Genotyping-by-Sequencing of Rice Genome

Extracted DNA samples were digested for 3 h at 75 °C with the restriction enzyme ApeKI (New England Biolabs, Ipswich, MA, U.S.A), and genotyping-by-sequencing (GBS) was performed based on the methods outlined by Vigueira et al. [26 (link)] using the Illumina Hiseq 2000 platform (Illumina Inc., San Diego, CA). Raw sequence reads in a FASTQ format were processed using a standard TASSEL-GBS pipeline version 5.0 (http://www.maizegenetics.net/) [27 (link)]. Briefly, filtered reads were aligned to the rice genome MSU 6.0 (http://rice.plantbiology.msu.edu) using the Burrows–Wheeler alignment (BWA) tool [28 (link)]. This allowed a maximum of four mismatches and no gap within 5 bp at the end of each read. Loci with more than 30% of missing data and monomorphic data were discarded. Individuals with more than 95% of missing data were removed and not included in this study. After filtering, a total of 32,053 SNPs were retained for GWAS analysis. The mixed linear model (MLM) in the TASSEL-GBS program was used to generate a Manhattan plot for association analysis.