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为了建立桤木属植物AFLP体系,筛选扩增条带丰富的引物,本研究以欧洲桤木(A.glutinsoa)为材料,对AFLP反应过程中的关键因素(DNA质量和浓度,酶切,扩增体系等)进行了研究。结果表明:预扩增稀释产物3.0μL,酶切时间以5 h,连接16 h,连接产物稀释10倍用于预扩增,预扩增产物稀释20倍为宜。确定桤木AFLP-PCR 20μL的反应体系:模板DNA 300 ng,10×Taq DNA聚合酶Buffer(含Mg2+)2.0μL,dNTPs0.3μL,引物1.0μL,Taq DNA聚合酶0.6 U,加ddH2O至20μL,并利用建立的体系筛选出7对扩增条带多、条带清晰、多态性好的引物。本研究建立了适用于桤木属植物的AFLP反应体系,并筛选出适合桤木属植物的引物,为今后利用AFLP标记技术进行桤木属遗传多样性分析提供了一定的实验依据。
In order to establish the AFLP system of Alnus spp. And to screen primers with abundant amplification bands, this study took A.glutinsoa as material, and studied the key factors (DNA quality and concentration, digestion, amplification Increase system, etc.) were studied. The results showed that pre-amplification of 3.0 μL diluted product, digestion time of 5 h, ligation 16 h, ligation product diluted 10 times for pre-amplification, pre-amplification products diluted 20 times is appropriate. Determine the alder AFLP-PCR 20μL reaction system: template DNA 300 ng, 10 × Taq DNA polymerase Buffer (containing Mg2 +) 2.0μL, dNTPs 0.3μL, primers 1.0μL, Taq DNA polymerase 0.6 U, plus ddH2O to 20μL, Seven pairs of primers with multiple bands, clear bands and good polymorphism were screened by using the established system. In this study, the AFLP reaction system applied to the genus Alnus was established, and the primers suitable for the genus Alnus were screened out, which provided some experimental basis for the future analysis of the genetic diversity of Alnus through AFLP marker technology.