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Pdf (674.91 K) Egyptian Journal of Zoology (EJZ), Vol. 75: 1-13 (June, 2021) p-ISSN: 1110-6344 Publisher: The Zoological Society of A. R. Egypt e-ISSN: 2682-3160 DOI: 10.12816/ejz.2020.44427.1041 RESEARCH ARTICLE GENETIC RELATIONSHIPS AMONG FOUR MUTELID SPECIES (BIVALVIA: UNIONIDA) IN EGYPT REVEALED BY RAPD-PCR TECHNIQUE Mona F. Fol1; Mostafa Y. Morad2; Irene S. Gamil2; Salwa F. Sabet1; Reda M. Mansour2* 1Zoology Department, Faculty of Sciences, Cairo University, Giza, Egypt 2Zoology and Entomology Department, Faculty of Science, Helwan University, Cairo, Egypt ABSTRACT Article History: Random amplified polymorphic deoxyribonucleic acid- Received: 28 September 2020 polymerase chain reaction (RAPD-PCR) is considered as one Revised: 27 November 2020 Accepted: 30 November 2020 of the simple and quick methods that are used to resolve the genetic relationship among species of taxa that are Published Online: difficult to identify on the basis of morphological characters. 1 December 2020 Bivalves show great morphological variations that couple Keywords: with relatively few constant characters rendering them Chambardia letourneuxi a systematically difficult group, and many of them have Chambardia rubens numerous subspecies and several synonyms. Due to the Mutela dubia high degree of morphological variability within Order Mutela rostrata RAPD-PCR Unionoida, four species: Mutela dubia, M. rostrata, Chambardia rubens, and C. letourneuxi were collected from *Correspondence: Benha region (Qaluobiya Governorate, Egypt) to resolve Reda Mansour their degree of genetic similarity depending on RAPD-PCR Zoology and Entomology technique. Out of six primers used, only five primers Department, Faculty of Science Helwan University “UBC476, UBC477, UBC478, UBC483, and UBC486” Cairo, Egypt worked successfully. The study revealed that C. rubens and E-mail: C. letourneuxi are very closely related species, as they [email protected]. showed closely similar bands on using the primers edu.eg UBC477, UBC483, and UBC486. INTRODUCTION affect these characters[2]. In Egypt, the Recent molecular approaches in taxonomy species of family Mutelidae have showed have led to a steady increase in the so variable shell morphology due to identification of cryptic species. Prior to environmental changes and geographical molecular techniques, evolutionary relation- distribution leading to confusion in their ships among bivalves were determined nomenclature and classification[3-5]. Ibrahim using morphological species concepts[1]. et al.[3] reported that within Mutelidae there The advent of molecular techniques has are only two genera found in Egypt revealed that some morphological characters, namely, Mutela and Chambardia. Mutela such as shell structures in bivalves, are was reported to have three species; Mutela highly polymorphic and any change due to dubia, M. rostrata, and M. singularis; environmental and population density can while genus Chambardia has two species, 1 Genetic relationships of four mutelid species by RAPD-PCR Chambardia rubens and C. letourneuxi. Extraction of DNA and RAPD-PCR On the other hand, Harper et al.[4] and technique El-Assal et al.[5] have separated each of The four samples under investigation were Mutela and Chambardia into only two dissected and preservation of their soft species. Based on the morphological parts was carried out in absolute ethanol characters, shells of Mutela spp. devoid at 20°C until utilization. The extraction of the umbonal sculpture, while Chambardia total genomic DNA from frozen alcohol- spp. have shells with umbonal sculpture. preserved foot was achieved using Qiagen Shells of M. dubia and C. rubens are ovate- DNeasy tissue kit (Valencia, Santa Clarita, elongate, while those of M. rostrata and CA, USA) following the manufacturer’s C. letourneuxi are elongate. In addition, shell instruction. Six primers were used in the of M. dubia has posterior dorsal margin present investigation in RAPD-PCR[8-11]: showing the largest height, while M. rostrata 476: 5`-TTGAGGCCCT-3`, 477: 5`-TGTT has dorsal and ventral margins that are GTGCCC-3`, 478: 5`-CGAGCTGGTC-3`, almost parallel. On the other hand, C. rubens 479: 5`-CTCATACGCG-3`, 483: 5`-GCA has shell length/height (L/H) ratio equals CTAAGAC-3`, 486: 5`-CCAGCATCAG-3` to 1.6, while that of C. letourneuxi has L/H (Midland Certified Reagent Company, ratio measuring 1.8-2.0[3]. Midland, TX, USA). One of the DNA features that were used In the preliminary experiments, only to analyse the population genetic diversity five primers worked successfully “UBC476, is random amplified polymorphic DNA- UBC477, UBC478, UBC483, and UBC486”. polymerase chain reaction (RAPD-PCR) Amplifications were practiced following technique. RAPD-PCR standard method Williams et al.[12] with some modifications. uses short-single oligonucleotide (10-12 PCR mixture (50 µL) contained 2.0 µL bases) with the random order as a primer sample DNA, 1.0 µL primer, 25 µL master to amplify DNA genes in nanograms in mix and 22 µL distilled water (PCR low annealing temperature[6]. Therefore, the MyTaq™ HS Red Mix, Bioline, Memphis, present study aimed to use RAPD-PCR TN, USA). By utilization of each primer, method to determine the degree of each amplification reaction was repeated genetic similarity and evaluate the genetic three times to verify band autosimilarity[13]. relationship among the four mutelid species, Amplifications were produced in T-personal M. dubia, M. rostrata, C. rubens, and thermal cycler (TC-3000G, Techne Inc., C. letourneuxi from the River Nile in Egypt. Burlington, NJ, USA), programmed for 45 cycles of 94°C for 1.0 minute, 35°C MATERIAL AND METHODS for 1.0 minute, and 72°C for 1.0 minute. Collection of samples An initial denaturation step for 3.0 minutes Samples of the four mutelid species, at 94°C and a final extension holding for M. dubia, M. rostrata, C. rubens, and 10 minutes at 72°C were included in C. letourneuxi (n = 5, for each species) the first and last cycles, respectively. were collected from Benha (Qaluobiya The reaction products (10 µL) were resolved Governorate) along River Nile in Egypt by 2.0% agarose gel electrophoresis at 85 V (Figure 1) using a special net made of in 1.0 Tris-acetate-EDTA (TAE) buffer. hard metallic frame. Mussels were collected The gel was visualized using ethidium during spring 2017 and transferred to bromide stain and then photographed the laboratory of Invertebrates. Sorting with gel documentation system (SynGene, and maintenance of samples were done GeneTools - File version: 4.02.03, France). under the standard conditions of food Species -specific fragments were revealed and temperature. Identification of mussels using GeneRuler 1.0 kb Plus DNA Ladder followed the keys of Ibrahim et al.[3] and (Fisher Scientific, Toronto, Canada) to Graf and Cummings[7]. compare the amplified products. 2 Fol, M. F. et al. Figure 1: Photographs showing the morphology of external shell surface of the four mutelid species: (a) Mutela dubia shell; (b) M. rostrata shell; (c) Chambardia rubens shell; (d) C. letourneuxi shell. GA: growth annuli, PS: posterior slope, U: umbo. Scale bar = 2 cm. Molecular data analysis and estimation of similarity index (D-value) Molecular analysis was executed using gel of each primer among the studied mutelid documentation system for the dendogram species. RAPD-PCR amplification products 3 Genetic relationships of four mutelid species by RAPD-PCR took the score 1/0 for presence/absence as the mean and overall value for all of homologous bands[14] and analyses species and each primer. were accomplished using the NTSYSpc 2.2 software[15]. For RAPD markers, RESULTS similarity coefficient matrix was calculated DNA was successfully extracted from using Jaccard similarity algorithm[16]. the foot of the four studied species; Construction of dendograms was done Mutela dubia, M. rostrata, Chambardia using the unweighed pair-group method rubens, and C. letourneuxi. RAPD-PCR was with arithmetical algorithms averages carried out using six primers of which, (UPGMA)[17]. Genetic diversity was also primer (UBC 479) gave no reproducibility revealed as the percentage of polymorphic for all studied species, while the other bands. The percentage of polymorphism five primers provided strongly amplified was calculated for each species, as well fragments (Figures 2-6). Figure 2: RAPD-PCR profile of the mutelid species using UBC476 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC476 primer demonstrating the relationships of the species under study. Figure 3: RAPD-PCR profile of the mutelid species using UBC477 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC477 primer demonstrating the relationships of the species under study. 4 Fol, M. F. et al. Figure 4: RAPD-PCR profile of the mutelid species using UBC478 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC478 primer demonstrating the relationships of the species under study. Figure 5: RAPD-PCR profile of the mutelid species using UBC483 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC483 primer demonstrating the relationships of the species under study. Figure 6: RAPD-PCR profile of the mutelid species using UBC486 primer. (a) Gel electrophoresis showing amplification profile of samples, M: 1.0 kb DNA marker, (b) dendrogram of UBC486 primer demonstrating the relationships of the species under study. 5 Genetic relationships of four mutelid species by RAPD-PCR Each primer produced 3-6 polymorphic (Table 1). Some RAPD-PCR fragments were bands (Table 1). The number of amplified found to be unique; four unique bands in M. bands (monomorphic, polymorphic, and dubia, one unique band in M. rostrata, one unique) of the DNA of each species with unique band in C. rubens, and six unique different primers is given, and the genetic bands in C. letourneuxi, while the total diversity is also given as the percentage of polymorphic bands were found to be 90% the polymorphic bands for each primer among the four studied species (Table 2).
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