2023年5月31日 星期三
铁线莲属植物ISSR-PCR反应体系优化及遗传多样性分析
Optimization of ISSR-PCR reaction system and analysis on genetic diversity of Clematis plants
2019年 第28卷 第2期 页码[42-48]    下载全文[2.9MB]  
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25.0 μL,包括40 ng·μL-1 模板DNA 1.0 μL、0.4 μmol·L-1引物1.0 μL、0.15 mmol·L-1 dNTPs 2.0 μL、10×Ex Taq Buffer(Mg2+ Plus) 2.0 μL、TaKaRa Ex Taq酶0.2 μL和ddH2O 18.8 μL。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。

Abstract

 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 (Mg2+ Plus), 0.2 μL of TaKaRa Ex Taq polymerase, 18.8 μL of ddH2O. 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.

关键词铁线莲属; ISSR-PCR; 反应体系优化; 遗传多样性
Key wordsClematis Linn.; ISSR-PCR; reaction system optimization; genetic diversity
作者余伟军1, 姚红1, 孙瑞琦1, 王桂青1, 钟淮钦2, 黄敏玲2, 曾黎辉1
所在单位1. 福建农林大学园艺学院, 福建 福州 350002; 2. 福建省农业科学院作物研究所, 福建 福州 350013
点击量1692
下载次数746
基金项目福建农林大学科技创新项目(KFA17352A); 福建省自然科学基金项目(2016J01109)