Genetic Diversity Analysis Of Tibetan Poplar(Populus Szechuanica Var. Tibetica) Population Collected From Different Altitudes And Phenotype Of Seedlings Association Analysis

Tibetan poplar is an important tree species in the southwestern China, with the highest altitude habitat and excellent adaptation to environment of plateau, it possesses the characteristic of tolerance to low temperature, strong illumination, easy to breed, long life, is an important part of genetic pool in Populus and could provide lots of seeding and genetic source for breeding of stress resistance. Before the conversation and collection of germplasm, the genetic base should be detected and estimated clearly. To protect the special tree, related work should be preceded immediately. To this end, based on the investigation of distribution of Tibet poplar, the SEJILA Mountain was selected to be the study area for its sharply altitude falling. We collected469genotypes totally along the SEJILA Mountain. SSR and SNP markers were applied to analysis population structure and genetic diversity of population from different altitudes to reveal the relationship between altitude and genetic variation, therefore to provide the theoretical basis for the conservation of Tibetan poplar germplasm. The LD mapping was used to identify the locus associated with growing phenotype like height, ground diameter branches and leaf shape. The results of LD provided theoretical basis for the selection of clone excellent individuals, innovation germplasm resources, and outstanding locus identification. The following is main results:(1) In this study, we selected populations of Populus szechuanica var. tibetica which were living in Sejila Mountain in southeastern Tibet as study material to analysis the influence of altitude on genetic diversity and population structure. In the molecular analysis, we chose24primer pairs to process genotyping after screening.126alleles were amplified in total469individuals,5.25alleles per locus; PPL (polymorphism percentage level) was100%, expected heterozygosity (He) of high and low altitude, both at a high level in population, were0.48and0.49, respectively. The analysis of molecular variance (AMOVA) showed that the differentiation between populations was6.38%to total variation and the most variation was among the individuals. That estimation of Fst was0.02also supported the conclusion that the differentiation between populations was in low level. The value of gene flow (Nm) was9.89which was in a very high level. In conclusion, the genetic diversity and population structure of populations sampled from Sejila Mountain showed no evident pattern along altitude gradient. The strategic conservation and utilization of Tibetan poplar could be facilitated on the base of understanding of differentiation and distribution patterns of the species. The study about mechanism of adoption of high altitude could be processed smoothly.(2) The method of genotyping by sequencing (GBS) was used to excavate SNPs in the Tibetan poplar population composed by60individuals collected from different altitudes. After data filtering, quality control, a total of6520SNPs were identified and genotyped in the all individuals. The short sequences were used to mapping the genome of black cottonwood, the result showed that the mapping rate was low, it may means that the genome of black cottonwood and Tibetan poplar varied significantly. The comparison results of genetic diversity analysis between SNP and SSR showed that the cluster result showed that the two clustered groups are consistent with the sampling location, but the result of SSR had no clear differentiation.(3) The total of469samples were cloned and planted in two location with different altitude, Sichuan province and Tibet. The growing phenotypes like height, ground diameter, branch et al were measured for annual seeding. The influence of genetic and environment was analyzed, using different clone from reciprocal transplantation design. A novel mathematic model was developed to proceed the data analysis obtained in this experiment.(4) Package fgwas2which was developed on the R software was suitable for the big data and GWAS, the advantage is that it could consider all the loci at the same time. The Plink is free software used to do the GWAS and SNP data summary. The two ways was taken to do the association with growing phenotypes like height, ground diameter and branches. The results of fgwas2analysis showed that3loci were identified with height the heritability of0.11,locus was106346, the highest,2loci were identified with ground diameter, the heritability of0.109, locus was112578, the highest,3loci were associated with number of branch, the heritability of0.09locus was109890; The results of Plink analysis showed that17loci were identified with height, the most significant p-value was6.09×10-5for locus111604,17loci were identified with ground diameter, the most significant p-value was7.87×10-5 for locus106346,4loci were associated with number of branch, the most significant p-value was5.29×10-5for locus130018. The result of BLAST showed that most loci were predicted gene for the genome of Poplar had not been annotated. The two models was tested by comparison of result. It showed that the loci founded based on two different method existed overlap, it meaned that the two ways to identify loci associated with important traits were available.