DNA Fingerprinting of Some Mediterranean and Red Sea Starfish Genomes

Atif M. El- Naggar

Women's College, for Arts, Science and Education Ain Shams University, Cairo, Egypt

Abstract: The discriminative power of DNA markers used as a tool to characterize the Starfish which is very important because they can be used to assess the genetic diversity among all the Echinodermata. During the characterization of starfish of the Red and Mediterranean seas, we evaluated the discriminative power of primers for RAPD and for the first time, ISSR marker systems using AMOVA and building dendrogram , A total number of 130 RAPD bands were obtained. Of these 94 bands were polymorphic (72.3%) and only 36 were monomorphic (27.7%) while, the total number of 178 ISSR bands were obtained. of these 145 bands were polymorphic (81.46%) and only 30 were monomorphic (18.5%) these means that the ISSR markers is highly convenient and the similarities detected with ISSR are greater than the similarities observed with RAPD

Key words: Starfish-DNA fingerprinting-Molecular Marker-RAPD-ISSR

INTRODUCTION with 5 equal arms, and a pluriradiate form with 3 to 8 arms of different length. Another difference between The Asteroidea is one of the largest and most these forms is the number of madreporites. Whereas familiar classes within the Phylum Echinodermata. the pentaradiate specimens have only 1 madreporite, These animals, commonly known as sea stars or the pluriradiate specimens have a variable number of starfishes, form a diverse and species group. There are madreporites. Their number is sometimes, but not approximately 1600 extant species[1,2,3] which are found always, equal to that of the arms. Clark,[9] suggested throughout the world's oceans. Following the that the 2 morphological forms of the starfish A. classification of Blake[4], these species are grouped into burtoni belong to the same species and that the seven orders: Brisingida, Forcipulatida, Notomyotida, pluriradiate form represents young specimens that Paxillosida, Spinulosida, Valvatida and Velatida. reproduce by fission. Molecular markers can be used to reveal Like other echinoderms, asteroids are important polymorphisms at the DNA level between and within members of many marine benthic communities. They populations of different taxa[11]. Changes in nucleotide can be voracious predators, having significant impacts sequences are used in molecular evolutionary studies on community structure. For example, Paine[5] used both for estimating the rate of evolution and for Pisaster ochraceus to illustrate his concept of the role reconstructing the evolutionary history of organisms[12]. keystone species play in community ecology. The Some of these techniques can be applied for crown-of-thorns starfish, Acanthaster planci, is comparisons between populations and between particularly well-known because it can cause extreme individuals in the same populations. The random detrimental effects to coral reefs, particularly during amplified polymorphic DNA (RAPD) and inter simple population outbreaks[6]. sequence repeat (ISSR) techniques and[13,14] respectively Like other asterozoans, asteroids have a has been recognized as a useful tool for population and characteristic star-shaped body plan consisting of a systematic studies[15]. central disc and multiple (typically 5) radiating arms. Asteroids are most easily distinguished from other Considering the potentials of the DNA marker asterozoans (the Ophiuroidea) by the structure of the based genetic diversity analysis, the present study arms[7]. aimed to evaluate the usefulness of molecular markers Tortonese[8] recorded the presence of this Indo- viz. RAPD and ISSR, in assessing and analyzing the Pacific starfish in the Mediterranean, where it has been nature and the extent of genetic phylogeny to several found along the coasts of Lebanon. In the Red Sea, 2 samples of starfish (Asteroidea) species collected from forms of A. burtoni are known[9,10]: a pentaradiate form both the Mediterranean and the Red Sea.

Corresponding Author: Dr.Atif Mohamed El-Nagaar,Women's College for Arts,Science and Education,Ain Shams University, Cairo, Egypt E-mail: [email protected], 39 Res. J. Cell & Mol. Biol., 3(1): 39-48, 2009

MATERIALS AND METHODS estimated by comparing the DNA bands with a 1 Kb DNA ladder (Bioron, Germany) using GelWorks 1D Starfish specimens were collected subtidally by advanced (UVP, UK) scuba diving or by hand in the intertidal zone. The selected species were collected form the Red Sea and Table 1: The Six Asteroidean species, obtained from different the Mediterranean coast of Egypt (Fig. 1). Upon sources were used Serial No. Species Source collection, specimens were cleaned in seawater, 1 Fromia ghardaqana RED SEA immediately frozen on dry ice, and subsequently stored 2 Gomophia egyptiaca RED SEA at -20°C. Identifications were based on original 3 Marthasterias glacialis Mediterranean Sea descriptions of Blake[4]. 4 Astropecten irregularis Mediterranean Sea 5 Asterina Burtoni Red Sea 6 Asterina gibbosa Mediterranean Sea DNA Extraction and Polymerase Chain Reaction (PCR): The soft tissue, stomach and pyloric caecae, of Each DNA fragment generated was treated as a each starfish (Table 1) was removed and separated separate character and scored as a discrete variable, from the skeleton. The DNA was extracted using using 1 to indicate presence, and 0 for absence. AxyPrep multisource Genomic DNA Mini-Prep Kit Accordingly, a rectangular binary data matrix was (Axygen Bioscience, USA, cat. No. Ap-MN-MS- obtained and statistical analysis was performed using GDNA-50) according to manufacturer manual. An the NTSYS-pc version 2.1[17] statistical package. A initial screening of 58 RAPD decamer and 30 ISSR pairwise similarity matrix was generated using simple primers (suitable for the analysis of the starfish matching coefficient (by means of SIMQUAL Asterina burtoni) was performed in order to test their procedure of NTSYS-pc) for RAPD and ISSR data. readability and amplification profiles for polymorphism. Then, cluster analysis was performed (by means of After this screening procedure, 15 RAPD and 18 ISSR SAHN procedure of NTSYS-pc) via unweighted pair- primers were selected (Table 2) For RAPD group method using arithmetic average (UPGMA) to amplification[16] and for ISSR, approximately 10 ng of develop a dendrogram for RAPD and ISSR results. genomic DNA were amplified in a 25 ìl reaction Also, for the correspondence of the similarity matrices –1 mixture containing 2.5 mM ìl dNTPs, 1 U Taq DNA of RAPD and ISSR data were performed (by means of Polymerase (Bioron, Germany), 10 mM Tris HCl (PH MXCOMP procedure of NTSYS-pc) for the null = 7.5), 50 mM KCl, 1.5 mM MgCl , 0.1 % Tritonn100 2 hypothesis that there is no association between the –1 and 0.2 mg ml BSA, and 10 pmol primer. The similarity matrices of RAPD and ISSR.data To obtain samples were subjected to 1 cycle of denaturation at significance level, 5000 permutations were performed. 94°C for 3 min, followed by 40 cycles at 36°C (for In addition, a genetic similarity matrix was calculated RAPD) or 40°C (for ISSR) for 2 min (annealing step), according to Nei[18]. Analysis of molecular variance 72°C for 2 min (extension step), and 94°C for 1 min (AMOVA) was performed using GENALEX 6[19] in (denaturation step). The program was completed by 1 RAPD and ISSR to partition the total molecular min at 36°C and 3 min at 72°C. To visualize the PCR variance between and within populations (clusters). products, 15 µl of each reaction was loaded on 1.8% agarose gel. The gel was run at 90 V for about 1 hr RESULTS AND DISCUSSIONS and visualized with UV transilluminator and photographed using UVP gel documentation system, for RESULTS: In this work, the utility of RAPD and each amplification, a negative control reaction without ISSR markers in the genetic identification analysis of DNA template was included. PCR reaction, that Asteroidea was studied. From the different populations, generated high level of polymorphism across both types 8 starfish belong to six species were collected from of analyses, was repeated twice in order to verify the different locations in the Red and the Mediterranean reproducibility of scored polymorphic bands. This Sea (Fig. 1). Their DNA was isolated and used for procedure allowed only those bands present in all both RAPD and ISSR analysis. replicated experiments to be scored as markers. Amplicon size were estimated using 1 Kbp DNA Identification of RAPD Markers: A high level of standards (Bioron, Germany) polymorphism was generated utilizing the 15 RAPD primers (Table 3). A total number of 130 RAPD bands Data Analysis: Only the clear, unambiguous and were obtained. Of these 94 bands were polymorphic reproducible bands were considered for scoring. Each (72.3%) and only 36 were monomorphic (27.7%). The band was considered to be a single locus. Data were highest number of amplicons was generated from scored as ‘1’ for the presence and ‘0’ for the absence species 1 (12 amplicons), while species 2 generated the of a DNA band of each specimen. DNA band size was lowest (2 amplicons) and at the primer leve, the

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Table 2: Names and sequences of RAPD and ISSR primers RAPD Primers ISSR Primers ------Name* Sequence Name* Sequence B-02 TGATCCCTGG 814 (CT)8TG ------B-03 CATCCCCCTG 844A (CT)8AC ------B-06 TGCTCTGCCC 844B (CT)8GC ------B-10 CTGCTGGGAC 17898A (CA)6AC ------C-02 GTGAGGCGTC 17898B (CA)6GT ------C-06 GAACGGACTC 17899A (CA)6AG ------C-11 AAAGCTGCGG 17899B (CA)6GG ------C-14 TGCGTGCTTG HB8 (GA)6GG ------O-03 CTGTTGCTAC HB9 (GT)6GG ------O-07 CAGCACTGAC HB10 (GA)6CC ------O-09 TCCCACGCAA HB11 (GT)6CC ------O-10 TCAGAGCGCC B2 (AG)8T ------Z-04 AGGCTGTGCT B3 (GA)8A ------B5 (GA)8T ------B10 (AG)8G ------P3 (GT)8C ------UBC-825 (AC)8T ------UBC-827 (AC)8G

Table 3: Number of amplified fragments markers of six starfish species based on RAPD –PCR analysis. strains A19 B02 B03 B06 B10 C02 C06 C11 C14 O02 O03 O07 O09 O10 Z 04 Total 1 AF65 442 5 1566 42 5 6 2 63 ------SM00 110 0 2000 61 0 1 0 12 2 AF66 243 7 2566 93 5 6 1 71 ------SM00 000 1 0000 10 0 0 0 2 3 AF76 422 5 4656 73 6 5 8 76 ------SM00 000 0 1000 01 0 0 1 3 4 AF85 442 5 3647 58 7 5 6 79 ------SM02 010 0 1001 02 0 0 0 7 5 AF66 234 5 2646 57 7 5 1078 ------SM02 000 0 0000 01 0 0 5 8 6 AF85 424 5 2635 55 9 6 3 72 ------SM10 100 0 1000 12 2 0 1 9 TSM14 220 1 5001 87 2 1 7 41 TAF 10 9 6 6 4 7 7 7 6 7 13 12 13 8 15 130 ------PB57 442 3 7232 1312114 1594 TAF = Total amplified fragment, PB = Polymorphic bands, AF = Amplified fragment, SM =specific marker, including either the presence or absence of a band, TSM = Total no. of specific markers across starfish species. 1 = Fromia ghardaqana, 2= Gomophia egyptiaca, 3= Marthasterias glacialis,4= Astropecten irregularis, 5= Asterina Burtoni, 6= Asterina gibbosa.

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Fig. 1: Egypt map showing the sampling stations in the Red and Mediterranean Sea highest number of amplicons was generated from in each (approximately 560,500, 300, 200, 2750, 930, primers Z 04 (13 amplicons), while the lowest was 990 and 590 bp long). In addition, primer B02 (1670, generated from primer B10 (4 amplicons). A number 868), C06 (200 bp), O02 (930 bp), O03 (1100, 570 & of 41 amplicons were a specific markers in which 25 315bp) and O07 (1335 & 635bp) yielded clear and of them were scored for the presence of a unique band characteristic negative markers. Other wise markers are for a given starfish species (positive markers), while 9 a positive as shown in table (4) were scored for the absence of a common band (negative marker). The highest number of the specific Identification of ISSR Markers: ISSR is a class of markers (12) was scored Species 1, while the lowest molecular markers based on inter-tandem repeats of number of the specific markers (two) was scored for short DNA sequences. These inter repeats are highly species 2. In comparison among the six starfish species polymorphic, even among closely related genotypes, (Asteroidea), fifteen primers revealed the power of due to the lack of functional constraints in these RAPD in distinguishing among selected samples of the nonfunctioning regions. Similarly, a high level of Red and Mediterranean Sea. A moderate number of polymorphism was generated utilizing the eighteen RAPD amplicons per primer was sufficient to produce ISSR primers (Figure 3 and Table 5). A total number useful fingerprints for the starfish samples of 178 ISSR bands were obtained. Of these 145 bands discrimination. Based on the amplification products, were polymorphic (81.46%) and only 30 were fifteen individual primers were selected and the monomorphic (18.5%). The highest number of amplification polymorphism against DNA samples from amplicons was generated from species 2 (104 the six starfish was examined. Of the fifteen primers, amplicons), while species 6 generated the lowest (90 exact 12 primers yielded clear and characteristic amplicons). The highest number of amplicons was amplification markers as shown in Table 4. These generated from primers 17898b (20 amplicons), while bands were consistent and present in all six selected the lowest was generated from primer B05 (1 species while primers B10, C11 and C14 did not yield amplicons). A number of 56 amplicons were specific any characteristic amplification markers. Moreover, markers in which 39 of them were scored for the primers O10, A19, and C02 yielded a clear and presence of a unique band for a given species. characteristic one marker in each (approximately 1200, (Positive markers), while 18 were scored for the 630 and 1850 bp long), while primers B03, B06, O02, absence of a common band (negative marker). The and O09 yielded a clear and characteristic two markers highest number of Starfish (Asteroidea) markers

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Table 4: Starfish species markers resulting from RAPD- PCR analysis. Starfish species Positive marker Negative marker total Fromia ghardaqana 7512 ------Gomophia egyptiaca 202 ------Marthasterias glacialis 213 ------Astropecten irregularis 617 ------Asterina Burtoni 718 ------Asterina gibbosa 819 Total 25 9 41

Table 5: Number of amplified fragments markers of six starfish species based on ISSR –PCR analysis strains 17898a 17898b 17899a 17899b 814 844a 844b B10 B02 B03 B05 HB10 HB11 HB08 HB09 P03 UBC-825 UBC-827 Total 1 AF671154382755 107 6 68 8 99 ------SM112000010002 02 1 00 0 10 2 AF777154362552 1010 12 64 8 104 ------SM000000000000 01 4 00 0 5 3 AF867254372374 88 5 38 8 98 ------SM000000000210 11 1 00 0 6 4 AF10104274362445 45 7 48 6 95 ------SM030000000002 01 1 00 4 11 5 AF761274442657 39 6 38 9 93 ------SM020000010002 01 0 01 0 7 6 AF10101174452753 14 4 59 8 90 ------SM230000200301 30 0 02 1 17 TSM392000220517 46 7 03 5 56 TAF 11 20 8 2 7 4 5 13 2 11 7 11 11 16 15 7 14 14 178 ------PB 6 19 8 1 2 0 3 12 0 10 3 11 11 15 14 5 12 13 145 TAF = Total amplified fragment, PB = Polymorphic bands, AF = Amplified fragment, SM =specific marker, including either the presence or absence of a band, TSM = Total no. of specific markers across starfish species. 1 = Fromia ghardaqana, 2= Gomophia egyptiaca, 3= Marthasterias glacialis,4= Astropecten irregularis, 5= Asterina Burtoni, 6= Asterina gibbosa

Fig. 3: ISSR profile of the starfish amplified with 814 ISSR primer. M: 1 Kb ladder marker, 1 = Fig. 2: RAPD profile of the starfish amplified with Fromia ghardaqana, 2= Gomophia egyptiaca, 3= B02 RAPD primer. M: 1 Kb ladder marker, 1 Marthasterias glacialis, 4= Astropecten = Fromia ghardaqana, 2= Gomophia egyptiaca, irregularis, 5= Asterina Burtoni, 6= Asterina 3= Marthasterias glacialis, 4= Astropecten gibbosa irregularis, 5= Asterina Burtoni, 6= Asterina gibbosa. In conclusion, all ISSR primers used in the present study allowed for enough distinction among the six (Seventeen) was scored Species 6, while the lowest species of Asteroidea. Based on the amplification number of Asteroidea -specific markers (five) was products, seven primers were selected and the scored for Species 2 (Table 5). amplification polymorphism against DNA samples from

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Asteroidea was examined. Of the 18 primers, exact 14 Genetic Relationship as Revealed by RAPD Data: primers 17898a, 17898b, 17899a, 844b, B10, B03, B05, The genetic similarity between the six Asteroidea HB10, HB11, HB08, HB09, P03, UBC-825 and UBC- species based on nei method (18) was performed by 827 yielded clear and characteristic amplification RAPD analysis. The highest genetic similarity was markers. These bands were consistent and present in all between Species 3 and species 4 (78.7%), while the six selected species while primers 17899b, 814, 844a lowest genetic similarity was between species 1 and and B02 did not yield any characteristic amplification species 6 (61.2%) as shown in table (9) markers. Moreover, primers 17898a, 17899a, 844b, B05 and HB11 yielded a clear and characteristic one Cluster Analysis as Revealed by RAPD Data: The positive for species marker (approximately 800, 720, dendrogram developed based on nei similarity matrix 770, 1865 and 540 bp long respectively). In addition grouped the six Asteroidea species into two clusters primer 17898a (1000bp), 17898b (1000bp), 17899a (Figure 4). The first cluster branched into two (740 bp), 844b (800 bp), B03 (565& 550bp), HB10 subclusters, the first subcluster grouped species 1 and (970& 740 bp), HB11 (1370, 790 & 490bp, HB09 species 2 together which have the same linkage (1160& 715bp), UBC-827 (1330bp) and UBC- 827 distance (0.761), the second cluster formed two; one (1420, 1085& 695 bp) yielded clear and characteristic containing only one Asteroidea species; species 6 and negative markers. On the other hand, the rest of the other contains two subclusters, the first contains markers are a positive as shown in table (6). species 5 and the other contains two branches for species 3 and species 4 which revealed the highest AMOVA Analysis: Only the clear, unambiguous and genetic relationship (0.787). reproducible bands were considered for scoring. Each band was considered to be a single locus. Data were Genetic Relationship as Revealed by ISSR Data: The scored as ‘1’ for the presence and ‘0’ for the absence genetic similarity between the six Asteroidea species of a DNA band of each accession. DNA band size was based on nei method[19] was performed by ISSR estimated by comparing the DNA bands with a 1 Kb analysis The highest genetic similarity was between DNA ladder (Bioron, Germany) using GelWorks 1D Species 2 and species 3 (79.2%), while the lowest advanced (UVP, UK) genetic similarity was between species 3 and species 6 Results from AMOVA analysis of RAPD data (53.2%) as shown in table (10) indicated that 0% of the genetic variation is attributable to differences among groups while 100% of the genetic Cluster Analysis as Revealed by ISSR Data: The variation is attributable to within groups in RAPD data. dendrogram developed based on nei similarity matrix Sum of squares in were found to be 1.37 and 190.7, grouped the six Asteroidea species into two clusters respectively. Detailed results from AMOVA were given (Figure 5). Species 1 and species two was found in the in Table 7. Results from AMOVA indicated that 0% of same group attached with species 1 within the same the genetic variation is attributable to differences cluster. The later connected with a cluster containing among groups while 100% of the genetic variation is species 4 and species 5. All of the previous clusters are attributable to within groups in ISSR data. Sum of connected together with another branch containing squares in were found to be 0.68 and 264.42, species 6 that revealed the highest genetic relationship. respectively. Detailed results from AMOVA were given The genetic relationships among Asteroidea, in RAPD in Table 7. and ISSR were presented in Fig4 and Fig5. Results of analysis of RAPD and ISSR via cluster analysis were Findings from AMOVA supported these results by slightly different. having estimated variances as 0.00 (with 5 degrees of freedom) and 0.246 (with 774 degrees of freedom) for Discussion: DNA fingerprinting is a routine method RAPD respectively, while in ISSR having estimated employed to study the extent of genetic phylogeny. The variances as 0.00 (with 5 degrees of freedom) and first molecular phylogenetic analyses of animal 0.248 (with 1062 degrees of freedom) as shown in phylogeny date back 20 years[21] and since this time Table (8) respectively Results from AMOVA indicated our understanding of animal relationships has that 0% of the genetic variation was attributable to undergone a series of revolutions[22]. differences among accessions in ISSR groups while it Comparative studies in Plant or animal species was 0% in RADP which mean that the ISSR technique involving RAPD and ISSR marker systems were is same powerful as RAPD at this study. successfully used by very limited researchers[23,24,25 ,26].

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Table 6: Starfish species markers resulting from ISSR- PCR analysis. Starfish species Positive marker Negative marker total Fromia ghardaqana 73 10 ------Gomophia egyptiaca 50 5 ------Marthasterias glacialis 42 6 ------Astropecten irregularis 74 11 ------Asterina Burtoni 52 7 ------Asterina gibbosa 10 7 17 ------Total 38 18 56

Table 7: Summary AMOVA of RAPD Source df SS MS Est. Var. % Among Pops 5 1.369 0.274 0.000 0% ------Within Pops 774 190.677 0.246 0.246 100% Total 779 192.046 0.520 0.247

Table 8: Summary AMOVA of ISSR Source df SS MS Est. Var. % Among Pops 5 0.683 0.137 0.000 0% ------Within Pops 1062 264.421 0.249 0.249 100% Total 1067 265.104 0.386 0.248

Table 9: Starfish species similarity matrix resulting from RAPD- PCR analysis. Fromia Gomophia Marthasterias Astropecten Asterina Asterina ghardaqana egyptiaca glacialis irregularis Burtoni gibbosa Fromia ghardaqana 1 ------Gomophia egyptiaca 0.761 1 ------Marthasterias glacialis 0.691 0.776 1 ------Astropecten irregularis 0.662 0.693 0.787 1 ------Asterina Burtoni 0.652 0.725 0.714 0.726 1 ------Asterina gibbosa 0.612 0.676 0.707 0.693 0.711 1

Table 10: Starfish species similarity matrix resulting from ISSR - PCR analysis Species Fromia Gomophia Marthasterias Astropecten Asterina Asterina ghardaqana egyptiaca glacialis irregularis Burtoni gibbosa Fromia ghardaqana 1 ------Gomophia egyptiaca 0.76 1 ------Marthasterias glacialis 0.69 0.78 1 ------Astropecten irregularis 0.66 0.69 0.79 1 ------Asterina Burtoni 0.65 0.73 0.71 0.73 1 ------Asterina gibbosa 0.61 0.68 0.71 0.69 0.711 1

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Fig. 4: Cluster analysis (similarity) of molecular fingerprint (RAPD) data from six starfish species. Loc1 = Fromia ghardaqana, Loc2= Gomophia egyptiaca, Loc3= Marthasterias glacialis, Loc4= Astropecten irregularis, Loc5= Asterina Burtoni, Loc6= Asterina gibbosa.

Fig. 5: Cluster analysis (similarity) of molecular fingerprint (RAPD) data from six starfish species. Loc1 = Fromia ghardaqana, Loc2= Gomophia egyptiaca, Loc3= Marthasterias glacialis, Loc4= Astropecten irregularis, Loc5= Asterina Burtoni, Loc6= Asterina gibbosa.

The discriminative power of DNA markers used as tool six Asteroidea species into clusters. First cluster had to characterize the Starfish is very important because the Fromia ghardaqana and Gomophia egyptiaca, they can be used to assess the genetic diversity among while, Gomophia egyptiaca and Marthasterias glacialis all the Echinodermata. Bulk analyses are useful to appears highly similar using the RAPD marker systems. obtain information on genetic variability between On the other hand, the subcluster of Marthasterias different populations[27]. During the characterization of glacialis and Astropecten irregularis revealed that they starfish of the Red and Mediterranean seas, we are much similar in RAPD marker system. The high evaluated the discriminative power of primers for similarity between Astropecten irregularis and Asterina RAPD and ISSR marker systems. A high level of Burtoni was noticed in ISSR analysis indicating that polymorphism was generated utilizing the 15 RAPD these genotypes are closely related. The pattern of primers (Table 5). A total number of 130 RAPD bands subclustering of the cluster, which included six were obtained. Of these 94 bands were polymorphic genotypes varied over different marker systems. In (72.3%) and only 36 were monomorphic (27.7%) RAPD analysis, the Starfish genotypes were grouped while, the total number of 178 ISSR bands were into two subclusters revealing sufficient amount of obtained. Of these 145 bands were polymorphic diversity within the cluster. In our study, ISSR markers (81.46%) and only 30 were monomorphic (18.5%) grouped six starfish genotypes in three subclusters. these means that the ISSR markers is highly convenient Recently, ISSR markers also have been employed as Crossland et al[27]. The dendrogram developed from for genetic diversity analysis in plant and animal RAPD data based on nei similarity matrix grouped the species[29,23]. These data indicated that RAPD and ISSR

46 Res. J. Cell & Mol. Biol., 3(1): 39-48, 2009 markers were slightly difference effective for phylogeny 11. Richardson, T., S. Cato, J. Ramser, G. Kahl, K. studies Starfish. However,[29,30] concluded that ISSR Weising, 1995. Hybridization of microsatellites to would be a better tool than RAPD for phylogenetic RAPD: a new source of polymorphic markers. studies. Nagaoka and Ogihara[31]. Have also reported Nucleic Acids Res., 23: 3798–3799. that the ISSR primers produced several times more 12. Li, W.H., D. Graur, 1991. Fundamentals of information than RAPD markers. molecular evolution. Sinauer, Sunderland, MA Moreover, the similarities detected with ISSR are 13. Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. greater than the similarities observed with RAPD[32]. It Rafalski, S,V. Tingey, 1990. DNA polymorphism may be due to highly polymorphic, abundant nature of amplified by arbitrary primers are useful as genetic the microsattelites due to slippage in DNA markers. Nucleic Acids Res., 18: 6531–6535. [33] replication . 14. Awasthi, A.K., G.M. Nagaraja, G.V. Naik, S. During this work, Using ISSR markers system Kanginakudru, K. Thangavelu and J. Nagaraju, would be the first time in studying the phylogenetic 2004. Genetic diversity and relationships in relationship between the six starfishes belonging to the mulberry (genus Morus) as revealed by RAPD and Red and Mediterranean Sea. Moreover, it would be ISSR marker assays. BMC Genet., 5(1): 1. more reliable than RAPD one. 15. Caetano-Anollés, G., 1993. Amplifying DNA with The present results of the phylogenetic dendrogram arbitrary oligonucleotide primers. PCR Meth Appl., of starfish (level of orders and families) are similar to 3: 85–94. [34] [35] those of wada et al knott and Wray and 16. Williams, J., K. Hanafey, J. Rafalski, S. Tingey, Matsubara et al.[36]. On the other hand, it differs with 1993. Genetic analysis using random amplified those of lafay et al.[37]. polymorphic DNA markers. Methods Enzymol., 218: 705–741 REFERENCES 17. Rohlf, F.J., 2000. NTSYSpc: Numerical taxonomy and multivariate analysis system. Version 2.1. 1. Hyman, L.H., 1955. The Invertebrates: Exeter Publications, New York, USA. Echinodermata. Volume IV. McGraw-Hill, New 18. Nei, M. and W. Li, 1979. Mathematical model for York, New York. studying genetical variation in terms of restriction 2. Clark, A.M., 1977. Starfishes and related endonucleases. Proceedings of the National echinoderms. T. F: H. Publications, London. Academy of Sciences USA, 76(10): 5269-5273. 3. Clark, A.M. and M.E. Downey, 1992. Starfishes of 19. Peakall, R. and P.E. Smouse, 2006. genetic the Atlantic. Chapman and Hall, London. 4. Blake, D.B., 1987. A classification and phylogeny analysis in Excel. Population genetic software for of post-Paleozoic sea stars (Asteroidea: teaching and research. Molecular Ecology Notes, Echinodermata). Journal of Natural History, 21: 6(1): 288-295. 481-528. 20. Nei, M., 1978. Estimation of average 5. Paine, R.T., 1966. Food web complexity and heterozygosity and genetic distance from a small species diversity. American Naturalist, 100: 65-75. number of individuals. Genetics, 89: 583-590. 6. Moran, P.J., 1988. The Acanthaster phenomenon. 21. Field, K.G., G.J. Olsen, D.J. Lane, S.J. Australian Institute of Marine Science Monograph Giovannoni, M.T. Ghiselin, E.C. Raff, N.R. Pace, Series, 7: 78. R.A. Raff, 1988. Molecular phylogeny of the 7. Dean, J., 1999. What makes and ophiuroid? A animal kingdom. Science, 239: 748– 753. morphological study of the problematic Ordovician 22. Bourlat, S.J., C. Nielsen, A.D. Economou, M.J. stelleroid Stenaster and the paleobiology of the Telford, 2008. Xenoturbella is a deuterostome that earliest asteroids and ophiuroids. Zoological eats molluscs. Nature, 424: 925–928. Journal of the Linnean Society, 126: 225-250. 23. Souframanien, J. and T. gopalakrishna, 2004. A 8. Tortonese, E., 1960. Echinoderms from the Red comparative analysis of genetic diversity in Sea. I. Asteroidea. Bull Sea Fish Res Stn., 29: blackgram genotypes using RAPD and ISSR 17–23. markers. Theoretical and Applied Genetics, 109(8): 9. Clark, A.M., 1952. The ‘Manihine’ expedition to 1687-1693. the Gulf of Aqaba. VII. Echinodermata. Bull Br 24. Gillaspie, A.G., M.S. Hopkins and R.E. Dean, Mus (Nat Hist) Zool., 1: 203–214. 2005. Determining genetic diversity between lines 10. Tortonese, E., 1960. Echinoderms from the Red of Vigna unguiculata subspecies by AFLP and Sea. I. Asteroidea. Bull Sea Fish Res Stn., 29: SSR markers. Genetic Resources and Crop 17–23. Evolution, 52(3): 245-247.

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