Heredity 73(1994) 117—123 Received 25 November 1993 Genetical Society of Great Britain Application of the RAPD technique in tilapia fish: species and subspecies identification F. BARDAKCI* & D. 0. F. SKIBINSKI Molecular Biology Research Group, School of Biological Sciences, University of Wales, Singleton Park, Swansea SA2 8PP, U.K. RandomAmplified Polymorphic DNA (RAPD) analysis was applied to three species of the tilapia genus Oreochromis and four subspecies of 0. niloticus. Thirteen random lO-mer primers were used to assay polymorphisms within and between populations. Different RAPD fragment patterns were observed for different species, although not always for different subspecies. Evidence is presented that RAPD markers might be useful for systematic investigation at the level of species and subspecies. Keywords:DNA,Oreochromis, polymorphism, RAPD, systematics, tilapia. fully to identify the subspecies of 0. niloticus (Capili, Introduction 1990; Seyoum & Kornfield, 1992) but little effort has Tilapiaare cultured extensively throughout the world, yet been devoted to the analysis of nuclear DNA for especially in Africa and the Far East. Most of the this purpose. important tilapiine species used in aquaculture are The development of random amplified polymorphic members of the Oreochromis genus, as many members DNA (RAPD) markers, generated by the polymerase of this taxon grow well under diverse culture condi- chain reaction (PCR), allows the examination of tions (Fryer & lIes, 1972; Pullin & Capili, 1989). genomic variation without prior knowledge of DNA Despite its commercial importance, resources have sequences (Williams et a!., 1990, 1993; Welsh & only recently been devoted to the development of McClelland, 1990; Hadrys, 1992). The number and improved strains of tilapia. Moreover few efforts have the size of amplified fragments depend on length and been made to assess the relative value of different sequence of short, single and arbitrary primers. methods for genetic characterization of tilapia germ- Priming sites are randomly distributed throughout a genome and polymorphisms in such sites result in plasm. The most recent classification of tilapiine species is differing amplification products, detected by the based on reproduction, development, feeding, struc- presence and absence of fragments. Such polymorph- tural characteristics and biogeography (Trewavas, isms are inherited in a Mendelian fashion and can be 1983). However, such characters are of limited value used as genetic markers. The method has been success- for identification purposes because they show con- fully used to detect variation between strains of siderable interpopulation variation and differences bacteria and rice (Welsh & McClelland, 1990), mice between species are small (Fryer & Iles, 1972; Abban, (Welsh et al., 1991), Gliricidia (Chalmers et al., 1992), 1988). Protein electrophoresis has been extensively closely related species of black Aspergilli (Megneg- used to discriminate species of tilapia (Kornfield at al., neau et al., 1993), and cocoa (Russell et a!., 1993) and 1979; McAndrew & Majumdar, 1983, 1984; Abban, between species of parasitic protozoa (Tibayrenc et a!., 1988; Sodsuk & McAndrew, 1991) and their hybrids 1993). (Macaranas et a!., 1986) but this technique could not This paper provides evidence that RAPD markers discriminate subspecies of 0. niloticus (Seyoum, 1989, can be used to discriminate between three widely culti- cited in Seyoum & Kornfield, 1992; Seyoum, 1990). vated species of tilapia and between several subspecies Mitochondrial DNA markers have been used success- of Oreochromis niloticus, probably the species with greatest commercial importance. An assessment is also made of the utility of RAPD analysis for systematic *Correspondence analysis of tilapia. 118 F. BARDAKCI & D. 0. F. SKIBINSKI Materials and methods some modifications. Amplification reactions were per- formed in 500 ifiM KC1, 100 mvi Tris (pH 9.0 at 25°C), 1 per cent Triton X-100, 2.5 mtvi MgCl, 100 MM each of Species and subspecies stud/ed dATP, dTTP, dGTP and dCTP (Pharmacia), 5 pmoles Thespecies and subspecies used in this study are heldof 10-base primer, 0.5 units of Taq DNA polymerase in aquaria at the University College of Swansea, Wales. (Promega Biotec.), and 20 ng of genomic DNA in a Their sources are given in Table 1. 0. niloticus final volume of 25 ML. The mixture was overlaid with (baobab) is a commercial strain of 0. niloticus with the same volume of mineral oil. DNA amplification possible hybrid ancestry. It is not recognized as a sub- was performed in a thermal cycler (Hybaid, U.K.). For species but is designated as one for convenience in this the first cycle, denaturation, annealing and extension study. were 94°C for 2 mm, 35°C for 1 mm and 72°C for 2 Their sources are given in Table 1. 0. niloticus mm, respectively. Denaturation time was decreased to (baobab) is a commercial strain of 0. niloticus with 30 s for the following 44 cycles. One negative control possible hybrid ancestry. It is not recognized as a sub- (absence of template DNA) was performed for each set species but is designated as one for convenience in this of amplifications. study. Approximately 10 ML of amplification products were separated on 5 per cent vertical nondenaturing DNA extraction polyacrylamide gels in TBE buffer (0.89 M Tris, 0.89 M boric acid and 0.11 M EDTA, pH 8.3). Gels were fixed DNAwas prepared from fin tissue following the with 10 per cent ethanol and 0.5 per cent acetic acid method described by Hillis & Moritz (1990) with some solution twice for 3 mm, stained with 0.1 per cent silver modifications. Approximately 50 mg of the caudal fin tissue was cut into small pieces and suspended in 500 jcL STE (0.1 M NaC1, 0.05 M Tris and 0.01 M EDTA, Table 2 Sequence and operon codes of the random primers pH 8). After adding 30 ,uL SDS (10 per cent) and 30 used to study variation in Oreochromis species ML proteinase K (10 mg mL 1), the mixture was Primer codes incubated at 50°C for 30 mm. DNA was purified by Sequence (5'to3') successive extraction with phenol, phenol:chloro- 0PA03 AGTCAGCCAC form:isoamyl alcohol (25:24:1) and chloroform:isoamyl OPA 04 AATCGGGCTG alcohol (24:1), respectively. DNA was precipitated with OPA 05 AGGGGTCTTG ice-cold absolute ethanol and washed with 70 per cent OPA 07 GAAACGGGTG ethanol. The pellet was dried and resuspended in 150 OPA 08 GTGACGTAGG ,uL TE (10 mr'i Tris-HC1, 1 mtvi Na2EDTA.H20, pH OPA 10 GTGATCGCAG 7.2). OPA 12 TCGGCGATAG OPA 13 CAGCACCCAC OPA 17 GACCGCTTGT Amplificationconditions and electrophoresis OPA 19 CAAACGTCGG Aset of 13 decamer primers from Operon Technolo- OPB 08 GTCCACACGG OPC 02 GTGAGGCGTC gies was used in this study (Table 2). The amplification OPC 11 conditions were based on Williams et al. (1990) with AAAGCTGCGG Table 1 Species and subspecies used in this study and their source Species and subspecies Abbreviation Source Oreochromis aureus Aur Lake Manzala, Egypt Oreochromis mossambicus Mos Aquarist stock Oreochromis niloticus 0. n. vulcani (vulcani) Vul Lake Vulcani, Kenya 0. n. baringoensis (baringo) Bar Lake Baringo, Kenya 0. n. niloticus (manzala) Nil Lake Manzala, Egypt 0.n. (baobab) Bao Baobab Fish Farm, Kenya Common names of 0.niloticus subspecies are given in brackets. Nomenclature according to Trewavas (1983). RAPD ANALYSIS OF TILAPIA 119 nitratesolution for 10 mm, rinsed twice with distilled cnn0C') LL)r fiNfl LO0) W0) water, and then developed in an alkaline solution (1.5 040 (OLE) -cn (N per cent NaOH, 0.1 per cent NaBH4 and 0.15 per cent — r CH2O). A total of four individuals per species and sub- L11 B fl species (two males and two females) were analysed. Silver staining of polyacrylamide gels was found to be far superior to ethidium bromide staining of agarose 111 1I gels in terms of the number of fragments that could be III Ii resolved clearly. Analysis .3 TheRAPD patterns of individuals were compared within and between populations. Fragments were scored as 1 if present or 0 if absent. The index of similarity between individuals was calculated using the >Jflifl1 formula: :rm !IL np), where is the number of fragments shared by fill individuals x and y and n and n are the number of iirujj I fragments scored for each individual (Lynch, 1990). ) Within population similarity (S) is calculated as the Zfl:r.njI average of across all possible comparisons between individuals within a population. m. gjjp Between population similarity, corrected for within TffJJJ population similarity, is: a S= 1 + S— 0.5(S1+ S1), (00 Ofi cnN-C where S1 and S1 are the values of S for populations i (0 nfl LOLOO) 0) and j,respectivelyand S, is the average similarity N NO (ØLOfl N between randomly paired individuals from populations i and! (Lynch, 1990). It is possible for the value of S to I exceed 1. j H II S was also converted to a measure of genetic distance (D,1) using the equation: L-_HffrL1 D1 =— 1n[S/,J(S1S1)], (Lynch, 1991). D values were used to construct dendrograms using the unweighted pair-group method of analysis (UPOMA) (Sneath & Sokal, 1973). nfl The statistics can be calculated for each primer separately or for the combined data for all primers. F PP Results 'Tjp p. Allthe primers examined produced different RAPD fragment patterns (Fig. 1). The number of fragments generated per primer varied between six and 17. All =SWift primers gave species-specific RAPD patterns. Sub- z I Fig. I RAPD patterns from each of six populations using (a) up primer OPA 04 and (b) OPA 07. Samples (reading from the right hand lane): size markers; negative control (no template), t, ,fl 0. aureus, 0.mossambicus, 0. n. vulcani, 0. n. baringoensis, 0. n. niloticus, 0. n. (baobab). 120 F. BARDAKCI & D. 0. F. SKIBINSKI 0 species-specific patterns were obtained for some but CO 0 0 0 00.t- r) t rN 0 0O\ N - 0 'I00 C\ 0 not all primers.