Comparative study of single nucleotide polymorphisms (SNPs) of a candidate growth gene (IGF-I) in Oreochromis niloticus and Sarotherodon melanotheron

In teleost fishes, the regulation of growth performance by the GH and IGF system also seems to be highly conserved. Adult Oreochromis niloticus (Nile tilapia) could reach up to 60 cm maximum length while Sarotherodon melanotheron (black chin Tilapia) has a maximum length of 28 cm. This study describes the analysis of Single Nucleotide Polymorphism in isolated, amplified, and sequenced DNA from two common Tilapia species (O. niloticus and S. melanotheron) with the aim of identifying genetic variation and single nucleotide polymorphisms (SNPs) in one of the main genes, Insulin like growth Factor-1 (IGF-I), related to growth in the Tilapia species. The extracted DNA from the clipped caudal fins of the Tilapia species samples using Sambrook and Russell’s modified chlolorophenol/isoamyl alcohol DNA extraction method were further amplified in a thermal cycler with designed IGF-I forward and reverse primers of 447 bp which were subsequently sequenced with an automated analyzer. The PCR product was separated on 1.5% ethidium stained agarose gel electrophoresis and the bands revealed on the gel were all of the same length (447 bp). The Sequence alignment revealed a total of five single nucleotide polymorphisms which were detected in the forward reaction at the positions 181, 199, 328, 362 and 369 of the sequences and in the reverse reaction at positions 18, 20, 54, 183 and 201. A total of 138 amino acid sequence was translated from the DNA sequence with variations sequence at positions 1, 3, 4, 61, 67, 121 and 123. These results showed variations among these two fish species which could explain differential growth performance between them.


INTRODUCTION
Single nucleotide polymorphism (SNP) markers are the method of choice for genetic analyses including diversity and quantitative trait loci studies (Thu et al., 2017).
Determination of the genetic variability was described by Lupchinski Jr et al. (2011) as an essential step for the implementation of genetic improvement programs that are focused on the selection of faster growing fish with lower feed conversion rates and resistant to diseases. Studies of genetic diversity at DNA level represents in expansion field in aquaculture aimed at finding out those DNA variations associated with productive phenotypes, so as to use them as tools for assisting the offspring selection at early stage and possibly predict their performance (Na-Nakorn and Moeikum, 2009). This strategy is known as gene-assisted selection (GAS) (De-Santis and Jerry, 2007). Growth performance is often used as an indicator of the status of individuals and populations in culture and the wild, and therefore, major effort has been applied towards garnering a more comprehensive understanding of how multiple components of the GH (growth hormone) and IGF (insulin-like growth factor) system interact to control growth and metabolism (Picha et al., 2008;Beckman, 2011). To improve growth and growth efficiency in aquaculture, an advanced understanding of the physiological mechanisms that regulate growth in fishes are needed. The growth hormone/insulin-like growth factor (GH/IGF) in the endocrine axis regulates growth in all vertebrates, including fishes as described by Davis et al. (2008).
Tilapia is the common name for nearly a hundred species of cichlids from the tilapinne cichlid tribe. Tilapia are mainly freshwater fish inhabiting shallow streams, ponds, rivers and lakes and less commonly found living in brackish water. The survey carried out by Oguntade et al. (2014) shows that some fish species including Tilapia are fast disappearing in Nigerian water bodies such as Brass and Nun River of Niger Delta.
In 2017, according to FAO statistics, Nile tilapia (Oreochromis niloticus) culture alone was ranked fourth among the most cultured in the world, in terms of both production and value with a total aquaculture production of 4.1 million tonnes (FAO, 2019). The other top four species were silver carp, grass carp, common carp and other Cyprinids (FAO, 2019). Nile tilapia represents approximately 86% of total global tilapia production (FAO, 2019). In 2017, it is anticipated that global Nile Tilapia production will reach nearly 4 million tonnes (FAO, 2017). Adult Oreochromis niloticus (Nile tilapia) reach up to 60 cm maximum length while Sarotherodon melanotheron (black chin Tilapia) has a maximum length of 28 cm (Olaosebikan and Raji, 1998) when subjected to the same environmental condition. This justifies the higher value and demand for Nile tilapia, hence higher production of other Tilapia species is needed to meet the demand for Tilapia. Black chin Tilapia on the other hand thrive well in high salinity region but are constrained by their growth and as such do not meet market value. Since IGF-I also regulate growth in fishes; the need to study its Sokenu et al. 547 variation in the two Tilapia species arises with the aim of identifying a possible growth factor that will promote a higher production of S. melanotheron from saline environment to complement the production of O. niloticus from fresh water bodies to meet global tilapia demand. This would be achieved by detecting the genetic variations and single nucleotide polymorphisms (SNPs) in one of the main growth genes, Insulin like growth Factor-1 (IGF-I) in two Tilapia species.

DNA extraction and amplification
DNA was isolated from the caudal fin tissue of the ten sampled fish using modified chlorophenol/ isoamyl/alcohol protocol according to Sambrook and Russell (2001) on bench at biotechnology laboratory of NIOMR, Badore outstation, Nigeria. The integrity of the DNA was checked on 1% ethidium bromide stained agarose gel electrophoresis and the isolate was stored at -20°C prior PCR amplification.
The PCR amplification was run with the specific primers (IGF-I forward-5'-CTTGGACGAGTAGGAGGCAAATG-3' and IGF-I reverse-3'-GAAATACAAGCAAGCGATAAGAA-5') of 447 bp designed to amplify coded regions (exons) of the IGF-I gene sequences (IGF-I, GenBank accession AF033797) which was resequenced and used. The DNA amplification was carried out by polymerase chain reaction (PCR) in a with 20 ng of genomic DNA using Thu et al. (2017) protocol, 20 ul reactions containing 0.2 uM of each primer, 200 uM of dNTPs, 50 mM KCL, 10 mM Tris HCL (pH 8.3), 1.5 mM MgCl2 and 0.5 units of Taq DNA polymerase with Eppendorf thermocycler with an amplification profile of initial denaturation at 95 o C for 10 min, followed by 35 cycles with 95 o C for 30s, annealing temperature at 60 o C for 45s, extension at 72 o C for 45s and final extension at 72°C for 5 min. The product was checked on 1.5% agarose gel electrophoresis at 70v for 1.5 h in 1x TBE buffer and the gel was stained with ethidium bromide for visualization through Fisher Scientific UV transilluminator.

DNA sequencing
Purified PCR products from the amplification of the ten Tilapia fishes, O. niloticus (5) and S. melanotheron (5) were bidirectional sequenced in an automatic sequencer (ABI 3500XL Genetic Analyzer).
Nucleotide sequences obtained were edited and aligned using clustal O (version1.2.4) multiple sequence alignment software, the Single nucleotide Polymorphisms (SNPs) were discovered by visual analysis and dendogram was also created while translation of the DNA sequence of each species was done using biolign alignment software (version 4.0.6.2).

DNA extraction and amplification
The IGF-I amplified PCR product of the extracted DNA run on 1.5% ethidium bromide stained agarose gel demonstrated that IGF-I genes had the same bands which demonstrated equal fragment length of the ten Tilapia fishes, O. niloticus (5) and S. melanotheron (5) as shown on Plate 1, where M is the known 50 bp-10 kb DNA ladder.

DNA sequencing and analyses
The sequence alignment generated for the forward and reverse primers of IGF-1 of the ten tilapia fish (5 Oreochromis niloticus and 5 Sarotherodon melanotheron) were shown in Figures 1 and 2  The dendogram revealed lower similarities between SM1-SM5 and ON1-ON5 and higher similarities among SM1-SM5 and among ON1-ON5 (Figure 3). The lower similarity between SM and ON might imply a high genetic variation and could be due to the fact that they are different species and do not have common ancestry. This finding is on the contrary with the report of Usman et al. (2013) who obtained a high similarity coefficient of 78% between T. guineensis and S. melanotheron from the wild.
A total of 138 amino acid sequence was translated from the DNA sequence of O. niloticus and S. melanotheron as shown in Figures 4 and 5. The alignment of these sequences revealed seven (7) Variations at positions 1, 3, 4, 61, 67, 121 and 123 as shown in Figure 6.
The variations observed are as follows; position 1 R (arginine) in SM to V (valine) in ON, position 61 Q (glutamine) in SM to *(stop codon) in ON (Table 3).
This study described the use of Single Nucleotide Polymorphism (SNP) markers to validate genetic variation in a candidate growth gene (IGF-I) in O. niloticus and S. melanotheron. The DNA sequences gotten from primer used in this study was about 447 bp in agreement with the result obtained by Cuevas-Rodríguez et al. (2016).
Among the growth genes, IGF-I is said to contribute in a variety of physiological processes, such as growth, metabolism, reproduction and osmoregulation (Reinecke et al., 2005) in teleosts. Thus variation of IGF-I might be a good reason for the growth difference between O. niloticus and S. melanotheron.