Molecular Identification Of Chachiensis And Relative Species

ObjectiveChachiensis belongs to(Rutacae)as the Rutaceae citrus plants(Citrus),the skin dried 3 years used as pericarpium citri reticulatae chachiensis,It can facilitate the spleen,and stomach anti-nausea,dry wet phlegm,liver,bravery,solution knot carbuncle and so on.Its pulp is rich in sugars,organic acids,natural vitamins and other nutrients and bioactive substances,at the same time,it can be used as orange peel tea,additives,and spices,etc,has high edible value.Pericarpium citri reticulatae chachiensis as the genuine medicinal material of Guangdong,it is the first choice of clinical medicine,and has broad prospects for development.But because of pericarpium citri reticulatae chachiensis and general tangerine peel in the market price is different,so the market exist the general phenomenon that ordinary tangerine peel instead of pericarpium citri reticulatae chachiensis,while them can be distinguished by the main producing area,pericarpium citri reticulatae chachiensis is made in Guangdong and Sihui,while general tangerine peel are produced in Fujian,Hunan,Jiangxi,Chongqing,Hubei,Sichuan and other places.While microscopic identification and detection method of orange peel index components,not suitable for fast accurate identification broadly pericarpium citri reticulatae chachiensis and ordinary tangerine peel.So as to ensure the stable quality of pericarpium citri reticulatae controllable,safe and clinical treatment effective,to establish a rapid identification of Chachiensis and other citrus varieties is a problem to be solved.MethodsThis study use the ISSR molecular markers to distinguish Chachiensis and relative species 38 materials;comparison the advantage of SCoT and ISSR molecular markers,and to explore the feasibility of clonal species identification with SCoT;search for the barcode of Chachiensis and relative species identification;and attempts at the molecular point of view distinguish between two grafted of Chachiensis;Finally,optimization the extraction methods of DNA,try comparing the DNA content of different years of Chachiensis peel,compare Chachiensis peel and leaves gene sequence,in order to circulate on the market in the future of all kinds of dried tangerine peel accurate identification.Results1.L16 orthogonal design method is adopted for the Chachiensis ISSR reaction system was optimized,the final optimization results is 1×PCR buffer,1.5mmol/L Mg2+,0.2mmol/L dNTPs,0.3 ?mol/L primer,0.5UTaq enzyme,50 ng template DNA.After verification through the screening of annealing temperature and polymorphism,screened 11 ISSR primers,amplified the 2126 rules,polymorphism of 100%,by UPGMA cluster analysis to Chachiensis and relatives 38 materials on the level of similarity coefficient is 0.54?0.94 to be distinguished.2.Comparing the advantage of SCoT and ISSR molecular markers combined with analysis results.By L25 orthogonal method to fabrics in reaction system is optimized,finally get the best system for 1×PCR buffer,2mmol/L Mg2+,0.25mol/LdNTPs,0.625?mol/Lprimer,0.5UTaq enzyme,20 ng template DNA.Finally chose 12 primers for 38 genetic polymorphism analysis,it amplified the 2846 rules,percentage of polymorphic loci was 98.7%.At the similarity coefficient of 0.54?0.91 level that all material can be distinguished.Thus both SCoT and ISSR molecular markers could separate 38 materials,although have certain differences,but the overall results are roughly the same.SCoT and ISSR molecular markers are the effective tools of the Chachiensis and relatives identification.3.The SCoT molecular markers combine with the cloning sequencing for the Chachiensis and relatives are analyzed in sequence alignment,homology was 63.840%,while the genetic distance between 0.001?0.654,based on Kimura genetic distance analysis between Phylogenetic Tree,each selected materials to save one or more of the mutations can be used as a specific site and other selected materials.Thus verified the SCoT combined with the cloning sequencing DNA molecular markers in the Chachiensis and relatives of molecular identification is effective.4.Through four international barcode on the Chachiensis and relatives screening,Sequencing the success rate of 50%of matK could identify sequences,rbcL sequence mutation loci is 0,the BLAST found ITS2 and psbA-trnH identification efficiency is 100%,so finally chose ITS2 and psbA-trnH sequence as the barcode,material identification by combining sequence analysis is superior to the single barcode identification efficiency,therefore recommend ITS2+psbA-trnH sequence as the Chachiensis and relatives in the identification of sequence.5.Use four international barcode selection to distinct tow kind of cultivated Chachiensis,the ITS2,psbA trnH and matK identify found the loci were 0,and the level of rbcL sequence two kinds of cultivated variety fully distinguished,provides selected seedling planting on the Chachiensis with broad prospects and application value.6.Set up cCTAB method as an Chachiensis peel DNA effective extraction method,10 times DNA production more than qiagen method,and different year of dried tangerine DNA content and time,there is no clear correlation.With Chachiensis peel and leaves psbA-trnH sequence alignment found completely consistent,further verify the DNA barcode identification of advantage.Conclusion:ISSR combined with SCoT molecular markers have high genetic polymorphisms of molecular markers,Chachiensis and relatives in the UPGMA clustering tree almost entirely to differentiate.SCoT molecular markers combined with cloning sequencing get multiple mutation sites,can be used as material distinguish loci.ITS2 combining psbA-trnH sequence for barcode identification of Chachiensis and relative species.RbcL sequence can completely different between two kinds of cultivated Chachiensis.cCTAB method can effectively extract more than 10 years wide dried Chachiensis peel DNA,at the same time through the psbA-trnH sequence alignment,validate the universality and accuracy of the barcode.