| The oriental armyworm Pseudaletia separata(Walker) is one of the most important agricultural pests in China and other Asian countries, and occurs throughout China except for Xinjiang and Tibet provinces. It has the habits of migration and gluttony, frequently leading to a large-area outbreak. Great efforts have been made by Chinese scientists in the study of its migration regularity, migratory behavior mechanism and the relationship between flight and reproduction, however molecular genetics of oriental armyworm was seldom involved. In this work some polymorphic microsatellite loci of this Lepidopteran insect were characterized which can be used as markers for genetic study. we have firstly construct six microsatellite-enriched libraries, then screen microsatellite markers. Furthermore, the extraction methods of genomic DNA from Lepidoptera adults were optimized, and the features and variations of microsatellite sequences were analyzed.CTAB-based method was combined with routine phenol extraction (called "CTAB-combined method" below) to obtain high-quality and high-yield DNA from various populations of ethanol-preserved adult oriental armyworms collected in the field. 10-20 mg tissue is appropriate to extract DNA in a 2 ml microcentrifuge tube, with a qualified DNA products proportion of 68.42%-95.28% and a DNA yield of 5.59-10.04 mg/g, significantly greater than 7.69%-40% and 2.83-5.78 mg/g of DNA products prepared with a route method (tissue digestion with SDS and proteinase K, DNA purification with phenol:chloroform:isoamyl alcohol (25:24:1) and DNA precipitation with alcohol). Simultaneously, hot NaOH solutions were successfully used to isolate genomic DNA which is suitable for temples in PCRs when abdominal or thoracic tissue was heated for 20 min, or cephalic tissue was heated for 40 min. It was demonstrated that two kinds of DNA samples were both good enough for microsatellite genotyping, but DNA preparations extracted with NaOH-based approach failed in microsatellite isolation. Six microsatellite-enriched libraries were constructed with a magnetic-bead enrichment method, in which 5'biotin-labelled (CA)15, (GA)15, (ATG)12, (GAT)12, (TAGA)8 and (GTGA)8 were used as probes. This involved a series of successive steps, i.e.enzyme restriction of genomic DNA, the recovery of 300-700bp fragments, adapter ligation, purification and denaturation of ligation products, capture of target fragments with magnetic beads combined with probes, elution of target fragments, PCR amplification, vector ligation and transformation into DH5a competent cells, blue-white screening. Finally six microsatellite-enriched libraries were achieved containing 8531 clones. In 866 clones randomly sampled from libraries and sequenced, microsatellite-positive clones account for 35.8%, greater than results of similar research in cotton bollworm Heliothis armigera and masson pine moth Dendrolimus punctatus.369 microsatellites were found from 866 sequenced clones by Repeatmasker software and manual analysis. In addition,46 low complexity regions,6 mobile elements and 1 tRNA were recognized from them. These microsatellites were divided into 21 types of repeat motif, of which TTG/CAA motif is the most common with a proportion of 37.13%, secondly CA/TG (30.89%), thirdly CAT/ATG (15.72%), only 16.26% from other types. Among all microsatellites, short (repeat sequence length<50bp) type or imperfect (existence of mutations among repeat sequences) type or AT-rich type is in the majority, and the compound type is small in number. Statistical results of imperfect microsatellites show that the rate of base substitution, base deletion and base insertion are 0.6%-26.1%, 0.5%-10.6% and 0.3%-11.3%, respectively.Further analysis showed that mutations in repeat sequences of oriental armyworm genome have other characteristics as follow:base substitution holds overwhelming superiority in imperfect microsatellites with a proportion of 92.38%, microsatellites containing deletion or insertion only account for 27.62% and 25.24%, respectively; the proportions of microsatellites containing substitution+insertion and substitution+deletion are both a little more 20%, deletion+insertion and substitution+deletion+insertion types have similar ratios, a little more 12%; the occurance rate of transition is greater than transversion in two most abundant types in this study; microsatellites with 101-150 bp and 151-200bp repeat sequences possess greater mutation rate and more mutational bases than those with<50 bp and> 200 bp repeats; compared with TG/CA type, TTG/CAA type has more imperfect members, but a less percentage of mutational bases; in a mutation-containing microsatellite repeat sequence, mutations mainly occur in 5'proximal sequences, secondly in middle segment, least in two extremes. But no microsatellite family was found in comparison of all microsatellites, only 11 types of multicopy microsatellites were discovered involving 24 (0.6%) microsatellite loci. These features above are mostly consistent with that of 5 species which have been whole-genome sequenced.Among DNA fragments containing microsatellites,8 polymorphic microsatellite loci (GenBank Acc. no. FJ896055-FJ896062) were finally identified after three rounds of screening:primary screening of 24 DNA samples with touchdown PCRs, optimization PCRs of 24 DNA samples and the third-round selection on ABI Prism 3100 genetic analyzer. Genotypic data of 48 adult samples were analysed with GenePop 4.0. No significant deviation from Hardy-Weinberg proportions was detected after Bonferroni correction, and exact tests for gametic disequilibrium between loci were all nonsignificant. The number of alleles per locus ranged from 11 to 31, and the expected heterozygosity ranged from 0.747 to 0.931 (Cervus version 3.0.3). The availability of these 8 informative loci for P. separata will provide useful markers for studies of population genetics.The major innovations of this study were as follows:the extraction methods of genomic DNA were improved from Lepidoptera adults for microsatellite marker isolation, characterization and large-scale genotyping.For the first time 6 microsatellite-enriched libraries of oriental armyworm were constructed which were of a higher average rate of microsatellite-positive clones.The situations of motif, abundance and mutation of microsatellite loci of oriental armyworm were first analyzed enriching the information of Lepidoptera microsatellites.8 polymorphic microsatellite markers were identified, filling a gap in microsatellite genetic markers of oriental armyworm.
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