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Multiple Origins of Chalcone Synthase Short Interfering RNAs in Glycine max Revealed by Genome Assemblies of Mutant Alleles

ABSTRACT: With long-read assemblies, we delineate three origins of short interfering RNAs for chalcone synthase (CHS siRNAs) in Glycine max. Mutations from ii (yellow seed coat with black hilum) to i (fully pigmented) lack either a single antisense CHS1 gene by deletion or the partial 5’ subtilisin fragment-CHS1 gene region by a large inversion mutation. On the other hand, we show that the dominant I allele, with fully yellow seed coats, arose from the i black, wild soybean genome by an inverted duplication that places a partial 5’ DnaJ fragment next to slightly truncated CHS genes. In a direct I to i mutation, the two CHS genes are further truncated, destroying their capacity to produce siRNAs. The two-color saddle pattern of the ik allele arose by an inverted duplication event that places a partial 5’ P450 fragment in front of two inverted repeat CHS genes. Despite the importance of the three different 5’ promoters that abut inverted repeat CHS genes as drivers of precursor expression, the profiles of cognate gene family members for subtilisin, DnaJ, and P450 are not always limited to the seed coat as tightly as CHS siRNAs, implying that transcriptional or processing events for CHS dsRNA or siRNAs differ in other tissues.

ORGANISM(S): Glycine max

PROVIDER: GSE239923 | GEO | 2025/07/31

REPOSITORIES: GEO

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The Transition from Primary siRNAs to Amplified Secondary siRNAs that Regulate Chalcone Synthase During Development of Glycine max Seed Coat

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Endogenous, tissue-specific short-interfering RNAs silence the chalcone synthase gene family in Glycine max seed coats

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Project description:The soybean (Glycine max) seed coat has distinctive, genetically programmed patterns of pigmentation and the recessive k1 mutation can epistatically overcome the dominant I and i-i alleles, which inhibit seed color by producing small interfering RNAs (siRNAs) targeting chalcone synthase (CHS) mRNAs. Small RNA sequencing of dissected regions of immature seed coats demonstrated that CHS siRNA levels cause the patterns produced by the i-i and i-k alleles of the I locus, which restrict pigment to the hilum or saddle region of the seed coat, respectively. To identify the K1 locus, we compared RNA-Seq data from dissected regions of two Clark isolines having similar saddle phenotypes mediated by CHS siRNAs but different genotypes (homozygous i-k K1 versus homozygous i-i k1). By examining differentially expressed genes, mapping information, and genome resequencing, we identified a 129-bp deletion in Glyma.11G190900 encoding Argonaute5 (AGO5), a member of the Argonaute family. Amplicon sequencing of several independent saddle pattern mutants from different genetic backgrounds revealed independent lesions affecting AGO5, thus establishing Glyma.11G190900 as the K1 locus. Non-functional AGO5 from k1 alleles leads to altered 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 i-i alleles.

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