Characterisation of Faba Bean (Vicia faba L.) Transcriptome Using RNA-Seq: Sequencing, De Novo Assembly, Annotation, and Expression Analysis

Abstract

RNA sequencing (RNA-Seq) is a deep sequencing method used for transcriptome profiling. RNA-Seq assemblies have successfully been used for a broad variety of applications, such as gene characterisation, functional genomic studies, and gene expression analysis, particularly useful in the absence of a well-studied genome reference sequence. This study reports on the development of reference unigene sets from faba bean using RNA-Seq. Two Australian faba bean cultivars (Doza and Farah) that differ in terms of disease resistance, breeding habit, and adaptation characteristics, and have been extensively used in breeding programs, were utilised in this study. The de novo assembly resulted in a total of 58,962 and 53,275 transcripts with approximately 67 Mbp (1588 bp N50) and 61 Mbp (1629 bp N50) for Doza and Farah, respectively. The generated transcripts have been compared to the protein and nucleotide databases of NCBI, as well as to the gene complements of several related legume species such as Medicago truncatula, soybean, and chickpea. Both assemblies were compared to previously-published faba bean transcriptome reference sets for the degree of completeness and utility. Annotation of unigenes has been performed, and patterns of tissue-specific expression identified. The gene complement derived from this comprehensive transcriptome analysis shows that faba bean, despite its complex 13 Gbp genome, compares well to other legumes in expressed gene content. This study in faba bean represents the most comprehensive reference transcriptomes from two different Australian cultivars available to date and it provides a valuable resource for future genomics-assisted breeding activities in this species.

Generation of a Comprehensive Transcriptome Atlas and Transcriptome Dynamics in Medicinal Cannabis

“…Transcripts that displayed an inconsistent expression pattern between RNA-Seq and qRT-PCR data could be due to relevant primer pairs’ lack of specificity resulting in non-specific amplification, or detection of paralogous gene sequence’ expression profile. The validation results were found to be comparable with other published plant transcriptome studies 36,37 .…”

“…A higher N50 is likely due to the presence of untranslated regions (UTRs) in this study as compared to previous datasets. In terms of functional characterisation of the transcripts, 64% of Cannbio’ transcripts were assigned at least one GO term, which is comparable with other published plant transcriptome studies 32,36,37 .…”

“…The sequence data was processed, and de novo assembly was generated as described previously 36,37 . Briefly, raw sequence reads were filtered by using Cutadapt v. 1.9 44 and a custom perl script.…”

“…The F-Box gene was used as an internal reference gene. The qRT-PCR, melting curve analysis and normalisation of the obtained data against the internal control was conducted as detailed previously 36,37 . Average of normalised qRT-PCR data and normalised read count from each of the different samples; leaf, root and reproductive female floral buds (Stage 1 and Stage 4) was used for calculation of Pearson’s correlation coefficient.…”

Recent Advances in Faba Bean Genetic and Genomic Tools for Crop Improvement

“…cloning was delayed both due to its genome complexity (e.g., abundance of transposable elements) and its being a minor crop. In the absence of a reference genome assembly for this species, high-throughput approaches such as transcriptome analysis have been efficient tools for enrichment of genomic resources (e.g., Ray et al, 2015;Arun-Chinnappa & McCurdy, 2015;Ocaña et al, 2015;Braich et al, 2017;Gao et al, 2020;Yang et al, 2020). However, from these reported transcriptome datasets, only limited DNA sequence data are available in public databases (Mokhtar et al, 2020).…”