Annals of Human Biology, January –February 2012; 39(1): 80–83 Copyright q Informa UK, Ltd. ISSN 0301-4460 print/ISSN 1464-5033 online DOI: 10.3109/03014460.2011.630678 HUMAN BIOLOGICAL SURVEY Allele frequencies for 15 autosomal STR markers in the Libyan population Houssein Khodjet-el-Khil1, Karima Fadhlaoui-Zid1, Leonor Gusmão2, Cı́ntia Alves2, Amel Benammar-Elgaaied1 & António Amorim2,3 1 Laboratory of Molecular Genetics, Immunology and Human Pathologies, Faculty of Sciences, University of Tunis El Manar, Tunis, Tunisia, 2Institute of Pathology and Molecular Immunology (IPATIMUP), University of Porto, Porto, Portugal, and 3Faculty of Sciences, University of Porto, Porto, Portugal Cherni et al. 2008; Ennafaa et al. 2009 and Pereira et al. 2010b). However, little is known about the genetic landscape of the Libyan population which until recently remained the only state of the Maghreb without genetic evolution investigations. Indeed, most existing studies have focused on Libyan Jews (Rosemberg et al. 2001) or Libyan Tuareg (Ottoni et al. 2009, 2011 and Pereira et al. 2010a). Only two recent investigations have concerned the general Libyan population based on ancestry informative SNP markers (Khodjet el khil et al. 2011) and mitochondrial DNA markers (Fadhlaoui-Zid et al. 2011). The present work is the first to describe STR markers polymorphism in the general Libyan population in order to contribute to the analysis of its genetic diversity for forensic purposes. Background: Until recently Libya remained the only state of the Maghreb without genetic evolution investigations of the genetic landscape of its population. Apart from some studies of Libyan Jews and Libyan Tuareg, only two recent investigations, based on autosomal ancestry informative SNP and mitochondrial DNA markers, have concerned the general Libyan population. Aim: The present work is the first to describe STR markers polymorphism in the general Libyan population in order to contribute to the analysis of its genetic diversity for forensic purposes. Subjects and Methods: Allele frequencies for 15 STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, VWA, D2S1338, D19S433) included in the AmpFlSTR Identifiler kit were determined in a sample of 99 unrelated individuals originating from the general Libyan population. Results: No deviations from Hardy –Weinberg equilibrium were observed, with the exception of CSF1PO. Genetic parameters of forensic interest such as combined power of discrimination (PD) and combined probability of exclusion (PE) showed values higher than 0.999. Comparisons with data from other North African populations showed significant differences between Libyans and Tunisians, Moroccans and Egyptians. Conclusions: The high informativity observed for these 15 STRs in a Libyan population demonstrates their usefulness for forensic and parental purposes. SAMPLE Libya is located in the North of Africa, bordered by the Mediterranean Sea to the north and sharing borders with Egypt to the east, Algeria and Tunisia to the west and Sudan, Chad and Niger to the south. It was occupied, as was the case for all North African populations, by Phoenicians, Romans, Vandals, Byzantines, Islamic dynasties (Ummayad, Abbasid, and Fatimid) and the Ottoman Empire. Italians were the last invaders of Libya before its independence in 1951. At the 2006 census, Libya had a population of 5,670,688. Two-thirds of the population are settled in coastal areas. Small tribal groups in southern Libya are nomadic or seminomadic. The indigenous population of Libya is mostly Berber and Arab in origin (http://en.wikipedia.org/wiki/ Libya). Since our analysis was based on STR markers that are frequently used in forensic cases, and aimed to better evaluate the level of heterogeneity in the Libyan population, we decided to recruit individuals that were as representative Keywords: Libya, Population Genetics, North Africa, DNA, STRs, BACKGROUND The genetic diversity of North African populations has been the subject of many studies (Bosch et al. 1997, 2000; Cruciani et al. 2004; Olivieri et al. 2006; Adams et al. 2008; Correspondence: Houssein Khodjet-el-Khil, Laboratory of Molecular Genetics, Immunology and Human Pathologies, Faculty of Sciences, University of Tunis El Manar, Tunis, Tunisia. E-mail: housseinkek@yahoo.fr (Received 2 April 2011; revised 3 August 2011; accepted 4 October 2011) 80 ALLELE FREQUENCIES FOR 15 AUTOSOMAL STR MARKERS IN THE LIBYAN POPULATION 81 Table I. Allele Frequencies, Observed and Expected Diversity Parameters, and Probability Values for Conformation with Hardy-Weinberg Equilibrium at 15 STR Loci in Libyan Population (N ¼ 99). Ho: Observed heterozygosity; He: Expected heterozygosity; P: uncorrected probabilities for exact test of Hardy-Weinberg equilibrium; in bold, significant P value after Bonferroni correction (a ¼ 0.05/15 ¼ 0.0033). Allele D8S11 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TP0X D18S51 D5S818 FGA 6 7 8 9 9.3 10 10.2 11 12 12.2 13 13.2 14 14.2 15 15.2 16 16.2 17 18 19 20 21 22 22.2 23 24 25 25.3 26 27 28 29 30 30.2 31 31.2 32.2 33.2 34.2 35 36 Ho He P 0.010 0.010 0.121 0.136 0.010 0.005 0.020 0.010 0.076 0.338 0.293 0.253 0.197 0.177 0.288 0.066 0.020 0.101 0.051 0.035 0.056 0.056 0.076 0.121 0.141 0.273 0.106 0.348 0.263 0.258 0.359 0.333 0.323 0.217 0.010 0.045 0.141 0.162 0.222 0.015 0.005 0.035 0.010 0.030 0.020 0.005 0.434 0.172 0.005 0.005 0.076 0.005 0.157 0.338 0.005 0.040 0.242 0.061 0.005 0.283 0.081 0.025 0.278 0.091 0.141 0.192 0.051 0.040 0.020 0.136 0.005 0.222 0.030 0.258 0.061 0.146 0.066 0.025 0.030 0.066 0.076 0.116 0.015 0.005 0.172 0.202 0.364 0.167 0.167 0.086 0.121 0.010 0.121 0.101 0.268 0.061 0.247 0.152 0.106 0.020 0.121 0.081 0.071 0.020 0.030 0.025 0.247 0.045 0.040 0.051 0.030 0.015 0.061 0.106 0.146 0.197 0.005 0.162 0.187 0.076 0.005 0.005 0.015 0.808 0.843 0.032 0.010 0.091 0.242 0.283 0.025 0.071 0.116 0.071 0.056 0.010 0.015 0.005 0.848 0.829 0.856 0.798 0.770 0.504 0.687 0.725 0.002 0.677 0.742 0.678 0.768 0.783 0.995 0.707 0.772 0.122 as possible of the overall population. The sample studied in this work comprises 99 unrelated individuals selected from the general population of the west region of Libya, mainly from the capital Tripoli and its surrounding villages. 0.798 0.752 0.652 0.879 0.851 0.549 0.020 0.005 0.015 0.005 0.808 0.838 0.254 0.828 0.657 0.815 0.716 0.737 0.156 0.869 0.893 0.320 0.818 0.779 0.273 0.889 0.861 0.122 DATA COLLECTION The analyses were conducted on a sample of 99 healthy male individuals. They were collected in Tunisia in collaboration with medical biology laboratories that receive Libyan Table II. Forensic parameters obtained in a Libyan population sample, analysed for 15 autosomal loci. MP: matching probability; PD: power of discrimination; PE: power of exclusion; TPI: typical paternity index; PIC: polymorphic information content. D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TP0X D18S51 D5S818 FGA MP PD PIC PE TPI 0.058 0.942 0.816 0.614 2.605 0.061 0.939 0.805 0.692 3.300 q Informa UK, Ltd. 0.094 0.906 0.731 0.595 2.475 0.139 0.861 0.670 0.408 1.597 0.112 0.888 0.693 0.393 1.547 0.084 0.916 0.743 0.540 2.152 0.087 0.913 0.736 0.439 1.707 0.107 0.893 0.709 0.595 2.475 0.051 0.949 0.828 0.752 4.125 0.054 0.946 0.813 0.614 2.605 0.068 0.932 0.784 0.653 2.912 0.134 0.866 0.670 0.364 1.456 0.032 0.968 0.875 0.732 3.808 0.087 0.913 0.745 0.633 2.750 0.048 0.952 0.841 0.773 4.500 82 H. KHODJET-EL-KHIL ET AL. Table III. Exact test of differentiation between Lybian, Tunisian, Moroccan and Egyptian populations. In bold significant p values after Bonferroni correction (p , 0.05/6 ¼ 0.0083). Tunisians (102) (khodjet el khil et al. 2008), Moroccans (209) (Coudray et al. 2007a) and Egyptians (297)(Coudray et al. 2007b). D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 vWA TP0X D18S51 D5S818 FGA Lybians vs. Tunisians Lybians vs. Moroccans Lybians vs. Egyptians Tunisians vs. Moroccans Tunisians vs. Egyptians Moroccans vs. Egyptians 0.3654 0.1343 0.0105 0.0044 0.0000 0.0000 0.0223 0.1700 0.0006 0.0000 0.0000 0.0020 0.0089 0.3197 0.0023 0.0291 0.0000 0.0003 0.1053 0.1494 0.0412 0.0035 0.0000 0.0000 0.0183 0.0078 0.0000 0.0118 0.0000 0.0000 0.0618 0.0882 0.0000 0.0000 0.0000 0.0006 0.0001 0.0714 0.7037 0.0000 0.0000 0.0010 patients coming from Libya. Information about their four grandparents originating from Libya was obtained and all participants provided their written informed consent. Our data was compared to other North African populations collected from the literature: Tunisians (102) (Khodjet el khil et al. 2008), Moroccans (209) (Coudray et al. 2007a) and Egyptians (297) (Coudray et al. 2007b). 0.0035 0.1352 0.0699 0.0000 0.0000 0.0000 0.0022 0.4517 0.0052 0.0804 0.0000 0.0000 0.0086 0.0347 0.2119 0.0401 0.0000 0.0000 0.2244 0.0002 0.2994 0.0001 0.0011 0.0002 0.0034 0.0000 0.1174 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0012 0.0000 0.0021 0.0085 0.0008 0.2434 0.0883 0.0628 0.0000 0.0099 0.1416 0.0103 0.0687 0.0000 0.0000 Population substructure analysis was performed using STRUCTURE software ver 2, distributed in http://pritch. bsd.uchicago.edu (Pritchard et al. 2000; Falusch et al. 2003). RESULTS Observed allele frequencies at the analysed 15 autosomal STR loci are presented in Table I. No deviations from HardyWeinberg equilibrium were observed, except at the CSF1PO locus (significant p value after Bonferroni correction) due to an excess of homozygotes. Forensic parameters for the 15 studied loci are presented in Table II. The combined power of discrimination (PD) and the combined power of exclusion (PE) for the 15 studied loci were higher than 0.999. Locus by locus comparisons with available published data on other North African populations are presented in Table III. Significant differences were found between Libyans and Tunisians (Khodjet-el-khil et al. 2008) at 5 loci; Moroccans (Coudray et al. 2007a) at 5 loci; Egyptians (Coudray et al. 2007b) at 6 loci. In order to better analyse the genetic relationships between Libyans and compared North African populations, we used the STRUCTURE program that considers individual genotypes and assigns them to clusters (K) that are initially defined and can be varied across independent runs. Different simulations were done with K from 1 to 5 assuming admixture model and considering correlation between allele frequencies. All runs were computed with burning length and a Markov chain of 100,000 iterations. Simulation with K ¼ 4 clusters is presented in Figure 1 and clearly shows the absence of population clustering and genetic substructure between these compared North African populations based on these 15 STR markers. DATA MANAGEMENT AND STATISTICAL ANALYSIS Genotyping DNA from fresh blood was extracted using a standard phenol-chloroform method (Sambrook et al. 1989). Approximately 10 –20 ng target DNA was amplified by multiplex PCR for 16 Loci: 13 autosomal STR loci (CODIS core STR loci CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and VWA); two additional STR loci, D2S1338 and D19S433, and Amelogenin locus. Amplification was performed in a GeneAmp PCR system 2720 using the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems, Foster City, California) according to the manufacturer’s instructions. PCR products generated mixed with internal size standard (GS-500 LIZ; Applied Biosystems, Foster City, CA) were electrophoresed in an ABI 310 Genetic Analyzer (Applied Biosystems Foster City, CA) and analyzed with GeneScan 3.1.2 and GeneMapper 3.2 software using the supplied allelic ladders (Applied Biosystems). Statistical approach Allele frequencies, Hardy– Weinberg equilibrium, expected heterozygosity (He); observed heterozygosity (Ho) and exact test of population differentiation were carried out with the Arlequin Software Version 3.1.1 (Excoffier et al. 2005). Matching probability (MP), power of discrimination (PD), polymorphism information content (PIC), probability of exclusion (PE) and typical paternity index (TPI) were calculated with Powerstats Version 1.2 (http://www.promega.com/geneticidtools/powerstats/). COMMENT High informativity and conformity with Hardy-Weinberg equilibrium in the majority of loci suggest that the AmpFlSTR Identifiler is an appropriate STR markers system 1.00 0.80 0.60 0.40 0.20 0.00 1 2 3 4 Figure 1. North African population genetic structure based on 15 STR markers. Run is performed assuming K ¼ 4. 1: Moroccan (209). Coudray et al. 2007; 2: Tunisian(102). Khodjet el khil et al. 2008; 3: Lybian (99) present study; 4: Egyptian (297). Coudray et al. 2007. k ¼ 4. Annals of Human Biology ALLELE FREQUENCIES FOR 15 AUTOSOMAL STR to be used for identification and paternity purposes in Libyan population. This STR data could also be considered in evolutionary comparison studies. ACKNOWLEDGEMENTS We are grateful to the DNA donors who made this study possible. This work was partially supported by the Tunisian Ministry of High Education, Scientific Research and Technology and approved by the national ethical committee. 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