Index Introduction Population Methods Samples Y-typing and data analysis Results and Discussion Quality control Acknowledgments References

Introduction

The non-recombining portion of the human Y-chromosome provides anthropologists and geneticists with an extremely powerful tool for historical and demographic studies (Cavalli-Sforza et al., 1994). The number of studies about Short Tandem Repeats on the Y chromosome (Y-STR) increases greatly every year, but to evaluate their efficiency in forensic and anthropological sciences, it is necessary to investigate a large number of different populations (Turrina et al., 2006). Y-STR markers can be used for paternity testing, male kinship analysis, for evolutionary studies, and for studies of human migration (Butler, 2003).

There is also considerable interest in determining the Y-chromosome haplogroup, a group or a family of Y-chromosomes related by descent. Determination of the Y-haplogroups by direct testing of single nucleotide polymorphisms (SNPs) can sometimes be an expensive process. Therefore, there is much interest in predicting the haplogroups from a set of Y-STR markers (Athey, 2005). We estimated frequency of the major Y-chromosome haplogroups from acquired Y-STR data by use of the web-accessible program, Whit Athey’s Haplogroup Predictor, which is based on a Bayesian-allele-frequency approach. The program computed the probability that a Y-STR haplotype was in a haplogroup (Athey, 2006). Identification of Romany Y-chromosome haplogroups yields the ancestry of the paternal line of the population being studies.

Population

Approximately 350,000–380,000 Romanies live in Slovakia according to the official statistical list. The highest concentration (54%) of the Romany population is located in eastern Slovakia (Vano, 2001). The Romany population of Slovakia represents a genetically isolated population with a high frequency of consanguinity and inbreeding, which is about 10–100 times higher than in the European population of the same region (Ferák et al., 1987). Several studies of various genetic markers in different Romany samples from Slovakia showed that their gene pool differs from the Slovak majority population and neighboring European populations (Nagy et al., 2006; Soták et al., 2008; Malyarchuk et al., 2008). The population differentiation analysis based on autosomal STR markers between Slovak Romanies and Europeans showed significant differences in spite of their geographical proximity (Soták et al., 2008). The Romany population is characterized by nomadism, a caste structure and its position in numerous countries as an underprivileged ethnic minority (Morar et al., 2004).

The first reference to Romany people in Slovakia is from the 14th century in the region of Spiš. The origin of the Romany population living in Slovakia has not yet been satisfactorily explained (Bernasovský and Bernasovská, 1999). Cultural anthropology, linguistics, and limited historical records from the surrounding majority population describe the ‘gypsies’ as a population of Indian origin, with their exodus from India dating approximately from the 5th–10th centuries AD, their arrival in Byzantium dating from the 11th or 12th centuries, and their dispersal throughout Europe documented at the end of the 15th century (Fraser, 1992; Marushiakova and Popov, 1997).

Methods

Samples

Genomic DNA of 200 healthy and unrelated Slovak Romany males was extracted from buccal swabs using the Jet Quick DNA tissue kit according to the manufacturer’s instructions (Genomed). Written informed consent was obtained from all participants.

Y-typing and data analysis

The multiplex polymerase chain reaction (PCR) reaction was performed for each DNA sample by use the of PowerPlex^® Y System according to the supplier’s protocol (Promega). This kit coamplifies 12 STRs of the Y chromosome (Y-STRs), including the markers defined as the ‘European minimal haplotype’ (Gill et al., 2001) (DYS19, DYS385 a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393), plus two loci (DYS438 and DYS439) added to this panel by the Scientific Working Group on DNA Analysis Methods (SWGDAM) (Lee et al., 2004) and DYS437. PCR products were detected by a MegaBACE 1000 genetic analyzer. MegaBACE Genetic Profile software was used to collect data and to analyze fragment sizes. Y-STR alleles were named according to the number of repeat units they contain according to the recommendations of the DNA Commission of the International Society for Forensic Genetics (Bär et al., 1997). The discrimination capacity was calculated as the number of the different haplotypes divided by the number of individuals. Prediction of Y-chromosome haplogroups from Y-STR values was performed using Whit Athey’s Haplogroup Predictor Program, version 5, which is based on a Bayesian-allele-frequency approach (Athey, 2006). Twelve Y-STR markers were input and the Bayesian probability for each haplogroup was estimated. The Y-haplogroup nomenclature used in this paper was according to the recommendations of the Y-chromosome consortium (YCC) (2008).

Results and Discussion

The genetic analysis of 200 Romany samples showed 83 different haplotypes; 61 (30.5%) occurred in only one copy. The most frequent haplotype 10-12-12-28-10-16-14-11-13-22-14/14 was found in 22 copies. The gene diversity in the analyzed population was 0.9623 ± 0.0057 and the discrimination capacity was 41.5%.

We predicted Y-chromosome haplogroups from Y-STR data by the use of Haplogroup Predictor program. Table 1 and Figure 1 show the distribution of Y-haplogroups in the Slovak Romany population. The observed haplotypes, predicted haplogroups of the Romany population and the Bayesian probability are reported in Table 2. We found only eight haplogroups from the core 15 haplogroups in the studied population. Four haplogroups—namely H, E1b1b, J2, I1a—occurred in high frequencies (>10%) and together accounted for ~90% of all Y-chromosomes. Haplogroups R1a, R1b, I2a, N1 were observed in small numbers (representing 0.5–4.5%). The remaining haplogroups, E1b1a, I1b2a, J1, G2a, G2c, L, and Q, were not detected in the samples studied. The Bayesian probability was greater than 60% in all of the samples.

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Figure 1. Graphical presentation of Y-haplogroup frequencies from Table 1.

Haplogroup H was found to be the most prevalent Y-lineage and represented 40% (80 individuals) of all the samples. According to the International Society of Genetic Genealogy, haplogroup H is typical of the Indian subcontinent area. Y-haplogroups were analyzed in three tribal populations from Andhra Pradesh in southern India and haplogroup H was the most prevalent with frequencies of 0.30 (Thanseem et al., 2006). Studies of Bulgarian, Macedonian, and Vlax Romanies showed that 44.8%, 59.6%, and 73%, respectively, obtained haplotypes belonging to Y-haplogroup H (Gresham et al., 2001; Kalaydjieva et al., 2001b; Pericic et al., 2005). According to these results, the Romany population in Slovakia shares a common ancestry with Romany populations from neighboring countries. Haplogroup H has been found very rarely in non-Romany populations and populations outside the Indian subcontinent (Wells et al., 2001). The Romany populations in Europe are the main source of haplogroup H. The ratio of haplogroup H in the European populations is less than 5% (Wells et al., 2001; Minárik et al., 2008).

Haplogroup E1b1b (formerly E3b) was the second most frequent haplogroup in the Slovak Romany population. Y-STR analysis identified 16 haplotypes within this haplogroup. Haplogroup E1b1b was found in 42 Romany males (21%) and has a distribution spread from Africa around the Mediterranean into Europe and the Middle East (Cruciani et al., 2004). In the European population, a high frequency of this haplogroup was found in Romania (21.4%), Bulgaria (20.7%), Albania (31.6%), and Italy (11.5%) (Cruciani et al., 2004).

The third most frequently found haplogroup in the Slovak Romany population was J2 with a frequency of 16.5% (33 Romanies). Haplogroup J2 is presented in different countries such as Turkey, Iraq, Kurdistan, Lebanon, Syria, Armenia, Georgia, Italy, and many Balkan states. Both haplogroups H and J2 have been found in the south and east of India with a frequency ranging from 20 to 30% (Bamshad et al., 2001; Kivisild et al., 2003).

We observed the most frequent haplotype 10-12-12-28-10-16-14-11-13-22-14/14 (DYS391, DYS389I, DYS439, DYS389II, DYS438, DYS437, DYS19, DYS392, DYS393, DYS390, DYS385a/b) within I1a haplogroup in 22 males. Haplogroup I1a was found in 28 individuals (14%). A high frequency of haplogroup I1a has been reported in several Slavic populations (Slovaks, Czechs, Poles, Estonians, Ukrainians, Slovenes, Bosnians, and Macedonians) according to International Society of Genetic Genealogy.

The most prevalent haplogroup in Western Europe is R1b, accounting for almost 70% of all lineages (Campbell, 2007). We found only nine individuals (4.5%) with this haplogroup in the Romany population. The distribution of the next subclade, R1a, shows an increasing west–east frequency and variance gradients with peaks among Finno-Ugric and Slavic speakers (Pericic et al., 2005). Its frequency increases eastwards and reaches a peak in Poland (56.4%), Hungary (60%), and Ukraine (54%) (Semino et al., 2000). We determined six (3%) haplotypes in which haplogroup R1a was predicted. Semino et al. (2000) grouped together more than 95% of European Y chromosomes into 10 phylogenetically distinct haplogroups, where 70–80% of the Y-chromosome gene pool was presented by R1a, R1b, I, and N.

Research into mitochondrial DNA haplogroups presented haplogroup M with a high frequency in the Slovak Romany population and the Romany populations from neighbouring countries (Gresham et al., 2001; Cvjetan et al., 2004; Malyarchuk et al., 2006; Malyarchuk et al., 2008). Haplogroup M is rare in Europe (Richards et al., 1998; Simoni et al., 2000) and is classified as a haplogroup characteristic mainly of Asian populations (Quintana-Murci et al., 1999). Fifteen autosomal STR loci of the eastern Slovak majority population and the Romany population were described (Soták et al., 2008). This study supported the theory that the Romany population was a conglomerate of a genetically isolated founder population and the coexistence of the non-Romany and Romany populations has been influenced by a spontaneous or forced assimilation for many decades and has taken place in spite of the high level of social isolation of Romany populations.

The genetic data about the Slovak Romany population obtained in this study provide significant evidence of their Asian origins, especially Indian origins. Distributions of haplogroups can explain a migration of Romany ancestors from the Indian subcontinent through the Middle East and Balkans to Central Europe. Kalaydjieva’s research has shown that the original group appeared in India some 32–40 generations ago and was small, probably under 1000 people (Kalaydjieva et al., 2001a). Consecutive population fissions have occurred after the arrival of the ‘gypsies’ in Europe, giving rise to numerous sub-isolates.

The achieved results indicated that the Slovak Romany population lives in socially isolated groups. It is possible to reveal their origin by following their genetic structure. The influences of the genetic drift and different degrees of admixture with non-Romany populations have shaped a peculiar mosaic of paternal lineages of the Slovak Romany population.

Quality control

The laboratory has previously participated in the Y-STR haplotype reference database (http://www.yhrd.org) quality assurance exercise in 2008, where five quality control samples were typed correctly with the with Power-Plex Y System.

Acknowledgments

This work was supported by Grant No.: AV4/0011/07. We would like to thank all staff members who helped to collect the samples.