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Exclusion of Two Microsatellite Markers in the Sexlinkage Study of Nile Tilapia (oreochromis Niloticus L.)

Marufa Sultana Mitu
Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Antima Gani
Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md Bakhtiar Abid
Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Sadia Nusrat Sharna
Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Farzana Yesmin
Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Sajib Banik
Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md Moudud Islam
Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. Present address: Department of Fish Biology and Biotechnology, Chattogram Veterinary and Animal Sciences University, Chattogram, Bangladesh

Mohd Golam Quader Khan
Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Monosex Nile tilapia (Oreochromis niloticus) is highly preferred in semi-intensive and intensive culture systems to prevent uncontrolled reproduction and to obtain fast growing male. Production of all male tilapia is being practiced by the hatcheries of Bangladesh mainly by administering androgen hormones (particularly 17-α-methyl-testosterone) with feed in a mixture of undifferentiated fry for about a month. The direct application of hormone to such food chain often arises question in respect to public health and safety. The alternative to this is the production of putative super males, a rather safe but longer procedure to obtain all male progeny. However, sex determination system in tilapia is fairly complex. Recent developments have resulted in a linkage map and genetic markers that can be used to analyze the sex determination system. For genetic analysis of different genotypes of fish, microsatellite DNA marker ARO120 and ARO121 were used for studying the inheritance pattern for possible sex linkage using Polyacrylamide gel electrophoresis. In case of ARO120, it was observed that the Dam XX was heterozygous; 11 out of 22 female progeny and 10 out of 22 male progeny were found to be heterozygous. In case of ARO121, it was observed that the Dam XX was heterozygous; 16 out of 22 female progeny and 20 out of 22 male progeny were found to be heterozygous. Though the marker polymorphisms were observed in this study, these were excluded from the sexlinkage study due to limited extent of information as sex-linked markers in Nile tilapia BFRI strain. This study provides a baseline for further research using other suitable polymorphic markers for assisting marker-assisted selection

Keywords: Nile tilapia, Microsatellite marker, Sex linkage

Location: Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh.

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Tilapia

Objectives:

To establish the suitability of these markers as sex linked polymorphic or monomorphic marker and identify the inheritance pattern of alleles in both sex of Nile tilapia.

Methodology:

The experimental sites were Field Laboratory Complex, Bangladesh Agricultural University (BAU), Aquarium Facilities of Faculty of Fisheries, (BAU) and Fish Genetics and Biotechnology Laboratory of Department of Fisheries Biology and Genetics, (BAU), Mymensingh. The study was conducted for one year. One day old spawns of first feeding stage of tilapia were previously collected from Agro-3 fish farm and hatchery, situated at Boilor of Trisal in the district of Mymensingh and stocked in three different ponds of Fish Field Laboratory Complex, (BAU) having an area of three decimals each. Initially a mixture of fingerlings (N=200) of Nile tilapia O. niloticus containing normal males (XY) and normal females (XX) were previously collected. Ponds were previously prepared using 3 kg calcium carbonate per decimal and rotenone. The fingerlings were fed crumble feed daily at the rate of 4% body weight four times in a day up to thirty days. For another ninety days they were reared up to sex determination stage using pelleted feed twice a day. The sex of experimental species was identified by visual observation of genital papilla. For the purpose of breeding 15 pairs of male and female were selected after six months and kept in individual hapas ( with 1:1 sex ratio (Male: Female). Among them offspring from five pairs of parents were collected after accomplishment of breeding. Twelve glass aquaria (each with 25L water holding capacity) were set to keep the fish fry out of the cross between XY males and XX females and also YY supermales and XX females. After crossing five pairs of XY males and XX females, the hatchlings were brought and kept at the aquaria after eleven days of fertilization. The fry were then fed DES hormone with the feed with different doses viz. T₁ (100mg DES hormone /kg feed), Treatment T₂ (200mg DES hormone/kg feed), Treatment T₃ (300mg DES hormone/kg feed) and T₄ (without hormonal feed). There were three replications for each of these three treatments that contained 300 fry in each aquarium. The hormonal treatment was conducted for a period of one month. The fry were released in cisterns after the completion of the hormonal trial. Normal feed had been provided to grow them up to sexual maturity in pond. Three putative YY supermales (originally brought from the owner of the Agro-3 private hatchery fish hatchery Boilor, Trisal, Mymensingh) were successfully crossed with XX females (with 1:1 ratio of Male: Females). After incubation and yolk sac absorption period, they were stocked in glass aquaria each (N=100) under four hormone treatments viz. T₁ (200mg DES hormone/kg feed), T₂(300mg DES hormone/kg feed), T₃ (400mg DES hormone/kg feed), T₄ (500mg DES hormone/kg feed) and a control (without hormonal feed). There were two replications for each of these four treatments that contained 100 fry in each aquarium. Hormonal treatment was done up to one month and the released in five different cisterns for rearing using normal feed up to maturity. Fin clips were collected from Family HRT 25 and dam samples of different genotypes (XX, XY, “XY” and YY) and preserved in separate eppendorfs containing 95% ethanol and stored at -180C. Before taking each sample, all scientific procedures were rigorously maintained. Genomic DNA was extracted from fin clip tissues according to the method described by Islam and Alam (2005). Phenol-chloroform protocol (Sambrook and David, 2001) was used for this extraction. All DNA samples were tested qualitatively (presence of RNA or degradation of DNA) and quantitatively on 1% agarose gel. The software DNA FRAG version 3.03 was used to estimate marker length and allelic length to identify any variation between the male and female haplotypes with microsatellite ARO120 and ARO121.

Source of Information: Res. Agric. Livest. Fish. Vol. 6, No. 1, April 2019 : 143-151.

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The sex reversal rates of the survived progeny were determined upon sexual maturity after 6 months (five months after the first sampling on survivability). Although for one month, the survival rate was determined for each of the replication group, the replicates were merged afterward for observation of sex reversal after becoming sexual maturity in 6 months. The PAGE with ARO120 resulted in marker heterozygosity in the Dam of family HRT 25 in 11 out of 22 family progeny and 10 out of 22 family progeny. The rest were homozygous for one or the alternative allele. The genotyping with ARO121 resulted in marker heterozygosity in the Dam of family HRT 25 in 16 out of 22 family progeny and 20 out of 22 family progeny. The rest were homozygous for one or the alternative allele. In the current study, two microsatellite markers were used separately to assess the genotype to discriminate the polymorphic allele and monomorphic allele. At first 22 normal female, 22 normal male and one parent (mother) were genotyped using ARO120, where 11 out of 22 female progeny and 10 out of 22 male progeny were found to be heterozygous or having polymorphic allele. It seemed the mother and father contains same allele at heterozygous state. While another genotype analysis using ARO121 shows mother in heterozygous, where 16 out of 22 female progeny and 20 out of 22 male progeny were found to be heterozygous or having polymorphic allele. Lee et al. (2004) found that sex determining locus lies within a few centimorgans of microsatellite markers GM354, UNH168, GM271 and UNH131 in Blue tilapia (O. aureus) which were all polymorphic. Future work can be performed based on the observation on the allelic segregation with other primers, including primer ARO120. The allelic variation in the XX/XY model could demonstrate some alleles could be stronger in effect (producing close to all male) while some others are weaker giving intermediate sex ratios in the progeny. However this study excludes both the markers from being important sex-linked markers due to limited extent of genetic information.

Knowledge Management: Journal