摘要
梨属植物是蔷薇科(Rosaceae)苹果亚科(Maloideae)或梨亚科(Pomoideae)中的一个重要属。一般认为梨属植物的基本种有20个左右,被中国植物分类学家认可的原产中国大陆的梨属种(包括非基本种)有13个。由于梨属植物种间和种内杂交非常普遍,种间可以区别的性状特征有时不明显,因此单独基于传统形态学的分类方法很难确切判断种和分析种间的系统关系。化学成分、同工酶、花粉超微结构等研究手段对解明梨属植物种间关系也做出了重要贡献,这些信息虽然不易受环境影响但标记数量非常有限。目前还没有建立起完整的梨属植物内系统关系,许多可疑杂种或栽培种的起源问题仍存在争议。
本研究广泛选取了东亚原产为主的梨属种和代表品种为样本,应用不同来源的多种DNA序列及cpSSR分子标记试图从分子系统学的角度为阐明梨属内的系统关系、梨属植物分化过程中可能经历的进化史及杂种起源提供证据。主要结果如下:
1通过PCR优化在包括44个梨样本和3个外类群(苹果属)的所有供试样本中得到了128条功能性ITS序列;而在普通PCR条件下,在若干样本中得到了42条ITS假基因序列。由于功能性拷贝序列分化度低(avr=2.6%),个体内存在不同程度的多态性和可疑重组子,且个体内多态序列在系统树上并非单源,因此导出的系统结果复杂,支持率普遍较低,并没有预期地解决梨属植物种间关系,根据个体内功能性ITS序列多态性程度的高低仍揭示了不同种或品种的杂合背景,暗示了梨属栽培种的分化过程中存在网状进化和其它更复杂的进化史。在梨属植物中得到的ITS假基因序列因GC含量低、二级结构遭破坏,在普通PCR条件下被优先扩增。其序列与相应的功能拷贝序列差异度很大(15%),在系统树上也独立组成单源群,因此推测其起源时间较早。根据不同起源可以将所得假基因分为两个谱系(lineage),即、ψa、ψb、ψc、ψd和ψe、ψf、ψg。其中ψb、ψc、ψd为共同起源,进化速率较快,且基于这些序列导出的系统结果关系明确、支持率高,具有很大的潜力来重建梨属内系统关系。这些假基因序列呈中性进化,是应用分子钟进行分化时间估计的很好的资源。此外,一些“古老孑遗”的ITS假基因序列为阐明一些较原始的种的演化历程提供了宝贵证据。基于功能性ITS功能序列和ITS假基因序列的系统结构中,西方梨都为单源,与东方梨显著分开,支持东西方梨为独立分化的观点。
2梨属植物Adh基因中存在长度各异的6个内含子区,我们仅对包括内含子1和内含子2区的5'端650 bp长的序列进行了分析。经大量克隆测序发现,梨属植物中至少存在4种主要的Adh位点:Adh1、Adh2、Adh3及Adh4和一种可疑的Adh5。Adh内含子1区长度变异较大,各Adh位点在此区分别共享了其特有的长度变异。不同Adh位点间的序列分化度较高,为8%-20%,而各位点内的序列分化度也较低(<2%)。在大多数样本个体内,每个不同Adh位点都存在序列多态性,即存在多个同类Adh拷贝。其中属于Adh1、Adh2和Adh3的差异拷贝大多为单源;而来自Adh4(拷贝数最多)的个体内差异拷贝大多数为多源,因此导出的系统树结构与功能ITS序列相似,关系复杂,可能存在谱系筛选及基源拷贝未鉴定准确的原因。四个位点中Adh2因起源较晚,拷贝数较单纯,显示了较好的应用价值。从系统树结构和支持率看,低拷贝核基因初步显示了比功能性ITS序列更好的系统学潜力
3两个叶绿体非编码区,trnL内含子及trnL-F,在梨属植物中高度保守,未能预期解决梨属内系统关系。从系统树结构看,东西方梨两大组再次显著分开,而西方梨样本仅存在一种trnL-F序列的长度类型,多样性简单且与苹果属关系更近。而东方梨存在三种序列长度类型,多样性较丰富。这两个叶绿体非编码区的长度突变和核苷酸突变为一些杂种的母系提供了依据,如‘库尔勒香梨'的母本非西洋梨;褐梨、河北梨及部分秋子梨间关系密切;中国白梨品种并非起源于P.breteschneideri1(罐梨),两者不存在直接联系。在东方梨中秋子梨显示了最丰富的多样性。
4应用12对cpSSR引物对供试梨样本进行分析发现仅4对引物的PCR产物在梨属中存在多态性。最后共获得14个多态位点,基于这些位点构建的UPGMA系统树结构与两种叶绿体非编码区序列的系统结果基本相似。
The genus Pyrus L.belongs to the subfamily Pomoideae(Maloideae),and the family Rosaceae.It is generally accepted that there are about 20 stock species in Pyrus,and thirteen species originating from China have been classified and extensively accepted by Chinese botanical taxonomists.However,all species of Pyrus are inter-crossable and inter and intra-specific hybridisation have been thought to be the major mode of evolution in this genus,which makes it difficult to define the species and estimate exact genetic relationships of Pyrus species based on morphological characteristics.Other biological markers including chemical compounds,pollen ultrastructure and isozymic patterns have also been used to distinguish pear species and cultivars.However,these markers are quite number-limited and/or vulnerable to environmental effects.The phylogeny of the Pyrus have not been completely constructed and the origin of several putative hybrids and cultivated species remains unclear or controversial.
In this paper,we selected Pyrus species,cultivars and wild types mainly native to East Asia as samples and tried to elucidate the phylogenetic relationships among Pyurs sepecies and understand the evolutionary process involved in the species diversification in Pyrus based on molecular markers including several DNA fragments and cpSSR marker.The main results were as follows:
1 A total of 128 funtional ITS sequences were obtained in all investigated accessions including 44 Pyrus speceis or cultivars and three outgroup species(Malus Mill.) under the modified PCR conditions,and 42 putative ITS pseudogenes were discovered in several accessions under normal PCR.Functional ITS copies led to confused and poorly resolved phylogeny as a result of low sequence divergence (avr=2.6%),existence of unidentified ancient recombinants and a high degree of intra-individual functional ITS polymorphism which were mostly polyphyletic in the tree,suggesting reticulate evolution and other complex evolutionary process such as rapid ancient radiation have been involved in the diversification of Pyrus species.The genetic background of different Pyrus species or cultivars were elucidated by their degree of intra-individual functional ITS polymorphism.ITS pseudogenes displaying lower GC content and unstable secondary structure were preferentially amplified under normal PCR conditions.All pseudogenes were highly divergent from their corresponding functional copies(15%) and formed a monophyletic group in the phylogenetic tree based on all paralogs,indicating they were of relatively early origin. All of the pseudogenes found in Pyrus could be divided into two lineages according to their separate orgin.One linegae includedψaψb,ψc andψd,and the other includedψe,ψf andψg.Certain types of pseudogenes(ψb,ψc andψd) were of common origin and have been evolving with high substitution rate.Phylogenetic relationships based on these pseudogenes were unambiguous and highly supported.Therefore,these types of ITS pseudogenes have great potential to reconstruct the phylogeny of Pyrus. Evolution of ITS pseudogenes discovered in this study was under neutral selection and will be good resources to evaluate the divergence time of species using 'molecular clock'.Besides,some relict pseudogenes provided credible clues for the evolutionary history of some relict and primary Pyrus species.The Occidental species were monophyletic in the phylogenetic trees based on both functional ITS sequences and ITS pseudogenes,apparently separating from Oriental pears,which supports the view that Oriental pears geographically and genetically distinct from the East Asian pears.
2 There were six different-sized introns in the Adh gene in Pyrus sepecies,and only partial sequence of the 5' portion including intron 1 and intron 2(about 650 bp) were sequenced.As a result,four major Adh loci,i.e.Adh1,Adh2,Adh3,and Adh4,in addition to a putative Adh5 were identified and all of these five Adh loci respectively shared particular length variation in intron 1 region.Sequence divergence among different Adh loci ranged from 8%to 10%,while that within Adh loci was quite low (<2%) and could not provide enough information to resolve the phylogeny of Pyrus. Besides,divergent copies representing each Adh loci could be obtained in most pear individuals.Divergent copies found in one individual presenting Adh1,Adh2 and Adh3 were monophyletic in the tree,while those representing Adh4 which was the most frequently cloned in this study were mostly polyphyletic in the tree,which may caused by hybridisation as well as lineage sorting.Besides,there might be unidentifiable paralogs among Adh4.
Among the four major Adh loci,Adh2 was of relatively late origin and showed potential as another powerful phylogenetic tool for Pyrus.Low copy nuclear gene was more powerful than ITS and cpDNA to resolve the phylogenetic relationships in Pyrus.
3 trnL intron and trnL-F were highly conserved among Pyrus speceis and failed to resolve the phylogeny of Pyrus.Occidental species displayed only one type of trnL-F region,suggesting they were genetically simple compared to that of Oriental pears.Besides,the Occidental speceis were monophyletic in the tree and more closely related to species of Malus species.Informative length variation and nucleotide substitution in these two cpDNA region also provided evidence of the maternal origin of several species and cultivar.It was found that the maternal parent of 'Korlaxiangli' could not be P.communis,the putative hybrids P.phaeocarpa,P.hopeiensis were closely related to some accessions of P.ussuriensis.Chinese white pear cultivars did not originate from P.breteschneideri.It was also found that P.ussuriensis was the most diversified species among Oriental pears.
4 In the study of cpSSR analysis,only four from 12 primers showed length polymorphism in all of the Pyrus accessions including outgroup ones from Malus.As a result,only 14 polymorphic loci were examined and the phylogeneitc tree based on UPGMA methods was similar to that based on the above two cpDNA non-coding regions.
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