Analysis of sex determination in Nile tilapia (Oreochromis niloticus L.): a molecular genetics approach

Abstract

Seven families of XX and YY homozygous Oreochromis niloticus were produced by mitotic gynogenesis from XY neofemales and their genetic status was verified by multilocus DNA fingerprinting and progeny testing. Two of these gynogenetic families and their corresponding diploid controls were used with 64 AFLP primer combinations in different levels of screening (XX/YY grand pool; XX/YY family pool; XX/YY gynogenetics and XX/XY control individuals) to search for sex-linked or sex-specific markers. Grand pool screening did not reveal any sex-linked markers. Subsequent family pool and individual level screening identified four sex-linked AFLP markers from two primer combinations, three Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420). Two of these (OniX420, OniY425) were shown to be allelic. Single locus PCR markers were developed for all of those markers. Linkage analysis of these markers and the sex locus within the source families revealed tight linkage, with estimated map distances of 13cM, 17cM and 20cM for OniY382, OniY227 and OniX420/OniY425 respectively. However, these sex-linked AFLP markers failed to consistently identify sex in unrelated individuals. To develop an effective system for parentage analysis in normal and gynogenetic progeny, AFLPs and multiplexed polymorphic microsatellite loci were investigated. Both were found to be effective, but microsatellites were more appropriate since they are codominant and some loci showed high gene-centromere recombination rates, suitable for discriminating meiotic from mitotic gynogenetics, while AFLPs are dominant markers. Spontaneous diploidization of the maternal chromosome set (SDM) was observed in gynogenetic progeny of one XY neofemale. Maternal inheritance and ploidy status were verified by multilocus DNA fingerprinting and chromosome karyotyping. Close genetic linkage between the red gene and an autosomal sex-reversal gene(s) in gynogenetic progeny and influences of autosomal sex-reversal gene(s) producing males in a fully inbred XX clonal line were previously reported in O. niloticus. To test if the same autosomal sex-reversal locus was responsible in both cases, a series of test crosses was carried out involving XX clonal neomale(s) and homozygous red females. The results indicated the involvement of more than one autosomal sex-reversal locus, one of which is linked to red body colour.