhao, C., Wang, L., Wang, X., Li, S., Bao, Y., Li, X., Feng, D., Kong,

L., Wang, H. (2011). Conditional QTL mapping for plant height with respect to the length of the spike and internode in two mapping populations of wheat. Theoretical and Applied Genetics, 122(8), 1517-1536.

Deletić, N., Stojković, S., Gudžić, S., Djurić, V., Aksić, M. (2012). Genotypic specificity of some winter wheat traits and their effect on grain yield. Genetika, 44(2), 249-258.

Deletić, N., Stojković, S., Biberdžić, M., Lalević, D., Gudžić, S. (2013). Individual and joint effect of yield components on grain yield of triticale and barley. Proceedings of the Fourth International Conference: „Research People and Actual Tasks on Multidisciplinary Sciences“ 12-16 June 2013, Lozenec, Bulgaria, Vol. I, 36-40.

Hill, T., Lewicki, P. (2007). Statistics: Methods and Applications. StatSoft, Tulsa, OK, USA. Madić, M., Paunović, A., Djurović, D., Knežević, D. (2005). Correlations and “path” coefficient

analysis for yield and yield components in winter barley. Acta Agriculturae Serbica, 10(20), 3-9.

Peter, H. (2003). The genes of the green revolution. Trends in Genetics, 19(1), 5-9. Saleem, U., Khaliq, I., Mahmood, T., Rafique, M. (2006). Phenotypic and genotypic correlation

coefficients between yield and yield components in wheat. Journal of Agricultural Research, 44(1), 1-6.

Sharma, S.N., Sain, R.S., Sharma, R.K. (2003). Genetics of spike length in durum wheat. Euphitica, 130(2), 155-161.

31

BREEDING OF EUROPEAN WHITE ELM IN SERBIA – SELECTION AND PROGENY TEST

Jovana DEVETAKOVIĆ1, Vladan IVETIĆ1, Mirjana ŠIJAČIĆ-NIKOLIĆ1, Vladan POPOVIĆ2,

Boris IVANOVIĆ3

1 Faculty of Forestry University of Belgrade, Kneza Višeslava 1, 11030 Belgrade

2 Institute of Forestry, Kneza Višeslava 3, 11030 Belgrade 3 SE “Srbijašume”, Bulevar Mihaila Pupina 113, 11070 Belgrade

Corresponding author: jovana.devetakovic@sfb.bg.ac.rs

Abstract

A breeding program of European white elm is started with aim of species reintroduction on previously abandoned natural sites. By mass selection at population level, autochthonous population at Veliko ratno ostrvo island at the confluence of the Sava and Danube in Belgrade was selected. From the base population, consisting of 56 genotypes, 14 genotypes were selected for a progeny test. Half-sib lines from two stock types were tested during three years. Seedlings produced in poly bags were planted in the field after the second year, while bare-roots seedlings were planted at the field site in first year. Based on morphological attributes and calculated genetic gain six half-sib lines (families) were selected for further improvement program. Key words: European white elm, breeding, genotypes, selection, progeny test Introduction

Conservation of European white elm (Ulmus laevis Pall.) on the one side, and breeding on the other side are recognized goals in management (Koskela et al. 2003, Šijačić-Nikolić, Milovanovic 2010). European white elm is recognized as rare and endangered species in some countries, for example in Serbia (Banković et al. 2009) and Denmark (REFORGEN 2003).

Dutch elm disease (DED) is considered to be the main cause of decreasing of individuals and populations of the species within genus Ulmus, but disappearance of wetlands and modification of species natural habitats is more often considered to be the cause of European white elm extinction (Collin 2003). Unlike other European elms, wild (Ulmus minor Mill.) and mountain (Ulmus glabra Huds.) which are classified in section Ulmus, within the genus Ulmus, European white elm belongs to section Blepharocarpus (Machon et al. 1995, Wiegrefe et al. 1994), as confirmed by genetic studies (Witheley 2004). European white elm does not easily hybridize with other elm species, and it is self-incompatible (Mittempergher and LaPorta 1991).

32

Studies on breeding of this species as in other elms are focused mainly on resistance to the Dutch elm disease (DED) (Mittempergher and Santini, 2004).

In Serbia, European white elm was subjected to studies of morphological attributes (Isajev, 1978) and conservation methods (Aleksić, Orlović 2004).

The aim of this study was to select European white elm trees from autochthonous population at Veliko ratno ostrvo island for future breeding program of this species in Serbia.

Material and methods

A natural population of European white elm consisting of 56 trees at the Veliko ratno ostrvo, at the confluence of the Sava and Danube in Belgrade, was selected for this study. The trees are spatially divided into three subpopulations:

The first subpopulation (I) consists of 24 trees located in the inshore area. The second subpopulation (II) consists of 22 marked trees in the internal part of the

island, close to the canal Galijaš. The third subpopulation (III) consists of 10 trees individually dispersed throughout the

forest edge or in a field (Šijačić-Nikolić, Milovanović, 2012). All trees of the base population consisting of 56 trees were measured and georeferenced.

The coordinates are specified in the field, using GPS devices Trimble® GeoExplorer® series, which was in connection with a laser distancer TruPulse 360 B, which were measured by the height of the trees. The collected data were transferred to the GPS device to a computer for further processing using the GPS Pathfinder® Office ver. 4.20.

Marked trees diameter on the breast height was measured using a caliper, with precision to 1 mm and at the same height age was determined using Preslers borer.

Individual selection was based on the taxation data, abundance and quality of seed crop. From the base population a total of 12 best trees by taxation data and seed quality which make 20% were selected (Table 1), and their seeds were collected in May 2011.

Table 1. Position, size and age of selected mother trees

Subpopulation

Mark of tree on the field

Gaus-Kruger Coordinates Height

(m)

Diameter (cm) Age

Easting

Northing

I 13 7456215 4965040 23,1 48,7 26 14 7456215 4965038 7,7 15,65 13 32 7456352 4964955 20,2 38,3 23

II

19 7456054 4965270 18,4 36,5 16 21 7456046 4965285 9,5 31,5 16 33 7456138 4965226 13,7 29,35 20 34 7456170 4965254 21 31,3 20 35 7456150 4965257 20,9 30,4 20 36 7456136 4965262 13,9 37,8 23 37 7456170 4965258 7,5 17,65 15

III 29 7455281 4965700 13 47,75 24 31 7455137 4965585 12 23,5 17

33

Seed from each family was sown and bare-root and container seedlings were produced from each individual family with maintaining family identity. Bare-root seedlings were produced in nursery Manić, in raised seedbeds on brown soil type. Container seedlings were produced in the Nursery of the Faculty of Forestry, in poly bags filled with mixture of peat and perlite (2:1). Seedlings were irrigated and weeding on need, fertilization not applied. After the first growing season, a sample of 20 seedlings per maternal line was planted on the field on Veliko ratno ostrvo island. After the second growing season, a total sample of 720 seedlings (20 seedlings x 12 families x 3 stock types) were measured for height (HT) and diameter (D).

Measured values were used for variance analysis (one-way ANOVA) and separation of homogenous groups (LSD test). Based on analysis of variance, single tree (h2

ST) and family (h2F)

heritability was calculated for height and diameter (formulas 1 and 2). Expected genetic gain (∆G) was calculated based on family heritability (formula 3). Formula 1: Calculation of single tree heritability (h2

ST)

VfVfs

Vf4h

2

ST +

⋅=

Formula 2: Calculation of family heritability (h2

F)

Vfs

Vfs

Vfh

2

F

+

=

Formula 3: Calculation of expected genetic gain (∆G)

∆G = i · h2 ·σp

Where: Vf – family variance, Vfs – family x stocktype variance, s – number of stocktypes, i – selection intensity and σp -

phenotypic standard deviation of attribute

Results and discussion The mean values of the diameter and height observed per maternal lines clearly show the

separation of the average values of the lines 31, 33, 36 and 37 for height, and lines 29, 14, 33, and 37 in diameter, as confirmed by the results of LSD test (table 2).

The mean value of the heights are in the range from 61,74cm to 79,21cm, and mean values of the diameter are from 7,53mm to 10,34mm, which is significantly lower than the values reported by Cicek el al. (2010).

Table 2. Descriptive statistical parameters for measured traits and LSD test of 12 half-sib lines of

European White Elm

N=720 HEIGHT DIAMETER LINE Mean Std.Dev. Std.err -95% +95% Mean Std.Dev. Std.err -95% +95% 13 70,28abc 23,05 3,56 63,10 77,47 9,40abc 3,48 0,54 8,31 10,49 14 70,00abc 24,90 2,81 64,38 75,61 9,73ab 4,84 0,55 8,63 10,82 19 60,56c 27,31 4,37 51,71 69,42 7,53d 3,16 0,51 6,51 8,56 21 70,51abc 34,03 4,63 61,23 79,81 9,22abc 4,31 0,59 8,05 10,40 29 69,50ac 25,41 2,93 63,66 75,35 9,58ab 3,39 0,39 8,80 10,36 31 76,72ab 24,77 2,65 71,44 82,00 9,54ab 4,28 0,46 8,63 10,45 32 61,74c 25,81 3,51 54,70 68,78 7,99cd 3,31 0,45 7,09 8,90 33 74,97ab 26,76 4,46 65,92 84,03 9,88ab 4,07 0,68 8,50 11,25 34 70,64abc 25,89 2,82 65,02 76,26 8,82acd 4,02 0,44 7,94 9,69 35 71,95abc 29,48 4,25 63,40 80,52 9,55abc 4,39 0,64 8,27 10,82 36 72,83ab 26,70 3,85 65,08 80,59 9,21abcd 3,47 0,50 8,19 10,20 37 79,21b 31,61 4,18 70,83 87,60 10,34b 4,40 0,58 9,17 11,51

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A calculation of the heritability of traits and of genetic gain was done based on 6 selected lines: 14, 29, 31, 33, 36 and 37. Table 3. Analysis of variance (one-way ANOVA) regarding height and diameter of 12 half-sib lines of

European white elm and results of trait’s heritability and genetic gain

HEIGHT DIAMETER

EFFECT df SS MS F p df SS MS F p

STOCKTYPE 2 238526 119263 356,51 0,00 2 6253,61 3126,81 547,13 0,00 FAMILY 11 16628 1512 4,52 0,00 11 336,65 30,60 5,35 0,00 STOCKTYPE*FAMILY 22 24514 1114 3,33 0,00 22 372,96 16,95 2,97 0,00 ERROR 666 222798 335 666 3806,13 5,71 h2

ST 0.42 0.85 h2

F 0.26 0.45 ∆G 2.958 0.6

Heritability (family) is in the range from 0, 26 for height, to 0, 45 for diameter, while values of heritability for individual trees have somewhat higher values, from 0,42 for height to 0,85 for diameter. The values obtained are less than the values of heritability for the same traits of some other tree species, but the values are dependent on the age during the tested (Mora and Saavedra 2012; Kajba et al. 2011, Xie and Yanchuk 2002). The selection on the basis of height, as attributes with a higher heritability than the diameter, was the base for many breeding programs (Lambeth et al. 1983), but some other studies found that heritability estimates for height and diameter were comparable, and that values of diameter heritability can be higher than height heritability (Kowalczyk 2005, Rink and Kung 1991, Yeh and Heaman 1982 ). Reasons for high values of diameter heritability can be found in the fact that more factors affect the height then diameter.

The main goal of this breeding program is conservation and reintroduction, but the recorded genetic gain has an important role because in the condition of frequent flooding like on Veliko ratno ostrvo island, rapid initial growth is advantage.

A total of six plus trees were selected on the basis of backward selection, while the other family will be used forward selection.

The selected number of six genotypes is not enough for the establishment of seed orchards because of problems with an effective population size being so low (Hansen and Kjær 2006, Burgarella et al. 2008, Lai et al. 2010), which requires the need for additional selection in other autochthonous populations, so as to satisfy the minimum number of clones for the establishment of seed orchards of this species which present minimum population sizes range from tens to hundreds (Johnson and Lipow 2002). Conclusion

- Obtained values of heritability and genetic gain are within the expected range and sufficient for separation of plus trees.

- Insufficient number of selected trees indicates a need for further selection. - Implemented method can be successfully applied in future.

35

Acknowledgement This article is an output from a research project funded by Ministry of Educations and

Science of the Republic of Serbia TR37008. References Aleksić J., Orlović S. (2004). Ex situ conservation of genetic resources of field elm (Ulmus

minor Mill.) and european white elm (Ulmus laevis Pall.). – Genetika, Vol. 36, No. 3, 221-227.

Banković S., Medarević M.,Pantić D., Petrović N., Obradović S., (2009). Šumski fond Republike Srbije-stanje i problemi, Glasnik Šumarskog fakulteta, Beograd, br. 100, 7-30.

Burgarella C., Navascués M., Soto Á., Lora Á., Fici S. (2007).Narrow genetic base in forest restoration with holm oak (Quercus ilex L.) in Sicily. Annals of Forest Science 64 (7) 757-763.

Collin E. (2003). EUFORGEN Technical Guidelines for genetic conservation and use for European white elm (Ulmus laevis). International Plant Genetic Resources,Institute, Rome, Italy. 6 pages.

Çiçek E., Tilki F., Özbayram A.K., Çetin B. (2010). Three-year growth comparison between rooted cuttings and seedlings of Fraxinus angustifolia and Ulmus laevis. Journal of Applied Sciences Research, 6(3): 199-204.

Hansen O.K., Kjær E.D. (2006). Paternity analysis with microsatellites in a Danish Abies

nordmanniana clonal seed orchard reveals dysfunctions. Canadian Journal of Forest Research, 36(4): 1054-1058.

Isajev V. (1978). Proučavanje unutarvrsne promenljivosti veza (Ulmus leavis Pallas) i njen značaj za oplemenjivanje ove vrste. Magistarski rad, Univerzitet u Beogradu-Šumarski fakultet, Beograd: (1-77).

Johnson R., Lipow S. (2002). Compatibility of breeding for increased wood production and long term sustainability: the genetic variation of seed orchard seed and associated risks. In:

Proceedings Wood compatibility initiative workshop, No 18: 169–179. Kajba D., Katičić I., Bogdan S. (2011). Procjena genetskih parametara u testovima polusrodnika

hrasta lužnjaka (Quercus robur L.) iz sjemenskih zona Posavine, Podravine i Podunavlja. Croatian Journal of Forest Engineering, 32, 177-192.

Koskela J., Vries S.M.G., Gil L., Mátyás C., Rusanen M. and Paule L. (2003). Conservation of forest genetic resources and sustainable forest management in Europe. Proceedings of the Symposium of the North American Forest Commission,Forest Genetic Resources and Silviculture Working Groups, and the International Union of Forest Research Organizations (IUFRO), Canada.

Kowalczyk J. (2005). Comparison of phenotypic and genetic selections in Scots pine (Pinus sylvestris L.) single tree plot half-sib progeny tests. Dendrobiology, vol. 53, 45–56.

Lai B.S., Funda T., Liewlaksaneeyanawin C., Klápště J., Van Niejenhuis A., Cook C. Stoehr M.U., Woods J., El-Kassaby Y. A. (2010). Pollination dynamics in a Douglas-fir seed orchard as revealed by pedigree reconstruction. Annals of forest science, 67(8), 808.

Lambeth C., Van Buijtenen, J. P., McCollough R. B., Duke S. D. (1983). Early selection is effective in 20-year old genetic tests of loblolly pine. Silvae Genetica 32:210-215.

Machon N., Lefranc M., Bilgeri I. And Henry J.-P. (1995). lsoenzymes as an aid to clarify the taxonomy of French elms. Heredity 74, pages 39—47.

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Mittempergher L., La Porta N. (1991). Hybridization Studies in the Eurasian Species of Elms (Ulmus spp.), Silvae Genetica 40, 5/6.

Mittempergher L., Santini A.(2004). The history of elm breeding. Invest Agrar: Sist Recur For 13 (1), 161-177.

Mora F., Saavedra J. (2012). Combining genetic gain and diversity under an individual selection method in a selected provenance of Eucalyptus cladocalyx. Ciencia e Investigacion Agraria, 39(1):177-184.

REFORGEN, 2003. http://foris.fao.org/reforgen/factSheet.jsp

Rink G., F.H. Kung (1991). Height and diameter variation in twelve white ash provenance/progeny tests in eastern United States. L.H. McCormick and K.W. Gottshalk (eds.), Proceedings of the 8th Central Hardwoods Forest Conference. Pennsylvania State University, University Park, PA, March 3-6, 1991, pp. 179-187.

Šijačić-Nikolić M., Milovanović J. (2010). Konzervacija i usmereno korišćenje šumskih genetičkih resursa. Šumarski fakultet Univerziteta u Beogradu, Beograd 1-200.

Šijačić-Nikolić M. (2012). Program genetičke konzervacije drvenastih vrsta Velikog ratnog Ostrva. Univerzitet u Beogradu – Šumarski fakultet, Beograd . Yeh Francis C., Heaman Chris (1982). Heritabilities and genetic and phenotypic correlations

for height and diameter in coastal Douglas-fir, Canadian Journal of Forest Research,

1982, 12(2): 181-185. Wiegrefe S., Sytsma K., Guries R. (1994). Phylogeni of Elms (Ulmus, Ulmaceae): Molecular

Evidence for a Sectional Classification. Systematic Botany, Vol.19, No.4, pages 590-612. Witheley R. (2004). Quantitative and molecular genetic variation in Ulmus laevis Pall.. Doctoral

thesis, Swedish University of Agricultural Sciences, Uppsala. Xie C.-Y., Yanchuk A. D. (2002). Genetic parameters of height and diameter of interior spruce

in British Columbia. Forets Genetics, 9(1):1-10.

37

SELECTION OF MAZE INBRED LINES FOR IMPROVED RATIO BETWEEN PHYTIC ACID AND Mg, Fe, Mn AND Zn CONTENT IN GRAIN

Vesna DRAGIČEVIĆ1*, Milomir FILIPOVIĆ1, Natalija KRAVIĆ1, Milovan STOJILJKOVIĆ2,

Snežana MLADENOVIĆ DRINIĆ1

1 Maize Research Institute “Zemun Polje”, Slobodana Bajića 1, 11185 Zemun Polje, Serbia,

*E-mail: vdragicevic@mrizp.rs 2 Vinča Institute of Nuclear Sciences, P.fah 522, 11001 Belgrade, Serbia

Abstract

The aim of this investigation was to test 40 maize inbred lines for increased potential availability of Mg, Fe, Mn and Zn content from grain. The analysis was based on their ratio with phytate and β-carotene. Significant and positive correlations were found between kernel weight, β-carotene and Zn content, as well as between phytate, β-carotene and Mg content. Among inbred lines with decreased ratio between phytic and inorganic phosphorus (L1, L26, L32), lower phytate/Mg, phytate/Zn and phytate/Mn ratios were observed, too. Moreover, in lines with increased ratio between phytic and inorganic phosphorus (L3, L4, L10, L23 and L31), decreased phytate/β-carotene, phytate/Mg, phytate/Fe and phytate/Mn ratios were found. This could indicate that improved Mg, Fe, Mn and Zn availability might exist even in grain rich in phytate, due to high β-carotene content. Key words: β-carotene, phytate, mineral elements, Zea mays L. Introduction

Malnutrition, resulting from deficiency of mineral nutrients in food, could cause serious health problems. These deficiencies can be overwhelmed by the increase of mineral nutrients in food, including supplementation, food fortification or plant breeding (Lönnerdal, 2003). The highest importance was given to Fe and Zn in vegetarian diets, since elimination of meat and increased intake of legumes and whole grains rich in phytate, decreases the Fe and Zn absorption (Hunt, 2003). This could lead to anemia (Fe deficiency), as well as to problems with immune system or abnormal blood losses (Zn deficiency). On the other hand, low Mg status has been associated with numerous pathological conditions, like chronic inflammatory stress component, widely connected with obesity, atherosclerosis, hypertension, osteoporosis, diabetes mellitus and cancer (Nielsen, 2010). Manganese is also essential for humans; it is a part of the antioxidant

38

system in mitochondria, and is involved in metabolism, bone development and wound healing (Underwood and Suttle, 1999).

Variability of mineral elements in maize grain is significant (Mladenović Drinić et al., 2013; Dragičević et al., 2013), and their availability depends on numerous factors. Some substances, such as antinutrients (e.g. phytate, polyphenolics, etc.), interfere with the absorption or utilization of nutrients, and there are also enhancing substances - promoters (i.e. ascorbic acid, S-containing amino acids, β-carotene, etc.), which promote micronutrient bioavailability or decrease antinutrient activity (Welch and Graham, 2004).

One of the most important factors which disturb the absorption of mineral nutrients is phytic acid - Phy (myo-inositol 1,2,3,4,5,6-hexakisphosphate), the major phosphorus storage compound in grains (containing up to 80% of total P in grain). Due to high density of negatively charged phosphate groups, phytic acid forms very stable complexes with mineral ions, making them unavailable for intestinal absorption. As the phytic acid content in diet increase, the intestinal absorption of Zn, Fe and other mineral nutrients decreases (Walter Lopez et al., 2002). Some plant species, like maize, could have normal levels of total P, but significantly reduced levels of phytic acid, which in turn increases the level of inorganic phosphorus (Lönnerdal, 2003). According to the Phy/Zn and Phy/Fe molar ratios, it is possible to determine maize lines or foods with potentially high ability of Fe and Zn utilization (Ma et al., 2007).

β-carotene could be considered as promoter, positively affecting absorption of mineral nutrients. Luo and Xie (2012) found that addition of food, rich in β-carotene or pure β-carotene, can significantly enhance the bioavailability of Fe and Zn from the grains.

According to those findings, breeding for biofortification of mineral elements in cereals is possible (White and Broadley 2005) through the increase of their concentration and concentration of promoters, along with decrease of anti-nutrients. From that point, the experiment was conducted on forty maize inbred lines, in order to evaluate chemical composition of grain and to determine the relations between phytic acid and β-carotene, as factors affecting the absorption of Mg, Fe, Zn and Mn. Material and methods Plant material

The set of forty maize inbred lines (marked with letter L and appropriate number) from Maize Research Institute Zemun Polje were the objectives of the present study. Genetic background of the examined lines was: dent BSSS - L2-L5, L10, L12, L13, L16, L24-L28, L31-L34, L40; dent BSSS Lancaster x tropical white - L21; dent BSSS independent source - L17, L23, L39; dent independent source - L7, L14; dent Lancaster independent source - L22; dent Lancaster - L8, L15, L29, L35-L38; semident Lancaster - L18; flint - L1; semident BSSS independent source - L30; domestic dent unrelated - L6, L9, L11; white dent unrelated - L19, L20. Field trial and laboratory experiments

The experiment was carried out in 2011 in Zemun Polje, Serbia (44°52´N, 20°19´E, 81 m asl). The soil was slightly calcareous chernozem (low in investigated micro-elements). A randomized block design with two replications was used in the experiments. Inbreds were

39

manually self-pollinated and harvested. After drying to 14% of moisture content, 1000 kernel weight was measured.

Chemical composition of grain was determined colorimetrically. Phytic (Pphy) and inorganic phosphorus (Pi), were determined by the method of Dragičević et al. (2011) on spectrophotometer (Biochrom Libra S22, United Kingdom). β-carotene was determined according to AACC (American Association of Cereal Chemists Method) procedure (1995). Mineral elements (Mg, Fe, Mn and Zn) were determined after wet digestion with HNO3 + HClO4, by Inductively Coupled Plasma - Optical Emission Spectrometry (Spectro Flame, Spectro Analytical Instruments, Germany).

Correlation analyses were performed using Pearson’s correlation coefficient. Molar ratios betweenphytic and inorganic P (Pphy/Pi), as well as between phytic acid (Phy) and observed parameters (Phy/β-carotene, Phy/Mg, Phy/Fe, Phy/Mn and Phy/Zn) were presented as means ± standard deviation (SD). Results and discussion

Improved availability of mineral elements mainly depends on lowering of their ratio with phytic acid (Walter Lopez et al., 2002; Lönnerdal, 2003; Welch and Graham, 2004). Decrease in Pphy content could also mean Pi increase in grain and is measurable through decrease of Pphy/Pi ratio. Group of inbred lines with Pphy/Pi < 7 (Table 1) mainly had the lower Phy/β-carotene values (L7, L8, L26 and L27 with values < 800). The only exception was inbred L1, with Pphy/Pi and Phy/β-carotene ratios being 4.51 and 952.1, respectively. Also, the same line exhibited low Phy/Mg, Phy/Fe and Phy/Mn ratios (which is about 40%, 53% and 200% lower compared to the highest ratio values, respectively). The lowest Phy/β-carotene ratio was observed in L25 line, with relatively high Pphy/Pi, and Phy/Fe ratios (of 1.1 and 597.1, respectively).

Some inbred lines with low Phy/Fe ratio (i.e. L3, L4, L10, L23 and L31) also displayed low Phy/Zn ratio. It is obvious that they belong to the group with Pphy/Pi ratio > 7 and even > 10 (inbreds L4 and L10), thus representing a potential source for increased Fe and Zn absorption (White and Broadley, 2005). Moreover, inbreds L4 and L10 could not be considered as potentially low phytic acid (lpa) genotypes (Raboy et al., 2001). Among genotypes with Pphy/Pi < 7, L32, with Phy/Fe ratio of 114.7 and L26, with Phy/Zn ratio of 2237, which is the lowest value among all of the tested genotypes, could be considered as favorable inbred lines. Although white kernel inbred line, with the highest Phy/β-carotene ratio, L21, belongs to the group with low Pphy/Pi ratio, having also the lowest Phy/Mn ratio. This could make it desirable as source for increased Mn availability.

In regard that 1000 kernel weight was not present in Table 1, generally significant and positive correlations between 1000 kernel weight and β-carotene, Fe and Zn, indicate potentially higher Fe and Zn availability from larger maize kernels (Table 2). This could be important, since increased kernel weight, as the main parameter of yield, is highly dependent on good availability of assimilates (Borrás et al., 2004). Irrespectively to kernel size, β-carotene positively correlated with Mg and Mn, but also with phytate. In regard that any reduction of phytic acid content in food is likely to result in improved Fe, Zn and Mn status (Underwood and Suttle, 1999; Lönnerdal, 2003), high and positive correlations between phytate and Mg and Mn is undesirable and could linger further breeding process for increased availability of mineral nutrients, based on phytate decrease and β-carotene increase.

40

Table 1. Molar ratios between phytic/inorganic phosphorus, phytic acid/β-carotene, phytic acid/Mg,

phytic acid/Fe and phytic acid/Zn

Pphy/Pi Phy/β-carot. Phy/Mg Phy/Fe Phy/Mn Phy/Zn

L1 4.5 ± 0.1 952 ± 13.1 2.34 ± 0.1 300.6 ± 9 180.0 ± 26 4235 ± 10 L2 11.2 ± 0.7 908 ± 9.0 3.32 ± 0.6 571.4 ± 9 285.7 ± 25 5515 ± 96 L3 7.6 ± 0.3 821 ± 13.8 2.75 ± 0.3 69.5 ± 1 332.9 ± 31 4360 ± 10 L4 10.3 ± 0.8 873 ± 6.2 2.63 ± 0.2 77.0 ± 1 309.7 ± 43 3101 ± 5 L5 8.2 ± 1.5 782 ± 19.2 3.11 ± 0.3 252.9 ± 20 357.1 ± 13 3696 ± 38 L6 7.6 ± 0.3 993 ± 9.3 2.78 ± 0.2 572.4 ± 7 255.2 ± 20 3641 ± 52 L7 6.3 ± 0.1 767 ± 13.0 3.30 ± 0.1 576.0 ± 17 295.0 ± 35 5846 ± 64 L8 7.0 ± 0.4 735 ± 17.2 3.04 ± 0.6 596.3 ± 16 234.6 ± 29 3306 ± 10 L9 8.7 ± 0.7 815 ± 9.8 2.91 ± 0.4 240.7 ± 15 333.3 ± 44 5484 ± 128 L10 11.3 ± 0.4 1300 ± 21.6 3.00 ± 0.2 45.3 ± 1 299.4 ± 50 7311 ± 155 L11 13.2 ± 0.6 1717 ± 48.9 3.33 ± 0.2 565.9 ± 23 337.2 ± 47 4823 ± 38 L12 8.2 ± 0.6 1272 ± 33.8 3.56 ± 0.8 579.1 ± 20 388.4 ± 121 1148

± 149

L13 10.4 ± 0.5 786 ± 10.0 3.87 ± 0.3 636.3 ± 4 391.2 ± 45 6936 ± 103 L14 11.6 ± 0.2 1077 ± 4.0 3.56 ± 0.1 586.5 ± 19 368.8 ± 68 3899 ± 42 L15 11.1 ± 0.4 870 ± 22.7 3.09 ± 0.1 620.1 ± 20 269.2 ± 35 8954 ± 139 L16 10.2 ± 0.4 646 ± 2.7 2.49 ± 0.1 590.4 ± 20 178.5 ± 23 5726 ± 21 L17 8.5 ± 0.2 559 ± 1.9 2.92 ± 0.1 118.8 ± 2 242.5 ± 16 5224 ± 15 L18 7.4 ± 0.1 612 ± 2.4 3.20 ± 0.1 310.8 ± 2 276.0 ± 26 8707 ± 68 L19 10.3 ± 0.2 1620 ± 23.3 3.00 ± 0.2 621.5 ± 30 281.2 ± 40 8268 ± 18 L20 7.7 ± 0.2 3398 ± 15.3 3.66 ± 0.4 591.0 ± 24 256.1 ± 23 4818 ± 72 L21 6.0 ± 0.1 3139 ± 40.5 2.99 ± 0.2 636.2 ± 10 167.1 ± 8 4701 ± 72 L22 7.7 ± 0.1 781 ± 23.3 3.01 ± 0.1 634.3 ± 4 225.8 ± 8 4893 ± 50 L23 9.3 ± 0.3 486 ± 15.6 3.16 ± 0.1 56.7 ± 0 260.5 ± 8 2941 ± 4 L24 6.8 ± 0.2 860 ± 6.3 3.17 ± 0.3 623.9 ± 26 234.3 ± 29 6219 ± 67 L25 11.1 ± 0.6 408 ± 3.1 3.02 ± 0.2 597.1 ± 14 244.1 ± 41 5060 ± 7 L26 6.0 ± 0.2 761 ± 14.3 3.05 ± 0.2 205.5 ± 4 257.5 ± 13 2237 ± 7 L27 6.0 ± 0.1 696 ± 3.0 3.42 ± 0.3 584.4 ± 5 263.7 ± 9 2897 ± 14 L28 8.7 ± 0.2 747 ± 10.4 3.31 ± 0.2 561.2 ± 7 390.0 ± 28 8633 ± 18 L29 9.4 ± 0.1 700 ± 12.3 3.59 ± 0.2 568.2 ± 13 305.3 ± 25 9864 ± 81 L30 8.4 ± 0.2 647 ± 6.3 3.50 ± 0.1 612.1 ± 7 274.5 ± 8 3973 ± 2 L31 9.0 ± 0.1 561 ± 9.3 2.90 ± 0.4 69.2 ± 3 216.4 ± 40 4039 ± 17 L32 6.4 ± 0.2 833 ± 24.7 3.13 ± 0.8 114.7 ± 10 315.1 ± 112 5374 ± 44 L33 8.9 ± 0.1 665 ± 11.0 2.71 ± 0.6 136.0 ± 16 248.9 ± 36 4387 ± 13 L34 10.2 ± 0.5 854 ± 6.4 3.07 ± 0.2 635.6 ± 10 320.7 ± 34 2808 ± 9 L35 9.4 ± 0.1 741 ± 3.7 3.02 ± 0.1 633.5 ± 12 286.9 ± 9 4442 ± 22 L36 7.9 ± 0.3 608 ± 6.0 3.55 ± 0.6 608.4 ± 20 251.3 ± 61 7045 ± 99 L37 7.1 ± 0.2 474 ± 7.8 2.93 ± 0.2 615.2 ± 19 228.7 ± 46 5706 ± 68 L38 11.1 ± 0.1 478 ± 3.2 2.89 ± 0.2 171.6 ± 2 168.8 ± 19 1044

± 204

L39 7.5 ± 0.3 762 ± 19.4 3.39 ± 0.3 557.5 ± 12 272.0 ± 38 4814 ± 21 L40 9.5 ± 0.2 686 ± 2.1 3.10 ± 0.1 592.0 ± 21 217.5 ± 6 4233 ± 3 Mean 8.7 ± 0.3 935 ± 13.2 3.12 ± 0.3 443.4 ± 12 275.5 ± 33.4 5501 ± 51

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Table 2. Correlations between 1000 kernel weight, β-carotene, phytate, Mg, Fe and Mn content in tested

maize inbred lines

1000 grain weight β-carotene Phytate Mg Fe Mn

β-carotene 0.340* Phytate 0.117 0.449*

Mg -0.078 0.292* 0.421* Fe 0.259* 0.159 0.066 0.287* Mn 0.149 0.301* 0.419* 0.626* -0.026 Zn 0.375* 0.046 -0.221 0.091 0.187 -0.008

* significant values at the 0.05 probability level.

Conclusion

Based on phytate decrease and β-carotene content increase, it could be concluded that some of investigated maize inbred lines can be considered as valuable source for further breeding programs related to increased Mg, Fe, Mn and Zn availability. Based on low Pphy/Pi ratio, the inbreds L1, L26, L32 (each with standard β-carotene concentration) could represent potential source for increased Mg, Fe, Mn and Zn availability, and L21, as white kernel maize inbred, for increased Mn availability. It can also be concluded that kernels with higher weight present a good source for higher Fe and Zn availability, whereas further decrease in phytate could be related to Mg, Mn and β-carotene decrease, thus negatively affecting breeding programs for improved availability of mineral elements. Acknowledgements

This study was supported by Project TR31068 "Improving the quality of maize and soybean by conventional and molecular breeding" from the Ministry of Education, Science and Technological Development, Republic of Serbia, and by COST Action FA 0905 “Mineral Improved Crop Production for Healthy Food and Feed”. References American Association of Cereal Chemists Method (1995). Approved Methods of the AACC, The

association: St. Paul, MN, USA, AACC Method, Pp. 14-50. Borrás L., Slafer G.A., Otegui M.E. (2004). Seed dry weight response to source–sink

manipulations in wheat, maize and soybean: a quantitative reappraisal. Field Crop Res 86: 131-146.

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Luo, Y.W., Xie, W.H. (2012). Effects of vegetables on iron and zinc availability in cereals and legumes. Internat Food Res J 19: 455-459.

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INCREASED AVAILABILITY OF Mg, Fe, Mn AND Zn IN DROUGHT TOLERANT MAIZE INBRED LINES

Vesna DRAGIČEVIĆ1, Natalija KRAVIĆ1, Milovan STOJILJKOVIĆ2, Violeta

ANĐELKOVIĆ1, Jelena VANČETOVIĆ1

1 Maize Research Institute “Zemun Polje”, Slobodana Bajića 1, 11185 Zemun Polje, Serbia,

*E-mail: vdragicevic@mrizp.rs 2 Vinča Institute of Nuclear Sciences, P.fah 522, 11001 Belgrade, Serbia

Abstract

After two-year of field experiment, potential Mg, Fe, Mn and Zn availability was conducted on the set of 10 inbred lines from MRIZP Gene bank drought tolerant mini-core collection. Potential availability in micro-elements was determined through their ratio with phytate as inhibitor and β-carotene as promoter. After harvesting and drying to 14 % of moisture content, measurement of 1000 kernel weight, inorganic P (Pi), phytate (Pphy), β-carotene, Mg, Fe, Mn and Zn content in maize grain were determined. Inbred line with the highest Pi content also had the lowest Pphy content, as well as Fe and Zn content. This inbred could be considered as valuable source for improved Mg, Fe, Mn and Zn availability, due to the lowest ratio between phytate and minerals. Moreover, inbred lines with β-carotene about 20 µg g-1, could also be considered as favourable for increased Mg, Fe, Mn and Zn availability, due to low phytate/β-carotene ratio. Key words: β-carotene, micro-elements, inorganic phosphorus, phytate, Zea mays L. Introduction

Human body needs more than 22 mineral elements, which can be provided by the nutrition. Intake of mineral elements could be limited by the inadequate diet or their low level in staple food. Accumulation of mineral elements in seeds and grains is controlled by a number of processes including root-cell uptake, root-shoot transfer, and the ability of leaf tissues to load these nutrients into the phloem elements which are ultimately responsible for delivering these nutrients to developing seeds and grains via the phloem sap (Welch, 2003). Increase of the concentration of mineral elements in edible parts of plants is called biofortification and it could be attained by fertilization and breeding (White and Broadley, 2005).

From one side, it is important to increase concentration of mineral elements in grain and from the other, it is important that mineral elements are bio-available from digested food. There

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are substances which interfere with the absorption or utilization of mineral elements, called inhibitors (e.g. antinutrients), like phytate, polyphenolics etc., as well as enhancing substances - promoters (e.g. ascorbic acid, S-containing amino acids, β-carotene, etc.) that promote micronutrient bioavailability or decrease activity of inhibitors (Welch and Graham, 2004).

According to present data, breeding for biofortification of mineral elements in cereals is possible (White and Broadley, 2005) through the increase of their concentration, along with concentration of promoters and decrease in concentration of inhibirors. Some plant species, like maize, could have normal levels of total P but significantly reduced levels of phytic acid, which in turn increases the level of inorganic phosphorus (Lönnerdal, 2003). According to the Phy/Zn and Phy/Fe molar ratios, it is possible to determine maize lines or foods with potentially high ability of Fe and Zn utilization (Ma et al., 2007). It is also important to identify genetic resources with high levels of the targeted mineral elements, to consider the heritability of the targeted traits and to investigate genotype x environment interactions (Ortiz-Monasterio et al., 2007).

From this aspect, the experiment was conducted on ten maize inbred lines tolerant to drought, in order to evaluate chemical composition of grain and to determine the relations between phytic acid, inorganic phosphorus and β-carotene, as factors affecting the absorption of micro-elements, i.e. Mg, Fe, Zn and Mn. Material and methods Plant material

Based on the field trials´ results and general combining ability, obtained after two-year of screening under controlled drought in Egypt, as well as under temperate climate conditions in Serbia and Macedonia, a drought tolerant mini-core collection of 41 accessions (15 inbred lines, 13 local and 13 introduced landraces) was established (Anđelković et al. 2010; Babic et al. 2011). The ten drought tolerant maize inbred lines were the objective of the present study. Genetic background of the investigated inbred lines is as follows: independent source Lancaster - L1 (semiflint); independent source - L2, L4 and L6 (dents); independent source BSSS - L7 (flint); Lancaster - L3 and L8 (semiflints); Lancaster BSSS - L9 and L10 (semidents); unknown - L5 (dent). Field trials and laboratory experiments

For this study, the experiment was carried out in 2010 and 2011 in Zemun Polje, Serbia (44°52´N, 20°19´E, 81 m asl). The soil was slightly calcareous chernozem (low in investigated micro-elements). A randomized block design (RCBD) with two replications was used in the experiments. Inbreds were manually self-pollinated and harvested. After drying to 14% of moisture content, 1000 kernel weight was measured.

Chemical composition of grain was determined colorimetrically. Phytic (Pphy) and inorganic phosphorus (Pi), were determined by the method of Dragičević et al. (2011). β-carotene was determined according to AACC procedure (1995). Mineral elements (Mg, Fe, Mn and Zn) were determined after wet digestion with HNO3 + HClO4, by Inductively Coupled Plasma - Optical Emission Spectrometry. Statistical analysis

All analyses were performed in four replicates (n=4) and the results were presented as mean ± standard deviation (SD). The data were subjected to two-way analyses of variance

45

(ANOVA). F-test was used for means comparison at the 0.05 probability level. Correlation analyses were performed using Pearson’s correlation coefficient. Results and Discussion

In this study has been shown that effect of year, genotype and their interaction had significant impact on variation in 1000 kernel weight, Pphy, Pi, β-carotene, Mg, Fe, Mn and Zn content in grain, as presented in Table 1. Only for Fe content, effect of the year was statistically non-significant. Average 1000 kernel weight varied in range from 195.7 (L6) to 291.2 g (L1). For the same inbred line (i.e. L1) was found the highest β-carotene content in grain (21.20 mg kg-1). It can be supposed that high level of β-carotene content in grain could be related to increased availability of investigated mineral elements (Brinch-Pedersen et al., 2007). The lowest β-carotene content of 6.91 mg kg-1 was observed for drought tolerant inbred L8. The lowest Pphy, as desirable trait for improved availability of mineral elements (Walter Lopez et al., 2002; Lönnerdal, 2003; Welch and Graham, 2004), was found in L2 (2.99 g kg-1), the same line with the highest Pi (0.97 g kg-1), Fe (15.02 mg kg-1) and Zn (20.67 mg kg-1) content. Observed Pi, Fe and Zn content were two times higher compared to the lowest values obtained for L9 and L7, respectively. The highest Mg and Mn content were found in drought tolerant L8 inbred line. Table 1. 1000 kernel weight (KW) and chemical composition of grain for chosen drought tolerant maize

inbred lines

Lines KW (g)

Pphy Pi β-carot Mg Fe Mn Zn (g kg-1) (mg kg-1)

L1 291.2 3.80 0.47 21.20 426.7 12.94 2.52 11.02 L2 206.4 2.99 0.97 10.53 475.3 15.02 3.38 20.67 L3 231.0 3.41 0.59 11.90 448.9 10.25 2.15 16.11 L4 215.9 3.43 0.39 13.95 451.6 11.28 2.33 14.03 L5 248.8 3.56 0.65 19.43 463.0 9.80 2.33 13.53 L6 195.7 3.34 0.47 16.31 499.5 8.95 1.75 12.72 L7 264.5 3.46 0.43 12.31 283.9 7.72 1.38 9.91 L8 249.9 3.68 0.69 6.91 524.5 11.53 3.55 12.19 L9 221.4 3.53 0.37 15.81 480.3 10.91 2.53 10.44

L10 217.2 3.73 0.39 17.52 469.5 11.53 2.06 11.13 F-test

Year F 0.941 23.38 0.83 0.65 2.77 0.23 1.61 5.18 p 0.01 0.00 0.37 0.42 0.10 0.63 0.21 0.03

Genot. F 0.695 13.06 66.65 39.80 22.78 26.35 32.61 6.51 p 0.21 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Y x G F 0.950 42.84 294.53 447.28 1395.66 100.40 100.42 12.62 p 0.545 0.00 0.00 0.00 0.00 0.00 0.00 0.00

According to results presented in Table 2, required decrease in Pphy/Pi, Phy/Mg, Phy/Fe,

Phy/Mn and Phy/Zn ratios, which contributes to increased availability of Pi, Mg, Fe, Mn and Zn (Underwood and Suttle; 1999; Lönnerdal, 2003) was mainly presented in grain of drought tolerant inbred L2. Moreover, the lowest value of Phy/β-carotene ratio, along with relatively low Phy/Fe ratio in L1, could indicate improved Fe availability due to the positive impact of β-carotene (Brinch-Pedersen et al., 2007).

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Table 2. Molar ratios between phytic/inorganic phosphorus, phytic acid/β-carotene, phytic acid/Mg,

phytic acid/Fe and phytic acid/Zn

Pphy/Pi Phy/β-carotene Phy/Mg Phy/Fe Phy/Mn Phy/Zn L1 8.12 ± 0.82 518 ± 161 2.68 ± 0.40 88 ± 10.0 445 ± 220 104 ± 55 L2 3.08 ± 0.39 821 ± 264 1.89 ± 0.08 60 ± 5.9 262 ± 6 44 ± 7 L3 5.81 ± 0.63 827 ± 145 2.28 ± 0.27 100 ± 5.9 468 ± 23 64 ± 1 L4 8.84 ± 0.09 710 ± 39 2.28 ± 0.03 91 ± 5.6 435 ± 2 73 ± 42 L5 5.52 ± 0.65 529 ± 7 2.31 ± 0.19 109 ± 9.3 452 ± 29 79 ± 12 L6 7.11 ± 0.89 592 ± 47 2.01 ± 0.07 112 ± 0.45 564 ± 87 79 ± 16 L7 8.03 ± 1.99 811 ± 127 3.66 ± 2.14 135 ± 65.9 743 ± 364 105 ± 23 L8 5.32 ± 0.18 1540 ± 91 2.11 ± 0.25 96 ± 8.6 307 ± 22 91 ± 39 L9 9.66 ± 0.38 646 ± 118 2.21 ± 0.16 97 ± 12.5 413 ± 25 102 ± 48 L10 9.59 ± 1.01 615 ± 103 2.39 ± 0.21 97 ± 1.4 535 ± 30 101 ± 15

Statistical analyses presented in Table 3 have shown that significant and positive

correlations between Pphy/Pi and Phy/Mg, Phy/Fe, Phy/Mn and Phy/Zn could indicate positive relation between enhanced phytate decrease along with Pi, Mg, Fe, Mn and Zn increase in maize grain in further breeding process, including the investigated inbred lines (Ma et al., 2007; Queiroz et al., 2011). However, significant and negative correlation between Pphy/Pi and Phy/β-carotene could underline that improved β-carotene in grain of the drought tolerant inbreds is related to higher phytate content. Considering the positive impact of β-carotene on availability of mineral elements, the negative influence of phytate on availability could be suppressed, irrespectively to non-significant correlation between Phy/β-carotene and Phy/Mg, Phy/Fe, Phy/Mn and Phy/Zn. Table 3. Pearson’s correlation coefficients between molar ratios of observed chemical parameters (phytic

and inorganic phosphorus, phytic acid, β-carotene, Mg, Fe and Zn) in chosen drought tolerant

inbred lines

Pphy/Pi Phy/β-carotene Phy/Mg Phy/Fe Phy/Mn Phy/β-carotene -0.343*

Phy/Mg 0.401* 0.039 Phy/Fe 0.421* 0.005 0.837* Phy/Mn 0.545* -0.192 0.868* 0.876* Phy/Zn 0.608* 0.049 0.445* 0.390* 0.479*

* significant values at the 0.05 probability level

Conclusion Based on obtained results, it could be concluded that among investigated drought tolerant maize inbred lines, inbred L2 could be considered as valuable source for improved Mg, Fe, Mn and Zn availability (particularly for Fe and Zn), based on low Pphy and moderate β-carotene content. Moreover, L1, as inbred line with improved β-carotene content and relative low Phy/Fe ratio, could be considered as a potential source for higher Fe availability.

Acknowledgements This study was supported by Project TR31068 "Improving the quality of maize and soybean by conventional and molecular breeding" from the Ministry of Education, Science and

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Technological Development, Republic of Serbia, and by COST Action FA 0905 “Mineral Improved Crop Production for Healthy Food and Feed”.

References American Association of Cereal Chemists Method (1995). Approved Methods of the AACC, The

association: St. Paul, MN, USA, AACC Method, Pp. 14-50. Anđelković V., Božinović S., Pavlov M., Vančetović J. (2010). Combining abilities for grain

yield of the most drought tolerant maize accessions from the MRI gene bank. Genetics, Plant Breeding and Seed Production, 45th Croatian & 5th International Symposium on Agriculture. Proceedings, 368-371.

Babic M., Andjelkovic V., Mladenovic Drinic S., Konstantinov K. (2011). The conventional and contemporary technologies in maize (Zea mays L.) breeding at Maize Research Institute, Zemun Polje. Maydica 56: 155-164.

Brinch-Pedersen H., Borg S., Tauris B., Holm P.B. (2007). Molecular genetics approaches to increasing mineral availability and vitamin content of cereals. J Cereal Sci 46: 308-326.

Dragičević V., Sredojević S., Perić V., Nišavić A., Srebrić M. (2011). Validation study of a

rapid colorimetric method for the determination of phytic acid and norganic

phosphorus from grains. Acta Periodica Technologica 42: 11-21. Lönnerdal B. (2003). Genetically modified plants for improved trace element nutrition. J

Nutr 133: 1490S-1493S. Ma G., Li Y., Jin Y., Zhai F., Kok F.J., Yang X. (2007). Phytate intake and molar ratios of

phytate to zinc, iron and calcium in the diets of people in China. Eur J Clin Nutr 61:

368-374. Ortiz-Monasterio J.I., Palacios-Rojas N., Meng E., Pixley K., Trethowan R., Peña R.J. (2007).

Enhancing the mineral and vitamin content of wheat and maize through plant breeding. J Cereal Sci 46: 293-307.

Queiroz V.A.V., Guimarães P.E.O., Queiroz L.R., Guedes E.O., Vasconcelos V.D.B., Guimarães

L.J., Ribeiro P.E.A., Schaffert R.E. (2011). Iron and zinc availability in maize lines.

Ciência e Tecnologia de Alimentos 31: 577-583. Underwood E.J., Suttle N.F. (1999). Manganese. In: The Mineral Nutrition of Livestock, CABI

Publishing, USA, Pp. 397-420. Walter Lopez H., Leenhardt F., Coudray C., Remesy C. (2002). Minerals and phytic acid

interactions: is it a real problem for human nutrition? Internat J Food Sci Technol 37: 727-739.

Welch R.M. (2003). Farming for Nutritious Foods: Agricultural Technologies for improved

Human Health. IFA-FAO Agriculture Conference “Global Food Security and the Role of

Sustainable Fertilization” Rome, Italy, 26-28 March 2003. Pp. 1-24. Welch R.M., Graham R.D. (2004). Breeding for micronutrients in staple food crops from a

human nutrition perspective. J Exp Bot 55: 353-364. White P.J., Broadley M.R. (2005). Biofortifying crops with essential mineral elements. Trends

Plant Sci 10: 586-593.

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49

EVALUATION OF SELECTION INDICES FOR DROUGHT TOLERANCE IN MAIZE (Zea mays L.) HYBRIDS

Zoran DUMANOVIĆ, Vojka BABIĆ, Života JOVANOVIĆ, Milomir FILIPOVIĆ,

Violeta ANĐELKOVIĆ*

Maize Research Institute Zemun Polje, Slobodana Bajića 1, 11185 Belgrade, Serbia *avioleta@mrizp.rs

Abstract

Drought strongly affects maize productivity worldwide. In order to detect drought tolerant maize hybrids, experiments with eight maize hybrids (ZP341, ZP427, ZP434, ZP555, ZP560, ZP600, ZP606, and ZP666) were conducted in 2012 and 2013, at six locations under rainfed and irrigated conditions. Drought tolerant indices were evaluated to test the ability to separate more drought tolerant hybrids from those with less stress tolerance. Five stress tolerance indices including stress tolerance index (STI), mean productivity (MP), geometric mean productivity (GMP), stress susceptibility index (SSI), and tolerance index (TOL) were calculated for each genotype. The maximum value of STI (0.776), MP (9.71), and GMP (9.51) were recorded for hybrid ZP555. Correlation coefficients revealed that STI had the highest value in both conditions with grain yield. Three dimensional graphs, based on the stress tolerance index and grain yield in rainfed and irrigated conditions, as well as biplot analysis separated groups of hybrids with different level of drought tolerance and grain yield.

Key words: biplot, grain yield, maize, stress tolerance indices. Introduction

Maize is the cereal with the largest worldwide grain production in five year period (2008-2013, http://faostat3.fao.org/download/Q/QC/E) and final grain yield is highly affected by drought. In 2012, as implication of drought, maize yields and production in the United States were reduced by 21% and 15% respectively, compared to 2009-11 mean levels (http://quickstats.nass.usda.gov). In Serbia, maize is the most important crop grown mainly without irrigation. In 2012, both maize total production and average grain yield were reduced about 48%, and in 2013, maize grain yield was reduced by 9%, compared to 2009-2011. One of possibilities to reduce the yield losses is breeding of maize hybrids that tolerate drought and, at the same time have stable yield across environments. Improvement in drought tolerance is a long term and not easy to obtain due to strong genotype x environment interaction. Various methods

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were proposed for measurement of drought tolerance, and application of different indices based on mathematical relation between stress and optimum condition were recommended as a very suitable (Rosielle and Hamblin, 1981; Fernandez, 1992). The aim of present study was to evaluate efficiency of different selection indices in identification of maize hybrids that can be recommended for growing in both, drought prone areas and non-stress conditions. Material and methods

The study was carried out in 2012 and 2013 at six locations (Becej, Deronje, Kurjace, Leskovac, Loznica and Svilajnac) in two independent experiments. In non-stress conditions, irrigation was applied seven times based on crop water requirements during growth periods. Maize hybrids in the rainfed experiments were not additionally irrigated (water was received only from precipitations). The drought tolerance of eight maize hybrids (ZP341, ZP427, ZP434, ZP555, ZP560, ZP600, ZP606, and ZP666), belonging to different FAO maturity groups, was evaluated in experiments using randomized complete block design (RCBD) with two replications. The hybrids were grown in three-row plots, with the distance between rows of 0.75 m and different distance within the row, according to FAO maturity group. Thus, the final planting densities were as follows: 69000 plants ha-1 (for hybrids ZP341, ZP427 and ZP434), 64900 plants ha-1 (for hybrids ZP555 and ZP560) and 59000 plants ha-1 (for hybrids ZP600, ZP606 and ZP666).

Standard agricultural practice according to maize hybrid requirements for achieving genetic potential was applied.

For every genotype drought tolerance indices were calculated based on their grain yield in both conditions, using the following equations:

Tolerance index TOL= /0 * / (Rosielle and Hamblin, 1981)

Mean productivity index MP= 12314+ (Rosielle and Hamblin, 1981)

Stress tolerance index STI= 14512�162�7 (Fernandez, 1992)

Geometric mean productivity GMP= 8�/0��/� (Fernandez, 1992)

Stress susceptibility index SSI= ��9:9;<= SI=1->164162? (Fisher and Maurer, 1978)

Yp = potential yield of genotype in non-stress conditions (I-irrigated in the present study). Ys = yield of genotype in stress conditions /60 = average yield of all genotypes under non-stress conditions /6 = average yield of all genotypes under stress conditions.

Correlation analysis was also carried out to determine the associations between obtained grain yield of hybrids and indices. The differences among hybrids and drought indices were evaluated by means of Principle Component Analysis (PCA), Statistical analysis was performed by using SPSS 15.0 for Windows Evaluation. Results and discussion

Optimal precipitation for maize growing in Serbia is 459 mm (Vasic and Kerecki, 1988), but besides total amount, distribution during season is very important. The most critical stages for final grain yield are flowering and grain filling periods (Grant et al., 1989). Average

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temperatures and precipitation for vegetative period, as well as deficit in precipitation according to Vucic (1991) are presented in Table 1 (a and b). Table 1. Average temperatures (tavg), number of days with maximum temperatures above 30ºC

(ND>30ºC), and above 35ºC (ND>35ºC), precipitation (mm), optimal precipitation for maize

(according to Vucic, 1991), during growing season in 2012 (a) and 2013 (b)

a) Month tavg ND>30ºC ND>35ºC precipitation

(mm) optimal

precipitation deficit of

precipitation April 13.6 1.0 0.0 54.9 35 (-19.9) May 16.6 3.3 0.0 108.6 75 (-33.6) June 22.5 18.7 3.2 26.8 80 53.2 July 25.2 22.0 13.0 69.8 100 30.2

Avgust 23.5 22.0 9.2 3.5 95 91.5 September 20.3 12.3 0.7 23.4 40 16.6

Σ 79.3 26.0 286.9 425 138.1 b)

Month tavg ND>30ºC ND>35ºC precipitation (mm)

optimal precipitation

deficit of precipitation

April 13.2 3.2 0.0 23.9 35 11.1 May 18.2 5.0 0.0 97.6 75 (-22.6) June 20.2 9.3 2.8 57.9 80 22.1 July 22.3 15.8 2.3 29.4 100 70.6

Avgust 23.8 19.3 8.3 34.2 95 60.8 September 16.6 0.7 0.0 66.1 40 (-26.1)

Σ 53.3 13.5 322.1 425 102.9

Figure 1. Biplot based on first two principal component axes (PC1 and PC2) of maize hybrids and

drought tolerance indices

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Biplot analysis (Figure 1), revealed that the PCA1 explained 79.3% for drought stress conditions (with Yd, MPd, STI, and GMP) of total variation (data not presented) and could be labeled as yield potential and drought tolerance. Considering high and positive value of this PCA on biplot, hybrids ZP606 and ZP666 will be superior under both, rainfed and irrigated conditions, with high PC1 and low PC2. The PCA2 explained 20.7% of total variation (with SSI and TOL) and represent the dimension of tolerance and low susceptibility. In opposite direction to them are hybrids ZP600 and ZP434 which exhibited lowest level, both for PCA1 and PCA2. There were no hybrids with low score of PCA1 and high level of PCA2, that could be classified as low-yielding and unstable, according to Kaya et al., 2002. Biplot analysis for separation of genotypes according to drought tolerance was applied in different species: maize (Khodarahmpour and Hamidi, 2011), sunflower (Ghaffari et al., 2012), wheat (Farshadfar et al., 2012), grass pea (Basaran et al., 2012), etc.

Among indices and grain yield under rainfed and irrigated conditions, the strongest correlations were with STI. In many studies (Fernandez, 1992; Khodarahmpour and Hamidi, 2011) STI is recommended as the most appropriate measure of drought tolerance. Although a correlation analysis is very useful in interpreting relation between traits, it is not valuable for performance estimation of individual genotype. Therefore, three-dimensional analysis between STI and grain yields is performed and presented on Figure 2.

Table 1. Correlation coefficients between drought indices and grain yield under drought stress

Index GYI GYD STI MP GMP TOL SSI GYI 1 GYD 0.660 1 STI 0.953*** 0.859** 1 MP 0.971*** 0.821* 0.998*** 1

GMP 0.951*** 0.827* 0.999*** 0.997** 1 TOL 0.916** 0.304 0.752* 0.794* 0.748* 1 SSI 0.813* 0.100 0.598 0.650 0.594 0.978*** 1

STI-stress tolerance index, MP-mean productivity index, GMP-geometric mean productivity index, TOL-tolerance index, SSI-stress susceptibility index, GYI- grain yield in irrigated conditions, GYD-grain yield in drought stress, *,** and *** refer to level of significance, P<0.05, P<0.01 and P<0.001 respectively.

Three-dimensional plot using Yi (x-axis), Yd (y-axis) and STI (z-axis) presented relations between index and yield in rainfed and irrigated conditions and separating hybrids into groups (Fernandez, 1992). It was previously confirmed that in breeding for drought tolerance the most important is group A, with hybrids ZP555, ZP666 and ZP560, which show superiority in both non-stress and stress condition. Hybrids belonging to group D (ZP427, ZP434 and ZP600) are with weak performance, e.g. low yield under stress and non-stress conditions (Fernandez, 1992). In our case they could not be considered as drought sensitive, particularly ZP427 and ZP434. Those hybrids are with shorter vegetation and they are finishing flowering and grain filling before terminal drought (in July), and by drought “escape” achieving stable, but lower yield across environments. Also, correlations between grain yield and SSI were low and non-significant and none of tested hybrids could be classified as drought sensitive. According to our results, there were no hybrids in group B (high yield only in non-stress conditions) that is very desirable because of small percentage of irrigated fields in Serbia. It has been stated (Blum, 1997) that under stress conditions only genotypes with stress adaptive genes performed stable yield.

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Figure 2. Three dimensional plot of yield in irrigated (Yi) and drought stress (Yd) conditions with stress tolerance index (STI)

Conclusion

Among different indices that have been evaluated in two years study STI, GMP and MP have similar relations with grain yield under both, irrigated and non-irrigated conditions. Based on the highest and most significant correlation, STI was the most appropriate for breeders usage. STI could be recommended as very useful to separate hybrids with better tolerance to drought (either because of lower water requirements or less yield reduction in drought conditions).

Acknowledgments

This work was supported by Ministry of Education, Science and Technological Development, Republic of Serbia, through the project TR31068 “Improving the quality of maize and soybean by conventional and molecular breeding”.

References Basaran, U., Acar Z., Karacan M.and Onar A.N. (2012). Variation and correlation of morpho-

agronomic traits and biochemical contents (protein and -odap) in Turkish grass pea (Lathyrus sativus L.) landraces. Turk. J. Field Crops. 18:166-173.

Farshadfar, E., Jamshidi B. and Aghaee M. (2012). Biplot analysis of drought tolerance indicators in bread wheat landraces of Iran. Int. J. Agr. Crop Sci. 4:226-233.

Fernandez, G.C.J. (1992). Effective selection criteria for assessing plant stress tolerance. In: Proceedings of the Int. Symposium on Adaptation of Vegetables and Other Food Crops in Temperature and Water Stress, 13-18 August, p.257-270. Tainan, Taiwan.

Fischer, R.A. and Maurer R. (1978). Drought resistance in spring wheat cultivars. I. Grain responses. Aust J Agr Res. 29:897-912.

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Ghaffari, M., Toorchi M., Valizadeh M. and Shakiba M.R. (2012). Morpho-physiological screening of sunflower inbred lines under drought stress conditions. Turk. J. Field Crops. 17: 185-190.

Grant R.S., Jackson B.S., Kiniry J.R., Arkin G.F. (1989). Water deficit timing effects on yield components in maize. Agron. J. 81: 61-65.

http://faostat3.fao.org/download/Q/QC/E (Accessed June 2014) http://quickstats.nass.usda.gov (Accessed June 2014) Kaya Y., Palta C., Taner S. (2002). Additive main effects and multiple interactions analysis of

yield performance in bread wheat genotypes across environments. Turk. J. Agric. Hort. 26:275-279.

Khodarahmpour Z. and Hamidi J. (2011). Evaluation of drought tolerance in different growth stages of maize (Zea mays L.) inbred lines using tolerance indices. Afr. J. Agric. Res. 10:13482-13490.

Rosielle, A.A. and Hamblin J. (1981). Theoretical aspects of selection for yield in stress and non-stress environments. Crop Sci. 21 (6): 943-946.

Vasić,G. and Kerečki B. (1988). Drought and effect of irrigation on maize production. Suša i efekat navodnjavanja na proizvodnju kukuruza.. Improvement of maize production and utilization – Maize 88. Book of papers 103-116. (in Serbian)

Vučić. N. (1991). Reduction patterns in yield variations in Vojvodina. Book of papers Institute for field and vegetable crops Novi Sad. (Putevi redukcije magnitude oscilacija prinosa u Vojvodini. Zbornik radova Naučnog instituta za ratarstvo i povrtarstvo, Novi Sad), Sv. 9: 5-8. (in Serbian).

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WHEAT GENOTYPE RESPONSE ON BACTERIAL AUXIN TREATMENT

Snežana ĐORĐEVIĆ1, Dragana STANOJEVIĆ2, Dušan KOVAČEVIĆ1, Željko DOLIJANOVIĆ1, Zvonko RADAN3

1Faculty of Agriculture, University of Belgrade, Nemanjina 6, Zemun-Belgrade, Serbia

2Biounik, Research and Development Center, Šimanovci 22310, Serbia 3Fermopromet, Majške Međe-Bolman, Croatia

Corresponding author: stanojevicd78@gmail.com

Abstract

Biostimulator (based on bacterial auxin) applied at wheat seeds had very strong influence on root and shoot length of all 7 genotypes tested but there was no significant difference in seed germination percentage between control seeds and seeds treated with different auxin concentration, shown by analysis of variance. Most effective on shoot height and root length was auxin concentration from 0.5 µg/ml. This influence clearly demonstrates importance of auxin concentration on steam height and root length in different wheat genotypes. Results of this study showed that auxin had positive effects in relation to stimulate all wheat genotype tested (different length of the growing season; early, medium early and late variety). Key words: Bacillus spp., auxin, wheat seeds, genotype. Introduction

Wheat (Triticum aestivum L.) is the most important grain that is grown around the world, while the sown areas in Serbia annually range between 500 000 and 600 000 ha. Wheat is used for human food, animal feed and for other purposes. Wheat is second by the amount of the total production of field crops in Serbia, behind corn (Jeftić, 1986).

The main role of wheat as food is derived from the properties of its proteins, which are unique among agricultural plants. Wheat products, above all bread, represent the basic elements of human nutrition, because wheat bread is in diet of over 70 % of the population on the planet. The presence of wheat and other small grains in the human diet ranges from 25 % in developed and 80 % in developing countries, which means that for a large part of the human population

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small grains, are only source of carbohydrates, protein, B vitamins and minerals. By spending wheat products Serbia occupies one of leading places in Europe (Roljevic et al., 2011).

In front of selection of field crops are big challenges; in the classical production with the creation of genotypes for the intensive conventional production and in organic production where some specific criteria are important (Murphy et al., 2007). Excessive use of nitrogen fertilizers in top-dressing of spring crops can have negative effects on the environment. Biostimulators are becoming more interesting in non-leguminous crops such as wheat and corn, because they can satisfy the need of these crops for nitrogen in phase of top-dressing (Bahrani and Hagh, 2010). Biostimulators are defined as compounds of biological origin and act by increasing natural capabilities of plants to grow and develop, especially under unfavorable soil and climatic conditions.

Aim of this work was to establish influence of bacterial auxin, on different wheat genotypes (different origin and the length of the growing season-late, medium late and early varieties); following parameters such as root length, steam height and seed germination percentage. Materials and methods Identification of the bacterial isolates

Bacterial strains (Bacillus subtilis, B. megaterium and B. circulans) were isolated from maize rizosphera. The 3 isolates were tentatively identified following Bergey’s Manual of determinative bacteriology (Holt et al, 1994). Identification was carried out by subjecting the isolates to cultural, morphological (colony morphology and pigmentation), microscopic (Gram staining and motility), biochemical (utilization of carbon sources and enzymatic activity) and physiological characteristics (temperature, pH, salt and sugar tolerance) and finally identification was done by DB BBL Crystal Gram positive identification system. Assay for IAA production

The production of Indole acetic acid (IAA) was assayed by using Salkowski method (Gordon and Weber, 1951; Glickman and Dessaux, 1995). The bacteria were inoculated in to the nutrient broth. After 48 h of growth at 180 rpm/28°C, the bacterial culture was centrifuged (6000 rpm/ 30 min.) and 1 ml of supernatant was mixed with 2 ml of Salkowski reagent (2.0 ml 0, 5 M FeCl3 + H2SO4). The reaction mixture was incubated at room temperature for 30 min and then light absorbance was measured immediately at 535 nm. The amount of IAA produced was calculated using the standard curve prepared with known concentration of IAA (0.5, 1, 2, 5, 7.5, 10 µg/mL filtrated IAA).

Avena straight growth test

Oat seed, (Avena sativa L.) was used in bioassay Avena straight-growth (Sirois, 1966; Nitsch and Nitsch, 1956).Coleoptiles (5 mm length) of 3 days old seedlings were dipped in 2 ml of tested solutions and were incubated at 28°C/24h at 20 rpm. Length of section was measured before and after the experiment. A standard activity curve relating coleoptile growth to the dilutions of pure synthetic concentration of IAA was developed as a control. Final length for oat coleoptiles was measured after the tissues were floated in solutions containing different

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concentrations of each bacteria tested and their mixture. The concentration of the auxin was calculated from the Avena straight-growth test by interpolation on the IAA curve of the controls. Seed germination

Certified wheat seeds (Triticum aestivum L.), were obtained from Istitute Tamis, Pancevo, Serbia. Seeds were disinfected with 2 % NaClO, rinsed thoroughly in sterile distilled water and then inoculated with bacterial suspension. Two days old cultures were centrifuged at 6.000 rpm/ 30 min. Treatment was consisting a combination of three bacteria. Seeds were germinated 8 days at 20°C (16/8h), in germination chamber. Germination test was conducted in four replications of 100 seeds each by adopting between paper methods as described by ISTA procedures (ISTA, 2009), while 4×5 randomly selected normal seedlings were used to measure the root and shoot length. Seeds treated with commercial fungicide Mankogal S were used as control. The final count of germination and the measurement of the root and shoot lengths were estimated on the 8th day. We tested different concentration of bacterial auxin in relation to 7 different wheat genotypes; early variety: Nikol, Dika, Solehio; medium early: Zvezdana, Sirtaki, Farineli Yunta Quatro; late variety: Balaton. Statistical analysis

For all experiments, the data were subjected to statistical analysis using software SPSS 18 program. Data were subjected to analysis of variance (ANOVA). Results and discussion

The analysis of variance was calculated according to randomize complete block design with two factors: genotype and different auxin concentration and their interaction in relation to steam height, root length and seed germination percentage. Steam height (Table 1), root length (Table 2) and seed germination percentage (Table 3) were significantly influenced by genotypes tested. Statistical data analysis showed significant differences between almost all genotypes tested; only difference between Farinely Yunta Quatro and Solehio wasn’t statistically significant in relation to tested characters. All auxin concentrations significantly influenced steam height as well as root length (Table 1 and 2). Auxin concentration had very little effect in relation to seed germination percentage (Table 3). Control (seeds without auxin) had significantly lower steam height and root length in relation to all concentration of auxin tested (Table 4). In addition to the effect of the main factors, their interactions (Genotype*Auxin,) were also highly significant in relation to these characteristics tested. Differences in variance between control seeds and seeds treated with different auxin concentrations of all 7 genotypes tested clearly demonstrate auxin influences in relation to steam height and root length (Table 4.). Table 1. Analysis of variance for steam height (cm)

Source Type III Sum of

Squares df Mean Square F Sig. Corrected Model 5752,792(a) 34 169,200 32,542 ,000 Intercept 116980,301 1 116980,301 22498,707 ,000 Genotype 4038,127 6 673,021 129,441 ,000 Auxin 999,539 4 249,885 48,060 ,000 Genotype * Auxin 715,126 24 29,797 5,731 ,000 R Squared = ,625 (Adjusted R Squared = ,605)

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Table 2. Analysis of variance for root lenght (cm)

Source Type III Sum of

Squares df Mean Square F Sig. Corrected Model 6763,535(a) 34 198,928 10,294 ,000 Intercept 233304,378 1 233304,378 12072,331 ,000 Genotype 1340,760 6 223,460 11,563 ,000 Auxin 4083,933 4 1020,983 52,831 ,000 Genotype * Auxin 1338,842 24 55,785 2,887 ,000 R Squared = ,345 (Adjusted R Squared = ,311) Table 3. Analysis of variance for seed germination (%)

Source Type III Sum

of Squares df Mean Square F Sig. Corrected Model 1437,243(a) 34 42,272 9,138 ,000 Intercept 1303908,007 1 1303908,007 281853,506 ,000 Genotype 1266,443 6 211,074 45,626 ,000 Auxin 46,743 4 11,686 2,526 ,045 Genotype * Auxin 124,057 24 5,169 1,117 ,339 R Squared = ,747 (Adjusted R Squared = ,666)

All 7 genotypes were found to be improved in growth parameters (shoot length, root length) significantly (p < 0.05) under auxin treatments as compared to the control seedlings, under laboratory condition. The highest increases in steam height were exhibited by auxin concentration in range from 0.5 µg/ml. Genotype Dika had highest increase, while the smallest had genotype Nikol. Highest root increase was recorded in genotype Solehio, smallest in Zvezdana. The most stimulating concentration for root length was 0.5µg/ml (Table 4.). Control seeds (without auxin) in all genotypes showed lower increase of root length and steam height compared to auxin treatments. Seed germination percentage was significantly affected by wheat genotype. There were no significant differences between different auxin concentration and control in relation to this character tested. Auxin concentration as well as their interaction (genotype*concentration auxin) didn’t show statistically significant influence. Similar to our results Akbari G. et al., (2007), found that auxin increased hypocotyl length, seedling fresh and dry weight and hypocotyl dry weight, but did not find differences in germination percentage and radicle length. Auxin biosynthesis by B. amyloliquefaciens showed the potential importance of Auxin derived by Rhizobacteria to stimulate root exudation in Triticum aestivium (Talboys et al., 2014). Improvement of root and shoot length observed in this study is due to hormone auxin, which triggers cell division, elongation and differentiation (Hussain, 2010). The proposed mechanism for the auxin effect on cell elongation is triggered by the hormone induced opening of calcium channels in the plasmalemma which, in turn, might cause changes on calcium homeostasis in the cytosol (Didonet and Magalhães, 1993). Experimental evidence which supports the idea that auxin contributes root elongation was Chen and Xiong study, (Chen and Hyong, 2005) in which they showed that pdx1 mutants are unable to produce pyridoxal phosphate, vitamin B6, which in turn impairs the capacity of plants to convert tryptophan to IAA.

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Table 4. Mean values (interaction effect; genotype*auxin) for steam height (cm), root length (cm) and

seed germination (%) Steam height(cm) Root length(cm) Seed germination (%)

Genotype Auxin(µg/ml)

Std. Mean Deviation N

Std. Mean Deviation N

Std. Mean Deviation N

Dika 0.05

0.1 0.25 0.5

Control Total

17,28 3,47 20 17,29 2,79 20 17,84 3,43 20 18,36 2,62 20 15,9 8,41 20 17,3 4,65 100

18,49 3,69 20 16,90 3,30 20 20,54 18,30 20 16,20 3,57 20 11,65 3,83 20 16,75 9,11 100

92,75 3,09 4 86,25 4,57 4 90,50 3,87 4 88,75 4,57 4 88,75 2,36 4 89,40 4,03 20

Solehio 0.05 0.1

0.25 0.5

Control Total

14,37 1,28 20 13,67 1,02 20 14,02 1,37 20 14,19 1,27 20 10,78 1,98 20 13,40 1,93 100

20,99 3,16 20 22,22 2,42 20 22,41 2,33 20 23,28 2,67 20 13,79 2,64 20 20,54 4,34 100

98,00 2,70 4 97,75 1,89 4 99,00 ,81 4 99,00 ,81 4 97,75 1,2 4 98,30 1,5 20

Sirtaki 0.05 0.1

0.25 0.5

Control Total

13,28 2,46 20

15,75 1,20 20 16,96 1,59 20 17,87 2,57 20 10,26 1,65 20 14,82 3,37 100

18,20 3,17 20 18,06 2,55 20 17,37 2,11 20 17,16 1,17 20 14,79 2,09 20 17,11 2,58 100

98,00 2,70 4 97,75 1,89 4 99,00 ,81 4 98,75 ,95 4 99,50 ,57 4 98,60 1,56 20

Nikol

0,05 0.1

0.25 0.5

Control Total

10,67 1,51 20 11,50 1,74 20 11,21 ,977 20 10,07 ,898 20 9,52 1,19 20 10,59 1,47 100

17,22 2,95 20 18,42 2,06 20 21,33 2,55 20 22,72 1,74 20 12,76 2,72 20 18,49 4,23 100

97,25 1,70 4 97,50 1,91 4 98,25 ,95 4 97,75 ,95 4 95,75 2,0 4 97,30 1,65 20

Balaton 0.05 0.1

0.25 0.5

Control Total

12,51 1,33 20 11,15 1,13 20 12,57 1,44 20 12,83 1,08 20 7,30 1,31 20 11,27 2,42 100

17,22 2,95 20 18,42 2,06 20 21,33 2,55 20 22,72 1,74 20 12,76 2,72 20 18,49 4,23 100

97,25 ,95 4 97,25 1,70 4 96,50 2,51 4 98,25 ,95 4 95,50 1,29 4 96,95 1,70 20

Zvezdana 0.05 0.1

0.25 0.5

Control Total

10,32 1,62 20 11,26 ,97 20 10,06 1,41 20 10,50 1,62 20 7,60 3,10 20 9,95 2,23 100

15,76 4,06 20 18,73 3,62 20 18,78 3,83 20 16,56 6,01 20 13,61 3,58 20 16,69 4,66 100

96,00 1,82 4 96,00 2,82 4 96,50 2,08 4 98,50 1,73 4 95,00 3,36 4 96,40 2,47 20

Farineli Yunta Quatro 0.05

0.1 0.25 0.5

Control Total

12,74 1,08 20 14,86 1,57 20 12,39 1,30 20 12,74 1,40 20 12,74 1,08 20 13,09 1,56 100

20,36 3,66 20 21,95 4,07 20 20,18 2,52 20 20,14 4,14 20 4,45 20 19,70 4,27 100

98,00 2,16 4 98,75 ,50 4 99,00 ,81 4 99,25 ,50 4 98,00 1,41 4 98,60 1,23 20

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These plants had a short primary root with a reduced root meristem size. Higher IAA and cytokinin contents in the grain at the early grain filling stage may promote the division of endosperm cells thus constitute a powerful sink (Jenner and Rathjen, 1978) and enhance assimilate transport and its accumulation in the developing grains. Results of Houshmandfar and Asli (2011) suggest that cytokinins and IAA levels of grains during the early phase of grain development play an important role in regulating grain filling pattern and dry matter accumulation. It might be possible to improve grain weight by increasing cytokinins and IAA levels in the grain, especially at the early filling stage. Results from this study showed that shoot and root height of different wheat genotypes can be effectively enhanced by auxin from bacterial strains of genus Bacillus. Auxin treatment had equal positive effect no matter of the length of the growing season of wheat genotypes tested. References Akbari, G., Mohammad S.A., Sanavy M., Yousefzadeh S. (2007). Effect of Auxin and Salt

Stress (NaCl) on Seed Germination of Wheat Cultivars (Triticum aestivum L.). Pakistan Journal of Biological Science, (10) 15: 2557-2561.

Bahrani, A. and Hagh M. (2010). Flag Leaf Role in N Accumulation and Remobilization as Affected by Nitrogen in a Bread and Durum Wheat Cultivars. American-Eurasian Journal of Agriculture & Environmental Science, 8 (6): 728-735.

Chen, H. and Xiong L. (2005). Pyridoxine is required for post-embryonic root development and tolerance to osmotic and oxidative stresses. The Plant Journal, 44(3):396-408.

Didonet, A. and Magalhães (1993). The role of auxin-like compounds in plant growth promoting rhyzobacteria: the wheat-azospirillum association. Revista Brasileira de Fisiologia Vegetal, 5(2):179-183.

Glickman, E. and Dessaux Y.A. (1995). Critical examination of the specificity of the salkowski reagent for indolic compounds produced by phytopathogenic bacteria. Applied Environmental Microbiology, 61: 793-796.

Gordon S.A. and Weber R.P. (1951). Colorimetric estimation of indolacetic acid. Plant Physiology, 2: 192-195.

Holt, J.G., Krieg N.R., Sneathm P., Staley J.T., Williams S.T. (1994). Bergey’s Manual of Determinative Bacteriology, 9th edn. Baltimore, MD: Williams and Williams.

Houshmandfar, A., D.E. Asli (2011). An Appraisal of the Yielding Ability and its Attributes among Different Cultivars of Bread Wheat under Field and Pot Conditions. Advances in Environmental Biology, 5(6): 1367-1369.

Hussain, A (2010). Phytohormones cytokinin and iaa from microbial origin and their impact on plant growth. Phd thesis. Department of microbiology and molecular genetics, university of the pubjab

ISTA (2009). Handbook of Seedling Evaluation, 3rd Ed. ISTA Germination Committe, Zurich, Switzerland.

Jeftić S. (1986). Wheat (Pšenica), Naučna knjiga. Jenner, C.F., A.J. Rathjen (1978). Physiological basis of genetic differences in the growth of the

grains of six varieties of wheat. Australian Journal of Plant Physiology, 5: 249-262. Murphy, K. M., Campbell K. G., Lyon S. R., Jones S. S. (2007). Evidence of varietal adaptation

to organic farming systems, Field Crops Research, Vol. 102, pp. 172–177.

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Nitsch, J.P. and Nitsch C. (1956). Studies on the Growth of Coleoptile and First Internode Sections. A New, Sensitive, Straight-Growth Test for Auxins. Plant Physiol., 31 (2):94–111.

Roljević, S., Cvijanović D., Sarić R. (2011). Genetički resursi pšenice u svetu i Srbiji. Zbornik naučnih radova Instituta PKB Agroekonomik, Vol. 17, br. 1-2, str. 27-33.

Sirois, J.C. (1966). Studies on Growth Regulators. I. Improved Avena Coleoptile Elongation Test for Auxin 1Plant Physiol.;41(8): 1308–1312.

Talboys, J., Owen W., Healey R., Withers P. Jones D. (2014). Auxin secretion by Bacillus

amyloliquefaciens FZB42 both stimulates root exudation and limits phosphorus uptake in Triticum aestivium. BMC Plant Biology, 14:51.

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HIGH INCIDENCE OF RADIAL CHROMOSOMES IN CIRCULATION LYMPHOCYTES PREDICTS BONE MARROW FAILURE IN FANCONI ANEMIA

PATIENT – A CASE REPORT

Jelena FILIPOVIĆ1, Andreja LESKOVAC1, Ana VALENTA ŠOBOT, Sandra PETROVIĆ1, Dragana VUJIĆ2, Gordana JOKSIĆ1

1Vinča Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia 2School of Medicine, University of Belgrade, Belgrade, Serbia

Corresponding author: Gordana Joksić, Vinča Institute of Nuclear Sciences, Mike Petrovića

Alasa 12-14, 11001 Belgrade, Serbia, tel: +381113408565, fax:+3813408787 , e-mail: gjoksic@vinca.rs

Abstract

Fanconi anemia is a genetic disorder characterized by bone marrow failure, increased rate of chromosomal breaks and radial formation. In this study, we compared the lymphocyte findings in FA-D2 patient obtained during the two routine checkups performed six months prior to bone marrow transplantation (BMT) and four years before BMT. Apart from the results of the analysis of the baseline and DEB induced chromosomal aberrations in peripheral lymphocytes, we analyzed the involvement of telomeres in radial formation and in complex chromosomal aberrations. At the second examination time, six months prior to BMT, among 821 metaphases analyzed, 7.06% radials were found, a considerably more compared to the previous state observed four years before (3%). Two types of radials were observed; the first type (50%) arose as a result of the simple exchange between two non-homologous chromosomes, whereas the second type of radials was a consequence of either exchange between telomeres of one and non-telomeric sequences of another chromosome (31.03%) or telomere fusions (18.97%). The 8.62% of all radials were complex and consisted of three or more chromosomes with fused telomeres. In conclusion, the high percentage of radials mostly arises as a consequence of extremely shortened telomeres and their appearance in peripheral blood lymphocytes should be considered as an early indication for BMT requirement.

Key words: Fanconi anemia, incidence of radials, telomeres, bone marrow failure Introduction

Fanconi anemia (FA) is a rare recessive disorder characterized by congenital anomalies, progressive pancytopenia that leads to bone marrow failure, hematological malignancies and

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solid tumors (Fanconi, 1967). It belongs to one of the chromosomal instability syndromes with an incidence of 1 per 350,000 births (Auerbach et al., 1989). Great clinical variability of disease is due to the existence of several complementation groups. Up so far 15 Fanconi anemia complementation groups have been reported (FA-A, B, C, D1, D2, E, F, G, I, J, L, M, N, O and P) with corresponding genes named FANCA-FANCP. They are all involved in the FA/BRCA DNA damage repair pathway and play a pivotal role in the cellular response to stress induced by DNA alkylating agents (Kee and D'Andrea, 2010). The first molecular investigation of Fanconi anemia in Serbia revealed that the most frequent complementation group is FA-D2, which is rarely present in European population (Vujic et al., 2014). Up so far it has been described that FA-D2 patients display an earlier onset of hematological manifestations compared to most other FA complementation groups (Kalb et al., 2007), and a broad spectrum of developmental abnormalities clinically described as major and minor abnormalities. FANCD2 is a central player in a DNA repair network and checkpoint responses to DNA damage as it serves as a link between the “upstream” and “downstream” proteins in this pathway (Bogliolo and Surrallés, 2000). Thus, it is not surprising that dysfunction of this protein can cause a wide-range spectrum of inherited abnormalities. On the cellular level FA cells show increased sensitivity to interstrand cross-linking (ICL) agents such as mitomycin C (MMC) and diepoxybutane (DEB) which are used for the diagnosis of Fanconi anemia (Auerbach, 2009). When exposed to these agents FA cells show increased frequency of chromosomal and chromatid breakages, chromosomal radial formation, prolonged cell-cycle arrest in G2/M phase of the cell cycle and reduced cell survival (Kaddar and Carreau, 2012; Petrovic et al., 2013). Additionally, there is increasing number of evidence that the telomeres in FA cells display accelerated shortening rate accompanied with a disturbed capping function (Adelfalk et al., 2001; Joksic et al., 2012).

Chromosomal radial figures are structural chromosome aberrations described in many chromosomal instability syndromes (Cohen and Grey, 1989). They are one of the main cytogenetic hallmarks of FA cells. Depending on their appearance and the number of chromosomes involved in their formation, they can be classified as triradials, quadriradials and multiradials. There are several proposed manners of their formation, as it shall be discussed later on.

However, a precise mechanism of chromosomal radial figures formation, especially complex radials, has not yet been precisely determined. Since telomere shortening and telomere erosion in leukocytes are considered as prognostic signs that directly correlate with the severity of the bone marrow failure, the probability of developing aplastic anemia and with the age of onset for myelodysplasia and tumors (Li et al., 2003), our aim was to investigate the role of telomeres in radials formation. Furthermore, there is no literature data considering the telomere involvement in radials formation or prediction of the clinical outcome based on the frequency and structure of the radials.

In this study, we compared the data obtained from FA-D2 patient six months prior to bone marrow transplantation (BMT) with the data collected four years before BMT when we actually confirmed that patient belongs to the FA-D2 complementation group.

Materials and methods

The Ethical Committee of Mother and Child Health Care Institute of Serbia provided samples and approved lymphocytes analysis. Patient’s parents signed an informed consent regarding this investigation.

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Blood cultures During a routine control, the patient’s peripheral blood was collected into heparinized

vacutainers. Aliquots of heparinized whole blood (0.5 mL) were set up in cultures containing PBmax-karyotyping medium (Invitrogen-Gibco, Paisley, UK) and treated with DEB (Sigma Chemicals Co., Germany) (final concentration 0.1µg/mL) 48 hours after the culture initiation. Cells were harvested 72 h after initiation of the cultures with addition of colchicine (Sigma-Aldrich, Munich, Germany) during the last 3 h (final concentration 2.5 µg/mL). Cells were collected by centrifugation and treated with hypotonic solution (0.56% KCl). Cell suspensions were fixed in methanol/acetic acid (3:1), washed three times with fixative and dropped onto clean slides.

Inverse DAPI staining

Slides were incubated overnight at the room temperature, afterwards dehydrated in series of ethanol (70%, 95% and 100%, respectively, five minutes each), and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI)-containing vectashield solution (Vector Laboratories Ltd, Peterborough, UK). Metaphase analysis

Metaphase spreads were analyzed according to the International System for Human Cytogenetic Nomenclature (ISCN, 2005) using the epifluorescent AxioImager A1 microscope (Carl Zeiss, Jena, Germany) and the IKAROS software (MetaSystems, Germany). The baseline level of chromosomal aberrations and DEB–induced chromosomal aberrations were examined. At least 500 metaphase spreads were analyzed each time. The latest analysis revealed results obtained on 821 metaphase spreads. Telomere fluorescent in situ hybridization (FISH)

After metaphase spreads analysis DAPI was removed, and after subsequent appropriate wash steps slides were hybridized with a telomere PNA oligonucleotide probe (TTAGGG)n directly labeled with Cy5 (Panagene, Korea) and left in a dark humidified chamber for 2 hours at the room temperature. Afterwords, the slides were washed in 70% formamide in 20xSSC and stained with DAPI. Analysis was performed using AxioImager A1 microscope (Carl Zeiss), IKAROS software (MetaSystems) and the computer software ImageJ version 1.44. Results

The results of lymphocyte analysis in the samples obtained from FA-D2 patient six months before BMT and four years before BMT are shown in Figure 1. Representative metaphases with radial formation are presented in Figure 2. Telomere involvements in radials formation are presented in Figure 3.

Among 821 metaphases analyzed six months before BMT 7.06% radials were found, a considerably more compared to the previous examination-3% (Figure 1.).

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Figure 1. Percentage of radials found before bone marrow transplantation and four years before

Figure 2. Metaphases with radials formation (A) and chromosome fusion (B) at first examination

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Figure 3. Types of radials depending on telomere involvement; 1- simple exchange between two non-

homologous chromosomes, no telomere involvement, 2a- exchange between telomeres of one and non-telomeric sequences of another chromosome and 2b- telomere fusions

Lymphocyte findings in FA-D2 patient six months prior to BMT are presented in Figures 3. and 4.

Figure 4. Involvement of telomeres in radial formation: (A) telomere-telomere fusion between

one chromatid of non-homologous chromosomes, (B) complex radial arising from telomere-telomere fusion of several chromosomes, (C) complex radial arising from telomere-telomere fusions and simple exchange between several chromosomes

Discussion

Longitudinal study of the incidence and types of chromosomal aberration in this patient showed that at the first examination time, all observed radials were a consequence of a simple exchange between two non-homologous chromosomes, and telomeres were no involved in their formation. No complex radials were found. However chromosomal fusions were observed, in a very low incidence of 0.1%, i.e. typical example of chromosomal fusion is dicentric chromosome unaccompanied with acentric fragment (Figure 2. B).

Lymphocyte findings 4 years later revealed more than doubled incidence of radials. Combining inverse DAPI staining technique and FISH with PNA telomere probe (Figure 4.), two types of radials were found: first type (50% of all radials) emerged as a result of simple exchange

50%

31%

19%1

2a

2b

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between two non-homologous chromosomes (tri- and quadriradials), whereas second type (another 50%) was a consequence of either exchange between telomeres of one and non-telomeric sequences of another chromosome (31.03%) or telomere fusions (18.97%). 8.62% of all radials were complex (consisted of 3 or more chromosomes with telomere fusions). Six months latter patient needed bonne marrow transplantation.

Previous studies proposed several possible mechanisms of radials formation. Quadriradials are considered to be a consequence of interchromosomal recombination, accompanied by crossing-over, while triradials arise from illegitimate intrachromosomal recombination or from an unrepaired double strand break (DSB) in a single chromatid (`chromatid break') (Scully et al., 2000). On the other hand, symmetrical triradials might be a consequence of partial endoreduplication (Kuhn and Therman, 1982). However, telomere involvement in their formation has not been previously reported. Our results suggest that 50% of radials are a consequence of shortened telomeres. Telomeres, which identify chromosome ends, have been implicated in the control of both genomic stability and cell proliferation capacity (Hemann et al., 2001). Short telomeres in peripheral blood lymphocytes from FA patients might reflect accelerated hematopoiesis as a compensation for an increased rate of apoptosis and cell cycle delays in FA. Such stress-induced replication leads to shorter telomeres even with a constant rate of shortening per cell division (Leteurtre et al., 1999).

Increased radials incidence 6 months before bone marrow transplantation suggest that radials formation is not a normal event in ICL repair, but aberrant structures arising from either dysfunctional telomeres or unsuccessful attempt to resolve chromosomal damage.

Assuming all those findings we can conclude that complex radial formation in peripheral blood reflects that significant number of bone marrow cells that carry extremely shortened telomeres, whose dysfunction is displayed by their involvement in radials formation. For clinicians this finding could be very important enouncing bonne marrow failure and need for transplantation.

Acknowledgement

The authors are thankful to the patient, parents and medical staff of the Institute of Mother and Child Health Care in Belgrade. This work is financially supported by Serbian Ministry of Education, Science and Technological Development, Project No. 173046. References Adelfalk, C., Lorenz M., Serra V., Von Zglinicki T., Hirsch-Kauffmann M. and Schweiger M.

(2001). Accelerated telomere shortening in Fanconi anemia fibroblasts--a longitudinal study. FEBS Lett. 506: 22-26.

Auerbach, A. D. (2009). Fanconi anemia and its diagnosis. Mutat. Res. 668: 4-10. Auerbach, A. D., Rogatko A.and Schroeder-Kurth T. M. (1989). International Fanconi Anemia

Registry: relation of clinical symptoms to diepoxybutane sensitivity. Blood 73: 391-396. Bogliolo, M. and Surrallés J. (2000). The Fanconi Anemia/BRCA Pathway: FANCD2 at the

Crossroad between Repair and Checkpoint Responses to DNA Damage. In Madame Curie Bioscience Database [Internet]. Landes Bioscience, Austin (TX).

Cohen, M. and Grey H. (1989). Chromosome Instability Syndromes. In Adv. Hum. Genet., pp. 43-149. Ed. by H. Harris and K. Hirschhorn. Springer US.

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Fanconi, G. (1967). Familial constitutional panmyelocytopathy, Fanconi's anemia (F.A.). I. Clinical aspects. Semin. Hematol. 4: 233-240.

Hemann, M. T., Strong M. A., Hao L.-Y.and Greider C. W. (2001). The Shortest Telomere, Not Average Telomere Length, Is Critical for Cell Viability and Chromosome Stability. Cell 107: 67-77.

Joksic, I., Vujic D., Guc-Scekic M., Leskovac A., Petrovic S., Ojani M., Trujillo J. P., et al. (2012). Dysfunctional telomeres in primary cells from Fanconi anemia FANCD2 patients. Genome Integr 3: 6.

Kaddar, T. and Carreau M. (2012). Fanconi Anemia Proteins and Their Interacting Partners: A Molecular Puzzle. Anemia: 11.

Kalb, R., Neveling K., Hoehn H., Schneider H., Linka Y., Batish S. D., Hunt C., et al. (2007). Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype. Am. J. Hum. Genet. 80: 895-910.

Kee, Y. and D'Andrea A. D. (2010). Expanded roles of the Fanconi anemia pathway in preserving genomic stability. Genes Dev. 24: 1680-1694.

Kuhn, E. M. and Therman E. (1982): Origin of symmetrical triradial chromosomes in human cells. Chromosoma 86: 673-681.

Leteurtre, F., Li X., Guardiola P., Le Roux G., Sergere J. C., Richard P., Carosella E. D., et al. (1999). Accelerated telomere shortening and telomerase activation in Fanconi's anaemia. Br. J. Haematol. 105: 883-893.

Li, X., Leteurtre F., Rocha V., Guardiola P., Berger R., Daniel M. T., Noguera M. H., et al. (2003). Abnormal telomere metabolism in Fanconi's anaemia correlates with genomic instability and the probability of developing severe aplastic anaemia. Br. J. Haematol. 120: 836-845.

Petrovic, S., Leskovac A., Joksic I., Filipovic J., Vujic D., Guc-Scekic M. and Joksic G. (2013). Leukocyte apoptosis as a predictor of radiosensitivity in Fanconi anemia. Curr. Sci. 105: 56-60.

Scully, R., Puget N. and Vlasakova K. (2000). DNA polymerase stalling, sister chromatid recombination and the BRCA genes. Oncogene 19: 6176-6183.

Vujic, D., Petrovic S., Lazi E.c, Kuzmanovic M., Leskovac A., I. Joksic, D. Micic, et al. (2014). Prevalence of FA-D2 rare complementation group of Fanconi anemia in Serbia. Indian J. Pediatr. 81: 260-265.

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SSR ANALYSIS OF GENETIC DIVERSITY AND RELATIONSHIPS AMONG MAIZE DROUGHT TOLERANT AND SUSCEPTIBLE INBRED LINES

Dragana IGNJATOVIC-MICIC, Ana NIKOLIC, Sofija BOZINOVIC, Jelena VANCETOVIC

Maize Research Institute “Zemun Polje” Slobodana Bajica 1, 11185 Belgrade, Serbia

Abstract

SSR markers were used to analyze twelve drought tolerant inbreds from drought tolerant mini core collection (Maize Research Institute gene bank) and five commercial drought susceptible inbreds. Thirty primer pairs revealed 143 alleles and average genetic distance of 0.64. Genetic relationships of the inbreds determined by cluster and principal component analyses (PCA) were partially based on heterotic affiliations determined by conventional methods, whereas PCA was more accurate. The combination of SSR markers used could differentiate all inbred lines and high genetic distances detected indicated high level of diversity among them. The lack of pedigree data complicates the interpretation of discrepancies in heterotic grouping and probably a combination of both conventional and molecular results could give the least erroneous classification.

Key words: maize, SSR, genetic diversity Introduction

Massive use of uniform commercial hybrids in maize production that warrants high grain yields has led to the loss of its genetic variability - only 5% of maize variability is in commercial use (Carena et al. 2009). This is detrimental for maize adaptation to different stresses and inadequate to resolve the problems related to climate changes and population growth.

Genotypes from gene bank collections not used in commercial production could present novel sources of drought tolerance, increase maize variability and improve its adaptability (Meseka et al. 2013). New sources of drought tolerance were identified within Maize Research Institute (MRI) gene bank collection (Vancetovic et al. 2010). Forty-one accessions with good general combining ability were used to create drought tolerant mini core collection, which consists of landraces from former Yugoslavia, introduced landraces and introduced inbred lines. One of the goals in MRI breeding programs is to use the inbred lines from drought tolerant mini core for improvement of commercial genotypes. Efficient use of inbred lines requires knowledge on their genetic diversity and relationships, and simple sequence repeat (SSR) markers have proven valuable for this type of analysis (Gupta and Varnshney 2000). The

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research presented herein refers to the SSR analysis of drought tolerant mini core inbred lines with the aim to (1) assess their genetic diversity and relationships with drought susceptible lines and (2) verify the accuracy of their classification into heterotic groups previously done by line x tester method. Material and methods

The analyzed plant material was comprised of twelve inbred lines from MRI drought tolerant mini core collection and five drought susceptible inbred lines – one from MRI gene bank collection and four commercial lines (Table 1). Pedigree data on drought mini core tolerant inbred lines were not available and heterotic groups were previously determined based on the general combining ability testing in the field trials (Vancetovic et al. 2014). Table 1. Identification and available information on maize inbred lines assayed for genetic diversity using

SSR markers

No. Identification Donor countryb†

Heterotic groupc

Kernel type

Drought toleranced Abbreviation A/CILa Name†

1 TL-A A5962 TVA1415-1 CSK L semiflint T 2 TL-B A5050 727574 USA LB dent T 3 TL-C A5147 TVA912-1 CSK IL flint T 4 TL-D A5500 PZS61 SUN LB semident T 5 TL-E A5700 ČK674/78-2 SUN L flint T 6 TL-F A5933 TVA810-1 CSK I semident T 7 TL-G A5974 RC109 CSK I dent T 8 TL-H A6062 Vir44 PEP SUN IL dent T 9 TL-I A6078 UČ23 SUN I dent T

10 TL-J A6122 S49 POL L flint T 11 TL-K A6500 TVA1736-1 CSK I flint T 12 TL-L A6655 TVA303-1 CSK L semiflint T 13 SL-A CIL - - B dent S 14 SL-B CIL - - B dent S 15 SL-C CIL - - L dent S 16 SL-D CIL - - B dent S 17 SL-E A207 V-395 YUG - dent S

a A- accession; CIL – commercial inbred line b country from which the inbred line was donated: CSK- Czechoslovakia, USA – United States of America, SUN – Soviet Union, Pol – Poland, Yug - Yugoslavia c L-Lancaster, I – Iowa Dent, B – (BSSS) Iowa Stiff Stalk Synthetic d T- drought tolerant; S – drought susceptible † information on commercial inbred lines is protected by MRI

DNA was isolated from maize flour obtained by grinding five seeds per inbred line by the

standard CTAB method. Choice of SSR primers was based on bin locations to provide adequate coverage of all chromosomes (www.maizegdb.org). A total of 30 informative SSR markers were chosen after initial screening of 65 SSR loci on five inbred lines. After PCR, the amplified fragments were separated on 8% polyacrylamide gels with 20bp ladder as a marker on a small format vertical gel system (Mini Protean Tetra-Cell BioRad) and stained with ethidium bromide.

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The gels were phographed using BioDocAnalyze (BDA) gel documentation system (Biometra, Germany) and SSR profiles for each primer were determined.

Allele frequencies were scored as pixel total percentage of individual bands within the sample, using UN-SCAN–IT gel 6.1 program package. Genetic distances (GD) between inbred lines were evaluated by the Modified Rogers Distance coefficient. Unweighted pairgroup method (UPGMA) analysis was applied for cluster analysis. The co-phenetic coefficients values were computed and the significance of the co-phenetic correlation observed was tested using the Mantel matrix correspondence test. Principal component analysis (PCA) was calculated from the GD matrix. GD, cluster and PCA were done with NTSYSpc2 program package (Rolf 2000). Results and discussion

Thirty SSR primers produced a total of 143 alleles among the 17 inbred lines and the average allele number per locus was 4.77. GD ranged from 0.42 (TL-E/TL-F) to 0.74 (SL-A/SL-C, SL-D/TL-E and SL-D/TL-I), with the average value of 0.64. The average distance was relatively high, indicating high level of diversity among inbred lines. Similar diversity among maize inbred lines was reported in Vaz Patto et al. (2004) and Legesse et al. (2007). Considering GD between different heterotic groups (average GD of 0.63), the highest diversity was observed between B, on one side and L, I and IL on the other side (0.67, 0.67 and 0.66, respectively). The lowest GD was obtained between I and IL inbred lines (0.58). The average GD for SL-E inbred line, not assigned to any heterotic group, and all the other inbred lines was 0.65. This line was most distant with B inbred lines (average GD of 0.70) and least distant with I and IL inbred lines (average GD of 0.61). Although the GD pattern was obvious the differences were not high, probably due to the small sample of lines analyzed.

Cluster analysis based on the GD matrix is presented in the form of dendrogram (Fig. 1). The co-phenetic correlation coefficient was 0.758 and can be considered as a sufficiently good extent to which the clustering of inbred lines was accurate in representing the estimates of GD (Legesse et al. 2007). The first cluster includes two drought susceptible commercial inbred lines from B heterotic group and was clearly separated from all the other analyzed inbred lines. Cluster II had both susceptible and tolerant inbred lines from different heterotic groups. However, both susceptible lines were attached to the grouped TL-B, TL-E and TL-F lines in subcluster Ia, with SL-B from B hetereotic group being at the outmost position. Also, two lines (TL-B and TL-D) with BL germplasm positioned in cluster II. Cluster III consisted of two sub-clusters, with three and two lines in IIIa and IIIb, respectively. It contained one drought susceptible inbred line (SL-E) from the undefined heterotic group, two lines with IL germplasm (TL-C and TL-H), and one line each from L (TL-L) and I (TL-I) heterotic groups. Finally, TL-A was loosely joined to cluster II and TL-K to both clusters II and III.

The genetic relationships between the analyzed inbred lines determined using PCA analysis of the GD estimates are presented in Fig. 2. The first (PC1) and the second (PC2) principal components explained 16% and 22% of the total variation, respectively. PC1 separated the inbred lines with B germplasm on the positive side of the axis. All lines with L and/or I germplasm had negative PC1 (with exceptions of TL-A and TL-K), but these two groups were not clearly separated.

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Figure 1. Dendrogram constructed with UPGMA cluster analysis based on MRD genetic distances among drought susceptible and tolerant inbred lines. Boxes indicate heterotic groups: white – BSSS (B), dark gray – Iowa Dent (I), light gray – Lancaster (L), white/light gray - BSSS and Lancaster (BL), dark gray/light gray - Iowa Dent and Lancaster (IL), black– unknown.

Figure 2. Principal component analysis (PCA) of the five drought susceptible and 12 drought tolerant

inbred lines performed on the SSR-based estimates of MRD genetic distances. Boxes indicate heterotic groups: white – BSSS (B), dark gray – Iowa Dent (I), light gray – Lancaster (L), white/light gray - BSSS and Lancaster (BL), dark gray/light gray - Iowa Dent and Lancaster (IL), black– unknown.

Coefficient0.42 0.49 0.55 0.61 0.67

SL-AMW

SL-A

SL-D

SL-B

SL-C

TL-B

TL-E

T L-F

TL-D

TL-G

TL-J

TL-A

SL-E

TL-H

TL-I

TL-C

TL-L

TL-K

I

IIIIa

IIb

III

IIIa

IIIb

Coefficient0.42 0.49 0.55 0.61 0.67

SL-AMW

SL-A

SL-D

SL-B

SL-C

TL-B

TL-E

T L-F

TL-D

TL-G

TL-J

TL-A

SL-E

TL-H

TL-I

TL-C

TL-L

TL-K

I

IIIIa

IIb

III

IIIa

IIIb

C1-0.20 -0.05 0.10 0.25 0.40

C2

-0.23

-0.11

0.02

0.15

0.28

SL-A

SL-BSL-C

SL-D

SL-E

TL-A

TL-B

TL-C

TL-D

TL-E

TL-F

TL-G

TL-H

TL-I

TL-J

TL-K

TL-L

C1-0.20 -0.05 0.10 0.25 0.40

C2

-0.23

-0.11

0.02

0.15

0.28

SL-A

SL-BSL-C

SL-D

SL-E

TL-A

TL-B

TL-C

TL-D

TL-E

TL-F

TL-G

TL-H

TL-I

TL-J

TL-K

TL-L

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It is known that genetic groupings are not necessarily the same as the assignment of heterotic groups (Li et al. 2002, Zheng et al. 2008).Our results revealed that both cluster and PCA analyses grouped the inbred lines partially according to the heterotic groups. However, grouping of inbreds from cluster II was in better accordance with heterotic pattern in PC analysis. In cluster analysis, one B (SL-B) and two BL (TL-B and TL-D) inbreds were separated into two subclusters and grouped with L and I inbreds. On the contrary, in PCA they positioned on the same side of PC1 axis (SL-B and TL-D) or near zero (TL- B), as the other two inbreds with B germplasm. Position of TL- B on negative PC1 side but near zero can be explained by the larger amount of L than B germplasm in this genotype. For TL-D, the proportion of L and B germplasm would be the opposite. Conclusion

Neither cluster nor PCA could clearly distinguish between L and I inbreds. Several factors such as selection and/or erroneous pedigree records may play a role in this observation. The pedigree data of the analyzed 12 tolerant inbred lines are unknown and their assignment to heterotic groups was performed on yield trials for the general combining ability. It could be possible that some of these lines are genetically related and have mutual good combining ability with B heterotic group. Nevertheless, it can be concluded that PCA and cluster analyses were partially consistent with the previous classification of inbred lines into heterotic groups based on their breeding behavior. Acknowledgement: This research was supported by the Ministry of Education, Science and Technological Development of Republic of Serbia through the project TR31028 Exploitation of

maize diversity to improve grain quality and drought tolerance. References Carena MJ, Bergman G, Riveland N, Eriksmoen E and Halvorson M (2009). Breeding maize for

higher yield and quality under drought stress. Maydica 54: 287-296. Gupta PK and RK Varnshney (2000). The development and use of microsatellite markers for

genetic analysis and plant breeding with emphasis on bread wheat. Euphytica 113: 163-185.

Legesse BW, Myburg AA, Pixley KV and Botha AM (2007). Genetic diversity of African maize inbred lines revealed by SSR markers. Hereditas 144:10-17.

Li Y, Du J, Wang T, Shi Y, Song Y and Jia J (2002). Genetic diversity and relationships among Chinese maize inbred lines revealed by SSR markers. Maydica 47: 93-101.

Meseka S, Fakorede M, Ajala S, Badu-Apraku B and Menkir A (2013). Introgression of alleles from maize landraces to improve drought tolerance in an adapted germplasm. Journal of Crop Improvement 27:96-112.

Rohlf FJ (2000). NTSYSpc Numerical taxonomy and multivariate analysis system, v. 2.1. Exerter Publications, New York.

Vancetovic J, Mladenovic Drinic S, Babic M, Ignjatovic-Micic D and Andjelkovic V (2010). Maize genebank collections as potentially valuable breeding material. Genetika 42: 9-21.

Vancetovic J, Ignjatovic-Micic D, Bozinovic S, Babic M, Filipovic M, Grcic N and Andjelkovic V (2014). Grain quality of drought tolerant accessions within the MRI Zemun Polje maize germplasm collection. Spanish Journal of Agricultural Research 12: 186-194.

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Vaz Patto MC, Pêgo S, Satovic Z and Fevereiro P (2004). Assessing the genetic diversity of Portuguese maize germplasm using microsatellite markers. Euphytica 137: 63-72.

Zheng DH, Van K and Lee SH (2008). Molecular diversity and relationships among elite maize inbreds from US and CIMMYT populations and current heterotic groups in China. Hereditas 145:182-193.

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DETERMINATION OF KTI IN SOYBEAN GENOTYPES BY MOLECULAR MARKERS

Dragan KOVAČEVIĆ, Vesna PERIĆ, Mirjana SREBRIĆ, Aleksandra SUDARIĆ, Snežana MLADENOVIĆ DRINIĆ

Maize Research Institute “Zemun Polje”, Belgrade, Serbia

Agricultural Institute Osijek, Croatia Abstract

DNA markers tightly linked to the Ti locus controlling presence and absence of kunitz trypsin inhibitor protein was used for screening of 80 soybean genotypes. Also F2 population derived from a cross between variety Afrodita, with KTI, and a variety Laura, lacking KTI, was analyzed with specific PCR primer to select seeds which lacked these protein and by PAGE. The analysis resulted in 21 TiTi genotypes, 62 heterozygous and 27 with titi genotype. The polypeptide band at 21.5 kDa position corresponding to the kunitz trypsin inhibitor protein is present in the parent cultivar Afrodita but absent in Laura. Out of the 110 F2 seeds, 83 exhibited presence of kunitz trypsin inhibitor polypeptide while remaining 27 lacked kunitz trypsin inhibitor polypeptide. All titi genotypes which showed no 21.5 kDa protein band had allele 2 amplified by Satt228 marker. Indirect selection based on DNA marker tightly linked to Ti locus is an easier and more efficient method that allows identification of all three genotypic classes for KTI and will be extremely useful in breeding programs. Key words: soybean, kunitz trypsin inhibitor, SSR markers, PAGE Introduction

Raw soybean cannot be used for monogastric animal feeding because of the presence of factors that decrease its nutritional value. Among the antinutritional factors present in the soybean seed, the main ones are the protease inhibitors, which affect the growth and/or basal metabolism of different animal species. The protease inhibitors from soybeans fall into two categories: (1) those that have a molecular weight of 20000 to 25000 Da with relatively few disulfide bonds and a specificity directed primarily toward trypsin (Kunitz inhibitor, Kunitz 1945), and (2) those that have a molecular weight of only 6000 to 10000 Da with a high proportion of cystine residues and are capable of inhibiting chymotrypsin as well as trypsin at independent binding sites (Bowman-Birk inhibitor, Birk 1961). Approximately 80% of the trypsin inhibition is caused by KTI. Soybean kunitz trypsin inhibitor (KTI) is a small,

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monomeric and non-glycosylated protein containing 181 residues first isolated and crystallized from soybean seeds by Kunitz (1945). Proper heat processing is required to destroy KTI protein. However, excessive heat treatment may lower amino acid availability. The genetic removal of the KTI protein will improve the nutritional value of soybean. Molecular markers tightly linked to desired genes are a valuable tool to detect genotypes of interest, saving time and resources. To date, detection of the KTI protein free genotypes has been based on SDS-PAGE gel electrophoresis analysis of crude protein from mature seeds, however, with this method, test samples are restricted to proteins from mature soybean seeds. This is a time-consuming process, which is not possible in the early seedling stages of the corresponding population. Plant breeders can use molecular markers to select indirectly individuals in segregating populations that carry a gene for a favorable trait if a tight linkage exists between a marker locus and the genetic locus controlling that trait. The Ti locus has been located on linkage group 9 in the classical linkage map of soybean (Kiang, 1987), which is integrated in molecular linkage map A2 (chromosome number 8) of the USDA/Iowa State University soybean molecular linkage map (Cregan et al., 1999). Kim et al. (2006) were identified DNA marker, Satt228, tightly linked to the Ti locus controlling presence and absence of kunitz trypsin inhibitor protein. Materials and methods

80 soybean genotypes were selected from Soybean collection of Maize research institute. An F2 population was derived from a cross between soybean genotype Afrodita, with kunitz trypsin inhibitor, and a variety Laura, lacking KTI. Parents and one hundred ten F2 genotypes were analyzed electrophoretically and by Sat 228 molecular markers to determine the presence or absence of kunitz trypsin inhibitor.

The protein (KTI) extraction from soybean flour was performed by the procedure of Hymowitz and Hadley (1972) and protein extracts was analysed by SDS PAGE. Wide-Range SDS-PAGE molecular mass standard (Sigma) containing the 21.5kDa soybean trypsin inhibitor protein, was used to aid recognition of samples lacking the kunitz trypsin inhibitor.

DNA extraction was performed by Kamiya and Kiguchi (2003). The sequence of Satt228 marker was 5'-TCATAACGTAAGAGATGGTAAAACT-3 (forward) and 5'-CATTATAAGAAAACGTGCTAAAGAG-3'(reverse), (Kim et al. 2006). The DNA was amplified in a reaction mixture of 25 µl containing: 30 ng of genomic DNA, 1.0 unit of Taq

DNA polymerase, 1X PCR buffer (DreamTaq™ Green Buffer, Fermentas), 2.5 mM MgCl2, 0.8 mM dNTPs and 0.5µM of each one of the specific primers pairs for amplification of the alleles kti. PCR conditions were as follows: 95oC for 5 min followed by a 15-step touchdown decreasing by 0.5oC each step: 95oC for 30 s, 63.5 to 56oC for 1 min and 72oC for 1 min followed by 25 cycles of 95oC for 30 s, 56oC for 1 min and 72oC for 1 min. The amplification products were separated in 2.5% agarose gel immersed in buffer 1X TBE (Tris-borate 0.09 M, 1mM EDTA). Results and discussion

DNA marker, Satt 228, tightly linked to Ti locus at distance 0 cM, designed to amplify portions of the recessive alleles kti, was used to screen set of 80 soybean variety (Fig1.). TiTi genotypes had allele1, however, titi genotypes had allele 2. Out of 80 genotypes 73 had allele 1. Three variety Kunitz, Laura, Lana lacking KTI had allele 2. Four genotypes with KTI have allele 1 but also weak band in position of allel 2. These genotypes was check by PAGE and all of them have 21.5 kDa protein band (Fig 2). Co segregation between KTI protein (21.5 kDa size)

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and allele 1 of Satt228 marker was observed and this result indicates that Satt228 marker may effectively utilized to select the plant with titi genotype.

M 1 2 3 4 5 6 7 8 9 10 Fig 1. Satt 228 marker analysis of soybean genotypes Line 1, 9, 10 ti genotypes, 2,3,4,5,6,7,8 Ti genotypes

M 1 2 3 4 5 6 7 8 9 Fig 2. PAGE analysis of soybean genotypes Line 1 ,3, 5, 6, 7, 8 KTI, 2.Laura, 4 Kunitz, 9 Lana

Soybean genotype Laura (derived in Maize Research Institute) has the ti allele and lacks a soybean kunitz trypsin inhibitor. Variety Afrodita have kunitz trypsin inhibitor protein band (TiTi). An F2 population derived from a cross between variety Afrodita, and a variety Laura was analyzed by SDS electrophoresis and with specific PCR primer to select seeds which lacked this protein. Parents and 110 F2 genotypes were analyzed through SDS PAGE to determine the presence or absence of kunitz trypsin inhibitor. TiTi genotypes had 21.5 kDa band that indicates KTI protein, however titi genotypes did not have the band in protein gel electrophoresis from the mature seed (Fig. 3). The observed data for F2 genotypes were 83 genotypes with KTI protein

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band (21.5KDa) and 27 genotypes with no KTI protein band. These observations fit the expected 3:1 ratio for the presence or absence of the KTI protein band. That was in agreement with results of Kim et al, (2008) who obtained the segregation ratio of 3:1 in the F2 seed and the Chi-square values that strongly suggest that kunitz trypsin inhibitor protein band is controlled by a single recessive gene.

1 2 3 4 5 6 7 8 9 10 Fig 3. Polyacrylamide gel of protein extracted from parents and F2 seeds. Line 1.Laura, 2. Afrodita, 3-10 F2 plants Genotypes of F2 population obtained by cross of Afrodita and Laura were analysed by Satt 228 marker. Amplification patterns obtained from Satt228 marker using genomic DNA of parent Laura and F2 genotypes with titi genotype (Kunitz trypsin inhibitor protein absent) and parent Afrodita and F2 genotypes with TiTi genotype (Kunitz trypsin inhibitor protein present), as well as heterozygots are shown in Fig 4. The analysis resulted in 21 TiTi genotypes, 62 heterozygous and 27 with titi genotypes. The selection assisted by specific molecular markers for the absence of KTI allowed the identification of heterozygous individuals.

Fig 4. Satt 228 marker analysis of F2 soybean genotypes Line 1 Afrodita, line 2. Laura, lines 3,5,6,7,8,9 TiTi genotype, line 4. titi genotype, lines 10,11,12 heterozygots

Molecular markers tightly linked to desired genes are a valuable tool to detect genotypes of interest, saving time and resources (Tanksley et al., 1989). From the comparison of gel electrophoresis for Kunitz trypsin inhibitor protein and banding pattern amplified by Satt228

21,5KDa

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marker, there was a strong agreement between protein band (21.5 kDa) for Kunitz trypsin inhibitor protein and banding pattern by Satt228 marker. All TiTi genotypes which showed 21.5 kDa protein band in protein electrophoresis had the allele 1 amplified by Satt228 marker from the genomic DNA. However, all titi genotypes which showed no 21.5 kDa protein band had allele 2 amplified by Satt228 marker. This result indicates that Satt228 marker may be effectively utilized to select the plants with the titi genotype. Indirect selection based on DNA marker tightly linked to Ti locus is an easier and more efficient method that allows identification of all three genotypic classes for KTI and will be extremely useful in breeding programs. Reference Birk Y. (1961). Purification and some properties of a highly active inhibitor of trypsin and

chymotrypsin from soybeans. Biochem Biophys Acta 54: 378-381 Cregan, P.B., Jarvik T., Bush A.L., Shoemaker R.C., Lark K.G., Kahler A.L., Kaya N., VanToai

T.T., Lohnes D.G., Chung Jongil & Specht J.E. (1999). An integrated genetic linkage map of the soybean genome. Crop Sci. 39:1464-1490.

Hymowitz, T. & Hadley H.H. (1972). Inheritance of a trypsin inhibitor variant in seed protein of soybeans. Crop Sci. 12:197-198.

Kiang, Y.T., (1987). Mapping three protein loci on a soybean chromosome. Crop Sci. 27: 44-46. Kim M.S., Park M.J., Jeong W.H., Nam K.C., Chung J.I. (2006). SSR marker tightly linked to

the Ti locus in soybean [Glycine max (L.) Merr.]. Euphytica 152: 361-366. Kim, MS, Hur MR, Jeong WH, Park MS, Lee KJ, Shim SI, Kim MC, Jung WS, Lee JH, and

Chung JI. (2008). Confirmation of Satt228 marker tightly linked to the Ti locus in four different soybean populations. Genes &Genomics 30(4):329-336.

Kamiya, Ku Motokazu and Kiguchi, Tadahiko. (2003). Rapid DNA extraction from soybean seeds. Breeding Science 53 (3): 277-279.

Kunitz, M., (1945). Crystallization of a soybean trypsin inhibitor from soybean. Science 101:668-669.

Tanksley S.D. (1989). RFLP mapping in plant breeding: New tools for an old science. Biotechnology 7: 257-264.

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ASSESSMENT OF GENETIC DIVERSITY AMONG DROUGHT TOLERANT MAIZE LOCAL LANDRACES USING SSR MARKERS

Natalija KRAVIC*, Violeta ANDJELKOVIC, Jelena VANCETOVIC, Ana NIKOLIC, Danijela

RISTIC, Dragana IGNJATOVIC-MICIC

Maize Research Institute “Zemun Polje”, Slobodana Bajića 1, 11185 Belgrade, Serbia *e-mail: nkravic@mrizp.rs

Abstract

Maize landraces represent potential sources of favorable traits for current and future breeding programs. Genetic diversity among thirteen local maize landraces from MRIZP gene bank drought tolerant mini-core collection was studied using a set of 18 SSR markers. A total of 155 alleles were recorded with an average of 8.6 per primer pair. The polymorphism information content (PIC) values ranged from 0.50 (bnlg557) to 0.82 (umc1944) with a mean of 0.66, indicating high discriminating ability of the SSR markers used. Jaccard’s similarity coefficients, ranging from 0.15 to 0.58, and cluster analysis revealed substantial diversity among the genotypes. Also, assigning of investigated maize landraces into distinct heterotic groups (BSSS, Lancaster and independent), based on their general combining ability with used inbred testers, was in accordance with SSR marker-based molecular characterisation of the accessions. Key words: cluster analysis, gene bank, heterotic groups, microsatellites, Zea mays L. Introduction

Strong commercial pressures resulted in evident gap between available genetic resources stored within gene banks and breeding program activities. Use of a narrow range of tested elite germplasms during the development of modern hybrids, and the substitution of landraces by elite cultivars, inevitably leads to the loss of genetic diversity (Pollack 2003). In this context, previous studies have shown that breeding mainly leads to qualitative rather than quantitative shifts in registered cultivars (Le Clerc et al. 2006). Thereby, maize breeders have become more aware of the needs for maintaining genetic diversity among hybrid varieties and improving the management of genetic resources through the conservation and utilisation of traditional populations, i.e. landraces. Maize Research Institute „Zemun Polje“ (MRIZP) gene bank maintains the collection of 2217 maize local landraces, collected from different agro-ecological sites of the former Yugoslavian territories, as well as the collection of 3589 introduced genotypes (inbred lines, synthetics, composites, and landraces) from about 40 countries worldwide. Local

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landraces are considered to be the most significant genotypes, since they represent the original biological material created by the process of natural selection and adapted to local growing conditions, thus offering the great opportunities for different breeding purposes (e.g. drought tolerance) and contributing to yield increase (Mladenovic Drinic et al. 2012).

Knowledge of germplasm diversity among local landraces and breeding stocks is expected to have a significant impact on the improvement of crop plants. In maize, this information is known to be useful in planning crosses for hybrid development, assigning genotypes to heterotic groups, maintaining genetic variability of landraces, and protecting inbreds and varieties. It can be obtained by surveying both qualitative and quantitative morphological traits or using molecular markers for investigating polymorphisms at the DNA sequence level (Dubreuil et al. 2006; Nikolic et al., 2013).

Besides providing useful information on genetic diversity and relationships between accessions, advantages of DNA-based markers include independence from environmental and pleiotropic effects, a potentially unlimited number of available markers and selectively neutral nature of many molecular markers (Kumar et al. 2009). Simple sequence repeat (SSR) markers are highly informative, and easy detectable with PCR. They occur frequently in plant genomes, showing an extensive variation in different individuals and accessions. Since SSR markers are co-dominant, multi-allelic, highly polymorphic and randomly distributed throughout the genome, they are widely used for assessing maize genetic diversity (Varshey et al. 2005).

The objectives of this study were: (i) to assess the genetic diversity among drought tolerant maize local landraces using simple sequence repeat (SSR) markers and (ii) to investigate whether the grouping of the accessions according to SSR markers used was in consistence to their assigning into heterotic groups based on general combining ability with related testers. Material and methods Plant material

After testing of the entire MRIZP gene bank collection (5806 accessions) under controlled drought in Egypt, as well as under temperate climate conditions in Serbia and Macedonia, a drought tolerant mini-core collection of 41 accessions (15 inbred lines, 13 local and 13 introduced landraces) was established (Babic et al. 2011). The subset of thirteen maize local landraces was the objective of the present study. DNA extraction and amplification

DNA-pooled sampling strategy (bulk analysis) was used for SSR analysis. Each landrace was presented with 30 plants. Genomic DNA was isolated from seed, using a modified CTAB procedure (Saghai-Maroof et al., 1984). A total of 18 informative SSR markers were chosen for the analysis. PCR amplification reaction was carried out in 25 µl reaction volume containing DreamTaq™. Green PCR Master Mix (2X), 0.5µM primers and 250 ng of DNA. Amplification was conducted using the following cycling profile: an initial denaturation at 95°C/5 min, followed by 15 cycles each of denaturation at 95°C/30 s, annealing at 63.5°C/1 min (-0.5°C/cycle) and extantion at 72° C/1 min; another 22 cycles of 95°C/30 s, 56°C/1 min and 72°C /1 min were performed. Final elongation was at 72°C for 4 min. Amplified fragments were separated on 8% polyacrylamide gels with 100bp DNA marker ladder on a vertical gel system (Mini Protean Tetra-Cell BioRad) and stained with ethidium bromide. The gels were phographed

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using BioDocAnalyze (BDA) gel documentation system (Biometra, Germany) and SSR profiles for each primer were determined. Data analysis and genetic diversity estimation

SSR profiles were scored as presence versus absence of alleles in each sample and the data were assembled into a binary matrix. Polymorphic information content (PIC) was used to determine allele diversity at each locus and was calculated according to Roldan-Ruiz (2001) as PICi = 2fi (1-2fi), where fi is the frequency of the amplified allele (band present) and (1-2fi) is the frequency of null allele (band absent) of marker i. Correlations between allele number and PIC values were calculated using Pearson's correlation coefficient.

Genetic similarities (GS) between the accessions were calculated according to Jaccard's coefficient (Jaccard 1908). Unweighted pair-group method with arithmetic mean (UPGMA) was applied for cluster analysis and relationships between the landraces were visualized as dendrogram. The co-phenetic coefficients values were computed and the significance of the co-phenetic correlation observed was tested using the Mantel matrix correspondence test (Mantel 1967). Statistical analysis was performed using NTSYSpc2 software package (Rolf 2000). Results and discussion

Eighteen SSR primers produced a total of 155 alleles, measuring approximately 100 to 400bp. The number of alleles per locus varied from three (phi085) to fourteen (umc1019, umc1006 and bnlg1526), with average number of 8.6, as shown in Table 1. Similar results have been reported by Yang et al. (2011) in a study in which 82 SSR primers produced an average of 8.2 alleles per locus, and by Eschholz et al. (2008), who obtained an average of 10.8 alleles per locus with just 10 primers in Swiss flint maize germplasm. Also, Xiang et al. (2010) have investigated diversity across 22 maize landrace varieties with 41 SSR primers and found an average of 6.9 alleles per locus using polyacrylamide gel electrophoresis (PAGE). Notably different result has been reported by Molin et al. (2013) where genotyping with 47 SSR primers revealed an average of 2.9 alleles per locus using agarose gel electrophoresis.

In present study, average rate of polymorphism was 98.8%, i.e. 17 primers used were 100% polymorphic, whereas one primer (umc1109) displayed 80% polymorphism. Our finding was consistent to that reported by Yao et al. (2007). In regard to the number of polymorphic alleles per primer, Molin et al. (2013) reported the value of 2.2 with 40.3% of an average rate of polymorphism.The differences in the number of alleles and the rate of polymorphisms compared with those in the literature may be associated with the type of matrix (agarose or polyacrylamide) used to separate the alleles. This possibility is highlighted in the studies of Yao et al. (2007) and Xiang et al. (2010), in which a higher number of alleles per locus with SSR analysis was reported using polyacrylamide gels. In addition, Kostova et al. (2006) have observed that the use of SSRs with dinucleotide repeats might result in a greater number of alleles, which is in agreement with Yang et al. (2011) and Eschholz et al. (2008), who used 44 and 50% of primers to sample these regions, respectively. In the present study, only 39% of the SSR primers sampled repetitive dinucleotides, which may be reflected in the lower number of amplified alleles per locus.

The polymorphism information content (PIC) values ranged from 0.50 (bnlg557) to 0.82 (umc1944) with a mean of 0.66 (Table 1). Since a PIC value of greater than 0.7 is considered to

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be highly informative, and a value of 0.44 is considered to be moderately informative, obtained results indicated high discriminating ability of the SSR markers used. Our results are in accordance with reported average PIC value of 0.61 (Yang et al. 2011), but lower compared to Xiang et al. (2010) and Eschholz et al. (2008). Seven SSR loci had PIC values higher than 0.7 indicating their informativeness and potential to detect differences among the investigated drought tolerant maize local landraces. The highest average PIC was found for SSRs with di-nucleotide repeats (0.70). The SSRs with tri- and penta-nucleotide repeats had average PIC values of 0.63 and 0.59, respectively. Similar results were reported by Legesse et al. (2007). The only SSR marker with tetra-nucleotide repeats (umc2047) had PIC of 0.73. Moreover, correlations between allele number and PIC values were highly significant and positive (r = 0.790; P ≤ 0.01). Correlations between the same parameters (r = 0.848; P ≤ 0.001) were also found in study of Portuguese maize germplasm diversity using standard sequencing gels (Vaz Patto et al. 2004). In our study, detection was performed using polyacrylamide gels, so the differences between correlations significance might refer to higher resolution capabilities of electrophoresis type.

Table 1. Characteristics of 18 SSR markers used in the genetic diversity analysis, including their repeat

type, bin number, number of alleles and PIC values

No. Primer Repeat type Bin no.* No. of alleles PIC value 1 umc1013 (GA)9 1.08 10 0.57 2 umc2047 (GACT)4 1.09 7 0.73 3 phi036 AG 3.04 5 0.71 4 umc1109 (ACG)4 4.10 5 0.60 5 bnlg589 AG 4.10 5 0.54 6 bnlg557 NA** 5.03 4 0.50 7 umc126 AG 5.06 10 0.65 8 umc1019 (CT)17 5.06 14 0.81 9 phi085 AACGC 5.06 3 0.59 10 umc1006 (GA)19 6.02 14 0.81 11 umc1393 (GTC)4 7.02 5 0.57 12 umc1324 (AGC)5 7.03 8 0.69 13 umc1944 NA** 7.04 13 0.82 14 phi116 ACTG/ACG 7.06 12 0.71 15 phi080 AGGAG 8.08 8 0.59 16 phi033 AAG 9.01 10 0.68 17 umc1492 (GCT)4 9.04 8 0.60 18 bnlg1526 (AG)15 10.04 14 0.82 Total 155 Average 8.6 0.66

* Location of allele in the chromosome of maize genome**NA - not available on the site MGDB (URL: http://www.maizegdb.org)

Jaccard’s similarity coefficients and cluster analysis revealed substantial diversity among

the genotypes. The average genetic similarity estimate with the SSR markers used was 0.31; the comparison between the landraces K2 and K10 exhibited the highest divergence, with an index of 0.85, whereas maximal similarity (0.58) was observed in the comparison between K5 and K6, as well as between K6 and K8 (Fig 1).

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Fig 1. Dendrogram of 13 drought tolerant maize local landraces (bulked landrace samples) constructed

using UPGMA cluster analysis of Jaccard similarity values obtained by SSR data

As expected, GS was higher within than among heterotic groups (Fig. 1), ranging from 0.26 (BSSS heterotic group) to 0.42 (Independent source), with the average GS of 0.34. Considering GS between different heterotic groups (average GS of 0.27), the lowest and equal GS of 0.24 was observed between BSSS and the two other heterotic groups (Lancaster and Independent source). The highest GS was obtained between Independent source and Lancaster heterotic group (0.33).

Cluster analysis based on the GS matrix is presented in the form of dendrogram (Fig. 1). The co-phenetic correlation coefficient was 0.844 and can be considered as a sufficiently good extent to which the clustering of landraces was accurate in representing the estimates of GS. Cluster A was divided into two sub-clasters: sub-claster A1 which includes local landraces genetically related to Lancaster type of germplasm (K1, K3, K4, , K9, K12) and sub-claster A2 by comprising the genotypes related to Independent source (K5, K6, K8, K10, K11). On the other hand, cluster B consisted of local landraces genetically related to BSSS type of germplasm (K2, K7, K13) and was clearly separated from all the other analyzed local landraces. Grouping of the accessions according to SSR markers used was in line with previously determined heterotic patterns of the investigated genotypes (Table 2), based on their general combining ability with related testers (Andjelkovic et al. 2010). Table 2. Combining abilities (CA) and the supposed genetic relatedness with observed heterotic sources

of 13 drought tolerant maize local landraces

Supposed genetic relatedness with Local landraces Good CA with

BSSS K2, K7, K13 Lancaster, Independent source

Lancaster K1, K3, K4, K9, K12 BSSS, Independent source

Independent source K5, K6, K8, K10, K11 BSSS, Lancaster

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Conclusion SSR markers used in this study exhibited high level of polymorphism, indicating their

informativeness and potential to detect differences among the investigated genotypes. The low similarity values among the observed drought tolerant local landraces, revealed high level of genetic diversity among them. Assigning of the landraces into distinct heterotic groups, as a decisive factor for their utilisation by different crosses for developing hybrids with better performances, was consistent with their grouping based on SSRs molecular characterisation. Hence, the diverse genotypes could be used for future identification of QTLs for drought tolerance. Acknowledgments

This work was supported by Project TR31028 “Exploitation of maize diversity to improve grain quality and drought tolerance” from the Ministry of Education, Science and Technological Development, Republic of Serbia. References Andjelkovic V., Bozinovic S., Pavlov M., Vancetovic J. (2010): Combining abilities for grain

yield of the most drought tolerant maize accessions from the MRI gene bank. Genetics, Plant Breeding and Seed Production, 45th Croatian & 5th International Symposium on Agriculture. Proceedings, 368-371.

Babic M., Andjelkovic V., Mladenovic Drinic S., Konstantinov K. (2011): The conventional and contemporary technologies in maize (Zea mays L.) breeding at Maize Research Institute, Zemun Polje. Maydica 56: 155-164.

Dubreuil P., Warburton M., Chastanet M., Hoistington D., Charcosset A. (2006): More on the introduction of temperate maize into Europe: large scale bulk SSR genotyping and new historical elements. Maydica 51: 281-291.

Eschholz T.W., Peter R., Stamp P., Hund A. (2008): Genetic diversity of Swiss maize (Zea mays

L. ssp. mays) assessed with individuals and bulks on agarose gels. Genet Resour Crop Ev 55: 971-983.

Jaccard P. (1908): Nouvelles rescherches sur la distribution floral. Bull Soc vaud sci nat 44: 223-270.

Kostova A., Todorovska E., Christov N., Sevov V., et al. (2006): Molecular characterization of Bulgarian maize germoplasm collection via SSR markers. Biotechnol Biotec Eq 20: 29-36.

Kumar P., Gupta V.K., Misra A.K., Modi D.R., Pandey B.K. (2009): Potential of Molecular Markers in Plant Biotechnology. Plant Omics J 2(4): 141-162.

Le Clerc V., Cadot V., Canadas M., Lallemand D., Guèrin D., Boulineau F. (2006): Indicators to assess temporal genetic diversity in the French Catalogue: no losses for maize and peas. Theor Appl Genet 113: 1197-1209.

egesse B.W., Myburg A.A., Pixley K.V., Botha A.M. (2007):. Genetic diversity of African maize inbred lines revealed by SSR markers. Hereditas 144: 10-17.

Nikolić A., Andjelković V., Dodig D., Mladenović Drinić S., Kravić N., Ignjatović-Micić D. (2013): Identification of QTL-s for drought tolerance in maize, II: yield and yield components. Genetika 45(2): 341-350.

Mantel N. (1967): The detection of disease clustering and a generalized regression approach. Cancer Research 27: 209-220.

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Mladenovic Drinic S., Andjelkovic V., Ignjatovic-Micic D. (2012): Genetic Diversity of Maize Landraces as Sources of Favorable Traits, The Molecular Basis of Plant Genetic Diversity, Prof. Mahmut Caliskan (Ed.), ISBN: 978-953-51-0157-4, InTech, Available from: http://www.intechopen.com/books

Molin D., Coelho C.J., Máximo D.S., Ferreira F.S., Gardingo J.R., Matiello R.R. (2013): Genetic diversity in the germplasm of tropical maize landraces determined using molecular markers. Genet Mol Res 12(1): 99-114.

Pollack L.M. (2003): The history and success of the public-private project on germplasm enhancement of maize (GEM). Adv Agron 78: 45-87.

Roldan-Ruiz I., Van Euwijk F.A., Gilliland T.J., Dubreuil P., Dillmann C., De Loose M., Baril C.P. (2001): A Comparative study of molecular and morphological methods of describing relationships between perennial rye grass (Lolium perenne L.) varieties. Theor Appl Genet 103: 1138-1150.

Rohlf F.J. (2000): NTSYS-pc Numerical taxonomy and multivariate analysis system, Version 2.0 Exeter Software, Setaket, NY

Saghai-Maroof M., Soliman K., Jorgensen R., Allard R. (1984): Ribosomal DNA spacer length polymorphism in barley; Mendelian inheritance, chromosomal location and population dynamics. Proceedings of the National Academy of Sciences of the United States of America 91: 8014-8018.

Varshney R.K., Graner A., Sorrells M.E. (2005): Genic microsatellite markers in plants: features and applications. Trends Biotechnol 23(1): 48-55.

Vaz Patto M.C., Satovic Z., Pêgo S. (2004): Assessing the genetic diversity of Portuguese maize germplasm using microsatellite markers. Euphytica 137: 63-72.

Xiang K., Yang K.C., Pan G.T., Reid L.M., et al. (2010): Genetic diversity and classification of maize landraces from China’s Sichuan basin based on agronomic traits, quality traits, combining ability and SSR markers. Maydica 55: 85-93.

Yang X., Xu Y., Shah T., Li H., et al. (2011): Comparison of SSRs and SNPs in assessment of genetic relatedness in maize. Genetica 139: 1045-1054.

Yao Q., Yang K., Pan G., Rong T. (2007): Genetic diversity of maize (Zea mays L.) landraces from southwest China based on SSR data. J Genet Genomics 34: 851-859.

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91

VARIATION OF AVAILABILITY IN MINERAL ELEMENTS CONTENT FROM GRAIN IN MAIZE LANDRACES

Natalija KRAVIĆ1, Vesna DRAGIČEVIĆ1, Milovan STOJILJKOVIĆ2, Mile SEČANSKI1,

Jelena VANČETOVIĆ1, Violeta ANĐELKOVIĆ1

1 Maize Research Institute “Zemun Polje”, Slobodana Bajića 1, 11185 Zemun Polje, Serbia

2 Vinča Institute of Nuclear Sciences, P.fah 522, 11001 Belgrade, Serbia * e-mail: nkravic@mrizp.rs

Abstract

The aim of this study was to examine possible availability of mineral elements (i.e. P, Fe, Mg and Zn) through their ratio with phytate as inhibitor and with β-carotene as promoter for their absorption. Two-year experiment was conducted on twenty-six maize landraces from MRIZP Gene Bank drought tolerant mini-core collection. After harvesting, parameters such as 1000 kernel weight, content of phytate, β-carotene, Mg, Fe and Zn in grain were determined. Phytate/β-carotene ratio ranged from 350 up to 1000, with exception of white kernel maize, being higher than 2000. Mg and Zn content significantly increased with decrease in β-carotene content, which could have negative impact on their availability. Principal Component Analysis (PCA) indicated that phytate and β-carotene mostly contributed to different principal component axis than Fe and Zn, giving the opportunity to their increase in grain, independently of variations in inhibitor and promoter contents. Key words: β-carotene, mineral elements, PCA, phytate, Zea mays L. Introduction

Mineral elements such as phosphorus (P), iron (Fe), magnesium (Mg) and zinc (Zn) perform a variety of functions in plant cells, and are essential for growth and development in plants as well as in animals and humans (White and Broadley, 2005; Menkir, 2008). It is estimated that over three billion people are currently malnourished because of lack of minerals, especially iron and zinc, in their diet (Welch and Graham, 2004). Moreover, modern plant breeding has been historically oriented toward high agronomic yield rather than the nutritional quality (Morris and Sands, 2006), although increased yield may have resulted in a lower density of minerals in grain (Graham et al., 1999). Biofortification, aimed to enhance mineral elements concentrations and/or bioavailability in edible plant tissues, either agronomically or genetically using both conventional breeding and modern biotechnology, is considered to be the most promising and cost-effective approach to alleviate mineral malnutrition (Cakmak, 2008). The

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accumulation of minerals in seeds is a complex phenomenon, which is most likely controlled by a number of genes. The movement of mineral elements from soils to seeds involves their mobilization from soils, uptake by roots, translocation to the shoot, redistribution within the plant and deposition in seeds (White and Broadley, 2009).

Certain organic compounds interfere with the absorption or utilization of mineral elements. Namely, antinutrients (inhibitors), like phytate, polyphenolics etc., limit the absorption of Fe, Zn and Mg, whereas promoters (enhancing substances), such as ascorbat (vitamin C), β-carotene (pro-vitamin A), S-containing amino acids etc., stimulate mineral nutrients bioavailability or decrease the activity of inhibitors (Welch and Graham, 2004).

Since exploring natural biodiversity, as a source of novel alleles to improve the productivity, adaptation, quality and nutritional value of crops is of prime importance in 21st century breeding programs (Ortiz-Monasterio et al., 2007), the aim of this study were (i) to evaluate chemical composition of grain in investigated maize landraces and (ii) to determine relations between phytic acid, inorganic phosphorus and β-carotene, as factors affecting the absorption of important mineral elements, i.e. Fe, Zn and Mg. Material and methods Plant material

Thirteen local (P1-P13) and thirteen introduced (P14-P26) maize landraces from Maize Research Institute “Zemun Polje” (MRIZP) gene bank drought tolerant mini-core collection (Babic et al. 2011), were the objective of the present study. Genetic background of the investigated landraces is as follows: BSSS - P2 (dent); Lancaster - P6, P7, P9, P14, P19, P23, P26 (semiflints); independent source - P5 (semident), P10 (dent), P11 (flint); BSSS Lancaster - P8, P16, P24, P25 (dents); independent source Lancaster - P1, P12, P17, P22 (semiflints), P3 (dent), P13, P18 (flints); unknown - P4, P15, P20, P21 (semiflints). Kernel color of the investigated landraces is as follows: white kernel maize (P1, P4, P8, P10, P21 and P26); yellow kernel maize (P2, P5, P6, P9, P11, P12, P13, P18, P22 and P23); orange kernel maize (P3, P7, P14, P15, P16, P17, P19, P20, P24 and P25).

Field trials and laboratory experiments

For this study, the experiment was carried out in 2010 and 2011. in Zemun Polje, Serbia (44˚52´N, 20˚19´E, 81 m asl). The soil was slightly calcareous chernozem (with low content of investigated mineral elements). A randomized block design with two replications was used in the experiments. Inbreds were manually self-pollinated and harvested. After drying to 14% of moisture content, 1000 kernel weight was measured.

Chemical composition of grain was determined colorimetrically. Phytic (Pphy) and inorganic phosphorus (Pi) were determined by the method of Dragičević et al. (2011). β-carotene was determined according to American Association of Cereal Chemists Method (1995). Mineral elements (Mg, Fe and Zn) were determined after wet digestion with HNO3 + HClO4, by Inductively Coupled Plasma - Optical Emission Spectrometry (Spectro Analytical Instruments, Germany). Statistical analysis

Analyses of molar ratios were performed in four measurements (n=4) and the results were presented as mean ± standard deviation (SD). After two-way analyses of variance

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(ANOVA), the differences among landraces, based on mean values of observed parameters, were evaluated using F-test at the 0.05 probability level and Principle Component Analysis (PCA). Statistical analysis was performed by SPSS 15.0 for Windows Evaluation version. Results and Discussion

Effects of genotype, year and year by genotype interaction had significant impact on the variation of Pi and β-carotene content, as well as on Mg, Fe and Zn content in grain (Table 1), which was in accordance to Ortiz-Monasterio et al. (2007). Among investigated maize landraces, genotype and genotype by year interaction had significant influence regarding 1000 kernel weight, whereas effect of year and genotype by year interaction were significant for Pphy content. Average 1000 kernel weight varied from 304.3 g (P15) to 510.6 g (P6). It was noticed that landraces with the lower values of kernel weight (particularly P1) also had lower level of Pi content, as well as the higher level of Mg, Fe and Zn content in grain. Our findings are in agreement with reported inverse correlation between grain yield components and concentrations of grain minerals in maize (Bänziger and Long, 2000) and wheat (Oury et al., 2006). Based on results obtained, the landrace P1 could be considered as potentially valuable source in breeding for increased content of mineral elements, although their availability could be suppressed due to the low β-carotene content (only 2.96 mg kg-1).

In this study, genotypic variations of Pphy and Pi were presented in lesser extent, i.e. from 3.15 to 3.76 g kg-1 and from 0.30 to 0.46 g kg-1, respectively. This could be considered as negative trait for availability of investigated mineral elements (Walter Lopez et al., 2002; Lönnerdal, 2003). However, there is considerable intra-specific variation in phytate concentration in edible portions that is independent of variation in Fe and Zn concentrations (Erdal et al., 2002). Accordingly, increased level of β-carotene in grain (up to 26.75 mg kg-1 in landrace P20) could be considered as the main factor leading to improved availability of mineral elements (Brinch-Pedersen et al., 2007).

Previously it was reported that the Phy/Zn and Phy/Fe molar ratios are sufficiently relevant in determination of maize genotypes with potentially high ability of Fe and Zn utilization (Ma et al., 2007). Based on the results presented in Table 2, required decrease in Phy/Mg, Phy/Fe and Phy/Zn ratios, which contributes to increased availability of Mg, F, and Zn (Underwood and Suttle 1999; Lönnerdal 2003), were exhibited in grain of landraces P1, P13 and P21, respectively. This is important, since P1 and P21 belong to the group of landraces with relatively high Phy/β-carotene ratio (> 2000), which could be considered as limiting factor for Mg and Zn availability.

Principal component analysis (PCA) of investigated drought tolerant maize landraces revealed that Fe, Mg and Zn content in grain contributed to the first axis (PCA1), which explained 35.604% of the total variability. The second axis (PCA2), which explained 18.422% of the variation, was defined with 1000 kernel weight and Pphy content, whereas the third axis (PCA3), defined with Pi and β-carotene content, explained 16.360% of the total variability (Fig 1). According to the results, it could be concluded that factors which induce variation in phytate content didn’t affect variations of mineral elements and β-carotene content in grain. Also, inverse correlation between β-carotene content and 1000 kernel weight, as well as positive correlation between Pphy and 1000 kernel weight were observed. Such results could indicate that further increase in Mg, Fe and Zn content in grain by breeding may not be negatively affected by the parallel increase in phytate content in inbred lines derived from the investigated landraces (Ma et

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al., 2007; Šimić et al., 2012). In this study, positive influence of β-carotene content increase on improved availability of examined mineral elements has to be considered carefully, since the landraces with the highest Mg, Fe and Zn values in grain are low in β-carotene content (Table 1). Table 1. Weight of 1000 kernels (KW) and chemical composition of grain for chosen drought tolerant

maize landraces

Landraces 1000 KW Pphy Pi β-carot. Mg Fe Zn

(g) (g kg-1) (mg kg-1)

P1 325.8 3.49 0.30 2.96 490.3 15.25 23.72

P2 340.6 3.43 0.35 10.85 400.0 12.42 12.19

P3 345.2 3.34 0.36 21.98 403.0 10.70 12.41

P4 365.6 3.48 0.43 9.61 412.3 11.81 14.11

P5 327.4 3.50 0.40 17.22 428.3 11.09 13.17

P6 510.6 3.45 0.40 18.43 436.4 14.70 13.95

P7 373.3 3.51 0.42 19.01 385.8 12.92 13.91

P8 459.0 3.46 0.46 2.95 445.8 11.17 13.23

P9 362.0 3.48 0.42 14.89 440.3 11.14 13.42

P10 386.2 3.15 0.40 3.41 420.9 9.75 11.95

P11 419.9 3.41 0.41 15.72 412.7 11.11 10.86

P12 372.7 3.46 0.46 15.05 419.8 10.20 11.44

P13 337.6 3.43 0.36 15.72 440.8 16.33 22.58

P14 331.8 3.44 0.36 19.64 426.7 10.80 15.41

P15 304.3 3.37 0.46 19.04 448.8 10.67 11.44

P16 361.0 3.32 0.37 19.64 411.9 10.11 11.47

P17 321.7 3.51 0.41 21.20 417.0 10.59 16.34

P18 350.3 3.76 0.40 12.43 408.3 9.84 16.38

P19 372.8 3.41 0.39 25.54 454.4 11.33 18.09

P20 390.5 3.59 0.37 26.75 427.8 12.61 13.52

P21 378.3 3.45 0.36 4.04 448.9 13.75 23.48

P22 322.8 3.49 0.38 12.12 427.3 10.59 9.41

P23 330.7 3.48 0.42 19.84 386.1 8.30 7.41

P24 406.8 3.74 0.42 20.30 408.4 12.31 12.05

P25 343.7 3.47 0.37 21.07 415.6 12.69 9.91

P26 311.7 3.25 0.36 4.28 402.8 9.59 11.95

F-test

Year F 0.37 236.0 1.67 7.30 54.06 1.68 3.62

p 0.55 0.00 0.20 0.01 0.00 0.20 0.06

Genot. F 6.24 0.36 6.12 17.02 2.00 2.55 2.85

p 0.00 1.01 0.00 0.00 0.01 0.01 0.00

Y x G F 1.88 18.73 51.25 183.12 163.84 36.22 9.36

p 0.02 0.00 0.00 0.00 0.00 0.00 0.00

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Table 2. Molar ratios between phytic and inorganic phosphorus, phytic acid and β-carotene, and phytic

acid and observed mineral elements in grain of investigated maize landraces

Pphy/Pi Phy/β-carot. Phy/Mg Phy/Fe Phy/Zn

P1 11.54 ± 0.85 3407.9 ± 2.7 2.14 ± 1.23 68.84 ± 4.45 44.26 ± 1.64

P2 9.87 ± 0.22 912.1 ± 10.2 2.57 ± 0.30 82.90 ± 0.21 84.49 ± 0.59

P3 9.17 ± 1.08 439.4 ± 17.3 2.49 ± 1.35 93.84 ± 6.22 80.96 ± 2.68

P4 8.12 ± 0.80 1045.7 ± 8.8 2.54 ± 1.03 88.52 ± 0.97 74.11 ± 1.90

P5 8.69 ± 0.48 586.7 ± 18.1 2.45 ± 0.69 94.74 ± 0.83 79.79 ± 1.19

P6 8.58 ± 0.95 540.8 ± 15.7 2.38 ± 1.22 70.52 ± 1.02 74.31 ± 2.05

P7 8.32 ± 0.81 532.8 ± 18.5 2.73 ± 0.91 81.56 ± 1.16 75.79 ± 1.98

P8 7.58 ± 0.76 3394.6 ± 3.6 2.33 ± 1.12 93.12 ± 2.12 78.61 ± 1.75

P9 8.33 ± 0.69 675.9 ± 13.4 2.38 ± 0.99 93.97 ± 1.31 78.00 ± 1.60

P10 7.95 ± 0.70 2665.4 ± 2.1 2.25 ± 1.09 97.04 ± 1.50 79.15 ± 1.90

P11 8.34 ± 0.74 626.3 ± 10.3 2.48 ± 1.02 92.16 ± 2.50 94.29 ± 1.88

P12 7.51 ± 0.81 663.9 ± 13.6 2.48 ± 1.07 101.85 ± 2.14 90.86 ± 1.92

P13 9.51 ± 0.94 631.3 ± 15.0 2.34 ± 1.29 63.21 ± 1.76 45.71 ± 2.14

P14 9.50 ± 0.58 506.1 ± 14.7 2.42 ± 0.79 95.76 ± 2.56 67.11 ± 1.38

P15 7.34 ± 0.87 511.9 ± 15.1 2.26 ± 1.32 95.00 ± 3.31 88.64 ± 2.04

P16 8.88 ± 0.83 488.8 ± 16.2 2.42 ± 1.09 98.77 ± 3.86 87.07 ± 2.05

P17 8.58 ± 0.47 477.8 ± 11.7 2.53 ± 0.56 99.49 ± 3.39 64.49 ± 1.07

P18 9.47 ± 0.51 873.8 ± 8.3 2.77 ± 0.64 114.79 ± 1.17 69.00 ± 1.24

P19 8.73 ± 0.47 385.8 ± 19.2 2.26 ± 0.74 90.49 ± 3.11 56.65 ± 1.17

P20 9.84 ± 0.98 387.9 ± 23.3 2.52 ± 1.31 85.59 ± 5.74 79.85 ± 2.32

P21 9.54 ± 0.74 2463.6 ± 3.8 2.31 ± 1.11 75.36 ± 3.49 44.13 ± 1.80

P22 9.09 ± 0.56 832.4 ± 10.9 2.46 ± 0.73 99.10 ± 0.83 111.61 ± 1.24

P23 8.22 ± 0.39 506.9 ± 15.7 2.71 ± 0.52 126.10 ± 1.90 141.27 ± 1.11

P24 8.94 ± 0.26 532.7 ± 17.7 2.75 ± 0.35 91.37 ± 0.82 93.39 ± 0.66

P25 9.35 ± 0.25 475.9 ± 15.7 2.51 ± 0.36 82.20 ± 1.29 105.28 ± 0.66

P26 9.13 ± 0.37 2188.2 ± 1.5 2.42 ± 0.44 101.65 ± 0.41 81.58 ± 0.88

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Fig 1. Principal component analysis (PCA) for Pphy, β-carotene and mineral elements in the grain of 26

investigated drought tolerant maize landraces (synthetic variables: PCA1 - principal component axis 1, PCA2 - principal component axis 2 and PCA3 - principal component axis 3)

Conclusion

Investigation of drought tolerant maize landraces revealed relatively higher level of Pphy, which could be considered as inhibiting factor for improved availability of Fe, Zn and Mg. In the majority of landraces, lower level of β-carotene content was also observed. However, consistently positive and highly significant correlations (P ≤ 0.001) between contents of all investigated mineral elements showed that simultaneous selection for these mineral nutrients could be highly effective. Based on the performance of the landraces per se, genotypes P1, P13 and P21 could be considered as potential sources of favorable genes for further breeding programs for improved nutritional quality, such as enhanced availability of mineral elements (i.e. P1 as a source for Mg and Zn, P13 as a source for Fe and Zn, and landrace P26 as a good source for Zn). Acknowledgements

This work was supported by Project TR31028 “Exploitation of maize diversity to improve grain quality and drought tolerance” from the Ministry of Education, Science and Technological Development, Republic of Serbia. References American Association of Cereal Chemists Method (1995): Approved Methods of the AACC,

The association: St. Paul, MN, USA, AACC Method, pp. 14-50. Babic M., Andjelkovic V., Mladenovic Drinic S., Konstantinov K. (2011): The conventional and

contemporary technologies in maize (Zea mays L.) breeding at Maize Research Institute, Zemun Polje. Maydica 56: 155-164.

Bänziger M., Long J. (2000). The potential for increasing the iron and zinc density of maize through plant-breeding. Food Nutr Bull 21: 397-400.

Brinch-Pedersen H., Borg S., Tauris B., Holm P.B. (2007): Molecular genetic approaches to increasing mineral availability and vitamin content of cereals. J Cereal Sci 46: 308-326.

Cakmak I. (2008): Enrichment of cereal grains with zink: agronomic or genetic biofortification? Plant Soil 302: 1-17.

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Dragičević V., Sredojević S., Perić V., Nišavić A., Srebrić M. (2011): Validation study of a rapid colorimetric method for the determination of phytic acid and norganic phosphorus from grains. Acta Periodica Technologica 42: 11-21.

Erdal I., Yilmaz A., Taban S., Eker S., Torun B., Cakmak I. (2002): Phytic acid and phosphorus concentrations in seeds of wheat cultivars grown with and without zinc fertilization. J Plant Nutr 25: 113-127.

Graham R., Senadhira D., Beebe S., Iglesias C., Monasterio I. (1999): Breeding for micronutrient density in edible portions of staple food crops: conventional approaches. Field Crop Res 60: 57-80.

Lönnerdal B. (2003): Genetically modified plants for improved trace element nutrition. J Nutr 133: 1490S-1493S.

Ma G., Li Y., Jin Y., Zhai F., Kok F.J., Yang X. (2007): Phytate intake and molar ratios of phytate to zinc, iron and calcium in the diets of people in China. Eur J Clin Nutr 61: 368-374.

Menkir A. (2008): Genetic variation for grain mineral content in tropical-adapted maize inbred lines. Food Chem 110: 454-464.

Morris C.E., Sands D.C. (2006): The breeder’s dilemma - yield or nutrition? Nat Biotechnol 24: 1078-1080.

Ortiz-Monasterio J.I., Palacios-Rojas N., Meng E., Pixley K., Trethowan R., Peña R.J. (2007): Enhancing the mineral and vitamin content of wheat and maize through plant breeding. J Cereal Sci 46: 293-307.

Oury F.-X., Leenhardt F., Rémésy C., Chanliaud E., Duperrier B., Balfourier F., et al. (2006): Genetic variability and stability of grain magnesium, zinc and iron concentrations in bread wheat. Eur J Agron 25: 177-185.

Šimić D., Mladenović Drinić S., Zdunić Z., Jambrović A., Ledenčan T., Brkić J., Brkić A., Brkić I. (2012): Quantitative trait loci for biofortification traits in maize grain. J Hered 103: 47-54.

Underwood E.J., Suttle N.F. (1999): Manganese. In: The Mineral Nutrition of Livestock, CABI Publishing, USA, Pp. 397-420.

Walter Lopez H., Leenhardt F., Coudray C., Remesy C. (2002): Minerals and phytic acid interactions: is it a real problem for human nutrition? Internat J Food Sci Technol 37: 727-739.

Welch R.M., Graham R.D. (2004): Breeding for micronutrients in staple food crops from a human nutrition perspective. J Exp Bot 55: 353-364.

White P.J., Broadley M.R. (2005). Biofortifying crops with essential mineral elements. Trends Plant Sci 10: 586-593.

White P.J., Broadley M.R. (2009): Biofortification of crops with seven mineral elements often lacking in human diets – iron, zinc, copper, calcium, magnesium, selenium and iodine. New Phytol 182: 49

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RESPONSE OF WINTER TRITICALE GENOTYPES ON NITROGEN FERTILIZATION

Dragana LALEVIĆ, Milan BIBERDŽIĆ

Faculty of Agriculture, University of Priština (Kosovska Mitrovica), Serbia

Kopaonička bb, 38 219 Lešak Corresponding author: dragana.lalevic@gmail.com

Abstract

In this paper, we studied the impact of different variants of fertilization on the yield and grain quality in five cultivars of winter triticale (Kg-20, Triumph, Rtanj, Tango and Odisej). The experiment, which was carried out in the period 2009 – 2012, in the area of Bijelo Polje (highland area of Montenegro), was set up in a randomized block system with three replications. Unfertilized plot (the control), the lowest rate of nitrogen (60 kg N ha-1) alone and three steps of N fertilization (60 + 90 + 120 kg N ha-1) on same level of P and K (80 P2O5 + 80 K2O kg ha-1) were applied.

The analysis of the obtained data has shown significant dependence of grain yield and its quality on genotype and mineral nutrition. Tango had the highest average grain yield (6.7 t ha-1) while Kg-20 had the lowest (4.5 t ha-1). Also, Tango had the highest value of the 1000 grain mass, while Triumph had the highest value of hectoliter weight. The application of fertilizers has led to a very large and significant increase of yield compared with the control. Accordingly, all studied cultivars had the highest yield when the tree nutritious elements, N, P and K, were used (120 kg ha-1 N, 80 kg ha-1 P2O5 and 80 kg ha-1 K2O).

Considering that triticale has manifested a great adaptability in these agro-ecological conditions and that grain yield per unit area is one of the most important which influences profitability cost-effectiveness of production, the area under this crop should be increased. Key words: triticale, genotype, fertilization, yield, grain quality Introduction Triticale, as a new highly successful species of small grains created by crossing wheat and rye, in recent years has become increasingly important, both for our producers and in the world. Thanks to a number of good agronomic characteristics, this species is becoming more attractive and takes a larger area (Denčić and Kobiljski, 2004; Milovanović, 1993; Milovanović et al. 1995; Milovanović et al. 2007). Triticale has high resistance to diseases and pests, and extremely good endurance to drought, acid soils and poor quality soils. Because of its high adaptability, triticale is suitable for cultivation in mountainous and hilly areas, where, even with the

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application of lower cultivating technology, satisfactory yields are achieved. The number of areas for triticale cultivation in the world is increasing, indicating that due to the increase of population, triticale will be as important as other types of cereals such as, wheat and barley. According to (Milovanović et al, 2007), there are over 4 million acres under triticale all over the world. In addition to high yield, newer types of triticale are characterized by extremely good nutritious values, which is why this grain is recommended as a nutrient for all species of domestic animals (Đekić et al. 2009b). Material and Methods

In the experiment which was carried out in the period 2009-2012, in the highland area of Montenegro, in the vicinity of Bijelo Polje – Sutivan (43º 01' 45" north latitude and 19º 44' 44" east longitude) on the alluvial type of soil, five cultivars of winter triticale were tested (Odisej, Kg-20, Triumph, Rtanj and Tango). The experiment was set out in randomized block system with three replications and experimental plot size of 6 m2. Unfertilized plot (the control), the lowest rate of nitrogen (60 kg N ha-1) alone and three steps of N fertilization (60 + 90 + 120 kg N ha-1) on same level of P and K (80 P2O5 + 80 K2O kg ha-1) were applied. Common agronomical practices were used in the experiment. Sowing was carried out by manual method in optimal term (October). Phosphorus and potassium were used in equal amounts (80kg ha-1) before the sowing period, while nitrogen was used in small amount before the sowing period, and the rest of the planned amount was used as a fertilization at the end of March. The average results of yield of dry grain, hectoliter weight and 1000 grain mass, are presented in this paper, for analyzed period of three years. The obtained results were statistically processed using method of variance analysis, whereby the significance of average treatments was tested with LSD test, with significance threshold of 1 and 5 %. Weather and Soil Conditions The soil on which the experiment was performed is weakly calcareous, the total content of carbonate being 2.4 – 2.44 %. Based on pH value in saline extract the tested soil is of acid-based reaction. The soil is quite humic: 3.35-3.96 % with low phosphorus 5.12 – 4.24 mg/100g soil) and potassium content (7.5 – 3.8 mg/100g soil). Data in Table 1. indicates the difference in average monthly temperatures between years in which the research was conducted. Table 1. Average monthly air temperature and precipitation amount (Podgorica Weather Bureau)

Year Months Average X XI XII I II III IV V VI VII

Monthly rainfall (mm) 2009-10 135 94 94 101 80 70 78 80 63 86 881 2010-11 65 131 147 36 76 31 46 121 33 79 765 2011-12 36 7 55 79 183 57 47 46 34 8 552 1961-90 80 115 91 87 68 60 70 76 72 64 783

Average monthly temperatures (°C)

2009-10 9.77 5.95 4.06 1.31 2.4 6.39 10.93 15 18.11 20.95 9.5 2010-11 10.12 8.54 2.05 -0.65 0.94 6.03 10.54 14.5 18.9 21.23 9.2 2011-12 9.3 3.25 2.17 -1.72 -3.52 5.96 10.8 15.02 20.67 24.63 8.7 1961-90 9.4 4.7 0.2 -1.3 0.7 4.9 9.0 13.3 16.3 17.9 7.5

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During season 2009/10, from October to July, the rainfall was 881.5 mm, while in the same period in 2010/11 and 2011/12 the amount of rainfall was lower (765 and 552 mm, respectively). From the data in the table it can be noted that conditions for germination and autumn plant development were considerably favorable during first two years compared to the third year of research. Also, the amount of rainfall in the period from April to June in the first year of research were higher compared to the other two years of research. Considering that the amount of rainfall and temperature in these months are extremely important for development of small grains, the first year of research can be characterized as the most optimal in terms of weather conditions for growing triticale in this area. Results and Discussion Grain yield, a 1000 grain mass and hectoliter weight are complex quantitative properties conditioned by activity of large number of genes strongly influenced by external environment (Mladenov et al., 1998).

Table 2. Impact of nitrogen fertilization on properties of winter triticale cultivars Winter triticale properties ( 3-year means: 2010-2012)

Cultivar (A) Nitrogen fertilization (kg ha-1)* - the factor B a b c d e

N 0 N60 N60+PK N90+PK N120+PK Average A Grain yield (kg ha-1)

Odisej 3954 4886 5917 6236 6718 5542 Kg-20 3342 4306 4784 4928 5135 4499

Trijumf 4601 5253 5988 6282 6889 5803 Rtanj 4864 5395 6539 6931 7334 6213 Tango 5345 6405 6712 7218 7840 6704

Average B 4421 5249 5988 6319 6783 5752 LSD A B AB 0,05 89.60 89.60 200.46 0,01 127.06 127.06 284.29

Mass of 1000 grain (g) Odisej 40.52 43.49 43.6 42.8 44.46 43.23 Kg-20 29.94 33.08 35.64 33.78 34.98 33.51

Trijumf 41.75 45.09 42.61 42.55 44.77 43.64 Rtanj 42.84 46.98 45.67 45.8 46.28 45.50 Tango 45.07 49.33 48.39 49.15 50.53 48.67

Average B 40.02 43.59 43.18 42.82 44.2 42.91 LSD A B AB 0,05 1,63 1,63 3,64 0,01 2,31 2,31 5,17 Hectoliter weight (kg)

Odisej 64.99 69.87 70.43 70.15 70.87 69.26 Kg-20 61.75 63.53 63.78 62.87 65.09 63.4

Trijumf 68.4 71.56 70.82 71.22 71.66 70.73 Rtanj 63.77 66.98 66.38 66.75 65.78 65.93 Tango 65.66 69.08 67.41 67 68.9 67.61

Average B 64.91 68.2 67.77 67.6 68.46 67.39 LSD A B AB 0,05 1,54 1,54 3,44 0,01 2,18 2,18 4,87

* PK = 80 kg P2O5 and 80 kg K2O per hectare

Average yield of winter triticale for all cultivars and fertilization variants during the three years of research period was 5752 kg ha-1. In average for all cultivars the highest yield (6.78 t ha-

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1) was achieved with variant (e) of fertilization. In average for all variants, Tango achieved the highest yield (6.7 t ha-1), whereas the lowest yield was achieved with Kg-20 (4.5 t ha-1). Our results are in accordance with the results of Dodig et al. (2006) which highlighted Tango as a cultivar of high genetic potential for yield with ability of its manifestation on different localities. Differences in the yield between cultivars in our research are largely determined by genetic factor. Specific response of cultivars to mineral nutrition, especially nitrogen, was the subject of interest for other scientist as well (Jelić et al. 2004; Bojović, 2010). Complete application of all three elements (NPK) in the form of fertilizer had very positive influence on the yield. Research indicates that the winter triticale achieves high yields when nitrogen is used in the amount of 120 kg ha-1 and phosphorus and potassium in the amount of 80 kg ha-1. Noticeable positive effect of the complete application of fertilizer is the result of lower pH value of the soil, as well as low content of available phosphorus and potassium in this soil which, for this reason, must be added in the form of fertilizer. Our results are in accordance with other authors (Gibson et al. 2007; Lestingi et al. 2010, Bojović, 2010). The climate factors in the years of research also had a significant impact on the grain yield of triticale. Thanks to the suitable climate conditions, the average yield in the first year (6.2 t ha-1) was significantly higher compared to the second and third year of research (5.7 t ha-1 i.e. 5.3 t ha-

1). A 1000 grain mass is cultivar characteristic which is the reason for bigger variations between different genotypes than between variants of mineral nutrition (Jelić et al. 2002; Lalević et al. 2012). Data given in Table 2. indicate that the average value of absolute mass for all cultivars and fertilization variants for three-period research was 42.91 g. Cultivar Kg-20 has the lowest value of absolute mass with the control variant of fertilization, while cultivar Tango the highest value with the (e) variant of fertilization. Application of fertilizer had a significant effect of absolute mass value in all cultivars. Accordingly, a 1000 grain mass was considerably higher with all variants of fertilization in comparison to control. Our results are in accordance with the results of Milošev et al (2006) i Jaćimović et al. (2008) who found that a 1000 grain mass was significantly higher with more intensive fertilization treatments, especially in cases when nitrogen was used. Hectoliter weight is a complex feature controlled by great number of genes. Also, environmental conditions have a significant effect on this feature, making breeding difficult. Average value of hectoliter weight for all cultivars and fertilization variants for three-year period was 67.39 kg. Each cultivar had the lowest hectoliter weight value in the control. The application of fertilizers made the hectoliter weight grow significantly and it reached its highest level with the (e) variant of fertilization (68.48 kg). Cultivar Kg-20 had the lowest hectoliter weight in the control (61.74 kg), while the cultivar Triumph had the highest hectoliter weight with (e) variant of fertilization (68.48 kg). There is a strong dependence of hectoliter weight from dose of nitrogen applied. The tested cultivars had mostly positive reaction to the application of the complete nutrient, as well as to growing doses of nitrogen, which is in accordance with the results previously presented Lalević et al, 2012. Conclusion Based on our research on achieved yield and quality of winter triticale following conclusions can be drawn:

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- The highest grain yield with all variants of fertilization and in average was achieved with cultivar Tango, and the lowest with the cultivar Kg-20. The highest average grain yields was achieved with (e) variant of fertilization and it was significantly higher compared to

the yield achieved with other variants of fertilization. - The highest value of a 1000 grain mass had cultivar Tango (50.53 g) with (e) variants of

fertilization and the lowest had cultivar Kg-20 (29.94 g) in the control. - The lowest hectoliter weight had cultivar Kg-20 (61.75 kg) in the control, and the highest had cultivar Triumph (71.66 kg) with (e) variant of fertilization. - The application of fertilizers significantly increased values of all tested parameters

compared to the control. - For the tested area winter triticale cultivar Tango and (e) variant of fertilization are

recommended considering that this cultivar, with quantity and quality of its yield, suits best the given climatic conditions. References Bojović, B. (2010):.Uticaj mineralne ishrane na neke parametre produktivnosti sorata pšenice i

tritikalea, Doktorska disertacija, Univerzitet u Kragujevcu, Prirodno-matematički fakultet. Gibson, L. R., Nnance C. D., Karlen D. L. (2007). Winter triticale response to nitrogen

fertilization when grown after corn or soybean. Agronomy Journal, 99: 49-58. Denčić, S., Kobiljski, B. (2004). Pšenica i tritikale kao stočna hrana. Acta agriculturae Serbica,

Vol. 9, br. spec. br., str. 485-492. Dodig, D., Stanković, S., Milićević-Nikodijević Slađana, Jović Miroslava (2006). Novopriznate

zaječarske sorte strnih žita, Selekcija i semenarstvo, Vol. XII, No. 1-2, p. 49-54, Novi Sad. Đekić, V., Glamočlija, Đ., Staletić, М., Perišić, V. (2009b). Prinos i komponente prinosa zrna

Kg sorti ozimog tritikalea. Zbornik izvoda IV Simpozijuma sa međunarodnim učešćem „Inovacije u ratarskoj i povrtarskoj proizvodnji“, 23-24. oktobar, 2009., Beograd, 132-133.

Đekić, V. Staletić M., Glamočlija, Đ., Perišić, V. (2009/3). Varijabilnost prinosa i komponenata prinosa zrna kragujevačkih ozimih sorti tritikalea. Journal og Agriculture Science, 70, str. 55-60.

Jaćimović, G., Malešević, М., Marinković, B., Crnobarac, Ј., Latković, D., Šeremešić, S., Milošev., D. (2008). Komponente prinosa jare pšenice u zavisnosti od nivoa đubrenja azotom, fosforom i kalijumom. Letopis naučnih radova, Poljoprivredni fakultet Novi Sad, Vol. 32, I, str. 57-63.

Jelić, M., Stojanović, J., Stojanović, S., Živanović, S. (2002). Optimalna tehnologija proizvodnje kragujevačkih sorti strnih žita, Agroinovacija, br. 3, 163-171.

Jelić, M., Dugalić, G., Milivojević Jelena, Nikolić Olivera, Živanović-Katić Snežana (2004). Uticaj sistema mineralne ishrane na prinos zrna ozimog tritikalea, Acta Agriculturae Serbica, vol. IX, 17, 493-499.

Lalević Dragana, Biberdžić, М., Jelić, M., Barać, S. (2012). Some characteristics of triticale cultivated in rural areas, Agriculture & Forestry, Vol. 58 Issue 2, p27-34. 8p.

Lestingi, A., Bovera, F., De Gorgio, D., Ventrella, D. and Tateo, A. (2010). Effect of tillage and nitrogen fertilisation on triticale grain yield, chemical composition and nutritive value. J. Sci. Food Agric. 90: 2440-2446.

Milovanović, M. (1993). Investigation of yield and technological traits of grain of the intergenus hybrids triticale (X Triticosecale Wittmack). Rew. Of Res. Work at the Faculty of Agriculture, 38, 2,71-82.

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Milovanović, M., Perišić, V., Vera Đekić, Vesna Stevanović (2007). KG Rubin-new triticale cultivar. A Collection of Articles from the Technical College in Pozarevac, N° 1, p. 19-23, Pozarevac.

Milovanović, M., Kuburović, M., Stojanović, S., Ognjanović, R. (1995). Some recent results of winter triticale breeding in Kragujevac. 1st Balcan Symposium „Breeding and cultivation of wheat, sunflower and legume crops“, Albena-IWS, Proceedings, 125-129, Bulgaria.

Milošev, D., Bogdanović, D., Jarak, M., Šeremešić, S. (2006). Uticaj azota iz različitih izvora na prinos i komponente prinosa pšenice. Zbornik radova, Institut za ratarstvo i povrtarstvo Novi Sad, sv. 42, 195-202.

Mladenov, N., Mišić, T., Pržulj, N., Hristov, N. (1998). Year effects on wheat seed quality. International Symposium Breeding of Small Grains, Kragujevac, November 24-27, 1998. Book of Proceedings, 343-349.

Đekić Vera, Staletić Mirjana, Glamočlija, Đ., Perišić, V. (2009/3). Varijabilnost prinosa i komponenata prinosa zrna kragujevačkih ozimih sorti tritikalea. Journal og Agriculture Science,

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PRENATALLY DETECTED TRISOMY 20 MOSAICISM- A SINGLE CASE REPORT

Ana MILOŠEVIĆ-ĐERIĆ1, Boško RISTANOVIĆ1, Milena AĆIMOVIĆ1, Gordana ŠOŠIC2, Tanja NOVAKOVIĆ2

1Department of Gynaecology and Obstetrics, Hospital Centre Uzice, Uzice, Serbia

2Clinic of Gynaecology and Obstetrics, Clinics Centre Kragujevac, Kragujevac, Serbia

Corresponding author: Ana Milosević-Đerić, Department of Gynecology and Obstetrics, Hospital Uzice, Milosa Obrenovica 17, 31 000, tel +381 31562 848, fax +381 31561 861, Uzice,

Serbia, mail: ana.mdjeric@gmail.com Abstract

The risk of appearance of single and multiple anomalies in cases of trisomy 20 mosaicism detected by amniocentesis still remains uncertain. The phenotype of the individuals with mosaic chromosome 20 trisomy differs from normal to full manifestations of the trisomy. Nonetheless, the presence of phenotype abnormalities depends on percentage of trisomic cells line detected prenatally. Results. In this work prenatally cytogenetic analysis showed the trisomy 20 mosaicism, with the extra chromosome in 53% of amniotic cells. The fetal blood karyotype as well as detailed ultrasonic scan of the baby was normal. Parents decided to continue the pregnancy. On clinical examination the newborn male, born at term, showed normal phenotype. This case together with all other known cases of trisomy 20 mosaicism may contribute to the formation of a database in order to better understand the biological significance of this chromosome finding prenatally as well as necessity of further postnatal diagnosis and long time follow-up regardless of the normal clinical signs at birth. Key words: trisomy 20, mosaicism, amniocentesis

Introduction

The mosaic or mosaicism denotes the presence of two or more cell lines with different genetic constitution in one individual who has developed from a single fertilized egg (Turnpenny and Ellard, 2007). The most common form of chromosomal mosaicism found through prenatal diagnosis involves trisomies. Trisomy 20 mosaicism is one of the mosaic form prenatally diagnosed via amniocentesis. Complete trisomy 20 is not viable, the usual phenotype findings are severe and only few cases in the literature involved fetuses surviving past the first trimester. For genetic counseling mosaic trisomy 20 is much challenging than its nonmosaic form (Bianca

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et al., 2005). Phenotype of prenatally detected trisomy 20 mosaicism is in 90-95% of cases observed as normal. Abnormal outcomes include unexplained fetal demise, intrauterine growth restriction, and multiple congenital anomalies (Robinson et al., 2005). Nevertheless, it is known that there is a clear association between the percentage of trisomic cells detected on amniocentesis and abnormality. Risk for abnormal outcome in cases with less than 50% trisomic cells has been estimated on 4,5% versus estimated risk of 20% for more than 50% trisomic cells (Wallerstein et al., 2000).

Case presentation

Here we report case of prenatally detected trisomy of chromosome 20 mosaicism in male fetus during amniocentesis. A 36 year-old woman presented in her third pregnancy; she had 2 vaginal deliveries of term infants. Both her familial and personal genetic histories were unremarkable. Amniocentesis was performed in the 18th gestational week for advanced maternal age. Metaphase chromosome preparation was obtained from amniotic fluid cells, using standard technique. Conventional cytogenetic analyses were carried out using GTG banding. Sample analysis revealed a normal male karyotype in 14 cells, while in 16 cells were observed trisomy 20 (46,XY [14] / 47,XY,+20 [16]). Chromosomes isolated from trisomic cells line stained by G banding techniques are shown in Figures 1 and 2 (Fig.1,Fig.2). The parents were informed that high level trisomy 20 mosaicism was detected in the amniotic fluid cells and fetal cordocentesis was suggested. Ultrasonography performed showed normal phenotype and mosaic cells were not observed in the cord blood sample. After genetic counselling parents elected continue pregnancy.

Fig.1 Chromosomes isolated from trisomic cell line (G banding pattern). Chromosomes 20 are marked

with the circles. Arrow indicates extra chromosome 20.

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Fig.2 Chromosomes isolated from trisomic cell line and arranged by pairs. Arrow indicates extra

chromosome 20. Discusion

Trisomy 20 is one of the most common mosaic trisomy detected on prenatal diagnosis, during standard screening by amniocentesis or chorionic villus sampling (Mavromatidis et al., 2010). Despite this fact clinical significance of trisomy 20 mosaicism detected during prenatal diagnosis still is a problem for genetic counseling. Main reason is rarity of live born cases with inconsistent clinical findings to make general data base of possible clinical outcome (Reiss et al., 1998). We report an interesting case of the normal live born male after prenatal diagnosis of the high percentage of trisomy 20 mosaic cells (53%) during amniocentesis. Based on a review of the literature, the level of mosaic cells found is associated with risk of abnormal outcome. In published cases with more than 50% mosaic cells detected prenatally chance for abnormalities were about 20%. When less than 50% mosaic cells were detected chance for abnormal outcome is about 4,5% (Wallerstain et al., 2000). Interesting is that a higher levels of trisomy were observed in male fetuses as compared to female fetuses (HSU et al., 1991). To exclude possibility of abnormal outcome we performed detailed ultrasonic scan and fetal cord karyotype. Ultrasonography of fetus showed normal development without severe congenital anomalies and intra-uterine growth retardation (IUGR). Trisomic cells were not found in cord blood. This is not unexpected findings since in most cases trisomic cells do not appear in blood even in cases with abnormal outcome (Bianca et al., 2005, Heljic et al., 2008). However, the origin of these abnormal cells and their causative role is still unclear. Confirmation of trisomy 20 in blood has been described in one case of prenatally detected 79% mosaic trisomy 20. 17% of foetal cord cell also showed trisomy 20. Phenotype of a born male baby was normal with no dysmorphology

(Brothman et al., 1992). According published cases mosaicism may be confined to extraembrionic tissue or other various fetal tissues. In the work of Reish et al. (1998) they report 46,XX chromosome constitution in white blood cells, while skin fibroblasts demonstrated trisomy 20 mosaicism (54%) by fluorescence in situ hybridization (FISH) analysis. On clinical exam baby was with bilateral epicanthal folds, delayed closure of fontanel and no other gross anomalies, but demonstrated a considerable developmental delay in gross and fine motor skills along with hypotonicity (Reish et al., 1998). Velissariou and coworkers published trisomy 20 mosaicism detected in amniotic fluid (98%) and was confirmed in the term placenta (100%), as

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well as in the blood (10%) and urine sediment (100%) of the newborn girl. There was intrauterine and postnatal growth retardation. Newborn at age nine month showed moderate psychomotor retardation, central hypotonia with peripheral hypertonia, numerous minor morphogenetic variants, marked kyphosis, and extensive Mongolian spot (Velissariou et al., 2002).

Strømme and coworkers (2005) describe a case of 16 1/2–year-old girl with multiple anomalies including cleft lip and palate and a normal karyotype in blood lymphocyte. Her karyotype in skin fibroblasts was examined, because of hyperpigmentation along the lines of Blaschko, and showed mosaicism for trisomy 20. This was the first report of this karyotipe associated with both hyperpigmentation and facial clefting (Strømme et al., 2005). In our presented case a normal male baby was born at term. After birth, clinical examination of baby was normal without growth retardation, hypotonia, structural central-nervous system (CNS) abnormalities and seizures, facial dysmorphism, failure to thrive, and development delay. No further information on the psychomotor development of the child was available. Some authors show that over a number of years of follow-up of this patients it has apparent that there are some striking similarities among them including spinal abnormalities, hypotonia, lifelong constipation, sloped shoulders and significant learning disabilities despite despite normal intelligence (Willis et al., 2008).

Conclusion

We report a case of the phenotypicaly normal live born male after prenatal diagnosis of a high percentage of trisomy 20 mosaicism in amniocytes. This case demonstrates that even a high percentage of trisomic cells are detected a phenotype of baby is compatible with normal psychomotor development and phenotype. However, as studies up to now showed it is important to follow up phenotype of patient. Sometimes some problems are overlooked during standard pediatric exams. Child diagnosed with mosaic trisomy 20 should be assessed or followed even in the presence of an apparently normal physical exam at bird. Our results can contribute for collections of data on all cases cases of trisomy 20 mosaicism diagnosed prenatally, in order to provide more accurate information about it. References Bianca, S., C. Ingegnosi, C. Tetto, A. Cataliotti, G. Ettore (2005). Prenatally detected trisomy 20

mosaicism and genetic counseling. Prenat. Diagn. 25(8):725-6. Brothman, A.R., K. Rehberg, P.D. Storto, S.E. Phillips, R.T Mosby (1992). Confirmation of true

mosaic trisomy 20 in a phenotypically normal liveborn male. Clin. Genet. 42(1):47-9. Heljic, S., H. Maksic, J. Dizdarevic, S. Uzicanin, I Kalkan (2008). Trisomy 20 mosaicism

detected prenatally: A case report of phenotypically normal male liveborn. Ultraschall in Med. 10.1055.

Hsu, L.Y, S. Kaffe, T.E. Perlis (1991). A revisit of trisomy 20 mosaicism in prenatal diagnosis--an overview of 103 cases. Prenat. Diagn. 11(1):7-15.

Mavromatidis G, Dinas K, Delkos D, Vosnakis C, Mamopoulos A, Rousso D (2010). Case of prenatally diagnosed non-mosaic trisomy 20 with minor abnormalities. J. Obst. Gyn. Res. 36(4):866-868.

Reish, O., B. Wolach, A. Amiel, I. Kedar, T. Dolfin, M. Fejgin (1998). Dilemma of trisomy 20 mosaicism detected prenatally: is it an innocent finding? Am. J. Med. Genet. 77(1):72-5.

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Robinson, W.P., B. Mcgillivray, M.E. Lewis, L. Arbour, I. Barrett, D.K (2005). Prenatally detected trisomy 20 mosaicism. Prenat. Diagn. 25(3):239-44.

Strømme, P., C.B. Van Der Hagen, M. Haakonsen, K. Risberg, R. Hennekam (2005). Follow-up of a girl with cleft lip and palate and multiple malformations: trisomy 20 mosaicism. Scand. J. Plast. Reconstr. Surg. Hand. Surg. 39(3):178-9.

Turnpenny, P. And S. Ellard (2007). Emers base of medical genetics (in serbian Emerijevi osnovi medicinske genetike) 13. editions. Belgrade Datastatus.

Velissariou, V., T. T. Antoniadi, J. Gyftodimou, K. Bakou, M. Grigoriadou, S. Christopoulou, A. Hatzipouliou, J. Donoghue, P. Karatzis, E. Katsarou, M. B. Petersen (2002). Maternal uniparental isodisomy 20 in a foetus with trisomy 20 mosaicism: clinical, cytogenetic and molecular analysis. Eur. J. Hum. Gen. 10(11)694-698.

Wallerstein, R., M.T. Yu, R.L. Neu, P. Benn, C. Lee Bowen, B. Crandall, C. Disteche, R. Donahue, B. Harison, D. Hershey, R.R. Higgins,L.S. Jenkins, C. Jackson-Cook, E. Keitges, G. Khodr, C.C. Lin, F.W. Luthardt, L. Meisener, G. Mengden, S.R. Patil, M. Rodrigez, L.J. Sciorra, L.G. Shaffer, G. Stetten, D.L. Van Dyuke, H. Wang (2000). Common trisomy mosaicism diagnosed in amniocytes involving chromosomes 13, 18, 20 and 21: karyotype-phenotype correlations. Prenat. Diagn. 20(2):103-22.

Willis, M.J., L.M. Bird, M. Dell'aquilla, M.C. Jones (2008). Expanding the phenotype of mosaic trisomy 20. Am. J. Med. Genet. A. 146(3):330-6.

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SSR ALLELE FREQUENCY DIFFERENCES AMONG DROUGHT TOLERANT AND SUSCEPTIBLE MAIZE INBRED LINES

Ana NIKOLIC, Dragana IGNJATOVIC-MICIC, Sofija BOZINOVIC, Violeta ANDJELKOVIC, Jelena VANCETOVIC

Maize Research Institute “Zemun Polje”, Slobodana Bajica 1, 11185 Belgrade, Serbia

Abstract

Seventeen maize inbred lines – 12 drought tolerant inbreds from drought tolerant mini core collection (Maize Research Institute gene bank) and five commercial drought susceptible inbreds were evaluated by SSR markers. The aim of the research was to test the significance of frequency difference for each allele at a marker locus between drought tolerant and susceptible inbred lines. Allele frequency difference at SSR loci identified six regions on chromosomes 1, 6, 7 and 10 that are known to be involved in drought tolerance response. Most of the frequency differences were significant at p‹ 0.05, except at bin 1.09 (p‹ 0.01) and bin 7.04 (p‹ 0.001). These results suggest that markers closely linked to the candidate genes for drought tolerance could be used for screening inbreds for the presence of desirable allele.

Key words: maize, SSR, allele frequencies, drought tolerance

Introduction

Drought is one of the most important and complex environmental stresses. In maize, drought alone causes an average yield loss of about 17 – 60% (Edmeades et al. 1999). Conventional breeding for drought tolerance was quite successful in improving yield potential of crops (Carena 2009, Vancetovic et al. 2014) while development of molecular marker techniques enabled quantitative trait loci (QTL) mapping and dissection of genetic and physiological components of drought stress response. Although marker assisted selection (MAS) was a promising approach for breeding drought tolerant genotypes, it has contributed very little to the release of improved cultivars due to the complexity of QTL (Collins et al. 2008, Xu and Crouch 2008).

Drought tolerance of maize genotypes could be improved by the use of available genetic resources (Hayano-Kanashiro et al. 2009, Meseka et al. 2013). Maize Research Institute (MRI) gene bank provided to be a rich source of desirable alleles for improving drought tolerance. Drought tolerant mini core collection consisting of 41 accessions with good combining ability

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was created from more than 6000 gene bank maize genotypes with good performance under drought conditions (Vancetovic et al. 2010). Landraces from former Yugoslavia, introduced landraces and introduced inbred lines are constituents of this collection.

In order to implement inbred lines with desirable traits/alleles in the process of commercial hybrid improvement it is necessary to get the information about their genetic structure. Nowadays, with a number of different molecular genetic methods available, such as SSR (simple sequence repeat) marker technique, it is possible to get the valuable data about the presence, number and frequency distribution of desirable alleles.

The objective of this research was (1) to analyze inbreds with SSR markers and to calculate the frequency of the scored alleles and (2) to test the significance of frequency difference for each allele at a marker locus between drought tolerant and drought susceptible inbred lines.

Material and methods

The analyzed plant material was comprised of twelve inbred lines from MRI drought tolerant mini core collection and five drought susceptible inbred lines – one from MRI gene bank collection and four commercial lines. The susceptible lines were incorporated into the experiment in order to test if any allele differences could be detected in comparison to tolerant lines.

DNA was isolated from maize flour obtained by grinding five seeds per inbred line. The extraction method used was based on the protocol described by Saghai-Maroof et al. (1984), with some modifications. Choice of SSR primers was based on bin locations to provide adequate coverage of all chromosomes (www.maizegdb.org). A total of 30 informative SSR markers (Table 1) were chosen after initial screening of 65 SSR loci on five inbred lines. Amplified fragments were separated on 8% polyacrylamide gels with 20bp ladder as a marker on a small format vertical gel system (Mini Protean Tetra-Cell BioRad) and stained with ethidium bromide. The gels were phographed using BioDocAnalyze (BDA) gel documentation system (Biometra, Germany) and SSR profiles for each primer were determined.

Allele frequencies were scored as pixel total percentage of individual bands within the sample, using UN-SCAN–IT gel 6.1 program package. The information content was calculated for each marker using the polymorphism information content – PIC (Lynch and Walsh 1998). PIC values were calculated as PIC = 1- Σ fi2, where fi2 is the frequency of the i

th allele. Correlations between allele number and PIC values were calculated using Pearson's correlation coefficient. χ2 test was used to test the significance of frequency difference for each allele at a marker locus between drought tolerant and susceptible inbred lines. Results and discussion

Thirty SSR primers produced a total of 143 alleles among the 17 inbred lines, and the allele number per locus varied from two (umc1652, umc1969, umc1109 and umc1006) to nine (umc1482), with average number of 4.77 (Table 1). The mean PIC value of the SSR loci was 0.57, ranging from 0.17 (umc1969) to 0.75 (umc1695). Eighteen SSR loci had PIC values higher than 0.60 indicating their potential informativness to detect differences among inbred lines. Correlations between allele number and PIC values were significant (r=0.654, p‹0.01). Although these results were in accordance with other authors (Heckenberger et al. 2002, Vaz Patto et al. 2004) their values were smaller. This most probably can be explained by the choice of SSR

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markers and polyacrylamide electrophoresis. In the research of the listed authors detection was performed on automated sequencing gels that have much higher resolution capabilities.

Table 1. SSR markers used in the genetic diversity analysis and their genomic location (bin), repeat

class, allele size, number of alleles and PIC values

Locus Bin Repeat class Allele size

(~bp)* Number of

alleles PIC

umc1269 1.01 (CCT)4 300-400 3 0.65 umc1568 1.02 (TAG)4 110-300 6 0.52 umc1278 1.07 (CT)12 80-180 5 0.73 umc2047 1.09 (GACT)4 110-140 5 0.48 umc1605 1.12 (GGC)4 140-160 3 0.48 umc1845 2.03 (AG)8 130-150 5 0.56 umc1465 2.04 (ACACA)4 110-1360 4 0.38 phi036 3.04 AG 60-80 6 0.71

umc1489 3.07 (GCG)5 120-140 7 0.66 umc1594 3.09 (TA)10 120-150 8 0.70 umc1652 4.04 (CCG)5 120-140 2 0.46 umc1969 4.05 - 90-100 2 0.17 umc1109 4.10 (ACG)4 110-120 2 0.21 umc1707 4.11 (AT)6 130-160 7 0.63 phi085 5.06 AACGC 220-260 3 0.53

umc1482 5.05 (AGC)5 60-180 9 0.67 umc1006 6.02 (GA)19 100-120 2 0.50 umc1887 6.03-6.04 (CGA)4 90-150 5 0.67 umc1857 6.04 (TAA)6 140-180 6 0.63 umc1520 6.06 (GA)8 170-190 4 0.64 umc1695 7.00 (CA)8 110-180 7 0.75 umc1782 7.04 (GAC)4 130-150 6 0.65 umc1933 8.08 (CCA)6 80-100 3 0.20 umc1638 8.09 (CTCCGG)5 140-150 5 0.61 umc1040 9.01 CT11 70-100 4 0.67 umc1492 9.04 (GCT)4 130-220 7 0.64 umc1657 9.05 (GACGG)4 140-160 3 0.60 umc1336 10.03 (ACCAG)4 50-150 6 0.72 umc1993 10.06 - 80-100 4 0.64 umc1645 10.07 (CT)10 140-160 4 0.56 Average 4.77 0.57

*~bp – approximate allele size (base pairs) determined in comparison with 20bp ladder marker

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Identified SSR loci with significant allele frequency difference between drought susceptible and drought tolerant inbred lines are given in Table 2, while allele frequency differences between these loci are depicted in Figure 1.

Possible candidate genes for drought tolerance identified in the consensus QTL regions were reported in Hao et al. (2010). Phytosulfokine peptide precursor 1 (psk1) was found at bin 7.00 and ribosome-inactivating protein 2 (rip2) at bin 7.04. Umc1695 and umc1782 SSR used in our research are located within psk1and rip2 genes, respectively (http://www.maizegdb.org). Moreover, allele frequency difference at bin 7.04 was with the highest significance level (p‹ 0.001).

At another region (bin 10.03) identified for allele frequency differences among drought tolerant and susceptible inbred lines, three candidate genes (abp4, ppo1 and nac1 – auxin binding protein homolog 4, polyphenol oxidase 1 and NaCl stress protein 1) were found in the meta-analysis.

However, 12 regions with drought tolerant QTL (nine of them being adaptive) found in the meta-analysis were not detected in our study. This could be due to several reasons, such as different genetic material and different markers used. Also, the number of inbred lines analyzed was small and results should be confirmed on a greater number of both susceptible and tolerant genotypes. Moreover, the results in Hao et al. (2010) were obtained on comprehensive experiments aimed at identification of QTL involved in drought tolerance, whereas drought susceptible and tolerant lines were only screened for SSR polymorphisms. But the results on allele frequency differences point to the possibility of using linked markers, especially the ones inside the gene sequences, to identify QTL in inbred lines and thus facilitate MAS for drought tolerance.

Table 2. Identified SSR loci with significant allele frequency difference between drought susceptible and

drought tolerant inbred lines

SSR

bin

Allele χ2 test positiona sizeb

(~bp) CHI

statistics P value p‹

umc2047 1.09 3 140 8.743 0.0031 0.01 umc1857 6.04 2 150 5.236 0.0221 0.05

3 160 4.408 0.0358 0.05 umc1520 6.06 1 170 6.199 0.0128 0.05 umc1695 7.00 3 130 5.236 0.0221 0.05

5 160 5.236 0.0221 0.05 umc1782 7.04 3 150 12.986 0.0003 0.001

4 160 4.958 0.026 0.05 umc1336 10.03 4 130 5.236 0.0221 0.05

aposition of the allele on the gel b~bp – approximate allele size (base pairs) determined in comparison with 20bp ladder marker

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Figure 1. SSR loci with significant allele frequency difference (at least p‹ 0.05) between drought tolerant

and drought susceptible inbred lines. All loci – average allele frequency for all 30 SSR within drought tolerant and susceptible lines; a1, a2 – different alleles at the same locus.

Conclusion

The results of allele frequency difference between drought tolerant and susceptible inbred lines at SSR loci suggest that the use of markers closely linked or within the sequences of candidate genes involved in drought tolerance could be used for screening inbred lines for the presence of desirable alleles, with the aim to facilitate MAS.

Acknowledgement

This research was supported by the Ministry of Education, Science and Technological Development of Republic of Serbia through the project TR31028 Exploitation of maize diversity

to improve grain quality and drought tolerance. References Carena MJ, Bergman G, Riveland N, Eriksmoen E and Halvorson M (2009). Breeding maize for

higher yield and quality under drought stress. Maydica 54: 287-296. Collins NC, Tardieu F and Tuberosa R (2008). Quantitative trait loci and crop performance

under abiotic stress: where do we stand? Plant Physiology 147: 469-486. Edmeades GO, Bolanos J, Chapman SC, Lafitte HR and Banziger M (1999). Selection improves

drought tolerance in tropical maize populations. I. Gain in biomass, grain yield, and harvest index. Crop Science 39: 1306 – 1315.

Hao Z, Li X, Liu X, Xie C, Li M, Zhang D and Zhang S (2010). Meta-analysis of constitutive and adaptive QTL for drought tolerance in maize. Euphytica 174:165-177.

Hayano-Kanashiro C, Calderón-Vázquez C, Ibarra-Laclette E, Herrera-Estrella L and Simpson J (2009). Analysis of Gene Expression and Physiological Responses in Three Mexican Maize Landraces under Drought Stress and Recovery Irrigation. PLoS ONE 4(10): e7531.

Heckenberger M, Bohn M, Ziegle JS, Joe LK, Hauser JD, Hutton M and Melchinger AE (2002). Variation of DNA fingerprints among accessions within maize inbred lines and

0.30

0.60

0.63

0.60

0.20

0.60

0.60

0.00

1.00

0.60

0.33

0.00

0.17

0.17

0.75

0.08

0.08

0.92

0.42

0.08

0.00 0.20 0.40 0.60 0.80 1.00

All loci

umc2047

umc1520

umc1857a1

umc1857 a2

umc1695a1

umc1695a2

umc1782a1

umc1782a2

umc1336

Allele frequency (%)

Tolerant

Susceptible

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implications for identification of essentially derived varieties. I. Genetic and technical sources of variation in SSR data. Molecular Breeding 10:181-191.

Lynch M and Walsh JB (1998). Genetics and Analysis of Quantitative traits. Sinauer Assocs., Inc., Sunderland, MA.

Meseka S, Fakorede M, Ajala S, Badu-Apraku B and Menkir A (2013). Introgression of alleles from maize landraces to improve drought tolerance in an adapted germplasm. Journal of Crop Improvement 27:96-112.

Saghai-Maroof M, Soliman K, Jorgensen R and Allard R (1984). Ribosomal DNA Spacer Length Polymorphism in Barley: Mendelian Inheritance, Chromosomal Location and Population Dynamics. Proceedings of the National Academy of Sciences of the USA, 91: 8014–8018

Vancetovic J, Mladenovic Drinic S, Babic M, Ignjatovic-Micic D and Andjelkovic V (2010). Maize genebank collections as potentially valuable breeding material. Genetika 42: 9-21.

Vancetovic J, Ignjatovic-Micic D, Bozinovic S, Babic M, Filipovic M, Grcic N and Andjelkovic V (2014). Grain quality of drought tolerant accessions within the MRI Zemun Polje maize germplasm collection. Spanish Journal of Agricultural Research 12: 186-194.

Vaz Patto MC, Pêgo S, Satovic Z and Fevereiro P (2004). Assessing the genetic diversity of Portuguese maize germplasm using microsatellite markers. Euphytica 137: 63-72.

Xu Y and Crouch JH (2008). Marker-assisted selection in pant breeding: from publications to practice. Crop Science 48:391-407.

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GENOTOXIC AND CYTOTOXIC EFFECT OF CAMPHOR, EUCALYPTOL AND THUJONE IN REPAIR PROFICIENT AND MISMATCH REPAIR DEFICIENT

EUKARYOTIC CELLS

Biljana NIKOLIĆ*, Dragana MITIĆ-ĆULAFIĆ, BrankaVUKOVIĆ-GAČIĆ, Jelena KNEŽEVIĆ-VUKČEVIĆ

Department of Microbiology, University of Belgrade - Faculty of Biology, Studentski trg 16,

11000 Belgrade, Serbia *biljanan@bio.bg.ac.rs

Abstract

Genotoxic and cytotoxic effect of plant monoterpenes camphor (C), eucalyptol (E) and thujone (T) was compared in repair proficient human fetal lung fibroblasts (MRC-5) and mismatch repair (MMR) deficient colorectal carcinoma cell line (HCT116). The results obtained in comet assay showed that genotoxic effect was weak in MRC-5, and considerably stronger in HCT116 cells. We also noted that the baseline level of DNA damage was significantly higher in HCT116 cells. Additionally, HCT116 cells were more sensitive to cytotoxic effect of monoterpenes in MTT assay. The obtained IC50 values were approximately 10 mM and 5 mM for C, 10 mM and 3.5 mM for E, and 3 mM and 0.75 mM for T, in MRC-5 and HCT116 cells, respectively. Furthermore, the rate of proliferation in the presence of monoterpenes was significantly lower for HCT-116 cells than for MRC-5 cells. Our results emphasize the importance of MMR for prevention of DNA lesions induced by C, E and T, and may indicate higher sensitivity of cancers with MMR deficiency to monoterpenes. Keywords: monoterpenes, genotoxicity, mismatch repair, human fetal lung fibroblasts,

colorectal carcinoma cell line Introduction

Terpenes are plant secondary metabolites formed from five-carbon isoprene units (C5H8) whose main function is to provide a chemical defense against environmental stress, such as protection against infections and parasites, as well as a repair mechanism for wounds and injuries (Bakkali et al., 2008; Salminena et al., 2008). However, they are endowed with many beneficial health effects and can be used to treat different health disorders (Paduch et al., 2007). It has been shown that terpenes are important cancer chemopreventive and chemotherapeutic agents (Rabi and Bishayee, 2009). Although numerous studies indicate protective capacity of terpenes against induction of DNA lesions, some reports indicate their genotoxic and even mutagenic effects (Di Sotto et al., 2011; Kocaman et al., 2011; Sebastiàa et al., 2012).

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The monoterpenes investigated in this study: camphor (C), eucalyptol (E), and thujone (T) are widely distributed in the essential oils of many medicinal and aromatic plants. As it is postulated in our previous work, high doses of C, E and T were mutagenic in E. coli reversion assay, and genotoxic in comet assay on Vero cells; however, their lower doses could stimulate DNA repair mechanisms including nucleotide excision repair (NER) and postreplicative recombination repair (PRR), and consequently prevent genotoxicity induced by potent mutagens, such as UV-C and 4NQO (Nikolić et al., 2011).

Taking into account the importance of mismatch repair (MMR) pathway for the mutation avoidance and maintenance of the genomic stability, as well as the observed modulatory effect of C, E and T on the DNA repair and mutagenesis processes (Nikolić et al., 2011) in this work we further studied their genotoxic effect in repair proficient human fetal lung fibroblasts (MRC-5 cell line) and MMR deficient human colorectal carcinoma cells (HCT116 cell line). In order to assess the chemotherapeutic potential of monoterpenes, their cytotoxic and antiproliferative effects against MRC-5 and HCT116 cells were also investigated. Genotoxic effect was monitored in comet assay, while cytotoxic and antiproliferative effects were monitored in MTT assay. 2Material and Methods

The human cell lines used were fetal lung fibroblasts MRC-5 (ECACC No. 84101801) and colorectal carcinoma cells HCT116 (ATCC CCL-247). The MMR deficiency of HCT116 cells is due to lack of hMLH1 expression (Hawn et al., 1995).The human cells were grown in DMEM medium with 4.5% glucose and 2 mM L-glutamine, supplemented with 10% fetal bovine serum, penicillin G (100 U/ml) and streptomycin (100 µg/ml) at 37°C with 5% CO2 and 100% humidity. Both cell lines grow attached to the surface, and single cell suspensions were obtained using 0.1% trypsin.

To determine genotoxicity of monoterpenes, MRC-5 and HCT116 cells were inoculated into 12-well plates at a density 2x105 cells/well and incubated 4 h to attach. The medium was replaced with the fresh one containing different concentrations of each monoterpene and the incubation continued for 20 h. The monolayer was harvested and cells were centrifuged at 100g

for 10 min and resuspended in PBS buffer to obtain 3x105 cell/ml. After second centrifugation at 100g for 10 min, pellet was resuspended in 60 µl of PBS buffer and used for alkaline comet assay, performed as described by Singh et al. (1988).

To determine cytotoxicity of monoterpenes, MRC-5 and HCT116 cells were grown in 96-well microtiter plates until they form monolayer and then treated with each monoterpene for 20h at 37°C with 5% CO2 and 100% humidity. The cell viability was monitored by MTT reduction assay, as previously explained by Hansen et al. (1989).To determine antiproliferative effect of monoterpenes, cells were inoculated into 96-well plates in the medium containing different concentrations of each monoterpene and incubated for 4 days. Cell growth was monitored every 24 by MTT reduction assay. Results and Discussion

Genotoxic effect of C, E and T in repair proficient MRC-5 and MMR deficient HCT116 cells was investigated in the range of non-toxic concentrations (viability was 80% or higher), as previously determined by MTT assay. The statistically significant increase of comet tail intensity was obtained for all applied concentrations of monoterpenes with weak response in MRC-5 cell line and much stronger in HCT116 cells (Fig. 1). The extent of DNA damage was considerably higher in HCT116 cells, indicating possible involvement of MMR in repair of lesions induced by

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monoterpenes. Moreover, increased baseline level of DNA damage observed in untreated HCT116 is consistent with the importance of MMR in maintaining the genome stability. The MMR system has been highly conserved throughout evolution; the eukaryotic MMR is essentially similar to prokaryotic one, but significantly more complicated due to the presence of distinct, partially redundant MutS and MutL homologues. The MutS complexes contain the MSH2 protein and one of the other five binding partners, while the MutL complexes contain the MLH1 protein and one of the other four binding partners. Moreover, there is no MutH homologue in eukaryotic cells and it seems that instead specific MutH endonuclease activity nicks and gaps that are left behind the progressing replication fork are used (Jiricny, 2006). As a response to genomic insult eukaryotic cells trigger a signal transduction pathway, known as DNA damage checkpoint, whose role is to overcome the damage by coordinating cell cycle progression, DNA replication and DNA repair processes. At the top of the checkpoint cascade are ATM and ATR protein kinases, whose activation requires recognition and initial processing of a lesion to a unique common structure, an ssDNA region, that serves as the checkpoint triggering signal (for review see Lazzaro et al., 2009). It is especially important to note that hMLH1 interacts with ATM at the very top of DNA damage checkpoint cascade (Adamson et al., 2002; Brown et al., 2003) and that in cells lacking hMLH1 expression, such as HCT116 cells, not only MMR but other DNA repair processes, as well as DNA replication and cell cycle progression might be disturbed. In the light of this fact, higher genotoxicity of monoterpenes in the MMR deficient hMLH1- cells could be expected.

A.MRC-5

B. HCT116

Figure 1. Genotoxic effect of monoterpenes in MRC-5 (A) and HCT116 (B) cells. TI – comet tail intensity;.EtOH – Ethanol, used as solvent control; 4NQO – Positive control; cells were

treated for 60 min with 5 µM 4NQO. Median 25%-75% Non-Outlier Min-Max; * p˂0.05

Increased genotoxicity, as well as higher baseline level of DNA damage detected in

HCT116 cells, indicated that this cancer cell line could be more susceptible to monoterpenes. In order to assess their chemotherapeutic potential, in further work we determined their cytotoxic and antiproliferative effects. The obtained results indicated that C, E and T significantly reduced cell viability, especially in HCT116 cell line. The IC50 concentrations estimated from the graphs

were about 10 mM and 5 mM for C, about 10 mM and 3.5 mM for EmM for T, in MRC-5 and HCT116 cells, respectively (Fig. confirmed stronger effect of monoterpenes in HCT116 cell line (Fig.

Figure 2. IC50 values of monoterpenes in MRC

Figure 3.Antiproliferative effect of monoterpenes in MRCMTT reduction corresponds to cell viability. EtOH

Our finding that HCT116 c

since the incidence of colorectal cancer, as well as its mortalitycolorectal cancers to chemotherapy is frequent and limits the effectiveness of current cancer therapies (Longley et al., 2006). 5the standard treatment for colorectal cancer, but the adequate response was achieved only in 1015% of patients. Moreover, since MMR is involved in repair of 5deficiency could be considered as an important cause cell lines (Johnston and Kaye, 2001; Meyers recommend C, E and T for further chemotherapeutic study, especially against cancers with MMR deficiency.

In conclusion, monoterpenes C, E and T, used in high doses,in human cells. Pronounced effects induced with MMR deficiency show the involvement of this mechanism in the repair of lesions induced by monoterpenes. Increased sensitivity of the

120

ere about 10 mM and 5 mM for C, about 10 mM and 3.5 mM for E, and about 3 mM and 0.75 5 and HCT116 cells, respectively (Fig. 2). Results of antiproliferation assay

confirmed stronger effect of monoterpenes in HCT116 cell line (Fig. 3).

values of monoterpenes in MRC-5 and HCT116 cells.

3.Antiproliferative effect of monoterpenes in MRC-5 and HCT116 cells.

MTT reduction corresponds to cell viability. EtOH – Ethanol; used as solvent control.

HCT116 cells are more susceptible to C, E and T is of special interest ncidence of colorectal cancer, as well as its mortality, is high. The resistance of

colorectal cancers to chemotherapy is frequent and limits the effectiveness of current cancer 2006). 5-Fluorouracil-(5-FU) based chemotherapeutic

the standard treatment for colorectal cancer, but the adequate response was achieved only in 1015% of patients. Moreover, since MMR is involved in repair of 5-FU-induced DNA lesions, its deficiency could be considered as an important cause of resistance of MMR deficient cancers cell lines (Johnston and Kaye, 2001; Meyers et al., 2001). In the light of

for further chemotherapeutic study, especially against cancers with

noterpenes C, E and T, used in high doses, are genotoxic and cytotoxic in human cells. Pronounced effects induced with MMR deficiency show the involvement of this mechanism in the repair of lesions induced by monoterpenes. Increased sensitivity of the

d about 3 mM and 0.75 Results of antiproliferation assay

is of special interest is high. The resistance of

colorectal cancers to chemotherapy is frequent and limits the effectiveness of current cancer FU) based chemotherapeutic regimens were

the standard treatment for colorectal cancer, but the adequate response was achieved only in 10-induced DNA lesions, its

of resistance of MMR deficient cancers this, our results

for further chemotherapeutic study, especially against cancers with

are genotoxic and cytotoxic in human cells. Pronounced effects induced with MMR deficiency show the involvement of this mechanism in the repair of lesions induced by monoterpenes. Increased sensitivity of the

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colorectal cancer MMR- cell line to C, E and T encourages further study of their chemotherapeutic potential. Acknowledgement This work was supported by the Ministry of Science of Republic of Serbia, Project No. 172058. We thank Dragana Ćetojević-Simin, Oncology Institute of Vojvodina, Sremska Kamenica, Serbia, for providing the MRC-5 cells, and Snežana Marković, Laboratory of Cell and Molecular Biology, Faculty of Science, University of Kragujevac, Serbia, for providing the HCT116 cells. References Adamson AW, Kim W-J, Shangary S, Baskaran R, Brown KD (2002). ATM is activated in

response to N-methyl-N′-nitro-N-nitrosoguanidine-induced DNA alkylation, J. Biol. Chem., 277 38222–38229.

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Brown KD, Rathi A, Kamath R, Beardsley DI, Zhan Q, Mannino JL, Baskaran R (2003)The mismatch repair system is required for S-phase checkpoint activation, Nat. Genet., 33, 80–84.

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OPPOSITE EFFECT OF CAMPHOR, EUCALYPTOL AND THUJONE ON DNA REPAIR AND MUTAGENESIS: A QUESTION OF DOSE

Biljana NIKOLIĆ, Dragana MITIĆ-ĆULAFIĆ, BrankaVUKOVIĆ-GAČIĆ, Jelena KNEŽEVIĆ-

VUKČEVIĆ

Department of Microbiology, University of Belgrade - Faculty of Biology, Studentski trg 16, 11000 Belgrade, Serbia *biljanan@bio.bg.ac.rs

Abstract Genotoxic/antigenotoxic effect of monoterpenes camphor (C), eucalyptol (E) and thujone

(T) were studied in different DNA repair mutants of E. coliK12. Application of low doses induced antimutagenic effect against UV and 4NQO in repair proficient bacteria, which was lost with nucleotide excision repair (NER) deficiency. However, low doses of monoterpenes increased SOS induction and intrachromosomal recombination following UV, indicating their genotoxicity. Additional evidence of genotoxicity included: (1) decreased antimutagenic effect with increased concentrations; (2) co-mutagenic response in NER deficient cells; (3) mutagenic response to high doses in mismatch repair (MMR) deficient strain; (4) decreased survival of DNA repair mutants in the presence of monoterpenes. In comet assay on Vero cells we confirmed the antigenotoxic effect of low doses and genotoxic effect of high doses. We propose that by making small amounts of DNA lesions low concentrations of monoterpenes stimulate error-free DNA repair and reduce genotoxicity, while high concentrations are genotoxic. This hypothesis explains contradictory data concerning genotoxicity/antigenotoxicity of C, E and T.

Keywords: monoterpenes, E. coli K12, Vero cells, DNA repair, genotoxicity/antigenotoxicity

Introduction

Terpenes (C5H8)n are the largest group of natural substances abundantly found in fruits, vegetables and aromatic and medicinal plants. They are endowed with many beneficial health effects, including antimicrobial, antiparasitic, spasmolytic, hypoglycemic, anti-inflammatory, anti-allergenic and immune-modulatory (Paduch et al., 2007). It has been shown that terpenes are important cancer chemopreventive and chemotherapeutic agents (Lampe, 2003). Numerous studies indicate protective capacity of terpenes against endogenous sources of DNA lesions, as well as against environmental genotoxic agents, indicating their possible use in primary prevention of many mutation related diseases (Kaefer and Milner, 2008).

The monoterpenes camphor (C), eucalyptol (E), and thujone (T), widely distributed in essential oils of many medicinal and aromatic plants, possess strong antimicrobial effect

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(Soković et al., 2010), while E also possesses cytotoxic, anti-inflammatory, analgesic, antiexudant, gastroprotective and hepatoprotective properties (Moteki et al., 2002; Santos et al., 2004). Literature data indicate their genotoxicity or antigenotoxicity, depending on the cell type, experimental setup, genetic background and concentrations applied (Hikiba et al., 2005; Mitić-Ćulafić et al., 2009; Miyamoto et al., 2009; Moteki et al., 2002; Nishimura et al., 2008; Pavlidou et al., 2004; Ribeiro et al., 2007; Vuković-Gačić et al, 2006). Taking into account this controversy, in this work we investigated the effect of C, E and T on DNA repair and mutagenesis in prokaryotic and eukaryotic models, and searched for underlying molecular mechanism(s).

Material and Methods The E. coli K12 strains used in this study are AB1157 (repair proficient), AB2470 (as

AB1157, but recB21), DL131 (as AB1157, but recF143), SY252 (argE3, repair proficient), IB103 (as SY252, but mutS), IB105 (as SY252, but uvrA), IB111 (as SY252, but λp(sfiA::lacZ) cIind1PHOc), IB127 (as IB111, but uvrA), GY8281 (lacMS286 Ф80dIIlacBK1∆recA306

srl::Tn10/miniFrecA+) and GY8252 (lacMS286 Ф80dIIlacBK1∆recA306 srl::Tn10/ miniFrecA730). The used mammalian cell line is Vero, obtained from the kidney of a normal adult of African green monkey (ECACC No: 88020401). Media and growth conditions for both bacteria and Vero cells were as previously described (Nikolić et al., 2011).

To monitor toxicity of C, E and T in bacteria, exponential cultures(OD600 ~ 0.4) of AB1157, AB2470, DL131, SY252, IB103 and IB105 were treated with monoterpenes for 120 min and cell viability was determined in regular time intervals. E coli K12 reversion assay on SY252, IB103 and IB105, performed as described by Nikolić et al. (2011), was used to monitor mutagenic effect, as well as antimutagenic effect of post-treatment with monoterpenes against UV and 4NQO. To monitor antimutagenic effect of pre-treatment of SY252 and IB105 with monoterpenes, some modified E coli K12 reversion assay, described by Nikolić et al. (2012) was performed. To evaluate effect of monoterpenes on SOS induction and general protein synthesis in IB111 and IB127 strains, method of Quillardet and Hofnung (1993), in details explained by Nikolić et al. (2011), was used. Effect of monoterpenes on recombination was determined in strains GY8281 and GY8252 by monitoring Lac+ recombinants on MacConkey-lactose plates containing different concentrations of monoterpenes, as described in Nikolić et al. (2011).

Comet assay, performed as described by Singh et al. (1988) was used to determine antigenotoxic effect of monoterpenes against 4NQO-induced genotoxicity in Vero cells. Vero cells were treated as monolayer, for 20h with serial concentrations of monoterpenes, and for 1h with 5 µM 4NQO, both at 37°C, in 5% CO2. Two different protocols were performed: pre-treatment with monoterpenes (treatment with C, E or T, followed by treatment with 4NQO), as well as post-treatment with monoterpenes (treatment with 4NQO, followed by treatment with C, E or T).

Results and Discussion Antimutagenic effect of C, E and T against UV and 4NQO was determined in E. coli K12

reversion assay. Reversions were monitored in repair proficient and nucleotide excision repair (NER) deficient isogenic strains, using monoterpene pre-treatment and post-treatment protocols. Monoterpenes reduce mutagenicity in both experimental protocols, but only in repair proficient strain (Fig. 1), while in NER deficient counterpart antimutagenic response was diminished; moreover T was slightly mutagenic (data not shown). Co-mutagenic effect with UV was

observed when NER deficient cells were pre2A).

Figure 1. Antimutagenic effect of monoterpenes in repair proficient Results are presented as % of mutagenesis

A common feature of the dose-dependent manner; on the contrary,Uindicating mutagenicity at higher concentrations.mutagenic response in NER deficient strain, indicates the importance of NER mechanism for maintaince of antimutagenic properties of C, E and T. Additionally, mutagenic effect of C, E and T were also obtained when high doses were applied in MMR deficient mutant o(Fig. 2B).

Figure 2. Co-mutagenic effect (with UV) of monoterpenes in NER deficient strain (A) and mutagenic effect of monoterpenes in MMR deficient strain (B) of In NER deficient strain C and E induced coeffect in post-treatment experiment.

The effect of monoterpenes on UVcontrolled β-galactosidase gene (deficient IB127 strains. Clear evidence of positive effect was obtained with C and E, since level of UV-induced β-galactosidase wasDNA damage. However, T significantly reduced protein synthesis and consequently did not increase the level of UV-induced

The effect of monoterpenes on two different recA alleles: GY8281 activated RecA protein are induced following DNA damaging treatments, while

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en NER deficient cells were pre-treated with C and E, and post-treated with T (

Figure 1. Antimutagenic effect of monoterpenes in repair proficient E. coli K12 strain

Results are presented as % of mutagenesis - %M.

A common feature of the monoterpenes was that they did not reduce mutagenicity in a n the contrary,U-shaped concentration-response curves were obtained

mutagenicity at higher concentrations. The absence of antimutagenicity, as well as conic response in NER deficient strain, indicates the importance of NER mechanism for

maintaince of antimutagenic properties of C, E and T. Additionally, mutagenic effect of C, E and T were also obtained when high doses were applied in MMR deficient mutant o

mutagenic effect (with UV) of monoterpenes in NER deficient strain (A) and mutagenic

effect of monoterpenes in MMR deficient strain (B) of E. coli K12. In NER deficient strain C and E induced co-mutagenic effect in pre-treatment experiment, and T induced co

treatment experiment.

The effect of monoterpenes on UV-induced SOS response was monitored using SOS galactosidase gene (sfiA::lacZ fusion) in repair proficient IB111 and in NER

deficient IB127 strains. Clear evidence of positive effect was obtained with C and E, since galactosidase was higher and/or persisted longer, indicating additional

T significantly reduced protein synthesis and consequently did not induced β-galactosidase (data not shown).

The effect of monoterpenes on homologous recombination was determined alleles: GY8281 (recA

+) is recombination proficient, and high levels of activated RecA protein are induced following DNA damaging treatments, while

treated with T (Fig.

monoterpenes was that they did not reduce mutagenicity in a response curves were obtained,

The absence of antimutagenicity, as well as co-nic response in NER deficient strain, indicates the importance of NER mechanism for

maintaince of antimutagenic properties of C, E and T. Additionally, mutagenic effect of C, E and T were also obtained when high doses were applied in MMR deficient mutant of E. coli K12

mutagenic effect (with UV) of monoterpenes in NER deficient strain (A) and mutagenic

treatment experiment, and T induced co-mutagenic

induced SOS response was monitored using SOS fusion) in repair proficient IB111 and in NER

deficient IB127 strains. Clear evidence of positive effect was obtained with C and E, since the , indicating additional

T significantly reduced protein synthesis and consequently did not

recombination was determined in strains with ) is recombination proficient, and high levels of

activated RecA protein are induced following DNA damaging treatments, while GY8252

(recA730) is partially recombination deficient, but constitutive for SOS induction, constitutively high levels of activated Rrecombination in both recA

+ and with constitutively high level of RecA, which could be related to their inhibitory effects on the general protein synthesis (data not shown).

The results obtained in bacterial assays,tmonoterpenes at higher concentrations. mutants of E. coli K12, we measured their survival in the presence of high doses of monoterpenes. The results confirmed that NER, MMR and recombination are involved in the repair of lesions induced by C, E and T (data not shown).

Oposite effects of different doses of monoterpenes was confirmed in eukarytic model using comet assay. When Vero cells were treated with high doses of C, E and T, genotoxic response was obtained (Fig. 3A). However, when Vero cells, treated with 4NQO, were pretreated with low doses of monoterpenes, antigenotoxic respons was detected (

We propose that C, E and Tstimulate error-free DNA repair the hormesis phenomenon, defined as a low dose beneficial response to a stressor agent. Hormesis is now generally accepted as a reproducible biological phenomenon, being highly generalized and independent of biological model, chemical/physical stressor applied point measured (Calabrese, 2010).andsimilar hypothesis was also postulated by al., 2003;Moteki et al., 2002; Őzkan and Erdoal., 2012).

Figure 3. Effect of monoterpenes in comet assay on Vero cells: genotoxic (A) and antigenotoxic against 4NQO (B). Results are presented as % of tail intensity for antigenotoxicity of both post- and pre

In conclusion, monoterpenes

mutagenesis, depending on the applied dose. Inand consequently act as antimutagens

Acknowledgement This work was supported by the Ministry of Science of Republic of Serbia, Project No. 172058.

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) is partially recombination deficient, but constitutive for SOS induction, f activated RecA protein. Obtained results indicated that C

and recA730 strains, while E and T were effective only in the with constitutively high level of RecA, which could be related to their inhibitory effects on the general protein synthesis (data not shown).

obtained in bacterial assays,taken together, pointed at genotoxic properties of er concentrations. To evaluate the sensitivity of different DNA repair K12, we measured their survival in the presence of high doses of

monoterpenes. The results confirmed that NER, MMR and recombination are involved in the ions induced by C, E and T (data not shown).

Oposite effects of different doses of monoterpenes was confirmed in eukarytic model using comet assay. When Vero cells were treated with high doses of C, E and T, genotoxic response

ver, when Vero cells, treated with 4NQO, were pretreated with low doses of monoterpenes, antigenotoxic respons was detected (Fig. 3

C, E and Tin low doses induce small amounts of DNA lesions, which and reduce UV- and 4NQO-induced genotoxicity. T

hormesis phenomenon, defined as a low dose beneficial response to a stressor agent. Hormesis is now generally accepted as a reproducible biological phenomenon, being highly

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postulated by other authors (Kocaman et al., 2011; Őzkan and Erdoğan, 2013; Shaughnessy et al., 2006

Figure 3. Effect of monoterpenes in comet assay on Vero cells: genotoxic (A) and antigenotoxic against

presented as % of tail intensity - TI(%). Concentration ranges: (1) for genotoxicity the range 5and pre-treatment the range 0.5-100 µM for C and T, and 0.05-10

In conclusion, monoterpenes C, E and Tcould induce opposite effect on DNA repair and mutagenesis, depending on the applied dose. In low concentrations they stimulate

antimutagens, but in high concentrations they are genotoxic.

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) is partially recombination deficient, but constitutive for SOS induction, with Obtained results indicated that C stimulated

s, while E and T were effective only in the strain with constitutively high level of RecA, which could be related to their inhibitory effects on the

pointed at genotoxic properties of To evaluate the sensitivity of different DNA repair

K12, we measured their survival in the presence of high doses of monoterpenes. The results confirmed that NER, MMR and recombination are involved in the

Oposite effects of different doses of monoterpenes was confirmed in eukarytic model using comet assay. When Vero cells were treated with high doses of C, E and T, genotoxic response

ver, when Vero cells, treated with 4NQO, were pre-treated or post-Fig. 3B).

in low doses induce small amounts of DNA lesions, which induced genotoxicity. This indicates

hormesis phenomenon, defined as a low dose beneficial response to a stressor agent. Hormesis is now generally accepted as a reproducible biological phenomenon, being highly

lized and independent of biological model, chemical/physical stressor applied and end-The hormesis phenomenon is not rare in genotoxicology

2011; Labieniec et

2006; Sebastiàa et

Figure 3. Effect of monoterpenes in comet assay on Vero cells: genotoxic (A) and antigenotoxic against

TI(%). Concentration ranges: (1) for genotoxicity the range 5-500 µM; (2) 10 µM for E.

opposite effect on DNA repair and ncentrations they stimulate DNA repair

, but in high concentrations they are genotoxic.

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CORRELATION BETWEEN GENETIC DISTANCE, SPECIFIC COMBINING ABILITY AND HETEROSIS IN MAIZE

Jovan PAVLOV1 , Nenad DELIĆ1, Nikola GRČIĆ1, Milan STEVANOVIĆ1,

Zoran ČAMDŽIJA1, Miloš CREVAR1 and Sofija BOŽINOVIĆ1

1Maize Research Institute, “Zemun Polje”, Slobodana Bajića 1, 11185 Belgrade, Serbia *e-mail: jpavlov@mrizp.rs

Abstract

Six maize inbred lines as well as fifteen hybrid combinations derived from them were used in this work in order to analyze genetic relationship involving inbred lines and to predict heterosis between crosses. The trial was set up according to RCB design at two locations in two repetitions in 2010. Nineteen SSR primers were applied in order to obtain genetic distances between inbred lines. Analysis of specific combining ability was done using Griffing (1956), method 2, model 1. Heterosis was estimated as a high-parent (HP) heterosis. Values of genetic distance ranged from 0,08 to 0,65. The lowest value of specific combining ability for grain yield was found in combination L1xL2 (-0,43), while the lowest value of heterosis for grain yield had combination L1xL3 (32,32*). The highest values of specific combining ability and heterosis for grain yield were determined in combination L4xL5 (2,20**; 115,96** respectively). Values of genetic distance were positively correlated with values of specific combining ability and heterosis (r=0,41 and r=0,53* respectively), so it could be concluded that using SSR markers we can reliably predict heterosis in F1 generation. Key words: maize, genetic distance, heterosis, specific combining ability Introduction

Developing of maize hybrids is costly and long term process, which needs crosses between a lot of inbred lines and evaluation of hybrids in field trials. Therefore, only a limited number of hybrids among all possible crosses can be tested (Gvozdenovic et al, 2009). The efficiency of hybrid breeding programs could be increased if the inbred lines per se could be screened and the superior crosses predicted before field evaluation (Melchinger et al. 1990). The study of genetic divergence can assist in the choice of genotypes to be used in breeding programs for the development of new populations (Cruz and Regazzi, 1997), as well as new hybrids. Utilization of genetic distance for predicting hybrid heterosis is of great interest to maize breeders. Many authors propose use of SSR molecular markers for determination of genetic

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distance between inbred lines and classifiying inbreds into heterotic groups (Reif, 2003, Xu, 2004, Balestre, 2008). SSR molecular markers represent powerful tool in the assessment of the genetic diversity between maize inbred lines, so field trials can be planed more efficiently based on prior information based on SSR molecular markers (Reif et. al, 2003, Mladenovic-Drinic et

al, 2012). The objective of this study was to determine the association between SSR based genetic

distance of six ZP maize inbred lines and values of specific combining abilities and high-parent heterosis for grain yield. Material and methods

Six inbred lines were crossed according to incomplete diallel design (without reciprocals). As a result of these crosses 15 hybrid combinations were obtained. Based on knowledge about examined inbreds we assumed that they belong to the same heterotic group. Inbred lines and hybrid combinations were used in order to analyze genetic relationship between inbred lines and to predict heterosis between crosses. Two separate trials (one for inbreds and other for hybrid combinations) were set up according to RCB design at two locations in two repetitions in 2010. Inbred lines as well as hybrid combinations were planted in density of 70175 plants per hectare. Nineteen SSR markers were used in order to determine genetic distance between examined inbred lines. Table1. SSR markers used in work and their location

SSR locus Bin no. umc 2235 1.1 umc 2248 2 bnlg 198 2.1

bnlg 1350 3.1 umc 1288 4 bnlg 557 5 phi 085 5.1 phi 126 6

umc 1006 6 bnlg 1443 6.1 umc 1859 6.1 umc 1393 7 umc 1426 7 umc 1695 7 umc 1414 8 umc 1040 9 bnlg 1506 9.1 umc 1507 10 umc 1827 10

Isolation of DNA was done according to the modified protocol to Saghai and Maroof

(1984). Genetic similarity between inbred lines was determined using Simple matching coefficient (SM). Тhe presence or absence of strips on the gels was determined visually and

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converted into binary data. Based on these data, genetic similarities between inbred lines were calculated, using Simple matching coefficient: Gsij=a+d/a+b+c+d, where: a- presence of strips in both of the genotypes i and j (1.1), b- presence of strip in the genotype i and absence in genotype j (1.0) c- presence of strip in the genotype j and absence in genotype i (0.1) d- absence of strips in both of genotypes i and j (0.0) Analysis of specific combining ability was done using Griffing (1956), method 2, model 1.

Heterosis was estimated as a high-parent (HP) heterosis. High-parent heterosis (HPH) was calculated as HPH= [(F1-HP)/HP] x 100; where, F1 is the mean of the F1 hybrid performance and HP is the mean of the best parent, where, F1 is the mean of the F1 hybrid performance and HP is the mean of the best parent.

The correlation between SCA and GD, as well as, HPH and GD for grain yield were calculated using Spearman’s rank correlation coefficient. Test of significance for Spearman’s rank correlation coefficients were calculated using following formula (Hadživuković, 1973):

t � r@n * 21 * r+

r – value of Spearman’s rank correlation coefficient n – number of examined genotypes Results and discussion

Values of genetic distance between pairs of inbred lines are presented in table 2. In this study we used six inbred lines for which we assumed that belong to the same heterotic group. Table 2. Values of genetic distances between examined inbred lines

Values of genetic distance obtained using SSR markers showed that first three inbred

lines are very closely related, concerning their origin. Moreover, inbred lines L4 and L5 are more distant with other inbred lines, but it could be considered that they belong to the same heterotic group with first three inbreds. However, obtained results showed high values of genetic distance between inbred line L6 and all other inbred lines used in this study. Our results are partially in accordance with the result obtained by Xia et al (2004) who found agreement in results obtained using of SSR markers with pedigree information of examined inbred lines.

Inbred L1 L2 L3 L4 L5

L1 L2 0,15 L3 0,20 0,08 L4 0,36 0,30 0,27 L5 0,30 0,29 0,30 0,39 L6 0,61 0,65 0,61 0,64 0,58

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Table 3. Grain yield (t/ha) for examined inbred lines and their crosses

Grain yield L 1 L 2 L 3 L 4 L 5

L1 3,355 L2 3,882 5,124 L3 3,943 5,309 5,632 L4 5,198 9,486 8,850 8,202 L5 4,855 8,676 8,642 8,542 11,220

L6 4,521 8,387 6,924 7,549 9,295 9,900

Grain yield of inbred lines and hybrid combinations used in this study are presented in table 3. Grain yields of inbred lines were in range from 3,355 t/ha (L1) to 5,198 t/ha (L4). When it comes to hybrid combinations, the lowest grain yield was observed for hybrid combination L1xL2 (5,124 t/ha), while the highest grain yield had hybrid combination L4xL5 (11,220 t/ha). This results are in accordance with results obtained by Ali et al. (2007) who found that the differences in yields of different hybrids are possible in hybrids of various genetic backgrounds.

Table 4. Values of specific combining abilities for examined crosses

Inbred L 1 L 2 L 3 L 4 L 5

L 1

L 2 -0,43 L 3 -0,28 0,13 L 4 2,09** 1,55* 0,86

L 5 1,40 1,46* 1,32 2,20**

L 6 1,80* 0,44 1,03 0,97 1,70*

Values of specific combining abilities for grain yield are presented in table 4. The lowest value of specific combinig ability had hybrid combination L1xL2, while the highest value was observed for hybrid combination L4xL5, which is in accordance with the results obtained for values of grain yield for hybrid combinations. For hybrid combination L4xL5 also is observed highest value of heterosis for grain yield (115,96**), while lowest value is observed for combination L1xL3 (32,21*), which is presented in table 4. Intensity of heterosis and values of combining abilities depend directly on genetic divergence between the parents (Ferreira et al., 1995; Paterniani et al., 2008). Table 5. Values of high parent heterosis for examined crosses

Inbred L 1 L 2 L 3 L 4 L 5 L 1 L 2 32,26* L 3 32,21* 40,24* L 4 82,53** 70,30** 57,84** L 5 78,60** 77,90** 75,84** 115,96** L 6 85,48** 53,12** 66,96** 78,86** 103,79**

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Values of Spearman’s rank correlation coefficients reveal moderate positive correlation between genetic distance and SCA (0,41), as well as significant positive correlation between genetic distance and high parent heterosis (0,51*). Obtained results are in agreement with results of other studies, which also obtained middle or strong positive correlation between genetic distance and heterosis for grain yield, as well as between genetic distance and values of specific combining abilities (Radojčić et al., 2008, Xia et al., 2004, Mladenović-Drinić et. al, 2012).

Conclusions

Based on the results obtained in this study it could be concluded that use of SSR markers could help breeders in classifying inbred lines in heterotic groups. Furthermore, it could be concluded that inbred line L6 has different origin from other five lines used in this study, which could be classified into the same heterotic group. In this study we determined moderate positive correlation between genetic distance and SCA, as well as significant positive correlation between genetic distance and high parent heterosis. Based on these results it could be concluded that using SSR markers we can reliably predict heterosis in F1 generation, although values of correlations was not so high. References Ali, W., Rehman, H., Ahmad, K., Munir, I., KhaN, A. (2007). Genetic variability among maize

hybrids for yield and yield components. Sarhad Journal of Agriculture 23 (1): 75-80. Balestre, M., Machado, J.C. , Lima, J.L. , Souza, J.C. And Nóbrega Filho L. (2008). Genetic

distance estimates among single cross hybrids and correlation with specific combining ability and yield in corn double cross hybrids. Genetics and Molecular Research 7 (1), 65-73.

Cruz, C. D.; Regazzi, A. J. Modelos (1997). biométricos aplicados ao melhoramento genético. 2nd ed. Viçosa: UFV.

Ferreira, D. F.; Oliveira, A. C.; Santos, M. X.; Ramalho, M. A. P. (1995). Métodos de avaliação da divergência genética em milho e suas relações com os cruzamentos dialélicos. Pesquisa Agropecuária Brasileira, 30 (9), 1189-1194.

Griffing, B.A. (1956). Concept of general and specific combining ability in relation to diallel crossing systems. Aust. J. Biol. Sci. 9: 463-493.

Gvozdenović, S., Saftić, Panković, D., Jocić, S., Radić, V. (2009). Correlation between heterosis and genetic distance based on SSR markers in sunflower (Helianthus annuus L.). Journal of Agricultural Sciences, (1), 1-10.

Hadživuković, S. (1973). Statistički metodi s primenom u poljoprivrednim i biološkim istraživanjima, izdavač Radnički univerzitet Radivoj Ćirpanov.

Melchinger, A. E., Lee, M., Lamkey, K. R. et al. (1990). Genetic diversity for restriction fragment length polymorphisms and heterosis for two diallel sets of maize inbreds. Theor. Appl. Genet. 80: 488-496.

Mladenović Drinić, S., Kostadinović, M., Ristić, D., Stevanović, M., Čamdžija, Z., Filipović, M., Kovačević D. (2012). Correlation of yield and heterosis of maize hybrids and their parental lines with genetic distance based on SSR markers. Genetika, 44 (2), 399-408.

Paterniani, M. E. A. G. Z.; Guimarães, P. S.; Lüders, R. R.; Gallo, P. B.; :ouza, A. P.; Laborda, P. R.; Oliveira, K. M. (2008). Capacidade combinatória, divergência genética entre linhagens de milho e correlação com heterose. Bragantia, v. 67, n. 3, p. 639-648.

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Radojčić, A., Drinić, G., Drinić Mladenović, S. (2008). Prediction of combining ability and heterosis using RAPD markers. Proceedings. 43rd Croatian and 3rd International Symposium on Agriculture. Opatija. Croatia (335-339).

Reif Jc, Melchinger Ae, Xia Xc, Warburton M.L., et al. (2003). Genetic distance based on simple sequence repeats and heterosis in tropical maize populations. Crop Sci. 43: 1275-1282.

Saghai-Maroof M.A., Soliman K.M, Jorgensen R.A. and Allard R.W. (1984): Ribosomal Dna Spacer-Length Polymorphisms In Barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 81: 8014-8018.

Xu, S.-X, Liu, J. And Liu, G.S. (2004). The use of SSRs for predicting the hybrid yield and yield heterosis in 15 key inbred lines of Chinese maize. Hereditas 141: 207-215.

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POLLEN GRAIN DEFORMATION ASSAY ON Galanthus FOR VARIABILITY ASSESSMENT AND ENVIRONMENTAL BIOMONITORING

Jasna PARADIŽ1, Jože BAVCON2, Danijel VRHOVŠEK3

1Plant Cytogenetics, Private Research Investigating Office, Hudovernikova 4, 1000 Ljubljana,

Slovenia; jasna.paradiz@gmail.com 2University Botanic Gardens Ljubljana, Department of Biology Biotechnical Faculty, Ižanska

cesta 15, 1000 Ljubljana, Slovenia 3Limnos, Company for Applied Ecology, Podlimbarskega 31, 1000 Ljubljana, Slovenia

Abstract

Pollen grain deformation (PGD) assay on Galanthus varieties of Ljubljana Botanical Garden Collection was made to assess their cytogenetic balance, as well as of native flora at different localities in Slovenia. In specimens originated from the Dinaric region the highest PGD ratio was established, indicating highly unbalanced pollen grains of tall variants. Ploidy estimation based on pollen size revealed intraspecific Galanthus variability, being of a great adaptive importance to plants at their high level habitats with harsh weather conditions. PGD data are particularly valuable in diagnosing decorative forms of horticultural desirability from extreme biotopes. In naturally growing plants, however, PGD ratio at Ljubljana environs was slightly changed. Somewhat higher PGD in Galanthus at Slavnik natural habitats; and even higher PGD were established at Dolenjska area with significant anthropogenic contamination effects of intensive agricultural activity. Biomonitoring plants by PGD have a high application value in the ecosystem management, and involve ecoremediation measure for nature conservation and protection to improve the quality of life and sustainable development of culture. Key words: Galanthus nivalis L., pollen grains, cytogenetic change, biodiversity, monitoring bioassay Introduction

Common snowdrop (Galanthus nivalis L.) is the only representative in Slovenia, native and widespread, and beloved of all. Although less diversified than typical of Amaryllidaceae (Kandemir, 2010; Senel, et al., 2002), special Galanthus varieties were identified in their natural habitats, and sampled systematically at different localities for the Botanical Garden Collection in Ljubljana (Bavcon, 2009). Both chromosome counts 2n=24, 18 on indigenous Galanthus were

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reported (Löve, 1973; Druškovič and Lovka, 1995), and B chromosomes at Ljubljana Moor (Lovka et al., 2003; Paradiž and Bavcon, 2012). SAT chromosomes, other records of 2n=24-28, 36 on elsewhere material (Davis et at., 1996; D'Amato and Bianchi, 1999), triploids and polyploid cultivars (Lledo et al., 2004; Zonneveld et al., 2003) suggest a ploidy variation of local populations in varying environments of their distribution area.

Variable levels of ploidy estimations are traditionally based on clearly identifiable deviations of pollen grain size. Sterile pollen as abortion criteria is used in biomonitoring environmental genotoxicity (Mičieta and Murin, 1996; Paradiž and Lovka, 1999, 2004; Mišik et

al., 2006; Paradiž, 2011; Paradiž and Bavcon, 2012) and native flora in bioindication of environmental changes (Vrhovšek et al., 1984) for a long time. But diverse ploidy levels of plants and increased pollen grain sterility in different ecological conditions have not yet been subjected to the test for their interdependence. In the present study, by using pollen grain deformation (PGD) assay on Galanthus varieties Collection, intraspecific ploidal variation under field conditions of the Botanical Garden in Ljubljana was identified, and cytogenetic diversity and disturbance were compared to PGDs in naturally growing plants at different localities of Slovenia. Material and methods

From different localities in Slovenia specimens of Galanthus nivalis differing somehow from the common type were transferred from their habitats, and planted in a mixture of soil from the site, compost and leaf mould, then cultivated in experimental fields of the Botanical Garden in Ljubljana. In 2008 to 2014, for pollen grain deformation (PGD) assay 18 Galanthus varieties of the Collection were chosen. They originated from 3 localities (Gameljne, Dol, Zminec) of the central part of Slovenia, and 4 (Sortež, Podkraj, Col, Črni Vrh) of the Dinaric region, all being of tall habit with big flower, but a single small variant from Črni Vrh.

In situ PGD assay was performed in 21 Galanthus nivalis populations, and 20 species of local flora at 15 localities from environs of the central part of Slovenia (Ljubljana City, Ljubljana Moor), Slavnik, and Dolenjska (Tržišče, Pijavice). Plant bioindicators were: Bellis perennis L., Cornus mas L., Corydalis solida (L.) Sw., Corylus avellana L., Crocus albiflorus Kit. ex Schult., Crocus napolitanus Mord. & Loisel., Dentaria enneaphyllos l., Erica herbacea L., Erythronium dens-canis L., Hacquetia epipactis (Scop.) DC., Helleborus multifidus Vis., Helleborus niger L., Helleborus viridis L., Hepatica nobilis Schreber, Leucojum vernum L., Mercurialis perennis L., Petasites albus (L.) Gaertn., Primula vulgaris Huds., Pulmonaria

officinalis L., and Tussilago farfara L. For the PGD assay young flowers and buds were sampled, the slides were prepared from

fully developed anthers in aceto-carmine, and in at least 500 pollen grains per slide, nonstainable pollen grains, and deviation in pollen size and shape were scored. The processing is described (Paradiž and Lovka, 1999, 2004). Results and discussion

In 18 Galanthus varieties of the Botanical Garden Collection, a high pollen grain deformation (PGD) ratio of deformed (D), i.e. bigger size and irregular shape to sterile (S) pollen pointed to variable levels of ploidy in 11 plants (Table 1). Unbalanced pollen grains were indicated in 1 plant from Zminec, and plants from the Dinaric region, except in one plant from Sortež and another one from Col.

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Table 1. Pollen grain deformation (PGD) assays on Galanthus nivalis varieties of Botanical Garden

Collection in Ljubljana, originating from different localities in Slovenia

Galanthus varieties Altered pollen grain frequency % Locality Plant Year Ab tot sD Sterile Size Shape D/S Dol 1 2012 3,00 0,91 1,75 1,00 0,25 0,71 Gameljne 2 2008 2,25 0,69 1,75 0,00 0,50 0,29 Zminec 3 2014 10,58 2,36 9,50 0,75 0,33 0,11

4 2014 10,33 1,52 3,33 6,92 0,08 2,10 5 2014 10,50 3,23 9,75 0,33 0,42 0,08 6 2014 8,83 2,40 7,33 1,00 0,50 0,20

Sortež 7 2009 18,35 4,23 2,39 15,96 0,00 6,69 8 2014 78,87 2,30 63,87 8,79 6,22 0,23 9 2014 44,89 0,56 14,96 25,91 4,01 2,00

Podkraj 10 2012 30,51 8,91 7,81 9,47 13,23 2,91 Col

11 2014 1,92 0,52 1,25 0,42 0,25 0,53 12 2014 4,17 1,76 1,08 2,50 0,58 2,85 13 2014 14,04 4,22 3,95 8,07 2,02 2,56

Črni Vrh

14 2014 15,93 2,09 6,26 0,49 9,19 1,55 15 2014 3,27 1,01 1,06 1,77 0,44 2,08 16 2014 6,63 2,07 1,33 4,70 0,60 4,00 17 2014 2,83 0,84 1,17 1,42 0,25 1,43 18 2014 26,44 10,10 2,37 22,20 1,86 10,14

1-17 = tall variant, 7 = giant flower, 2 = spathe variant, 18 = small variant Year = transplantation from habitat to Botanical Garden Ab tot = total pollen grain aberrations D/S = deformed (D) of bigger size and altered shape to sterile (S) pollen grains

High PGD ratio in majority of taller Galanthus varieties originated from Sortež, Podkraj,

Col, and Črni Vrh (the Dinaric region), and Zminec (Škofja Loka) was found, in a contrast to those from Ljubljana environs (Dol, Gameljne) with low PGD ratio. The highest PGD ratio of taller Dinaric varieties in a giant flower is presented 3 years after plant transplantation from Sortež. But even higher PGD ratio in a single small variant in the year of its transplantation from Črni Vrh was established.

Aberration frequency in Galanthus varieties from the central part of Slovenia (Dol, Gameljne, Zminec) was almost 3-times lower than that from the Dinaric region (21%). But sterile pollen frequencies, comparing them with the yearly average of up to 5% (Paradiž and Lovka, 1999, 2004; Paradiž, 2011; Paradiž and Bavcon, 2012) were higher in plants from Zminec (central part of Slovenia) and the Dinaric region for 2,5% and 4%, respectively. In increased pollen sterility harsh growth conditions of their habitats at higher levels could be a reflected.

PGD results in Graph 1 pointed to in situ higher ploidy variation in Galanthus nivalis than in other bioindicators at Ljubljana, Slavnik, and Dolenjska, but lower than in Galanthus varieties of the Botanical Garden Collection. PGD ratio in Galanthus plants at Slavnik was higher than at Ljubljana environs, but lower than at Dolenjska. At lowlands of Dolenjska the highest PGD ratio in Galanthus could at least partly be attributed to an additional environmental pollution.

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Graph 1. Deformed to sterile pollen grains frequency in Galanthus varieties of the Botanical Garden, and

naturally growing plants in their habitats. Legend 1 = Ljubljana / Botanical Garden: Galanthus varieties 2 = Škofja Loka / Botanical Garden: Galanthus varieties 3 = Dinaric region / Botanical Garden: Galanthus varieties 4 = Ljubljana: 14 plant species 5 = Ljubljana: Galanthus nivalis 6 = Slavnik: 6 plant species 7 = Slavnik: Galanthus nivalis 8 = Dolenjska: 2 plant species 9 = Dolenjska: Galanthus nivalis

Large pollen clusters in response to environmental stresses are not common, but observed in Galanthus nivalis at Dolenjska (5%) and Slavnik (2%), and in transplanted varieties from the Dinaric region (8%). Occurrence of multipollen aggregate can provide critical PGD signals of a potential damage owing to different factors in the environment. Conclusions

Results of the pollen grain deformation (PGD) assay on Galanthus varieties of the Botanical Garden Collection have shown that size variations are linked with polyploidization processes. Tall forms with big flowers exhibit variable levels of ploidy, and particularly diversified is a giant Dinaric form. But gigantism is not always an attending feature of polyploidy, as it was established in a single small Dinaric variant.

PGD ratio of deformed to sterile pollen grains, showed a high Galanthus intraspecific ploidy variation in the Dinaric region, but low in the central part of Slovenia. Such a diverse ploidal status enables a wide tolerance to complex environmental impact in adaptation plant response at higher altitude habitats. In situ biomonitoring plants, however, indicated a high PGD ratio in Galanthus nivalis at Dolenjska agricultural environs, and unbalanced cytological situation of survival reasons in plant response to environmental pollution.

0%

50%

100%

1 2 3 4 5 6 7 8 9

Fre

quen

cy

Plant habitat / Botanical Garden

Sterile pollen grains Deformed pollen grains

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PGDs unmask environmental clastogenic effects, enable early warnings of genotoxic risks for all plants, and the endangered species, and promote the necessary protection ecoremediations (Vrhovšek and Vovk Korže, 2009), as well as cost-effective control strategies (Hansen, 2008) and precautionary measures regarding European directives on biodiversity.

Biomonitoring PGDs can benefit in diagnosing decorative varieties not sufficiently distinct in nature as to make it easily recognizable. PGDs in polluted environments can verify an array of relevant ecological endpoints of clear consequence for the abundance and persistence of plant populations, such as growth, reproduction, and survival. References Bavcon, J. (2009). Common Snowdrop (Galanthus nivalis L.) and Its Diversity in Slovenia,

Botanični vrt, Oddelek za biologijo, Biotehniška fakultata, Ljubljana. D'Amato, G. F., Bianchi G. (1999). The chromosome banding of some Italian Amaryllidaceae,

Caryologia, 52 (1-2), 87-92. Davis, A. P., Mordak H., Jury S. L. (1996). Taxonomic Status of Three Caucasian Snowdrops:

Galanthus alpinus Sosn., G. bortkewitschianus Koss and G. caucasicus (Baker) Grossh., Kew Bulletin, 51 (4), 741-752.

Druškovič, B., Lovka M. (1995). Pregled določitev kromosomskih števil praprotnic in semenk v Sloveniji, Biološki vestnik, 40 (3-4), 151-168.

Hansen, P. D. (2008). Advanced Environmental Monitoring, Earth and Environmental Science, Biosensors for Environmental and Human Health, Springer, Netherlands.

Kandemir, N. (2010). A karyological investigation on the two varieties of Galanthus fosteri Baker (Amaryllidaceae), Biological Diversity and Conservation, 3 (2), 20-25.

Lledó, M. D., Davis A. P., Crespo M. B., Chase M. W., Fay M. F. (2004). Phylogenetic analysis of Leucojum and Galanthus (Amaryllidaceae) based on plastid matK and nuclear ribosomal spacer (ITS) DNA sequences and morphology, Plant Systematics and Evolution, 246 (3-4), 223-243.

Löve, A. (1973). IOPB Chromosome Number Reports XLI, Taxon, 22 (4), 459-464. Lovka, M., Krušnik C., Kosi G., Paradiž J. (2003). Natural heritage in wetland habitats,

Nacionalni inštitut za biologijo, Ljubljana. Mičieta, K., Murin G. (1996). Microspore analysis for genotoxicity of a polluted environment,

Environmenal and Experimental Botany, 36 (1), 21-27. Mišik, M., Solenska M., Mičieta K., Mišikova K., Knasmüller S. (2006). In situ monitoring of

clastogenicity of ambient air in Bratislava, Slovakia using the Tradescatia micronucleus assay and pollen abortion assays, Mutation Research, 605, 1-6.

Paradiž, J. (2011). Environmental biomonitoring plants for sustainable nature protection, Journal for Geography, 6 (2), 143-151.

Paradiž, J., Bavcon J. (2012): Karyological differentiation of Galanthus nivalis L. cytotypes in Slovenian wetlands, 6th Congress of the Genetic Society of Slovenia with International Participation, Genetika 2012: Book of abstracts, Genetic Society of Slovenia, Maribor, 70.

Paradiž, J., Lovka M. (1999). Pollen Grain Bioassay for Environmental Pollution Screening, Phyton, 39 (3), 175-180.

Paradiž, J., Lovka M. (2004)_ Pollen grain bioassay for environmental contamination biomonitoring, Acta Biologica Slovenica, 47 (2), 75-81.

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Senel, G., Ozkan M., Kandemir N. (2002). A karyological investigation on some rare and endangered species of Amaryllidaceae in Turkey, Pakistan Journal of Botany, 34 (3), 229-235.

Vrhovšek, D., Druškovič B., Batič F., Bricelj M., Kosi G., Kralj M., Paradiž J. (1984). Biološki indikatorji onesnaženosti voda in kopnega v mestu Ljubljana: raziskovalna naloga 1983-1984, Inštitut za biologijo, Univerza v Ljubljani, Ljubljana.

Vrhovšek, D., Vovk Korže A. (2009). Ekoremediacije, Univerza v Mariboru, Filozofska fakulteta Maribor, Mednarodni center za ekoremedaicije in Limnos, Podjetje za aplikativno biologijo, Maribor in Ljubljana.

Zonneveld, B. J. M., Grimshaw J. M., Davis A. P. (2003). The systematic value of nuclear DNA content in Galanthus, Plant Systematics and Evolution, 241, 89-102.

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GENETIC RELATIONSHIP BETWEEN SOYBEAN CULTIVARS FROM DIFFERENT BREEDING INSTITUTIONS

Vesna PERIC, Ana NIKOLIC, Mirjana SREBRIC, Snezana MLADENOVIC DRINIC

Maize Research Institute “Zemun Polje”, Slobodana Bajica 1, Zemun, Belgrade;

Corresponding author: vperic@mrizp.rs Abstract The objective of the study was to determine genetic diversity among eighteen soybean cultivars developed in three different breeding programs in Serbia and Croatia. The genetic relationships were estimated using 10 SSR markers. Genetic similarity (GS) between cultivars was estimated with Jaccard coefficient. Cluster analysis was performed according to UPGMA (Unweighted pair group method with arithmetic mean). Total number of fragments amplified was 33, of which 21 (63%) was polymorphic. A number of bands per primer were 2-5. The greatest genetic similarity according to Jaccard coefficient was determined for pair of genotypes Lana/Laura (GSJacc=1,00) and Nena/Laura (GSJacc=0,96), while the most distant ones were Backa and Balkan, with GSJacc=0,47. Clustering method based on SSR data similarity matrices classified genotypes in two major clusters. Eighteen soybean cultivars were assigned into groups showing partial correspondence with their origin. Five soybean cultivars released in Maize Research Institute “Zemun Polje” were grouped into the same cluster, while genotypes from Institute of Field and Vegetable Crops, Novi Sad and Agricultural Institute Osijek were mostly allocated in both clusters. Cluster analysis revealed that separation of varieties was in partial accordance with their origin.

Key words: soybean, cultivar, genetic diversity, SSR analysis

Introduction Cultivated soybean is one of the most important crops used for animal feed and human

consumption (Popovic et al, 2012). The main problem in developing high yielding soybean cultivars in breeding programs worldwide is very narrow genetic base, reported in numerous studies on diversity of soybean in USA (Gizlice et al., 1994; Narvel et al., 2000), South America (Priolli et al., 2002; Giancola et al., 2002; Bonato et al., 2006), Canada (Fu et al., 2007), Asia (Liu et al., 2011) and Europe germplasm collections (Baranek et al., 2002). Soybean is largely self pollinated species with relatively small number of ancestors contributing to the genetic background of commercial varieties. Additionally, considerable loss of genetic diversity of soybean appeared as the consequence of making crosses between elite lines, which lead to

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limited genetic variability in segregating generations and reduces possibility for development of superior genotypes in breeding process. Traditional approach in diversity studies combines pedigree data and measures of morphological variability (Giancola et al., 2002; Liu et al., 2011), though this information has limited importance. Pedigree data are often incomplete, while classical morphological characteristics have limited number and are influenced by different factors (growth stage; polygenic control of quantitative traits; expression depends on environmental conditions). Use of molecular markers in diversity studies may overcome mentioned problems, since they are easy for observation and scoring, available in great number and free from genotype-environment interaction (Lombard et al., 2001). Application of SSR (simple sequence repeat) markers in soybean breeding proved to be suitable for genetic diversity studies (Tavaud-Pirra et al., 2009, Ristova et al., 2010), breeding for specific traits (Shi et al., 2010), determining relationship between ancestors and introduced germplasm (Gizlice et al., 1994; Braun-Guedira et al., 2000) and variety identification (Tantasawat et al., 2011).

In order to determine the level of genetic diversity and analyze relationships between eighteen soybean cultivars developed in three breeding institutions, SSR analysis was conducted Examined cultivars are assumed to be similar, due to the practice of selection within the material existing in local germplasm collections (adapted to similar environmental conditions; similar sellection targets) and exchange between them. Materials and methods

The experimental material included eighteen soybean genotypes of good agronomic performance (high genetic potential for seed yield, resistance to lodging and seed shattering, tolerance to stress conditions) developed in three breeding institutions: Maize Research Institute “Zemun Polje”, Serbia (MRIZP), Institute of Field and Vegetable Crops, Novi Sad, Serbia (IFVCNS) and Agricultural Institute Osijek, Croatia (AIOS) (Table 1).

Table 1. List of genotypes for SSR analysis

Genotype Institution Maturity group Origin 1 Laura MRIZP I Serbia 2 Lana MRIZP II Serbia 3 Olga MRIZP II Serbia 4 Lidija MRIZP II Serbia 5 Nena MRIZP II Serbia 6 ZPS015 MRIZP 0 Serbia 7 Balkan IFVCNS I Serbia 8 Kolubara IFVCNS 0 Serbia 9 Afrodita IFVCNS 0 Serbia 10 Ravnica IFVCNS I Serbia 11 Vojvodjanka IFVCNS II Serbia 12 Backa IFVCNS 0 Serbia 13 Korana AIOS 00 Croatia 14 Lucija AIOS 00-0 Croatia 15 Julijana AIOS 0 Croatia 16 Vita AIOS 0 Croatia 17 Ika AIOS 0-I Croatia 18 OS 101 AIOS I Croatia

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DNA was extracted from soybean seeds as described by Kamiya andKiguchi (2003). Seed powder was incubated at 55oC for 20 min in 0.2 ml of digestion buffer containing 16 µg proteinase K. After digestion, DNA extraction was done with one volume of phenol/chlorophorm/isoamyl alcohol. The mixture was then centrifuged at 14000 rpm for 5 min. Precipitation with 0,2 ml of isopropanol was used to purify DNA, followed by centrifugation at 14000 rpm for 5 min and drying. The DNA concentration was determined spectrophotometrically at 260 nm.

Molecular analysis is performed using 10 SSR primers Metabion Int. AG (Satt 009, Satt 122, Satt 173, Satt 308, Satt 167, Satt 127, Satt 192, Satt 225, Satt 232, Satt 406). The reaction mixture for DNA amplification contained: 20 ng of genomic DNA, 1.0 unit of Taq DNA polymerase, 1xPCR buffer (DreamTaq™ Green Buffer, Fermentas), 2.4 mM MgCl2, 0.8 mM dNTPs and 0.5µM of primer pairs. PCR conditions were as follows: 95oC for 5 min followed by a 15-step touchdown decreasing by 0.5oC each step: 95oC for 30 s, 63.5 to 56oC for 1 min and 72oC for 1 min followed by 25 cycles of 95oC for 30 s, 56oC for 1 min and 72oC for 1 min. The amplification products were separated by polyacrilamide gel electrophoresis prepared in 1xTBE buffer, stained with ethidium bromide and photographed with BDA live system, Biometra.

Results were visually scored and presence/absence of DNA band for each primer was transformed in binary code (0, 1). Genetic similarity (GS) between cultivars was estimated with Jaccard coefficient according to SSR data. Cluster analysis was done by NTSYS-pc version 2.1 software (Rohlf, 2000), using UPGMA (Unweighted pair group method with arithmetic mean). Results and discussion

The SSR polymorphism of 18 soybean genotypes showed variation in 6 of 10 analyzed loci. A total number of fragments amplified was 33, of which 21 (63%) was polymorphic. The number of alleles per primer varied from 2 to 5, with average 3,3 bands per primer (Table 2). Amplification products for 5 primers were monomorphic No unique fragments were found. Polymorphic information content (PIC) values for all polymorphic primers ranged from 0,673 to 0,741 with mean value of 0,720. The matrix of pairwise GSJacc calculations showed minimum values of 0,47 to 1,00, with average genetic similarity of 0,70, indicating the close genetic relationships of the material used in this study. Similar values for average GS were reported by Ristova et al. (2010), while other authors detected higher level of diversity revealed by SSR markers (Narvel et al., 2000; Giancola et al., 2002).

Table 2. Polymorphic information content (PIC) values, position on chromosomes, and number of

detected alleles for 10 SSR loci

Primer Chromosome No. of alleles Polymorphic information content (PIC) Satt 009 3 4 0.732 Satt 122 14 4 0.741 Satt 173 10 4 0.722 Satt 308 7 5 0.730 Satt 167 9 4 0.673 Satt 127 20 2 monomorphic Satt 192 12 3 monomorphic Satt 225 5 2 monomorphic Satt 232 19 2 monomorphic Satt 406 16 3 monomorphic Average 3.3 0.720

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As expected, the greatest genetic similarity coefficient based on the similarity by Jaccard was found between varieties Lana and Laura (GSJacc=1,00), since they are half-sib lines. High level of similarity was determined for genotypes Nena and Laura (GSJacc = 0,96) and Nena and Lana (GSJacc = 0,96). The lowest genetic similarity was calculated for a pair Backa-Balkan (GSJacc= 0,47). Cluster analysis was performed on Jaccard similarity matrix by using the UPGMA method of grouping. The constructed dendrogram is presented in the Figure 1.

Figure 1. Cluster analysis of 18 soybean genotypes based on Jaccard coefficients of similarity for SSR

data The dendrogram divided genotypes in two main groups (A and B). Classification of

genotypes in clusters showed partial agreement with their origin. Cluster B consisted of five soybean varieties developed in MRIZP (ZPS 015, Laura, Lana, Nena i Olga), variety Balkan from IFVCNS and cultivar Lucija from AIOS. Remaining genotypes from IFVCNS and AIOS were allocated in the cluster A, showing weak congruence with their origin, with exception of Julijana and Vita, genotypes from the same breeding program, which were grouped into the same subcluster.

Conclusion

The genetic similarity of investigated elite genotypes developed in three breeding institutions in the region of Serbia and Croatia is relatively high, confirming the assumption about the exploiting the similar breeding stocks in the selection process. High value of average genetic similarity indicated the close genetic relationships of investigated cultivars. Cluster analysis revealed that separation of varieties was not in total accordance with the origin. Agreement between grouping pattern and origin was found for genotypes from MRIZP. The

Coefficient0.63 0.72 0.82 0.91 1.00

VojvodankaMW

Korana

Afrodita

Julijana

Vita

Backa

Kolubara

Ika

Lidija

Ravnica

OS101

Vojvodanka

Lucija

Balkan

ZPS015

Laura

Lana

Nena

Olga

A

B

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moderate level of association between divergence and origin of cultivars found in this study could be enhanced by the implementation of greater number of SSR markers. Furthermore, introgression of new desirable sources of germplasm is requirement for creating variability and enhancing efficiency of breeding programme.

Acknowledgement

This study is a part of a scientific project Reg. No. TR 31068, supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia.

References Baranek, M., Kadlec, M., Raddova, J.,Vachun, M. and Pidra, M. (2002). Evaluation of genetic

diversity in 19 Glycine max (L.) Merr. accessions included in the Czech National Collection of Soybean Genotypes. Czech J.Genet.Plant Breed., 38, 69–74, ISSN 1212-1976.

Bonato Ana Lidia, Eberson, C.S., Isaias, O.G and C.A. Arias (2006). Genetic similarity among soybean (Glycine max L. Merill.) cultivars released in Brazil using RFLP markers. Genet. Mol. Biol. 29, 4, 692-704.

Brown-Guedira, G.L, Thompson, J.A., Nelson, R.L. and M.L. Warburton (2000). Evaluation of genetic diversity of soybean introductions and North American ancestors using RAPD and SSR markers. Crop Sci.,40, 815-823.

Fu, Y.-B., Peterson,G.W. and M.J. Morrison. (2007). Genetic diversity of Canadian soybean cultivars and exotic germplasm revealed by simple sequence repeat markers. Crop Sci. 47:1947–1954.

Giancola, S.S., Poltri, M., Lacaze, P. and H.E. Hopp (2002). Feasibility of integration of molecular markers and morphological descriptors in a real case study of a plant variety protection system for soybean. Euphytica, 127:95-113.

Gizlice, Z., Carter, T.E. and J.W. Burton (1994). Genetic base of North American public soybean cultivars released between 1947 and 1988. Crop Sci., 27, 885-892.

Kamiya, Ku Motokazu and Kiguchi, Tadahiko (2003). Rapid DNA extraction from soybean seeds. Breeding Science 53 (3): 277-279.

Liu, M., Zhang, M., Jiang, W., Sun, G., Zhao, H. and Hu, S. (2011). Genetic diversity of Shaanxi soybean landraces based on agronomic traits and SSR markersAfr. J. Biotechnol. 10 (24), 4823-4837.

Lombard, V., Dubreuil, P., Dilmann, C. and C. Baril (2001). Genetic distance estimators based on molecular data for plant registration and protection: A review. Acta Hort. 546:55–63.

Narvel, J.M., Fehr, W.R., Chu, W., Grant, D. and R.C. Shoemaker (2000). Simple sequence repeat diversity among soybean plant introductions and elite genotypes. Crop. Sci., 40, 1452-1458.

Tantasawat, P., Trongchuen, J., Prajongjai, T., Jenweerawat, S. and W. Chaowiset (2011). SSR analysis of soybean (Glycine max (L.) Merr.) genetic relationship and variety identification in Thailand. Aust J Crop Sci. 5(3):283-290.

Tavaud-Pirra, M., Sartre, P., Nelson, R., Santoni, S., Texier N. and P. Roumet (2009). Genetic Diversity in a Soybean Collection. Crop Sci, 59(3), 895-902.

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Popovic, V., Vidic, M., Jockovic, Dj., Ikanovic, J., Jaksic S., Cvijanović G. (2012). Variability and correlations between yield components of soybean [Glycine Max (L.) Merr.]. Genetika, Belgrade, Vol. 44, No.1, 33-45.

Priolli, R.H.G., Mendes Jr, C.T., Arantes, N.E., Contel, E.P.B. (2002). Characterization of Brazilian soybean cultivars using microsatellite markers. Genet Mol Biol., 25, 185-193.

Ristova, D., Šarcevic, H., Šimon, S., Mihajlov, L. and I. Pejic, (2010). Genetic diversity in southeast European soybean germplasm revealed by SSR markers. Agriculturae Conspectus Scientificus, 75 , 21-26.

Rohlf, F.J. (2000). NTSYS-pc Numerical taxonomy and multivariate analysis system. Version 2.1 Exeter publications, New York, USA

Shi, A., Chen, P., Zhang, B. and A. Hou (2010). Genetic diversity and association analysis of protein and oil content in food-grade soybeans from Asia and the United States. Plant Breeding, 129 (3), 250-256.

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UTILIZATION OF MAIZE GENETIC RESOURCES IN GRAIN QUALITY IMPROVEMENT

Danijela RISTIC, Marija KOSTADINOVIC, Dragana IGNJATOVIC-MICIC, Jelena

VANCETOVIC

Maize Research Institute “Zemun Polje”, Slobodana Bajica 1, 11185 Belgrade, Serbia Abstract

Maize can be used in three ways: in the human diet, as feed and as a raw material in industry. In underdeveloped and developing countries, maize production has enormous significance because this cereal is basic food for human consumption. A lot of favorable alleles that can contribute to higher yield, abiotic stress tolerance, disease and pests resistance or nutritional quality improvement, are dispersed within maize landraces. The recessive opaque-2

(o2) mutation increases lysine and tryptophan content in maize grains. Maize with increased levels of these essential amino acids, hard endosperm and good agronomic performances is called quality protein maize (QPM). The fifteen landraces were analyzed for total protein and tryptophan content, as well as for the presence of o2 mutation, the basis for developing QPM. The genotypes chosen based on the high tryptophan level and the presence of opaque-2 mutation can be used in plant breeding as potential source of improved quality. Key words: landraces, maize, opaque-2 gene, protein quality, SSR Introduction

Maize is one of the most important crops cultivated around the world. Europe is the third largest maize producer and consumer after the United States and China (USDA National Agricultural Statistics Service Data, 2007). Maize can be used in three ways: in human diet, as animal feed and as feedstock in industry. About 80% of world production of maize is used for animal feed. In underdeveloped and developing countries, particularly in Africa and South America, maize production is of great importance since it is the main cereal food in the human diet and the only source of protein.

However, maize nutritional value is very low due to deficiency of two essential amino acids - lysine and tryptophan, which are essential for building proteins in humans and monogastric animals (Bressani, 1991). The opaque-2 (o2) mutation which increases lysine and tryptophan content (Mertz et al., 1964) is closely associated with some undesirable traits such as

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low yields (Singh and Venkatesh, 2006), soft and chalky kernels that renders by seeds susceptible to storage pest and ear rots (Toro et al., 2003). These undesirables traits have been avoided in genetically improved, hard endosperm Quality Protein Maize (QPM), created by research team of International Maize and Wheat Improvement Center (CIMMYT), Mexico (Vivek et al., 2008).

Given the current climate change, human population growth and an increasing demand for food, creating hybrids with improved performance is one of the main goals of modern agriculture. Narrow genetic base of commercial maize varieties indicate the necessity for conservation, characterization and utilization of maize genetic resources (Carena et al., 2009). Broadening the genetic base of maize and breeding high yielding cultivars adaptable to diverse agro-ecologies conditions will certainly depend on the efficient and rapid identification and introgression of novel and/or favourable alleles (Prasanna, 2010). In this sense, using maize landraces in the process of selection and breeding aims to expand the genetic base of existing elite germplasm, as well as increase to the quality of grain for human and animal consumption (Uarrota et al., 2011), by introduction of new alleles that do not exist in commercial varieties.

Maize Research Institute (MRI) gene bank collection maintains about 6000 accessions. This offers great opportunities for breeding, considering its size and content – it is among the ten largest maize gene banks in the world. The objective of this study was to analyze fifteen landraces for total protein and tryptophan content, as well as for the presence of o2 mutation. Material and methods

A sample of 15 maize landraces which belongs to diferent agro-ecological groups was analyzed for grain quality characteristics.

Biochemical analysis

Each genotype was represented by 60 randomly chosen kernels, divided into two sub-samples consisting of 30 kernels each. Kernels were dried in a controlled oven at 65°C over night (16-18 hours), and milled in a Cyclone sample mill - Simmons Fastener, USA. The flour was defatted by hexane treatment for 4 hours in Soxhlet extractor.

Tryptophan content was determined using the colorimetric method of Nurit et al. (2009). The color was developed in the reaction of flour hydrolysate (obtained by overnight digestion with papain solution at 65°C) with a reagent containing glyoxylic acid and ferric chloride dissolved in sulfuric acid. After incubation at 65°C/30 min, absorbance was read at 560 nm. Tryptophan content was calculated using a standard calibration curve, developed with known amounts of tryptophan, ranging from zero to 30 µg/µl.

The protein content was determined by the standard Kjedahl method based on nitrogen determination as explained in Vivek et al (2008). The protein was estimated from the nitrogen value as: % protein = % nitrogen x 6.25 (conversion factor for maize).

Quality index (QI), defined as tryptophan to protein ratio in the sample, was calculated as: QI = 100 x (tryptophan content in the sample / protein content in the sample).

SSR analysis

Genomic DNA was isolated from seed bulk using the CTAB procedure according to Saghai-Maroof et al. (1984). Bulks were prepared by pooling an equal amount of flour obtained by grounding 30 seeds per landrace. Seeds were taken from 30 different plants. Simple sequence

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repeat analysis was done with three primers specific for opaque-2 gene (phi057 and umc1066). Polymerase chain reaction was carried out in 20 µl reaction volume containing: DreamTaq™ Green PCR Master Mix (2X), 0.25 µM primers and 20 ng of DNA. Amplifications were performed in thermocycler Biometra TProfessional Standard 96 with the following program: an initial denaturation at 94ºC /2min, followed by 40 cycles each of denaturation at 94ºC/1min, annealing at 60ºC/2min and extension at 72ºC/2min, with final extension at 72ºC/10min. The amplified fragments were resolved by electrophoresis on 2.5% agarose gel in 0.5 x TBE buffer, with 100bp ladder as a marker. After staining with 0.5 µg/µl ethidium bromide they were visualized under UV transilluminator and documented in gel documentation system (BioDocAnalyze, Biometra). Allele designations were made and approximate size range for the amplification products for each SSR locus was determined based on the positions of the bands relative to the 100 bp molecular weight ladder.

Results and discussion

Modern and future maize breeding depends upon genetic sources of desirable traits, which are maintained favorable within that existing gene banks. Plant genetic resources of wild relatives of cultivated crops and landraces contain a wide set of alleles that are lost by creating elite varieties through the selection process. These alleles are invaluable in dealing with the sustainable development of agriculture and food production (Varshney et al., 2011). Chemical composition of maize accession from MRI was analyzed on drought tolerant core collection (Andjelkovic et al., 2012).

Protein and tryptophan contents and QI of analyzed accessions are presented in Table 1. All the values were compared with threshold limits for quality protein maize given in Vivek et al. (2008). The landraces showed a considerable, range for protein content, with the average value od 12.36%. Kjedahl method revealed high levels of protein content (from 9.58% to 14.46%), meaning that these genotypes could be a good source of proteins in quality breeding programs. Table 1. Tryptophan and protein contents and quality index (QI) of analyzed landraces

No Accession Tryptophan content (%)

Protein content (%)

QI

1 int4 0.065 11.46 0.37 2 int408 0.059 12.73 0.46 3 int20 0.048 13.17 0.56 4 int33 0.064 14.46 0.44 5 int419 0.061 12.97 0.47 6 int641 0.063 12.73 0.49 7 int132 0.059 11.24 0.53 8 int606 0.058 12.29 0.47 9 int785 0.063 12.05 0.52 10 int186 0.057 11.28 0.50 11 int190 0.058 9.58 0.60 12 int79 0.057 11.74 0.49 13 int902 0.069 12.95 0.53 14 int121 0.050 12.81 0.39 15 int558 0.064 13.91 0.46

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Because of the high correlation of lysine and tryptophan (Nurit et al., 2009), for the

purpose of assessing grain quality only tryptophan content was analyzed, due to the simplicity of the method. Tryptophan content higher than 0.075 (level specific for QPM germplasm) was not obtained in the analyzed accessions. Landraces int4, int33, int558 and int902 had the highest values for tryptophan (0.064%, 0.065% and 0.069%, respectively), which is more than the value obtained in standard hybrid (0.06), as shown in our earlier work (Kostadinovic et al., 2012; Ignjatovic-Micic et al., 2013). Quality index was low (below 0.6) for almost all landraces, while the highest QI of 0.06 was revealed in landrace int190. The amplified homozygous and heterozygous accessions with opaque-2 specific phi057 SSR marker are presented in Figure 1. Recessive opaque-2 allele was found in all accessions, except in int408, int20 and int641. The presence of recessive opaque-2 allele found in the 12 genotypes was heterozygous (O2o2). Low levels of tryptophan could thus be explained by the heterozygous form of the opaque-2 gene. Nevertheless, the presence of recessive o2 allele classifies these accessions as potential sources of the recessive mutation for high tryptophan content.

Figure 1. Electrophoregram of SSR analysis with phi057. o2 –positive control for opaque-2 allele, N –

negative control for opaque-2 allele, 1 to 15 – analyzed landraces. Conclusion

Analyzed landraces showed large variability for the grain quality traits, which can be exploited for genetic improvement. Biochemical analysis determined the genotypes with high protein content. Further study should involve a larger number of landraces that show good agronomic traits, such as biotic and abiotic stress and high yield.

Acknowledgements This work was supported by the Ministry of Education, Science and Technological

Development of Republic of Serbia, through the project TR31028 “Exploitation of maize diversity to improve grain quality and drought tolerance”. References Andjelkovic, V. (2013). Biochemical and agronomic performance of quality protein maize

hybrids adapted to temperate regions. Maydica, vol 58 (3-4), pp 311-317. Andjelkovic, V., Ignjatovic-Micic D., Mladenovic Drinic S., Vancetovic J. (2012).

Implementation of maize genetic resources in drought tolerance and grain quality improvement at Maize research institute „Zemun Polje“. Book of proceedings Third International Scientific Symposium "Agrosym Jahorina 2012", pp: 429-434.

Bressani, R. (1991). Protein quality of high lysine maize for humans. Cereal Foods World, 36: 806-811.

o2 N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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Carena, M.J., Yang J., Caffarel C., Mergoum M., Hallauer A.R. (2009). Do different production environments justify separate maize breeding programs?, Euphytica, vol. 169, pp. 141-150.

Ignjatovic-Micic, D, M. Kostadinovic, G. Stankovic, K. Markovic, J. Vancetovic, S. Bozinovic, Kostadinovic, M., Ignjatovic-Micic D., Stankovic G., Mladenovic Drinic S. (2012). Protein quality analysis of F2 maize kernels. Plant Breeding and Seed Production, vol.XVIII, No 1, 33-39.

Mertz, E.T., Bates L.S., Nelson O.E. (1964). Mutant gene that changes protein composition and increase lysine content of maize endosperm. Science, 145: 279-280.

Nurit, E., Tiessen A., Pixley K., Palacios-Rojas N. (2009). Reliable and inexpensive colorimetric method for determining protein-bound tryptophan in maize kernels. J. Agric. Food Chem. 57: 7233-7238.

Prasanna, B.M. (2010). Phenotypic and molecular diversity of maize landraces: characterization and utilization. The Indian Journal of Genetics and Plant Breeding, vol. 70, pp. 315-327.

Saghai-Maroof, M., Soliman K., Jorgensen R., Allard R. (1984). Ribosomal DNA spacer length polymorphism in barley; mendelian inheritance, chromosomal location and population dynamics. Proceedings of the National Academy of Sciences of the United States of America, vol. 91, pp. 8014-8018.

Singh N.N., Venkatesh S. (2006). Development of quality protein maize inbred lines. In: Heterosis in Crop Plants. Ed. Kaloo G, Rai M, Singh M, Kumar S. Res. Book Center, New Delhi, pp. 102-113.

Toro A, Medici L, Sodek L, Lea P, Azevedo R (2003). Distribution of soluble amino-acids in maize endosperm mutants. Scientia Agricola 60: 91-96.

Varshney, RK, Bansal K.C., Aggarwal P.K., Datta S.K., Craufurd P.Q. (2011). Agricultural biotechnology for crop improvement in a variable climate: hope or hype? Trend Pl Sci.; 16:363–71.

Vivek, B.S., Krivanek A.F., Palacios-Rojas N., Twumasi-Afriyie S. and Diallo A.O. (2008). Breeding Quality Protein Maize (QPM): Protocols for Developing QPM Cultivars. Mexico, D.F.: CIMMYT.

Uarrota, V.G., Schmidt E.C., Bouzan Z.L., Maraschin M. (2011). Histichemical analysis and protein content of maize landraces (Zea mays L.), Journal of Agronomy, 10: 92-98.

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153

GENETIC GAIN FROM SELECTION IN PROGENY OF SOYBEAN SISTER CROSSES

Mirjana SREBRIC1, Vesna PERIC1, Gordana SURLAN MOMIROVIC2

1Maize Research Institute "Zemun Polje", Slobodana Bajica 1,11185 Zemun Polje, Belgrade,

Serbia 2Faculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Zemun, Belgrade,Serbia

Abstract

Segregating population was created by crossing the Kunitz x Kador. After testing the grain yield, three F3 lines were used in two sister cross combination. Progenies of chosen lines and their sister crosses were tested together with the initial parents, in a two-year experiment on two locations. Genetic gain from selection, of the investigated characteristics was calculating for both progeny types. Mother’s progenies mostly have shown higher values of genetic gain from selection in addition to the respective full sib progenies. Key words: soybean, sister crossing, genetic gain from selection Introduction

Seed yield is of great importance for all cultivated plant species, including soybean. Yield and yield components of soybean are quantitative traits and their expression are strongly influenced by environmental conditions (Paul et al. 2003). Number of pods, number of grains and 1000 grain yield of the most important components of soybean yield (Padney and Torrie, 1973), as well as the number of pods and number of seeds per unit area (De Bruin and Pedersen, 2009). Grain yield per plant and number of seeds per plant are effective criterion for breeding soybean for increased grain yield (Sudarić et al., 2002).

The estimate of the expected genetic gain from selection, as an indicator of the success of the applied selection method and the means of the study population, is of great importance in practical selection. Size of genetic gain from selection depends on the current population variability, heritability of observed traits, selection intensity and the number of generations required for one cycle of recurrent selection (Eberhart, 1980). Genetic gain from selection can be used to compare new developed cultivars to the conventional varieties (Sudaric et al., 2001), by comparing the performance of different selection methods (Miladinovic et al. 2011) and different materials in the selection process (Stankovic, 2002 ).

154

Materials and methods Soybean lines investigated in this study were developed from the cross between Kunitz

and Kador. All individual plants whose vegetation length didn’t exceed the vegetation of the earlier parent (variety Kador) were sorted out from F2 population. Forty eight individual F2 plants with a sufficient amount of seed were selected for further analysis. Part of seed of each F2 plant was used for field experiments, whereas the rest was left as a reserve. After yield testing, selected lines are marked as L6 and L30, and used for sister-crossing. The crossings were performed on F3 plants originating from seed reserves. Total seeds from maternal plants, including potentially hybrid seeds and seeds obtained by selfing, were preserved. Fifteen plants where the sister-cross was successfully completed were grown (FSF1). Their seed was produced during the next season, as long as the seed of maternal plants. This providied a sufficient quantity of seed at the same level of homozygosity (87.5%) in the progeny of mothers (F3:4) and the progeny of sister crosses (FSF1:2) for field experiment. This was in agreement with Falconer (1960).

The field experiment design was a randomized complete block (RCB), with three replicates at two locations (Zemun Polje and Indjija) for a period of two years (2006 and 2007). The elementary plot consisted of two rows five meters long, with a row spacing of 0.5 m and spacing between plants of 4 cm. The experiment included the offspring of mothers, sister-crosses progeny and initial parents - variety Kunitz and Kador. The following yield components were observed: seed yield per plant, plant height, the height of the lowest formed pod, number of nodes on the main stem, number of branches per plant, number of pods per plant, number of seeds per plant and 1000 seed weight. All measurements were performed on a sample of 40 plants per replication.

The data for each type of progeny are processed by analysis of variance for split plot experiment with three factors.

Genetic gain and relative genetic gain by categories of progenies for grain yield per plant and other traits were calculated. Genetic gain of selection is calculated through a procedure by Allard, 1960, with assumed intensity of selection of 10% (x = 1.76), as recommended by Vratarić et al. (2010) and Miladinovic et al. (2011).

B4 � C x D2E x F+ C – coefficient for assumed selection intensity D2E - phenotypic standard deviation F+ – broad sense heritability Genetic gain for studied traits in each progeny was compared across relative genetic gain from selection (R), which is expressed as a percentage of the average value, and calculated as follows: R =

G:5 H – mean value of the studied trait. Results and discussion

Values of studied traits of soybean F3:4 progenies of mothers and FSF1:2 progeny from a cross L6xL30 are shown in Table 1. Significant and very significant differences between the tested progeny of mothers, as well as progenies full sib crosses were found.

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Significant and highly significant differences were found among the mother’s progenies and corresponding progenies of sister lines. There were no significant differences between the mean values for the F3:4 lines and FSF1:2 progeny, both for grain yield per plant, and other examined traits. Table 1. Mean values of studied traits of soybean F3:4 lines from a cross L6xL30 (mothers and FSF1:2

progeny) Trait

Mot

her’

s pr

ogen

ies

Genotype Seed yield/plant (g)

Plant height (cm)

The lowest pod height (cm)

Number of nodes/plant

Number of branches/plant

Pod number/plant

Seed number/plant

1000 seed weight (g)

1 11,08 116,1 26,0 17,42 1,52 31,75 71,75 153,8 2 11,79 107,9 26,1 17,50 1,64 32,30 71,95 164,4 3 10,38 105,8 25,8 16,15 1,21 30,84 67,06 153,6 4 11,08 109,6 25,0 16,28 1,13 32,45 72,04 152,5 5 12,57 111,3 21,3 17,42 2,42 36,82 77,01 154,3 6 11,54 113,3 27,2 16,26 1,58 33,61 73,27 154,1 7 11,29 106,2 26,3 16,08 1,04 26,58 59,27 168,9 8 10,95 115,2 26,7 17,31 1,37 31,41 68,10 160,8 9 10,70 110,3 27,8 16,58 1,35 27,75 60,21 160,7 10 11,01 109,6 24,7 17,16 1,61 30,37 65,62 168,2 11 12,01 106,6 25,2 16,85 1,46 34,05 78,09 155,7 12 11,68 110,7 26,0 17,36 1,64 33,40 72,65 160,7 13 11,32 108,3 26,2 16,35 1,32 30,44 71,43 157,1 14 12,49 117,8 27,7 17,77 1,60 35,65 75,90 165,4 15 12,55 110,3 26,5 17,37 1,48 34,51 75,22 163,8 HI 11,54 110,6 25,9 16,89 1,49 32,03 70,64 159,60

FS

pro

geni

es

16 10,44 104,0 24,1 15,91 1,44 27,60 66,21 155,6

17 11,82 108,1 24,2 17,59 1,47 32,61 72,05 165,2 18 11,65 97,2 24,5 16,75 1,40 32,51 69,33 165,8 19 11,60 117,8 25,6 17,62 1,60 33,81 76,29 155,9 20 12,60 110,6 25,1 17,34 1,37 31,92 71,24 157,0 21 12,79 120,7 25,2 16,13 1,93 35,54 77,22 164,7 22 13,23 113,8 23,0 17,21 1,39 35,67 77,14 158,9 23 13,94 112,2 22,0 16,53 1,47 36,68 78,98 163,5 24 12,76 111,7 26,0 17,07 1,36 32,86 73,08 175,6 25 12,35 104,8 25,0 17,86 1,11 32,35 71,97 173,1 26 10,84 105,3 26,4 16,55 1,53 28,90 64,14 166,3 27 12,04 111,7 25,4 16,84 1,34 34,12 73,52 163,0 28 14,44 101,6 21,8 16,30 1,67 37,74 82,53 172,3 29 11,28 117,7 27,7 16,90 1,79 32,67 69,70 161,4 30 13,71 118,1 25,7 17,55 1,43 36,84 75,25 175,5 HI 12,30 110,1 24,8 16,90 1,48 33,53 73,24 164,92

Kunitz 8,99 105,7 26,1 17,00 1,11 23,65 51,21 175,5

Kador 12,97 112,3 26,0 16,56 1,38 34,71 74,49 160,4

Lsd 0.05 0,770 2,44 1,13 0,498 0,240 1,951 3,882 2,467

Lsd 0.01 0,949 3,22 1,49 0,656 0,316 2,572 5,116 3,252

Genetic gain from selection for progeny developed from the cross between two sister

lines (L6 and L30) is shown in Table 2. Genetic gain for grain yield per plant was the same for both categories of progenies (0.75 g and 0.76 g), also confirmed by relative genetic gain from selection (6.47% and 6.21%). Genetic gain from selection for other studied traits varied more. Genetic gain in the progenies of L6 x L30 for plant height (7.46 cm) and the height of the lowest pod (1.47 cm) was higher than the plant height (2.08 cm) and a height of pod (0.72 cm) in the

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progeny of their mothers. Realized genetic gain in progeny of mothers for the number of nodes (0.80), number of branches (0.52), number of pods per plant (3.66), number of seeds per plant (8.81) and 1000 seed weight (7.94 g), was higher than the one achieved for the number of nodes (0.58), number of branches (0.04), number of pods (1.43) and 1000 seeds weight (3.39 g) in the full sib progenies. By comparing the calculated values of the relative genetic gain from selection, the highest profit was achieved for the number of branches in the progeny of mothers (35.4%), although this does not make a whole branch per plant. The lowest genetic gain was found for the height of the tree progenies of mothers (1.88%). Table 2. Genetic gain from selection for offspring of mothers (L6 F3:4), and the progeny of sister cross (L6

x L30 FSF1:2)

Trait Progeny Gs R (%) Means

Seed yield/plant (g) L6 F3:4 0,75 6,47 11,54 L6 x L30 FSF1:2 0,76 6,21 12,30

Plant height (cm) L6 F3:4 2,08 1,88 110,6 L6 x L30 FSF1:2 7,46 6,78 110,1

The lowest pod height (cm) L6 F3:4 0,72 2,78 25,9 L6 x L30 FSF1:2 1,47 5,94 24,8

Number of nodes/plant L6 F3:4 0,80 4,78 16,89 L6 x L30 FSF1:2 0,58 3,45 19,90

Number of branches/plant L6 F3:4 0,52 35,4 1,49 L6 x L30 FSF1:2 0,04 2,45 1,48

Pod number/plant L6 F3:4 3,66 11,41 32,03 L6 x L30 FSF1:2 1,43 4,26 33,53

Seed number/plant L6 F3:4 8,81 12,78 70,64 L6 x L30 FSF1:2 2,20 3,01 73,24

1000 seeds weight (g) L6 F3:4 7,94 4,98 159,60

L6 x L30 FSF1:2 3,39 2,06 164,92

Sudarić et al. (2001) have obtained a low values for genetic gain for seed yield (from 0.038 to 0.045 t/ha) and accordingly, the relative genetic gain from selection of only 0,98-1.2%. Tested material was comprised of elite lines F4-F6 and the standard varieties. Genetic gain from selection achieved in the progeny tested in this study is consistent with results published by Miladinovic et al. (2011), who tested soybean genotypes of different genetic background and from different sources. The results presented in this paper are within the wide range of values for genetic gain published by previous authors: for grain yield Gs = 17.6-362 kg/ha, R = 0.84 to 11.8%; for the number of pods per plant Gs = 0.4 to 16.3 and R = 1.2 to 37.9%; for the number of seeds per plant Gs = 2.2-40.6 and R = 2.8 to 39.2%; for 1000 seeds weight Gs = 5.9 to 34.8 g and R = 3.7 to 22.2%. Conclusion

There were significant and highly significant differences within all studied traits both among progeny of mothers and between the progeny of sister crosses.

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No significant differences were found between the progeny of mothers and corresponding cross with its sister unit.

The calculated values of the least significant differences were very low, which is expected in the way of applied statistical data processing.

In addition to the 1000 seed weight (L6 x L30) there was no significant difference between the average values of grain yield and other studied traits in progeny of mothers and progeny of sister crosses.

The calculated values of genetic gain from selection were very different for all traits and offspring.

For most of the studied traits greater genetic gain was achieved in progeny of mothers. Acknowledgement

This study is a part of a scientific project Reg. No. TR 31069 and TR31068, supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia References Allard, R. W. (1960). Principles of Plant Breeding. Ed. by John Wiley and Sons., Inc. New York,

1-485. De Bruin, J. L., Pedersen P. (2009). Growth, yield and yield component changes among old and

new soybean cultivars. Agronomy Jour 101(1): 124-130 Eberhart, S.A., (1980). Factors effecting efficiencies of breeding methods. African Soils, 15:

669-680. Falconer, D. S. (1960). Introduction to quantitative genetics. Edinburgh, Oliver & Boyd, Second

edition 1981. Miladinović. J., Burton J. W., Balešević Tubić S., Miladinović D., Đorđević V., Đukić V.

(2011). Soybean breeding: comparison of the efficiency of different selection methodes. Turk J. Agric For 35: 469-480

Padney. J. P., Torrie J. H. (1973). Path coefficient analysis of seed yield components in soybeans (Glycine max L. Merr.). Crop Sci 13: 503-505

Paul,. K., Alam M. S. E., Rahman L., Hassan L., Paul S. K. (2003). Genotype x enviroment interaction in soybean (Glycine max L.). Journal of Biological Sciencies. 3(2): 204-214

Stanković, G. (2002). Genetička varijabilnost i genetička dobit od selekcije kod sintetičke populacije kukuruza (Zea mays L.). Poljoprivredni fakultet Univerziteta u Novom Sadu. Magistarska teza.

Sudarić, A., Vratarić M., Duvnjak T. (2002). Quantitative analysis of yield components and grain yield for soybean cultivars. Poljporivreda. 8(2):11-15

Sudarić, A., Vratarić M., Duvnjak T., Sudar R. (2001). Genetski napredak u kvantitativnim svojstvima uroda i kakvoće zrna OS-linja soje I grupe zriobe, poljoprivreda 7(2):8-15

Vratarić, M., Sudarić A., Duvnjak T., Šunjić K. (2010): Agronomska vrijednost vrlo ranih sorata soje. Sjemenarstvo 27(1-2): 5-17

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INFLUENCE OF BRASSINOSTEROID BASED FERTILIZER ON THE GERMINATION OF TWO MAIZE HYBRIDS

Hadi WAISI1, Bogdan NIKOLIĆ2, Vesna DRAGIČEVIĆ3, Danijela PAVLOVIĆ2, Milorad

VUJIČIĆ4, Sanja ĐUROVIĆ2, Lana ĐUKANOVIĆ2

1 The Jaroslav Černi Institute for the Development of Water Resources, J. Černog 80, Belgrade

2 Institute of plant protection and environment, Teodora Drajzera 9, Belgrade 3 Maize Research Institute “Zemun Polje”, Slobodana Bajića 1 11185, Belgrade 4 Faculty of Biology, University of Belgrade, Studentski trg 16 11000 Belgrade

Abstract

The aim of this study was to reveal the growth potential of two different maize hybrids. Seedlings of ZP434 and ZP704 hybrids were tested and their response to fertilizer 24-Epin-Extra (based on 24-epibrassinolid phytohormone). Seedlings were germinated by the ISTA rules and measurements were conducted after 7 days. Concentration of the fertilizer’s solution was from 5.2x10-6 to 5.2x10-15 M. The growth of the maize genotypes produces various changes in germination percentage and weight of seedlings, which can be of great importance in choosing the right genotypes in relation to their response to phytohormone application. Concentration range between 5,2x 10-10 to 5, 2x10-12 M gave the best potential for growing of hybrid seedlings and this should be used in future.

Key words: Maize hybrids, germination, brassinosteroid Introduction

Brassinosteroids (BRs) are important regulators of plant growth and development. They share structural similarities with animal steroids. Brassinosteroids are phytohormones that have important roles in plant development such as sex determination, internode elongation, and leaf development (Clouse and Sasse, 1998). Research on Arabidopsis and rice revealed a general conservation in the key components of BRs signalling. However, knowledge of a BRs biosynthesis and signalling in maize are still poor. BRs stimulate growth through enhanced cell divisions and cell elongation (Mandava, 1988; Marquardt and Adam, 1991). In several systems they act synergistically with auxins (Yopp et al, 1981; Katsumi, 1985; Kim et al, 1990) and additively with gibberellins (Mandava et al, 1981; Katsumi, 1985). Further observed responses to BRs application include enhancement or retardation of root growth, unrolling of leaves, differentiation of xylem vessels, enhanced ethylene production, membrane hyperpolarization,

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increased ATPase activity, enhanced DNA, RNA and protein synthesis, stimulation of photosynthetic activity, and changes in the balance of other endogenous phytohormones (Arteca et al., 1988)

An important goal of modern agriculture is to minimize production inputs with simultaneous maximizing of yields. Growth inhibitors have been used extensively to study the underlying mechanisms of height regulation in plants (Hola et al, 2000; Richards, 2010). Some physiological active substances, like 2,4-D, when it is applied in low concentrations could facilitate germination and starting growth of root and shoot enhancing vigour and high yielding ability (Dragicevic et al, 2004; Dragicevic et al, 2007). From this point, the aim of experiment was to examine effects of range of concentrations of 24-epibrassinolide on germination and starting stage of growth of two different maize hybrids.

Materials and methods

The influence of different concentrations of 24-epibrassinolide (product „Epin-Extra“, Russia; in concentrations 5.2x10-6 – 5.2x10-15 M) on germination and starting growth stage was examined. Two maize hybrids, ZP 434 (new generation hybrid, drought tolerant) and ZP 704 (older generation hybrid, drought sensitive) were tested.

Seeds (4x50, which weight was previously measured) were germinated in plastic boxes, on filter paper sheets topped at the beginning of experiment with 60 ml of different concentrations of 24-Epin-Extra (“Epin extra”) solution and under greenhouse conditions at 27°C (day) and 21°C (night), with an 12 h light (110-160 µmol photons m-2 s-1)/ 12 h dark regime.

After seven days, germination percentage was evaluated together with fresh weight of root and shoot. Seedling parts (root, shoot and seed rest) were separated, dried at 60°C and then weighted for determination of dry biomass, hydrolysis (Hy) and biosynthesis (Bs), according to the following formula’s (Dragicevic et al., 2007):

Hy (g) = Dry weight of Seed (g) – Dry weight of seed rest (g) [1] Bs (g) = Dry weight of root (g) + Dry weight of shoot (g) [2]

Statistical analysis comprehended determination of standard deviation for germination

test, hydrolysis and biosynthesis; analysis of the variance (ANOVA) and LSD-test (at the 0.05 probability level) were used to analyse variations in fresh substance of root and shoot and regression analysis was used for interdependence of germination percentage and hydrolysis and biosynthesis. Results and discussion

Main difference between examined hybrids was present on germination level, with 86% and 99% for ZP 434 and ZP 704, respectively (Figure 1). ZP 434 also had greater response to application of 24-epibrassinolide, with increased germination percentage by concentrations 5.2x10-11 – 5.2x10-15 M (up to 6.5%, with 5.2x10-15 M), as well as germination decrease by concentrations 5.2x10-7 – 5.2x10-10 M (up to 44.5%, with 5.2 x 10-7 M), compared with control. Germination of ZP 704 slightly varied under the influence of different concentrations of 24-epibrassinolide (±4%), with the highest drop of 13.5%, induced by concentration of 5.2x10-7 M, compared to control.

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Figure 1. Effects of different concentrations of 24-epibrassinolide (“Epin extra” preparation) on

germination percentage of ZP 434 and ZP 704 hybrids. Table 1. Effect of different concentrations of 24-epibrassinolide on fresh weight (g seedling

-1) of the root

and the shoot of ZP 434 and ZP 704 seedlings

Fresh weight of the root (g seedling-1) Fresh weight of the shoot (g seedling-1)

ZP434 ZP704 Aver. ZP434 ZP704 Aver.

Control 0.173cd 0.136de 0.154ab 0.134c 0.102d 0.118ab

5.2 X 10-7 0.046f 0.073e 0.060c 0.029g 0.038f 0.033c

5.2 X 10-8 0.102e 0.102e 0.102bc 0.051ef 0.036g 0.044bc

5.2 X 10-9 0.120de 0.102e 0.111bc 0.062ef 0.049ef 0.056bc

5.2 X 10-10 0.141d 0.126de 0.133b 0.088de 0.048ef 0.068bc

5.2 X 10-11 0.226bc 0.097de 0.162ab 0.122cd 0.038f 0.080b

5.2 X 10-12 0.275a 0.139de 0.207a 0.200a 0.066e 0.133a

5.2 X 10-13 0.193c 0.121de 0.157ab 0.137b 0.058ef 0.097ab

5.2 X 10-14 0.219bc 0.097de 0.158ab 0.166bc 0.059ef 0.112ab

5.2 X 10-15 0.228b 0.113de 0.171ab 0.172b 0.081de 0.127a

Aver. 0.172a 0.111b

0.116a 0.058b

LSD 0.05 Concentr. Genotype C x G Concentr. Genotype C x G

0.054 0.057 0.032 0.047 0.048 0.028 The values followed by same letters are not significantly different at the P=0.05 level.

According to results presented in Table 1, ZP 434 was characterised by significantly

higher root and shoot fresh weight, in average, while 5.2x10-12 M concentration of 24-epibrassinolide expressed the highest influence on average increase in fresh weight of root and shoot and 5.2x10-7 M concentration significantly decreased average fresh weight of root and shoot. This stimulation could be attributed to activation of BRs-induced genes, which are implicated in cell elongation or expansion, of which 5 of the BL-induced genes are either known

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to be early auxin induced or share homology to auxin induced genes (Li and Chory, 1999; Darley et al, 2001). Interaction between genotype and concentration also showed the significant impact on variation of root and shoot fresh weight, with the highest values for root and shoot obtained on ZP 434 by 5.2x10-12 M concentration. The same concentration of 24-epibrassinolide had the highest impact on ZP 704 root, while its shoot had the highest fresh weight in control. Exogenous application of BRs inhibits primary root extension and lateral root formation, with occasional promotions of elongation or adventitious rooting seen with (Closse and Sasse, 1998), what is in accordance with results achieved by application of 5.2x10-7 M concentration of 24-epibrassinolide.

Hydrolysis and biosynthesis, as parameters which point up utilization of seed substance by young plants also had different trend in seedlings of examined hybrids (Figure 2). ZP 704 was characterised by the more intensive average hydrolysis than ZP 434. In both hybrids, decreasing trend of hydrolysis was present with increase in 24-epibrassinolide concentration, what was particularly underlined in seedlings of ZP 434. Intensification of hydrolysis in vigorous seed is present under the influence of some substances applied in low concentrations – hormetics (Dragicevic et al., 2004). There was no particular difference between hybrids in average biosynthesis, with relative lower values realised in ZP 434 under the influence of higher 24-epibrassinolide concentrations (≥5.2x10-10 M) and the highest value obtained by 5.2x10-7 M concentration.

Figure 2. Effects of different concentrations of 24-epibrassinolide on hydrolysis and biosynthesis of ZP 434 and ZP 704 maize seedlings.

When connection between germination, hydrolysis and biosynthesis was considered, the

significant and positive correlation between germination percentage and hydrolysis was present in both examined hybrids (R2 = 0.783 for ZP 434 and R2 = 0.329 for ZP 704). They mainly differ on biosynthesis level, with significant drop of biosynthesis, parallel with germination increase at ZP 434 and slight parallel increase of biosynthesis and germination at ZP 704.

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Figure 3. Correlation between germination percentage and hydrolysis and biosynthesis of two maize

genotypes

Conclusion

Based on obtained data, it could be concluded that two different hybrids react divergently to applied 24-epibrassinolide concentrations. ZP 434 as genotype with lower germination percentage in control reacted positively to low 24-epibrassinolide concentrations (≤5.2x10-12 M) by germination increase, together with increasing trend of root and shoot fresh weight. Such poorer germination of ZP 434 seeds could be linked with more intensive hydrolysis and relative low biosynthesis. Irrespective to lower values of root and shoot fresh weight of ZP 704, germination, hydrolysis and biosynthesis remained stable, under the influence of different 24-epibrassinolide concentrations. This could refer to requirement of 24-epibrassinolide usage in improving of germination ability and starting growth of maize genotypes with problematic germination. Acknowledgements

This study was financially supported by the Serbian Ministry of Education, Science and Technological Development under the projects number TR 31080 and TR 31037. References Arteca R.N., Bachman J.M., Mandava N.B. (1988). Effects of indole-3-acetic acid and

brassinosteroid on ethylene biosyntthesis in etiolated mung bean hypocotyl segments. Journal of Plant Physiolology, 133: 430-435.

Clouse S.D., Sasse J.M. (1998). Brassinosteroids: Essential, Regulators of Plant Growth and Development. Annual Review of Plant Physiology and Plant Molecular Biology, 49: 427–51.

Darley C.P., Forrester A.M., McQueen-Mason S.J. (2001). The molecular basis of plant cell wall extension. Plant Molecular Biology 47: 179–195.

Dragicevic V., Sredojevic S., Djukanovic L., Srebric M., Pavlov M., Vrvic M. (2007). The stimulatory effects of 2,4-D as hormetic on maize seedling’s growth. Maydica 52: 307-310.

Dragicevic V., Sredojevic S., Vrvic M., Djukanovic L., Todorovic M. (2004). The mass and water partitioning as growth factors of maize seedlings influenced by ageing and 2,4-D. Fresenius Environmental Bulletin, 13 (4): 336-340.

R² = 0,783

R² = 0,409

0

0,02

0,04

0,06

0,08

0,1

0,12

30 50 70 90

g s

ee

dli

ng

-1

ZP 434

R² = 0,329

R² = 0,31

0,05

0,06

0,07

0,08

0,09

0,1

85 90 95 100

Hy Bs

ZP 704

Germination (%)

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Hartwig T., Best N., Bishop B., Budka J., Brown E., Weizbauer R., Osborne R., Potluri D., Schulz B. (2011). Interactions with growth media affect the efficacy of a potent brassinosteroid biosynthesis inhibitor in maize. ASPB Midwest Section Annual Meeting. 2011, Purdue University, West Laffayet, IN, USA, Poster No. 43.

Hartwig, T., Chuck G., Fujioka Sh. , Weizbauer R., Chintamanani S., Choe S., Johal G., Schulz B. (2011). Brassinosteroid control of sex determination in maize. Proceedings of the

National Academy of Sciences of the United States of America, 108 (49): 19814-19819. Hola D., Rothova O., Kočova M., Kohout L., Kvasnica., M. (2010). The effect of

brassinosteroids on the morphology, development and yield of field-grown maize. Plant

Growth Regulation, 61: 29-43. Jianming L., Joanne C. (1999). Brassinosteroid actions in plants. Journal of Experimental

Botany, 50 (332): 275–282. Katsumi M. (1985). Interaction of a brassinosteroid with IAA and GA3 in the elongation of

cucumber hypocotyl sections. Plant Cell Physiology, 26: 615-625. Kim S., Abe H., Little C.H.A., Pharis R. (1990). Identification of two brassinosteroids from the

cambial region of Scots pine (Pinus silvestris) by gas chromatograpy-mass spectrometry, after detection using a dwarf rice lamina inclination bioassay. Plant Physiology, 94: 1709-1713.

Mandava N.B. (1988). Plant growth promoting brassinosteroids. Annual Review of Plant

Physiology and Plant Molecular Biolology, 39: 23–52. Mandava N.B., Sasse J.M., Yopp J.H. (1981). Brassinolide, a growth-promoting steroidal

lactone. II. Activity in selected gibberellin and cytokinin bioassays. Physiologia

Plantarum, 53: 453-461. Marquardt V., Adam G. (1991). Recent advances in brassinosteroid research. In: Chemistry of

Plant Protection, Herbicide Resistance—Brassinosteroids, Gibberellins, Plant Growth

Regulators (ed. H. Boerner, DMartin, VSjut, HJ Stan, J Stetter), 7: 103–39. Berlin: Springer-Verlag

Richards, R.A. (2000). Selectable traits to increase crop photosynthesis and yield of grain crops. Journal of Experimental Botany, 51 (GMP Special Issue): 447-458.

Yopp J.H., Mandava N.B., Sasse J.M. (1981). Brassinolide, a growth-promoting steroidal lactone. I. Activity in selected auxin bioassays. Physiologia Plantarum 53: 445-452.

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PROBIOTIC POTENTIAL OF LACTOBACILLI OF DIFFERENT ORGIN

Milica ZIVKOVIC1, Ewelina STEFANOVIC2, Marija MILJKOVIC1, Jovanka LUKIC1, Maja TOLINACKI1, Jelena BEGOVIC1, Natasa GOLIC1, Milan KOJIC1, Ivana STRAHINIC1*

1Laboratory for Molecular Microbiology, Institute of Molecular Genetics and Genetic

Engineering, University of Belgrade, Vojvode Stepe 444/a, P.O. Box 23, 11010 Belgrade, Serbia 2Department of Food Biosciences, Teagasc Food Research Centre, Moorepark, Fermoy, Co.

Cork, Ireland.

*Correspondung author Ivana Strahinic, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444/a, P.O. Box 23, 11010 Belgrade,

Serbia, E-mail address: strahi@imgge.bg.ac.rs

Abstract

The aim of this study was to test health promoting potential of natural isolates of lactobacilli of dairy and human origin. Dairy natural isolates of lactobacilli as well as the lactobacilli isolates from the newborn feces, which showed the ability to survive in the simulated gastrointestinal conditions, were tested for bile salt hydrolase (BSH) activity, the ability to reduce the cholesterol level, adhesion to human enterocyte-like Caco-2 cells and proliferation of gut-associated lymphoid tissue (GALT) cells. The results showed that Lactobacillus brevis BGHI5 and all tested lactobacilli of dairy origin had high potential to reduce the cholesterol level. L. paracasei subsp. paracasei BGGR2-66-88 was highly adhesive to Caco-2 cells. In addition, all selected strains decreased the proliferation of GALT cells suggesting the possible immunomodulatory potential of the isolates. Key words:Lactobacilli, probiotics, cholesterol, Caco-2, GALT Introduction

Lactobacilli are prominent members of the normal microflora of the gastrointestinal tract (GIT) and have been proven to exert positive effects on human health. In vivo and in vitro studies showed that lactobacilli can lower levels of cholesterol. Also, lactobacilli demonstrate significant adhesion to gut mucosa and prominent immunomodulatory ability, either by immunostimulation or immunosuppression, which is strain dependent (Nikolic et al., 2012). For this study we have selected lactobacilli strains isolated from dairy products and breast-fed infant feces to study their

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probiotic properties including cholesterol-assimilating and bile salt hydrolyzing activity, with both activities having cholesterol lowering effects (Liong and Shah, 2005). Additionally, adhesion of selected strains to Caco-2 cells and their influence on proliferation of gut-associated lymphocytes (GALT) were tested. Materials and methods Isolation and identification of strains, media and growth conditions

Strains used in this study are shown in Table 1. The strains were grown in De Man-Rogosa-Sharpe (MRS) medium (Merck GmbH, Darmstadt, Germany) at 30°C. The strain identification was performed by 16S rDNA sequencing (Jovcic et al., 2009) and/or by SDS-PAGE, AFLP and REP-PCR methods.

Table 1. List of strains used in this study.

Strain Origin Relevant characteristics Reference L. brevis BGHI5 newborn feces Bac–, BacS, Prt+, EPS-, Agg– This study L. gasseri BGHI11 newborn feces Bac–, BacS, Prt+, EPS-, Agg– This study L. fermentum BGHI14

newborn feces Bac–, BacS, Prt+, EPS-, Agg– Lukic et al., 2013

L. rhamnosus BGHI22

newborn feces Bac–, BacS, Prt+, EPS+, Agg– This study

L. paracasei subsp. paracasei BGSJ2-8 artisanal cheese BacSJ+, BacSJIm, Prt+, Agg+ Topisirovic et al., 2006 BGSJ2-82 Derivative of the strain BGSJ2-8, BacSJ– Lozo et al., 2007

BGSJ2-83 Derivative of the strain BGSJ2-8, BacSJ–, BacSJIm, Prt+, Agg–

Lozo et al., 2007

BGGR2-66 artisanal cheese Bac+, BacIm, Prt+ Tolinacki et al., 2012 BGGR2-66-88 Derivative of the strain BGGR2-66, Bac– This study

BGHN14 artisanal cheese BacSJ–, BacSJS, Prt+, Agg– Kojic et al., 1991 L. paraplantarum BGCG11 artisanal cheese Exopolysaccharide (EPS) producer Kojic et al., 1992 NB1 Derivative of the strain BGCG11,

EPS-CG11– Nikolic et al., 2012

Bac+ - bacteriocin producer; Bac– - absence of bacteriocin production; BacSJ+ - bacteriocin BacSJ producer; BacSJ– - absence of bacteriocin BacSJ production; BacSJIm – immunity to bacteriocin BacSJ; BacSJS – sensitivity to bacteriocin BacSJ; Prt+ – proteolytic activity; Agg+ – autoaggregation phenotype; Agg– – absence of autoaggregation phenotype, Prt+ – proteolytic activity;

EPS-CG11– absence of EPS-CG11 production.

Assimilation of cholesterol and quantitative BSH Assay

The assay for assimilation of cholesterol was performed as described by Ziarno et al., 2007 with minor modifications. Minimal medium for lactobacilli was prepared as described in Cerning et al., 1994, substituting Tween 80 with 4 mg/mL of cholesterol (Cholesterol-PEG 600, Sigma-Aldrich). Cholesterol level was analyzed using commercial kit (CHOL-PAP, Bioanalytica, Belgrade, Serbia). The bile salt hydrolase (BSH) assay was based on measuring the release of glycine by hydrolysis of sodium glycocholate by Lactobacillus cells (Kumar et al., 2011).

Adhesion of lactobacilli to intestinal cell line

The colonocyte-like cell line Caco-2 (ECACC No. 86010202) was used for adhesion assays. The culturing and maintenance of the cell lines as well as adhesion of lactobacilli were carried out as described previously (Nikolic et al., 2012).

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Proliferation of GALT lymphocytes in the presence of non-viable lactobacilli strains

Overnight lactobacilli cultures were inactivated by UV light (Nikolic et al., 2012). The isolation of lymphocytes from GALT and the quantification of the GALT response were performed as described by Hidalgo-Cantabrana et al., 2014. Proliferation of GALT-lymphocytes was determined by Cell proliferation assay kit (Millipore Corporation, Billerica, MA, USA). Study was approved by the Animal Experimentation Ethical Committee of the University of Belgrade (Serbia). Four female Wistar rats (6-8 weeks old) were used in experiments. Animals were sacrificed using the increase of CO2 concentration.

Statistical analysis

The growth rate, survival in simulated GIT conditions and hydrophobicity are presented as mean ± SD. The means were measured by one-way ANOVA followed by Dunnet’s Test. Results and Discusion

Assimilation of cholesterol

Lactobacilli from cheese showed significantly higher cholesterol assimilation compared to fecal lactobacilli. Among fecal lactobacilli, BGHI5 showed the highest assimilation. However, there were no significant differences among dairy lactobacilli (Fig 1).

Figure 1.Cholesterol assimilation of lactobacilli isolated from newborn feces and cheese. Columns that do

not share the same letter (a,b, c, or d) are significantlly different (p <0.01).

High concentration of cholesterol in human blood stream has been recognized as a risk

factor for the coronary heart disease. Some reports showed that lactobacilli were able to assimilate cholesterol in vitro and in vivo (Hashimoto et al., 1999). Considering the results we have obtained, both lactobacilli from feces and those from cheese could be tested in vivo since they exerted very high assimilation properties.

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Quantitative BSH Assay

Figure 2. BSH activity of lactobacilli isolated from newborn feces and cheese.

Columns with distinct letters are significantly different All tested lactobacilli possess bile salt hydrolase activity. Among lactobacilli from feces,

BGHI5 and BGHI11 showed significantly higher BSH activity than BGHI14 and BGHI22 (Fig 2). Lactobacilli from cheese showed significantly higher BSH activity than fecal lactobacilli. Enzyme BSH has important role in overall cholesterol metabolism. The fecal loss of bile salts may cause an increased requirement for cholesterol as a precursor for new bile-salt synthesis, thereby resulting in a decreased level of cholesterol. Due to formation of toxic secondary bile acids after transformation of deconjugated bile acids by gut microflora, strains with lower BSH activity (BGHI14 and BGHI22) may be beneficial in pathological conditions (i.e. colon cancer) (Tan et al., 2007).

Adhesion of lactobacilli strains to Caco-2 intestinal cell line

Adhesion experiments demonstrated that among fecal lactobacilli BGHI5 showed the highest adhesion while BGHI11 and BGHI22 showed the lowest adhesion (Fig 3). Lactobacilli from cheese showed higher adhesion than lactobacilli from feces. Adhesion to epithelial cells is a prerequisite for antagonistic activity against enteropathogens (Coconnier et al., 1993) or stimulation of the immune system (Schiffrin et al., 1995). Neither of the tested derivatives showed ability to aggregate or produce EPS, which are phenotypes commonly associated with increased adhesion to gut mucosa (Nikolic et al, 2012; Tompa et al., 2010) Thus, additional work is required to find potential molecules responsible for observed differences in adhesion of tested strains.

Figure 3. Adhesion to Caco-2 cells. Columns with asterisks are significantly different (*: p< 0.05, **: p<

0.01) from the control strain BGHN14.

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Proliferation of GALT cells from rats in the presence of lactobacilli

All tested UV-treated lactobacilli significantly lowered GALT derived lymphocytes proliferation (Fig 4). We assumed that UV-inactivated lactobacilli possess immunomodulatory potential regardless their origin or surface characteristics.

Figure 4. Proliferation of GALT cells from rats in the presence of lactobacilli. Columns with asterisks are

statistically different (*: p< 0.05, **: p< 0.01, ***: p< 0.001) from the control (GALT cells without lactobacillus stimuli).

Conclusions Lactobacilli strains tested in this work demonstrated significant cholesterol lowering

effect, mild BSH activity, distinct adhesion ability to Caco-2 cell line and showed immunomodulatory potential. All obtained results support strong indications that selected strains could be considered as potential probiotic strains and should be further studied for treatments of specific disease conditions. Acknowldgements

This work was funded by The Ministry of Education, Science and Technological Development of the Republic of Serbia, grant ON 173019. References Cerning, J., Renard, C.M.G.C., Thibault, J.F., Bouillanne, C., Landon, M., Desmazeaud, M.,

Topisirovic, L. (1994). Carbon source requirements for exopolysaccharide production by Lactobacillus casei CG11 and partial structure analysis of the polymer. Appl Environ Microbiol 60, (3914-3919)

Coconnier, M.H., Bernet, M.F., Kernéis, S., Chauvière,G., Fourniat, J., Servin, A.L. (1993). Inhibition of adhesion of enteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiol Lett, 110, (299-305)

Hashimoto, H., Yamazaki, K., He, F., Kawase, M., Hosoda, M., Hosono, A. (1999). Hypocholesterolemic effects of Lactobacillus casei ssp. casei TMC 0409 strain observed in the rats fed cholesterol contained diets, Anim Sci J 72, (90-97)

Hidalgo-Cantabrana, C., Nikolic M., López, P., Suárez, A., Miljkovic, M., Kojic, M., Margolles, A., Golic, N., Ruas-Madiedo, P. (2014). Exopolysaccharide-producing Bifidobacterium

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