【作者】 张景荣；

【导师】 刘军；

【作者基本信息】 四川农业大学 ， 森林培育， 2006， 硕士

【摘要】 本研究选取具有一定的地理代表性、观赏价值极高的名贵茶花品种为试材，摸索适合茶花基因组DNA的提取方法，筛选用于种质鉴定的合适引物并构建指纹图谱，同时进行种质资源遗传多态性、亲缘关系的分析，探讨了RAPD用于茶花品种资源分析的可行性，获得了以下结果： 1．采用改进的CTAB法抽提茶花基因组DNA，研磨时加入了适量PVP粉末，在2％的CTAB抽提液中临时加入了1％β-巯基乙醇，以防止了酚氧化成醌，避免了褐变；用CTAB/NaCl溶液去除多糖等措施，经A₂₆₀和A₂₈₀比值的测定，以及电泳检测和PCR扩增，结果表明改进的CTAB法所提取的DNA无论在纯度上还是在在完整性上都比其它方法要好。 2．用正交设计法建立了茶花RAPD-PCR扩增体系：反应总体积25μl，模板DNA80ng，10碱基随机引物的浓度为0.3μmol/L，dNTPs的浓度为0.2 mmol/L，MgCl₂的浓度为2mmol/L，Taq-DNA聚合酶1U。PCR扩增程序为：94℃预变性5 min，94℃变性50s，36℃退火50s，72℃延伸90s，共40个循环，最后72℃延伸8min。 3．从100个随机引物中筛选出的20个有效引物共产生136条DNA片段，大小分布在0.25～2.0Kb之间，遗传多态性带120条占总数88.2％，每个引物扩增的DNA带数平均为6.8条。23个茶花品种的遗传相似性分析表明，各基因型间的Nei氏相似系数分布在0.4386～0.8936之间，平均相似性系数为0.7668。 4．通过非加权算术平均数聚类（UPGMA）法，绘制了23个茶花品种的遗传关系树状图。当以相似系数0.75为分割线时，23个品种可划分为4个类群。类群A为‘白尼姑’、‘花展时节’、‘波谱’；类群B为‘拉拉罗克’、‘香神’、‘南斑柯’、‘南极星’、‘樱桃节’、‘红台阁’、‘烈香’、‘凡丽’、‘贞洁’、‘惠勒’、‘红绒贝蒂’、‘达婷’、‘高帽子’、‘塔莉尔小姐’、‘复色明天’、‘秋牡丹’、‘魔术城’、‘凯蒂’；类群C为‘哈罗德’；类群D为‘春节’。RAPD分析结果与传统分类学结果基本相符。 5．用引物SBS-X5，SBS-X6和SBS-S15扩增的12条多态性条带构建了23个茶花品种的DNA指纹图谱，可用于茶花的种质鉴定。 6．实验发现，茶花株型紧凑这一性状可能与RAPD特异标记SBS-S15-1000片段上的某些基因连锁。更多还原

【Abstract】 In this paper,a set of methods suitable for total DNA extraction of Camellia plants were put forward. Appropriate primers were screened and the fingerprints of 23 camellia cultivars from home and abroad were generated using RAPD method. In addition, the relationship between Camellia cultivars and their genetic diversity were studies with the RAPD technique. The feasibility of using RAPD method to analysis on the germplasm resources of Camellia Cultivars was discussed.The main results were as follows:1. The total DNA from the fresh young leaves of camellia cultivars was successfully extracted by using the developed CTAB method .In light of the influence of secondary compounds, such as polyphenols in camellia cultivars on the quality of extracted total DNA , some measures were taken to eliminate them including a proper amount of PVP was added during grinding to prevent oxidation of phenolic compounds,1%β-mercaptoe-thanol added to the extracte solution to separate the phenolic compounds, using CTAB/NaCl solution to separate polysaccharides. The results of ratio of A₂₆₀ and A₂₈₀ ,electrophoresis and amplification suggested that the developed CTAB method could obtain high quality DNA with better purity and integrity compared with other methods.2. A steady system of PCR reaction for camellia cultivars was built with orthogonal design. The optimal PCR system for RAPD analysis was as follows: 80ng DNA template, 0.3μmol/ L random primer,0.2 mmol/L dNTPs, 2mmol/L MgCl₂, 1U Taq polymerase in 25 μl reaction solution. The reaction program was devised for one cycle at 94℃ 5 minutes and followed by 40 cycles, each with 50 seconds at 94℃, 50 seconds at 36℃,90 seconds at 72℃, and a final extension at 72℃ for 8 minutes.3. 20 primers were screened from 100 ten-bp arbitrary primers. A total of 136 DNA fragments ranging from 0.20- 2.0kbp were amplified, among which 120 （ 88.2% ） was polymorphic,using these 20 primers. The average number of DNA band produced by each primer was 6.8. The result of genetic similarity analysis for 23 cultivars showed that the Nei’s coefficient ranged from 0.4386～0.8936, and the average Nei’s coefficient was 0.7668.更多还原