De novo assembly of a genome-wide transcriptome map of Vicia faba (L.) for transfer cell research

Abstract

Vicia faba (L.) is an important cool-season grain legume species used widely in agriculture but also in plant physiology research, particularly as an experimental model to study transfer cell (TC) development. TCs are specialized nutrient transport cells in plants, characterized by invaginated wall ingrowths with amplified plasma membrane surface area enriched with transporter proteins that facilitate nutrient transfer. Many TCs are formed by trans-differentiation from differentiated cells at apoplasmic/symplasmic boundaries in nutrient transport. Adaxial epidermal cells of isolated cotyledons can be induced to form functional TCs, thus providing a valuable experimental system to investigate genetic regulation of TC trans-differentiation. The genome of V. faba is exceedingly large (ca. 13 Gb), however, and limited genomic information is available for this species. To provide a resource for future transcript profiling of epidermal TC differentiation, we have undertaken de novo assembly of a genome-wide transcriptome map for V. faba. Illumina paired-end sequencing of total RNA pooled from different tissues and different stages, including isolated cotyledons induced to form epidermal TCs, generated 69.5 M reads, of which 65.8 M were used for assembly following trimming and quality control. Assembly using a De-Bruijn graph-based approach generated 21,297 contigs, of which 80.6% were successfully annotated against GO terms. The assembly was validated against known V. faba cDNAs held in GenBank, including transcripts previously identified as being specifically expressed in epidermal cells across TC trans-differentiation. This genome-wide transcriptome map therefore provides a valuable tool for future transcript profiling of epidermal TC trans-differentiation, and also enriches the genetic resources available for this important legume crop species.

Keywords: RNA-Seq; Vicia faba; de novo transcriptome assembly; transcription factors; transfer cell; wall ingrowths.

Figure 1

Distribution of total number of V. faba contigs vs. contig length. Most contig lengths were in the range of 500–800 bp (50%) and above 1500 bp (20%). Total number of contigs decreased as contig length increased.

Figure 2

Top BLASTX hits of V. faba contigs against the Viridiplantae database. Contig sequences of V. faba are highly conserved with closely related and sequenced legume species, with 85% of the contigs showing similarity with Cicer arietinum (chick pea) and Medicago truncatula.

Figure 3

Functional annotation of V. faba contigs. Blast2GO characterization of GO terms in the three categories of Biological Process, Molecular Function, and Cellular Component.

Figure 4

Distribution of V. faba transcripts into different transcription factor families. Pie chart showing families of transcription factors and total number of members identified for each family. Homeobox family members (HB) contributed 19% of the total transcription factors identified and classified, followed by WRKY (8%) and AP2-EREBP (8%) sequences. The number listed after each family name represents the total number of transcription factors assigned to that family.