SSR Fingerprinting Of Aconitum Carmichaeli Debx.and Its Relatives

Aconitum carmichaeli Debx.is a perennial herb in genus Aconitum L.family Ranunculaceae.There are about 350 species in the world in the genus,among them about200 species distributed in China,76 of them can be used as medicine.A.carmichaeli is cognate originate and morphologically similar to their relatives in genus Aconitum.Because many other plantes in genus Aconitum can also be used as the substitute of A.carmichaeli,which brings many adulterants for this medicinal herb,and resultes in unsafe and ineffective treatment unavoidably.What’s more,the chemical contiment of different A.carmichaeli germplasm differs from each other because their different environments,which bring great challenge in cultivate and improved varirety breeding.However,traditional identification which based on morphological or anatomic characteristics can’t distinguish A.carmichaeli effectively from their relatives in the same genus.Therefore,it is urgent to establish a more effective method for A.carmichaeli germplasm and adulterants.In this study,the development of novel microsatellite markers and genetic diversity study A.carmichaeli were carried out with the aims to set up an accurate identification for its germplasm and adulterants,chich also provide scientific guarantee for high-quality germplasm conservation,development and follow-up research resources for A.carmichaeli.This study was established by extracting DNA genomic library for A.carmichaeli.High-throughput sequencing using Illumina sequencing technology A.carmichaeli,get a lot of short sequences of microsatellites 100bp fragment by fragment screening microsatellite to established microsatellite library A.carmichaeli.Using Illumina second-generation high-throughput sequencing total of 38,942,660 125bp pairing sequence for quality trim pieces and then assembled to give 200,386 splice sequence.After filtration,screening,and ultimately get more than 299 bp fragment sequence 12,095 pieces of microsatellite library.Select the repeat segment longer fragments A.carmichaeli microsatellite library to microsatellite primer design,received a total of 55 microsatellite primers.Primer pairs were designed for microsatellite fragments（loci）,PCR amplification and electrophoresis were carried out for 80 individuals from 4 wild populations to detected the genetic polymorphism and genetic diversity of A.carmichaeli.After testing screened14 pairs of strips clear,rich polymorphic for microsatellite primers A.carmichaeli,14 loci with high polymorphism average number of alleles within the examined population（A=4.85）,the average observed heterozygosity（H_o=0.613）,the average expected heterozygosity（H_e=0.583）,the average polymorphism information content（PIC=0.52）.Four related species of A.carmichaeli were employed to investigated the cross-taxa transferability of the microsatellite markers.Found of 11 microsatellite loci were successfully amplified,accounting for 78.57%screening of microsatellite primers,and most of them were proved amplifiable in the 4 related species of A.carmichaeli.Heritage Diversity analyzed using 4 populations of A.carmichaeli,which of showed high genetic diversity at the species level,the average number of alleles（A=3.25）were selected from A.carmichaeli populations,the average expected heterozygosity（H_e=0.493）,the average Shannon’s information index（I=0.851）,the average Nei’s genetic diversity index（h=0.4927）;gene flow between populations was limited（N_m=1.43）,genetic differentiation(F_st=0.149)was also revealed.The results show,The novel developed microsatellite markers in this study were of high polymorphism and good cross-taxa transferability.The genetic diversity of A.carmichaeli was high at species level.In this study,SSR markers were employed to analyses 51 germplasm of A.carmichaeli.sampled from 17 populations and its 65 relatives from 13 species in the same genus.Genetic diversity,genetic differentiation and phylogenetic relationship of the germplasm and species were revealed.As the results,11 SSR primer pairs used in this study are stable and polymorphic,109 alleles were screened out totally,with the average of 9.91 per pair.A.carmichaeli germplasms and its relatives performed high polymorphism（A=9.9091,I=1.5262,h=0.6905）,pronounced genetic differentiation(G_st=0.437)and limited gene flow among species（N_m=0.4518）were detected.Cluster analysis revealed that most of the A.carmichaeli.germplasm sampled from the same location clustered together formed 17 small clades,and then formed a clade;as for those relatives,samples of same species clustered together and form different clades individually;14 species of genus Aconitum formed 5 big clades,which is consistent with the result of traditional taxonomy.6 primer pairs（Tchin03、Tchin04 Tchin20、Tchin26、Tchin29、Tchin30）,which performed higher polymorphic and more abundance genotype were chosen to construct the fingerprinting for A.carmichaeli germplasms and its relatives.The study provides reliable molecule basis for variety classification and identification of A.carmichaeli and its relative in the same genus.