Population Genetics Structure Of Verbena Officinalis In China

Verbena officinalis Linn,is a perennial herb.It belongs to the genus of Verbena in the family Verbenaceae.V.officinalis is a traditional medicinal plant in China,and it is mainly studied in the aspects of pharmacological action and chemical components,but few studies of its lineage structure and genetic pattern.In this study,Verbena tenera was selected as the external group,and the phylogeography and genetic structure of 22 V officinalis populations in China were analyzed using three molecular markers?cpDNA,nrDNA and SSR?,so as to explain the characteristics of V.officinalis in terms of the level of genetic diversity,historical dynamics and distribution pattern in China.1.Phylogeography of V officinalisThe cpDNA and ITS were used to analyze the evolution of V officinalis in China.Two cpDNA intergenic spacer?trnL-trnF,psbd-trnt?were selected from the universal chloroplast primers which were used for our study.There were two haplotypes of trnL-trnF in domestic populations,with a percentage of variation sites of 0.11%,haplotype diversity Hd=0.087 and nucleotide diversity ?=0.00010.There were only 3 haplotypes of psbD-trnT in the population in China,with a percentage of mutation sites of 1.8%,haplotype diversity Hd=0.345,nucleotide diversity ?=0.00046.Two haplotypes were produced in ITS sequence,with a percentage of variation sites of 0.15%,haplotype diversity Hd=0.236,nucleotide diversity ?=0.00035.After combining chloroplast sequence and ITS sequence,there were 6 haplotypes in China.Haplotype polymorphism?Hd?and nucleotide polymorphism???were 0.537 and 0.00029.Hap₁ is the widespread haplotype.Hap₂ was closest to the outer class group and the ancestor haplotype,and Hap₃ is the closest to Hap₄.Population dynamics analysis showed that V officinalis experienced rapid expansion events in its history.2.RNA sequencing and SSR site analysis of V.officinalisIn this study,57689 Unigenes sequences were obtained from V.officinalis based on RNA-seq,from which 12730 SSR sites were identified,containing 128 motif types.In the repeat types of SSR sites,single-base repeat and two-base repeat accounted for the largest proportion.And three-base repeat was the second.AG/CT was the most abundant in the two-base repeat,and AAG/CTT was the most abundant in the three-base repeat.In conclusion,the SSR sites have high frequency,rich types and high density in V.officinalis.3.Genetic diversity based on SSR molecular markers11 polymorphic SSR makers were chioced from 90 SSR makers randomly selected,and 68 alleles were detected in 22 populations,with the average number of alleles K=6.2.The three parameters of observed heterozygosity?Ho?,expected heterozygosity?He?and polymorphism information content?PIC?were 0.781,0.565 and 0.512.The null frequency of SSR14 was 0.6337,which was too high to meet the requirements of the experiment,so it was eliminated.The population of V.officinalis was amplified with 10 SSR makers.The results showed that the genetic diversity of population was high?Ho=0.847,He=0.495,PPL=92.27%?.Inbreeding coefficient?F?of all populations was negative,and the heterozygote surplus in the populations was very obvious,which may be related to the breeding mode and growth environment of V.officinalis.PcoA,STRUCTURE and cluster analysis showed that populations of V.officinalis were most likely to be clustered into 2 group,and the gene flow between the two lineages?Nm=1.1679?were small.There was greater gene flow within lineages?lineages 1:Nm=2.2366,lineages 2:Nm=1.9015?.According to the AMOVA,7.81%of the variance came from between lineages,9.82%from the difference between populations,and 82.37%from within populations.The genetic differentiation coefficient Fst between the two lineages was 0.1763,which was at the intermediate differentiation level.Bottleneck inspection found that all populations were experienced the bottleneck.IBD test showed that there was a significant correlation between genetic distance and geographic distance?r=0.273,p=0.01?,indicating that one of the reasons for genetic variation oof populations of V officinalis was related to isolation caused by geographical distance.