基于铁皮石斛EST-SSR标记开发的兰科植物遗传多样性分析
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摘要
兰科植物有着极高的观赏价值和经济价值,一直都深受人们的喜爱,兰花产业已成为国际花卉业的重要组成部分。我国兰花资源中一些种不但具有很高的观赏价值,而且具有极高的药用价值。由于过度开采,生境破坏而导致野生兰花资源已濒临灭绝,因此对兰科植物的研究和保护工作已刻不容缓。开展对对兰科植物的系统进化研究,直接关系到对现有资源的有效评价和合理利用,也有利于优质新品种的培育,关乎整个产业的发展。
为了能在从分子水平对兰科植物进行较全面的系统进化研究,本研究首先对兰科植物石斛属中的代表种铁皮石斛进行SSR标记的开发,将标记用于石斛属中验证其通用性和多态性,然后将石斛EST-SSR标记扩展到兰科植物材料中,为兰科植物的遗传多样性等相关研究奠定基础。
(1)本研究首先对杭州师范大学“浙江省药用植物种质改良与质量控制重点实验室”构建的铁皮石斛cDNA文库进行分析,对4075条EST序列进行搜索,得到了含有838个SSR位点的706条有效EST序列,最终成功开发110对EST-SSR引引物。在所有EST序列中的SSR位点中,二、三核苷酸重复类型最多,分别为37.83%和42.96%,四核苷酸重复类型其次(12.53%)；六核苷酸重复类型最少,仅占2.98%。
(2)成功开发的110对铁皮石斛EST-SSR引引物,位于108个EST序列上,其中99条序列在GenBank数据库中搜索到了基因功能注释。将110对引物用于在31种石斛属植物,进行了通用性及多态性分析,研究结果表明,石斛EST-SSR在石斛属通用性和多态性都很高,分别为47.93%和82.58%。
(3)将本实验室前期开发的金钗石斛EST-SSR与本研究开发的铁皮石斛EST-SSR用于兰科植物的研究,选择其中在石斛属中通用性>50%的引物,对兰科的15个属44个材料进行通用性与多态性的分析,结果表明通用性偏低,为25.05%,多态性较好,为55.38%。；将其中有代表性的10对引物扩增结果进行测序,共获得223条序列,对EST-SSR标记在兰科植物中从序列水平上进行分析,最后探讨了兰科植物各属间的系统进化关系。
实验结果表明：EST-SSR标记的开发有助于对兰科各属间遗传多样性和亲缘关系的研究,也为保护和开发宝贵的兰花种质资源提供科学的依据。
The Orchidaceae family has many Chinese traditional precious medical herbals and valuable ornamental plants,.The orchid industry has become an important part of international flower industry. Our country has some wild orchid resources with very high ornamental value and extremely high medicinal value. Due to excessive mining, this species is on the verge of extinction. The study and protection of the plants is urgently needed. Phylogenetic studies of Orchidaceae is not only directly related to the effective evaluation and rational use of existing resources but also more conducive to the cultivation of high-quality new varieties and the development of the orchid industry.
In order to conduct a more comprehensive phylogenetic study of orchids at molecular level, this study developed SSR markers from Dendrobium officinale, which is the representative species of the genus Dendrobium of orchids. The EST-SSR markers used to verify the transferability and polymorphism of Dendrobium, can be applied to the orchid plants to lay foundation for genetic diversity study of orchids.
(1)The study analyzed cDNA library of Dendrobium qfficinale established by Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants.4075EST sequences were screened and706EST sequences cont aining838microsatellite loci were obtained. Finally,110primer pairs for EST-SSRswere designed.Di-nucleotide repeats and Tri-nucleotide repeats were more t han other types(37.83%and42.96%respectively), followed by the type of Tetr a-nucleotide repeats(12.53%) and the Hexa-nucleotide types just accounting for2.98%of all the primers.
(2)110primer pairs fromthe Dendrobium officinale cDNA library are in108EST sequences.And among the108EST sequences, the gene functions of99sequences have been annotated in GenBank Database.110primer pairs were used to test the genomic DNA of31species of Dendrobiumto assess the transferability and polymorphism of the Dendrobium.The results showed that, the Dendrobium EST-SSR in the Dendrobium has high transferability and polymorphism of47.82%and88.56%, respectively.
(3)We took all the EST-SSR primers from Dendrobium nobile and Dendrobium officinale cDNA library constructed by our laboratory, which have high transferability and polymorphism to test genomic DNA of44individuals from15genera of orchid. Result from genera of orchid other than Dendrobium showed low transferability of25.05%and high polymorphism of55.38%. Then the polymorphism loci of10representative pairs of primers were sequenced,and a total of223sequences were received. Differences among the genera orchid were analyzed at sequence level.Finally intergeneric relationships of orchid were studied through clustering.
These results prove that EST-SSR markers are effective to reveal genetic diversity and relationship across Orchidaceae.
为了能在从分子水平对兰科植物进行较全面的系统进化研究,本研究首先对兰科植物石斛属中的代表种铁皮石斛进行SSR标记的开发,将标记用于石斛属中验证其通用性和多态性,然后将石斛EST-SSR标记扩展到兰科植物材料中,为兰科植物的遗传多样性等相关研究奠定基础。
(1)本研究首先对杭州师范大学“浙江省药用植物种质改良与质量控制重点实验室”构建的铁皮石斛cDNA文库进行分析,对4075条EST序列进行搜索,得到了含有838个SSR位点的706条有效EST序列,最终成功开发110对EST-SSR引引物。在所有EST序列中的SSR位点中,二、三核苷酸重复类型最多,分别为37.83%和42.96%,四核苷酸重复类型其次(12.53%)；六核苷酸重复类型最少,仅占2.98%。
(2)成功开发的110对铁皮石斛EST-SSR引引物,位于108个EST序列上,其中99条序列在GenBank数据库中搜索到了基因功能注释。将110对引物用于在31种石斛属植物,进行了通用性及多态性分析,研究结果表明,石斛EST-SSR在石斛属通用性和多态性都很高,分别为47.93%和82.58%。
(3)将本实验室前期开发的金钗石斛EST-SSR与本研究开发的铁皮石斛EST-SSR用于兰科植物的研究,选择其中在石斛属中通用性>50%的引物,对兰科的15个属44个材料进行通用性与多态性的分析,结果表明通用性偏低,为25.05%,多态性较好,为55.38%。；将其中有代表性的10对引物扩增结果进行测序,共获得223条序列,对EST-SSR标记在兰科植物中从序列水平上进行分析,最后探讨了兰科植物各属间的系统进化关系。
实验结果表明：EST-SSR标记的开发有助于对兰科各属间遗传多样性和亲缘关系的研究,也为保护和开发宝贵的兰花种质资源提供科学的依据。
The Orchidaceae family has many Chinese traditional precious medical herbals and valuable ornamental plants,.The orchid industry has become an important part of international flower industry. Our country has some wild orchid resources with very high ornamental value and extremely high medicinal value. Due to excessive mining, this species is on the verge of extinction. The study and protection of the plants is urgently needed. Phylogenetic studies of Orchidaceae is not only directly related to the effective evaluation and rational use of existing resources but also more conducive to the cultivation of high-quality new varieties and the development of the orchid industry.
In order to conduct a more comprehensive phylogenetic study of orchids at molecular level, this study developed SSR markers from Dendrobium officinale, which is the representative species of the genus Dendrobium of orchids. The EST-SSR markers used to verify the transferability and polymorphism of Dendrobium, can be applied to the orchid plants to lay foundation for genetic diversity study of orchids.
(1)The study analyzed cDNA library of Dendrobium qfficinale established by Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants.4075EST sequences were screened and706EST sequences cont aining838microsatellite loci were obtained. Finally,110primer pairs for EST-SSRswere designed.Di-nucleotide repeats and Tri-nucleotide repeats were more t han other types(37.83%and42.96%respectively), followed by the type of Tetr a-nucleotide repeats(12.53%) and the Hexa-nucleotide types just accounting for2.98%of all the primers.
(2)110primer pairs fromthe Dendrobium officinale cDNA library are in108EST sequences.And among the108EST sequences, the gene functions of99sequences have been annotated in GenBank Database.110primer pairs were used to test the genomic DNA of31species of Dendrobiumto assess the transferability and polymorphism of the Dendrobium.The results showed that, the Dendrobium EST-SSR in the Dendrobium has high transferability and polymorphism of47.82%and88.56%, respectively.
(3)We took all the EST-SSR primers from Dendrobium nobile and Dendrobium officinale cDNA library constructed by our laboratory, which have high transferability and polymorphism to test genomic DNA of44individuals from15genera of orchid. Result from genera of orchid other than Dendrobium showed low transferability of25.05%and high polymorphism of55.38%. Then the polymorphism loci of10representative pairs of primers were sequenced,and a total of223sequences were received. Differences among the genera orchid were analyzed at sequence level.Finally intergeneric relationships of orchid were studied through clustering.
These results prove that EST-SSR markers are effective to reveal genetic diversity and relationship across Orchidaceae.
引文
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