【作者】 刘万勃；

【导师】 宋明； 刘富中；

【作者基本信息】 西南农业大学 ， 蔬菜学， 2002， 硕士

【摘要】 本实验采用RAPD和ISSR两种分子标记技术研究了甜瓜种质遗传多样性，并利用RAPD标记进行了快速鉴定甜皿杂种的研究。实验结果如下： 通过试验确定了适合甜瓜的最佳PCR反应成分(包括dNTP、引物、Mg²⁺、TaqDNA聚合酶等的浓度组合)，并通过对变性、退火、延伸时间，退火温度和循环次数的优化试验确定了省时的扩增程序，最终得到了一个重复性好、清晰度高、较理想的RAPD-PCR和ISSR-PCR技术体系。 在供试的37份甜瓜材料中筛选到具有多态性的RAPD引物21个，ISSR引物10个（其中ISSR-2与ISSR-4等量混合组成ISSR-10引物对）。其中，RAPD引物共扩增出多态性带106条，多态性条带比率（PPB）为58.62％，平均多态信息量（PIC）为0.47；ISSR引物共扩增出多态性带73条，PPB值为65.51％，平均PIC值为0.53。根据两种标记的结果，采用UPGMA聚类分析，将供试材料分为两大类群：野生甜瓜和栽培甜瓜；栽培甜皿又分为厚皮甜瓜和薄皮甜瓜两大亚群，各野生甜瓜种质之间的遗传距离较大，这与其分类地位基本一致。两种分子标记的分析结果呈极显著正相关(r=0.62＞r₀₀₁)。RAPD和ISSR标记对“2002-31”（编号7）的聚类结果存在较大的差异，这可能与“2002-31”来源于薄皮甜瓜和厚皮甜瓜的杂种，在后代自交纯化过程中仍具有两者的遗传特性而产生的一种中间类型有关，从这一点来说，薄皮甜瓜与厚皮甜瓜在分子水平上没有严格的界限。在亚群内，RAPD与ISSR标记的聚类结果差异较大，这也说明了甜瓜种质的变异方式较为复杂。研究表明，RAPD和ISSR标记可用于甜瓜种质遗传多样性的研究。 建立了快速鉴定甜瓜杂种的RAPD技术体系。通过试验，提出了直接以萌动的甜瓜种子为材料快速提取基因组DNA的方法，将这种方法提取的DNA与CTAB法提取的DNA同时进行RAPD-PCR扩增，两者的带型无明显变化，主带均相同，结果表明SDS快速提取法可用于PCR反应，非常适合于甜瓜杂种的快速鉴定。通过对亲本和杂种间的RAPD分析，从80个随机引物中选出了5个可进行3个甜瓜杂种纯度鉴定的引物（即在各自父本和杂种单株上都出现母本不具有谱带的引物），他们分别是，中蜜1号：OPN11-850bp、OPM17-830bp；中蜜2号：OPA02-830bp、OPM16-550bp、OPM17-700bp；中蜜3号：OPN06-750bp、OPN11-600bp、OPM17-700bp。其中，OPN11可用于中蜜1号和中蜜3号的纯度鉴定，OPM17可同时用于3个杂种的纯度鉴定。使用OPMl7进行纯度鉴定时，由于中蜜2号、中蜜3号的父本相同，出现的特征谱带在中蜜2号、中蜜3号两个杂种中恰好均为OPM17-700bp。以上引物经各自的20个杂种单株检验，均能出现各自父本的特征谱带，然后进行田间抽样检验，结果与实际相符，表明实验结果具有可靠性。更多还原

【Abstract】 Random amplified polymorphic DNA (RAPD) and Inter-simple sequence repeat (ISSR) molecular markers were used to detect the genetic diversity of melon(C玞w/w/s melo L.) germplasms, and melon hybrid were determinated by using RAPD. The result are the following:Experiment was made to investigate the amplification efficiency of different amplification conditions, Optimum combinations of amplification conditions (dNTP density, primer density, Mg2+ density, Taq DNA polymerase density, etc.) were identified rapidly and effectively. Time-saving reaction procedures for RAPD-PCR and ISSR-PCR were determined through experiments. As a result, a satisfactory RAPD and ISSR techique systems for melon (Cucumis melo L.) with desirable repeatability and resolution was estblished.A total of 21 RAPD primers and 10 ISSR primers (ISSR-10 primers is evenly composed of ISSR-2 and ISSR-4) were identified with polymorphism among the entries. For RAPD markers, 106 polymorphic bands were produced, percentage of polymorphic bands (PPB) was 58.62%, mean polymorphism information content (PIC) was 0.47. For ISSR markers,73 polymorphic bands were generated, PPB was 65.51%, mean PIC was 0.53. UPGMA cluster analysis based on two molecular markers data divided these melons into two groups: wild melon group and cultivated melon group. The distance of each wild melon germplasms are far, cultivated melon group was classified into two sub-groups, which are thick-peel melon and thin-peel melon. They are consistent with the grouping based on taxology systematic botany. For accession "2002-3l"(No.7), the result of cluster analysis is different based on RAPD and ISSR, maybe it was related to "2002-31" was a hybrid of thick-peel melon and thin-peel melon, and it maintained the genetic traits of its parent in course of inbreeding. According to that, there hasn’t absolute bounds between thick-peel melon and thin-peel melon. In sub-groups,the different of cluster analysis between RAPD and ISSR was distinct, it showed melon variation was complicated. Genetic similarity matrices revealed that the estimates of correlation coefficients of RAPD and ISSR were significantly correlated. Based on the results, RAPD and ISSR can be applied to detectgenetic diversity analysis among melon germplasm.RAPD technique system was established to determinate quickly melon hybrid. A DNA extraction method was posed that extracted DNA quickly by bourgeoning melon seed, by comparison with CTAB, the amplified patterns hadn’t distinct difference, the result indicated that the quick DNA extraction method could be applied to PCR reaction, and it was suited to hybrid identification of melon, Eight primers were picked out for genetic purity determination from eighty arbitrary primers by RAPD analysis between parent and hybrid, for ZmOl, they were OPN11, OPM17; for Zm02, they were OPA02, OPM16, OPM17; for Zm03, they were OPN06> OPN1K OPM17. The primers OPN11 could be used for purity determination in zmOl and Zm03; the primer OPM17 could be used for genetic purity determination in all these three hybrids, because male parent of Zm02 and Zm03 were the same one, the haracteristic band of Zm02 and Zm03 was OPMl 7-700.All the primers picked out amplified respectively the characteristic band of male parent in 20 hybrid individuals, the result of sampling test in field conformed to reality. It showed hybrid determination in melon by using RAPD was reliable.更多还原