RNA-Seq using second-generation sequencing technologies permits generation of a reference unigene set for a given species, in the absence of a well-annotated genome sequence, supporting functional genomics studies, gene characterisation and detailed expression analysis for specific morphophysiological or environmental stress response traits. A reference unigene set for lentil has been developed, consisting of 58,986 contigs and scaffolds with an N50 length of 1719 bp. Comparison to gene complements from related species, reference protein databases, previously published lentil transcriptomes and a draft genome sequence validated the current dataset in terms of degree of completeness and utility. A large proportion (98%) of unigenes were expressed in more than one tissue, at varying levels. Candidate genes associated with mechanisms of tolerance to both boron toxicity and time of flowering were identified, which can eventually be used for the development of gene-based markers. This study has provided a comprehensive, assembled and annotated reference gene set for lentil that can be used for multiple applications, permitting identification of genes for pathway-specific expression analysis, genetic modification approaches, development of resources for genotypic analysis, and assistance in the annotation of a future lentil genome sequence.

Field pea (Pisum sativum L.) is a cool-season grain legume that is cultivated world-wide for both human consumption and stock-feed purposes. Enhancement of genetic and genomic resources for field pea will permit improved understanding of the control of traits relevant to crop productivity and quality. Advances in second-generation sequencing and associated bioinformatics analysis now provide unprecedented opportunities for the development of such resources. The objective of this study was to perform transcriptome sequencing and characterisation from two genotypes of field pea that differ in terms of seed and plant morphological characteristics. Transcriptome sequencing was performed with RNA templates from multiple tissues of the field pea genotypes Kaspa and Parafield. Tissue samples were collected at various growth stages, and a total of 23 cDNA libraries were sequenced using Illumina high-throughput sequencing platforms. A total of 407 and 352 million paired-end reads from the Kaspa and Parafield transcriptomes, respectively were assembled into 129,282 and 149,272 contigs, which were filtered on the basis of known gene annotations, presence of open reading frames (ORFs), reciprocal matches and degree of coverage. Totals of 126,335 contigs from Kaspa and 145,730 from Parafield were subsequently selected as the reference set. Reciprocal sequence analysis revealed that c. 87 % of contigs were expressed in both cultivars, while a small proportion were unique to each genotype. Reads from different libraries were aligned to the genotype-specific assemblies in order to identify and characterise expression of contigs on a tissue-specific basis, of which 87 % were expressed in more than one tissue, while others showed distinct expression patterns in specific tissues, providing unique transcriptome signatures. This study provided a comprehensive assembled and annotated transcriptome set for field pea that can be used for development of genetic markers, in order to assess genetic diversity, construct linkage maps, perform trait-dissection and implement whole-genome selection strategies in varietal improvement programs, as well to identify target genes for genetic modification approaches on the basis of annotation and expression analysis. In addition, the reference field pea transcriptome will prove highly valuable for comparative genomics studies and construction of a finalised genome sequence.

RNA sequencing (RNA-Seq) is a deep sequencing method used for transcriptome profiling. RNA-Seq assemblies have successfully been used for a broad variety of applications, such as gene characterisation, functional genomic studies, and gene expression analysis, particularly useful in the absence of a well-studied genome reference sequence. This study reports on the development of reference unigene sets from faba bean using RNA-Seq. Two Australian faba bean cultivars (Doza and Farah) that differ in terms of disease resistance, breeding habit, and adaptation characteristics, and have been extensively used in breeding programs, were utilised in this study. The de novo assembly resulted in a total of 58,962 and 53,275 transcripts with approximately 67 Mbp (1588 bp N50) and 61 Mbp (1629 bp N50) for Doza and Farah, respectively. The generated transcripts have been compared to the protein and nucleotide databases of NCBI, as well as to the gene complements of several related legume species such as Medicago truncatula, soybean, and chickpea. Both assemblies were compared to previously-published faba bean transcriptome reference sets for the degree of completeness and utility. Annotation of unigenes has been performed, and patterns of tissue-specific expression identified. The gene complement derived from this comprehensive transcriptome analysis shows that faba bean, despite its complex 13 Gbp genome, compares well to other legumes in expressed gene content. This study in faba bean represents the most comprehensive reference transcriptomes from two different Australian cultivars available to date and it provides a valuable resource for future genomics-assisted breeding activities in this species.