Phylogenetic Analysis Of Pyrus L. Based On Multiple DNA Fragments And CpSSR Markers

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.