Computational protocol: DNA Barcodes of Arabian Partridge and Philby’s Rock Partridge: Implications for Phylogeny and Species Identification

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Protocol publication

[…] The blood samples were collected from 3 specimens of Arabian partridge and 2 specimens of Philby’s rock partridge. The taxonomic classification of these birds is as follows: Kingdom-Animalia, Phylum-Chordata, Class-Aves, Order-Galliformes, Family-Phasianidae, Genus-Alectoris, Species- Alectoris melanocephala (Arabian partridge) and Alectoris philbyi (Philby’s rock partridge). All these 5 birds belonged to the captive breeding program of the National Wildlife Research Center (NWRC) at Taif, Saudi Arabia. These birds were brought to NWRC from the local animal market so their primary origin is unknown. The DNA was extracted from the blood samples using DNeasy Blood and Tissue Kit (Qiagen GmbH, Germany) according to manufacturer’s instructions. The extracted DNA was finally dissolved in 200 μl of elution buffer and stored at −20 °C.COI sequences were amplified using the primer pair of BirdF1 and BirdR1 and FideliTaq PCR master mix (GE Healthcare) in a reaction volume of 30 μl. The PCR conditions included a denaturation step (1 min at 94 °C) followed by six cycles of 1 min at 94 °C, 1.5 min at 45 °C, and 1.5 min at 72 °C, followed in turn by 35 cycles of 1 min at 94 °C, 1.5 min at 55 °C, and 1.5 min at 72 °C, and a final extension for 5 min at 72 °C. The PCR products were electrophorsed on a 1% agarose gel stained with ethidium bromide. The PCR products were purified using MicroSpin S300 columns (GE Healthcare) before being sequenced using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) on 3130XL genetic analyzer (Applied Biosystems). For each sample, two sets of sequencing reactions were performed using the forward and reverse primers for high accuracy. All the nucleotide sequences obtained in this study have been deposited in the GenBank with the following accession numbers: Arabian partridge specimens 1 to 3 (HQ168027 to HQ168029) and Philby’s rock partridge specimens 1 and 2 (HQ168030 and HQ168031).For comparative evaluation of barcode sequences of this study with previously published sequence data of other species form Alectoris genus, we searched the GenBank nucleotide database and found only 6 barcode records of a single species, Alectoris chukar. Of these, we omitted the short sequences of 2 records (606 and 679 bp) and downloaded 4 sequences (Accessions: GQ481313 to GQ481316) with 694 nucleotides.We also compared the COI-based phylogenetic inference with another suitable mitochondrial gene, control region (CR), which often evolves faster than rest of the mitochondrial genome and is highly variable in birds. This variability has led to the expanding usage of CR sequences to examine questions ranging from population structure to phylogenetic relationship. We downloaded 9 records (3 for each species) of mitochondrial control region (CR) genes of Alectoris melanocephala (GenBank Accessions: AJ222734–AJ222736, all 1155 bp), Alectoris philbyi (AJ005574, AJ222737, AJ222738, all 1153 bp) and Alectoris chukar (FM203234, 1154 bp; FM203235, 1152 bp; FM203236, 1154 bp).The sequences were aligned by ClustalW and the alignment file was saved in appropriate formats (MEGA and PHYLIP). The aligned sequence data were subjected to unweighted pair group method with arithmetic mean (UPGMA) and maximum likelihood (ML) methods for looking into phylogenic inference. The UPGMA analysis was performed using MEGA4 software and the bootstrap consensus trees inferred from 1000 replicates were taken to represent the evolutionary history of the taxa analyzed., The software, Tree Puzzle was used for ML analysis and the resulting phylogenetic trees were viewed by TreeView software. […]

Pipeline specifications

Software tools Clustal W, MEGA, PHYLIP, TREE-PUZZLE, TreeViewX
Application Phylogenetics
Organisms Alectoris chukar