Wine Authenticity – a genomic approach
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2016-01-28
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A globalização do sector alimentar disponibiliza aos consumidores uma grande diversidade de produtos, aumentando a sua preocupação em relação à sua origem. As vantagens associadas às metodologias baseadas em DNA, fiabilidade e reprodutibilidade, têm potenciado a sua aplicação nesta temática. Na última década, as ferramentas bioinformáticas, surgiram como uma necessidade para analisar o crescente volume de dados obtidos em biologia molecular. A vitivinicultura é uma actividade economicamente sustentável, com necessidade de mão de-obra especializada e com relevância para a sociedade, não só em termos económicos, como também ambientais e sociais. O valor e qualidade dos vinhos devem-se, entre outros factores, às castas envolvidas na sua produção. As regiões vínicas com a Denominação de Origem (DO) contemplam apenas a utilização de um número restrito de castas. No entanto, é permitido por lei a introdução de outras castas, em percentagens legalmente definidas, constituindo uma oportunidade para práticas fraudulentas. Com o presente estudo pretende-se desenvolver um sistema para autenticidade, de vinhos e mostos, eficiente no controle de prácticas fraudulentas e detecção de rotulagem incorrecta, visando a valorização dos vinhos DO. Numa primeira abordagem, DNA genómico extraído de Vitis vinifera L. (folhas e mostos monovarietais) de 10 castas portuguesas e uma referência (Cabernet Sauvignon) foi analisado utilizando marcadores moleculares microssatélites (SSR) e intermicrossatélites (ISSRs). Os perfis obtidos para as amostras de folhas e mostos, com marcadores ISSRs não foram coincidentes. Por outro lado, a utilização de seis marcadores SSRs (estabelecidos pela International Organization of Vine and Wine - OIV), revelou a presença de 42 alelos coincidentes, permitindo a discriminação das amostras de mosto baseado no perfil alélico da respectiva casta. Este estudo demonstrou a eficiência dos marcadores moleculares SSRs, com aplicação na autenticação varietal dos mostos. Na autenticidade de vinhos, a extracção de DNA a partir de vinho é a principal limitação quando se pretende aplicar metodologias baseadas no DNA. Neste estudo foi desenvolvido um protocolo de extracção de DNA considerando as características específicas da matriz do vinho. O protocolo desenvolvido permitiu a extracção de grandes quantidades de DNA e de elevada qualidade. As concentrações de DNA variaram entre 260 e 465 ng/µL e as razões qualitativas, A260nm/A280nm e A260nm/A230nm, entre 1,71 - 1,81 e 1,66 - 1,98, respectivamente. O DNA obtido de amostras de vinho foi amplificado por PCR utilizando os marcadores SSRs, com total correspondência entre o perfil do vinho monovarietal (tinto e branco) e a respectiva casta. O mesmo protocolo foi aplicado a vinhos de lotes comerciais e os resultados mostraram que o tamanho dos alelos obtidos era o esperado, considerando as castas referidas no rótulo. Assim, este método optimizado pode ser a base para a implementação de um sistema de autenticidade fidedigno. Atualmente, os Single Nucleotide Polymorphism (SNPs) são os marcadores moleculares mais aplicados em plantas e referem-se à identificação de polimorfismos baseados na alteração de um único nucleótido. Devido às suas características inerentes apresentam um enorme potencial para fins de autenticidade. A técnica de High Resolution Melting (HRM) é presentemente utilizada para a deteção de SNPs e eventos de Inserções-Deleções (INDELs) no genoma de Vitis. Esta técnica baseiase na variação da temperatura média de desnaturação do DNA na presença de um fluoróforo saturado que se intercala na cadeia dupla de DNA. O perfil antociânico é específico de cada casta, sendo um factor potencialmente interessante para a identificação varietal e, portanto, para a detecção de SNPs. Com base neste pressuposto, foi realizado um estudo molecular com a finalidade de detetar SNPs em genes que codificam as enzimas da via biossintética das antocianinas (i.e., Chalcona isomerase - CHI e UDP-glucose:flavonóide-3-Oglucoil-transferase - UFGT), a fim de diferenciar um conjunto específico de castas. A sequência do gene CHI apresentou apenas 5 SNPs. Por sua vez, o gene UFGT mostrou ser muito polimórfico, com um total de 58 SNPs e 1 INDEL para um grupo de 22 castas estudadas. Um ensaio de HRM foi estabelecido com base em um fragmento de 704 bp, pertencente ao gene UFGT, permitindo a discriminação de 18 haplótipos, tendo em conta a forma da curva de desnaturação. Um outro estudo foi realizado para avaliar a futura aplicação da técnica de HRM, como método de análise na autenticidade de mostos e vinhos. Para validar os diferentes ensaios utilizaram-se três tipos de amostras: folha, mosto e vinho monovarietal, tendo em consideração o tamanho do fragmento amplificado (Vv1-704 bp, Vv2-375 bp e Vv3-119 bp). O ensaio Vv1-HRM, baseado no gene UFGT e considerando 32 variações nucleotídicas, foi bem-sucedido quando aplicado a amostras de mostos, permitindo a sua total discriminação. Os fragmentos Vv2 (F3H) e Vv3 (UFGT) foram amplificados com sucesso em todas as amostras testadas, permitindo a discriminação dos genótipos. A análise HRM baseou-se na temperatura média de desnaturação (Tm) para o fragmento Vv2 e na forma da curva de desnaturação para os fragmentos Vv1 e Vv3. O presente estudo descreve um ensaio HRM sensível, rápido, eficiente e promissor, aplicado pela primeira vez em amostras de vinhos, com excelentes perspectivas de aplicação na autenticidade de vinhos.
Food market globalization offers consumers a great diversity of food products, increasing their concern in relation to the products’ origin. The development of food authenticity/traceability systems in the last decades have aimed to re-establish consumers’ confidence. The use of DNA-based systems for this particular purpose has widely increased in the last years, as DNA technology has become more precise and robust, along with bioinformatics’ tools and databases available. Vine-growing and wine production is an essential economic and labour intensive activity and plays a major role to society in socioeconomic, environmental and societal terms. Wine quality and market value greatly depend on the grapevine varietal composition. The wine producing region, specified by the Denomination of Origin (DO), can only use a limited number of grapevine varieties. However, other varieties can be included in wine production under certain legally defined percentages which is by itself an attraction for fraudulent practices. With the present study we intend to contribute for the establishment of an efficient wine authenticity system targeting the valorization of DO wines, fraud and mislabeling detection, considering must and wine samples. Primarily, DNA extracted from Vitis vinifera L. (leaf and monovarietal must) of ten different Portuguese grapevine varieties and one of reference (Cabernet Sauvignon) were screened using microsatellite (SSR) and inter-microsatellite (ISSRs) marker systems. The ISSR markers did not produce coincident results between reference leaf and must samples. Concerning the 6 SSRs markers used (established by the International Organization of Vine and Wine – OIV), a total of 42 alleles were detected, allowing the discrimination of the must samples according to the respective leaf sample. This study indicates that SSR markers may be successfully applied to must varietal authentication. The DNA extraction from wine samples is the main bottleneck when aiming to apply DNAbased methodologies to wine authenticity. In this study, a DNA extraction protocol was established considering the particular characteristics of the wine matrices. The developed protocol allowed the extraction of large amounts of high-quality DNA from wines (ranges from 260 to 465 ng/µL) with optimal A260nm/A280nm and A260nm/A230nm ratios ranging from 1.71 to 1.81 and from 1.66 to 1.98, respectively. All DNA extracted from wine samples were amplifiable with a specific Vitis vinifera L. SSR markers, with a total correspondence between monovarietal wine (red and white) and its respective variety. The same protocol was applied to commercial blended wines producing amplicons with the expected size range, taking into consideration the varieties referred on the label. This enhanced method can be the basis of a successful authenticity system. Single Nucleotide Polymorphisms (SNPs) have become the most popular genetic marker system in plants and are interesting for authenticity purposes due to their ability to identify small genetic variations. High Resolution Melting (HRM) is a recent advance technique for the detection of SNPs and Insertion-Deletions (INDELs) in the Vitis genome and depends on DNA melting in the presence of saturating intercalating double-stranded DNA binding dyes. The anthocyanin profile is specific of each variety, being potentially interesting for varietal identification and therefore for SNP detection. Based on these assumptions, a molecular study was conducted aiming to detect SNPs within the genes encoding the enzymes of the anthocyanin pathway (e.g., chalcone isomerase - CHI and UDP-glucose:flavonoid 3-O-glucosyl-transferase - UFGT) in order to differentiate a selection of grapevine varieties. The CHI gene sequence presented a low polymorphism rate, 5 SNPs. Whereas the UFGT gene was highly polymorphic, with a total of 58 SNPs and 1 INDEL, allowing the discrimination of 18 different genotypes within the 22 grapevine varieties studied. A HRM assay was designed based on a large UFGT fragment, 704 bp, allowing the discrimination of 18 haplotypes based on the melting curve shape. Another study was performed to evaluate the application of this HRM assay, as a screening method for must and wine authenticity purposes. Three sample types (leaf, monovarietal must and wine) were used to validate the different HRM assays according to the amplified fragment length (Vv1–704 bp, Vv2–375 bp and Vv3–119 bp). The Vv1 HRM assay, based on UFGT, was only successful applied to monovarietal must samples, allowing the discrimination of the must varietal composition considering 32 nucleotide differences. The Vv2 (F3H) and Vv3 (UFGT) fragments were successfully amplified in all the sample types, allowing the discrimination of the genotypes. The HRM analysis was based on the melt temperature (Tm) (Vv2 fragment) and in the melting curve shape (Vv1 and Vv3 fragments). This current study reports on a sensitive, rapid, efficient and promising HRM assay applied for the first time to wine samples with excellent prospects for wide application in wine authenticity.
Food market globalization offers consumers a great diversity of food products, increasing their concern in relation to the products’ origin. The development of food authenticity/traceability systems in the last decades have aimed to re-establish consumers’ confidence. The use of DNA-based systems for this particular purpose has widely increased in the last years, as DNA technology has become more precise and robust, along with bioinformatics’ tools and databases available. Vine-growing and wine production is an essential economic and labour intensive activity and plays a major role to society in socioeconomic, environmental and societal terms. Wine quality and market value greatly depend on the grapevine varietal composition. The wine producing region, specified by the Denomination of Origin (DO), can only use a limited number of grapevine varieties. However, other varieties can be included in wine production under certain legally defined percentages which is by itself an attraction for fraudulent practices. With the present study we intend to contribute for the establishment of an efficient wine authenticity system targeting the valorization of DO wines, fraud and mislabeling detection, considering must and wine samples. Primarily, DNA extracted from Vitis vinifera L. (leaf and monovarietal must) of ten different Portuguese grapevine varieties and one of reference (Cabernet Sauvignon) were screened using microsatellite (SSR) and inter-microsatellite (ISSRs) marker systems. The ISSR markers did not produce coincident results between reference leaf and must samples. Concerning the 6 SSRs markers used (established by the International Organization of Vine and Wine – OIV), a total of 42 alleles were detected, allowing the discrimination of the must samples according to the respective leaf sample. This study indicates that SSR markers may be successfully applied to must varietal authentication. The DNA extraction from wine samples is the main bottleneck when aiming to apply DNAbased methodologies to wine authenticity. In this study, a DNA extraction protocol was established considering the particular characteristics of the wine matrices. The developed protocol allowed the extraction of large amounts of high-quality DNA from wines (ranges from 260 to 465 ng/µL) with optimal A260nm/A280nm and A260nm/A230nm ratios ranging from 1.71 to 1.81 and from 1.66 to 1.98, respectively. All DNA extracted from wine samples were amplifiable with a specific Vitis vinifera L. SSR markers, with a total correspondence between monovarietal wine (red and white) and its respective variety. The same protocol was applied to commercial blended wines producing amplicons with the expected size range, taking into consideration the varieties referred on the label. This enhanced method can be the basis of a successful authenticity system. Single Nucleotide Polymorphisms (SNPs) have become the most popular genetic marker system in plants and are interesting for authenticity purposes due to their ability to identify small genetic variations. High Resolution Melting (HRM) is a recent advance technique for the detection of SNPs and Insertion-Deletions (INDELs) in the Vitis genome and depends on DNA melting in the presence of saturating intercalating double-stranded DNA binding dyes. The anthocyanin profile is specific of each variety, being potentially interesting for varietal identification and therefore for SNP detection. Based on these assumptions, a molecular study was conducted aiming to detect SNPs within the genes encoding the enzymes of the anthocyanin pathway (e.g., chalcone isomerase - CHI and UDP-glucose:flavonoid 3-O-glucosyl-transferase - UFGT) in order to differentiate a selection of grapevine varieties. The CHI gene sequence presented a low polymorphism rate, 5 SNPs. Whereas the UFGT gene was highly polymorphic, with a total of 58 SNPs and 1 INDEL, allowing the discrimination of 18 different genotypes within the 22 grapevine varieties studied. A HRM assay was designed based on a large UFGT fragment, 704 bp, allowing the discrimination of 18 haplotypes based on the melting curve shape. Another study was performed to evaluate the application of this HRM assay, as a screening method for must and wine authenticity purposes. Three sample types (leaf, monovarietal must and wine) were used to validate the different HRM assays according to the amplified fragment length (Vv1–704 bp, Vv2–375 bp and Vv3–119 bp). The Vv1 HRM assay, based on UFGT, was only successful applied to monovarietal must samples, allowing the discrimination of the must varietal composition considering 32 nucleotide differences. The Vv2 (F3H) and Vv3 (UFGT) fragments were successfully amplified in all the sample types, allowing the discrimination of the genotypes. The HRM analysis was based on the melt temperature (Tm) (Vv2 fragment) and in the melting curve shape (Vv1 and Vv3 fragments). This current study reports on a sensitive, rapid, efficient and promising HRM assay applied for the first time to wine samples with excellent prospects for wide application in wine authenticity.
Descrição
Esta dissertação foi expressamente elaborada com vista à obtenção do Grau de Doutor em Genética Molecular Comparativa e Tecnológica pela Universidade de Trás-os-Montes e Alto Douro.
Palavras-chave
Vitis vinifera L. , Vinho