The Transition of From Endogenous Primary to Secondary siRNAs that Silence Soybean Chalcone Synthase is Revealed by Small RNA-Seq of Developing Seed Coats

In soybean, dominant alleles of the I locus inhibit pigmentation of the seed coat while the homozygous recessive i allele results in fully pigmented seed. The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down regulation of CHS7 and CHS8 target mRNAs during the mid-maturation phase of seed development leading to the yellow seed coat color (Tuteja et al., Plant Cell, 2004 & 2009).  Here, we report that high throughput sequencing clearly reveals the transition from primary to secondary siRNAs occurring during development. The normalized total counts of CHS siRNAs gradually increased in very young seed beginning at 4 days after flowering (DAF), then dramatically increased from the 5-6 mg seed weight range and peaked at the 50-75 mg stage before decreasing during the remainder of seed maturation and desiccation. Interestingly, the siRNA populations in the young seed had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did 50-75 mg seed which had higher abundance of 21-nt siRNAs representing CHS7 and CHS8. These data demonstrate that 22-nt primary CHS siRNAs originating from the CHS1-3-4 cluster were present very early in seed development. We propose that the 22-nt primary CHS siRNAs trigger amplification of secondary CHS siRNAs by RNA-dependent RNA polymerase (RdRP) from the CHS7 and CHS8 target mRNAs, thus enabling the total levels of CHS siRNAs to rise dramatically.