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[…] for 3 min, 33 cycles of 95°C for 45 sec, 59°C for 1 min 15 sec, 72°C for 2 min, and final extension at 72°C for 10 min. For the COI portion, the initial denaturation was set at 95°C for 3 min, followed by 33 cycles of 95°C for 45 sec, 50°C for 1 min 15 sec, 72°C for 2 min, and final extension at 72°C for 10 min. The amplified products were loaded onto a 1.5% agarose gel and visualized using GelRed™ (Biotium, San Francisco, California, United States.). PCR products were purified using magnetic beads and sequenced in both directions with the ABI 3730 capillary sequencer (Applied Biosystems) in the DNA Laboratory (School of Life Sciences) at Arizona State University., Contigs were assembled in Sequencher 5.1 (Genes code corp., Ann Arbor, MI). All sequences were checked and edited manually, and chromatograms were inspected to confirm base calling and to identify recombination sites. Consensus DNA sequences were then aligned using ClustalW (Thompson et al. ) including three out-group taxa: H. columbus sequences obtained from this study and sequences of Rotylenchus robustus (JX015440) and Rotylenchus paravitis (JX015415) from GenBank. The original alignment for ITS consisted of 1050 bp, but several indels were detected. Therefore, divergent and ambiguously aligned positions were removed and conserved blocks selected using the software Gblocks v0.91b (Castresana ) with default values. The resulting dataset comprised 550 bp of the ITS1 portion of the gene. For the mitochondrial region, the alignment consisted of 347 bp. The new generated haplotypes for both genes were deposited in GenBank (Table)., For the COI marker, the aligned sequences were well defined and chromatograms had no double peaks, ambiguous positions or indels. Nonetheless, we tested for the occurrence of stop codons that could denote the presence of nuclear copies of mitochondrial-derived genes (numts) or COI pseudogenes (Zhang and Hewitt ; Song et al. ; Moulton et al. ). Numts are copies of mitochondrial genes moved to the nuclear genome that become nonfunc […]

Pipeline specifications

Software tools Sequencher, Clustal W, Gblocks