BackgroundSclerotinia 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.ResultsGenotyping-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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4160-1) contains supplementary material, which is available to authorized users.

RESUMO A ferrugem asiática da soja (FAS), causada pelo fungo Phakopsora pachyrhizi, é uma das doenças mais severas na cultura. Dentre as estratégias para seu manejo propõe-se a utilização de genótipos com resistência horizontal ou parcial, ainda restritos no mercado, mas promissores pela durabilidade da resistência. O objetivo deste trabalho foi avaliar a resistência de diferentes genótipos de soja à ferrugem asiática para futura utilização em programas de melhoramento. Na safra 2015/2016, foram avaliados 11 genótipos de soja provenientes do LAGER-UFU e a cultivar Desafio 8473 (padrão de suscetibilidade) frente à infecção artificial e natural de P. pachyrhizi no campo. Com os dados de severidade da FAS, calculou-se a área abaixo da curva de progresso da doença (AACPD). O percentual de área foliar afetada e os valores da AACPD dos genótipos avaliados oscilaram de 1,56 a 80,75 e de 380,62 a 975,62, respectivamente. Os genótipos L-210, L-166, L-268, L-203, L-279, L-224, L-216 e L-218 apresentam menores valores de AACPD, indicando resistência parcial, enquanto os genótipos L-144, L-104, L-266 e a cv. Desafio 8473 apresentaram maiores valores de AACPD, portanto, com menor resistência parcial ou suscetíveis. Ainda assim, os genótipos L-144, L-104 e L-266 apresentaram coeficientes de progresso da severidade da doença de 1,8 a 2,0 vezes menores comparativamente à cv. Desafio 8473. Conclui-se que os genótipos do Programa de Melhoramento Genético da UFU (LAGER-UFU), apresentam potencial de utilização no desenvolvimento de cultivares de soja resistentes à P. pachyrhizi.

White mold is a disease of great importance in Brazil. Selection for white mold resistance would be greatly facilitated by the use of molecular markers and the discovery of genes involved with the disease. Soybean do not have complete resistance, but instead have differing levels of partial resistance that could be successful in slowing disease progression enough to allow the plants to recover, especially if the environment favors more the plant and less the pathogen. Molecular markers of the SNP type were identified from soybean genotypes, with the purpose of associating molecular markers with the resistance to fungus S. sclerotiorum in the wide genome by the GBS technique. We collected soybean genotypes reported as partially resistant to disease, as well as many from three different breeding programs in Brazil. We inoculated the plants with the isolate Jataí at the V2 stage using the straw test method and scored disease progress down the plant stems at four days post inoculation.

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 andHaberer 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.

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