Cloning and characterization of two repeated sequences in the symbiotic fungus Tuber melanosporum Vitt. Free

Abstract

Two repeated DNA sequences of European strains of the symbiotic fungus Tuber melanosporum were isolated and characterized. One of these, SS14, representing about 0.05% of the fungal genome, was shown to be a T. melanosporum-specific sequence by Southern and dot-blot hybridization. The second one, named SS15, is about 0.0025% of the entire genome, and it is specific not only to T. melanosporum but also to the Asian black truffle Tuber indicum. Neither of these two fragments hybridizes with any of the other European truffle species tested. By sequence analysis of these two fragments, PCR primers were designed and used to selectively amplify DNA from T. melanosporum ascocarps and ectomycorrhizae by simple and multiplex PCR. No amplification products were obtained with DNA from either mycorrhizal roots or fruit bodies of other ectosymbiotic fungi. The two identified genomic traits also provided useful information for a better understanding of the phylogenetic relationships among black truffle species and for testing T. melanosporum intraspecific variability.

1 Introduction

Truffles are ascomycetous fungi belonging to the order Pezizales, which establish a symbiotic association, ectomycorrhizae, with the roots of gymnosperm and angiosperm trees and shrubs. This mutualistic symbiosis plays a pivotal role in the growth and nutrition of forest trees [1,2]. However, the specificity of mycorrhizal interaction varies widely, and may be responsible for various patterns of efficacy under different ecological conditions [3]. Unlike other ectosymbiotic fungi, many species of the genus Tuber produce economically valuable edible fruit bodies. In addition to beneficial effects of mutualistic association, the flourishing worldwide truffle market has primarily encouraged a large production and commercialization of trees inoculated with Tuber species. Thus, large-scale programs for growing truffle species have been developed both within and outside reforestation projects mainly in Europe, where the most marketable species are indigenous. The first step of these programs is the nursery inoculation of host plants. A clear and unambiguous identification of truffle mycorrhizae by morphological analysis is often virtually impossible because of the marked similarity in many morphological traits existing among species. The problem is all the more serious as morphologically similar Tuber spp. may have widely different ecological growth requirements, as well as very unequal organoleptic qualities and therefore commercial value [4]. As a consequence, the control of inoculated seedlings for the presence of the symbiotic truffle agent, the monitoring of fungal development in open field conditions and the assessment of relationships between the introduced fungal species and a preexisting microbial community, cannot be performed exhaustively by morphological controls alone [5]. In addition, these fungal species are often difficult to grow in pure culture and no anatomical trait allows the typing of truffle mycelia.

Tuber melanosporum Vitt. (Perigord truffle, or ‘tartufo nero pregiato di Norcia e Spoleto’) is the black truffle which, because of its distinctive taste, has a higher commercial value than other black truffles such as Tuber brumale Vitt., Tuber aestivum Vitt., Tuber uncinatum Chatin, Tuber mesentericum Vitt. and Tuber indicum Cooke and Massee.

The increasing economic importance of truffles is stimulating studies on these species. In particular, the lack of sufficient macro- and micromorphological diagnostic traits in these fungi throughout their life cycles has led research workers to look for molecular markers allowing a reliable species characterization. Molecular techniques providing more efficient and reliable identification tools than any morphological character have recently been described to identify these ectosymbiotic fungi in the vegetative and symbiotic phases [5–11]. The present study reports a rapid amplification of polymorphic DNA (RAPD) analysis for differentiating between two morphologically similar, sympatric truffle species, T. melanosporum and T. brumale, in order to identify T. melanosporum species-specific fragments and test for the presence of T. melanosporum intraspecific variability.

2 Materials and methods

2.1 Sample source

The truffle species used are listed in Table 1. For each species, ascocarps from at least three different natural areas in Italy were processed, and for each collection site 1–5 samples were studied. T. melanosporum and T. brumale were also collected from natural truffle areas in France and Spain, so that the total numbers of collection sites tested for these two species were 22 and 15, respectively. Ectomycorrhizae were collected from mycorrhizal plants growing either in open field conditions or in nursery pots. Single inoculated apical root-tips and pooled samples of ectomycorrhizae were processed, obtained by collecting ectomycorrhizae from single root masses grown in pots and/or different host plants. All the truffles and ectomycorrhizae were first examined macro- and microscopically as a preliminary species determination [12–14].

2.2 DNA isolation

For RAPD analysis, DNA from fruit bodies was isolated basically as described by Henrion et al. [5]. For dot-blot, SCAR and multiplex PCR analyses of ascocarps, leaves or roots of host plants, DNA was isolated either as above or as described by Paolocci et al. [11]. For the preliminary dot-blot analysis of ectomycorrhizae, DNA was isolated and purified by a CsCl gradient [4]. As a routine procedure, however, the more rapid and less tissue-demanding DNA isolation protocol reported by Paolocci et al. [11] was followed. For Southern analyses on ascocarps, DNA was isolated and purified by a CsCl gradient purification step and for each sample two independent DNA isolations and blottings were performed.

2.3 RAPD analysis and cloning of specific T. melanosporum bands

Twenty primers of arbitrary sequences obtained from Severn Biotech Ltd. were tested on T. melanosporum and T. brumale DNA and, as control, on host plant Corylus avellana. The reaction mix contained 10 mM Tris–HCl (pH 8.8), 50 mM KCl, 0.1% Triton X-100, 5 mM MgCl₂, 200 μM of each dNTP, 20 pmol of primer, 2 U of DynaZyme 2 recombinant Taq-polymerase (Fynnzyme) and 20 ng of DNA target in a final volume of 25 μl. The cycling parameters carried out in a 2400 Gene Amp (Perkin Elmer) thermal cycler were as follows: an initial denaturation step at 94°C for 5 min; 40 cycles consisting of 30 s at 94°C, 30 s at 37°C, 45 s at 72°C; a final extension step at 72°C for 5 min. A negative control (no DNA template) was included in all PCR experiments. PCR products (20 μl) were size-fractionated on 1.2% agarose gels. A putative specific T. melanosporum product generated by amplification with primer 15 (5′-GCTTGTGAAC-3′) was cut, recovered from agarose gel and submitted to a second round of PCR amplification to obtain enough product to be cloned into pGMT-Easy vector (Promega). Two different clones, pGMSS14 and pGMSS15, were identified (see Section 3).

2.4 Probe preparation, sequence analysis and primer designing

For the first screening of fungal DNA by dot-blot and Southern analysis, the fragments used as probes were excised from plasmids using the enzymes EcoRI for pGMSS14 and BstXI for pGMSS15, respectively. Double strand DNA sequencing was carried out on both clones using the Big Dye terminator kit (Perkin Elmer) according to the supplier's instructions, and reaction products were run on an ABIPRISM 310 Genetic Analyzer automated sequencer (Perkin Elmer).

By sequence analysis, two primer pairs: SS14Fw (5′-CTGTTGAGATACTGTGTTGTAC-3′)/SS14Bk (5′-CAGGAATGTAGGAGCTTACCTGA-3′) and SS14MelS (5′-CCCTATGACTGTCAATGTCC-3′)/SS14MelSpe23 (5′-GAACGAGGAGAGTCAGATGGAG-3′) were designed for pGMSS14. Similarly, the primer pair SS15Fw (5′-ACGCATATGAGGGCCAAAGTGC-3′)/SS15Bk (5′-AGAAGGATGCTGGGTTCCATTAC-3′) was chosen for pGMSS15. The SS14Fw/SS14Bk and SS15Fw/SS15Bk primer pairs yielded 631- and 767-bp long amplicons, respectively. These two primer pairs were used for probe preparation for dot-blot and Southern blot experiments, as well as for SCAR analyses (see below). The primer pair SS14MelS/SS14MelSpe23, yielding a 262-bp long amplicon on SS14, was selected to be used in multiplex PCR amplification.

T. brumale and T. melanosporum ITS fragments obtained as described by Paolocci et al. [6] were used as probes for control experiments in dot-blot and/or Southern hybridizations.

The SS14 and SS15 sequences are registered in GenBank under the following accession numbers: AF210077 and AF210078, respectively.

2.5 Dot-blot hybridization

Fifty–100 ng of ascocarp and ectomycorrhizal DNA was used. However, when samples consisting of only 1–10 root-tips were processed, all the DNA was used. The DNA was heat-denatured at 100°C for 15 min and transferred to a N+ membrane (Hybond) by means of a dot-blot apparatus (Bio-Rad) according to the manufacturer's instructions, in the presence of 0.4 N NaOH as transfer solution. Pre-hybridization, hybridization and high stringency post-hybridization washes were carried out as described for Southern analysis (see below).

2.6 Estimation of the repetitiveness of SS14 and SS15 fragments by dot-blot analysis

T. melanosporum and T. indicum genomic DNA were serially diluted in stepwise concentrations ranging from 100 ng per 10 μl to 100 pg per 10 μl. Similarly, the SS14 and SS15 PCR fragments were diluted from 100 pg per 10 μl to 0.5 pg per 10 μl, as controls. Genomic DNA and PCR fragment dilutions were carried out in duplicate and spotted on Hybond N+ membranes (Amersham). The blotting, hybridization and washing procedures were as used for Southern analysis. The membranes were exposed to X-ray films for 4, 12, 24 and 48 h. After a 24-h exposure, the dot-blot signals displayed on autoradiograms by positive controls were still not saturated, therefore dot intensity was measured, using the Phoretix 1D software after scanning the autoradiograms.

2.7 Southern analysis

Genomic DNA (4 μg) isolated from all truffle species listed in Table 1 was digested with either EcoRI or HindIII (Biolabs), run on a 1% agarose gel and blotted onto a Hybond N+ membrane (Amersham). Pre-hybridization and hybridization were performed at 65°C according to the filter supplier's instructions. Post-hybridization high stringency washes were performed at 65°C (0.1×SSPE, 0.1% w/v SDS, three changes of 15 min each).

2.8 SCAR and multiplex PCR analysis

SCAR analysis using either the primer set SS14Fw/SS14Bk or SS15Fw/SS15Bk was conducted on host plant, ascocarp and ectomycorrhizal DNA through the following PCR cycles: a first denaturation step at 94°C for 3 min; 25 cycles consisting of 30 s at 94°C, 30 s at 62°C and 45 s at 72°C; and a final extension step for 7 min at 72°C. Amplifications were carried out in a final aqueous volume of 50 μl containing 50 mM KCl, 2.5 mM MgCl₂, 10 mM Tris–HCl (pH 9), 10 pmol of each primer, 200 μM of each dNTP, in the presence of 2.5 U of Taq-polymerase (Pharmacia Biotech).

Multiplex PCR analysis was carried out on fungal and mycorrhizal samples using the SS14MelS/SS14MelSpe23 and SS15Fw/SS15Bk primers. The multiplex PCR reaction buffer was the same as for SCAR single locus amplification. Cycling conditions were as follows: a first denaturation step at 94°C for 3 min, 25 cycles consisting of 30 s at 94°C, 10 s at 62°C and 45 s at 72°C; and a final extension step for 7 min at 72°C.

As a control for DNA quantity and amplificability, all samples processed in SCAR and multiplex PCR were also amplified with the universal ITS1/ITS4 primer pair [15] as described by Paolocci et al. [11].

2.9 Restriction fragment length polymorphism (RFLP) analysis of SS14 and SS15 SCAR markers

RFLP analyses were performed on both SS14 and SS15 fragments obtained as reported in Section 2.8, from DNA isolated from a total of 40 T. melanosporum ascocarps collected in France, Spain and Italy. The endonucleases tested, derived by sequence analysis, were: AluI, MseI and DpnII for SS14; AluI, EcoRI, TaqI and BglI for SS15. The enzymes were all from Biolabs, and reaction conditions were as suggested by the supplier. Each analysis was carried out on 15 μl of PCR product and the resulting fragments were run on 2% agarose gels.

3 Results

3.1 RAPD analysis and cloning of T. melanosporum DNA fragments

Genomic DNA from ectosymbiotic European black truffle species T. melanosporum and T. brumale and from C. avellana, a host plant common to T. melanosporum and T. brumale, were amplified using 20 arbitrary single primers. For each truffle species considered, five samples harvested in five different regions were initially analyzed. A comparison of the RAPD profiles allowed the selection of a major T. melanosporum PCR product of about 700–800 bp, yielded by the primer 15. This product was always present in this species, irrespective of the collection site, but virtually absent in T. brumale and host plants (data not shown). This amplicon was recovered from gel, re-amplified and the product of the second amplification was ligated into the pGMT-Easy vector. The screening of transformed E. coli colonies showed that two PCR fragments of about the same size, approximately 800 bp, were cloned. These two fragments were not resolved by the electrophoretic conditions used to separate the initial RAPD products. However, they were dissimilar for the presence of an EcoRI recognition site that was missing in the shortest fragment. The clone carrying a 723-bp product (SS14) was named pGMSS14. The other clone, carrying a 810-bp product (SS15), was designated pGMSS15.

No appreciable T. melanosporum intraspecific polymorphism was revealed by this analysis, as already reported by other authors [16,17].

3.2 Southern analysis

Southern analysis of truffle genomic DNA was carried out to check for T. melanosporum species-specificity and repetitiveness of SS14 and SS15 fragments. The SS14 probe on EcoRI-digested genomic T. melanosporum DNA showed a ladder-like hybridization signal, where three major bands (of about 7 kbp, 3 kbp and 2 kbp) were clearly distinguishable from all the others, and intraspecific polymorphism was never displayed by ascocarps from different places of origin (Fig. 1a). No hybridization signal was displayed by the DNA of any of the Tuber spp., whether it was digested with EcoRI or HindIII, with the only exception of T. melanosporum (data not shown).

The SS14 probe revealed intraspecific polymorphism on HindIII-restricted T. melanosporum DNA. Fig. 1b shows the two patterns revealed by this probe on T. melanosporum genotypes collected in Italy, France and Spain, the countries where this truffle is mainly found. All six Spanish truffles tested showed the same hybridizing pattern, while polymorphism was detected in French and Italian truffles, for which six samples per country were also tested (Fig. 1b and data not shown). Since all the truffles processed were at approximately the same stage of maturity, and HindIII enzyme is relatively insensitive to DNA methylation, no epigenetic modifications can explain the band polymorphism observed among the fruit bodies. This polymorphism is therefore likely to result from a different distribution of HindIII recognition sites in the flanking regions of the repeated fragment SS14. On the contrary, by probing the filters with the SS15 fragment, polymorphism was not displayed on EcoRI- nor on HindIII-digested T. melanosporum DNA. In the latter case, an approximate 6-kbp band was the most repetitive signal, along with two other minor bands of about 10 and 8 kbp, respectively. The SS15 fragment was, however, not specific to T. melanosporum only, since T. indicum also hybridized with this probe. Conversely, the Asian truffle was clearly distinguishable from T. melanosporum by the presence of small fragments, ranging in size from 1.5 kbp to about 300 bp, that were specific to T. indicum alone (data not shown).

As control, all the filters were also hybridized using the T. brumale ITS fragment as a probe, and all the species tested showed hybridizing bands (data not shown).

3.3 Dot-blot analysis

Dot-blot experiments were performed on DNA isolated from ascocarps and host plant species in order to verify the specificity of the two selected probes and test for their reliability as T. melanosporum markers throughout its life cycle. The SS14 genomic fragment confirmed T. melanosporum specificity, as no signal was evident on DNA isolated from all the other species tested and from host plants (Fig. 2a). As expected, the SS15 fragment was also present in T. indicum (Fig. 2b), irrespective of the Asian genotypes tested [18]. Fig. 2c shows truffle and host plant DNA hybridized with the T. brumale ITS probe used as control for the amount of DNA loaded. Due to incomplete T. melanosporum specificity, the SS15 probe was no longer used for screening of ectomycorrhizal DNA.

For the first screening of inoculated root-tips, CsCl gradient-purified DNA from T. brumale and T. melanosporum ectomycorrhizae were tested, and the SS14 probe hybridized only with T. melanosporum DNA (data not shown). A similar result was achieved on DNA isolated from ectomycorrhizal samples omitting the CsCl purification step. The SS14 probe not only proved its T. melanosporum specificity on mycorrhizal root-tips, but also confirmed its repetitiveness by showing a hybridizing spot on DNA from as little as a single ectomycorrhiza (Fig. 3a, row A), whose estimated total amount of DNA spotted on the filter was less than 5 ng. Fig. 3a (row C) also shows the positive identification of a single T. melanosporum ectomycorrhiza when processed in the presence of a variable number of host root-tips inoculated with the competing fungus T. brumale. This, however, cannot be a quantitative assay, since the amount of DNA yielded by each mycorrhiza is highly dependent on the physiological stage of the samples processed. Therefore, the intensity of hybridizing spots may not be proportional to the number of mycorrhizae analyzed, as will be clear from Fig. 3a (row C).

3.4 Evaluation of repetitiveness of SS14 and SS15 loci in T. melanosporum

Dot-blot analyses were performed on serially diluted genomic DNA to calculate and compare the repetitiveness of the two T. melanosporum RAPD probes. The signal intensities produced by SS14 probes on T. melanosporum genomic DNA dilutions were compared with those produced on serial dilutions of the SS14 PCR fragment, used as standard. Phoretix software analysis showed that the intensity of signals produced by 50, 20 and 10 ng of total T. melanosporum DNA spots corresponded to that obtained with 25, 10 and 5 pg of the SS14 control, respectively (data not shown), that is 0.05% of the T. melanosporum genome. Similarly, the analysis conducted with the SS15 probe revealed that this locus is about 0.0025% of the entire T. melanosporum genome since 100, 50 and 20 ng of genomic DNA corresponded to 2.5, 1 and 0.5 pg of probe. In addition, the SS15 locus is repeated in T. indicum as much as it is in T. melanosporum, confirming the results from dot-blot analyses in Fig. 2b.

All these results are in agreement with those obtained by both Southern and dot-blot hybridization, which display more intense bands, or a higher number of hybridizing bands, on membranes when probed with SS14 rather than SS15.

3.5 Sequence analysis and PCR-based screening of truffles and ectomycorrhizae

SS14 and SS15 sequence analyses neither displayed open reading frames nor revealed, by BLAST searching, similarities with any sequence present in the databases. The nucleotide sequences allowed selecting SCAR primers and endonuclease enzymes to carry out the RFLP analysis on both SS14 and SS15 amplicons. Two primer pairs, SS14MelS/SS14MelSpe23 and SS14Fw/SS14Bk, yielding a 262-bp long fragment and a 631-bp long fragment, respectively, were designed for SS14. The primers 15SSFw and SS15Bk, yielding a 767-bp product, were selected on the SS15 sequence and tested for their specificity also versus T. indicum fruit bodies and ectomycorrhizae. Both SS14Fw/SS14Bk and SS15Fw/SS15Bk primer pairs clearly and highly amplified only T. melanosporum fruit bodies and ectomycorrhizae, but did not show any amplicons on the genomes of other truffle species or host plants (data not shown). The SS14MelS/SS14MelSpe23 primers were designed for use in a multilocus PCR amplification in the presence of SS15Fw/SS15Bk primers to develop a diagnostic model based on the simultaneous amplification of these two different and independent loci. Fig. 4a shows the amplification pattern resulting from multiplex PCR amplification on DNA isolated from morphologically similar black truffle ectomycorrhizae and from ectomycorrhizal species frequently co-existing or competing with truffle species such as Hebeloma spp., Hymenogaster spp., Cenococcum spp. and other undetermined ectomycorrhizal forms such as ‘AD type’[12]. Only T. melanosporum exhibited the two expected bands, with the 262-bp amplicon yielded by the SS14 locus and the 767-bp amplicon by the SS15 locus. On the contrary, the other species tested did not have any PCR products, thus confirming the specificity of the primers designed and the absence of any primer false interaction. The SS14 and SS15 primers permitted T. melanosporum to be differentiated from all the other truffle species in any test condition, irrespective of the physiological stage of the samples, whether performing a single or a multilocus PCR amplification. Fig. 4b shows the pattern resulting from PCR amplification of the same samples as in Fig. 4a using the universal ITS1/ITS4 primers. As these primers anneal to a wide range of target DNAs [6], their use often prevents a reliable identification of the species. Therefore, since other microorganisms are likely to be present when processing open field ectomycorrhizal samples, the use of species-specific primers rather than universal ribosomal primers is strongly recommended.

3.6 RFLPs of PCR-amplified T. melanosporum-specific fragments

RFLP analyses were performed on the SS14 and SS15 amplified regions from a total of 40 T. melanosporum fruit bodies collected in Spain, Italy and France. Tables 2 and 3 report the fragments with their relative lengths resulting from RFLP analysis on the two SS14 and SS15 amplicons. None of the restriction enzymes identified any different T. melanosporum genotype, since all the truffles examined displayed identical restriction patterns (data not shown). The results confirmed the highly conservative nature of both repeated fragments on different genotypes across the T. melanosporum geographical distribution range.

4 Discussion

Two repeated T. melanosporum fragments, SS14 and SS15, were identified and characterized. Repeated DNA sequences other than rDNA can be used as markers within or among species, depending on their rate of divergence [19]. Certain sequences, having gone through a rapid evolution, are only conserved at the species level, and hence considered species-specific; others are shared by related species and, therefore, are useful for studying the evolution and phylogenetic relationships among species [20]. The T. melanosporum SS14 sequence belongs to the species-specific group of repeated genomic fragments, while the SS15 sequence is shared with T. indicum, indicating a close relationship between these two black truffle species.

Southern and dot-blot analyses showed that SS14 is, in fact, conserved only at the species level and that it is quite a repetitive DNA fragment (0.05% of total genome). No other species-specific probes have so far been identified in this species, and though other species-specific probes have been isolated in other truffle species [21], SS14 is the only one which allows identification by DNA hybridization using as little as a single mycorrhizal root-tip. Unlike SS14, the SS15 fragment did hybridize with T. indicum spp., in which its repetitiveness is much the same as in T. melanosporum. These low-priced Asian black truffles have recently been the object of morphological and molecular studies, as their massive importation into Europe has been raising economic and ecological questions [11]. The Asian species actually bears a closer morphological similarity to T. melanosporum, at the mycorrhizal level [14], than does T. brumale. The lack of morphological markers to distinguish T. indicum from T. melanosporum mycorrhizae may facilitate the introduction into Europe, by planting nursery inoculated plants, of the Asian species which may compete with the more valuable indigenous one(s).

The alignment of the ITS region of the most important truffle species confirmed, at the molecular level, the marked morphological resemblance between T. indicum and T. melanosporum, with the nucleotide homology between the two species averaging 93%[18]. No other European species, whether white or black, has a comparable percentage of homology with T. melanosporum [22,23]. The fact that the SS15 locus was found to be specific to no truffle species other than T. melanosporum and T. indicum only adds to evidence from ITS analysis supporting the hypothesis that these two species, although evolving independently, share a closer common origin than does T. melanosporum with sympatric species such as T. brumale. Since no presence of spontaneous T. indicum ascocarps in Europe, or T. melanosporum in Asia have ever been reported, the evolutionary scenario which has caused these two species to split must have been a complex one. Hypogeic and ectomycorrhizal fungi such as Tuber spp. rely on animals for spore dispersal and have evolved along with their host plants. Thus, barriers (e.g. high mountain ranges, climatic changes) both to the migration of mycophagists and to the diffusion of host plants have limited fungal diffusion and gene flow between relatively distant populations [24]. It is conceivable that the arising, over Earth's history, of such barriers may have favored diversification between T. indicum and T. melanosporum as two distinct endemic species in Asia and Europe, respectively. Within the order Pezizales, the development of a robust phylogenetic framework has proven difficult because of character convergence and extreme paucity of fossil records [25]. Therefore, molecular data may represent a promising tool for elucidating the evolutionary history within this group of fungi.

Previous studies have found limited to non-existent intraspecific diversity in T. melanosporum, corroborating the hypothesis of a closed mating system, such as homothallism or even exclusive selfing in this species [16]. These considerations suggested that the organoleptic differences among ascocarps collected from different sites may be explained by environmental variation rather than genetic factors [17]. The data reported here are in agreement with the results mentioned above: neither RAPD profiles nor SCAR/RFLP patterns of the SS14 and SS15 sequences revealed any substantial variation in T. melanosporum across its geographical distribution range. Nevertheless, two different hybridization patterns of the SS14 probe were identified within this species, and despite the limited number of samples processed, the analysis permitted a first, rough intraspecific discrimination. Studies using other markers, such as AFLP and microsatellites, rather than RAPD or species-specific probes, are therefore in progress in our lab to assess the genetic variability within this species.

Finally, we are attempting to develop the simplest possible molecular procedures, based on multiplex PCR amplification using species-specific primers designed on repeated regions, such as ITS, to differentiate among the more economically and ecologically important mycorrhizal species. The aim is to provide reliable diagnostic tools to control and certify mycorrhizal plants, and to monitor the inoculated fungi when the plants are in open field conditions and the new introduced species have to compete with preexisting microbial communities. The possibility of using discriminating genomic traits other than ribosomal rDNA in some truffle species can enormously facilitate the selection of ITS-specific primers in others. The ITS region is, in fact, relatively short (about 650–700 bp) and contains the 5.8S gene, which is approximately 150-bp long, and highly conserved (about 99% nucleotide identity) on all truffle species. The SS14 and SS15 loci and the SCAR primers designed here not only allow T. melanosporum identification, but also facilitate the designing of species-specific ITS primers for other truffle species.

Acknowledgements

The Authors are grateful to Dr. G. Chevalier (Station d'Agronomie, Unité de Mycologie, INRA, Clermont-Ferrand, France) and Prof. A. De Miguel (Universidad de Navarra, Departamento de Botanica, Pamplona, Spain) who helped with collection of some samples in France and Spain, respectively. The research was funded by the Italian National Council for Research, Progetto strategico: ‘Biotecnologia dei funghi ectomicorrizici: dalle applicazioni agro-forestali a quelle agro-alimentari’.

References