Study On Genetic Diversity Of Panax Japonicus(T.Nees) C.A.Meyer

Panax japonicus?T.Nees?C.A.Meyer,a perennial herb in family Araliaceae,is the most widely distributed species in genus Panax L.The plant was used as Chinese Traditional Medicine with long history,thus it is of high medicinal and economic value.However,due to large demand in market and relative long cultivation and breeding cycle,its wild resource is nearly exhausted and need urgent protection.In this study,Simple Sequence Repeats?SSR?molecular markers for P.japonicus were developed based on second-generation sequencing technology,the population genetic analysis of the species was carried out to reveal the genetic background aiming to provide a theoretical reference for the conversation of the species.Illumina second-generation sequencing technology was used to establish a genomic library and microsatellite library of P.japonicus.The characteristics of SSR composition types were analyzed.The genomic sequencing returned and assembled,20 051 consensus sequences were obtain.A total of 1 998 SSR loci were found in 1 537 sequences,and SSR-containing fragments accounted for 7.67%of the middle sequence.Among all SSR fragments,single nucleotide repeats accounted for 39.99%of the total number of SSR loci,and dinucleotide repeats accounted for 36.93%.Among the single nucleotide repeat types,A/T repeats dominated?87.23%?;AT/AT dominates the dinucleotide repeat type?accounting for 51.76%?,and the variation in repeat length is negatively correlated with SSR abundance.33 pairs of primers were designed and synthesized.PCR amplification and polyacrylamide gel electrophoresis were carried out to detect the polymorphism of 5 P.japonicus populations.As the results,20 pairs of primer were polymorphic and performed clear bands.The number of alleles?N_A?ranged from 3 to 10 per locus,with the average of 5.1.The polymorphic information content PIC ranged from 0.42 to 0.85.The observed heterozygosity?H_O?for each pair of primer is 0.0192-1.0000,with an average of 0.5923;the expected heterozygosity?H_E?is 0.1876-0.8755,with an average of 0.6430.Population genetic analysis of five populations of P.japonicus revealed that the primers developed in this study performed good polymorphism.Examination of the universality across the outer taxa showed that all of the primer pairs had good universality in genus Panax,however the uninversity is poor intergenus.In addition,20 pairs of SSR primer developed for P.japonicus were employed to detect the genetic structure of the species on 273 individuals sampled from 14 populations of 3 variants collected from Yunnan,Sichuan,Hubei,and Shaanxi.As the results,20 primer pairs were of high polymorphism,202 polymorphic alleles were detected in total,the number of alleles at each locus was between 4 and 16,with an average of 10.1 per locus;the genetic diversity in species level was high?H_O=0.4799,H_E=0.8393,I=2.0513,h=0.8378?;AMOVA analysis showed that the genetic variation of P.japonicus mainly happened between populations,with strong genetic differentiation between populations(G_st=0.6092,F_st=0.61622),in line with the limited gene flow among populations?N_m=0.1557?.The results of the clustering analysis divided the 14 populations into three groups,populations with short genetic distances were relatively close in geographical distribution.The Mantel Test results?R=0.617,P=0.01?confirmed that the genetic distance and the geographical distance of P.japonicus had a significant correlation.The high genetic diversity of P.japonicus may be resulted from its relative wide distribution rang and multiple remained small populations,different isolated population accumulated genetic variation respectively in the long evolution,which made the species persist abundant genetic diversity.The strong genetic differentiation among population was resisted in population genetic drift that resulted from geographical isolation and limited gene flow.Based on the wide distribution and high genetic diversity of P.japonicus,we propose a conservation strategy that focuses on in situ conservation.