Molecular Identification Of Fengdou Dendrobium Based On Chloroplast Genome And RAD-seq Technology

Dendrobium is the second largest genus of Orchidaceae.There are about 1500 species in the world,and about 100 species are distributed in China(including Taiwan),of which more than 40 species are Medicinal Dendrobiums.Dendrobium species of “Fengdou”(DSFs)are a very important group of Medicinal Dendrobiums in Dendrobium,including about 20 species.D.officinale and D.huoshanense are the most precious species in the group.Due to the variety and complexity of the group,including many closely related and recently diverged species,authentication of the botanical origin and medicinal materials of group have not been effectively resolved,which is adverse for conservation of germplasm resources and the safe use of medicinal materials.In this study,we employed plastome data to accurately identify 23 DSFs,and explored the mutational dynamics of Dendrobium plastome.In the complete plastome level,we screened specific nucleotide sites of D.officinale,and explored a simple and efficient method for identification of “Tiepi Fengdou” based on ARMS-q PCR technology.In addition,in order to develop origin traceability method for D.huoshanense,RAD-seq and the plastomes were also used to identify the populations of D.huoshanense.Here,we investigated 101 complete plastomes from 23 DSFs,comprising 72 newly sequenced and 29 documented,and analyzed the structure and sequence characteristics of the plastomes.The results showed obvious expansion or contraction of the IRs in the plastomes of DSFs.In line with other groups,we found SNPs and indels co-occurred in the plastomes of Dendrobium,and further verified three existing hypotheses to explain this phenomenon.According to the results of this study,we only support both the repeat-associated and the indel-associated mutation hypotheses to explain this phenomenon;the regional difference hypothesis could not fully explain this phenomenon.In this study,101 plastomes of DSFs were used to identify the botanical origin of DSFs.In order to compare discriminatory power of the complete plastome,its different regions(LSC,IR and SSC)and the commonly used DNA markers for DSFs,and to assess the potential impact of different filtering strategies(no,light and strict)of sequence alignment on species identification,this study constructed ML trees based on 24 datasets.The results showed that relaxed filtering strategy(no,light)did not have a negative impact on the results of species identification.IR,SSC and the commonly used DNA markers datasets could not fully identify all of DSFs,while the complete plastome and LSC region could effectively identified all the tested DSFs with maximum support values.Considering the advantages of LSC region in cost and efficiency of identification,LSC region is recommended as a powerful barcode for accurate identification of DSFs.We further identified D.officinale and its “Fengdou” medicinal materials(“Tiepi Fengdou”).In this study,132 complete plastomes(72 newly sequenced and 60 documented)of D.officinale and its 25 adulterant Dendrobium species were compared.A total of 11 nucleotide sites were screened for the design of specific primers of D.officinale.Four pairs of ARMS specific primers were designed for D.officinale based on the principles of ARMS primer.Subsequently,these four pairs of primers were employed to amplify the genomic DNA extracted from the fresh materials of D.officinale and its 25 adulterant species.Of which two pairs of primers(Dot1/Dot2,Doa1/Doa2)were preliminary selected for identification of “Tiepi Fengdou” according to the amplification results,and then they were used for q PCR amplification of the pre-amplified products from“Tiepi Fengdou” and its adulterants.The results showed that the amplification curves of “Tiepi Fengdou” were the first appeared,with the highest amplification efficiency;the Ct value of “Tiepi Fengdou” was significantly lower than that of its adulterants.Therefore,the identification system based on ARMS-q PCR technology can be used for the authentication of D.officinale and its “Fengdou” medicinal materials.Finally,the population identification of D.huoshanense was carried out based on RAD-seq and plastomes data.A total of 471,018 SNP markers among 26 individuals from 5 populations were obtained by RAD sequencing.In addition,12 plastomes(10 newly sequenced and 2 documented)of D.huoshanense were employed to screen intraspecific hypervariable regions.The 6 relatively hypervariable fragments(mat K-5’trn K,rps16-trn Q,trn S-trn G,clp P-psb B,ndh F-rpl32,ycf1)were obtained.The cluster analyses of different populations of D.huoshanense were carried out based on RAD-seq data,intraspecific hypervariable regions and the complete plastomes.The results showed that the resolution of intraspecific hypervariable regions and the complete plastomes among D.huoshanense populations were low,yet RAD-seq data could cluster all individuals from Huoshan well;hence,the technology could be used for the origin traceability of D.huoshanense from Huoshan population.However,RAD-seq data still can not cluster the populations of Longhushan,Huangshan and Liuyang well respectively,which indicates that the genetic differentiation level among populations of D.huoshanense is low,which may be related to the fact that D.huoshanense is a recently diverged species with narrow distribution,and its natural habitats were destroyed and disturbed by human activities.