The Genetic Diversity And Differentiation Of Galeruca Daurica Populations In Inner Mongolia

Galeruca daurica (Joannis) is a new pest causing damages seriously in Inner Mongolia grasslands in recent years. By using both microsatellites and cytochrome oxidase subunit I (COI), we analyzed the genetic diversity, gene flow and genetic differentiation among eight populations of G. daurica to investigate the genetic diversity and differentiation among different geographic populations of G. daurica in Inner Mongolia. The main results are as follows:1. Four levels of five factors (Mg2+, dNTPs, primers, DNA and Taq DNA polymerase) in PCR system were selected by L16(45) orthogonal design. The primers and an optimum reaction system were screened. A suitable PCR reaction system (20μL) included:2μL 10×Buffer (Mg2+ free),1.2μL 25mmol/L Mg2+,1.6μL 2.5 mmol/L dNTP, 1.6μL 10μmol/L primers, 1μL 50ng/μL template DNA,0.2μL 5U/μL Taq DNA polymerase, and 12.4μL ddH2O. By screening,5 primer with stable augmentation were obtained:Dviz13, DbaO8, Dviz12, Mv-MS11 and Ls-A121.2. By using five microsatellite primer pairs, we analyzed the genetic diversity among eight populations of G. daurica. The number of alleles and effective number of alleles per locus ranged from 10 to 18 and 9.2796 to 16.0388, respectively. The polymorphic information content per locus ranged from 0.6760 to 0.8985 and the expected heterozygosity ranged from 0.3430 to 0.5284, demonstrating the high degree of polymorphism. Moreover, the expected heterozygosity of the eight populations ranged from 0.2216 to 0.3701, with the average of 0.2680. The genetic differentiation index (FSr) ranged from 0.1244 to 0.4116, with the average of 0.2521, and the gene flow (Nm) ranged from 0.3574 to 1.7596, with the average of 0.9622. Eight populations were clustered into three groups according to their genetic distances, and the genetic distance between populations showed a highly significant correlation with the geographical distance (r= 0.4854, P= 0.0180). The results shows that the G. daurica populations are characterized by low genetic diversity. The gene exchange seldom exits and high genetic differentiation occurs between different geographic populations. The lower migration ability of this leaf beetle and geographic obstacles might be the main reason for the low gene flow and high genetic differentiation.3. Based on the 417 bp segments of mtDNA COI gene of 197 individuals from 8 geographic populations of G. daurica in Inner Mongolia,43 variable sites were detected and 62 haplotypes were defined based on the 197 COI sequences, including two haplotypes shared by all the 8 populations and three or more haplotypes unshared by different populations. The total haplotype diversity of all populations was 0.9466, and the range of haplotype diversity of different populations was 0.8889-0.9600. The total FST andd Nm were 0.03151 and 15.37, respectively. A neutral test was not significant (Tajima’s D=-1.6300,0.10>P>0.05), indicating that there might be no population expansion in recent history. Molecular variance analysis (AMOVA) showed no obvious genetic differentiation among populations with a high proportion of the total genetic variance attributable to variation within populations (96.34%). There was no significant correlation between the genetic differentiation and geographic distance among different populations. The haplotype network showed that the haplotypes were distributed in different clades and no obvious geographic structure had been formed.