Computational protocol: Coexistence of sexual individuals and genetically isolated asexual counterparts in a thrips

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Protocol publication

[…] T. tabaci were sampled from a leek crop field in Kuriyama town and Nanporo town, Hokkaido prefecture, Japan on July 30, 2009 and August 10, 2011, respectively. After the sex of each adult was determined under a microscope, genomic DNA was extracted from adult females using a modified Chelex method. Dried individual thrips were crushed in a mixture comprising 5 μl proteinase K (20 mg/ml) and 200 μl 5% Chelex solution (10 mM Tris-HCl; pH 8.0) and incubated at 55°C for more than 12 h in 1.5-ml micro centrifuge tubes. Subsequently, the mixture was boiled at 98°C for 10 min to inactivate proteinase K. Water layer nearby Chelex layer was used as a template DNA.The reproductive mode of each adult female was determined using a PCR-SSP method. The primer set comprised one primer shared by the lineages (TCOR, 5′-attgcgtaaattattcctaaaagtcca-3′) and two primers (sexual lineage-specific primer TCOS, 5′-aacagcTattctCcttcttttatctC-3′; asexual lineage-specific primer TCOC, 5′-gaacagtatatccacctttatcaacG-3′; the capital letters indicate the lineage-specific nucleotides) that amplified mtDNA of different fragment lengths for each lineage. The composition of the reaction mixture for the PCR-SSP was as follows: 10 μl reaction mixture comprised 5 pmol consensus primer and 2.5 pmol lineage-specific primers, 5 μl 2 × MightyAmp Buffer, 0.1 μl MightyAmp DNA Polymerase (TaKaRa, Otsu, Japan; 1.25 U/μl), and 0.5 μl template DNA. PCR-SSP was performed using a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA) by the following temperature cycles: initial denaturation for 2 min at 98°C, followed by 35 cycles of denaturation and annealing of 10 sec at 98°C and 1 min at 60°C, respectively, and the final extension for 1 min at 68°C. The fragment length of PCR products was detected by 1% agarose gel electrophoresis using 5 μl of 100 bp DNA Ladder (TaKaRa, Otsu, Japan) and ethidium bromide staining.After identification of the lineage, 20 randomly chosen adult females from each lineage were genotyped at nine microsatellite loci. To amplify these microsatellite loci, a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA) was used for PCR that was performed as follows: initial denaturation of 2 min at 96°C followed by 35 cycles of denaturation for 5 sec at 98°C, annealing for 30 sec at temperature specific for each primer pair, extension for 1 min at 68°C, and final extension of 1 min at 68°C. The primer sequences and annealing temperature for these microsatellite loci were described previously. Each 10 μl reaction mixture comprised 2 pmol primer, 5 μl 2 × MightyAmp Buffer, 0.1 μl MightyAmp DNA Polymerase (TaKaRa, Otsu, Japan; 1.25 U/μl), and 1 μl template DNA. One-μl PCR product was electrophoresed with 0.5 μl size standard 400 (Beckman-Coulter, Fullerton, CA) on a CEQ-8000 Genetic Analyzer (Beckman-Coulter, Fullerton, CA). Data of allele frequencies were analyzed using the program Arlequin version microsatellite analysis data were used to infer phylogenetic relationships between the females of both lineages. As a measure of pairwise genetic differences among females, we used the following allele-sharing distance: where L is the total number of loci and d(i, j),l = 0, if the individuals i and j have identical genotypes at the locus l, d(i, j),l = 0.5 (if they share a single allele), and d(i, j),l = 1.0, if they have no common allele. A neighbor-joining tree was constructed from the pairwise distance matrix using the software MEGA version 5. […]

Pipeline specifications

Software tools Arlequin, MEGA
Applications Phylogenetics, Population genetic analysis