Bulk segregation analysis for seed size and foliar disease resistance

The QTL-seq analysis was conducted from two RIL populations using the multiseason phenotyping data for three traits, namely, pod weight, rust resistance and LLS resistance. In brief, the bulks were made by pooling DNA from selected RILs with extreme phenotypes for these traits. For pod weight, the DNA from 54 RILs possessing low pod weight and 54 RILs with high pod weight were pooled from the population (Yueyou 92 × Xinhuixiaoli). Similarly, DNA from 25 RILs each for resistance and susceptible RILs (TAG 24 × GPBD 4) were pooled to constitute four bulks, that is, resistant bulk for rust and LLS, and susceptible bulk for rust and LLS, respectively. The resistance parent GPBD 4 was an interspecific derivative of A. cardenasii, that is, the resistance source for both of the diseases. Together with four parents, a total of ten DNA samples were sequenced on Illumina HiSeq 2500. The sequencing data were analyzed using the QTL-seq pipeline⁸⁶ (http://genome-e.ibrc.or.jp/home/bioinformatics-team/mutmap) for calculating the SNP-index using the tetraploid genome assembly developed and reported in this article. The ∆SNP-index for each trait was then calculated by subtracting the SNP-index of one bulk from that of another bulk. The candidate-gene discovery was performed in the genomic regions with the highest ∆SNP-index.