Analysis Of Genetic Diversity And Hybrids Identification Of Dendrobium Officinale Based On Target Region Amplification Polymorphism(TRAP)

Dendrobium officinale Kimura et Migo,belongs to the genus of Dendrobium,Orchidaceae,is traditional Chinese medicine and its promising medicinal constituents mainly are polysaccharide and alkaloid.However,the wild resources of D.officinale are extremely in danger of extinction due to low germination rate,slow-growing,habitat deterioration and over-exploitation.In order to protect the endangered species and meet the great market demands,plant breeders have raised the size of artificial cultivation,which has become the main way to solve the shortage of wild resources of D.officinale.However,the complex germplasm source during the process of artificial cultivation resulted in a large difference on the quality of D.officinale.Therefore,establishing an effective and credible method,which can use to analyze the genetic diversity,population structure and identification of hybrid,is important for protecting wild resources and breeding fine varieties of D.officinale.Firstly,three pairs of fixed primers,which were designed based on the related genes(PEPC,UGP and PAL)of polysaccharide and alkaloid of D.officinale,combined with 18 arbitrary primers to develop TRAP molecular markers.Orthogonal design was used to optimize TRAP-PCR amplification conditions.The results showed that the advanced reaction system for TRAP-PCR is total 20 p.L reaction volume including 1.0 ?L of 2.5 mmol·L-1 dNTPs,1.2 ?L of 25 mmol?L-1 MgCl2,0.9 U Taq DNA polymerase,1.5 ?L of 2 mmol·L-1 arbitrary primers,2 p.L of 2 mmol·L-1 fixed primers and 20 ng of template DNA.In addition,eight pairs of TRAP primer were selected to assess the genetic diversity of D.officinale.Secondly,eight pairs of TRAP primers were used to assess the genetic diversity and population structure in nine wild populations of D.officinale.The results showed that a total of 148 fragments were scored in all samples,including 130(87.84%)polymorphic fragments.The genetic analysis revealed the high level of genetic diversity in cultivated populations of D.officinale(H=0.4125,I=0.5985),especially in population SD with the highest genetic diversity.Based on analysis of genetic structure,there was a moderate variation(Gs,=0.4706)and lower gene flow(Nm=0.5625)among populations.Moreover,the UPGMA dendrogram indicated that nine populations were divided into four clusters,which was consistent with the results of principal coordinate analysis(PCA).Mantel test revealed that no significant positive correlation was found between genetic distances and geographic distances(r=0.5496;p>0.05).Finally,hybrids and their parents were as materials for identification by TRAP markers.One common parent of reciprocal hybrids(H1 and H2)was D.officinale from population GN,the other parent was from populations NF or SD.Seven pairs of TRAP primers were selected to identify hybrids from their parents.The results showed that hybrids had female polymorphic bands,male polymorphic bands and heterozygous bands,which indicated that seven TRAP markers could identify hybrids from their parents.Furthermore,the UPGMA dendrogram revealed that reciprocal hybrids grouped with one parent of GN in first,and then grouped with the other parent(NF or SD).The results indicated hybrids were closer to D.officinale from Guangnan population.Therefore,this study analysised the genetic diversity of wild populations and identified hybrids from their parents by TRAP markers,which will be useful for selecting good varieties for artificial cultivation and provide an effective basis for the early identification of hybrids.And this study will be helpful for controlling the stability of germplasm and improving the quality of D.officinale.