蒙古高原特有植物蒙古莸ISSR-PCR反应体系优化及引物筛选

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英文篇名：Optimization of ISSR-PCR Reaction System and Primer Screening of C. mongolica, an Endemic Plant of Mongolia Plateau 作者：吴敏 ; 贺晓 ; 贺一鸣 ; 李琪 ; 金鑫 英文作者：Wu Min;He Xiao;He Yiming;Li Qi;Jin Xin;College of Grassland, Resources and Environment, Inner Mongolia Agricultural University; 关键词：蒙古莸 ; ISSR ; 体系优化 ; 引物筛选 英文关键词：Caryopteris mongolica;;ISSR;;System optimization;;Primers screening 中文刊名：FZZW 英文刊名：Molecular Plant Breeding 机构：内蒙古农业大学草原与资源环境学院; 出版日期：2019-03-14 出版单位：分子植物育种 年：2019 期：v.17 基金：内蒙古自然科学基金(2011MS0515)资助 语种：中文; 页：FZZW201905027 页数：6 CN：05 ISSN：46-1068/S 分类号：209-214

摘要

建立并优化适用于蒙古莸的ISSR-PCR扩增反应体系并筛选出稳定的ISSR引物,进一步研究蒙古莸群体的遗传多样性水平。采用单因素试验设计方法,以蒙古莸基因组DNA为扩增模板,对dNTPs、引物、Mg~(2+)、Taq DNA聚合酶、模板DNA进行优化。以此为基础,筛选出扩增条带清晰且多态性好的有效引物,并确定各引物最佳退火温度,最后用呼和浩特大青山、乌海渡口村、赛罕塔拉柄扁桃保护区的24个蒙古莸野生品种为材料验证体系的稳定性。最佳反应体系为:在总体积20μL的反应体系中,含10×PCR Buffer 2.5μL,Mg~(2+)2 mmol/L,Taq DNA聚合酶1.5 U,dNTP 1.5 mmol/L,引物0.5μmol/L,模板DNA 15 ng。此外,从100条ISSR引物中筛选出15条清晰稳定的引物,并确定了引物各自的最佳退火温度。首次建立了蒙古莸ISSR-PCR最适反应体系,这一体系的建立可应用于后续蒙古莸遗传多样性的研究。
        It is to establish and optimize the amplification reaction system of ISSR-PCR adaptable to Caryopteris mongolica and screen out ISSR primers so as to further research the genetic diversity level of the Caryopteris mongolica group. This research adopts the single factor experimental design method, taking the DNA from Caryopteris mongolica genome as the amplification template of ISSR-PCR, and optimizing dNTPs, primers, Mg~(2+),Taq DNA polymerase, primersand template DNA. On the basis of it, screens out the effective primers with clear amplified bands and good polymorphism, determine the optimal annealing temperature of primers, and uses 24 wild Caryopteris mongolica breeds from Daqing Mountain, Wuhai, Saihan as materials to verify the stability of the system. The results revealed that the optimal reaction system is the reaction system of total volume of 20 μL,containing 10 ×PCR Buffer 12.5 μL, 2 mmol/L Mg~(2+), 1.5 U Taq DNA polymerase, 1.5 mmol/L dNTP, 2 mol/L primers, and 15 ng template DNA. Except that, 15 clear and stable primers are screened out from 100 ISSR primers and the optimal annealing temperatures of primers are determined respectively. It establishes the most suitable reaction system for Caryopteris mongolica ISSR-PCR, which can be applied to the successive research on Caryopteris mongolica genetic diversity.

引文

Arcade A.,Anselin F.,and Rampant P.F.,Lesage M.S.,P覾ques L.E.,and Prat D.,2000,Application of AFLP,RAPD and IS-SR markers to genetic mapping of European and Japanese larch,Theor.Appl.Genet.,100(2):299-307
    Fang D.,Krueger R.R.,and Roose M.L.,1998,Phylogenetic relationships among selected citrus germplasm accessions revealed by inter-simple sequence repeat(ISSR)markers,J.Am.Soc.Hortic.Sci.,123(4):612-617
    Feng F.J.,Wang F.Y.,and Liu T.,2004,The influence of the IS-SR-PCR experiment system on Pinus koraiensis Sieb.et Zucc,Zhiwuxue Tongbao(Chinese Bulletin of Botany),21(3):326-331(冯富娟,王凤友,刘彤,2004,红松ISSR-PCR实验系统影响因素,植物学通报,21(3):326-331)
    Liu B.L.,1985,Pollen morphology of the family verbenaceae in China,Zhiwu Yanjiu(Bulletin of Botanical Reseach),5(4):23-62(刘炳仑,1985,中国马鞭草科植物的花粉形态,植物研究,5(4):23-62)
    Wang X.J.,Li A.P.,Ning M.S.,and Zhang J.G.,2006,Study on the biological and ecological characteristics and economic value of the ecological shrub of Caryopteris mongolica,Ganhanqu Ziyuan Yu Huanjing(Journal of Arid Land and Environment),20(2):191-194(王晓江,李爱平,宁明世,张纪刚,2006,生态灌木蒙古莸的生物生态学特性及其经济价值评价,干旱区资源与环境,20(2):191-194)
    Wu M.,2018,The ISSR genetic diversity analysis on the Caryopteris mongolica an endemic plant in Mongolian Plateau,Thesis for M.S.,Inner Mongolia Agricultural University,Supervisor:He X.,pp.3-42(吴敏,2018,蒙古高原特有植物蒙古莸的ISSR遗传多样性研究,硕士学位论文,内蒙古农业大学,导师:贺晓,pp.3-42)
    Xie Y.H.,Xia D.A.,Jiang J.,and Lin P.,2005,Optimization for ISSR-PCR system of Fraxinus mandshurica Rupr.using orthogonal design,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),3(3):445-450(谢运海,夏德安,姜静,林萍,2005,利用正交设计优化水曲柳ISSR-PCR反应体系,分子植物育种,3(3):445-450)
    Yu Y.,Chen H.S.,and Ge X.J.,2003,Optimization of experiment condition sand primer screening with ISSR markers,Redai Yaredai Zhiwu Xuebao(Journal of Tropical and Subtropical Botany),11(1):15-19(余艳,陈海山,葛学军,2003,简单重复序列区间(ISSR)引物反应条件优化与筛选,热带亚热带植物学报,11(1):15-19)
    Zhao Y.Z.,1995,On floristic geographical distribution of Caryopteris mongholica,Actaentiarum Naturalium Universitatis Neimongol,26(2):195-197
    Zietkiewicz E.,Rafalski A.,and Labuda D.,1994,Genome fingerprinting by simple sequence repeat(SSR)-anchored polymerase chain reaction amplification,Genomics,20(2):176-183