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

Abstract

Viola 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 de novo genome assembly and transcriptomic analyses of the non-model species Viola pubescens using short-read DNA sequencing data and RNA-Seq from eight diverse tissues. First, V. pubescens genome size was estimated through flow cytometry, resulting in an approximate haploid genome of 455 Mbp. Next, the draft V. pubescens 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 ab initio gene models which were functionally annotated based on homology to Arabidopsis thaliana 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 V. pubescens’ mixed breeding system. Orthologous clustering with the proteomes of A. thaliana and Populus trichocarpa revealed 8,531 sequences unique to V. pubescens , 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 V. pubescens cyclotides and published cyclotide sequences from other Violaceae and Rubiaceae species. Collectively, this work provides the most comprehensive sequence resource for Viola , offers valuable transcriptomic insight into V. pubescens , and will facilitate future functional genomics research in Viola and other diverse plant groups.

A revised phylogenetic classification forViola(Violaceae)

“…Thus far, reference sequence genome has been published for the diploid Viola (sect. Chamaemelanium) pubescens [192] and the octoploid V. (sect. Himalayum) kunawurensis (as V. "kunawarensis"; NCBI accession PRJNA805692), but numerous Viola genomes are planned sequenced by the Earth Biogenome Project during the next decade [193].…”

“…Thus far, reference sequence genome has been published for the diploid Viola (sect. Chamaemelanium ) pubescens [192] and the octoploid V . (sect.…”

Transcriptomic profiling of the medicinal plant Clitoria ternatea: identification of potential genes in cyclotide biosynthesis

“…The figure highlights the ER signal peptide, immediately followed by the cyclotide mature domain, linked by a 10-residue linker and co-occurring with the albumin-1a chain, and a short ~ 10 residue C-terminal propeptide tail. Characterization of 'cliotide' genes from various transcriptomic approaches suggest a unique architecture in Fabaceae, that differs greatly from Violaceae and Rubiaceae cyclotide genes 15,20,21,69,71,72 . The cyclotide gene arrangement in C. ternatea is very similar to that of albumin gene of many legume species, wherein a signal peptide is followed by an albumin-1b domain, a linker region, and an albumin-1a domain.…”

Exploring the Sequence Diversity of Cyclotides from VietnameseViolaSpecies

“…32,33 Of the 397 cyclotides discovered in dicots so far, 212 have been isolated from the Violaceae family. 32 However, as it is estimated, on the basis of genomic identification, that more than 89,000 cyclotides could exist within the Violaceae family, 34 there are many more members yet to be characterized.…”

“…To date, a diverse range of cyclotides has been discovered in the Rubiaceae, Violaceae, Fabaceae, Solanaceae, Poaceae, and Cucurbitaceae families, with a comprehensive collection of sequence information available in the CyBase web-database (). , Of the 397 cyclotides discovered in dicots so far, 212 have been isolated from the Violaceae family . However, as it is estimated, on the basis of genomic identification, that more than 89 000 cyclotides could exist within the Violaceae family, there are many more members yet to be characterized.…”