论文部分内容阅读
为建立并优化适合花生的EcoR Ⅰ和Mse Ⅰ内切酶组合的AFLP标记技术体系,本试验对花生基因组DNA大样提取方法、双酶切反应、连接体系、预扩增和选择性扩增等影响因素进行反复调试与优化,并对适合花生AFLP分析的引物组合(E/M)进行多态性筛选。结果表明,高质量的模板DNA提取采用改进的SDS-CTAB法,DNA样品的浓度在150~200 ng·μL~(-1),37℃双酶切3.0 h,接头浓度为50pmol·μL~(-1),T4-DNA连接酶浓度为10 U·μL~(-1),16℃连接10 h。以2×Es Taq MasterMix(含有Es Taq DNA Polymerase,3 mmol·L~(-1)MgCl_2和400μmol·L~(-1)d NTP)作为PCR反应原料,其中,预扩增反应体系为50μL,预扩增引物(E00和M00)的浓度为50 ng·μL~(-1),预扩增产物最适稀释倍数为20倍;选择性扩增反应体系为20μL,扩增产物中加入10μL Loading Buffer,经95℃变性10 min后,立刻转移到冰浴中冷却备用。最终,从225对AFLP引物组合中,筛选出稳定且多态性丰富的42对引物组合,可用于后期作图群体基因型检测和种质资源遗传多样性分析。本研究结果为下一步构建花生高密度遗传连锁图谱和开展分子标记辅助育种奠定了基础。
In order to establish and optimize the AFLP labeling system for EcoR Ⅰ and Mse Ⅰ endonuclease combinations suitable for peanut, this experiment studied the extraction method of peanut genomic DNA, double enzyme digestion reaction, ligation system, pre-amplification and selective amplification The influencing factors were repeatedly debugged and optimized, and the primer combinations (E / M) suitable for AFLP analysis of peanut were screened for polymorphism. The results showed that high quality template DNA extraction using improved SDS-CTAB method, DNA sample concentration of 150 ~ 200 ng · μL ~ (-1), double digestion at 37 ℃ 3.0 h, the linker concentration of 50 pmol·L ~ ( -1). The concentration of T4-DNA ligase was 10 U · μL ~ (-1) and was connected at 16 ℃ for 10 h. 2 × Es Taq MasterMix (containing Es Taq DNA Polymerase, 3 mmol·L -1 MgCl_2 and 400 μmol·L -1 dNTPs) was used as the PCR reaction material, in which the pre-amplification reaction system was 50 μL, The concentration of pre-amplification primers (E00 and M00) was 50 ng · μL -1, the optimum dilution of pre-amplification products was 20-fold, selective amplification reaction was 20 μL, and 10 μL Loading Buffer, after denaturation at 95 ℃ 10 min, immediately transferred to the ice bath to cool standby. Finally, from 225 pairs of AFLP primer combinations, 42 pairs of primer combinations with stable and abundant polymorphisms were screened for genotyping of late mapping population and analysis of genetic diversity of germplasm resources. The results of this study laid the foundation for further construction of high-density genetic linkage map of peanut and molecular marker-assisted breeding.