| Gypsy moth, Lymantria dispar (Linnaeus), is a worldwide forest defoliator, usually divided into two groups:European gypsy moth and Asian gypsy moth. In 2009, The North American Plant Protection Organization (NAPPO) has officially approved NO.33 Regional Standards for Phytosanitary Measures, the regional standard requires special quarantine measures for 30 special-focused ports from epidemic area of Asian gypsy moth included China which brings a great inconvenience for export trade.However, the major ports of export trade between China and the United States locate in low risk area with false report on Asian gypsy moth occurrence because of morphological similarity between L. dispar asiatica and its related species. Basiclly, the infection risk of Asian gypsy moth should exclude the risk of its related species. Therefore, study on genetic differences and rapid detection technology of Asian gypsy moth and its related species would be essential for resolving the trade barrier on China.Asian gypsy moth and two related species:L. monacha and L. xylina were used in this study. Genetic diversity was calculated with ISSR technique and mitochondrial DNA (mtDNA) gene Coxl sequences. Four mtDNA sequences were used in rapid identification. The results were as follows:(1) The reaction conditions and reaction program of ISSR-PCR were optimized, and the optimal reaction conditions for three related species as follows:a 20 μl PCR reaction mixture contained final concentrations of 2 μl 10×PCR buffer,0.2 mmol/L dNTP,1.75 mmol/L MgCl2,0.75 U TaqDNA polymerase,10 ng genomic DNA template,2.4 μmol/L primer, and pure water for each reaction.7 primers were screened from 106 conserved ISSR primers which produced reproductive bands.65 polymorphic loci were obtained across 110 individual samples, the proportion of polymorphic bands was 78.46%. The genetic diversity and the genetic distance was calculated by POPGENE32 software. Clustering analysis (using an unweighted pair-group method with arithmetic mean and multidimensional scaling) showed that geographic populations of the same species were clustered into the same group, and five geographic populations of L. dispar asiatica were divided into two groups, populations from Inner Mongolia and Heilongjiang province were clustered into the one group, while populations from Jilin and Liaoning province were clustered initially and then clustered with Guizhou population. L. monacha was clustered initial with L. xylina, and finally L. dispar asiatica was clustered with L. xylina and L. monacha.(2) Mitochondrial DNA was analyzed between Asian gypsy moth and its related species.20 conserved primer pairs that suitable for Lepidoptera were screened initially from 70 conserved primer pairs, and the sequences from Asian gypsy moth and related species were obtained with 4 conserved primer pairs. Based on these sequences,10 specific primer pairs were designed and screened, which 3 specific primer pairs were designed for L. dispar asiatica,4 specific primer pairs were designed for L. monacha, and 3 specific primer pairs were designed for L. xylina. The optimal annealing temperature of 10 specific primers were obtained. The minimum detection limits were tested by six DNA concentration gradient ranged from 50 ng to 500 fg, the result showed that primer pairs GM-3, NM-1 and nm-3 could detect minimum 50 pg target DNA, primer pairs GM-1, GM-2, CM-2, CM-3 could detect minimum 500 pg target DNA, and other primer pairs could detect 5 ng DNA. Sequence characteristics were analyzed based on Coxl gene, the genetic distance within and among species was calculated by Tamura 3-parameter model, the mean genetic distance within species for three related species was 0.004, revealed the low genetic differences within species. The mean genetic distance among species for three related species was between 0.090-0.136. Phylogenetic tree was constructed by neighbor-join and UPGMA method respectively based on Coxl gene sequence, the basic structure of two phylogenetic trees were the same. |
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