Egyptian Journal of Zoology (EJZ), Vol. 74: 22-29 (December, 2020) p-ISSN: 1110-6344 Publisher: The Zoological Society of A. R. Egypt e-ISSN: 2682-3160 DOI: 10.12816/ejz.2020.33134.1035

SHORT COMMUNICATION

EVALUATING THE USE OF THE MULTILOCUS GENETIC MARKER “5S RDNA” FOR THE DISCRIMINATION OF DIFFERENT OF FAMILY IN RED SEA, EGYPT

Ghada Mohammed-ElSherif1; Asmaa Galal-Khallaf1; Alaa G. M. Osman2; Sobhy El-Sayed Hassab El-Nabi1; Khaled Mohammed-Geba1* 1Zoology Department, Faculty of Science, Menoufia University, Menoufia, Egypt 2Zoology Department, Faculty of Science, Al-Azhar University (Assiut Branch), Egypt

ABSTRACT Article History: Family Lethrinidae (Sho´our fishes, Shaari fishes) is one of Received: 23 June 2020 the key families in the Red Sea and Indian Ocean fisheries. Revised: 30 June 2020 Accepted: 1 July 2020 Some of its species were recorded in the International Union for Conservation of Nature (IUCN) Red List of species as Published Online: endangered. In order to develop simple genetic marker that 3 July 2020 aid their rapid identification, fin clips of four lethrinid Keywords: species were collected from local fish markets in Hurghada city. 5S rDNA These species were lentjan, L. harak, L. microdon, 12S rDNA and L. mahsena. DNA was extracted from these samples and Genetic marker subjected to polymerase chain reaction (PCR) amplification Lethrinidae PCR for the mitochondrial 12S ribosomal RNA (rRNA) and the nuclear 5S rRNA genes. The 12S rRNA PCR amplicons were *Correspondence: sequenced. GenBank comparison and phylogenetic analysis for Khaled Mohammed-Geba the 12S rRNA gene sequences resulted in clear identification Zoology Department for the sampled species. Meanwhile, a simple, 2% agarose gel Faculty of Science Menoufia University electrophoresis for the 5S rDNA ran in a low voltage for 2 hours Menoufia, Egypt resulted in variable intraspecific patterns. These patterns appeared E-mail: as 3 PCR bands of approximately 500 base pair (bp) in length Khaled.mohammed@icman. for L. lentjan; 2 PCR bands at 500 and 400 bp for L. harak; csic.es single PCR band at 500 bp for L. microdon; and 3 bands at about 750, 500, and 400 bp for L. mahsena. Band separation was clear enough. 5S rDNA exhibited an accurate, interspecific, and easy- to-use genetic marker for identification of different lethrinid species covered by the current study.

INTRODUCTION identification, especially for roughly treated Several protocols have been described food stuffs where only very short, recover- for marine species identification due to able DNA fragments are still present[3,4]. the growing interest for healthy seafood. Those small-sized, leftover DNA fragments Molecular techniques are providing easily permitted a new era of DNA fingerprinting accessible surveillance tools for many through sequencing of short, informative biological applications, including the certi- genomic locations that are considered as bar- fication of the species present in commercial codes[5,6]. These barcodes provided valuable food products[1,2]. These markers provided tools for authenticating important market great success for finfish and shellfish species species, monitoring biodiversity, detecting

22 5S rDNA as a molecular marker for Egyptian lethrinids the biological invasions[7-9], and many other of interspecific genetic variations[15-17]. applications. This may lead to a serious problem for The 12S ribosomal RNA (12S rRNA) monitoring the real standing situation for mitochondrial gene (i.e. 12S rDNA) has the species conservational status, as well been applied extensively as a species marker as for their proper fisheries management. due to its interspecific sequence variability, Therefore, this study aimed to evaluate mutation rates, and lower, yet adequately the efficiency of application of the nuclear, conclusive, degeneracy in comparison to multilocus gene 5S rDNA for discrimination the protein-coding mitochondrial genes[3,10]. of the key species belonging to the Family The nuclear 5S rRNA gene exhibits a more Lethrinidae from the Red Sea in Egypt, complex array in comparison to the mito- and validating such efficiency with other chondrial 12S rDNA. It consists of a highly marker, i.e. the mitochondrial 12S rDNA, conserved, 120 base pair (bp) long trans- that proved a success in such purpose cribed area that plays a role in both ribo- before[10]. somal structure stability and peptidyl trans- [11] ferase enhancement . This transcribed area MATERIAL AND METHODS is followed by a hypervariable, tandemly Ethical considerations arranged, non-transcribed spacer whose [11-13] The samples were obtained from fishes length varies among species . For the after being fished, landed, and sold best of the authors’ knowledge, the for human consumption. According to the 5S rDNA was not tested for discrimination Committee for Ethics of our Faculty, among Egyptian fish species before. no permits are required to conduct this In Egypt, marine capture fisheries market-based study. contribute 127,821 tons annually, of which 2,157 are coming from a single fish Collection of samples family, which is Lethrinidae (emperors)[14]. Freshly caught, five samples of each of This fish group exhibits some morpho- Lethrinus lentjan, L. harak, L. microdon, logical similarities that basically emerge as and L. mahsena were obtained from adaptation to the surrounding environment local fish markets in Hurghada, the Red and feeding habits, and not only based Sea (Figure 1).

Figure 1: Different emperor species (Lethrinidae) collected from Hurghada city of the Red Sea in Egypt in the current study. (a) Lethrinus harak, (b) Lethrinus lentjan, (c) Lethrinus microdon, and (d) Lethrinus mahsena.

23 Mohammed-ElSherif, G. et al.

Fish samples were thoroughly checked applied consisted of an initial denaturation for the absence of skin lesions or external step at 95ºC for 10 min; 35 cycles of parasites. Pectoral fin clips (100 mg) were amplification (1 min at 95ºC for denaturation, removed from each fish, placed in 1.5 mL 1.0 min at 57ºC for annealing and 1.0 min sterile tubes filled with 70% ethyl alcohol at 72ºC for extension); and a final for preservation, and transferred to our extension step of 10 min at 72 ºC[10]. The laboratory for genetic analyses. PCR products were electrophoresed in 1% agarose gel, and visualized using DNA extraction UV-light (TransillumintorTi1, Biometra). DNA extraction was carried out using The positive, specific amplicons were sent the method described in Mohammed-Geba to Macrogen Inc. (Seoul, South Korea) et al.[18]. Briefly, 200 μL of TNES-urea -1 for conventional Sanger chain termination buffer with 2.4 U mL proteinase K solution sequencing method. (ThermoFischer Scientific, Waltham, MA, USA) were added to the individually lysing PCR amplification of 5S rRNA gene fins, with incubation at 65ºC. Then, 54 μL For amplifying the 5S rDNA, the of 5 mol NaCl were added, mixed, and universal primers described by Perez the tubes were centrifuged at 4000 xg for and García-Vázquez[20] were applied, that 10 minutes (min). The aqueous supernatant were 5SC: 5´- AAGCTTACAGCACCT was transferred to a new, sterile tube, and GGTATT-3´, and 5SMD: 5´-TTCAACATG 200 μL of freeze-cold isopropyl alcohol GGCTCCGACGGA-3´. The amplification were added. After 11000 xg centrifugation reaction was the same as for 12S rDNA, for 10 min, the supernatant was replaced by but using the following program[20]: initial 400 μL of 70 % ethanol. The tubes were denaturation at 95ºC for 5 min, 30 cycles centrifuged for 5 min at 11000 xg, and then of amplification (20 seconds at 95ºC the ethanol was completely removed. Finally, for denaturation, 20 seconds at 65ºC for 30 μL of Tris-ethylenediaminetetraacetic annealing and 20 seconds at 72ºC for acid buffer (TE, pH = 8) were added for extension) and a final extension step of DNA pellet resuspension. DNA quality was 7 min at 72ºC. The PCR products were checked by running in a 1.0 % agarose gel electrophoresed in a 2% agarose gel, electrophoresis stained with 0.5 μg/mL visualized using UV-transillumination, and ethidium bromide (ThermoFisher Scientific). photographed.

Polymerase chain reaction (PCR) ampli- fication of 12S rRNA gene RESULTS A hypervariable fragment, 400 bp long, Sequencing the 12S rDNA resulted in good of the mitochondrial 12S rDNA was quality sequence chromatograms. GenBank amplified by PCR in each sample. comparisons and ML phylogenetic analysis The universal primers described by exhibited close proximity for the sampled Palumbi[19] were applied in these PCRs species with their references from different that are: 12SA: 5´-AAACTGGGATTAGAT areas in the world (Figure 2). L. lentjan ACCCCACTAT-3´, and 12SF: 5´-GAG (accession number: MT648947) exhibited GGTGACGGGCGGGCGGTGTGT-3´. The high sequence identity (Seq. ID) with amplification reaction for each sample was the ones from Egypt (accession number: set to a total volume of 25 μL, using 50 ng KU680994, 99.21% Seq. ID) and China of template DNA, 1X MyTaq™ Red ( accession number: AY484981, 99.21% Seq. Mix (Meridian Life Science, Inc., Memphis, ID). L. harak (accession number: MT648948) TN, USA), 0.4 μmol of each primer, and was 99.61% identical to the one from deionized waters. PCRs were carried out Egypt (accession number: KU680995.1). in the thermal cycler Tpersonal (Biometra, L. microdon (accession number: MT648949) Göttingen, Germany). The PCR program exhibited 100% Seq. ID with its reference

24 5S rDNA as a molecular marker for Egyptian lethrinids

Figure 2: 12S rDNA-based maximum likelihood phylogenetic tree for the four studied Lethrinus species (blue color), their GenBank references (black color), and Plectropomus areolatus (: Serranidae) as outgroup (red color). Bootstrap values (only above 50% = 0.5) are shown as green circles above the clades. whose origin was also Egyptian (accession the size of 500 bp. L. mahsena exhibited number: KU680996.1). Finally, L. mahsena 3 different bands, the largest of which had (accession number: MT648946) was 100% the size of 750 bp, and 2 smaller bands at identical to its GenBank reference (Egypt, 500 and almost 400 bp (Figure 3). accession number: KU680997.1). Phylo- genetic analysis, whose best substitution DISCUSSION model was Tammura-Nie, with G-value Development of molecular markers for of 0.25, resulted also in perfect clustering of identification and authentication of seafood the assessed species with their GenBank products has key impacts on conservation references, with bootstrap values of > 50 % and human health[21]. In the current study, in all cases (Figure 2). 4 species from a key fish family in the Agarose gel resolution for the 5S rDNA Egyptian fisheries, i.e. Lethrinidae, could be resulted in a conserved band pattern in all successfully authenticated using 2 different samples, regardless to the degree of markers that are the 12S rDNA and the DNA damage or varying the annealing 5S rDNA. The 12S rDNA exhibited success temperatures. Such pattern was highly in authentication of many fish groups before. species-specific. L. lentjan exhibited 3 PCR In case of lethrinids and related families, bands, one of which was 500 bp, and the 12Sr DNA sequencing could previously others were one below and one above the elucidate success in Egyptian Lethrinus 500 bp. L. harak exhibited double bands species discrimination[10]. It was also applied that were at 400 and 500 bp. L. microdon for identifying the phylogenetic relationships samples exhibited a single band close to among Chinese species from the Family

25 Mohammed-ElSherif, G. et al.

Figure 3: Agarose gel (2%) electrophoresis for examples for 5S rDNA gene of an example from each of the four Lethrinus species analyzed in the current study. Order from left to right is M: DNA size marker, Ll: Lethrinus lentjan, Lh: Lethrinus harak, Lm: Lethrinus microdon, La: Lethrinus mahsena. Band sizes in base pairs (bp) are shown in the left side of the photo.

Lutjanidae[22]. It was applied as a marker to Macruronus magellanicus. Also, Pinhal detect the prevalence of fishes from the et al.[12] successfully generated 5S rDNA- Family Lethrinidae and other families in the based, interspecific electrophoretic patterns subsistence practices of the historic coastal from 8 shark species, i.e. Sphyrna lewini, community in southwest Madagascar[23]. Galeocerdo cuvier, Carcharhinus obscurus, Moreover, it was applied for biodiversity C. leucas, C. limbatus, C. achronotus, monitoring and tracking the lethrinids of the Alopias superciliosus, and Isurus oxynchus. Australian waters through their environ- De Luna Sales et al.[25] also identified mental DNA[24]. Hence, this gene exhibited the same 5S rDNA patterns for the in several cases a good discriminative power squids Loligo surinamensis, L. sanpaulensis, for this group of fishes and many others. Lolliguncula brevis, Sepiotheuthis sepioidea, In addition to the 12S rDNA, application Ornithoteuthis antillarum, and Illex of the 5S rDNA resulted in clear, poly- argentines. However, the application of morphic, and species-specific electro- banding variations in species discrimination phoretic patterns. Application of this may necessitate preliminary trials, as not protocol for discrimination of Lethrinus all species show such interspecific banding. spp. exhibited more simple and economic For instance, and in contrast to our potentials than using a 12S rDNA-based findings in the current study and those protocol. This is directly as the 5S rDNA previously mentioned for other seafood produced its discriminative pattern after only species, Veneza et al.[13] identified intra- a simple gel electrophoresis, in contrast to specific variations in the electrophoretic the 12S rDNA-based discriminative protocol bands of the 5S rDNA in species from the that could not be completed without Family Lutjanidae. sequencing. For small scale food and/or In conclusion, the 5S rDNA nuclear fisheries-specialized inspection laboratories, gene could be proven as a successful it may be recommended to apply such marker for discrimination the commonest markers that reduce time and costs 4 Lethrinus species in Egypt. The inter- for species and identification. In this specific multiple bands’ pattern identified sense, Perez and García-Vázquez[20] applied in the agarose gel electrophoresis can such one-step, electrophoresis-based dis- recommend the application of this molecular criminative 5S rDNA-based protocol to marker in rapid and cost-effective species characterize several fish species of the identification. Despite coupling this marker Family Merlucciidae, that were Merluccius with 12S rDNA gene sequencing results paradoxus, M. senegalensis, M. australis, in a more validated methodology, the M. gayi, M. bilinearis, M. hubbsi, and variable patterns identified for the

26 5S rDNA as a molecular marker for Egyptian lethrinids

5S rDNA can suggest it as a stand-alone in Egyptian aquafeed formulations marker for the species tested herein in the using pyrosequencing and metabar- current work. coding. Fisheries Research, 174: 143- 150. ACKNOWLEDGMENTS [5] Kress, W. J. and Erickson, D. Authors are grateful to the Lab of Natural L. (2008). DNA barcodes: genes, Resources, leaded by Prof. Eva García- genomics, and bioinformatics. PNAS, Vázquez (University of Oviedo, Asturias, 105(8): 2761-2762. Spain) that inspired us with the idea of [6] B hme, . Calo-Mata, . Barros- the current work. This research received no Vel zquez, . et al. (2019). Review of specific grant from any funding agency in recent DNA-based methods for main public, commercial, or not-for-profit sectors. food-authentication topics. J Agric Food Chem, 67(14): 3854-3864. CONFLICT OF INTEREST [7] Zhao, L.; Dong, J.; Sun, C. et al. The authors declare that they have no (2019). Phylogenetic analysis of sooty conflict of interest with the results of the grunter and other major freshwater present research. fishes in the suborder Percoidei based on mitochondrial DNA. Mitochondrial AUTHORS’ CONTRIBUTIONS DNA Part A, 30(2): 234-248. GM-E: investigation, methodology, writing [8] Rüber, L.; Tan, H. H. and Britz, original draft; AG-K: conceptualization, R. (2020). Snakehead (Teleostei: methodology, data curation, writing original Channidae) diversity and the Eastern draft; AGMO: resources, reviewing original Himalaya biodiversity hotspot. Journal draft; SEHE: supervision, reviewing original of Zoological Systematics and draft; KM-G: supervision, conceptualization, Evolutionary Research, 58: 356-386. software, reviewing original draft. [9] Borrell, Y. J.; Miralles, L.; Do Huu, H. et al. (2017). DNA in a bottleRapid REFERENCES metabarcoding survey for early alerts [1] Pérez, M.; Cabado, A. G.; Vieites, of invasive species in ports. PloS J. M. et al. (2004). Experimental One, 12(9): e0183347 (DOI: 10.1371/ assessment of a new rDNA-based journal.pone.0183347). method for the identification of [10] Galal-Khallaf, A.; Mohammed-Geba, Merluccius capensis and Merluccius K.; Osman, A. G. et al. (2017). SNP paradoxus in commercial products. based PCR-RFLP, T-RFLP and FINS Journal of Aquatic Food Product methodologies for the identification Technology, 13(2): 49-57. of commercial fish species in Egypt. [2] Duran, C.; Appleby, N.; Edwards, Fisheries Research, 185: 34-42. D. et al. (2009). Molecular genetic [11] Castro, S. I.; Hleap, J. S.; Cárdenas, H. markers: discovery, applications, data et al. (2016). Molecular organization storage and visualization. Current of the 5S rDNA gene type II in Bioinformatics, 4: 16-27. elasmobranchs. RNA Biol, 13(4): 391- [3] Rasmussen, R. S. and Morrissey, 399. M. T. (2008). DNA-based methods for [12] Pinhal, D.; Gadig, O. B.; Wasko, A. P. the identification of commercial fish et al. (2008). Discrimination of shark and seafood species. Comprehensive species by simple PCR of 5S rDNA Reviews in Food Science and Food repeats. Genet Mol Biol, 31: 361-365. Safety, 7(3): 280-295. [13] Veneza, I.; Felipe, B.; Oliveira, J. [4] Galal-Khallaf, A.; Osman, A. G.; et al. (2014). A barcode for the Carleos, C. E. et al. (2016). A case authentication of the snappers study for assessing fish traceability (Lutjanidae) of the western Atlantic:

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How to cite this article: Mohammed-ElSherif, G.; Galal-Khallaf, A.; Osman, A. G. M.; Hassab El-Nabi, S. E. and Mohammed-Geba, K. (2020). Evaluating the use of the multilocus genetic marker “5S rDNA” for the discrimination of different species of Family Lethrinidae in Red Sea, Egypt. Egyptian Journal of Zoology, 74: 22-29 (DOI: 10.12816/ejz.2020.33134.1035).

28 الوجلت الوصريت لعلن الحيواى الوجلذ 47 )ديسوبر 0202(

تقيين استخذام جيي الوحذة الريبوسوهيت "5S rDNA" هتعذد الوواقع لتوييز األًواع الوختلفت لفصيلت أسواك الشعور بالبحر األحور٬ هصر

غادة هحوذ الشريف ٬ 1أسواء جالل خالف 1٬ عالء جاد الكرين عثواى٬0 صبحي السيذ حسب الٌبي٬1 خالذ هحوذ جبت1 1لسُ عٍُ اٌحٛ١اْ، و١ٍت اٌعٍَٛ، جاِعت إٌّٛف١ت، إٌّٛف١ت، جّٛٙر٠ت ِصز اٌعزب١ت 0لسُ عٍُ اٌحٛ١اْ، و١ٍت اٌعٍَٛ، جاِعت االس٘ز (فزع أسٛ١ط)، جّٛٙر٠ت ِصز اٌعزب١ت

تعذ فص١ٍت أسّان اٌشعٛر ِٓ اٌفصائً اٌسّى١ت اٌزئ١س١ت فٟ ِصا٠ذ اٌبحز األحّز ٚاٌّح١ط إٌٙذٞ. تُ تسج١ً بعط أٛٔاعٙا فٟ اٌمائّت اٌحّزاء ٌألٛٔاع عٍٝ أٙٔا ِٙذدة باالٔمزاض. ِٚٓ أجً رصذ ُِعٍّاث ج١ٕ١ت بس١طت تساعذ عٍٝ اٌتعزف اٌسز٠ع عٙ١ٍا، تُ جّع ع١ٕاث ِٓ اٌشعأف ألربعت أٛٔاع ِٓ تٍه اٌفص١ٍت ِٓ أسٛاق األسّان اٌّح١ٍت فٟ ِذ٠ٕت اٌغزدلت، ٚتُ تعز٠فٙا ِبذئ١ا عٍٝ أٙٔا تٕتّٟ إٌٝ األٛٔاع "L. mahsena ،L. microdon ،L. harak ،Lethrinus lentjan". تُ استخالص اٌحّط إٌٞٚٛ ِٓ ٘ذٖ اٌع١ٕاث ِٓ أجً استعّاٌٗ فٟ تم١ٕت تفاعً اٌبٍّزة اٌّتسٍسً فٟ اٌىشف عٍٝ ج١ٓ اٌٛحذة اٌز٠بٛس١ِٛت ا١ٌّتٛوٛٔذر٠ت "ٚ "12Sج١ٓ اٌٛحذة اٌز٠بٛس١ِٛت اٌىزِٛٚس١ِٛت "5S"، ثُ تحذ٠ذ تتابع اٛ١ٌٕوٛ١ٍت١ذاث ٌٍٛحذة "ٚ ،"12Sتح١ًٍ تٍه اٌتتابعاث بٛاسطت ِمارٔتٙا بماعذة اٌب١أاث اٌج١ٕ١ت "ٚ ،"GenBankوذٌه بٛاسطت اٌتحا١ًٌ إٌَ َسب١ت، ب١ّٕا تُ تح١ًٍ ٔتائج تفاعً اٌبٍّزة اٌّتسٍسً اٌخاصت باٌٛحذة اٌز٠بٛس١ِٛت اٌىزِٛٚس١ِٛت "5S" بٛاسطت اٌفصً اٌىٙزبائٟ ِٕخفط اٌجٙذ فٟ ٘الَ األجارٚس اٌبس١ط بتزو١ش %2. أسفزث ٔتائج تح١ًٍ اٌٛحذة اٌز٠بٛس١ِٛت "12S" عٓ تطابك تتابعٚ َٟٔ َسبٟ واًِ ب١ٓ اٌع١ٕاث اٌ ُّجّعت ٚاألٛٔاع اٌتٟ تُ ٚصفٙا ِٛرفٌٛٛج١ا أثٕاء اٌتج١ّع، ٚأظٙزث ٔتائج تح١ًٍ اٌٛحذة اٌز٠بٛس١ِٛت "5S" أّٔاط ِتطابمت عٍٝ ِستٜٛ إٌٛع اٌٛاحذ، ٌىٓ ِتغ١زة ب١ٓ األٛٔاع اٌّختٍفت، بح١ث أظٙز "L. lentjan" ثالثت ٛٔاتج حٛي اٌحجُ ٚ ،055 pbأظٙز "L. harak" ٔاتج١ٓ باٌحج١ّٓ pb 055 ٚ ،055 pb ٚأظٙز "L. microdon" ٔاتجا ِٕفزدا بحجُ ٚ ،055 pbأظٙز "L. mahsena" ثالثت ٛٔاتج بأحجاَ ٚ .055 pb ٚ 055 pb ٚ 005 bpباٌتاٌٟ فإْ اٌج١ٓ اٌىزِٛٚسِٟٛ ِتعذد اٌّٛالع "5S rDNA" ٠عتبز َِ ْعٍَُ جٟٕ١ دل١ك ٚبس١ط ١ِّٚش ٌألٛٔاع اٌّختٍفت اٌتٟ تُ دراستٙا فٟ أسّان اٌشعٛر اٌتٟ تٕاٌٚتٙا اٌذراست اٌحا١ٌت.

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