Endogenous, tissue-specific short-interfering RNAs silence the chalcone synthase gene family in Glycine max seed coats
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ABSTRACT: We present results from deep sequencing of small RNA populations from several genotypes of soybean and demonstrate that the CHS siRNAs accumulated only in the seed coats of the yellow varieties having either the dominant I or i-i alleles and not in the pigmented seed coats with homozygous recessive i genotypes. However, the diagnostic CHS siRNAs did not accumulate in the cotyledons of genotypes with the dominant I or i-i alleles thus demonstrating the novelty of an endogenous inverted repeat region of CHS genes driving RNA silencing in trans of non-linked CHS family members in a tissue-specific manner. The phenomenon results in inhibition of a metabolic pathway by siRNAs in one tissue allowing expression of the flavonoid pathway and synthesis of secondary metabolites in other organs as the chalcone synthase small RNAs are found in the seed coats of yellow seeded soybean varieties but not in the cotyledons of the same genotype.
Project description:We present results from deep sequencing of small RNA populations from several genotypes of soybean and demonstrate that the CHS siRNAs accumulated only in the seed coats of the yellow varieties having either the dominant I or i-i alleles and not in the pigmented seed coats with homozygous recessive i genotypes. However, the diagnostic CHS siRNAs did not accumulate in the cotyledons of genotypes with the dominant I or i-i alleles thus demonstrating the novelty of an endogenous inverted repeat region of CHS genes driving RNA silencing in trans of non-linked CHS family members in a tissue-specific manner. The phenomenon results in inhibition of a metabolic pathway by siRNAs in one tissue allowing expression of the flavonoid pathway and synthesis of secondary metabolites in other organs as the chalcone synthase small RNAs are found in the seed coats of yellow seeded soybean varieties but not in the cotyledons of the same genotype. In order to compare the population of chalcone synthase related small RNAs, we sequenced 3 to 6 million small RNAs using the Illumina Genome Analyzer from the following four soybean cultivars and tissues with specific genotypes at the I locus: Richland immature seed coats (homozygous for the dominant I allele that specifies yellow seed coat); Williams immature seed coats (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum) Williams (i-i/i-i yellow) immature cotyledons (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum); Williams 55 immature seed coats (a Williams isogenic line homozygous for the recessive i allele that specifics pigmented seed coats. All seed coats and cotyledons were dissected from green stage immature seeds within the fresh weight range of 50-75 mg.
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: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:The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat.
Project description:The I locus is a 27-kb inverted repeat cluster of chalcone synthase genes CHS1-3-4 that mediates siRNA down-regulation of CHS7 and CHS8 target mRNAs during seed development leading to yellow seed coats lacking anthocyanin pigments. Here, we report small RNA sequencing of ten stages of seed development from a few days post fertilization through maturity, revealing the amplification from primary to secondary short interfering RNAs (siRNAs) occurring during development. The young seed populations had a higher proportion of siRNAs representing the CHS1-3-4 gene family members, consistent with this region as the origin of the primary siRNAs. More intriguingly, the very young seed had a higher proportion of 22-nt CHS siRNAs than did the mid-maturation seed. We infer that the primary CHS siRNAs increase during development to levels sufficient to trigger amplification of secondary CHS siRNAs from the CHS7/8 target mRNAs, enabling the total levels of 21-nt CHS siRNAs to rise dramatically. Further, we demonstrate that the soybean system exhibits tissue-specific CHS siRNA production because primary CHS siRNA levels are not sufficient to trigger secondary amplification in tissues other than the seed coat. High-throughput sequencing using Genome Analyzer II and Illumina HiSeq 2000 was performed with two biological replicates (Some stages don't have replicate).
Project description:Background: To understand gene expression networks leading to functional properties of the soybean seed, we have undertaken a detailed examination of soybean seed development during the stages of major accumulation of oils, proteins, and starches, as well as the desiccating and mature stages, using microarrays consisting of up to 27,000 soybean cDNAs. Results: It was discovered that genes related to cell growth and maintenance processes, as well as energy processes like photosynthesis, decreased in expression levels as the cotyledons approached the mature, dry stage. Genes involved with some storage proteins had their highest expression levels at the stage of largest fresh weight. However, genes encoding many transcription factors and DNA binding proteins showed higher expression levels in the desiccating and dry seeds than in most of the green stages. Conclusions: Data on 27,000 cDNAs have been obtained over five stages of soybean development, including the stages of major accumulation of agronomically-important products, using microarrays. Of particular interest are the genes found to peak in expression at the desiccating and dry seed stages, such as those annotated as transcription factors, which may indicate the preparation of pathways that will be needed later in the early stages of imbibition and germination. Five stages of development of soybean cotyledons were studied. The first three, with fresh weights of 25-50mg, 75-100mg, and 400-500mg, were green in color. The last two, yellow-colored cotyledons with a fresh weight of 200-300mg and dry whole seeds approximately 100-200mg in weight, were desiccating and yellow/brown in color. The green stage of 100-200mg fresh weight cotyledons was used as a reference for all other stages. Four replicates, including two dye swaps, were made of each of the first three (green) stages. Two replicates, including one dye swap, were made of each of the last two (yellow/dry) stages. The results from three different platforms (GPL229, GPL1012, and GPL1013) were combined.
Project description:In a previous study, seed coat and cotyledon tissues of Williams, Richland and T157 soybean lines were investigated to show tissue specificity of CHS siRNA expression (Tuteja et al., 2009). Here, we investigated more tissues such as leaf, root and germinating cotyledon to ascertain the tissue specificity of CHS siRNAs in Williams. Data from multiple small RNA libraries were sequenced deeply by the Illumina high-throughput sequencing technology. The total numbers of small RNA reads were from three million to thirty million, providing sufficient data to show the tissue specificity of CHS siRNA.
Project description:Five degradome libraries were constructed from three different seed developmental stages. Separate degradome libraries were constructed for seed coat and cotyledons to identify the tissue specific miRNAs and their potential targets. Sequencing and analysis of degradome libraries gives identification of 183 different targets for 80 known soybean miRNAs. We found 30 cotyledon specific, 18 seed coat specific and 32 miRNAs found in both tissues irrespective of the developmental stages. One interesting observation is that we found more miRNA targets in late seed developmental stages than earlier stages. Additionally, we have validated four different auxin response factor genes as targets for gma-miR160 via RNA ligase mediated 5? rapid amplification of cDNA ends (RLM-5?RACE). GO analysis indicated the enrichment of miRNA target genes in seed development. Construction of degradome libraries using cotyledons and seed coats from 3 different developmental stages
Project description:In a previous study, seed coat and cotyledon tissues of Williams, Richland and T157 soybean lines were investigated to show tissue specificity of CHS siRNA expression (Tuteja et al., 2009). Here, we investigated more tissues such as leaf, root and germinating cotyledon to ascertain the tissue specificity of CHS siRNAs in Williams. Data from multiple small RNA libraries were sequenced deeply by the Illumina high-throughput sequencing technology. The total numbers of small RNA reads were from three million to thirty million, providing sufficient data to show the tissue specificity of CHS siRNA. High-throughput sequencing using Genome Analyzer II and Illumina HiSeq 2000 was performed.
Project description:What methylation changes are occurring in different parts of early maturation stage seed largely remains unknown. To uncover the possible role of DNA methylation in different parts of early maturation stage seed, we characterized the methylome of seed coats,cotyledons, and the embryonic seed axis using Illumina sequencing. seed coats, cotyledon, and axis