International Journal of Advancements in Research ... · heteromorphic sex chromosomes which can be identified by fluorescence in situ hybridization (FISH) and cytogenetic stud-ies

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

Copyright © 2013 SciResPub.

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 popula-tions 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-ing a diversity of species (27 977 species) unmatched by other classes [2], [3]. The group exhibits a large variety of

mechanisms of sexual determination. Sexual determination refers to the event that compromises a bipotential gonad to develop as an ovary or a testicle [4]. The processes of sexual determination and differentiation in fish are highly plastic, even on top of a given genetic background, as it were, certain environmental factors, such as pH, temperature, and social conditions, may wield a strong influence [5], [6], [7], [8].

Sex identification is an important issue for studies includ-ing ecology, behavior, conservation and genetics of many spe-cies for especially fish species [3]. Genetic sex determination (GSD) and environmental sex determination (ESD) models are available for the identification of sex in fishes.

Genetic sexual determination (GSD) may involve monogen-ic and polygenic systems, with factors localized on autosomes or on sex chromosomes [9]. In polygenic systems, sex is de-termined by the accumulative genetic action of all factors in-volved in its determination, and not by a single genetic locus [10]. In monogenic systems, sex is determined by a gene locat-ed on a certain chromosome and genes on other chromosomes have little effect [8].

Many species of teleosts do not present morphologically differentiated sex chromosomes. However, some species have heteromorphic sex chromosomes which can be identified by fluorescence in situ hybridization (FISH) and cytogenetic stud-

ies. Fishes have both XX/XY and ZZ/ZW heterogametic modes (XY, male, ZW, female) of sex determination. Though the medaka (Oryzias) follows an XX/XY heterogametic mode of sex determination, the X and Y chromosomes are homo-morphic, unlike in mammals. [8], [11].

Most fish species exhibit reduced sexual dimorphism and usually lack heteromorphic sex chromosomes [12]. Sexing is thus difficult and alternative techniques to those based on karyotyping are required for the determination of sex. DNA-based tests can solve this problem but sex specific markers need to initially be isolated [13], [14].

DNA markers provide useful tools for examining sex link-age in fish since DNA structure is not anticipated to change with altered physiology or environments. Moreover, examina-tion of DNA sequence organization on sex chromosomes can provide useful insights into the evolutionary processes that are operating to influence sex-chromosome structure, and ul-timately, can yield information on the conservation (or lack thereof) of sex-determination processes among species [9].

Sex-specific DNA sequences have been identified in several fish species using different assays, including RAPDs, RFLPs, AFLPs and microsatellite markers [13], [14], [15], [16].

The center of divercity of the killifish genus Aphanius [17] is in Turkey [18]. Of the 19 described species in the genus, six species and four subspecies occur in Anatolia. Sex determina-tion markers was not identified for Aphanius species that are lack in heteromorphic sex chromosomes [19], [20], [21], [22].

F



In this article, we focus on the use of the amplified frag-ment length polymorphism (AFLP) technique [23] for the identification of sex-specific markers in Aphanius anatoliae ana-toliae [1] based on pooled DNA samples from known male and female individuals which were previously phenotypically sexed.

2 MATERIAL AND METHODS 2.1 Genomic DNA Isolation Total of 18 Aphanius anatoliae anatoliae fishes were collected from two populations. Four male and 4 female were collected from Güneşli (G), Eşmekaya region of Turkey. Five male and 5 female were collected from south of Tuz Gölü (T) (Fig. 1). Fish samples were stored in 95 % ethanol immediately after collec-tion for DNA analysis.

Fig. 1. Sample locations (G=Güneşli, T= Tuz Gölü). Genomic DNA was isolated from muscle tissue with a

standart proteinase K, phenol-chloroform method [24]. Ap-proximately 100 mg muscle tissue was digest in 500 μL STE buffer (0.1 M NaCl, 0.05 M Tris and 0.01 M EDTA, pH 8), 20 μL proteinase K (10 mg/mL) and 50 μL SDS (10 %). Following, DNA was extracted by a standard phenol-chloroform proce-dure and precipitated with absolute ethanol. Precipitated DNA was dissolved in distilled water and quantified at wave-length of 260 nm by a spectrophotometer.

Pooled DNA samples each population were prepared using 100 ng/μL of genomic DNA from 4 (Güneşli, 4 ♂ and 4 ♀) to 5 (Tuz Gölü, 5 ♂ and 5 ♀) males and femals to do 2 pools/each sex (n= 4-5 fish/pool). Pooled DNA were initially analyzed using a total of 15 different primer combinations.

2.2 AFLP Reaction AFLP analysis was carried out as described by [23] with some modifications. DNA samples were cutted with EcoRI and MseI (Fermentas, MBI) restriction enzymes. The oligonucleotid adoptors were ligated to cutted to the ends of DNA fragments to generate template DNA for amplification. In preamplifica-tion step, the genomic DNAs were amplified with two AFLP primers (EcoRI-A and MseI-C) both having a single selective nucleotide. PCR (Stuart, UK) amplifications were performed 26 cycles with the following cycle profile: a 1 min DNA dena-turation step at 94 oC, a 1 min annealing step at 56 oC and a 1 min extension step at 72 oC. The 5 times diluted PCR product DNA’s were used as templates for selective amplification. PCR (Stuart, UK) amplifications were performed with the following

cycle profile: a 30 s DNA denaturation step at 94 oC, a 30 s an-nealing step (see below) and a 2 min extension step at 72 oC. The annealing temperature in the first cycle was 60 oC, was subsequently reduced each cycle by 0.7 oC for the next 12 cy-cles and was continued with a 30 s DNA denaturation step at 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 used were as follows:

E-AGC+M-CGC (a1) E-ATC+M-CGA (b2) E-AGC+M-CGA (a2) E-ATC+M-CAC (b3) E-AGC+M-CAC (a3) E-ATC+M-CTG (b4) E-AGC+M-CTG (a4) E-ATC+M-CTA (b5) E-AGC+M-CTA (a5) E-ACA+M-CGC (c1) E-ATC+M-CGC (b1) E-ACA+M-CAC (c2) 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 tech-nique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA.] As demonstrated in this document, the numbering for sections upper case Arabic numerals, then upper case Arabic numerals, separated by periods. Initial paragraphs after the section title are not indented. Only the initial, introductory paragraph has a drop cap.

AFLP is relatively cheap, easy, fast and reliable method to generate hundreds of informative genetic markers. [23], [25], [26]. Pre-knowledge about DNA sequence is not required for AFLP method. Many different DNA regions can be screened simultaneously with this technique. AFLP markers are useful tools for identification of near relations like twin species and hybrid species, structure and diversity of population at the levels of species and subspecies. For a wide range of taxa, in-cluding plants, fungi, animals and bacteria, AFLP markers have been used to uncover cryptic genetic variation of strains, or closely related species, which had been impossible to re-solve with morphological or other molecular systematic char-acters [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37]. At the same time it is showed that AFLP is a usefull toll for the development of sex markers for both animals ([38], [39], [40], [41], [42], [14], [43] and plants ([44], [45], [46].

In this study, 18 individuals, 9 male and 9 female from two different populations of Aphanius anatoliae anatoliae species were from Turkey collected. Sex associated markers were ex-amined with using AFLP technique by 15 different primer combinations. AFLP-PCR products were separated on 6 % denaturing polyacrilamide gels and analyzed manually based on the presence/absence of DNA fragments. The 20 bands from produced 36 bands were appeared clearly. For three



primer combinations (a4, a5 and c5 ) of these clearly bands, sex-independent polymorphic bands were observed for the populations. The observed different fragments are in the range of 350-400 bp. For primer combinations of a4, a5 and c5 , ob-served different bands were found for the males of Tuz Gölü populations while they were found for the females of Güneşli populations (Fig. 2).

Fig. 2. AFLP band profiles of male and female individuals (G: Güneşli, T: Tuz Gölü).

The small number of primer combinations (n=15 ) used this study seems to be disadvantage, but sex specific markers had been developed for some studies with conducting rela-tively small numbers of primer combinations {([46], n=16); ([43], n=64); ([47], n=64)} and besides many studies had failed to identify sex specific markers with using higher primer combinations {([48], n=570); ([49], n=128-396); ([14], n=486)}. On the other hand, according to some researchers [16]; [50]; [14], the success of the identification of sex-spe cific mark-ers has depended largely on the presence of a sex chromosome or non-chromosomal genetic sex-determining mechanisms [39] in the target species. The failure to identify sex-associated markers in the present study may be arises from the absence of sex chromosomes for Aphanius a. anatoliae. Another sugges-tion of failure to development sex markers is suggested by [49], according to [49], in order to explain the failure of sex-marker identification, a sex determining system based on a gene dosage effect has to be considered. Based on these facts, to obtain precise results, for future investigations, we recom-mend to use the number of primer combinations for the identi-fication of sex-specific marker of Aphanius a. anatoliae.

4 CONCLUSION In this study, sex dependent markers were not developed as insufficient numbers of individuals or primer combinations were used. It is noteworthy that the inter-population sex inde-pendent polymorphic bands were different between females and males of same populations, Sex markers can be developed with conducting AFLP-PCR technique. Further studies with AFLP technique, sex dependent markers can be developed with using many more primer combinations, populations and individuals. Consequently, this study will be a guide to re-searcher for the choice of primers and primer combinations.

ACKNOWLEDGMENT The authors wish to thank Ass. Prof. Dr. Esra Bağda for lan-guage editing.

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♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ ♀ ♂ a4 a5 c5

M G T G T G T



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International Journal of Advancements in Research ... · heteromorphic sex chromosomes which can be identified by fluorescence in situ hybridization (FISH) and cytogenetic stud-ies