藏茴香ISSR-PCR反应体系的优化

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英文篇名：The Optimization of ISSR-PCR Reaction System for Carum carvi 作者：田霞 ; 周仙莉 ; 沈宁东 ; 朱惠琴 英文作者：Tian Xia;Zhou Xianli;Shen Ningdong;Zhu Huiqin;College of Agriculture and Animal Husbandry, Qinghai University; 关键词：藏茴香 ; ISSR-PCR ; 体系优化 ; 正交设计 ; 单因素试验 英文关键词：Carum carvi;;ISSR-PCR;;System optimization;;Orthogonal design;;Single factor tests 中文刊名：FZZW 英文刊名：Molecular Plant Breeding 机构：青海大学农牧学院; 出版日期：2019-04-28 出版单位：分子植物育种 年：2019 期：v.17 基金：国家自然基金项目(31560083);; 青海省科技厅基础项目(2018-ZJ-752)共同资助 语种：中文; 页：FZZW201908023 页数：8 CN：08 ISSN：46-1068/S 分类号：180-187

摘要

本次试验以藏茴香叶片为试验材料,采用单因子设计和L_(16)(4~5)正交设计试验相结合的方法,对影响藏茴香ISSR-PCR扩增结果的Taq酶、引物、Mg~(2+)、dNTPs和DNA用量进行优化,并在此基础上,筛选出最适的循环次数和退火温度。结果表明正交试验各因素显著性为Mg~(2+)浓度>dNTPs浓度>Taq酶>DNA用量>引物浓度。在25μL的反应体系中,Taq酶1.0 U、引物0.6μmol/L、Mg~(2+)1.5 mmol/L、dNTPs 0.20 mmol/L、DNA 40 ng为最佳用量,最佳退火温度为55℃,最佳循环次数为30。用不同的引物和不同的藏茴香种质对优化的ISSR-PCR反应体系进行验证,表明确定的优化体系具有稳定性高、重复性好等特性。该反应体系的建立为藏茴香ISSR标记的开发、种质资源的鉴定、遗传多样性分析和分子标记辅助选择育种等提供科学依据。
        In this study, the leaves of Carum carvi were used as the materials, and the single factor design and L_(16)(4~5)orthogonal design were used to optimize the amount of Taq enzyme, primers, Mg~(2 +), dNTPs and DNA used in ISSR-PCR amplification of Carum carvi. On this basis, the optimum cycle times and annealing temperature were selected. The results showed that the significance of the factors in orthogonal test were Mg~(2+)>dNTPs>Taq DNA polymerase>template DNA>primer. The optimized 25 μL ISSR-PCR system included Mg~(2+)1.5 mmol/L, dNTPs0.20 mmol/L, Taq DNA polymerase 1.0 U, primer 0.6 μmol/L and template DNA 40 ng. The best annealing temperature was 55℃ and the best cycle times was 30. The optimized system was verified by different primers and template DNA from different germplasm resources of Carum carvi, which indicated that the system was stable and reproducible. The ISSR-PCR optimized in this study could provide theoretical basis for ISSR marker development,germplasm identification, evaluation of genetic diversity and molecular assisted breeding for Carum carvi.

引文

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