Analysis Of Genetic Diversity And DNA Fingerprinting On Bletilla Striata Rchb.f.

Bletilla striata Rchb.is a perennial herb in genus Bletilla Rchb.f.,family Orchidaceae.There are 4 species in genus Bletilla in China,including B.striata,B.ochracea Schltr.,B.formosana Schltr.and B.sinensis Schltr.The Rhizome of B.striata can be used as a kind of medicine which has high value in the clinical application.Due to personally destructive excavation of wild B.striata and low germination rate of seed,the resource of wild B.striata rapidly decline in recently years.B.striata has been enlisted as a protectional plant at the second class in China that need urgent protection.As an endangered plant with important medicinal value,B.striata has many adulterants,while its wild resources was exhausted.In this study,the genomic sequence library and microsatellite library of B.striata was set up based on Illumina sequencing.SSR primers were designed and carried out to test their polymorphism with 80 individual from 4 wild populations.Four related species of B.striata were uesed to investigate their cross-taxa transferability.As the results,microsatellite sequences were abundance in the genome of B.striata,17 841 loci were detected in total.35 pair of primers were designed,20 of them performed high polymorphism after PCR and polyacrylamide gelelectrophoresis,the alleles ranged from 2 to 6 with average of 3.85 per locus.The cross-taxa transferability was also examined in 4 related species,most of 20 loci were proved amplifiable in these species.To study the population genetics of B.striata,20 pairs of selected primers were used to test 320 individuals sampled from 16 main distribution region.As the results showed:(1)The polymorphism of primers was high and lead to 196 stripes,the alleles varied from 5 to 13 per primer average per primer has 9.8 alleles.The genetic diversity of B.striata was high at species level(He = 0.8198,I = 1.9271,h = 0.8185),this may be resulted by its wide distribution rang,insect pollination and late origin;(2)AMOVA analysis showed the genetic differentiation of B.striata was strong(Fst = 0.47),genetic drift should be mainly responsible for it;Clusting analysis showed that 16 populations were divided into 4 major groups.There was a certain genetic differentiation among the 4 groups.Populations with nearer geographic distance performed a tighter genetic relationship.The Mantel Test showed genatic distance and geographical distance were remarkable dependency(r = 0.3312,p = 0.01),which indicated that the distribution pattern of B.striata fit to the Isolation-by-Distance model.Geographical isolation,habitat fragments and genetic drift are the major factors leading to strong genetic differentiation in B.striata.Furthermore,the genetic structure and evolutionary potential were revealed by analysis of genetic diversity,and the protection strategy provides a theoretical basis for the protection of B.striata.In this study,48 individuals of B.striata sampled from 16 populations,and 4 individuals from its each 3 relatives,including B.ochracea,B.formosana and B.sinensis,were analysed with SSR markers aiming to reveal their genetic diversity and relationship,to construct their SSR Fingerprinting.The results show that:(1)20 pairs of primer performed high polymorphism,the alleles ranged from 4 to 9,in summary of 127 and average of 6.35 per primer;all of 20 pairs of primer were proved amplifiable in the 4 species;(2)7 pairs of primers,BJSSR01,BJSSR14,BJSSR15,BJSSR16,BJSSR18,BJSSR19 and BJSSR22,that performed higher polymorphism,higher polymorphism information content(PIC)and higher polymorphism information content,were used to build the SSR fingerprinting of B.striata and its adulterants.(3)The study revealed that the genetic diversity of B.striata was high(Na = 6.35,I = 1.4291,h = 0.7068),genetic differentiation was prominent among species(Fst = 0.44),the gene flow among species was limited(Nm = 0.4753).(4)4 major groups were generated from 60 accessions tested by UPGMA clustering analyses,samples from same species were cluster together firstly,which is matched with their morphological taxonomy.Significant differences was reveled not only among the species,but also among the germplasms from different regions,those with shorter geographic distance performed a tighter genetic relationship.The selection and protection of the excellent varieties of B.striata were also discussed.The SSR fingerprinting of Bletilla was established to provide technical support for the selection of germplasms and the safety of medication.