Oriental armyworm enriched microsatellite library construction and screening genetic markers

Title: Oriental armyworm enriched microsatellite library construction and screening genetic markersAuthor: Zhang GuoyanDegree-granting units: Nanjing Agricultural UniversityKeywords: Oriental armyworm;; microsatellite;; DNA extraction;; microsatellite enriched library;; mutation;; genetic markersSummary:Oriental armyworm Pseudaletia separata (Walker) is a food crop in China and other Asian countries, one of the major pest, widely distributed all over the country. Oriental armyworm has a migratory, overeating and other features, easy to outbreaks of disaster. Scientists in China have their macro-migration law, migration behavior of the mechanism, the relationship between flight and reproductive conducted in-depth study, but its population is rarely involved in molecular genetics.Rare earth magnets  This study focused on microsatellite markers Oriental armyworm separation and identification, ready for future research tool for population genetics; the same time, the genome of the species in this study features and microsatellite variation for a more in-depth analysis and optimization of the Lepidoptera adult technical methods of DNA extraction.The traditional proteinase K + SDS + phenol extraction method and CTAB methods combined selected from natural populations of adult Oriental armyworm specimens preserved in ethanol to extract high-quality, high-yield method of genomic DNA, microsatellite markers for the preparative separation DNA samples required. Comparative study showed that traditional methods of extraction with CTAB + DNA, the middle of the abdomen to take 10-20 mg tissue samples to experiment with different populations of DNA products 68.42% -95.28% pass rate, the amount of DNA extraction qualified 5.59-10.04 mg / g, significantly higher conventional proteinase K + SDS lysis and http://www.chinamagnets.biz/faq.php phenol extraction method of 7.69% -40% and 2.83-5.78 mg / g. In addition, with hot NaOH method for rapid preparation for large-scale genotyping of DNA samples results show that the tissue samples for the chest and abdomen, with hot NaOH solution for 20 min, we can extract the required micro-satellite PCR amplification reaction DNA samples; for head tissue samples, treatment with hot NaOH solution 40-60 min is more appropriate. Comparative study showed that, from the hot NaOH method of DNA samples enriched microsatellite library in the build process fails, but the two samples as a template for PCR amplification of microsatellite genetic analyzer products in the same test results.The use of biotin-5 'labeled (CA) 15, (GA) 15, (ATG) 12, (GAT) 12, (TAGA) 8 and (GTGA) 8 as a probe, using the bead enrichment to build Oriental armyworm microsatellite enriched library. After genomic DNA digested ,300-700bp fragment recovered, connectors, purification, ligation products degeneration, has been combined with magnetic beads to capture target probe fragments, target fragments elution, PCR amplification, connected pGEM-T vector and transformed into DH5α and efficient competent cells, blue-white screening and other processes, and finally build a successful cloning of the armyworm contains 8531 microsatellite enriched library. From 866 randomly selected library clones were sequenced and analyzed in the presence of microsatellite sequences, statistical results show that the average micro-satellite built library clones rates 35.8% higher than in the past on the cotton bollworm, pine caterpillars and other Lepidoptera the results of similar studies insects.Software and manual analysis with REPEATMASKER combination has been selected for this study, a group of oriental armyworm microsatellite types, types of abundance variations in depth analysis and so on, and with InSatDb database of five whole-genome sequencing insects (black Drosophila melanogaster, Anopheles gambiae, Tribolium castaneum, the Italian bee, silkworm) genome features and microsatellite variation were compared. This study has found 369 microsatellite sequences Oriental armyworm, 46 low-complexity regions, six transposable elements and a tRNA. From micro-satellite belonging to 21 different repeat motifs, to TTG / CAA motif of the micro- satellite up, accounting for 37.13% of total micro-satellite, followed by the CA / TG ​​class and CAT / ATG category, accounting for 30.89% and 15.72%. Together, these three micro-satellites accounted for 83.74% of micro-satellites. Also has a micro-satellite sequences from short ones, incomplete type (mutation) ones, AT-enriched ones, less complex and so on. These features with five full-genome sequencing of microsatellite insects are basically the same results. Not entirely based on micro-satellite repeats variation statistics, the proportion of nucleotide substitution is generally 0.6% -11.7%, 15.6% -26.1% individual; missing base percentage of 0.5% -10.6%, the proportion of 0.3% into the base - 11.3% on average at the insertion / deletion base 1-1.8, 3-6 to reach the individual bases. Further analysis found that Oriental armyworm microsatellite repeat variations still exist the following characteristics: nucleotide substitutions in the mutant an advantage, in all microsatellite loci in the proportion of 92.38% occurred, while the proportion of deletion and insertion were only micro-satellite 27.62% and 25.24%; replace + insert, replace + ratio close to the occurrence of missing, are little more than 20%, while the lack of + insert, replace, + missing + insertion is relatively low, 12% were slightly more; number two most of the micro-satellite TTG / CAA (137 个), TG / CA (114 unit) of the repeat sequence variation in conversion and transversion frequency of occurrence is not the same, the conversion to more than transversion; repeat length of 101-150bp and the sequence 151-200bp microsatellite mutation probability of a larger, more variation of the number of bases, but less than 50bp and 200bp microsatellite variation greater than the proportion and the proportion of nucleotide variation were significantly reduced; TTG / CAA microsatellite variation in class higher than TG / CA class, and variation in base ratio was significantly lower than the latter; microsatellite repeats variations near the 5 'end (a repeated sequence is divided into five sections, namely the 5' end, near the 5 'end, the middle , near the 3 'end, 3' end) the number of bases of variation, followed by the middle of the poles are relatively less common variation, and variation in the region up to near the 5 'end of the highest number of micro-satellites. In addition, various types of microsatellite loci flanking sequence comparison, only 11 found in the same class of multi-copy sequences flanking microsatellite loci involving 24 microsatellite loci, accounting for all microsatellite loci found in 6.50%. But did not find highly similar flanking microsatellites family.In the presence of microsatellite clone sequences, through sequence selection, primer design and synthesis, Touchdown PCR primaries (24 genomic DNA samples), optimized PCR optimization (24 genomic DNA samples), Genetic Analyzer re-election (48 genome DNA samples) and three screening were screened out eight polymorphic microsatellite markers (GenBank accession number FJ896055-FJ896062). for eight microsatellite loci in 48 samples of the allele distribution statistics, and use GenePop software for testing, the Bonferroni correction, are in line with Hardy - Weinberg equilibrium (Hardy-Weinberg proportions), between microsatellite loci did not detect significant phase unbalanced gametes (gametic phase disequilibrium). Statistics calculated using Cervus software, various loci have 11-31 alleles, expected heterozygosity 0.747-0.931, showing a good all loci are polymorphic, can be used as oriental armyworm population studies of genetic markers.The innovation of this study is mainly manifested in the following areas: First, the improvement of the Lepidoptera adult genomic DNA extraction methods, to make it suitable for microsatellite enriched library construction, SSR markers and genotyping needs; Second, the domestic build the first successful microsatellite enriched library Oriental armyworm, sampling a higher rate of microsatellite positive clones; third, the first genome of Oriental armyworm microsatellite sequence features and mutation analysis of the genome enriched microsatellite Lepidoptera sequence information; four, the first Oriental armyworm selected 8 microsatellite markers, to fill the Oriental armyworm microsatellite markers blank.Degree Year: 2009