【作者】 陈芳；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2007， 硕士

【摘要】 小麦是世界上重要的粮食作物，运用生物技术加快小麦育种进程、提高小麦产量、改善小麦品质已是育种学家和生物技术工作者共同面临的重大使命。然而自然环境下生长的小麦，要面临各种各样不利的内外环境的胁迫，尤其是盐、旱环境。目前世界上土壤盐渍化问题相当严重，由于小麦对盐渍环境敏感，往往造成其产量和品质的大幅度下降。因此，研究植物的耐盐机理、克隆耐盐基因、培育耐盐新品种已成为全世界范围内研究的主要目标。由于小麦拥有庞大的基因组，使得分子标记技术在小麦中的应用落后于大麦、玉米、水稻等作物。但小麦基因组中含80％以上的DNA重复序列，可以利用SSR分子标记构建高密度的遗传连锁图。近年来，随着分子标记技术及检测系统的发展与完善，分子标记技术在小麦耐盐中的应用已有了很大进展。本实验是以耐盐的不对称体细胞杂交新品种山融3号为母本，盐敏感的常规品种济南17为父本，配置杂交组合，建立F₂代分离群体。在应用SSR-BSA（bulked segregant analysis）方法对F₂代分离群体耐盐性进行鉴定，结合小麦SSR图谱分析，认为该杂交组合中耐盐性状可能由一个主效耐盐基因控制，并将其定位于5A染色体上。在上述基础之上，为了进行更进一步的定位，使用重新建立的含有500单株的F₂代群体。将小麦5A染色体上的所有能查到序列的97对SSR引物对耐盐亲本和敏感亲本进行PCR扩增，筛选出17对引物扩增的微卫星DNA呈现多态性，多态性指数为17.5％。然后用这17对引物继续对耐盐池和敏感池进行PCR扩增，其中barc180，barc117，gwm304，gwm666在耐盐池中扩增出与耐盐亲本一致的条带，在敏感池中出现与敏感亲本一致的扩增带。对F₂代群体的500个单株的耐盐性状以及上述4对引物的扩增情况进行统计，并应用JoinMap3.0软件进行连锁分析，发现定位所用的4对SSR标记与已经公布的连锁图中的标记位点基本符合，只是遗传距离存在部分差异。4对SSR标记在5A染色体上的顺序为barc180，barc117，gwm304和gwm666，遗传距离为11.6cM，6.8 cM和65.8 cM。已发表的高密度连锁图的顺序与其一致，遗传距离为6.0 cM，6.0 cM，44.0 cM。JoinMap3.0软件分析得出耐盐主效基因位于gwm304和gwm666之间，距离两标记分别是30.2 cM和35.6 cM。结合小麦SSR高密度图谱分析，将该主效耐盐基因于5A染色体长臂距着丝点15-25cM处，使我们对该基因的进一步定位缩小到10cM之间。更多还原

【Abstract】 Common wheat （Triticum asetivum L.） is one of the most important cereals in the world. Therefore, it is important to accelerate wheat breeding, such as to increase its yield and quality, and to improve its stress tolerance, by using biotechnology. The edaphic salinization has turned to a very serious problem in the world recently. Thus, it is a main target in the world for scientist to work on the botanic salt-tolerant mechanisms; to clone salt-tolerant genes and to breed new salt-tolerant species. Because of the huge genome, the application of molecular marker technologies in the wheat drops is behind other crops, e.g. barley, maize and rice. Meanwhile, there are above 80% repeated DNA sequence in wheat genome, so that a high density genetic linkage map can be made by SSR molecular marks. Recently, the molecular marker technologies have a great application in the salt- tolerance of wheat because of the development of the molecular marker numbers and technologies.A novel salt-tolerant wheat variety Shanrong No.3（SR3） created by asymmetric somatic hybridization between wheat cultivar Jinan 177 and Agropyron elongatum, and salt-sensitive cultivar Jinan 17 was used as experimental material. The hybrid F₂ population between Shanrong No.3 and Jinan 17 has been constructed from sexual crossing. Salt-tolerances of the F₂ population were determined by microsatellite （SSR） and BSA （bulked segregant analysis） techniques in combination with the SSR map of wheat. It has primarily presented that salt-tolerance of this hybridization is likely controlled by a major gene which located on chromosome 5A by only one SSR locus in our early research. In this work, a F₂ population of 500 lines was constructed for further localization of the major gene of salt resistance. Among all of the 97 SSR marker pairs, 17 show polymorphism, with a polymorphic index 17.5%. Then PCR amplification has been carried out among the salt tolerance pond and sensitive pond with this 17 marker pairs. Of them, Xbarc 180, Xbarc 117, Xgwm 304, Xgwm 666 showed coherence to the parents. Analyzing by JoinMap3.0, we found that 4 marker pairs match to the linkage map published, but the genetic distances exhibit a few differences. The rank of the 4 SSR marker pairs on chromosome 5A is barcl80,barc117,gwm304 and gwm666, and the genetic distances are 11.6cM, 6.8 cM and 65.8 cM respectively, comparing with the published high density genetic linkage map with the similar rank and the genetic distances 6.0cM, 6.0 cM and 44.0 cM. And the major-gene was located on the position between gwm304 and gwm666 by software JoinMap3.0. In combination to the SSR high density genetic linkage map of wheat, the major gene for salt tolerance has been located on the 5A long arm of wheat chromosome, and the distance to the centromere is 15-25cM. This result makes us to localize the major gene in 10cM in the further study.更多还原