【作者】 陈晓红；

【导师】 胡宝忠； 牛永春；

【作者基本信息】 东北农业大学 ， 植物学， 2003， 硕士

【摘要】 小麦条锈病是一种世界性病害，严重威胁着小麦生产。在抗病育种中应用分子标记辅助选择有利于实现抗条锈病多基因积累，加速持久抗病品种的培育进程。 以小麦抗条锈病基因Yr5的供体亲本Triticum spelta album作为对照，对近等基因系Yr5／6×Avocet S和感病亲本Avocet S进行RAPD分析。扩增产物用4％变性PAGE分离，银染显色。可以检测到50～100条带，是琼脂糖凝胶电泳的5倍以上。本研究筛选了240个随机引物，发现23条稳定的多态性DNA片段。可见，该方法显著提高了小麦RAPD分析的多态性水平。而且，由于PAGE能够稳定检出低拷贝的扩增片段，也改善了RAPD分析的重复性。 对23条多态性DNA片段与Yr5基因的遗传连锁性进行初步检测，确定了分别由5条引物扩增的S1320₂₀₇、S1348₃₆₃、S1476₃₉₅、S1476₄₀₁、S1397₈₃₀、S1380₃₅₉共6条多态性片段与Yr5基因有连锁性。将其中连锁性好的多态性片段S1320₂₀₇、S1348₃₆₃、S1476₄₀₁进行克隆、测序，并根据序列设计特异性引物。用121株Avocet S和Yr5／6×Avocet S杂交制备的F₂代分离群体进一步进行遗传连锁性检测。特异扩增产物SC-S1320₁₅₆与Fr5基因完全连锁；标记SC-S1348₃₅₇与Yr5基因紧密连锁（交换率0.008），表明已经成功转化为Yr5基因的SCAR标记。 对小麦抗条锈病基因Yr5的近等基因系材料，提取接种后12h、24h、48h叶片的总RNA，制备三个时间段的cDNA模板。根据已克隆R基因普遍具有的NBS结构域的P-loop、Kinase-2a、Kinase-3a、EGF区设计5条RGA简并引物，组成5对引物组合。用相同的模板与引物，在低严谨条件下扩增，获得4条多态性片段；在高严谨条件下扩增，发现1条多态性片段D-4。将引物组合N₁-E₁扩增抗病和感病材料的产物分别回收作为模板，用引物组合K_2a-E₁和Touch down程序再扩增，检测到1条550bp左右差异DNA带，记为RD-1；将引物组合K_2a-E扩增抗病和感病材料的产物分别回收作为模板，用引物组合K_3aS-E₁和Touch down程序再扩增，检测到1条470bp左右差异DNA带，记为RD-2。用巢式PCR两次扩增目的DNA片段，提高了特异性，更有利于目的DNA片段检出。更多还原

【Abstract】 Stripe rust (yellow rust), caused by Puccinia striiformis West. f. sp. tritici, is one of the most important diseases on wheat throughout the world. It is an effective way to breed resistant cultivars conferring major resistance gene by maker-assisted selection, which would accelerate breeding program.RAPD analysis was performed between a near-isogenic line (NIL,) 7r5/6+Avocet S carrying the resistance gene Yr5 against wheat stripe rust and its susceptible parent Avocet S, using the YrS gene donor parent Triticum spelta album as control. Amplified DNA fragments were separated with 4% denaturing PAGE (polyacrylamide gel electrophoresis) and displayed by silver staining. Fifty to 100 bands were detected, 5 folds more than that revealed on agarose gels. A total of 240 random primers were screened, and 23 reproducible polymorphic DNA fragments were found. The results suggested that using denaturing PAGE-silver staining could remarkably increase the level of DNA polymorphism detected in wheat, and could improve the repeatability of RAPD analysis because of effective detection of low copy DNA fragments.Of 23 polymorphic DNA fragments, 6 fragments (S1320207, S1348363, S1476395, S1476401, S1397830 and S1380359) respectively derived from 5 primers, were confirmed to be linked to Yr5 gene by preliminary genetic linkage analysis. The fragments S1320207, S1348363 and S1476401, closely Gnked to the target gene, were recovered from polyacrylamide gel and cloned in pGEM-T easy vector, then sequenced from two ends. According to the sequences, two pairs of specific primers were designed. Genetic linkage between the PCR products and the target gene was tested on 121 segregating F2 plants derived from a cross between Avocet S and YrJ/6+Avocet S. It was shown that the polymorphic DNA fragment SC-S1320156 was completely linked to Yr5 gene, and SC-S1348357 was closely linked to Yr5 gene.The total RNA was extracted from wheat leaves respectively collected 12h, 24h and 48h after inoculation and cDNA were prepared. Five degenerated primers were designed according to the P-loop, Kinase-2a, Kinase-r3a, EGF domain of NBS which universally exist in cloned R genes. PCR amplifications were performed with the designed RGA primers and cDNA as templates. Four polymorphic DNA fragments were obtained when anneal temperature was low, only one polymorphic DNA fragment D-4 was found with the same templates and primers when annealtemperaturewas high.PCR products from primer pair N1-E1 were excised from PAGE and used as template. Reamplification was performed with primer pair K2a-E1 and Touch Down PCR was employed. Aspecific DNA band of 550bp was detected, named as RD-1. PCR products from primer pair K2a-E1 were excised from PAGE and used as template. Reamplification was performed with primer pair K3a-E1 and Touch Down PCR was employed. A specific DNA band of 470bp was detected, named as RD-2. Both RD-1 and RD-2 had expected molecular weight. Employing semi-nest primer to amplify target fragment twice, it could improve specificity and be in favor of detecting target fragment.Postgraduate: Chen Xiaohong Mjaor: Plant Molecular BiologySupervisor: Prof. Hu Baozhong Prof. Niu Yongchun更多还原