Genome-wide association mapping of resistance to a Brazilian isolate of Sclerotinia sclerotiorum in soybean genotypes mostly from Brazil.

BACKGROUND: Sclerotinia Stem Rot (SSR), caused by the fungal pathogen Sclerotinia sclerotiorum, is ubiquitous in cooler climates where soybean crops are grown. Breeding for resistance to SSR remains challenging in crops like soybean, where no single gene provides strong resistance, but instead, multiple genes work together to provide partial resistance. In this study, a genome-wide association study (GWAS) was performed to dissect the complex genetic architecture of soybean quantitative resistance to SSR and to provide effective molecular markers that could be used in breeding programs. A collection of 420 soybean genotypes were selected based on either reports of resistance, or from one of three different breeding programs in Brazil, two commercial, one public. Plant genotype sensitivity to SSR was evaluated by the cut stem inoculation method, and lesion lengths were measured at 4 days post inoculation. RESULTS: Genotyping-by-sequencing was conducted to genotype the 420 soybean lines. The TASSEL 5 GBSv2 pipeline was used to call SNPs under optimized parameters, and with the extra step of trimming adapter sequences. After filtering missing data, heterozygosity, and minor allele frequency, a total of 11,811 SNPs and 275 soybean genotypes were obtained for association analyses. Using a threshold of FDR-adjusted p-values <0.1, the Compressed Mixed Linear Model (CMLM) with Genome Association and Prediction Integrated Tool (GAPIT), and the Fixed and Random Model Circulating Probability Unification (FarmCPU) methods, both approaches identified SNPs with significant association to disease response on chromosomes 1, 11, and 18. The CMLM also found significance on chromosome 19, whereas FarmCPU also identified significance on chromosomes 4, 9, and 16. CONCLUSIONS: These similar and yet different results show that the computational methods used can impact SNP associations in soybean, a plant with a high degree of linkage disequilibrium, and in SSR resistance, a trait that has a complex genetic basis. A total of 125 genes were located within linkage disequilibrium of the three loci shared between the two models. Their annotations and gene expressions in previous studies of soybean infected with S. sclerotiorum were examined to narrow down the candidates.

Optimization of soybean dna extraction under different storage and development periods / Otimização de extração de dna de soja sob diferentes períodos de armazenamento e desenvolvimento

DNA extraction of plants with high quality is very important to researches in molecular biology. Several extraction protocols have been used to obtain soybean DNA; however, there is a lack of papers about extraction protocols optimization and the best developmental stage of the plant to collect them. Therefore, the main purpose of the study was to extract high quantity and quality of DNA from fresh or frozen soybean samples, using different protocols. Moreover, we analyzed the best developmental stage of the plant to do the extraction. Fresh leaves or leaves kept for two years in the ultra-freezer were submitted to the DNA extraction protocols: Haberer et al., 1996 (modified); second modification from Haberer et al., 1996; Murray & Thompson, 1980 (modified) e Doyle & Doyle, 1990 (modified). Modified protocol of Doyle & Doyle was used to value the best stage to collect the leaves to do the DNA extraction. The samples were collected in the stages of development VC, V1, V2, V3, V4 and R5. The experiments were conducted in completely randomized design with 10 samples per treatment. The data underwent variance analysis and the averages were compared by the Tukey test (p<0.05). Through Doyle & Doyle, 1990 and Haberer et al., 1996 modified protocols, for both fresh and frozen samples, it was possible to obtain a higher total DNA concentration if compared to the other tested protocols. However, the quality of DNAs extracted by the protocol Doyle & Doyle, 1990 (modified) was superior, due to a minor molecular degradation. Besides that, the extractions made with these protocols have shown to be more efficient using frozen leaves' tissue. Higher DNA concentrations were obtained analyzing VC samples; however, there were no statistical differences between the stages VC, V2 and V3. It is suggested thereby to use modified of Doyle & Doyle for DNA extraction from soybean leaves in V2 and V3 stages of development from frozen samples, providing the collect of a large number of samples and its storage until the analysis.

A extração de DNA de plantas com alta qualidade é de suma importância para pesquisas em biologia molecular. Diversos protocolos de extração vêm sendo utilizados para a obtenção de DNA de soja; contudo, há uma carência de trabalhos de otimização de protocolos de extração e de escolha do melhor estádio de desenvolvimento da planta para a coleta. Desta forma, o objetivo do estudo foi extrair DNA com alta quantidade e qualidade a partir de amostras frescas ou congeladas de soja, utilizando diferentes protocolos de extração. Além disso, foi analisado o melhor estádio de desenvolvimento da planta para a extração. Folhas frescas e armazenadas por cerca de dois anos em ultrafreezer foram submetidas aos protocolos de extração de DNA: Haberer et al., 1996 (modificado); segunda modificação de Haberer et al., 1996; Murray & Thompson, 1980 (modificado) e Doyle & Doyle, 1990 (modificado). Para a avaliação do melhor estádio de coleta das folhas para a extração de DNA foi utilizado o protocolo de Doyle & Doyle modificado. As coletas de amostras foram realizadas nos estádios de desenvolvimento VC, V1, V2, V3, V4 e R5. Os experimentos foram conduzidos em delineamento inteiramente casualizado com 10 amostras por tratamento. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste de Turkey (p<0,05). Através dos protocolos modificados de Doyle & Doyle, 1990 e Haberer et al., 1996, tanto para amostras frescas como para congeladas, foi possível obter uma maior concentração de DNA total se comparado aos demais protocolos testados. Porém, a qualidade dos DNAs extraídos pelo protocolo Doyle & Doyle, 1990 (modificado) foi superior, devido a menor degradação da molécula. Além disso, as extrações efetuadas com estes protocolos se mostraram mais eficientes quando foram utilizados tecidos foliares congelados. Maiores concentrações de DNA foram obtidas quando amostras em VC foram analisadas; porém, não houve diferença estatística entre os estádios VC, V2 e V3. Assim, sugere-se a utilização do protocolo modificado de Doyle & Doyle para extração de DNA de folhas de soja nos estádios de desenvolvimento V2 e V3 a partir de amostras congeladas, viabilizando a coleta de um grande número de amostras e o seu armazenamento até a análise.