【作者】 倪胜利；

【导师】 李唯；

【作者基本信息】 甘肃农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 春小麦是我国北方主要的小麦种植类型，创造抗旱抗盐新种质是春小麦育种的重要目标。利用生物技术进行种质改良是目前公认的最为有效的手段和方法，其中分子标记技术不仅因为能够有效而准确地进行杂种鉴定而成为重要的辅助育种手段，而且可以通过物种多样性测定和聚类分析比较，确定标记目标基因而受到学术界广泛重视。本研究结合具体研究条件，采用A-PAGE技术和RAPD标记技术对22个不同类型春小麦与小黑麦品种进行多态性分析和聚类比较，以建立小麦分子标记辅助育种体系，为进一步开展小麦抗性改良提供研究基础和必需的技术支撑。研究结果如下：1.利用A-PAGE技术进行麦醇溶蛋白谱带分析表明，供试的22品种具有20种醇溶蛋白带型，所有带型共分离出30条蛋白带，其中26条呈现多态性，说明供试品种具有丰富的醇溶蛋白位点等位变异，品种之间存在较大的遗传差异性。聚类分析发现，遗传相似系数范围为：0.005～0.996，平均值为：0.501。供试品种在相似系数为0.501时可聚为四类，即旱作春小麦、水地春小麦、旱作小黑麦、水地小黑麦，表现出供试品种间极高的遗传多样性。2.RAPD测定稳定性研究中，分别对模板DNA、MgCl2、dNTPs、引物和TaqDNA聚合酶等5个方面进行浓度梯度筛选，最终得到一套适合且相对稳定的PCR扩增体系其具体指标为：在20μl体系中，模板DNA25ng、MgCl25.0mmol、dNTPs 4.0mmol、引物和TaqDNA聚合酶1.25U。3.利用单引物RAPD标记分析结果表明：100个随机引物中的27个扩增产物具有多态性。27个引物共扩增出156条带，其中91条带具有多态性，每个引物可扩增出1～10条多态性带，平均3.4条。RAPD标记遗传相似系数变化范围为：0.235～0.994，平均值为0.615，表明品种间在分子水平上也存在较大遗传变异，遗传多样性较高。聚类分析中，除8360的差异显著独立成类外，三个旱作品种（小红麦、会宁老芒麦、旱地大青芒）聚在了一起，两个外源品种6-90与7015也聚在一类，其它品种均根据亲缘关系远近被聚5类：第Ⅰ类：包括两亚类（7个水地春小麦与4<WP=5>个旱作春小麦）；第Ⅱ类：8360；第Ⅲ类：15-3-106与小品20-1528；第Ⅳ类：1533和两个水地小黑麦；第Ⅴ类：包括两亚类（旱作小黑麦与水地小黑麦）。4.利用双引物RAPD标记分析结果表明：20对双引物中的18对（占90％）扩增产物具有多态性。18对引物共扩增出132条带，其中69条具有多态性，每对引物可扩增出1～12条多态性带，平均3.5条。RAPD标记遗传相似系数变异范围为：0.20～0.997，平均值为0.599。聚类分析中，除类似单引物RAPD聚类分析以外，许多姊妹系品种也被区分开来（小品20-1528与小品33-1528），这表明双引物RAPD的标记遗传多样性更精确，更清晰。更多还原

【Abstract】 The spring wheat(Triticum aestivum L.) was the main cultivated type of wheat in north of China.Creating new character wheat of resistant drought and salt is the main objective of spring wheat breeding. It is the most efficient means and method that using the technology of biology improved the character. Using the molecular marked technology not only appraised cross varieties effectively, but also analysised genetic diversity. 22 different types spring wheat and triticale were carried through RAPD analysis and A-PAGE analysis in embody condition in order to establish the molecular marked-assisted selection system of wheat and offer the necessary technology in improving resistant character of wheat. The results showed as follows:1.There were 20 gliadin genotypes in the 22 cultivars by acid polyacrylamide gel electrophoresis (A-PAGE) analysis. 30 gliadin bands were separated by electrophoresis and 26 of them polymorphism (amount to 86.67%). The genetic similarity coefficient (GS) based on gliadin ranged from 0.005 to0.996, with an average of 0.501.It was showed that there was abundant aberrance in these varities. Cluster analysis showed that the studied cultivars could be classified into 4 clusters at the level of GS 0.501,they are respectively the spring wheat in dry land, the spring wheat in humid land, triticale in dry land and triticale in humid land. Obviously, the studied cultivars had abundant variation and high genetic diversity.2.Some problems about the repeat ability of RAPD, which include template concentration, MgCl2 concentration, dNTPs concentration, primer concentration and Taq DNA polymerase concentration were studied separately. A set of PCR system, which was fit for our lab and has a good repeat ability was obtained.There were templet DNA 25ng, MgCl25.0mmol, dNTPs 4.0mmol, primer10μmol and Thermas aquaticus DNA polumerase1.25U in this system.3.100 arbitrary primers (10-mers) were used for the PCR amplification of random <WP=7>genomic DNA fragments. Some 27% (amount to 27) of the primers could amplify polymorphic bands. 91 amplified bands among 156 were polymorphic. Every primer could amplify 1 to 10 polymorphic bands, with an average of 3.4 bands. The mean GS based on RAPD makers among the 22 cultivars was 0.615, with a range from 0.235 to 0.994. Cluster analysis showed that the studied cultivars could be classified into 5 clusters at the level of GS 0.618. The results showed as follows: Ⅰ:include 4 spring wheat cultivars in dry land and 7 spring wheat cultivars in humid land; Ⅱ:8360; Ⅲ: 15-3-106 and Xiaopin20-1528; Ⅳ: 1533 and two triticale cultivars in humid land; Ⅴ: triticale in dry land and triticale in humid land.4.Twenty pairs two-primer through random combined 27 arbitrary primers were used for the PCR amplification of random genomic DNA fragments. Some 90% (amount to 18 pairs) of the two-primers could amplify polymorphic bands. 69 amplified bands among 132 were polymorphic. Every primer could amplified 1 to 12 polymorphic bands, with an average of 3.5 bands. The GS based on RAPD markers ranged from 0.20 to 0.997, with an average of 0.599. Besides the majority of varieties with same consanguinity were clustered together in cluster analysis, a few of sister department varieties were divided. They were Xiaopin20-1528 and Xiaopin33-1528.It showed that the genetic variation and genetic diversity were relatively high in the studied wheat cultivars.更多还原