中国兰ISSR-PCR反应体系优化及引物筛选

Optimization and primer screening of ISSR-PCR reaction system for Chinese Orchids

【目的】优化中国兰的ISSR-PCR反应体系,并筛选适用于中国兰ISSR分析的候选引物,为ISSR分子标记在中国兰的辅助育种及亲缘关系和遗传多样性分析等提供技术参考。【方法】以6个中国兰品种为材料,采集其叶片样品,分别用研钵法和研磨仪法进行破碎研磨,比较两种方法提取DNA的效果,利用L25(53)正交试验和单因素试验对DNA模板量、引物浓度、2×Taq Master Mix添加量、循环数和退火温度进行优化,建立最佳ISSR-PCR反应体系,并从ISSR分子标记通用引物中筛选适用于中国兰的ISSR分析候选引物。【结果】研磨仪法提取的DNA浓度明显高于研钵研磨法,但二者提取的DNA质量均较好(OD_(260)/OD_(280)为1.7~2.0)。对ISSR-PCR反应体系扩增结果的影响程度排序为DNA模板量>引物浓度>2×Taq Master Mix添加量。综合考虑成本和DNA模板量,最佳ISSR-PCR反应体系(20.0μL):DNA模板10.0 ng、引物0.8μmol/L和2×Taq Master Mix 9.0μL。最佳循环数为35,最佳退火温度为49.6℃。基于上述优化结果,从100条引物中共筛选出42条适用于中国兰的ISSR候选引物。【结论】研磨仪法可有效提高中国兰基因组DNA的提取率和质量,且利用优化后的ISSR-PCR反应体系和扩增程序及筛选出的引物,扩增获得的条带清晰、稳定,多样性好,可用于中国兰的遗传多样性和亲缘关系等分析研究。

【Objective】The ISSR-PCR reaction system of Chinese orchids was optimized,and the candidate primers suitable for ISSR analysis in Chinese orchids were screened,which could provide technical reference for the analysis of ISSR molecular markers in auxiliary breeding,genetic relationship and genetic diversity of Chinese orchids.【Method】Six Chinese orchids cultivars were as experimental materials.The leaf samples were collected,then crushed and ground by the mortar grinding method and the grinding instrument method respectively.The effects of the two methods for extracting genomic DNA were compared,and the amount of DNA template,primer concentration,2×Taq Master Mix amount,the number of cycles and the annealing temperature were determined by L25(53)orthogonal test and single factor test.Then the optimal ISSR-PCR reaction system was established.The candidate primers for ISSR analysis of Chinese orchids were screened from ISSR molecular marker universal primers.【Result】The DNA concentration extracted by the grinding instrument method was higher than that of the mortar grinding method,but the DNA quality by the two extract methods was fine(OD260/OD280 was 1.7-2.0).The degree of influence on the amplification results of ISSR-PCR reaction system was ranked as:DNA template amount>primer concentration>2×Taq Master Mix amount.Considering the cost and amount of DNA template,the optimal ISSR-PCR reaction system(20.0μL):DNA template 10.0 ng,primer 0.8μmol/L,2×Taq Master Mix 9.0μL.The optimum number of cycles was 35 and the optimum annealing temperature 49.6℃.Based on the above optimization results,42 candidate primers suitable for the Chinese orchid ISSR analysis were screened from 100 primers.【Conclusion】The mortar grinding method can effectively improve the extraction rate and quality of genomic DNA,the amplified bands of the optimized ISSR-PCR reaction system,amplification program,and the selected primers are clear,stable and diverse.It can be applied in analytical research on genetic diversity and genetic relationship of Chinese orchids.