Genetic Characterization of Four Fish Species of Genus Synodontis Using RAPD Marker

Indian Journal of Geo Marine Sciences Vol. 47 (12), December 2018, pp. 2395-2406

Genetic characterization of four fish species of Genus Synodontis using RAPD Marker

*Abu-Almaaty, A. H.1; Abdel-Basset M. Ebied2& Mohammad allam2 1Zoology Department, Faculty of Science, Port Said University, Egypt. 2Cytogenetic Laboratory- Zoology Department- Faculty of Science (Qena) - South Valley University. - Egypt.

[E-mail: [email protected]]

Received 5 April 2017; revised 02 June 2017

Twenty Random amplified polymorphic (RAPD) primers, produced DNA bands, wereused for the construction of the dendrogram and a similarity matrix. A total of 326 bands were obtained; 50 of them were monomorphic bands (15%), 184 polymorphic bands and 92 unique bands with polymorphism 85%.Similarity values among the studied samples ranged from 51.8% to 66.4%. High similarity values were obtained between Synodontisfrontosus andSynodontisclarias. (66.4%) and the low similarity values were obtained betweenSynodontisfrontosus andSynodontisserratus(51.8%). RAPD analysis confirmed that the four Synodontis species under study are genetically different from each otherand a genetic variation was found between and within the four species tested in this study.

[Keywords: Genetics – Molecular- Taxonomy – Fishes –Mochokidae- Synodontis - RAPD- DNA.]

Introduction interpopulation breeding, DNA detection and so on, The genus Synodontis is the most species rich and as it is a quick, sensitive and easy technique7-10,11. widespread genus of mochokid catfishes. As currently RAPD-PCR method is now widely employed in recognized the genus contains approximately 120 genetic research and is a powerful asset when trying valid species distributed throughout most of the to identify species. This technique involves the freshwaters of sub-Saharan Africa and the Nile River amplification of random segments of genomic DNA system. Larger Synodontis species are important food using a single primer of arbitrary nucleotide sequence. fishes in many parts of Africa and are commonly The polymorphisms detected in the nucleotide known as “squeakers” because they readily produce sequence can then be used as genetic markers12. sounds by stridulating their pectoral spines when Random amplified polymorphic DNA (RAPD) was handled or disturbed. Furthermore, many Synodontis used to profile the genetic diversity of population13. species are prized ornamental fishes because they The RAPD offered a quick and inexpensive molecular have striking pigmentation patterns or display unusual tool that assisted to distinguish, monitor and manage behaviors like an upside down swimming posture1-4. the genetic diversity of natural populations of fish The molecular genetics and the recombinant raised in fish hatcheries fish14. RAPD analysis is techniques had profound impact on the understanding useful since different number and size of fragments in of biological systems, which are foundation of different fish species can be obtained. Different modern biotechnology. In fact no subject area has a fish species were given different number and size 15-18 more sustained impact on shaping our knowledge of of bands . living systems than genetics5. RAPD is one of the It is noteworthy, molecular markers such as simplest molecular tools for genetic and systematic Random Amplified Polymorphic DNA (RAPD) have analyses of various organisms and has provided regularly been employed to examine relationship at important applications in catfish6.Random amplified the intra and inter-populations of various organisms polymorphic DNA (RAPD) analysis has been widely because it has been shown to have a high power of used in genetic diversity studies, identification of fish resolution, especially in detecting cryptic pairs of species and in confirming close relationships between species, genetic differentiation in intra- or 19,20 ————— species . The RAPD method is a PCR-based *Corresponding author technique that amplifies random DNA fragments with 2396 INDIAN J. MAR. SCI., VOL. 47, NO. 12, DECEMBER 2018

the use of single short primers of arbitrary nucleotide sequence under low annealing conditions; this technique has been extensively used for species classification and microorganism strain determination21,22. The main reason for the success of RAPD analysis is the gain of a large number of genetic markers that require small amounts of DNA without the requirement for cloning, sequencing or any other form of the molecular characterization of the genome of the 21 species in question . RAPD markers can be sensitive Fig. 2 — The external features of Synodontisclarias. to changes in reaction conditions, resulting in low reproducibility and inconsistencies in amplification efficiencies among samples23. In spite of the fact there have been considerable development of molecular genetic studies in the last ten years, very little information are available on the molecular genetic characteristics of fresh water fishes especially in the upper Egypt and especially (RAPD) analysis on four species of fresh-water fishes in Upper Fig. 3 — The external features of Synodontis shall. Egypt namely; Synodontisfrontosus, Synodontisclarias, Synodontis shall and Synodontisserratus (Family: Mochokidae - Order: Siluriformes). So the aim of the present study is fill this gap and also to indicate the molecular differences and taxonomic and phylogenetic relationship between these species under this study.

Materials and Methods Fig. 4 — The external features of Synodontisserratus. In the present study, four fresh water fish species, (Synodontisfrontosus, Synodontisclarias, Synodontis Table 1 — The sequence of 20 primers using for RAPD shall andSynodontisserratus) figures (1, 2, 3 and 4) analysis of family: Mockokidae family: Mochokidae, were collected from different S. No. Name of primer Nucleotide Sequence (5'-3') localities of upper – Egypt (Qena and Aswan). The length Identification and classification of species were 1 OPA – 4 10-mer AATCGGGCTG carried out using descriptions and keys provided 2 OPA -9 10-mer GGGTAACGCC 24,25 3 OPA -15 10-mer TTCCGAACCC by . 4 OPA -17 10-mer GACCGCTTGT Muscles tissue from fishes immediately after 5 OPB -6 10-mer TGCTCTGCCC capture, were put on tubes and stored in a freezer until 6 OPB -9 10-mer TGGGGGACTC processed for RAPD-PCR analysis. 7 OPB -10 10-mer CTGCTGGGAC Twenty primers table (1) (Operon) were used to 8 OPB -16 10-mer TTTGCCCGGA amplify genomic DNA. Operon 10-mer kits contained 9 OPB -17 10-mer AGGGAACGAG 10 OPC -1 10-mer TTCGAGCCAG 11 OPC -11 10-mer AAAGCTGCGG 12 OPC -18 10-mer TGAGTGGGTG 13 OPD-5 10-mer TGAGCGGACA 14 OPE-10 10-mer CACCAGGTGA 15 OPF-6 10-mer GGGAATTCGG 16 OPG-2 10-mer GGCACTGAGG 17 OPM-2 10-mer ACAACGCCTC 18 OPM-5 10-mer GGGAACGTGT 19 OPM-17 10-mer TCAGTCCGGG 20 OPZ-18 10-mer AGGGTCTGTG Fig. 1 — The external features of Synodontisfrontosus. ALMAATY et al.: GENETIC CHARACTERIZATION OF FISH 2397

10 base oligonucleotide primers (Table 1) used in this The similarity matrix was used in the cluster study. DNA extraction using the QiagenDNeasy analysis. Cluster analysis was employed to organize (Qiagen Santa Clara, CA). the observed data into meaningful structures to DNA concentration was determined by diluting develop taxonomies. At the first step, when each accession represents its own cluster, the distances the DNA 1:5 in H2O. The DNA samples were electrophoresed in 1% agarose gel against 10 ng of a between these accessions are defined by the chosen DNA size marker. This marker covers a range of distance measure (Dice coefficient). However, once concentration between 95 ng and 11 ng. Thus, several accessions have been linked together, the estimation of the DNA concentration in a given distance between two clusters is calculated as the sample was achievedby comparing the degree of average distance between all pairs of accessions in the fluorescence of the unknown DNA band with the two different clusters. This method is called different bands in the DNA size marker. A set of Unweighted Pair Group Method using Arithmetic 26 twenty primers RAPD (Tables 1) was used. The Average (UPGMA) . amplification reaction was carried out in 25 μl reaction volume containing 1X PCR buffer, 1.5 mM Results MgCl2, 0.2 mMdNTPs, 1 μM primer, 1 U Taq The genetic analysisand taxonomic phylogenetic DNA polymerase and 25 ng templates DNA. relationship of four fresh water species of fishes PCR amplification was performed in a Perkin- Synodontisfrontosus, Synodontisclarias, Synodontis Elmer/GeneAmp® PCR System 9700 (PE Applied shall and Synodontisserratus (Family: Mochokidae- Biosystems) programmed to fulfill 40 cycles after an Order: Siluriformes) by using RAPD- PCR technique initial denaturation cycle for 5 min at 94ºC. Each were investigated. The following are the amplification cycle consisted of a denaturation step at 94ºC for 1 results of the four species obtained by the examined min, an annealing step at 36ºC for 1 min, and an primers: elongation step at 72ºC for 1.5 min. Primer extension The RAPD analysis using twenty primers showed segment was extended to 7 min at 72ºC in the final the number of bands in the Synodontisfrontosus cycle. Amplification products were resolved by species was 164 bands. The large number of bands 15 electrophoresis in a 1,5 % agarose gel containing generated by the primers OPC – 11, OPB-10 and ethidium bromide (0.5 ug/ml) in 1X TBE buffer at 95 OPB - 17, while the small number of bands 1 volts. PCR products were visualized on UV light and generated by the primer OPB - 04. Size of bands photographed using a Polaroid camera. Amplified ranged from 150 bp by the primer OPC– 11, OPB-17 products were visually examined and the presence or and OPB-10 to 1400 bp. by the primers OPZ – 18, absence of each size class was scored as 1 or 0, OPM – 02 and OPF - 06. respectively. Random amplified polymorphic DNA (RAPD) Banding patterns generated by RAPD-PCR marker technique was used to examine the genetic variability analyses were compared to determine the genetic on a fish species, Synodontisclarias produced relatedness of the. Clear and distinct amplification different RAPD band patterns of number of 141 bands products were scored as ‘1’ for presence and ‘0’ for ranged approximately from150 bp. to 1200 bp. Small absence of bands. Bands of the same mobility were size of bands generated by the primers OPC – 11, scored as identical. OPB – 17, OPB - 09 and OPB – 10, while the largest The genetic similarity coefficient (GS) between size of bands generated by the primers OPB – 16 and two genotypes was estimated according to Dice OPB – 09. The generated bands ranged in number 26 coefficient . from 1 by the primer OPB – 04 to 13 by the primers OPB - 09 and OPB – 10. Dice formula: GSij = 2a/(2a+b+c) The RAPD analysis using twenty primers showed Where GSij is the measure of genetic similarity the number of bands in the Synodontisshall species between individuals i and j, a is the number of bands was 228 fragments. Large number of fragments 17 shared by i and j, b is the number of bands present in i generated by the primer OPB – 10, while the and absent in j, and c is the number of bands present small number of fragments 5 generated by the primer in j and absent in i. OPE - 10. 2398 INDIAN J. MAR. SCI., VOL. 47, NO. 12, DECEMBER 2018

The size of fragments ranged from 150 bp by the primer OPB – 17 and OPB - 10 to 1800 bp. by the primers OPA – 09 and OPC – 01. All the twenty OPERON primers produced 199 bands in the species of Synodontisserratus. The number of band amplified per primer varied from 2 bands by the primer OPF – 6 to 18 bands by the primer OPB – 10 and had a size arranged from 150 bp. by the primer OPB – 10 to 2000 bp. by the primer OPG – 02. The short ten-nucleotide primers generated discrete DNA amplified fragments of varying lengths and revealed RAPD variation among the Fig. 6 — The Agarose gel electrophoresis products generated species. All twenty primers (table 1) amplified with. primers OPA-15, OPG-2 and OPZ-18. successfully on the genomic DNA extracted from all studied fish species. The twenty primers yielded amplification products in the four species of the family Mochokidae. The number of fragments amplified per primer varied between 6 (OPE-10) and 24 (OPC-11) (16.3 bands/ primer) and had a size range from 150 bp (OPC - 11, OPB - 9, OPB – 10 and OPB – 17) to 2000 bp (OPG-02). The DNA fragments generated by the twenty primers from the genomic DNA of the four species were separatedusing Agarose gel electrophoresis and illustrated in figs (5, 6, 7, 8, 9, 10, 11 and 12). The banding patterns of these DNA fragments were analyzedby Gene profiler computer software program and summarized in Fig. 7 — The Agarose gel electrophoresis products generated table (2). The number and positions of the with. primers OPM-2, OPM-5 and OPM-17. bands depended on species and primer as shown in this table. A total of 326 DNA bands were generated by all primers in all species, out of these DNA bands 50 The number of bands was variable in (15%) were conserved among all four species, each species. The number of amplified bands while 276 bands were polymorphic and unique with in Synodontisfrontosus was 164, and in polymorphism 85 % table (3). Synodontisclarias 141, while in Synodontisshall 228 bands and inSynodontisserratus 199 bands. The RAPD bands profile for these species as seen in the Figures (5, 6, 7, 8, 9, 10, 11 and 12) indicated specificity of the DNA patterns for a given species. We have found that the primers of same length but with different sequences generated different DNA patterns among fishes. Data of the presence / absence of DNA fragment of Synodontisfrontosus, Synodontisclarias, Synodontis shall andSynodontisserratuswere used to calculate the genetic similarity. Then based on the calculated genetic similarity presented in table (3) and figure (13), an estimation of the relationship between the Fig. 5 — The Agarose gel electrophoresis products generated with.primers OPA-4, OPA-17 and OPB-6.(1- Synodontisfrontosus, above species was concluded where the lowest 2- Synodontisclarias 3-Synodontis shall 4-Synodontisserratus). genetic similarity 51.8 % was observed between, ALMAATY et al.: GENETIC CHARACTERIZATION OF FISH 2399

Fig. 8 — The Agarose gel electrophoresis products generated with. primers OPC-11, OPA-9 and OPB-16.

Fig. 11 — The Agarose gel electrophoresis products generated with. primers OPB-9.

Fig. 9 — The Agarose gel electrophoresis products generated with. primers OPF-6 and OPE-10.

Fig. 12 — The Agarose gel electrophoresis products generated with. primers OPG-5, OPB-10.

Fig. 10 — The Agarose gel electrophoresis products generated Discussion with. primers OPB-17, OPC-1 and OPC-18. All the primers which amplified successfully on the genomic DNA from the samples Synodontisfrontosus andSynodontisserratus, while (Synodontisfrontosus, Synodontisclarias, Synodontis the highest value 66.4 % was found between shall and Synodontisserratus) yielded distinct RAPD Synodontisfrontosus andSynodontisclarias. pattern. The number of fragment amplified per primer The results of the (RAPD-PCR) DNA analysis varied between (6 - 24) fragments and had a size were compared with those obtained from the classical arranged from (150 – 2000 bp.). Although several methods in taxonomy using morphological and amplified fragments were shared by all four Species anatomical characters alone. (50 bands), clearly distinguishable bands were evident 2400 INDIAN J. MAR. SCI., VOL. 47, NO. 12, DECEMBER 2018

Table 2 — Survey of RAPD Markers using twenty primers. (1- Synodontisfrontosus, 2- Synodontisclarias 3-Synodontis shall 4-Synodontisserratus), where (1) means present and (0) means absence. OPA-4 OPA-15 N M 1 2 3 4 N M 1 2 3 4 1 1000 0 0 1 0 1 1600 0 0 0 1 2 750 0 0 1 1 2 1400 0 0 0 1 3 720 0 0 1 1 3 1200 0 0 1 0 4 630 1 0 1 1 4 1100 1 0 0 1 5 500 0 0 1 1 5 1050 1 0 0 1 6 450 0 0 0 1 6 1000 1 0 0 1 7 400 0 0 1 1 7 900 1 0 0 0 8 320 0 1 1 1 8 800 1 1 1 1 OPA-17 9 720 1 0 0 1 N M 1 2 3 4 10 650 0 0 1 1 1 1500 0 0 0 1 11 570 0 1 0 0 2 1400 0 0 0 1 12 550 1 0 1 1 3 1300 0 0 1 0 13 500 1 1 1 1 4 1200 0 0 1 1 14 450 0 1 1 1 5 950 0 0 1 1 15 400 0 1 1 1 6 850 0 0 1 1 16 350 0 1 1 0 7 750 1 1 0 0 17 270 1 0 0 0 8 700 1 1 1 1 18 250 0 0 1 0 9 650 0 0 1 0 OPG-2 10 600 1 1 1 0 N M 1 2 3 4 11 520 1 1 0 0 1 2000 0 0 0 1 12 470 0 0 1 1 2 1500 0 0 0 1 13 420 1 1 1 0 3 1300 1 0 0 0 14 350 1 1 0 1 4 1200 0 1 1 0 15 330 0 0 1 0 5 1100 0 1 1 0 16 300 1 1 1 1 6 1000 1 1 1 1 17 250 0 0 0 1 7 900 0 0 1 0 OPB-6 8 750 0 0 1 1 N M 1 2 3 4 9 700 1 1 1 1 1 1500 0 0 0 1 10 650 0 0 0 1 2 1100 0 0 1 0 11 600 1 1 1 1 3 1000 0 1 1 1 12 550 1 1 1 1 4 900 0 1 1 1 13 500 1 1 1 0 5 850 0 0 1 0 14 450 1 1 0 1 6 800 0 0 1 1 15 400 0 1 1 0 7 730 0 1 1 1 16 330 1 1 0 1 8 680 0 0 1 0 17 310 0 0 1 0 9 630 1 1 1 0 18 280 0 1 1 0 10 600 0 0 1 0 19 250 0 0 1 1 11 520 1 1 1 0 20 200 0 0 1 1 12 480 0 1 0 1 OPZ-18 13 450 1 0 1 0 N M 1 2 3 4 14 430 0 1 0 0 1 1400 1 0 0 0 15 400 0 1 1 1 2 1000 1 0 1 0 16 350 1 0 1 1 3 900 0 0 1 0 OPM-2 4 850 1 0 1 0 N M 1 2 3 4 5 800 0 1 0 0 1 1400 1 0 1 1 6 750 1 1 1 1 2 1050 1 0 0 1 7 700 1 1 1 0 3 950 1 0 0 1 8 650 1 0 1 0 4 750 1 0 1 1 9 600 1 1 0 1 5 700 1 1 1 1 10 580 0 1 1 0 6 650 1 1 0 1 11 550 0 0 1 0 7 600 1 0 0 1 12 500 1 1 1 1 8 450 1 1 1 1 13 460 1 0 0 0

(Contd.) ALMAATY et al.: GENETIC CHARACTERIZATION OF FISH 2401

Table 2 — Survey of RAPD Markers using twenty primers. (1- Synodontisfrontosus, 2- Synodontisclarias 3-Synodontis shall 4-Synodontisserratus), where (1) means present and (0) means absence. (Contd.) OPM-2 OPZ-18 9 400 1 0 1 1 14 420 1 0 1 1 10 340 1 0 0 1 15 400 1 1 1 1 11 320 1 1 1 0 16 380 1 0 0 0 12 290 0 0 1 0 17 320 0 1 0 0 13 270 1 1 0 0 18 300 1 0 1 1 14 250 1 0 0 1 19 250 1 0 1 0 OPM-5 OPM-17 N M 1 2 3 4 N M 1 2 3 4 1 1400 0 0 0 1 1 1500 0 0 0 1 2 780 0 0 1 1 2 1100 0 0 1 1 3 730 0 0 1 0 3 1000 0 0 1 1 4 700 0 0 1 1 4 900 0 0 1 0 5 620 0 0 1 1 5 820 1 0 1 1 6 580 0 0 1 1 6 750 1 1 1 1 7 530 0 0 1 0 7 720 1 1 1 1 8 510 0 0 1 0 8 690 0 0 1 0 9 490 0 1 0 0 9 650 1 0 0 0 10 470 0 0 1 1 10 530 0 0 1 1 11 450 0 1 1 0 11 480 0 0 1 1 12 400 1 1 1 1 12 450 0 1 1 0 13 350 0 1 1 0 13 400 0 0 1 0 14 300 1 1 1 1 14 350 0 1 1 1 15 280 0 0 0 1 15 290 0 0 1 0 16 250 0 0 1 1 16 250 0 0 1 0 17 240 0 1 1 0 17 200 1 0 1 0 18 230 0 1 1 1 OPA-9 19 200 0 1 1 1 N M 1 2 3 4 OPC-11 1 1800 0 0 1 1 N M 1 2 3 4 2 1400 0 0 1 1 1 1200 0 0 0 1 3 1100 0 0 0 1 2 1000 0 0 1 1 4 950 1 0 1 1 3 900 1 0 1 1 5 850 1 1 1 0 4 800 1 0 0 1 6 800 0 0 0 1 5 750 1 0 0 0 7 750 0 0 1 0 6 700 1 1 1 1 8 720 1 1 0 0 7 630 1 1 1 1 9 680 1 0 1 1 8 600 1 0 1 1 10 600 1 1 1 1 9 530 1 0 0 1 11 550 1 0 0 1 10 500 1 0 1 0 12 500 1 1 1 1 11 450 1 1 0 0 13 450 1 0 0 1 12 400 1 1 1 1 14 430 1 1 1 1 13 390 1 1 1 1 15 390 1 0 1 1 14 370 0 1 1 1 16 350 1 1 1 1 15 350 0 0 0 1 17 300 1 0 1 1 16 320 0 0 1 0 18 250 0 1 1 0 17 300 1 0 1 1 19 230 1 0 1 0 18 290 1 1 0 0 OPB-16 19 280 0 1 1 1 N M 1 2 3 4 20 270 0 0 1 0 1 1200 0 1 0 1 21 250 1 0 0 0 2 1000 1 0 1 0 22 230 0 1 0 0 3 900 1 0 1 0 23 200 0 1 1 1 4 750 1 0 1 0 24 150 1 0 0 0 5 730 1 1 1 1 OPF-6 6 600 1 0 1 0 N M 1 2 3 4 7 500 1 1 1 1 1 1400 1 0 1 0 8 420 1 0 1 0 2 1100 1 1 1 1 9 300 1 1 1 1

(Contd.) 2402 INDIAN J. MAR. SCI., VOL. 47, NO. 12, DECEMBER 2018

Table 2 — Survey of RAPD Markers using twenty primers. (1- Synodontisfrontosus, 2- Synodontisclarias 3-Synodontis shall 4-Synodontisserratus), where (1) means present and (0) means absence. (Contd.) OPF-6 OPB-16 3 900 1 1 1 1 10 240 1 0 1 0 4 800 1 0 1 0 OPE-10 5 700 1 0 0 0 N M 1 2 3 4 6 600 0 0 1 0 1 1100 0 1 1 1 7 400 0 0 1 0 2 1000 0 1 1 1 OPB-17 3 850 0 1 1 0 N M 1 2 3 4 4 700 1 1 1 1 1 1100 0 0 1 1 5 600 0 1 1 0 2 1000 1 0 1 0 6 550 1 0 0 0 3 900 0 0 1 1 OPC-1 4 790 0 0 1 0 N M 1 2 3 4 5 750 0 0 1 1 1 1800 0 0 1 1 6 700 1 0 0 0 2 1500 0 0 1 1 7 600 1 1 1 1 3 1100 0 1 1 1 8 550 1 0 0 1 4 1000 0 0 0 1 9 500 0 1 1 0 5 800 0 0 1 1 10 490 1 1 1 1 6 750 0 1 0 1 11 470 1 0 1 1 7 720 1 0 1 1 12 440 1 1 1 0 8 700 0 0 0 1 13 420 1 1 1 0 9 650 0 0 0 1 14 400 1 0 0 1 10 580 0 1 1 1 15 370 1 1 1 1 11 520 0 0 1 0 16 350 1 0 0 1 12 450 0 1 1 1 17 300 1 0 0 1 13 400 1 0 0 1 18 280 0 1 0 1 14 380 1 1 0 1 19 250 1 1 1 0 15 350 1 0 1 0 20 200 1 1 1 1 16 320 0 1 1 1 21 150 1 1 1 0 17 300 0 0 0 1 OPC-18 18 290 0 1 1 0 N M 1 2 3 4 19 250 0 1 1 1 1 1200 0 0 0 1 20 220 1 1 1 1 2 1100 0 0 1 0 OPB-9 3 1000 0 0 1 0 N M 1 2 3 4 4 750 1 1 1 1 1 1400 0 0 0 1 5 600 1 1 1 1 2 1200 0 1 1 0 6 530 1 0 0 0 3 1100 1 0 1 0 7 500 1 1 1 0 4 900 1 0 0 1 8 450 1 1 1 1 5 800 1 1 1 1 9 420 0 1 1 0 6 700 1 1 1 0 10 400 0 1 1 0 7 650 1 1 0 1 11 370 0 0 1 0 8 580 0 1 1 0 12 350 0 0 1 0 9 550 1 1 1 1 13 330 0 0 1 0 10 500 1 1 0 0 14 300 1 1 1 0 11 470 0 1 1 1 OPG-5 12 420 0 1 1 1 N M 1 2 3 4 13 350 1 0 1 0 1 1500 0 0 1 0 14 310 1 0 0 1 2 1200 0 0 1 1 15 300 0 1 1 0 3 900 0 0 1 1 16 290 0 0 0 1 4 800 0 0 1 1 17 260 1 1 0 1 5 750 0 0 0 1 18 200 1 0 0 1 6 600 0 0 1 0 19 180 0 1 0 1 7 570 1 0 0 1 20 150 0 1 0 0 8 540 0 0 0 1 OPB-10 9 500 1 1 1 1 N M 1 2 3 4 10 470 1 0 0 0 1 1800 0 0 0 1 11 420 0 0 1 1 2 1400 0 0 0 1

(Contd.) ALMAATY et al.: GENETIC CHARACTERIZATION OF FISH 2403

Table 3 —Number of amplified and polymorphic DNA – fragments in1- Synodontisfrontosus, 2-Synodontisclarias, 3- Synodontis shall and 4- Synodontisserratus. S. NO. Primer Total of No. of amplified bands No. of No. of No. of Poly Polymorph code amplified 1 2 3 4 Monomorp Unique morphicband ism% bands hic bands s bands 1 OPA - 4 8 1 1 7 7 0 2 6 100% 2 OPA-17 17 7 7 11 10 2 6 9 88% 3 OPB- 6 16 4 8 13 8 0 6 10 100% 4 OPA-15 18 9 6 9 12 2 7 9 89% 5 OPG - 2 20 8 11 14 12 4 6 10 80% 6 OPM- 5 19 2 8 16 12 2 6 11 89% 7 OPM -2 14 13 5 7 11 2 1 11 86% 8 OPM-17 17 5 4 15 9 2 7 8 88% 9 OPC- 11 24 15 10 14 15 4 8 12 83% 10 OPB- 10 22 15 13 17 18 8 4 10 64% 11 OPE- 10 6 2 5 5 3 1 1 4 83% 12 OPF- 6 7 5 2 6 2 2 3 2 71% 13 OPA - 9 19 13 7 14 14 4 3 12 79% 14 OPZ- 18 19 14 8 13 6 3 7 9 84% 15 OP - 18 14 6 7 12 4 3 7 4 79% 16 OPB- 16 10 9 4 9 4 3 0 7 70% 17 OPC- 1 20 5 9 13 17 1 5 14 95% 18 OPB- 17 21 15 10 15 14 4 2 15 81% 19 OPB - 9 20 11 13 10 12 2 3 15 90% 20 OPG - 5 15 5 3 8 9 1 8 6 93% Total 326 164 141 228 199 50 92 184 85% from high number of polymorphic marker and optimize RAPD amplified condition and ascertain the appearance of unique bands betweenthe four species reproducibility of RAPD markers for individual taxa (Synodontis frontosus, Synodontisclarias, Synodontis prior to apply RAPD fingerprinting to any genetic shall and Synodontisserratus). RAPD technology is a analysis27. useful tool for identifying DNA polymorphism, In the present study, four Synodontis species can be estimation of genetic diversity and difference of successfully differentiated as well as characterized related species in fish. However, it is essential to on the basis of 163 species-specific RAPD markers 2404 INDIAN J. MAR. SCI., VOL. 47, NO. 12, DECEMBER 2018

Table 4 — Genetic similarity values calculated from the Synodontiscatfishes.Using RAPD-PCR is very useful DNA fragments amplified from Synodontisfrontosus, in determination genetic molecular variations and Synodontisclarias, Synodontis shall andSynodontisserratus relationship degree between the species whichbelong using twenty OPERON primers. to same family. Randomly amplified polymorphic S. serratus S. shall S. clarias Synodontis DNA-PCR (RAPD-PCR) is a technique widely used frontosus for studying the DNA polymorphism between closely 100 1 related species without the requirement of prior 100 66.4 2 28-30 100 52 59.5 3 knowledge of the genome . PCR technique is 100 55.9 60.7 51.8 4 basically a primer extension reaction for amplifying specific nucleic acids in vitro. The use of a thermo stable polymerase referred to as taq allows a short stretch of DNA to be amplified to about a million fold so that one can determine its size, nucleiotide sequence, etc.31. Polymerase chain reaction (PCR) technology has become a widespread research technique and has led to the development of several novel genetic assays based on selective amplification of DNA32. RAPDs have gained considerable attention 33 particularly in population genetics for species and subspecies identification, for gynogenetic fish Fig. 13 — Dendrogram demonstrating the relationship among identification34,35 and for gene mapping studies in Synodontis species.(1- Synodontisfrontosus,2- Synodontisclarias 36,37 3-Synodontis shall 4-Synodontisserratus). fish . The presence of more numbers of fragments might be due to the presence of more priming site at the template DNA with the particular series of Operon primers employed in their study. It is further suggested that the use of more numbers of random primers from different Operon series in more numbers of samples might be helpful to achieve more reliable results in the genetic studies38. Using RAPD fingerprinting on fish has been limited so in the current study, this technique was applied to analyze the genetic relationships among Synodontis species. Using a RAPD analysis, the intrapopulation variation was detected with different primers in tilapia7. This technique was more sensitive than the mtDNA analysis which failed to reveal the 39,40 Fig. 14 — Demonstrating the relationship degree among variation within the tilapia populations . RAPD has Synodontis species. Based on data recorded from polymorphism been used to detect genotoxic-induced DNA damage of RAPD markers. and mutations in different organisms, including fish41. generated. RAPD assay was found to be useful for The RAPD method was successfully used to detect establishing genetic relationship, genome specificity the variation between the different species of fish. The and phylogeny among wild species. So the results results obtained in this study showed that RAPD demonstrate that RAPD technology represents a could be usedto generate useful fingerprints useful and reliable tool for species discrimination and characteristic of fish species and for genotyping of for detecting genetic relationships in Synodontis individuals within the species. Thus, it provides an species, and for detecting genetic relationships in efficient and sensitive method which can be used to Synodontis species. The results are relevant for their estimate genetic variability, relatedness, inbreeding conservation as well as aid in the understanding levels, pedigree analyses, detection of economic traits of the phylogeny and biogeography of these and in other maker based studies in fishes42. ALMAATY et al.: GENETIC CHARACTERIZATION OF FISH 2405

The present study indicated that least genetic 7 Bardacki, F. and Skibinski, D. O. F., Applications of the distance was observed in Schilbemystus and RAPD technique in tilapiafish: species and subspecies identification. Heredity 73: (1994) 117–123. Schilbeuranoscopus these two species fell into a 8 Ding, S.X.; Wang, J.; Quan, C.G. and Su, Y.Q., Gentic distinct cluster from other species Siluranodonauritus. diversity in population of cultured Nibeamiichthioides. This points out that these two species are genetically Bulletin of Science 43(21): (1998) 2294–2299. closer and show divergence as compared to other 9 Liu, Z.H.; Shi, T.; Liu, X.Z.; Chen, C. and Kong, J., species of Siluranodonauritus. These two species are Identification of three populations (Chinese Fugurubripes, Japanese Fugurubripes and Fugupseudommus) usingRAPD considered morphologically closer in contrast to the markers. Transactions of Oceanology and Limnology 4: other species of Schilbe. 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