Short Report

Genetic Profiling of the Azores Islands (Portugal): Data from10 X-Chromosome STRS

FRANCISCA SILVA,1,2 RUI PEREIRA,3 LEONOR GUSMAO,3 CRISTINA SANTOS,4 ANTONIO AMORIM,3,5 MARIA JOAO PRATA,3,5

CONCEICAO BETTENCOURT,1,2 PAULA LOURENCO,1,2 AND MANUELA LIMA1,2*1Center for Research in Natural Resources (CIRN) and Department of Biology, University of the Azores, 9501-855, Ponta Delgada, Portugal2IBMC—Institute for Molecular and Cell Biology, University of Porto, 4150-180 Porto, Portugal3IPATIMUP, University of Porto, 4200-465, Porto, Portugal4Unitat Antropologia Biologica, Dep. Biologia Animal, Biologia Vegetal i Ecologia, Facultat Biociencies, Universitat Autonoma de Barcelona,08193 Cerdanyola del Valles, Barcelona, Spain5Faculty of Sciences, University of Porto, 4099-002, Porto, Portugal

ABSTRACT The populations from the Azores islands have been the target of several genetic studies, using dataderived from monoparental and recombining genetic systems. These studies have provided a complex picture of thegenetic landscape of the three groups of Azorean islands, and further data are required to assess its genetic profile. Wepresent a study of the polymorphism in 10 X-chromosome STR loci (DSXS8378, DXS9898, DXS7133, GATA31E08,GATA172D05, DXS7423, DXS6809, DXS7132, DXS9902, DXS6789) conducted on a total of 304 chromosomes (97females and 110 males) of unrelated individuals with Azorean ancestry. Average gene diversity was 74.47%, rangingfrom 66.21% (DXS7133) to 81.19% (GATA172D05). No shared haplotypes were found. Genotype frequencies amongfemales displayed conformity with Hardy-Weinberg expectations for all loci. Pairwise linkage disequilibrium tests didnot reveal evidences of association between the studied markers. Significant differences in allelic frequencies betweenthe Western and the Eastern group of islands are in agreement with previous results from mitochondrial DNA and Ychromosome studies, providing further evidence that the Azores cannot be considered an homogeneous population.Moreover, differences between the Western group and the North of Portugal are also reported, supporting the perti-nence of a specific database for the Azores populations, on what concerns the genetic markers analyzed. Am. J. Hum.Biol. 22:221–223, 2010. ' 2009 Wiley-Liss, Inc.

The Azores archipelago (Portugal), with a total area of2.344 Km2, is formed by nine volcanic islands, clustered inthree geographical groups (Eastern, Central, and West-ern). The archipelago is located in the Atlantic Ocean,1,500 Km from mainland Portugal and has presently atotal population of 237,315 inhabitants, distributed in avery asymmetric way among islands (INE, 2001). Theislands were uninhabited when discovered by Portuguesenavigators in the early 15th century; the settlement pro-cess was initiated in 1439 with individuals from variousregions of mainland Portugal and from Madeira island.Moreover, Spanish, French, Italian, English, German, andFlemish, as well as African and Moorish slaves also con-tributed to the peopling of the archipelago (Mendonca,1996). The populations from the Azores islands have beenthe target of several genetic studies, using data derivedfrom monoparental and recombining genetic systems (Bet-tencourt et al., 2006; Branco et al., 2006, 2008a,b,c; Fer-nando et al., 2005; Montiel et al., 2005; Neto et al., 2007;Santos et al., 2003). These studies have provided noncon-sensual interpretations concerning the degree of homoge-neity of the global Azorean population, prompting thestudy of additional genetic systems. The recent interest,both from a forensic as well as a population geneticsstandpoint, in the information derived from the X chromo-some (see, among others, Aler et al., 2007; Asamura et al.,2006; Catanesi et al., 2007; Hou et al., 2007; Tariq et al.,2008) is justified by the fact that it combines favorable fea-tures of both the autosomes (presence of recombination infemales) and of the monoparental systems (mitochondrialDNA and Y chromosome), such as the presence of a sex-biased pattern of inheritance and direct haplotyping in

males (Schaffner, 2004). The recent availability of X-chro-mosome STR data for Mainland Portugal (Gusmao et al.,2009; Pereira et al., 2007) provides the adequate frame-work against which allelic frequencies obtained for theAzores could be tested. In this work we analyzed 10 STRsof the X-chromosome, aiming to contribute to the geneticprofiling of the Azores islands.

MATERIALS AND METHODS

Population samples

Blood samples and buccal swabs were collected from207 unrelated individuals (97 females and 110 males)from the Azores islands (117 chromosomes from the East-ern, 140 from the Central, and 47 from Western group).All subjects signed an informed consent form and filled ina genealogical questionnaire, which confirmed their Azor-ean ancestry until the third generation. Samples were dis-tributed considering the island of origin of the parents of

Contract Grant sponsor: Construindo uma bio-regiao Europeia- Biopolis(European Region-PIC Interreg III B, Azores—Madeira—Canarias); Con-tract grant number: 05/MAC/2.3/C14; Contract Grant sponsor: IPATIMUP(Programa Operacional Ciencia e Inovacao); Contract grant number: POCI2010; Contract Grant sponsor: Fundacao para a Ciencia e a Tecnologia;Contract grant numbers: SFRH/BD/21875/2005, SFRH/BD/30039/2006;Contract Grant sponsor: Direccao Regional da Ciencia e da Tecnologia(DRCT); Contract grant number: M311/I/015/2005.

*Correspondence to: Manuela Lima, Department of Biology, Universityof the Azores, 9501-855, Ponta Delgada, Portugal. E-mail: mlima@uac.pt

Received 2 February 2009; Revision received 2 June 2009; Accepted 3June 2009

DOI 10.1002/ajhb.20971

Published online 10 July 2009 in Wiley InterScience (www.interscience.wiley.com).

AMERICAN JOURNAL OF HUMAN BIOLOGY 22:221–223 (2010)

VVC 2009 Wiley-Liss, Inc.

the individuals sampled. Genomic DNA was extractedusing the JETQUICK blood and cell DNA purification kit(Genomed), according to manufacturer’s specifications.

Genotyping

Ten X-chromosome STR loci (DXS8378, DXS9898,DXS7133, GATA31E08, GATA172D05, DXS7423,DXS6809, DXS7132, DXS9902, DXS6789) were coampli-fied following the methodology described in detail byGusmao et al. (2009).

Statistical analysis

Haplotype and gene diversities were calculated usingArlequin 3.1.1 (Excoffier et al., 2005). Allele frequencies,as well as conformity with the Hardy-Weinberg equilib-rium (HWE) expectations, this last tested in the femalessubsample (n 5 97), were determined with Genepop 3.4(Raymond and Rousset, 1995). Pairwise tests of linkagedisequilibrium were determined with Arlequin 3.1.1(Excoffier et al., 2005). Exact tests of population differen-tiation were applied to compare the allelic frequenciesbetween groups of islands, using Genepop 3.4. (Raymondand Rousset, 1995) Furthermore, population differentia-tion was tested using the pairwise genetic distance FST

and analysis of molecular variance (AMOVA), conductedfor the total sample (304 chromosomes), after inferringthe females haplotype, using the ELB algorithm (Excoffieret al., 2005). Data available in Gusmao et al. (2009) forNorth and Centre Portugal were used in comparisonswith data produced in this study, using Arlequin 3.1.1.(Excoffier et al., 2005).

TABLE 1. Allelic frequencies for the 10 STRs analyzed, in the threegeographic groups of the Azores (OR- Eastern, CE- Central, OC-

Western); GD-average gene diversity

OR CE OC

DXS83789 0.009 0.007 0.021

10 0.256 0.379 0.29811 0.308 0.314 0.40412 0.376 0.264 0.21313 0.051 0.029 0.06414 – 0.007 –GD-0.702

DXS71339 0.402 0.421 0.532

10 0.171 0.171 0.12811 0.385 0.350 0.29812 0.026 0.029 0.04313 0.017 0.021 –14 – 0.007 –GD-0.662

GATA172D056 0.188 0.214 0.2138 0.145 0.136 0.2139 0.051 0.114 0.021

10 0.316 0.243 0.19111 0.197 0.193 0.23412 0.103 0.100 0.128GD-0.811

DXS680927 0.009 0.007 –28 0.009 0.007 0.04329 0.009 0.007 –30 0.034 0.014 –31 0.188 0.143 0.10632 0.128 0.179 0.21333 0.333 0.271 0.27734 0.179 0.221 0.21335 0.085 0.121 0.08536 0.026 0.021 0.06437 – 0.007 –GD-0.810

DXS71329 – 0.007 –

11 0.017 0.021 –12 0.111 0.086 0.02113 0.368 0.271 0.14914 0.316 0.364 0.53215 0.154 0.179 0.23416 0.034 0.036 0.06417 – 0.021 –18 – 0.014 –GD-0.739

DXS99029 0.026 – –10 0.034 0.021 –11 0.265 0.407 0.29812 0.316 0.271 0.46812.1 0.060 0.021 –13 0.291 0.243 0.21313.1 – 0.014 0.02114 0.009 0.014 –14.1 – 0.007 –GD-0.720

DXS678914 0.009 0.007 –15 0.077 0.029 0.02116 0.009 0.043 –17 – 0.007 –19 0.017 – –20 0.436 0.457 0.44721 0.205 0.221 0.27722 0.188 0.129 0.17023 0.051 0.107 0.06424 0.009 – 0.021GD-0.718

TABLE 1. (continued)

OR CE OC

DXS98987 – 0.007 –8.3 0.256 0.207 0.44710 0.009 0.014 –11 0.162 0.207 0.14912 0.316 0.279 0.23413 0.205 0.214 0.10613.3 – 0.007 –14 0.034 0.057 –15 0.017 0.007 0.064GD-0.778

GATA31E089 0.162 0.129 0.170

10 – 0.036 –11 0.154 0.150 0.06412 0.197 0.286 0.27713 0.274 0.264 0.23414 0.197 0.121 0.23415 0.017 0.014 0.021GD-0.801

DXS742313 0.034 0.100 0.23414 0.385 0.329 0.25515 0.436 0.386 0.29816 0.128 0.171 0.17017 0.017 0.014 0.043GD-0.700

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RESULTS AND DISCUSSION

The simultaneous analysis of the 10 X-STRs in malesrevealed the absence of shared haplotypes. Male andfemale samples were pooled to calculate allelic frequencies(Table 1), since the exact test of differentiation, performedfor the 10 loci, failed to reveal significant differencesbetween them. Gene diversity was calculated for each ofthe 10 markers (Table 1). Average diversity was of 0.7447,a value within the range of reports for mainland Portugal(Gusmao et al., 2009; Pereira et al., 2007).

In the female’s subgroup, all loci were in conformitywith Hardy-Weinberg equilibrium expectations, after theBonferroni correction for multiple tests.

Linkage disequilibrium tests for all pair of loci, inmales, did not reveal evidence of association between thestudied markers, after the Bonferroni correction. Thislack of LD suggests absence of significant associations dueto genetic substructure, being in accordance with the dis-tances between the selected X chromosome loci (>5 Mb),except for DXS6809 and DXS6789 (Gusmao et al., 2009).AMOVA results considering the three groups of islandsdisplayed significant P values for markers DXS9898 (P 50.04790) and DXS7132 (P 5 0.00684). The weighted aver-age over the set of 10 STRs analyzed, produced a signifi-cant P value (FST 5 0.00497; P 5 0.01369).

The exact test of population differentiation (combininginformation from the 10 X-STRs) revealed significant dif-ferences between the Western and the Eastern groups (P5 0.00306), after the Bonferroni correction. A locus bylocus analysis showed that differences found concernedmarkers DXS7423 (P 5 0.00085) and DXS7132 (P 50.00649).

Allelic frequencies in each group of islands were com-pared with available data for Mainland Portugal (Northand Centre) (Gusmao et al., 2009). Significant genetic dif-ferentiation (after corrections for multiple analyses) wasdetected between the subsample of the Western group andthe sample from North Portugal.

CONCLUDING REMARKS

Previous results on mtDNA (Santos et al., 2003)and Y chromosome (Montiel et al., 2005) had denotedthe presence of a distinct haplogroup distribution inthe Western group of Azorean islands, leading to theidea that, from the nonrecombining genomes perspec-tive, the Azores cannot be considered as an unstruc-tured population, since it displays not only inter butprobably also intra-group heterogeneity. Resultsreported in this article indicate that differences arealso present considering the X chromosome STRs usedin this work. Thus, both adequate data sets, as wellas information derived from several genetic systemsare necessary to fully ascertain the variability of theAzorean gene pool. Genetic differentiation betweenthe Western group and the Eastern group or North ofPortugal obtained with the markers used supports theneed of specific forensic databases for different groups

of islands of the Azores archipelago on what thesemarkers are concerned.

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