Establishment Of Paulownia AFLP And MSAP Reaction Systems And Primers Screening

Paulownia originated from China. Distribution in 25 provinces, municipalities and autonomous regions of China, recent years,they were also introduced in other parts of the world. With fast growth, well materials, suitable for farming by intercropping etc, Deeply loved by the masses, alleviation the shortage of timber, improvement of ecological environment, increasing farmers’ income and people’s living standards, this have important ecological significance and economic significance and social significance for our country to Largely planting Paulownia , But, paulownia witches ’broom bring huge economic losses, because the paulownia witches ’broom is an infectious disease whitch caused by phytoplasma, it can caused paulownia DNA methylation decrease, so it is necessary to make clear the changes of the structure of paulownia DNA and methylation patterns after phytoplasma who invased paulownia, due to the growth cycle of paulownia is very long, this paper we established AFLP and MSAP two molecular Reaction systems with the tissue culture of Paulownia tomentosa×Paulownia fortunei, and screened their primers, For further analysis the background of same paulownia between amphidiploids genetic, the genetic relationship between different kinds paulownia, the relationship between paulownia mutants, the changes of paulownia DNA base sequences and DNA methylation level between paulownia witches ’broom and health, eventually reveal the mechanism of witches broom and screen the effective drugs: The main results are as follows:1 The establishment of Paulownia AFLP Reaction System and Its Primer Screening were investigated through study on the main factors which affected the system quality 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 I respectively in 20μL reaction volume at 37℃for 3 h,and inactivated at 80℃for 20 min;The ligation reaction 20μLwith 15μL the digestion product, 0.25μmol·L-1 Pst I adapter, 2.5μmolL-1 Mse I adapter, 1μL 10×T4 buffer,2 U T4 ligation enzyme at 22℃for 18 h was the optimum condition; The preamplification reaction 20μLwith 5μL the ligation fragment which was diluted 10 times, 100μmol·L-1 dNTP,2 U Taq enzyme, Pst I and Mse I primer respectively 0.25μmol·L-1,2μL 10×PCR buffer was the optimum condition; The selective amplification reaction 20μL with 5μL preamplification product which was diluted 20 times, 100μmol·L-1dNTP,2UTaq enzyme,Pst I and Mse I primer respectively 0.35μmol·L-1 (P+AGT/M+AGT),2μL 10×PCR buffer. Finally, 97 pairs of primers were chosen for the AFLP analysis of paulownia plants.2. The establishment of Paulownia MSAP Reaction System and Its Primer Screening were investigated through study on the main factors which affected the system quality. The results indicated that the optimum conditions for restriction enzyme digestion of the genomic DNA from the paulownia plant were as follows:300 ng DNA template, 16 UEcoR I andHapⅡ(Msp I )respectively in 25μL reaction volume at 37℃for 8 h, and inactivated at 80℃for 20 min;The ligation reaction 25μL with 20μL the digestion product, 0.16μmol·L-1EcoR Iadapter, 1.6μmolL-1 HapⅡ(Msp I )adapter,2.5μL 10×T4 buffer,2 U T4 ligation enzyme at 22℃for18 h was the optimum condition; The preamplification reaction 20μL with 5μL the ligation fragment which was diluted10 times, 100μmol·L-1 dNTP,0.5U Taq enzyme, EcoR I andHapⅡ(Msp I ) primer respectively 0.25μmol·L-1,2.5μL 10×PCR buffer was the optimum condition; The selective amplification reaction 20μL with 5μL preamplification product which was diluted 30 times, 100μmol·L-1 dNTP,0.5 U Taq enzyme, EcoR I andHapⅡ(Msp I ) primer respectively 0.3μmol·L-1 (E+AAA,H/M+AAA),2.5μL 10×PCR buffer. Finally, 60 pairs of primers were chosen for the MSAP analysis of paulownia plants.