摘要
遗传多样性是物种或群体抵抗环境变化的重要因素,也是生物多样性的核心组分,对于野生果树资源而言,遗传多样性更是砧木筛选的重要依据,对生产具有重要的意义。原产于我国的野生梨属种豆梨(Pyrus calleryana Dcne)和川梨(P. pashia D. Don)是我国南方地区的主要砧木资源。另外,豆梨被认为是最古老的梨属种之一,川梨则被认为是东、西方梨分化过程中的过渡种。因此,研究豆梨和川梨群体遗传多样性对解决梨属植物的起源演化过程也具有重要的意义。迄今为止,我国野生梨属资源遗传多样性和遗传结构的相关研究还非常有限。本研究利用cpDNA单倍型和nSSR的方法,结合对果实表型性状的分析,以野生豆梨和川梨群体为材料,对这两个野生种的遗传多样性和群体遗传结构进行评价,主要的研究结果如下：
1. cpDNA单倍型和nSSR的结果表明目前浙江省豆梨保持着较高的遗传多样性水平(cpDNA:Hd=0.719,π=0.00105, nSSR:Hc=0.639). AMOVA分析表明豆梨的遗传变异主要存在于群体内,cpDNA和核基因水平上群体内的遗传变异分别占总遗传变异的77.04%和91.14%。Mantel检验表明群体间的遗传距离与其地理距离呈现显著的正相关(cpDNA单倍型：r=0.332,p=0.048;nSSRs:r=0.592, p=0.008)。SSR的结果表明群体间出现了一定程度的遗传分化(Fst=0.089)。本研究中花粉流／种子流仅为0.86,说明群体间的遗传物质交换主要依靠种子传播进行。STUCTURE的结果表明所有豆梨群体可以分为两个类群,根据不同类群在各群体中的比例可以将所有群体分为两个组,豆梨群体cpDNA和nSSR的N-J聚类树均表明所有群体可以分为东北和西南两组。浙江省的8个豆梨群体中,群体FY的遗传变异程度最高,而且群体规模也较大,因此在开展浙江省豆梨资源的保护工作时,群体FY应该优先得到保护。
2.川梨群体保持着较高水平的遗传多样性(cpDNA:Hd=0.718, π=0.00085; nSSR:He=0.741). AMOVA分析表明川梨的遗传多样性主要存在于群体内,群体内的遗传变异在cpDNA和核基因水平上分别为59.61%和91.24%。cpDNA单倍型的Mantel检验表明群体间的遗传距离与其地理距离无显著相关(r=0.139,p=0.09),但是nSSR的Mantel检验的结果则显示群体间的遗传距离与其地理距离呈显著正相关(r=0.783,p=0.000)。在叶绿体水平上,群体间的遗传分化较大,遗传分化系数为0.404,核基因水平上群体间仅出现了中等程度的遗传分化,遗传分化系数为0.087。cpDNA单倍型的结果表明群体间的遗传分化较大,特有单倍型较为普遍,单倍型的网络图呈辐射状分布,推测川梨群体经历了近期的快速分化。川梨群体存在着亲缘地理结构(Nst>Gst, p<0.05),对cpDNA单倍型的亲缘地理学分析表明川梨群体经历了连续的扩张事件.NCA分析表明支系1-2、2-1和总支系经历了连续的群体扩张,总体的扩张时间大约发生在621000到209000年前之间。根据Ennos的花粉流和种子流比值的公式得到,花粉流是种子流的6.05倍,表明川梨群体之间的遗传物质交换主要通过花粉的传播进行。川梨的资源保护和利用工作应该集中于遗传变异丰富的群体以及包含特有基因的群体,群体YN1和YN8分别代表了最高水平的cpDNA和nSSR的遗传变异水平,应该优先得到保护。
3.通过对本研究所用的豆梨和川梨样品的cpDNA单倍型结果进行比对,发现豆梨和川梨共享2个单倍型(单倍型S1和S2),这两个单倍型分别为豆梨和川梨的祖先单倍型。豆梨和川梨在单倍型网络图上并不能清晰的分为两枝,说明两者在叶绿体水平上的分化程度并不是很高,存在较近的共同祖先。
4.本研究对豆梨和川梨6个果实表型性状的变异进行分析的结果表明单果重在群体内和群体间的变异程度最高(P. calleryana:26.37%; P. pashia:37.06%)。 UPGMA聚类图和主坐标分析的散点图表明利用6个果实表型性状可以将豆梨和川梨区分开来。
Genetic diversity is a critical factor for the long-term survival of a species or a population. To wild fruit tree species, understanding the genetic diversity is the primary task of rootstock selection. Pyrus calleryana Dcne and P. pashia D. Don are wild pear species which originate in China and widely used as rootstocks in Southern China. P. calleryana is one of the most ancestral pear species while P. pashia is thought to be the linkage between Oriental and Occidental Pear. Therefore, these two species also play important roles in the evolution of Pyrus. However, lack of sufficient research on P. calleryana and P. pashia limits the utilities of these two important pear species. In this study, a combined approach using cpDNA haplotypes and nSSRs was taken to study the genetic diversity and population structure of P. calleryana and P. pashia. Considering that P. calleryana and P. pashia are morphologically similar to each other, the phenotypic variation of them was also studied using six fruit phenotypic characters. The main results are listed as follows:
1. Two cpDNA intergenic regions (accD-psaI and trnL-trnF) and14nSSR makers were used to study77individuals from8populations. A high level of genetic diversity was detected in P. calleryana (cpDNA:Hd=0.719,π=0.00105, nSSR: He=0.639). AMOVA analyses revealed that the majority of the genetic diversity occurred within population. Mantel tests showed that the genetic distance was significantly correlated with the geographic distance (cpDNA haplotype:r=0.332, p=0.048; nSSRs:r=0.592,p=0.008). STRUCTURE analysis exhibited that two clusters existed among the8populations. All the populations could be divided into two groups, correlating with geographic regions (northeast and southwest). Geographic distance was a key factor in shaping current genetic structure of P. calleryana. Pollen/seed flow (0.86) indicated that seed dispersal among populations was more frequent and pollen dispersal was limited to some extent. Population FY was the most diverse population and suitable population to be protected under in situ conservation strategy.
2. In this study, we investigated the genetic diversity and population structure of P. pashia using accD-psaI and trnL-trnF intergenic spacers and13nSSR makers for a total of327individuals over22populations. P. pashia contained a relatively high level of genetic diversiy (cpDNA:Hd=0.718, π=0.00085; nSSR:He=0.741). AMOVA analyses of molecular variance indicated that the genetic variation mainly existed within populations, representing59.61%and91.24%of the total variation on the cpDNA and nuclear DNA level, respectively. Mantel test of cpDNA did not show a correlation between the genetic and geographic distances (r=0.139, p=0.09) while the nSSR-based Mantel test showed that geographic distance was an important factor to shape the current population genetic structure (r=0.783,p=0.000). According to the cpDNA haplotype results, genetic differentiation among populations was high (Gst=0.404). However, nSSR only revealed moderate genetic differentiation (Fst=0.087). Nst (0.420) was significantly higher than Gst (0.402)(p<0.05), which was reflected on the phylogeographic structure in P. pashia. NCA analyses of clade1-2, clade2-1, and the total cladogram inferred contiguous range expansion events in P. pashia. The overall population expansion of P. pashia was estimated to occur between621,000and209,000years ago. NCA analyses of clade2-2showed that restricted gene flow existed among populations. STRUCTURE analysis showed that four clusters formed the current genetic structure of P. pashia. According to the Ennos' formula, pollen flow was6.05times more than seed flow. Populations YN1(cpDNA) and YN8(nSSR) represented the highest genetic diversity of P. pashia and should have the priority when conservation strategy is implemented.
3. All the cpDNA haplotypes of P. calleryana and P. pashia were used to construct Neighbor-Net network. Two haplotpyes were shared by P. calleryana and P. pashia (haplotype S1and S2), which were the most ancestral haplotypes of P. calleryana and P. pashia, respectively. Shared haplotypes and non-monophyly of these two species on the Neighbor-Net network indicated that they had a recent ancestor.
4. Six phenotypic variations were analyzed in three P. calleryana populations and four P. pashia populations. Results showed that single fruit weight was the most diverse phenotypic character (P. calleryana:CV=26.37%; P. pashia:CV=37.06%). Four clusters were detected from UPGMA clusters, and all the samples of P. calleryana were grouped into one cluster. PCoA analysis also distinguished P. calleryana from P. pashia.
引文
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