Genetic Variation In Staphylea Bumalda In Anhui Province Based On ISSRs

Staphylea bumalda DC is a perennial deciduous shrub or small tree. It is rare and special woody oil trees in China. This thesis is composed of two parts:(1) establishment of Inter-simple sequence repeats-polymerase chain reaction (ISSR-PCR) system and polymorphic primer screening for S. bumalda; (2) investigation of genetic diversity of S. bumalda in Anhui province based on ISSRs.ISSR-PCR system was established through orthogonal test and optimized through single factor experiment. The results showed that the optimal ISSR-PCR amplification system was in a volume of 20.0 uL containing template DNA (5-10 ng·μL-1) 1.0 vL, dNTP (10 mmol·L-1) 0.3 μL, primer (10 μmol·L-1) 0.5 μL, Mg2+(25 mmol·L-1) 2.5μL, Taq DNA polymerase (5 U·μL-1) 0.3 μL,10×PCR buffer 2.5 μL and HPLC water 12.9 μL. Temperature profile of ISSR-PCR is:initial denaturation for 5 minutes at 94 ℃; 42 cycles of denaturation for 45 seconds at 94 ℃, annealing for 30 seconds at 54.0-55.3 ℃, and extension for 90 seconds at 72 ℃; a final extension for 7 minutes at 72 ℃. Fourteen polymorphic ISSR primers were selected from 80 ISSR primers using established ISSR-PCR reaction system for further diversity research of S. bumalda.The genetic variation of 6 populations with a total of 138 individuals of S. bumalda in Anhui province was assessed using 14 ISSR primers at 221 polymorphic loci. The results showed the mean values at the species level for the percentage of polymorphic loci (PPLS) of 100.00%, the effective number of alleles (nes)of 1.210, the Nei’s gene diversity (Hes) of 0.157, and the Shannon’s information index (7S) of 0.276; At the population level, PPLP= 38.76%, nep= 1.185,Hep= 0.114, and Ip= 0.176; Genetic differentiation (Gst= 0.276) indicated that 27.6% of total genetic diversity were attributed to the differences among populations; Analysis of molecular variance (AMOVA) revealed that genetic variation among and within S. bumalda populations accounted for 43.07% and 56.93% of the total genetic variation, respectively, showing the most genetic variation was found within populations; The UPGMA cluster analysis separated the six populations clearly, and showed no correlation between population grouping and geographic distribution. The results of present study will provide important information for conservation,exchange, and sustainable utilization of genetic resources, and tree breeding of S. bumalda.