Phylogeography And Genetic Diversity Of Sanicula Orthacantha

Sanicula orthacantha S.is an herbaceous perennial plant,belongs to the genus of Sanicla in the family of Apiaceae/Umbelliferae.S.orthacantha is widely distributed in southern China and is a tertiary relic plant.It is a good material for studying phylogeography and genetic diversity.In this study,237 samples from 23 populations were collected,and the epidermal micromorphology of different populations was studied.For the first time,chloroplast DNA and n SSR molecular markers were used to analyze the phylogeography,genetic diversity,genetic structure and historical dynamics of S.orthacantha population.The reasons for the present geographical distribution pattern of S.orthacantha were discussed,which provided new evidence for the origin and migration path of Arctic-Tertiary relic plants in China.In addition,the comparative genomic method was used in this study to compare the chloroplast genomes of S.orthacantha and S.lameligera,which may provide more classification evidence for confusing species.The main research results are as follows:The micro-morphological characteristics of leaf epidermis from 19 populations of S.orthacantha were observed to be stable.Adaxial epidermis have no stomata,and the outline of the cells is mostly unclear.Primary waxy ornamentations are present in dense or sparse stripe patterns,and there are a small or large number of granular epidermal appendages.Abaxial epidermis have stomata,the stomata are oval in shape.Most stomatal ornamentations are radial,and particulates are present outside ornamentation.The habitat of S.orthacantha is relatively single,and changes in geographical location may have little effect on the leaf epidermal characteristics or even morphological characteristics of each population of S.orthacantha.By comparing the chloroplast genomes of the two confusing species,it is found that both chloroplast genomes annotation identified a total of 129 genes,the gene function,gene sequence,and GC content are extremely conservative.GC content in IRs region is generally higher than LSC and SSC regions.Repeats of S.orthacantha and S.lamelligera chloroplast genomes are mostly forward repeats and palindrome repeats,and the size is concentrated at 30-39 bp.There are most monouncleotide in SSR,especially(A/T)n type,SSR fragments may be used in subsequent molecular marker studies of Sanicula plants.Most codons have a preference,and A/T bases are preferred as codon endings.The non-coding region has more abundant variability than the coding region.The regions with large differences ycf1,rps16 intron,trn N-trn R,trn I intron,trn I-rrn16,trn S-ycf3,and rpo C1 intron can be used as molecular fragments for phylogenetic research in S.orthacantha.The IR boundaries of the two chloroplast genomes are generally conservative,with only slight differences in IRa/LSC.Based on the chloroplast DNA non-coding region fragments trn L-trn F,rps16 intron,and rpl32-trn L,176 individuals from 18 natural populations of S.orthacantha were analyzed.A total of 37 haplotypes were detected,of which H5 occurred most frequently and was located at the center of the haplotype network.The population of S.orthacantha has a high level of genetic diversity,among them,haplotype diversity Hd=0.931,nucleotide diversity ? = 0.00225,and overall genetic diversity HT=0.962.The AMOVA results revealed that the genetic differentiation of the S.orthacantha population mainly exists in this population.Nst>Gst,P>0.05,the difference is not significant,indicating that the S.orthacantha population has no obvious geographic structure.The results of mismatch distribution analysis show that the S.orthacantha population has not experienced a large-scale rapid expansion event recently.Based on the haplotype network map and the haplotype geographic distribution results,it is speculated that the southwestern region may be the origin center of S.orthacantha,and the migration route of S.orthacantha is from west to east.Sichuan and Zhejiang regions with high levels of genetic diversity may be glacial refuges for S.orthacantha.Based on five pairs of n SSR molecular markers,genetic diversity and genetic structure of 207 individuals in 18 populations of S.orthacantha were analyzed.Based on orthogonal experiments and single factor design experiments,the optimal S.orthacantha SSR-PCR system was established: 2×Taq Mix 10?L,primer 0.5?L,and template DNA 1?L.The optimized system was used to screen primers,five primers were selected from 50 primers with good polymorphism,stable amplification and few non-specific bands.Data analysis showed that the overall genetic diversity of S.orthacantha was high(Na=2.475,Ne=1.9406,I=0.6511,Ho=0.4168,He=0.4177).AMOVA analysis showed that the genetic differentiation of S.orthacantha mainly existed in the population(76%).STRUCTURE bayesian cluster analysis showed that the 18 populations of S.orthacantha could be divided into two groups,but these two groups have no obvious geographic structure.