International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
14
Determination of Sex Markers by Amplified Fragment Length
Polymorpism (AFLP) in Killifish Aphanius anatoliae anatoliae
(Leidenfrost, 1912)
Efkan BAĞDA1*, Fevzi BARDAKCI2, Z. Deniz INAN3
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: efkanbagda@hotmail.com
1*
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
F
ISHES are the most abundant vertebrates on Earth, showing 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 including ecology, behavior, conservation and genetics of many species 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 monogenic and polygenic systems, with factors localized on autosomes
or on sex chromosomes [9]. In polygenic systems, sex is determined by the accumulative genetic action of all factors involved in its determination, and not by a single genetic locus
[10]. In monogenic systems, sex is determined by a gene located 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 studCopyright © 2013 SciResPub.
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 homomorphic, 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. DNAbased tests can solve this problem but sex specific markers
need to initially be isolated [13], [14].
DNA markers provide useful tools for examining sex linkage in fish since DNA structure is not anticipated to change
with altered physiology or environments. Moreover, examination of DNA sequence organization on sex chromosomes can
provide useful insights into the evolutionary processes that
are operating to influence sex-chromosome structure, and ultimately, 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 determination markers was not identified for Aphanius species that are
lack in heteromorphic sex chromosomes [19], [20], [21], [22].
International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
In this article, we focus on the use of the amplified fragment length polymorphism (AFLP) technique [23] for the
identification of sex-specific markers in Aphanius anatoliae anatoliae [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 collection for DNA analysis.
15
cycle profile: a 30 s DNA denaturation step at 94 oC, a 30 s annealing 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 cycles 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 visualized with silver staining. AFLP markers were analyzed
manually based on the presence/absence of DNA fragments.
3 RESULTS AND DISSCUSSION
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]. Approximately 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 procedure and precipitated with absolute ethanol. Precipitated
DNA was dissolved in distilled water and quantified at wavelength 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 preamplification 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 denaturation 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
Copyright © 2013 SciResPub.
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
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, including 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 resolve with morphological or other molecular systematic characters [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 examined 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
International Journal of Advancements in Research & Technology, Volume 2, Issue4, April-2013
ISSN 2278-7763
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 , observed different bands were found for the males of Tuz Gölü
populations while they were found for the females of Güneşli
populations (Fig. 2).
M G T
G T
♀♂♀♂ ♀♂♀♂
a4
a5
G T
♀♂♀♂
c5
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 relatively 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 markers 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 suggestion of failure to development sex markers is suggested by
[49], according to [49], in order to explain the failure of sexmarker 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 recommend to use the number of primer combinations for the identification 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 independent 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 researcher for the choice of primers and primer combinations.
ACKNOWLEDGMENT
The authors wish to thank Ass. Prof. Dr. Esra Bağda for language editing.
Copyright © 2013 SciResPub.
16
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