A Mutation in an Argonaute Protein Explains the Epistatic Interactions of the K and I Loci which Control Soybean Seed Color Patterns through Levels of Chalcone Synthase siRNAs

Four alleles of the I (inhibitor) locus in soybean control distribution of pigment over soybean seed coats via a naturally-occurring and tissue-specific form of RNA interference. The recessive k1 mutation acts epistatically to overcome the effect of the dominant I and iⁱ alleles that normally inhibit seed color by producing short-interferring RNAs (siRNAs) targeting chalcone synthase (CHS) mRNAs. Using small RNA sequencing of dissected regions of the immature seed coats, we demonstrated that CHS siRNA levels are the cause of patterns produced by the iⁱ and i^k alleles of the I locus, which restrict pigment to the hilum or saddle region of the seed coat, respectively. In order to identify the unknown K1 locus, we compared RNA-Seq data from dissected regions of two Clark isolines having similar saddle phenotypes mediated by CHS siRNAs but with different genotypes of homozygous i^k, K1 versus homozygous iⁱ, k1. By examining differentially expressed genes, map position information, and genome resequencing, we identified the k1 mutation to be a 129 bp deletion in Glyma.11G190900 encoding AGO5, a specific member of the Argonaute gene family. The small deletion leads to alternative splicing by exon skipping and premature termination of its translation product. The mutated AGO5 from the k1 locus participates in changing the patterned distributions of CHS siRNAs, thus explaining how the k1 mutation reverses the phenotype of the seed coat regions from yellow to pigmented, even in the presence of the normally dominant I or iⁱ alleles.