Study On Genetic Diversity Of Meconopsis Integrifolia Using ISSR And SSR Markers

185 individuals from 16 wild populations of M.integrifolia collected from four provinces of Qinghai,Tibet,Sichuan and Yunnan.The genetic diversity,genetic structure and genetic distance were examined and analyzed by using molecular markers of Inter-Simple Sequence Repeat(ISSR)and Simple Sequence Repeat(SSR).The aim is to provide reference for the conservation and orderly development and utilization of the entire germplasm resources of M.integrifolia.This study used Popgen1.32 to calculate genetic similarity coefficient(GS),Shannon index,Nei’s index first.And genetic variation analyzed by AMOVA.Then software MEGA4.0 and MVSP was used to cluster analysis of 16 populations and 185 individuals,and to build dendrograms by UPGMA method.Moreover,MVSP was used to do principal component analysis(PCA).Finally,the mental test between the genetic distance and geographic distance between populations was tested by using TFPGA software.The main conclusions were as followed:(1)A total of 279 bands were detected by 21 primers of ISSR.On average,3.7 bands were amplified per primer,of which 77 bands were polymorphic,and bands were distributed between 150 and 2000 bp.Percentage of genetic diversity at the species level was 99.64%.The comparison result of the PPB at the population level was JZWS > DDS > CD > SJLS(=BMXS)> YS(=CS=BLS)> NMC > BYQ > YLXS > HY > DR > MDK > QJYS > BM.(2)ISSR genetic diversity results showed that,Nei’s gene diversity index(He)was 0.0520-0.2083,and Shannon’s diversity information index(I)was 0.0795-0.3134.The average number of effective alleles(Ne)was 1.6769,accounting for 84.00% of the mean number of observed alleles(Na)of 1.9964.It was indicated that ISSR was feasible as a molecular marker for analyzing the genetic diversity of M.integrifolia.Comparing genetic parameters,the genetic diversity of population BLS was highest,and the genetic diversity of population BM was the lowest.The genetic differentiation coefficient(Gst)was 0.6197,indicating that the genetic variation mainly existed between the populations,which was 61.97%.The results of molecular variance(AMOVA)also showed that the major genetic variation of M.integrifolia was found among the population(59.949%).(3)In the UPGMA clustering of different populations of Meconopsis integrifolia,using the genetic distance obtained by ISSR calculation,the population(BM)was first isolated as one cluster,and clustered with other populations as category IV.The population(DDS)and population(CD)clustered into a branch,and with population(JZWS)clustered together as category Ⅱ.The population(YS)and population(CS)clustered into a branch,and with population(SJLS)clustered together as category Ⅲ.The population(NMC)and population(MDK)clustered into a branch,population(BLS)and population(DR)clustered into a branch.And with population(BYQ),population(HY),population(BMXS),population(QJYS),population(YLXS)clustered together as category I.(4)A total of 84 bands were detected by 23 primers of SSR.On average,13.3 bands were amplified per primer,of which 278 bands were polymorphic,and bands were distributed between 100 and 220 bp.Percentage of genetic diversity at the species level was 91.67%.The comparison result of the PPB at the population level was BLS > BMXS > MDK > DR > JZWS > BYQ > SJLS > HY > DDS(=CD)> QJYS > YS > NMC > YLXS > CS > BM.(5)SSR genetic diversity results showed that,Nei’s gene diversity index(He)was 0.0226-0.1603,and Shannon’s diversity information index(I)was 0.0334-0.2436.The average number of effective alleles(Ne)was 1.5037,accounting for 78.45% of the mean number of observed alleles(Na)of 1.9167.It was indicated that SSR was feasible as a molecular marker for analyzing the genetic diversity of M.integrifolia.Comparing genetic parameters,the genetic diversity of population JZWS was highest,and the genetic diversity of population BM was the lowest.The genetic differentiation coefficient(Gst)was 0.6902,indicating that the genetic variation mainly existed between the populations,which was 69.02%.The results of molecular variance(AMOVA)also showed that the major genetic variation of M.integrifolia was found among the population(63.38669%).(6)In the UPGMA clustering of different populations of Meconopsis integrifolia,using the genetic distance obtained by SSR calculation,the population(BM)was first isolated as one cluster,and clustered with other populations as category IV.The population(BYQ)and population(YLXS)clustered into a branch,population(MDK)and population(QJYS)clustered into a branch,and with population(NMC)clustered together as category I.The population(HY)and population(BLS)clustered into a branch,and with population(DR)clustered together as category Ⅱ.The population(DDS)and population(CD)clustered into a branch,and with population(BMXS),population(SJLS),population(JZWS),population(YS),population(CS)clustered together as category Ⅲ.(7)The Nm calculated by ISSR and SSR markers were 0.3069 and 0.2244,respectively,which were all less than 1,indicating that the genetic drift plays a more important role in the genetic differentiation among the 16 populations of M.integrifolia.Using TFPGA,based on the ISSR and SSR marker,doing mental test between the genetic distance and the geographical distance.The results of the two markers showed that the P value was greater than 0.05,indicated that there was no correlation between genetic distance and geographic distance.There was no significant effect on the genetic differentiation between the two groups.