RNA-seq based high throughput sequence and expression analysis of selected root architecture genes in wheat

Abstract

Drought is a multigenic trait that has a major global impact on crop productivity. The aim of this research is to reveal the gene variants involved in wheat drought tolerance through transcriptomic analysis based on RNA-seq. In this study 6 wheat cultivars namely, Batis, Blue Silver, Local White, UZ-11-CWA-8, Chakwal 50 and Synthetic S22 were transcriptomically analyzed for identification of root growth genes in the research. The International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v1 by HISAT2 was aligned with the default parameters for the identification of their genomic positions (version 2.0.5). HISAT2 gave overall alignment rate for Batis 92.95%, Blue Silver 92.16%, Local White 92.92%, UZ-11-CWA-8 93.95%, Chakwal-50 94.43% and Syn-22 92.49%, respectively. After alignment, StringTie an RNA-seq abundance estimator assembled and quantified transcripts from alignments generated by HISAT2; isoforms producing primary results in the GTF file comprising assembled and fully covered transcripts that match the reference annotation with the gene abundance files. StringTie produced 6 GTF files for all cultivars with full covered, end-to-end transcripts in the reference annotation, with reads showing total 608 fully covered transcripts for Batis, 617 Blue Silver, 574 Local White, 567 UZ-11-CWA-8, 551 Chakwal-50 and 542 Syn-22, respectively. Similarly, 6 GTF transcript files containing information of the transcripts produced by StringTie from RNA-seq information display a total of 147974 transcripts assembled for Batis, 149383 Blue Silver, 147715 Local White, 148850 UZ 11-CWA-8, 147711 Chakwal-50 and 147802 Syn-22. Ballgown package identified ARD4, DRO1, HKT, PIN2, PSTOL, and RPK1 genes which have major role in root architecture and their differential expressions in wheat homoeologous genomes. RNA seq studies, showed that expression variations in genes in both drought sensitive and drought tolerant varieties. AgriGO tool used Singular Enrichment Analysis to produce cellular and biological gene ontology analysis which showed gene interaction pathways. Our findings unveiled genes to be tested through functional genomics studies under the impact of drought stress loci. These findings will have future consequences for plant genetic improvement against abiotic stress by editing root architecture genes to improve plant yield