【作者】 唐娟娟；

【导师】 朱睦元； 范义荣；

【作者基本信息】 浙江大学 ， 遗传学， 2002， 硕士

【摘要】 采用RAPD分子标记来构建黄山松单株树(仙居×磐黄)的分子遗传连锁图谱。通过236个10 bp的随机引物或组合对6粒黄山松种子胚乳组织DNA进行PCR扩增，共筛选出33个多态引物用于作图。其中以S1427与其它引物等量混合来进行扩增多态性较高。利用筛选出的33个多态性引物(组)对60个个体进行RAPD分析，共有52个标记进入连锁分析，其中包含10个偏分离位点。采用作图软件3.0分析，52个标记中有39个标记构成9个连锁群，其余13个标记未进入连锁群。连锁群总图距为770.7cM，标记间的最大图距为40.OcM，最小图距为1.7cM。标记平均间距为20.2cM，最长的连锁群达306.3cM，包含有13个RAPD标记：最短的连锁群长1.7cM，包含2个标记。本研究首次构建黄山松分子标记连锁框架图，可作为黄山松基因组研究的基础。 利用RAPD分子标记分析了黄山松10个家系的遗传多样性，为育种提供依据，从206个随机引物中筛选出17个引物，获得38个多态位点。Shannon多样性指数平均为4.5251，Shannon多样性值范围在0.0102-0.0504之内。家系(余姚×文石)遗传多样性水平最高，其Shannon多样性指数为7.9658。根据这38个多态位点计算遗传距离，进行聚类分析。在遗传距离0.42处可将10个家系聚类分成3组。 采用整合微流控芯片系统分析了黄山松RAPD多态性。发现基于芯片的检测方法比琼脂糖凝胶电泳方法灵敏度更高，所需的样品量要少得多，所需的时间仅为其1／4。并且能自动对DNA片段进行定性、定量分析。它是一种高效、灵敏、迅速、重复性好的检测新技术。更多还原

【Abstract】 Random amplified polymorphic DNA (RAPD) markers were used to construct molecular genetic linkage map of a single tree (P.Taiwanensis Hayata.) from a cross 18 X 28 (each from the superior tree from Xianju county and Pan ’ an county in Zhejiang province). 236 random oligonucleotide primers were screened and 33 primers were selected to generate RAPD markers within a sample of 60 megagametophyte DNAs. Specially, amplification of the mix primers of SI427 and the other primer appeared to be effective. A total of 52 segregating loci were identified, including 10 loci which appeared to be experencing segregation distortion. Using mapmaker version 3.0, the 39 markers formed 9 main linkage groups and 13 markers were not assigneded to any linkage group. The resulting linkage map of P. Tarwanensis Hayata (including 39 markers) spanned 770.7 cM with an average distance of 2fl.ZcM between markers, linkage groups in size from 1.7 cM (2 markers included )to 306.3 cM(13 markers included),adjacent markers map disance 1.7 cM to 40.0 cM. This map should be the first framework and provide a basis for analysis of genome in P. Tarwanensis Hayata.The analysis of genetic diversity of 10 families in P.Taiwanensis Hayata. was carried out by RAPD molecular markers, which may provide the theoretical base for breeding. 17 primers were screened from 206 random oligonucleotide primers and 38 polymorphic loci were obtained from PCR of 17 primers. The mean Shannon Wevieis index was 4.5251,and Shannon Weveis value of phenotypic diversity varied from 0.0102 to 0.0504. The level of genetic diversity of the family (yuyao X wenshi) was highest,and its Shannon Wevieis index was 7.9658.The genetic distance was caculated according to the 38 polymorphic loci and the cluster map was constructed. The cluster analysis indicated that 10 families could be divided into three groups at the point of genetic distance 0.42.Meanwhile we have proposed the use of an integrated microfluidic chip-based system as a new tool in the analysis of polymorphic RAPD fragments in P.Taiwanensis Hayata.. The chip-based method was found to be very sensitive, requiring much less sample and only quarter the time compared to the agarose gel method. The automated data analysis sizes and quantitates the DNA fragments, thus yielding a more thorough, reproducible, sensitive, and rapid analysis.更多还原