Project description:Background: To elucidate features of seed development, we investigated the transcriptome of a soybean isoline from the germplasm collection that contained an introgressed allele known as minute hilum (mi) which confers a smaller hilum region where the seed attaches to the pod and also results in seed coat cracking surrounding the hilum region. Results: RNAs were extracted from immature seed from an extended hilum region (i.e., the hilum and a small ring of tissue surrounding the hilum in which the cracks form) at three different developmental stages:10-25, 25-50 and 50-100 mg seed fresh weight in two independent replicates for each stage. The transcriptomes of these samples from both the Clark isoline containing the mi allele (PI 547628, UC413, ii R t mi G), and its recurrent Clark 63 parent isoline (PI 548532, UC7, ii R T Mi g), which was used for six generations of backcrossing, were compared for differential expression of 88,648 Glyma models of the soybean genome Wm82.a2. The RNA sequence data obtained from the 12 cDNA libraries were subjected to padj value <0.05 and at least two-fold expression differences to select with confidence genes differentially expressed in the hilum-containing tissue of the seed coat between the two lines. Glyma.09G008400 annotated as encoding an ethylene forming enzyme, ACC oxidase (ACO), was found to be highly overexpressed in the mi hilum region at 165 RPKMs (reads per kilobase per million mapped reads) compared to the standard line at just 0.03 RPKMs. Evidence of changes in expression of genes downstream of the ethylene pathway included those involved in auxin and gibberellin hormone action and extensive differences in expression of cell wall protein genes. These changes are postulated to determine the restricted hilum size and cracking phenotypes. Conclusions: We present transcriptome and phenotypic evidence that substantially higher expression of an ethylene-forming ACO gene likely shifts hormone balance and sets in motion downstream changes resulting in a smaller hilum phenotype and the cracks observed in the minute hilum (mi) isoline as compared to its recurrent parent.
2020-10-16 | GSE146287 | GEO
Project description:Glycine max genome resequencing of UC153, i-i k3 black saddle mutation found in x-rayed Clark
Project description: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.
Project description:The seed coat of black (iRT) soybean with the dominant R allele begins to accumulate cyanic pigments at the transition stage of seed development (300 – 400 mg fresh seed weight), whereas the brown (irT) nearly-isogenic seed coat with the recessive r allele lacks cyanic pigments at all stages of seed development. We used microarrays to determine global gene expression differences between black (iRT) and brown (irT) soybean seed coats at the transition stage of seed development (300 – 400 mg fresh seed weight). To identify the complete set of gene transcripts that are differentially expressed between the seed coats of black (iRT) and brown (irT) Clark isolines, seed coats were dissected at the transition stage of seed development (300 – 400 mg fresh seed weight) for microarray analysis using the Affymetrix Soybean GeneChip. To ensure seed coats were of the same stage of development, the days post anthesis, pod length, pod color, embryo morphology, and transcript levels of the developmental marker gene Gm-r1083-1191, a putative cutin biosynthesis gene, and DFR1 were ensured to be equivalent between black (iRT) and brown (irT) isolines.