The Research On Genetic Diversity And Molecular Phylogenetic Population Of D.involucrata

Davidia involucrate Baill, an endemic plant species in china and the relict species of the Tertiary ancient tropical flora, is the world famous ornamental woody plants, listed as one of China's first class of protected plants, which has high research value in plant phylogeny and the change of geological history. In this study, the author analyzed population genetic diversity and molecular phylogegraphy of11natural D.involucrata populations were analyzed by ISSR (Inter Simple Sequence Repeat) molecular markers and cpDNA non-coding sequences markers, aiming to explore the level of genetic diversity and the spatial distribution patterns of Davidia involucrate Baill and the information of geological history change and providing the basic information of population genetics for designing the genetic resource conservation stratagy of D.involucrata. The major findings are as follows:(1) The results of ISSR.-PCR reaction system optimized by single factor and orthogonal experiments were:25μL reaction system, including Mg2+1.8mM, DNA60ng, dNTP0.40mM, primers0.52μM, Taq enzyme1.25U.The response program was devised as followings: a initial denaturation94℃for4min,36cycles of94℃for45s,48℃~58℃for45s,72℃for90s, and a final extension at72℃for7min; preserved at4℃.(2) The genetic diversity of240individuals from11natural populations of this plant was analyzed using ISSR markers, the results were:Using the ISSR-PCR reaction system and procedures optimized,5primers (UBC829. UBC84,UBC846,UBC848,UBC864), generated stable, distinct and highly polymorphic bands were selected from the100primers. The results showed that77bands were amplified with five primers, in which there were74polymorphic bands. The percentage of polymorphic bands was96.10%. At the population level with the percent of polymorphic bands ranged from37.66%to63.64%, the average value was54.07%. The Shannon's information index(HSP) within species was0.4849, its value within population (Hpop) were from0.1886to0.3274, and the mean value was0.2774. This result showed high level genetic diversity both at population and species level. Analysis of molecular variance (AMOVA) revealed that among and within-population genetic variation accounted for46.22%and53.78%of the total genetic variation, respectively. A high degree of genetic differentiation was also found among populations(φst=0.4170). Population genetic differentiation (φst) was positively correlated with geographical distances (r=0.546,P=1.619e-05). These11populations were divided into3major groups, based on the UPGMA cluster analysis obviously:Sangzhi,Yongshun in Hunan,Xingshan,Changyang in Hubei as a group, Shimen in Hunan,Shennongjia in Hubei,Fangjingshan and Songtao in Guizhou as a group, Emeishan,Yaan in Sichuan and Zhijin in Guizhou as a group.(3) The cpDNA non-coding sequences PCR reaction system optimized were:25μL volume,50ng DNA,10Taq Buffer(Mg2+free),2.5mM MgCl2,0.4mM dNTPs,0.2μM primer1,0.2μM primer2,1.25U Pfu DNA Polymerase. The PCR procedure was: initial denaturizing94℃for5min,35cycles of94℃for1min, annealing time for45s, and72℃annealing temperature extension, a variety of primers and products were different, a final extention at72℃for7min, and preserved at4℃.(4) The phylogegraphy of240individuals from11natural populations of this plant was analyzed by cpDNA non-coding sequences markers:Utilizing optimized reaction system and the program,3pairs(trnSGCU and trnGUUC,rps16-F and rps16-R,atpB-49and rbcL-188) from9pairs of primers were screened out. The results of sequence alignment were:According to2kinds of haplotypes with significant geographical features,11natural Davidia involucrata populations formed2haplotypes, with2variants,11D. involucrata populations were divided into2branchs. One branch included3populations of Emeishan,Yaan in Sichuan,Zhijin in Guizhou located in southwest of china, and the other one included8populations(3populations of Yongshun in Huna,Sangzh,Shimen,3populations of Shennongjia in Hubei,Xingsha,Changyang,2populations of Fanjingshan in Guizhou and Songtao), which located in central south of china. The above were consistent with the results of ISSR UPGMA (unweighted pair-group method with arithmetic-means) cluster analysis generally. The information revealed that the extant populations of D. involucrata may be originated from the respective last glacial refuge populations, the founder effect playing a major role in the process. After the Quaternary ice age, the shelter population expanded rapidly, forming single-haplotype populations of involucrate.(5) Based on the analysis of ISSR molecular markers and cpDNA non-coding sequences markers, the author proposed the strategies of protecting D.involucrata resources: Protecting all natural populations in situ conditions possible, prohibiting the cutting and digging excessively in order to protect the genetic variation as much as possible, and taking priority to save high genetic diversity populations of Emeishan in Sichuan,Sangzhi in Hunan and Fanjingshan in Guizhou. In addition, strengthening the research about introduction, domestication and artificial propagation technique of D.involucrara natural populations, not only alleviating the own reproductive capacity, as well as man-made excavation which cause a threat to the survival, but also ensuring the introduction and plantation in an area of flowering, fruiting, sustainable survival and reproduction.