DNA Barcode Identification And Germplasm Resources Genetic Diversity Analysis Of Amomum Villosum Lour.

Object iveAmomum villosum is one of China’s famous "Four Great Southern Medicines",which was included in the 2015 edition of the Chinese Pharmacopoeia.The source is dry and ripe fruit of Amomum villosum Lour.,Amomum villosum Lour.Var.Xanthioides T.L.Wu.et Senjen or Amomum longiligulare T.L.Wu.A.villosum belong to a large variety of traditional Chinese medicines,and it is easy to confuse clinical medicines.It is difficult to distinguish the authenticity of authentic medicinal materials.The quality of authentic medicinal materials is difficult to guarantee,which seriously affects the efficacy of traditional Chinese medicines and is not conducive to improving the quality of medicinal materials.In this study,A.villosum,the traditional Chinese medicinal material,was used as the main research object.DNA barcode technology based on molecular biology and ISSR molecular marker technology were used to obtain DNA genetic information differences.DNA barcode database of A.villosum in Guangdong Province was constructed.The identification abili ty of DNA barcodes to Amomum sp.and use molecular markers to explore the genetic diversity of A.villosum to set up effective barcodes for identification of Amomum sp.,and establish recommendations for population cultivation of A.villosum vulgaris,laying a foundation for further research on A.villosum.Methods1.Analysis of genetic information differences of Amomum villosum by DNA barcode technology:Collected 141 samples of the original plant of A.villosum Lour.and 49 adulterations of A.villosum as experimental materials.The total DNA of the samples was extracted by the plant DNA extraction kit method and the improved method of the kit,mainly from the commonly used plants currently identified five sequence bands(nucleotide spacer ITS2,nuclear gene ITS,chloroplast gene spacer psbA-trnH,chloroplast gene coding region matK and rbcL)for analysis:PCR amplification efficiency,sequencing success rate,candidate species within species intervariation,sequence characteristic structure,BLAST analysis,phylogenetic tree clustering analysis,etc.are used to identify A.villosum and its adulterations.2.Analysis of genetic giversity of Amomum villosum Lour.from different populations based on ISSR molecular marker technology:Inter-Simple Sequence Repeats(ISSR)molecular marker technology was used to detect the genetic diversity of A.villosum Lour.samples from a total of 141 individuals in 7 populations in Guangdong province,and in-depth analysis of the genetic structure of A.villosum Lour.to construct a cluster dendrogram.3.Based on the above experimental research results,a comprehensive identification method of A.villosum and planting suggestions for A.villosum are summarized.Results1.In this study,five candidate sequence bands(ITS2,ITS,psbA-trnH,matK and rbcL)of 141 samples of Amomum villosum Lour.plant and 49 samples of Amomum genus were successfully extracted and amplified by sequencing.Screening and identification of 13 species of Amomum genus.Among them,the ITS2 sequences has the best identification result,with more mutation sites,larger interspecies distance,and higher species identification rate,combined with the secondary structure of the ITS2 sequence characteristics,can effectively distinguish A.villosum Lour.,A.villosum Lour.var.Xanthioides T.L.Wu et Senjen,A.longiligulare T.L.Wu and their adulterations.There are no mutation sites in each species of the ITS sequence,the intradistance is 0,and the interdistance is the largest among the five barcodes.The success rate of matK sequence is the lowest,and the rbcL sequences is more conservative.The average inter-species genetic distance of the five fragments is greater than the intra-species genetic distance.ITS2 secondary structure can distinguish Amomum sp..The NJ phylogenetic tree constructed based on a single sequence uses the nuclear gene spacer ITS2,psbA-trnH and ITS2 sequences to identify efficiency best,so ITS sequence,ITS2 sequence combined with ITS2 sequence secondary structure information,and psbA-trnH sequence are recommended as effective barcodes for identifying A.villosum Lour..2.Genetic diversity analysis of Amomum villosum from different populations based on ISSR molecular marker:In this experiment,6 ISSR primers were used to amplify A.villosum DNA,and 66 bands were amplified,among which 56 were polymorphic,with a polymorphism ratio of 84.84%.On average,11 bands were amplified from each primer,and 6-14 polymorphic bands were amplified from each primer.The number of alleles was 1.2879-1.7121,with an average of 1.4834.The number of effective alleles ranged from 1.1848 to 1.4240,with an average of 1.3115.The average heterozygous degree ranged from 0.1117 to 0.2536,with an average of 0.1820.Shannon’s diversity index is between 0.1658 and 0.3816,with an average of 0.2689.The average heterozygosity H of A.villosum was 0.1820 and the average value of Shannon’ s diversity index was 0.2689,indicating that the level of genetic diversity of species was low.A.villosum maintains a certain genetic diversity,and the genetic level of each population from high to low is ZY>ZJD>GY>MM>YC>XFC>TK.And most genetic variations are distributed within populations,and there is no correlation between geographic distance and genetic distance between populations.The principal component analysis and cluster analysis of A.villosum samples from 7 different regions were performed by GenAlEx6.502 and NTSYS 2.10e software.The results showed that they could be divided into three groups while the genetic similarity coefficient is 0.84.In this study,according to Nei’s analysis of gene diversity,the coefficient of genetic differentiation(Gst)among A.villosumpopulations equaled 0.4487,indicating strong genetic differentiation among the populations.This was also confirmed by the gene flow among populations(Nm=0.6143),indicating difficulties in genetic exchange among populations.3.Based on the above research results,the following conservation measures are proposed for Amomum villosum:priority should be given to protecting the populations along the roads of G325(ZY),which are rich in genetic diversity,the ZJD population,and the population located in Yangchun National Geopark(GY),with a view to more genetic diversity may be conserved.At the same time,in view of the significant genetic differentiation among A.villosum populations,in situ conservation should be stepped up for each existing population,and it is recommended to add a protected area in Yangchun City.ConclusionIn this study,the molecular biology-based DNA barcode technology and ISSR molecular marker technology were used to determine the identification method of Amomum villosum samples.By using five candidate sequence bands ITS2,psbA-trnH,ITS,matK,and rbcL for amplification and sequencing,confirmation the accuracy of the DNA barcode.The ITS2 sequence combined with its secondary structure characteristics can be used for identification of A.villosum Lour.,A.villosum Lour.var.Xanthioides T.L.Wu et Senjen,A.longiligulare T.L.Wu and Amomum mixed fakes.It is recommended that the ITS2 sequence be combined with the ITS2 sequence secondary structure information and psbA-trnH sequence and the ITS sequence are effective barcodes for identifying A.villosum Lour..In the analysis of genetic diversity,it is necessary to take appropriate in-situ conservation measures for the population of A.villosum Lour.in Yangchun City,increase genetic communication between populations,and improve the genetic diversity of A.villosum Lour..