Genetic Relationship Analysis And Molecular Identity Construction Of Different Cultivars (Lines) Of Angelica Sinensis

Objective: To analyzed the genetic diversity,genetic structure and genetic variation of 11cultivars(lines)and 7 wild Angelica sinensis populations in Gansu Province based on fluorescent labeled SSR markers and chloroplast genes(cp DNA),and constructed their molecular identity cards.The results of the studies provided a reference for germplasm identification and breeding of new cultivars.Methods:1.Identification and genetic variation analysis of wild and cultivated germplasm of A.sinensis based on cp DNA: Three pairs of cp DNA primers were used for PCR amplification and sequencing of A.sinensis samples.Mega X software was used to perform statistics on sequence characteristics and to calculate mean genetic distances between A.sinensis populations.Unweighted pair-group method with arithmetic means(UPGMA)clustering tree based on genetic distance was constructed using NTSYS 2.10 e software.Dan SP v6 software was used to calculate sequence polymorphism and Tajima’s test.PERMUT software was used to calculate the structure of populations.Finally,TCS haplotype network plots were constructed using Pop ART 1.7 software.2.Genetic relationship analysis and molecular identity construction of different cultivars(lines)of A.sinensis based on fluorescent labeled SSR markers: Ten pairs of fluorescent labeled SSR primers were screened out for PCR amplification of the test material,the calculation of genetic parameters was carried out by using Popgen 1.31 and Crevus 3.0software,the population structure analysis was carried out by using Structure 2.3.4 software,the Nei’s genetic distance UPGMA cluster diagram of each population was constructed by NTSYS software,and the molecular variance analysis and principal coordinates analysis were analyzed by Gen ALEx software.3.Molecular identity construction: the amplification band types were coded with Arabic numerals and English letters and sorted from small to large.The primers were sorted according to the Shannons information index(I)to construct DNA molecular identity in the form of strings.After removing duplicate ID numbers,the molecular identities of each sample were classified according to cultivars(lines).The molecular identities of each sample were strung into online website(http://barcodemore.wiicha.com/,https://cli.im/),to get bar code and qr code in the form of molecular identity.Results:1.Three pairs of cp DNA primers were amplified,sequenced,compared,and combined to give a sequence length of 1759 bp.One variable site was detected in the wild and 480 variable sites were detected in the cultivated,including 97 singleton variable sites,383 parsimony informative sites,and 152 insertion-deletion sites.In the three regions of mat K,psb A-trn H,and rbc L of cp DNA in the wild group and the cultivated group,mat K was the region with the highest polymorphism.Tajima’test of all the combined sequences of A.sinensis were not significantly negative,but psb A-trn H and rbc L genes of the cultivated were significantly negative,indicating that the whole A.sinensis followed neutral evolution,while psb A-trn H and rbc L genes had undergone selection.The degree of genetic differentiation between wild populations(Fst=0)was lower than that between cultivated populations(Fst=0.11419,P<0.05).The degree of genetic differentiation between wild and cultivated A.sinensis was high(Fst=0.94255,P<0.01).Genetic variation in the cultivated was mainly found within populations(89%).UPGMA cluster tree based on genetic distance showed that the wild and the cultivated were clustered into one branch respectively,and their genetic relationship was far away.The TCS haplotype network map consisted of 15 haplotypes and 4unknown haplotypes.It was divided into 3 parts,with a high number of variants between them.Shared haplotypes were only found within wild or cultivated groups,and there were no shared haplotypes between groups.2.Ten pairs of fluorescent SSR primers showed high polymorphism,the average Shannon’s information index was 1.5304,the average effective number of alleles was 3.4800,the average Nei’s expected heterozygosity was 0.6606,the average heterozygosity was 0.5702,and the average polymorphic information content was 0.629.A total of 55 alleles were detected in the 11 populations,and the average effective number of alleles,Shannon’s information index and Nei’s expected heterozygosity were 2.8745,1.1333 and 0.5703,respectively,indicating that the overall genetic diversity of the 11 A.sinensis cultivars(lines)was high.The results of F-statistic and gene flow showed that there was moderate degree of genetic differentiation and a lot of genetic exchange among populations.Population structure analysis showed that the 275 samples contained 4 genotypes.The heterozygosity of the population was high,and the phenomenon of genetic admixture within the population was serious.AMOVA and PCo A analysis showed that the variation mainly came from individuals,the differentiation between cultivars(lines)was not significant,and gene exchange was frequent.The genetic distance of the 11 populations ranged from 0.0639 to 0.4272,and the genetic identity ranged from 0.6523 to 0.9381.The most distant relatives were "Mingui No 1" and " Mingui No 3",and the closest relatives were " Mingui No 4" and " Mingui No 5".The new lines with half purple and half green stems had the farthest genetic distance and the lowest genetic identity with the six A.sinensis cultivars.3.A total of 275 amplification band types were generated from the selected 10 pairs of fluorescent SSR,and 275 molecular identities of A.sinensis were generated.Bar code and qr code DNA molecular identities were successfully constructed.Conclusion:1.The genetic diversity of A.sinensis was low.The population diversity of the cultivated was higher than that of the wild.The degree of genetic differentiation between the wild and the cultivated was high,but the degree of genetic differentiation within the wild and the cultivated were low.Genetic variation in the cultivated was mainly found within populations.2.The heritability of cultivated A.sinensis was high,and the differentiation coefficient of each variety(line)was low.Gene exchange was frequent,and the phenomenon of genetic interbreeding in each cultivar(line)was serious.3.The 275 molecular identities of A.sinensis were different from each other.The molecular identity could distinguish different A.sinensis cultivars(lines).