| The Glehnia littoralis Fr.Schmidt ex Miq is a perennial herbaceous plants of the Glehnia in Apiaceae.Its dry root is a traditional Chinese medicinal material called Beisha ginseng,which has important application value.In China,the wild resources of Glehnia littoralis are mainly distributed on the eastern beaches.In recent years,with the encroachment of sand embankment and the intensification of human activities,the wild resources of G.littoralis become endangered and have been listed as a second-class protected plant.In view of this,G.littoralis were called pandas of plant world,and its protection is urgently needed.Genetic diversity is the premise and basis for the protection and utilization of species germplasm resources,but thus far,reports on the genetic diversity of G.littoralis are limited.In this study,we assessed the genetic diversity of G.littoralis in China based on AFLP molecular markers and nrDNA ITS gene fragments,hoping to reveal its genetic diversity and genetic structure and explore the main factors affecting the genetic variation among the population,and then provide theoretical guidance for the protection of germplasm resources of G.littoralis.The main results of the study are as follows:1.A total of 127 individuals from 9 wild populations were detected by ten pairs of selective AFLP primers.Ten primers combinations produced a total of 1929 DNA polymorphic loci with the polymorphic frequency of 100%.The number of polymorphic loci in 9 populations ranged from 918 to 1343,the percentage of polymorphic loci ranged from 45.31%to 69.41%.The average percentage of genetic diversity polymorphic loci was 60.80%.The genetic diversity was assessed by software AFLP-SURV1.0 and GenAlEx 6.5.The results showed that:the overall genetic diversity Ht was 0.182,the average genetic diversity Hw was 0.168,and the average Shannon index I was 0.238.Compared with other endangered medicinal plants,G.littoralis showed higher genetic diversity.The amova analysis using GenAlEx 6.5software showed that the average genetic diffentration coffcfent(Gst)between populations was 0.158,indicating that genetic variation mainly occurred within the population.The gene flow（Nm）between the populations was 2.665,revealing more gene exchanges between the populations,this reduced genetic differentiation between populations.Clustering analysis was conducted using NTSYS based on genetic distance,the dendrogram revealed nine G.littoralis populations were divided into two branches:1)The first one included the population of HY,QHD,CHII and CHI,of which HY and QHD grouped together,CHIIand CHIgrouped together.2)The second branch includes WFD,ZH,ZS,RZ and XC,among which WFD and ZH grouped together,ZS,RZ and XC clustered together.We found there was no positive correlation between genetic distance and geographical distance,such as CHII and CHI,there were also geographically distant clusters,such as HY and QHD,which show a close genetic relationship.Principal component analysis（PCoA）based on genetic distance matrix also divided nine populations into two groups,well supporting the above clustering analysis results.In addition,the results of Mantel test also supported that there was no significant correlation between genetic distance and geographical distance of the G.littoralis population（r=-0.0898,P=0.259,P>0.05）.2.The genetic diversity and genetic structure of 135 individuals in 11 populations were analyzed by nrDNA ITS.The results showed that three haplotypes H1-H3 were found in 135 individuals,among which the haplotype H1 was the most widely distributed.According to the haplotype Network analysis by Network software,haplotype H1 is in the middle of the Network,while H2 and H3 are at two ends respectively,indicating that H1 is the ancestral haplotype.DnaSP analysis showed that at the species level,haplotype diversity Hd was 0.582,the nucleotide diversity index Pi was 0.00107.According to the results calculated by software PERMUT,both the population total genetic diversity index（Ht=0.576）and the population genetic diversity index（Hs=0.512）revealed high genetic diversity,mainly provided by by genetic variation within the population.The differentiation coefficient(Gst)between populations was 0.111,which also verified the above results.AMOVA analysis showed that the genetic differentiation coefficient(Fst)between populations was 0.1526,84.74%of the genetic differentiation occurred within the population,and 15.26%of the genetic differentiation occurred within the population,indicating that the genetic variation mainly occurred within the population.The gene flow（Nm）between populations was 4.005,indicating that frequent gene exchange exists between populations,which reduces the genetic differentiation between populations,these also supports the analysis results of PERMUT and AMOVA.All the results based on nr DNA ITS are consistent with that of AFLP.The UPGAM tree diagram was constructed based on the genetic distance.The results showed that the 11 populations of Glehnia littoralis were divided into two branches:The first group includes the population groups CHII,CHI,and ZH,the remaining 8 populations include HY,QHD,CHIII,WFD,ZS,BYQ,RZ and XC were clustered into one clade,closer geographically populations do not showed a closer genetic relationship,such as the CHI,CHII and CHIII in Liaoning Province.The results supports that there is no obvious positive correlation between geographic distance and genetic distance.Mantel test results（r=0.3296,p=0.067）also confirmed this.3.The results of AFLP and ITS analysis are consistent,indicating that the G.littoralis has high genetic diversity,and the reason for the endangered status of this species may be the damage of ecological environment,including Marine invasion and human disturbance.The high level of genetic diversity of G.littoralis provides the genetic basis for the recovery and re-expansion of this endangered species.In view of this,it is suggested to carry out in-situ conservation or ex-situ conservation for wild germplasm resources populations of G.littoralis and establish germplasm resource Banks to restore the scale of wild population. |
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