Molecular Biological Identification Of Four Kinds Of Dendrobium In Dabie Mountain

Objectives:(1)Based on the analysis of ITS sequences,the specific PCR primers were designed for the identification of D.huoshanense and its adultments of D.officinale and D.henanense.(2)The microsatellite loci of D.huoshanense were developed to enrich the microsatellite library.The aim was to develop a specific microsatellite primer for the identification of D.huoshanense and D.moniliforme,and to esTab.lish a rapid detection method of PCR for D.huoshanense.And provide the basis for the later study on the genetic structure and diversity of D.huoshanense.(3)The SNPs between D.huoshanense and D.moniliforme were developed based on resequencing,which were used to identify D.huoshanense and D.moniliforme.Medthods:(1)The ITS sequences of four Dendrobium species were obtained by sequencing,and combined sequences were downloaded from GenBank.The sequences were alignmented analysis by BioEdit software.The specific primers were designed according to the sequences mutation sites of D.huoshanense,D.officinale and D.henanense,which were analyzed by primer5 and olige6 software.After PCR reaction and electrophoresis,to screen primers which positive reaction of D.huoshanense(with bright band),while D.officinale and D.henanense showed negative reaction(no band)as target primers.And sample verification.(2)Based on the high polymorphism of microsatellite loci,the microsatellite loci of D.huoshanense were developed by magnetic beads enrichment method using(CT)12 and(GAA)8 as hybridization probes.The primers were designed at the two ends of the microsatellite loci,and the PCR reaction between D.huoshanense and D.moniliforme was carried out to screen the primers which can distinguish D.huoshanense and D.moniliforme.(3)The whole genome sequence of D.officinale was used as the reference gene,and the SNP loci were reconstructed to D.huoshanense and D.moniliforme,and D.huoshanense and D.moniliforme were identified.Results:(1)Based on the mutation sites of ITS sequences,among the designed primers,the primers SH-CP9s/SH-CP9a showed a bright band in the DNA samples of D.huoshanense when the annealing temperature was 58 ? and on a 2%agarose gel.The DNA samples of D.officinale and D.henanense had no band.In addition,the same results were found during the sample validation process.(2)A total of 200 microsatellite loci were collected by magnetic bead enrichment method,and 100 SSR primers were designed to remove loci that could not be designed at both ends or designed with lower score.However,SSR primers that could differentiate D.huoshanense and D.moniliforme were not obtained in the present identification study.(3)A total of 8,342,377 SNPs were obtained in the genome of D.officinale,0(0.00%)were located in the coding area,and there were 3,201,895 SNPs between D.huoshanense and D.officinale,and SNP was obtained between D.officinale and D.moniliforme.And there were 3,155,447 loci,while the SNPs in the samples of D.huoshanense were 987,414,and there were 997,621 SNPs in the samples of D.moniliforme.Conclusion:(1)The specific primers SH-CP9s/SH-CP9a based on ITS sequences could effectively distinguish D.huoshanense and its adultments of D.officinale and D.henanense,and the sTab.ility of the results was confirmed by sample verification.(2)Based on the developed microsatellite loci,D.huoshanense and D.moniliforme could not be distinguished under agarose electrophoresis.But in addition to identification,SSR locus primers obtained by magnetic bead enrichment method can be used in molecular markers research of D.huoshanense of "Daodi",and population genetic and biological protection research.(3)A total of 8,342,377 SNPs were obtained in the genome of D.officinale.Therefore,in the subsequent analysis experiments,the SNPs of D.huoshanense and D.moniliforme were compared,and the SNPs between D.huoshanense and D.moniliforme were screened,and the primers were designed based on these sites.D.huoshanense and D.moniliforme were identified.