Dissecting seed pigmentation-associated genomic loci and genes by employing dual approaches of reference-based and k-mer-based GWAS with 438 Glycine accessions.

The soybean is agro-economically the most important among all cultivated legume crops, and its seed color is considered one of the most attractive factors in the selection-by-breeders. Thus, genome-wide identification of genes and loci associated with seed colors is critical for the precision breeding of crop soybeans. To dissect seed pigmentation-associated genomic loci and genes, we employed dual approaches by combining reference-based genome-wide association study (rbGWAS) and k-mer-based reference-free GWAS (rfGWAS) with 438 Glycine accessions. The dual analytical strategy allowed us to identify four major genomic loci (designated as SP1-SP4 in this study) associated with the seed colors of soybeans. The k-mer analysis enabled us to find an important recombination event that occurred between subtilisin and I-cluster B in the soybean genome, which could describe a special structural feature of ii allele within the I locus (SP3). Importantly, mapping analyses of both mRNAs and small RNAs allowed us to reveal that the subtilisin-CHS1/CHS3 chimeric transcripts generate and act as an initiator towards 'mirtron (i.e., intron-harboring miRNA precursor)'-triggered silencing of chalcone synthase (CHS) genes. Consequently, the results led us to propose a working model of 'mirtron-triggered gene silencing (MTGS)' to elucidate a long-standing puzzle in the genome-wide CHS gene silencing mechanism. In summary, our study reports four major genomic loci, lists of key genes and genome-wide variations that are associated with seed pigmentation in soybeans. In addition, we propose that the MTGS mechanism plays a crucial role in the genome-wide silencing of CHS genes, thereby suggesting a clue to currently predominant soybean cultivars with the yellow seed coat. Finally, this study will provide a broad insight into the interactions and correlations among seed color-associated genes and loci within the context of anthocyanin biosynthetic pathways.