Analysis Of Genetic Relationship Of D.huoshanense Population And Its Identification Based On Chloroplast Genome

Dendrobium huoshanense C.Z.Tang et S.J.Cheng,also call 'Mihu',belonging to Dendrobium species,Orchidaceae,is a rare and endangered perennial herb.D.huoshanense mainly distribute in Anhui Province,Hubei Province,Jiangxi Province and Henan Province.Owing to its strong healthcare effect,such as nourishing the kidney,benefting the stomach,enhancing the body's immunity,resisting cancer,and prolonging life,D.huoshanense have been extensively used as Traditional Chinese Medicine(TCM)for hundreds of years.The stem of D.huoshanense can be made into 'Jin Huodou'.To maximise the impact of conservation and management efforts,it is urgent for us to insight into the genomics,phylogeograpy and population genetics.Because of its great medicinal value,the 'Jin Huodou'is sold at the price as high as 70-100 thousand yuan per kg,which is much higher than other Dendrobium products.There is a pressing need to develop an accurate,sensitive and rapid method for authenticating D.huoshanense and 'Jin Huodou' from other Dendrobium.This research focused on three aspects as follows:1.The sequence of complete chloroplast genomes of D.huoshanense and the study of comparative chloroplast genomics.Here,we reported the complete chloroplast genomes(cp)of D.huoshanense.Comparing with other Dendrobium genomes,they showed similarities in structure as well as gene order and content,but differences in the organization of the inverted repeat/small single-copy junction and ndh genes.Phylogenetic analyses based on complete chloroplast genomes showed that:D.moniliforme was sisiter to D.huoshanense.Comparative studies of plastomes have also found that loss of ndh genes has occurred independently among Dendrobium.Four types of junctions in the Dendrobium cp genomes were characterized on the basis of the variability of genes flanking the IRB/SSC junction.2.Mutational hotspot selection and the study of genetic relationship of D.huoshanense at population level.The cp genomes of three D.huoshanense and D.moniliforme were divided into 372 small fragments(400bp)and compared.The hotspots among them were selected and used to access genetic relationship of D.huoshanense at population level.Our results showed that:all pairwise correlation analysises on SNP,InDel,SSR were dramaticlly positive correlation,all correlation analysises on GC vs.SNP,GC vs.InDel and GC vs.SSR were dramaticlly negative correlation.Sequence variability(SV)of intergenic region between trnV and rps12 is maximum.The clustering tree showed that:The Huoshan pupulations(HS,H1,H2),which had the closest relationship,grouped together,while the relationship among the Longhushan population(LHS1,LHS2),Funiushan population(FNS)and Yingshan population(YS)were not clear.3.The identification of D.huoshanense and its 'Fengdou'.A novel method,combining SYBR Green ?-based real-time PCR(rt-PCR)with amplification refractory mutation system(ARMS),was established.The alignment of the mutational hotspot of twenty Dendrobium species using MEGA 5.0 led to the identification of D.huoshanense-specific nucleotides.Using Primer Premier 6.0 software,allele-specific ARMs primers were designed to target the mutational hotspot containing D.huoshanense-specific nucleotides.By performing RT-qPCR amplification with specific ARMs primer pairs,90-370bp fragment was specifically amplified from the mutational hotspot of D.huoshanense,but not from that of any reference species.Six pairs of specific ARMS primer,including NL1/NL4,NL1/NL5,NL4/NL6,TP1/TP2,NL1/NL3,NT0/NT12 were proved good specificity and amplification efficiency.Amplification curves of D.huoshanense were appeared fastest among all Dendrobiums,amplification efficiency of D.huoshanense were the highest among all Dendrobiums.The average of Ct value of D.huoshanense is lower than the other species that presented significantly higher Ct values.There is a statistically significant difference between them(P<0.001).The results indicated that the established system can be able to rapidly and unequivocally discriminate D.huoshanense and 'Jin Huodou' from its adulterants.