10.6092/issn.2531-7342/19004
Truffles represent a resource of great value all over the world. The various species are widespread and occupy various habitats. Methods to identify their value in terms of marketing, cultivation, and the ecological–forestal field are needed. The traditional classification of Tuber species is based on their morphology; nevertheless, the macroscopic and microscopic characteristics of the fruiting body, spores, and ectomycorrhizae may be absent or insufficiently discriminating. This problem is amplified when dealing with valuable truffle species, which are often replaced by truffles with lower organoleptic qualities and commercial value. Today, molecular biology can help to correctly identify them through several widely used methods that are fast, specific, and sensitive. Molecular methods are mostly PCR-based, with primers designed on various targets, genes, or regions widely variable between species but sufficiently conserved within the same species. The DNA regions analyzed for identifying truffles include ribosomal internal transcribed spacer (ITS), ribosomal large subunit (LSU), β-tubulin, and EF-α elongation factor. This review summarizes the main and most reliable DNA-based molecular methods for identifying Tuber species. The methods are listed schematically and clearly for quick consultation and use in all areas of study in which monitoring, and verification of the species is essential.
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Overview of molecular methods for quick identification of <em>Tuber</em> species
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Zampieri E, Murat C, Cagnasso M, Bonfante P, Mello A (2010) Soil analysis reveals the presence of an extended mycelial network in a Tuber magnatum truffle-ground: mycelial network in a T. magnatum truffle-ground. FEMS Microbiology Ecology 71:43–49. https://doi.org/10.1111/j.1574-6941.2009.00783.x
Overview of molecular methods for quick identification of <em>Tuber</em> species
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                     IJM - Italian Journal of Mycology
                     ISSN 2531-7342 - Vol. 53 (2024): 45 - 64
                     Journal homepage: https://italianmycology.unibo.it/

Review


       Overview of molecular methods for quick identification of
                            Tuber species
                                     Antonella Amicucci1, Bianca Ranocchi1

1
    Department of Biomolecular Sciences, Urbino University; via Saffi 2, 61029 Urbino, Italy


Corresponding author e-mail: antonella.amicucci@uniurb.it
______________________________________________________________________________

ARTICLE INFO
Received 30/01/2024; accepted 23/02/2024
https://doi.org/10.6092/issn.2531-7342/19004

Abstract
Truffles represent a resource of great value all over the world. The various species are widespread and occupy various
habitats. Methods to identify their value in terms of marketing, cultivation, and the ecological–forestal field are needed.
The traditional classification of Tuber species is based on their morphology; nevertheless, the macroscopic and
microscopic characteristics of the fruiting body, spores, and ectomycorrhizae may be absent or insufficiently
discriminating. This problem is amplified when dealing with valuable truffle species, which are often replaced by truffles
with lower organoleptic qualities and commercial value. Today, molecular biology can help to correctly identify them
through several widely used methods that are fast, specific, and sensitive. Molecular methods are mostly PCR-based, with
primers designed on various targets, genes, or regions widely variable between species but sufficiently conserved within
the same species. The DNA regions analyzed for identifying truffles include ribosomal internal transcribed spacer (ITS),
ribosomal large subunit (LSU), β-tubulin, and EF-α elongation factor. This review summarizes the main and most reliable
DNA-based molecular methods for identifying Tuber species. The methods are listed schematically and clearly for quick
consultation and use in all areas of study in which monitoring, and verification of the species is essential.

Keywords
Truffle, ectomycorrhizae, processed food, ITS region, PCR

________________________________________________________________________________

Introduction

Truffles are the fruiting bodies of ectomycorrhizal fungi belonging to the genus Tuber, order
Pezizales, and division Ascomycota (Læssøe and Hasen, 2007; Trappe et al., 2009). They form an
ectomycorrhizal symbiosis on the roots of different tree species, including beech, oak, birch, poplar,
hazel, hornbeam, and pine (Harley and Harley, 1987). Some Tuber species are edible and appreciated
for their intense and superb aroma (Pelùsio et al., 1995; Mello et al., 2006) acting as cue for animals
which are thus able to spread their spores (Splivallo et al., 2011).


                                                      ______________
    Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
    The journal is hosted and maintained by AlmaDL. © 2024 The Authors. This work is licensed under CC BY 4.0
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      Amongst the known 200 Tuber species (Bonito et al., 2013), the most expensive species is
Tuber magnatum Picco, whose ascomata retail prices vary from EUR 1000/kg to EUR 6000/kg in
Italy but can reach higher prices outside of Italy (Hall et al., 2007). For these reasons, truffles
represent a notable economic resource for Italy. In 2018 (latest data available), the economic value
of truffle exports was around 49.2 million euros for fresh or frozen truffles and around 13.7 million
euros for preserved truffles (Istat, 2019).
      Moreover, valuable edible truffles have a fundamental role in various economic fields,
including gastronomy, tourism, retail markets, and truffle cultivation (Thomas, 2014; Büntgen et al.,
2017) and Europe accounts for 85% of the world truffle export market (Hamzić Gregorčič et al.,
2020). Another important aspect is the conservation of natural forests; for example, the European
Tuber melanosporum Vittad. can grow in natural areas without the utilization of machinery and
chemicals, and their plantations represent a firebreak thanks to low plantation densities, soil tilling,
and, therefore, the herbicidal effect of the fungus (Büntgen et al., 2017).
      The unequivocal identification of the species is fundamental, as it guarantees all phases of
mycorrhizal seedlings production, cultivation, trade, and the research experimental models setting up
(Zambonelli et al., 2021). In the production of mycorrhized seedlings, it is essential to verify the
identity of the truffles used for inoculation, the presence of the selected species, and to minimize the
contamination risk of mycorrhized roots by competitive fungi. It is important to monitor the presence
of specific Tuber mycelium in the greenhouse before and after transplanting in the field of
mycorrhized seedlings to verify the presence of competitive fungi. For example, Pulvinula
constellatio Berk. and Br. Boud. are ascomycete fungi that can create ectomycorrhizal infections in
plants with Tuber spp. in greenhouses (Amicucci et al., 2001). Regarding commerce, T.
melanosporum may be exchanged for the Chinese truffle Tuber indicum Cooke & Massee because it
is morphologically very similar, but the latter has very low quality (Lazzari et al., 1995). T. magnatum
can be the subject of fraud or confused with similar species, especially when the salient morphological
features for identification are missing and at the mycorrhizal level where it is typically contaminated
by other species (Iotti et al., 2012; Mello et al., 2001). Over the years, numerous researchers have
defined the distinctive morphological characters of the species (Amicucci et al., 1998; Pacioni et al.,
1999; Mello et al., 2000; Paolocci et al., 2004; Kovács et al., 2006; Marozzi et al., 2020). Although
morphological methods are fundamental, taxonomically and morphologically similar species may
have different organoleptic and commercial value. Moreover, sometimes some distinctive
morphological features are absent: immature ascomata lack spores, mycelia are poorly defined among
the many species, mycorrhizae can lose their typical ornamentations, and cooking or other food
preparation processes can alter their distinctive characteristics (Iotti et al., 2012).
      For these reasons, for decades, the ever-growing interest in this precious fungus has prompted
many researchers to carry out increasingly refined research to find molecular markers capable of
recognizing truffles in all phases of their life cycle. Numerous molecular markers have been
developed to identify the most economically important truffle species to date (Gardes et al., 1991;
Mello et al., 1999; Paolocci et al., 1999). The molecular methods have become indispensable to
complement the traditional methods, overcome the lack of distinctive elements, increase the
reliability of identification, and speed up the analysis.
      This review aims to illustrate and list the molecular methods used for identification. The
development of the PCR technique enabled the molecular identification of truffles, starting with the


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work by Gardes et al. in the 1990s (Gardes et al., 1991). With the PCR technique, many researchers
screened target DNA sequences conserved within a species, but variable among different species
(Mello et al., 1999).
       The genome sequencing of different Tuber species has also provided various advantages by
expanding the distinctive target DNA’s availability, hypervariable regions, and specific mating types
of genes. One of the regions most used for evolutionary, diagnostic, and taxonomic studies is the
ribosome internal transcribed spacer region (ITS) of nuclear rDNA. The ITS region is located
between ribosomal rRNA 18S and 28S genes and includes the gene for the 5.8S rRNA (Jeandroz et
al., 2008). ITS regions are present in multiple copies within the same genome, and the intraspecific
homogeneity in Tuber is highly conserved; these characteristics made this target suitable for
designing species-specific primers for several years (Ciarmela et al., 2022), although intragenomic
variability has recently emerged in some fungi (Bradshow et al., 2023).
       Furthermore, β-tubulin is a good barcoding region for designing Tuber-genus-specific primers
(Zampieri et al., 2009). At the same time, elongation factor 1-α was employed for designing new
primer pairs specific for each Tuber phylogenetic lineage and for phylogeographic analyses (Leonardi
et al., 2021). Finally, the microsatellite regions have also been studied to find species-specific
markers. However, these targets have proved to be more versatile for characterizing intraspecific
variability and have no longer been considered for these purposes (Amicucci et al., 2002; Murat et
al., 2011; Molinier et al., 2013; Molinier et al., 2016).

Molecular methods based on ITS region

ITS (Internal transcribed spacer) is a non-coding ribosomal DNA region interposed to ribosomal
genes 18S e 28S, as shown in Figure 1, and repeated in clusters in all eukaryote genomes.




                                        Fig. 1. – Scheme of the ITS region of fungi.



      The repeated units of transcribed rDNA regions are separated by intergenic spacers (IGS)
(Ciarmela et al., 2002; Jeandroz et al., 2008). An IGS separates the large subunit (LSU) rRNA gene
(encoding 5.8S and 28S rRNA) in one repeat from the small subunit (SSU) rRNA gene (encoding
18S rRNA) in the adjacent downstream repeat (Ciarmela et al., 2002; Jeandroz et al., 2008). This IGS
region, expressing extremely intraspecies variability, allows studies of intraspecies and taxonomic
variability but is not a suitable target for developing species-specific markers (Ciarmela et al., 2002).

Restriction Length Fragment Polymorphism Analyses (RFLP)
ITS may present polymorphisms of length and sequence and is amplifiable by using universal primers
(White et al., 1990; Bruns et al., 1991; Gardes et al., 1993). The length of the ITS region is quite
similar between the different species, while the restriction fragment length polymorphism analysis
(RFLP) produces characteristic profiles. The ITS amplicons are digested with specific restriction
endonucleases selected from the restriction maps of the ITS sequences of each species to detect


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  polymorphisms. The digested DNA is separated by gel electrophoresis, from which the pattern RFLP
  is produced (Bertini et al., 1998; Paolocci et al., 1999; Bertini et al., 2006). The variable lengths of
  cleavage fragments are characteristic of a sequence of DNA. The restriction analysis of the ITS region
  allows obtaining species-specific patterns for different species of truffles: electrophoretic separation
  allows the comparison of restriction profiles produced by different species. The results of this method
  are obtained quickly and are easy to interpret. Table 1 summarizes the enzymes that can be used to
  identify white truffle species.


Table 1 - Enzymes specific for white truffle species.
Species              Enzymes
                   MspI       RsaI     AvaII      EcoRI      NcoI      HinfI      TaqI     MboI      ClaI     AluI      KpnI     StuI     SfiI    ScaI
T. magnatum          +        +        +          +          +         +          +        -         +        -         +        +        +       -
T. borchii           +        +        +          +          -         +          +        -         +        +         -        -        -       -
T. puberulum         +        +        +          -          -         -          -        -         -        -         -        -        -       +
T. dryophilum        +        +        +          -          +         -          -        -         -        -         -        -        -       -
T. maculatum         +        +        +          -          -         +          -        +         -        +         -        -        -       -
Species-specific enzyme = +; non-species-specific enzyme = -


        As regards the species T. melanosporum and T. indicum, the research of Paolocci et al. reports
  the possibility of distinguishing these two species through ITS restriction analysis using the RsaI or
  SspI enzymes (Paolocci et al., 1997). The sizes of the restriction fragments, which are useful for
  recognition, are shown in Table 2.



              Table 2 - ITS size fragments by RsaI and SspI
               Species                  Size fragments (bp) by enzyme RsaI               Size fragments (bp) by enzyme SspI
               T. melanosporum          191/429                                          Not cut
               T. indicum B1            123/497                                          418/202
               T. indicum B2            Not cut                                          418/202
               T. indicum A             153/276/195                                      376/248



  ITS specific primers
  The ITS regions of the different Tuber species were sequenced, analyzed, and aligned through a
  dedicated computer program. The sequence alignment analyses allowed the detection of species-
  specific regions, and with the support of web platforms, specific primer pairs were selected,
  respecting the specificity of pairing, the length, the GC content, and avoiding inverted repeated
  sequences. Table 3 lists the specific primer pairs available for Tuber species to date.




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     Table 3 - List of specific primer pairs available for Tuber species and conditions used for PCR analysis.
      Tuber species                                 Primer name         Primer sequence (5’-3’)                            Frag. length       Thermocycling             Accession No.        Reference
                                                                                                                           (bp)               parameters                GenBank
      T. magnatum                  SCAR             TAR I               CCCCAACGCGTAAAAGAGTA                               145                25 cycles: 94 °C          ---                  Amicucci et al., 1997
                                   (RAPD)           TAR II              CGTATCGGTGTCTTGCCAGTG                                                 45 s, 53 °C 45 s
                                   ITS-             TmagI               GGATGCGTCTCCGAATCCTGAA                             173                25 cycles: 94 °C          AF003911             Amicucci et al., 1998
                                   rDNA                                 T                                                                     20 s, 62 °C 15 s
                                                    TmagII              TCGGGCCCTTTCTCAGACTGCTG

                                   ITS-             P7                  TCCTACCAGCAGTCTGAGAAAG                             434                40 cycles: 94 °C          ---                  Mello et al., 1999;
                                   rDNA                                 GGC                                                                   45 s, 65 °C 45 s,                              Mello et al. 2006
                                                    M3                  TGAGGTCTACCCAGTTGGGCAG                                                72 °C 45 s
                                                                        TGG
                                   ITS-             MG09 I              TCACTTGGGGCGTCCGAGAT                               158                30 cycles: 94 °C          ---                  Rossi et al., 2000
                                   rDNA             MG09 II             ACTGCTGGAAACGGGGAATG                                                  15 s, 52 °C 15 s
                                   ITS-             TmgITS1for          GCGTCTCCGAATCCTGAATA                               106                25 cycles: 95 °C          ---                  Iotti et al., 2012
                                   rDNA             TmgITSrev           ACAGTAGTTTTTGGGACTGTGC                                                20 s, 60 °C 30 s,
                                                                                                                                              72 °C 40 s
                                   ITS-             Tmag3               TTAACTGTTTAAGTTTGTCAGGC                            151                35 cycles: 94 °C          FM205629             Rizzello et al., 2012
                                   rDNA             Tmag4               CCTGAATATCTCCTGTGTACCAT                                               30 s, 56 °C 30 s,
                                                                                                                                              72 °C 2 min
                                   ITS-             ITSMAGN             GTCACTGAAAACCCACTCACG                              230                25 cycles: 95 °C          AF325174             Rubini et al., 2001
                                   rDNA             ITSBACK3            TGAGGTCAACCCAGTTGGACAG                                                30 s, 63 °C 30 s,
                                                                        T                                                                     72 °C 45 s
                                   ITS-             TmagI               GGATGCGTCTCCGAATCCTGAA                             635                30 cycles: 94 °C          AF003911             Amicucci et al., 2000
                                   rDNA                                 T                                                                     20 s, 62 °C 15 s,
                                                    ITS4                TCCTCCGCTTATTGATATGC                                                  72 °C 30 s
                                   β-tubulin        Tubmagnf            CCTCCCAATTTGCAATACAC                               282                25 cycles: 94 °C          FN252812             Zampieri et al., 2010
                                                    Elytubr             AAAGACGAAGTTATCTGGCCTG                                                30 s, 61 °C 30 s,
                                                                        A                                                                     72 °C 30 s
      T. borchii                   ITS-             TboI                TGTATGGGATGCCCTATCGGAC                             397                30 cycles: 94 °C          AF003920             Amicucci et al., 1998


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                                   rDNA                                 T                                                                     20 s, 55 °C 15 s,
                                                    TboII               CTATTACCACGGTCAACTTC                                                  72 °C 30 s
                                   ITS-             TBA                 TGCCCTATCGGACTCCCAAG                               432                40 cycles: 94 °C          ---                  Mello et al., 1999
                                   rDNA             TBB                 GCTCAGAACATGACTTGGAG                                                  45 s, 65 °C 45 s,
                                                                                                                                              72 °C 45 s
                                   ITS-             rTboII              GAAGTTGACCGTGGTAATAG                                                  30 cycles: 94 °C          AF003920             Amicucci et al., 2000
                                   rDNA             ITS4                TCCTCCGCTTATTGATATGC                               185                20 s, 62 °C 15 s,
                                                                                                                                              72 °C 30 s

                                   SCAR             TB I                AGAGAATACATGCGGTAGTGT                              363                30 cycles: 94 °C          U94390               Bertini et al., 1998
                                   (RAPD)           TB II               CTCGCTTGGAGATAATAACAG                                                 30 s, 58 °C 30 s,
                                                                                                                                              72 °C 40 s
                                   SCAR             TBF1for             TCACCAAGCACTAACTCCTCTCT                            699                30 cycles: 94 °C          U83996.1             De Bellis et al., 1998
                                   (TBF gene)                                                                                                 30 s, 53 °C 30 s,
                                                                                                                                              72 °C 45 s
                                                    TBF2rev             CCAAACCGAATCAAACAACTAA
                                                                        T
      T. maculatum                 ITS-             TmacI               GACACAGGCTCCCGATAAAACA                             407                25 cycles: 94 °C          AF003909             Amicucci et al., 1998
                                   rDNA                                 C                                                                     20 s, 62 °C 15 s,
                                                                                                                                              72 °C 30 s
                                                    TmacII              CAGCAGCACTGATAGCCCCG
                                                    rTmacII             CGGGGCTATCAGTGCTGCTG                               225                30 cycles: 94 °C          AF003909             Amicucci et al., 2000
                                                    ITS4                TCCTCCGCTTATTGATATGC                                                  20 s, 62 °C 15 s,
                                                                                                                                              72 °C 30 s
      T. dryophilum                ITS-             TdryI               ATCGGGCTCCCAAGCAAAACA                              510                30 cycles: 94 °C          AF003917             Amicucci et al., 1998
                                   rDNA                                                                                                       20 s, 62 °C 15 s,
                                                    TdryII              TCTACTACCATGGTTCACTTT                                                 72 °C 30 s

      T. puberulum                 ITS-             TpuI                TCTGTTACCAGGGTCCACATT                              368                30 cycles: 94 °C          AF003918             Amicucci et al., 1998
                                   rDNA             TpuII               GGCTTCTGGGTTGAGGTGTTT                                                 15 s, 55 °C 30 s
                                   ITS-             TpuI                TCTGTTACCAGGGTCCACATT                              560                30 cycles: 94 °C          AF003918             Amicucci et al., 2000
                                   rDNA             ITS4                TCCTCCGCTTATTGATATGC                                                  20 s, 62 °C 15 s,
                                                                                                                                              72 °C 30 s
      T. rufum                     ITS-             Ru1f                TGCTTTCCCAGGTGGTTGG                                566                30 cycles: 94 °C          AF106892,            Iotti et al., 2007
                                   rDNA             ITS4                TCCTCCGCTTATTGATATGC                                                  20 s, 63 °C 20 s,         AY112894


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                                                    Ru2f                TTGC TTTCCCAGGGAATTGG                              572                72 °C 30 s
                                                    ITS4                TCCTCCGCTTATTGATATGC
      T. macrosporum               ITS-             Tmacr For           CGTCGCTCATCAAAGCAGTC                               248                33 cycles: 94 °C          AF106885,            Benucci et al., 2011
                                   rDNA                                                                                                       20 s, 61 °C 20 s,         FM205663,
                                                                                                                                              72 °C 20 s                FM205664,
                                                    Tmacr Rev           CCGCCAGTACCACCAGGAG                                                                             AY112895
      T. melanosporum              ITS-             ITSML               TGGCCATGTGTCAGATTTAGTA                             436                23 cycles: 94 °C          U89359               Paolocci et al., 1997
                                   rDNA             ITS4LNG             TGATATGCTTAAGTTCAGCGGG                                                30 s, 63 °C 30 s,
                                                                                                                                              72 °C for 45 s
                                                    Mela-fw             ACGACGGACTTTATAAACGGTT                             141                30 cycles: 95 °C          U89359               Schelm et al., 2020
                                                                        ATAA                                                                  20 s, 60 °C 60 s
                                                    Mela-rv             AGCGGGTATCCCTCCCTGATT
                                   ITS-             T.mel_for           TTGCTTCCACAGGTTAAGTGA                              351                30 cycles: 94 °C 2        GQ917052             Bonito, 2009
                                   rDNA             T.mel_rev           TAAAGTCCGTCGTTCATGC                                                   min, 55 °C 30 s,
                                                                                                                                              72 °C 30 s
      T. brumale                   ITS-             ITSB                CAATGTCAGAGCCAATCTAATG                             700                23 cycles: 94 °C          AF001010             Paolocci et al., 1999
                                   rDNA                                 C                                                                     30 s, 63 °C 30 s,
                                                    ITS4LNG             TGATATGCTTAAGTTCAGCGGG                                                72 °C 45 s
      T. indicum                   ITS-             ITSCHCH             AACAACAGACTTTGTAAAGGGT                             140                23 cycles: 94 °C          U89360,
                                   rDNA                                 TG                                                                    30 s, 63 °C 30 s,         U89361,              Paolocci et al., 1999
                                                    ITS4LNG             TGATATGCTTAAGTTCAGCGGG                                                72 °C 45 s                U89362
                                                    Indi-fw             AACAACAGACTTTGTAAAGGGT                             146                30 cycles: 95 °C          U89359               Schelm et al., 2020
                                                                        T                                                                     20 s, 60 °C 60 s
                                                    ITS4LNG             TGATATGCTTAAGTTCAGCGGG
      T.                           ITS-             UncI                TGGGCCGCCGAAAACTTG                                 405                27 cycles: 95 °C 1        AJ492199-            Mello et al., 2002
      aestivum/uncinatum           rDNA             UncII               CTGACGAGATGCCCCGGA                                                    min, 59 °C 45 s,          210
                                                                                                                                              72 °C 45 s
                                                    Tu1sekvF            AGAGCACCAAACCACAG                                  496-502            34 cycles: 95 °C          AJ492216             Grindler et al., 2011
                                                                                                                                              60 s; 52, 59 or           AJ888120




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                                                    Tu2sekvR            ACCACAGCGTCTACCAA                                                     63.5 °C 45 or 40s,
                                                                                                                                              72°C 45 or 40 s


      Multiplex:                   ITS-             TmagI               GGATGCGTCTCCGAATCCTGAA                             635                30 cycles: 94 °C          AF003911,            Amicucci et al., 2000
                                   rDNA                                 T                                                                     20 s, 62 °C 15 s,         AF003918,
                                                                                                                                              72 °C 30 s
      T. magnatum,                                  TpuI                TCTGTTACCAGGGTCCACATT                              560                                          AF003909,
      T. puberulum,                                 rTmacII             CGGGGCTATCAGTGCTGCTG                               225                                          AF003920
      T. maculatum,                                 rTboII              GAAGTTGACCGTGGTAATAG                               185
      T. borchii                                    ITS4                TCCTCCGCTTATTGATATGC
                                                    (Reverse)
      Multiplex:                   ITS-             ITSML               CGTCGCTCATCAAAGCAGTC                               440                23 cycles: 94 °C          U89359,              Paolocci et al., 1999
                                   rDNA             ITSB                CAATGTCAGAGCCAATCT                                 700                30 s, 63 °C 45 s,         AF001010
                                                                                                                                              72 °C 45 s
      T. melanosporum,                              ITSCHCH             AACAACAGACTTTGTAAAGGGT                             140                                          U89360-62
      T. brumale,                                                       TG
      T. indicum                                    ITS4LNG             TGATATGCTTAAGTTCAGCGGG
                                                    (Reverse)




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              In some cases, such as in soil analyses, the PCR reaction, albeit with specific primers, may not
       show the presence of the species of interest due to the high amount of different species in the soil. In
       this case, Nested PCR can be used. A first amplification can be carried out with ITS universal primers,
       and subsequently, a second reaction can be performed using as a template a small amount of the first
       reaction product and a pair of primers internal to the previous target (Green and Sambrook, 2019).

Table 4 - List of specific primers and probes for qPCR for truffles.
 Species                  Primer/Probe         Sequence 5’ – 3’                                     Product      Cycles                      Reference
                                                                                                    size
 T. melanosporum          Mela – fw            ACGACGGACTTTATAAACGGTTA                              141          95 °C 4 min; 30             Schelm et al.,
                                               TAA                                                               cycles: 95 °C 20 s,         2020
                          Mela – rv            AGCGGGTATCCCTCCCTGATT                                             60 °C 60 s; 72 °C 10
                                                                                                                 min
                          probe                Cy5–
                                               GACCTGGATCAGTCACAAGTCTT
                                               GTCTGGT-BHQ2


                          Tmelfwd              TCTCTGCGTATCACTCCATGTTG                              61           95 °C 30 s; 40              Parladé et al.,
                          Tmelrev              TCCCACAGGTGCCAGCAT                                                cycles: 95 °C 5 s, 60       2013
                                                                                                                 °C 34 s
                          STQTmel              6FAM-TTCCACAGGTTAAGTGAC-
                                               MGB


                          T.mel_for*           TTGCTTCCACAGGTTAAGTGA                                351          95 °C 10 min; 45            Zampieri et al.,
                                                                                                                 cycles: 95 °C 30 s,         2012
                          T.mel_rev*           TAAAGTCCGTCGTTCATGC                                               56 °C 30 s, 72 °C 30
                                                                                                                 s, 95 °C 15 s, 60 1
                                                                                                                 min, and 95 °C 15 s


 T. magnatum              TmgITS1for           GCGTCTCCGAATCCTGAATA                                 106          95 °C 6 min; 25             Iotti et al., 2012
                                                                                                                 cycles: 95 °C 20 s,
                          TmgITS1rev           ACAGTAGTTTTTGGGACTGTGC                                            60 °C 30 s, 72 °C for
                                                                                                                 40 s; 72 °C 7 min
                          TmgITS1prob          TGTACCATGCCATGTTGCTT


                          TmgITS2for           AAACCCACTCACGGAATCAC                                 99           95 °C 6 min; 25             Iotti et al., 2012
                          TmgITS2rev           CGTCATCCTCCCAATGAAA                                               cycles: 95 °C 20 s,
                                                                                                                 60 °C 30 s, 72 °C for
                          TmgITS2prob          GTACCAAGCCACCTCCATCA                                              40 s; 72 °C 7 min


                          Tmag3*               TTAACTGTTTAAGTTTGTCAGGC                              151          95 °C 10 min; 40            Rizzello et al.,
                                                                                                                 cycles: 95 °C 30 s,         2012
                          Tmag4*               CCTGAATATCTCCTGTGTACCAT                                           56 °C 30 s, 72 °C 30
                                                                                                                 s


 T. indicum/              Indi – fw            AACAACAGACTTTGTAAAGGGTT                              146          95 °C 5 min; 30             Schelm et al.,
 T.himalayense                                                                                                   cycles: 95 °C 20 s,         2020
                          ITS4LNG              TGATATGCTTAAGTTCAGCGGG
                                                                                                                 60 °C 60 s; 72 °C 10
                          probe                HEX-                                                              min
                                               GGACCTAGATCAGTCACAAGTCA
                                               TGTCTGG-BHQ2
       * SYBR Green Reaction


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       In the context of identification, quantitative Polymerase Chain Reaction (qPCR) is therefore
convenient for studying the truffle distribution in the soil to clarify the unknown aspects of their
biology, the unresolved problems in productivity, and the factors leading to the spreading and
development of the mycelium in the soil (Suz et al., 2006; Zampieri et al., 2010). The quantification
of a specific species of Tuber is even more helpful to achieve such purposes. Table 4 lists the specific
primers and probes for truffles designed to date.
       The specific primers selected can be employed to set up a multiplex PCR. A reaction is carried
out with a set of primers to analyze complex samples in which DNA mixtures from different species
may be present simultaneously. The multiplex PCR allows the simultaneous identification of different
truffle species in a single amplification reaction. This method has the advantage of saving time,
reagents, and amount of target DNA. In addition, at the same time, it is possible to identify truffles
and related ectomycorrhiza in a single amplification (Paolocci et al., 1999; Amicucci et al., 2000).
Therefore, multiplex PCR is useful for studying the ecology of different species of Tuber and
symbiotic relationships (Paolocci et al., 1999). The selection of primers must, however, be very
accurate to work with the optimal amplification conditions; in particular, primers must have similar
melting temperatures and must not hybridize with each other.

RAPD technique and SCAR markers

RAPD (Random Amplified Polymorphic DNA) is a modified PCR technique in which a single and
short (generally 10 bp) random oligonucleotide is used. The annealing temperature is very low to
allow the binding of the primer to the genomic DNA (Williams et al., 1990; Babu et al., 2021). The
result of this amplification is a specific pattern of the individual that depends on the distribution of
complementary sequences to those of the primer along the genome. RAPD is cost-effective, quick,
and easy, and no information about the target organism’s genome is needed. Through random-primed
amplification, nucleotide sequence polymorphisms are detected simultaneously for multiple samples.
Despite its great potential, this method has significant criticalities (Williams et al., 1990). These
include, for example, the high risk of contamination and the low reproducibility. Moreover, RAPD
markers are not locus-specific because they are all dominant. For this reason, the nucleotide profiles
obtained with random primers are used to identify species-specific markers; once they have been
detected, they are purified from the agarose gel, and sequenced, and primers are drawn from them to
be used in specific PCR. The markers obtained from RAPD fragments are named SCARs (Sequence
Characterized Amplified Region) and are locus-specific PCR-based markers (Potenza et al., 1994;
Amicucci et al., 1997; Bertini et al., 1998; Paolocci et al., 2000; Rossi et al., 2000; Mello et al., 2006).
The developed primer pairs specific for Tuber spp. are listed in Table 3.
      RAPD analysis was among the first to be used to study the genetic diversity among Tuber spp.
because of its advantages. Thanks to the use of specific markers, the technique becomes strategic for
the identification of Tuber spp. in all stages of the biological cycle, so it allows the detection of
possible commercial frauds and guarantees certified products.




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Methods for DNA extraction-free analysis

A DNA isolation procedure must precede all the methods described. Generally, the methods are
laborious and time-consuming. In recent years, direct PCRs have been developed to skip the isolation
phase and immediately apply the selected PCR.
       In particular, in 2005, Iotti et al. developed a new technique for identifying ectomycorrhizae
directly, quickly, and precisely by PCR omitting the DNA extraction. The process consists of adding
BSA to stabilize the PCR reactions and prevent the adhesion of enzymes. The mycorrhized tips of
each sample were separated from other tissues and cleaned. The ECM manipulation was done in Petri
dishes containing sterile distilled water to prevent PCR contamination. A small portion of each
morphotype was then transferred directly to the PCR tube containing sterile water. Analysis was
performed on fresh and frozen (-80 °C) mycorrhizae (Iotti et al., 2005). This method minimizes
contamination risks and doesn’t use harmful substances.
       Furthermore, for successful truffle cultivation, it is crucial to use the appropriate species of
truffle for the inoculum. The inoculum used for truffle cultivation comes from truffle fruitbodies. It
is a spore substrate, so it is necessary to have a molecular method for identifying spores.
       The protocol of Bonito consists in a direct amplification from fruitbodies of T. melanosporum
(Bonito, 2009). Fresh fruitbodies were cut and dried, then they were amplified by direct PCR using
the T.mel_for – T.mel_rev primer set (Bonito, 2009).
       These methods speed up the identification process and also require only minimal quantities of
starting material, facilitating analysis when only a few mycorrhizae, or a few spores, are available. It
also has the advantage of safeguarding the carpophore which will then eventually be placed on the
market. Preserving the original structure of the truffle preserves its quality and improves
environmental sustainability.

Applications in processed truffle food

To ensure high-quality Tuber species that are used in food products, molecular techniques are
necessary for accurate identification. These applications can be used in truffle traceability, from
harvest to table. The ability to record the origin and route of each truffle allows for greater
transparency in the supply chain. This not only ensures product quality but also promotes ethical and
sustainable business practices. The combined use of molecular markers and advanced analytical
techniques allows truffles to be traced at the molecular level in food, ensuring the quality and
authenticity of the final product. It is important in the food sector to combat counterfeiting and ensure
that consumers receive authentic, high-quality products. The applications of molecular methods to
identify truffle species in food products are exposed in Amicucci et al. (2002), in Rizzello et al.
(2012), and also in Tejedor-Calvo et al. (2023). These papers show specific cases of identification in
processed truffle-based food products: cream, fresh pasta, truffle-flavored rice, oil, salt, and cheese.
In one specific case, a species of Chinese truffle was detected, rather than T. melanosporum, as
incorrectly reported on the package (Amicucci et al., 2002).
      Commercial fraud is a big problem in the marketing of truffles and mycorrhizal seedlings.
However, it is even more prevalent in truffle-based food products because the temperatures and
treatments involved in the processing of truffles can damage the elements that are important for their



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identification, which represent a fundamental and unique tool in the truffle industry. Truffle-based
food products represent a significant portion of the overall truffle market.

Whole-genome sequencing

Despite the complexity and high cost, genomics has become an applied science in the fungal world
as well, aimed at better understanding the biology and characterizing the species in a more definite
way. The genomic sequencing technique aims at predicting and annotating the set of genes present in
the organism and also the non-coding sequences (Pareek et al., 2011). In fact, genome sequencing
includes the sequencing of the entire exome (i.e., all coding regions of each gene) and all intergenic
regions, which represent the majority of DNA (Kuo et al., 2014). The knowledge of the genome
determines its structure and provides useful information to understand the function of genes (Pareek
et al., 2011; Köser et al., 2012). In addition, it lays the foundations for understanding the interactions
between the organism and the environment in which it lives and other organisms, as well as the
biochemical and transcriptomics properties, which is why it represents a turning point in
understanding the biology of mycorrhizal fungi such as truffles.
        The first sequences were based on the application of the method based on Sanger biochemistry,
which, over the years, has undergone changes and innovations, resulting in the achievement of a
large-scale automated sequencing method. The first fungal genomes were sequenced via the Sanger
method but with the Whole-Genome Shotgun approach (Heather and Chain, 2016; Gryganskyi et al.,
2023). The WGS approach is a more advantageous method than clone-by-clone because it greatly
reduces sequencing times and costs. DNA is initially fragmented into several random segments of
defined sizes, which are then sequenced using the Sanger method (Köser et al., 2012). Many
fragmentations and sequencing cycles are performed to obtain high overlapping DNA fractions that
allow the reconstruction of the entire genome through alignment and assembly programs. This
strategy has the advantage of generating sequence reads from both insert ends and it produces highly
redundant sequence coverage (Edwards et al., 1990). The first fungal genome to be completely
sequenced was Saccharomyces cerevisiae in 1996; the sequencing of the first filamentous fungus,
Neurospora crassa, was completed in 2003 (Galagan et al., 2003; Otero et al., 2010).
        The interest in sequencing the genomes of truffles was born out of simple basic knowledge, but
it has had and will continue to have enormous importance in deepening knowledge of its biology. It
has provided and will provide fundamental knowledge for the study of processes related to its
biological cycle: the establishment of symbiosis with the plant, carpophore formation, and diffusion
of mycelium in the soil. Furthermore, whole-genome sequencing will allow the identification of new
targets useful for identification. It will also be able to provide information on the process of synthesis
of aromas, which are highly appreciated and distinctive among the different species. Table 5 provides
the main information on the truffle genomes characterized to date, with the related links to access
them.
        More organisms belonging to Tuber are being sequenced, in addition to the already published
genomes which started with that of T. melanosporum (Martin et al., 2010). As part of the "1000
Fungal Genomes Project (1KFG): Deep Sequencing of Ecologically-relevant Dikarya" (CSP1974),
the genome and transcriptome of Tuber canaliculatum Gilkey (a type of truffle native to eastern North
America) and Tuber gibbosum Harkn. (Oregon white truffle, California, Washington and British
Columbia) have been sequenced (Bonito et al., 2010; Bonito et al., 2013; Benucci et al., 2016).

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Table 5 - Tuber genomes sequenced.
 Tuber species             Size         Predicted       Repeated                Web site                                          Reference
                           Mb           protein-        sequences %
                                        coding          or Transposable
                                        gene            elements
                                        number
 T. melanosporum           125          7496            57.73                   www.genoscope.cns.fr/tuber                        Martin et al.,
                                                                                                                                  2010


 T. magnatum               192          9,433           58                      https://mycocosm.jgi.doe.gov/Tubma                Murat et al.,
                                                                                1/Tubma1.home.html                                2018b

 T. borchii                97.18        12,346          47,12                   http://genome.jgi.doe.gov/Tubbor1                 Murat et al.,
                                                                                                                                  2018a
 T. aestivum               145          9,344           49.5                    https://mycocosm.jgi.doe.gov/Tubae1               Murat et al.,
                                                                                /Tubae1.home.html                                 2018b
 T. brumale                171.44       12,380          61,5                    https://mycocosm.jgi.doe.gov/Tubbr1               Morin et al.,
                                                                                _1/Tubbr1_1.home.html                             2021

 T. indicum                110.49       11,870          47,1                    https://mycocosm.jgi.doe.gov/Tubbr1               Morin et al.,
                                                                                _1/Tubbr1_1.home.html                             2021



Conclusions and perspectives

Truffles belong to the genus Tuber and are among the most prized mushrooms in the world. Trade,
agriculture, and research sectors have developed around this valuable product, and due to its
economic value, it is important to guarantee its origin, safety, traceability, and authenticity. Many
researchers are studying to understand the unknown aspects of truffle biology and to develop effective
cultivation methods, especially for T. magnatum, which is among the most precious species. The
application of molecular biology techniques is useful not only for these purposes but also for
certifying seedlings, guaranteeing the marketed products, and monitoring the presence of the species
of interest.
       This review presents the molecular targets and methods for rapidly identifying and quantifying
the most important Tuber species. It is a scheme of molecular tools that researchers and others can
apply to unambiguously identify mycorrhizae, mycelial cultures, and carpophores. It is essential to
guarantee the reliability of the study models and the mycorrhization systems and to evaluate the
presence of the precious Tuber in food products. All this is fundamental for evolutionary and
ecological studies, studies on the mechanisms underlying the biological cycle, to understand the
interactions with other microorganisms and the reactions to biotic and abiotic factors.
       We reported the validated and most cited protocols in scientific identification papers, but in
perspective, the emerging technique of digital PCR has recently begun to be applied to environmental
studies; it can provide a very sensitive identification and quantification method, and many researchers
are already working on it, using the same targets and primers, but with much greater sensitivity. The
principle of high-level sample partitioning ensures that dPCR results are precise and accurate, even
when starting from very low target copy numbers (Whale et al., 2013). The proposed methods



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summarized in this review may not be exhaustive for taxonomic studies, as some species, other than
Tuber, have shown intraspecific variability of some targets used.
      Moreover, new sequencing technologies will make it easier to discern the genomes of many
other species of Tuber in addition to those already sequenced, providing new knowledge useful for
identifying and characterizing the species. There will also be new fascinating strategies with the
advent of artificial intelligence (AI) and machine learning through software applications capable of
quickly and accurately recognizing species-specific patterns in DNA sequences (Picek et al., 2022).
This provides significant potential for studying and understanding mycology and opening up new
applications in agriculture, industry, and medicine (Picek et al., 2022).


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