Identification And Mapping Of Candidate Genes Associated With Broad Spectrum And Durable Resistance To Rice Blast And Study Of Its Mechanism Of Resistance

SHZ-2, an indica cultivar with broad spectrum and durable resistance to multiple races of the blast pathogen, has been recognized as a cultivar for long time in Guangdong, China. However, it has been difficult to transfer its broad spectrum and durable resistance into other cultivars due to the poor understanding of the molecular genetic mechanism of its resistance. In order to understand its molecular genetic basis of the broad spectrum and durable resistance of SHZ-2, it was crossed to TXZ-13, a blast susceptible variety, to produce a BC3 line (BC-10) that exhibited strong to moderate blast resistance over eight cropping seasons in the field. To identify and dissect the QTL responsible for durable blast resistance, 244 BC4F3 lines were evaluated for blast resistance in the greenhouse and blast nursery. To fine mapping the QTLs, two populations of 343 F2 derived from SHZ-2×TXZ-13 and 600 F2 from SHZ-2×TXZ-13 were developed by single seed descent. A single feature polymorphism microarray, Golden Gate chips, SSR markers and gene-specific primers were used to identify the introgressions of SHZ-2 in BC-10, to decide the QTLs associated with the durable resistance to multiple races in Philippines and fine mapping the QTLs. The main results obtained are as followings:1. Chromosomal introgressions from SHZ-2 were identified by using a single feature polymorphism microarray, SSR markers and gene-specific primers. Segregation analysis of BC4F3 population indicated that three regions (QTLs) on chromosome 2, 6, and 9, designated as qBR2.1, qBR6.1 and qBR9.1 were associated with blast resistance and contributed to the reduction of diseased leaf area (DLA) by 16.2%, 14.9 and 22.3% respectively.2. We defined three QTLs using pairs of near-isogenic lines extracted from heterogeneous inbred families (HIF). Pairwise comparison of these lines enabled the dissection of the relative contributions of individual QTL. The qBR9.1 conferred strong resistance as expected because of the presence of NBS-LRR genes. Under field condition, qBR2.1 or qBR6.1 individually did not reduce disease but when combined together, they reduced disease by 19.5%, suggesting that small effect QTLs could be effective in reducing epidemics. The qBR6.1 and qBR9.1 region contains NBS-LRR sequences, whereas the qBR2.1 did not. The expression pattern of candidate genes within the QTL regions suggested functional roles of these genes in response to blast infection.3. A new major QTL of qBR9.1 conferring durable resistance to rice blast was finely mapped into a 69.1 kb region on the chromosome 9 that has been inherited from the resistant line SHZ-2 into backcross 3 lines BC-10. By annotation analysis, only one predicated disease resistance gene with NBS-LRR domain was found within this region. Using gene specific marker analysis, we found this gene co-segregated with blast resistance in F2 and F3 populations derived from SHZ-2. We tentatively designate it as Pi46(t).4. Sequence analysis revealed that the Pi46(t) encodes a nucleotide binding site and leucine-rich repeat (NBS–LRR) protein which is composed of 743 amino acid polypeptide, Pi46 was highly induced by blast infection in resistant lines SHZ-2 and BC-10.5. Three new gene specific markers, CRG4-1 CRG4-2 and CRG4-3, have also been developed, which are co-segregated with the Pi46(t) and have been used for MAS breeding and the introduction of the durable resistant gene Pi46(t) into IR64 and the development of near isogenic lines.6. To identify the genes involved in blast resistance to blast in rice and understand the molecular mechanism of plants resistance to blast, a comparative transcriptomic analysis were did among the backcross line BC-10 and its parental lines SHZ-2 and TXZ-13 using 44k microarray method.We found the number of genes responding to blast infection in resistance lines BC-10 and SHZ-2 was twice that in susceptible line TXZ-13 at 24 HAI and 48 HAI. Cluster analysis reveals that the blast resistance in SHZ-2 and BC-10 may relate to those DEGs which response to blast infection both in SHZ-2 and BC-10 at 48 HAI. The function analysis of the DEGs suggests that the JA signaling pathway plays important role in the rice resistance to blast. The expression of some of the transcription factors, such as WRKY, AP2 and ZIM were significantly up-regulated only in resistant line BC-10 and SHZ-2 by blast infection. And some of the defense related genes were activated only in resistant lines BC-10 and SHZ-2.