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Accession IconSRP045669

Methylation Affects Transposition and Splicing of a Large CACTA Transposable Element From a MYB Transcription Factor Regulating Anthocyanin Synthase (ANS) Genes in Soybean Seed Coats (RNA-Seq)

Organism Icon Glycine max
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Technology Badge IconIllumina HiSeq 2000

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We determined the molecular basis of three soybean lines that vary in seed coat color at the R locus which is thought to encode a MYB transcription factor. RM55-rm is homozygous for a mutable allele (rm) that specifies black and brown striped seeds; RM30-R* is a stable black revertant isoline derived from the mutable line; and RM38-r has brown seed coats due to a recessive r allele shown to translate a truncated MYB protein. Using long range PCR, 454 sequencing of amplicons, and whole genome re-sequencing, we determined that the variegated RM55-rm line had a 13 kb CACTA subfamily transposon insertion (designated TgmR*) at a position 110 bp from the beginning of Intron2 of the R locus, Glyma09g36983. Although the MYB encoded by R was expressed at only very low levels in older seed coats of the black revertant RM30-R* line, it upregulated expression of anthocyanidin synthase genes (ANS2, ANS3) to promote the synthesis of anthocyanins. Surprisingly, the RM30-R* revertant also carried the 13 kb TgmR* insertion in Intron2. Using RNA-Seq, we showed that intron splicing was accurate, albeit at lower levels, despite the presence of the 13kb TgmR* element. As determined by whole genome methylation sequencing, we demonstrate that the TgmR* sequence was relatively more methylated in RM30-R* than in the mutable RM55-rm progenitor line. The stabilized and more methylated RM30-R* revertant line apparently lacks effective binding of a transposae to its subterminal repeats, thus allowing intron splicing to proceed resulting in sufficient MYB protein to stimulate anthocyanin production and thus black seed coats. In this regard, the TgmR* element in soybean resembles McClintock’s Spm-suppressible and change-of-state alleles of maize. This comparison explains the opposite effects of the TgmR* element on intron splicing of the MYB gene in which it resides depending on the methylation state of the element. Overall design: Differential RNA expression of anthocyanin pathway genes in the seed coats of two seed color lines: RM30-R* (black) and RM38-r (brown). The multiple gene expression analysis was performed at five different stages of seed development which were chosen based on the fresh weight (fwt) of entire seeds: 100-200, 200-300, 300-400 and 400-500 mg with green cotyledons, and a later stage in which seed desiccation had initiated judging by the yellowing of cotyledons and a lower fresh weight of 300-400 mg
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