栽培种花生AFLP标记体系的优化及多态性引物筛选

Screening for Polymorphic Primer Pairs and Optimization of AFLP Marker System in Peanut

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 AFLP marker reaction system using double-digestion of EcoR I and Mse I endonuclease in peanut, the extracting method of genomic DNA, the DNA digestion and ligation system and several key factors affecting the PCR pre-amplifieation and selective amplification were optimized in this study. In addition, primer combinations （E/M） suitable for AFLP analysis were also screened in peanut. The results showed that high quality of genomic DNA was extracted with the improved SDS-CTAB method, the concentration of DNA sample was 150 - 200 ng·μL-1, the DNA was digested 3.0 hours under 37℃, the concentration of EcoR I and Mse I adapter was 50 pmol·μL-1, the concentration of T4 -DNA ligase was 10 U· μL-1, DNA ligation was under 16℃ for 10 hours. 2 × Es Taq MasterMix was used as a PCR reaction material, which including Es Taq DNA Polymerase, 3 mool· L-1 MgCl2 and 400 umol· L-1 dNTP. The total volume of pre-amplification reaction system were 50 μL, the concentration of pre- amplification primer（E00 and M00） were 50 ng·μL-1, the products of pre-amplification for selective amplification were diluted to 20 times, and the total volume of selective amplification reaction system were 20 μL, 10μL Loading Buffer were aaded into the products of selective amplification, then denaturalized 10 minutes under 95℃, the products were transferred into ice-bath immediately, then conserved it. Finally, we screened 42 stable and polymorphic primer pairs from 225 AFLP primer combinations, which will be used for detecting the genotype of mapping population and analyzing genetic diversity of peanut germplasm resources in the future. The results of this article provide technical support for construction of genetic linkage map and molecular marker assisted breeding in peanut.