Single nucleotide polymorphism (SNP) discovery in cultivated and wild alfalfa (Medicago sativa L.) using Illumina transcriptome sequencing

Abstract Background Alfalfa is a perennial, outcrossing species, and a widely planted forage legume producing highly nutritious biomass. Currently, improvement of cultivated alfalfa mainly relies on recurrent phenotypic selection. Marker assisted breeding strategies can enhance alfalfa improvement efforts, but these strategies require a large number of markers. Next generation sequencing strategies enable high-throughput discovery of SNP markers efficiently for a complex polyploid species. Result Transcriptome sequencing of stems from 27 alfalfa genotypes (23 tetraploid and 4 diploid) was used to identify genetic variation and increase the number of SNP markers in alfalfa. De novo assembly of quality-filtered reads generated 25,183 contigs with a total length of 26.8 Mbp and an average length of 1065 bp, with an average read depth of 55.9X for each genotype. Overall, 21,954 (87.2%) of the 25,183 contigs represented 14,878 unique protein accessions. Gene ontology (GO) analysis suggested that a broad diversity of genes was represented in the resulting sequences. The realignment of reads to the contigs enabled the detection of 872,384 SNP and 31,760 InDels. High Resolution Melting (HRM) analysis was used to validate 85% of the 192 SNP evaluated. Structure analysis of the 27 alfalfa genotypes clearly separates individuals from the subspecies falcata, the diploid subspecies caerulea, and the tetraploid subspecies sativa (cultivated tetraploid alfalfa). Conclusion Transcriptome sequencing is an efficient way to identify SNP in alfalfa, a polyploid species with a complex and un-sequenced genome. The EST and SNP markers generated from this study are publicly available and can contribute to future alfalfa research and breeding applications.