Genetic polymorphisms of 17 X-STR loci in two Tunisian populations from Sousse and Makthar

Abstract Background Tunisia has a complex demographic history of migrations from within Africa, Europe, and the Middle East. However, only one population study based on X-STR markers has been reported so far. Aim To investigate the genetic polymorphisms of 17 X-STRs in two Tunisian populations from the cities of Sousse and Makthar, and to reveal the genetic relationships with other reference populations. Subjects and methods A total of 194 unrelated healthy individuals were analysed for 17 X-STR markers. Results Our results indicate that DXS6809 is the most polymorphic locus, whereas DXS6807 is the least informative marker in the populations of Sousse and Makthar. In addition, forensic statistical parameters, such as the power of discrimination in males and females, as well as the mean of exclusion in duos and trios, reveal that the panel of 17 X-STRs is highly informative and useful in different forensic applications. Overall, pairwise genetic distances (Fst) and non-metric MDS plots demonstrate clustering of different populations according to their geographic locations and their historical relationships. Conclusion Overall, the study of X-STR markers of the Tunisian populations can help to promote the establishment of a forensic DNA reference database in Tunisia and provide reference for future anthropological research.


Introduction
Genetic analysis of global populations has proven to be very effective in enabling better understanding and alignment of events that have happened in the past.
North African populations hold enormous linguistic, cultural, and economic diversity. Reconstructing the patterns of population interaction, migration, admixture, and replacement that contributed to this diversity has been the main purpose of numerous genetic studies. Our research aims to reconstruct the genetic pattern of some Tunisian populations.
Tunisia is a Mediterranean country located in North-Central Africa and bounded on the north and the east by an extensive coastline (1300 km). Its strategic location and abundant resources have played a major role in promoting population admixture and genetic diversity (Leroy-Beaulieu 1886; Hodges 2020). Numerous invaders, mainly from the Mediterranean basin, have been attracted to Tunisia for its natural and human resources (Camps 1974;Amine and Carlson 2012;Boular es 2012). Present day Tunisian populations are ethnically heterogeneous, with the presence of the indigenous Berbers in addition to descendants from various civilisations such as the Phoenicians, Greeks, Romans, Vandals, Byzantines, Arabs, Spanish, Ottomans, and French.
The Tunisian territory is divided into 24 governorates, and each is characterised by its diversity, such as the city of Sousse, which is located on the northern coast of Tunisia ( Figure 1). Sousse was founded by the Phoenicians in the 11th century BC under the name of Hadrumetum and it was influenced by neighbouring towns such as Carthage, which would later come under the reign of the Romans (146 BC and 410 BC) (Trousset 2002). Soon after the capture of Carthage by Genseric in 439 (Lee 2013), Hadrumetum passed into the hands of the Vandals (Merrills and Miles 2009). Afterwards, the Byzantines ruled the area for 135 years. The seventh century witnessed the Arabian conquest of the city; it was renamed Susa and the Islamic religion and Arabic language and culture were subsequently introduced (Edis 1999). Following the Arab rule, Sousse was invaded by the Normans of Sicily who remained in power from 1145 to 1160. In the 12th century, the city was reconquered by the Almohads (Azzopardi 2002). This was followed by the reign of the Ottomans (1574-1881) and the French domination, during which Sousse became the second commercial port of the country.
The population diversity was not limited to only the coastal area of Tunisia, cities in the centre of the Tunisian territory were also under the influence of different invaders. In this study, we focussed on the city of Makthar; which is located 162.4 km from the city of Sousse, (Figure 1). Makthar is of Libyan-Phoenician origin (Selin 2008) and was founded during the Numidian reign (Ghaki 2012). In the third century BC, as part of the Punic Carthage, the city experienced economic growth (Charles-Picard 1958) which attracted the Roman Empire and resulted in the conquest of the city (146 BC). The city was occupied by the Vandals in 439 under the name of Mactaris, until the Byzantine Empire conquered it in 533. The Arab invasion followed in the middle of the seventh century and changed its name into Makthar. The city was totally abandoned by the 11th century until it regained its glow under the French colonisation that started in 1881, after which it became the nucleus of other nearby townships (the National Institute of Heritage -Tunisia, 2020).
Over the past decade, the field of human population genetics has enhanced our understanding of the origins, evolutionary history, and migration patterns of different populations. Previous studies have been conducted on Tunisian populations using different genetic marker systems such as Y-chromosome and mitochondrial DNA analyses (Fadhlaoui-Zid et al. 2004. Xchromosome STRs (X-STRs) analysis is a novel approach to the study of Tunisian population genetics. Only one study using X-STR markers has previously been performed on a Tunisian population from Nabeul (Messoussi 2019; Figure 1).
The X-chromosome is one of the two chromosomes determining sex in humans. Females inherit one of their two Xchromosomes from their mother and the other from their father, while males receive only their X-chromosome from their mother. The forensic potential of this chromosome is mainly due to its inheritance pattern, as well as the easy access to its haplotypes. As a result, the interest in studying genetic markers located on the X-chromosome such as X-STRs has increased (Tillmar et al. 2017). This has resulted in the creation of X-STR typing panels (Diegoli 2015;Prieto-Fern andez et al. 2016a). Due to their characteristics, X-chromosome markers, especially X-STRs, have gained recognition as powerful tools to complement the information provided by autosomal STRs and mitochondrial DNA. X-STRs are especially informative in kinship analyses where the majority of the profiles to compare correspond to second-or third-degree relatives, ie grandparents-grandchildren, paternal half-sisters, paternal aunt/uncle nieces and maternal uncle-nephews (Pinto et al. 2011).
Given that these markers are located on the same chromosome, two or more closely located markers may be inherited together as a haplotype. Yet, even if the physical distance between loci is minimal, recombination might occur in case of existing hotspots of recombination between them (Krawczak 2007).
In this investigation, we assess, for the first time, the genetic variation of 17 X-STR loci in 194 unrelated individuals from the Tunisian cities of Sousse (96 individuals) and Makthar (98 individuals), with the purpose of complementing our previous Tunisian X-STR database (Messoussi et al. 2019) for anthropological and forensic purposes.

Sampling and DNA extraction
Blood samples were collected from two Tunisian populations from the cities of Sousse (N ¼ 96; 49 males and 47 females) and Makthar (N ¼ 98; 32 males and 66 females). The donors were unrelated and healthy. All samples were obtained from volunteer donors under informed consent, following the ethical standards of the Helsinki Declaration. This study was approved by the local Ethics Committee of Charles Nicolle Hospital in Tunis. The DNA extraction from blood samples was done using the standard phenol-chloroform method. The quantification and degree of purity were determined using a Scientific NanoDrop TM 1000 spectrophotometer (Thermo Fisher Scientific Inc. Wilmington, DE).

Reference populations
In the present study, we used genotype data corresponding to 17-X-STR loci from 14 previously published populations (Spanish, French, Moroccan, Tunisian (Nabeul), Guinea and Malawi, Portugal and Ireland) (Supplementary Table S1), to carry out the population analysis and elucidate the genetic relationships between them and the populations from Sousse and Makthar.

Statistical analyses
The software Arlequin 3.5 (Excoffier 2007) was employed to perform the following statistical analyses: allele frequencies, exact chi-square test for Hardy-Weinberg equilibrium (HWE) per locus for female data, exact test of differentiation (100.000 Markov steps) between male and female allele frequencies, pairwise test of linkage disequilibrium (LD) between pairs of markers for male subsample, haplotype frequencies for the cluster DXS7132-DXS10075-DXS10079, gene diversity (GD), and population pairwise Fst genetic distances between the 16 populations. The population pairwise Fst analysis was conducted using allele frequencies from the male and female combined samples. A three-dimensional Non-Metric Multidimensional Scaling (3D-NMDS) was performed using R software statistical package 3.6.2 (R Development Core Team 2006).
To assess the forensic efficiency of each locus, mean exclusion chance (MEC) for trios consisting of a mother, a daughter, and a putative father (Kishida's formula) (MECT) (Kishida 1997), MEC for duos consisting of a daughter and a putative father (Desmarais' formula) (MECD) (Desmarais 1998) and power of discrimination for males (PDM) and for females (PDF) were performed using the online application at the Forensic ChrX Research web page (http://www.chrx-str.org) (Szibor 2006).

Genetic portrait of the Tunisian populations from Sousse and Makthar
Allele frequencies, HWE and GD for the populations of Sousse and Makthar are displayed in Supplementary Tables S2a and S2b, respectively. The allele distribution of X-STR loci between the male and female subsamples inside each population showed no significant differences when examined with the exact test after Bonferroni correction for multiple testing. Consequently, female and male subsamples were combined, and allele frequencies were calculated for the 17 X-STR markers for Sousse and Makthar. A total of 120 and 129 alleles were observed among the 17 loci in Sousse and Makthar, respectively. The number of alleles per loci for both populations is between 5 and 10 for the 17 markers.
For the two studied populations, the DXS6809 marker exhibits the highest variability with 10 alleles and GD values of 0.8397 and 0.8388 in Sousse and Makthar, respectively. As for Nabeul, a previously studied Tunisian population (Messoussi et al. 2019), DXS6809 is the most informative STR marker, both in Sousse and Makthar population samples. Conversely, DXS6807 was the least polymorphic loci with seven and five alleles and a GD value of 0.6091 and 0.6265 for Sousse and Makthar, respectively. In the population of Makthar, a variant allele of DXS6803 such as allele 10.2 was detected in one individual.
The pairwise p values of the linkage disequilibrium tests of the two populations are shown in Supplementary Table  S3. No detectable evidence of LD was found between loci after Bonferroni correction (p > .0004) in Sousse and Makthar populations. Nonetheless, the cluster DXS10075-DXS1079-DXS7132 was considered as a haplotype according to a previous study, where an extensive linkage disequilibrium analysis was performed for these markers (Prieto-Fern andez et al. 2016b). Haplotype frequencies of this cluster are shown in Supplementary Table S4.

Parameters of forensic interest
Along with the allele frequency and GD, statistical parameter values of forensic interest (PD M , PD F , MEC T and MEC D ), which are useful in kinship tests, are provided in Supplementary Table S2a for the population of Sousse and Table S2b for  For the population of Sousse, the PD ranges from 0.6058 to 0.8353 for males and from 0.7991 to 0.9537 for females. For the population of Makthar, the PD ranges from 0.6240 to 0.8345 for males and from 0.8138 to 0.9519 for females. In both populations, the marker with the highest power of discrimination is DXS6809. Therefore, these results indicate that DXS6809 is highly discriminant as was previously found in the Nabeul population (Messoussi et al. 2019).

Population comparison
Pairwise genetic distances (Fst) were calculated among the 2 studied populations (Sousse and Makthar) and 14 other reference populations from the literature: 2 North African populations (Nabeul and a Moroccan population from Casablanca), a sub-Saharan population (Guinea and Malawi), and 11 Western European populations (Prieto-Fern andez et al. 2016a, 2016b which were previously analysed with the same 17 X-STR panel (Supplementary Table S5). We found that the two populations studied here are significantly different from each other with p values of .05. Subsequent to the application of the Bonferroni correction (p ¼ 0.05/ 121 ¼ .0004), pairwise Fst genetic distance between the two Tunisian populations became statistically insignificant (p ¼ .0121).
After applying the Bonferroni adjustment, pairwise Fst genetic analysis indicates statistically significant differences between the Sousse and the populations of Guinea and Malawi, Barcelona and Brittany (France), whereas the Makthar showed differences with the populations of Guinea and Malawi, autochthonous Basques, and Brittany (France).
The obtained Fst genetic distances among the investigated populations are illustrated in the 3 D-NMDS plot in Figure 2. In the 3D-NMDS projection, the two Tunisian populations cluster tightly with other North African populations, namely Tunisians from Nabeul and Morocco (Casablanca). In addition, the plots reveal a clear differentiation of the North African populations from the rest of the Western European and Sub-Saharan African populations.

Discussion
Occupying a strategic geographical position in the Mediterranean, Tunisia has always been the centre of maritime and commercial activities, which participated in its prosperity and attracted a lot of populations. Thus, it was conquered several times by different cultures (such as the Phoenicians, Romans, Arabs and the French, and many more) during different periods of time. The numerous interactions between these different populations and the Berbers which are the native inhabitants of the Tunisian territory have caused the diversification of its population.
As a consequence, researchers are interested in analysing the Tunisian genetic background. Several studies were conducted to fulfil this purpose but only a few of them targeted chromosome X. Contrary to these studies, our study focuses on the analysis of 17 X-STRs of a coastal Tunisian city: Sousse, and the city of Makthar, which is located in the centre of the country.
The GD analysis showed that the DXS6809 marker is the highest variable marker, which is in agreement with the literature (Edelmann et al. 2003), where it was proven that DXS6809 is a highly informative marker in a number of populations. On the other hand, the DXS6807 marker is the least variable among all the tested markers.
Afterward, a linkage disequilibrium (LD) test was conducted on the Sousse and Makthar populations. The LD refers to the non-random association of alleles at different loci and assesses any deviation of population-specific haplotype frequencies. The LD pattern in a population is generally shaped by selection, mutation rate, recombination rate, consanguinity, genetic drift, and other factors (Chakravarti 1999).
The outcome of the LD test showed there are no significant differences between the two populations.
The pairwise genetic distances (Fst) analysis exerted on our samples alongside other Mediterranean populations has exhibited the existence of a significant distinction between the Sousse and the populations of Guinea and Malawi and Brittany (France). On the other hand, the population of Makthar showed differences with the populations of Guinea and Malawi, Barcelona, and Brittany.
The NMDS presentation of the populations illustrates the two Tunisian populations clustering tightly with the other North African populations: Tunisian (Nabeul) and Moroccan (Casablanca). On the contrary, the plots illustrate the differentiation of the North African cluster of populations from the Western European and Sub-Saharan African populations.
Our results are in accordance with those of the study based on 17 X-STR for the Tunisian population from Nabeul. In fact, the lack of differentiation between both Tunisian populations analysed in this study and the North African population of Morocco (Casablanca) and certain Western European populations was also observed when the Tunisian population from Nabeul city was examined (Messoussi et al. 2019).
These results indicate that the genetic relationships among the 16 populations are related to their geographic distribution. Our results agree with prior analyses of uniparental Y-chromosome markers, which were conducted on the population of Sousse by Fadhlaoui-Zid et al. (2015). This analysis indicates a distinctive North-African cluster that separates from the rest of the Sub-Saharan Africa and Western Europe. Therefore, the X-STR results provided here have proved to be complementary to other studies, eg based on the Y-chromosome, on these populations. Furthermore, our findings are in accordance with the autosomal analysis of the populations of Sousse and Makthar published by Al- Haj-Taib et al. (2021), that has proven the differentiation of North-African cluster from different European populations.
The difference shown between the North-African and the European clusters proves the genetic differences among them and common genetic features between the different North African populations. This differentiation may be the consequence of the different historical background of North-African populations from European populations.