| Rice blast is one of the most threatening fungal diseases of rice production..This study first Mainly identificating and analyzing to rice blast resistant genes in the distribution in rice germplasm resources of Liaoning Province;Secondly,New gene Pi65(t) rice resistance to rice blast for precise positioning and CRISPR/Cas9 knock out the construction of the carrier,which can guide breeding and cultivating of disease resistant rice varieties.1,To understand the distribution of rice blast resistant genes and their Contribution to rice blast disease in Liaoning Province,we selected 176 rice germplasm,and identified the distribution of rice blast resistance genes pi21、Pi36、Pi37、Pita、Pid2、Pid3、Pi5 and Pib in these germplasm.Meanwhile,the phenotypes of rice blast resistance of these germplasm were studied by inoculating.The results showed that 83 germplasm were resistant to rice blast disease,among them,the rates of cultivars,weedy rice and landraces were 41.48%、1.14%and 4.54%,respectively.Rice blast resistance genes pi21、Pi36 and Pi37 were not detected in all rice germplasm,and 49、47、74、52 and 89 germplasm carried Pita、Pid2、Pid3、Pi5 and Pib,respectively.Most of the genes were found in cultivars,but they were rare in landraces and weedy rice.Most of the germplasm that have no any rice blast resistant genes and carry only single rice blast resistant gene were susceptible to rice blast.Gene pyramiding could improve the resistant to some extent.In this test,about 32 germplasm did not carry any rice blast resistance genes that we identified,and only 21.87%of which were resistant to rice blast;about 52 germplasm carried single rice blast resistance gene,and only 17.31%of which were resistant to rice blast,too;about 39 germplasm carried two rice blast resistance genes,and 69.23%of which showed resistant to this disease.Within them,14 germplasm that carried Pita and Pi5 all showed resistant to rice blast;among 31 germplasm carried three rice blast resistance genes,the germplasm that were resistant to rice blast accounted for 77.42%,and the resistance of germplasm that carried Pita,Pid3 and Pi5 simultaneously are signally good;about 22 germplasm carried four rice blast resistance genes,with 72.73%of which were resistant to rice blast;the germplasm carried five rice blast resistance genes were not found.2、In previous research,by new generation of sequencing technology,using a DH group of gangyu 129/liaoxingl,a new rice blast resistance gene Pi65(t)with broad spectrum was mapped to chromosome 11 between 30.42 to 30.85 Mb.Using the software QTL IciMapping,Pi65(t)was further mapped to a locus between InDel-1 and SNP-4 with genetic distances of 0.11 and 0.98 cM,respectively.Within this region,4 predicted R genes were found with nucleotide binding site and leucine-rich repeat(NBS-LRR)domains,including Os11g0694500,Os11g0694600,Os11g0694850 and Os11g0695000.In this research,the sequence of 4 candidate genes in gangyu 129 and liao xing 1 were tested.The sequence of Os11g0694500 is same to the sequence of its allele in Nipponbare genome,and the resistant and susceptible parents could express the gene normally,which showed that Osllg0694500 might not related to the rice blast resistant of Gangyu 129.The CRISPR/Cas9 vectors of Os11g0694600,Os11g0694850,Os11g0695000 were constructed and were tranfered to Gangyu129 by agrobacterium-mediated transformation method.After genetic transformation,we got 12 transgenic seedlings(T0)that the allele of Os11g0694850 was knocked out in Gangyu 129,and the genotypes identification showed that the sequences of Os11g0694850 alleles in 9 strains were edited successfully.As for the To generation transformants were heterozygous and Pi65(t)is a dominant gene,the function of the gene can not be determined according to the resistant phenotype of T0 transgenic plants yet,and it is necessary to screen the homozygotes from transgenic seedlings (T1) to study further. |
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