Classic and CRISPR/CAS9-based genetic mapping of the TFA loci in barley

The utilization of wild gene alleles and genome editing approaches may sound like an oxymoron in the study of gene function since the first seeks to look at naturally occurring DNA variation and the latter relies mostly on knocking-out genes. Cultivated barley (Hordeum vulgare subsp. vulgare) is ranked as the fourth most important cereal crop and therefore it is imperative to develop a state-of-the-art molecular toolbox to allow its improvement. We aim at inducing mitotic recombination within quantitative trait loci (QTL) regions with agricultural importance. The RECAS9 approach is conducted by applying CRISPR/CAS9 to heterozygous calli which is expected to lead to loss-of-heterozygous recombinants (Sadhu et al., 2016; Lazar et al., 2020). In this study, we target the Transformation Amenability (TFA) loci that control the ability of barley cells to regenerate into plants- an imperative trait for efficient genome editing efforts (Hisano & Sato, 2016). Following the classic genetic mapping of a complex locus affecting regeneration levels on chromosome 2, we target RECAS9 to further dissect several QTL. We compare between consequences of delivering CAS9/sgRNA ribonucleotide proteins (RNP) or Agrobacterium with CRISPR/CAS9 binary vectors on the mitotic recombination levels. The recessive mode of inheritance of the TFA locus we observe in F2::3 analysis is expected to select for LOH events during RECAS9 on the telomeric end of chromosome 2, promoting gene discovery. In addition, a comparative RNAseq analysis is conducted between relevant genotypes to shed more light on the genetic network underlying the TFA locus effects on regeneration.