DISRUPTION OF GENOME INTEGRITY TO CREATE GENETIC VARIABILITY BY EDITING (USING CRISPR CAS9) THE GENES ASSOCIATED WITH DNA MISMATCH REPAIR AND CHARACTERIZATION OF THEIR RELEVANCE IN CROP IMPROVEMENT
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Date
2022-08-03
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University of Agricultural Sciences, Bangalore
Abstract
Genetic variation is crucial for crop improvement because of narrow variability in many species. We adopted a gene editing approach to create variations in the rice genome by targeting the mismatch repair genes (MMR), Mut S Homolog 1 (MSH1), Mut S
Homolog 2 (MSH2) and Mut L Homolog 1 (MLH1). The hypothesis is that any disruptionin these genes leads to altered DNA mismatch repair that create indels and reprogrammed genome, resulting in altered phenotypes. The guide RNA with high specificity and least off targets were designed, cloned to a suitable binary vector pRGEB 32. Transgenic plants were developed by both in vitro Agrobacterium tissue culture approach and modified in planta agrobacterium mediated direct embryonal axis transformation
approach. Putative transformants were identified by selection against hygromycin. Sequencing analysis confirmed 4 edited plants in MSH1 gene and 1 edited plant in MSH2and 3 edited lines in MLH1 gene. These gene edited lines exhibited morphological
variation with respect to plant height, tiller number, biomass etc., compared to the wild type plants. The flanking sequence analysis confirms that the observed phenotype is not due to the interference of transgene integration in the rice genome but due to the mutation in the MMR genes. The existing mutant plants have to be stabilized by crossing with its wild type counterpart and such stabilized lines could be a potential source to create more segregating population or act as donor lines in breeding programmes.