【作者】 石磊；

【导师】 陈秀蓉； 郑晓鹰；

【作者基本信息】 甘肃农业大学 ， 植物病理学， 2007， 硕士

【摘要】 大白菜(Brassica campestris L. ssp. pekinensis)原产我国,又名结球白菜、白菜、黄芽菜。是一种具有中国特色的大众蔬菜,我国拥有最丰富的大白菜种质资源。本论文对667份大白菜种质资源材料的抗病毒病进行评价并建立了大白菜核心种质SSR指纹库技术,旨在为大白菜种质资源的保护和利用、抗病毒病微核心种质DNA指纹库的建立以及抗病育种的亲本选择提供重要的理论依据。主要研究结果如下:抗病性评价结果:表现高抗的材料有26份,占鉴定总数的3.9%;抗病材料78份,占鉴定总数的11.7%;耐病材料205份,占鉴定总数的30.7%;感病材料222份,占鉴定总数的33.3%;高感材料份136份,占鉴定总数的20.3%。形态性状调查发现,叶片深绿色、叶脉粗的直筒型品种比较抗病。仅根据主要形态特征,并不能把抗、感品种分开,部分品种的形态相似,但抗性表现差异很大。此外,利用RT-PCR技术,通过获得包含芜菁花叶病毒(TuMV)CP基因的cDNA片段,大小约为980bp,可以对田间大白菜病样进行快速检测。建立一套适用于大白菜SSR分析标准体系:即在10μL反应体系中,1×buffer;2.50mmol/L MgCl2;0.10mmol/L dNTPs;0.35μmol/L SSR引物;1.00 ng/μL模板DNA和0.40U Taq酶。扩增程序为72℃热启动3min,94℃预变性2min后进行20个迫降循环:94℃变性60s,68℃退火60s,72℃延伸45s(-1℃/2 cycles);再进行20个循环:94℃变性60s,58℃退火60s,72℃延伸45s,于72℃延伸10min。应用SDS-聚丙烯酰胺凝胶(银染)电泳检测。利用芸薹属SSR引物数据库和NCBI-EST数据库信息得到核心SSR引物52对。研究表明:EST-SSR标记是最高效、便捷、低廉的标记。并根据27对大白菜SSR引物扩增片段范围组成不同的组合进行多重PCR反应初步研究,在单一PCR相同的反应条件下,所有组合中能够正常扩增的有27个,其中18个两重PCR、8个三重PCR和1个四重PCR组合扩增稳定,表现稳定的多重组和可以作为复合标记应用于大白菜核心DNA指纹库构建研究中。用筛选到的部分引物在5份不同栽培地区的大白菜品种间的扩增结果,初步分析了SSR标记的多态性及用于大白菜品种指纹鉴定的潜力。其中引物Ra3-D04、Ra2-G09的鉴别能力最强,单独引物就可以鉴别出供试的5个品种;引物Ra3-H10、Bn-38A分别可以鉴别出2个和3个品种。依据形态标记和SSR分子标记对20份抗性不同的大白菜品种的遗传相似性分析结果和聚类树系图表明:整个试验成对品种的GS变幅分别在0.08～0.85和0.42～0.87范围内,大部份供试品种间的遗传基础比较远;有些在形态标记上距离很远的品种,在SSR分子标记上聚在一起;大多数抗性一致的品种的亲缘关系较近,但是也有抗性差异大的品种间的GS很大,表现出高度的相似性;不同抗性品种在基于形态性状和SSR标记建立的聚类树系图中分布情况不同,抗、感品种都呈现交叉分布;品种间的遗传差异与其抗性表现的关系并不十分明显。SSR技术是分析大白菜品种亲缘关系和遗传多样性的较理想方法。更多还原

【Abstract】 Chinese cabbage (Brassica campestris L. ssp. Pekinensis) is native to China and has other names, such as heading cabbage, cabbage and huangyacai. It is rich in Chinese resources. In this study, 667 Chinese cabbage accessions were identified on the resistance to turnip mosaic virus and research on technique of simple sequence repeats for Chinese cabbage. The result of study has offered the important data materials for establishing core databases, for selecting and mating parents in the resist disease breed engineering and for rational utilization and preservation potentializing gene resources of Chinese cabbage germplasms. The results were shown as following:The result showed that 26 accessions were highly resistant (HR) to TuMV, making up 3.9% of the total accessions identified, 78 were resistant (R) to TuMV 11.7%, 205 were medium resistant (T) to TuMV 30.7%, 222 were susceptible (S) to TuMV 3.3%, 136 were highly susceptible (HS) to TuMV 20.3%. And the investigation of morphological characters indicated that some resistant accessions had the same characters, such as, deep color in leaf, wide leaf vein and straight type. It is difficult differentiate the type of resistant only based their mainly morphological characters. Some accessions had the same on the morphological characters but they had the different on the type of resistance.A simple rapid and useful method was developed and a procedure of detecting turnip mosaic virus was set up by RT-PCR. According to the published, the primer pair of coat protein gene was synthesized. The reverse transcription step was performed with total RNA extracted from infected Chinese cabbage tissue. The 980 bp nucleotide fragments were amplified, which were the same as what we had expected. And it will give us an important preparation virus diagnosis and quarantine on Chinese cabbage in the field.A standard SSR system fitting for identification of Chinese cabbage cultivars was established finally. In 10μL reaction solution, contained 1×buffer, 2.50mmol/L MgCl2, 0.10mmol/L dNTPs, 0.35μmol/L SSR primers, 1.00 ng/μL DNA template and 0.40U Taq polymerase. Amplification was carried out in two steps. After one hot starting step of 3 minutes at 72℃and one denaturing step of 2 minutes at 94℃, DNA was denatured for 60 seconds at 94℃, annealing temperature for 60 seconds at 68℃for 2 cycles and subsequently was dropped 10℃every 2 cycles until a final temperature of 58℃was reached. For the last 20 cycles of the amplification, an annealing temperature of 58℃was employed. After all the cycles, amplification was done for 10 minutes at 72℃. The SDS- polyacrylamide gel electrophoresis and silver staining was applied as the new and effective way.In this study, laborious work was conducted in primer selection, 52 SSR primer pairs with over one bands were screened from Brassica database and cabbage-EST database. After study, EST-SSR marker is most effective, convenient and low-priced then other markers. Based on this experience, 27 pairs of Chinese cabbage SSR primers were used for building various multiplex-PCR-sets according to their amplified bands’size range. Under the same conditions as single PCR reaction, 45 two-PCR-sets appeared six different amplification behaviors, of which 36 were amplified normally. 18 two-PCR-sets, 8 three-PCR-sets and 1 four-PCR-sets were amplified normally. These screened normally amplified multiplex-PCR-sets could be applied in Chinese cabbage DNA fingerprinting database.The polymorphisms of SSR marker and the potentiality of SSR fingerprint were analyzed through the screened SSR markers in 5 Chinese cabbage cultivars. Primer Ra3-D04 and Ra2-G09 were more effective and they can be identified 5 varieties separately. Primers Ra3-H10 and Bn-38A can be identified 2 and 3 varieties separately.Genetic similarity and cluster analysis based on morphological marker and SSR molecular all indicated that genetic similarities of paired varieties ranged from 0.08 to 0.85 and from 0.42 to 0.87, respectively. And genetic basis of most varieties was dissimilar. Some varieties were similar by SSR molecular, but their different on the morphological. Most of varieties were similar and has the same type to TuMV. But some resistance and susceptible varieties have highly genetic similarities, and all could cluster together based two markers. There was no remarkable relationship between genetic difference and their showed resistance or susceptible to TuMV.更多还原