Targeted Editing Of Pi21 And OsBadh2 Genes For Rice Improved Blast Disease Resistance And Fragrance Quality Of Kongyu131

Kongyu-131 has been planted large area as one of the conventional rice varieties in Northeast China, it is susceptible to blast disease under the condition of low temperature and high humidity. Previous studies showed that Pi21 and OsBadh2 locus were recessive rice possessed blast resistance and fragrance character, but Pi21 and OsBadh2 genes were dominant in variety kongyu-131. We want to improve blast resistance and fragrance quality of kongyu-131 by editing Pi21 and OsBadh2 genes through CRISPR-Cas9 system. The main results are showed as follows:1. Four target sites(sgRNA1-4) were designed for specific combination of Pi21 gene. We obtained two and six heterozygous mutants for sgRNA1 and sgRNA2 targets of Pi21 gene, respectively, and 13 heterozygous mutants were obtained for sgRNA3 and sgRNA4 targets of Pi21 gene. Five single copy lines were identified from eight heterozygous mutants for sgRNA1 and sgRNA2. We obtained 12 Cas9-free homozygous plants in T1 generation. QRT-PCR and RT-PCR results showed that a highest expression of Pi21 gene was detected in leaf sheath, and then was detected in stem. The expression level of Pi21 in leaf sheath and stem of the three homozygous lines(62-2, 61-3 and 18-23) were all reduced compared with the wild type kongyu-131.2. Blast resistance identification showed that kongyu-131 was susceptible to the five inoculation strains. Pi21 homozyous mutants 62-2(deletion C) and 36-18(deletion 4-bp) were resistant to blast strain 14-7303-1, but 17-1(deletion A) was susceptible to 14-7303-1. The three homozygous lines were all susceptible to the other four inoculation blast strains. Co-segregation analysis results showed that the blast resistance phenotype and the mutation of pi21 were co-segregation, which suggested that the blast resistance reaction was caused by editing of Pi21. Agronomic traits investigation results showed that the main agronomic traits of the three homozygous lines(62-2, 17-1 and 36-18) were similar with the wide type kongyu-131. Comparative sequencing results showed that the sequence of quality gene LOC_Os04g32890(linkage with Pi21 gene) in the three homozygous lines were the same with the wild type, which suggested that the edited of Pi21 gene without changing the sequence of adjacent gene LOC_Os04g32890.3. We obtained two heterozygous mutants for sgRNA1 target and seven heterozygous mutants for sgRNA3 and sgRNA4 targets of OsBadh2 gene.