耐冬山茶基因组DNA的提取及CDDP引物的筛选
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摘要
根据山茶叶片次生代谢产物较多的特点,使用改良CTAB法提取耐冬山茶基因组DNA,所得基因组DNA纯度较高,满足分子实验要求。使用21条CDDP引物,在20μL包含2×Es Taq Master Mix(含染料)酶10μL、10 pmol·μL-1引物1μL、30 ng·μL-1DNA模板2μL及dd H2O 7μL的反应体系下对随机选择的样品进行扩增,得到11条多态性好,条带清晰的引物,为CDDP分子标记在山茶遗传多样性研究中的应用奠定了基础。
According to the characteristics of the camellia leaves witch are rich in secondary metabolites, using the modified CTAB method to extract its genomic DNA.The purity of genomic DNA is high enough to meet the requirements of molecular experiments. Twenty-one CDDP primers are screened under the system with 10μL of 2×Es Taq Master Mix ferment(Containing Dyes),1μL of 10 pmo L·μL-1 primer, 2μL of 30 ng·μL-1 DNA template and 7μL of dd H2 O. Eleven primers with rich polymorphism and clear bands were obtained. This study laid a foundation for the application of CDDP molecular markers in the study of genetic diversity of camellia.
According to the characteristics of the camellia leaves witch are rich in secondary metabolites, using the modified CTAB method to extract its genomic DNA.The purity of genomic DNA is high enough to meet the requirements of molecular experiments. Twenty-one CDDP primers are screened under the system with 10μL of 2×Es Taq Master Mix ferment(Containing Dyes),1μL of 10 pmo L·μL-1 primer, 2μL of 30 ng·μL-1 DNA template and 7μL of dd H2 O. Eleven primers with rich polymorphism and clear bands were obtained. This study laid a foundation for the application of CDDP molecular markers in the study of genetic diversity of camellia.
引文
[1]王仁卿,张淑萍,石竹,李力.青岛耐冬山茶多样性特征及其保护[J].山东林业科技,1998,116(3):1-4.
[2]刘婵,王博,段青,黄海泉.滇山茶基因组DNA不同提取方法效果比较[J].江苏农业科学,2010,166(6):49-50.
[3]陈林杨,宋敏舒,查红光,李志敏.一种改良的植物基因组DNA通用提取方法[J].植物分类与资源学报,2014,36(3):375-380.
[4]熊发泉,蒋菁,钟瑞春,韩柱强,贺梁琼,李忠,庄伟建,唐荣华.两种新型目标分子标记技术——CDDP与PAAP.植物生理学通讯[J].2010,46(9):871-875.
[5]COLLARDB C Y,MACKILL D J.Conserved DNAderived polymorphism(CDDP):a simple and novel method for generating DNA markers in plants[J].Plant Molecular Biology Reporter,2009,27(4):558-562.
[6]POCZAI P,VARGA I,BELL N E,et al.Genetic diversityassessment of bittersweet(Solanum dulcamara,Solanaceae)germplasm using conserved DNA-derived polymorphism and intron-targeting markers[J].Annals of Applied Biology,2011,159(1):141-153.
[7]李莹莹,郑成淑.利用CDDP标记的菏泽牡丹品种资源的遗传多样性[J].中国农业科学,2013,46(13):2739-2750.
[8]王小文.基于CDDP标记的不同花色牡丹的遗传关系分析[D].泰安:山东农业大学,2015:1-36.
[9]李田,郭俊娥,郑成淑,孙霞,孙宪芝.菊花品种的遗传多样性分析及CDDP指纹图谱构建[J].北京林业大学学报.2014,36(4):95-100.
[10]林立,胡仲义,李纪元,祝志勇,倪穗.10个山茶岛屿天然居群的遗传多样性分析[J].园艺学报,2012,39(8):1531-1538.
[11]王奎玲,牟少华,刘庆超,唐启和,刘庆华.部分耐冬山茶栽培品种的AFLP分析[J].中国农业科学,2011,44(3):651-656.
[12]李力,王仁卿,王中仁,王少萍,李书韦华,青岛耐冬山茶的多样性(II)——居群的遗传多样性分析[J].生物多样性,1996,4(1):1-6.
[13]Matasyoh L G,Wachira F N,Kinyua M G,et al.Leaf storage conditions and genomic DNA isolation efficiency in Ocimum gratissimum from Kenya[J].African Journal of Biotechnology,2008,7(5):557-564.
[14]李承哲,刘占林,杨红超,黄安玲,刘芳.改良CTAB法提取鸢尾属植物DNA[J].现代农业科技,2011,(8):31-34.
[2]刘婵,王博,段青,黄海泉.滇山茶基因组DNA不同提取方法效果比较[J].江苏农业科学,2010,166(6):49-50.
[3]陈林杨,宋敏舒,查红光,李志敏.一种改良的植物基因组DNA通用提取方法[J].植物分类与资源学报,2014,36(3):375-380.
[4]熊发泉,蒋菁,钟瑞春,韩柱强,贺梁琼,李忠,庄伟建,唐荣华.两种新型目标分子标记技术——CDDP与PAAP.植物生理学通讯[J].2010,46(9):871-875.
[5]COLLARDB C Y,MACKILL D J.Conserved DNAderived polymorphism(CDDP):a simple and novel method for generating DNA markers in plants[J].Plant Molecular Biology Reporter,2009,27(4):558-562.
[6]POCZAI P,VARGA I,BELL N E,et al.Genetic diversityassessment of bittersweet(Solanum dulcamara,Solanaceae)germplasm using conserved DNA-derived polymorphism and intron-targeting markers[J].Annals of Applied Biology,2011,159(1):141-153.
[7]李莹莹,郑成淑.利用CDDP标记的菏泽牡丹品种资源的遗传多样性[J].中国农业科学,2013,46(13):2739-2750.
[8]王小文.基于CDDP标记的不同花色牡丹的遗传关系分析[D].泰安:山东农业大学,2015:1-36.
[9]李田,郭俊娥,郑成淑,孙霞,孙宪芝.菊花品种的遗传多样性分析及CDDP指纹图谱构建[J].北京林业大学学报.2014,36(4):95-100.
[10]林立,胡仲义,李纪元,祝志勇,倪穗.10个山茶岛屿天然居群的遗传多样性分析[J].园艺学报,2012,39(8):1531-1538.
[11]王奎玲,牟少华,刘庆超,唐启和,刘庆华.部分耐冬山茶栽培品种的AFLP分析[J].中国农业科学,2011,44(3):651-656.
[12]李力,王仁卿,王中仁,王少萍,李书韦华,青岛耐冬山茶的多样性(II)——居群的遗传多样性分析[J].生物多样性,1996,4(1):1-6.
[13]Matasyoh L G,Wachira F N,Kinyua M G,et al.Leaf storage conditions and genomic DNA isolation efficiency in Ocimum gratissimum from Kenya[J].African Journal of Biotechnology,2008,7(5):557-564.
[14]李承哲,刘占林,杨红超,黄安玲,刘芳.改良CTAB法提取鸢尾属植物DNA[J].现代农业科技,2011,(8):31-34.