| 苹果SSR-PCR反应体系的建立与优化 |
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| 引用本文： | 刘琳,孙晓帅,李乾,范书臣,时鸿迪,马钧,刘丹丹. 苹果SSR-PCR反应体系的建立与优化[J]. 中国农学通报, 2021, 37(1): 61-66. DOI: 10.11924/j.issn.1000-6850.casb20200100068 |
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| 作者姓名： | 刘琳 孙晓帅 李乾 范书臣 时鸿迪 马钧 刘丹丹 |
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| 作者单位： | 1.云南大学农学院,昆明 650091;2.西藏大学理学院,拉萨 850000;3.云南省农业科学院园艺作物所,昆明 650091 |
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| 基金项目： | 云南省科技计划“云南特色水果品质提升与优势品牌创建”(2019ZG002);国家自然科学基金“苹果砧木平邑甜茶PcG蛋白MhMSI1调控单性生殖的分子机理”(31660566) |
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| 摘 要： | 为建立苹果属植物SSR-PCR反应优化体系,采用L16(45)正交实验和退火温度梯度实验,分析反应体系中使用的模板、引物、Mg2+、dNTPs以及Top Taq酶浓度对扩增产物的影响,并对这5个因素进行4个水平不同浓度梯度的筛选和优化.结果表明,引物浓度对SSR-PCR反应体系的影响最大,通过综合分析最终确定SSR-P...
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| 关 键 词： | 苹果 SSR-PCR最佳反应体系 正交设计试验 种质资源 单因素试验 |
| 收稿时间： | 2020-01-20 |
Establishment and Optimization of SSR-PCR Reaction System of Apple Plants |
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| Liu Lin,Sun Xiaoshuai,Li Qian,Fan Shuchen,Shi Hongdi,Ma Jun,Liu Dandan. Establishment and Optimization of SSR-PCR Reaction System of Apple Plants[J]. Chinese Agricultural Science Bulletin, 2021, 37(1): 61-66. DOI: 10.11924/j.issn.1000-6850.casb20200100068 |
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| Authors: | Liu Lin Sun Xiaoshuai Li Qian Fan Shuchen Shi Hongdi Ma Jun Liu Dandan |
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| Affiliation: | 1.College of Agriculture, Yunnan University, Kunming 650091;2.College of Science, Tibet University, Lhasa 850000;3.Institute of Horticultural Crops, Yunnan Academy of Agricultural Sciences, Kunming 650091 |
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| Abstract: | To establish an optimal SSR-PCR system for apple plants, the L16(45) orthogonal test and the annealing temperature gradient experiment were used to analyze the effect of template concentration, primer concentration, Mg2+concentration, dNTPs concentration and Top Taq enzyme concentration in the SSR reaction system on the amplification products. Additionally, the above five factors were screened and optimized at 4 different levels. The results showed that the primer concentration factor had the greatest impact on the SSR-PCR reaction system. Though analysis, the optimal reaction system of 25 μL for SSR-PCR was finally determined to be as follows: 30-60 ng DNA template, 1.2 μL forward and reverse primer (5 μmol/L), 0.5 μL dNTPs (2.5 mmol/L), 0.5 μL Top Taq enzyme (2.5 U/μL) and 2.5 μL 10×Top Taq buffer (containing Mg2+). Finally, optimized reaction system was used for PCR amplification using 21 different apple plants, and the amplification products were about 100-200 bp displaying 2-3 alleles. The optimal SSR-PCR reaction system can provide a preliminary basis for the genetic variation research and molecular identification of genetic relationships in different populations of apple plants. |
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| Keywords: | apple SSR-PCR optimal reaction system orthogonal design experiment germplasm resource single factor test |
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