【作者】 王士伟；

【导师】 张明方；

【作者基本信息】 浙江大学 ， 蔬菜学， 2010， 硕士

【摘要】 甜瓜链孢菌性枯萎病（Fusarium oxysporum f.sp.Melonis,Fom）是由尖孢镰刀菌（Fusarium oxysporum f.sp.melonis Snyder and Hansen）导致的一种最难控制的土传病害,成为影响甜瓜品质和产量的主要因素。本研究以两个厚皮甜瓜（Cucumis melo）品种为实验材料,分别为美国甜瓜（Cantaloupe）和仙果027-5（xianguo 027-5）。通过基因克隆和序列比对获得3个单核苷酸多态性位点。在它们的F₁与F₂代群体中,采用限制性内切酶酶切（CAPS）和等位基因特异性PCR（AS-PCR）两种DNA分子标记验证这些单核苷酸多态性位点的有效性和可靠性,进而将验证的标记转化为功能性分子标记,应用于常规品种的筛选。我们克隆得到了Fom-2 LRR区DNA序列,长为1311 bp。采用生物信息学软件对其进行比对分析,发现高抗和高感枯萎病材料的第281、1076和1216碱基处存在3个单核苷酸多态性位点,并且这些碱基的突变导致了相应氨基酸的改变。以高抗和高感枯萎病材料杂交得到的F₁（30株）与F₂（100株）代群体为研究材料,采用限制性内切酶酶切方法对其单核苷酸多态性位点进行分析。筛选得到特异性的扩增引物对CAPS-2F与CAPS-2R,CAPS-3F与CAPS-3R,特异性PCR后经限制性内切酶酶切,结果发现SNP2与SNP3两个位点在F₂群体中纯合抗病类型、杂合抗病类型与纯合感病类型呈现1∶2∶1的分离比例,符合孟德尔单基因遗传学规律,成功将CAPS标记转化为功能性分子标记。依据CAPS方法获得的结果,我们设计筛选得到AS-PCR特异性引物2-3F1与2-3R1引物对,在退火温度为62℃,35个循环条件下进行特异性PCR,F₂群体中抗性材料和感病材料呈现3∶1的孟德尔单基因分离比例。因此CAPS和AS-PCR两种方法都证明了SNP2与SNP3的有效性与可靠性。采用CAPS与AS-PCR方法对34份常规栽培品种进行分析,筛选得到2个纯合枯萎病抗病材料和9个杂合抗病材料,为甜瓜枯萎病抗性育种提供了良好的种质资源。更多还原

【Abstract】 Fusarium wilt of melon （Fusarium oxysporum f. sp. Melonis, Fom） caused by the fungus Fusarium oxysporum f. sp. melonis Snyder and Hansen, has become one of the most destructive diseases of melon. It severely limited the melon yeild production and quality. This research focused on the development and utilization of the SNP （single nucleotide polymorphism）-based FM associated with resistance of fusarium wilt in melon. The DNA moleculer marker, CAPS （Cleaved amplified polymorphic sequences, CAPS） and AS-PCR （Allele-specific PCR, AS-PCR）, were employed to validate the effectiveness and reliability of the markers in F₁ and F₂ population, which derived from a cross between Cantaloupe and xianguo 027-5. Then the candidate markers were converted into the functional marker and used in the selection of cultivars.A 1311bp leucine-rich repeat （LRR） region of the Fom-2 gene was cloned. Three single nucleotide polymorphism （SNP） sites were found and located at 281st, 1076th and 1216th of the cloned LRR region, respectively. These three single nucleotide polymorphism sites were analysed by CAPS method in F₁ （30 plants） and F₂ populations （100 plants） derived from cross between wilt resistant and susceptible germplasms. According to CAPS method, we used CAPS-2F and CAPS-2R, CAPS-3F and CAPS-3R as two pairs of specific primers to amplify fragments of interest, then the PCR products were digested by EcoR I and Xba I. The result showed the 1:2:1 Mendelian single-dominant segregation pattern for site 2 and 3 in the F₂ population. The CAPS makers were sucessfully converted into the fuctional markers. As for the AS-PCR method, the specific primers 2-3F1 and 2-3R1 were used to amplify fragments of interest. When the annealing tempreture was 62℃and cycle number was 35, it could be easily distinguished the resistant and susceptible plants. The genetic segregation ratio in F₂ population was 3:1 among the resistant and susceptible plants. The results of CAPS and AS-PCR methods showed that site 2 and 3 were reliable and effective.The further screening of 34 cultivars were conducted by CAPS and AS-PCR markers. Among the 34 tested varieties, 11 were identified as resistant genotypes including 2 homozygous and 9 heterozygous genotypes. These resistant plants can be used as good germplasm resources in resistant breeding.更多还原