Eggshell membrane for DNA sexing of the endangered Maleo (Macrocephalon maleo)
Here we present the first demonstration of the use of eggshell membrane for research on endangered Maleo (Macrocephalon maleo). We used 24 post-hatched eggshell membranes collected from two different sites, Tambun and Tanjung Binerean, in North Sulawesi, 12 samples in each. Two different DNA extraction methods: alkaline lysis method and gSYNCTM DNA Extraction Kit were applied. To determine the sex of Maleo, we utilized PCR-based DN A sexing using CHD genes, with the primer set 2550F/2718R. We successfully extracted all samples; mean sample concentration was 267.5 ng/µl (range 47–510.5 ng/µl) and samples were of high purity (A 260/280 ratio 1.85±0.03). All samples were used to successfully identified sexes, 9 females and 15 males. Our research clearly illustrates that eggshell membranes can be used for DNA sexing and open the possibility to build noninvasive DNA collections over large spatial scales for population study of endangered birds. The data consist of A. Electrophoresis photos of (1). DNA extraction of Maleo from eggshell membrane, collected from Tambun and Tanjung Binerean, North Sulawesi, from 4th April until 1st May 2018. We used gSYNCTM DNA Extraction Kit (Genaid) followed the provided user manual with little modifications to isolate the DNA frm the eggshell membrane. The eluded DNA (1 µl) was quantified using NanoVue Plus™ (Biochrom, Harvard Bioscience, Inc), at A260 nm. The 260/280 nm absorbance ratio was measured to give an indication of purity of the DNA. (2). PCR products of molecular sexing, using lysate and extracted DNA. We applied PCR based DNA sexing by using CHD genes, with the primer set 2550F/2718R (28). PCR used a 10 µl total volume containing template DNA (genomic DNA or lysate), 1.2 µl sterile dH2O, 5 µl 2x PCR buffer KOD FX Neo, 2 µl dNTPs (2 mM), (TOYOBO Co. Ltd.), 0.3 µl Primer 2550F (10 µM; 5'-GTT ACT GAT TCG TCT ACG AGA-3'), and 0.3 µl Primer 2718R (10 µM; 5'-ATT GAA ATG ATC CAG TGC TTG-3', (28)), and 0.2 U KOD polymerase enzyme. PCR was carried out in a Veriti™ 96-well thermal cycler (Applied Biosystems™). For genomic DNA templates, the following profile was used: 1 cycle at 94℃ for 2 min followed by 35 cycles of 98℃ for 10 sec, 53℃ for 30 sec and 68℃ for 45 sec;, and a final extension at 68℃ for 7 minutes. For lysate as DNA template, the PCR profiles was the same for DNA genome, except that it was run for more cycles (40x). B. Sequences of CHD-W and CHD-Z genes of Maleo The electrophoresis gels were cut on upper and lower bands for female sample and single band for male sample, then purified for sequencing. The sequence reactions were carried for both direction in sequencing services laboratory provided by 1st BASE Laboratories (Apical Scientific Sdn Bhd), Malaysia.