Genetic Diversity Of In Vitro Culture Of Pyrus Detected By Molecular Markers

Applying three different varieties of Pyrus 'Jinshui', 'Jinhua' and 'Shenbuzhi' cultivated in vitro for 3 to 5 years and their plants in vivo as experimental materials. Two kinds of DNA molecular marker techniques such as RAPD and ISSR were used to detect the genetic diversity between the in vitro and in vivo plants. The results showed that:1. The optimum reaction system of RAPD-PCR and program for pear were established. In a total volume of 20uL, containing template DNA 30ng, 1 xbuffer, Mg²⁺2.0 mmol· L^-1, RAPD primer 0.5μmol· L^-1, dNTPs0.5 mmol· L^-1, Taq-DNA polymerase1.0U. Amplified procedure was programmed for 5min at 94 ℃; 1min at 94℃ , 1min at annealing temperature 36℃, extension at 72℃ for 2min, 40 cycles; then a final extension at 72℃ for 10min and then keep at 4℃. Amplified products were detected by electropho-resis in 1.5% agarose gel.2. The optimum reaction system of ISSR-PCR and program for pear were established. In a total volume of 20uL, containing template DNA 30ng, 1 xbuffer, Mg²⁺2.0 mmol· L^-1, ISSR primer 0.4 μmol· L^-1, dNTPs0.5 mmol· L^-1, Taq-DNA polymerase1.0U. Amplified procedure was programmed for 3min at 94℃; 1min at 94℃, 1min at annealing temperature 52℃(50℃,55℃), extension at 72℃ for 2min, 40 cycles; then a final extension at 72℃ for lOmin and then keep at 4℃. Amplified products were detected by electrophoresis in 1.5% agarose gel.3. The percentage of polymorphic bands (PPB) of group A, B and C were 1.48%, 1.29% and 2.65 by RAPD analysis, while 2.78%, 2.60% and 3.42 by ISSR analysis. The PPB of group A and B which cultivated in vitro for 34 mouths were lower than that of...