Genetic Diversity Of Rhus Chinensis Based On ISSRs

Rhus chinensis are deciduous shrubs or small trees,belonging to the family Anacardiaceae and the genus Rhus.It is a host plant of Chinese gall and an important economic tree species in China.The thesis comprises two parts:?1?establishment and optimization of inter-simple sequence repeats-polymerase chain reaction?ISSR-PCR?system and polymorphic primer screening for Rh.chinensis;?2?investigation of genetic diversity of Rh.chinensis in Anhui province based on ISSRs.ISSR-PCR amplification system was established through orthogonal test and optimized through single factor analysis for Rh.chinensis.The results showed the optimal ISSR-PCR system with a total volume of 20.0?L,containing template DNA?5-10 ng/?L?2.0?L,primer?10?mol/L?0.6?L,d NTP?10 mmol/L?0.2?L,Mg²⁺?25 mmol/L?2.8?L,Taq DNA polymerase?5 U/?L?0.3?L,10×PCR buffer 2.5?L,and HPLC water 11.6?L.The temperature profile of ISSR-PCR consists of initial denaturation for 5 min at 94?;40 cycles including denaturation for 40 s at 94?,annealing for 40 s at 54.0?,and extension for 1.5 min at 72?;a final extension for 2 minutes at 72?;store at 4?.Fourteen polymorphic ISSR primers with clear and repeatable band patterns were selected from 80 ISSR primers using established ISSR-PCR system for further study.The genetic diversity of six populations with 150 individuals of Rh.chinensis in Anhui province was investigated based on ISSR markers using 14 selected primers.A total of 201 polymorphic loci were obtained.The results revealed the mean values at the species level for the percentage of polymorphic loci?PPL_S?of 100.00%,the effective number of alleles(n_es)of 1.324,the Nei's gene diversity?expected heterozygosity?(H_es)of0.225,and the Shannon's information index?I_s?of 0.370;At the population level,PPL_p=67.83%,n_ep=1.282,H_ep=0.178,and I_p=0.280;Genetic differentiation(G_st=0.21)indicated that 21.0%of total genetic diversity were attributed to the differences among populations;Analysis of molecular variance?AMOVA?indicated that genetic variation among and within Rh.chinensis populations accounted for 31.47%and 68.53%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.Provenance-specific ISSR bands were found in the present study,which can be used for provenance identification in the future.In conclusion,the present research revealed the moderate-level genetic diversity and differentiation within and among populations of Rh.Chinensis at in Anhui,and suggested some conservation strategies of the species.Results of the present study will provide theoretical basis and practical guidance for archive building,conservation,and sustainable utilization of genetic resources,and the selection of parents in cross breeding of Rh.chinensis.