RNA-Seq analysis of soft rush (Juncus effusus): transcriptome sequencing, de novo assembly, annotation, and polymorphism identification

Abstract

Background: Juncus effusus L. (family: Juncaceae; order: Poales) is a helophytic rush growing in temperate damp or wet terrestrial habitats and is of almost cosmopolitan distribution. The species has been studied intensively with respect to its interaction with co-occurring plants as well as microbes being involved in major biogeochemical cycles. J. effusus has biotechnological value as component of Constructed Wetlands where the plant has been employed in phytoremediation of contaminated water. Its genome has not been sequenced. Results: In this study we carried out functional annotation and polymorphism analysis of de novo assembled RNA-Seq data from 18 genotypes using 249 million paired-end Illumina HiSeq reads and 2.8 million 454 Titanium reads. The assembly comprised 158,591 contigs with a mean contig length of 780bp. The assembly was annotated using the dammit! annotation pipeline, which queries the databases OrthoDB, Pfam-A, Rfam, and runs BUSCO (Benchmarking Single-Copy Ortholog genes). In total, 111,567 contigs (70.3%) were annotated with functional descriptions, assigned gene ontology terms, and conserved protein domains, which resulted in 30,932 non-redundant gene sequences. Results of BUSCO and KEGG pathway analyses were similar for J. effusus as for the well-studied members of the Poales, Oryza sativa and Sorghum bicolor. A total of 566,433 polymorphisms were identified in transcribed regions with an average frequency of 1 polymorphism in every 171 bases. Conclusions: The transcriptome assembly was of high quality and genome coverage was sufficient for global analyses. This annotated knowledge resource can be utilized for future gene expression analysis, genomic feature comparisons, genotyping, primer design, and functional genomics in J. effusus.