油梨基因组DNA提取、SSR-PCR反应体系优化及引物筛选

摘要

旨在建立稳定可靠的油梨（Persea americana Mill）叶片DNA的提取方法和SSR-PCR反应体系及筛选出稳定的油梨SSR多态性引物，为开展油梨种质SSR分子标记提供遗传研究的基础。以油梨叶片为试材，比较3种油梨叶片DNA提取方法；利用L16（45）正交实验设计对油梨SSR-PCR反应体系进行优化；利用优化的反应体系筛选引物；同时，选取5对多态性引物对45份油梨种质进行PCR扩增，进一步检测该优化体系的稳定性。结果表明，常规2×CTAB法、改良2×CTAB法和植物DNA提取试剂盒法等3种DNA提取方法中，改良2×CTAB法对油梨基因组DNA的提取效果最佳；获得最优反应体系为：20μL总反应体系中，含约40 ng DNA模板、1.5 mmol/L Mg2+、0.15 mmol/L dNTPs、0.5 U Taq DNA聚合酶、0.5μmol/L引物；以此体系为基础进行引物筛选，从73对油梨SSR引物中筛选出了30对扩增条带清晰的多态性引物，说明该反应体系可用于油梨SSR标记的进一步研究；稳定性检测获得的谱带清晰，表明该优化反应体系是稳定可靠的。由此可见，改良的2×CTAB法可用于油梨叶片DNA的大量样本提取，优化后的SSR-PCR反应体系及筛选出的30对多态性引物可用于油梨SSR标记的进一步研究。%This study is to establish a stable and reliable DNA extraction method,optimize the SSR-PCR reaction system,and screen the stable polymorphism primers for avocado(Persea americana Mill)SSR for providing the genetic foundation to conduct the SSR molecular marker of germplasm in avocados. Taking avocado’leaves as the study material,3 avocado DNA extraction methods were compared,based on the L16(45)orthogonal experiment design,the SSR-PCR reaction system in avocados was optimized,and then by optimized reaction system the SSR primers were screened. To further test the stability of the optimized SSR-PCR system,the germplasms in 45 pieces of avocados were amplified by PCR using 5 pairs of polymorphism primers. The results showed that:among 3 DNA extraction methods of conventional 2×CTAB method,improved 2×CTAB method,and plant DNA kit method,the improved 2×CTAB method was the best regarding the extraction effect of avocado genomic DNA. The optimal SSR-PCR reaction system in avocados was:a total volume of 20μL containing 40 ng of genomic DNA,1.5 mmol/L Mg2+,0.15 mmol/L dNTPs,0.5 U Taq DNA polymerase,0.5μmol/L primer. Based on the above optimized reaction system,30 pairs of polymorphism primers with clear bands were screened from 73 SSR primers of avocados,indicating that the reaction system can be used for the further study of SSR markers in avocados. The bands of stability test were clear,showing that the optimized system was stable and reliable. Thus,improved 2×CTAB method can be used in DNA extraction of plentiful samples,and the optimized SSR-PCR reaction system and the 30 polymorphism primers can be utilized for the further study of SSR markers in avocados.