| Summary: | Puccinia graminis f. sp. tritici (Pgt) is the causal agent of the devastating stem rust disease in wheat. In recent years, new super virulent races of the fungus have emerged causing large scale epidemics. In an attempt to clone the stem rust resistance gene Sr44, we generated and screened an ethyl methane sulphonate (EMS) mutant population of an Sr44 wheat-alien introgression line. We identified twelve independent susceptible mutants from 1171 M2 families and sequenced the nucleotide binding leucine-rich repeat (NLR) genes in ten of the mutants and the wild-type. However, sequence comparison did not reveal a clear candidate. To investigate meristem cell fate in wheat, we phenotyped the sister spikes of ten Sr44 M2 families which segregated for susceptibility in the main spike. Ninety-two percent of the tested spikes were found to be resistant suggesting that they are genetically distinct from the main tiller. To improve the immunity of barley against wheat stem rust, we transformed the previously cloned wheat Sr22, Sr33, and Sr45 genes into barley. The resultant transgenic lines expressed high-level resistance to Pgt indicating wheat Sr genes can be transferred into barley. Nucleotide sequence analysis of the Sr22 locus revealed that some alleles have undergone historical sequence exchange in the LRR region. We also generated and phenotyped wheat transgenics to confirm the gene postulation of two previously identified Sr22 alleles. Stacking of multiple Sr genes at a single transgene locus is expected to result in more durable resistance. We have attempted to use CRISPR/Cas9 to repair the hygromycin phosphotransferase II (HPTII) gene as a proof-of-concept to in vivo sequential stacking of multiple Sr genes. Super transformation of barley T0 and T1 transgenics containing a landing pad did not yield positive transformants. However, we identified one deletion event out of twenty-four calli of T0 transgenics, indicating functional CRISPR/Cas9 activity.
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