水稻细胞质雄性不育基因的起源研究

The chimeric mitochondrial genes result in the cytoplasmic male sterility (CMS). mtDNA is maternal inherit for the CMS plant, thereby the CMS gene can be maintained from generation to generation after the hybridization between sterile line and maintainer line. Then the origin of the mitochondrial chimeric genes result in the CMS formed an secret, when and how did the chimeric gene resulting in CMS occur, this problem relate to inheritance and evolution of the mtDNA and the origin of the CMS source. We traced back nearly all the CMS source parentage and the related chimeric genes reported，and then foud that the distant hybridization is the main way to obtain CMS source, also both the other two ways (Spontaneous sterile plant and Somatic hybridization) are related to distant hybridization.It is interisting that the parents used to creat CMS source by distant hybridization is normal phenotype. Therefore that the CMS genes exist before distant hybridization or arise during hybridization is the key for the origin of the cytoplasm.CMS genes have been screened respectively from sunflower,radish. None in full accord with CMS gene sequence was found.The CMS gene will be screened from the local old varieties and three different species wild rice distributing in china by the primer (designed aiming to rice CMS gene) and sequence base on population genetics principle, and then the frequency among the population will be statisticed too. Once the CMS gene was found, the revelent restorer gene will be screened in the relevant individual and population, and the maintain of the CMS gene problem will be discussed.The distant hybridization between the individual carrying the CMS gene and the individual without CMS gene,and the distant hybridization between the individual both without CMS gene will be porformed respectively to create the new CMS source, and then verify the origin of the CMS gene base on comprehensive comparison the CMS gene locus between prior to hybridization and after distant hybridization. The chimeric mitochondrial CMS-associated genes downloaded from the Genebank will be analysised by TOPALi，EMBOSS for interpreting the molecular mechanism of the chimeric mitochondrial gene occurring. The molecular theory of CMS sterile line breeding and the molecular mechanisms of the chimeric mitochondrial occuring will be completed once this project was carried out.

远缘杂交是产生细胞质雄性不育胞质的三种途径（天然不育株、远缘杂交、体细胞杂交）中的主要途径。不育基因几乎都是嵌合的线粒体DNA，它们是在杂交前就存在还是杂交时产生？这个问题是不育胞质起源的关键所在。拟通过居群遗传学原理，广泛采集分布于我国的野生稻及地方老品种，用已设计好的多个水稻不育基因引物扩增并测序来检测不育基因在野生稻群体及老品种中是否存在,并统计及其出现的概率；通过具、不具不育基因个体间及都不具不育基因个体间的多对远缘杂交创造新不育胞质，再联合杂交前后mtDNA比较，验证不育基因的起源；用TOPALi，EMBOSS等针对嵌合基因的分析软件、系统分析Genebank中细胞质雄性不育基因及与不育密切相关的嵌合mtDNA序列是否存在特异性嵌合点，或其它嵌合机制，以期解释不育基因产生的分子机理。本项目对细胞质雄性不育系育种分子理论的完善、嵌合线粒体DNA产生的分子机理的完善具有重要意义

水稻细胞质雄性不育基因是杂交水稻应用的基石，不育基因起源的研究是研究线粒体DNA遗传与进化及与核DNA互作的重要切入点。本项目采集24个野生稻居群，共561个个体，分布于5个省。（1）用配子体不育基因及与配子体不育基因共转录的B-atp6特异性引物分别对每个个体检测，仅在5个居群中分别检测到扩增产物，能检测到配子体不育基因特异性引物扩增产物的个体均能检测到B-atp6引物的扩增产物，2对引物均在每个居群分别随机选取15个个体扩增产物进行测序，YJ居群仅有5个个体，对5个个体均分别进行测序。 配子体不育基因变异序列共形成5种单倍型，5种单倍型对应氨基酸序列之间及与配子体不育基因对应氨基酸序列之间互不相，对配子体不育基因及B-atp6的变异序列组成7种单倍型，AMOVA联合分析显示5个居群间存在较强的遗传分化，居群间遗传分化系数FST为0.86816，居群间的变异占86.82%，居群内的变异占13.18%。对配子体不育基因变异序列的分析及联合B-atp6的联合分析均验证红莲型不育胞质源自我国海南，与红莲型不育基因产生的实际情况相吻合；胞胎型不育基因胞质也可能存在于我国大陆南部，配子体不育胞质可能源自我国南部热带与亚热带气候交替的区域，不育基因可能是特殊气候及地理条件下的产物。实验还证明对Batp6的检测不能替代配子体不育基因的，其同源区段存在差异。携带类似配子体不育基因的居群结实率变化较大: 0%-80%；花粉育性正常，仅检测一株为半不育。（2）仅在24个居群的2个居群中检测到孢子体不育基因orf352的高同源区段，在另外2个居群中检测到孢子体不育基因AY295770高同源区段，其中来宾居群仅有一个个体携带AY295770的变型。孢子体不育基因也可能来源于热带亚热带气候交替的区域。（3）收集34个水稻地方老品种，每个品种分别提取10株，共340个个体检测不育基因。西盟的地方老品种中检测到orf352约600bp的同源区段，其它地方老品种中均未检测到不育基因的同源序列。杂交实验证明了不是所有的远缘杂交都会产生不育胞质。追踪亲本及子代的线粒体DNA的实验证明线粒体DNA在远缘杂交时确实存在子代个体与母本在某些位点上发生了变异，子代个体间存在多样性的情况。（4）收集到特殊材料——彝良红花米，该材料携带3个不育基因，但在3月底播种的基本能正常结实，在4月底播种则结实率明显下降(