European Journal of Biomedical and Pharmaceutical Sciences

A RAPID PCR ASSAY FOR IDENTIFICATION OF MALES IN WIDOW TETRA, GYMNOCORYMBUS TERNETZI (BOULENGER, 1895)

Clifton Justin David* and T. J. Pandian

ABSTRACT

Non-invasive, early sex determination using molecular markers have potential application in aquaculture and human genetics. Double Sex/Male abnormal Protein (mab3) related transcription factors (Dmrt) family genes may be useful as Y chromosome-specific markers for determining maleness in fishes and humans. Objectives: To develop a rapid PCR assay for early identification of androgenetic clones (Y2Y2) and unexpected females (X1Y2) with male genotype in widow tetra (WT), Gymnocorymbus ternetzi larvae using Dmrt1, a male-specific marker. Methods: Genomic DNA isolated from larval tail fins of 8 androgenetic males (Y2Y2) and 4 unexpected female progenies (X1Y2), produced by progeny testing of androgenetic males were selected. A robust PCR protocol was optimized using primers for Dmrt1 specific genes (Dmrt1F –AAGTGCTCCCGCTGCCGGAA; Dmrt1R -GCTGGTACTGCTGGTAGTTG). The amplified genomic products were identified using agarose gel (2.5%) electrophoresis, cloned and sequenced. Results: Dmrt1 specific primer amplified a Y chromosome-specific 300 bp and 237 bp amplicons in WT males which was absent in females. Sequencing of the 237 bp amplicon confirmed homology (84%) with previously reported medaka and Buenos Aires tetra (BA tetra) Dmrt sequences. Conclusion: Y chromosome-specific markers have profound applications in human foetal sex determination and early identification of males in genetically manipulated fishes.

Keywords: Dmrt1, Y chromosome-specific markers, PCR assay, genomic DNA, androgenetic clones, Human.