The Selfing Genetic Differentiation Analysis Of Lilium Formolongi Based On RAPD Markers

Lilium formolongi was an interspecific hybridization between Lilium formosanum and Lilium Longiflorum with the characteristics of producing cut-flower by seedlings. For strong compatibility between lines and preservable bulbs, Lilium formolongi was a good material for genetic diversity and genetic differentiation analysis. The trend of genetic diversity and genetic differentiation of selfed lines was studied based on RAPD markers. The results could be summarized as follows.1. By optimal experimental design, the conditions of RAPD were confirmed as follows.Single primer reaction: dNTPsl50 mol/L, Mg2+1.5mmol/L,Taq polymerase 1U, primer0.3 mol/L,DNA template 50ng,total volume 20 u 1 .Double prime reaction: dNTPs200 u mol/L, Mg2+2mmol/L, Taq polymerasel.5U, primer0.4 mol/L, DNA template 50ng, total volume 20 1 Concentrations of double primer were higher than those of single primer, except that of DNA template, for best effect.Reaction procedure parameters: (1)pre-denatured DNA at 94 C for 5 min,1 cycle; (2) denatured DNA 94 C for 30sec,annealed primer at 37C for 50sec,extended primers at 72 C for 1min ,35 cycles; (3)re-extended reaction at 72 C for 10min,l cycle; (4) Stored product at 4 C.2. A total of 163 polymorphic bands out of 198 reproducibleproducts were amplified from the selected 11 single primers and 8 double primers, corresponding to 81.8% polymorphism of the amplification bands. The bands generated by double primer had no obvious relationship with single primer. The bands generated by single primer had no relation with double primer either.3. To evaluate genetic diversity of generations, percentage of polymorphic bands, Shannon genetic diversity index and relative polymorphism frequency were studied. The percentage of polymorphic bands of primer S19, S303+S1366, S19+S210, S469+S19, S55+S30 were 100%. The percentage of polymorphic bands of F2, F3, F4 generations were 59.1%, 56.6%, 48.0% respectively, which showed reduction trend.4. The genetic diversities of three generations were 1.749, 1.530, 1.292, which also showed reduction trend. Among total variation, genetic diversity intrapopulation accounted for 76.2%, interpopulation for 23.8%. This result showed that genetic diversity of intrapopulations was higher than that of interpopulations.5. UPGMA cluster analysis could reveal original strain relationships. The data set from double primers revealed more accurately. RAPD markers could accurately reveal small change of genetic diversity and genetic differentiation among near genetic relationship.