Provided for non-commercial research and education use.

Not for reproduction, distribution or commercial use.

Vol. 10 No. 1 (2018)

Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Physiology & molecular biology journal is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers that elucidate important biological, chemical, or physical mechanisms of broad physiological significance. www.eajbs.eg.net

Citation :Egypt. Acad. J. Biolog. Sci. ( C. physiology and Molecular biology ) Vol.10(1)pp9-21 (2018)

Egypt. Acad. J. Biolog. Sci., 10(1): 9- 21 (2018) Egyptian Academic Journal of Biological Sciences C. Physiology & Molecular Biology ISSN 2090-0767 www.eajbs.eg.net

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

Mohammed Bassyouni M.EL-Mahdi Laboratory of Molecular Genetics and Molecular Biology, Zoology Department Faculty of Science, South Valley University, Qena, Egypt E-mail address: [email protected] (M. ELMahdi)

ARTICLE INFO ABSTRACT Article History Genetic discrepancies among three River Nile catfish; Received: 25/12/2017 mystus Linnaeus, 1758 (), Bagrus bajad Accepted: 30/1/2018 Forsskål, 1775 (Bagaridae) and Clarias gariepinus Burchell, 1822 ______(Claridae) was evaluated by RAPD-PCR technique using 8 deca Keywords: oligonucleotides (A-03, A-04, A-05, A-06, A-09, A-10, A-11, and Genetic polymorphism, A-12). A total of 52 amplified bands were produced, from them 5 Catfish, Siluformes, bands were common with level of monomorphism of % 9.62, and RAPD-PCR, Qena, 47 bands were polymorphic with level of polymorphism of 90.38 River Nile, Egypt %. An instructive RAPD fingerprint profile was generated with various band size lengths ranging from 200 to 2900 base pair. According to Nei-72 distance matrix of genetic and the unweighted pair group method average (UPGMA), the three studied catfish species are related to each other and likely have the most sharing common ancestor. However, Schilbe mystus (Schilbeidae) and Bagrus bajad (bagaridae) are genetically more close to each other than to Clarias gariepinus. The subset of Clarias gariepinus as sister clade to Schilbe mystus and Bagrus bajad suggested monophyly of the family Clarridae. The study suggested occurrence of genetic variations among the investigated River Nile catfish, with high level of genetic convergence/relation between Schilbe mystus and Bagrus bajad. This probably be useful for enhancing their potential use in aquaculture breeding programs, as well as providing insights on their taxonomic status within the Siluriformes.

INTRODUCTION Genetic variation (polymorphisms) caused by mutational alterations are necessitated for species survival and adaptations in diverse environmental conditions (Fisher, 1930).The patterns of genetic discrepancy to species level can be exposed using tools of molecular genetic markers (Linda and Paul, 1995). Such information of molecular markers-based on decipherable polymorphism at DNA and protein levels are source of knowledge for several research applications including molecular

Citation :Egypt. Acad. J. Biolog. Sci. ( C. physiology and Molecular biology ) Vol.10(1)pp9-21 (2018)

10 Mohammed Bassyouni M.EL-Mahdi

phylogeny, conservation, McClelland, 1990). RAPD markers have breeding, management of been improved significantly to observe populations, and monitoring of farmed genetic variations and been widely used species stocks (Perkins and Krueger, in fish population genetic studies, wild 1993; Ferguson et al., 1995; Liu and and cultured fish stocks and Cordes, 2004). inter/intraspecific fish genetic variation The catfish (whisker-like barbells) (You et al, 2007; Mamuris et al., are a diverse group of containing 1999a; Islam et al., 2007; Abdul ,.׽4100 species, and represent ׽12% of Muneer et al., 2009; Rahman et al teleosts counting for ׽6.3% of known 2009; Danish et al., 2012; Popoola et vertebrates species. The catfish al., 2015; Verma and Serajuddin, 2017). (Siluriformes) have a tremendous There are various fish species to economic value for being an important be considered for evaluation as dietary proteins source (Ferraris, 1999; aquaculture potentials. Nevertheless, the Eschmeyer and Fong, 2014; Wilson and successful conservation and management Reeder, 2005) and are significantly of species will be based on important species for aquaculture comprehensive knowledge of their purpose worldwide (Kim, 1997; USDA, genetic structure, relatedness and pattern 2002; Buton, 1979; Aremu and Ekunode, of genetic variation (Allendorf and 2008). Phelps, 1981).Thus, using diverse Clarias gariepinus Burchell, 1822 molecular genetic tools will be important (Claridae) is omnivorous predatory fish for understanding and management of a widely distributed throughout African particular fish species for farming and aquatic systems and possess diverse culturing purpose. The current work habitat preferences, rapid growth as well aimed to investigate the genetic as withstanding to severe water polymorphisms and relations among conditions (Welcome, 1979; Bruton, three River Nile catfish (Siluriformes) 1986; Nwadukwe, 1995). Schilbe mystus species; Schilbe mystus Linnaeus 1758, Linnaeus, 1758 (Schilbeidae) is Bagrus bajad Forsskål 1775, and Clarias omnivorous surface feeders native to gariepinus Burchell 1822 using RAPD- African fresh water lakes possessing PCR fingerprint analysis. rapid growth rate and becoming commercially important fish species MATERIALS AND METHODS (Omondi, and Ogari 1994; Adedolapo Fish Sampling : 2007; Amer et al., 2008; Ayoade et al., The three River Nile catfish 2008; Kareem et al., 2015). Bagrus bajad (Siluriformes) species (Fig. 1) used in Forsskål, 1775 (Bagaridae) is this study were collected by the author carnivorous fish widely distributed in from fish market (Elsehreg), Qena, Egypt African freshwater systems (Risch, 1986) during August/October 2017, brought to being commercially importance and the laboratory and were placed down to potential aquaculture candidate (El- species level (Bailey, 1994; Bishai and Drawany and Elnagar, 2015; Alhassan Khalil, 1997; FishBase 2017). Tissue and AnsuDarko, 2011). samples (muscle, fins and scales) were RAPD is a PCR-based assay individually preserved at -20°C for utilizing a single short deca- genomic DNA extraction and future oligonucleotide sequence to amplify a analyses. complementary arbitrary fractions Fish Genomic DNA Extraction: (markers) of a particular genome Genomic DNA from each fish (Williams et al., 1990; Welsh and sample (~30mg of muscle tissues) was RAPD-evaluated Genetic Polymorphisms and Relations among three River Nile Catfish 11 extracted using EZ-10 spin column 8), stained with ethidium bromide (0.5 genomic DNA extraction kit for animal µg/ml) and imaged under UV light tissue (Bio Basic Inc., Canada) according (Elttrofor M20 SaS Photo-Gel System, to the manufacturer’s guidelines. The Italy) with Nikon Coolpix LB40 digital concentrations and clarity of DNA were camera. Products size was compared with approximated under spectrophotometric 100 bp DNA ladder (0.1 µg/µl, Solis UV absorption at A260 and A280 and BioDyne, Estonia). 1% agarose gel. DNA samples were Data Analysis: stored at −20 °C. For RAPD valuation, RAPD RAPD-PCR Assay: images were analysed for polymorphic RAPD assay was performed based DNA bands using the PyElph gel image on Williams et al., (1990). Each RAPD- analysis program (Pavel and Vasile, PCR reaction was accomplished in a 2012). DNA fingerprints were scored for final volume of 25 µl of 1.0× pre-mixed the presence/absence (1/0) of RAPD OnePCRTM 2X (GeneDireX Inc, USA), bands of identical molecular sizes in a ~50 ng genomic DNA of each sample, binary matrix. The POPGENE version and 10 pM of each deca-nucleotide 1.32 (Yeh et al., 1999) was used to primer alone. Eight (8) deca-nucleotide analyse the binary matrix data to primers were used for the DNA compute the Nei's original measures of amplification [A-03, A-04, A- 05, A-06, genetic identity and genetic distance A-09, A-10, A-11, and A-12] (Bio Basic (Nei, 1972). A dendrogram was Inc, Canda). constructed based on Nei’s original PCR amplifications were measure of genetic distance using the performed in a thermocycler (Primus 25 unweighted pair group method average advanced, PEQLAB Biotechnologie ‘UPGMA’ clustering method (Sneath and GmbH) under cycling sittings fitted an Sokal, 1973), and displayed with the initial denaturation at 95°C for 2 min, 45 Molecular evolutionary genetics analysis cycles (94°C for 1 min, 36°C for 1 min Version 6.0 (MEGA6) software (Tamura and 72°C for 2 min), then a 10 minutes et al., 2013). For each primer used, the cycle of final extension at 72°C. total number of scored bands, number of Amplification products (15 µl) were polymorphic bands, and number of electrophoresed using 1.5% (w/v) monomorphic bands were recorded. agarose gels in TAE buffer (0.40 mM Tris, 0.20 mM acetate, 2 mM EDTA pH

Fig. 1: Photographs of the three River Nile catfish (siluriformes) included in this study. These are Schilbe mystus Linnaeus 1758 (family: Schilbeidae), Bagrus bajad Forsskål, 1775 (family: Bagaridae) and Clarias gariepinus Burchell, 1822 (family: Claridae)

12 Mohammed Bassyouni M.EL-Mahdi

RESULTS 200 to 2900 base pair comparing to a RAPD evaluated genetic 100 bp DNA Ladder (Solis Bio Dyne). polymorphism : As shown in Table 1, out of 8 A set of 8 deca-nucleotide primers used; primers A-06, A-05, and primers were monitored for the A-11 produced higher number of 9, 8 capability to generate fingerprint and 7 bands respectively compared to banding pattern and to assess other primers. The highest number of polymorphism among three catfish polymorphic bands (9) was obtained species (Table 1). All primers produced with primer A-06 while the lowest ones a total of 52 amplified bands with an (2) was obtained with primer A-12. The average of 6.5 bands per primer from band frequency per species were 0.4615, which 5 bands were common exhibiting 0.4423 and 0.5000 for Schilbe mystus, low level of monomorphism of % 9.62, Bagrus bajad and Clarias gariepinus and 47 bands were polymorphic respectively while band frequency for displaying high level of polymorphism each primer ranged from 0.0577 to of 90.38 % with an average number of 0.1731 as depicted (Fig. 2). The RAPD polymorphic fragments/primer of 5.9. genotyping profile obtained for catfish An instructive RAPD fingerprint profile (Siluriformes) under study with 8 tested was generated by the 8 primers with primers is shown in Fig.3. various band size lengths ranging from

Table 1. Features of primers used to generate RAPD fingerprint profile of three River Nile catfish (Siluriformes). Number of amplified bands per species (NABands/Species), Band frequency for each primer (Band Freq/Primer), Band frequency per species (Band Freq/Species), Total number of amplified bands (TNABands), Number of polymorphic bands (NPBands), Number of monomorphic bands (NMBands), Polymorphism percentage (POL%), and Range of amplified band in base pair (RAB [bp] ).

RAPD-evaluated Genetic Polymorphisms and Relations among three River Nile Catfish 13

Fig. 2: Band frequencies computed for the three River Nile catfish species, Schilbe mystus, Bagrus bajad and Clarias gariepinus (Above) and for every used deca- nucleotide primers (below).

Fig. 3: RAPD fingerprint profiles for three River Nile catfish species using 8 different primers. MW: Molecular weight (100-3000 base pair), Sch: Schilbe mystus, Bb: Bagrus bajad, and Cg: Clarias gariepinus 14 Mohammed Bassyouni M.EL-Mahdi

Phylogentic and genetic relationship on Nei’s original measures of genetic analyses: distance was constructed (Fig. 4), Nei's original measures of genetic accordingly revealed that the studied identity and genetic distance was used to catfish (Siluriformes) segregated into two construct a genetic distance and identity main clusters distinguishing both Schilbe matrix (Table 2). As shown in the table, mystus and Bagrus bajad in single cluster the lowest distance values of 0.8157 as sister group that possess a closer (high identity of 0.4423), between position and share a common node (node Schilbe mystus and Bagrus bajad, while B). While, the Clarias gariepinus split up the highest value of 1.0068 (low identity in a separate group forming sister clade to of 0.3654), between Bagrus bajad and Schilbe/Bagrus group. The phylogenetic Clarias gariepinus. Accordingly, Schilbe tree shows that Schilbe mystus is more mystus and Bagrus bajad are closest to closely related to Bagrus bajad than to each other, while Bagrus bajad and Clarias gariepinus and all the three Clarias gariepinus are distant to each species share common ancestor other.The UPGMA phylogentic tree (node A). based

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). Schilbe Species Bagrus bajad Clarias gariepinus mystus Schilbe mystus **** 0.4423 0.3846 Bagrus bajad 0.8157 **** 0.3654 Clarias 0.9555 1.0068 **** gariepinus

Fig. 3: Dendrogram demonstrating the genetic relationships among studied catfish (Siluriformes) based on estimation of Nei's genetic distance and identity using RAPD data (Table 2) and clustered using the UPGMA method. The branch lengths and tree nodes A, and B are shown.

RAPD-evaluated Genetic Polymorphisms and Relations among three River Nile Catfish 15

DISCUSSION The band/marker frequency per Herein, RAPD-PCR assay with species were values of 0.4615, 0.4423 deca-oligonucleotide primers was applied and 0.5000 for Schilbe mystus, Bagrus to amplify diverse DNA fragments bajad and Clarias gariepinus (RAPD markers/loci) of genomic DNA respectively. Among them, Clarias from three River Nile catfish species. gariepinus recorded highest band These loci are inherited in Mendelian frequency of 0.5000 which suggested its manner (Rothuizen and Van Wolferen, higher rate of heterozygosity. This could 1994). The presence or absence of a be a reason for its adaptable phenotype, particular DNA fragment signify the rapid growth, broad habitat diversity and dominant or recessive allele fashions globally distributed as reported (e.g (Williams et al., 1990; Welsh and Winemiller and Kelso-Winemiller, McClelland, 1990). Results obtained here 1996; Thakur, 1998; Alves et al., 1999; demonstrated the capability of the used Yalçin et al., 2001b; Senanan et al., randomly primers to generate an 2004; Vitule et al., 2006). instructive RAPD banding patterns and to The RAPD fingerprint obtained assess polymorphism among catfish for the thee River Nile catfish (from species under study. Because of PCR natural populations) under study arbitrary primers are of valuable in displayed various band size lengths spotting polymorphic genetic markers ranging from 200 to 2900 base pair which (Hunt and Page, 1992). could be reasonable with observations by From a total of 52 amplified other studies applied on cultured and wild markers/bands produced, 5 bands were fish population (Liu et al.,1999; Ambak common exhibiting low level of et al., 2006; Abd El-Kader et al., 2013; monomorphism (%9.62). While 47 bands Mostafa et al., 2009; Popoola et al., were polymorphic displaying high level 2014). of polymorphism (90.38 %) which The RAPD fingerprints of studied suggest existence of interspecies high fresh water catfish would be of help genetic variations among studied catfish revealing their interspecies genetic species. Some studies reported that a variation and genetic relationship based higher polymorphism symbolize higher on presence or absence of identical size genetic variation, while the low RAPD markers. Result of the UPGMA polymorphism indicate the opposite tree-based Nei’s genetic distance (Fig. 3) (Yoon and Park, 2002; Rahman et al., revealed two segregated main clusters 2009; Yusuf et al., 2017). where Schilbe mystus is genetically more The highest primer band frequency close to Bagrus bajad forming sister of 0.1731 was recorded for primer A-06, group (node B), than to Clarias while the lowest one of 0.0577 with gariepinus that is separately positioned as primer A-12 which suggested high and sister clade sharing common ancestor to low occurrence of both primers binding them (node A).This may indicate more sites receptively within genomes of the genetic makeup resemblance between studied catfish. Data showed detection of Schilbe mystus and Bagrus bajad. For high molecular size fragment of ~ 2900 that Schilbe mystus is more closely bp that was recorded only for Clarias related to Bagrus bajad than to Clarias gariepinus with primer A-06. This may gariepinus. Studies reported the consider primer-specific band for Clarias capability of RAPD assay to evaluate gariepinus indicating its internal various genomic loci and facilitate genotype-specific variation. analysis of genetic distance and phylogenetic relationships (Clark and 16 Mohammed Bassyouni M.EL-Mahdi

Lanigan, 1993), as well as the genetic Abdul Muneer, P.M., Gopalakrishnan, variation and level of similarity among A., Musammilu, K.K., Mohindra, fish species (Barman et al., 2003). The V., Lal, K.K., Basheer, V.S., and separation of Clarias gariepinus as sister Lakra, W,S. (2009): Genetic clade to both Schilbe mystus and Bagrus variation and population structure bajad may point to the monophyly of of endemic yellow catfish, family clarridae which supported by other Horabagrus brachysoma studies (Agnese and Teugels, 2005; (Bagridae) among three populations Sullivan et al., 2006; Pouyaud et al., of Western Ghat region using 2009; Yu and Quilang 2014). RAPD and microsatellite markers. In conclusion, results showed the Mol Biol Rep 36: 1779. consistency and informative efficiency of Adedolapo, A. (2007). Age and Growth RAPD PCR fingerprint technique in fish of the African Butter Catfish, phylogenetic studies down to the species Schilbe mystus (Linnaeus, 1758) in level and produced characteristic DNA Asejire and Oyan Lakes, South- fingerprint profiles for the three studied Western Nigeria. Journal of River Nile catfish species. The three Fisheries and Aquatic Science, 2: species are closer to each other, however 110-119. Schilbe mystus (Schilbeidae) and Agnese, J.F., and Teugels, G.G. (2005): Bagrus bajad (Bagaridae) are genetically Insight into the phylogeny of more close. The subset of Clarias African Clariidae (Teleostei, gariepinus as sister clade to Schilbe Siluriformes): Implications for their mystus and Bagrus bajad may be a sign body shape evolution, to the monophyly linage of the family biogeography, and . Mol Clarridae. The study outcomes suggested Phylogenet Evol 36:546-553. occurrence of genetic polymorphisms Ailendorf, F.W. and Phelps, S.R. (1981): (variations) among the investigated River Use of allelic frequencies to Nile catfish, with high level of genetic describe population structure. Can. convergence/relation between Schilbe J. Fish. aquat. Sci. 38: 1407-1514. mystus and Bagrus bajad which probably Alhassan, E. H. and Ansu-Darko, M. be useful to improve their potential use in (2011): Food and feeding habits of aquaculture breeding programs, as well as a potential aquaculture candidate, providing insights on their taxonomic the black Nile Catfish, Bagrus status within the siluriformes. bajad in the Golinga Reservoir. ACKNOWLEDGEMENT Australian Journal of Basic and This work has been funded by the South Applied Sciences, 5 (5): 354 - 359. Valley University (research grant), Qena, Alves, C.B.M., Vono, V. and Vieira, F. EGYPT. Author is thankful to U.M (1999): Presence of the walking Mahmoud (Professor of fish biology, catfish Clarias gariepinus Zoology Dept., Faculty of Science, (Burchell) (Siluriformes, Clariidae) Assiut University) for help in checking in Minas Gerais state hydrographic samples morphology. basins, Brazil. Revista Brasileira de REFERENCES Zoologia 16: 259-263. Abd El-Kader, H.A.M., Abd El-Hamid Ambak, M.A., Bolong, A.A., Ismail, P., Z.G., and Mahrous, K.F. (2013): and Tam, B.M. (2006): Genetic Genetic diversity among three variation of snakehead fish species of Tilapia in Egypt detected (Channa striata) populations using by random amplified polymorphic random amplified polymorphic DNA marker. J. Applied Biol. Sci., DNA. Biotechniques. 5: 104–110. 7(2): 57-64. RAPD-evaluated Genetic Polymorphisms and Relations among three River Nile Catfish 17

Amer F.I., Naguib S.A. A., and Abd El species of the sub genus Clarias. Ghafar F.A. (2008): Comparative Trans Zool Soc., 35: 1-45. study on the intestine of Schilbe Clark, A.G., and Lanigan, C.M.S. (1993): mystus and Labeo niloticus in Prospects for estimating nucleotide correlation with their feeding divergence with RAPDs. Mol. Biol. habits. Egypt. J. Aquat. Biol. fish. Evol. 10 (5): 1096-1111. 12 (4), 275-309 . Danish, M., Singh, I.J., Giri, P. and Aremu, M.O. Ekunode, O.E. (2008): Singh, C.P.(2012): Molecular Nutritional evaluation and characterization of two populations functional properties of Clarias of catfish Clarias batrachus L. lazera (African catfish) from river using random amplified Tammah in Nasarawa State, polymorphic DNA (RAPD) Nigeria. Am J Food Technol 3(4): markers. African Journal of 264-74. Biotechnology, 11: 14217-14226. Ayoade, A., Fagade S., Adebisi, A., El-Drawany, M. A. and Elnagar, W. G. Adedolapo, A., Solomon, Fagade., (2015): Growth, food and feeding and Abiodun A. (2008): Diet and habits of Bagrus bayad and Bagrus dietary habits of the fish Schilbe docmac inhibiting Muess channel, mystus (Siluriformes: Schilbeidae) Sharkia province, Egypt. Journal of in two artificial lakes in Research and Development, 6 (7): Southwestern Nigeria Rev Biol 348pp. Trop. 56: 4, 1847-55. Eschmeyer, W.N. and Fong, J.D. (2017): Bailey, R.G. (1994): Guide to the fishes Species by Family/Subfamily. of the River Nile in the Republic of (http://researcharchive.calacademy. the Sudan, Journal of Natural org/research/ichthyology/catalog/S History, 28:4, 937-970. peciesByFamily.asp). Electronic Barman, H.K., Barat, A., Yadav, B.M., version accessed (6/ 2017). Banerjee, S., Meher, P.K., Reddy, Ferguson, A., Taggart, J.B., Prodohl, P., and Jana, R.K. (2003): Genetic P.A., McMeel, O., Thompson, C., variation between four species of Stone, C., McGinnity, P. and Indian major carps as revelled by Hynes, R.A. (1995): The random amplified polymorphic application of molecular markers to DNA assay. Aquaculture 217: 115- the study and conservation of fish 123. populations with special reference Bishai, H.M. and M.T. Khalil, (1997): to Salmo. Journal of Fish Biology, Freshwater fishes of Egypt. Publ. 47(A), 103-126. Nat. Biodiv. Unit 9:229 p. Ferraris, C.J.Jr. and De Pinna, M.C.C. Bruton, M.N. (1986): The life history (1999): Higher-level names for styles of invasive fishes in southern catfishes (: Africa. In: Macdonald I.A.W, Ostariophysi: Siluriformes). Kruger F.J, Ferrar, A.A eds. The Proceedings of the California Ecology and Management of Academy of Sciences 51:1–17. Biological Invasions in southern Fisher, R.A. (1930): The Genetical Africa, Oxford University Press, Theory of Natural Selection. Cape Town, 201-209. Oxford University Press, UK. Buton, M.N. (1979): The breeding Froese, R. and Pauly, D. Editors. (2017): biology and earl development of FishBase. World Wide Web Clarias gariepinus (Pisces: electronic publication. Clariidae) in Lake Sibaya, South www..org, version Africa, with a review of breeding in (06/2017). 18 Mohammed Bassyouni M.EL-Mahdi

Hunt, G.J., Page, R.E. (1992): Patterns DNA (RAPD) analysis. Heredity of inheritance with RAPD 83: 30–38. molecular markers reveal novel Mostafa, M.G., Ishtiaq Ahmed, A.S., types of polymorphism in the Mustafa, M.G., Rabbane, M.G., honey bee. Theoretical and Applied Islam, M.N., and Rafiquzzaman, Genetics 85: 15-20. S.M. (2009): Genetic diversity of Islam, M.N., Islam, M.S., and Alam, wild and farmed Kalibaus (Labeo M.S. (2007): Genetic structure of calbasu, Hamilton, 1822) by RAPD different populations of walking analysis of the genomic DNA. catfish (Clarias batrachus L.) in Croatian J. Fish.: Ribarstvo, 67(2): Bangladesh. Biochem Genet 45: 41-52. 647. Nei, M. (1972): Genetic distance between Kareem O.K., Olanrewaju A.N., and populations. Am Nat 106: 283–292. Orisasona, O. (2015): Length- Nwadukwe, F.O. (1995): Analysis of weight Relationship and Condition production, early growth and factor of Chrysichythys survival of Clarias gariepinus nigrodigitatus and Schilbe (Burchell), Heterobranchus mystus in Erelu Lake, Oyo State, longifilis (Val.) (Pisces: Clariidae) Nigeria. J Fisheries Livest Prod and their F1 hybrids in ponds, 3:150. doi:10.4172/2332- Netherlands Journal of Aquatic 2608.1000150. Ecology 29: 2, 177. Kim, I. S. (1997): Illustrated Omondi, R., and Ogari J. (1994): Encyclopedia of fauna and flora of Preliminary study on the food and Korea. Academy publishing feeding habits of Schilbe mystus company. Korea, Seoul. pp. 187- (Linn., 1762) in River Nyando" . In 190. Okemwa, E., Wakwabi, E.O., and Linda, K.P. and Paul, M. (1995): Getabu, A. (Ed.) Proceedings of the Developments in molecular genetic Second EEC Regional Seminar on techniques in fisheries. In: G.R. Recent Trends of Research on Lake Carvalho and T.J. Pitcher, Eds., Victoria Fisheries, NAIROBI : Molecular Genetics in Fisheries, ICIPE SCIENCE, p. 115-119. Chapman and hall, London, 1-28. Pavel, A.B and Vasile, C.I (2012): Liu, Z.J. and Cordes, J.F. (2004): DNA PyElph-a software tool for gel marker technologies and their images analysis and phylogenetics. applications in aquaculture BMC Bioinforma 13:9. genetics. Aquaculture, 238, 1-37. Perkins, D.L. and Krueger, C.C. (1993): Liu, Z.J., Li, P., Argue, B.J., and Heritage brook trout in northeastern Dunham, R.A. (1999): Random USA: Genetic variability within amplified polymorphic DNA and among populations. markers: usefulness for gene Transaction of American Fisheries mapping and analysis of genetic Society, 122, 1515-1532. variation of catfish. Aquaculture, Popoola O.M., Fasakin, E.A., and 174: 59–68. Awopetu, J..I (2014): Genetic Mamuris, Z., Stamatis, C. and variability in cultured and wild Triantaphyllidis, C. (1999a): population of Clarias gariepinus Intraspecific genetic variation of (Osteichthys: Clariidae) using striped red mullet (Mullus random amplified polymorphic surmuletus L.) in the Mediterranean DNA (RAPD) marker. Croatian sea assessed by allozyme and Journal of Fisheries 72: 5 – 11. random amplified polymorphic RAPD-evaluated Genetic Polymorphisms and Relations among three River Nile Catfish 19

Pouyaud, L., Sudarto, . and Paradis, E. Phylogenetics and Evolution (2009): The phylogenetic structure 41:636–662. of habitat shift and morphological Tamura, K., Stecher, G., Peterson, D., convergence in Asian Clarias Filipski, A. and Kumar, S. (2013): (Teleostei, Siluriformes: Clariidae). MEGA6: Molecular Evolutionary Journal of Zoological Systematics Genetics Analysis Version 6.0. and Evolutionary Research, 47:344- Molecular Biology and Evolution 356. 30: 2725-2729. Rahman, S.M.Z., Khan, M.R., Islam, S., Thakur, N.K. (1998): A biological Alam, S. (2009): Genetic variation profile of the African of wild and hatchery populations of Catfish Clarias gariepinus and the catla Indian major carp (Catla impacts of its introduction in Asia, catla Hamilton 1822: pp. 275–292. In: Ponniah, A.G., Cypriniformes, Cyprinidae) Das, P., and Verma, S.R. revealed by RAPD markers. (eds). Fish Genetics and Genetics and Molecular Biology. Biodiversity Conservation. Natcon 32:197-201. Publications, Muzzafarnagar (UP) Risch L. M. (986). Bagridae: in Check- India. List of the freshwater fishes of USDA (U.S. Department of Agriculture) Africa. Daget, J. ;Gosse, J. P. and (2002): Catfish Production,February D. F. E. Thys van denAudenaerde 2, 2002 http://www.usda.gov/nass (eds.). ISNB, Brussels; MRAC, (National Agricultural Statistics Tervuren; and ORSTOM, Paris., 2: Service (NASS), Agricultural 2-35. Statistics Board, U.S. Department Rothuizen, J. and Van Wolferen, R. of Agriculture). (1994): Randomly amplified DNA Verma, J. and Serajuddin, M. (2017): polymorphisms in dogs are Intra-specific and inter-generic reproducible and display Mendelian phylogenetic relationships in transmission. Anim.Genet., 25: 13- endangered catfish (Clupisoma 18.. garua and Eutropiichthys vacha) of Senanan, W., Kapuscinski, A.R., Na- family schilbeidae. Journal of Nakoran, U. and Miller, L.M. Entomology and Zoology Studies (2004): Genetic impacts of hybrid 5: 198-202. catfish farming (Clarias Vitule, J.R.S., Umbria, S.C., and Aranha, macrocephalus X Clarias J.M.R. (2006): Introduction of the gariepinus) on native catfish African Catfish Clarias gariepinus farming in central (BURCHELL, 1822) into Southern Thailand. Aquaculture 235: 167– Brazil, Biological Invasions 8: 184. 677–681 Sneath, P.H.A and Sokal, R.R. (1973): Welcome, R.I. (1979): The inland Numerical Taxonomy-the fisheries of Africa. FIFA Occ. Principles and Practice of Paper 7, pp: 1-3. Numerical Classification. W. H. Welsh, J. and McClelland, M. (1990): Freeman, San Francisco, USA. fingerprinting genomes using PCR Sullivan, J.P., Lundberg, J.G., and with arbitrary primers. Nucleic Hardman, M. (2006): A Acids Research 18 (24): 723-7218. phylogenetic analysis of the major Williams, J.G.k., Kubelik, A.R., Livak, groups of catfishes (Teleostei: K.J., Rafalski, J.A. and Tingey, Siluriformes) using rag1 and rag2 S.V. (1990): DNA polymorphisms nuclear gene sequences. Molecular amplified by arbitrary primers are 20 Mohammed Bassyouni M.EL-Mahdi

useful as genetic markers. Nucleic Carp (Carassius carassius) Acids Research 18 (22):6531-6535. Estimated with Random Amplified Wilson, D.E. and Reeder, D.M. (2005): Polymorphic DNA. Asian Mammal Species of the World: A Australasian Journal of Animal Taxonomic and Geographic Sciences 15(4): 470-476. Reference JHU Press. You, F., Zhang, P.J., Wang, K.L. and Winemiller, K.O., and Kelso-Winemiller, Xiang, J.H. (2007): Genetic L.C. (1996): Comparative ecology variation of natural and cultured of catfishes of the Upper Zambezi stocks of Paralichthys River floodplain. Journal of Fish olivaceus by allozyme and Biology 49: 1043-1061. RAPD. Chinese Journal of Yalçin, S., Akyurt, I. and Solak, K. Oceanology and Limnology, 25: (2001b): Certain reproductive 78–84. characteristics of the catfish Yu, S.C.S., and Quilang, J.P. (2014): (Clarias gariepinus Burchell, 1822) Molecular phylogeny of catfishes living in the River Asi, (Teleostei: Siluriformes) in the (Turkey). Turkish Journal of Philippines using the mitochondrial Zoology 25: 453-460. genes COI, Cyt b, 16S rRNA, and Yeh, F.C., Yang, R.C., and Boyle, T. the nuclear genes Rag1 and Rag2. (1999): POPGENE version 1.32: Philippine Journal of Science Microsoft Windows–based 143(2):187-198. freeware for population genetic Yusuf, N.O., Yisa A.T., and Sadiku, analysis, a quick user guide. Center S.O.E. (2017): Genetic Variation for International Forestry Research, Between Cultured and Wild University of Alberta, Edmonton, Populations of Oreochromis Alberta, Canada. niloticus deduced from Randomly Yoon, J.M., and Park, H.Y. (2002): Amplified Polymorphic DNA Genetic Similarity and Variation in (RAPD) Markers. Asian Journal of the Cultured and Wild Crucian Biotechnology, 9: 43-49.

RAPD-evaluated Genetic Polymorphisms and Relations among three River Nile Catfish 21

ARABIC SUMMARY

التغايرات الوراثية والعالقة بين ثالثة أنواع من آسماك نھر النيل القطية من قنا، مصر باستعمال مؤشرات التضاعف العشوائي متعدد األشكال لسلسلة الدنا (RAPD)

محمد بسيونى محمد المھدى معمل الوراثة الجزيئية و بيولوجيا الجزيئيات - قسم علم الحيوان- كلية العلوم - جامعة جنوب الوادى - قنا - جمھورية مصر العربية

أخضعت المادة الوراثية (DNA) لثالثة أنواع من آسماك نھر النيل القطية وھى الشلباية Schilbe) (mystus Linnaeus, 1758 البياض (Bagrus bajad Forsskål, 1775) و القرموط ( Clarias gariepinus Burchell, 1822) للتنميط الوراثى بواسطة التضاعف العشوائي (RAPD) باستخدام البادئات العشوائية اآلتية : (A-11 ، A-10 ، A-09 ، A-06 ، A-05 ، A-04 ، A-03 و A-12 ). أظھرت البادئات تعددية شكلية بين انواع آسماك نھر النيل القطية قيد الدراسة و سجلت كل البادئات حزم مؤشرة فريدة مختلفة تتراوح من ٢٠٠ -٢٩٠٠ زوج قاعدة. أعطت البادئات الثمانية عدد اثنين و خمسون (٥٢) حزمه منھا ( ٥) حزم أُ ِحاد ﱡي ﱠالش ْكل (Monomorphic) بمعدل تباين احادى بنسبة 9.6 % و سبع واربعون (٤٧) كانت متعددة الشكل (polymorphic) مع معدل تباينى متعدد بنسبة 90.38% .كان أعلى معدل للتردد الحزمى لسمكة القرموط مقارنة بالشلباية و البياض بينما سجل البادئ A-06 اعلى معدل للتردد الحزمى بالنسبة للبادئات اآلخرى المستخدمة. وفقا لمصفوفة معامـــــل البعـــــد الـــــوراثي (معامـل ٧٢ Nei) والتحليـل العنقـودي باســـتخدام طريقـــة المجموعـــة الزوجيـــة غيـــر الموزونـــة مـــع المتوســــــط الحســــــابي (UPGMA)، وضح اآلرتباط الوراثى بين آسماك نھر النيل القطية المدروسة بعضھا لبعض ولھا سلف مشترك، لكن كل من سمكتى الشلباية و البياض اقرب وراثيا. ان انشقاق سمكة القرموط مكونأ مجموعة مستقلة و شقيقة لمجموعة سمكتى الشلباية و البياض ربما يعزى الى نمطھا األحادى فى نسبھا التطورى. اقترحت الدراسة وجود تغايرات وراثية بين آسماك نھر النيل القطية الثالثة مع ارتفاع درجة التقارب الوراثى بين سمكتى الشلباية و البياض و الذى ربما يكون مفيدأ لبرامج تربيتھم وتنمية استزراعھم السمكى وكذلك تقديم رؤى حول الوضع التصنيفى لھم ضمن اآلسماك القطية.