Computational protocol: Wild Gazelles of the Southern Levant: Genetic Profiling Defines New Conservation Priorities

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Protocol publication

[…] For each sample three mitochondrial regions were amplified: 12s rRNA (167bp) [], control region (CR) (328bp) (F: 3′-GCCATAGCCCCACTATCAA CAC-5′, R: 3′-TTTTGACTTAAATGTGCTATGTACG-5′) and a region of the Cytochrome B gene together with tRNA-Thy and tRNA-Pro (CytB) (790bp) (F: 3′- ATGAGGACAAATATCAT TTTGAGG-5′, R: 3′-GTTTAAGTAGAATTTCAGCTTTGGGT-5′). The latter two primer sets were designed using the online primer design software Primer3Plus []. PCR amplifications were conducted using the high-fidelity AmpliTaq Gold DNA Polymerase (Applied Biosystems) to minimize polymerase errors. Reactions had a final volume of 25μl with 1.25 units Taq-Gold, 3.5mM MgCl2, 0.3mM dNTPs, 0.4μM of each primer and 1x buffer. Touchdown PCR cycling conditions were as follows: initial denaturation at 95°C for 10 min followed by a total of 35 cycles of 15 sec at 95°C, 30 sec annealing for 3 cycles each at 60°C, 58°C, 56°C, 54°C, 52°C and 50°C, and 17 cycles at 48°C, and elongation at 72°C for 45 sec; after the 35 cycles there was a final extension step of 10 min at 72°C.PCR products were visually examined on a 1.5% agarose gel. PCR products of the expected size were purified using Exonuclease I—shrimp alkaline phosphatase (HDV Pharmacia) prior to Sanger sequencing. Sequencing reactions were performed on an ABI PRISM 3730xl DNA Analyzer at the Center for Genomic Technologies at the Hebrew University of Jerusalem. Each sample was Sanger sequenced for the sense and the anti-sense strands of each marker and a consensus sequence was determined. CR and Cytb sequences have been submitted to GenBank (accession numbers KM523344-KM523547), 12S sequences are available as supporting information (). [...] Sequence chromatograms were visually inspected using Sequencher 5.0 software (Genecodes, USA) for ambiguities and errors. Final consensus sequences were aligned using Geneious Pro 5.6.6 (BioMatters, New Zealand). Published sequences of gazelles from localities outside of Israel were obtained from GenBank, including those published by [,,,]. To assess the level of population differentiation, nucleotide diversity (π) and haplotype diversity (Hd) were calculated for the CR and Cytb fragments using DNASP v5 []. AMOVA and Fst calculations were executed in Arlequin 3.5 []. Sequences of the 12S fragment were primarily used for validation of species identification (mainly of the road kills), as gazelles are known to harbor intraspecific morphological differences. Due to the low variation in the 12S fragment it was not used in population differentiation analyses. […]

Pipeline specifications

Software tools Primer3, Sequencher, Geneious, DnaSP, Arlequin
Applications Population genetic analysis, qPCR
Organisms Gazella gazella, Gazella dorcas, Homo sapiens