【作者】 沈登锋；

【导师】 邬荣领；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2014， 博士

【摘要】 藏川杨(Populus szechuanica var. tibetica)是我国西南地区重要的乡土树种,是杨属中生活环境海拔最高的树种,对于高原环境具有良好的适应性,如耐低温、耐强光照、易繁育、寿命长等,是我国杨树育种宝贵的基因资源库,能够为杨树的遗传育种提供大量抗逆性强的种苗和基因资源。在进行种质资源的保护、收集之前需要对该物种的遗传背景进行有效评估。为保证藏川杨资源保护和育种工作的顺利开展,迫切需要开展藏川杨群体遗传多样性方面的研究。为此,在对藏川杨基因资源进行踏查的基础上,选择了西藏林芝地区的色季拉山脉沿线进行了种质资源的收集,共收集保存469个基因型个体。在此基础上,利用SSR, SNP两种分子标记对不同海拔分布的藏川杨群体进行了遗传多样性的分析,揭示了海拔变化与遗传变异之间的关系,为藏川杨遗传资源的保存保护提供了理论依据。同时利用基于自然群体的连锁不平衡作图方法,进行了SNP位点与表型生长数据包括苗高、地径、分枝数、叶片形态等的关联分析,研究结果为重要性状相关位点的发掘、创新种质资源、无性系优株的苗期选择等提供了科学理论依据,具有一定的应用价值,主要研究结果如下：(1)利用24个基于胡杨(Populus euphratica)EST序列开发的SSR标记位点对不同海拔的藏川杨群体进行了遗传多样性的研究,中性检验结果显示24个位点中有两个处于自然选择压力下,在进行遗传结构、遗传分化等由非中性进化引起的指标分析时,应进行中性位点的筛选,保证结果可靠性,使用24对微卫星标记进行遗传分析。在469个个体中共检测到126个等位位点,每个位点的平均等位位点数为(Na)5.25；位点多态率(PPl)为100%,期望杂合度(He)在高、低海拔都处于较高水平,分别为0.48和0.49；分子方差分析(AMOVA)的分析结果表明种群间分化占总变异的6.38%,遗传变异主要集中在不同个体之间；基因分化系数(FsT)的结果为0.02,也证实群体分化处于较低水平,检测到群体间的基因流(Nm)为9.89,处于较高水平。基于上述结果认为,海拔因素未对生长在色季拉山高低海拔的藏川杨群体造成地理隔离从而产生种群分化,藏川杨种质资源的保存无需考虑海拔的差异,在此地区不同海拔生长的藏川杨群体遗传背景一致,为研究高海拔的适应机制的材料选择提供了很大的便利。(2)使用GBS(genotyping by sequencing)的方法在由60个藏川杨基因型个体组成的群体中进行SNP位点开发,共得到6520个位点。利用群体的SSR标记与SNP标记两种标记遗传多样性结果进行对比发现：1)聚类结果有着显著差异,SNP的聚类结果与两个不同群体来源相一致,而SSR的结果显示两个群体来源的个体并无明显的分离。(3)对采集的469个种质资源进行了无性系化,并将其分别定植于不同海拔高度的试验点。对一年生的试验苗进行了苗高、地径、分枝数、叶形等表型性状的测定。并将环境及遗传因素对于表型变异的影响进行了细致剖析,且开发了适用于本次实验设计的模型,发现13个与地径相关联的位点及10个与分枝数相关联的位点。(4)利用R软件包fgwas2和PLINK软件对60个藏川杨群体的苗高、地径、分枝数、叶形等表型性状进行了SNP位点的关联分析。经R软件包fgwas2分析共发现3个位点与苗高性状相关联,其中106346位点表型变异的贡献率最高为11%,共发现2个位点与地径性状相关联,其中112578位点表型变异的贡献率最高为10.4%,共发现3个位点与分枝数性状相关联,其中109890位点表型变异的贡献率最高为10.9%；PLINK的分析结果如下：与株高性状进行关联的结果共得到17个位点(P<0.001),与其关联最紧密的SNP位点是111604,显著性达到6.09×10-5,与地径性状进行关联分析的结果共得到17个位点(p<0.001),其中显著性最高的为位点106346,为7.87×10-5,对分枝数性状进行了关联分析,共得到4个位点与该表型具有显著关联(p<0.001),其显著性最高的是位点130018,其显著值达到5.29×10-5。并对显示与性状相关的位点进行了BLAST,由于杨树基因组的注释还未完善,所以大部分位点显示与预测基因相关联。两个方法所检测到的与表型相关联的位点存在着一定的重叠,这表明两个方法均具有良好的检验效力。更多还原

【Abstract】 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.更多还原