Phylogeography Of Lilium Pumilum Redouté In Southeast Of Qinghai-tibetan Plateau

Qinghai-Tibetan Plateau(QTP)is one of the world's biodiversity focuses and has abundant plant resources.The geological accident and environmental fluctuation during the Quaternary glacitial and interglacial period had significant effect on biology distribution and genetic diversity in QTP,but its' influence mechanism is not clear.In this study,after laboratory researchers' s 7 years of collection,we collected25 populations(217 individuals)of Lilium pumilum from southern of QTP,combined chloroplast intergenic spaces(cpDNA)with two nuclear gene(ITS),to study the genetic diversity,population demography history and phylogeography of Lilium pumilum.The maining results and conclusions are as following:(1)To obtain an optimal cpDNA-PCR system for Lilium pumilum DC.,single-factor test and L16(45)orthogonal test were used to 6 populations which collected from southeast region of Qinghai.Concentration of Mg2+,dNTPs,primers,Taq polymerase and DNA template were screened at four levels.As a result,the best cpDNA-PCR amplification system for L.pumilum was Mg2+2 mmol/L,dNTPs 0.2mmol/L,Primer 0.2 ?mol/L,Taq polymerase 0.25 U,and DNA template 50 ng in a 25?L reaction system.Annealing temperature was 53.1?.The optimized cpDNA-PCR system was validated on 6 populations,by which the high tability and repeatability of this system was confirmed.The establishment of the reaction conditions could be further used in study of genetic diversity and phylogeography of L.pumilum DC.(2)The differences between the ITS and cpDNA sequences were analyzed by PCR direct sequencing.After compared,the length of ITS4-ITS5 sequence was696 bp,of which 4 were variable sites with a percentage of 0.57%,the(G+C)content was 60.5%-60.8%,there were totally 7 kind of haplotypes.Compared with ITS1-ITS4,the length was 680 bp,of which 4 were variable sites with a percentage of0.59%,the(G+C)content was 60.3%-60.7%,there were totally 8 kind of haplotypes.The cpDNA fragments could not provide effective variable sites,therefore,it was not fit to the phylogeographic study of this species.The ITS sequences could provide a few variable sites,and we can analyze the phylogeographic of L.pumilum with the help of population genetic structure study,neutrality test and mismatch analysis,all this results can judge whether the population experience population expansion.(3)ITS sequence AMOVA analysis revealed that genetic variation was very rich,and genetic variation among population was significantly smaller than genetic variation within population.Nm>1,which means the number of immigration was large,gene flow was high.NST>GST,and P >0.01,which means the populations were not full of apparent phylogeograpgical structure.Phylogenetic analysis showed that H1 and T1 were relatively old haplotypes,while other haplotypes were derived from the old haplotypes.The MDA and neutral test shows that the population of L.pumilum may experience a small range of expansion.At the same time,the IBD analysis between genetic distance and geographic distance were proved that there have significant correlation between them.