Identification of genomic region and candidate genes underlying carotenoid accumulation in soybeans using next generation sequencing-based bulk segregant analysis

The improvement of soybean seed carotenoid contents is indispensably important owing to its beneficial role to human health and nutrition. However, the genetic architecture underlying soybean carotenoid biosynthesis remains largely unknown. In the present study, we employed the next generation sequencing-based bulked-segregant analysis for identifying new genomic regions governing seed carotenoids in 1551 natural soybean accessions. The genomic DNA samples of individual plants with extreme phenotype were pooled to form two bulks with high (50 accessions) and low (50 accessions) carotenoid contents for Illumina sequencing. A total of 125.09 Gbp of clean bases and 89.82% of Q30 were obtained and the average alignment efficiency was 99.45% with average coverage depth of 62.20× and 99.75% genome coverage. Based on the G’ method analysis, a total of 16 candidate genomic loci with a total length 20.41 Mb were found to be related to the trait. Of these loci, the most significant region displaying the highest elevated G’-value was found in chromosome 06 at a position of 18.53-22.67 Mb, and chromosome 19 at a genomic region intervals of 8.36-10.94, 12.06-13.79 and 18.45-20.26 Mbs, and were preferably taken to identify the key candidate genes. In these regions, 250 predicted genes were found and analyzed to get 90 significantly enriched (P<0.05) gene ontology (GO)-terms. Based on ANNOVAR analysis, 50 genes with non-synonymous and stopgained mutations were preferentially selected as potential candidate genes. Of which, following their gene annotation function and high significant haplotype variation in various environments, five genes were identified as the most promising candidate genes regulating soybean seed carotenoid accumulation, and suggested for further functional validation studies. Collectively, understanding the genetic bases of carotenoid pigments and identifying genes underpinning carotenoid accumulation via bulked-segregant analysis sequencing (BSA-seq) approach provide new insight for exploring future molecular breeding of high carotenoid content in soybean cultivars.