Pigmentation of soybean seed coats via a mutation that abolishes production of multiple-phased siRNAs of chalcone synthase genes.

Lack of pigmentation in seed coats of soybean is caused by natural RNA silencing of chalcone synthase (CHS) genes. This phenomenon is an evolutionary consequence of structural changes in DNA that resulted in the production of double-stranded RNAs (dsRNAs) that trigger RNA degradation. Here we determined that a mutant with pigmented seed coats derived from a cultivar that lacked the pigmentation had a deletion between DNA regions ICHS1 and a cytochrome P450 gene; the deletion included GmIRCHS, a candidate gene that triggers CHS RNA silencing via production of CHS dsRNAs. We also characterized CHS short interfering RNAs (siRNAs) produced in the wild-type seed coats that had CHS RNA silencing. Phased 21-nt CHS siRNAs were detected in all 21 phases and were widely distributed in exon 2 of CHS7, which indicates commonality in the pattern of RNA degradation in natural CHS RNA silencing between distantly related species. These results with the similarities in the rearrangements found in spontaneous mutants suggest that the structural organization that generates dsRNAs that trigger phased siRNA production is vulnerable to further structural changes, which eventually abolish the induction of RNA silencing.

Frequency of cytosine methylation in the adjacent regions of soybean retrotransposon SORE-1 depends on chromosomal location.

Mobilization of transposable elements (TEs) is suppressed by epigenetic mechanisms involving cytosine methylation. However, few studies have focused on clarifying relationships between epigenetic influences of TEs on the adjacent DNA regions and time after insertion of TEs into the genome and/or their chromosomal location. Here we addressed these issues using soybean retrotransposon SORE-1. We analyzed SORE-1, inserted in exon 1 of the GmphyA2 gene, one of the newest insertions in this family so far identified. Cytosine methylation was detected in this element but was barely present in the adjacent regions. These results were correlated, respectively, with the presence and absence of the production of short interfering RNAs. Cytosine methylation profiles of 74 SORE-1 elements in the Williams 82 reference genome indicated that methylation frequency in the adjacent regions of SORE-1 was profoundly higher in pericentromeric regions than in euchromatic chromosome arms and was only weakly correlated with the length of time after insertion into the genome. Notably, the higher level of methylation in the 5' adjacent regions of SORE-1 coincided with the presence of repetitive elements in pericentromeric regions. Together, these results suggest that epigenetic influence of SORE-1 on the adjacent regions is influenced by its location on the chromosome.

Frequent generation of mutants with coincidental changes in multiple traits via ion-beam irradiation in soybean.

Ion beams are powerful mutagens that can induce novel mutants in plants. We previously established a system for producing a mutant population of soybean via ion-beam irradiation, isolated plants that had chlorophyll deficiency, and maintained their progeny via self-fertilization. Here we report the characterization of the progeny plants in terms of chlorophyll content, flowering time and isoflavone content in seeds. Chlorophyll deficiency in the leaf tissues was linked with reduced levels of isoflavones, the major flavonoid compounds accumulated in soybean seeds, which suggested the involvement of metabolic changes associated with the chlorophyll deficiency. Intriguingly, flowering time was frequently altered in plants that had a reduced level of chlorophyll in the leaf tissues. Plant lines that flowered either earlier or later than the wild-type plants were detected. The observed coincidental changes were presumed to be attributable to the following origins: structural changes of DNA segments leading to the loss of multiple gene functions, or indirect effects of mutations that affect one of these traits, which were manifested as phenotypic changes in the background of the duplicated composition of the soybean genome.