FOLIA BIOLOGICA ET GEOLOGICA 61/2, 229–238, LJUBLJANA 2020 PCR PRIMERS COMPARISONS FOR A SUCCESSFUL TUBER SPP. DNA REGION AMPLIFICATION IN ROUTINE IDENTIFICATIONS PRIMERJAVA PCR ZAČETNIH OLIGONUKLEOTIDOV ZA USPEŠNO POMNOŽEVANJE DNA REGIJE TUBER SPP. PRI RUTINSKI IDENTIFIKACIJI Tina UNUK NAHBERGER 1 , Hojka KRAIGHER 1 & Tine GREBENC 1 http://dx.doi.org/10.3986/fbg0076 ABSTRACT PCR primers comparisons for a successful Tuber spp. DNA region amplification in routine identifications Since late 20 th century DNA sequencing became the method of choice method in precision species identification. The ITS region is one of the official fungal barcoding DNA markers, although in some cases sequencing of the ITS re- gion may, due to misidentification, mislabeling or nomen- clature errors in public databases, lead to incorrect or insuf- ficient identification, as is currently a case in the genus Tuber. The aim of this study was to test, which ITS primer pairs are most appropriate and optimal for Tuber species DNA region amplification. Thereby we (1) compared ampli- fication success for different Tuber species using fungal spe- cific primer pair ITS1f and ITS4 and (2) compared amplifi- cation success using different ITS primer pair combinations in amplifying DNA region an example species Tuber aesti- vum. Based on results, Tuber aestivum was one of the most reluctant Tuber species in this study and in most cases failed to amplify with the above primer pair. After comparing dif- ferent ITS primer pairs, we conclude that the primer pair ITS5 and ITS7 is the most appropriate primer pair for ampli- fication DNA region of T. aestivum as it resulted in high am- plification success from ectomycorrhizal root tips. Based on sequences, gained from public databases, we found that ITS1f and ITS6 primers have a mismatch in one base pair compared to the target sequence of Tuber aestivum, thus re- sulting in poor or no amplification success. Although prim- er pair ITS5 and ITS7 in our study was proven to be the most appropriate primer pair in amplifying DNA region Tuber aestivum species, further analysis about appropriateness of it for a general barcoding and identification of ectomycorrhiza in complex community samples is needed. 1 Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana Correspondence: Tina Unuk Nahberger, tina.unuk@gozdis.si, Hojka Kraigher, hojka.kraigher@gozdis.si, Tine Grebenc, tine.grebenc@gozdis.si IZVLEČEK Primerjava PCR začetnih oligonukleotidov za uspešno pomnoževanje DNA regije Tuber spp. pri rutinski identi- fikaciji Od konca 20. stoletja je določanje nukleotidnega zapo- redja DNA postalo ena izmed pogosteje uporabljenih metod za določanje vrst. ITS regija je edna izmed uradnih glivnih DNA markerjev, čeprav lahko določanje nukleotidnega za - poredja le-te, v nekaterih primerih, predvsem zaradi napač- ne določitve, označevanja oziroma napak v nomenklaturi v javnih bazah podatkov, privede do napačne oziroma nena- tančne določitve vrst, kar je trenutno težava pri določitvi vrst iz rodu Tuber. Namen te študije je bil testirati kateri pari ITS začetnih oligonukleotidov so najbolj primerni in opti- malni za pomnoževanje DNA regij gliv iz rodu Tuber. S tem namenom smo v študiji (1) primerjali uspešnost pomnože- vanja DNA regije različnih vrst iz rodu Tuber, z uporabo glivno specifičnih začetnih oligonukleotidov ITS1f in ITS4 ter hkrati (2) primerjali uspešnost pomnoževanja DNA regi- je vrste Tuber aestivum z uporabo različnih ITS začetnih oli - gonukleotidov. Na podlagi rezultatov ugotavljamo, da je vrsta T. aestivum izmed vseh analiziranih gliv iz rodu Tuber, bila najtežavnejša vrsta v naši študiji, saj je v večini primerov pomnoževanje DNA regije te vrste z uporabo glivno speci- fičnih začetnih oligonukleotidov ITS1f in ITS4 bilo neuspe - šno. Po primerjavi uspešnosti pomnoževanja z različnimi ITS začetnimi oligonukelotidi ugotavljamo, da sta bila v naši študiji ITS začetna oligonukleotida ITS5 in ITS7 najprimer - nejša za pomnoževanje DNA regije vrste T. aestivum, saj je bila uspešnost pomnoževanja iz ektomikoriznih vršičkov v tem primeru največja. Na podlagi T. aestivum nukleotidnih zaporedij pridobljenih iz javnih podatkovnih baz ugotavlja- mo, da je za začetna oligonukleotida ITS1f in ITS6 značilno UNUK NAHBERGER, KRAIGHER, GREBENC: PCR PRIMERS COMPARISONS FOR A SUCCESSFUL TUBER SPP. DNA 230 FOLIA BIOLOGICA ET GEOLOGICA 61/2 – 2020 Keywords: Tuber spp., ITS region, PCR amplification, ITS primers neujemanje s tarčnim nukleotidnim zaporedjem (T. aesti- vum) v enem baznem paru, kar se lahko odraža bodisi v slabšem pomnoževalnem uspehu ali v nepomnoževanju na splošno. Kljub temu, da v naši študiji ugotavljamo, da sta začetna oligonukleotida ITS5 in ITS7 najprimernejša za po- množevanje DNA regije glive T. aestivum, so potrebne na- daljnje analize, s katerimi bi potrdili splošno primernost omenjenega para ITS5/ITS7 za pomnoževanje DNA regije ne samo vrst iz rodu Tuber, temveč za določanje ektomikori - znih glivnih združb na splošno. Ključne besede: Tuber spp., ITS regija, PCR pomno- ževanje, ITS začetni oligonukleotidi 1. INTRODUCTION Functioning of forest ecosystems depends on the inter- actions between roots of vascular plants and mycor- rhizal fungi, which’s central role is capturing and re- translocation of soil nutrients and water, and conse- quently sustaining above-ground vegetation (Smith & Read , 2008). Association between mycorrhizal fungi and vascular plants is one of the key players in soil ecology (Dahlberg, 2001; Allen et al., 2002). Besides the importance of mycorrhizal fungi for functioning and nutrient transport in forest ecosystems, at least 400 mycorrhizal fungal species produce edible sporo- carps (fungi) thus representing an important ecosys- tem service with a high economic interest (Boa, 2004; Bakker et al., 2019). Among edible fungi, truffles are the most appreciated and expensive (Amicucci et al., 1998; Bohannon , 2009). Truffles are ectomycorrhizal fungi, belonging to the genus Tuber. There are at least 180 known species of truffles (Bonito et al., 2010; Gryndler et al., 2011), with new being described fre- quently (Milenković et al., 2016; Grupe et al. 2018), for this reason there is a high interest for their timely and accurate identification. DNA based methods have in recent decades be- came a critical research tool in fungal taxonomy, as DNA sequencing in many cases represents the most reliable tool for unequivocal species identification (Kang et al., 2010). Since early 1990 the internal tran- scribed spacer (ITS) region had been among most fre- quently sequenced genetic markers for identification of fungi (White et al., 1990) and for analyzing composi- tion and dynamics of ectomycorrhizal communities (Gardes et al., 1991; Kraigher et al., 1995; Horton & Bruns , 2001; Begerow et al., 2010; Schoch et al., 2012). The ITS region is a molecular marker with high power for species-level identification. Due to its wide- spread use and ease to amplify it, it was selected as one of official fungal barcoding DNA markers (Raja et al., 2017). The average size of the ITS region in fungi is about 550 base-pairs, but may vary considerably among lineages (Feibelman et al., 1994; Schoch et al., 2012). The ITS region is composed of the two variable spacers, namely ITS1 spacer and ITS2 spacer, and of a highly conserved 5.8S ribosomal gene (White et al., 1990). The ITS region molecular marker shows high probability of correct identification at the species level for a broad group of fungi, except in some highly spe- cific genera where its separating power is low (Schoch et al., 2012) or in cases where an intraspecific ITS re- gion variation may lead to fail in a species identifica- tion (Linder et al., 2011; Chen et al., 2016; Li et al., 2013). For the genus Tuber the current identification based on BLAST analysis of the ITS region sequences (Altschul et al. 1990) may be challenging, due to spe- cies misidentification, mislabeling or nomenclature er- rors in the public databases, and due to insufficiently representation of some taxa in databases (Trappe 2004; Halasz et al., 2005; Iotti et al., 2007). The ten- dency the Tuber ITS region amplification went in di- rection of designing and using species-specific ITS primer-pairs that may not give satisfactory amplifica- tion results over all species in the genus. Thereby, the aim of this study was to estimate an efficient universal fungal ITS primer pair (ITS 1f, ITS4; sensu Gardes & Bruns , 1993) to amplify DNA from various Tuber spe- cies. In species where these primers did not yield suf- ficient amplification, other available ITS primer pair combinations were tested, both on sporocarps’ and ectomycorrhizas’ isolated DNA samples. UNUK NAHBERGER, KRAIGHER, GREBENC: PCR PRIMERS COMPARISONS FOR A SUCCESSFUL TUBER SPP. DNA 231 FOLIA BIOLOGICA ET GEOLOGICA 61/2 – 2020 2.1 Biological material Sporocarps for testing (Table 1) were selected from the collection at the Slovenian Forestry Institute and sev- eral other collections, so as to represent most frequent- ly collected morphological Tuber species (Grebenc et al. 2010). For testing the amplification from ectomyc- orrhizal samples, Tuber aestivum root tips from pot- planted and inoculated silver fir seedlings were used (Unuk Nahberger et al., 2020; in prep.). In total, 38 sporocarps were tested in the first step, and 113 ecto- mycorrhizal roots of T. aestivum on silver fir were test- ed in the second step. Ectomycorrhizal root tips were randomly chosen from silver fir root systems. The identity of T. aestivum ectomycorrhiza was confirmed following the methodology and identification key of Agerer (Agerer , 1987-2012). 2.2 DNA extraction DNA from sporocarps and from ectomycorrhiza was extracted using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) protocol, following manufacturer’s instructions. 2.3 PCR amplification Amplifications were performed in GeneAmp PCR Sys- tem 9700 (Applied Biosystems, USA) in a total volume 25 μl of the PCR amplification mixture, with volume of extracted DNA 1 ng/μl. The PCR reactions with the pairs of the barcoding primers ITS1f/ITS4 were per- formed as reported in Sulzbacher et al. (2016). The PCR reactions with the pairs of primers ITS5/ITS7, ITS5/ITS6 (Bertini et al., 1999) were performed using the protocol by Bertini et al. (1999). Based on the test of the barcoding primer (ITS1f/ITS4) with DNA extracts from sporocarps, the primer pairs ITS1f/ITS4, ITS1f/ ITS2, ITS5/ITS6 and ITS5/ITS7 were used in amplify- ing sporocarps’ DNA samples that fail to amplify in the barcoding test. The same selection of primers was also used in ectomycorrhizal DNA amplification from Tuber aestivum root tips. All PCR reactions were per- formed five times on all samples to demonstrate the optimality of individual primer pair for amplification. 2.4 Sequencing PCR products were run on 1.5% agarose gels in 0.5x TBE buffer and visualized with Gel Doc EQ System, PC (Biorad, USA). Amplified DNA fragments were cut out of agarose gels and purified with innuPREP DOU- BLEpure Kit (Analytik Jena AG, Jena, Germany) fol- lowing manufacturer’s instructions. Purified DNA fragments were sequenced at a commercial sequencing laboratory (Macrogen Inc., Seoul, South Korea). Sam- ples were sequenced in both directions either with the pairs of primers ITS1f/ITS4 (White et al., 1990; Gardes & Bruns , 1993) ITS1f/ITS2 or ITS5/ITS7 (Bertini et al., 1999). The obtained sequences were processed in Geneious version 11.1.4 (https://www.ge- neious.com, Kearse et al., 2012). Nucleotide base calls with an error probability greater than 5% were trimmed from read ends to improve read quality, while reads were assembled into contigs at 90% base pair similarity. BLASTN algorithm from NCBI website (National Center for Biotechnology Information; https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to assess the similarity of obtained ITS sequences to se- quences in GenBank. 2.5 Primers annealing position and mismatch analysis Sequences with amplified complete ITS region molec- ular marker, including partial 18S rRNA gene, com- plete ITS1, 5.8S rRNA and ITS2 genes, and partial 28S rRNA gene, were obtained from GenBank database at the National Centre for Biotechnology (NCBI). Se- quences from the database and our newly obtained se- quences were analyzed with Geneious version 11.1.4. For alignment, MAFFT alignment program as plugin available for Geneious was used. ITS primers mis- matches and annealing position with Tuber aestivum sequences from GenBank database were analyzed in Geneious, where all forward and reversed primers were tested with maximum mismatches set at 5 base pairs. 2. MATERIAL AND METHODS UNUK NAHBERGER, KRAIGHER, GREBENC: PCR PRIMERS COMPARISONS FOR A SUCCESSFUL TUBER SPP. DNA 232 FOLIA BIOLOGICA ET GEOLOGICA 61/2 – 2020 All available Tuber collections except T. aestivum and one collection of T. mesentericum yielded sufficient amplification with a fungal barcoding primer pair ITS 1f and ITS4 (Table 1) for downstream applications (e.g. Sanger DNA sequencing) without additional steps. For collections that failed amplification with a fungal barcoding primer pair ITS 1f and ITS4 a fur- ther selection of primers for amplification of the par- tial or complete ITS region of fungi primer pairs ITS1f/ITS4, ITS1f/ITS2, ITS5/ITS6 and ITS5/ITS7 were used to amplify DNA from sporocarps and T. aestivum ectomycorrhiza. As T. aestivum showed to be the most difficult one for amplifying with the ITS1f and ITS4 primer pair, further analyzes using different primer pairs were con- ducted to find the most appropriate and optimal prim- er pair for amplification of this species. Based on all together 113 tested T. aestivum mor- photypes, 27 Tuber morphotypes were successfully amplified and sequenced using primer pair ITS1f/ ITS4, 61 morphotypes were amplified and sequenced using primer pair ITS5/ITS7 and 25 morphotypes using primer pair ITS1f/ITS2, while primer pair ITS5/ ITS6 completely failed in amplification of T. aestivum 3. RESULTS Table 1: Collections with name, herbarium code, GenBank accession number and reference for each Tuber sporocarp samples used for testing the ITS1f and ITS4 barcoding primer pair. + amplification yielded enough DNA for down- stream applications, (+) amplification was successful but weak, - amplification failed. MA Fungi – The Herbarium at the Real Jardín Botánico, Madrid, Spain; MES – personal collection of Mattew E. Smith, USA; MS – personal collection of Marcelo Sulzbacher, Brazil; FHS – collection of the Institute for Multidisciplinary Research in Belgrade, Serbia; AP – personal collection of Andrej Piltaver, Slovenia. Morphological species name Herbarium code GenBank accession number Amplification with ITS1f & ITS4 Reference Tuber aestivum Vittad. MA Fungi 54693 FM205622 (+) Grebenc et al. 2010 Tuber aestivum Vittad. TUBAES/270211 - this study Tuber aestivum Vittad. TUBAES/060811A - this study Tuber aestivum Vittad. TUBAES/180812A (+) this study Tuber aestivum Vittad. TUBAES/060714A (+) this study Tuber aestivum Vittad. TUBAES/251014B - this study Tuber anniae W.Colgan & Trappe TUBsp/241013A + this study Tuber borchii Vittad. TUBBOR/100108 FM205630 + Marjanović et al. 2010 Tuber brumale Vittad. TUBBRU/150309 FN433128 + Grebenc et al. 2010 Tuber brumale var. moschatum (Bull.) Hall, Buchanan, Wang & Cole TUBBRUfoMOS/250109A FN433130 + Grebenc et al. 2010 Tuber excavatum Vittad. TUBEXC/070309G FN433148 + Grebenc et al. 2010 Tuber excavatum Vittad. TUBEXC/110812A + this study Tuber floridanum Grupe, Sulzbacher & M.E. Sm. MES654 (Holotype) MF611781 + Grupe et al. 2018 Tuber floridanum Grupe, Sulzbacher & M.E. Sm. MS475 MF611782 + Grupe et al. 2018 Tuber foetidum Vittad. FHS-Tmes FM205704 + Marjanović et al. 2010 Tuber fulgens Quél. TUBFUL/221008 FN433154 + Grebenc et al. 2010 Tuber fulgens Quél. TUBFUL/041008B FN433150 + Grebenc et al. 2010 Tuber himalayense B.C. Zhang & Minter AP-T71 FM205589 + this study Tuber indicum Cooke & Massee AP-T50A FM205590 + this study Tuber macrosporum Vittad. FHS-455 FM205663 + Marjanović et al. 2010 Tuber macrosporum Vittad. FHS-449 FM205664 + Marjanović et al. 2010 Tuber maculatum Vittad. MA Fungi 57008 FM205560 + Grebenc et al. 2010 Tuber maculatum Vittad. FHS-399 FM205644 + Marjanović et al. 2010 Tuber maculatum Vittad. FHS-426 FM205645 + Marjanović et al. 2010 Tuber magnatum Pico TUBMAG/141207 FM205633 + Marjanović et al. 2010 UNUK NAHBERGER, KRAIGHER, GREBENC: PCR PRIMERS COMPARISONS FOR A SUCCESSFUL TUBER SPP. DNA 233 FOLIA BIOLOGICA ET GEOLOGICA 61/2 – 2020 root tips. Primers pairs showed different concentration of amplified DNA as shown in Figure 1 for selected representative samples. Primer pair ITS5/ITS7 showed the strongest intensities on the agarose gel for most of amplified DNA. Ectomycorrhizal DNA was in most cases difficult to amplify with primers pairs ITS1f/ ITS4, ITS1f/ITS2 or with ITS5/ITS6 yielding low or no amplification at all. 3.1 Primers annealing position and mismatch analysis From the GenBank database, sequences with partial 18S rRNA gene, complete ITS1 spacer, 5.8S rRNA gene, ITS2 spacer and partial 28S rRNA gene sequence, were downloaded and analyzed for primers annealing posi- tion and nucleotide mismatches. Primers pairs ITS1f/ ITS2, ITS1f/ITS4, ITS5/ITS6 and ITS5/ITS7 were in- cluded in analysis. The alignment of used sequences, showed that ITS1f primer is located 14 nucleotides up- stream ITS5 primer, where several sequences of suita- ble lengths, showed on ITS1f primer mismatch on the 12 th primer nucleotide, with oligo base A and target base G. For primer ITS5 no mismatches were observed. The primers ITS4 and ITS6 are located 35 nucleotides and 8 nucleotides upstream regarding primer ITS7. With the primer ITS2, only complete internal tran- scribed spacer 1 can be amplified, as it is located in 5.8S rRNA gene. The primer ITS6 had also a mismatch on the 5 th primer nucleotide, with oligo base G and target base A, which was confirmed for 42 of 45 ana- lyzed sequences. The amplification product obtained by using the primer pair ITS1f/ITS2 is the smallest, while the product obtained by using the primer pair ITS5/ITS7 is the biggest PCR fragment. Tuber magnatum Pico FHS-465 FM205651 + Marjanović et al. 2010 Tuber mesentericum Vittad. TUBMES/060811A - this study Tuber mesentericum Vittad. TUBMES/020912 (+) this study Tuber mesentericum Vittad. TUBMES/110114B + this study Tuber oligospermum (Tul. & C. Tul.) Trappe FHS-XX13 FM205683 + Marjanović et al. 2010 Tuber oligospermum (Tul. & C. Tul.) Trappe MA Fungi 39553A FM205505 + Grebenc et al. 2010 Tuber petrophilum Milenković BEO 20600 HG810883 + Milenković et al. 2016 Tuber petrophilum Milenković BEO 20601 HG810884 + Milenković et al. 2016 Tuber rufum Pollini TUBRUF/070911B + this study Tuber rufum fo. nitidum (Vittad.) Montecchi & Lazzari) FHS-XX1 FM205677 + Marjanović et al. 2010 Tuber rufum fo. apiculatum E. Fisch FHS-353 FM205669 + Marjanović et al. 2010 Tuber rufum fo. ferrugineum (Vittad.) Montecchi & Lazzari TUBRUFvarFER/041008 FN433160 + Grebenc et al. 2010 Tuber rufum fo.lucidum (Bonnet) Montecchi & Lazzari FHS-471 FM205665 + Marjanović et al. 2010 Tuber rufum Pollini TUBRUFvarRUF/070908B FN433168 + Grebenc et al. 2010 4. DISCUSSION Due to its variability in length and nucleotide se- quence among different fungi, the internal tran- scribed spacer (ITS) region has been frequently re- ported as a convenient target region for species de- limitation in fungi and molecular identification of ectomycorrhizal fungi (Gardes & Bruns, 1993; Simon et al., 1992; Henrion et al. 1994; Lanfranco et al., 1993; Amicucci et al., 1998; Bertini et al., 1999, Benucci et al., 2011a). Although its broad use- fulness for many taxa, repeated fails in amplification with universal barcoding ITS primers were reported for some. Besides technical reasons, such as interfer- ence of inhibitors in PCR, or a non-optimal PCR pro- tocols, Bertini et al. (1999) suggested that most often, the problem lies in the primer sequences, as they in their study confirmed a significant interference of high base pair coupling degree with the annealing ef- ficiency. A fungal barcoding primer pair ITS1f and ITS4 in our study amplified most of the Tuber species selected among European, North American and Asian species, with an exception of a broadly distributed European UNUK NAHBERGER, KRAIGHER, GREBENC: PCR PRIMERS COMPARISONS FOR A SUCCESSFUL TUBER SPP. DNA 234 FOLIA BIOLOGICA ET GEOLOGICA 61/2 – 2020 Figure 1. Partial or complete ITS region amplification success in representative DNA extracts from Tuber aestivum ectomycor- rhiza. Primer pairs for partial ITS amplification ITS1f/ITS2 (lines 1-6), the fungal ITS barcoding primer pair ITS1f/ITS4 (lines 7-12), and two alternative primer pairs ITS5/ITS7 (lines 13-18) and ITS5/ITS6 (lines 19-24) were used. Amplified ITS regions obtained with different primers pairs were evaluated and grouped based on intensities of bands (concentrations of DNA after amplification) in: ++ strong intensity; + moderate intensity, (+) weak intensity, and - unsuccessful amplification of the ITS region. species T. aestivum. This primer pair repeatedly failed to amplify both T. aestivum DNA extracted from spo- rocarps and from ectomycorrhiza, suggesting a nega- tive influence of the primer mismatch in the ITS1f primer sequence as a potential cause of its uselessness in sporocarp and ectomycorrhiza barcoding. Similar lack of amplification was also seen in other primer pair combinations. Primer pair ITS5/ITS6 was not an appropriate primer pair in our study, since the T. aes- tivum amplification using this specific primer pair was not successful. ITS6 primer also showed a mis- match between oligo and target base in comparison to UNUK NAHBERGER, KRAIGHER, GREBENC: PCR PRIMERS COMPARISONS FOR A SUCCESSFUL TUBER SPP. DNA 235 FOLIA BIOLOGICA ET GEOLOGICA 61/2 – 2020 Figure 2: Schematic view of the complete ITS region in fungi, with marked mismatching positions for individual primers in Tuber aestivum sequence. Tuber spp. annealing site sequence, as we have shown in the primers mismatch analy- sis. T. aestivum amplification using primer pair ITS1f/ITS2 also resulted in poor, or no amplification, despite shorter sequences which, such as in primer pair ITS1f/ITS2, should be amplified easier and with better success. Also, in primer pair ITS1f/ITS2 we assume primer mismatch in ITS1f to be the reason for poor amplification outcome. As previously reported by Bertini et al. (1999), less efficient amplification using primer pairs ITS1f/ITS4, ITS1f/ITS2 or ITS5/ITS6 can also be a result of primer to primer in- teraction, which may have a significant ef- fect on annealing and finally on amplifica- tion efficiency of the PCR reaction run under the same reaction conditions. On the other hand, the primer pair ITS5/ITS7 successfully amplified DNA of T. aestivum DNA both from clean sporocarps and from over 70% of all mixed DNA sam- ples of ectomycorrhizal roots. The primer pair ITS5/ITS7 was already reported to be suitable for PCR amplification of Tuber spe- cies, as allowed amplification even at low DNA quality and concentration (in our study in case of sample E18/26). The primer ITS5 was suggested to be more appropriate than the ITS1 primer, as it forms less nucleo- tide interactions (Bertini et al., 1999). In general, designing and optimization of the most efficient pairs of primers for Tuber species detection and characteriza- tion is of high interest for many biotechno- logical applications. Appropriate molecular techniques, as is in this case the use of ap- propriate primers pairs, are very important in the food industry (Strojnik et al. 2020, Šiškovič et al. 2020), in the in vitro propa- gation of mycelia to verify presence of truf- fles of high economic, or in testing and cer- tifying natural truffle-grounds and truffle plantations (Amicucci et al., 1998, Benucci et al. 2011b). 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New Phy- tologist 150: 555-562. https://doi.org/10.1046/j.1469-8137.2001.00142.x ACKNOWLEDGEMENTS The study was funded by the Young Researcher Scheme (Slovenian Research Agency) for Tina Unuk and co- financed by The Research Programme P4-0107 Forest Biology, Ecology, and Technology (Slovenian Research Agency), a basic research project J4-1766 “Methodolo- gy approaches in genome-based diversity and ecologi- cal plasticity study of truffles from their natural distri- bution areas” (Slovenian Research Agency), bilateral cooperation with Montenegro (BI-ME/18-20-017) and Bosnia and Herzegovina (BI-BA/19-20-027), and the LIFEGENMON project (LIFE ENV/SI/000148). We would also like to thank to dr. Marìa P. Martín and dr. Žaklina Marjanović for access to their Institutional herbaria (MA-Fungi and FHS, respectively) and to prof. Matthew E. Smith, dr. Marcelo Sulzbacher, and dr. Andrej Piltaver, for contributing samples or DNA sequences for this study. 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