Recombining and Fine-Mapping Resilience Alleles in Barley by Genome Editing

Use of wild germplasm in plant breeding has been aimed to increase crop production under climate change. A major challenge to this precise breeding is the identification of causal allele of a trait under different environment. This results from rare, non-uniform meiotic recombination between the wild and cultivated alleles and suppressed recombination at certain chromosomal parts such as the pericentromeric region. Here we modified a recently demonstrated CRISPR-directed mitotic recombination approach in yeast for genome recombining of HsDry2.2, a wild barley quantitative trait locus (QTL), which resides within a low-recombinogenic region in chromosome 2 and show drought-conditioned increasing effects on grain number and additional traits. Nevertheless, the location of the QTL is delimited to 5.5 cM which corresponds to app. 400 Mbp on the physical map. Therefore, generating recombinations every 100 Kbp for finer-mapping will require at least 4000 F2 plants, assuming the recombination landscape is uniform. Our novel approach to manipulate the mitotic rather than meiotic recombination machinery of this locus is by initiating double-strand-breaks via introducing CAS9 and gRNA molecules as ribonucleoprotein (RNP) complex into tissue culture of the dividing cells carrying the heterozygous QTL loci. The loss of heterozygosity generated will be confirmed by digital droplet PCR (ddPCR) and long-range sequencing of regenerated recombinant plants. The successful outcome of this work will serve as a model for genome recombining of wild-derived QTL underlying crop resilience, and for better understanding of their mode of action.