Abstract:
There is a demand by field biologists to establish molecular techniques to identify
the sex of elephants using non-invasive samples. The present study applied a
molecular method, Amplification Refractory Mutation System (ARMS), for sex
identification of Asian elephants (Elephas maximus) using their dung samples.
This technique focused on allele specific identification, targeting the zinc finger
(zf) region in X and Y-chromosomes. Based on the unique nucleotide present in the
zfx and zfy gene sequences in the intron zf region of Asian elephants, two sets of
primers were designed manually. DNA was extracted from dung samples collected
from 12 males and 16 females and PCR method was performed to amplify DNA
using designed primers. To confirm the results, one step RT-PCR (Applied
Biosystems) was performed using SYBR Green dye (QuantiTech-Qiagen).
The PCR method yielded 118bp and 130bp products for X and Y genes
respectively. For male DNA samples both 118bp and a 130bp bands were observed
while it was only 118bp band for female DNA samples. The Melt curve analysis
(MCA) of one step RT-PCR method support the above results by resulting two
separate peaks plotted at 74°C Tm for male samples and at 77.6°C Tm for female
samples. The result concludes that the designed primers based on the Zinc finger
protein encoding region of X and Y-chromosomes are highly specific for sex
identification of Asian elephants using their dung as non- invasive sample.
