【作者】 邵安；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2010， 硕士

【摘要】 世界上土壤盐渍化问题十分严重,而中国的土壤盐渍化更为突出。小麦是世界上重要的粮食作物,但它对盐渍环境敏感,土壤盐渍化已造成其产量和品质的大幅下降。因而,研究小麦耐盐机理、克隆小麦耐盐基因,并运用生物技术加快小麦育种进程、提高小麦产量、改善小麦品质显得格外重要。20世纪以来,各种DNA分子标记技术相继建立,并成功应用于基因图位克隆和分子标记辅助育种。但小麦是六倍体,基因组庞大,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。普通小麦起源的相对较近和它的多倍体性质既给其遗传研究带来了问题,同时也带来了特殊的机遇。本实验前期工作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,并利用SSR-BSA法初步将山融三号耐盐主效基因(QTL)定位于5A染色体长臂分子标记xgwm304与xgwm666之间。本研究通过两端分子标记辅助选择建池,并对更多的SSR、EST-SSR进行筛选及耐盐性相关分析,发现了6对差异引物,包括WMS410和Xcfa2141两个远端连锁引物。结合所有差异引物进行连锁分析,并整合到最新公布的小麦5A SSR连锁图谱,得出目的基因相对准确的位置,发现主效基因大致位于5A染色体长臂12-0.35-0.57缺失段处。利用区段内已知EST序列进行区段内差异STS引物的设计及筛选,分离出相关候选基因,以便于进一步研究。在本实验结果的基础上,可以继续从水稻、短柄草的对应EST开发设计引物,利用即将构建完成的重组自交系群体及回交群体,可对基因进行进一步精确定位,缩短耐盐相关分子标记与基因之间的距离,循环饱和目标区段直至逼近耐盐基因,最终期望完成耐盐主效基因的克隆。更多还原

【Abstract】 Solid degradation, especially aridity and edaphic salinization, has been a very serious problem around the world. Common wheat(Triticum asetivum L.),one of the most important cereals, is very sensitive to salt-stress.Therefore, it is important to breed new salt-tolerance wheat cultivars by biotechnological methodologies. This urges us to elucidate the botanic salt-tolerant mechanisms as well as to map and clone salt-tolerant genes based on molecular marker technologies.Because of its hexaploidy and huge genome, the application of molecular marker technologies in wheat is behind other crops, e.g. barley, maize and rice. Recently, however, such application in the salt-tolerance of wheat has been largely accelerated following the development of the molecular marker numbers and technologies examination systems.A novel salt-tolerant, drought-resistant and high productive wheat variety Shanrong No.3(SR3)was generated via asymmetric somatic hybridization between common wheat Jinan 177 and Agropyron elongatum in our lab.Specifically, several pieces of chromatins from Argopyron elogatum were intrgressed into the Jinan 177 genome, which may offer the excellent traits of SR3.In our early research, salt-tolerance of the F2 population originating from a cross between SR3 and Jinan17, a salt-sensitive wheat cultivar, 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 SR3 is likely controlled by a major QTL which locates on chromosome 5AL between markers xgwm304 and xgwm666.In this work, in order to gain a higher resolution mapping of the major QTL governing SR3’s salt-tolerance, F2 population with 340 lines of SR3 and Jinan 17 was re-constructed.Besides, we improved the BSA techniques with molecular marker assisted selection. Genetic and SSR, EST-SSR, STS analysis were performed among individuals in F2 segregated population, using 59 SSR marker pairs,83 EST-SSR marker pairs and 31 rice SSR marker pairs. Among all of these marker pairs,27 showed polymorphism, with a lymorpolymorphic index of 15%.Then PCR amplification was carried out among the salt-tolerant pond and salt-sensitive pond with these marker pairs.Of them, Xcfa2141,WMS410, xgwm304, xgwm666, BE-5 and TC245679 showed coherence to the parents.Comparing with the high density genetic linkage map published, the major-QTL was located on the position of 5AL12-0.35-0.57 with the same similar rank and the genetic distances calculated by software Mapmaker 3.0.Based on this result, we searched all EST sequences in this interval and designed 96 STS primers. By the same PCR amplification, we found some polymorphic marker pairs which are related with salt-tolerance. Then EST sequences amplified with these polymorphic marker pairs were analyzed, and some of them were found to be salt-tolerance candidate genes for further study. Summarily, this work provides a solid foundation for the next map-basic cloning.更多还原