RAPD技术在苦瓜、辣椒一代杂种种子纯度鉴定上的应用研究
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摘要
本文分析了国内外关于分子标记鉴定蔬菜作物种子纯度的研究进展，RAPD技术以方便快捷，反应灵敏，标记数量多，所用引物没有种属限制，所需模板数量少等优点，较适合在种子纯度检测中应用。
以碧绿2号、碧绿3号两个苦瓜品种为材料，研究了含糖量较高的植物材料的DNA提取方法，采用四种CTAB法对苦瓜的基因组DNA进行提取，四种CTAB法都适合苦瓜DNA的提取，提取的DNA具有天然DNA的特性，可成功进行RAPD-PCR扩增，尤以改良CTAB法最为简便、快速，适合商业化苦瓜杂种纯度鉴定，并且建立了适合苦瓜RAPD分析的优化体系，即在25μL反应体系中，DNA模板的量为20ng，Mg~(2+)浓度为1.5mmol／L，dNTP浓度为0.2mmol／L，Taq DNA聚合酶的最适用量为1.0U。并以此优化体系为基础对碧绿3号苦瓜两个亲本的基因组DNA进行了RAPD分析，发现引物OPQ-01可在碧绿3号及其亲本的RAPD图谱中形成多态性差异，可应用于碧绿3号苦瓜种子纯度的检测，对1份种子样品进行了RAPD检测，其结果与田间种子纯度检测结果完全一致。
利用RAPD技术，通过对60个随机引物的筛选，获得能区分粤椒4号及其亲本的引物OPS-01，并得到单株验证。
The achievements in the studies of vegetable seed purity test at broad and home were analysed. The results showed that RAPD technique in suited to the test of seed purity because of its convenient, sensitive reaction more markers ,limitless primer and a few templates.
A handy way extract DNA was studied by two balsam pear varities (No.3 Bilv and No.2 Bilv), i.e improved CTAB method .An optimum reaction system for RAPD analysis was set up. In the 25μL reaction system ,the concentration of DNA,Mg2+,Taq DNA polymerase and dNTPs is 20ng, 1.5mmol/L,1.0U,0.2mmol/L respectively.Based it on, the Genomic DNA of two parents of "No.3 Bilv" were tested by RAPD analysis. The results showed that primer OPQ-01 could form varied differences in RAPD maps of No.3 Bilv and their parents. It could also be appliedto the test of seed purity of No.3 Bilv. The results of RAPD test on a seed sample were completely consistant with that of seed purity testing in the fields.
Primer OPS-01 which can distinguish F1 hybrid of N0.4 Yuejiao and their parents were screened from 60 random primers by RAPD method . Its ability to test F1 variety purity was proved by single plant test.
以碧绿2号、碧绿3号两个苦瓜品种为材料，研究了含糖量较高的植物材料的DNA提取方法，采用四种CTAB法对苦瓜的基因组DNA进行提取，四种CTAB法都适合苦瓜DNA的提取，提取的DNA具有天然DNA的特性，可成功进行RAPD-PCR扩增，尤以改良CTAB法最为简便、快速，适合商业化苦瓜杂种纯度鉴定，并且建立了适合苦瓜RAPD分析的优化体系，即在25μL反应体系中，DNA模板的量为20ng，Mg~(2+)浓度为1.5mmol／L，dNTP浓度为0.2mmol／L，Taq DNA聚合酶的最适用量为1.0U。并以此优化体系为基础对碧绿3号苦瓜两个亲本的基因组DNA进行了RAPD分析，发现引物OPQ-01可在碧绿3号及其亲本的RAPD图谱中形成多态性差异，可应用于碧绿3号苦瓜种子纯度的检测，对1份种子样品进行了RAPD检测，其结果与田间种子纯度检测结果完全一致。
利用RAPD技术，通过对60个随机引物的筛选，获得能区分粤椒4号及其亲本的引物OPS-01，并得到单株验证。
The achievements in the studies of vegetable seed purity test at broad and home were analysed. The results showed that RAPD technique in suited to the test of seed purity because of its convenient, sensitive reaction more markers ,limitless primer and a few templates.
A handy way extract DNA was studied by two balsam pear varities (No.3 Bilv and No.2 Bilv), i.e improved CTAB method .An optimum reaction system for RAPD analysis was set up. In the 25μL reaction system ,the concentration of DNA,Mg2+,Taq DNA polymerase and dNTPs is 20ng, 1.5mmol/L,1.0U,0.2mmol/L respectively.Based it on, the Genomic DNA of two parents of "No.3 Bilv" were tested by RAPD analysis. The results showed that primer OPQ-01 could form varied differences in RAPD maps of No.3 Bilv and their parents. It could also be appliedto the test of seed purity of No.3 Bilv. The results of RAPD test on a seed sample were completely consistant with that of seed purity testing in the fields.
Primer OPS-01 which can distinguish F1 hybrid of N0.4 Yuejiao and their parents were screened from 60 random primers by RAPD method . Its ability to test F1 variety purity was proved by single plant test.
引文
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