Studies On Breeding Of Parents Of Hybrid Rice With Durable Disease Resistance Using Molecular Marker-assisted Selection

On the basis of previous studies,breeding of rice varieties with durable disease resistance using molecular markers was studied:1)new intra-genic markers were developed;2)the SSR polymorphisms among 65 cultivars were surveyed and a database of polymorphisms between hybrid rice parents and resistance gene donors was established;3)dominant rice blast resistance genes Pi-1,Pi-2,Pi-9 and Pi-K^h with broad spectrums were introgressed into hybrid rice parentsâ…¡-32B and HuaziB by marker-assisted selection and near-isogenic lines carrying single or multiple blast resistance genes were created;4)the technology of multiple polymerase chain reaction (MPCR)was optimized and applied in the breeding program.Main results are as follows:1.A segment containing seven SNPs and one indel was identified by comparing the Pi-9 gene sequence with the genome sequences of cultivars Nipponbare and 93-11. According to the principle of FLDAS-PCR,two forward primers FP 1 and FP2 and one shared reverse primer RP were designed using the segment as template.PCR analysis detected single bands with different sizes in the two homozygous SNP genotypes and two bands in the heterozygous SNP genotype as expected.Linkage analysis using a BC₃F₂ population showed that there was no recombination between this SNP marker and Pi-9 gene,suggesting that selection of Pi-9 gene with the help of this marker would be efficient.2.Establishment of the polymorphism database of hybrid rice parents and resistance donors.1)The polymorphisms between the main parents and anti-source are surveyed by molecular markers,initially establish the parents SSR database,79 pairs of primers can be used to reveal the polymorphism between of the parents,in which seven primers (PTA248,SRM231(RM206),RM206,MRG4766,FPI+FP2+RP(AP22)and SRM24 (AP22))that closely linked with the anti-sources Xa-21,Xa-23,Pi-k^h,Pi-1,Pi-9 and Pi-2 are recommended for the preferred choice markers firstly,in which 47 primers with high value of PIC(>0.5)and more evenly distributed throughout the genome,are recommended for genetic background selection markers for breeders.2)Two methods of cluster analysis based on phenotypic characters and SSR molecular markers were compared to study the diversity of 65 the test material,while on the basis of SSR cluster analysis,the entries were classified into two groups(i.e. Indica and Japonica two groups)and nine sub-groups.The Indica group consisted of maintainer line group and restorer line group.The Japonica line group consisted of the north and the south Japonica subsets,while the result which based on phenotypic character cluster analysis is basically the same with the result based on SSR markers clustering.3.Pi-1,Pi-2,Pi-9 and Pi-K^h genes are introgressed to receptor parents by marker-assisted backcross breeding,and respectively polymerize Pi-1 and Pi-2,Pi-1 and Pi-9,Pi-1 and Pi-k^h,Pi-k^h and Pi-9,Pi-k^h and Pi-2 to the same species by near-isogenic lines which were crossing respectively.We also can access to the pyramid line which contain three homozygous resistance genes at the same time,so it is respectively obtained which 8 near-isogenic lines ofâ…¡-32B and test crossing sterile lines,22 near-isogenic lines of HuaziB and test crossing sterile lines carried Pi-1 gene, which 5 near-isogenic lines ofâ…¡-32B and test crossing sterile lines carried Pi-2 gene, which 8 near-isogenic lines ofâ…¡-32B and test crossing sterile lines,12 near-isogenic lines of HuaziB carried Pi-9 gene,which 8 near-isogenic lines ofâ…¡-32B and test crossing sterile lines carried Pi-k^h gene.Theâ…¡-32B near-isogenic lines carrying different resisting genes were crossing respectively.8 individuals carrying Pi-2Pi-2Pi-1Pi-1,12 individuals carrying Pi-1Pi-1Pi-k^hPi-k^h,3 individuals carrying Pi-1Pi-1Pi-9Pi-9,8 individuals carrying Pi-2Pi-2Pi-k^hPi-k^h,4 individuals carrying Pi-9Pi-9Pi-k^hPi-k^h were obtained respectively,and also 8 pyramid lines carrying Pi-9Pi-9Pi-1Pi-1Pi-k^hPi-k^h were obtained.And the HuaziB near-isogenic lines carrying different resisting genes were crossing respectively,we obtain 5 individuals carrying Pi-9pi-9Pi-1pi-1.These NILs which contain resistance gene were identified by inoculating Magnaporthe grisea isolates in the field/greenhouses,the result shows that the resistance of pyramid lines contained 3 resistance genes are stronger in the pyramid lines of double and single-gene,the pyramid lines of double-gene are stronger than single-gene,it implies that gene pyramiding can increase the spectrum and the strength of resistance to fungi blast.It can be concluded that gene pyramiding is an effective approach for the improvement of rice varieties with durable resistance to blast.4.Optimize the reaction system of MPCR in SSR primers,and make it sue to the polymerization of Pi-1 and pi-1 breeding.Successfully detecte 5 individuals carrying Pi-2pi-2Pi-1pi-1.And make up 15 groups which contain four PCR primer combinations through 59 pairs SSR primer analysis of results showed that the genetic background of Jinkang1B,its genetic similarity coefficients is 0.93 with the Jin23 B's. The result is the same with the result of PCR amplification with a single(r=0.99).