The Research Of Chloroplast Genomes And Phylogenetic Relationships Of Salicaceae

Salicaceae is a widely distributed flowering plant family.Traditionally,Salicaceae consists of two genera,Populus L.and Salix L.,which is Salicaceae sensu stricto.With the advancement of molecular biology and sequencing technology,Salicaceae has undergone a series of taxonomy revisions by the Angiosperm phylogeny group.Most species of Flacourtiaceae and all species of Scyphostegiaceae were classified into Salicaceae.According to the priority principle of the nomenclature,the combined new family adopted the name Salicaceae.In the past,most studies focused on the phylogenetic relationships between Populus and Salix,while the phylogenetic relationships of other genera were less studied,and the chloroplast genome data of other genera were rare.Therefore,this study used whole-genome sequencing and bioinformatics technology to sequence and assemble the chloroplast genomes of 4 species from different genera of Salicaceae,analyzing the genome structures,genome size,gene order,gene numbers and GC contents.In order to clarify the phylogenetic relationships of some genera in genome levels,we used the assembled chloroplast genomes and the published chloroplast genomes of several other Salicaceae genera,with Hevea brasiliensis(Willd.ex A.Juss.)Muell.Arg.(Euphorbiaceae)as the outgroup to construct the phylogenetic tree.Secondly,based on the 15 pairs of single-copy nuclear loci of Salicaceae sensu stricto.Single-copy nuclear gene primers were developed for 17 genera of Salicaceae for subsequent phylogenetic analysis.Finally,based on 9 chloroplast fragments and 5 single-copy nuclear gene fragments,3 species of Achariaceae(Hydnocarpus anthelminthicus Pierre ex Laness.,Hydnocarpus borneensis Sleumer,and Gynocardia odorata Roxb.)as outgroups.Phylogenetic trees of 17 genera of salicaceae were constructed by the maximum likelihood method,the maximal parsimony method and the bayesian inference method,respectively.Then compared and analyzed chloroplast gene tree and nuclear gene tree.The main results of this study are as follows:(1)With the chloroplast genome of Flacourtia indica(Burm.f.)Merr.as the reference genome,the chloroplast genome of 4 species(Homalium cochinchinense(Lour.)Druce.,Bennettiodendron leprosipes(Clos)Merr.,Xylosma congesta(Lour.)Merr.,Flacourtia jangomas(Lour.)Raeusch.)were assembled and analysed.Genome structures were all typical quadripartite structure,including a large single copy,a small single copy and two inverted repeat regions.The 4 chloroplast genomes were similar in size,number of genes,gene order,and GC contents.In the phylogenetic tree of chloroplast genome constructed by the neighboring method,B.leprosipes and Idesia polycarpa Maxim.were clustered into a clade,which was verified close relatives of Populus-Salix.(2)Based on the 15 pairs of single copy nuclear loci of Salicaceae sensu stricto,5 pairs of single-copy nuclear gene primers were developed with high amplification ability and versatility in 17 genera of Salicaceae.Twenty individuals of Scolopia chinensis(Lour.)Clos(Scolopia Schreb.)were used to evaluate the application of these primers in terms of diversity indices and neutral test.The number of haplotypes(H)was 3-9,the separation sites(S)was 2-9,the average nucleotide difference(κ)was 0.644-2.278,π and θw were 0.00104-0.00451,0.00121-0.00475,respectively.The results showed that these primers had the high genetic diversity in S.chinensis.Neutral test results indicated that all loci were consistent with neutral evolutionary assumptions,which are suitable for subsequent phylogenetic analysis.(3)The phylogenetic trees generated from the 3 methods had similar topology to each other with a few differences in some clades.The 2 phylogenetic trees showed that Idesia Maxim.,Bennettiodendron Merr.,Poliothyrsis Oliv.,Itoa Hemsl.and Olmediella Baill.were closely related to Populus-salix.Bennettiodendron,Idesia and Olmediella were most closely relatives of Populus-Salix.Carrierea Franch.did not show a close relationship with Populus-salix in the nuclear gene tree,It may be related to the lack of phylogenetic information of nuclear dataset or introgression,and further verification of its phylogenetic relationship with Populus-salix is needed.The species of Salicoideae Arnott,Scyphostegioideae Revea and Samydoideae Reveal were on different branches.By combining morphological and molecular data,we support the classification of Salicaceae into three subfamilies.The species of Populus and Salix were clustered to different clades,showing a sister group relationship.Which proved that both Populus and Salix are the monophyletic origin.The species of Flacourtieae were clustered to 3 different clades,suggesting that this tribe is not the monophyletic origin.So we suggest to reclassify the species of Flacourtieae.