Development of Transgenic Line System for Mass Production of Hybrid Seeds

Abstract

The generation of male sterility mutants and identification of novel male fertility genes are of immense importance in agriculture sector for hybrid production. In the present study, an efficient transgenic male sterility/fertility system is generated using CRISPR-Cas9 technology. Further, various sgRNAs features for giving rise to functional gRNAs against ineffective genomic regions are determined using various statistical tests and a novel male fertility gene is identified and subjected to computational analysis for functional annotation. For this purpose, first a SM CRISPR-Cas9 vector with egg cell specific promoter was developed and 12- oxophytodienoate reductase3 (OPR3) gene in various natural accessions of Arabidopsis thaliana was successfully knockout. The deletions and indels in T1 and T2 mutants were revealed in the sequencing results. In the mutant plants, anthers remained close at the anthesis stage and were identical to wild type plants. The treatment of C24-T2 male sterile mutant inflorescences with jasmonic acid (JA) resulted in siliques elongation. The cross-pollination of mutants with wild type pollens resulted in the F1 hybrid generation. Besides C24, the successful knocking out of OPR3 gene in Col-0 and Ler also established the SM CRISPR-Cas9 vector functioning in these natural accessions. The statistical tests on experimentally validated plant sgRNAs data targeting ineffective genomic regions demonstrated that preference of nucleotide in protospacer adjacent motif (PAM) proximal region, PAM proximal seed region GC content, integral RAR and 3rd stem-loop structures, and nucleotides free availability in sgRNAs seed and tracrRNA regions are important factors related with sgRNAs high on-target cleavage efficiencies. The computational analysis of 3p.AtFBP113 showed that it is localized on chromosome 3 small arm and encodes a protein product that is soluble, thermostable, and hydrophilic in nature with xxi the likelihood of localization in different subcellular compartments. The anticipation of immunoglobin-like fold in the F-box associated (FBA) domain at C-terminal region suggested its function in protein binding. Functional annotation using gene ontology and digital gene co-expression investigation further supported the 3p.AtFBP113 role in protein binding and ubiquitination pathway. Further, the predicted tertiary structure showed two kinds of domains and folds like features. Thus, an efficient transgenic male sterility/fertility system for hybrid breeding is rapidly generated using an efficient SM CRISPR-Cas9 vector that is capable of functioning across diverse natural accessions. The study also illustrates sgRNAs features associated with their on-target high cleavage efficiency against ineffective regions of Arabidopsis genome that were previously described to generate ineffective sgRNAs and indicates the development of an efficient plant-based sgRNA designing tool considering the whole genomic regions for highly effective genome editing. Further, the study outcomes will provide a better understanding of 3p.AtFBP113 functions during male gametogenesis, which can serve as a potential target for hybrid breeding by manipulating male fertility.