Fine Mapping Of A Panicle Blast Resistance Gene Pb-bd1 In Japonica Landrace Bodao And Functional Analysis Of Transgenic Lines

Rice blast caused by Magnaporthe oryzea is the most widely distribute and most devastating rice disease.Rice can be attacked by rice blast in its whole grouth period,especially at seedling stage and booting stage.The rice production caused by rice blast is reduced by 10-30%every year in the world,which poses a huge threat to the global food security.So it is important to excavation and identification of broad-spectrum,high and long-lasting resistance to rice blast for controlling the occurrence and damage of rice blast.Bodao is a japonica rice landrace from the Taihu Lake Region and it has broad-spectrum resistance to rice blast.It is an excellent germplasm resource for studying rice-blast interaction and breeding of new rice varieties.On our previous reasearch,we mapped a major effective QTL Pb-bd1 for panicle blast resistance in rice chromosome 11.So,based on the previous reasearch,this study carried out fine mapped the broad-spectrum resistance gene Pb-bd1;nucleotide sequence difference analysis of candidate genes in Bodao and Suyunuo;tissue-specific expression profiles of candidate genes;candidate genes identified by genetic modification.Main research findings are as follows:1.Fine mapping of Pb-bd1.In this study,the recombinant was screened in 6000 strains of BC4F5 by expanding population.Pb-bd1 was finally mapped in marker BS83 and BS98 in a 40.6Kb region,6 candidate genes identified within this region:P1 encoding NAC domain-containing protein,P2 encoding unknown expression proteins,P3,P4 encoding NBS-LRR type disease resistance proteins,P5,P6 encoding vWA domain containing proteins.2.Candidate sequence alignment.After designing primers,amplification,sequencing,the candidate gene coding sequences are compared between the parents:P1 has six SNPs of nucleotide substitutions and two Indels in exon leading to amino acid changes;P2 encodes an unknown protein in Bodao,but there is no candidate gene P2 in Suyunuo;P3 sequence does not differ between the two parents;P4 has eight SNPs of nucleotide substitutions and two Indels in exon leading to amino acid changes;P5 has three SNPs of nucleotide substitutions in exon leading to amino acid changes,P6 sequences vary greatly between the two parents.3.Tissue-specific expression profiles of candidate genesAnalysis of tissue expression characteristics of candidate genes in rice using public databases GENEVESTIGATOR and RiceXPro.The expression results showed that the expression patterns of P3 and P4 in different tissues of rice were similar,and their expression in roots was higher;the expression patterns of P5 and P6 in different tissues of rice were similar,and their expression in roots and embryo was higher.4.Validation of transgenic functions of candidate genesThe CRISPR/Cas9 vectors of P1-P6 were constructed and were transferred to Bodao by agrobacterium-mediated transformation method.Now we have got T1 generation transformation of P1、P2、P3、P4 and T0 generation transformation of P5、P6.Screening the pure mutant lines in T1-generation transgenic lines from P1-P4,and verifying by Magnaporthe oryzae.The transgeniclines obtained by P5 and P6 is the T0 generation,and the target sequence analysis is carried out,and the successfully edited mutant is subjected to inoculation verification.