DIVERSITY and GENETIC RELATEDNESS AMONG GENOTYPES of Vitis Spp

799 DIVERSITY AND GENETIC RELATEDNESS AMONG GENOTYPES OF Vitis spp. USING MICROSATELLITE MOLECULAR MARKERS1 PATRÍCIA COELHO DE SOUZA LEÃO2, COSME DAMIÃO CRUZ3 SÉRGIO YOSHIMITSU MOTOIKE3 ABSTRACT - The purpose of this research was to study the genetic diversity and genetic relatedness of 60 genotypes of grapevines derived from the Germplasm Bank of Embrapa Semiárido, Juazeiro, BA, Brazil. Seven previously characterized microsatellite markers were used: VVS2, VVMD5, VVMD7, VVMD27, VVMD3, ssrVrZAG79 and ssrVrZAG62. The expected heterozygosity (He) and polymorphic information content (PIC) were calculated, and the cluster analysis were processed to generate a dendrogram using the algorithm UPGMA. The He ranged from 81.8% to 88.1%, with a mean of 84.8%. The loci VrZAG79 and VVMD7 were the most informative, with a PIC of 87 and 86%, respectively, while VrZAG62 was the least informative, with a PIC value of 80%. Cluster analysis by UPGMA method allowed separation of the genotypes according to their genealogy and identification of possible parentage for the cultivars ‘Dominga’, ‘Isaura’, ‘CG 26916’, ‘CG28467’ and ‘Roni Redi’. Index terms: Grape, multivariate analysis, genealogy, SSR. DIVERSIDADE E RELAÇÕES GENÉTICAS ENTRE GENÓTIPOS DE Vitis spp. UTILIZANDO MARCADORES MOLECULARES MICROSSATÉLITES RESUMO- O presente trabalho teve como objetivo estudar a diversidade genética e as relações de parentesco de 60 genótipos de videira procedentes do Banco de Germoplasma da Embrapa Semiárido, Juazeiro-BA. Sete marcadores microssatélites previamente caracterizados foram utilizados: VVS2, VVMD5, VVMD7, VVMD27, VVMD3, ssrVrZAG79 e ssrVrZAG62. Foram calculadas a heterozigosidade esperada (He), conteúdo de informação polimórfica (PIC), e as análises de agrupamento foram processadas para gerar um dendograma, utilizando-se do algoritmo UPGMA. A He variou de 81,8 a 88,1%, com média de 84,8%. Os lócus VrZAG79 e VVMD7 foram os mais informativos, com PIC de 87 e 86%, respectivamente, enquanto VrZAG62 foi o menos informativo, com um valor de PIC de 80%. A análise de agrupamento pelo método UPGMA permitiu separar os genótipos de acordo com sua genealogia e identificar possíveis parentais para as cultivares ‘Dominga’, ‘Isaura’, ‘CG 26916’, ‘CG28467’ e ‘Roni Redi’. Termos para indexação: Uva, análise multivariada, genealogia, SSR. 1(Trabalho 233-12). Recebido em: 28-08-2012. Aceito para publicação em: 18-06-2013. 2Researcher (D.Sc.) - Embrapa Semi-Árido, BR 428, Km 152, Zona Rural, CEP:56300-970, Petrolina-PE, Caixa-Postal: 23 Phone: +55 (87) 38621711 Fax: (87) 38621744. E-mail: [email protected] 3Professor (D.Sc.) - Universidade Federal de Viçosa, Câmpus Universitário, Centro, CEP: 36570000 – Viçosa-MG, Brasil Phone: +55(31) 38992906. E-mails: [email protected]; [email protected] Rev. Bras. Frutic., Jaboticabal - SP, v. 35, n. 3, p. 799-808, Setembro 2013 800 P. C. de S. LEÃO et al. INTRODUCTION MATERIAL AND METHODS The development of new grapevine cultivars Plant matter adapted to Brazilian tropical and subtropical A group of 60 grapevine accessions were conditions has been one of the main objectives assessed, including 33 hybrids, 22 accessions of of genetic breeding programs in the country. At the species Vitis vinifera L., and five accessions EMBRAPA Semiárido, grapevine breeding studies of unknown origin. The accessions are part of the are in an initial phase, and, in most recent decades, Germplasm Bank of Embrapa Semiárido, located in research efforts have been concentrated on morpho- Juazeiro, state of Bahia, Brazil (9o24’S, 40o26’W, and agronomic enrichment and characterization of the 365.5m of altitude) and they are selected based on genotypes of the Germplasm Bank (BORGES et their importance for grape breeding and on previous al. 2008; LEÃO et al., 2010; LEÃO et al., 2011). studies (LEÃO et al., 2010; 2011). This collection has begun in 1965 with cultivars collected in the Northeast region and later expanded DNA extraction and amplification conditions with others imported from FAO in Italy; Instituto Agronômico de Campinas, in São Paulo; and Genomic DNA was extracted from young Embrapa Uva e Vinho, in Rio Grande do Sul. It expanded leaves collected from the apex of shoots, as currently has 261 accessions, with most of the species described by Lodhi et al. (1994). Vines were 8 years being Vitis vinifera L.. at the time to collect the samples. The leaves were Most of the grapevine cultivars are ancient homogenized by means of a mechanical homogenizer and were derived from different processes, such as Homes 6 (Bioreba, Longmont, CO). In the final stage, differentiation from wild grapevines, spontaneous the pellets were suspended in a 1X Tris EDTA buffer crosses between wild grapevines and cultivars solution and stored at -20oC. The integrity of the or crosses between cultivars. Ease of asexual DNA was verified in 0.8% agarose gel stained with propagation gave rise to an estimated number ethidium bromide. of 14,000 cultivars, for different purposes: fresh Seven previously characterized microsatellite consumption, raisins, juices and wine, with the markers were used: VVS2 (Thomas e Scott, 1993), number of grapevine cultivars maintained in VVMD5, VVMD7, VVMD27, VVMD31 (BOWERS germplasm collections being estimated at around et al. 1996; BOWERS et al. 1999), ssrVrZAG79 and 10,000 (ALLEWELDT and DETTWEILER, 1994). ssrVrZAG62 (SEFC et al. 1999). They were chosen Older wild cultivars involved in natural crosses were as being most recommended for characterization of not able to be identified since they no longer exist cultivars in the grapevine germplasm collections naturally, but many parent species are still cultivated (THIS et al., 2004). One of the primers of each pair or are preserved in collections. Through the use of was labeled at the 5’ end with fluorescent dyes such molecular methods, these parent cultivars and their as 6-FAM (blue), HEX (yellow) or NED (green) descendents may be recognized, allowing the study (Applied Biossystems). of their geographic origin and evolutionary history. Amplifications were performed in a final The use of microsatellite markers has reaction volume of 10 µl containing 2.5 ng/µl DNA, allowed reconstruction of pedigrees of innumerable 10 pmoles of each primer, 2.5 mM of deoxynucleotides economically important grapevine cultivars, such mix (Applied Biossystems); 1 µL of Gold 10X as the discoveries that ‘Cabernet Sauvignon’ is a buffer (Applied Biossystems); 2mM MgCl2 (Applied descendent of ‘Cabernet Franc’ and ‘Sauvignon Biossystems) and 0.5 units of AmpliTaq Gold DNA Blanc’ (BOWERS and MEREDITH, 1997), ‘Durif’, polymerase (Applied Biossystems). known in California as ‘Petit Syrah’ (MEREDITH et The PCR (polymerase chain reaction) al., 1999), resulted from the crossing of ‘Peloursin’ X was performed in a thermocycler PTC-100 (MJ ‘Syrah’, and ‘Muscat de Hamburgo’ is a descendent Researcher) using a single program for all the of ‘Moscato de Alexandria’ and ‘Schiava Grossa’ primers. The amplification program consisted of an (CRESPAN, 2003). initial stage of 5 minutes at 95oC, followed by 35 The purpose of this paper was to study the cycles of 30 seconds at 95oC, 45 seconds at 60oC genetic diversity and genetic relatedness of 60 and 1 minute at 72oC and a final extension stage of genotypes belonging to the grapevine Germplasm 7 minutes at 72oC. Bank of Embrapa Semiárido in Petrolina, PE. The amplifications were confirmed in 2% agarose gel stained with ethidium bromide (10 mg/ mL). The PCR products were denatured at 94oC for Rev. Bras. Frutic., Jaboticabal - SP, v. 35, n. 3, p. 799-808, Setembro 2013 DIVERSITY AND GENETIC RELATEDNESS AMONG GENOTYPES 801 2 minutes and then applied in 6% acrylamide gel, from different breeding programs. The largest group performing electrophoresis in a DNA sequencer is composed by Brazilian cultivars from the breeding model ABI 377 (PE/Applied Biosystems). programs of Instituto Agronômico de Campinas- The sizes of the alleles were assessed based IAC (‘A Dona’, ‘Aurora’ or ‘IAC 77526’, ‘Isaura’, on comparison with the known internal size standard ‘Juliana’, ‘Patricia’, ‘Paulistinha’ and the root stock Genescan Rox 500 (Applied Biosystems) and four cultivars ‘IAC 313’ and ‘IAC 766’) and Embrapa grapevine cultivars (Carignane, Riesling, Thompson Uva e Vinho (‘Moscato Embrapa’, ‘BRS Rubea’, Seedless and Chardonnay), whose allelic profiles ‘BRS Clara’, ‘BRS Linda’, ‘BRS Morena’ and ‘BRS are known as database of Foundation Plant Service/ Lorena’). USDA and University of California at Davis, to All the loci used amplified in a satisfactory allow comparison of the allelic size profiles obtained. manner in almost all the cultivars and were The individuals were genotyped using the software multiallelic. Eighty-nine alleles were obtained, GeneScanTM version 3.1 and GenotyperTM version ranging from 11 (VVS2) to 14 (VVMD31 and 2.5.2. (PE/Applied Biosystems). VVMD7), with an average and effective number of 12.7 alleles per locus (Table 1). This value is higher Statistical analyses than those already mentioned by other authors The expected heterozygosity (He) was working with grapevine accessions of diverse 2 calculated according to the formula: He = 1 - ∑pi , geographic origins. Almadanim et al. (2007) obtained where pi is the frequency of the allele ith for the locus a mean of 8.17 alleles per locus, Vouillamoz et al. studied (Nei, 1987). The polymorphic information (2006) observed a mean value of 11.9 alleles per 2 2 content (PIC) was calculated as 1 - ∑ pi - ∑∑ 2 pi locus and Martín et al. (2003) found 11 alleles per 2 pj , where pi is equal to the frequency of the ith locus. Lamboy and Alpha (1998), analyzing the allele and pj is the frequency of the (i + 1)th allele diversity of 110 accessions belonging to 21 species (BOTSTEIN et al., 1980). Dissimilarity matrices of Vitis and 4 hybrids found 24.4 alleles per locus, were obtained using the arithmetic complement of a greater quantity than that observed in this study. the weighted index given by: The most frequent alleles in each locus were VVMD5 – 238 (25.8%), VVMD7 – 239 (21.6%), VVMD27 – 185 (26.7%), VVMD31 – 212 (29.2%), VVS2 -133 (20.0%), VrZAG62 – 189 (35.0%) and VrZAG79 – 255 (21.7%) (Figure 2).

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