# First identification of polymorphic microsatellite markers in the Burgundy truffle, Tuber aestivum (Tuberaceae)

Virginie Molinier, Claude Murat-Furminieux, Emmanuelle Morin, Armelle

Gollotte, Daniel Wipf, Francis Martin

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Virginie Molinier, Claude Murat-Furminieux, Emmanuelle Morin, Armelle Gollotte, Daniel Wipf, et al.. First identification of polymorphic microsatellite markers in the Burgundy truffle, Tuber aestivum (Tuberaceae). Applications in Plant Sciences, 2013, 1 (2), 4 p. 10.3732/apps.1200220. hal-01268457

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# First Identification of Polymorphic Microsatellite Markers in the Burgundy Truffle, *Tuber aestivum* (Tuberaceae)

Author(s): Virginie Molinier, Claude Murat, Emmanuelle Morin, Armelle Gollotte, Daniel Wipf, and Francis Martin

Source: Applications in Plant Sciences, 1(2) 2013.

Published By: Botanical Society of America

URL: http://www.bioone.org/doi/full/10.3732/apps.1200220

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PRIMER NOTE

FIRST IDENTIFICATION OF POLYMORPHIC MICROSATELLITE MARKERS
IN THE BURGUNDY TRUFFLE, TUBER AESTIVUM (TUBERACEAE)<fnref n="1" />

VIRGINIE MOLINIER²,<fnref n="5" />,<fnref n="6" />, CLAUDE MURAT³,<fnref n="5" />, EMMANUELLE MORIN³, ARMELLE GOLLOTTE⁴, DANIEL WIPF², AND FRANCIS MARTIN³

²UMR Agroécologie INRA 1347/AgroSup/Université de Bourgogne, Pôle Interactions Plantes Microorganismes ERL 6300 CNRS, BP 86510, 21065 Dijon CEDEX, France; ³UMR "Interaction Arbres Microorganismes", Centre INRA Nancy, 54280 Champenoux, France; and ⁴Inoplant, 13 rue des Souhait s, 21110 Aiserey, France

* *Premise of the study:* *Tuber aestivum*, the most common truffle in Europe, plays an important role in the commercial truffle market. For the first time, microsatellite primers were developed to investigate polymorphism within this species.

* Methods and Results: Using direct shotgun pyrosequencing, 15 polymorphic microsatellites were identified out of the 7784 perfect microsatellites present in the 534 620 reads obtained. Tested on 75 samples, these microsatellites were highly polymorphic. The number of alleles varied from four to 15, and the expected heterozygosity ranged from 0.266 to 0.620. A multilocus analysis allowed the identification of 63 genotypes over the 75 samples analyzed.

* Conclusions: Direct shotgun pyrosequencing is a fast and relatively low-cost technique allowing identification of microsatel- lites in nonmodel species. The microsatellites developed in this study will be useful in population genetic studies to infer the evolutionary history of this species.

**Key words:** direct shotgun pyrosequencing; polymorphism; truffle; *Tuber aestivum*; Tuberaceae.

Truffles are edible ectomycorrhizal fungi belonging to the genus *Tuber* P. Micheli ex F. H. Wigg. and living in symbiosis with different host trees. More than 100 *Tuber* species exist in the world but only a few, such as *T. magnatum* Picco, *T. melanosporum* Vittad., and *T. aestivum* Vittad., have interesting organoleptic qualities. The first two species have a natural distribution restricted to limited European countries, but *T. aestivum* has a natural distribution across Europe. Previous studies utilizing random-amplified polymorphic DNA (RAPD) or intersimple sequence repeat (ISSR) markers and sequencing of a few genes suggested the existence of genetic diversity between *T. aestivum* populations (Gandeboeuf et al., 1997; Mello et al., 2002; Weden et al., 2004). However, the absence of codominant molecular markers, such as microsatellites or simple sequence repeats (SSRs), precluded the investigation of *T. aestivum* population genetics.

Recently, next-generation sequencing techniques have provided
new prospects to develop microsatellite markers in nonmodel

species, quickly and for relatively low costs (Abdelkrim et al.,
2009; Csencsics et al., 2010; Perry and Rowe, 2011). Direct
shotgun pyrosequencing (DSP) has been successfully used for
the identification of microsatellites in animals or plants but,
to our knowledge, this approach has never been used for ecto-
mycorrhizal fungi. Recently the sequencing of the black truf-
fle genome revealed a substantial richness in repeated sequences
such as microsatellites (Murat et al., 2011). This result sug-
gested that a DSP approach could be used for identifying mic-
rosatellites in *Tuber* species. The aim of our study was therefore
to use DSP to identify, for the first time, polymorphic microsat-
elites in the economically important truffle *T. aestivum*.

METHODS AND RESULTS

Total genomic DNA was isolated from an ascocarp of *T. aestivum* harvested in Haute-Saône (France) (MD4) (Appendix 1, group A). DNA was extracted from frozen tissues with a cetyltrimethylammonium bromide (CTAB) protocol available at the MYCORWEB website (http://mycor.nancy.inra.fr/). A total of 150 µg of genomic DNA was sent to Genoscope (Evry, France). Direct shotgun sequencing occurred in a 1/2 plate optimized run of 454 GS-FLX Titanium and produced 534 620 reads. The fasta and quality files generated were trimmed using the program "TrimSeq.pl" to trim raw sequencing reads at both ends based on moving averages of quality scores (phred = 20) and to remove reads smaller than 300 bp. After trimming and editing, 411 374 reads corresponding to 195 Mbp were retained for microsatellite identification. MISA (http://pgrc.ipk-gatersleben.de/misa/download/misa.pl) was used to identify di-, tri-, tetra-, penta-, and hexanucleotide simple sequence repeat markers. Using this software, a total of 7784 perfect microsatellites were identified (2519 di-, 3568 tri-, 1248 tetra-, 324 penta-, and 125 hexanucleotide repeat motifs). Using the MISA output files, only microsatellites beginning between 100 and 300 bp from the 5' ends of the reads were selected. After this step, 181 microsatellites with more than 10 repeats were obtained. WebSat (Martins et al., 2009) and Primer3

<footnote n="1">¹Manuscript received 4 May 2012; revision accepted 5 August 2012.</footnote>

<footnote>The authors thank Genoscope for financing the *Tuber aestivum* genome sequencing in the TUBEREVOL project. The work presented was also supported by the Conseil Régional de Bourgogne (Program Jeune Chercheur Entrepreneur; grant 20100112095254682-1). The authors thank G. Chevalier, H. Frochot, P. Déquéant, and D. Besson for providing *T. aestivum* samples. The authors thank the strategic platform "GENTYANE" INRA, Ibisa 2009, and its group leader C. Poncet. The authors thank Krista Plett for English editing of this manuscript.</footnote>

<footnote n="5">⁵These authors contributed equally to this work.</footnote>

<footnote n="6">⁶Author for correspondence: virginie.molinier@dijon.inra.fr</footnote>
<table><caption>TABLE 1. Characteristics of the primers developed in *Tuber aestivum* for the 15 selected microsatellites.</caption><thead><tr><th>Locus</th><th>5' end-labeled dye</th><th>Primer sequences (5'-3')</th><th>Repeat motif</th><th>Size range (bp)</th><th>T<sub>a</sub> (°C)</th><th>EMBL accession no.</th></tr></thead><tbody><tr><td>aest1</td><td>FAM</td><td>F: AAATAACGCTCCAGATCCCTTT<br>R: TAGAGAGGTTCTAGCCGTCAGG</td><td>(CCACTC)<sub>10</sub></td><td>236-296</td><td>60</td><td>HE793313</td></tr><tr><td>aest6</td><td>FAM</td><td>F: CGTTTATTGTTCGCCTTTTCTC<br>R: TAAGCTACTGAGCGACAGGTTG</td><td>(AGTAAT)<sub>6</sub></td><td>212-236</td><td>60</td><td>HE793314</td></tr><tr><td>aest7</td><td>FAM</td><td>F: GAGAACGTGATGTGTGATGGTT<br>R: ATGAGATGCAAGCACTGTAGGA</td><td>(ACAGC)<sub>6</sub></td><td>256-286</td><td>60</td><td>HE793315</td></tr><tr><td>aest10</td><td>VIC</td><td>F: AATAAAGCACCAGATCACCACC<br>R: GACTAGCGAAAGGGGATGAGTA</td><td>(AGTAC)<sub>6</sub></td><td>280-310</td><td>60</td><td>HE793316</td></tr><tr><td>aest15</td><td>VIC</td><td>F: GACTTTCCGTCCACTTGTTTC<br>R: GATCCATTCATCCATCCATACC</td><td>(GGATG)<sub>6</sub></td><td>297-322</td><td>60</td><td>HE793317</td></tr><tr><td>aest18</td><td>FAM</td><td>F: TATTCCTATCCCACACGACCAC<br>R: AACATTGCTGTTTACGGCTCTT</td><td>(ACTG)<sub>11</sub></td><td>136-156</td><td>60</td><td>HE793318</td></tr><tr><td>aest24</td><td>VIC</td><td>F: TGAAAGTGTTAGAGATCGGCAA<br>R: GTACTTCGCCAGACAGAGAGT</td><td>(TCA)<sub>18</sub></td><td>292-319</td><td>60</td><td>HE793319</td></tr><tr><td>aest25</td><td>FAM</td><td>F: AGTTGTTTGTATGATGATGCCG<br>R: ACTGGTATGACACGCTCCAAAT</td><td>(ATT)<sub>10</sub></td><td>122-140</td><td>60</td><td>HE793320</td></tr><tr><td>aest26</td><td>FAM</td><td>F: CAAGACAGAGACGCTAATCCCT<br>R: GAGTATGCAATAATGGGTCCGT</td><td>(TAT)<sub>11</sub></td><td>124-178</td><td>60</td><td>HE793321</td></tr><tr><td>aest27</td><td>VIC</td><td>F: AAGTGTTAGAGATCGGCAAAGC<br>R: CCTCACTCCAGACTTATCCCAC</td><td>(TCA)<sub>18</sub></td><td>314-341</td><td>60</td><td>HE793322</td></tr><tr><td>aest28</td><td>VIC</td><td>F: ACCTTCTTTTCCTGTGGCAAT<br>R: AAATAGTCTGAGGGGAGTCGTG</td><td>(TTA)<sub>18</sub></td><td>344-452</td><td>60</td><td>HE793323</td></tr><tr><td>aest29</td><td>FAM</td><td>F: GAGTGGGGAGAATCACGTCTAC<br>R: TATCCAAATGTTCCTGCACTTC</td><td>(AGTAC)<sub>6</sub></td><td>186-226</td><td>60</td><td>HE793324</td></tr><tr><td>aest31</td><td>VIC</td><td>F: TAGATACAGCACCGTAGCACCA<br>R: TTTCGTAATTGTCTGTCGTTG</td><td>(CCTAC)<sub>6</sub></td><td>285-325</td><td>60</td><td>HE793325</td></tr><tr><td>aest35</td><td>FAM</td><td>F: GTATGCGGTAGGTGGGTTTACT<br>R: CTTGTGCTCGTACTCCACTTGT</td><td>(TTTC)<sub>10</sub></td><td>124-160</td><td>60</td><td>HE793326</td></tr><tr><td>aest36</td><td>VIC</td><td>F: ACAATACTCACCGACCTTTGCT<br>R: CATGATACAACAGCATCCCG</td><td>(GACT)<sub>13</sub></td><td>310-350</td><td>60</td><td>HE793327</td></tr></tbody></table>

Note: EMBL = European Molecular Biology Laboratory; T<sub>a</sub> = annealing temperature.

(Rozen and Skaletsky, 2000) were respectively used to check repeats and to design primers for 40 microsatellites randomly selected among the 181 microsatellites previously obtained. The PCR feasibility was checked using OligoAnalyzer 3.1 (http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/Default.aspx). Primers were tested in silico using AmplifX software (http://crn2m.univ-mrs.fr/pub/amplifx-dist).

To evaluate the microsatellite primers in a PCR reaction, two *T. aestivum* ascocarps were used. DNA was extracted with a DNasy Plant Mini Kit (QIA-GEN, Hilden, Germany) according to the manufacturer's instructions. All PCR amplifications were performed using 30 ng of template DNA in a final volume of 20 µL containing 10× PCR buffer (Invitrogen, Carlsbad, California, USA), 1 U of Taq DNA polymerase (Invitrogen), 0.125 mM each of dNTPs, 0.5 µM each of forward and reverse primers, and sterile water to adjust to the final volume. PCRs were conducted in a Mastercycler Gradient S PCR system (Eppendorf, Hamburg, Germany) after an initial denaturation of 3 min at 94°C, 30 cycles at 94°C for 25 s, 58°C for 25 s, and 72°C for 40 s, followed by 72°C for 15 min. PCR products were checked on a 1.5% agarose gel. According to Paolocci et al. (2006), the gleba of *Tuber* ascocarps is mainly composed of haploid maternal tissue and the DNA extracted from ascocarps therefore corresponds to a haploid tissue. For this reason, only the microsatellites producing a single band signal were selected for future investigations. Twenty-five primer pairs (62.5%) resulted in good amplification (i.e., one band for both ascocarps).

To identify polymorphic microsatellites, the 25 retained primer pairs were tested with 14 *T. aestivum* samples from different European countries (Appendix 1, group B). Genomic DNA from the 14 ascocarps was isolated from 20 mg of dried gleba as described above. PCR conditions were the same as those described above. PCR products were separated on a 4% agarose gel. Fifteen out of the 25 microsatellites (60%) showed size polymorphisms on 4% agarose gel. To assess more precisely the level of polymorphism, 15 microsatellites were used to perform a genotyping analysis using 5' end-labeled primers (FAM or VIC) (Table 1). Seventy-five samples from seven European populations (Appendix 1, group C) were chosen to perform preliminary genotyping. PCR products were assayed, using 500 LIZ as a size standard, on an ABI 3730XL sequencer (Applied Biosystems, Foster City, California, USA) at the "Plateforme de Génotypage GENTYANE" (Clermont-Ferrand, France). All amplifications

were carried out individually. Allele sizes were analyzed with GeneMapper (Applied Biosystems). Expected heterozygosity ($H_e$) was calculated using GenAIEx (Peakall and Smouse, 2006) (Table 2). As expected, due to the haploid nature of the ascocarp (Paolocci et al., 2006), each locus gave only one peak for all 75 samples. The number of different alleles per locus ranged from four to 15, and $H_e$ ranged from 0.266 to 0.620. Finally, 63 different multilocus genotypes were obtained from the 75 *T. aestivum* isolates.

## CONCLUSIONS

A DSP approach on a single *T. aestivum* ascocarp allowed for the identification of 7784 perfect microsatellites for 195 Mbp (nearly 40 microsatellites per Mbp). To our knowledge, our study is the first report of DSP to identify nuclear polymorphic microsatellite markers in an ectomycorrhizal fungal species. Indeed, until now, the DSP approach has only been performed on organisms belonging to the Animalia and Plantae kingdoms. This method is very quick and relatively inexpensive compared to the enriched library methods commonly used to develop polymorphic microsatellites. Indeed, with only 1/2 plate of a pyrosequencing run, we were able to identify several thousand microsatellites. DSP is therefore a useful approach to characterize polymorphic markers for nonmodel species. The markers developed in this study will enable future population genetic studies for this truffle species. Moreover, from a commercial point of view, population genetic studies could be useful to label some remarkable ecotypes showing particular alleles. Indeed, because the price of this truffle continues to increase and varies depending on geographical origin, a certificate of "true geographical origin" could be used to avoid fraud in the commercial truffle market.
<table><caption>TABLE 2. Genotyping of the 15 microsatellites for 75 truffle samples belonging to seven *Tuber aestivum* populations.*</caption><thead><tr><th rowspan="2">LOCUS</th><th colspan="3">"Daix" (n = 23)</th><th colspan="3">"Andal" (n = 9)</th><th colspan="3">"Bedr" (n = 9)</th><th colspan="3">"Drome" (n = 9)</th><th colspan="3">"Tarsul" (n = 11)</th><th colspan="3">"Sweden" (n = 6)</th><th colspan="3">"Valsuz" (n = 8)</th></tr><tr><th>A</th><th>A<sub>c</sub></th><th>H<sub>c</sub></th><th>A</th><th>A<sub>c</sub></th><th>H<sub>c</sub></th><th>A</th><th>A<sub>c</sub></th><th>H<sub>c</sub></th><th>A</th><th>A<sub>c</sub></th><th>H<sub>c</sub></th><th>A</th><th>A<sub>c</sub></th><th>H<sub>c</sub></th><th>A</th><th>A<sub>c</sub></th><th>H<sub>c</sub></th><th>A</th><th>A<sub>c</sub></th><th>H<sub>c</sub></th></tr></thead><tbody><tr><td>aest1</td><td>5</td><td>3.599</td><td>0.722</td><td>3</td><td>1.976</td><td>0.494</td><td>4</td><td>2.613</td><td>0.617</td><td>3</td><td>2.314</td><td>0.568</td><td>4</td><td>3.667</td><td>0.727</td><td>2</td><td>1.385</td><td>0.278</td><td>4</td><td>2.286</td><td>0.563</td></tr><tr><td>aest6</td><td>5</td><td>3.058</td><td>0.673</td><td>2</td><td>1.528</td><td>0.346</td><td>3</td><td>1.588</td><td>0.370</td><td>2</td><td>1.528</td><td>0.346</td><td>2</td><td>1.198</td><td>0.165</td><td>2</td><td>1.800</td><td>0.444</td><td>2</td><td>1.280</td><td>0.219</td></tr><tr><td>aest7</td><td>5</td><td>3.245</td><td>0.692</td><td>2</td><td>1.246</td><td>0.198</td><td>3</td><td>2.314</td><td>0.568</td><td>3</td><td>1.976</td><td>0.494</td><td>3</td><td>2.373</td><td>0.579</td><td>2</td><td>1.385</td><td>0.278</td><td>3</td><td>2.133</td><td>0.531</td></tr><tr><td>aest10</td><td>5</td><td>3.503</td><td>0.715</td><td>2</td><td>1.246</td><td>0.198</td><td>4</td><td>3.000</td><td>0.667</td><td>2</td><td>1.246</td><td>0.198</td><td>4</td><td>3.270</td><td>0.694</td><td>2</td><td>1.800</td><td>0.444</td><td>3</td><td>2.667</td><td>0.625</td></tr><tr><td>aest15</td><td>3</td><td>1.924</td><td>0.480</td><td>2</td><td>1.246</td><td>0.198</td><td>2</td><td>1.246</td><td>0.198</td><td>2</td><td>1.800</td><td>0.444</td><td>2</td><td>1.198</td><td>0.165</td><td>1</td><td>1.000</td><td>0.000</td><td>2</td><td>1.600</td><td>0.375</td></tr><tr><td>aest18</td><td>3</td><td>2.232</td><td>0.552</td><td>2</td><td>1.246</td><td>0.198</td><td>2</td><td>1.528</td><td>0.346</td><td>1</td><td>1.000</td><td>0.000</td><td>3</td><td>1.754</td><td>0.430</td><td>3</td><td>2.571</td><td>0.611</td><td>3</td><td>2.667</td><td>0.625</td></tr><tr><td>aest24</td><td>5</td><td>2.658</td><td>0.624</td><td>4</td><td>2.077</td><td>0.519</td><td>5</td><td>3.522</td><td>0.716</td><td>2</td><td>1.246</td><td>0.198</td><td>5</td><td>4.172</td><td>0.760</td><td>2</td><td>1.800</td><td>0.444</td><td>4</td><td>3.556</td><td>0.719</td></tr><tr><td>aest25</td><td>4</td><td>3.023</td><td>0.669</td><td>3</td><td>1.976</td><td>0.494</td><td>3</td><td>2.455</td><td>0.593</td><td>1</td><td>1.000</td><td>0.000</td><td>3</td><td>1.754</td><td>0.430</td><td>2</td><td>2.000</td><td>0.500</td><td>4</td><td>2.286</td><td>0.563</td></tr><tr><td>aest26</td><td>6</td><td>3.206</td><td>0.688</td><td>3</td><td>1.588</td><td>0.370</td><td>4</td><td>3.240</td><td>0.691</td><td>2</td><td>1.246</td><td>0.198</td><td>5</td><td>3.457</td><td>0.711</td><td>2</td><td>1.800</td><td>0.444</td><td>5</td><td>4.000</td><td>0.750</td></tr><tr><td>aest27</td><td>5</td><td>2.606</td><td>0.616</td><td>4</td><td>2.077</td><td>0.519</td><td>5</td><td>3.522</td><td>0.716</td><td>2</td><td>1.246</td><td>0.198</td><td>4</td><td>3.457</td><td>0.711</td><td>2</td><td>1.800</td><td>0.444</td><td>3</td><td>2.909</td><td>0.656</td></tr><tr><td>aest28</td><td>8</td><td>5.136</td><td>0.805</td><td>2</td><td>1.246</td><td>0.198</td><td>7</td><td>5.400</td><td>0.815</td><td>3</td><td>1.976</td><td>0.494</td><td>4</td><td>3.667</td><td>0.727</td><td>3</td><td>2.571</td><td>0.611</td><td>4</td><td>3.200</td><td>0.688</td></tr><tr><td>aest29</td><td>4</td><td>2.989</td><td>0.665</td><td>3</td><td>2.455</td><td>0.593</td><td>4</td><td>3.000</td><td>0.667</td><td>2</td><td>1.246</td><td>0.198</td><td>3</td><td>1.754</td><td>0.430</td><td>3</td><td>2.000</td><td>0.500</td><td>4</td><td>2.286</td><td>0.563</td></tr><tr><td>aest31</td><td>3</td><td>2.108</td><td>0.526</td><td>3</td><td>1.588</td><td>0.370</td><td>5</td><td>4.263</td><td>0.765</td><td>3</td><td>2.314</td><td>0.568</td><td>2</td><td>1.658</td><td>0.397</td><td>3</td><td>2.571</td><td>0.611</td><td>2</td><td>1.882</td><td>0.469</td></tr><tr><td>aest35</td><td>4</td><td>2.372</td><td>0.578</td><td>2</td><td>1.246</td><td>0.198</td><td>3</td><td>1.588</td><td>0.370</td><td>2</td><td>1.246</td><td>0.198</td><td>2</td><td>1.198</td><td>0.165</td><td>2</td><td>1.385</td><td>0.278</td><td>2</td><td>1.600</td><td>0.375</td></tr><tr><td>aest36</td><td>3</td><td>1.426</td><td>0.299</td><td>3</td><td>1.976</td><td>0.494</td><td>4</td><td>2.613</td><td>0.617</td><td>3</td><td>2.455</td><td>0.593</td><td>3</td><td>1.458</td><td>0.314</td><td>1</td><td>1.000</td><td>0.000</td><td>4</td><td>2.909</td><td>0.656</td></tr></tbody></table>

Note: A = number of alleles; A_c = number of effective alleles; H_c = expected heterozygosity; n = sample size.

*See Appendix 1 for locality information.

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<table><caption>APPENDIX 1. Locality information of the different samples and populations of *Tuber aestivum* used in this study.</caption><thead><tr><th>Group</th><th>Sample/Population ID</th><th>Country, District*</th><th>Latitude*</th><th>Longitude*</th></tr></thead><tbody><tr><td rowspan="2">Group A</td><td>MD4</td><td>France, Haute-Saône</td><td>47°55'12"N</td><td>6°4'48.9"E</td></tr><tr><td>E38</td><td>Hungary, Northern Great Plain (Debrecen)</td><td>47°31'47.906"N</td><td>21°38'21.6852"E</td></tr><tr><td rowspan="10">Group B</td><td>E43</td><td>Luxembourg (Rumelange)</td><td>49°28'00"N</td><td>6°02'00"E</td></tr><tr><td>E58</td><td>Switzerland (Lauzanne)</td><td>46°31'11.862"N</td><td>6°38'0.948"E</td></tr><tr><td>E60</td><td>Turkey</td><td>ND</td><td>ND</td></tr><tr><td>E62</td><td>China (Beijing)</td><td>39°54'15.1704"N</td><td>116°24'26.6"E</td></tr><tr><td>E70</td><td>United Kingdom, Bedfordshire</td><td>52°8'9.5208"N</td><td>0°27'59.943"W</td></tr><tr><td>E98</td><td>Romania (Bucharest)</td><td>44°26'15.7596"N</td><td>26°5'50.520"E</td></tr><tr><td>E104</td><td>Sweden, Gotland (Roma)</td><td>57°30'28.944"N</td><td>18°27'1.116"E</td></tr><tr><td>F1</td><td>France, Corsica (Santa Lucia di Moriani)</td><td>42°23'11.04"N</td><td>9°31'49.08"E</td></tr><tr><td>F23</td><td>France, Drôme (Montjoyer)</td><td>44°28'38"N</td><td>4°51'10"E</td></tr><tr><td>F38</td><td>France, Lozère (Montbrun)</td><td>44°20'17.02"N</td><td>3°30'15.98"E</td></tr><tr><td>F68</td><td>France, Var (Ampus)</td><td>43°36'26.193"N</td><td>6°22'59.7"E</td></tr><tr><td>F166</td><td>France, Aube (Vailly)</td><td>48°22'9.976"N</td><td>4°7'45.637"E</td></tr><tr><td rowspan="6">Group C</td><td>UMF1</td><td>United Kingdom, Norfolk (Watton)</td><td>52°34'15.0522"N</td><td>0°49'40.882"E</td></tr><tr><td>"Daix"</td><td>France, Côte d'Or (Daix)</td><td>47°21'6.192"N</td><td>4°59'57.4542"</td></tr><tr><td>"Andal"</td><td>Spain, Andalusia (Grenada)</td><td>37°10'35.3532"N</td><td>3°35'52.544"W</td></tr><tr><td>"Bedf"</td><td>United Kingdom, Bedfordshire (Bedford)</td><td>52°8'9.5208"N</td><td>0°27'59.943"W</td></tr><tr><td>"Drome"</td><td>France, Drôme (Montjoyer)</td><td>44°28'38"N</td><td>4°51'10"E</td></tr><tr><td>"Tarsul"</td><td>France, Côte d'Or</td><td>47°31'57"N</td><td>4°59'3.4506"E</td></tr><tr><td></td><td>"Sweden"</td><td>Sweden, Gotland (Roma)</td><td>57°30'28.944"N</td><td>18°27'1.116"E</td></tr><tr><td></td><td>"Valsuz"</td><td>France, Côte d'Or (Val Suzon)</td><td>47°24'29"N</td><td>4°53'39"E</td></tr></tbody></table>

Note: ND = GPS coordinates not determined.

*Due to the confidentiality between authors and truffle providers, only GPS coordinates of the closest town or village are indicated instead of the precise location.