Phylogenetic Analyses And SSR Mining Of Genus Gleditisa

With the important ecological,medical and economic values,the exploration of the genus Gleditsia evolution relationship and the development of molecular markers will be a basis for evaluation of its resources.Genus Gleditsia may have experienced positive selection in the long evolution process.To explore the phylogenetic relationship of genus Gleditsia in family Leguminosae and detect the high resistance and secondary metalbolism gene under positive selection,we used phylogenomic method to construct one-to-one orthologous groups,and then conduct multiple sequence alignments to construct phylogenomic species tree.Consequently,we annotated the function of the genes under positive selection to explore the evolutionary of genus Gleditsia.Meanwhile,sequence analysis of three cpDNA was applied to examine taxon relationship of genus Gleditsia.MatK,PsbA-trnH and TrnL-F sequences were selected and amplificated by PCR and sequenced to construct phylogenetic tree.Furthermore,molecular markers for Gleditsia were still in lack for further genetic resources evaluation and the mining of EST-SSR markers would provide a molecular method for genetic diversity studies.1.Based on the four complete genome sequences of Leguminosae(Medicago truncatula,Lotus japonicus,Glycine max and Phaseolus vulgaris),we obtained 3278 core orthologous groups,which were the one-to-one gene orthologs present in all four Leguminosae species.Based on the core orthologous group,we construct core-ortholog database to search the corresponding orthologs in rest 20 species.In the 24 species,98one-to-one orthologous group were obtained,and for each group of ortholog,multiple sequence alignment was conducted separately.Finally,all the multiple sequence alignments were connected to form the super alignment matrix with 16,326 amino acids.From the phylogenetic tree,genus Gleditsia and Glymnocladus have a vevy close relationship,and the two genus together constitute a big branch with Desmanthus illinoiensis,Chamaecrista fasciculate and Senna hebicarpa,indicating that the two branches have a close relationship.Meanwhile,except for Cercis Canadensis,the rest species constitute a branch,and are independent with the other branches.Obviously,genus Gleditsia has separated from other Leguminosae species in the early stage.And Cercis Canadensis form an independent branch in the long evolutionary.2.A total of 2616 one-to-one Ortholog Group was firstly identified from two Gleditsia species.These Ortholog Groups were annotated by Gene Ontology annotation with the GOterms of “Metabolic process”,”Response to stimulus”,”Catalytic activity”,”Macromolecular complex”,etc.And for the KEGG pathway annotation,the pathway of “Biosynthesis of secondary metabolite”,“Metabolic pathways”,“Plant-pathogen interaction” were highly present.Within the 2616 one-to-one OGs,we detected 177 OGs show strong positive selection,and functional enriched GO and KEGG functional terms of these postion selection genes were strongly assocated with the resistance pathway and secondary metabolic process.3.Sequences of MatK,PsbA-trnH and TrnL-F were concatenated and aligned to examine taxon relationships among genus Gleditsia with the Gymnocladus chinensis and Gymnocladus dioica as the outgroup species.The total length of combined sequence was1,871 bp,and the number of variation sites was 285(15.19%).Accoding to the tree of Leguminosae and Gleditsia,genus Gleditsia is monophytic and forming a sister group with genus Glymnocladus,which is in accordance with other study in the;G.japonica var.velutina.and G.japonica var.delavayi are close to G.japonica,indicating a relative relationship.4.Based on the transcriptome RNA-seq data from NCBI,a total of 41003 Unigenes were obtained by assembling the RNA-seq data of G.sinensis.The total length,average length and N50 of the Unigenes were 70.4 Mb,1716 bp and 2533 bp,respectively.8494EST-SSR loci were detected in 7009 Unigenes,among which 1200 Unigenes contained two or more SSRs and 369 Unigenes contained compound SSRs.For all EST-SSR loci,6494 primers were designed and 60 from them were randomly selected to validate their effectiveness.Consequently,44 primer pairs can amplify target products,among which 17 primer pairs showed polymorphism and most polymorphism markers were identified to be located in UTR region.Furthermore,The PIC value ranged from 0.195 to 0.742,with an average of 0.501.Finally,we validated whether the primers could amplified target products in relatives of G.sinensis,among the 44 effective primers,32,31,23,24,7,40,25,18,and18 primer pairs can amplified target products in G.tricanthos,G.japonica,G.japonica var.velutina,G.japonica var.delavayi,G.fera,G.australis,G.microphylla,Gymnocladus chinensis,Gymnocladus dioica,indicating the high transferability of our developed primers.The result demonstrated that mining EST-SSR loci using transcriptome data is one of the efficient ways for developing molecular markers in tree species,with the advantages of steady ampilification,high polymorphism and high transferability.