Identification of induced mutations in hexaploid wheat genome using exome capture assay

Wheat is a staple food crop of many countries. Improving resilience to biotic and abiotic stresses remain key breeding targets. Among these, rust diseases are the most detrimental in terms of depressing wheat production. In the present study, chemical mutagenesis was used to induce mutations in the wheat variety NN-Gandum-1. This cultivar is moderately resistant to leaf and yellow rust. The aim of mutagenesis was to improve resistance to the disease as well as to study function of genes conferring resistance to the disease. In the present investigation, a 0.8% EMS dose was found optimum for supporting 45-55% germination of NN-Gandum-1. A total of 3,634 M2 fertile plants were produced from each of the M1 plant. Out of these, 33 (0.91%) and 20 plants (0.55%) showed absolute resistance to leaf and yellow rust, respectively. While 126 (3.46%) and 127 plants (3.49%) exhibited high susceptibility to the leaf and yellow rust, respectively. In the M4 generation, a total of 11 M4 lines (nine absolute resistant and two highly susceptible) and one wild type were selected for NGS-based exome capture assay. A total of 104,779 SNPs were identified that were randomly distributed throughout the wheat sub genomes (A, B and D). Induced mutations in intronic sequences predominated. The highest total number of SNPs detected in this assay were mapped to chr.2B (14,273 SNPs), which contains the highest number of targeted base pairs in the assay. The average mutation density across all regions interrogated was estimated to be one mutation per 20.91 Mb. The highest mutation frequency was found in chr.2D (1/11.7 kb) and the lowest in chr.7D (1/353.4 kb). Out of the detected mutations, 101 SNPs were filtered using analysis criteria aimed to enrich for mutations that may affect gene function. Out of these, one putative SNP detected in Lr21 were selected for further analysis. The SNP identified in chimeric allele (Lr21) of a resistant mutant (N1-252) was located in a NBS domain of chr.1BS at 3.4 Mb position. Through computational analysis, it was demonstrated that this identified SNP causes a substitution of glutamic acid with alanine, resulting in a predicted altered protein structure. This mutation, therefore, is a candidate for contributing to the resistance phenotype in the mutant line. Based on this work, we conclude that the wheat mutant resource developed is useful as a source of novel genetic variation for forward-genetic screens and also as a useful tool for gaining insights