Endogenous short interfering RNAs of chalcone synthase genes associated with inhibition of seed coat pigmentation in soybean

Abstract

Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The inhibition of seed coat pigmentation is due to naturally occurring posttranscriptional gene silencing (PTGS) of the chalcone synthase (CHS) genes. In the present study, RNA gel blot analysis in different tissues indicated that endogenous CHS siRNAs are accumulated only in the seed coat, supporting the suggestion that CHS PTGS is seed coat-specific. The probe region of a CHS gene was divided into several parts, each of which was used for RNA gel blot analysis in the seed coat. Interestingly, endogenous CHS siRNAs were only detected with probes corresponding to the exon 2 region. The results were confirmed by deep sequencing analysis of CHS siRNAs in the seed coat, i.e., CHS siRNAs were predominantly derived from the 3′-half of exon 2 of the CHS gene. Mapping of CHS siRNA sequences on CHS genes and an inverted repeat region of the CHS pseudogene (pseudoCHS3) suggested that primary CHS siRNAs may be generated from double-stranded RNA of pseudoCHS3, and subsequently secondary CHS siRNAs may be produced according to the two-hit model, which has been proposed to explain siRNA amplification by the RNA-dependent RNA polymerase.