Genetic analysis of EMS-induced AGO5 mutants affecting seed coat pigmentation in soybean (Glycine max L.)

In soybean, seed coat pigmentation is regulated by post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes at the I locus. Although several components of the PTGS pathway have been implicated in this process, genetic evidence linking specific RNA silencing factors to seed coat pigmentation remains limited.

We identified three independent brown seed coat mutants from an EMS-mutagenized population of the soybean cultivar Pungsannamul, which normally produces yellow seeds. Genetic segregation analysis demonstrated that the brown seed coat phenotype in all three mutants is controlled by a single recessive locus. Complementation tests revealed that the three mutants are allelic. Genetic mapping by bulk segregant analysis (BSA) using a SNP genotyping array localized the causal locus to a 2.83-Mb interval on chromosome 11 containing the ARGONAUTE5 (AGO5) gene. Sequence analysis identified distinct mutations in AGO5 in each mutant line, including a splice-site mutation and two missense substitutions within the conserved P-element Induced Wimpy Testis (PIWI) domain. Co-segregation analysis using derived cleaved amplified polymorphic sequence (dCAPS) markers confirmed a complete association between AGO5 mutant alleles and the brown seed coat phenotype. Expression analysis revealed significantly elevated CHS7 transcript levels in the seed coats of all three AGO5 mutants compared with the wild type.

Our results provide genetic evidence that AGO5 plays a critical role in PTGS-mediated regulation of CHS expression and seed coat pigmentation in soybean, consistent with phenotypes of previously reported PTGS component mutants such as dicer-like2 (dcl2a/dcl2b) double mutants.