De novo assembly of red clover transcriptome based on RNA-Seq data provides insight into drought response, gene discovery and marker identification

Background: Red clover (Trifolium pratense L.) is a versatile forage crop legume, which can tolerate a variety of soils and is suitable for silage production for winter feed and for grazing. It is one of the most important forage legumes in temperate livestock agriculture. Its beneficial attributes include ability to fix nitrogen, improve soil and provide protein rich animal feed. It is however, a short-lived perennial providing good biomass yield for two or three years. Improved persistency is thus a major breeding target. Better water-stress tolerance is one of the key factors influencing persistency, but little is known about how red clover tolerates water stress. Results: Plants from a full sib mapping family were used in a drought experiment, in which the growth rate and relative water content (RWC) identified two pools of ten plants contrasting in their tolerance to drought. Key metabolites were measured and RNA-Seq analysis was carried out on four bulked samples: the two pools sampled before and after drought. Massively parallel sequencing was used to analyse the bulked RNA samples. A de novo transcriptome reconstruction based on the RNA-Seq data was made, resulting in 45181 contigs, representing 'transcript tags'. These transcript tags were annotated with gene ontology (GO) terms. One of the most striking results from the expression analysis was that the drought sensitive plants were characterised by having approximately twice the number of differentially expressed transcript tags than the tolerant plants after drought. This difference was evident in most of the major GO terms. Before onset of drought the sensitive plants overexpressed a number of genes annotated as senescence-related. Furthermore, the concentration of three metabolites, particularly pinitol, but also proline and malate increased in leaves after drought stress. Conclusions: This de novo assembly of a red clover transcriptome from leaf material of droughted and non-droughted plants provides a rich source for gene identification, single nucleotide polymorphisms (SNP) and short sequence repeats (SSR). Comparison of gene expression levels between pools and treatments identified candidate genes for further analysis of the genetic basis of drought tolerance in red clover.