Genetic Variation Of Natural Populations Of Pinus Tabulaeformis Carr. And Its Systematic Position

Chinese pine,Pinus tabulaeformis Carr.has a broad distribution across northeastern to southwestern China.It has a strong adaptability to dry and barren habitat,meanwhile,it is a fast-growing tree species and possesses a strong ability to develop roots.Therefore it also has the function of conserving soil and water,enriching headwaters and improving soil fertility.The species therefore can be a main species for afforestation in the semiarid and waste mountains in northeast of China.Shanxi Plateau has long been known as its distribution center,where the tree predominate the forest landscape.However,natural Chinese pine forests have long been affected by natural processes and anthropogenic perturbation without systematic management,leading to serious ecological hazards and partial loss of ecological functions.Understanding of the genetic diversity and genetic structure of species is necessary for the conservation of species and populations,especially the conifer species.Many studies on the levels of morphology,chromosome,isozyme from Chinese pine have been conducted in recent decades.However,the genetic diversity and structure of the Chinese pine populations on the genomic level remains unknown.Little is known about how genetic variation is distributed within and among populations,despite the species considerable ecological importance.On the base of analysis of geographical distribution of Chinese pine and of the field investigation,the Lishan in Zhongtiao Mountains,Zituan Shan in Taihang Mountains,Lingkong Shan in Taiyue Mountains,Luya Shan in Guanqin Mountains,and Guandi Shan in Lvliang Mountains were selected as investigated fields,which represent the natural distribution of Chinese pine in Shanxi Loess Plateau.Neutral molecular markers such as random amplified polymorphic DNA(RAPD) and inter simple sequence repeats(ISSR) are well established in population genetics and have proven to be useful tools for the detection of plant population variability and differentiation.In our study,both ISSR and RAPD markers have been used to survey the genetic structure at different levels in P.tabulaeformis(within and among populations) and the correlations between genetic variation and environmental system and the mechanism of ecological fitness and molecular evolution.Two pairs of primers were used to amplify noncoding region of chloroplast trnS-trnG and trnT-trnL from genome of chloroplast,respectively.The resultant sequences were analyzed by alignment and the phylogram trees were constructed using a series of softwares to inference the systematics relationship of the Pinaceae and Pinus,and to provide the significant information for the comparison of genome and systematic evolution of Pinus.The findings of this research are as follows:RATIO OF POLYMORPHIC LOCI AND INDEX OF DIVERSITYA total of 125 reproducible bands were obtained from 140 individuals of P. tabulaeformis using 15 RAPD primers.Ninety-nine polymorphic loci were identified from 125 reproducible RAPD bands(79.2%).Nei's analysis of genetic diversity ad Shannon's information index are 0.2853 and 0.4232, respectively,which varies in five populations of P.tabulaeformis range from 0.2243～0.2626 and 0.3352～0.3860,respectively.A total of 35 reproducible bands were obtained from 140 individuals by using 5 ISSR primers. Twenty-eight polymorphic loci(80%) were identified.Nei's analysis of genetic diversity and Shannon's information index are 0.3085 and 0.4468,respectively. The genetic diversity of five populations was compared and ranked by using the diversity index measured by two markers.The results indicated that the taxis of diversity of five populations base on RAPD and ISSR were similar except for the LS population.The genetic diversity of P.tabulaeformis is high on Lingkong Mountain and Guandi Mountain and low on Guanqin Mountain.The genetic diversity of P.tabulaeformis is relatively in the middle to high level compared with that of species in Pinus.THE GENETIC VARIATIONS AND DIFFERENTIATIONSThe majority of genetic variations were found within populations and some genetic differentiation occurred among populations of P.tabulaeformis.Base on the RAPD marker,the total genetic diversity(Ht) and genetic diversity within populations(Hs) of P.tabulaeformis are each 0.2583 and 0.2428,and the corresponding differentiation among populations(Gst) are 0.1491.Base on ISSR marker,the Ht and Hs of P.tabulaeformis are 0.3805 and 0.2667, respectively,and Gst is 0.1356.The data from two molecular markers suggest the existence of genetic variation and differentiation in population of P. tabulaeformis in Shanxi.The majority variation(85%) was found within populations.Though genetic variation among population(15%) is not large,it should not be negligible.Base on the analysis combining with natural geographic environmental factors,we concluded that the effects on the development of pattern of genetic structure of P.tabulaeformis population involved geographic location,natural habitat and human perturbation.GENETIC IDENTITY AND GENETIC DISTANCE AMONG POPULATIONS OF P.TABULAEFORMISThe genetic identity and genetic distance among five P.tabulaeformis populations were calculated using Nei's original measure.The largest genetic identity(0.9716) and the smallest genetic distance(0.0288) were found between LY population in Guanqin Mountains and XW population in Guandi Mountains, and similarity of two populations is highest.Meanwhile,the least genetic identity(0.8999) and biggest differentiation(0.1054) were found between ZT population in Taihang Mountains and XW population in Guandi Mountains.The UPGMA dendrogram tree was constructed by using genetic distance of five populations to further confirm the genetic relationship among populations in Shanxi.THE PHYLOGENETIC RELATIONSHIP OF PINUS INFERRED FROM CHLOROPLAST DNAChloroplast DNA(cpDNA) has been considered as an important tool for inferring phylogenetic relationships at many taxonomic levels corresponding to different development rate of the encoding and nonencoding regions in genome of chloroplast.In this study,two pairs of specific primers were used to amplify noncoding region of chloroplast trnS-trnG and trnT-trnL from chloroplast genome,respectively.Alignment of the sequences of P.tabulaeformis from different population indicates that these two DNA sequences are identical and conserved among five populations even within genus and varies among genera of Pinaceae,suggesting the two DNA sequences are not fit to being used to analyses the systematic relationship within species and could be used for that among species within familia.In this study,we constructed the phylogenic tree by combining the chloroplast trnS-trnG and trnT-trnL sequences amplified from five P.tabulaeformis populations and downloaded from GenBank (http://www.ncbi.nlm.nih.gov).The phylogenic relationship between P. tabulaeformis and its relative species were analyzed combining with the morphology,which will provide much more information for elucidating the genetic relationship among species and genera of Pinaceae.CONSERVATION OF PINE FORESTThe genetic diversity of pine populations in Shanxi plateau mainly rests on the different allelic frequency rather than on the composition of alleles,indicating that the disappearance of some individuals does not lead to a loss of genes. However,a decrease in individuals may cause genetic drift and inbreeding leading to a decline in fitness with environmental or disease-related reductions in productivity for Chinese pine populations.Furthermore it will affect the evolution potential of the pine population.We should protect the natural pine forest with scientific consciousness.The conservation of pine forest should be focus on the protection of environment and decrease the artificial perturbation, which may cause the fragments level of the pine forest.