Polymorphism in a sex‐linked
 DNA
 marker located on
 LG23
 in Hainan strain of Nile tilapia (
 
 Oreochromis niloticus
 
 )

Marker-5, which is linked to the major sex-determination gene amhy on LG23, has been used for sex genotyping of various strains of Nile tilapia. Marker-5 specifically amplifies fragments of approximately 1,422 and 982 bp of the X and Y chromosomes, respectively. Herein, we evaluated Marker-5 polymorphism in Nile tilapia of Hainan strain, China. Agarose gel electrophoresis showed that in 13 out of 85 female fish, Marker-5 amplified a larger band, distinct from the standard X-specific band. Sanger sequencing showed that this larger band was 1,688 bp in length and showed high similarity with both 1,422-bp X-specific and 982-bp Marker-5 is useful for its utilization in sex-controlled breeding in farmed Nile tilapia. Y fragments' length would make sex genotyping difficult by Marker-5. The application of markers to new populations is expected to reveal new polymorphisms. Understanding polymorphisms in the sex-linked marker will provide a basis for the development of GMT in more strains via marker-assisted selection. chromosomal locations of new alleles Marker-5, evaluate the possibility of results that band by female-specific of tilapia. and in the Hainan identified by Marker-5. The results of this study will help to control the sex-genotype of farmed Nile tilapia using reported sex-linked marker. Our will help to use GMT technology in different Nile tilapia strains. at 50 days after hatching. The fish were weighed after 100 days of culture and candidate broodstock were selected. The selected broodstock was randomly paired and inbreeding was avoided. From 2012 2020, one generation was established per year, labeled G1-9. In the present study, 50, 118, and 130 fish were sampled from G7, G8, and G9, respectively. Tail fins of the Hainan strain of Nile tilapia sampled and at (cid:3) in for DNA extraction. and generations specific band in the XX/XX while no 2414-bp amhy- specific bands were Marker- amhy -5608 amplified the amhy-specific bands DNA Marker after one week of rearing (Khater, Ali, & Mohamed, 2017). In the present study, the offspring from families of G9 of the Hainan strain were cultured at 30 ± 1 (cid:3) C. A relatively low proportion of males (2 of 13, 15.4%) was found in XX L offspring from family 200516 of G9. The sex-reversed XX L offspring from this family might be caused by water temperature. In family 200539, progeny testing showed that XY L offspring are all male, suggesting that Y L -linked amhy can control the male sex. In contrast, agarose gel electrophoresis could not distinguish between XX and XY L fish. The XY L fish identified by Marker-5 could be mistakenly identified as XX fish. Thus, marker polymorphism is the third explanation for the existence of XX male fish. This implies that Marker-5 could not be used alone to genotype Nile tilapia of different strains. Taken together, three explanations were proposed for the identification of XX male fish by Marker-5. Marker polymorphism, other major male determination loci, and sex reversal should explain for all male, balanced, and female-skewed sex ratios, respectively, in XY L /XX/XX L fish identified by Marker-5 in Nile tilapia. Female sex-linked marker might be identified in the XY/X L Y fish in our future study. In summary, female XY fish identified by Marker-5 could also be attributed to environmental factors, mutations related to amhy , and other female sex-determination alleles. In addition, XX L fish, which showed two bands by Marker-5, could be empirically mistaken as XY. Marker-5 also identified XY female fish in farmed Nile tilapia from China (Sun et al., 2014). Some of which were a result of wrong genotyping because of polymorphism. Thus, the results of the present study should contribute to more accurate identification of the sex genotype of Nile tilapia. These studies also imply that the strains with natural sex-reversed XY female fish, identified by Marker-5, should not be used directly to develop GMT technology. A female ratio of progeny of YY males of <2% is still acceptable, as the male ratio is usually controlled at over 98% by methyltestosterone in large-scale production in fish farms. However, a higher proportion of XY females from YY males should be the main constraint against the use of GMT. In families 200516 and 200539, all fish possessing amhy were male, indicating that amhy is a strong male determination locus in these families. In addition, the XY/XY/XY L male ratio reached 89.1% ( N = 55) in randomly sampled fish from G7 and G8 of the Hainan strain. These results suggested that amhy can control the male sex type in some families in the Hainan strain, providing a basis for the development of GMT with amhy -linked markers. Future studies should investigate the mechanisms underlying the formation of naturally sex-reversed XY/X L Y female Nile tilapia. Continuous studies of sex determination in tilapia will help to resolve these issues, which limited the utilization of GMT. - and Y-specific bands, which was a heteroduplex. Marker-5 could not distinguish between XX and XY L fish or between XX L and X L Y L fish alone. The results of molecular sexual identification by the Marker-5 in Hainan strain are complex, and it is inappropriate as one strain for GMT development at present. X L and Y L are rare cases in some strains of Nile tilapia. In most cases, Marker-5 is applicable in aquaculture as reported by Sun et al., 2014. The results of the present study can improve the utilization of Marker-5 for the development of GMT technology in farmed Nile tilapia strains.