Population Genetic Variation Of Neolamarckia Cadamba (Rubiales) Based On Cytonuclear Markers

Neolamarckia cadamba（Roxb.）Bosser belongs to the genus Neolamarckia of the family Rubiales,which is an evergreen deciduous tree.It is naturally distributed in Guangdong,Guangxi and Yunnan Provinces in China,and in Vietnam,Indonesia,Malaysia,and Myanmar in tropical regions.Because of its rapid growth,straight texture and even structure,the species is an important timber tree for industrial purpose in South China.This study is based on the sample collections of germplasm resources of N.cadamba,and analyzes genetic diversity,population genetic differentiation and the phylogeographical pattern of populations across natural distribution of the species in China,with aims at providing a scientific basis for genetic improvement,breeding and conservation of genetic resources of N.cadamba.Two types of molecular markers were selected in this study.One was the sequence of ITS（internal transcribed spacer）from nuclear DNA（nDNA）,and the other was two SNP（single nucleotide polymorphisms）sites of mt DNA.Ten populations were sampled across natural distribution of the species in China,including Longzhou,Fangchenggang,Nanning,Guangzhou,Yunfu,Baoshan,Dehong,Jinghong,Mangshi,Mengla,and one population was sampled from Indonesia.Two hundred and fifty-six individuals in total were sequenced with these two markers.The whole mitochondrial genome of N.cadamba was sequenced and characterized.Its genomic structure consists of two circles of segments.The smaller circle of mt DNA is109,836bp in length,and encodes 14 genes that exhibit a pattern of AT-bias.The larger circle of mt DNA is 305,144 bp in length and encodes 69 genes that exhibit a GC-bias.Analysis from two mt DNA markers showed that population genetic diversity was low（π=0.0010）and phyogeographical structure was absent in distribution of N.cadamba in China(N_st=G_st=0.7121).Neutrality tests indicated that these two mt DNA markers were selectively neutral,with estimates of 0.6488 of Fu and Li’s D（P-value>0.10）,1.1504 of Fu and Li’s F（P>0.10）and 1.6907 of Tajima’s D（P>0.10）.The results might also imply that populations of N.cadamba likely did not experience historical population shrinkage or expansion.Neutrality tests with both Tajima’s D and Fu and Li F（0.3740）statistics indicated that ITS sequences of nDNA were selectively neutral（all testing P-values>0.10）,which might imply that the eleven populations had not experienced contraction or expansion events in history.Estimates of two measures of population genetic differentiation(N_st=0.0271 Vs.G_ST=0.0252)were not significantly different from each other（P>0.10）,indicating that phylogeographical structure among populations was absent.Analysis of population genetic differentiation indicated that most of the genetic variation occurred within populations(F_st=2.25%),suggesting that extensive gene flow occurred among populations.There was no significant correlation between the genetic variation among populations and their geographical distances,indicating that genetic differentiation between populations was not caused by geographical isolation.Except for a few populations,most populations had different degrees of linkage disequilibrium among ITS markers.Analysis with STRUCTURE indicated that three groups of populations（K=3）were clustered with a maximum likelihood,and most individuals were highly heterozygous in genetic composition.Most individuals came from the cross of multiple ancestral subpopulations,but genetic differentiation among populations was small,which was consistent with the results in terms of F_st estimate.A new statistic method was developed to test cytonuclear linkage disequilibrium（LD）,including gametic and zygotic LDs.We pooled the available data from 11 populations of N.cadamba assayed with both ITS sequences and mt DNA markers to test cytonuclear LD.The results showed that about 3-5%of all 404 ITS-SNP sites of minor allele frequency greater than 0.05 had significant zygotic LDs with the two mt DNA markers（P value<0.01）,but no significant gametic cytonuclear LD was detected.This implies that extensive gene flow among natural populations（genetic admixture）and genetic drift（small sample sizes）could generate a certain level of cytonuclear LD.Further surveys of cytonuclear LDs are needed in populations that are scattered in natural distribution.At the species level,genetic variation of N.cadamba mainly existed within populations.The population genetic information revealed in this study could provide an important message for managing the genetic resources of N.cadamba.Concerning the narrow distribution of the species in China,it is suggested in conserving the genetic resources of N.cadamba that selection of populations with rich haplotype diversity and high genetic diversity is preferable,such as the populations in four regions of Dehong,Mangshi,Mengla,and Nanning in Yunnan Province.A strategy that combines both in situ and ex situ protection is suggested,and this study provides a theoretical basis for formulating such strategies of genetic conservation of N.cadamba germplasm resources in China.