Chloroplast Genome Sequences Of Diospyros Spp.:Phylogenetic And Genetic Variation Analyses

Diospyros is the largest genus in Ebenaceae,comprising more than 500 species.As the representative species of Diospyros for fruit cultivation,D.kaki Thunb.has traditionally been an important food resource in China,Korea,and Japan.D.kaki Thunb is hexaploid,thus the research progress in molecular biology is slow,and the progenitor,origin,and polyploidization mechanisms of D.kaki Thunb are still ambiguous.Therefore,identifying a closely related diploid species to be used as reference for future research has important scientific and theoretical value.In this study,by using high-throughput sequencing technology,the chloroplast(cp)genomes of five Diospyros species were sequenced.Gene maps of Diospyros chloroplast genomes were successfully mapped,and the genome structure and sequence variations among five Diospyros species were analyzed.Phylogenetic analyses based on different cpDNA sequences confirmed the station of Diospyros in plant phylogenetic evolution,and further clarified the genetic relations among Diospyros species.Based on the sequencing results,cpDNA markers were developed to study the interspecific genetic variations.The main results were showed as follows:1.Complete chloroplast genomes from D.kaki,D.lotus L.,D.oleifera Cheng.,D.glaucifolia Mete.,and Diospyros 'Jinzaoshi' were sequenced using Illumina sequencing technology.This is the first cp genome reported in Ebenaceae.The cp genome sequences of Diospyros ranged from 157,300 to 157,784 bp in length,presenting a typical quadripartite structure with two inverted repeats each separated by one large and one small single-copy region.For each cp genome,134 genes were annotated,including 80 protein-coding,31 tRNA,and 4 rRNA unique genes.In all,179 repeats and 283 single sequence repeats were identified.Four hypervariable regions,namely,intergenic region of trnQ rpsl6,trnV ndhC,and psbD trnT,and intron of ndhA,were identified in the Diospyros genomes.2.The global alignments between Ebenaceae and other published families in Ericales indicated that non-coding sequences exhibit a higher divergence across different species,especially the Vaccinium macrocarpon.Moreover,IRs were more conserved than SCs,and the IR/SC borders and lengths of Ebenaceae were also different from that in other families.In addition,Phylogenetic trees which reconstructed based on 61 protein-coding sequences shared by 19 angiosperm species indicated the basal position of Ericales in asterids and suggested that Ebenaceae is monophyletic in Ericales.3.Intergenic region of trnQ rpsl6 which varied most among five Diospyros species was used to study the phylogenetic relationship of 17 species.172 variable sites and 80 parsimony-informative sites were found,and two phylogenetic trees were constructed by MP and ML methods.As a result,eight related species of D.kaki were screened out in pilot study.Then,five special primer pairs of cpDNA hypervariable regions were used to amplify these regions of 8 Diospyros species,then the PCR products were digested by seven restriction enzymes.Total 105 polymorphic fragments were gathered,cluster analysis showed that D.'Jinzaoshi'?D.olei?D.oleifera and D.artrotricha had a near relationship with D.kaki,while D.glaucifolia and D.lotus were distantly related to D.kaki.After that,in order to find the closest relatives of D.kaki,cpDNA ndhA and nrDNA ITS sequences were used to analyze the phylogenetic relationship of 7 related species of D.kaki.ML tree constructed based on ndhA sequences indicated that D.kaki was close to D.artrotricha,and D.oleifera was close to D.'Jinzaoshi'.Moreover,the male and female plant of D.lotus and D.virginiana could be separate,and the female plants were closer to D.kaki.While ML tree based on ITS sequences indicated that D.oleifera were closer to D.kaki than D.artrotricha.Finally,Phylogenetic analyses based on the whole cp genome,80 protein-coding,and intergenic and intron sequences indicated that D.oleifera is closely related to D.kaki and could be used as a model plant for future research on D.kaki.4.Based on the sequencing results,four cpDNA markers were developed,and 16 Diospyros plants were used to verify their feasibility.For the four cpDNA markers,31?27?7?25 variable sites were detected,respectively;and the parsimony-informative sites were 14?5?2?13.Cluster analysis showed that CP1 marker could distinguish the five related species from 11 persimmon cultivars,indicating that CPI was suitable for analyzing the genetic variations between different Diospyros species.While,CP2?CP3?CP4 failed to make a distinction between persimmon cultivars and their related species.Nonetheless,several single base mutations and Indels were found across the 11 persimmon cultivars,and most of the cultivars showed specific variable sties,except for 'Fuyuu' and 'Zhongshi 1#',this result suggested that CP2?CP3?CP4 markers could be used to study the intraspecific genetic variations.In addition,the genetic diversity of selected 21 persimmon cultivars was analyzed based on ITS sequences variations.Four variable sites were found,and 21 persimmon cultivars were classified into ten types,which implied that the intraspecific variation could be analyzed according to ITS sequences variations.