Populational analysis of Saccharomyces cerevisiae populations from different appellations of origin and grape varieties by microsatellite analysis

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1 Populational analysis of Saccharomyces cerevisiae populations from different appellations of origin and grape varieties by microsatellite analysis L. Pereira *, R. Franco-Duarte *, P. Ramos *, F. Alemão *, P. Gomes *, S. Sousa * M. Santos,4, F. L. Duarte, M. Casal, D. Schuller Centro de Biologia Molecular e Ambiental, Universidade do Minho, Braga Estação Vitivinícola Nacional, Quinta da Almoínha, Dois Portos BIOCANT, Centro de Inovação em Biotecnologia, Cantanhede 4 Departamento de Biologia, Universidade de Aveiro, Aveiro

2 Introduction ti Fermentation technologies are deeply rooted in the history of most societies; Winemaking environments are characterized by a fascinating genetic diversity of S. cerevisiae strains, but population genetic variation is still poorly understood. Objective to evaluate populational l relationships among Saccharomyces cerevisiae strains isolated from some of the Portuguese most important grapevine varieties in different appellations of origin, using polymorphic microsatellites

3 Vinho Verde Materials and Methods - sampling sites - Bairrada Estremadura Palmela Alentejo (Évora) Vinho Verde Estremadura Alvarinho Loureiro Avesso Arinto Touriga Nacional Castelão Trincadeira Preta Aragonês Arinto Touriga Nacional Bairrada Palmela Bical Baga Maria Gomes Aragonês Touriga Nacional Castelão 5 Wine Regions 9 Grape varieties Vineyards 6 samples / vineyard grape samples Alentejo (sub-region Évora) Aragonês Trincadeira Preta Touriga Nacional

4 Materials and Methods - spontaneous fermentations ti - Weight loss of 7 g/l: Random collection of isolates per fermentatin Minho Bairrada Estremadura Palmela Alentejo Total Molecular typing Nº of samples Spontaneous fermentations Number of isolates Saccharomyces sp. % % % 9 % Total: 5 S. cerevisiae strains

5 Materials and Methods Mitochondrial DNA restriction analysis (mtdna RFLP) - molecular typing - Interdelta sequence amplification Preliminary screening Querol et al.99 Lopez et al. Ness et al. 99 Legras et al. ) In depth characterization Microsatelite analysis AAT AAT5 AAT6 AAT AAT4 AAT Microsatellite Chrom. Position/ORF Repeat Ref. ScAAT ScAAT ScAAT ScAAT 4 ScAAT 5 ScAAT 6 YPL9 ScYOR67C C4 C5 XIII ATT II YBL84c ATT IV YDR6w ATT VII ATT XVI TAA IX TAA XV YOR56c TAA XV YOR67c TGT XV 7-95 TAA+TAG VI 5-44 GT Pé érez and Gal lego, al., 5 Legras et

6 Results - S. cerevisiae strains involved in spontaneous fermentations - T. Nacional Alvarinho Loureiro Avesso taneous fermentatio ons Arinto T. Nacional Baga Bical Maria Gomes Aragonês T. Nacional Aragonês Arinto Cast. T. Preta Spon Minho Bairrada Estremadura Palmela Alentejo Fermentation by non-saccharomyces species j

7 Results - fermetation f t ti kinetics ki ti - S. cerevisiae - We eigt loss (g g/l) We eigt loss (g g/l) Nº of strains Day Nº of strains Day Non Saccharomyces species We eigt loss (g g/l) We eigt loss (g g/l) Aragonês Arinto Touriga Nacional Castelão Trincadeira preta Baga Alvarinho Loureiro Day Day 5 5

8 Results - distribution of the most frequent microsatellite alleles - Most frequent alleles AAT AAT6 AAT5 AAT AAT4 Locus Nº of alleles Allele (nº of repeats) ScAAT ScAAT ScAAT ScAAT ScAAT ScAAT C C YPL ScYOR Total: 9 alleles

9 Distribution of the most frequent alleles among vineyards of the Vinho Verde Region in consecutive years Allele freq quency Alvarinho Vinho Verde strains 4 strains 65 strains Allele frequen ncy Loureiro o Alle ele frequency strains strains strains Avesso A strains 4 strains ScAAT ScAAT ScAAT ScAAT4 ScAAT5 ScAAT6

10 Distribution of the most frequent alleles among S. cerevisiae strains from vineyards of the Vinho Verde and Bairrada Regions in 6 Vinho Verde strains 59 strains. Bairrada Allele frequen ncy ScAAT ScAAT ScAAT ScAAT4 ScAAT5ScAAT6 C4 C5 ScYPL9 ScYOR67 Most frequent allele per locus (Minho) Most frequent allele per locus (Bairrada)

11 Most frequent alleles among S. cerevisiae strains from grape varieties i ti off the th Vi Vinho h Verde V d and d Bairrada B i d Regions R i 7. Alvarinho ( strains) Avesso ( strains) Arinto ( strains) T Nacional (5 strains) T. (Minho) Allelle frequency y Lo reiro (7 strains) Loureiro Baga (8 strains) S AAT ScAAT (vineyar with unique grape variety ) Aragonês ( strains) T. Nacional ((4 strains)) (Bairrada) A Allele freque ency (vineyar with ( i ith severall S AAT ScAAT S AAT4 ScAAT4 S AAT5 ScAAT6 S AAT6 ScAAT5.5 grape variety ) Baga (4 strains) S AAT ScAAT C4 C5 ScYPL9 ScYOR67

12 Expected (He)) and observed (Ho)) heterozygosity Alvarinho Loureiro o Avesso Arinto l T. Nacional l T. Nacional Baga a Baga a Aragonês Genome renewal (Mortimer et al., 994) ScAAT ScAAT Environmental isolates of S. cerevisiae undergo mating among their progeny ScAAT ScAAT4 ScAAT5 ScAAT6 C4 Multiple heterozygote strain C5 ScYPL9 ScYOR67 He / Ho n.d. (homozygous locus) Heterozygous genotypes reduction relative to that expected under random mating is a consequence of populational substructuring homozygote strain

13 Similarity of S. cerevisiae populations from distinct wine regions and grape varieties Alvarinho 6QP6_ Avesso QCov_ Loureiro _QAm Euclidean distance r =.9 Baga Ba_4 Baga Ba_8 Aragonês TiRo_ T. Nacional TN_4 Arinto Ar_ T. Nacional TN_5 km Coefficient

14 Consensus tree of S. cerevisiae populations based on F ST genetic distances Alsace Galice USA Austria Italy Rhone Valley Burgundy Nantes Bordeaux Portugal (Minho) Montpellier Cognac Spain (miscellaneous) Bulgaria Romania South Africa Hungary 99 Germany 9 Lebanon French Indies French Indies Bread (Italy) Bread 46 China (rice wine) Beer (Ale) Nigeria Fermented milk China (distillery) Alsace Japan Sake ins Win ne stra. Legras et. al., Mol. Ecol. 7

15 Conclusions The finding of 5 S. cerevisiae strains reveals a fascinating genetic diversity in vineyard environments. Microsatellite analysis permitted a high resolution populational screen, showing that genetic differences among S. cerevisiae populations derived from both diagnostic vineyard-, specific alleles and the accumulation of small allele-frequency differences across ten microsatellite loci; each vineyard contains differentiated S. cerevisiae populations, hypothesizing the occurrence of specific native strains that can be associated with a terroir.

16 Acknowledgements Magda Silva Graça Hugo Alves Sofia Machado Barbara Dellinger Jean-Luc Legras INRA Colmar, France Adega Cooperativa de Cantanhede Comissão de Viticultura da Região dos Vinhos Verdes Companhia das Quintas Estação Vitivinícola Amândio Galhano PROVAM Produtores de Vinhos Alvarinho de Monção Quinta de Ameal Rui Cunha Quinta da Cancela Anselmo Mendes Quinta de Covela Euclides Rodrigues Quinta de Lourosa José Domingues Quinta da Pedra João Melícias Quinta da Soalheira Frederico Gomes Sociedade Agrícola Gabriel Francisco Dias & Irmãs Leonor Novais Solar de Bouças

17 Results - Allelic distribution Most frequent alleles AAT AAT5 AAT6 AAT AAT4 Locus Nº of alleles l Allele (nº of repeats) ScAAT ScAAT ScAAT ScAAT ScAAT ScAAT C C YPL ScYOR Distribution of the most frequent alleles among grape varieties of the Vinho Verde Region in consecutive years Vinho Verde Allele e frequency Alvarinho - 5 strains Loureiro - 96 strains Avesso - 64 strains ScAAT ScAAT ScAAT ScAAT4 ScAAT5 ScAAT6