黄瓜DAMD反应体系的建立及种质遗传资源研究

Establishment of Cucumber DAMD Reaction System and Identification of Germplasm Resource

通过L16（45）正交试验,研究Mg^2＋、dNTPs、Taq酶、引物和模板DNA对黄瓜DAMD（direct amplificationof minisatellite region DNA）反应的影响.结果表明,适宜的DAMD反应体系包括1×Taq酶缓冲液、1.5 mmol/LMg^2＋、0.25 mmol/L dNTPs、1.2 UTaq酶、2.0μmol/L引物和10-80 ng模板DNA,总体积20μL.采用该体系并结合温度梯度PCR,确定了15种小卫星核心DNA引物的适宜退火温度.利用这些引物对20份不同类型的黄瓜材料进行扩增,引物平均检测到10.2个位点,平均多态性位点7.3个,平均多态性百分率71.4%.PCA分析表明,20份材料明显可分为3个区域,第I区包含绝大部分华南型黄瓜,第II区包含绝大部分华北型黄瓜,第III区为野生型黄瓜.此结果与SSR标记的研究结果基本一致,证明本实验构建的DAMD反应体系的稳定性及DAMD标记的可行性.

The effects of Mg2＋,dNTPs,Taq DNA polymerase,primer and template DNA on DAMD（direct amplification of minisatellite region DNA） reaction were studied using orthogonal experiment L16（45）.The results showed that the optimal reaction system was established as follow：the 20 μL volume containing 1×Taq DNA polymerase buffer,1.5 mmol/L Mg^2＋,0.25 mmol/L dNTPs,1.2 U Taq DNA polymerase,2.0 μmol/L primers and 10-80 ng template DNA.The optimal annealing temperatures of the 15 minisatellite core DNA primers were defined using the reaction system and touch down PCR.Various types of 20 cucumber accessions were subjected to PCR amplification with the primers.As a result,a minisatellite core DNA primer detected a mean of 10.2 loci with a mean polymorphic loci of 7.3（mean 71.4% polymorphism）.All the accessions could be included in three regions based principal coordinate analysis.Region I contained the major South ecotype accessions and Region Ⅱ comprised of the major North ecotype accessions and the wild ecotype was included in Region III.This division agreed well with the finding with SSR（simple sequence repeat） marker data.These results verified the stability of DAMD reaction system established in present study and the practicability of DAMD markers.