【作者】 蒋云林；

【导师】 吴为人；

【作者基本信息】 福建农林大学 ， 作物遗传育种， 2008， 硕士

【摘要】 在前人研究的基础上,对利用分子标记技术培育持久抗瘟水稻品种进行了研究:1)开发新的基因内标记;2)检测65个亲本间的SSR多态性,建立抗源与亲本间多态性数据库;3)利用分子标记辅助育种的方法将广谱、完全显性的抗稻瘟病基因Pi-1、Pi-2、Pi-9和Pi-K^h导入杂交稻亲本Ⅱ-32B和花紫B中,培育出导入单个或多个抗稻瘟病基因的近等基因系;4)对多重PCR（multiplepolymerase chain reaction,MPCR）技术进行优化和应用。主要结果如下:1、通过比对Pi-9基因序列、日本晴和93-11基因组序列,找到1个聚集7个SNP与一个indel的区段。根据片段长度差异等位基因特异PCR（FragmentLength Discrepant Allele Specific PCR,FLDAS-PCR）的原理,在该区段设计了Pi-9基因内FLDAS反向引物FP1和FP2及通用正向引物RP。通过PCR检测表明,不同SNP纯合子为长度不同的单一谱带,杂合子则为两条带,其结果与设计完全一致。用BC₃F₂群体进行连锁分析表明,该SNP标记与Pi-9基因没有交换,说明用其对Pi-9基因进行选择是有效的。2、抗源与杂交稻骨干亲本间的多态性数据库建立。1)应用SSR标记检测了抗源与骨干亲本间的多态性,初步建立了这些亲本的SSR数据库,79个标记均能在亲本间揭示多态性。其中7个与抗源紧密连锁的标记（PTA248、SRM231/RM206、RM206、MRG4766、AP22和SRM24/AP22）可以分别作为抗病基因Xα-21、Xα-23、Pi-k^h、Pi-1、Pi-9和Pi-2前景选择的首选标记;47个SSR标记具有高PIC值（＞0.5）且较均匀地分布在整个基因组中,推荐作为背景选择的首选标记。2)分别以SSR标记和18个表型性状为指标,对65个供试材料进行了聚类分析。利用SSR分子标记将供试材料划分为籼、粳两大类群,9个亚群,籼稻群主要包含了恢复系亚群和保持系亚群,粳稻群主要包含了北方与南方粳稻亚群。表型聚类的划分结果与SSR标记聚类结果基本相同。3、通过标记辅助回交育种,分别将Pi-1、Pi-2、Pi-9和Pi-K^h基因导入到受体亲本中,并通过近等基因系间杂交,将Pi-1和Pi-2、Pi-1和Pi-9、Pi-1和Pi-k^h、Pi-k^h和Pi-9以及Pi-k^h和Pi-2分别聚合到同一品系中,还获得了三个抗性基因均纯合的累加系。1)将Pi-1基因导入Ⅱ-32B和花紫B中,分别得到8个和22个携带Pi-1基因的Ⅱ-32B和花紫B近等基因系及其对应的不育系;2)将Pi-2基因导入Ⅱ-32B中,得到携带Pi-2基因的Ⅱ-32B近等基因系5个及其对应的不育系;3)将Pi-9基因导入Ⅱ-32B和花紫B,得到携带Pi-9基因的Ⅱ-32B近等基因系8个及其对应的不育系以及花紫B近等基因系12个;4)将Pi-k^h基因导入Ⅱ-32B中,得到携带Pi-k^h基因的Ⅱ-32B近等基因系8个及其对应的不育系;5)通过将含不同抗性基因的Ⅱ-32B近等基因系两两杂交和复交,获得了Pi-2Pi-2Pi-1Pi-1抗性单株8株,Pi-1Pi-1Pi-k^hPi-k^h抗性单株12株,Pi-1Pi-1Pi-9Pi-9抗性单株3株,Pi-2Pi-2Pi-k^hPi-k^h抗性单株8株,Pi-9Pi-9Pi-k^hPi-k^h抗性单株4株,Pi-9Pi-9Pi-1Pi-1Pi-k^hPi-k^h的聚合系8个。将含不同抗性基因的花紫B近等基因系两两杂交,获得Pi-9pi-9Pi-1pi-1抗性单株5株。同时利用田间抗病鉴定与室内接菌显示,三基因聚合系抗性明显强于双基因聚合系和单基因株系,双基因聚合系抗性则强于单基因株系,说明基因聚合后抗谱增宽、抗性加强,是培育稻瘟病持久抗性品种的有效方法。4、优化了SSR标记的MPCR反应体系,将其用于Pi-1和Pi-2基因的聚合选育,能对Pi-1和pi-1、Pi-2和pi-2进行准确的分型,成功检测到含Pi-2pi-2Pi-1pi-1的单株5株;并且通过59对SSR引物构建4重PCR引物组合15组,对金抗1B的遗传背景分析结果表明,它与金23B的遗传相似系数为0.93,与单一PCR的扩增结果基本一致（r=0.99）。更多还原

【Abstract】 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 parentsⅡ-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 ofⅡ-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 ofⅡ-32B and test crossing sterile lines carried Pi-2 gene, which 8 near-isogenic lines ofⅡ-32B and test crossing sterile lines,12 near-isogenic lines of HuaziB carried Pi-9 gene,which 8 near-isogenic lines ofⅡ-32B and test crossing sterile lines carried Pi-k^h gene.TheⅡ-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）.更多还原