In soybean, seed color is determined by a specific class of small RNAs known as short- interfering RNAs (siRNAs). The dominant ii allele of the I (inhibitor) locus is composed of an inverted-repeat cluster of six chalcone synthase (CHS) genes on chromosome 8 whose unique arrangement generates CHS siRNAs that downregulate target CHS7 and CHS8 genes on non-linked chromosomes resulting in yellow seed coats. Chapter 1 presents a transition in size of the siRNAs from the primary 22-nt siRNAs representing the origin of the siRNAs from the ii allele to the predominantly 21-nt secondary siRNAs representing the target CHS7 and CHS8 genes. Chapter 2 determines the extent of naturally occurring deletions resulting in pigmented seed coats using genomic resequencing and copy number determination by digital PCR. Two size deletions (130 kb and 22 kb) were discovered that each eliminate part of the 27-kb inverted repeat CHS cluster resulting in black seed coats. This study demonstrates the importance of the correct representation of the target region in repetitive regions for determining structural variation since both of the current versions of the soybean reference genome (Wm82.a1 and Wm82.a2) have inversions and gaps and do not accurately represent the ii allele. Chapter 3 shows the interaction of the unlinked k1 mutation that modifies the distribution of CHS siRNAs in the seed coat resulting in a pigment pattern phenotype. Using RNA-Seq and genomic re-sequencing coupled with genetic marker information, a 129 bp deletion was discovered in a gene (Glyma.11G190900) encoding a member of the argonaute family of proteins (AGO5) that identifies the k1 mutation and leads to a non-functional protein.
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Structural variation at soybean loci regulating small RNAs and seed color
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