响应面法优化余甘子种质资源ISSR反应体系

王建超 何银莺 黄旭萍 沈朝贵 陈发兴 郭林榕

摘要：【目的】優化余甘子种质资源ISSR-PCR反应体系，为余甘子种质资源遗传多样性及亲缘关系研究提供基础。【方法】以缅甸、印度、广东、云南、福建等5份来源小同的余甘子种质资源的基因组DNA组成混合的DNA模板，综合单因素试验和响应面分析法，分析引物浓度、2×Taq Master Mix添加量、DNA模板量、退火温度等反应条件对IS SR-PCR反应体系的影响，优化建立余甘子IS SR-PCR反应体系。【结果】引物浓度和2×Taq MasterMix添加量对扩增效果有较大影响，DNA模板量影响较小;引物浓度和DNA模板量交互作用明显;余甘子ISSR反应体系为引物浓度0.4 umol L-l，2×Taq Master Mix添加量l3 uL， DNA模板量30 ng，扩增结果与响应面分析模型理论值相对误差仪为9.39%;退火温度为50.5- 52.7℃时，随着温度的升高，条带质量变好，数量变多，退火温度为52.7℃时可获得多样性好的清晰条带。【结论】获得ISSR-PCR反应体系为引物浓度0.4 umoI·L-1，2×Taq MasterMix添加量13 UL， DNA模板量30 ng，退火温度52.7℃，扩增循环数35循环，扩增获得的条带清晰、稳定，多样性好，该体系适于余甘子种质资源的遗传多样性和亲缘关系等分析研究

关键词：余甘子;ISSR-PCR;响应面;体系优化

中图分类号：S 682.310.36

文献标志码：A

文章编号：1008-0384（ 2021） 07-075907

Response Surface Optimization of ISSR-PCR Reaction for Genetic Study on

Phyllanthus Emblica

WANG Jianchao1.2. HE Yinying2. HUANG Xuping2， SHEN Chaoguj1， CHEN Faxing2. Guo Linrong1*

（l.Fruit Research Institute， Fujian Academy ofAgricultural Sciences. Fuzhou， Fujian 350013，China：

2.College ofHorticulture， Fujian Agriculture and Forestrv University. Fuzhou， Fujian 350002. China）

Abstract： 【Objective】 ISSR-PCR reaction system for genetic study on Phyllanthus emblica germplasms was optimized【Methods】 A mixed DNA template composed of genomic DNA ofP emblica germplasms came from Myanmar. India.Guangdong， Yunnan， and Fujian was obtained. The single factor test and response surface analysis were used to optimize theISSR-PCR reaction conditions including primer concentration. amount of 2×Taq Master Mix. DNA template amount， andannealing temperature.【Result】 The primer concentration and addition amount of 2×Taq Master Mix had a greater impacton the amplification than did the DNA template amount. A significant interaction between the primer concentration and DNAtemplate amount was found. The optimized system with a primer concentration of 0.4 ymoI·L-1.a 2×Taq Master Mix of13 UL， and a DNA template concentration of 30 ng was established that achieved a low relative error of 9.39% in predicting thetheoretical response. Within the annealing temperature between 50.5 ℃ and 52.7 ℃. increasing temperature improved thenumber and quality of bands. When the annealing temperature is 50.5-52.7.C，the number of strips increases. and the quality ofthe strips becomes better with the increase of temperature. The annealing temperature is 52.7℃to obtain good diversity andclear bands. 【Conclusion】 The optimized ISSR-PCR reaction system was established applying a primer concentration of0.4 umoI-L-1，a 2×Taq Master Mix ofl3L，aDNA template concentration of 30 ng at the annealing temperature of 52.7℃for35 amplification cycles. The methodology could be used to study the genetic diversity and relationship of P. emblicagermplasms .

1.3统计分析

采用Excel 2016、Design Expert 8.0.6等软件进行统计分析，其中Design Expert 8.0.6进行响应面试验设计、数据分析和模型的建立。

2结果与分析

2.1单因素对ISSR反应体系的影响

单因素试验的扩增产物经琼脂糖凝胶电泳结果如图2，可见，引物浓度与2×Taq Master Mix添加量对反应体系的影响较大。引物浓度偏高或偏低都会影响扩增结果，引物浓度过高会引起引物与DNA模板错配和非特异性产物扩增，增加产生二聚体的概率，引物浓度偏低则不易测出扩增位点，本研究引物浓度为0.30～0.40 umol.L-l时电泳效果较好。……