Eggshell membrane for DNA sexing of the endangered Maleo ( Macrocephalon maleo).

Background: Noninvasive DNA sampling has been applied across many avian genetic studies for a variety of purposes including conservation and management of endangered birds. However, its application in megapodes is still lacking. The previous genetic studies on megapodes used either blood or fresh tissue. Here we present the first demonstration of the use of eggshell membrane for research on endangered Maleo ( Macrocephalon maleo).  Methods: 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 gSYNC TM DNA Extraction Kit were applied.  To determine the sex of Maleo, we utilized PCR-based DNA sexing using CHD genes, with the primer set 2550F/2718R.  Results: We successfully extracted all samples; the mean sample concentration was 267.5 ng/µl (range 47-510.5 ng/µl) and samples were of high purity (A260/280 ratio 1.85±0.03). All samples were used to successfully identified sexes, 9 females and 15 males.  Conclusions: 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.