The Effect of Structural Variation at the I Locus on Regulation of Chalcone Synthase Short Interfering RNAs in Soybean

The soybean I locus has four alleles (I,  i^i,  i^k, i) that determine the spatial distribution of pigmentation on the seed coat. The dominant iⁱ allele of Williams (yellow seed coat) is a 27-kb inverted repeat cluster containing six chalcone synthase (CHS) genes that generates primary siRNA leading to down-regulation of CHS7 and CHS8 target mRNAs by amplification of secondary CHS siRNAs during seed development (Tuteja et al., 2009, Plant Cell; Cho et al., 2013, PLoS ONE). Here, we determined the extent of the deletion that occurs in a mutant black-seeded isoline of Williams, designated UC55, that is homozygous for a recessive i allele.  Using high throughput genome re-sequencing and verification by PCR, we demonstrate that i black seed have a deletion of about 122 kb that includes 19 coding regions, six of which are CHS genes. The deletion appears be a homologous recombination event between one of the CHS genes in the CHS I cluster region and a repeated CHS gene in a region 122 kb away. The deletion destroys part of the 27-kb inverted repeat cluster of the dominant iⁱ allele. These alleles are clear examples of naturally occurring inverted repeat duplications that spawn small RNAs at the locus (iⁱ allele) and a deletion that destroys the small RNA generating structure (i allele). Other than the seed coat color change, the isolines appear identical in most agronomic properties indicating that the 13 non-CHS genes that are deleted are not deleterious to plant development and performance.