Identificação de marcadores SNP em genes candidatos associados ao conteúdo e qualidade do óleo de soja

• Ano de defesa: 2012
• Autor(a) principal: Bueno, Rafael Delmond
• Orientador(a): Moreira, Maurílio Alves
• Banca de defesa: Piovesan, Newton Deniz , Teixeira, Arlindo Inês
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Viçosa
• Programa de Pós-Graduação: Doutorado em Bioquímica Agrícola
• Departamento: Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
• País: BR
• Palavras-chave em Português: Soja; SNP; Óleo
• Palavras-chave em Inglês: Soy, SNP; Oil
• Área do conhecimento CNPq: CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOTECNIA::MELHORAMENTO VEGETAL
• Link de acesso: http://locus.ufv.br/handle/123456789/320

The development of productive cultivars with significant increases in protein and/or oil, as well as improving the fatty acid composition of the oil fraction, are among the main objectives of soybean breeding programs. Identification and validation of SNPs (Single Nucleotide Polymorphism) in gene regions associated with these characteristics and their use in marker-assisted selection (MAS) represent a powerful tool for genetic improvement of soybean, when seeking to increase efficiency and provide greater genetic gains. The objective of this study was to identify SNPs in gene regions associated with the content and quality of oil for contrasting genotypes of the germplasm bank of the Soybean Quality Improvement Program (Programa de Melhoramento da Qualidade da Soja) of UFV, and validate polymorphisms as molecular markers for segregating populations and RILs. Twenty-two candidate genes were selected that are potentially related to the content and quality of soybean oil. The partial sequences of the 22 candidate genes or homologous genes were obtained from the GenBank. Complete gene sequences were obtained from the BLASTn algorithm in the PHYTOZOME database. The primers designed to amplify specific regions of each candidate gene in contrasting genotypes with respect to content and quality of soybean oil, yielded 33.91 kbp of high quality sequences, with Phred value greater than or equal to 30, where 88 were identified as SNPs and 26 as INDELs. In order to obtain information on expression of candidate gene transcripts, an alignment was performed using the BLASTn algorithm against ESTs libraries derived from seven soybean tissues deposited in the NCBI database. The results showed that all transcripts selected of the genes ADPRI and G3PDH were expressed constitutively, while transcripts of the genes ABI3, ACC, AMIP, ARAF, ASIL, CLPPR, CPT, DGAT, DOF4, FUS3, LEC1A, LEC1B, LPCAT, MDH and WRI showed organ-specific expression. Primers for TSPCR genotyping (Temperature-Switch Polymerase Chain Reaction) were used to discriminate the SNP alleles in five populations (RIL and F2). The polymorphisms identified by sequencing were first tested in their respective progenitors, and for this purpose 20 TSPCR primer sets were designed. Only the TSPCR primers designed in polymorphism of the genes AMIP-5, AMIP-3, ABI3, LEC1B, ARAF, LPCAT, PDAT, FAD3B and FAD3C showed polymorphism, without nonspecific bands and/or marking of weak bands in their respective progenitors. The TSPCR genotyping primers designed in the polymorphism of genes AMIP-5, AMIP-3, ABI3 and LEC1B were amplified in 176 F6 progenies of the cross PI371611 x CD222 (contrasting oil contents). TSPCR primers designed in polymorphism of genes ARAF, LPCAT, and LEC1B were genotyped in 236 progenies in the F6 generation, derived from the cross CD01RR8311 x SUPREMA (contrasting oil contents). The TSPCR primers designed to detect polymorphism present in genes ABI3 and LEC1B were amplified in 170 F6 progenies derived from a cross between A7002 x CD219 (contrasting oil contents). The TSPCR primers designed to discriminate the SNP alleles identified in the candidate genes ARAF and PDAT were amplified in 259 F6 progenies of the cross FA22 x CD219 (contrasting oleic acid content). TSPCR primers designed in the polymorphism of genes FAD3B, FAD3C and ABI3 were amplified in 185 progeny in generation F2 of the cross A29 x Tucunaré (contrasting linolenic acid content). Polymorphism of the gene ABI3 presented significant associations with protein concentration in the population F6 (A7002 x CD219) and with the characteristic palmitic acid content in population F2:3 of the cross between A29 and Tucunaré. In this same population significant associations were detected for the SNP marker present in gene FAD3B with the levels of stearic, oleic and linolenic fatty acids, and SNP marker of gene FAD3C with levels of linoleic and linolenic acids. The TSPCR genotyping methodology proved effective in discriminating SNP alleles in the five populations studied. Association of SNP markers present in genes ABI3, FAD3B and FA3C, which are related to the biosynthesis of fatty acids, allow the use of SAM in order to select individuals with the best fatty acid profile, optimizing the development of special soybean lines for agribusiness.