酥瓜(Cucumis melo var. conomon Group)基因组DNA提取及RAPD分子标记反应体系的建立

Extraction of Genomic DNA and Construction of RAPD Molecular Marker Reaction System in Cucumis melo var. conomon Group

本研究以改良的CTAB法提取酥瓜基因组DNA,利用正交设计L_(16)(4~5),对酥瓜RAPD-PCR反应体系的5个影响因素(引物,dNTPs,Taq DNA聚合酶,Mg^(2+)和模板DNA)在4个水平上进行优化试验,并在30~50℃范围内摸索退火温度,建立适合酥瓜RAPD-PCR反应体系。研究表明,在25μL反应体系中,含有引物0.8μmol/L、dNTPs 0.3μmol/L、Taq DNA聚合酶1 U、Mg^(2+)0.5 mmol/L、DNA模板30 ng为最佳反应体系,RAPD-PCR扩增程序中最佳退火温度为37.6℃。该体系有助于酥瓜种质资源的遗传多样性分析评价。

In this research, in order to establish optimized RAPD-PCR reaction system for crisp melon （Cucumis melo var. conomon Group）, the genomic DNA of crisp melon was extracted by modified CTAB method. Five influencing （primers, dNTPs, Taq DNA polymerase, Mg^2＋ and template DNA） factors in crisp melon RAPD-PCR reaction system were optimized on 4 levels by orthogonal design L16 （4^5） and the annealing temperature in the range of 30 - 50℃ was explored. The research showed that the optimal conditions for 25 μL RAPD system consisted of 0.8 μmol/L primer, 0.3 μmol/L dNTPs, 1 U Tat/DNA polymerase, 0.5 mmol/L Mg〉 and 30 ng DNA. Moreover, the optimal annealing temperature of RAPD-PCR amplification program was 37.6℃. This optimized RAPD-PCR reaction system would be helpful for analyzing germplasm classification and identification of crisp melon.