Genetic Similarity Levels in Nile Tilapia of Some Nigerian Water Bodies Using SSR Markers

MATERIALS AND METHODS

Study area and fish samples: Two hundred and four specimens identified as O. niloticus species were obtained from six states representing the geo-political zones in Nigeria (Fig. 1). The collection was done between January, 2013 and October, 2013 with the help of local fishermen. Fish were kept on ice until transferred to the lab and frozen at -80°C until analyzed.

Thirty-four individuals from two water bodies in each of the states were sampled: Adamawa (Gariyo and Kiri Lakes), Kebbi State (Birnin Kebbi/Rimer River), Kogi State (Lokoja confluence and Sarkin Noma River), Oyo State (Eleyele lake and Oba Dam), Anambra State (Igbariam and Otuocha Rivers) and Delta State (Warri- Umuochi/Ona River). The laboratory analysis was conducted at the Biotechnology Laboratory of the Nigerian Institute for Oceanography and Marine Research, Victoria Island, Lagos Nigeria.

DNA extraction and SSR-PCR: Genomic DNA was isolated using the salting out method from the caudal fin according to Lopera-Barrero et al.²⁸. Caudal fins (approximately 3.5-4.5 g) were placed in Eppendorf microtubes with 90% absolute ethanol and maintained in a freezer at -20°C. Lysis buffer (550 μL) containing 7 μL of 200 μg mL^–1 of proteinase K was added. The samples were incubated in a water bath at 50°C for 12 hrs. Sodium chloride (660 μL of 5 M) was added and centrifuged at 12,000 rpm for 10 mins. The supernatant was transferred to a new tube and 700 μL of cold absolute ethanol was used to precipitate the DNA. The samples were incubated at -20°C for 2 hrs. The DNA sample was centrifuged and washed with 700 μL of 70% v/v ethanol. The sample was re-suspended in 80 μL TE buffer (10 mM of Tris pH 8.0 and 1 mM of EDTA). Ribonuclease (30 μg mL^–1) was added and incubated at 42°C for 4 hrs. This was stored in the freezer at -20°C before PCR. The concentration and purity of the extracted DNA were determined by measuring absorbance at 260 and 280 nm with a Biorad Nano Spectrophotometer.

The PCR reactions were performed in a Biorad I-cycler with the following program. Initial denaturation step at 95°C for 5 min, 35 cycles of 30 sec at annealing temperature (45-60°C) and 1 min at 72°C, followed by a final elongation step of 10 min at 72°C. The amplified DNA fragments were electrophoresed using 1.5% (w/v) agarose gel and stained with ethidium bromide. The amplified products were visualized on a UV transilluminator and photographed. The SSR primers used were selected from the database at the National Center for Biotechnology Information (Table 1)²⁹.

Phylogenetic analysis: Phylogenetic analysis was done using alleles count, the alleles were scored and the distance (UPGMA) method was used to construct a dendrogram that showed the similarities and differences between the genes. The analysis was carried out using NYSYS software.

Fig. 1:

Map of Nigeria showing sampling sites

RESULTS AND DISCUSSION

Genetic variation in any species is a resource that is manifested at two different hierarchical levels. The first level is explicit and includes the genetic differences between individuals within a population. The second level of genetic variation is that which exists among the various populations^30-32. The SSR markers have become one of the most popular genetic markers because of their high⁹ polymorphism and PCR-based analysis^10,15. Microsatellite bands in this study were always unstable (i.e., strong, faint, fuzzy and sharp) bands generated with each primer because one or more copies of DNA may exist per genome or may be attributed to the varying annealing process between the primer and the DNA.

Plate 1:

Amplified SSR loci for individuals from North East, Nigeria (Adamawa State) Lane M is 1 kb DNA ladder, A, B, C are primers UNH 104, UNH143 and UNH 022 showing fragments sizes 138, 200 and 257 bp, respectively and NB: Electrophoresis of remaining 16 individuals for b was run on a different plate not shown here

This problem of mixed bands shows the well-known sensitivity of PCRs¹⁰. The SSR fragments generated by primers UNH 208,119,180 and 156 showed low polymorphism among the fishes studied. These primer sequences may have annealed to variable sequences, which might be of great utility at lower taxonomic levels, e.g., for the differentiation of closely related species. However, microsatellite fragments generated by three primers UNH 143, 104 and 022 showed both stable amplification and a high degree of polymorphism. The DNA fragment sizes of these primers range from 138, 200 and 257 bp (Plate 1), respectively. Their sequences may be considered as more conserved sequences, which are most useful in higher taxonomic levels and evolutionary relationships. Their high polymorphism and PCR-based analysis have made them one of the most popular genetic markers^10,15. Some microsatellite loci have very high numbers of alleles per locus (>20), making them very useful for applications such as parent-offspring identification in mixed populations, while others have lower numbers of alleles and may be more suited for population genetics and phylogeny^11,15,19,33.

Fig. 2(a-c):

Similarity coefficient of among O. niloticus (A unit is 0.03) SSD: South South Delta, SWO: South West Oyo, SEA: South East Anambra, NWK: North West Kebbi, NEA: North East Adamawa and NCK: North Central Kogi

The phylogenetic analysis generated a dendrogram’ that showed the similarities and differences between the genes. A total of 118 clusters with varying similarity coefficients ranging from 0.44 to 1 were observed (Fig. 2). Thus, for instance, at a similarity coefficient of 100%, 39 clusters occurred out of which only five were interesting: 1A, 1B, 1C, 1D and 1E clusters (Fig. 2a and b). They were made up of 15, 15, 23, 9 and 15 samples, respectively. Samples from three states in SSD, SWO and NWK were always present in each cluster at this similarity coefficient. A lot of migration is likely taking place among these fishes over a period. This was in conformity with previous observations of high genetic similarity of Oreochromis niloticus species within the Niger River Basin populations over genetically relevant timescales using SNP markers³⁴.

Seven major clusters of A, B, C, D, E, F, G and sub-clusters a, b, c, d, e, f, g and h were observed. The sub-clusters comprise 10, 6, 5, 3, 6, 10, 28 and 3 clusters at about 73, 77, 73, 79, 66, 62, 56 and 73% similarity coefficients indicating varying levels of similarity. The major clusters showed similarity coefficients of about 73, 67, 64, 59, 56, 48 and 44%, respectively (Fig. 2c). Hence, at cluster G, the level of genetic similarity (44%) within the population is low. Shared bands among O. niloticus in the six geopolitical zones were as high as 73%, hence, there was a high similarity among some strains although primers may differ³⁵. These high similarity levels may be a result of a major event like flooding which is common in Nigeria or escapes from restricted areas (farms). Oreochromis niloticus used in previous work to assess mitochondria diversity of some cichlids in Nigeria also showed varying levels of similarity³⁶. However, varying levels of similarity could be an indication that the species is hybridizing with other tilapia species. The Oreochromis niloticus like other cichlid species can easily hybridize even with other congeneric and produce fertile offspring that at times have the O. niloticus phenotype^37-39. The DNA fingerprints of mixed DNA samples have been reported to be useful in assessing the relationship between closely related populations due to the high-level genetic differentiation detected⁴.

These data supported the view that the very high similarity between tilapia leads to a high probability of hybridization between them, based on the genetic distance between strains we may deduce that there are varying levels of genetic variability among O. niloticus in Nigeria water bodies under study. Genetic variability among O. niloticus observed in this study was consistent with previous studies of this species using both mtDNA and nuclear markers^{17,22,33,35,40-42}.

CONCLUSION

Oreochromis niloticus collected from water bodies in six states representing the geopolitical zones of Nigeria show various levels of genetic similarity. The similarity of intensity may be due to the increased amount of hybridization that occurs in the wild among these species. This study findings recommend the use of a more robust genomic approach like the exon gene-capture method in addressing the issues of hybridization.

SIGNIFICANCE STATEMENT

Oreochromis niloticus is well utilized for aquaculture in Nigeria and globally with possibilities of imported strains even hybridizing in the wild when they escape from the farm or through flooding. Our findings using microsatellite markers confirms varying levels of genetic similarities and migration among wild Oreochromis niloticus species from the six geopolitical zones of Nigeria. Thereby proving that though pure individuals exist and are spread by events like flooding, introgression might be occurring. Aquaculture and fisheries are faced with challenges, including a lack of pure stock or good seeds to improve fish production. I suggest a robust molecular approach based on a gene capture to ascertain the introgressive hybridization or purity of this species before use in aquaculture or research.

ACKNOWLEDGMENTS

Mojekwu, T.O. appreciates the genomic mapping of Commercially Important Fishes in Nigerian Waters Project (CARGS project) with identification number RFA No. 3 (2) of the Agricultural Research Council of Nigeria (ARCN) for funding this project. Thanks to NIOMR, the Executive Director, the Head of the Department of Biotechnology, Dr. K. Oresegun and Dr. I. Megbowon, the project team leader for the support, time and advice given to T.O. Mojekwu during this work.