Background: The peanut (Arachis hypogaea L.) is an important oilseed crop in tropical and subtropical regions of the world. However, little about the molecular biology of the peanut is currently known. Recently, next-generation sequencing technology, termed RNA-seq, has provided a powerful approach for analysing the transcriptome, and for shedding light on the molecular biology of peanut. Results: In this study, we employed RNA-seq to analyse the transcriptomes of the immature seeds of three different peanut varieties with different oil contents. A total of 26.1-27.2 million paired-end reads with lengths of 100 bp were generated from the three varieties and 59,077 unigenes were assembled with N50 of 823 bp. Based on sequence similarity search with known proteins, a total of 40,100 genes were identified. Among these unigenes, only 8,252 unigenes were annotated with 42 gene ontology (GO) functional categories. And 18,028 unigenes mapped to 125 pathways by searching against the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG). In addition, 3,919 microsatellite markers were developed in the unigene library, and 160 PCR primers of SSR loci were used for validation of the amplification and the polymorphism. Conclusion: We completed a successful global analysis of the peanut transcriptome using RNA-seq, a large number of unigenes were assembled, and almost four thousand SSR primers were developed. These data will facilitate gene discovery and functional genomic studies of the peanut plant. In addition, this study provides insight into the complex transcriptome of the peanut and established a biotechnological platform for future research.

Background: Rapid advances in next-generation sequencing methods have provided new opportunities for transcriptome sequencing (RNA-Seq). The unprecedented sequencing depth provided by RNA-Seq makes it a powerful and cost-efficient method for transcriptome study, and it has been widely used in model organisms and non-model organisms to identify and quantify RNA. For non-model organisms lacking well-defined genomes, de novo assembly is typically required for downstream RNA-Seq analyses, including SNP discovery and identification of genes differentially expressed by phenotypes. Although RNA-Seq has been successfully used to sequence many non-model organisms, the results of de novo assembly from short reads can still be improved by using recent bioinformatic developments. Results: In this study, we used 212.6 million pair-end reads, which accounted for 16.2 Gb, to assemble the hexaploid wheat transcriptome. Two state-of-the-art assemblers, Trinity and Trans-ABySS, which use the single and multiple k-mer methods, respectively, were used, and the whole de novo assembly process was divided into the following four steps: pre-assembly, merging different samples, removal of redundancy and scaffolding. We documented every detail of these steps and how these steps influenced assembly performance to gain insight into transcriptome assembly from short reads. After optimization, the assembled transcripts were comparable to Sanger-derived ESTs in terms of both continuity and accuracy. We also provided considerable new wheat transcript data to the community. Conclusions: It is feasible to assemble the hexaploid wheat transcriptome from short reads. Special attention should be paid to dealing with multiple samples to balance the spectrum of expression levels and redundancy. To obtain an accurate overview of RNA profiling, removal of redundancy may be crucial in de novo assembly.

The male-sterile ms2 mutant has been known for 40 years and has become extremely important in the commercial production of wheat. However, the gene responsible for this phenotype has remained unknown. Here we report the map-based cloning of the Ms2 gene. The Ms2 locus is remarkable in several ways that have implications in basic biology. Beyond having no functional annotation, barely detectable transcription in fertile wild-type wheat plants, and accumulated destructive mutations in Ms2 orthologs, the Ms2 allele in the ms2 mutant has acquired a terminal-repeat retrotransposon in miniature (TRIM) element in its promoter. This TRIM element is responsible for the anther-specific Ms2 activation that confers male sterility. The identification of Ms2 not only unravels the genetic basis of a historically important breeding trait, but also shows an example of how a TRIM element insertion near a gene can contribute to genetic novelty and phenotypic plasticity.