AFLP) in Killifish Aphanius Anatoliae Anatoliae (Leidenfrost, 1912)

International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013 14 ISSN 2278-7763

Determination of Sex Markers by Amplified Fragment Length Polymorpism (AFLP) in Killifish Aphanius anatoliae anatoliae (Leidenfrost, 1912)

Efkan BAĞDA1*, Fevzi BARDAKCI2, Z. Deniz INAN3

1* Cumhuriyet University, Faculty of Science, Department of Biology, Sivas, Turkey, 2. Adnan Menderes University, Faculty of Science, Department of Biology, Aydın, Turkey, 3. Cumhuriyet University, Faculty of Medicine, Department of Histology, Turkey. Email: [email protected]

ABSTRACT Identification of sex in fishes is a difficult task, thus genetic and ecological factors involved in sex determination. Therefore, sex specific genetic markers can be developed with using molecular techniques. In this study, sex markers of Anatolian endemic Aphanius anatoliae anatoliae [1] is tried to identify with using AFLP (Amplified Fragment Length Polymorphism) technique. A total of 15 different primer combinations were analysed with using a sex-typed pool strategy for 18 individuals of two populations from Turkey. Three of clearly appeared bands were polymorphic between populations. Observed different bands were found for the males of Tuz Gölü populations while they were found for the females of Güneşli populations. The results of the study showed that the AFLP technique has a potential of solving sex identification problems of A. a. anatoliae and it is obvious that sex associated markers can be developed with AFLP technique. Between the populations of A. a. anatoliae, sex independent polymophic AFLP band profiles were observed, thus we suggest to study larger numbers of populations. From the results of this study, it is recommended to use larger number of primer combinations for the identification of sex-specific marker of A. a. anatoliae to obtain precise results.

Keywords : Sex Marker, Sex Determination, AFLP, Aphanius anatoliae anatoliae, Fish

1 INTRODUCTION ISHES are the most abundant vertebrates on Earth, show- ies. Fishes have both XX/XY and ZZ/ZW heterogametic Fing a diversity of species (27 977 species) unmatched by modes (XY, male, ZW, female) of sex determination. Though other classes [2], [3]. The group exhibits a large variety of the medaka (Oryzias) follows an XX/XY heterogametic mode mechanisms of sexual determination. Sexual determination of sex determination, the X and Y chromosomes are homo- refers to the event that compromises a bipotential gonad to morphic, unlike in mammals. [8], [11]. develop as an ovary or a testicle [4]. The processes of sexual Most fish species exhibit reduced sexual dimorphism and determination and differentiation in fish are highly plastic, usually lack heteromorphic sex chromosomes [12]. Sexing is even on top of a given genetic background, as it were, certain thus difficult and alternative techniques to those based on environmental factors, such as pH, temperature, and social karyotyping are required for the determination of sex. DNA- conditions, may wield a strong influence [5], [6], [7], [8]. based tests can solve this problem but sex specific markers Sex identification is an important issue for studies includ- need to initially be isolated [13], [14]. ing ecology, behavior, conservation and genetics of many spe- DNA markers provide useful tools for examining sex link- cies for especially fish species [3]. Genetic sex determination age in fish since DNA structure is not anticipated to change (GSD) and environmental sex determination (ESD) models are with altered physiology or environments. Moreover, examina- available for the identification of sex in fishes. tion of DNA sequence organization on sex chromosomes can Genetic sexual determination (GSD) may involve monogen- provide useful insights into the evolutionary processes that ic and polygenic systems, with factors localized on autosomes are operating to influence sex-chromosome structure, and ul- or on sex chromosomes [9]. In polygenic systems, sex is de- timately, can yield information on the conservation (or lack termined by the accumulative genetic action of all factors in- thereof) of sex-determination processes among species [9]. volved in its determination, and not by a single genetic locus Sex-specific DNA sequences have been identified in several [10]. In monogenic systems, sex is determined by a gene locat- fish species using different assays, including RAPDs, RFLPs, ed on a certain chromosome and genes on other chromosomes AFLPs and microsatellite markers [13], [14], [15], [16]. have little effect [8]. The center of divercity of the killifish genus Aphanius [17] is Many species of teleosts do not present morphologically in Turkey [18]. Of the 19 described species in the genus, six differentiated sex chromosomes. However, some species have species and four subspecies occur in Anatolia. Sex determina- heteromorphic sex chromosomes which can be identified by tion markers was not identified for Aphanius species that are fluorescence in situ hybridization (FISH) and cytogenetic stud- lack in heteromorphic sex chromosomes [19], [20], [21], [22].

In this article, we focus on the use of the amplified frag- cycle profile: a 30 s DNA denaturation step at 94 oC, a 30 s an- ment length polymorphism (AFLP) technique [23] for the nealing step (see below) and a 2 min extension step at 72 oC. identification of sex-specific markers in Aphanius anatoliae ana- The annealing temperature in the first cycle was 60 oC, was toliae [1] based on pooled DNA samples from known male and subsequently reduced each cycle by 0.7 oC for the next 12 cy- female individuals which were previously phenotypically cles and was continued with a 30 s DNA denaturation step at sexed. 94 oC, a 30 s annealing step at 56 oC and a 2 min extension step at 72 oC for the next 23 cycle. For the selective amplification, primer combinations 2 MATERIAL AND METHODS used were as follows: 2.1 Genomic DNA Isolation E-AGC+M-CGC (a1) E-ATC+M-CGA (b2) Total of 18 Aphanius anatoliae anatoliae fishes were collected E-AGC+M-CGA (a2) E-ATC+M-CAC (b3) from two populations. Four male and 4 female were collected E-AGC+M-CAC (a3) E-ATC+M-CTG (b4) from Güneşli (G), Eşmekaya region of Turkey. Five male and 5 E-AGC+M-CTG (a4) E-ATC+M-CTA (b5) female were collected from south of Tuz Gölü (T) (Fig. 1). Fish E-AGC+M-CTA (a5) E-ACA+M-CGC (c1) samples were stored in 95 % ethanol immediately after collec- E-ATC+M-CGC (b1) E-ACA+M-CAC (c2) tion for DNA analysis. E-AAT+M-CTG (c4) E-ACA+M-CTG (c3) E-AAT+M-CTA (c5 ). AFLP-PCR products were denatureted at 90 oC for 4 min. and separated on 6 % denaturing polyacrilamide gels and vis- ualized with silver staining. AFLP markers were analyzed manually based on the presence/absence of DNA fragments.

3 RESULTS AND DISSCUSSION The AFLP analysis involves three steps: (1) restriction of the genomic DNA and ligation of oligonucleotide adapters, (2) selective amplification of sets of restriction fragments, and (3) gel analysis of the amplified fragments. [23]. [The AFLP technique is based on the selective PCR amplification of restriction Fig. 1. Sample locations (G=Güneşli, T= Tuz Gölü). fragments from a total digest of genomic DNA.] Genomic DNA was isolated from muscle tissue with a As demonstrated in this document, the numbering for sections standart proteinase K, phenol-chloroform method [24]. Ap- upper case Arabic numerals, then upper case Arabic numerals, proximately 100 mg muscle tissue was digest in 500 μL STE separated by periods. Initial paragraphs after the section title buffer (0.1 M NaCl, 0.05 M Tris and 0.01 M EDTA, pH 8), 20 are not indented. Only the initial, introductory paragraph has μL proteinase K (10 mg/mL) and 50 μL SDS (10 %). Following, a drop cap. DNA was extracted by a standard phenol-chloroform proce- AFLP is relatively cheap, easy, fast and reliable method to dure and precipitated with absolute ethanol. Precipitated generate hundreds of informative genetic markers. [23], [25], DNA was dissolved in distilled water and quantified at wave- [26]. Pre-knowledge about DNA sequence is not required for length of 260 nm by a spectrophotometer. AFLP method. Many different DNA regions can be screened Pooled DNA samples each population were prepared using simultaneously with this technique. AFLP markers are useful 100 ng/μL of genomic DNA from 4 (Güneşli, 4 ♂ and 4 ♀) to 5 tools for identification of near relations like twin species and (Tuz Gölü, 5 ♂ and 5 ♀) males and femals to do 2 pools/each hybrid species, structure and diversity of population at the sex (n= 4-5 fish/pool). Pooled DNA were initially analyzed levels of species and subspecies. For a wide range of taxa, in- using a total of 15 different primer combinations. cluding plants, fungi, animals and bacteria, AFLP markers have been used to uncover cryptic genetic variation of strains, 2.2 AFLP Reaction or closely related species, which had been impossible to re- AFLP analysis was carried out as described by [23] with some solve with morphological or other molecular systematic char- modifications. DNA samples were cutted with EcoRI and MseI acters [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37]. At (Fermentas, MBI) restriction enzymes. The oligonucleotid the same time it is showed that AFLP is a usefull toll for the adoptors were ligated to cutted to the ends of DNA fragments development of sex markers for both animals ([38], [39], [40], to generate template DNA for amplification. In preamplifica- [41], [42], [14], [43] and plants ([44], [45], [46]. tion step, the genomic DNAs were amplified with two AFLP In this study, 18 individuals, 9 male and 9 female from two primers (EcoRI-A and MseI-C) both having a single selective different populations of Aphanius anatoliae anatoliae species nucleotide. PCR (Stuart, UK) amplifications were performed were from Turkey collected. Sex associated markers were ex- 26 cycles with the following cycle profile: a 1 min DNA dena- amined with using AFLP technique by 15 different primer o o turation step at 94 C, a 1 min annealing step at 56 C and a 1 combinations. AFLP-PCR products were separated on 6 % o min extension step at 72 C. The 5 times diluted PCR product denaturing polyacrilamide gels and analyzed manually based DNA’s were used as templates for selective amplification. PCR on the presence/absence of DNA fragments. The 20 bands (Stuart, UK) amplifications were performed with the following from produced 36 bands were appeared clearly. For three Copyright © 2013 SciResPub. International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013 16 ISSN 2278-7763 primer combinations (a4, a5 and c5 ) of these clearly bands, REFERENCES sex-independent polymorphic bands were observed for the [1] Leidenfrost, G., 1912. Kis–âzsiai halak (Fishes from Asia populations. The observed different fragments are in the range Minor). Allattani Közlemênyek, 11 (3): 130–131, figures. of 350-400 bp. For primer combinations of a4, a5 and c5 , ob- (Zoological Messages, Budapest). served different bands were found for the males of Tuz Gölü [2] Nelson, J. S., 2006. Fishes of the World. 4th edition, Pub- populations while they were found for the females of Güneşli lished by John Wiley & Sons Inc., Hoboken, New Jersey. populations (Fig. 2). [3] Luckenbach, J. A., Borski,R.J., Daniels, H.V. and Godwin,

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