Screening, Application Of Sex-specific Molecular Markers And Cloning Of Candidate Sex-determining Gene SdY In Nile Tilapia

The Nile tilapia (Oreochromis niloticus) is a worldwide farmed fish, China is the largest producer and exporter of Nile tilapia, in which males grow faster about 50% than females. Therefore, extensive studies have been conducted to elucidate its sex-determining mechanism and to achieve sex-control. The Nile tilapia is a gonochoristic fish with an XX/XY sex-determining system, short spawning cycle and sex is mainly controlled by genetic factors, and its genome sequence has been completed and opened. These advantages make Nile tilapia an excellent model for studying sex-determination and differentiation. To date, a number of studies have been conducted to identify the sex chromosome and the sex-determining gene. Sex-determining region in Nile tilapia were previously detected on linkage groups (LG) 1,3 and 23. But which LG corresponding to the sex chromosome is not chear. So far, the sex-determining gene has not been cloned in Nile tilapia. Sex manipulation has been performed in some farmed fish species because of significant growth differences between females and males. In Nile tilapia, several studies have been reported sex-linked DNA markers, while many of them were sex-linked AFLP, SSR or SNP markers, which are inconvenient in utiliziation and with low accordance rate. Therefore, a method of rapid identification of the genetic sex is urgently needed to resolve this disturbing question, and the technology of genetically male tilapia through molecular marker-assisted selection has not yet been established. In the present study, five X or Y-linked molecular markers were screened through random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and random PCR amplification of sex-determining region. Furthermore, sex chromosomes correspording to LG23 were identified in this study through fluorescent in situ hybridization (FISH) combined with map of the markers to the genome sequences. Based on the sex-specific markers, the sex-determining region was defined to a smaller region through genetic linkage analysis. Finally, the candidate sex-determining gene sdY(sex determining gene on Y chromosome) was cloned through positional cloning. The main results are as follows:1. Screening of sex-specific markers by RAPD combined with bulked segregant analysis (BSA) was performed in this study. A total of 1200 RAPD primers were employed to identify sex-specific markers in XX, XY and YY genomic DNA pools and only 152 primers produced genetic polymorphism fragments. Thirteen sex-linked markers were identified and further verified by individuals with different genetic sex. Sex-linked bands were amplified, recovered from the gels, cloned and sequenced. Analysis showed that most of the markers were repetitive sequences. The primer S1368 (Marker-1) which produced one X-linked fragment (1971 bp) was amplified from the XX females and XY males, but not from the YY supermales, and was named Marker-1. The X-specific band was cloned and sequenced. A blast search of with this sequence revealed that it was located on Scaffold29 of LG23. Primers based on the up and downstream sequences flanking Marker-1 produced two bands, one with 740 bp in XX, XY and YY individuals and another Y-specific band with 648 bp only in XY and YY individuals. The two bands were cloned and sequenced. Alignments of the two sequences showed a 92-bp deletion in the Y-linked band.2. AFLP approach with 160 primer combinations was employed to screen sex-specific markers in XX, XY and YY genomic DNA pools. Fifteen primer combinations which amplified X-or Y-specific band were selected for further study. M1X3 produced a Y-linked fragment, while M4X7 and M4X13 produced X-linked fragments. The X- and Y-linked bands were recovered from gels, cloned, and sequenced. A blast search of the genomic DNA database with these sequences demonstrated that M1X3 (Marker-2) and M4X7 (Marker-3) were located on Scaffold7 and M4X13 (Marker-4) on Scaffold29, with both Scaffolds located on LG23. The up- and downstream sequences of the three markers were obtained from the genome sequences. Specific primers based on genomic DNA sequence were designed to amplify fragments spanning AFLP markers using XX, XY and YY as templates. Primer Marker-2F/R amplified a 675 bp fragment in XX and XY individuals and a 669 bp fragment in XY and YY individuals. Primer Marker-3F/R amplified a 763 bp fragment in XX and XY individuals and a 770 bp fragment in XY and YY individuals. Primer Marker-4F/R amplified a 980 bp fragment in XX and XY individuals and a 620 bp Y-linked band in XY and YY individuals. The amplified fragments were sequenced and compared. The results showed that Marker-2 has oligo-guanine repeats in one position (P1) and nucleotide polymorphisms in another position (P2) (X-CT/Y-TC). Marker-3 has a 7 bp Y-specific insertion. Marker-4 has a 360 bp deletion in the Y-linked band compared with the X-linked band. Based on sequence characterized amplified region (SCAR), the three markers were all successfully converted into X or Y-linked SCAR markers.3. Recent studies have revealed that the sex-determining locus (SD) peak on Scaffold101 of LG23 in O. niloticus. Meanwhile, the four sex-linked markers isolated by RAPD and AFLP were located on Scaffold7 and 29 (both adjacent to Scaffold101) of LG23. In order to find more genetic differences between X and Y chromosomes, a total of 32 pairs of ordinary primers were designed based on sequences of Scaffolds 7, 101 and 29 and used for screening of sequence differences between X and Y. One primer combination, Marker-5F/R, produced an 1151 bp X-linked band in both XX females and XY males, and a 711 bp Y-linked band in both XY and YY males. These two bands were cloned and sequenced. Alignments of the two sequences revealed that the Y-specific band has an insertion of 36 bp at position 1, and two deletions of 4 and 472 bp at positions 2 and 3, respectively, compared with the X-linked band. A blast search of genomic DNA with these sequences demonstrated that Marker-5 was located on Scaffold101 of LG23. The two fragments were successfully converted into SCAR markers by X or Y-specific primers designed at the sequence difference sites.4. The five selected markers were used to test the genetic sex of the progeny between different crosses:XX (♀) × XX(♂), XX (♀) × XY (♂), XX (♀) × YY (♂), XY (♀) × XY (♂) and XY (♀) × YY (♂). Every cross showed a high concordance rate between identified genotypic and phenotypic sex. Besides applicability to tilapia from our laboratory, these markers were used to identify the genetic sex of 215 fish in 8 families from different fish farms in China. Only 29 individuals were mismatched between phenotypic and genetic sex, with 22 females identified as XY and 7 males as XX. For all families these markers were reliable for sex identification; for some families (HN, HN2, THS and HG) they worked well, while for some others (HN1, T1, TH and T2), these markers did not work well. The concordance rate between identified genetic sex and phenotypic sex ranged from 76% to 100%. These data indicated that the Nile tilapia reared in our laboratory and the eight families from different fish farms might share the same sex chromosome (LG23). Also, the sex-specific molecular markers screened in this study have good generality. In Nile tilapia, the inability to rapidly identify the genotype of XX neomales, XY males and YY supermales has hampered the development of monosex stocks. The MAS technique has been demonstrated to be an effective method for sex control in fish. Therefore, the sex-specific markers we isolated were used for the establishment of the MAS technique (for GMT reproduction) because they can be directly used for PCR identification of the genetic sex of Nile tilapia at an early stage of development without further test crosses. These markers provided a rapid, accurate and efficient selective method for identification of XX females, XY males, YY supermales, XX neomales, XY and YY neofemales. Linkage analysis was performed in three XX (♀) × XY (♂) normally crossed families (277 individuals). Assuming complete interference, these five markers were located at 8 (Marker-1),6 (Marker-2),8 (Marker-3),5 (Marker-4) and 3 cM (Marker-5) from the SD, which was between Marker-2 and Marker-4, according to their calculated recombinant rates. In family 2, these five markers were located at 6、4、6、3and 2cM away from SD, respectively. Taking physical map distance, linkage group distance and natural sex reversal into consideration the distance between the markers and SD might be even closer than the data initially suggested, likely signifying that Marker-5 is just adjacent to the SD. This predicted sex-determining region on ScaffoldlOl is consistent with the region reported by the Israeli group. Additionally, physical mapping of these markers to its genome sequences and fluorescence in situ hybridization (FISH) using fosmid clones containing Marker-1 and -3 confirmed that LG23, instead of the LG1 and LG3, is the sex chromosome.5. With the help of X- or Y-specific molecular markers and the microarrayed fosmid library constructed by our group, the candidate sex-determining gene sdY (sex-determining gene on Y chromosome) of Nile tilapia was identified by positional cloning. Sequence analysis revealed that it is a duplicated copy of sdX located on the sex chromosome. However, a 5 bp insertion was found in the 6th exon, resulting in frameshift mutation generating a truncated mutant of the sdX. In addition, there were one insertion and three deletions in the upstream promoter region. RT-PCR and RACE were performed to obtain the full length cDNA of sdY. Tissue distribution analysis indicated that it was expressed exclusively in the XY and YY testis but not in XX ovary, and its expression started from the critical period of molecular sex determination (5 days after hatching,5dah), while sdX was expressed in both ovaries and testis. The ontogeny analysis indicated that sdY started to express from 5dah and then gradually decreased and last to adult. In sex reversed adult gonads, sdY was highly expressed in XY (♀) gonads but not in sex reversed XX (♂) gonads. These results demonstrated that the expression pattern of sdY was determined by genotype rather than phenotype. Immunohistochemistry (IHC) analysis showed that sdX and sdY signals were detected in granulosa cells in the ovary, while they were detected in myoid cells and Sertoli cells in thr testis. Therefore, sdY might be the candidate sex determining gene. It is one of the few sex determining genes (mammalians SRY/Sry, avains Dmrtl, amphibians DM-W, medaka DMY, Patagonian pejerrey amhy and rainbow trout sdY) which were successfully cloned in nearly 50,000 species of vertebrates. Moreover, the function of sdY will be studied by transgenic overexpression and site specific knock-in of sdY in XX and knockout in XY by TALEN, respectively. The gonad morphology and histology, expression levels of key factors in female and male pathway, and estrogen synthesis of the transgenic fish will be analyzed. In short, both positive (gain of function) and negative (loss of function) aspects of experiments will be adopted, in order to verify the candidate gene sdY is the sex-determining gene.The discovery of tilapia sex determining gene sdY (master switch) is of great significance for the clarification of gene cascade and regulatory net works in its sex determination and differentiation, and for sex control of Nile tilapia. It is also helpful in elucidating the molecular mechanism of sex determination and differentiation in teleosts, or even vertebrates.