Study On Population Genetic Structure And Genetic Diversity Of Cymbidium Kanran Based On ITS And CpDNA PetB-petD Sequences

Orchidaceae is one of the largest families of flowering plants,and it is a flagship group in plant conservation.Cymbidium kanran(Cymbidium,Orchidaceae)has high ornamental and economic value,and the resources of C.kanran in China are very rich,but in recent years,with the excessive digging of people,the natural resources and habitats have been destroyed,protecting the C.kanran resources is a task which brooks no delay.Based on the investigation of C.kanran resources distribution in China,this study used ITS and cpDNA molecular markers to research the genetic diversity and genetic structure of 132 C.kanran individuals of 10 populations from 7 provinces in China,we analysed the characteristics of genetic differentiation,gene flow,population dynamics and put forward some corresponding protection strategy,so as to provide theoretical basis and references for the conservation and sustainable utilization of C.kanran resources.The main results are as follows:(1)After the sequence alignment,the total length of ITS sequence is 658 bp,and the content of G+C range from 57.60% to 68.39%,with an average of 65.99%;the content of G+C in ITS1 region range from 53.91% to 71.19%,with an average of 67.57%;the content of G+C in 5.8S region range from 56.31% to 60.74%,with an average of 57.69%;the content of G+C in ITS2 region range from 67.86% to 70.63%,with an average of 69.82%.After the sequence alignment,the total length of cpDNA petB-petD sequence is 536 bp,and the content of G+C range from 33.26% to 33.68%,with an average of 33.61%.The ITS sequences have 179 variable sites,of which 83 are parsimony informative sites;the cpDNA pet B-petD sequences have 10 variable sites(an insertion and deletion of 12 bp is included),of which 5 are parsimony informative sites.(2)Based on the ITS sequence data,18 ITS haplotypes were detected,all populations have more than one kind of haplotype;IHap5 and IHap6 are the main haplotypes;according to the TCS network,we speculate that IHap5 is the ancestral haplotype.The total haplotype diversity is 0.802 and the total nucleotide diversity is 0.04274.The level of genetic diversity of each population changes with a wide range(Hd=0.182-0.857,Pi=0.00077-0.07092),of which LN population has the highest haplotype diversity while JZ population has the lowest haplotype diversity;and CY population has the highest nucleotide diversity while YF population has the lowest nucleotide diversity.On the whole,the level of genetic diversity is high.The percent of genetic variation within populations(64.83%)is larger than the genetic variation among populations(35.17%).Fst= 0.3517,Nm=0.4608,gene flow is in middle level;Gst=0.28016,Nst=0.36120,without significant difference(P>0.05),which indicate that there is no obvious phylogeographic structure.Result of Mantel test showed that there is no significant correlation between genetic distance and geographical distance(r=-0.2725,P=0.4889>0.05).(3)Based on the cp DNA petB-petD sequence data,11 cpDNA haplotypes were detected,all populations have CHap1,which is widely distributed;according to the TCS network,we speculate that CHap1 is the ancestral haplotype.The total haplotype diversity is 0.609 and the total nucleotide diversity 0.00169.The level of genetic diversity of each population changes with a wide range(Hd=0-0.803,Pi=0-0.00329),of which YH population has the lowest genetic diversity(Hd=0,Pi=0),WYS population has the haplotype diversity and RJ population has the highest nucleotide diversity.On the whole,the level of genetic diversity is high.The percent of genetic variation within populations(88.01%)is larger than the genetic variation among populations(11.99%).Fst=0.1199,Nm=1.8351>1,the level of gene flow is high.Gst=0.13685,Nst=0.15436,without significant difference(P>0.05),which indicate that there is no obvious phylogeographic structure.(4)Synthesizeing the distribution of C.kanran resources in China and the characteristics of genetic diversity and genetic structure,we put forward some suggestions for the conservation of C.kanran resources:(1)Recording the distribution of C.kanran and establishing resources database;(2)Taking measures of in-situ conservation,such as seting up nature sanctuary and so on,moreover,the management should be strengthened and the construction of it should be improved;(3)Taking measures of ex-situ conservation for the C.kanran populations of which the habitats have been threatened;(4)Protecting the pollinating insects;(5)Combing artificial breeding with transplanting techniques,establishing and optimizing rapid propagation system,and cultivating excellent varieties,so as to meet the demand of the market.