The Function Analysis Of Pi-hk1 Candidates And Fine Mapping Of Pi-hk2 In Heikezijing

Rice is one of the most important crops in the world,which feeds almost half of people around the world.Every year the yield loss caused by rice blast influenced the staple food of about 60 million of population.Therefore,it is of great practical significance to study the infection mechanism of rice blast,mine rice blast resistance genes,and find an economic and environmentally method improving rice blast resistance.Pi-hk1 confers a broad-spectrum resistant rice blast resistance locus in japonica rice landrace Heikezijing,which was previously fine mapped by Zhang’s lab,including 9 candidate genes(P8-P16).P15 encodes NBS-LRR domain containing protein,and it was found that the complementary transgenes of P15 showed increased blast resistance.Pi-hk2 is a race-specific blast resistance locus located in the interval of 228 Kb between the molecular markers ID-9 and RM24048 on chromosome 9 in Heikezijing.In this study,transgenic materials were used to analysis the function of Pi-hk1 candidates,and the backcross population of one resistant line RIL84 and susceptible landrace Suyunuo were used to fine map Pi-hk2 locus.The main results are as follows:1.The function analysis of Pi-hk1 candidates.In this study,we obtained the CRISPR/Cas9 mutant materials of P15 candidate.Their phenotype indicated that the deletion of P15 caused resistant Heikezijing susceptible,proving that P15 is indeed in rice blast resistance process.The P15 in Heikezijing has extra two WRKY domains compared with the P15 in Suyunuo.The P15 with full length,the P15 with one WRKY domain removed,and the P15 with two WRKY domains removed,were merged with the green fluorescent protein,respectively,to transform the susceptible rice cultivar Suyunuo.And three types of transgenic plants were obtained.After inoculation with Magnaporthe oryzae,the results of fluorescence microscopy showed that these three fusion proteins accumulated in the vicinity of the cell wall of the rice blast fungus,indicating that they can respond to the infection of Magnaporthe oryzae.The full-length P15 protein transformed lines showed improved rice blast resistance than Suyunuo,and the deletion of the WRKY domain affected P15-mediated resistance.Besides,the phenotype of P8,P9,P10,P11,P12,P13,P14,P15,P16 and P12K transgenic materials were performed.The results showed that the resistance level of most of lines were similar with Suyunuo.However,27 lines of P9,P10,P11,P12,P13,P14 and P15 complementary transgenic materials showed less lesion number and lesion length.We supposed that the Pi-hk1 locus might work as a team.2.The fine mapping of Pi-hk2.In order to fine mapping Pi-hk2,12 recombinant individuals were screened from the population BC1F5(3,200 individuals)and BC1F6(4,100 individuals),which were the progenies of RIL84 and Suyunuo.6 pairs of molecular markers including ID-M15,ID-M44,ID-M23,ID-M5,ID-M66 and CAP-1 were developed.The genotypes and phenotypes of the recombinants indicated that Pi-hk2 locus were flanked by molecular markers ID-M5 and RM24048.There was 110 Kb in this region,including genes LOC＿Os09g16440,LOC＿Os09g16449,LOC＿Os09g16458,LOC＿Os09g16470,LOC＿Os09g16480,LOC＿Os09g16490,LOC＿Os09g16500,LOC＿Os09g16510,LOC＿Os09g16520,LOC＿Os09g16530,LOC＿Os09g16540,LOC＿Os09g16550 and LOC＿Os09g16560.