| Livestock Research for Rural Development 29 (1) 2017 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
Snapper (Lutjanus goreensis) and Grunter (Pomadasys jubelini) populations from a high (Lagos Lagoon) and low brackish lagoon (Badagry Lagoon) were studied to assess their genetic relationship using randomly amplified polymorphic DNA analysis (RAPD) technique. This involved the use of 10-mer OPAS primers to assay polymorphisms between the two populations of both species.
Varying RAPD fragment patterns were observed for different locations. Of the two primers utilized, only one produced distinct and consistent RAPD profiles for both species from the two locations. Forty-nine (49) and thirty-six (36) reproducible bands representing slight (25.8% and 20.0 %) DNA polymorphisms in L. goreensis and P. jubelini were uncovered in populations from both locations. Cluster analysis performed to create dendrograms using UPGMA by the Phyllip software gave no sufficient genetic divergence to discriminate the samples of different populations. The result of this research is very important because it presents baseline information on the genetic diversity of snappers and grunters from these two water bodies.
Keywords: analysis, bands, primer, RAPD, variation
In the tropics, Snappers and Grunters are valuable fishery reserves and are greatly prized as genuine protein source (Allen 1985). Badagry Lagoon and Lagos Lagoon; the principal lagoon in the Gulf of Guinea (FAO 1969; Emmanuel et al 2008; Soyinka and Kassem 2008) have been noted to constitute a significant proportion of artisanal fishing of the capture fisheries sector including the Snappers and Grunters. According to Solé-Cava (2001), various aggressions to the aquatic ecosystems have caused serious alterations in environment dynamics, jeopardizing the rich fish variety. The conservation of genetic variation has therefore been an essential component of several species management programmes.
According to Mohammadi and Prasanna (2003), genetic diversity can be revealed based not only on morphological and biochemical, but also molecular types of information. Essentially, the advantages of molecular markers by far outweigh other methods because they show in greater details, genetic differences devoid of intrusions from environmental factors. Scientists including Pante et al (1988) and Kuton and Kusemiju (2010) have approached such studies utilizing morphometric and meristic analysis. Ahmed et al (2004) used allozyme electrophoric analysis, Avtalion et al (1976) carried out serum protein analysis. Crosetti et al(1988) used karyotype analysis, Seyoum and Kornfield (1992) applied the mitochondria DNA restriction analysis and immunology/agglutination assays. In addition, El-Zaeem et al(2006) used DNA fingerprinting, whereas Lee and Kocher (1998); Bo-young et al (2005) applied DNA microsatellite analysis to detect genetic variations in various fish species.
Garcia et al (2004) highlighted that the past limitations associated with pedigree data and morphological, physiological and cytological markers for assessing genetic diversity in cultivated and wild species have largely been circumvented by the development of DNA markers such as restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs) and simple sequence repeats (SSRs, microsatellites). However, these molecular markers have technical differences in terms of cost, speed, amount of DNA needed, technical labour, degrees of polymorphism, precision of genetic distance estimates and the statistical power of tests.
According to Bardakci (2001), currently, the restriction fragment length polymorphism (RFLP) assay has been the choice for many species to measure genetic diversity and construct a genetic linkage map. However, an RFLP assay which detects DNA polymorphism through restriction enzyme digestion, coupled with DNA hybridisation, is, in general, time consuming and laborious. Over the last decade, polymerase chain reaction (PCR) technology has become a widespread research technique and has led to the development of several novel genetic assays based on selective amplification of DNA. This popularity of PCR is primarily due to its apparent simplicity and high probability of success. Unfortunately, because of the need for DNA sequence information, PCR assays are limited in their application. The discovery that PCR with random primers can be used to amplify a set of randomly distributed loci in any genome facilitated the development of genetic markers for a variety of purposes.
RAPD is a protocol which is based on PCR (arbitrary nucleotide sequence which is ten nucleotides long, using short single primers) to amplify arbitrary portions of the genome. The simplicity and applicability of the RAPD technique have captivated many scientists’ interests. Perhaps the main reason for the success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the requirement for cloning, sequencing or any other form of the molecular characterisation of the genome of the species in question (Bardakci 2001).
RAPD markers have found a wide range of applications in sex chromosomes differentiation (Iturra et al 1998), genetic inheritance identification (Elo et al 1997), gene mapping (Liu et al 1999), genotoxicity in fish (Sayed et al 2013) and fish conservation (Dioh et al 1997), RAPD has been widely applied. As a molecular instrument for investigating genetic interactions and divergence, the technique has been useful in studying several aquatic species such as Artemia species (Yi et al 1999) Anguilla species (Takagi and Taniguchi 1995), Penaeus monodon (Tassanakajon et al 1997) as well as fish species including Salmo salar (Elo et al 1997), Symphysodon species (Koh et al 1999). A study on Mugil cephalus was also reported by Suresh et al 2013. Asagbra et al (2014) used similar procedure for cichlids and mud catfishes, while Ambak et al (2006) studied the snakehead fish in line with this protocol.
Although the RAPD method is relatively fast, cheap and easy to perform in comparison with other methods that have been used as DNA markers, the issue of reproducibility has been of much concern since the publication of the technique. In fact, ordinary PCR is also sensitive to changes in reaction conditions, but the RAPD reaction is far more sensitive than conventional PCR because of the length of a single and arbitrary primer used to amplify anonymous regions of a given genome (Bardakci 2001). Despite the reproducibility problem, the RAPD method will probably be important due to the speed, cost and efficiency of the RAPD technique to generate large numbers of markers in a short period as long as other DNA-based techniques remain unavailable in terms of cost, time and labour.
The present investigation in a moderate laboratory, involves the assessment of genetic variation in Lutjanus goreensis and Pomadasys jubelini ppopulations from a high (Lagos Lagoon) and low (Badagry Lagoon) brackish water bodies using RAPD profiling as the molecular tool. The data would help to identify and detect if environment influences the genetic characteristics of the selected fish species thus supplying useful facts for better understanding of not only breeding and systematics, it would also provide baseline information in studies relating to conservation, evolution and ecology.
Badagry Lagoon (Figure 1), which is approximately 60 km long and 3 km wide, lies between longitude 3°0 and 3°45 E and latitude 6°25 and 6°30 N. It is part of a continuous system of lagoons and creeks lying along the coast of Nigeria from the border with the Republic of Benin to Niger Delta with depth of the water ranging from one meter to three meters (1 – 3 m). It is also characterized by freshwater and low brackish water situations for most of the year (Ezenwa and Kusemiju 1985). Soyinka and Ayo-Olalusi (2009) reported a maximum salinity of 8.0 ‰ in the lagoon. It is influenced by tides and floods from Lagos Lagoon and Cotonou harbour through Lake Nokue and Lake Porto-Novo. River Yewa is the major river emptying into the lagoon and has rivers Isalu and Ijomo as tributaries. Creeks connected to the lagoon include Bawa and Doforo. (FAO 1969).
The Lagos lagoon (Fig.1) is the largest of the four lagoon system of the gulf of Guinea and also the largest of the eight lagoons systems of Nigeria. It stretches for about 257km from Cotonou in the republic of Benin to the western edge of the Niger delta. Two factors, fresh water discharge from rivers and tidal sea water incursion influence the biological physical and chemical characteristics of the Lagos lagoon (FAO 1969). Soyinka (2011) recorded a maximum of 23.0 ‰ salinity in the lagoon.
Samples of L. goreensis and P. jubelini were collected from Lagos and Badagry Lagoons (Figure 1). They were obtained from the catches of artisanal fishermen in two locations. The specimens were chilled in an ice chest immediately after collection and were frozen as soon as possible after collection.
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| Figure 1. Map showing sampling sites Source: Soyinka et al (2010) |
Muscle tissue was aseptically collected from the dorsal part of the fish specimens with a sterile lancet after removal of fins and preserved in 95% ethanol, which was then stored at -20°C until DNA was extracted.
Extraction of DNA followed the protocol described by Miller et al (1988) which involved salting out to obtain DNA from each of the fish specimens.
DNA yield was determined with a nanodrop spectrophotometer (NANO 1000, China) based on maximum absorbance of DNA at 260 nm. 1 µL of the DNA sample was applied on the platform of the nanodrop spectrophotometer and a reading was taken after adjustment of absorbance to zero using water as blank. The yield was measured in ng/µL. The 260 nm / 280 nm ratio was obtained to give an analysis of the purity of the sample and the concentration of the extracted DNA was also found.
Amplification reaction was performed in 50 ml volume mixtures consisting of Polymerase Chain Reaction buffer (50 mM KCl, 0.1% Triton X-100,10 mm Tris-HCl pH 8.3, 1.5 mM MgCl2), 2.5 mM dNTP (BioBasic, Canada), 5.0 μm of each RAPD primers, 50 ng of template DNA and 3U Taq DNA polymerase. A single primer was used in each PCR reaction. Amplifications of DNA fragments were carried out by using a thermal cycler (Hamburg, Germany) with the following cycling profile: pre-denaturation at 94°C for 4 min, followed by 35 cycles of amplification (1 min denaturation at 94°C, 1 min annealing at 36°C and 1 min extension at 72°C). The process concluded with extension at 72°C for 10 min. analysis of the resultant amplification products was done at 100V for 4 h with 1.8% agarose gel electrophoresis (BioRAD, USA) using TBE 1× buffer (0.9 M Tris, 0.9 M Boric acid and 20mM EDTA, pH 8.3). Furthermore, a DNA size criterion of 100 bp molecular weight marker was used. In order to visualize the amplified products with a digital camera, ethidium bromide was used to stain them.
Agarose gel (1.5 gm /100ml) was prepared in pH 8.0 buffer which contained 89 mmol of Tris-borate, 2 mmol of EDTA and 89 mmol of boric acid. After mixing the DNA samples with loading buffer, they were electrophoresised at 50 volts for 1 hour. Afterwards, agarose gel was stained with ethidium bromide (0.5 µg/ml) for 30 minutes and then photographed on U.V light with digital camera. RAPD-PCR technique can often produce non-reproducible amplification product (Callejas and Ochando 2002).
The RAPD Polymerase Chain Reaction (PCR) banding patterns generated with the primer were analyzed using Phyllip software (version 2.1, USA). Existence or non-existence of amplicons in each lane of Agarose Gels was premised on scores recorded in binary format. Scores were exclusively allotted only to the intense and reproducible bands that ranged between 400 and 1200 bp. This was done to maintain consistency across the samples of different populations. A band that occurred was noted as “1” while the absent band was marked as “0”. Parallel comparison of the amplified products in the gel with standard molecular size marker (100 bp DNA ladder) gave an estimation of molecular sizes of the RAPD products. The program was fed with the resultant data to convert the polymorphic bands into dice distance. Dendrograms were thereafter produced by the unweight pair group method using arithmetic (UPGMA) average clustering. Finally, gel Images were used to analyze banding patterns.
DNA was eventually extracted from the total of 16 specimens L. goreensis and 9 specimens of P. jubelini. Purity of DNA extracted from the analyzed samples ranged between 1.52 and 1.98 (L. goreensis) and 1.68 and 1.75 (P. jubelini). Mean DNA yield and purity were 112.6 ± 9.31ng/µl and 1.61 ± 1.48 for L. goreensis and 133.52 ± 41.4ng/µl and 1.68 ± 0.74 for P. jubelini from Lagos and Badagry Lagoons respectively. These values were 115.2 ± 9.64 ng/µl and 1.67 ± 0.58 ng/µl for L. goreensis and 168.4 ± 41.24ng/µl and 1.69 ± 1.78 for P. jubelini from Lagos and Badagry Lagoons respectively. Therefore, the samples were in pure condition without contamination of protein and RNA.
The primer generated bands in the range of 500 to 2,000 bp for both species studied. However, only the repeatable major bands ranging between 400 and 1200 bp were scored for consistency. A total of 49 reproducible bands in L. goreensis and 36 in P. jubelini were obtained (Plates 1 and 2). This study revealed a minimal disparity of polymorphic loci (25.76% and 20%) in both species amid the two populations considered.
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| Plate 1. Banding pattern of the specimens of L. goreensis produced by RAPD using OPAS 13 primer. M = 100 bp marker; Lanes 1, 6 & 12 = Blank; Lanes 2 - 5, 7- 11 = SL1 - SL4, SL5 - SL8; Lanes 12- 19 = SB1-SB8 |
Plate 2. Banding pattern of the P. jubelini specimens of Grunters produced by RAPD using OPAS 13 primer. Lane10 = 100 bp marker; Lanes 1, 2, 3, 4 and 5 = GL 1-GL5; Lanes 6-9= GB1-4. |
Dendrograms were constructed after data generated from the RAPD primer band analysis. UPGMA cluster analysis of the similarity matrix based on RAPD primer analysis separated the Snappers into two major clusters (Figure 2), while the grunters were separated into three major clusters (Figure 3), which consists of minor clusters at various degree of co-efficient phylogenetic analysis. The 1st cluster in L. goreensis consist of SL2, SL1, SL3, SB5, SB3, SB4 and SL4 while the second cluster consists of SB5, SL5, SL4, SB6, SB1,SB4, SL8, SL7, SB2 and SB7. The 1st cluster in P. jubelini consists of GL1 and GB3, the second cluster consists of GL4 and GB2 while the third cluster consists of GL2, GB4, GB1 and GB3.
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| Figure 2. UPGMA dendrogram for two populations of Lutjanus goreensis from Lagos and Badagry Lagoons |
Figure 3. UPGMA dendrogram for two populations of Pomadasys jubelini from Lagos and Badagry Lagoons |
The extracted DNA from both species had high yield and purity. This is synonymous with the result of several studies carried out on fishes and shellfishes including Ambak et al (2006); Danish et al (2012) and Asagbra et al (2014). A degree of reliability is therefore established that the protinase k method is apposite for the extraction of DNA from samples of Grunters.
The use of randomly amplified polymorphic DNA (RAPD) procedure to analyse genetic dissimilarity and to produce DNA fingerprints of location-specific populations of Snappers and Grunters has not been attempted in Nigeria and this study will pave a way for future studies on analysis of the effect of different environments on the genome of this fish species. By extension, this can also be applicable to other economically important fishes and shellfishes.
The results of RAPD profiles showed slightly differentiated fingerprints of Snapper and Grunter populations. So discrimination among the tested populations was not easy. The RAPD procedure in this study did not establish a clear difference between the species from different locations. This lack of clearly established disparity may have resulted from the use of few primers or few numbers of specimens in the study. However, according to Bardakci (2001), the simplicity and applicability of the RAPD technique have captivated the interest of many scientists. Perhaps, the main reason for the success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the requirement for cloning, sequencing or any other form of the knowledge of the molecular characterisation of the genome of the species in question. But it is recommended that additional studies which will utilize copious newer and updated primers that can operate without a reference sequence such as GBS (Genotyping by Sequence) and RAD-seq (Restriction site Associated DNA Sequencing) as well as increased numbers of fish specimens in order to, more accurately, identify the similarities and variations in the populations of these species not only from Lagos and Badagry Lagoons but from other crucial locations, be employed. According to Bidochka et al (1994), the use of several RAPD primers can also diminish the risk of misinterpretation. Despite being based on the method of Miller et al (1988), results from this study agreed with several studies that used other methodology (Williams et al 1990).
One primer that presented no amplified fragments in both species, despite several repeats, was OPAS 14. This could be because it lacks complementary sequences among the genomic DNA extracted from the fish specimens (Devos and Gale 1992). The number of bands on the Agarose gel depends on the number of appropriately oriented and target sites present in DNA of the species. The result from this study on the number of polymorphic bands is compatible with that of Stacey et al (2007). Ferguson et al (1995) noted that these population-specific unique bands can be used to detect any probable population integration. Population-specific bands were reported by a number of researchers including Tassanakajon et al (1999); Mishra et al (2009) and Nagarajan et al (2006).
Cluster analysis did not exhibit sufficient genetic discrepancy to distinguish the samples of differing populations of the species studied. In this context the dendrograms indicated insignificant dissimilarity between the species of both locations. The present study revealed that the two different Snapper and Grunter populations were precisely similar species as shown in the RAPD molecular profiles. This result, according to Al-Hassan (1985) is congruent with many morphological and allozyme studies including Fakunmoju et al (2014) who also noted an absence in taxonomic variation among species of L. goreensis sampled from Badagry and Lekki Lagoon based on morphometric and meristic characters.
This also agreed with the report of Da Silva et al (2010) who concluded that populations of marine fish (Atherinella brasiliensis) from southern Brazil displayed minimal dissimilarity and genetic configuration. On the other hand, Bielawski and Pumo (1997) detected sufficiently high levels of variation among four populations of the Atlantic coast striped bass (Morone saxatilis) from the Atlantic coast of North America. The result from this study is also opposed to that of Welsh and McClelland (1990) and Hadreys et al(1992).
Result from this study concerning genetic variation among different populations of the species could be in line with the hypothesis of Chapman et al (1999), that there may be no relationship between genetic differences and geographical distances among sites. There is a possibility that analogous locations, despite geographic disparity, may show harmonized populations. Also, the isolation time and/or the number of generations may not be adequate to facilitate a possible dichotomy and genetic structuring between the specimens of these two places. Outcome of the present investigation is very important because it is probably a first approach to the genetic diversity of L. goreensis and P. jubelini in Lagos and Badagry Lagoons. Knowledge of the genetic variability is crucial because ignorance of the distribution pattern in the wild and variations between the individuals of a species could render conservation and preservation programs almost unachievable. This information is also useful to farmers in coastal communities and the government in the rational cultivation of these two species as pure population inspite of the differences in locations. However in future, more RAPD primers must be used in order to obtain greater basis for the assessment of genetic characteristics of the species.
The authors appreciate the assistance of Dr. Iwalomo in the laboratory of Nigerian Institute of Medical Research, Yaba, Lagos; Dr. Sifau, Department of Cell Biology and Genetics, University of Lagos, for constructive criticisms and reading of the proof.
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Received 15 November 2016; Accepted 23 November 2016; Published 1 January 2017