Genome-Wide Identification and Editing of Root Architecture Genes in Wheat

Abstract

Wheat is one of the main staple food crops and more than a quarter of population relies on it for feed. Roots are the “principal hidden organ” that has crucial function in vascular plants. Root architecture traits modulate the organization of roots structure and is associated with acquisition of nutrients and water from soil. The root characteristics play a dynamic role under drought stress and crops yield in water scarce environment. Slight alterations in root distribution within soil might lead to the changes in yield. The root system architecture is governed by the numerous genes and environment, and is studied in model grass rice, however, little is known in wheat. Therefore, there is a need to study and understand the genes and its mechanisms involved in regulating root architecture system in wheat. Receptor like protein kinase 1(RPK1) gene is a root architecture gene that plays vital role in regulation of abiotic stress, root system architecture and yield. RPK1 belongs to the subfamily of Leucine-rich receptor kinases (LRR-RLK) and the family of Receptor-like kinases (RLKs) and is well characterized in rice. The characterization of RPK1 gene family has not been reported in Wheat (Triticum aestivum) . Here, we demonstrate the genome-wide identification, and expression analysis of TaRPK1 gene family in wheat. Results confirmed set of 15 TaRPK1 genes that are categorized mainly into three sub-groups on the basis of phylogenetic tree. The TaRPK1 genes were localized on 1-3 chromosome, in respective A, B, and D genomes. Structure of genes, conservation of motif, prediction of collinearity, and synteny analysis were performed systematically. Gene ontology study displayed the role of TaRPK1 genes during molecular and biological processes. 18 putative miRNAs were also identified, targeting TaRPK1 genes, signifying their function in development, growth, and stress responses. The analysis of cis-regulatory elements exhibited the presence of hormone responsive elements, light related elements, and motifs related to abiotic stress in the promoter regions. The 3-dimensional structure of TaRPK1 proteins was predicted based on homology modelling. In-silico expression pattern in different stages and tissues suggested the highest expression of TaRPK1 genes in root tissues. Moreover, qRT-PCR further validated the increased TaRPK1 genes expression in root tissues of drought tolerant varieties, in comparison to drought-susceptible variety. DRSML QAU Abstract Genome-Wide Identification and Editing of Root Architecture Genes in Wheat IV CRISPR/Cas9, genome editing tool was efficiently applied for targeted mutagenesis of RPK1 gene. The two constructs, namely LR-1 and LR-2 constructs, each with two guide RNAs, were delivered in wheat through Inplanta agrobacterium mediated transformation. Sanger sequencing confirmed 7 CRISPR/Cas9 based mutated T0 lines of LR-1 constructs and 6 CRISPR/Cas9 based mutated T0 lines of LR-2 constructs, with overall mutation efficiency of 41.93%. The T0 plants displayed higher monoalleleic mutation compared to diallelic mutation. The proportion of deletions was higher as compared to insertions. The deletions were mainly short, but longer deletions such as 12d, 17d, 19d, and 20d were detected in T0-6, T0-10, T0-14, and T0-16 lines by gRNA2 of LR-1 construct. Genome edited T0 lines displayed significant alteration in morphological and root architectural traits with significant decrease in spike length, root diameter and root angle and increased root length, depth, volume and surface area in comparison to the control plants. The observed reduction in spike length, root diameter, and root angle and enhanced root length, root depth, root volume, and root surface area results in significant increase in grain weight and number of effective tillers, compared to control plants. This increase in grain weight and number of effective tillers suggested that edited lines compensate grain yield reduction caused by spike length reduction. This study reveals that TaRPK1 editing results in improvement of root architecture traits and hence yield enhancement. In conclusion, this work highlighted the genome-wide characterization, and potential application of CRISPR editing technology in a root architecture gene within wheat. The use of such a precise and targeted CRISPR technology might provide assistance for wheat improvement at a sufficient rate to ensure food security globally