Project description:Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. The application of genomic approaches would advance development of alfalfa as a cellulosic feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. These ESTs were de novo assembled into 132,153 unique sequences. By combining the de novo assembled ESTs (132,153 sequences) with our previously identified EST sequences (341,984 sequences, unpublished data), and the ESTs available from GenBank (12,371 sequences), we built the first Alfalfa Gene Index (MSGI 1.0). MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1, 294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Transcript profiling of stem internodes of genotypes 708 and 773 was conducted by quantifying the number of Illumina EST reads that were mapped to sequences in MSGI 1.0. We identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index (MSGI 1.0) assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a cellulosic feedstock.

Project description:In order to clarify the molecular mechanisms of drought tolerance between different maize (Zea mays L.) varieties at the protein level, iTRAQ (the isobaric tags for relative and absolute quantitation) quantitative proteomics were used for the comparative analysis of protein expression in the seedling roots of the drought-tolerant Chang 7-2 and drought-sensitive TS141 maize varieties under 20% PEG 6000 (polyethylene glycol 6000)-simulated drought stress. A total of 7723 proteins were identified using iTRAQ in the maize seedling roots. We detected 39371 peptides and 30148 unique peptides. The identified protein molecular mass was mainly distributed between 10–60 kDa. COG analysis divided these proteins into 24 categories, among which general function prediction contained the largest number of proteins (19.1%), followed by posttranslational modification, protein turnover, and chaperones (10.52%). In the identified differentially expressed proteins, 1552 of which were significantly differentially expressed. Of these, 1249 were differentially expressed in Chang 7-2 following drought stress, 577 of which were up-regulated and 672 were down-regulated. Four hundred and twenty five differentially expressed proteins were identified in TS141, 249 of which were up-regulated and 176 were down-regulated. Of these proteins, 32 demonstrated opposite expression levels in the two varieties, and there were 56 up-regulated proteins that were common between the two varieties. Comparing the Chang 7-2 treatment group and control group, the DEPS in the cellular component category were mainly enriched in cytoplasmic membrane-bounded vesicles, cytoplasmic vesicles, membrane-bound vesicles and vesicles; in the molecular function category, the DEPs were mainly enriched in cation binding and metal ion binding; and in the biological process category, the major enriched categories included phosphate-containing compound metabolic process, single-organism transport cellular component organization or biogenesis, carbohydrate metabolic process, and cellular component organization. Comparing the TS141 treatment group and control group, the DEPS in the cellular component category were primarily enriched in cytoplasmic membrane-bounded vesicles, cytoplasmic vesicles, membrane-bound vesicles, and vesicle; in the molecular function category, the DEPs were mainly enriched in cation binding and metal ion binding; and in the biological process category, the major enrichment categories included oxidation-reduction process and carbohydrate metabolic process. In Chang 7-2, the differentially expressed proteins were associated with ribosome pathway, glycolysis/gluconeogenesis pathway, and amino sugar and nucleotide sugar metabolism. In TS141, the differentially expressed proteins were associated with metabolic pathway, phenylpropanoid biosynthesis pathway, and starch and sucrose metabolism.

Project description:Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. The application of genomic approaches would advance development of alfalfa as a cellulosic feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling. Cell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. These ESTs were de novo assembled into 132,153 unique sequences. By combining the de novo assembled ESTs (132,153 sequences) with our previously identified EST sequences (341,984 sequences, unpublished data), and the ESTs available from GenBank (12,371 sequences), we built the first Alfalfa Gene Index (MSGI 1.0). MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1, 294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Transcript profiling of stem internodes of genotypes 708 and 773 was conducted by quantifying the number of Illumina EST reads that were mapped to sequences in MSGI 1.0. We identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes. Our results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index (MSGI 1.0) assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a cellulosic feedstock. Examination of 2 different tissue types at different developmental stages (Elongating vs. post-elongation stem internodes) in two alfalfa genotypes (708 and 773) with divergent cell wall composition in stems.

			Action	DRS
	GDA_ZHMC_4I_1_001_f1.mzML	Mzml
	GDA_ZHMC_4I_1_001_f10.mzML	Mzml
	GDA_ZHMC_4I_1_001_f11.mzML	Mzml
	GDA_ZHMC_4I_1_001_f12.mzML	Mzml
	GDA_ZHMC_4I_1_001_f2.mzML	Mzml

Dataset Information

ITRAQ-based labeling proteomics analysis of alfalfa roots in response to PEG-induced osmotic stress

Dataset's files

Publications

Physiological and Proteomic Responses of Contrasting Alfalfa (<i>Medicago sativa</i> L.) Varieties to PEG-Induced Osmotic Stress.

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