Study On Chloroplast Genome Of Schisandraceae,and Molecular Identification Of Kadsura Interior

Schisandraceae is a family of Austrobaileyales belonging to basal angiosperms.Schisandraceous plants has important medicinal value.Schisandra chinensis,S.sphenanthera and Kadsura interior has been recorded in Chinese Pharmacopoeia.However,there are a lot of problems in the systematic study of Schisandraceae,such as systematic position of Schisandraceae,monophyletic and species complex problem in Schisandra and kadsura,and species classification.These problems have a great effect on clinical medication.In this study,we hope to promote the development of the classification and identification of Schisandraceae by chloroplast genome,DNA barcoding and phylogeography.The chloroplast?cp?genome is semiautonomous and maternally inherited.The cp genome has a wealth of genetic variation and moderate rate of nucleotide evolution.It can be used for phylogenetic studies at different taxonomic levels.The total genomes of 16 species in Schisandraceae were completely sequenced by using the Illumina PE150 platform.According to length,these cp genomes were divided into two types,i.e.Long?L?type and Short?S?type.The L type which length range is 152.7-153.4 kb includes K.interior,K.heteroclite,K.longipedunculata,K.japonica and K.oblongifolia.S type which length range is 152.7-153.4 kb includes K.coccinea,S.propinqua,S.plena,S.chinensis,S.sphenanthera,S.rubriflora and S.henryi.113 unique genes,including 79 protein-coding genes,30 tRNA genes and four rRNA genes,were annotated in Schisandraceous cp genomes.A total of 145 simple sequence repeats?SSRs?,including 99 Mono-SSRs?29 Di-SSRs?6 Tri-SSRs?5 Tetra-SSRs?2 Penta-SSRs and 3 Hexa-SSRs,were detected in Schisandraceous cp genomes.The proportion of T,F and P was classified three types.Type 1 includes K.coccinea with 43-47%of T,26-29%of F and 26-29%of P.Type 2 includes species of L type with 28-29%of T,29-35%of F and 32-39%of P.Type 3 includes most species of Schisandra,except S.plena,with 40-45%of T,36-40%of F and 16-21%of P.By comparing each cp genome with S.chinensis,we found the cp genomes of S.plena and S.propinqua were similar to that of Kadsura species.A large IR expansion of about 8 kb has occurred in L type species.A phylogenetic analysis based on all Schisandraceous cp genome sequences supported that taxa Pleiostema and Sinoschisandra should be set as two sect.which belong to subgen.Schisandra.Species of L type grouped into one clade which involved in all species of subgen.Kadsura distributed in China.Remarkably,four samples of K.heteroclite did not group into one clade.DNA barcoding was used to identify K.interior.We analyzed five DNA barcodes?ITS,ITS2,psbA-trnH,matK and rbcL?in terms of distance-based,tree-based and character-based identification methods to distinguish K.interior and its adulterants.In distance-based and tree-based identification,K.interior could be distinguished easily from the species of Schisandra and K.coccinea.In character-based identification,there are two single nucleotide polymorphisms?SNPs?in ITS and one SNP in psbA-trnH which can be used to distinguish K.interior from K.heteroclita and K.longipedunculata.The results indicate that DNA barcoding can be used to identify the K.interior.The ITS and psbA-trnH sequence can be the most ideal DNA barcodes for discriminating K.interior and its adulterants by the combination analysis of distance-based,tree-based and character-based identification?SNPs?.Phylogeographical analyses were carried out for species identification in Kadsura.The result shown that K.interior owned two specific haplotypes of psbA-trnH and one shared haplotype of ITS with K.heteroclite which was collected in Pingbian Yunnan.Therefore,by this method we are able to identify K.interior from its affinities.After classifying all haplotypes of ITS into six types?C₁-C 6?,we also find out C l distributed in southwest China,C-2 in central China,C₃ in east China.It proved genetic basis for species delimitation and application of similar medicinal materials.