Comprehensive Stress-Based De Novo Transcriptome Assembly and Annotation of Guar (Cyamopsis tetragonoloba (L.) Taub.): An Important Industrial and Forage Crop

Abstract

The forage crop Guar (Cyamopsis tetragonoloba (L.) Taub.) has the ability to endure heat, drought, and mild salinity. A complete image on its genic architecture will promote our understanding about gene expression networks and different tolerance mechanisms at the molecular level. Therefore, whole mRNA sequence approach on the Guar plant was conducted to provide a snapshot of the mRNA information in the cell under salinity, heat, and drought stresses to be integrated with previous transcriptomic studies. RNA-Seq technology was employed to perform a 2 × 100 paired-end sequencing using an Illumina HiSeq 2500 platform for the transcriptome of leaves of C. tetragonoloba under normal, heat, drought, and salinity conditions. Trinity was used to achieve a de novo assembly followed by gene annotation, functional classification, metabolic pathway analysis, and identification of SSR markers. A total of 218.2 million paired-end raw reads (~44 Gbp) were generated. Of those, 193.5M paired-end reads of high quality were used to reconstruct a total of 161,058 transcripts (~266 Mbp) with N50 of 2552 bp and 61,508 putative genes. There were 6463 proteins having >90% full-length coverage against the Swiss-Prot database and 94% complete orthologs against Embryophyta. Approximately, 62.87% of transcripts were blasted, 50.46% mapped, and 43.50% annotated. A total of 4715 InterProScan families, 3441 domains, 74 repeats, and 490 sites were detected. Biological processes, molecular functions, and cellular components comprised 64.12%, 25.42%, and 10.4%, respectively. The transcriptome was associated with 985 enzymes and 156 KEGG pathways. A total of 27,066 SSRs were gained with an average frequency of one SSR/9.825 kb in the assembled transcripts. This resulting data will be helpful for the advanced analysis of Guar to multi-stress tolerance.

Identification of Key Metabolic Pathways and Biomarkers Underlying Flowering Time of Guar (Cyamopsis tetragonoloba (L.) Taub.) via Integrated Transcriptome-Metabolome Analysis

“…So far, several transcriptome assemblies for guar were reported in literature: among them one specific to roots [ 35 ] and two specific to leaves [ 21 , 22 ]. The latter studies focused on leaf tissue which was picked up from 21-day-old seedlings.…”

“…For Trinity, assemblers were tested with different k-mer values. Also k-mer values from the previous de novo guar leaf transcriptome assembly were tested (k-mer = 25, k-mer = 32) [ 21 , 22 ]. Trinity de novo and reference-guided assemblies and rnaSPAdes V. 3.13.0 assembly were compared using Transrate V. 1.0.3 [ 23 ] based on such assembly quality characteristics as total number of transcripts, total and average length of assembled transcripts and N50.…”

Identification of Differentially Expressed Drought-Responsive Genes in Guar [Cyamopsis tetragonoloba (L.) Taub]

“…Gene Quantification. In our previous works [36,37], we sequenced and assembled a comprehensive stress-based de novo transcriptome for guar, which was used here as a reference transcriptome. The reads were aligned to the reference transcriptome to estimate gene richness using RNA-Seq by Expectation-Maximization (RSEM; [38]).…”

Identification of Heat-Responsive Genes in Guar [Cyamopsis tetragonoloba (L.) Taub]

“…Gene Quantification. In our previous work [33], a comprehensive de novo guar transcriptome assembly was utilized as a reference based on the longest isoforms of the transcriptome 61508 transcripts. To estimate gene abundance, an alignment-based approach (aligning reads to the reference transcript assembly) was carried out via RSEM V1.1.13 (RNA-Seq by Expectation-Maximization; [34]).…”