Ploidy and chromosomal number in Tuber aestivum

FEMS Microbiology Letters 167 (1998) 101^105 Ploidy and chromosomal number in Tuber aestivum a Department of Basic and Applied Biology, Faculty of Sciences, University of L'Aquila, Via Vetoio Coppito, I-67010 L'Aquila, Italy Department of Environmental Sciences, Faculty of Sciences, University of L'Aquila, Via Vetoio Coppito, I-67010 L'Aquila, Italy b Received 25 June 1998; revised 19 August 1998; accepted 21 August 1998 Abstract Chromosome number is generally considered to be the most accurate and direct measure of ploidy. In fungi, however, counting of chromosomes is difficult and inaccurate because of their small size. To date truffles have been characterized using molecular approaches (analysis of DNA and PCR-based techniques) in order to find out differences among species, but not by a cytogenetical approach. Although the small size of truffle chromosomes is of some hindrance for a cytogenetic study, in the present work Tuber aestivum chromosome counts were determined in metaphase configurations from haploid nuclei of ascospores by aceto-orcein staining. Nuclear chromosome number and topography were also evaluated by propidium iodide DNA staining using confocal microscopy. Tuber aestivum was found to possess a basic number of 5 or 6 chromosomes, medium length 90.95 Wm. The karyology of ascospores during their developmental stages was also investigated. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Tru¥e; Chromosome ; Ascomycetes karyotype; Ascomycotina; Pezizales 1. Introduction Some tru¥es (Ascomycotina) of the genus Tuber Micheli ex F.H. Wigg. are economically important due to the organoleptic properties of some species (Tuber magnatum Pico, T. melanosporum Vittad., T. aestivum Vittad., T. borchii Vittad.). Tru¥es are also biologically interesting since they are hypogeous fungi living under microaerobic conditions and possess abnormal mitochondria [1,2] when grown under anaerobic conditions similarly to Saccharomyces cerevisiae and other yeasts in the Ascomycotina. Both basic and applied concerns suggest this work * Corresponding author. Tel.: +39 (862) 433264; Fax: +39 (862) 433273; E-mail: miranda@aquila.infn.it will provide a better understanding of the reproductive cycle of tru¥es of the genus Tuber, ¢rst by detecting chromosome number both in spores and hyphae. Karyotyping has often been used in the classi¢cation of animals and plants; moreover, the reproductive cycle of some species can be assessed only if the ploidy of somatic and reproductive cells is known. Chromosome number is considered to be the most accurate and direct measure of ploidy, but counting chromosomes of fungi is di¤cult and inaccurate because of their small size [3^5]. Electrophoretic karyotypes of several Ascomycetes are also known [6,7]. To date several tru¥es have only been characterized molecularly through the analysis of DNA, rRNA and other PCR-based techniques in order to discern 0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 3 7 8 - 4 Downloaded from https://academic.oup.com/femsle/article-abstract/167/1/101/705675 by guest on 10 June 2020 Anna Poma a , Giovanni Pacioni b , Roberta Ranalli a , Michele Miranda a; * 102 A. Poma et al. / FEMS Microbiology Letters 167 (1998) 101^105 di¡erences among species [8^10]; however, none of these species has been investigated using a cytogenetical approach. In the present work T. aestivum chromosomes were observed and measured in metaphases of haploid ascospores and dikaryotic or diploid hyphal metaphases by aceto-orcein staining, permitting counting of chromosomes. Nuclear chromosome number was also evaluated by propidium iodide DNA staining for confocal microscopy. We also investigated the karyology of ascospores during their developmental stages to better understand sporal karyological changes. 2. Materials and methods Immature T. aestivum specimens were collected close to L'Aquila or Teramo (Abruzzo, Italy) and used for study soon after collection or else stored at 325³C for 2^4 days prior to karyotypical analysis. 2.1. Staining of tru¥e chromosomes and nuclei for light microscopy Thin slices of the tru¥es (1^2 mm) were treated with asci wall lytic enzymes cellulase, protease and chitinase L2265 (Sigma) with 0.01% (w/v) colchicine overnight, ¢xed in ethanol/glacial acetic acid (3:1 (v/v), overnight), hydrolyzed in 1 M HCl for 30 min at R.T. and squashed in 45% (v/v) acetic acid and orcein solution. The cover slides were removed by dipping in liquid nitrogen and samples dehydrated in graded alcohol series, dipped in xylene; ¢nally the slides were mounted in Canadian balsam. 2.2. DNA staining by propidium iodide for confocal microscopy DNA staining was performed using a method [11] modi¢ed for T. aestivum specimens. Thin tru¥e slices were treated with 0.01% (w/v) colchicine overnight at 20³C, ¢xed in ethanol/acetic acid 20 (3:1 (v/v) overnight), dipped in 1 mg ml31 NaBH4 solution for 10 min. Lipids were removed using acetone treatment for 2 h. Slices were treated with block/permeabilization bu¡er (0.2% BSA, 0.1% Triton X-100, PBS) for 5 min, stained with 1 Wg ml31 propidium iodide (Sigma) for 15 min, rinsed 2 times with block/ permeabilization bu¡er for 5 min and mounted with Vectashield1 (Vector Laboratories, Burlingame, CA, USA) to prevent photobleaching. Slides were stored Downloaded from https://academic.oup.com/femsle/article-abstract/167/1/101/705675 by guest on 10 June 2020 Fig. 1. Light microscopy of aceto-orcein stained Tuber aestivum chromosomes (arrowed). a: Sporal chromosomes; b : hyphal chromosomes. Magni¢cation 1200U. Seven sporal and twenty hyphal metaphases were scored. A. Poma et al. / FEMS Microbiology Letters 167 (1998) 101^105 103 in the dark at 4³C before observation by confocal microscopy using a Sarastro 2000 microscope (Molecular Dynamics, CA, USA). 2.3. Reagents The reagents used were of the purest grade available and generally obtained from Sigma or Merck. 3.1. Light microscopy The orcein-staining procedure was su¤cient to make the chromosomes of both spores and hyphae visible. Treatment including asci wall lytic enzymes prior to squashing and staining was found to improve the results. Chromosomes and chromatin were strongly stained showing very little or no background. Five chromosomes (Fig. 1a and Table 1) could be observed in cells at metaphase during mitoses of sporal haploid nuclei. A chromosomal medium size of approximately 0.95 Wm (roundish in shape) was estimated. We have not observed di¡erTable 1 Chromosome number of Tuber aestivum Source Hyphal metaphases Sporal metaphases Monti della Laga, Teramo, Italy 9 9 9/10 9/10 10 10 10 10 11 11 12 5 5 6 6 8/9 9/10 9/10 10 10 10 10/11 10/11 10/11 5 5/6 5/6 San Marco, L'Aquila, Italy Fig. 2. Tuber aestivum sporal nuclei stained with propidium iodide. Scale bar = 1 Wm. More than 500 sporal nuclei were examined. ently shaped chromosomes. Hyphal chromosomes are very similar to spore chromosomes in shape and size (Fig. 1b) and as reported in Table 1 and Fig. 1b, 10 chromosomes/metaphase were scored. 3.2. Confocal microscopy The nuclear chromosome number and topography were evaluated by propidium iodide DNA staining for confocal microscopy of sporal metaphases. Fig. 2 shows chromosomes constituting a butter£y-shaped body. Auto£uorescence did not allow chromosome counting as clearly as by light microscopy. 3.3. Karyology During ascospore formation, the nuclei in the sporigenous area divide, originating ¢rst two and later 4^8 daughter nuclei (Fig. 3a,b,c). In the ripening spore, the wall is formed ¢rst, while reticulation is formed later (Fig. 3d). The dividing nuclei migrate close to the wall previous to occupying the center of the forming spores while the cytoplasm becomes vacuolate. Downloaded from https://academic.oup.com/femsle/article-abstract/167/1/101/705675 by guest on 10 June 2020 3. Results 104 A. Poma et al. / FEMS Microbiology Letters 167 (1998) 101^105 4. Discussion The numbers of spore and hyphal chromosomes, 5.4 þ 0.1 (n) and 10 þ 0 (2n) respectively, strongly suggest a haploid chromosome number of 5 for T. aestivum; this value of n is in the range of those of several Ascomycetes, for example Neurospora crassa (n = 7) [6] and also the mean bp content calculated from the mean metaphase chromosomal size. In fact by assuming that the metaphase chromosomes of T. aestivum undergo a contraction of the same order of magnitude of those of other metaphasic chromo- Downloaded from https://academic.oup.com/femsle/article-abstract/167/1/101/705675 by guest on 10 June 2020 Fig. 3. Tuber aestivum ascospores. a^c: Karyological variations; d: ripe ascospores. More than 500 ascospores were examined. A. Poma et al. / FEMS Microbiology Letters 167 (1998) 101^105 Acknowledgments This work was supported by a special grant from CNR (Consiglio Nazionale delle Ricerche, Italy) and Regional Administrations for `Tuber: Biotechnology of Mycorrhization'. 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Downloaded from https://academic.oup.com/femsle/article-abstract/167/1/101/705675 by guest on 10 June 2020 somes (about 5000U) [12] then 0.95 Wm (chromosomal length)U5000 would give 4.75 mm of linear DNA, equivalent to about 14 Mbp/chromosome, that is about in the range of some N. crassa chromosomes (4^12.6 Mbp), as estimated by CHEF [13]. This is in line with tru¥es as well as N. crassa being Ascomycotina. Our ¢ndings show that tru¥es share chromosomal dimensions with other fungi which are considerably smaller than those of plants and animals. This is consistent with fungi having smaller genomes compared to plants. However, a more direct measure of DNA content/genome is required to exactly assess T. aestivum genomic size. On the basis of the karyological observations in parallel to chromosome counts, the reconstruction of the `sporal stage' (when spores are produced within the asci) may be attempted from a cytogenetical point of view. From the diploid presporigenous hyphae (2n = 10) premeiotic (sporogonial cells) would arise, which after meiosis should give rise to haploid (n = 5) spores. This would suggest that hyphal dikaryotic cells [14] might give rise to diploid synkaryotic presporal hyphae. In situ hybridization to a nucleolar organizer probe [15,16] should mark haploid, dikaryotic and synkaryotic nuclei and may help to further clarify the nuclear cycle of T. aestivum. 105