RAPD-evaluated Genetic Polymorphisms and Relations among three River Nile Catfish (Siluriformes) Species from Qena, Egypt

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The catfish (whisker-like barbells) are a diverse group of fishes containing 4100 species, and represent 12% of teleosts counting for 6.3% of known vertebrates species.The catfish (Siluriformes) have a tremendous economic value for being an important dietary proteins source (Ferraris, 1999;Eschmeyer and Fong, 2014;Wilson and Reeder, 2005) and are significantly important species for aquaculture purpose worldwide (Kim, 1997;USDA, 2002;Buton, 1979;Aremu and Ekunode, 2008).
RAPD is a PCR-based assay utilizing a single short decaoligonucleotide sequence to amplify a complementary arbitrary fractions (markers) of a particular genome (Williams et al., 1990;Welsh and McClelland, 1990).RAPD markers have been improved significantly to observe genetic variations and been widely used in fish population genetic studies, wild and cultured fish stocks and inter/intraspecific fish genetic variation (You et al, 2007;Mamuris et al., 1999a;Islam et al., 2007;Abdul Muneer et al., 2009;Rahman et al., 2009;Danish et al., 2012;Popoola et al., 2015;Verma and Serajuddin, 2017).
There are various fish species to be considered for evaluation as aquaculture potentials.Nevertheless, the successful conservation and management of species will be based on comprehensive knowledge of their genetic structure, relatedness and pattern of genetic variation (Allendorf and Phelps, 1981).Thus, using diverse molecular genetic tools will be important for understanding and management of a particular fish species for farming and culturing purpose.The current work aimed to investigate the genetic polymorphisms and relations among three River Nile catfish (Siluriformes) species; Schilbe mystus Linnaeus 1758, Bagrus bajad Forsskål 1775, and Clarias gariepinus Burchell 1822 using RAPD-PCR fingerprint analysis.

MATERIALS AND METHODS Fish Sampling :
The three River Nile catfish (Siluriformes) species (Fig. 1) used in this study were collected by the author from fish market (Elsehreg), Qena, Egypt during August/October 2017, brought to the laboratory and were placed down to species level (Bailey, 1994;Bishai and Khalil, 1997;FishBase 2017).Tissue samples (muscle, fins and scales) were individually preserved at -20°C for genomic DNA extraction and future analyses.

Fish Genomic DNA Extraction:
Genomic DNA from each fish sample (~30mg of muscle tissues) was extracted using EZ-10 spin column genomic DNA extraction kit for animal tissue (Bio Basic Inc., Canada) according to the manufacturer's guidelines.The concentrations and clarity of DNA were approximated under spectrophotometric UV absorption at A260 and A280 and 1% agarose gel.DNA samples were stored at −20 °C.

Data Analysis:
For RAPD valuation, RAPD images were analysed for polymorphic DNA bands using the PyElph gel image analysis program (Pavel and Vasile, 2012).DNA fingerprints were scored for the presence/absence (1/0) of RAPD bands of identical molecular sizes in a binary matrix.The POPGENE version 1.32 (Yeh et al., 1999) was used to analyse the binary matrix data to compute the Nei's original measures of genetic identity and genetic distance (Nei, 1972).A dendrogram was constructed based on Nei's original measure of genetic distance using the unweighted pair group method average 'UPGMA' clustering method (Sneath and Sokal, 1973), and displayed with the Molecular evolutionary genetics analysis Version 6.0 (MEGA6) software (Tamura et al., 2013).For each primer used, the total number of scored bands, number of polymorphic bands, and number of monomorphic bands were recorded.

RAPD evaluated genetic polymorphism :
A set of 8 deca-nucleotide primers were monitored for the capability to generate fingerprint banding pattern and to assess polymorphism among three catfish species (Table 1).All primers produced a total of 52 amplified bands with an average of 6.5 bands per primer from which 5 bands were common exhibiting low level of monomorphism of % 9.62, and 47 bands were polymorphic displaying high level of polymorphism of 90.38 % with an average number of polymorphic fragments/primer of 5.9.An instructive RAPD fingerprint profile was generated by the 8 primers with various band size lengths ranging from 200 to 2900 base pair comparing to a 100 bp DNA Ladder (Solis Bio Dyne).
As shown in Table 1, out of 8 primers used; primers A-06, A-05, and A-11 produced higher number of 9, 8 and 7 bands respectively compared to other primers.The highest number of polymorphic bands (9) was obtained with primer A-06 while the lowest ones (2) was obtained with primer A-12.The band frequency per species were 0.4615, 0.4423 and 0.5000 for Schilbe mystus, Bagrus bajad and Clarias gariepinus respectively while band frequency for each primer ranged from 0.0577 to 0.1731 as depicted (Fig. 2).The RAPD genotyping profile obtained for catfish (Siluriformes) under study with 8 tested primers is shown in Fig. 3.   Nei's original measures of genetic identity and genetic distance was used to construct a genetic distance and identity matrix (Table 2).As shown in the table, the lowest distance values of 0.8157 (high identity of 0.4423), between Schilbe mystus and Bagrus bajad, while the highest value of 1.0068 (low identity of 0.3654), between Bagrus bajad and Clarias gariepinus.Accordingly, Schilbe mystus and Bagrus bajad are closest to each other, while Bagrus bajad and Clarias gariepinus are distant to each other.The UPGMA phylogentic tree based on Nei's original measures of genetic distance was constructed (Fig. 4), accordingly revealed that the studied catfish (Siluriformes) segregated into two main clusters distinguishing both Schilbe mystus and Bagrus bajad in single cluster as sister group that possess a closer position and share a common node (node B).While, the Clarias gariepinus split up in a separate group forming sister clade to Schilbe/Bagrus group.The phylogenetic tree shows that Schilbe mystus is more closely related to Bagrus bajad than to Clarias gariepinus and all the three catfishes species share common ancestor (node A).
Table 2. Nei's (1972) original measure of genetic distance and identity from three studied catfish species computed for RAPD data.Nei's genetic identity (above diagonal) and genetic distance (below diagonal).

DISCUSSION
Herein, RAPD-PCR assay with deca-oligonucleotide primers was applied to amplify diverse DNA fragments (RAPD markers/loci) of genomic DNA from three River Nile catfish species.These loci are inherited in Mendelian manner (Rothuizen and Van Wolferen, 1994).The presence or absence of a particular DNA fragment signify the dominant or recessive allele fashions (Williams et al., 1990;Welsh and McClelland, 1990).Results obtained here demonstrated the capability of the used randomly primers to generate an instructive RAPD banding patterns and to assess polymorphism among catfish species under study.Because of PCR arbitrary primers are of valuable in spotting polymorphic genetic markers (Hunt and Page, 1992).
From a total of 52 amplified markers/bands produced, 5 bands were common exhibiting low level of monomorphism (%9.62).While 47 bands were polymorphic displaying high level of polymorphism (90.38 %) which suggest existence of interspecies high genetic variations among studied catfish species.Some studies reported that a higher polymorphism symbolize higher genetic variation, while the low polymorphism indicate the opposite (Yoon and Park, 2002;Rahman et al., 2009;Yusuf et al., 2017).
The highest primer band frequency of 0.1731 was recorded for primer A-06, while the lowest one of 0.0577 with primer A-12 which suggested high and low occurrence of both primers binding sites receptively within genomes of the studied catfish.Data showed detection of high molecular size fragment of ~ 2900 bp that was recorded only for Clarias gariepinus with primer A-06.This may consider primer-specific band for Clarias gariepinus indicating its internal genotype-specific variation.
The RAPD fingerprint obtained for the thee River Nile catfish (from natural populations) under study displayed various band size lengths ranging from 200 to 2900 base pair which could be reasonable with observations by other studies applied on cultured and wild fish population (Liu et al.,1999;Ambak et al., 2006;Abd El-Kader et al., 2013;Mostafa et al., 2009;Popoola et al., 2014).
The RAPD fingerprints of studied fresh water catfish would be of help revealing their interspecies genetic variation and genetic relationship based on presence or absence of identical size RAPD markers.Result of the UPGMA tree-based Nei's genetic distance (Fig. 3) revealed two segregated main clusters where Schilbe mystus is genetically more close to Bagrus bajad forming sister group (node B), than to Clarias gariepinus that is separately positioned as sister clade sharing common ancestor to them (node A).This may indicate more genetic makeup resemblance between Schilbe mystus and Bagrus bajad.For that Schilbe mystus is more closely related to Bagrus bajad than to Clarias gariepinus.
Studies reported the capability of RAPD assay to evaluate various genomic loci and facilitate analysis of genetic distance and phylogenetic relationships (Clark and Lanigan, 1993), as well as the genetic variation and level of similarity among fish species (Barman et al., 2003).The separation of Clarias gariepinus as sister clade to both Schilbe mystus and Bagrus bajad may point to the monophyly of family clarridae which supported by other studies (Agnese and Teugels, 2005;Sullivan et al., 2006;Pouyaud et al., 2009;Yu and Quilang 2014).In conclusion, results showed the consistency and informative efficiency of RAPD PCR fingerprint technique in fish phylogenetic studies down to the species level and produced characteristic DNA fingerprint profiles for the three studied River Nile catfish species.

Fig. 3 :
Fig. 3: Dendrogram demonstrating the genetic relationships among studied catfish (Siluriformes) based on estimation of Nei's genetic distance and identity using RAPD data (Table2) and clustered using the UPGMA method.The branch lengths and tree nodes A, and B are shown.

Table 2 )
and clustered using the UPGMA method.The branch lengths and tree nodes A, and B are shown.