INTRODUCTION

Truffles are ectomycorrhizal fungi of eco-nomic value due to their organoleptic proper-ties. The mycelium of mycorrhiza and sporocarphave cells containing two nuclei of uncertain ori-gin (GRENTE et al. 1972; PACIONI and COMANDI-NI 1999). The ascocarp formation in truffles is aconsequence of the activation of the sexual phaseof the biological cycle; it has been hypothesizedthe formation of a dikaryotic secundary myceliumand the karyogamy in the ascal cell followed bymeiosis with ascospores formation (RAGNELLI etal. 1992; PACIONI et al. 1995; LANFRANCO et al.1995). Some doubts have been arised from theTuber cycle by considering that a series of abnor-malities have been pointed out in respect to theAscomycetes (GREIS 1939; MARCHISIO 1964;PARGUEY-LEDUC et al. 1987) such as the produc-tion of a variable number of spores in the asciwith a constant total volume and the fixation ofpopulations in homozygosis, a phenomenonexplainable but not immediately compatible with

an hypothetical somatogamy. Therefore Tuberreproduction system might be different from thatusually accepted for a dikaryotic mycelium pro-duced via heterothallic somatogamy (GRENTE etal. 1972). The absence of heterozigosity supportsthe hypothesis that the two nuclei of dikaryotichyphae of Tuber are the product of a self-fertil-ization process (PACIONI et al. 1993; URBANELLI etal. 1998).

In order to obtain a better information in theTuber taxonomy and understanding about thereproductive cycle, we have studied the kary-otypes of T. melanosporum, T. magnatum, T.borchii. Chromosome number is considered tobe the most accurate and direct measure ofploidy, but counting chromosomes of fungi is dif-ficult because of their small size (MCCLINTOK

1945; SAMSONE et al. 1975; SHAW 1983; BARRY

1996). The use of image analysis in karyotyping ofplant species allows the realization of detailedkaryotype also in species with difficult chromo-somes (FUKUI and IIJIMA 1991; FUKUI 1992;FUKUI et al. 1998; VENORA et al. 1991, 1999).Electrophoretic karyotypes of several Asco-mycetes are also known (ORBACH et al. 1988;BORBYE et al. 1992; NAGY and HORNOK 1994)

CARYOLOGIA Vol. 55, no. 4: 307-313, 2002

The karyotypes of three Tuber species(Pezizales, Ascomycota)A. POMA1, *, G. VENORA2, M. MIRANDA1 and G. PACIONI3

1 Dipartimento di Biologia di Base ed Applicata, Università dell’Aquila, Via Vetoio, Coppito I-67100 L’Aquila, Italy.2 Stazione Sperimentale di Granicoltura per la Sicilia, Caltagirone, Catania, Italy.3 Dipartimento di Scienze Ambientali, Università dell’Aquila, 67100 L’Aquila, Italy.

Abstract - The present work investigates the karyotypes of Tuber borchii Vittad.,Tuber magnatum Pico and Tuber melanosporum Vittad. The chromosome diploidnumbers were 8 for the three species examined as determined in metaphase con-figurations from hyphae nuclei by the squash method and aceto-orcein staining.We investigated the three species by means of an image analysis system to obtainmeasurements of the chromosomes. Range of chromosome length was from 1.26µm the longest in T. melanosporum, to 0.65 µm, the shortest in T. magnatum.

Key Words: karyotype, Tuber borchi, T. magnatum, T. melanosporum.

* Corresponding author: fax ++39 862 433273; e-mail:poma@univaq.it.

and pulsed-field gel electrophoresis allowed theanalysis of genomes of a number of fungi whereclassical genetic analysis is not possible because ofthe lack of a sexual stage and cytological studiesare difficult because of the small size of fungalchromosomes (MILLS and MCCLUSKEY 1990;ORBACH et al. 1996; TAGA et al. 1998). In order todiscern differences among species several truf-fles have been characterized molecularly throughthe analysis of DNA, rRNA and other PCR-basedtechniques (LANFRANCO et al. 1993; LAZZARI et al.1995; O’DONNEL et al. 1997; BERTINI et al. 1998);by a cytogenetical approach only T. aestivumchromosome number has been investigated(POMA et al. 1998).

In the present work T. melanosporum, T. mag-natum and T. borchii chromosomes were countedin hyphal metaphases for the first time by aceto-orcein staining. We also investigated the threespecies by means of an image analysis system,that allows to obtain measurements of the chro-mosomes (VENORA et al. 1991, 1995).

MATERIALS AND METHODS

SpecimensTuber magnatum, T. borchii and T. melanospo-

rum specimens were collected close to L’Aquila orChieti (Abruzzo, Italy) and used for study soonafter collection.

T. borchii mycelial strain ATCC 96540 wasgrown in modified Melin-Norkrans nutrient solu-tion (MMN) (MARX 1969). Mycelia were culturedin 50 ml tubes each containing 35 ml of mediuminoculated with fungus cultured on potato dextroseagar (PDA; Sigma, Milan, Italy) plugs and kept at24°C in the dark. Growth was evaluated accordingto SALTARELLI et al. (1998), by estimation of totalprotein content.

Cytological karyotypingThin slices of the truffles (1-2 mm) were treated

with 0.01% w/v colchicine overnight, fixed inethanol/glacial acetic acid (3:1 v/v, overnight),

hydrolyzed in 1 M HCl for 30 min at 25°c andsquashed in 45% (v/v) acetic acid and orcein solu-tion. The cover slices were removed by dipping inliquid nitrogen and samples dehydrated in gradedalcohol series, dipped in xylene; finally the slideswere mounted in Canadian balsam. T. borchiimycelium samples were fixed in 0.1 M phosphate-buffered 2.5% gluraraldehyde (pH 6.8) for 1 h atroom temperature, washed, stained with orcein andobserved with a Leitz Orthopan light microscope.

T. borchii mycelium samples for fluorescencelight microscope observations were fixed in Carnoyfor 60 min at room temperature, washed, dehy-drated in the ethanol series, stained with propidiumiodide (PI) (Sigma) 1µg/ml (15 min, in the dark atroom temperature). Slides were mounted with Vec-tashieldTM (Vector Laboratories, Burlingame, Cali-fornia) to prevent photobleaching and were storedin the dark at 4° C before observation with a Leitzfluorescence light microscope (TAGA et al. 1994).

Relevant images were digitally elaborated with aspecific macro (a succession of image analysis pro-cedures) developed on an image analysis systemKS400 software (Zeiss), that allows to measure thesurface (area) and length (max diameter) of eachchromosome in the three species of Tuber. The datawere submitted to Anova for statistical analysis andthe mean were grouped with the Cluster analysis ofSCOTT and KNOTT (1974).

RESULTS

The orcein-staining procedure was sufficientto make the chromosomes of hyphae visible.A treatment with asci wall lytic enzymes prior tosquashing and staining was found to improve theresults. Chromosomes and chromatin werestrongly stained showing very little or no back-ground.

Highly condensed chromosomes were chosenat metaphase stage. An average number of eightchromosomes (see Table 1) could be observed incells at metaphase during mitoses of T. borchii(Fig. 1 a), Tuber magnatum (Fig. 1 b) and

308 POMA, VENORA, MIRANDA and PACIONI

Table 1 – Hyphal chromosome number of T. borchii, T. magnatum, T. melanosporum.

Chromosome No. ofTaxon Sites complement, individuals Scored

2n/hyphal cytogenetically metaphasesmetaphase studied

T. borchii San Marco, L’Aquila, Italy 8 5 48T. magnatum Quadri, Chieti, Italy 8 8 45

L’Aquila, Italy 8 8 45T. melanosporum L’Aquila, Italy 8 6 50

T. melanosporum (Fig. 1 c) hyphal nuclei. Wepropose a new karyotype for species based onmeasurements of chromosome surface and lengthas achieved by image analysis, for each chromo-some, not pairs. The morphometric data areshowed in table 2 and idiogrammatic represen-tation of each species in Fig. 2 (differences amongstudied species are displayed).

In T. borchii mycelium at the in vitro level(T. borchii mycelial strain ATCC 96540, Fig. 3 a,b) the karyological observations by orcein andpropidium iodide have shown binucleatedhyphae (black arrows) prior to karyogamy.When karyogamy finally occurred the resultingnucleus was diploid (Fig. 1 a,b, c). The nucleimedium diameter was 1-1.2 µm, comparable toT. melanosporum hyphal nuclei (CERUTI et al.1964; FASOLO-BONFANTE and BRUNEL 1972).

DISCUSSION

The numbers of hyphal chromosomes, strong-ly suggest a chromosome number of 8 (2n) forT.borchii, T. magnatum and T.melanosporum; thesevalues are in the range of those of severalAscomycetes (BARRY 1996) and previouslyobserved for T. aestivum (10 ± 0.4, POMA et al.1998). The T. borchii mean bp (base-pairs) contentcalculated from the mean metaphase chromoso-mal size is equivalent to about 6-7 Mbp/chromo-

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Fig. 1 – Light microscopy of aceto-orcein stained T. borchii(a), T. magnatum (b) and T. melanosporum (c) sporocarphyphal chromosomes (arrowed and highly condensed).Eight chromosomes are usually counted at metaphases as inthis figures. Scale bar 2 µm.

aa

bb

cc

Table 2 – Chromosome morphometric data of three Tuberspecies.

Chrom. Suface ChromosomeNo. Area (µm2) Length (µm)

Tuber magnatum Pico1 2.61 ± 0.38 a 1.04 ± 0.07 a2 2.13 ± 0.19 b 0.96 ± 0.04 b3 1.78 ± 0.28 c 0.92 ± 0.10 c4 1.68 ± 0.26 d 0.89 ± 0.08 c5 1.59 ± 0.26 d 0.87 ± 0.03 c6 1.50 ± 0.23 d 0.84 ± 0.07 d7 1.25 ± 0.19 d 0.75 ± 0.03 e8 0.79 ± 0.34 e 0.66 ± 0.10 e

Tuber borchii Vittad.1 2.49 ± 0.52 a 1.13 ± 0.12 a2 2.32 ± 0.70 b 1.06 ± 0.17 b3 2.16 ± 0.67 b 0.95 ± 0.10 c4 1.93 ± 0.54 b 0.95 ± 0.17 c5 1.88 ± 0.59 b 0.97 ± 0.16 c6 1.80 ± 0.56 b 0.89 ± 0.11 c7 1.52 ± 0.34 b 0.85 ± 0.08 c8 1.44 ± 0.35 b 0.81 ± 0.07 c

Tuber melanosporum Vittad.1 4.32 ± 0.25 a 1.27 ± 0.04 a2 2.32 ± 0.12 b 0.97 ± 0.03 b3 2.13 ± 0.02 c 0.96 ± 0.04 b4 2.05 ± 0.07 c 0.96 ± 0.05 b5 1.84 ± 0.17 d 0.89 ± 0.02 d6 1.70 ± 0.13 d 0.90 ± 0.09 c7 1.56 ± 0.13 e 0.81 ± 0.04 d8 1.36 ± 0.11 e 0.78 ± 0.05 d

Values followed by the same letter are not significantly different,according to the Cluster analysis of SCOTT and KNOTT 1974 (P=0.05).

some, that is about in the range of some N. crassachromosomes (4-12.6 Mbp), as estimated byCHEF (MILLS and MCCLUSKEY 1990). This is inline with truffles as well as N. crassa being anAscomycete. Our findings show that truffles sharechromosomal dimensions with other fungi andconsiderably smaller than those of plants and ani-mals. This is consistent with fungi having smallergenomes compared to plants. From these dataan indirect measure of T. borchii DNA con-tent/genome (genomic size) is approssimativelycalculated as 0.30 pg.

As showed in Fig. 2 the precise measureachieved with image analysis evidenced some dif-ferences among the species. T. melanosporumshows the chromosome 1 significatively longerand larger in comparison to the other studiedspecies . The shortest chromosome was found inT. magnatum. In higher plants the ratio between

the longest and the shortest chromosome is anindication of the karyotype asymmetry that isrelated to evolution of the species group (STEB-BINS 1971). Among the three studied species themore asymmetric is T. melanosporum.

From karyotyping observations it appearsthat from the diploid presporigenous hyphae(2n= 8) premeiotic (sporogonial cells) wouldarise, that after meiosis should give rise to hap-loid (n= 4) nuclei. This suggests that hyphaldicaryotic cells might give rise to diploid syn-caryotic presporal hyphae. As ectomycorrhizalmantle and ascocarpal cells contain in generaltwo nuclei, the hypothesis of the presence of aprimary mononucleate mycelium and then of adycaryotic one could be confirmed. It is an openquestion if binucleated hyphal cells are derivedfrom the fusion of mononucleated cells belong-ing to mycelia characterized by different mating

310 POMA, VENORA, MIRANDA and PACIONI

Fig. 2 – Idiogrammatic representation of T. borchii, T. magnatum and T. melanosporum chromosomes. Total surface (µm2) andlenght (µm) are compared among the three species.

types or from automittic processes. Our data,related to the results obtained by electrophoret-ic analysis of gene-enzyme systems (PACIONI andPOMPONI 1991; BERTAULT et al. 1998; URBANEL-LI et al. 1998), seem to indicate Tuber as a genuswith homozygotic and automittic species. Thefixation of Tuber populations in homozygosisand the putative haploid-diploid cycle thatwould appear from the chromosomal hyphal andsporal numbers suggest that truffles of the genusTuber undergo automixis and subsequentlymeiosis.

Acknowledgments – This work was supportedby a special grant from CNR (Consiglio Nazionaledelle Ricerche, Italy) and Regional Administrationsfor “Biotecnologia dei funghi eduli ectomicorrizici:dalle applicazioni agro-forestali a quelle agro-ali-mentari”.

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Received March 26, 2002; accepted May 29, 2002

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