DNA Fingerprinting Of Gastrodia Elate BL Germplasms

Gastrodia elate BL.is a perennial herb in Genus Gastrodia R.Br.Family Orchidaceae.There are 13 species in genus G.elate can be divided into 5 forms according to the different inflorescence axis.They are G.elata Bl.f.elata,G.elata Bl.f.viridis Makino,G.elata Bl.f.glauca S.Chow,G.elata Bl.f.flavida S.Chow and Song and G.elata Bl.f.alba S.Chow.According to Pharmacopoeia of the People's Republic of China,the dry tuber of G.elate can be used as medicine called TIANMA?GASTRODIAE RHIZOMA?.It is an important traditional Chinese medicine with long history..The medicine is sweet and flat,and it belongs to the liver.Its main function is to stop the phlegm,calm the liver.It can be used for children to cure convulsions,headache,dizziness,limb numbness and other symptoms.However,the differences in the morphology of the Gastrodia tubers of different germplasms is tiny,however their medicinal components are of significant difference The germplasm resources are chaotic oberviously,an efficient and accurate identification system is needed urgently..In this study,a gene library for G.elata was established based on the second generation sequencing technology,microsatellite sequences were screened;the type,abundance,length and preference of the microsatellite loci were analyzed and compared;the primer for sequenceswith abundant microsatellite were designed.PCR amplificationand polyacrylamide gel electrophoresis were carried on 80 samples from 4 populations to detect the polymorphism of the each microsatellite locus.As the results:?1?61 048 gene sequences were obtained by genomic sequencing,12 107 microsatellite loci were detected,among which the dinucleotide repeats were the most and the length variation was the largest.?2?20 primer pairs among those 60 were detected amplifiable,stable,and polymorphic after PCR and electrophoresis;the number of complex alleles per site?N_a?is ranged from 4 to 14,with an average of 8.40.The polymorphism information content?PIC?averaged 0.77.The G.elata microsatellite molecular marker developed in this study laid the foundation for carrying out the research of G.elata genetics and identification of germplasm resources.With the aim to reveal the genetic background for G.elate,to provide scientific and technical basement for its conversation,germplasm selection and identification,simple sequence repeat?SSR?molecular markers were used in this study to analyze the population genetic structure and to construct the SSR identification fingerprinting for G.elata with its150 samples collected from 15 populations of its 4 forms?G.elata Bl.f.glauca S.Chow,G.elata Bl.f.elata,G.elata Bl.f.viridis Makino?.As the results shows,?1?Seven pairs of SSR primer were successfully used in the PCR amplification,and performed abundant polymorphism,the number of alleleper primer pair?N_a?ranged from 7 to 12 with the average of 9.8571,while the polymorphism information content?PIC?ranged from 0.6852 to 0.8345with the average of 0.7839.?2?Genetic diversity analysis of all samples of G.elata showed that the genetic diversity was high?species level:A=9.8571,H=0.8479,I=2.0548;form level:A=3.0565,H=0.5319,I=0.8916?.G.elata is widely distributed in China.The late origin of Orchidaceae plants and pollination methods may be the reasons for the rich genetic diversity of G.elata.?3?AMOVA analysis revealed strong genetic differentiation was happened among both populations and the forms,and the genetic differentiation between the forms was strong(F_st=0.5557,F_ct=0.3504).Habitat fragmentation and genetic drift are the main causes of strong genetic differentiation of G.elata.Conservation strategies are put forward accordingly.?4?Cluster analysis showed that the populations of the same form clustered together firstly,4 forms were separated completely,SSR fingerprinting had a good identification effect at the form level;138 genotypes were detected from the 150 samples in total,the Simpson index?D?was 0.9923,indicating the SSR fingerprinting has good ability of identification on the individual level.The G.elata SSR primer developed in this study established a fingerprint map system for the identification of G.elata germplasm resources,which provided important technical support for the breeding of G.elata germplasm resources and the safety of clinical drug use.