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

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the de novo assembled ESTs (132,153 sequences) with our previously identified EST sequences (341,984 sequences, unpublished data), and the ESTs available from GenBank (12,371 sequences), we built the first Alfalfa Gene Index (MSGI 1.0).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. Overall design: 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.