Computational protocol: Genetic analysis of the Hungarian draft horse population using partial mitochondrial DNA D-loop sequencing

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Protocol publication

[…] Based on the published horse mtDNA sequence (), primers were designed for amplifying a 398-bp fragment containing the most variable segment of horse mtDNA, Forward 5′-CCCCCACATAACACCATACC-3′, Reverse 5′-AGACAGGCATCCCCCTAGAT-3′. PCR amplifications were performed in 30 µL reaction volumes. Amplification mixture was as follows: 5 µl isolated genomic DNA, 8.8 µl dNTP (25 mM)/Fermentas, 1 µl GoTaq Flexi Buffer Promega, 8.2 µl MgCl2 (25 mM) Promega, 1 µl forward and 1 µl reverse primer (10 pmol/ µl) Sigma, 5 µl dH2O. The reaction mixture was incubated at 95 °C for 10 min, followed by 35 cycles each consisting of 20 s denaturation at 95 °C, 30 s annealing at 62 °C, 30 s of extension at 70 °C and then a final 10min extension at 72 °C. Sequencing was done by the Macrogen Company (The Netherlands, Amsterdam). Sequences were assembled and truncated to a length of 222 bp (between positions 15,531 and 15,752) to maximize sample size comparisons. The correct reading of nucleotides and the comparison of sequences were done with CodonCodeAlignerV.6.0.2., whereas statistical analysis was performed with two versions of Mega (Mega6 () and Mega7.0 (), with DnaSP5.1. () and Network 5.0. (). The DnaSP5.1. software was used for calculating the total number of haplotypes, haplotype (H ± SD) and nucleotide (π ± SD) diversities. Genetic distances among different mtDNA haplotypes were calculated by the two-parameter method of Kimura (). Molecular Phylogenetic analysis was done by Maximum Likelihood method based on the Tamura-Nei model (). The initial trees for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1234)). We used Arlequin 3.5.2.2. () software for calculating pairwise FST values (and 5% significance levels) and detecting shared haplotypes among populations. Median joining networks were constructed using NETWORK version 5.0.0.1 (). Calculations were performed under the following conditions: deletions or insertions were double weighted, transition/transversion ratio = 6,5, default setting of Epsilon (0) was chosen. […]

Pipeline specifications

Software tools MEGA, DnaSP, Arlequin
Applications Phylogenetics, Population genetic analysis
Organisms Equus caballus