Sequencing the Complete Mitochondrial Genome of the Giant Scops-Owl Otus Gurneyi

Undergraduate #12
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Raven Reed - Texas Southern University
Co-Author(s): Amanda Jackson and Hector Miranda, Texas Southern University, Houston, TX

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The Giant Scops-Owl Otus gurneyi is another enigmatic bird, that is found only in the islands of Mindanao, Philippines. It was formerly called the Mindanao Eagle-owl and had the scientific name Mimizuku gurneyi. However, recent molecular studies based on two mitochondrial genes found that the lineage is deeply embedded within the scosp-owl Otus clade rather than with the much larger Eagle-owl Bubo (Miranda et al 1997, Miranda et al 2010). The phylogenomics of the owls Strigidae as a whole is still unresolved. Investigators around the world are currently sequencing the complete mitochondrial genome of other birds but none has worked on many rare species such as the Giant Scops-Owl. The long-term goal of this project is to use the complete mitochondrial genomes of various groups of owls to infer their phylogenomics. To sequences the whole mitochondrial genome of the Giant Scops-Owl. Primers were designed using Primer3Plus and Geneious Pro 5.5 (Biomatters, New Zealand). The published complete mitochondrial genome of Passer montanus was used as the template to design the forward and reverse primers. Polymerase chain reaction (PCR) was carried out in a total volume of 25 uL containing 10-50 ng of template DNA, 4 µL of deionized water, 4 µL 10 mM of each dNTP, and 8 µL of AmpliTaq 360 Gold Polymerase Master Mix (Life Technologies). A total of 22-27 primer pairs was designed and synthesized thru Eurofisn/Operon (Alabama). In some cases where internal primer pairs failed, two PCR strategies were adopted; 1) PCR using the forward primer and the reverse primer of the adjacent 3’-end fragment, or the reverse primer of the target fragment and the forward primer of the 5’ adjacent fragment, and 2) designing new species-specific primers using the known flanking sequences for ‘two-way primer walk’. The PCR cycles were as followed: one cycle of 10 min at 95°C, 35 cycles of 20 s at 95°C, 20 s at 60°C, and 50 s at 72°C. The process was completed with a final elongation at 72°C for 10 min. These parameters were adjusted to optimize PCR product quality. All amplifications were performed on Veriti Thermal Cycler (Life Technologies). Sequences were aligned and assembled, and annotated using a sleuth of softwares such as CLUSTALW, finchTV, and Geneious Pro 5.5. Sequences of the whole genome will be submitted to Genbank and will be published in an appropriate journal once completed.

Funder Acknowledgement(s): National Science Foundation; Texas Southern University, Summer Undergraduate Research Program

Faculty Advisor: Hector Miranda, mirandahc@tsu.edu

Role: I had three duties in this research. My first duty was to carry out PCR on half of the genome. My second duty was to make agarose gel when needed and stain it. My last duty was to run gel electrophoresis on the DNA and upload images onto the computer.