Cytogenetic characterization and Molecular genetic variations of five marine species of Perciform fishes

The chromosomal analysis of five species of order Perciformes, species Cheilinus lunulatus, Cheilinus abudjubbe, Cheilinus mentalis, Cheilinus digrammus and Cheilio inermis were carried out; all samples were collected from Hurghada, on the Egyptian Red Sea coast. The diploid chromosome numbers and Fnumental numbers for five species under study were 2n=40 and FN=54, 2n=40 and FN=54, 2n=44 and FN=60, 2n=40 and FN=52 and 2n=48 and FN=66 respectively, they were different in their karyotypes,. The RAPD-PCR analysis was carried out on species under studies by using eight primers. All eight primers amplified successfully on the genomic DNA extracted from all studied fish species. Using RAPD-PCR is very useful in determination genetic molecular variations and relationship degree between the species which belong to same family. Introduction The Red Sea is home of unique fauna due to its almost isolated position near the Indian ocean, over ten percent of its fish species are endemic found exclusively between the Suez canal in the north and the Gate of Lamentations (Bab-el-Mandab) in the south Debelius, (2007). The genetic structure represents one of a very important elements for the taxonomy used by the modern cytotaxonomists, so the chromosomal analysis and karyotype studies have become an invaluable tool contributing to the solution of many systematic and evolution problems Rab et al. (1991). Cytogenetic is a branch of genetics that is concerned with study of the structure and function of the cell, especially the chromosomes Baltimore and Williams, (2006). Karyological studies have provided basic information on the number, size and morphology of chromosomes that is important to undertake chromosome manipulations in fish Khan et al. (2000). Furthermore, the karyotype might characterize a species through the determination of the details (number, size and structure) of its chromosomes Alcocer et al. (1999). Besides karyotype analysis helps to predict, with considerable certainly, the fertility or sterility of hybrids by comparing the number and morphology of the chromosomes of parental species Serebryakova, (1972). The diploid chromosome number varies from 2n=22—26, in some species of Nototheniidae, an Antarctic fish group, to 2n=240-260 in some anadromous Acipenseridae, which show several micro-chromosomes Ozouf-Costaz et al. (1997). The family Labridae (Wrasses) contains approximately 500 species distributed into 60 genera, and so far only 47 species (belonging to 21 genera) of them are cytogenetically studied (Arkhipchuk, (1999); Brum, (1996); Klinkhardt et al. 1995; Manna, 1989; Ojima and kashiwagi, (1989) and Vasil`ev, (1980) ). In the Labridae, although most species have been reported with 2n=48, the number of chromosome arms (FN) is often higher (FN=48-90), indicating a predominant occurrence of pericentric inversions within this group Alvarez et al. (1986). Random amplified polymorphic DNA (RAPD) has been widely used in the last decade in species identification program, and in assessing genetic variations among different taxa at DNA level because of its coast effectiveness and simple operation without requiring prior knowledge of species DNA sequences (Schnell et al. (1995) and Frankel et al. (1997). Atienzar et al. (2002) and Brahmane et al. (2006) and (2008), reported that , the main advantages in the use of the RAPD method lie in its rapidity, applicability to any organism without prior information on the nucleotide sequence, cell cycle, karyotype and potential detection of a variety of DNA damage and mutations at the whole genome level. Random amplified polymorphic DNA (RAPD) is one of the molecular techniques that has benefited from the advent of the PCR. According to Atienzar and Jha (2006) and Fouz et al. (2007), the RAPD method has been initially used to detect polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. In addition, the fact that the RAPD assay allows the visualization of a wide range of PCR products may explain why this assay is a preferred choice. Also the random amplified polymorphic DNA (RAPD) is a useful assay for the detection of genotoxininduced damage and mutations. In spite of the fact there have been considerable development of cytotaxonomic studies in the last twenty years, very little information are available on the cytogenetic characteristics of marine fishes in Egypt especially in the Red Sea. So the aim of the present study is fill this gap and also to indicate the taxonomic and phylogenetic relationship of five species of order Perciformes, Cheilinus lunulatus, Cheilinus abudjubbe, Cheilinus mentalis, Cheilinus digrammus, Cheilio inermis. Materials and Methods Five species of Cheilinus lunulatus, Cheilinus abudjubbe, Cheilinus mentalis, Cheilinus digrammus and Cheilio inermis, were collected from Red Sea (Egypt), transported to the laboratory of “National Institute of Oceanography and Fisheries (Red Sea Branch)” and kept alive until processed.


Introduction
The Red Sea is home of unique fauna due to its almost isolated position near the Indian ocean, over ten percent of its fish species are endemic found exclusively between the Suez canal in the north and the Gate of Lamentations (Bab-el-Mandab) in the south Debelius, (2007).
The genetic structure represents one of a very important elements for the taxonomy used by the modern cytotaxonomists, so the chromosomal analysis and karyotype studies have become an invaluable tool contributing to the solution of many systematic and evolution problems Rab et al. (1991). Cytogenetic is a branch of genetics that is concerned with study of the structure and function of the cell, especially the chromosomes Baltimore and Williams, (2006).
Karyological studies have provided basic information on the number, size and morphology of chromosomes that is important to undertake chromosome manipulations in fish Khan et al. (2000). Furthermore, the karyotype might characterize a species through the determination of the details (number, size and structure) of its chromosomes Alcocer et al. (1999). Besides karyotype analysis helps to predict, with considerable certainly, the fertility or sterility of hybrids by comparing the number and morphology of the chromosomes of parental species Serebryakova, (1972).
In the Labridae, although most species have been reported with 2n=48, the number of chromosome arms (FN) is often higher (FN=48-90), indicating a predominant occurrence of pericentric inversions within this group Alvarez et al. (1986).
Random amplified polymorphic DNA (RAPD) has been widely used in the last decade in species identification program, and in assessing genetic variations among different taxa at DNA level because of its coast effectiveness and simple operation without requiring prior knowledge of species DNA sequences (Schnell et al. (1995) and Frankel et al. (1997). Atienzar et al. (2002) and Brahmane et al. (2006) and (2008), reported that , the main advantages in the use of the RAPD method lie in its rapidity, applicability to any organism without prior information on the nucleotide sequence, cell cycle, karyotype and potential detection of a variety of DNA damage and mutations at the whole genome level. Random amplified polymorphic DNA (RAPD) is one of the molecular techniques that has benefited from the advent of the PCR.
According to Atienzar andJha (2006) andFouz et al. (2007), the RAPD method has been initially used to detect polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. In addition, the fact that the RAPD assay allows the visualization of a wide range of PCR products may explain why this assay is a preferred choice. Also the random amplified polymorphic DNA (RAPD) is a useful assay for the detection of genotoxininduced damage and mutations.
In spite of the fact there have been considerable development of cytotaxonomic studies in the last twenty years, very little information are available on the cytogenetic characteristics of marine fishes in Egypt especially in the Red Sea. So the aim of the present study is fill this gap and also to indicate the taxonomic and phylogenetic relationship of five species of order Perciformes, Cheilinus lunulatus, Cheilinus abudjubbe, Cheilinus mentalis, Cheilinus digrammus, Cheilio inermis.

Materials and Methods
Five species of Cheilinus lunulatus, Cheilinus abudjubbe, Cheilinus mentalis, Cheilinus digrammus and Cheilio inermis, were collected from Red Sea (Egypt), transported to the laboratory of "National Institute of Oceanography and Fisheries (Red Sea Branch)" and kept alive until processed.

ReseaRch PaPeR
Chromosome preparation and staining: Metaphases were obtained from cephalic kidney, spleen and gills after injection of 0.05% colchicines for approximately 2 hr, following the standard air-drying procedure Nirchio and Cequea (1998).chromosomal morphology was analysed through high quality spread photographs according to Leven et al. (1964).

Data analysis
The banding patterns generated by RAPD-PCR marker analyses were compared to determine the genetic relatedness of the 5 sample fish accessions. Clear and distinct amplification products were scored as '1' for presence and '0' for absence of bands. Bands of the same mobility were scored as identical.
The genetic similarity coefficient (GS) between two genotypes was estimated according to Dice coefficient Sneath and Sokal, (1973).

Dice formula: GSij = 2a/(2a+b+c)
Where GSij is the measure of genetic similarity between individuals i and j, a is the number of bands shared by i and j, b is the number of bands present in i and absent in j, and c is the number of bands present in j and absent in i.
The similarity matrix was used in the cluster analysis. The cluster analysis was employed to organize the observed data into meaningful structures to develop taxonomies. At the first step, when each accession represents its own cluster, the distances between these accessions are defined by the chosen distance measure (Dice coefficient). However, once several accessions have been linked together, the distance between two clusters is calculated as the average distance between all pairs of accessions in the two different clusters. This method is called Unweighted Pair Group Method using Arithmetic Average (UPGMA) Sneath and Sokal, (1973).

1-Cheilinus lunulatus
The chromosome number of Cheilinus lunulatus was scored, the diploid set consists of 2n=40 and fundamental number (FN) =54 as shown in (Fig 1). The karyotype consists of three groups; group A contains five metacentric pairs of chromosomes, group B which composed of two submetacentric and group C which consists of thirteen acrocentric pairs of chromosomes.

2-Cheilinus abudjubbe
The photographs of cell spread and karyotypes of this species was found to have a diploid chromosome number of 2n=40 and fundamental number FN=54, as shown in ( fig.  2). This number of chromosomes are allocated into four groups; group A consists of four metacentric pairs of chromosomes; group B is composed of one submetacentric pair ; group C has two subtelocentric pairs of chromosomes and group D which composed of thirteen pairs of acrocentric chromosomes.

3-Cheilinus mentalis
The diploid chromosome number of Cheilinus mentalis is 2n=44 and FN=60. For karyotyping, the chromosomes of 10 well-spread metaphase plates were cut out from photographs and paired on the bases of centromeric position and size as shown in fig. (3), and four different groups formed: group A has three metacentric pairs of chromosomes ; group B is composed of three subtmetacentric pairs of chromosomes; group C which consists of two subtelocentric pairs of chromosomes and group D which consists of fourteen pairs of acrocentric pairs of chromosomes.

4-Cheilinus digrammus
The chromosomal analysis of this fish indicated that the diploid chromosome number 2N=40 and the fundamental number is FN=52 ( fig. 4), the chromosomes are arranged in four groups: group A composed of three metacentric chromosome; group B consists of two submetacentric chromosomes; group C has only one subtelocentric chromosome and group D consists of fourteen pairs of acrocentric chromosomes.

5-Cheilio inermis
Chromosomal analysis of Cheilio inermis showed the diploid chromosome number of 2n=48 and FN=66. The mitotic metaphase spread and karyotype are shown in fig. (5). The chromosomes are allocated into four groups; group A consists of four metacentric pairs of chromosomes; group B is composed of one submetacentric chromosome group C composed of four subtelocentric chromosomes and group D contains fifteen acrocentric pairs of chromosomes.

RAPD-PCR analysis:
Eight single primers (OPA-12,OPA-14,OPA-15,OPA-17,OPA-18,OPA-19,OPA-20 and OPO-11) were used in the present investigation to determine the genetic differences among five species of family Labridae ( Cheilinus lunulatus, Cheilinus abudjubbe, Cheilinus mentalis, Cheilinus digrammus, and Cheilio inermis). All eight primers amplified successfully on the genomic DNA extracted from all studied fish species. The eight primers yielded amplification products in the five species of the family Labridae. The number of fragments amplified per primer varied between 4 (OPA-19) and 18 (OPA-18) (11.38 bands/ primer) and had a size range from 190 bp (OPO-18) to 2700 bp (OPA-12). The DNA fragments generated by the eight primers from the genomic DNA of the five species were separated using Agarose gel electrophoresis and illustrated in figs (6, 7, 8, 9,10,11,12 and13).. The banding patterns of these DNA fragments were analyzed by Gene profiler computer software program and summarized in tables (2, 3, 4, 5,6,7,8 and9). The number and positions of the bands depended on species and primer as shown in these tables.
The number of bands was variable in each species. Cheilinus lunulatus was the species that produced 46 bands, and in Cheilinus abudjubbe 44, while in Cheilinus mentalis 43 bands; 54 bands in Cheilinus digrammus; and 50 bands in Cheilio inermis. A total of 91 DNA bands were gener-ReseaRch PaPeR ated by all primers in all specimen, out of these DNA bands 65 (71.43%) were conserved among all specimens while 26 bands were polymorphic with percentage 28.57% of all the eight tested primers produced polymorphism in all specimens table10.
Following are the amplification results of the five species obtained by the examined primers:

Cheilinus lunulatus
All primers amplified yielded distinct RAPD pattern with Cheilinus undulates, the eight primers generated 46 fragments, ranging from 1 by the primer OPA-19 to 11 by the primer OPO-11 and the size of these fragments arranged from 250 bp by the primers OPA-14 and OPA-18 to 1900 bp by the primer OPA-17.

Cheilinus abudjubbe
The RAPD DNA analysis indicated that all amplified primers produced fragments with this fish, 44 are all the fragments varied from 1 by the primers OPA-15 and OPA-19 to 10 by the primer OPO-11, the size of these fragments varies from 250 bp by the primers OPA-14 and OPA-18 to 2500 bp by the primers OPA-14 and OPA-17.

Cheilinus mentalis
The RAPD analysis of C. mentalis illustrated that all primers except OPA-12 gave 43 fragments, ranging from 2 bands by the primer OPA-19 and 10 bands by the primers OPA-18 and OPO-11. The size of fragments ranged from 250 bp by the primer OPA-14 to 2100 bp by the primer OPA-17.

Cheilinus digrammus
Random amplified polymorphic DNA (RAPD) technique was used to examine the genetic variability on C. digrammus produced different RAPD band patterns of number of 54 bands ranged approximately from 190 bp by the primer OPA-18 to 2700 bp by the primer OPA-12. The generated bands ranged in number from 2 by the primer OPA-19 to 10 by the primer OPA-18.

Cheilio inermis
Random amplified polymorphic DNA (RAPD) technique was used to examine the genetic variability on Cheilio inermis produced different RAPD band patterns of number of 50 bands ranged approximately from 250 bp by the primer OPA-18 to 2100 bp by the primer OPA-17. The generated bands ranged in number from 2 by the primer OPA-19 to 11 by the primer OPA-18.
The Dice coefficient illustrated the genetic similarity between the five species, and the UPGMA clustering pattern are shown in Table (11) and Figure (14). The highest genetic distance was observed between Cheilinus lunulatus and Cheilinus abudjubbe (80%) and the lowest between Cheilinus lunulatus and Cheilinus mentalis (53%). The UPGMA dendrogram shows two groups, group A in which Cheilinus lunulatus and Cheilinus abudjubbe were clustered together, group B which consists of two clusters one contained Cheilio inermis.alone while the other contained Cheilinus mentalis and Cheilinus digrammus indicating the close genetic relationship among species.
To the best of author knowledge these results are reported for the first time in Egypt, this investigation confirmed that the polymerase chain reaction is very important nowadays to the taxonomists specially in sibling species in addition to the morphological and anatomical characters.

Discussion Cytogenetic analysis:
The present work is contribution to the karyological data on marine fish species, a group that has been relatively less studied than freshwater species. Considering the order Perci-formes, karyotypes of approximately 600 species have been analyzed so far, which correspond to 7% of the number of described species for this order Affonso, (2000).  2011)) who concluded that the diploid chromosome number of family Labridae ranged from 2n=22 to 2n=54.
In the family Labridae, although most species have been reported with 2n=48 the number of chromosome arms (FN) is often higher (48 -90), indicating a predominant occurrence of pericentric inversions within this group Alvarez, et al. (1986).

RAPD-PCR analysis:
This study reports on the use of RAPD markers for studying genetic similarity among five species (family, Labridae) of Red Sea coral reef fishes in Egypt for the first time. The RAPD assay has been used to construct phylogenetic trees for resolving taxonomic problems in many organisms (Chalmers et al. (1992;Bardakci and Skibinski, (1994); Greef and Triest, (1999); Barman et al. (2003);Soliman et al. (2003) and Ali, (2003)) RAPD bands in this study were always variant (i.e. strong, faint, fuzzy and sharp bands) generated with each primer because one or more copies of DNA may exist per genome or may be attributed to the varying of the annealing process between the primer and the DNA, this problem of mixed bands shows the well known sensitivity PCRs Bielawski et al. (1995). RAPD fragments generated by the all eight primers, there were high degree of polymorphism; their sequences may be considered as more conserved sequences which are most useful in higher taxonomic levels and evolutionary relationsh ips.
The description of this similarity coefficient is not simple, especially when more than one character is involved in the same cluster. Thus Cheilinus lunulatus and Cheilinus mentalis and Cheilinus digrammus are found to have a similarity coefficient of 80%, while between Cheilinus lunulatus and Cheilinus mentalis is 53%, between Cheilinus mentalis and Cheilinus digrammus is 63%, and between Cheilinus lunulatus and Cheilio inermis 63%. (The UPGMA dendrogram among the species under study is presented in fig (14).
According to Atienzar andJha (2006) andFouz et al. (2007), the RAPD method has been initially used to detect polymorphism in genetic mapping, taxonomy and phylogenetic studies and later in genotoxicity and carcinogenesis studies. In addition, the fact that the RAPD assay allows the visualization of a wide range of PCR products may explain why this assay is a preferred choice. Also the random amplified polymorphic DNA (RAPD) is a useful assay for the detection of genotoxininduced damage and mutations.