蒜头果ISSR-PCR反应体系建立及验证  

Establishment and verification of ISSR-PCR reaction system for Malania oleifera

作　　者：张鹏远 黄久妍 童海珍 胡博 余潇 雷小铃 王娟 ZHANG Peng-yuan;HUANG Jiu-yan;TONG Hai-zhen;HU Bo;YU Xiao;LEI Xiao-ling;WANG Juan(College of Life Sciences,Southwest Forestry University,Kunming,Yunnan 650224,China;Eco-development Academy,Southwest Forestry University,Kunming,Yunnan 650224,China)

机构地区：[1]西南林业大学生命科学学院,云南昆明650224 [2]西南林业大学绿色发展研究院,云南昆明650224

出　　处：《南方农业学报》2024年第3期834-845,共12页Journal of Southern Agriculture

基　　金：云南省重大科技专项(202002AA100007);云南省生物学质量工程项目(503190106);云南省万人计划“云岭产业技术领军人才”专项(2018-212)。

摘　　要：【目的】建立稳定的蒜头果ISSR-PCR反应体系,筛选出适用于蒜头果的多态性引物,为后续蒜头果遗传多样性研究及种质资源保护提供参考依据。【方法】采用单因素试验与L25(54)正交试验相结合的方法,对影响ISSR-PCR反应体系扩增效果的4个主要因素(模板DNA用量、引物浓度、d NTPs浓度、Taq DNA聚合酶)及反应循环数进行优化,确定最佳反应体系和条件。在此基础上筛选出适用于蒜头果的多态性引物,探究其最佳退火温度,并采集云南省境内3个野生居群10个蒜头果样品对优化的反应体系进行验证。【结果】蒜头果ISSR-PCR最佳反应体系(20.0μL):模板DNA 30 ng,ISSR引物0.3μmol/L,d NTPs 0.250 mmol/L,Taq DNA聚合酶1.00 U,dd H2O补足至20.0μL。ISSR最佳反应程序为30个循环。利用优化后的反应体系筛选出了8条引物(UBC825、UBC826、UBC827、UBC836、UBC844、UBC861、UBC834和UBC851)的最佳退火温度,分别为50.2、56.5、50.2、56.5、50.2、50.2、50.2和54.0℃,部分引物(如UBC827、UBC836、UBC861等)实测最佳退火温度与软件预测退火温度存在明显差异。采用优化后的ISSR-PCR反应体系及引物对10个蒜头果样品进行ISSR-PCR分子标记检测,结果显示建立的ISSR-PCR反应体系稳定可靠,不同采样点的蒜头果在遗传上相对保守,且10个蒜头果样品的遗传分化系数(Gst)为0.74,说明其74%的遗传多样性存在于居群间,且基因流(N_(m))为0.18,远小于1.00,说明发生遗传漂变的概率大,易发生遗传多样性降低及居群分化。【结论】通过优化蒜头果ISSR-PCR反应体系,建立可用于蒜头果ISSR-PCR分子标记扩增的稳定反应体系,可用于蒜头果种质资源保护与利用研究工作。【Objective】The purpose of the study was to establish a stable ISSR-PCR reaction system for Malania oleifera and to screen appropriate polymorphic primers for M.oleifera,so as to provide a reference basis for the subsequent study on the genetic diversity of M.oleifera and the conservation of germplasm resources.【Method】Single factor test combined with L25(54)orthogonal test was applied to optimize the four main factors(template DNA amount,primer concentration,dNTPs concentration,Taq DNA polymerase)and the number of reaction cycles affecting the amplification effect of the ISSR-PCR reaction system and to determine the optimal reaction conditions.On this basis,the polymorphic primers suitable for M.oleifera were screened,the optimal annealing temperature was explored,and the optimized reaction system was verified by collecting ten M.oleifera samples from three wild populations in Yunnan.【Result】The optimal ISSR-PCR reaction system for M.oleifera(in 20μL reaction system):30 ng of DNA,0.3μmol/L of primer,0.250 mmol/L of dNTPs,1.00 U of Taq DNA polymerase,and ddH2O replenished to 20.0μL.The optimal reaction program for ISSR was 30 cycles.The optimal annealing temperature of eight primers(UBC825,UBC826,UBC827,UBC836,UBC844,UBC861,UBC834 and UBC851)was screened out using the optimized reaction system at 50.2,56.5,50.2,56.5,50.2,50.2,50.2 and 54.0℃,respectively.The measured optimal annealing temperatures of some primers(such as UBC827,UBC836,UBC861)differed greatly from the predicted annealing temperatures of the software.Ten M.oleifera samples were tested for ISSR-PCR molecular markers using the optimized ISSR-PCR reaction system and primers.The results showed that the established ISSR-PCR reaction system was stable and reliable,and the samples of M.oleifera from different sampling sites were relatively conserved genetically.Additionally,the coefficient of genetic differentiation(Gst)of the ten M.oleifera samples was 0.74,indicating that 74%of the genetic diversity occurred among the populations,and the

关 键 词：蒜头果 ISSR-PCR 体系优化 验证 

分 类 号：S792.99[农业科学—林木遗传育种]