De-novo transcriptome assembly for gene identification, analysis, annotation, and molecular marker discovery in Onobrychis viciifolia

Background Sainfoin (Onobrychis viciifolia) is a highly nutritious tannin-containing forage legume. In the diet of ruminants sainfoin can have anti-parasitic effects and reduce methane emissions under in vitro conditions. Many of these benefits have been attributed to the tannin in sainfoin. A combination of increased use of industrially produced nitrogen fertilizer, issues with establishment and productivity in the first year and more reliable alternatives, such as red clover led to a decline in the use of sainfoin since the middle of the last century. In recent years there has been a resurgence of interest in sainfoin due to its potential beneficial nutraceutical and environmental attributes. However, genomic resources are scarce, thus hampering progress in genetic analysis and improvement. To address this we have used next generation RNA sequencing technology to obtain the first transcriptome of sainfoin. We used the library to identify gene-based simple sequence repeats (SSRs) and potential single nucleotide polymorphisms (SNPs).
Results One genotype from each of five sainfoin accessions was sequenced. Paired-end (PE) sequences were generated from cDNA libraries of RNA extracted from seven day old seedlings. A combined assembly of 92,772 transcripts was produced de novo using the Trinity programme. About 18,000 transcripts were annotated with at least one GO (gene ontology) term. A total of 59 transcripts were annotated as involved in the tannin biosynthesis pathway. We identified 3,500 potential SSRs. SNPs were identified by mapping the reads of the individual assemblies against the combined assembly. After stringent filtering a total of 77,000 putative SNPs were identified. A phylogenetic analysis of single copy number genes showed that sainfoin was most closely related to red clover and Medicago truncatula, while Lotus japonicus, bean and soybean are more distant relatives.
Conclusions This work describes the first transcriptome assembly in sainfoin. The 92K transcripts provide a rich source of SNP and SSR polymorphisms for future use in genetic studies of this crop. Annotation of genes involved in the tannin biosynthesis pathway has provided the basis for further studies of the genetic control of this important trait in sainfoin.