Transcriptomics Identifies Modules of Differentially Expressed Genes and Novel Cyclotides in Viola pubescens

Abstract

<i>Viola</i> is a large genus with worldwide distribution and many traits not currently exemplified in model plants including unique breeding systems and the production of cyclotides. Here we report <i>de novo</i> genome assembly and transcriptomic analyses of the non-model species <i>Viola pubescens</i> using short-read DNA sequencing data and RNA-Seq from eight diverse tissues. First, <i>V. pubescens</i> genome size was estimated through flow cytometry, resulting in an approximate haploid genome of 455 Mbp. Next, the draft <i>V. pubescens</i> genome was sequenced and assembled resulting in 264,035,065 read pairs and 161,038 contigs with an N50 length of 3,455 base pairs (bp). RNA-Seq data were then assembled into tissue-specific transcripts. Together, the DNA and transcript data generated 38,081 <i>ab initio</i> gene models which were functionally annotated based on homology to <i>Arabidopsis thaliana</i> genes and Pfam domains. Gene expression was visualized for each tissue via principal component analysis and hierarchical clustering, and gene co-expression analysis identified 20 modules of tissue-specific transcriptional networks. Some of these modules highlight genetic differences between chasmogamous and cleistogamous flowers and may provide insight into <i>V. pubescens'</i> mixed breeding system. Orthologous clustering with the proteomes of <i>A. thaliana</i> and <i>Populus trichocarpa</i> revealed 8,531 sequences unique to <i>V. pubescens</i>, including 81 novel cyclotide precursor sequences. Cyclotides are plant peptides characterized by a stable, cyclic cystine knot motif, making them strong candidates for drug scaffolding and protein engineering. Analysis of the RNA-Seq data for these cyclotide transcripts revealed diverse expression patterns both between transcripts and tissues. The diversity of these cyclotides was also highlighted in a maximum likelihood protein cladogram containing <i>V. pubescens</i> cyclotides and published cyclotide sequences from other Violaceae and Rubiaceae species. Collectively, this work provides the most comprehensive sequence resource for <i>Viola</i>, offers valuable transcriptomic insight into <i>V. pubescens</i>, and will facilitate future functional genomics research in <i>Viola</i> and other diverse plant groups.