Molecular identification of bats from Punjab-Pakistan

Abstract

Mitochondrial gene, COI has been identified as the standard barcode for molecular identification of many species. To date, no attempt has been reported for the molecular characterization of bats in Pakistan. The current study is therefore planned to design primers for the molecular marker COI through multi-species sequence alignment approach for Pakistani bat species. The COI sequences from major bat families were retrieved from NCBI and aligned on Mega 7 to find primers from most conserved regions. Based on the alignment results three primers with degeneracy were designed with one forward and two sets of reverse primers (PBCOIdF, PBCOIdR1 and PBCOIdR2). Touchdown PCR was carried out to amplify the COI gene from two major bats families i.e., Vespertilionidae, and Emballonuridae from the Punjab region. Primer pair (PBCOIdF and PBCOIdR1) yielded clean amplicons of size 579 and 576 bp for the bat specimens belonging to the Vespertilionidae family, whereas second primer pair (PBCOIdF and PBCOIdR2) gave good quality bands of size 673 and 664 for the Emballonuridae family. NCBI blast sequence analysis for specimens from Vespertilionidae and Emballonuridae revealed the identity score of 95% (Pipistrellus tenuis) and 97% (genus Taphozous), respectively. Group specific primers designed in the current study gave successfully satisfying results when applied to two large bat families. This work will certainly help to identify bats species across Pakistan and generate data regarding genetic diversity of this taxon parallel with other countries.