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Comparative analysis of agro-morphology, grain quality and aroma traits of
traditional and Basmati-type genotypes of rice, Oryza sativa L.
SOMNATH ROY1 , 2 , 6 , AMRITA BANERJEE
1 , 3 , B I JOY K. SENAPATI1 , 4 and GURUPADA SARKAR
1 , 5
1Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Nadia, West Bengal 741252, India; 2Present address: NBPGR RegionalStation – Shillong, Umiam, Meghalaya 793 103, India; 3Present address: ICAR Research Complex for NEH Region, Umaim,Meghalaya 793 103, India; 4Present address: Regional Research Station, New Alluvial Zone, BCKV, SC: Chakdaha, Nadia,West Bengal 741222, India; 5Present address: Regional Research Station, New Alluvial Zone, BCKV, Gayeshpur, Nadia,West Bengal, India; 6Corresponding author, E-mail: [email protected]
With 3 figures and 4 tables
Received December 5, 2011|Accepted February 22, 2012Communicated by R. Singh
Abstract
The purposes of this study were to assess the variation in agro-
morphological and grain quality traits among traditional and Basmati-
type aromatic/quality rices and to investigate plausible relationships
between the traits. A set of 12 cultivars, comprising ten traditional and
two Basmati types, were studied. Highest variation was observed for
grains/panicle followed by grain yield/plant. Cluster analysis grouped
all traditional cultivars except �Tulaipanji�, which clustered with
Basmati varieties. Selection for long grain with slender shape will
simultaneously increase amylose content and alkali spreading value or
gelatinization temperature. Aroma score categorized rice varieties as
mild and strongly aromatic, which also was similar to aroma
genotyping with gene-based marker for betaine aldehyde dehydrogenase
2 (BADH2). Sequence analysis of BADH2 revealed that all strongly
aromatic and two mild aromatic rice varieties contain characteristic
8-bp deletion and three SNPs in exon 7 of BADH2 gene. Multiple
alignment of the DNA sequences revealed the addition of AT in
�Gobindabhog� and a T/A SNP in �Gobindabhog� and �Tulsibhog�exon 8.
Key words: rice — aroma — grain quality — correlation —sequence analysis — BADH2
Introduction
Improving the rice grain quality has been a major concern in
rice breeding programme to meet the market demand. Ricequality is based on a combination of subjective and objectivefactors, the relative importance of which depends upon theparticular end-use. The quality components, common to all
users, include grain appearance, milling quality, cooking andprocessing quality and nutritional quality, of which cookingand eating qualities are the most important components for
Asian rice consumers. Cooking and processing qualities thatare important across the users include texture and stickiness.The two most important quality indicators for these charac-
teristics are amylose content (AC) and gelatinization temper-ature. AC is controlled by one major gene with severalmodifiers (McKenzie and Rutger 1983, Kumar and Khush1986, 1987). Gelatinization temperature, measured by alkali
spreading value (ASV), seemed to have complex nature ofinheritance (Heda and Reddy 1986). Genetic variability hasbeen the major driving force used by man to meet not only its
food needs but also to produce high-quality cultivars. Know-ledge on variability and association between the grain yield
components helps breeders in tailoring the hybridization
programme.Fragrance in rice is one of the important grain quality traits
in rice. It is a key factor in determining market price along withgrain shape and size (Fitzgerald et al. 2009). Aromatic rice
genotypes constitute a special group well known for theiraroma (Kumar et al. 1996, Singh et al. 2000a). There do manylocally adapted aromatic and quality rice genotypes (Richharia
et al. 1965, Richharia and Govindaswamy 1966, Siddiq andShobharani 1998, Khush 2000) comprise small-, medium- andlong-grain types with mild-to-strong aroma (Singh et al.
2000a,b). Khush (2000) classified Indian aromatic rice germ-plasm as indicas among which Basmati types have beenidentified as genetically distinct cluster based on both isozyme
and microsatellite data (Glaszmann 1987, Aggarwal et al.2002, Nagaraju et al. 2002, Garris et al. 2003, Jain et al. 2004).The aromatic rice cultivars are generally poor yielders becauseof their disease susceptibility and limited adaptation outside
their original geographical distribution. Aromatic rice acces-sions have been indentified within at least three geneticsubpopulation of rice, including Group V (i.e. �Basmati� and�Sadri� varieties), indica (i.e. �Jasmine� varieties) and tropicaljaponica.Many studies reported the genetic control of aroma in rice
(reviewed by Sakthivel et al. 2009a). The inheritance offragrance was reported to be controlled by one or two orthree dominant or recessive genes or by QTLs that found to be
cross-specific/genotype-specific. Reports indicated that at leastsix of twelve rice chromosomes (4, 5, 8, 9, 11 and 12) wereimplicated to harbour the gene/genes (Sun et al. 2008). Thishas further complicated the genetic basis of fragrance in rice.
Availability of high-density molecular marker maps andgenome sequences in rice has allowed mapping, fine mappingand positional cloning of gene for fragrance. An eight-base
pair deletion and three SNP in exon 7 of the gene encodingbetaine aldehyde dehydrogenase 2 (BADH2) on chromosome8 of Oryza sativa have been identified as the primary cause of
fragrance in Jasmine- and Basmati-like rice (Bradbury et al.2005a). Functional BADH2 is either responsible for metabo-lizing 2-acetyl-1-pyrroline (2-AP), which has been identified asthe main compound among over 100 volatile compounds
reported to be responsible for characteristic aroma in the aerialparts of Basmati- and Jasmine-like rice lines (Buttery et al.
Plant Breeding doi:10.1111/j.1439-0523.2012.01967.x� 2012 Blackwell Verlag GmbH
wileyonlinelibrary.com
1983, Paule and Powers 1989), or for activating the fragrancepathway that competes for substrate which would otherwise beused in the production of 2-AP and so a non-functionalBADH2 enzyme results in increased flux of substrate down the
pathway of 2-AP production. In contrast, non-fragrant ricevarieties possess a full functional copy of BADH2 gene whilefragrant varieties possess BADH2 containing 8-bp deletion
and three SNPs, resulting in a frame-shift that generates apremature stop codon which disables BADH2 enzyme. RNAi-mediated down-regulation of BADH2 gene reported to
enhance the accumulation of 2-AP in non-aromatic rice cv.�Nipponbare� (Niu et al. 2008). Recently, Kovach et al. (2009)reported that despite the multiple origin of fragrance trait,BADH2 is the predominant allele in virtually all fragrant rice
varieties.Identification of the gene for fragrance and development of
stable marker system for aroma genotyping have created
worldwide interest to look for allelic variants at this locus inthe rice gene pool. In addition to 8-bp deletion in exon 7,several variations including a 7-bp insertion in exon 8
(Amarawathi et al. 2008), a 7-bp deletion in exon 2 (Shi et al.2008), absence of miniature interspersed transposable element(MITE) in promoter (Bourgis et al. 2008), two new SNPs in
the central section of intron 8 (Sun et al. 2008), a TT deletionin intron 2 and a repeated (AT)n insert in intron 4 (Chen et al.2008) of BADH2 were reported in various fragrant varieties.Although Bradbury et al. (2005a) have identified the gene
and Chen et al. (2008) and Bradbury et al. (2008) elucidatedthe role of BADH2 in 2-AP biosynthesis, the molecular basisof fragrance remained unsolved. The presence of rice varieties
exhibiting elevated levels of 2-AP, but lacking any knownfunctional allele of BADH2, raised the possibility of existenceof additional fragrance-causing alleles of BADH2. Further, the
involvement of several other aromatic compounds (Widjajaet al. 1996) indicates that many other genes might play a rolein effecting unique or modified fragrance in rice.
In this study, 12 aromatic/quality rice cultivars includingtraditional rice and improved Basmati cultivars have beenstudied for agro-morphological and grain quality traits tounderstand the association between grain yield and its com-
ponents. In addition, a search has been made about thevariation in the region spanning the 8-bp mutation in exon 7through sequence analysis of part of BADH2 gene in the tested
rice cultivars.
Materials and Methods
This study was conducted at the Regional Research Station, New
Alluvial Zone, Bidhan Chandra Krishi Viswavidyalaya, Sub-Centre:
Chakdah, Nadia, West Bengal (23�04¢N latitude, 88�31¢E longitude,
6 m altitude). A set of 12 rice cultivars were taken for this study from
the germplasm evaluation trial in 2006 and 2007. These rices
represented 10 traditional aromatic/quality rice and two Basmati
varieties (Table 1). Thirty-day-old seedlings were transplanted in
randomized block design with two replications. Each entry consisted
of five rows of 6.0 m length with a plant spacing of 0.20 m in either
direction. The crop was rainfed, and other normal agronomic practices
were followed during crop growth period. The soil is a clay loam with
pH 7.7. The field was fertilized with 40 kg N/ha in three increments,
20 kg P2O5/ha and 20 kg K2O/ha applied by hand broadcasting. One-
third of N and full amount of phosphorus and potassium were applied
during final land preparation. The second and third increments of N
were applied at the tillering stage and prior to panicle initiation,
respectively.
The days required from sowing to 50% flowering (DF) and maturity
(DM) were recorded on plot basis. Panicles/plant (PP), harvest index
(HI) and grain yield/plant (GY) were measured as the average of
randomly selected five plants. Panicle length (PL), panicle weight (PW)
and grains/panicle (GP) were determined based on five individual
measurements of main stems in each entry. After harvesting, the grains
were dried in open sun until moisture content reached 140 g/kg and
used for recording grain quality traits. Grain length (GL), grain
breadth (GB) and grain length-to-breadth ratio (GLB) for brown rice
were determined from randomly sampled (twice) 10 grains. About
1000-grain weight (GW) was determined twice for each entry. Rice
grains (100 g) were de-husked and milled in Satake TM-05 grain
testing mill. The method of Little et al. (1958) was used to find out
ASV. For AC determination, milled rice grains were ground in mortar
and pestle and screened through 60 mesh sieve. AC was determined as
described in Juliano et al. (1981). Aroma was detected by sniffing of by
a panel of ten experts and was scored as mild, medium and strong
following KOH-based method (Nagaraju et al. 1991).
Data analysis The 2-year data on various traits were subjected to
analysis of variance, and the mean values, standard deviation (SD),
standard error (SE), minimum and maximum values (summary
statistics) were determined. Ward�s hierarchical clustering was used
to assess the phenotypic diversity in rice cultivars. Clustering was
performed using SPSS statistical software (version 16 for Windows;
SPSS Inc., Chicago, IL, USA). The Z values were calculated from
mean values and used for cluster analysis. Pearson�s correlation
coefficients (r) for the agro-morphological and grain quality traits were
calculated using SPSS software.
Genotyping for aroma Genomic DNA was extracted from leaf samples
using a SDS-based micro-Prep method, and the polymerase chain
reactions (PCR) were performed in a thermal cycler (Eppendorf,
Hamburg, Germany). The genotyping for aroma in twelve rice lines
was carried out following Bradbury et al. (2005b) using external sense
primer (ESP), internal fragrant antisense primer (IFAP), internal non-
fragrant sense primer (INSP) and external antisense primer (EAP).
PCR products were analysed by electrophoresis in 1.0% agarose gel
stained by ethidium bromide (0.5 ug/ml).
Sequencing of BADH2 gene fragment We amplified �580-bp fragment
spanning the 8-bp mutation described by Bradbury et al. (2005a) from
12 rice cultivars. Among the four primers reported by Bradbury et al.
(2005b), ESP and EAP were used. The PCR was performed using 1 ll(�20 ng) of genomic DNA, 1 ll of each primer (10 lM), 2.5 ll of 10·buffer containing 15 mM MgCl2, 1 ll of 2.5 mM dNTPs, 1 U of Taq
DNA polymerase in a total volume of 25 ll by adding dd H2O.
Cycling conditions were an initial denaturation (94�C) for 5 min, 35
cycles of 94�C for 30 s, 55�C for 30 s and 72�C for 1 min, followed by
final extension of 72�C for 7 min.
Table 1: List of the rice varieties used for analysis
Sampleno.
Name ofvariety Type
Aromatest
PCRanalysis
1 Basmati 385 Traditional Basmati Strong Fragrant2 Gobindabhog Aromatic/quality Strong Fragrant3 Kalikhasa Aromatic/quality Strong Fragrant4 Khaskani Aromatic/quality Strong Fragrant5 Madhuri Aromatic/quality Mild Fragrant6 Radhunipagal Aromatic/quality Strong Fragrant7 Taroari Basmati Premium Basmati Strong Fragrant8 Tulaipanji Aromatic/quality Mild Fragrant9 Tulsibhog Aromatic/quality Mild Fragrant10 Dudheswar Quality Mild Non-fragrant11 Masino Selection Mild Non-fragrant12 Shantibhog Quality Nil Non-fragrant
PCR, polymerase chain reaction.
2 S . Roy , A . Baner j e e , B . K . Senapat i e t a l .
PCR amplifications of expected fragments were confirmed by
electrophoresis in ethidium bromide–stained (0.5 lg/ml) 1.0% agarose
gels. The PCR products of the 12 rice varieties were commercially
sequenced from Bangalore Genei Ltd., Bangalore, India. The PCR
fragments were sequenced from both 5¢ and 3¢ ends by primer walking.
The final sequence for each entry was deduced from forward and
reverse sequences. The identity and homology of the sequences were
first checked using the BLAST N programme from the NCBI website.
The annotated sequences were registered under GenBank accession
numbers JN599151–JN599162. The BADH2 nucleotide sequence data
of twelve rice varieties were subjected to pair-wise multiple alignments
using the CLUSTAL W algorithm in MegAlignTM programme of
Lasergene at its default settings.
Results
The mean values for all the agro-morphological and grain
quality traits of tested rice cultivars are depicted in Tables 2and 3, respectively. There was significant variation amongthem for all traits except GB. DF varied from 107 days(�Dudheswar�) to 115 days (�Tulaipanji�). The average number
of panicles/plant ranged from 6.6 (�Basmati 385�) to 14.0(�Kalikhasa�). Maximum PL was recorded in �Tulsibhog�(30.0 cm) and minimum in �Gobindabhog� (19.0 cm). The
average number of filled grains/panicle showed wide variationand ranged from 50 (�Taroari Basmati�) to 210 (�Randhunipa-gal�). GL ranged from 4.2 (�Gobindabhog�) to 7.3 mm
(�Basmati 385�), GB from 1.7 (�Taroari Basmati�) to 2.1 mm(�Khaskani�) and GLB from 2.1 (�Khaskani�) to 4.12 (�TaroariBasmati�).
GW ranged from 15.3 (�Randhunipagal�) to 19.5 g (�Basmati385�) and HI from 0.30 (�Gobindabhog�) to 0.38 (�Kalikhasa�).GY was highest in �Tulsibhog� (29.45 g) and lowest in �TaroariBasmati� (7.08 g). ASV ranged from 2.35 to 3.35 with a mean
value of 3.1. All the rice varieties had low-to-intermediate ACand it ranged from 15.46 (�Randhunipagal�) to 20.2 (�Madhuri�).The aroma score categorized the genotypes as mild (�Madhuri�,�Tulaipanji�, �Tulsibhog�, �Dudheswar� and �Masino�) andstrongly aromatic (�Basmati 385�, �Gobindabhog�, �Kalikhasa�,�Khaskani�, �Randhunipagal� and �Taroari Basmati�). �Shantibhog�was recorded as non-aromatic based on aroma test (Table 1).Among the characteristics studied in this experiment, highest
variation was recorded for GP (%CV = 38.5) and GY(%CV = 33.6). Rest of the traits showed low-to-intermediatevariation.The cluster analysis basedon agro-morphological andgrain quality traits placed the genotypes into two major clusters
(Fig. 1). Cluster one included nine traditional cultivars, whichfurther formed three subclusters. The second major clustergrouped two basmati lines (�Basmati 385� and �Taroari Basmati�)and one traditional aromatic cultivar �Tulaipanji�. These geno-types recorded higher values for GL, GLB, GW and AC.The Pearson�s correlation coefficients among the 15 traits
measured in this study are shown in Table 4. GY had significantpositive association with PP, PW, GP and GB. GL, GLB andAC had significant negative association with GY. PW and HIrecorded significant negative correlation with DF. The GW had
significant negative correlation with DM and HI. PL showedsignificant negative correlation with PP and positive associationwith GB. GP was significantly correlated in positive direction
with PW andGB, and in negative direction with GL, GLB, GWand AC. GL recorded negative correlation with GB and GY.The AC exhibited significant positive association with GLB,
GW and ASV and negative correlation with GP. ASV, anindicator of gelatinization temperature, showed significantlypositive association with GW and AC.
The PCR analysis of twelve rice genotypes with four BADH2gene-based primers (Bradbury et al. 2005b) revealed amplifi-cation of 577/585-bp fragments that serves as positive controlin all rice lines. In addition, nine varieties amplified a 257-bp
fragment resulting from primer pair ESP/IFAP and threevarieties (�Dudheswar�, �Masino� and �Shantibhog�) revealed thepresence of a 355-bp fragment specific to non-aromatic rice
varieties (Fig. 2). The results of aroma test by sensory methodwere also at par with the PCR assay. Though, the rice varietiespossessed mild aroma they showed variable results in PCR
assay (Table 1).We sequenced the �580-bp PCR fragment amplified by ESP
and EAP primer pair from 12 rice cultivars for more precise
genotyping of the BADH2 alleles. This region of BADH2alleles contains three exons (exon 6–8) and three introns (6–8).The pair-wise alignment of the sequences revealed the presenceof 8-bp deletion and three SNPs in exon 7 (Fig. 3). These
characteristics were not observed in three non-aromatic rices
Table 2: Mean values of eight agro-morphological traits for 12 rice varieties
Variety DF DM PP PL PW GP HI GY
Basmati 385 108 138 7.4 27.0 2.18 135 0.33 15.90Gobindabhog 113.5 145.5 11.1 20.1 1.97 170.5 0.31 23.49Kalikhasa 108 138.5 13.0 24.0 2.10 78.5 0.37 17.38Khaskani 107.5 139 11.0 25.0 2.54 190 0.36 26.01Madhuri 111 139 12.0 22.5 1.93 136.5 0.32 24.83Randhunipagal 110 143 8.5 27.0 1.95 204 0.32 24.42Taroari Basmati 112 142 8.0 25.0 2.15 54.5 0.34 8.37Tulaipanji 114 143 9.5 26.0 1.11 66.5 0.34 10.82Tulsibhog 107.5 139.5 10.7 29.0 2.37 185 0.34 29.00Dudheswar 107 140.5 10.5 24.5 2.70 173 0.35 25.58Masino 108 137 9.2 23.0 1.68 90 0.34 14.06Shantibhog 111.5 144.0 11.0 21.5 2.16 120.5 0.34 21.11Mean 109.9 140.8 10.1 24.6 2.07 133.7 0.34 20.08SD 2.94 2.97 1.91 2.64 0.40 51.27 0.02 6.74SE 0.60 0.61 0.39 0.54 0.08 10.47 0.00 1.37Minimum 106.00 136.00 6.60 19.00 1.01 50.00 0.30 7.08Maximum 115.00 146.00 14.00 30.00 2.70 210.00 0.38 29.45P-value 0.048 0.018 0.038 0.001 0.000 0.000 0.001 0.000
DF, days to 50% flowering; DM, days to maturity; PP, panicles/plant; PL, panicle length; PW, panicle weight (g); GP, grains/panicle; HI, harvestindex; GY, grain yield/plant (g); SE, standard error.
Comparative analysis of agro-morphology, grain quality and aroma traits 3
as per the PCR assay. In addition to 8-bp deletion in exon 7,the �Gobindabhog� line had a T/A SNP and addition of AT in
intron 8. Similar T/A SNP was also observed in �Tulsibhog�.Among the three non-scented lines, �Dudheswar� and �Shan-tibhog� revealed a deletion of T in intron 6 while �Masino� hada G/T SNP in the same intron (Fig. 3).
Discussion
Improvement of grain quality is one of the most importantobjectives in rice breeding. Moreover, aromatic rices are
usually poor yielder. Aromatic rice varieties in general are tallstatured, have fewer number of panicles, lower grain yields andsusceptible to lodging. Morphological traits are useful forpreliminary evaluation and could be used as general approach
for assessing genetic diversity among morphologically distin-guishable aromatic rice cultivars (Hien et al. 2007). Glaszmann
Table 3: Mean values of seven grain quality traits for 12 rice varieties
Variety GL GB GLB GW ASV AC
Basmati 385 7.15 1.90 3.8 19.2 3.2 19.2Gobindabhog 4.35 1.90 2.3 15.7 2.8 18.3Kalikhasa 6.05 1.90 3.2 17.6 3.1 19.1Khaskani 4.60 2.05 2.2 18.1 3.3 19.7Madhuri 6.15 1.85 3.3 18.2 3.2 19.9Randhunipagal 4.85 2.00 2.4 15.7 2.5 15.8Taroari Basmati 6.80 1.75 3.9 18.7 3.2 19.8Tulaipanji 6.00 1.90 3.2 17.4 3.3 19.5Tulsibhog 5.50 2.05 2.7 17.3 3.2 16.9Dudheswar 6.10 1.85 3.3 17.7 3.2 17.0Masino 5.75 1.90 3.0 18.0 2.5 17.7Shantibhog 5.10 1.90 2.6 17.8 3.2 17.7Mean 5.70 1.91 3.05 17.62 3.14 18.43SD 0.85 0.12 0.56 1.07 0.30 1.36SE 0.17 0.02 0.11 0.22 0.06 0.28Minimum 4.20 1.70 2.10 15.30 2.35 15.46Maximum 7.30 2.10 4.12 19.50 3.35 20.20P-value 0.000 0.371 0.000 0.000 0.000 0.000
GL, grain length (mm); GB, grain breadth (mm); GLB, grain length/breadth; GW, 1000-grain weight (g); ASV, alkali spreading value; AC,amylose content; PP, panicles/plant; SE, standard error.
Fig. 1: Grouping of the twelve rice varieties on the basis ofstandardized squared Euclidean distance applied towards hierarchicalanalysis
Table 4: Pearson�s correlation between agro-morphological and grain quality traits
Traits
DF DM PP PL PW GP GL GB GLB GW HI ASV AC
DF –DM 0.779** –PP )0.081 )0.081 –PL )0.402 )0.289 )0.444* –PW )0.582** )0.157 0.177 0.102 –GP )0.348 0.071 0.099 0.182 0.539** –GL )0.161 )0.387 )0.387 0.321 )0.070 )0.580** –GB )0.392 )0.183 0.163 0.427* 0.177 0.674** )0.605* –GLB )0.032 )0.273 )0.374 0.172 )0.090 )0.650** 0.979** )0.750** –GW )0.287 )0.566** )0.211 0.130 0.186 )0.474* 0.752** )0.384 0.728** –HI )0.488* )0.457* 0.381 0.128 0.269 )0.339 0.222 )0.011 0.193 0.386 –ASV 0.020 )0.009 0.260 0.192 0.308 )0.109 0.302 )0.071 0.276 0.496* 0.393 –AC 0.300 )0.165 0.114 )0.225 )0.196 )0.548** 0.386 )0.403 0.430* 0.600** 0.178 0.519* –GY )0.379 )0.028 0.480* 0.036 0.578** 0.897** )0.575** 0.626** )0.639** )0.416 )0.136 0.059 )0.470*
AC, amylose content; ASV, alkali spreading value; DF, days to 50% flowering; DM, days to maturity; GB, grain breadth; GL, grain length;GLB, grain length-to-breadth ratio; GP, grains/panicle; GW, 1000-grain weight; GY, grain yield/plant; HI, harvest index; PL, panicle length; PP,panicles/plant; PW, panicle weight.*P = 0.05; **P = 0.01.
4 S . Roy , A . Baner j e e , B . K . Senapat i e t a l .
(1987), based on isozyme diversity, first reported that aromatic
rice varieties fall into separate group in comparison with indicaand japonica rice. The non-functional BADH2 interferes inpollen tube development and may be regarded as one of thereasons for low grain yield in aromatic rice varieties (Bradbury
et al. 2008). The clustering of the lines enables the selection ofparents based on wider intercluster distance (Mishra et al.2003, Chaturvedi and Mourya 2005). In addition, for improv-
ing the yield potential of aromatic rice varieties, the correla-tions between the grain yield and its components need to bewell understood.
The knowledge of the correlation among agro-morpholog-ical and grain quality traits is vital for choosing most efficientselection criteria. The correlation analysis indicated that
selection for longer grains would result in a negative responseto GB and would increase GLB. Koutroubas et al. (2004) alsoreported similar correlations. The relationship between GPand grain size/shape indicated that genes controlling grain size
also influence GP as suggested earlier by Chandraratna (1964).The negative correlations between GL and GB may resultfrom the linkage of length and width genes or pleiotropy
(McKenzie and Rutger 1983). Similar association between GL,GB, GLB and GW was recorded earlier in 28 aromatic/qualityrice cultivars by Roy et al. (2009). In this present study, AC
was positively correlated with GLB. Earlier, Koutroubas et al.(2004) also reported a similar correlation between AC andGLB, but a negative association between AC and GW. Wefound that AC had a positive correlation with GW. ASV, an
indicator of gelatinization temperature, showed significantlypositive association with GW and AC. The complex relation-ships between these traits indicated a breeder could expect
some concurrent increase in AC and ASV or gelatinizationtemperature when he selects long grains and slender shape.
All the aromatic rice included in this study had characteristic
8-bp deletion in BADH2. With an exception to the 8-bpdeletion reported as the genetic cause for aroma, Sakthivelet al. (2009b) did not found this deletion in some indigenous
aromatic rice genotypes of India. Similar exceptions were alsoreported in some fragrant varieties (Kuo et al. 2005, Navarroet al. 2007, Fitzgerald et al. 2008, Shi et al. 2008). Thesestudies indicated the existence of allelic/genetic diversity for
fragrance in aromatic rice gene pool. In our study, we foundthat four (�Dudheswar�, �Masino�, �Tulaipanji� and �Tulsibhog�)among the twelve rice cultivars had mild aroma level, and
according to PCR analysis along with sequence analysis, only
Fig. 3: Multiple alignment of the BADH2 gene sequences showing8-bp deletion and three SNPs in exon 7 of the aromatic rice varieties.Only variable positions are shown
Fig. 2: Agarose gel showing polymerase chain reaction amplificationof BADH2 using four gene-specific primers. Lane 1–3, aromatic ricevarieties viz. �Taroari Basmati�, �Gobindabhog� and �Tualipanji�; Lane4, a negative control (water); Lane 5–7, non-aromatic rice varieties viz.�Dudheswar�, �Masino� and �Shantibhog� M, molecular weight marker
Comparative analysis of agro-morphology, grain quality and aroma traits 5
two cultivars, that is �Tulaipanji� and �Tulsibhog�, were aromatic(Table 1). As BADH2 seems to explain the accumulation of2-AP in most (but not all) aromatic rice varieties, theinvolvement of other gene(s) in fragrance development and
their interaction with the environment could not be taken intoconsideration. Further characterization of other gene(s) infragrance development will lead to a complete understanding
of the variation in aroma level.The results of our study suggest that selection for grain
quality using conventional breeding methods could be opti-
mized by understanding the correlations among the yield andgrain quality components. The best strategy for aromatic/quality rice breeding would be to improve number of grainsper panicle. The primers reported by Bradbury et al. (2005b)
could be used effectively for aroma genotyping of bothBasmati and traditional aromatic/quality rice varieties. TheBADH2 mutation in exon 7 is the major cause of aroma in rice,
and this region is highly conserved among the rice genotypes,although the involvement of other genes also to be consideredfor the variation in the level of aroma in rice varieties.
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