Abstract
Yeast microsatellite loci consist of short tandem-repeated DNA sequences of variable length. The high mutational rate at these loci generates a remarkable repertoire of alleles, useful for strain differentiation and population genetic studies. In this work, we analyze the DNA sequences of thirteen alleles from each of ten microsatellite loci described for the yeast Starmerella bacillaris. Our results show that polymorphic variants of some informative alleles are dependent on SNPs and indels rather than on length variation at their originally defined tandem-repeated motifs. The analysis was extended to 55 previously described hypervariable microsatellite loci from a total of 26 sequenced genomes of yeast species that dominate the microbiota of spontaneously fermenting grape musts (i.e., Hanseniaspora uvarum, Saccharomyces cerevisae, Saccharomyces uvarum, and Torulaspora delbrueckii) or lead to wine spoilage (Brettanomyces bruxellensis and Meyerozyma guilliermondii). We found that allelic variants for some microsatellite loci of these yeast species are also dependent on SNPs and/or indels flanking their tandem-repeated motifs. For some loci, the number of units at their tandem repeats was found to be identical among the various characterized alleles, with allelic differences being dependent exclusively on flanking polymorphisms. Our results indicate that allele sizing of microsatellite loci using PCR, although valid for strain differentiation and population genetic studies, does not necessarily score the number of units at their tandem-repeated motifs. Sequence analysis of microsatellite loci alleles could provide relevant information for evolutionary and phylogeny studies of yeast species.
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Acknowledgements
MLRE held a fellowship from the National Research Council of Argentina (CONICET). ALR is a Career Research Investigator from CONICET. This work was supported by PICT-2014-3113 from Fondo para la Investigación Científica y Tecnológica (FONCyT) (Argentina) and SUV2015 (Universidad Católica de Córdoba) to ALR.
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This work was supported by PICT-2014-3113 from FONCYT (Argentina) and SUV2015 (UCC) to ALR.
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MLRE and ALR contributed to the study conception and design, material preparation, data collection, analysis, and writing of the manuscript. Both authors read and approved the final manuscript.
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Supplementary file2 Supplementary figure 1 Analysis of CZ11 alleles. DNA sequences of CZ11 alleles from strains MT017-029, L14, MT017-011, MT117-003 and L13 were obtained from PCR fragments amplified from genomic DNA using primers CZ11-FL and CZ11-RL (Fig. 1) or from the available genomes of strains NP2, CBS 9494, FRI751, PAS13 and PYCC 3044. Alleles include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), as well as insertions (arrows), deletions (dashes) and SNPs (asterisks), are indicated. The 16-nt tandem-repeated motifs observed in C. zemplinina type strain CBS 9494 are indicated (dark/light highlight). The CZ11 repeated motif (GT/GA/TA) is indicated with colored characters (PDF 217 kb)
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Supplementary file3 Supplementary figure 2 Analyses of CZ1, CZ4, CZ13, CZ15, CZ20, CZ33, CZ45 and CZ59 alleles. DNA sequences were obtained from the available genomes of strains NP2, CBS 9494, FRI751, PAS13 and PYCC 3044. Alleles include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), alternative primer binding sites at microsatellite loci CZ13 and CZ20 (black underlined), as well as insertions (arrows), deletions (dashes) and SNPs (asterisks), are indicated. Microsatellite TRMs are indicated with colored characters (PDF 229 kb)
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Supplementary file4 Supplementary figure 3 Analysis of CZ54 alleles. DNA sequences of CZ54 alleles from strains MT017-022, MT017-021, MT017-020, MT017-011 and MT017-029 were obtained from PCR fragments amplified from genomic DNA using primers CZ54-FL and CZ54-RL (Fig. 1) or from the available genomes of strains NP2, CBS 9494, FRI751, PAS13 and PYCC 3044. Alleles include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), as well as insertions (arrows), deletions (dashes) and SNPs (asterisks), are indicated. The CZ54 repeated motif (AGA) is indicated with colored characters. (PDF 210 kb)
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Supplementary file5 Supplementary figure 4 Analysis of H. uvarum HU292, HU440, HU508, HU593, HU467, HU620, HU409, HU853, HU68 and HU594 alleles. DNA sequences were obtained from the available genomes of strains DSM 2768, AWRI3581, 34-9, AWRI3580, CBA6001. Allele sequences include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), as well as insertions (arrows), deletions (dashes), SNPs (asterisks) and conserved single nucleotide changes in the repeated tandem motifs (underlined), are indicated. Microsatellite TRMs are indicated with colored characters. (PDF 257 kb)
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Supplementary file6 Supplementary figure 5 Analyses of S. cerevisiae C3, C4, C5, C6, C8, C9, C11, SCAAT1, SCAAT2, SCAAT3, SCAAT5, SCAAT6, YKR072CS, SCYOR267C, YKL172 and YPL009 alleles. DNA sequences were obtained from the available genomes of strains AWRI1631, JAY291, W303, M22, YPS163. Allele sequences include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), as well as insertions (arrows), deletions (dashes), SNPs (asterisks) and conserved single nucleotide changes in the repeated tandem motifs (underlined), are indicated. Microsatellite TRMs are indicated with colored characters. (PDF 254 kb)
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Supplementary file7 Supplementary figure 6 Analysis of S. uvarum SuARS409, SuYBR049C, SuYKL017C, SuYKR045C, SuYGC170W, SuYHR042-043, SuHTZ1PLB3, SuYHR102W and SuYIL130W alleles. DNA sequences were obtained from the available genomes of strains MCYC 623, U1, U2, U3, U4. Allele sequences include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), as well as deletions (dashes), SNPs (asterisks) and conserved single nucleotide changes in the repeated tandem motifs (underlined), are indicated. Microsatellite TRMs are indicated with colored characters. Large tandem duplications (gray highlight) and insertions (red underline) at loci SuYKL017C and SuYKR045C are indicated. (PDF 329 kb)
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Supplementary file8 Supplementary figure 7 Analysis of T. delbrueckii TD1A, TD1B, TD1C, TD2A, TD6A, TD7A, TD5A and TD8A alleles. DNA sequences were obtained from the available genomes of strains CBS 1146, SRCM101298. Allele sequences include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), as well as insertions (arrows), deletions (dashes), SNPs (asterisks) and conserved single nucleotide changes in the repeated tandem motifs (underlined), are indicated. Microsatellite TRMs are indicated with colored characters. (PDF 216 kb)
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Supplementary file9 Supplementary figure 8 Analysis of B. bruxellensis B101, B122, B135, B174, B22, B224, B273 and B301 alleles. DNA sequences were obtained from the available genomes of strains AWRI1499, LAMAP2480, CBS2796, CBS2499, UMY321. Allele sequences include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), as well as deletions (dashes), SNPs (asterisks) and conserved single nucleotide changes in the repeated tandem motifs (underlined), are indicated. Microsatellite TRMs are indicated with colored characters. (PDF 313 kb)
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Supplementary file10 Supplementary figure 9 Analysis of M. guilliermondii sc15, sc22, sc32 and sc72 alleles. DNA sequences were obtained from the available genomes of strains RP-YS-11, SO, W2, ATCC6260. Allele sequences include 5 additional base pairs, both 5’ and 3’, flanking the originally described microsatellite sequences (see Fig. 1). Originally described PCR primer sequences (bold characters; see Table S1), as well as insertions (arrows), deletions (dashes), SNPs (asterisks) and conserved single nucleotide changes in the repeated tandem motifs (underlined), are indicated. Microsatellite TRMs are indicated with colored characters. (PDF 307 kb)
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Raymond Eder, M.L., Rosa, A.L. Non-tandem repeat polymorphisms at microsatellite loci in wine yeast species. Mol Genet Genomics 295, 685–693 (2020). https://doi.org/10.1007/s00438-020-01652-2
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DOI: https://doi.org/10.1007/s00438-020-01652-2