Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

USDA-Agricultural Research Service, Plant Science Research Unit, St, Paul, MN 55108, USA.
BMC Genomics (Impact Factor: 3.99). 04/2011; 12(1):199. DOI: 10.1186/1471-2164-12-199
Source: PubMed


Alfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol 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. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. 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. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis 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 assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.

Download full-text


Available from: Carroll P Vance, Dec 20, 2013
  • Source
    • "Variation in cellulose content can confer changes in final cell size and shape (Fagard et al. 2000) as well as in the cell's (and the stem's) ability to withstand compressive or tensile forces (Spokevicius et al. 2007). Cellulose content is known to vary within stems (Yang et al. 2011) and between cells, with an extreme response observed in the development of tension wood cells that are almost entirely cellulose (Qiu et al. 2008). In E. globulus tension, wood is often characterised by the presence of gelatinous fibres in which the secondary wall is almost entirely cellulose (Washusen 2002). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The moderate to high levels of nucleotide diversity and low linkage disequilibrium found in many forest tree species make them ideal candidates for association mapping. Here, we report candidate gene-based association mapping results for complex wood quality and growth traits in Eucalyptus globulus Labill. ssp. globulus, the most widely grown eucalypt in temperate regions of the world. Ninety-eight single nucleotide polymorphisms (SNPs) from 20 wood quality candidate genes were assayed in a discovery population consisting of 385 trees sourced from a provenance-progeny trial. Twenty-five selected SNPs with significant associations (P .
    Tree Genetics & Genomes 12/2014; 10(6):1-18. DOI:10.1007/s11295-014-0787-0 · 2.45 Impact Factor
  • Source
    • "To perform a reliable gene expression analysis, accurate data normalization is mandatory, which prompted us to identify the most suitable reference genes for expression analysis. We chose as candidate reference genes a set of known and widely used genes which have been tested on M. truncatula [14], together with candidates proposed by Yang et al. [15] and Huis et al. [20]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Abiotic stress represents a serious threat affecting both plant fitness and productivity. One of the promptest responses that plants trigger following abiotic stress is the differential expression of key genes, which enable to face the adverse conditions. It is accepted and shown that the cell wall senses and broadcasts the stress signal to the interior of the cell, by triggering a cascade of reactions leading to resistance. Therefore the study of wall-related genes is particularly relevant to understand the metabolic remodeling triggered by plants in response to exogenous stresses. Despite the agricultural and economical relevance of alfalfa (Medicago sativa L.), no study, to our knowledge, has addressed specifically the wall-related gene expression changes in response to exogenous stresses in this important crop, by monitoring the dynamics of wall biosynthetic gene expression. We here identify and analyze the expression profiles of nine cellulose synthases, together with other wall-related genes, in stems of alfalfa plants subjected to different abiotic stresses (cold, heat, salt stress) at various time points (e.g. 0, 24, 72 and 96 h). We identify 2 main responses for specific groups of genes, i.e. a salt/heat-induced and a cold/heat-repressed group of genes. Prior to this analysis we identified appropriate reference genes for expression analyses in alfalfa, by evaluating the stability of 10 candidates across different tissues (namely leaves, stems, roots), under the different abiotic stresses and time points chosen. The results obtained confirm an active role played by the cell wall in response to exogenous stimuli and constitute a step forward in delineating the complex pathways regulating the response of plants to abiotic stresses.
    PLoS ONE 08/2014; 9(8). DOI:10.1371/journal.pone.0103808 · 3.23 Impact Factor
  • Source
    • "De novo transcriptomes have been successfully performed through the Illumina platform in a variety of non-model species, including Lupinus albus (lupin) [13], Cicer arietinum (chickpea) [14], Ipomoea batatas (sweetpotato) [15] and Medicago sativa (alfalfa) [16], to name a few. Zenoni et al. (2011) used 454 sequencing to generate de novo assembled transcriptomes separately for Petunia axillaris and Petunia inflata, parental species of Petunia hybrida, to develop microarray chips for transcriptomic analyses to study seed coat defects in a P. hybrida mutant [17]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Salinity and drought stress are the primary cause of crop losses worldwide. In sodic saline soils sodium chloride (NaCl) disrupts normal plant growth and development. The complex interactions of plant systems with abiotic stress have made RNA sequencing a more holistic and appealing approach to study transcriptome level responses in a single cell and/or tissue. In this work, we determined the Petunia transcriptome response to NaCl stress by sequencing leaf samples and assembling 196 million Illumina reads with Trinity software. Using our reference transcriptome we identified more than 7,000 genes that were differentially expressed within 24 h of acute NaCl stress. The proposed transcriptome can also be used as an excellent tool for biological and bioinformatics in the absence of an available Petunia genome and it is available at the SOL Genomics Network (SGN) Genes related to regulation of reactive oxygen species, transport, and signal transductions as well as novel and undescribed transcripts were among those differentially expressed in response to salt stress. The candidate genes identified in this study can be applied as markers for breeding or to genetically engineer plants to enhance salt tolerance. Gene Ontology analyses indicated that most of the NaCl damage happened at 24 h inducing genotoxicity, affecting transport and organelles due to the high concentration of Na+ ions. Finally, we report a modification to the library preparation protocol whereby cDNA samples were bar-coded with non-HPLC purified primers, without affecting the quality and quantity of the RNA-seq data. The methodological improvement presented here could substantially reduce the cost of sample preparation for future high-throughput RNA sequencing experiments.
    PLoS ONE 04/2014; 9(4):e94651. DOI:10.1371/journal.pone.0094651 · 3.23 Impact Factor
Show more