Genetic Diversity And Relationships Among Qinghai Spruce (Picea Crassifolia Kom.) Clones Based On SSRs

Qinghai spruce(Picea crassifolia Kom.)is an evergreen tree species,belonging to family Pinaceae.It is native to the northeast edge of Qinghai-tibet plateau,and important for afforestation and urban greening in northwest China,playing an irreplaceable role in ecological construction in Qilian Mountains and northwest area.To date,researches on seed germination,phenotypic variation and genetic variation based on isozyme for Qinghai spruce has been reported.However,application of SSR markers to this species is rarely reported.The thesis contains two parts:(1)establishment and optimization of SSR-PCR reaction system,and the primers selection for Qinghai spruce;(2)investigation of genetic diversity and relationships among Qinghai spruce clones based on SSRs.SSR-PCR reaction system was established through orthogonal test and optimized through single factor experiment.The results showed that the optimal SSR-PCR amplification system was in a volume of 20.0 ?L containing template DNA(5-10 ng·?L-1)1.0 ?L,dNTP(10 mmol·L-1)0.3 ?L,forward-and reward-primer(10 ?mol·L-1)0.25 ?L,respectively,Mg2+(25 mmol·L-1)2.5 ?L,Taq DNA polymerase(5 U·?L-1)0.3 ?L,10×PCR buffer 2.5 ?L and HPLC water 12.9 ?L.The temperature profile of SSR-PCR is: initial denaturation for 3 minutes at 94 ?;44 cycles of denaturation for 30 seconds at 94 ?,annealing for 30 seconds at 55.0-55.8 ?,and extension for 60 seconds at 72 ?;a final extension for 7 minutes at 72 ?.Using the established SSR-PCR reaction system,sixty SSR primer pairs developed in Norway spruce(Picea abies Karst.)were tested,and ten SSR primer pairs with clear,polymorphic,and repeatable band patterns were selected to Qinghai spruce for the further studies.Meanwhile,the cross-species transferability of SSR primer pairs within the genus Picea was proved.The genetic diversity of 109 Qinghai spruce clones in seed orchard in Longqu,Zhangye,Gansu province was assessed based on 10 SSR loci.The results showed the high genetic diversity of the clones with the mean values for the percentage of polymorphic loci(PPLS)of 100.00%,the effective number of alleles(ne)of 14.76,the Nei's gene diversity(He)of 0.92,and the Shannon's information index(I)of 2.77.UPGMA cluster analysis distinguished the 109 Qinghai spruce clones clearly,and showed the genetic relationships among all the clones.In conclusion,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,as well as for parents' apolegamy in cross breeding and the establishmet of advanced generation seed orchard.Furthermore,the results is also of ecological,economic and social benefit to water-soil conservation and development of specialty industry of the species in northwestern China.