Identification Of Rice Blast Resistance Gene Pie6(t) And Development Of The Pike-specific Markers

Rice(Oryza sativa L.)is a stable food consumed by nearly half of the world's population.Over the past decades,rice production has been doubled by introduction of high-yielding varieties and improved cultivation practices.In recent years,however,the production of rice have not increased due to the limiting factors,such as germplasm resource and biological stress.Among them,rice blast disease which was caused by filamentous ascomycete fungus Magnaporthe oryzae is one of the biggest threats to the rice production.Every year,about 10%to 30%of rice yield was lost because of this disease.The use of host resistance in breeding new rice cultivars which were conferred stable and high resistance to this destructive disease has proven to be the most economical and effective strategy.Unfortunately,a majority of genes that confer rice specific resistance to blast pathogens remain effective for only a few years when deployed at a larger scale.Due to this rapid adaptation of the pathogens,new varieties with different resistance genes are continuously needed to replace varieties which have become susceptible.In this study,we aimed to identify new blast resistance gene and develop the Pike-specific markers.The results are as follows:1.The tropical japonica line Ly9505 which was confer broad spectrum resistance to blast pathogens was used as donor parent.And the mapping population was constructed by hybriding Ly9505 with two susceptible cultivars Ejin B and R287 respectively,which were then used to map the blast resistance gene from Ly9505.By the Bulked segregation analysis(BSA)and recessive-class analysis(RCA),a blast resistance gene that was named as Pie6(t)in our study was identified from Ly9505.This gene was located near the centromere in Chr.12.It was finally mapped to a narrow region flanked by molecular markers ID4714 and RM7102 within a distance of about 2.76Mb.The identification of blast resistance gene Pie6(t)has provided a molecular basis to explain the broad spectrum resistance of rice line Ly9505.And the linked molecular markers which were developed in our study could be a useful tool in tracing the Pie6(t)in rice breeding program.2.The rice blast resistance gene Pike,a broad-spectrum blast resistance gene,was cloned through map-based strategy recently in our lab.In the current study,sequence alignment between Pike and the other Pik alleles(include Pik from cv.Nipponbare,cv.YuetaiB et al.)let us identify a specific SNP in the NBS region of Pike-1.The nucleotide 1328-G at the SNP site and the nucleotide sequence around the 1328-G in the Pike-1 was used as a diagnostic feature to distinguish Pike from other Pik alleles which were reported previously.Based on the SNP G1328C/T and the polymorphism around the SNP,three Pike-specific markers CP-G1328C,CP-G1328T and CP-G1328G' were developed.Using these markers,326 rice accessions collected from different parts of China and some other countries were screened.Of the 326 accessions,one cultivars named as 898B was found to carry the gene Pike except for Xiangzao143.The rice accession 898B could be used as an alternative resistance source for resistance breeding.The markers CP-G1328C,CP-G1328T and CP-G1328G' were shown codominant between Pike donor and most of cultivars.The newly developed markers may used to searching the R gene Pike from rice germplasms.And in rice breeding program,the new markers could be used to tracking the gene Pike effectively.3.The rice plants are believed to have evolved a mechanism for generating new R gene alleles through gene duplication or diversification that allows them to adapt rapidly to a broad spectrum of novel pathogen variants.To understand the evolutionary of Pike,we investigated the allelic diversity of Pike-1 partial NBS region in 48 breeding lines.The value of Tajima's D was positive in partial NBS region(Tajima's D=2.32634*),indicating balancing selection in Pike evolution.The phylogenetic tree was characterized by two deep bifurcations that separated three clades of the Pike allelic variants.These three clades were named as Clade 1,Clade 2 and Clade 3 respectively.And a low level of sequence similarity was observed between these clades.But within the clade,more than 99%nucleotide identity value was observed.All of the seven previously cloned Pik alleles(Pike,Pik,Pil,Pikm,Piks,Pikp,Pikh)were grouped into Clade 1 in the phylogenetic tree.The genomic sequence of Pike-1 allelic variants of the 22 breeding lines from Clade 1 were sequenced and analyzed.According to three positive-selection model M8,M5 and MEC,the frequency of the positive-selection sites was higher at the junction of CC and NBS domain.For the gene Pike,its LRR domain was more conserved than its CC and NBS regions.