GENETIC DIVERGENCE OF RABBITFISH SPECIES IN EGYPT USING ISSR-MARKERS
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J. Biol. Pham. Sci. Vol. 6, No. 1 July, 2008
GENETIC DIVERGENCE OF RABBITFISH SPECIES IN EGYPT
USING ISSR- MARKERS
By
Omaima E. Khafagy
From
Department of fish Resources and Aquaculture, Faculty of Environmental Agricultural
Sciences, Suez Canal University.
ABSTRACT
Genus Siganus is represented in Egypt by two species, Siganus rivulatus and
Siganus luridus. This study were, to studied the pattern of genetic variation in Siganus
species in several localities of Egypt including Red Sea , Alexandria and Arish, to expose
the potential of these markers in genetic studies requiring the detection of low
polymorphism; as well as using ISSR- PCR DNA markers. Restriction endonucleases of
the genomic DNA have been used to detect the genetic variability among and within fish
populations. The relative from, molecular weight (MW) and band frequency fingerprints
generated by the 6 primers revealed unique for each Siganus species in terms of number
and position of ISSR bands. The wide distribution of microsatellites in Siganus species
enables the analysis of inter-simple-sequence repeat (ISSR) markers. Very little of the
variation was attributed to the locality and most of the variance appeared within localities.
The variance components were significant (P50
(family Siganidae, genus Siganus) that comprises 27 marine species divided in two
subgenera, Siganus and Lo (Kuiter and Debelius, 2001).
Different techniques have been applied to study genetic differentiation in two
rabbitfish species (Siganus rivulatus and Siganus luridus), but to date not enough ISSR
studies have been published on this species Siganids (rabbitfish) are a relatively small
family of algaevorous fish widely distributed in the Indo-West Pacific Region (Woodland,
1983).
Since the opening of the Suez Canal in the 19th century, a growing number of
teleosts species have become permanently established in the Mediterranean Sea. As
environmental parameters in the Mediterranean Sea are very different from those in the Red
Sea, there is an opportunity to evaluate the gene flow associated with the acclimatization of
these species to their new environment. Initial colonization may have been restricted to
individuals with marginal genotypes compared to populations of the Red Sea, eventually
better adapted to the Mediterranean Sea environment (Papaconstantinou, 1990).
According to Ben-Tuvia (1978), the two main factors influencing the distribution of
organisms over large zoogeographical areas are temperature and salinity that are notably
different in both seas. Dominant currents in the Suez Canal are also an important factor
favouring the Lessepsian migration. Since the opening of the Suez Canal, the number of
migrant species into the eastern Mediterranean Sea continuously increased (Galil, 2000).
Choosing an effective method to assess genetic variability in a group of individuals
is of great interest to many researchers studying population genetics. In recent years,
different molecular markers based on PCR amplification have been developed and rapidly
have become essential tools in this field. Some of these markers are microsatellite-based,
such as the inter-simple sequence repeat (ISSR) markers (Zietkiewicz et al., 1994). ISSR
markers are generated from nucleotide sequences located between two microsatellite
priming sites inversely oriented on opposite DNA strands and near enough to be amplified
by PCR. This technique relies on the high polymorphism and wide distribution of
microsatellites to detect low differentiation levels.
The aim of this investigation was to assess genetic differentiation in samples
localities distributed in Red Sea and Mediterranean Sea to expose the potential of these
markers in genetic studies requiring the detection of low polymorphism and as a source of
sequences for developing microsatellite markers.
Research using ISSR markers has focused on evaluating genetic variation in terrestrial
ecosystems. Less attention has been paid to the application of these markers in marine
populations, where they have been used to evaluate the gene flow of two teleost species
between the Red Sea and the Mediterranean Sea (Hassan et al., 2003). Using different
molecular genetic techniques (mitochondrial DNA, Exon-Primed Intron-Crossing PCR
amplification (EPIC) and Inter Simple Sequence Repeats (ISSRs), patterns of genetic
differentiation on both sides of the Suez isthmus were tested on two Lessepsian rabbitfish
species (Siganus rivulatus and Siganus luridus).
In recent years, much of the research efforts have been focused in identifying the
factors that allow successful invasions, with the promising goal to predict the identity of future
invaders and vulnerable ecosystems (Goodwin et al., 1999 and Alcaraz et al., 2005). More
than 59 Lessepsian fish species were recorded in the Levant basin. Golani, (1999) supposes
that the existence of unsaturated niches in the eastern Levant basin, makes it more vulnerable
to colonization. The environmental conditions in the new habitat and the characteristics of
invaders play a prominent role in determining the success or failure of invasions (Reichard
and Hamilton 1997).
Such delay between the initial establishment of colonist and subsequent expansion
is a common feature of biological invasion (Lee, 2002 and Rilov et al., 2004) and can be
explained as an evolutionary phenomenon: the time needed for genetic adaptation to the
50J. Biol. Pham. Sci. Vol. 6, No. 1 July, 2008
new environment (Sakai et al., 2001). As a matter of fact, large scale changes in the
distribution pattern of fish species may reflect changes in the oceanographic climatic
conditions (Stephens et al., 1988) and recently many displacements of tropical affinity
species in the Mediterranean Sea have been related to environmental changes (Guidetti
and Boero 2001).
MATERIALS AND METHODS
Sample Collection and DNA Extraction
The analysis of ISSR markers was performed on 60 S.rivulatus and S.luridus
individuals were obtained from Mediterranean Sea (Alexandria and Arish) and Red Sea.
DNA was extracted as described by Winnepenninck et al.,(1993). Samples stored in
ethanol were rehydrated in PBS (0.137 M NaCl, 2.68 mM KCl, 10.1 mM Na2HPO4, 1.76
mM KH2PO4) and distilled water before DNA extraction.
PCR amplification and sequencing
Each PCR reaction of ISSR markers had a final volume of 25 µl, containing 20 ng
of DNA template, 1×PCR buffer (16 mM (NH4)2SO4; 67 mM Tris-HCl, pH 8.8;
0.1%Tween-20), 1µM primer, 0.2 mM each dNTP, 5.2 mM Mg2Cl, and 0.75 U Taq DNA
polymerase (Bioline). Amplifications were performed using a Touchdown PCR according
to (Don et al., 1991) in a PTC-100 thermal cycles under the following conditions: 94°C 20
S min, 66°C 30 S , 72°C 2 min, and the annealing temperature was dropped 1°C for each of
the subsequent 10 cycles followed by 30 cycles at 94°C 20 s, 55°C 30 s, and 72°C 2 min,
with a final extension at 72°C 5 min (Fisher et al., 1996). Six primer ISSR (GATC(TCTG),
HVH(TTCG)4, KRV(CT) 6, GATC(TCTG)7, YG(CT)9, YG(CA)9 (Table.1).
Microsatellite fragment were amplified with the following conditions: 94°C 2 min,
followed by 30 cycles of 92°C 1 min, Ta 1 min, and 72°C 30 S with a final extension step at
72°C 10 min. PCR reactions were carried out in a total volume of 25µl consisting of 20 ng
DNA template,1× Roche Taq PCR buffer (10mM Tris-HCl, pH 8.3; 50 mM KCl,0.2 µM
each forward and reverse primers, 0.2 mM each dNTP, 1.5 mM Mg2Cl, and 0.75 U Taq
DNA polymerase (Roche).
Electrophoresis and Band Scoring
Products obtained by DNA extraction and amplification of ISSR markers were
observed on 1.5% agarose gels stained with ethidium bromide using the UVP Gelworks
densitometry software, which quantifies DNA and assigns a fragment size to each band by
scoring against a molecular weight marker (Roche 100 bp ladder) (Fig. 2). A negative
control was added in each run to test for contamination. To choose scorable bands and
ensure reproducibility, a group consisting of 10 samples of each locality was amplified and
analyzed twice. Microsatellite markers were evaluated for consistent amplification and
polymorphism using according to Panaro et al.,( 2000).
RESULTS
The results of microsatellite analysis of muscle protein clear that each of the
examined species had its own characteristic pattern which could distinguish not only
between the two species and also between the same species. And the hybrid has more of its
protein pattern.
According to the Rp index, the primer ISSR(YG(CT)9) is the most appropriate to
differentiate individuals, and a 200 bp band obtained with the primer ISSR
(GATC(TCTG)8) is the most useful to differentiate sampling localities (Table.1).
5152
The results for the six primer pairs that yielded amplification products are shown in
(Table.3). One proved to be monomorphic 0.7627, and another) generated too many
nonspecific bands and was judged unacceptable for analysis. Two microsatellites (0.7717
and 0.8924) generated scorable and polymorphic products. Observed heterozygosities
(Ho)at YG(CA)9,GATC(TCTG)8 and KRV(CT)6 respectively.
Table.1 required a higher final annealing temperature, and as a result, only 6 step-
down cycles were needed, and the annealing temperature was kept at 59°C for 36 cycles.
Structure of ISSR markers and annealing of anchored primers.
Analysis of molecular variance also revealed a higher structure level between Red
Sea and Mediterranean Sea than within the Mediterranean Sea. However, structure in all
cases was very low, with 10% of the variance being attributable to the structure between
Red Sea and the Mediterranean Sea, and only 1.5%of the variance being attributable to the
structure between Alexandria and Arish (Mediterranean Sea). We noticed that there are
very little of the variation was attributed to the locality and most of the variance appeared
within localities. The variance components were significant (PJ. Biol. Pham. Sci. Vol. 6, No. 1 July, 2008
ISSR markers provides data that can be used to amplify single microsatellite loci. In
previous studies, microsatellites have been isolated from genomic libraries, and then these
sequences have been useful in natural populations (Astanei et al., 2005) or as a tool of
parentage identification (Walker et al., 2005).
The development of microsatellite markers is a long and expensive process,
however, and so some researchers examine the applicability of markers from related species
(Evans et al., 2001). This study was clearly related to genetic differentiation between
species. Furthermore, few loci are available in rabbitfish - related species, hence the
isolation and characterization of microsatellite markers from the sequences amplified by
anchored primers represents a feasible alternative as described previously (Keiper et al.,
2006 and Varela et al., 2007).
This technique provided a fast and cost-effective analysis of polymorphism in a
group of individuals, and at the same time, sequence data were gathered to perform more
studies using single microsatellite loci. Our findings uncovered a slight but detectable
lowering of the S.luridus genetic diversity in the (Red Sea). Invading population compared
with the parental one (Mediterranean Sea). These results stressed the importance to
encompass wide geographic samplings in this kind of studies, so far limited to the
Mediterranean Sea.
Data Analysis
Samples were collected in one locality in the Red Sea and two localities in the
Mediterranean Sea (Alxandria and Arish). The usefulness of ISSR markers, as
fingerprinting of the banding pattern obtained with each primer, was evaluated using the Rp
index of Prevost and Wilkinson (1999), who found a strong relationship between this
index and the ability of a primer to differentiate between individuals.
The Rp of a primer is Rp = Ib, where Ib (band informativeness) is equal to 1 - [ 2 ×
(0.5- p)], p being the proportion of individuals containing this band. This index could be
helpful as a primer choice criterion to perform fingerprinting analyses in a particular
species. The value of this index for each primer does depend on the number of individuals.
For these values to be comparable among different studies, however, the groups considered
must have similar levels of genetic diversity and the studies must apply the same analytical
procedure. The conditions in the Mediterranean, particularly in its region, are very different
than in the Red Sea, thus potentially resulting in strong selective pressure. These results
were Agreement with Papaconstantinou, 1990 and Golani 1993a).
Finally, 200 microsatellites were found in the sequences of 28 ISSR markers, and
two polymorphic microsatellite markers were developed may be located in a mosaic hybrid
zone (Bierne et al., 2003). Genetic diversity and structure among regions and populations
haplotype diversity (hd) was found to be higher in Red Sea than in any Mediterranean Sea
populations, as well as in the combined Mediterranean Sea samples. This result was
agreement with (Diamant, 1989). Structure of ISSR markers and annealing of anchored
primers in (Table.1) required a higher final annealing temperature, and as a result, only 6
step-down cycles were needed, and the annealing temperature was kept at 59°C for 36
cycles. Diagonal lines separate microsatellite priming sites inversely oriented on opposite
DNA strands and near enough to be amplified by PCR (Zietkiewicz et al., 1994) (Fig.1).
They concluded that the rabbitfish of Red sea were not significantly different from a pure
Siganus rivulatus locality, suggesting a high introgression of alleles of S. rivulatus in these
individuals. DNA diversity is maintained during the early invasions, thus avoiding
suboptimal assortments of genotypes and preserving the needed genetic plasticity to adapt
to the new habitat.
5354
They used the protein electrophoresis to confirm the identity of the two species and
their hybrids where there are few characters have been detected by morphological analysis.
This information accorded with the idea that Lessepsian migration involves many
individuals since its beginning and emphasized the need to explore the causes which trigger
migration itself, as for instance changes in the oceanographic climatic conditions (Keiper et
al. 2006).
Table (1): ISSR markers in two rabbitfish species
Primer a Size Bands (bp) b
Rp index
5' – YG(CA)9-3' 650,525, 450,250 1.57
5' – GATC(TCTG)8-3' 750, 800,950,550,200 3.14
5' – HVH(TTCG)4-3' 975,900,1025,1400 2.76
5' – KRV(CT) 6 - 3' 1425,1090,1050,1300 3.14
5' – GATC(TCTG)7-3' 1200,1350,1450,1525 256
5' – YG(CT)9-3' 1700,1250,950,750,650 1.59
a
H :A,T, or C ; V:C,G, or A ;Y: C or T; K:G or T; R:A or G
b
Rp : resolving power.
Table (2): Number of microsatellites which found in 19 ISSR markers
ISSR Primer a Repeat Motif b Size Accession no.
YG(CA)9 (CA)9 (ATTT)5(TA)7 (TG)8 683 AJ938137
(AC)7 (CAAC)3(CAAT)11 (CA)(TG)8 345 AJ938136
(CA)9 (AT) 5 (TG)28 237 AJ938135
GATC(TCTG)8 (TCTG)6 (TCCG)4(ACAG)6 750 AJ938129
(TCTG)8(ACAG)4(ACAG)6 775 AJ938128
(TCTG)6 (ACAG)6 650 AJ938127
(TCTG)6 (TCCG)4(ACAG)6 425 AJ938126
GATC(CTG)7 (CGT)7 (GAC)9 720 AJ938125
(CTG)6 (ACG)6 600 AJ938124
(TCG)6 (ACG)27 457 AJ938123
YG(CT)9 (CT)9 (AG)7 715 AJ938117
(TTCT)24 (CT)6 (GA)6 (AG)6 (GA)8 550 AJ938116
(TCTG)6 (TCCG)4(ACAG)6 730 AJ938115
KRV(CT)6 (CT)6 (TTG)4(AG)6 650 AJ938111
(TCTG)6 (TCCG)4(ACAG)6 450 AJ938110
(TTCG)4 (ACGA)11 200 AJ938109
HVH(TTCG)4 (TTCG)4 (ACAG)9 650 AJ938101
(TTCG)7(ACAG)6 350 AJ938100
(TTCG)6 (AT)5(AACG)6 250 AJ938199
a Y = C/T, R=A/G, V=A/C/G, H=A/C/T, K= G/T
b At least nucleotides randomly repeated twice or three times
54J. Biol. Pham. Sci. Vol. 6, No. 1 July, 2008
Table (3):Characterization of six microsatellite markers in two rabbitfish species
Locus Comments Ho Accession no
YG(CA)9 Polymorphic 0.8924 AJ938137
GATC(TCTG)8 Monomorphic 0.7627 AJ938129
GATC(CTG)7 Mosic _ AJ938125
YG(CT)9 Unscorable _ AJ938117
KRV(CT)6 Polymorphic 0.7717 AJ938111
HVH(TTCG) 4 Monomorphic _ AJ938101
Fig 1: Structure of ISSR markers and annealing of anchored primers. Diagonal lines separate
microsatellite priming sites inversely oriented on opposite DNA strands and near enough to
be amplified by PCR. Arrows indicate direction of DNA polymerization (5'-3'). N
represents an anchoring Nucleotide.
MW Marker A B C D E F G
(bp)
2000
1425
1350
1050
1000
950
725
600
500 525
475
250
Fig 2: ISSR markers ,Anchored primers generated polymorphic banding patterns in rabbitfish
species from three localities Alexandria , Arish, and Red Sea. Lane A ,B ,C represented
S.rivulatus of Alexandria; Arish; and Red Sea respectively and D ,E,F, represented S.
luridus of Alexandria; Arish; and Red Sea respectively. G negative control No DNA. MW
: DNA Molecular weight marker (Roche).
5556
CONCLUSION
The analysis of the populations of two rabbitfish species allowed concluding that the
rabbitfish species were extant in the Red Sea before the construction of the Suez Canal
based on the genetic similarity between all possible pairs of these populations. The
conditions in the Mediterranean Sea, particularly in its region, are very different than in the
Red Sea, thus potentially resulting in strong selective pressure. These results were
Agreement with Papaconstantinou, 1990 and Golani, 1993a).
ACKNOWLEDGMENTS
We would like to thank prof. Dr. Magdy T. Khalil Science Faculty, Ain shams
University, for his continues help in reading the mainscript of the article . And Special
thanks to the Genetic Engineering Center University Ain Shams.We wish to acknowledge
all fish farmers in collection fish samples in this study.
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دراسة االختالفات الوراثية ألسماك السيجان الموجودة فى مصر باستخدام
تحليل ISSR-PCR
أميمة خفاجى
قسم الثروة السمكية -كلية العلوم الزراعية البيئية ـ جامعة قناة السويس
تتمثل أسماك السيجان فـ مرـر والتـ تتبـ عائلـة Siganidaeبجـنس وادـه ـو Siganus
ويوجه منها نوعان فقط ف مرر ما Siganus rivulatus :و Siganus luridus
كان الههف من البحث و دراسة الفروق الوراثية واالختالفات بين المواق الثالث التـ أخـ ت
منها العينات العريش اإلسكنهرية والبحر األدمر ،أثبتت الهراسة أن ناك اختالفات صغيرة لكل موقـ
عل دهة وك لك ناك اختالفات عل مستوى الموق الواده .
كانت ناك اختالفات وراثية عل مستوى الـ . DNAوباستخهام ادهث االختبارات والتحاليل
الوراثية ISSRأمكن الكشف عن االختالفات الهقيقـة بـين كـل مجموعـة أو عشـيرة علـ دـهة وداخـل
المجوعة نفسها .بل االختالفات داخل المواق نفسها.وبين المواق الت تم اختيار العينات منها.
أمكن عن طريق المواق مقارنة الحزم المعزولة بالوزن الجزيئ و معرفة االختالفات الوراثية
الهقيقة بين النوعين.
كشف تحليل ISSRلعينات الـ DNAالمعزولة من البحر األدمـر والبحـر األبـيل المتوسـ ط
(اإلسكنهرية ،العـريش عـن وجـود اختالفـات وراثيـة بنسـبة عاليـة بـين المواقـ الـثالث بينمـا كانـت
االختالفات أقل داخل كل موق عل دهة ف العينات الت تمت دراستها.
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