Ext-BSA,extended Bulked Sample Analysis,and Its Validation By Identifying Resistance Loci For Turcicum Leaf Blight And Gray Leaf Spot In Maize

Bulked segregant analysis,which utilizes polymorphic markers and DNA bulks constructed using materials with extreme phenotypes,can be used to identify trait associated loci.With the development of sequencing technology,bulk DNA sequencing has been designed by using bulked segregant analysis.Then,bulked sample analysis?BSA?was proposed to improve bulked segregant analysis and plant materials selected from both segregant and natural populations could be used.For complex traits in plants,bulked sample analysis needs to be improved.In this study,an extended bulked sample analysis?Ext-BSA?was proposed and statistical power analysis was analyzed for conventional Ext-BSA,SNP-index and GWAS and using published genotype data and simulated phenotype data.Finally,Ext-BSA was used to identify resistant loci for tuicicum leaf blight in a maize natural population and gray leaf spot in a maize bi-parental population.The results are provided as follows:1.An extended-BSA method?Ext-BSA?was developed in this study,and the testing for each locus was extended to include five markers around the locus.The normalization value S-score was derived by subtracting the average value across the whole genome from the maximum value in extended region.The significant markers were declared with Bonferroni correction method.Furthermore,the t test for allele frequency,involved in this method,also improved the bulked sample analysis.2.Based on published genotyping by sequencing?GBS?data,375 tropical inbred lines were selected to simulate phenotype data for 1,000 times.Ext-BSA,SNP-index and GWAS were compared and the ratios for selection of extreme materials were adopted in SNP-index and Ext-BSA methods.The results indicate that the power for Ext-BSA ranging 51.6-98.1%was one fold larger than that for SNP-index.The false discovery rate?FDR?for SNP-index was close to 1,compared to about 0.5 for Ext-BSA.Compared to bulked sample analysis,genome-wide association study based on the mixed linear model performed best since it utilized the genotype and phenotype data from the entire population.In general,Ext-BSA was better than SNP-index,but the cost for bulked sample analysis was lower than GWAS.Therefore,bulked sample analysis was efficient in gene mapping.3.A total of 661 maize inbred lines were used to construct two bulked samples for turcicum leaf blight with 33 associated QTL identified.Gene annotation identified five candidate genes resistant to the disease.4.An F₂ population developed in our lab containing 659 lines was used for bulked sample analysis with 32 QTL identified for resistance to gray leaf spot.Gene annotation identified seven genes resistant to the disease.