De novo assembly and characterization of Camelina sativatranscriptome by paired-end sequencing

Chao Liang, Xuan Liu, Siu-Ming Yiu, Boon Leong Lim

BACKGROUND: Biofuels extracted from the seeds of Camelina sativa have recently been used successfully as environmentally friendly jet-fuel to reduce greenhouse gas emissions. Camelina sativa is genetically very close to Arabidopsis thaliana, and both are members of the Brassicaceae. Although public databases are currently available for some members of the Brassicaceae, such as A. thaliana, A. lyrata, Brassica napus, B. juncea and B. rapa, there are no public Expressed Sequence Tags (EST) or genomic data for Camelina sativa. In this study, a high-throughput, large-scale RNA sequencing (RNA-seq) of the Camelina sativa transcriptome was carried out to generate a database that will be useful for further functional analyses. RESULTS: Approximately 27 million clean "reads" filtered from raw reads by removal of adaptors, ambiguous reads and low-quality reads (2.42 gigabase pairs) were generated by Illumina paired-end RNA-seq technology. All of these clean reads were assembled de novo into 83,493 unigenes and 103,196 transcripts using SOAPdenovo and Trinity, respectively. The average length of the transcripts generated by Trinity was 697 bp (N50 = 976), which was longer than the average length of unigenes (319 bp, N50 = 346 bp). Nonetheless, the assembly generated by SOAPdenovo produced similar number of non-redundant hits (22,435) with that of Trinity (22,433) in BLASTN searches of the Arabidopsis thaliana CDS sequence database (TAIR). Four public databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG), Swiss-prot, NCBI non-redundant protein (NR), and the Cluster of Orthologous Groups (COG), were used for unigene annotation; 67,791 of 83,493 unigenes (81.2%) were finally annotated with gene descriptions or conserved protein domains that were mapped to 25,329 non-redundant protein sequences. We mapped 27,042 of 83,493 unigenes (32.4%) to 119 KEGG metabolic pathways. CONCLUSIONS: This is the first report of a transcriptome database for Camelina sativa, an environmentally important member of the Brassicaceae. We showed that C. savita is closely related to Arabidopsis spp. and more distantly related to Brassica spp. Although the majority of annotated genes had high sequence identity to those of A. thaliana, a substantial proportion of disease-resistance genes (NBS-encoding LRR genes) were instead more closely similar to the genes of other Brassicaceae; these genes included BrCN, BrCNL, BrNL, BrTN, BrTNL in B. rapa. As plant genomes are under long-term selection pressure from environmental stressors, conservation of these disease-resistance genes in C. sativa and B. rapa genomes implies that they are exposed to the threats from closely-related pathogens in their natural habitats.