Identification Of Rice Blast Resistance Genes In Early-season Indica Rice Varieties And Improving The Blast Resistance Of Zhongzao 39

Early-season indica rice plays a significant role in ensuring food security in China,rice blast is the most devastating diseases in rice production.Practice has proved that the most effective approach to control blast disease is creating,cultivating and planting resistant cultivars.In this study,phenotyping,genotyping,and genetic association analyses were used to evaluate 155 early-season rice cultivars and breeding lines.Here we reported the improvement of blast resistance of Zhongzao39 by introducing gene Pi25 into Zhongzao39 by molecular marker-assisted selection and editing transcription factor OsERF922 by CRISPR-Cas9 system.The main results are presented as follow:1.Phenotyping,genotyping and genetic association analyses were used to evaluate 155early-season rice cultivars and breeding lines?65 cultivars of early-season indica rice that were released from 1984–2020 as well as 90 breeding lines?.The presence of 18 blast resistance genes were screened in155 early-season rice cultivars and breeding lines,in which,all were positive for Pi36,Pik-p and Ptr,while none were positive for Pi25 and pi21.Rice cultivars and breeding lines possessing blast resistance genes varied from 7 to 14.According to phenotyping,the majority of the resistant rice cultivars and breeding lines were clustered together in Group III,which included cultivars released since 2011 and several breeding lines.Association analysis identified five markers?NBS2-Pi9,P9,G8900,Zt56591,and Z56592?that are significantly associated with the phenotyping results,indicating that they may play an important role in M.oryzae resistance in early-season indica rice breeding programs.2.R6,rice blast resistance gene Pi25 contained,was used as the donor parent,while the super early-season rice Zhongzao39 was used as the receptor and recurrent parent,to improve blast resistance through crossing,backcrossing,self-pollination and molecular marker-assisted selection.Meanwhile,rice blast resistance assay were conducted independently and respectively with spray inoculation in Greenhouse,injection inoculation in field and natural infection in disease nursery.Four resistance improved lines?i.e.FY82,FY90,FY125 and FY137?carrying blast resistant gene Pi25,which showed higher resistance to leaf and neck blast than Zhongzao 39 were finally obtained.3.OsERF922 negatively regulated rice blast resistance.We selected the 22 bases of OsERF922coding sequence 264-286 as the target sequence to construct editing vector,and obtained T₀ generation transgenic plants by Agrobacterium-mediated method into Zhongzao39.The mutation efficiency of OsERF922 was 85%,and the mutation types of OsERF922 including deletion,replacement and insertion of bases,among which the frequency of base deletion is the relatively large.Homozygous mutant T₁ line N14-8 without T-DNA components was obtained.