DNA Barcoding Of Orchidaceae Based On Chloroplast Gene Sequence

Orchidaceae plants are the most diverse monocotyledons,with high ornamental and medicinal value due to the highly specialized flower organs and the accumulation of medicinal secondary metabolites.However,with the excessive collection of resources and habitat destruction,wild orchids are critically endangered.All wild orchids are included in the protection scope of the convention on international trade in endangered species of wild fauna and flora(CITES).The accurate identification of orchid species not only plays an important role in the safe use of plant medicine,but also plays an essential role in the protection and utilization of germplasm resources.DNA barcoding is a fast,accurate,convenient and effective way to identify species by using a relatively short standard DNA sequence in living organisms.In this study,six chloroplast genes(MatK,rbcL,ndhF,rpoB,rpoC1,ycf1)based on DNA barcoding technology were used as the theoretical basis for species identification,seed resource protection and innovative utilization of orchids.The GC content of the sequence and the variation sites were analyzed.Among the single genes,MatK,ndhF and ycf1 provided relatively high mutation sites and relatively fast evolution rate in orchids.In the combined sequence,and MatK+ycf1,rbcL+ndhF and rpoC1+ycf1 provided relatively high mutation sites.Genetic diversity index was calculated and nucleotide pairing differential distribution was evaluated.The high haplotype diversity and relatively low nucleotide diversity indicated that single and combined genes had certain polymorphism differences in the population of orchidaceae,while the nucleotide difference distribution of multiple peaks showed no population expansion.Through the analysis of nucleotide replacement saturation of 6 single genes and 15 combined sequences,it was found that these sequences were all suitable for the analysis of phylogenetic relationship when they reached the saturation state.The phylogenetic relationship and the results based on genetic distance analysis showed that,among the individual genes,ndhF and ycf1 genes had relatively ideal combination sequences for the identification of orchidaceae at the level of genus and species,and matK+ycf1 and ndhF+ycf1 had relatively ideal identification ability at the level of genus and species.The ndhF,ycfl,matK+ycf1 and ndhF+ycf1 were recommended as candidate bar codes for orchidaceae.Species-specific barcodes of orchids were obtained based on SNP sites by using candidate genes,and generated DNA two-dimensional code id cards that could be quickly recognized by electronic devices.This study can provide important basic data and research methods for the efficient identification of Orchid species,and also provide researchers with standardized and accurate information on the barcode of orchid species.