香蕉（Musa spp.）遗传多样性及亲缘关系的RAPD分析
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摘要
香蕉(Musa spp．)是一种主要的热带、亚热带农业作物。虽然在国际商贸中占有重要地位，但人们对其遗传系统知之甚少。与其它农作物(水稻、玉米等)相比，香蕉的分子水平研究相对较少。本研究对24个不同国家、地区来源的香蕉进行的RAPD分析。结果如下：
1．暗处理两天，改良SDS和CTAB均可从半展的香蕉幼叶中提取高质量的DNA。
2．对RAPD条件优化分析，确定了香蕉RAPD分析的最佳方案，即PCR扩增程序为94℃预变性4min，94℃变性60S，37℃复性60S，72℃延伸105S，共40个循环，最后72℃延伸10min；25μl反应体系中包括：200 μmol／L dNTP，2.0 mmol／L Mg~(2+)，0.3 μmol／L引物，模板DNA20～50ng，Ex-Taq酶1U，2.5 μl 10×缓冲液。
3．利用18个引物对供试品种扩增的DNA谱带，进行了UPGMA聚类分析，在此基础上构建了24个香蕉种质的分子树状图。依据此图，供试香蕉种质分成三大类群：第一类群有20个种质，第二类群包括3个香蕉种质，第三类群只含广西鸡蕉(龙牙蕉类)一个种质。其中第一类群又可分为五个亚群：Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ。
4．用双引物RAPD评价三个巴西种质的亲缘关系，用双引物RAPD获得巴西变种与巴西香蕉间的相似系数为0.5714，低的相似系数说明巴西变种不大可能由巴西香蕉衍生而来。单、双引物扩增结果的比较表明：双引物RAPD扩增可产生更多的多态性片段。
Bananas(Musa spp.) is one of the major tropical and subtropical crops in the world. Despite the importance of banana in the international trade and comerce, little is known of its genetic system. Contrast to other crops such as rice and maize, there are fewer molecular researches on banana. In this study, twenty-four banana germplasm materials come from different countries and regions were surveyed by RAPD analysis. The results showed as follows : 1 . Using the improved methods of SDS and CTAB , high purity DNA
was obtained from half expanded leaves which had been kept in a
darkened chamber for two days .
2. Through optimal experiments, the best amplification system of RAPD analysis is set up ,that is : 94℃ initial denaturation for 4min, 40 cycles of 94℃ for 60s, 37℃ for 60s, 72℃ for 105s, and 72 ℃ for 10min. The 25 ul reaction mixture contained 20~50ng template DNA, 200umol/L dNTP, 2.0mmol/L Mg2+, 0.3umol/L primer, 2.5ul 10xEx-Taq polymerase buffer, and 1U of Ex-Taq polymerase .
3. Based on UPGMA cluster analysis of DNA bands which were amplified with eighteen RAPD primers , the molecular dendrogram of twenty-four banana germplasm materials were constructed . According to the gendrgram , the twenty-four germplasm materials were assigned to three major clusters . The first cluster included twenty genotypes, the second included three and the other only had one, that is Guangxijijiao(Longyajiao type) . The first cluster could be divided into five sub-groups : I, II , III , IV and V.
4. The two-primer RAPD was used to access the relationship of three bananas genotypes from Brazil. The similarity coefficient between Baxibianzhong and Baxixiangjiao obtained from two-primer RAPD was 0.5714 , the low similarity indicated that it is impossible that Baxibianzhong was derived from Baxixiangjiao . The comparison of polymorphism amplified with single-primer and two-primer in three genotypes showed that the two-primer RAPD could produce more polymorphic fragments than the single-primer RAPD .
1．暗处理两天，改良SDS和CTAB均可从半展的香蕉幼叶中提取高质量的DNA。
2．对RAPD条件优化分析，确定了香蕉RAPD分析的最佳方案，即PCR扩增程序为94℃预变性4min，94℃变性60S，37℃复性60S，72℃延伸105S，共40个循环，最后72℃延伸10min；25μl反应体系中包括：200 μmol／L dNTP，2.0 mmol／L Mg~(2+)，0.3 μmol／L引物，模板DNA20～50ng，Ex-Taq酶1U，2.5 μl 10×缓冲液。
3．利用18个引物对供试品种扩增的DNA谱带，进行了UPGMA聚类分析，在此基础上构建了24个香蕉种质的分子树状图。依据此图，供试香蕉种质分成三大类群：第一类群有20个种质，第二类群包括3个香蕉种质，第三类群只含广西鸡蕉(龙牙蕉类)一个种质。其中第一类群又可分为五个亚群：Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ。
4．用双引物RAPD评价三个巴西种质的亲缘关系，用双引物RAPD获得巴西变种与巴西香蕉间的相似系数为0.5714，低的相似系数说明巴西变种不大可能由巴西香蕉衍生而来。单、双引物扩增结果的比较表明：双引物RAPD扩增可产生更多的多态性片段。
Bananas(Musa spp.) is one of the major tropical and subtropical crops in the world. Despite the importance of banana in the international trade and comerce, little is known of its genetic system. Contrast to other crops such as rice and maize, there are fewer molecular researches on banana. In this study, twenty-four banana germplasm materials come from different countries and regions were surveyed by RAPD analysis. The results showed as follows : 1 . Using the improved methods of SDS and CTAB , high purity DNA
was obtained from half expanded leaves which had been kept in a
darkened chamber for two days .
2. Through optimal experiments, the best amplification system of RAPD analysis is set up ,that is : 94℃ initial denaturation for 4min, 40 cycles of 94℃ for 60s, 37℃ for 60s, 72℃ for 105s, and 72 ℃ for 10min. The 25 ul reaction mixture contained 20~50ng template DNA, 200umol/L dNTP, 2.0mmol/L Mg2+, 0.3umol/L primer, 2.5ul 10xEx-Taq polymerase buffer, and 1U of Ex-Taq polymerase .
3. Based on UPGMA cluster analysis of DNA bands which were amplified with eighteen RAPD primers , the molecular dendrogram of twenty-four banana germplasm materials were constructed . According to the gendrgram , the twenty-four germplasm materials were assigned to three major clusters . The first cluster included twenty genotypes, the second included three and the other only had one, that is Guangxijijiao(Longyajiao type) . The first cluster could be divided into five sub-groups : I, II , III , IV and V.
4. The two-primer RAPD was used to access the relationship of three bananas genotypes from Brazil. The similarity coefficient between Baxibianzhong and Baxixiangjiao obtained from two-primer RAPD was 0.5714 , the low similarity indicated that it is impossible that Baxibianzhong was derived from Baxixiangjiao . The comparison of polymorphism amplified with single-primer and two-primer in three genotypes showed that the two-primer RAPD could produce more polymorphic fragments than the single-primer RAPD .
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