Computational protocol: Allopatric speciation in ticks: genetic and reproductive divergence between geographic strains of Rhipicephalus (Boophilus) microplus

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Protocol publication

[…] Specimens of sixteen strains of R. microplus from America, Africa, Asia and Oceania and one strain of each R. annulatus and R. decoloratus from Africa were used for DNA extraction and sequencing of mitochondrial 12S and 16S rDNA (Table ). One to three specimens from each strain were analyzed. DNA was extracted from alcohol-preserved specimens and polymerase chain reaction (PCR) amplification of a fragment of the mitochondrial 16S rDNA was conducted as described []. The PCR conditions for 12S rDNA amplification were as described []. Amplified PCR products were purified using Wizard PCR Preps DNA Purification System (Promega Corporation, Madison, Wisconsin, USA). The purified DNA was directly sequenced at the IMyZA (Instituto de Microbiología y Zoología Agrícola, INTA, Castelar, Buenos Aires, Argentina). Both DNA strands were sequenced and assembled using BioEdit 7.05.3 software [].For phylogenetic analysis, the following tick mitochondrial 16S and 12S rDNA sequences available in the Genbank were also included: [...] R. annulatus (U95866), R. annulatus, Italy (AM410573), R. annulatus, Israel (AF133058), R. microplus, Nepal (AF150042), R. microplus, Australia (AF031847), R. decoloratus, Zimbabwe (AF150044), R. decoloratus, Kenya (AF031846), R. kohlsi, Jordan (AF150043), R. kohlsi, origin unknown (AY008686), Dermacentor albipictus, USA (AF150041), D. andersoni, USA (AF150040), D. reticulatus, France (AF150038), Haemaphysalis leachi, Zimbabwe (AF150035), H. punctata, Switzerland (AF150032), Hyalomma dromedarii, Morocco (AF150036), H. truncatum, Zimbabwe (AF150031), Rhipicephalus appendiculatus, Uganda (AF150028), R. bursa, Spain (AF150053), R. evertsi evertsi, Zimbabwe (AF150052), R. pusillus, France (AF150022), R. sanguineus, France (AF150020), and R. turanicus, France (AF150018).Multiple sequence alignments were done using Clustal W []. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4 []. Phylogenetic relationships between sequences were assessed by neighbored-joining (NJ) method []. The NJ topologies were examined using Tamura-Nei distances [] and relative support for the internal nodes was tested by bootstrapping over 1,000 replications []. Tamura-Nei distance measures were used because this model corrects for multiple hits, taking into account the differences in substitution rate between nucleotides and the inequality of nucleotide frequencies. It distinguishes between transitional substitution rates between purines and transversional substitution rates between pyrymidines. All positions containing alignment gaps were eliminated only in pairwise sequence comparisons. Ticks species of the genus Haemaphysalis were treated as out-group. [...] Microsatellite polymorphism was analyzed in tick strains from Argentina, Australia, Mozambique, India and New Caledonia (Table ). For Argentina, Australia and Mozambique strains, femalestrain 1 × malestrain 2 and femalestrain 2 × malestrain 1 homologous and heterologous crosses were used. For each cross, egg masses from two separate crosses were used for genotyping. Tick DNA was obtained from egg batches (ARG, AUS and MOZ strains), pooled larvae (New Caledonia strain) or pooled whole ticks (India and Indonesia strains). Tick tissues were homogenized in liquid N or with a 1 ml tuberculin syringe with a 25-G needle to extract DNA with Tri Reagent (Sigma, St. Louis, MO, USA) following manufacturer's recommendations. The purified DNA was dissolved in distilled water and the concentration determined using the NanoDrop 1000 (Thermo Fisher Scientific, Wilmington, DE, USA).Polymorphism was analyzed at microsatellite loci BmA12 (Genbank accession number DQ001904), BmA06 (DQ001905), BmB12 (DQ001906), BmC03 (DQ001907), BmC07 (DQ001909) and BmD12 (DQ001911) using oligonucleotide primers and PCR cycling conditions described []. The PCR was done with labeled forward primers (BmA12 and BmD12, 6-FAM; BmA06 and BmC07, NED; BmB12, VIC; BmC03, PET) in a 50-μl volume (1.5 mM MgSO4, 1X avian myeloblastosis virus (AMV) RT/Thermus flavus (Tfl) reaction buffer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 5 u Tfl DNA polymerase, 0.2 μM of each oligonucleotide primer) employing the Access RT-PCR system (Promega, Madison, WI, USA). Reactions were performed in an automated DNA thermal cycler (Techne model TC-512, Cambridge, England, UK). The reaction was terminated after a final extension at 68°C for 5 min. Control reactions were done using the same procedures, but without DNA added to check contamination of the PCR reaction. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 Kb Plus DNA Ladder, Promega). Fragments were separated using an ABI 3730 automated DNA sequencer (Applied Biosystems, Inc. Foster City, CA, USA) and sized relative to a ROX-labeled internal size standard (GeneScan-500LIZ, Applera, Norwalk, CT, USA). The data were analyzed using program Peak Scanner (Applied Biosystems).For statistical analysis, a binary matrix was constructed by scoring the different alleles from each microsatellite locus as presence (1) and absence (0) of the PCR bands for each cross. Based on the binary matrix, similarity matrixes were calculated using Jaccard's and Dice's coefficients [,]. The similarity matrix was subjected to Sequential Agglomerative Hieratical Nested Clustering (SAHN) and dendograms were constructed employing the Unweighted Pair Group Method of Arithmetic Averages (UPGMA) of Sneath and Sokal [] to group the progenies into clusters. All the analyses were performed using statistical software package NTSYSpc version 2.01e []. […]

Pipeline specifications

Software tools BioEdit, Clustal W, MEGA, NTSYSpc
Applications Phylogenetics, Population genetic analysis
Organisms Bos taurus, Rhipicephalus microplus, Rhipicephalus australis
Diseases Infertility