几种分子标记技术的建立及其在部分柿属植物亲缘关系研究中的应用
摘要
柿是柿科(Ebenaceae)柿属(Diospyros Linn. )植物，我国是柿属植物的分布中心和原产中心之一，捌有丰富的种质资源。但长期以来，对我国柿属植物的种类和分布还缺乏清楚的了解，对我国柿属植物的起源、演化和亲缘关系缺乏深入的探讨。本研究首次开发了柿属植物SSR分子标记技术，并同时采用SSR，SRAP，IRAP和REMAP四种DNA分子标记技术，分别对供试7种柿属植物(柿Diospyros kaki Thunb. ；君迁子D. Lotus L. ；浙江柿D. glaucifolia Metc. ；油柿D. oleifera Cheng. ；金枣柿；老鸦柿D. rhombifolia Herosl. ；美洲柿D. virginian L. ；)进行了亲缘关系分析，以期从DNA水平上明确柿种间及种下的亲缘关系和分类地位，为柿属植物种质资源的科学利用提供依据。主要结果如下：
1．利用数据库查询的方法开发柿属植物SSR引物。查询GenBank，EMBL和DDBJ数据库中的柿属植物核酸序列，采用Sputnik筛选包含SSR的序列，用Primer Primere 5.0设计SSR引物。从98条核酸序列中共计筛选到SSR引物7对，并建立起柿属植物SSR-PCR扩增技术体系。
2．利用ISSR技术结合抑制PCR技术开发SSR引物。使用ISSR引物8条，对基因组DNA扩增后，设计抑制PCR引物进行染色体步行，共计开发出12对SSR引物，其中9对表现较好。在GenBank上登录序列19条。
3．采用链亲和素(streptavidin)与生物素(biotin)之间具有很强的亲和能力的原理，用链亲和素磁珠富集法使用生物素标记的探针(GA)_(10)开发柿SSR引物6对，并在GenBank上登录6条。
4．利用自主开发的SSR标记，选用18对SSR引物对柿属植物的亲缘关系进行分析，实验中采用两种电泳方式进行检测。有关等位基因的相关信息从PAGE胶上获得，聚类图构建的数据则来源于琼脂糖凝胶电泳。结果通过琼脂糖凝胶电泳检测方式共得到158条多态性带，每个引物检测的带数从5到20条不等。6％的变性聚丙烯酰胺凝胶上检测到的等位位点数的变化从3到24不等，平均每个引物检测到15个位点。PAGE胶上所检测的扩增条带数明显比琼脂糖凝胶上的多。根据琼脂糖凝胶所获得的数据由NTSYS计算30份试材间的相似系数，结果表明供试材料的相似系数变化范围在0.42(浙江柿和老鸦柿)到0.93(富有和松本早生)间，平均值0.79。日本柿，中国柿和近缘种间相似系数平均值分别为0.83，0.81和0.71。期望杂合度和表观杂合度变化范围分别为0.42-0.77和0.27-0.59。
5．利用SRAP标记，使用文献中运用较多的7条正向引物，8条反向引物，通
Japanese persimmon (Diospyros kaki Thunb.) belongs to the Diospyros genus, Ebenaceae Family. China is one of the originating and distributing centres, there are abundant precious genetic resources in China. But the varieties, distribution, origin, evolvement and genetic relationships of Diospyros spp. are unclear for a long time. This study firstly developed the SSR molecular marker from the genus of Diospyros. Then, SSR, SRAP, IRAP and REMAP molecular markers were employed to evaluate the genetic relationships of Diospyros spp. including Diospyros kaki Thunb.; D. Lotus L.; D. glaucifolia Metc.; D.oleifera Cheng.; Jinzaoshi; D.rhombifolia Herosl.; D.virginian L. to elucidate the genetic relationships of cultivars and species of the Diospyros spp. at the DNA molecular level and provide the basis for the scientific and efficient utilization of the persimmon germplasm. The results were as follows:
1. SSR primers were developed based on repeated sequences in the public nucleic acids sequences database. A total of 98 sequences of Diospyros Linn., were examined in GenBank, EMBLand DDBJ. Seven SSR sequences were obtained which were all used to design SSR primers. The optimal SSR-PCR system were established
2. SSR primers developed using ISSR-suppression PCR technique. Suppression PCR primers were designed to conduct genome walking after eight ISSR primers amplified in genomic DNA. Of the 12 obtained SSR primers, three were monomorphic and the remaining nine showed high polymorphisms. Nineteen sequences were registered in GenBank.
3. Six SSR primers were developed using biotinylated (GA)10 and streptavidin-coated magnetic beads. Six sequences were registered in GenBank.
4. Eighteen SSR primer pairs were selected to evaluate the genetic relationships of Diospyros spp. from the developed primers. The dendrogram was constructed from the agarose gel data, while the information of alleles was from PAGE gel. Amplification was successful with the 18 SSR markers assayed, detected a total of 158 bands, ranging from 5 to 20 on agarose gel. The alleles collectively yielded unique genotypes for each of the 30 persimmon genotypes, which enabled the unambiguous discrimination of the 30 persimmon genotypes included in this study. The number of alleles per primer ranged
1．利用数据库查询的方法开发柿属植物SSR引物。查询GenBank，EMBL和DDBJ数据库中的柿属植物核酸序列，采用Sputnik筛选包含SSR的序列，用Primer Primere 5.0设计SSR引物。从98条核酸序列中共计筛选到SSR引物7对，并建立起柿属植物SSR-PCR扩增技术体系。
2．利用ISSR技术结合抑制PCR技术开发SSR引物。使用ISSR引物8条，对基因组DNA扩增后，设计抑制PCR引物进行染色体步行，共计开发出12对SSR引物，其中9对表现较好。在GenBank上登录序列19条。
3．采用链亲和素(streptavidin)与生物素(biotin)之间具有很强的亲和能力的原理，用链亲和素磁珠富集法使用生物素标记的探针(GA)_(10)开发柿SSR引物6对，并在GenBank上登录6条。
4．利用自主开发的SSR标记，选用18对SSR引物对柿属植物的亲缘关系进行分析，实验中采用两种电泳方式进行检测。有关等位基因的相关信息从PAGE胶上获得，聚类图构建的数据则来源于琼脂糖凝胶电泳。结果通过琼脂糖凝胶电泳检测方式共得到158条多态性带，每个引物检测的带数从5到20条不等。6％的变性聚丙烯酰胺凝胶上检测到的等位位点数的变化从3到24不等，平均每个引物检测到15个位点。PAGE胶上所检测的扩增条带数明显比琼脂糖凝胶上的多。根据琼脂糖凝胶所获得的数据由NTSYS计算30份试材间的相似系数，结果表明供试材料的相似系数变化范围在0.42(浙江柿和老鸦柿)到0.93(富有和松本早生)间，平均值0.79。日本柿，中国柿和近缘种间相似系数平均值分别为0.83，0.81和0.71。期望杂合度和表观杂合度变化范围分别为0.42-0.77和0.27-0.59。
5．利用SRAP标记，使用文献中运用较多的7条正向引物，8条反向引物，通
Japanese persimmon (Diospyros kaki Thunb.) belongs to the Diospyros genus, Ebenaceae Family. China is one of the originating and distributing centres, there are abundant precious genetic resources in China. But the varieties, distribution, origin, evolvement and genetic relationships of Diospyros spp. are unclear for a long time. This study firstly developed the SSR molecular marker from the genus of Diospyros. Then, SSR, SRAP, IRAP and REMAP molecular markers were employed to evaluate the genetic relationships of Diospyros spp. including Diospyros kaki Thunb.; D. Lotus L.; D. glaucifolia Metc.; D.oleifera Cheng.; Jinzaoshi; D.rhombifolia Herosl.; D.virginian L. to elucidate the genetic relationships of cultivars and species of the Diospyros spp. at the DNA molecular level and provide the basis for the scientific and efficient utilization of the persimmon germplasm. The results were as follows:
1. SSR primers were developed based on repeated sequences in the public nucleic acids sequences database. A total of 98 sequences of Diospyros Linn., were examined in GenBank, EMBLand DDBJ. Seven SSR sequences were obtained which were all used to design SSR primers. The optimal SSR-PCR system were established
2. SSR primers developed using ISSR-suppression PCR technique. Suppression PCR primers were designed to conduct genome walking after eight ISSR primers amplified in genomic DNA. Of the 12 obtained SSR primers, three were monomorphic and the remaining nine showed high polymorphisms. Nineteen sequences were registered in GenBank.
3. Six SSR primers were developed using biotinylated (GA)10 and streptavidin-coated magnetic beads. Six sequences were registered in GenBank.
4. Eighteen SSR primer pairs were selected to evaluate the genetic relationships of Diospyros spp. from the developed primers. The dendrogram was constructed from the agarose gel data, while the information of alleles was from PAGE gel. Amplification was successful with the 18 SSR markers assayed, detected a total of 158 bands, ranging from 5 to 20 on agarose gel. The alleles collectively yielded unique genotypes for each of the 30 persimmon genotypes, which enabled the unambiguous discrimination of the 30 persimmon genotypes included in this study. The number of alleles per primer ranged
引文
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