Physical and linkage mapping of genetic markers and genes associated with sex determination in tilapia (Oreochromis spp.)

ABSTRACT In order to combine previous observations from different sources on sex determination, and to identify sex chromosomes including the major sex determination locus in Nile tilapia, physical and genetic maps based on sex-linked markers and genes (such as sex-linked AFLPs, microsatellites, ovarian aromatase and DMO genes) were integrated and anchored. An accurate physical map using FISH techniques on mitotic cells was developed based on a previous map and 23 tilapia BAC clones previously assigned to linkage groups (LGs) 1, 3, 6, 7, 10 and 12; and on meiotic cells, 2 BAC clones containing the SLAM OniY227 and the dmrt4 gene were mapped. The six linkage groups were then assigned to different chromosomes, but surprisingly, the putative sex LG1 was located to a small submetacentric chromosome and not to the larger subtelocentric chromosome 1, where LG3 was assigned instead. The other LGs were assigned to different chromosomes and oriented with respect to the centromeres. A detailed comparison of the physical distribution of markers on chromosome 1 with respect to LG3 revealed a suppression in recombination in the subtelomeric region of the q arm between the marker GM354 (0 cM) and clcn5 (29 cM) and an abrupt increment of recombination between clcn5 (29 cM) and GM128 (77 cM) close to the centromere (Flpter=0.2). The unpairing region (20% of the total length) observed on the larger bivalents of XY fish during early pachytene in meiotic cells has been confirmed by DAPI staining and FISH to be at the terminal part of the q arm, opposite to the centromere. Comparison with six other tilapia species (2n=44) revealed a well conserved karyological distribution of the suspected LGs associated with sex determination (1 and 3). Besides, in O. karongae (2n=38) it was shown by SATA and UNH995/UNH104 marker hybridisation that LG1 has been re-arranged into the subtelomeric chromosome 2 as a result of a telomere-telomere fusion. A pool of 15 tilapia BAC clones previously localised on chromosome 1 and containing sex-linked AFLPs, dmrt1, dmrt4 and several SINEs were screened for new microsatellites; BACs were digested with SAU3AI and TC, GT, ATCT and CTGT probes radio-labelled with 32P. The high abundance of repetitive sequences in the BACs used led to only one useful polymorphic and co-dominant marker being obtained, associated to a BAC clone containing a copy of the dmrt1 gene on chromosome 1 (Flpter=0.85). Four linkage maps were constructed from an XY male, XY neofemale, XX neomale and XX female, mapping 4 and 8 markers on LG1 and LG3 (including the dmrt1 associated microsatellite) respectively. A specific sex-determination locus was identified on LG1 clearly linked with UNH995. However there appeared to be different allelic strengths for this sex determination locus, as shown by different sex ratios associated with different UNH995 genotypes. Additionally, one of the two XX fish mapped, showed the location of the recessive black blotching trait on LG3 (chromosome 1) between the markers GM128 and GM526, close to the centromere (Flpter=0.14). The results presented suggest a nascent Y chromosome in early stage of differentiation in Nile tilapia and with a functional master gene on LG1 close to the marker UNH995 (Flpter=0.67) located on the q arm of a small submetacentric chromosome. The potential influences of the autosomal LG3 (chromosome 1) in sex differentiation are also discussed