Genetic Variation And Relationships Among Qinghai Spruce (Picea Crassifolia Kom.) Clones Based On ISSRs

Qinghai spruce?Picea crassifolia Kom.?is an evergreen coniferous tree species endemic to China,belonging to family Pinaceae.It is important for afforestation and urban greening in northwest China.The thesis contains two parts:?1?establishment and optimization of ISSR-PCR reaction system,and the primers selection for Qinghai spruce;?2?investigation of genetic diversity and relationships of 125 Qinghai spruce clones in seed orchard in Longqu,Zhangye,Gansu province based on ISSRs.ISSR-PCR reaction system was established and optimized through orthogonal test and single factor analysis.The results exhibited the optimal ISSR-PCR amplification system in a total volume of 20.0?L,containing template DNA(5-10 ng·?L^-1)2.0?L,primer(10?mol·L^-1)0.8?L,dNTP(10 mmol·L^-1)0.3?L,Mg²⁺(25 mmol·L^-1)2.8?L,10×PCR buffer 2.8?L,Taq DNA polymerase(5 U·?L^-1)0.2?L,and HPLC water 11.1?L.The ISSR-PCR procedure consists of initial denaturation for 4 minutes at 94?;40 cycles of denaturation for45 seconds at 94?,annealing for 45 seconds at 49.5-50.5?,and extension for 60 seconds at 72?;and a final extension for 7 minutes at 72?.Twelve ISSR primers with clear,polymorphic,and repeatable band patterns were selected from 100 ISSR primers based on the established ISSR-PCR reaction system for Qinghai spruce.The genetic diversity of 125 Qinghai spruce clones in seed orchard in Longqu,Zhangye,Gansu province was assessed using 12 valid ISSR primers with 186 loci.The results indicated the high genetic diversity of the clones with the mean values for the percentage of polymorphic loci?PPL?of 100.00%,the effective number of alleles?n_e?of 1.2843,the Nei's gene diversity?H_e?of 0.2074,and the Shannon's information index?I?of 0.3502.The UPGMA cluster analysis clearly separated all the 125 clones of Qinghai spruce,and divided them into three major groups,revealing the genetic relationships among all the clones investigated.The results of the present research will provide theoretical basis and practical guidance for conservation of genetic resources,reasonable allocation of clones,and genetic improvement of seed quality in the clonal seed orchard of Qinghai spruce.In addition,the results are also of ecological,economic and social significance to water-soil conservation,reasonable development and utilization of the species,and promotion of local economy in northwestern area of China.