Genetic Characterization of the Arabic-Speaking Population from the Casablanca-Settat Region Using Autosomal STR Markers: Understanding the Interplay of Geography and Language in Moroccan Population History

Abstract

Background/Objectives: The Casablanca-Settat region of Morocco, located at the interface between Arab and Amazigh cultural zones, has only recently been investigated using autosomal short tandem repeat (STR) markers. The objective of this study was to characterize the genetic diversity and forensic efficiency of 15 autosomal STR loci in the Casablanca-Settat population and to evaluate its genetic relationships with other Moroccan populations. Methods: Fifteen autosomal STR loci were genotyped in 138 unrelated Arabic-speaking individuals from the Casablanca-Settat region. Allele frequencies, Hardy–Weinberg equilibrium, and standard forensic parameters were calculated. The genetic structure of the population was further examined through comparative analyses with 12 previously published Moroccan reference populations using multivariate and phylogenetic approaches. Results: A total of 146 distinct alleles were identified across the 15 loci. D18S51 was the most polymorphic marker (Ho = 0.9203), whereas D3S1358, TPOX, D5S818, and D16S539 exhibited lower allelic diversity. No statistically significant deviation from Hardy–Weinberg equilibrium was detected after correction for multiple testing. The combined power of discrimination exceeded 0.99, and the combined power of exclusion reached 0.99999965, demonstrating the high forensic efficiency of the STR panel. Population structure analyses positioned the Casablanca-Settat population within an intermediate genetic cluster, closely related to central Moroccan populations, consistent with historical gene flow and admixture. Conclusions: This study provides robust autosomal STR reference data for the Casablanca-Settat population, confirming the suitability of these markers for forensic identification in Morocco and offering valuable insights into regional population structure and genetic diversity.

1. Introduction

Genetic admixture among human populations has long been a subject of interest in anthropology, particularly in the context of the founder effect and genetic drift theories [1,2]. To assess genetic heterogeneity within and between populations [3,4], a range of molecular tools and statistical approaches are employed to better understand the underlying genetic structure and affinities among human groups. It is well established that geographically proximate populations tend to share greater genetic similarity, whereas this similarity typically decreases with increasing geographic distance [1,5].

Advancements in molecular biology, especially the use of highly polymorphic markers such as Short Tandem Repeats (STRs), have revolutionized the direct analysis of gene pools and gene flow among populations [6,7]. STR markers, due to their high mutation rates and codominant inheritance, are widely used in forensic investigations, kinship analysis, and population genetics [8,9]. Their utility in capturing both recent demographic events and deeper ancestral lineages makes them powerful tools for dissecting the complex genetic history of human populations [10].

This study focuses on the Casablanca-Settat region of Morocco, a geographically and ethnically diverse area with a rich history of migration and gene flow [11]. Located in the Atlantic coastal plain, this region has been shaped by multiple historical layers, including Arab, Amazigh (Berber), Sub-Saharan African, and European influences [12,13]. These historical migrations and socio-cultural interactions have blurred genetic boundaries between ethnic groups, making social and cultural factors often more distinguishing than biological differences.

Despite this complexity, certain practices such as endogamy and consanguinity have played a role in preserving local genetic signatures [11,14]. Recent studies have highlighted how these matrimonial behaviors influence genetic structure by increasing homozygosity and conserving lineage-specific alleles [14,15].

In this context, we present the first comprehensive autosomal STR dataset for an Arabic-speaking population from the Casablanca-Settat region. This study represents an ideal case to explore the relationship between linguistic affiliation (Arabophone) and genetic diversity, and to determine whether language correlates with distinct genetic signatures or whether genetic diversity transcends linguistic boundaries.

This study aims to investigate the genetic diversity of an Arabic-speaking population from the Casablanca-Settat region through the analysis of autosomal STR markers. We analyze allele distributions to understand the population’s genetic structure, historical admixture, and to test the usefulness of STRs for forensic and population studies in Morocco. This research helps expand knowledge of North African genetic diversity and supports the creation of regional forensic databases.

2. Material and Methods

2.1. Population Samples

Saliva samples were collected from 146 healthy, unrelated Arabophone individuals whose four grandparents originated from the localities in the region of Casablanca–Settat. All procedures were carried out in strict accordance with ethical standards, and written informed consent was obtained from all participants. This study included individuals from seven locations. El Jadida, Sidi Bennour, Zemmamra, Laounate, Hed Oulad Fraj, Sebt Saiss, and Beni Hellal. At the collection stage, 146 samples were obtained. After quality control, eight samples were excluded due to insufficient DNA quality or incomplete profiles, leaving 138 individuals for statistical analysis.

2.2. Ethics Statement

The study was conducted in accordance with the guidelines of the Biomedical Research Ethics Committee (CERBC) of Casablanca, Morocco, and complied with the principles of the Declaration of Helsinki (2008 version). Ethical approval was granted on 22 April 2022 (registration number N°6/19). Written informed consent was secured from all participants prior to sample collection.

2.3. DNA Extraction and STR Amplification

Genomic DNA was extracted from the collected buccal swabs using the manual phenol-chloroform-isoamyl alcohol method. The extracted DNA was quantified using NanoDrop™ 2000/2000c spectrophotometers. A DNA concentration of 1 ng was used for the amplification of 15 STR loci and the Amelogenin marker included in the Investigator IDplex Plus Kit (Qiagen, Hilden, Germany), covering the following loci: D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA.

Samples were amplified in multiplex using fluorescently labeled primers. The PCR products were then analyzed using the 3500 Genetic Analyzer System (Applied Biosystems, Foster City, CA, USA), with POP-4™ polymer for the 3500/SeqStudio™ Flex and the 3500 Genetic Analyzer 8-Capillary Array, 36 cm (Applied Biosystems, Foster City, CA, USA). The SST 550 BTO size standard (Qiagen) was used to determine the fragment sizes. Allelic designations were obtained by comparison with the allelic ladder, and genotyping was performed using the GeneMapper IDX software (v1.6, Applied Biosystems).

2.4. Forensic Parameter Estimation

Allele frequencies and Hardy–Weinberg equilibrium (HWE) were evaluated using GENEPOP software v.3.3 [16]. HWE was assessed by an exact test with an initial significance threshold of p < 0.05, and Bonferroni correction was applied to adjust for multiple testing across the 15 STR loci, resulting in a corrected significance level of p < 0.0033. Observed heterozygosity (Ho), expected heterozygosity (He), power of discrimination (PD), matching probability (PM), polymorphic information content (PIC), and power of exclusion (PE) were calculated using CERVUS software v.2.0.

2.5. Inter-Population Analysis

In order to determine the genetic relationships of our sample with other populations, we compared our data with previously published STR databases from various Moroccan regions and two global populations (Table S1). Central Morocco was represented by datasets from the central and central-western regions [17], Chaouia-Ouardigha [18], west-central Morocco [19], and the combined regions of Doukkala and Jbala [20]. The Middle Atlas Mountains were represented by the Azrou population [21]. Southern Morocco included samples from the far south [22] and the Souss region [23]. Nationwide data covering various Moroccan regions were obtained from broader studies [24,25]. For external comparison, we included reference populations from Spain [26] and China [27].

Neighbor-Joining (NJ) phylogenetic trees were constructed using MEGAX software (v.12.0.1). Nei’s genetic distances among populations were pairwise estimated using the dist.genpop function in the R package adegenet v.2.1.11. The principal component analysis (PCoA) was performed using Python v.3.12.4 (skbio library). To display the genetic relationship between populations visually, a two-dimensional Principal Coordinate Analysis (PCoA) based on Nei’s distance matrix was performed. The eigenvalues were calculated to determine the proportion of total genetic variation captured by each axis, ensuring the reliability of the spatial representation. Subsequently, the scatter plots of Principal Coordinate 1 (PCo1) and Principal Coordinate 2 (PCo2) were generated, representing the most significant dimensions of the genetic variance.

3. Results

3.1. Allele Frequency Distribution and Forensic Parameters

To characterize the genetic structure of 138 individuals from the Casablanca-Settat region of Morocco, 15 autosomal STR loci were analyzed. Across the 15 STR loci, 146 alleles were identified with frequencies ranging from 0.0068 to 0.4658. Supplementary File S1 summarizes the allele frequency data for 15 autosomal STR loci collected from 138 unrelated individuals.

Table 1. Characteristics of the Moroccan and global populations included for comparative analysis.

Sample Size	Abbreviation	Code	Ethnicity and Language Speaking	Location	References
209	Moroccan Berbers EC	P6	Berber from Asni and Bouhria.	Central and Eastern Morocco	[28]
201	Moroccan Berbers Azrou	P1	Berber-speaking of Azrou.	Central Middle Atlas region of Morocco.	[21]
75	Moroccan Berbers MA	P11	Berber-speaking	Middle Atlas of Morocco	[17]
150	Moroccan Berbers South	P5	Berber-speaking from Souss	Southern Morocco	[23]
204	Moroccan Arabs South	P12	Arabic-speaking	Southern Morocco	[22]
80	Moroccan Arabs NWC	P13	Arabic-speaking	North-Central and West-Central of Morocco.	[20]
71	Moroccan Arabs WC 1	P10	Arabic-speaking from Abda, Chaouia, Doukkali, and Tabdla	Central-West Coast of Morocco	[17]
150	Moroccan Arabs WC 2	P14	Arabic-speaking of Chaouia.	Western Center of Morocco	[15]
153	Moroccan Arabs WC 3	P8	Arabic-speaking of Chaouia-Ouardigha.	Western center of Morocco	[18]
138	This Study WC 4	P9	Arabic-speaking	Centre-ouest du Maroc	This study
425	Moroccan Mixed 1	P3	Berbers, Arabs, and Sahraouis.	Various parts of Morocco	[25]
320	Moroccan Mixed 2	P2	Arabs, Berbers, and Sahrawi.	Various parts of Morocco	[24]
342	Spain Caucasian	P4	Caucasian in Spain.	Spain	[26]
2975	China Qinghai	P7	Chinese.	China (Qinghai province)	[27]

provides an overview of the main forensic parameters. The most polymorphic marker was D18S51 (Ho = 0.9203), while the least polymorphic loci were D3S1358 and TPOX, each with only seven distinct alleles. Observed heterozygosity values varied between 0.7029 (TPOX) and 0.9203 (D18S51), with a mean of approximately 0.80, confirming a high level of genetic diversity. No statistically significant deviation from Hardy–Weinberg equilibrium was detected after Bonferroni correction. A positive correlation between allele number and PIC was observed, supporting the informativeness of highly polymorphic loci.

The power of discrimination (PD) ranged from 0.8573 (TPOX) to 0.9679 (D18S51). Additionally, the power of exclusion (PE) varied from 0.5741 to 0.9935 (

Table 2. Forensic parameters of 15 STR loci in the Moroccan population sample (N = 138).

	TH01	D3S1358	vWA	D21S11	TPOX	D7S820	D19S433	D5S818	D2S1338	D16S539	CSF1PO	D13S317	FGA	D18S51	D8S1179
Ho	0.7391	0.7319	0.8188	0.8551	0.7029	0.7826	0.8623	0.7464	0.8116	0.7754	0.8043	0.7609	0.8478	0.9203	0.8478
He	0.7911	0.7634	0.8176	0.834	0.6884	0.7683	0.8072	0.7556	0.8297	0.7811	0.7434	0.756	0.8406	0.866	0.8174
PE	0.7715	0.7062	0.8462	0.9088	0.5741	0.7309	0.8371	0.6999	0.9053	0.7623	0.66	0.7045	0.9131	0.9935	0.8531
PD	0.9243	0.905	0.9418	0.9538	0.8573	0.9124	0.9389	0.9025	0.9537	0.9212	0.8892	0.9036	0.9547	0.9679	0.943
PIC	0.759	0.7243	0.7926	0.8154	0.6428	0.7344	0.7832	0.7178	0.8124	0.7502	0.6985	0.7191	0.8207	0.8519	0.7938
p_HWE	0.1799	0.3707	0.1213	0.9915	0.9652	0.2893	0.0525	0.0824	0.0441	0.8611	0.9088	0.1917	0.9998	0.2854	0.3009

Ho, observed heterozygosity; He, expected heterozygosity; PD, power of discrimination; PE, power of exclusion; PIC, polymorphic information content; p-HWE, p-value of the Hardy–Weinberg Equilibrium exact test.

). The combined power of exclusion (CPE) was 0.99999965, and the combined matching probability (CMP) was 3.85 × 10⁻¹⁸, confirming the robustness of this marker set for forensic applications. Overall, the combined power of discrimination (CPD) is virtually 100%, highlighting the efficiency of these markers for both forensic and population genetics studies in the Moroccan population.

3.2. Interpopulation Analysis

Population relationships were examined using pairwise Nei’s genetic distances (

Table 3. Pairwise Nei’s genetic distances among 14 Moroccan and global populations.

	P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11	P12	P13	P14
P1	0
P2	0.03438	0
P3	0.034561	0.000192	0
P4	0.029068	0.047355	0.047341	0
P5	0.010162	0.032986	0.033263	0.02799	0
P6	0.026936	0.037678	0.037209	0.048953	0.022288	0
P7	0.098683	0.124181	0.123161	0.08881	0.097979	0.109046	0
P8	0.022007	0.03189	0.032046	0.033993	0.016364	0.018806	0.096576	0
P9	0.024809	0.038801	0.038639	0.037827	0.020945	0.026719	0.088831	0.01837	0
P10	0.028381	0.036317	0.036366	0.036014	0.024453	0.032932	0.103868	0.024574	0.024721	0
P11	0.035689	0.05087	0.050296	0.044481	0.028174	0.028966	0.096757	0.030051	0.030372	0.033371	0
P12	0.013	0.029154	0.029186	0.022648	0.014553	0.025969	0.091007	0.019546	0.027499	0.031338	0.026851	0
P13	0.021419	0.040558	0.040378	0.039304	0.018546	0.034092	0.098043	0.030699	0.035055	0.0287	0.030905	0.019013	0
P14	0.014985	0.029672	0.029819	0.029591	0.012032	0.024137	0.09055	0.019592	0.020627	0.026551	0.028066	0.009559	0.024609	0

Numerical values represent the genetic divergence between each pair of populations. Population abbreviations and labels (P1–P14) are defined in Table 1. Genetic distances were estimated pairwise using the dist.genpop function in the R package adegenet (v.2.1.11). Lower values indicate higher genetic similarity, while higher values reflect greater genetic differentiation.

), which were graphically represented by means of Principal Coordinates Analysis (PCoA) (Figure 1) and a Neighbor-Joining (NJ) phylogenetic tree (Figure 2). These complementary approaches allowed us to situate the Arabic-speaking Casablanca-Settat population (P9) within its broader geographical, linguistic, and historical context.

The PCoA analysis (Figure 1) proved highly robust, with the first two coordinates accounting for 81.13% of the total genetic variance (70.80% explained by Coordinate 1 and 10.33% by Coordinate 2). This high cumulative percentage, represented by the calculated eigenvalues, ensures a faithful representation of the population structure. The study population (P9) occupied a central position within the Moroccan cluster, particularly showing the lowest genetic distances with Moroccan Arabs WC1 (0.0184) and WC3 (0.0206). Similarly, the phylogenetic tree (Figure 2) revealed that P9 clusters tightly with various Moroccan groups, including both Berber and Arab populations from different regions of the country. This close clustering reflects a strong genetic relatedness likely shaped by shared ancestry and geographic proximity. The Spanish population was positioned on a neighboring branch, suggesting a moderate level of genetic affinity, possibly reflecting historical gene flow across the Mediterranean. In contrast, the Chinese population formed a distinct and distant branch, clearly separated along Coordinate 1, highlighting the significant genetic divergence between the Moroccan groups and this external reference population.

4. Discussion

In the present study, the analyzed STR loci demonstrated high forensic efficiency, as reflected by their strong discriminatory power across all markers (Table 1). This observation aligns well with previous reports on other Moroccan populations, including Chaouia [15]. Central Morocco [18], and Souss [23]. In particular, the Souss population exhibited remarkable allelic diversity at the D18S51 locus, a pattern also observed in our studied population. Moreover, loci such as D19S433. FGA, and D18S51 consistently showed high heterozygosity values (>0.85), underscoring their pronounced polymorphism and reinforcing their relevance for both forensic identification and population genetic studies throughout Morocco.

From a forensic perspective, the high levels of polymorphism and heterozygosity observed across the analyzed STR loci highlight the importance of generating region-specific allele frequency data for Morocco. The present dataset provides a reliable reference for the Casablanca-Settat region, supporting accurate statistical calculations in individual identification, kinship analysis, and paternity testing. Given the demographic complexity and internal migration characterizing this region, the use of localized population data is essential to avoid biased likelihood estimates that may arise from extrapolating frequencies from geographically or genetically distant groups. In this context, our results represent a valuable contribution toward the development and refinement of a national Moroccan STR database, enhancing both the reliability and equity of forensic genetic applications.

The genetic structure observed in the Casablanca-Settat population (P9), reflects both its historical and geographical context. Its central position in multivariate analyses (Figure 2) indicates a genetically intermediate profile relative to other Moroccan groups. consistent with the region’s role as a contact zone between Arab and Amazigh (Berber) communities [29]. This pattern aligns with the heterogeneous genetic character of North African populations. shaped by long-term migrations and cultural interactions [30,31].

The Arabic-speaking Casablanca-Settat population (study population) occupies an intermediate position within the Moroccan cluster, showing close genetic affinities with both Arab- and Berber-speaking groups from different regions of the country.

Phylogenetic analysis demonstrates that the population from the Casablanca-Settat region occupies a pivotal, intermediate position within the Moroccan genetic cluster. It exhibits its strongest genetic affinity with Arabic-speaking groups from West-Central Morocco, specifically “Moroccan Arabs WC 3” (P8) and “Moroccan Arabs WC 2” (P14). This proximity is consistent with the geographical (Figure 3) and linguistic profile of P9, suggesting a shared genetic heritage among Arabic-speaking populations in the West-Central Atlantic plains. Minor branching patterns observed in the NJ tree likely reflect local demographic histories, such as endogamy, tribal structure, or regional isolation. rather than deep genetic divergence [11,32].

Interestingly, Berber-speaking populations from Azrou (Middle Atlas) and from southern Morocco cluster in close proximity to the Arabic-speaking Casablanca-Settat population, indicating a transitional or admixed genetic core linking Arabophone and Amazigh groups. This close genetic affinity suggests substantial shared ancestry and long-term admixture despite linguistic differentiation. reinforcing the notion of limited genetic discontinuity among Moroccan populations [13,14,33]. This finding supports the hypothesis that Arabization in Morocco was predominantly cultural and linguistic rather than genetic [17,18,23].

The clustering of Moroccan Mixed (P2. P3) populations, which include individuals with diverse Moroccan origins. likely represents an average Moroccan genetic background shaped by multiple ancestral contributions [13,32]. The close positioning of the study population (P9) to these groups further supports its intermediate genetic status within the Moroccan genetic landscape [17,18,23].

The Spanish reference population branches adjacent to the Moroccan cluster, indicating a moderate level of genetic affinity between populations from Morocco and the Iberian Peninsula. This proximity is consistent with documented historical contacts across the Strait of Gibraltar, including trans-Mediterranean migrations and exchanges during different historical periods during the Islamic presence in Al-Andalus (8th–15th centuries) [34]. Similar signatures have been documented in Y-chromosome studies [35,36]. In contrast, the Chinese population forms a clearly distinct and distant branch, serving as an external outgroup and confirming the robustness of the genetic distance estimates and the overall tree topology.

The observed proximity between the Arabic-speaking Casablanca-Settat group and the Berber-speaking groups from Azrou and Souss likely reflects Morocco’s long history of population mobility and interaction shaped by migrations, trade routes, and tribal confederations (Figure 1). This close genetic affinity supports the existence of extensive historical gene flow between regions traditionally inhabited by Arab and Berber communities. Moreover. the gradient observed from the Berber mountain groups (Azrou) to the Arab-dominant plains (Chaouia) reveals a clinal pattern of genetic variation [4,37]. a well-documented feature in North African genetic structure [10,14]. Such clines generally result from continuous gene flow across geographic and cultural boundaries rather than sharp population divisions. In this context. Morocco’s genetic landscape appears to be shaped by gradual transitions and admixture, reflecting complex demographic dynamics rather than discrete, isolated clusters.

While the use of STR markers provides a robust and validated framework for assessing genetic diversity and is the gold standard for forensic identification, this approach has certain inherent limitations when exploring complex evolutionary histories. The current study, based on a standard STR panel, primarily captures recent demographic events and overall population affinities. However, the moderate mutation rate and limited number of loci compared to genome-wide assays may constrain the ability to detect more ancient or subtle admixture events. Therefore, our results should be viewed as a foundational genetic characterization of the “ThisStudyWC4” (P9) population within the current Moroccan forensic framework.

To refine the resolution of these findings, future research would benefit from the integration of high-density SNP data or Whole-Genome Sequencing (WGS). These high-resolution markers will allow for more sophisticated analyses, such as calculating ancestral proportions and dating gene flow events with greater precision. Furthermore, the current analysis could be strengthened by including sub-Saharan African reference populations to better quantify the historical southward gene flow that has shaped North African genomes.

Finally, as this study focuses on autosomal variation, it does not distinguish between maternal and paternal demographic histories. The future inclusion of uniparental markers (mtDNA and Y-STRs) will be essential to identify potential sex-biased migration patterns and to reconstruct the lineage-specific contributions of Arab and Berber groups. Such multi-marker approaches will ultimately provide a more comprehensive picture of Moroccan population dynamics and further enhance the national forensic genetic databases.

5. Conclusions

This study refines our understanding of Morocco’s genetic landscape by characterizing an Arabic-speaking population from the Casablanca-Settat region. The findings demonstrate that a shared linguistic identity does not necessarily imply genetic homogeneity.

Overall, the data suggest a clinal gradient extending from the Middle Atlas through southern Morocco to the Atlantic plains, shaped by migration, admixture and sociopolitical history. Additionally, both trans-Mediterranean affinities (linking Morocco and Iberia) and east west differentiation underscore Morocco’s role as both a crossroads and a barrier between Europe and sub-Saharan Africa.

These findings highlight the complex interplay of language, geography, and history in shaping North African genetic diversity. Future genome-wide and uniparental marker studies, integrated with linguistic, archaeological and historical evidence, will be essential to fully elucidate the demographic history and microevolutionary processes underlying this mosaic ancestry.