Strong intra- and inter-continental differentiation revealed by Y chromosome SNPs M269, U106 and U15

http://secher.bernard.free.fr/Articles/R1b_Cruciani.pdf
Fulvio Cruciani^a, Beniamino Trombetta ^a, Cheyenne Antonelli^a, Roberto Pascone ^b, Guido Valesini^c, Valentina Scalzi^c, Giuseppe Vona

^d, Bela Melegh^e, Boris Zagradisnik^f, Guenter Assum^g, Georgi D. Efremov^h, Daniele Sellittoⁱ, Rosaria Scozzari^a'

^a Dipartimento di Genetica e Biologia Molecolare, Sapienza Universita di Roma, Rome, Italy

^b Dipartimento di Pediatria, Sapienza Universita di Roma, Rome, Italy

^c Dipartimento di Clinica e Terapia Medica, Sapienza Universita di Roma, Rome, Italy

^d Dipartimento di Biologia Sperimentale, Universita di Cagliari, Cagliari, Italy

^e Department of Medical Genetics, University of Pecs, Pecs, Hungary

^f Laboratory of Medical Genetics, University Medical Centre Maribor, Maribor, Slovenia

^g Institut fur Humangenetik, Universitat Ulm, Ulm, Germany

^h Research Center for Genetic Engineering and Biotechnology, Macedonian Academy of Sciences and Arts, Skopje, The Former Yugoslav Republic of Macedonia ⁱ Istituto di Biologia e Patologia Molecolari, Consiglio Nazionale delle Ricerche, Rome, Italy

ARTICLE INFO

ABSTRACT

Article history:

Received 3 May 2010

Received in revised form 23 July 2010

Accepted 28 July 2010

Available online xxx

Keywords:

Y chromosome SNPs R1b haplogroup Phylogeography European populations

More than 2700 unrelated individuals from Europe, northern Africa and western Asia were analyzed for the marker M269, which defines the Y chromosome haplogroup R1b1b2. A total of 593 subjects belonging to this haplogroup were identified and further analyzed for two SNPs, U106 and U152, which define haplogroups R1b1b2g and R1b1b2h, respectively. These haplogroups showed quite different frequency distribution patterns within Europe, with frequency peaks in northern Europe (R1b1b2g) and northern Italy/France (R1b1b2h).

© 2010 Published by Elsevier Ireland Ltd.

1. Population

The populatio
n sample consisted of 2789 unrelated males from Europe (N = 2049; 27 populations), northern Africa (N =528; 5 populations) and western Asia (N =212; 5 populations). All samples were collected from volunteer donors with informed consent. The geographic location of the populations analyzed is shown in Fig. 1 (panel E) and terrestrial coordinates given in Table S1. The great majority of the DNA samples were previously analyzed as indicated in Table S1.

This study was approved by the ''Policlinico Umberto I, Sapienza Universita di Roma'' Ethical Committee.

2. DNA extraction

DNA was extracted according to the manufacturer's protocols either from peripheral blood using the QIAamp DNA blood Mini Kit (Qiagen, Hilden, Germany) or from saliva using the Oragene»DNA sample collection kit (DNA Genotek Inc., Kanata, Ottawa).

3. PCR

Markers M269 [1], U106 [2] and U152 [2] were amplified separately using the previously described primers [1,2] and a touch-down PCR protocol (annealing temperature decreasing from 63 °Cto56 °C in 14 cycles, and then 30 cycles at 56 °C).

4. Typing

M269, U106 and U152 PCR products were digested with the appropriate restriction enzymes (BssKI, NcoI and CviKI-1, respec­tively) following the manufacturer's protocols (New England Biolabs, Ispwich, MA). Restriction fragments were run on a low

Please cite this article in press as: F. Cruciani, et al., Strong intra- and inter-continental differentiation revealed by Y chromosome SNPs M269, U106 and U152, Forensic Sci. Int. Genet. (2010), doi:10.1016/j.fsigen.2010.07.006

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[1]Corresponding author at: Dipartimento di Genetica e Biologia Molecolare, Sapienza Universita di Roma, P.le Aldo Moro 5, 00185 Rome, Italy. Tel.: +39 06 49912826/49912924; fax: +39 06 4456866. E-mail address: rosaria.scozzari@uniroma1.it (R. Scozzari).

1872-4973/$ - see front matter © 2010 Published by Elsevier Ireland Ltd. doi:10.1016/j.fsigen.2010.07.006

G Model

Fig. 1. Frequency (%) maps of the haplogroup R1b1b2 (A), paragroup R1b1b2* (B), haplogroup R1b1b2g(C), and R1b1b2h(D). Locations of the sampled populations are shown in (E) as red circles, with areas proportional to the square root of the sample size. Numbers refer to populations in Table 1; frequency data from Austria reported in Niederstatter et al. [23] was also considered (location number 39).

range ultra agarose gel (Bio-Rad Laboratories, Hercules, CA) together with appropriate size markers and visualized by ethidium bromide. Sequenced alleles were used as controls of the length of allelic DNA fragments. ISFG recommendations on the analysis of DNA polymorphisms have been followed.

5. Data analysis

Haplogroup/paragroup frequencies were estimated by direct counting. An exact test of differentiation between all pairs of populations, based on the frequency distribution of Y*(xR1b1b2),

Please cite this article in press as: F. Cruciani, et al., Strong intra- and inter-continental differentiation revealed by Y chromosome SNPs M269, U106 and U152, Forensic Sci. Int. Genet. (2010), doi:10.1016/j.fsigen.2010.07.006

FSIGEN-620; No. of Pages 4

F. Cruciani et al. /Forensic Science International: Genetics xxx (2010) xxx-xxx

R1b1b2*(xR1b1b2g, R1b1b2h), R1b1b2g, and R1b1b2h, was performed using the Arlequin 3.11 software [3]. Haplogroup/ paragroup frequency maps were depicted on a grid of 44 x 60 lines using the Kriging procedure [4] through the use of the Surfer program (Golden Software Inc., Golden, CO). Haplogroup expan­sion time estimates were obtained by using the same tetranucleo-tide repeats as reported by Cruciani et al. [5,6] and applying the average square distance (ASD) method [7]. The ancestral haplotype was assumed to be the haplotype carrying the most frequent allele at each microsatellite locus. Confidence intervals (CIs) were calculated according to Cruciani et al. [5]. An evolutionary effective microsatellite mutation rate of 1.2 x 10~³ was used by considering the following. After calibrating for the specific microsatellites used in this study, a germ-line microsatellite mutation rate of 2.8 x 10~³ (instead of 2.0 x 10~³ [8]) was obtained. Then, following Zhivo-tovsky et al. [9] and considering a scenario of a constant rate of population growth (m = 1.01), this value was reduced by a factor 2.4. Estimates for haplogroups R-U106 and R-U152 were obtained by the analysis of 24 and 27 Y chromosomes respectively.

6. Results

Haplogroup/paragroup frequency distributions are shown in Fig. 1 and Table 1.

The results of the pair-wise population differentiation test are shown in Table S2.

7. Other remarks

SNPs are the most abundant class of human polymorphisms and have a number of distinguishing features which are useful in forensics. They can be amplified in short amplicons, they are amenable to large scale automation and are characterized by low mutation rates when compared to the routinely used short tandem repeats (STR) [10,11]. Recently, the analysis of SNPs from the human Ychromosome was introduced for special forensic applications [12], and Y haplogroup frequency data was integrated in the Y-STR haplotype reference database (YHRD, http://www.yhrd.org) [13,14].

The human Y chromosome haplogroup R1b1b2 (R-M269) displays an extremely wide geographic distribution within Europe, with a decreasing frequency cline from western Europe (frequencies up to 92%) towards the Balkans (usually <20%) [15,16].This haplogroup has also been detected at frequencies below 15% in western Asia [17,18] and northern Africa [6,19]. Previous studies had suggested that the observed R1b1b2 frequency cline in Europe is due to a population expansion from an Iberian Ice-Age refugium after the LGM [20,21 ]. In contrast, Balaresque et al. [16] recently argued that

Table 1

One R1b1b2 Y chromosome, previously found in sub-Saharan Africa [6], was typed for U106 and U152 in this study.

Please cite this article in press as: F. Cruciani, et al., Strong intra- and inter-continental differentiation revealed by Y chromosome SNPs M269, U106 and U152, Forensic Sci. Int. Genet. (2010), doi:10.1016/j.fsigen.2010.07.006

the geographical distribution of R1b1b2 microsatellite diversity is best explained by spread from the Near East during the Neolithic.

In the present study, we explore the phylogeography of the human Y-chromosomal haplogroup R1b1b2 by analyzing more than 2700 males from Europe, northern Africa and western Asia. The haplogroup-defining marker M269, and two additional markers (U106 and U152) which identify internal branches (R1b1b2g and R1b1b2h, respectively) were analyzed. Only four R1b1b2 chromo­somes were observed in northern Africa (average frequency 0.8%, range 0.0-1.2%), all belonging to the paragroup R1b1b2*.

This paragroup was also the most represented group of M269 chromo­somes in western Asia. Within Europe, a strong inter-population differentiation emerged, with 72% (253 out of 351) of the pair-wise population comparisons showing significantly different haplogroup frequencies (p < 0.05, Table S2). The haplogroups R1b1b2g and R1b1b2h showed quite different frequency distribution patterns, with frequency peaks in northern/central Europe and northern Italy/ France, respectively (Fig. 1).

This conforms to previous studies [22,23] which reported a high frequency of the haplogroup R1b1b2g in northern and central Europe (Austria 18.5-22.7%, Denmark 17.7%, England 21.7%, Germany 20.5%, Netherlands 37.2%).