| The diamondback moth,(Plutella xylostella), is a major insect pest of cruciferous plants. A high number of generations per year, wide dispersal and resistance to pesticides are characteristics of this insect pest. The damage caused by it amounts to at least one-billion dollars annually. In this paper, both mtCOI markers and ISSR markers are used to analyze genetic relationship in different geographic populations of Plutella xylostella in China. The biological information will help in the development of control strategies.(1) The length of the mtCOI gene fragment amplified was704bp, and the sequences of749samples were detected. Through comparison,1-26variable sites in each population were found, among which1-11parsimony-informative sites were existed. Among the COI gene of different geographic populations of p. xylostella, haplotype diversity (Hd), the diversity sites(S), nucleotide diversity (Pi), average number of nucleotide differences (k) were0.9132,103,0.00345,2.432respectively. The value of Tajima’s D was-2.37235(P<0.01), and the test were extremely significant. It was concluded that mtDNA evolution does not followed neutral models, suggesting that the results of sequence evolution was not random genetic drift of the natural populations. The value of Fu’s Fs test was all negative, so the population of p. xylostella was likely to expand in the past.(2) In our research,749samples analysed,207haplotypes were found. Forty-eight haplotypes were shared by at least two individuals. Twenty-five populations had their unique haplotypes, which showed that there was some level of genetic difference between the geographical populations. However, the genetic diversity was low concluded from the NJ phylogentic tree. The haplotype LX1and LX2are the most common haplotypes in China.(3)Network analysis based on statistical parsimony shows that haplotype LX1in the center of the network, the shared haplotypes near the center of the network, the unique haplotypes in the outer edge of the network.(4) Based on the genomic DNA of Plutella xylostella (L.), the factors influencing ISSR system were explored with the L16,(4) orthogonal design. The optimal conditions for ISSR reaction system (20μl) were determined as follows:1.5mmol-L-1of Mg2+,2.5U of Tag DNA polymerase,0.2μmolL-1of dNTPs mixture, and1.25μmol-L-1of each primers,20ng of template DNA. The reaction program was as:initial denaturation for5.0min at94℃,35cycles of denaturation for45s at94℃, annealing for1.0min at40-61, extension for1.5min at72℃,with a final extension of10.0min at72℃.The optimal annealing temperature for ISSR-PCR reaction was proposed by gradient PCR. The clear and reproducible amplification was obtained by the established ISSR-PCR reaction system.(5) In our study, these5primers with good repetition and high polymorphism have been selected from100ISSR primers. The DNA polymorphism of8different geographic populations of Plutella xytostella are analyzed by these5primers and124bands were amplified.The average percentage of polymorphic bands was100%. PopGenl.32analysis indicated that the genetic diversity of8populations (Gst) was0.0439, show that genetic variation of among populations is4.39%and the genetic variation within populations is95.61%, showing low levels of genetic differentiation among these populations; The gene flow of8populations (Nm) was10.8822, it showed a strong gene flow and high genetic similarity between these populations. The effective number of alleles (Ne) is1.3677±0.2308, the gene diversity (Ht) is0.2482±0.0157, the information index of Shannon (1) was0.3999±0.1664. |
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