泡桐AFLP反应体系的建立及引物筛选

以豫杂一号泡桐为材料,通过对影响AFLP技术体系的各主要因素的研究,建立了适于泡桐AFLP分析的技术体系.结果表明最佳酶切体系(20μL)为500 ng模板DNA,3 U的Pst 1和Mse I,在37℃下双酶切3 h;20μL最佳连接体系中为酶切产物15 μL,0.25 μmol·L-1Pst I接头,2.5 μmol·L-1 Mse I接头,1 μL 10×T4 Buff-er,2 U T4连接酶,22℃连接18 h;20 μL最佳预扩反应体系中5 μL稀释10倍的连接产物,100 μmol·L-1dNTP,2 U Taq酶,250 μmol.L-1 Pst I和Mse I引物(P+AGT/M+AGT),2 μL 10×PCR Buffer.20 μL最佳选择性扩增反应体系中5 μL稀释20倍预扩增产物,100 μmol·L-1dNTP,2 U Taq酶,350 μmol·L-1 Pst I和Mse I引物(P+AGT/M+AGT),2 μL 10×PCR Buffer.最后,筛选出了97对适宜于泡桐AFLP分析的引物.

Establishment of Paulownia AFLP reaction system and its primer selection

The establishment of Paulownia AFLP reaction system and its primer selection were investigated through study on the main factors affecting the system quality with Paulownia tomentosa × Paulownia fortune.The results indicated that the optimum conditions for restriction enzyme digestion of the genomic DNA from the Paulownia plant were as follows: 500 ng DNA template, 3 U Pst Ⅰ and Mse Ⅰrespectively in 20 μL reaction volume at 37 ℃ for 3 h;The ligation reaction (20 μL) with 15 μL the digestion product, 0.25 μmol·L-1 Pst I adaper, 2.5 μmol·L-1 Mse I adapter, 1 μL 10 ×T4 Buffer,2 U T4 ligation enzyme at 22 ℃ for 18 h was the optimum condition; the preamplification reaction (20 μL) with 5 μL the ligation fragment which was diluted 10 times, 100 μmol·L-1 dNTP,2 U Taq enzyme,250 μmol·L-1 Pst Ⅰ and Mse Ⅰ primer (P + AGT/M +AGT) ,2 μL 10 × PCR Buffer was the optimum condition; The selective amplification reaction (20 μL) with 5 μL preamplification product which was diluted 20 times, 100 μmol·L-1 dNTP,2 U Taq enzyme,350 μmol·L-1 Pst I and Mse Ⅰ primer (P + AGT/M + AGT) ,2 μL 10 × PCR Buffer.Finally,97 pairs of primers were chosen for the AFLP analysis of Paulownia plants.