Representational difference analysis for detection of sex-specific DNA markers from the Nile tilapia (Oreochromis niloticus)

Representational difference analysis from the Nile tilapia

Abstract

Sex-specific DNA markers are useful for identification of male and female fish even in the early stage of life which has many practical implications. Representational difference analysis (RDA) technique was applied to develop sex-specific DNA markers for the Nile tilapia Oreochromis niloticus. Genomic DNA ofYY male fish, XY male fish and XX female fish was restricted with BglII. We used oligonucleotide adaptors RBgl, JBgl and NBgl, RBam12/24 and RBgl12/24 adaptors and RBam24 and JBgl24 primers for preparing the amplicons from the genomic DNA. Three RDA experiments were performed: (1) YY tester with XX driver, (2) XX tester with YY driver and (3) XY tester with XX driver. A total of 146 RDA products from the male and female genomic DNA were cloned and sequenced from which 81 pairs of primers were designed and applied on three different genotypes to test specificity of the primers. One primer set designed from female RDA-derived clones (XX1_8F/R) yielded PCR products only in the tester female but not in the driver male. Another primer set designed male RDA-derived clones (YY1_14F/R) amplified one band in the male but two bands in the female DNA. However, the RDA analysis failed to detect unique fragments from the male and female genome in O. niloticus. Therefore, we suggest that the sex chromosomes are very weakly differentiated in tilapia.