Molecular Identification And Genetic Diversity Analysis Of Southern Medicine Citrus Grand Is ’Tomentosaand’ And Citrus Grand Is ’Tomentosa’

ObjectiveCitrus grandis’Tomentosaand’ and citrus grandis’Tomentosa’ are both medicinal plants of rutaceae citrus,which have significant clinical effects and are widely used as first-line Chinese medicinal materials.Because of its high price,some Citrus grandis’Tomentosaand’ and citrus grandis ’Tomentosa’ are gradually appearing in the market to deceive patients with false and true.The occurrence of counterfeit products not only affects the clinical efficacy,but also harms the patients’health.Therefore,the DNA barcodes of orange peel and tangerine peel were studied.The application of DNA barcode technology to the verification of citrus grandis ’Tomentosaand’and citrus grandis’ Tomentosa’ can quickly,effectively and accurately identify the authenticity of medicinal materials and protect the interests of patients.At the same time,molecular markers are used to study the genetic diversity of medicinal materials,providing a basis for the germplasm resource analysis of citrus grandis’ Tomentosaand’ and citrus grandis ’Tomentosa’.MethodsThe leaves of target plants were collected and preserved by silica gel.The genomic DNA of citrus grandis’ Tomentosaand’ and citrus grandis’Tomentosa’ was extracted by the extraction kit method,and the DNA barcodes of ITS2,psbA-trnH ITS,matKand rbcL were amplified.Sanger method was used for bidirectional sequencing,and the sequence after bidirectional sequencing was splicing.Uv spectrophotometry and Biodiet software were used to evaluate the quality of DNA barcode extraction.Mega6 was used for comparative analysis of citrus grandis ’Tomentosaand’ and citrus grandis ’Tomentosa’ sequences,as well as sequence comparison identification of 5 universal barcode sequences of other related species of citrus in rutaceae.Analyze the appraisal results.At the same time,8 pairs of primers suitable for these two medicinal materials were selected from 100 pairs of primers for ISSR molecular markers.The citrus grandis’Tomentosaand’ of 7 different populations and the citrus grandis’Tomentosa’ of 13 different populations were labeled by molecular markers.Bandscan software was used to check the gel electrophoresis gel map results,and the results were converted into a 0/1 matrix graph.The matrix graph was analyzed by POPGENE.The software NTsys 2.10e was used to construct UPGMA tree graph for cluster analysis to evaluate the genetic diversity of orange and orange peel.ResultspsbA-trnH and ITS2 sequences could cluster citrus grandis ’Tomentosaand’and citrus grandis ’Tomentosa’ of different populations,and other related species could also cluster into groups,and the sample plants could be clearly separated from their related species.In ITS sequence,citrus grandis’Tomentosaand’ and citrus grandis’Tomentosa’ also clustered together,but were not separated from stand orange.The citrus grandis ’Tomentosaand’ and sweet orange sequences in matK’ s sequence are still relatively similar.One sequence is not separated,but the others are generally well identified.The rbcL sequence performed least well and was clustered with several related species.Thirty-five citrus grandis’ Tomentosaand’ DNA samples were amplified by 8 pairs of primers.A total of 60 bands were obtained,of which 54 were polymorphic and the percentage of polymorphism was 90.0%.The genetic distance between seven populations was 0.0723 to 0.2959,the genetic consistency was 0.7439 to 0.9302,the genetic diversity index h*of population 6 was the highest,0.2347,Shannon index I*was the highest,0.3489,the polymorphic percentage was the highest,61.67%;the genetic diversity index h*of population 1 was the lowest,0.0720,Shannon index I*was the lowest,0.1087,the polymorphic percentage was the lowest,only.20%.The total genetic diversity of Citrus grandiflora in different populations was 0.2828.The genetic diversity of Citrus grandiflora in different populations was 0.1806 and 0.1022.The genetic differentiation coefficient Gst was 0.3616 and the gene flow Nm*was 0.8829 among different populations.The similarity coefficients of 35 citrus grandis’Tomentosaand’ individuals were almost separated between 0.57 and 0.98.With a threshold of 0.71,35 citrus individuals could be divided into six categories.Eight pairs of primers were amplified by PCR from 38 DNA samples of Pericarpium citrus grandis ’Tomentosa’,A total of 60 bands were obtained,of which 45 were polymorphic and the percentage of polymorphism was 81.8%.The genetic distance between 13 populations ranged from 0.0389 to 0.4864,and the genetic consistency ranged from 0.6373 to 0.9619.The genetic diversity index h*of population 5 was the highest,1.1616,Shannon index I*was the highest,0.2315,and the polymorphic percentage was the highest,36.36%.The genetic diversity index h*of population 10 was the lowest,0.0485,Shannon index I*was the lowest,0.0694,polymorphic percentage.It was also the lowest,only 10.91%.The total genetic diversity(Ht)of different populations was 0.2804,of which the genetic diversity(Hs)was 0.1150 in the population and 0.1654 in the population.The genetic differentiation coefficient Gst was 0.5898 and the gene flow Nm*was 0.3477 among different populations.The similarity coefficients of 38 individuals were almost separated from 0.65 to 0.96,and 38 individuals were divided into five categories at 0.72.ConclusionpsbA-trnHbarcodes and ITS2、ITS barcodes can be used to distinguish citrus grandis ’Tomentosaand’,citrus grandis ’Tomentosa’ and its related species.Both citrus grandis ’Tomentosaand’ and citrus grandis ’Tomentosa’ have rich genetic diversity.