铁线莲属植物ISSR-PCR反应体系优化及遗传多样性分析
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摘要
对铁线莲属(Clematis Linn.)植物基因组DNA的ISSR-PCR反应体系进行了优化,并采用ISSR分子标记对32个铁线莲属植物(包括8个野生种、1个变种和23个品种)及2个近缘属植物的遗传多样性进行研究。结果显示:铁线莲属植物基因组DNA ISSR-PCR最佳反应体系的总体积为25.0μL,包括40 ng·μL~(-1)模板DNA 1.0μL、0.4μmol·L~(-1)引物1.0μL、0.15 mmol·L~(-1)dNTPs 2.0μL、10×Ex Taq Buffer(Mg~(2+) Plus) 2.0μL、TaKaRa Ex Taq酶0.2μL和ddH_2O 18.8μL。对32个铁线莲属植物及2个近缘属植物基因组DNA的ISSR-PCR分析结果显示:共扩增出172个条带(位点),平均每条引物扩增出13.2个位点,其中,多态性位点168个,平均每条引物扩增出12.9个多态性位点,平均多态性位点百分率为98.2%。聚类分析结果显示:32个铁线莲属植物及2个近缘属植物的遗传距离在0.22～1.21之间。在遗传距离1.02处,将32个铁线莲属植物分为3个大组,‘杰出’(‘Superba’)单独聚为1个大组,‘斯托尔韦克’(‘Stolwijk Gold’)和‘柠檬之梦’(‘Lemon Dream’)聚为1个大组,其余植物聚为第3大组;在遗传距离0.92处,将第3大组进一步划分为3个亚组,第1亚组包括‘阿迪森’(‘Addisonii’)、‘帕斯卡’(‘Pascal’)和‘樱桃唇’(‘Cherry Lip’),第2亚组包括短柱铁线莲(C.cadmia Buch.-Ham. ex Wall.)、灰叶铁线莲[C.canescens(Turcz.) W. T. Wang et M. C. Chang]、毛蕊铁线莲(C.lasiandra Maxim.)、曲柄铁线莲(C.repens Finet et Gagnep.)、‘铃儿响叮当’(‘Jingle Bells’)和‘雀斑’(‘Freckles’),第3亚组包括吴兴铁线莲(C.huchouensis Tamura)等20个铁线莲属植物;在遗传距离0.77处,将第3亚组划分为7个小组。研究结果表明:ISSR分子标记能够应用于铁线莲属植物的遗传多样性分析。
ISSR-PCR reaction system of genomic DNA of Clematis Linn. plants was optimized, and genetic diversity of 32 plants of Clematis(including 8 wild species, 1 variety, and 23 cultivars) and 2 plants of related genera was researched by using ISSR molecular marker. The results show that total volume of optimal ISSR-PCR reaction system of genomic DNA from Clematis plants is 25.0 μL, including 1.0 μL of 40 ng·μL~(-1) template DNA, 1.0 μL of 0.4 μmol·L~(-1) primer, 2.0 μL of 0.15 mmol·L~(-1) dNTPs, 2.0 μL of Ex Taq Buffer(Mg~(2+) Plus), 0.2 μL of TaKaRa Ex Taq polymerase, 18.8 μL of ddH_2O. The result of ISSR-PCR analysis on genomic DNA from 32 plants of Clematis and 2 plants of related genera shows that 172 bands(loci) are amplified with average of 13.2 loci per primer, in which, there are 168 polymorphic loci with average of 12.9 polymorphic loci per primer and average percentage of polymorphic loci of 98.2%. The cluster analysis result shows that genetic distance of 32 plants of Clematis and 2 plants of related genera is 0.22-1.21. At the genetic distance of 1.02, 32 plants of Clematis are divided into three major groups, ‘Superba' is clustered into one major group, ‘Stolwijk Gold' and ‘Lemon Dream' are clustered into one major group, the rest are clustered into the third major group. At the genetic distance of 0.92, the third major group is further divided into three subgroups, the first subgroup includes ‘Addisonii', ‘Pascal', and ‘Cherry Lip', and the second subgroup does C. cadmia Buch.-Ham. ex Wall., C. canescens(Turcz.) W. T. Wang et M. C. Chang, C. lasiandra Maxim., C. repens Finet et Gagnep., ‘Jingle Bells', and ‘Freckles', the third subgroup does 20 plants of Clematis such as C. huchouensis Tamura. At the genetic distance of 0.77, the third subgroup is divided into seven small groups. It is suggested that ISSR molecular marker can be applied to the genetic diversity analysis on Clematis plants.
ISSR-PCR reaction system of genomic DNA of Clematis Linn. plants was optimized, and genetic diversity of 32 plants of Clematis(including 8 wild species, 1 variety, and 23 cultivars) and 2 plants of related genera was researched by using ISSR molecular marker. The results show that total volume of optimal ISSR-PCR reaction system of genomic DNA from Clematis plants is 25.0 μL, including 1.0 μL of 40 ng·μL~(-1) template DNA, 1.0 μL of 0.4 μmol·L~(-1) primer, 2.0 μL of 0.15 mmol·L~(-1) dNTPs, 2.0 μL of Ex Taq Buffer(Mg~(2+) Plus), 0.2 μL of TaKaRa Ex Taq polymerase, 18.8 μL of ddH_2O. The result of ISSR-PCR analysis on genomic DNA from 32 plants of Clematis and 2 plants of related genera shows that 172 bands(loci) are amplified with average of 13.2 loci per primer, in which, there are 168 polymorphic loci with average of 12.9 polymorphic loci per primer and average percentage of polymorphic loci of 98.2%. The cluster analysis result shows that genetic distance of 32 plants of Clematis and 2 plants of related genera is 0.22-1.21. At the genetic distance of 1.02, 32 plants of Clematis are divided into three major groups, ‘Superba' is clustered into one major group, ‘Stolwijk Gold' and ‘Lemon Dream' are clustered into one major group, the rest are clustered into the third major group. At the genetic distance of 0.92, the third major group is further divided into three subgroups, the first subgroup includes ‘Addisonii', ‘Pascal', and ‘Cherry Lip', and the second subgroup does C. cadmia Buch.-Ham. ex Wall., C. canescens(Turcz.) W. T. Wang et M. C. Chang, C. lasiandra Maxim., C. repens Finet et Gagnep., ‘Jingle Bells', and ‘Freckles', the third subgroup does 20 plants of Clematis such as C. huchouensis Tamura. At the genetic distance of 0.77, the third subgroup is divided into seven small groups. It is suggested that ISSR molecular marker can be applied to the genetic diversity analysis on Clematis plants.
引文
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[10] 孙诚,谢磊,李良千.铁线莲属尾叶铁线莲组(毛茛科)基于形态学证据的分支系统学[J].植物学通报,2007,24(1):87-98.
[11] TSUMURA Y,OHBA K,STRAUSS S H.Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)[J].Theoretical and Applied Genetics,1996,92(1):40-45.
[12] 张立荣,徐大庆,刘大群.SSR和ISSR分子标记及其在植物遗传育种研究中的应用[J].河北农业大学学报,2002,25(1):90-94.
[13] 赵孟良,韩睿,马胜超,等.菊芋基因组DNA提取方法的比较[J].中国蔬菜,2012(14):41-44.
[14] 米米童.铁线莲栽培12月计划[M].武汉:湖北科学技术出版社,2018:4-8.
[15] 韦荣昌,李虹,蒋建刚,等.多倍体无籽罗汉果及其亲本遗传背景的ISSR分析[J].园艺学报,2012,39(2):387-394.
[16] WANG W-T.A revision of Clematis sect.Viticella (Ranunculaceae)[J].广西植物,2007,27(1):1-28.
[17] 谢磊.铁线莲属尾叶铁线莲组的修订——兼论铁线莲属基于花粉形态学证据的系统发育[D].北京:中国科学院植物研究所,2005:29-35.
[18] WANG W-T,LI L-Q.A revision of Clematis sect.Fruticella (Ranunculaceae)[J].植物分类学报,2005,43(3):193-209.
[19] WANG W-T.A revision of Clematis sect.Clematis (Ranunculaceae)[J].植物分类学报,2003,41(1):1-62.
[2] 陈文允,普春霞.云南省铁线莲属药用资源调查[J].云南中医学院学报,2006,29(1):31-33.
[3] 王文采,李良千.铁线莲属一新分类系统[J].植物分类学报,2005,43(5):431-488.
[4] 江南,管开云,王仲朗.云南铁线莲属植物地理分布及区系特征[J].云南植物研究,2007,29(2):145-154.
[5] 中国科学院中国植物志编辑委员会.中国植物志:第二十八卷[M].北京:科学出版社,1999.
[6] 王璇,张玉华,蔡少青,等.来源于铁线莲属透骨草的鉴别研究[J].中国中药杂志,1998,23(4):198-254.
[7] YANG Q E.Cytology of ten species in Anemone,one in Anemoclema and six in Clematis (Trib.Anemoneae,Ranunculaceae) from China[J].植物分类学报,2002,40(5):396-405.
[8] 胡玉涛,周政昆.9种铁线莲属药用植物的比较鉴别[J].基层中药杂志,2002,16(4):36-37.
[9] SHI J H,LI L Q.Leaf epidermal feature in Clematis (Ranunculaceae) with reference to its systematic significance[J].植物学报,2003,45(3):257-268.
[10] 孙诚,谢磊,李良千.铁线莲属尾叶铁线莲组(毛茛科)基于形态学证据的分支系统学[J].植物学通报,2007,24(1):87-98.
[11] TSUMURA Y,OHBA K,STRAUSS S H.Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)[J].Theoretical and Applied Genetics,1996,92(1):40-45.
[12] 张立荣,徐大庆,刘大群.SSR和ISSR分子标记及其在植物遗传育种研究中的应用[J].河北农业大学学报,2002,25(1):90-94.
[13] 赵孟良,韩睿,马胜超,等.菊芋基因组DNA提取方法的比较[J].中国蔬菜,2012(14):41-44.
[14] 米米童.铁线莲栽培12月计划[M].武汉:湖北科学技术出版社,2018:4-8.
[15] 韦荣昌,李虹,蒋建刚,等.多倍体无籽罗汉果及其亲本遗传背景的ISSR分析[J].园艺学报,2012,39(2):387-394.
[16] WANG W-T.A revision of Clematis sect.Viticella (Ranunculaceae)[J].广西植物,2007,27(1):1-28.
[17] 谢磊.铁线莲属尾叶铁线莲组的修订——兼论铁线莲属基于花粉形态学证据的系统发育[D].北京:中国科学院植物研究所,2005:29-35.
[18] WANG W-T,LI L-Q.A revision of Clematis sect.Fruticella (Ranunculaceae)[J].植物分类学报,2005,43(3):193-209.
[19] WANG W-T.A revision of Clematis sect.Clematis (Ranunculaceae)[J].植物分类学报,2003,41(1):1-62.