| The history of picking and using medicinal plants in China can be pushed to the 6th century BC,that is,the compilation of the first collection of poems in China,the Book of Songs.Records of many medicinal plants,such as Caiying(Rumex acetosa),Caiai(Artemisia argyi),Tui(Leonurus artemisia),Caijuaner(Xanthium sibiricum),Caimeng(Fritillaria)and so on.In the long history of history,fake medicines and inferior medicines in traditional Chinese medicines have been submerged and mixed into non-medicinal parts,and the techniques of Chinese medicine identification have gradually grown in the struggle against counterfeit medicines.Today,molecular identification is the representative identification technology is the forefront of the Chinese medicine identification discipline.Its intersection with molecular systematics,phylogeography,population genetics,molecular biology and other disciplines has rapidly promoted the development of molecular identification of traditional Chinese medicine.As the traditional Chinese medicine of berberine,Coptis has always been controversial.Most of the species of Coptis in China are endangered,and their authenticity,quality grade and quality base are highly controversial.Identification of various species of genus berberine is of great significance,and molecular identification is necessary.Coptis chinensis var.brevisepala is both a famous historical drug and an endangered species.It has a relationship with the main base of Rhizoma coptidis and the evolutionary relationship of the original variant Coptis chinensis var.chinensis,which is related to the efficacy and quality of Rhizoma coptidis.Therefore,this study reconstructed the phylogenetic relationship of Coptis by complete chloroplast genome of Coptis,and clarified the evolutionary relationship and genetic relationship of Coptis,and established the basis for molecular identification of CoptisCoptis chinensis var.brevisepalais a variant of Coptis chinensis var.chinensis.In the field investigation,no transitional form was found,that is,the calyx of the Coptis chinensis var.brevisepala are 5-7 mm long,while the calyx of the Coptis chinensis var.chinensisis 1 cm or more.The calyx of the Coptis chinensis var.chinensis are droopy and soft.However,the taste reproduction ability is strong,the vitality is strong.and the environmental requirements are low.The Coptis chinensis var.brevisepala has a weak reproductive ability,requiring high air humidity and weak light,but it cannot be completely without light.If the current production area of Coptis chinensis var.brevisepala has been planted or distributed in the past,it should not be extinct before Coptis chinensis var.brevisepala.The whole genus of Coptis has certain pharmacological effects,but according to the results of the research on the past,it has a slight focus.However,due to the scarcity of resources of Coptis omeiensis and Coptis chinensis var.brevisepala,especially Coptis omeiensis,due to its narrow distribution area and its demanding habitat,it is on the verge of extinction.Since the Coptis in the previous work cannot be identified by the traditional barcodes,it is especially necessary to develop other barcodes of Coptis.The chloroplast genome is a single-line genetic gene in angiosperms,and is particularly suitable for identifying genus of Coptis,which is clearly distributed with the region.The chloroplast genome has been widely used in species identification,endangered plant protection and other fields.The mini-barcode developed by the chloroplast genome is often used as the most accurate identification fragment for the specialist.This study will sequence the chloroplast genome of two individuals in each population and develop a mini-barcode of the genus Coptis.1.Research on Resources of CoptisIn order to find out the "home" ofCoptis in China,this study first carried out wild in 12 provinces(Zhejiang,Fujian,Guangdong,Guangxi,Hunan,Hubei,Anhui,Jiangxi,Sichuan,Chongqing,Guizhou,Yunnan)south of the Yangtze River.And research on cultivation of berberine.The Coptis chinensis var.brevisepalaproduced in Huangshan City,Anhui Province,is adjacent to the Huangshan Scenic Area.It is the shade of the mountain.It is distributed in the same area as the local residents.It is distributed at an altitude of 783 meters.The number of population is more than 1000.and there are many perennial Coptis chinensis var.brevisepala.There are seedlings of Coptis chinensis var.brevisepala,and it can be seen that the local protection of Coptis chinensis var.brevisepala is better,and the reproduction rate of Coptis chinensis var.brevisepala is normal.The short-skinned berberine in Jianyang District of Fujian Province can be considered to have only one population,and the number is small,and the species is on the verge of extinction.Coptis deltoidea,Coptis omeiensisand Coptisteetaare also in an extremely endangered state.The number and distribution of populations are not optimistic.Coptis is suspected of being in a wild state after cultivation.The Coptis chinensis var.brevisepala is distributed in Zhejiang,Fujian,Guangxi,Guangdong,Anhui,Jiangxi and other provinces in China,while theCoptis chinensis var.chinensis(commonly known as "weilian")is distributed in Sichuan,Hubei,Hunan,Chongqing,Guizhou and other provinces.Coptisteeta is distributed in Yunnan Province.Coptis deltoidea(commonly known as "Yalian")are distributed in Whawushan,Ya'an City,Sichuan Province,and Emei Mountain,Leshan City,Sichuan Province.Coptis omeiensis is only distributed in Emei Mountain,Leshan City,Sichuan Province.These species have distinct geographical divisions and lay the foundation for molecular identification,especially based on the identification of chloroplast genomes.2.Reconstruction of phylogenetic relationship of CoptisXiang on the genus Coptis molecular phylogenetic analysis using a chloroplast 5-segment barcode(rbcL,trnLintron.trnL-F spacers,trnD-trnT,and trnH-psbA)and a nuclear gene barcode(ITS)Many of the remaining issues,especially the six species(variations)in our country,are not clearly explained.Therefore,this study reconstructed the phylogenetic relationship of Coptis by chloroplast genome of Coptis chinensis,and clarified the evolutionary relationship and genetic relationship of Coptis chinensis,and established the basis for molecular identification of Coptis.In this study,16 individuals of Coptis were selected for DNA extraction,chloroplast genome amplification,recovery and sequencing.The phylogenetic reconstruction of Phyllostachys pubescens can be successfully carried out by the neighboring method,the maximum likelihood method and the Bayesian method.The results of phylogenetic reconstruction of Coptis are quite different from those of previous studies.The phylogenetic tree of the genus Coptis,established by the adjacency method,shows that each species has evolved into a single line.Except for Coptis trifolia and Coptis groenlandica,these two species may not be fully evolved.Coptis chinensis var.chinensis,and its variant Copiis chinensis var.are the most controversial issues in the classification of Coptis.The species selection is slightly different,and two populations and four individuals are selected for the system.Developmental analysis.Coptis chinensis var brevisepala and Coptis chinensis var.brevisepalaare sister groups,but it can be considered that Coptis chinensis var.brevisepala has evolved into a single line and become an independent species.Coptis chinensis var.brevisepala is more closely related to Coptis chinensis var.brevisepala in the whole genus Coptis,providing a material basis for the development of new drug sources of berberine,and it is expected to develop new hybrid cultivars of berberine.Coptis omeiensis and Coptis deltoidea are sister groups.The above four species are more objectively constructed with Coptis teeta as reference.Coptis quinquesecta is distributed in Taiwan Province of China,and is closely related to mainland China,but has a close relationship with other genus genus in Japan and North America.Coptis ramosa is more closely related to Coptis trifoliolata,and is farther related to Coptis quinquefolia.It can be seen that there are two species supported on the molecule.Coptis asplenifolia is closer to the distribution of Coptis in China and TaiwanThe maximum likelihood method and Bayesian method successfully reconstructed the molecular phylogenetic tree,which is the same as the phylogenetic tree constructed by the neighboring method.The chloroplast genome data of Coptis is suitable for the adjacent method,the maximum likelihood method and the Bayesian method to construct the phylogenetic tree.Molecular phylogenetic trees were successfully established in all three methods,including phylogenetic tree,branch map.and circular branch map.The molecular systematic analysis of plants belonging to the genus Coptis was carried out.These results are inconsistent with the preliminary work of this study,Xiang et al.The Coptis chinensis var.brevisepala and Coptis chinensis var.chinensis are different varieties of the same species,and the difference in morphology is extremely small,only the shape and length of the bracts are different.In the preliminary work of this study,Xiang et al.'s classification results for Coptis chinensis var.brevisepala were considered to be farther in the molecule than the Coptis omeiensis and Coptis deltoidea.The relationship between Coptis omeiensisand Coptis deltoideawas close to Coptis chinensis var.chinensis which was the result of morphology.The results of the chloroplast genome support the combination of Coptis chinensis var.brevisepala and Coptis chinensis var.chinensis as a sister group,and the adjacent group of chlorosis and Emei huanglian.3.Molecular identification of products containing CoptisThe chloroplast genome of Coptis chinensis has been identified for intraspecific plants,and its molecular system relationship has been reconstructed with greater information and accuracy.However,the genes of the entire chloroplast genome are not only difficult to obtain,but also the extraction,splicing and proofreading are time-consuming and laborious.It is difficult to achieve in practice.There are many genes in the whole chloroplast genome that are highly discriminative.The development of these genes as a specific barcode for the identification of berberine is of great practical significance.In the interval of 125500-130000 bp,there is likely to be a hypervariable region of the genus Coptidis,and the hypervariable genes are ycfl,trnNGTT,trnRACG,rrn5s,rrn4,5S,rrn23S,and the like.In addition,there are many hypervariable genes in the vicinity of 120,000 bp,including ndhD,psaC,ndhG,ndhl,nghH and so on.This study developed the mini-barcode of Coptis,and identified Chinese patent medicines and other products containing Coptis.4.ConclusionThe genetic relationship and evolutionary relationship of various species of Coptis have been completely combed out,and a specific barcode of Coptis has been developed,as well as mini-barcode for proprietary Chinese medicines or other products containing Coptis,and these proprietary Chinese medicines or other products.Successfully identified. |
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