Cis-regulatory Mutations Drive Tissue-specific Subfunctionalization of sRNA loci Regulating Soybean Seed Color During Domestication

Gene duplication and structural rearrangements have played a pivotal role in plant genome evolution, enabling functional divergence and the development of novel traits. In soybeans (Glycine max), the I locus, which regulates seed color through tissue-specific subfunctionalization of sRNA loci, offers an example of these mechanisms. Using long-read sequencing, we investigated structural variations underlying four major alleles (I, ii, ik, and i) at the I locus. Our study revealed that large-scale rearrangements, including duplications, inversions, and deletions, within a 180-kb CHS repeat-rich region drive allele-specific RNA silencing through small interfering RNAs (siRNAs). The dominant I allele contains a cis-regulatory region of DnaJ upstream of CHS genes, while the ii and ik alleles feature subtilisin- and P450-driven siRNA loci, respectively. The recessive i allele lacks siRNA production due to deletions or inversions disrupting CHS gene clusters. Phenotypic analyses and RNA-seq confirmed allele-specific, tissue-dependent expression of siRNAs correlating with seed coat pigmentation patterns. This study highlights the evolutionary role of repeat-rich regions in generating regulatory innovations and phenotypic diversity. Our findings underscore the importance of structural rearrangements in domestication traits and demonstrate the power of long-read sequencing for resolving complex genomic regions, advancing both evolutionary biology and crop improvement. SIGNIFICANCE{square} Naturally occurring mutations in the soybean seed coat have revealed the evolution of sRNA genes and their subfunctionalization, resulting in tissue-specific gene expression. {square}Non-homologous recombination of repetitive sequences drives cis-regulatory region rearrangements, leading to the subfunctionalization of sRNA loci that regulates one of domestication traits.