海南油茶SCoT反应体系建立与多态性引物筛选  

Establishment of SCoT Reaction System and Screening of Polymorphic Primers for Tea-oil Camellia Resource in Hainan

作　　者：戚华沙 谢秋霞 林基忠 陈加利 王春梅 郑道君 王健[2] Qi Huasha;Xie Qiuxia;Lin Jizhong;Chen Jiali;Wang Chunmei;Zheng Daojun;Wang Jian(The Key Laboratory of Tropic Special Economic Plant Innovation and Utilization,National Germplasm Resource Chengmai Observation and Experiment Station,Institute of Tropical Horticulture Research,Hainan Academy of Agricultural Sciences,Haikou,571100;School of Horticulture,Hainan University,Haikou,571100)

机构地区：[1]海南省农业科学院热带园艺研究所,国家种质资源澄迈观测实验站,海南省特种经济植物种质资源创新利用重点实验室,海口571100 [2]海南大学园艺学院,海口571100

出　　处：《分子植物育种》2023年第20期6784-6792,共9页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31860082);海南省省属科研院所专项(KYYS-2021-27);海南省省属科研院所技术开发创新项目(SQKY2022-004);海南省农业科学院热带种质资源学重点学科建设项目(202002)共同资助。

摘　　要：SCoT-PCR是一种单引物扩增分子标记。为了将SCoT分子标记应用于海南油茶的分子鉴定和遗传多样性评价中,本研究采用单因素试验和正交试验结合方法,分析模板DNA、dNTPs、Taq DNA聚合酶及引物4种因素对海南油茶SCoT-PCR扩增结果的影响,构建海南油茶SCoT-PCR体系,并筛选多态性引物。单因素试验结果表明:DNA浓度对海南油茶扩增效率影响不大,高浓度dNTPs利于扩增产物,而Taq酶和引物扩增高效率偏于中浓度。正交试验结果表明:各因素对海南油茶SCoT-PCR扩增影响大小依次为引物>Taq DNA聚合酶>dNTPs>模板DNA;总体系为20μL时,最佳反应体系中模板DNA用量为30 ng,dNTPs浓度为0.30 mmol/L,引物浓度为0.60μmol/L,Taq DNA聚合酶用量为1.00 U。利用构建的SCoT-pcr体系对80条SCoT单引物进行筛选,最终筛选出16条SCoT条带清晰的多态性引物,多态性比率平均值达到76.25%。本试验结果可为海南油茶的遗传多样性分析和种质资源鉴定等研究提供参考数据。SCoT-PCR is a single-primer amplification molecular marker.In order to apply SCoT molecular markers in molecular identification and genetic diversity evaluation of tea-oil Camellia resource in Hainan,the effects of template DNA,dNTPs,Taq DNA polymerase and primers on SCOT-PCR amplification results of tea-oil Camellia resource in Hainan were analyzed by the combination of single factor test and orthogonal test.The SCoT-PCR system of tea-oil Camellia resource in Hainan was constructed and the polymorphic primers were screened.The single factor test results showed that the DNA concentration had little effect on the amplification efficiency of tea-oil Camellia resource in Hainan,high concentration of dNTPs was beneficial to the amplified product,while the high amplification efficiency of Taq enzyme and primers was biased towards the middle concentration.The results of the orthogonal experiment showed that the influences of various factors on the SCoT-PCR amplification of tea-oil Camellia resource in Hainan were in the following order:primers>Taq DNA polymerase>dNTPs>template DNA;when the total system was 20μL,the amount of template DNA in the optimal reaction system was 30 ng,the dNTPs concentration was 0.30 mmol/L,the primer concentration was 0.60μmol/L,and the amount of Taq DNA polymerase was 1.00 U.Using the constructed SCoT-PCR system,80 SCoT primers were screened to screen out 16 SCoT polymorphic primers with clear SCoT bands,and the average polymorphism ratio reached 76.25%.The results of this experiment can provide a reference for the analysis of genetic diversity and identification of germplasm resources of tea-oil Camellia resource in Hainan.

关 键 词：海南油茶 SCoT 反应体系 引物筛选 

分 类 号：S794.4[农业科学—林木遗传育种]