【作者】 黄琼；

【导师】 李冠；

【作者基本信息】 新疆大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 甜瓜(Cucumis melo L.)是新疆有特色的重要经济作物,新疆甜瓜以其特有的优良风味和品质享誉国内外,且种质资源丰富。对于新疆甜瓜生产来说,选育优良品种是最重要的环节。传统的常规育种方法育种周期长且工作量大,不能满足目前生产需要。而分子标记技术的产生和应用为加快育种进程提供了新的思路,利用分子标记技术对重要的农艺性状进行标记可更为准确快速,并对育种和基因克隆提供理论依据。建立合适的分子标记技术及辅助育种平台,对利用分子标记辅助育种,构建遗传图谱以及基因的定位克隆都具有重要的意义。目前国内在对甜瓜农艺性状进行分子标记方面的研究较少,本研究以新疆甜瓜为研究对象,通过对AFLP(扩增片段长度多态性)标记技术中的几个关键参数进行优化,建立了成熟的适合新疆甜瓜的AFLP技术体系,并初步筛选了果实绿色条纹性状的AFLP标记。主要研究结果如下:1.通过反复试验,采用改进后的CTAB法提取的新疆甜瓜基因组DNA质量好,符合AFLP反应要求;2.通过对酶切时间及各步扩增中模板的稀释倍数等进行研究,建立了适于新疆甜瓜分析的AFLP银染技术体系。双酶切必须完全彻底,基因组DNA300ng用EcoR I和Mse I各3U,37℃酶切5h;连接产物稀释10倍用于预扩增;预扩增产物的稀释75倍后进行选择性扩增;6%变性聚丙烯酰胺凝胶电泳和银染法检测多态性;3.利用64对选择性引物组合对两亲本新疆甜瓜自交系k4-13和k4-15进行AFLP多态性分析。64对引物组合扩增条带总数为3622条,其中多态性带881条,占总条带数的24.3%。本研究筛选出扩增带型清晰,多态性丰富的引物组合14对;4.以新疆甜瓜自交系k4-13和k4-15配置杂交组合,构建F2代分离群体,通过AFLP-BSA技术初步筛选到果实绿色条纹性状的6个AFLP标记。本研究填补了新疆在甜瓜分子标记研究方面的空白,为新疆甜瓜分子标记辅助育种创建了一个重要的技术平台,对新疆甜瓜优质品种选育具有指导意义,这将为新疆甜瓜功能基因的克隆和应用、分子遗传图谱的构建以及遗传多样性的研究奠定坚实基础,从而加快新疆甜瓜分子育种的进程,具有重要的理论意义与应用价值。更多还原

【Abstract】 Melon (Cucumis melo L.) is distinctive as a major economic crop in Xinjiang. Xinjiang melon enjoys a good reputation by its unique flavor and excellent quality in the world. Xinjiang has rich germplasm resources. The most important link in the production of Xinjiang melon is breeding fine varieties. Traditional breeding methods have long cycle and heavy workload, and can not meet the present production needs. But the formation and applications of molecular marker techniques provide a new way of thinking for speeding up the breeding process. It will be nicety and speediness using molecular markers linked to the genes of important agricultural traits, and it will help the work of breeding and gene cloning. The establishment of molecular marker techniques which are appropriate for Xinjiang melon and platform of molecular marker-assisted breeding has significance for breeding, genetic map constructing, gene localizating and gene cloning.Nowadays, the researches are less about molecular markers for agricultural traits in melon. In this paper, an AFLP (amplified fragment length polymorphism) analysis system for Xinjiang melon was established by optimizing some key factors, and then AFLP markers linked to green stripe traits of fruit in Xinjiang melon were screened preliminary. The main results were as follows:1. Via repeat experiments, the modified CTAB could be used for extracting genomic DNA which has high quality and is suitable for AFLP.2. By the research of digestion time and dilution times of each step, the AFLP silver staining analysis system for Xinjiang melon was established. The double enzyme digestion should be complete, 300ng genomic DNA was digested with 3U of EcoR I and 3U of Mse I at 37℃for 5 hours; the digested-ligated DNA fragments were diluted (10 folds) then used as templates for pre-amplification, and the products of pre-amplification should be diluted 75 times then used as templates for selective amplification. This analysis detected polymorphism with 6% denaturing poly-acrylamide gel electrophoresis and silver staining. 3. This study has analyzed AFLP polymorphism of two lines (k4-13 and k4-15) by using 64 pairs of selective primer combinations. By statistic there were 3622 bands in which there were 881 polymorphism bands. The rate of polymorphism was 24.3%. The result showed that 14 pairs have produced clear bands and represented rich polymorphism.4. Screening AFLP markers were studied among F2 segregated population originating from a cross between k4-13 and k4-15. Six AFLP markers linked to green stripe traits of fruit in Xinjiang melon were acquired by using BSA. This research has supplyed a gap of molecular markers for melon in Xinjiang, and has founded an important technology platform for molecular marker-assisted breeding in Xinjiang melon, has guiding significance to Xinjiang melon breeding. This would have laid a foundation for the clone and application of functional genes, the construction of genetic map and the research of genetic diversity. So it would be able to speed up the molecular breeding process and have had theoretical significance and important value.更多还原