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[目的]为快速和准确地对鹧鸪茶种质材料进行ISSR分析提供有效引物。[方法]基因组DNA的提取采用改良的CTAB法:先用0.8%琼脂糖凝胶电泳检测DNA完整性,EB染色,以Lambda DNA/HindⅢ+Eco RIMarkers为参照,电泳结果用Gel Doc XR型凝胶成像分析系统下照相并记录。再用Biophotometer紫外分光光度计分别测定OD_(260)和OD_(280),并计算出OD_(260)/OD_(280)值,每份样品的OD_(260)/OD_(280)比值均要求在1.8～2.0范围内。测定每份样品DNA浓度(ng/μl),并将其稀释至20 ng/μl,分装后置-20℃保存。利用99个ISSR引物,对来自海南岛境内的10个居群中共20份鹧鸪茶种质材料进行PCR扩增,以筛选出适合于所有鹧鸪茶种质材料ISSR分析的有效引物。[结果]从99个供试引物中共筛选出15个多态性丰富、条带清晰且可重复性良好的有效引物。用筛选出的15个引物对66份鹧鸪茶种质材料进行ISSR-PCR扩增,均可获得带型丰富和清晰可辨的DNA指纹图谱;15个引物共扩增出286条DNA谱带,其中231条为多态性带,占总扩增带数的80.77%,平均每个引物扩增出19.1条谱带。[结论]所筛选的15条引物可以有效地应用于鹧鸪茶种质资源材料的ISSR分析。
[Objective] The study aimed to provide effective primers for ISSR analysis of Partridge tea germplasm material rapidly and accurately. [Method] The genomic DNA was extracted by modified CTAB method. The DNA integrity was detected by 0.8% agarose gel electrophoresis and EB staining. Lambda DNA / HindIII + Eco RIMarkers was used as a reference. Gel Doc XR gel Imaging analysis and recording system. Then OD_ (260) and OD_ (280) were measured by Biophotometer UV spectrophotometer and OD_ (260) / OD_ (280) values were calculated. The OD_ (260) / OD_ (280) 1.8 ~ 2.0 range. Determine the DNA concentration (ng / μl) for each sample and dilute it to 20 ng / μl and store aliquots at -20 ° C. Ninety ISSR primers were used to amplify 20 parts of Partridge tea germplasm collected from 10 populations in Hainan Island to screen out effective primers for ISSR analysis of all Partridge tea germplasm materials. [Result] Fifteen effective primers with abundant polymorphism, clear bands and good reproducibility were screened out of 99 primers. Fifteen primers were screened for ISSR-PCR amplification of 66 parts of Parthenocarpic germplasm with abundant and clearly identifiable DNA fingerprinting bands. A total of 286 DNA bands were amplified by 15 primers , Of which 231 were polymorphic bands, accounting for 80.77% of the total number of bands amplified, with an average of 19.1 bands per primer. [Conclusion] The 15 primers screened could be effectively applied to ISSR analysis of Partridge tea germplasm resources.