Molecular characterisation of two cichlids populations (Tilapia guineensis and Sarotherodon melanotheron) from different water bodies in Lagos State, Nigeria

1 Department of Aquaculture and Biotechnology, Nigerian Institute for Oceanography and Marine Research, Victoria Island, Lagos, Nigeria. 2 Department of Aquaculture and Fisheries Management, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. 3 Department of Microbiology, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. 4 Department of Aquaculture and Biotechnology, Nigerian Institute for Oceanography and Marine Research, Victoria Island, Lagos, Nigeria. 5 Department of Zoology, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria.


INTRODUCTION
Nigeria is endowed with a lot of resources including fisheries resources that contribute immensely to the nutritional needs, economic growth and development of the nation.Fish provides essential amino acids like lysine *Corresponding author.E-mail: agbebi20@yahoo.com.Tel: +2348037045878, +2348072723153.and methionine which are limited in other animals.It is also rich in vitamins A and D as well as lipids which help to reduce thickness of blood thereby allowing it to flow easily in the body (Akande, 1990).Tilapia is a large genus in cichlid family -Cichlidae, which according to Nelson (2006) is the third largest fish in the world and one of the most diverse fish species.The species is chosen because of its relatively short generation time of about six months and its suitability for investigation of the application of genetics in aquaculture from conservation of genetic resources to breeding programmes (Eknath et al., 1993) and breed in captivity and they are enjoying wide acceptance as food fish because of their high palatability and history of use from inland fisheries.Information about the genetic diversity of wild fish populations and culture population is essential for effective management.However, genetic diversity has been measured indirectly and inferentially through controlled breeding and performances studies or by classical systematic analysis of phenotypic traits.
The widespread and mixing of two or more tilapia species in ponds and in natural water bodies makes the identification of tilapia to be difficult by mere traditional method.There is also need to address the taxonomic problems and also to identify the distinctness of both natural and aquaculture populations and inadequate expression of genetic strains.Therefore, this study is aimed to ascertain inter and intra-specific genetic heterogeneity among the wild and cultured both species (Tilapia guineensis and Sarotherodon melatheron) and examine phylogenetic relationship between the species.

Study sites
The fish culture at Akin Sateru Farms at Ajah and Nigerian Institute for Oceanography and Marine Research Fish Farm at Badore, both in Lagos State, Nigeria while fish sampling (wild) was at Lagos lagoon at Oworo and Badagry both along the coast of Nigeria, West Africa.However, the samples of cultured were also taken from the farm named earlier.The sizes of the samples ranged between 8.4 to 17.2 cm in standard length and between 26.1 to 133 g in weight (include their standard deviation 26.1 ± 36.66 to 133 ± 21.7).The samples were transported in ice chest to the laboratory at 20°C to Nigerian Institute for Oceanography and Marine Research Biotechnology laboratory.DNA was isolated from caudal fin clip using salt -out method according to Lopera- Barrero et al. (2008).Purity of the DNA was assessed by determining the value of absorbance of the DNA at 260 and 280 with value ranged between 1.63 to 2.10.DNA samples extracted from fish samples were amplified using five RAPD primers (OPC 04, 05, 10, OPR 02 and OPI 05, Operon Technologies Inc. USA).A total volume of 20 µl of the PCR ingredients which consists of 2 µl Solis Biodyne (SBD) 5x fire pol (master mix with 12.5 mM MgCl), 17.15 µl autoclaved water, 0.5 µl dNTP (0.2 mM; nucleotides), 0.1 µl of Taq polymerase, 0.25 µl of primers (25 p/mol) and 2 µl of DNA (10 ng) was run on Thermocycler.Initial denaturation of the PCR analysis was at 950°C for 5 min followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 350°C for 60 s and extension at 720°C for 2 min; a final extension at 72°C for 10 min was carried out.
The samples were cooled at 40°C.Horizontal electrophoresis was performed using a 1.5% agarose gel, at 70 V for 1.5 h in a 1 x TBE buffer.The gel was marked with ethidium bromide and the pictures taken on top of a UV transilluminator.The RAPD fragments were counted at different loci for the five primers and inputted into a software NTSYS to generate a dendrogram.

RESULTS
Purity of the DNA was carried out spectrophotometrically by measuring absorbance at 260 and 280 nm using TE buffer as blank which ranges between 1.63 to 2.10.RAPD analysis was performed on 80 genomic DNA samples of fishes from different water bodies using five 10 bp-oligo-nucleotide random primers (Table 1), which shows different numbers of stable amplification and polymorphism bands.

DISCUSSION
Random amplified polymorphic DNA (RAPD) bands in this study were always stable (that is strong, faint and sharp, except OPC 10 that had no amplification) bands generated with each primer because one or more copies of DNA may exist per genome or may be attributed to the varying of the annealing process between the primer and the DNA.However, the five random primers in RAPD-PCR analysis generated strong amplification profile with distinct bands (Plates 1 to 30) and some mixed bands.The problem of mixed bands shows the well known sensitivity of PCRs (Bielawski et al., 1995).These results are in agreement with Bardkci and Skibinski (1994) who stated that, patterns of similarities and differences between populations showed broad agreement across primers and the overall similarity level varied between primers.Thus, primer choosing is very important for this technique.Samples from wild water body are found to have a similarity coefficient of 100% from Figure 1 (TGW1 and TGW2) and Figure 2 (SW6, SW7, SW13 and SW12) showed that pure strain T. guineensis and S. melatheron.However, the cultured samples are close to that of the wild samples (Kuton and Kusemiju, 2010).The similarity between T. guineensis and S. melanotheron from the wild was 78%; between T. guineensis and S. melanotheron from the culture was 80%; and 100% of both wild and culture of samples; 82 and 88% of T. guineensis and S. melanotheron from wild and culture, respectively.
The dendrogram (Figures 3 and 4) indicates the relationship among the species which are of family Cichlidae.These findings indicated high probability of hybridization between the very closed species to improve the genetic characters; also high quality and quantity of DNA can be gotten in using of salt-out method for extraction and pure strain of the samples can be gotten from wild and culture in spite of the close morphology of body shape they all share.It can be concluded also that, RAPD-PCR could prove to be a useful tool for estimating the genetic variability and degree of similarity among fish species.

Figure 1 .
Figure 1.Dendrogram depicting the degree of relationship between Tilapia guineenis from wild and culture.

Figure 3
Figure 3 dendrogram showed depicting the degree of relationship between Tilapia guineesnsis and Sarotherondon melanotheron from the culture from both

Figure 2 .
Figure 2. Dendrogram depicting the degree of relationship between Sarotherodon melanotheron from wild and culture.

Figure 3 .
Figure 3. Dendrogram depicting the degree of relationship between Tilapia guineensis and Sarotherodon melanotheron from culture.

Figure 4 .
Figure 4. Dendrogram depicting the degree of relationship between Tilapia guineensis and Sarotherodon melanotheron from wild.

Table 1 .
Sequences of primers, GC% and number of bands.