【作者】 蔡英卿；

【导师】 赖钟雄； 潘东明；

【作者基本信息】 福建农林大学 ， 果树学， 2004， 硕士

【摘要】 本研究以福建省34份余甘子遗传资源为材料，摸索出适合余甘子叶片基因组DNA的提取方法；建立和优化了余甘子RAPD反应体系；筛选出具有品种（或单株）特异性的RAPD标记；采用RAPD标记，结合聚类分析，进行了福建省余甘子遗传资源的亲缘关系与遗传多样性研究。主要研究结果如下： 一、余甘子叶片DNA提取方法的建立。针对余甘子叶片富含多酚类、多糖、色素等物质，易与DNA结合成复合物，难溶解。又易抑制Taq酶活性，从而影响PCR扩增。本研究采用改良的SDS法，以新鲜幼嫩的余甘子叶片为材料，同时又在提取液中加入3％PVP，有效地克服消除了酚类物质的干扰，从余甘子叶片中提取了较高纯度和产量的DNA，适宜作为余甘子的RAPD分析。 二、余甘子RAPD反应体系的优化。在参考一般RAPD分析的反应程序的基础上，经过反复试验，确定适宜的PCR扩增程序为：94℃，预变性4min；94℃，1min→38℃，1.5min→72℃，2min，45个循环：72℃，10min。优化的余甘子RAPD反应体系为：在扩增反应总体积25μL中，包含0.6mmol·L^-1Mg²⁺、500μmol·L^-1dNTP、300 nmol·L^-1引物、25ng模板DNA、1.25U Taq DNA聚合酶。 三、余甘子RAPD标记的多态性程度分析。采用20个具特异扩增的多态性引物对福建省34份余甘子基因型进行扩增，20个引物，在34份供试材料中总共扩增出259个位点，且均是多态的，多态性程度达100％，平均每个引物扩增12.95个位点。各供试材料之间的遗传距离范围为0-1，其中栽培品种与野生资源之间遗传距离在0.43—1之间；莆田余甘资源与惠安余甘资源之间遗传距离为0.27—0.99。 四、品种（或单株）特异性RAPD标记的筛选与品种鉴别。采用20个具特异扩增的多态性引物对所有供试材料进行扩增，获得了19个引物对23份材料扩增的RAPD特征标记47个，17个引物在36对材料上共扩出36个共享特征RAPD标记。五、福建余甘遗传资源的RAPD标记的聚类分析。采用ED（欧氏距离）法进行聚类分析则可将福建34份余甘子遗传资源明显划分为惠安余甘和莆田余甘两大类.而采用Cosine距离法计算样品间的距离则全部供试的34份余甘子基因型可明显区分为三大类，栽培品种全部归为一大类，惠安野生余甘子为另一大类，莆田野生一号单独归为一类。更多还原

【Abstract】 In this experiment 34 accessions of Phyllanthus emblica L. genetic resources in Fujian Province were used as materials. The method of leaf DNA extraction was developed; and then the RAPD reaction system was established and optimized; the cultivar (or line) specific RAPD markers were screened; and the genetic relationship and genetic diversity of Phyllanthus emblica L. germplasm in Fujian Province were discussed by RAPD markers and clustering analysis. The obtained results were as follows:I .The development of the method of leaf DNA extraction in emblicPolyphenols, carbohydrate, and pigments etc. were rich in emblic leaves, which were subject to combine with DNA to form insoluble compounds, and which inhibited the activity of Tag enzyme, therefore which affected the PCR. In this study, in order to overcome the disturb of polyphenols effectively, the improved SDS method was developed, in which the fresh and tender leaves were used as the materials and 3% PVP was added in the extraction buffer. By the improved method, the pure and high-quality DNA were extracted from emblic leaves, which was suitable for RAPD amplification.II. The optimization of RAPD reaction system in emblicBased on the common RAPD reaction program and adjusting experiments, the optimized amplification program for emblic RAPD-PCR was: 94? for 240 s; 45 cycles at 94癈 for 60s, 38癈 for 90s and 72癈 for 120s; 72癈 for 600s. The RAPD amplification system was in a 25 ?L reaction mixture containing 0.6mmol ?L-1 Mg2+, 500 ?mol ?L-1 dNTP, 300n mol ?L-1 primer, 25 ng DNA and 1.25 unit Taq DNA polymerse.HI. The analysis of RAPD polymorphic degree in emblic 34 accessions of emblic germplasm in Fujian Province were analyzed by RAPD technique. 259 sites were totally detected by 20 random 10-mer oligo-mucleotide primers, all of which belonged to polymorphic sites. The polymorphic degree was up to 100%. On the average, one primer produced 12.95 sites. The genetic distances among materials were between 0 and 1, among of which the genetic distances were 0.43~1 between cultivars and the wild, and 0.27~0.99 between Putian and Huian emblic resources.IV. The screen of specific RAPD markers of cultivars (or lines) and cultivar434 accessions of Phyllanthus emblica L. germplasm were used for amplification with 20 random 10-mer oligo-nucleotide primers. The results indicated that 47 RAPD characteristic markers were amplified in 23 accessions with 19 primers and 36 common characteristic RAPD markers within two cultivars or lines were amplified in 34 accessions with 17 primers.V. Clustering analysis of RAPD markers in emblic genetic resources in Fujian provinceThe 34 accessions of Fujian emblic germplasm were divided into 2 groups by clustering analysis with ED (Euclidean distance) method-Group of emblic from Huian and Group of emblic from Putian, and 3 groups with Cosine method-the cultivars, the wild from Huian and the wild No.1 from Putian.更多还原