Population Genetics Of Rhododendron Dauricum L. And R.Mucronulatum Turcz.

Rhododendron L.is the largest genus in the family Ericaceae,and one of the major genera in the Flora of China-Himalaya.Many species in the genus have of great horticultural value.The genus contains about 1,000 species worldwide,producing 571 species in China,of which 409 species are endemic to China and are centrally distributed in southwest and south China.R.dauricum and R.mucronulatum,R.dauricum is a semi-evergreen shrub,and it is a common species in the Changbai flora and the Greater Hinggan Mountains flora.Compared with R.mucronulatum,R.dauricum is more adaptable to a colder environment,so the two species are also suitable for studying adaptation.In this study,chloroplast genes and simplified genome(SLAF-seq)sequencing were used on 27 populations of R.dauricum and 12 populations of R.mucronulatum to reveal the genetic diversity,genetic structure,divergence time and differentiation level of the two species.It is helpful to reveal the formation of species diversity and the mechanism of environmental adaptation.SSR marker has always been a common molecular marker in population genetics research,but the number of SSR markers will affect the results of genetic diversity analysis.In order to study the influence of the number of SSR markers on the results of parameters of population genetics,I generated the data set containing different number of SSR loci based on population SLAF-seq sequencing data of genome-wide level.In addition,I also designed 26 SSR markers with high amplification efficiency and polymorphism in R.dauricum,R.mucronulatum and even R.aureum Georgi.The main results in the study are as follows:(1)Based on the chloroplast gene fragments,15 polymorphic sites and 10 haplotypes were detected in R.dauricum.The haplotype polymorphism(Hd)and nucleotide diversity(π)were 0.6610 and 0.0018,respectively.However,only one haplotype was detected in R.mucronulatum.Moreover,based on the genomic SNPs,the nucleotide diversity of R.mucronulatum population is 0.2450 x 10-3,and the nucleotide diversity of R.mucronulatum population is 0.1850 x 10-3,indicating the nucleotide diversity of R.mucronulatum population is lower than that of R.dauricum.(2)The haplotype network constructed based on chloroplasts genes did not separate R.mucronulatum from R.dauricum.STRUCTURE analysis based on SLAF-seqs showed that the best K value was 2.When K=2,R.dauricum populations and R.mucronulatum populations diverged,but some R.dauricum populations(SL,LTS,MH,HC,LJ,CB,WT,JA)collected from the Changbai Mountains appeared mixed genetic background.(3)The ML tree constructed based on chloroplast genes is inconsistent with the phylogenetic tree constructed based on SLAF-seqs.Although the phylogenetic tree constructed based on the chloroplast gene divides all individuals into two large branches,the individual of R.dauricum collected from the Changbai Mountains and R.mucronulatum were assigned to a clade.And gene flow from R.mucronulatum was detected in these R.dauricum populations using Tree Mix.(4)AMOVA analysis based on SLAF-seqs showed that the main differentiation were between species(57.98%),indicating that the level of differentiation between the two species was obvious.In addition,the Fst values ??between species based on chloroplast and SLAF-seqs were 0.3541 and 0.2886,respectively.Based on the SNPs of the SLAF-seqs,we inferred the differentiation time of R.dauricum and R.mucronulatum was32.41 MYA(95% HPD interval: 17.54-53.11 MYA),which was in the early Oligocene.(5)A total of 292 selected candidate genes were identified among 634,881 SNP loci,which involved in multiple plant metabolic pathways,such as Cysteine and methionine metabolism(KO00270).Citrate cycle(TCA Cycle,KO00020);Glycolysis/Gluconeogenesis(KO00010);Pyruvate metabolism(KO00620);Sucrose and Starch metabolism(KO00500);Biosynthesis of Amino acids(KO01230);Carbon metabolism(KO01200);N-glycan biosynthesis(KO00510);Steroid biosynthesis(KO00100);Alpha-linolenic acid metabolism(KO00592);Cyanoamino acid metabolism(KO00460);Glutathione metabolism(KO00480)and Taurine and hypotaurine metabolism(KO00430).These genes may be related to adaptation to cold environments.(6)A total of 265 SSR loci were screened based on SLAF-seq data.Then,different number SSR loci were selected randomly from these loci and related population genetic parameters were calculated.When the number of microsatellite loci was less than 11,the absolute errors of Ar and Hs were significantly different from the actual values(p <0.01).When there were only 5 or 6 microsatellite loci,the probability of population clustering error reached 50%.(7)Among 265 microsatellite loci,26 pairs of primers were designed and verified by PCR.Among them,23 SSR markers had repeat units of dinucleotides and the rest were trinucleotide and tetraconucleotide.The results of this study revealed the genetic diversity and genetic structure of R.dauricum and R.mucronulatum,which not only clarified the genetic differentiation between the two species,provided a case for revealing the historical causes of the distribution pattern of seed plants in northeast China,but also provided a theoretical basis for the conservation of wild resources of R.dauricum and R.mucronulatum.At the same time,I also developed mass polymorphic SSR loci in these two species based on the second-generation sequencing technology.It makes the batch development of polymorphic SSR loci in non-model species(without reference genome)more convenient,which will promote the research of population genetics.