Study On DNA Barcode And Chloroplast Genome Of Herba Anoectochili

In Orchidaceae plants,Herba Anoectochili is a precious medicinal material and widely cultivated in Fujian,Guangzhou,Zhejiang and other areas of our country.Herba Anoectochili needs a special growth environment and is extremely difficult to breed.Most of the Herba Anoectochili sold on the market is artificial tissue cultured and often mixed with other medicinal plants such as Ludisia discolor and Goodyera schlechtendaliana,which are very similar to Herba Anoectochili,so it is very difficult to distinguish them by external shape alone.DNA barcode technology generally refers to the technology of quickly and accurately identifying species with a specific DNA fragment.In recent years,with the rapid development of molecular biology and bioinformatics,this emerging technology for identifying and classifying biological species has been widely used.In this study,we extracted the DNA of 32 Herba Anoectochili,6 Ludisia discolor,and 3Goodyera schlechtendaliana,and amplified their ITS2 sequences.The results showed that the lengths of ITS2 sequences of Anoectochilus roxburghii,Anoectochilus formosanus,Ludisia discolor and Goodyera schlechtendaliana were 253 bp,253 bp,250 bp,and 252 bp,respectively.Compared with Anoectochilus roxburghii,there are27 and 28 Single Nucleotide Polymorphism in Ludisia discolor and Goodyera schlechtendaliana,respectively.Using ITS2 sequence to construct a phylogenetic tree,it was found that all four plants can be distinguished well.In addition,the secondary structure of ITS2 of four plants was also drawn in this study.It was found that the secondary structure of ITS2 of Herba Anoectochili and its adulterations were significantly different.Based on the ITS2 sequence,a Herba Anoectochili-specific primer was designed in this study and verified by RT-PCR.This primer can quickly and correctly identify and distinguish Herba Anoectochili and its adulterations.The chloroplast genome has a moderate evolution rate,and the genes are conserved and single copies.Compared with shorter DNA barcodes,the chloroplast genome contains more genetic information,higher resolution and better versatility.Relying on these characteristics,the chloroplast genome is widely used in various research fields such as species identification and phylogeny.In this study,the chloroplast genomes of 5 species of Anoectochilus spp.(One of which has been sequenced),1 species of Ludisia sp.,and 1 species of Goodyera sp.were sequenced and assembled.The 7 chloroplast genome sequences were annotated with plann and uploaded to the NCBI database after corrected with Apollo.Similar to the chloroplast genome of most advanced plants,the Herba Anoectochili chloroplast genome has a typical four-segment structure.In addition,the performance of these 7 chloroplast genomes is highly consistent in many terms,such as genome size(151,414～153,814bp),number of genes,gene category,gene sequence,codon usage,and distribution of repeat sequences,reflecting the high conservation of the Herba Anoectochili chloroplast genome.Based on the entire chloroplast genome sequence,a phylogenetic tree of Herba Anoectochili was constructed in this study.The result indicates that the classification of Anoectochilus spp.is kind of complicated.The taxonomic status of some species still need to be discussed.The number of samples should be expanded,which is to be further verified by further work.