Project description:The plant cell wall performs a number of essential functions including providing shape to many different cell types and serving as a defense against potential pathogens. The net pattern mutation creates breaks in the seed coat of soybean (Glycine max) because of ruptured cell walls. Using RNA-Seq, we examined the seed coat transcriptome from three stages of immature seed development in two pairs of isolines with normal or defective seed coat phenotypes due to the net pattern. The genome-wide comparative study of the transcript profiles of these isolines revealed 364 differentially expressed genes in common between the two varieties that were further divided into different broad functional categories. Genes related to cell wall processes accounted for 19% of the differentially expressed genes in the middle developmental stage of 100-200 mg seed weight. Within this class, the cell wall proline-rich and glycine-rich protein genes were highly differentially expressed in both genetic backgrounds. Other genes that showed significant expression changes in each of the isoline pairs at the 100-200 mg seed weight stage were xylem serine proteinase, fasciclin-related genes, auxin and stress response related genes, TRANSPARENT TESTA 1 (TT1) and other transcription factors. The mutant appears to shift the timing of either the increase or decrease in the levels of some of the transcripts. The analysis of these data sets reveals the physiological changes that the seed coat undergoes during the formation of the breaks in the cell wall. Examination of soybean isolines in two different genetic background at three different seed weight stages: Seed coats of Clark standard (CS, wild type) & Clark defective (CD, seed coat mutant), Harosoy Standard (HS) & Harosoy defective (HD) at 50-100mg, 100-200mg and 400-500mg.
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.
Project description:Molecular characterization of leaf development has not been well studied in soybean. Studies have shown that genomic regions controlling multifoliate leaf morphology in Glycine max also regulates important agronomic characters including yield, seed weight, seed number, shattering, plant growth, and flowering. Two soybean isolines that differed in leaf phenotype were profiled by high throughput RNA and small RNA (sRNA) sequencing. A Clark isoline, homozygous for a dominant mutant allele, Lf1, that specifies a five-foliate compound leaf was compared to wild type Clark that is homozygous for the standard allele that produces trifoliate leaves. Although Lf1 is dominant, it presents variable expressivity as the young plantlets with the Lf1Lf1 genotype initially have trifoliate leaves in the first few weeks, after which they transition to five-foliate leaves. At later developmental stages, they begin to produce four-foliate or trifoliate leaves. In RNA-Seq experiments, a total of 91 and 95 million reads were generated in each lane of Illumina sequencing for the shoot tip of wild type Clark standard (CS) and mutant Clark five-foliate (CF) libraries, respectively. Of these, ~70% million reads aligned to the 78,743 target Glyma models from the reference soybean genome (cv. Williams 82) with maximum of 3 mismatches and up to 25 alignments. Where as in vegetative bud, 56 (CS) to 59 (CF) million reads were produced and of these ~80% aligned to the soybean reference genome. The comparative studies of the transcript profiles of the wild-type versus mutant line revealed a number of differentially expressed genes. A total of 1,296 and 2,083 genes were up-regulated in the shoot tip of CS and CF, respectively that showed ≥2-fold expression difference. On the contrary in the vegetative bud, much smaller number of genes 14 (CS) and 94 (CF) showed increased transcript abundance. In sRNA analysis, a collection of 200,447 and 268,508 unique sRNA sequences isolated from shoot tip tissue of CS and CF were aligned to the soybean reference genome and their target glyma models were predicted using bioinformatics. This sRNA analysis at genome level reveals differences in size distribution of classes in the CS and CF. This study provides insight into the initial understanding of leaf development in soybean by revealing a number of genes and sRNAs differentially expressed between the CS and CF.
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.