Chloroplast Genome Characteristics And Phylogeny Of The Genus Daphne

Daphne is a large genus of the Thymelaeaceae.There are about 95 species in the world,about 52 species in China,mainly distributed in southwest and northwest regions,and the rest scattered all over the country.At present,the researches on the genus Daphne mainly focus on the chemical composition analysis,ornamental ultilization,medical resources and morphological studies,while molecular systematics research is few and limited to the nuclear DNA and chloroplast DNA fragments of a few species,and lacks the relatively complete phylogenetic relationship analysis of the genus and its close genera.At present,the morphological classification of the family Daphne needs to be identified according to a sereirs of characters.However,some charactores are across and transitional in the genus of Daphne and Wikstroemia,and the systematic relationship between the two genera has been controversial.Based on the structural characteristics of chloroplast genome and its advantages in phylogenetic analysis,28 species of genus Daphne are selected in this study.Among them,plastomes of 21 species are the first reported in this paper.The following studies are conducted on the chloroplast genome information of 14 genera and 23 plants of the family Thymelaeaceae(plastomes from 10 genera and 10 species are the first reported).The basic characteristics of chloroplast genome sequence of Daphne showedhowed a typical quadripartite structure,and the genome size,gene length,content and arrangement sequence were relatively conservative.While D.genkwa was special,the whole genome sequence length of which was the shortest and the coding genes were the least.Our result suggested that the contraction of inverted repeat region and the loss of fragment might be the main reaso;the total gene number vary in a few species of other genus of Daphne,which was possibally caused by the early termination of translation due to the occurrence of terminator in ndh F,ndh G,ndh K and rpl32 genes.The relative synonymous codon usage of the genus Daphne is largely consistent.The most of the repeat sequences of the genus Daphne are located in inverted repeat region,which is different from most angiosperms.It's related possiblely to the obvious expansion of inverted repeat region of the genus Daphne.Simple sequence repeats are mainly distributed in large single-copy region,with the majority of mono-nucleotide SSRs,the least number of hexo-nucleotide SSRs,and a rich variety of tri-nucleotide SSRs,tetra-nucleotide SSRs and pent-nucleotide SSRs,which can be further developed as molecular markers for phylogenetic analysis,species identification and population diversity analysis.Through comparative analysis of chloroplast genome of Daphne,it was found that the sequence of Daphne and the family of Thymelaeaceae had obvious inverted repeat region expansion.The expansion affects the length and number of coding genes of chloroplast genome,and also affects the number and distribution of repeat sequences.The mechanism for inverted repeat region expansion remains unexplored.This study provides more basic data for the study of inverted repeat region expansion and contraction.There was no gene rearrangement in chloroplast genome sequence of Daphne.The results of multi-alignment showed that small single-copy region and inverted repeat region of D.genkwa could not be aligned with other species.The coding region of chloroplast genome of this species was more conservative than non-coding region,and the less conservation region was mainly distributed in gene interval,and concentrated in large single-copy region and four borders region.The results of nucleotide polymorphism(PI)analysis were consistent with multiple alignment,and the coding region was more conservative than the non-coding region.The detected variation hotspots could be further developed as DNA barcode markers for phylogenetic studies.By using 31 chloroplast genome sequences and 35 internal transcribed spacers sequences annotated by sequencing carried out in this study,and 23 chloroplast genome sequences and 19 ITS sequences downloaded from the NCBI database,phylogenetic analyses based on chloroplast whole genome sequences,shared coding sequences of chloroplast genomes and Internal transcribed spacers sequences of the genus Daphne were carried out,and the results showed that(1)Based on the current circumscription of the genus Daphne,the genus Daphne is a polyphyletic taxon rather than a monophyletic taxon;(2)The genus Daphne and the genus Wikstroemia have independent phylogenetic positions,and some species in the genus Daphne should transfer its phylogenetic positions to the genus Wikstroemia.The two genera do not form a monophyletic lineage and cannot be combined together;Among them,D.aurantiaca,D.genkwa,D.holosericea and D.rosmarinifolia clustered together with species of the genus Wikstroemia and should be transfer to the genus Wikstroemia,while D.championii was more closely related to species of the genera Edgeworthia,Eriosolena and Rhamnoneuron;(3)24 species of the genus Daphne formed monophyly,the species distribution may affect their systematic relationship;The phylogeny based on the chloroplast genome sequence display that species distributed in China diverged later than those distributed in Europe;(4)The chloroplast genome has certain advantages in solving the inter-species phylogenetic relationship among those genera in Thymelaeaceae.The two data sets based on chloroplasts and the different analysis methods of the inter-genus and intra-genus branches are highly supported;(5)The phylogenetic analysis of Daphne,the inconsistency between the analysis results of chloroplast DNA and nuclear ITS sequence is mainly due to the lack of enough informative sites in ITS sequence with short sequence length and the low support rate of the analysis results.At the same time,it can't be reject that the existence of interspecific hybridization of the species.This study obtained the chloroplast genome characteristic information of the genus Daphne,which provided a data basis for the classification and identification,phylogeny,genetic evolution,and resource development and utilization of the species.