Correlation Analysis Between Chemical Components And Genetic Diversity Of Ziziphus Jujuba Var.Spinosa From Different Areas In Shanxi Province

Objective:In this study,the resources of Ziziphus jujuba var.spinosa from different producing areas in Shanxi were studied,and the genetic background was preliminarily analyzed.The ISSR-PCR reaction system of Ziziphi spinosae semen(ZSS)was established,and the genetic diversity of Ziziphus jujuba var.spinosa resources from different producing areas was analyzed by using ISSR molecular marker technology.The contents of five main chemical components in ZZS from each producing area were determined,including magnoflorine,spinosin,6′′′-feruloyl spinosin,Jujuboside A and Jujuboside B.The correlation analysis between the content of chemical components and genetic diversity of ZZS from different areas was carried out.The aim of this study was to understand the genetic background of Ziziphus jujuba var.spinosa germplasm resources from different areas in Shanxi Province,and to preliminarily reveal the relationship between five main chemical components of ZZS and ISSR molecular markers.Methods:1.Genetic analysis of Ziziphus jujuba var.spinosa from different habitats based on ITS2 molecular markersDNA was extracted from ZSS from different areas.ITS2 universal primers and suitable conditions were used for amplification and sequencing.The obtained ITS2 sequences were analyzed by sequence alignment and phylogenetic analysis,and the differences(K2P,kimum 2-parameter)among the samples were calculated.2.Genetic diversity of Ziziphus jujuba var.spinosa from different habitats based on ISSR MarkersTo establish the best reaction system of ISSR-PCR of ZSS,the parameters such as DNA template dosage,primer concentration and 2×Taq master mix content were optimized by orthogonal test.Based on the optimization results,primers were screened,and the optimal annealing temperature of each primer was determined by gradient method.Genetic diversity analysis and cluster analysis of 130 DNA samples from 26 Ziziphus jujuba var.spinosa from different areas of Shanxi were carried out by using the established reaction system and the selected primers.3.Study on Chemical Constituents of ZZS from Different OriginHPLC-UV/ELSD liquid phase method was used to determine the contents of five main chemical components in ZZS from different areas,including magnoflorine,spinosin,6′′′-feruloyl spinosin,Jujuboside A and Jujuboside B,and the differences were analyzed.4.Correlation analysis between the contents of five main chemical components and molecular markersThe 26 mixed populations of Ziziphus jujuba var.spinosa germplasm resources corresponding to the 5 chemical contents were amplified by ISSR.Structure 2.3.4 software was used to analyze the genetic structure of Ziziphus jujuba var.spinosa germplasm resources from 26 producing areas and determine the best K value.Tassel 5.0 software was used to analyze the association between the loci amplified by 12 primers and five major components,magnoflorine,spinosin,6′′′-feruloyl spinosin,Jujuboside A and Jujuboside B.Results:1.Sequencing results of ITS2 from 26 germplasm resources of Ziziphus jujuba var.spinosa were compared.The results showed that the sequence consistency was 99.55%.The length of ITS2 region was relatively stable,with a total length of 220 bp.There were four sequence types in total,with two SNP sites,which were different at 88 and 94 loci,respectively,with little difference in GC content.The maximum K2 P distance was 0.009,and the minimum K2 P distance was 0.005.2.The results showed that 20 μL ISSR-PCR reaction system for ZSS contained DNA template 50.0 ng,primer 0.6 μmol/L and 2×Taq Master Mix 11.0 μL.The amplification procedure was pre-denaturation at 95°C for 3 min;denaturation at 94°C for 25 s,annealing at different annealing temperatures for 25 s,extension at 72°C for 1 min,35 cycles;extension at 72°C for 5 min,preservation at 4°C.Based on the optimization results,12 ISSR primers with good amplification results and clear bands were selected from 100 primers,and the optimal annealing temperature of each primer was determined by gradient method.A total of 123 polymorphic bands were amplified by 12 primers,and the percentage of polymorphism was 97.62%.The observed number of alleles,effective number of alleles Ne,Nei’s gene diversity index H and Shannon’s Information index I were2.0000,1.5947,0.3560 and 0.5341,respectively.The Nei’s gene diversity index ranged from 0.1866 to 0.3176,and the Shannon information index ranged from 0.2800 to 0.4649.The coefficient of gene differentiation(GST)was 0.2960,the gene flow(Nm)value was1.1892,and the genetic consistency ranged from 0.7272 to 0.9498.By cluster analysis,the germplasm resources of Ziziphus jujuba var.spinosa from 26 producing areas were divided into three groups.The 13 producing areas in Luliang,Taiyuan,Jinzhong and Changzhi were grouped into two categories,of which the 10 producing areas were grouped into one category alone,and the 13 producing areas in Xinzhou,Linfen,Yuncheng and Jincheng were grouped into the third category.3.HPLC-UV/ELSD liquid phase method was used to determine the chemical constituents of ZZS from different areas.The results showed that the linear ranges of magnoflorine,spinosin,6′′′-feruloyl spinosin,Jujuboside A and Jujuboside B were0.0249～4.98、0.021～0.42、0.0145～0.29、0.0255～0.51、0.0103～0.206 μg/m L,respectively.The recoveries were 96.8%、97.3%、97.2%、98.0%、97.7%,respectively.The precision,stability and repeatability met the requirements,indicating that the method was stable and scientific.The contents of five chemical components in ZZS from different areas were as follows: magnoflorine 0.745 mg/g ～1.537 mg/g,spinosin 0.358 mg/g ～ 0.815 mg/g,6′′′-feruloyl spinosin 0.122 mg/g ～ 0.571 mg/g,Jujuboside A 0.265 mg/g ～ 0.424 mg/g,Jujuboside B 0.100 mg/g ～ 0.226 mg/g.4.19 loci of 12 ISSR primers were associated with 5 major components,and UBC 89 could be associated with the most sites,with a total of 5 loci.There were 3 loci associated with magnoflorine content,and the rate of explanation ranged from 17.59% to 25.42%.There were 7 loci associated with spinosin content,and the rate of explanation ranged from15.35% to 28.45%.There were 4 loci associated with 6′′′-feruloyl spinosin content,and the rate of explanation ranged from 15.54% to 33.87%.There were 1 loci associated with Jujuboside A content,and the rate of explanation was 20.25%.There were 7 loci associated with Jujuboside B content,and the rate of explanation ranged from 15.69%-27.40%.At the same time,there was one site associated with spinosin content and Jujuboside B content,one site associated with spinosin content and 6′′′-feruloyl spinosin content,and one site associated with magnoflorine content and 6′′′-feruloyl spinosin content.Conclusion:Genetic analysis of Ziziphus jujuba var.spinosa from different areas in Shanxi Province based on ITS2 molecular marker technology showed that although there were some differences among the samples,the homology was relatively high,reaching 99.55%,indicating that the experimental samples were the same species,so there were only intra-specific differences.Then ISSR Molecular marker was selected to study the genetic diversity of Ziziphus jujuba var.spinosa from different regions of Shanxi Province.Based on the established ISSR-PCR reaction system and reaction conditions,12 primers were screened and 123 polymorphic bands were amplified.which indicated that the germplasm resources of different populations of Ziziphus jujuba var.spinosa in Shanxi Province were rich in genetic diversity.Genetic diversity of different populations of Ziziphus jujuba var.spinosa in Shanxi Province was different,and there were some genetic differentiation within and among populations,and the variation within populations was the main factor.The results showed that geographical location was not the main factor affecting gene exchange among populations of Ziziphus jujuba var.spinosa.The method for the determination of magnoflorine,spinosin,6′′′-feruloyl spinosin,Jujuboside A and Jujuboside B was feasible and the results were reliable.The coefficients of variation of the 5 main components were 20.33%、23.52%、23.73%、11.58% and 20.26%,respectively,indicating that the content of 5 main chemical constituents of ZZS in different habitats in Shanxi was significantly different.According to the content determination standard of ZZS in Chinese Pharmacopoeia,the quality of ZZS in Shanxi province is good on the whole,and the failure is mainly due to the content of spinosin,which should be further discussed.In addition,the contents of magnoflorine,6′′′-feruloyl spinosin and Jujuboside B were also studied,which can provide reference for their quality evaluation and clinical application in the later stage.After the ISSR loci were associated with chemical components,a total of 19 of the 12 primers were found to be associated with 5 major components,and 3 sites were associated with 2 major components.These sites can be selected to develop new molecular markers,and then become a new method to regulate the content of chemical components of Ziziphus jujuba var.spinosa,and provide a theoretical basis for the identification of ZZS varieties and the effect of species conservation on genetic differentiation and the study of genuine medicinal materials.