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Keywords:

  • mitochondrial DNA;
  • Slovak population;
  • Roma;
  • molecular phylogeography

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To gain insight into the mitochondrial gene pool diversity of European populations, we studied mitochondrial DNA (mtDNA) variability in 207 subjects from western and eastern areas of Slovakia. Sequencing of two hypervariable segments, HVS I and HVS II, in combination with screening of coding region haplogroup-specific RFLP-markers, revealed that the majority of Slovak mtDNAs belong to the common West Eurasian mitochondrial haplogroups (HV, J, T, U, N1, W, and X). However, a few sub-Saharan African (L2a) mtDNAs were detected in a population from eastern part of Slovakia. In addition, about 3% of mtDNAs from eastern Slovakia encompass Roma-specific lineages. By means of complete mtDNA sequencing we demonstrate here that the Roma-specific M-lineages observed in gene pools of different Slavonic populations (Slovaks, Poles and Russians), belong to Indian-specific haplogroups M5a1 and M35. Moreover, we show that haplogroup J lineages found in gene pools of the Roma and some Slavonic populations (Czechs and Slovaks) belong to new subhaplogroup J1a, which is defined by coding region mutation at position 8460.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Despite the divergent views on the ethnic history and origin of Slavs, it is generally accepted that their prehistory begins in 2000 B.C. from the Central European community of Urnfield cultures (Sedov, 1979; Šavli et al. 1996). Later, in the middle of the first millennium B.C. the Proto-Slavs were formed on the basis of Lusatian (Lausitz) culture. This emerged in Central Europe and spread over a region that reached from the central basin of the Oder River and the Bohemian mountain ridge, as far east as the Ukraine, and as far north as the shores of the Baltic Sea (Gimbutas, 1971; Sedov, 1979). In Roman times, the territory of Przeworsk culture, which is identified as Slavonic, expands to the south-east (Upper Dniester, Ukraine) and to the south (northern-eastern Slovakia) (Sedov, 1979). During the 5th-6th centuries A.D., the formation of the early medieval Slavonic cultures took place, and among them the Prague-Korchak culture is thought to have developed on the basis of Przeworsk culture (Gimbutas 1971; Sedov, 1979). According to anthropological and archaeological data, the homeland of the Slavs is located on the broad territories encompassing the eastern part of the Czech Republic, Slovakia, southern Poland and western Ukraine (Sedov, 1979; Alekseeva & Alekseev, 1989). At present these territories are mainly inhabited by Western (Czechs, Slovaks and Poles) and Eastern Slavs (Ukrainians).

Modern molecular genetic approaches are widely used for reconstruction of ethnic history of different peoples. Among these approaches, analysis of the maternally inherited mitochondrial DNA (mtDNA) seems to be the most appropriate tool for characterization of gene pools and for tracking maternal gene flow. Previous mtDNA studies of European populations have shown that Slavonic populations share a common genetic substratum characteristic of Central and Eastern European populations, such as German, Baltic and western Finno-Ugric populations (Malyarchuk et al. 2002; Pliss et al. 2006). It has been found that this genetic substratum also penetrates southeastern European populations (such as the Bosnians and Slovenians), thus reaching territory as far as the Western Balkans (Malyarchuk et al. 2003). Meanwhile, previous genetic data sets for Slavonic populations were incomplete because some ethnic groups of Slavs remain unexplored. Little is known about the mtDNA structure and diversity in Slovak populations inhabiting the region that is very important in respect of understanding the ethnic history of the Slavs as a whole. Recent mtDNA variability study in Czechs, the neighbors of Slovaks, has shown that they are genetically similar with adjacent European populations, but characterized by a small frequency of East Eurasian (2.8%) and Roma-specific (2.8%) mtDNA lineages (Malyarchuk et al. 2006b). Therefore, the aim of the present study was to characterize the mtDNA variation in Slovaks from western and eastern areas of Slovakia, based on variation of the HVS I and HVS II sequences, followed by a hierarchical survey of mtDNA haplogroup-specific restriction fragments length polymorphism (RFLP) markers. In addition, to determine the phylogenetic status of some unclassified mtDNA lineages (mostly Roma-specific) found in the gene pool of Slovaks, complete sequencing of mitochondrial genomes was performed.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Population Samples

A population sample of 207 individuals from two areas of Slovakia, western (n = 70) (area of Bratislava) and eastern (n = 137) (area of Kosice), was studied. These samples were represented by randomly chosen unrelated Caucasians from Slovakia. All individuals studied were maternally unrelated and originated from the area considered for this study. Appropriate informed consent was obtained from all participants in this study.

mtDNA Sequencing and RFLP Analysis

The total DNA was isolated from blood samples with a QIAamp DNA Blood Mini kit (QIAGEN, Hilden, Germany) according to manufacturer's protocol. To generate control region mtDNA templates for sequencing, the temperature profile for 32 cycles of amplification was: 94°C for 20 sec, 50°C for 30 sec, and 72°C for 1 min 30 sec, with initial denaturation at 94°C for 2 min (GeneAmp PCR System 9700; Applied Biosystems, Foster City, CA). The primer sets L15997/H16401 and L29/H408 were used to amplify the hypervariable segments I and II (HVS I and II), respectively. PCR products were purified by ultrafiltration (Microcon 100; Amicon) and sequenced directly from both strands using the BigDye Terminator v. 3.1 sequencing kit (Applied Biosystems, Foster City, CA). The nucleotide sequences of HVS I from position 15999 to 16400 and HVS II from position 30 to 407 were determined on ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA) and compared with the revised Cambridge reference sequence (rCRS) (Andrews et al. 1999). DNA sequence data were analyzed using SeqScape v. 2.5 software (Applied Biosystems, Foster City, CA). Nucleotide positions showing point indels and transversions located between positions 16180–16193 and 303–315 were excluded from further analysis.

Complete sequencing of mitochondrial genomes representing M5a and M35 lineages in one Slovak and one Russian individual and J1 lineages in three Slovak individuals was performed using the methodology described in details by Torroni et al. (2001).

All Slovak samples were subjected to RFLP analysis of coding region sites that were diagnostic of all major Eurasian clusters (haplogroups and subhaplogroups), on the basis of the hierarchical mtDNA RFLP scheme as described elsewhere (Malyarchuk et al. 2002; Malyarchuk et al. 2006b). RFLP typing was performed by restriction endonuclease analysis of PCR amplified mtDNA fragments, using the same primer pairs and amplification conditions as described by Torroni et al. (1996) and Finnilä et al. (2000).

Phylogenetic and Statistical Analysis

All available published data on HVS I mtDNA variability or RFLPs in Slavonic-speaking populations were used for comparative purposes: Bulgarians (Calafell et al. 1996; Richards et al. 2000), Russians (Malyarchuk et al. 1995; Orekhov et al. 1999; Richards et al. 2000; Malyarchuk et al. 2002; Belyaeva et al. 2003; Malyarchuk et al. 2004); Ukrainians (Malyarchuk & Derenko, 2001), Belorussians (Belyaeva et al. 2003), Poles (Richards et al. 2000; Malyarchuk et al. 2002), Czechs (Richards et al. 2000; Vanecek et al. 2004; Malyarchuk et al. 2006b), Bosnians (Malyarchuk et al. 2003; Cvjetan et al. 2004), Slovenians (Malyarchuk et al. 2003; Zupanic Pajnic et al. 2004), Croatians (Tolk et al. 2000; Cvjetan et al. 2004), Serbians, Herzegovinians and Macedonians (Cvjetan et al. 2004).

Genetic variation was analyzed using methods implemented in the Arlequin 2.0 software (Schneider et al. 2000). The statistical significance of FST-values was estimated by permutation analysis using 10000 iterations. Chi-square analysis of haplogroup frequencies in populations was performed by means of the program CHIRXC, which estimates the probability of homogeneity using Monte Carlo simulation (1000 runs) (Zaykin & Pudovkin, 1993).

Multidimensional scaling (MDS) analysis of pairwise interpopulation FST values was performed by means of the software package STATISTICA (StatSoft, Inc., Tulsa, OK, USA). For MDS analysis, data from the following populations were used: 200 Southern Germans (Lutz et al. 1998), 101 Austrians (Parson et al. 1998), 150 Western Germans (Baasner et al. 1998; Baasner & Madea, 2000), 179 Czechs from western Bohemia (Malyarchuk et al. 2006b), 104 Slovenians and 144 Bosnians (Malyarchuk et al. 2003), 436 Poles and 201 Russians (Malyarchuk et al. 2002), 50 Finns, 47 Estonians and 83 Karelians (Sajantila et al. 1995).

Most parsimonious trees of the mtDNA haplogroups were reconstructed manually, following a parsimony approach, and confirmed by use of the program Network 4.2. Sequence classification into mtDNA subclusters was based on the nomenclatures of Palanichamy et al. (2004) and Carelli et al. (2006) for subhaplogroup J1 and Sun et al. (2006) for haplogroups M5a and M35. Five completely sequenced mitochondrial genomes were submitted to the GenBank under accession numbers EF583175-EF583179.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The analysis of HVS I and II variation, in combination with RFLP typing of the coding region haplogroup-diagnostic sites allowed detection of 187 different mtDNA haplotypes in a total sample of 207 Slovak individuals (Table 1). Out of these 187 haplotypes, 126 were revealed in eastern Slovakia (for n = 137) and 69 haplotypes were found in western Slovakia (for n = 70). Only 8 haplotypes were shared by two Slovak samples studied (Table 1). The overwhelming majority of Slovak mtDNA haplotypes were classified into West Eurasian haplogroups (such as H, HV0, HV2, HV3, J, T, U, I, W, X, N1b) (Table 2). Only two distinct haplotypes belonging to African-specific haplogroup L2a were detected in eastern Slovakia, this finding was not unexpected because low frequencies of African mtDNAs were previously found in different European populations (Salas et al. 2002; Malyarchuk et al. 2004; Malyarchuk & Czarny, 2005; Pereira et al. 2005). In eastern Slovaks, two haplotypes belonging to macrogroup M were also detected (Table 1). However, in contrast to the previously studied Czech population from western Bohemia (Malyarchuk et al. 2006b), samples from Slovakia do not display any East Eurasian mtDNAs. One of the Slovak M-haplotype belongs to subhaplogroup M1b and is identical to M1b1a-haplotypes revealed in Italians and Bedouins from southern Israel (Olivieri et al. 2006) as well as in Saudi Arabs (Abu-Amero et al. 2007). A second M-lineage detected in Slovaks is defined by variants at positions 16129–16223-16230–16233-16304–16344. This lineage is identical to those revealed previously in gene pools of the Bulgarian Roma at frequency of 3.6% (Gresham et al. 2001). Based on the presence of the 16129 variant, Gresham et al. (2001) suggested that this lineage belongs to Indian-specific haplogroup M5. Nevertheless, to determine its exact phylogenetic status we completely sequenced our Slovak sample (Slv227) and compared it with Indian M-haplotypes published by Sun et al. (2006) (Fig. 1). As a result, we have found that our sample belongs to haplogroup M35 due to mutations at positions 199 and 12561. Moreover, it shared transition at 15928 with the South Indian sample T17 (from Andhra Pradesh) that allowed us to define a new Indian/Roma branch called as M35b.

Table 1.  mtDNA haplotypes in Slovak populations
HVS I (minus 16000)HVS IIHGN
  1. Number of subjects (N) is shown in parentheses; region belonging to eastern and western Slovakia is indicated by letters “E” and “W”, respectively. Mutations are shown indicating positions relative to the revised mtDNA CRS (Andrews et al. 1999). The nucleotide positions in HVS I and II sequences correspond to transitions; transversions are further specified. Haplogroup names (HG) are given in large letter according to the mtDNA classification (Richards et al. 2000; Palanichamy et al. 2004; Achilli et al. 2005; Olivieri et al. 2006; Sun et al. 2006; Torroni et al. 2006). The presence of insertions or deletions is referred by “i” and “d”, respectively, following the nucleotide position.

85 129152 263 309iC 315iCH*E(1)
93 129 291 316263 315iCH*W(1)
104 183AC 189 193iC 317AT263 309iCC 315iCH*E(1)
129263 309iCC 315iCH*E(1)
129263 315iCH*E(1)
129 184146 263 315iCH*E(1)
153 346 355 399146 195 263 309iC 315iCH*E(1)
174 362152 263 309iC 315iCH*E(1)
189 193dC 256152 195 263 315iCH*E(1)
218143 195 263 315iCH*E(1)
234152 263 309iCH*E(1)
234152 263 309iC 315iCH*E(1)
311152 200 263 315iCH*E(1)
311263 315iCH*E(1)
311 355263 315iCH*W(1)
324204 263 309iC 315iCH*W(1)
362152 263 309iCCH*E(1)
366263 309iC 315iCH*W(1)
CRS146 152 195 263 309iC 315iCH*E(1)
CRS183 263 309iC 315iCH*W(1)
CRS207 263 309iC 315iCH*W(1)
CRS262 263 309iC 315iCCH*W(1)
CRS263 309iC 315iCH*E(1)
CRS263 315iCH*E(2)
51 270263 315iCH1W(1)
93152 263 309iC 315iCH1E(1)
179 311263 309dC 315iCH1E(1)
18493 263 315iCH1W(1)
189152 263 309iC 315iCH1E(1)
189263 315iCH1E(1)
189 295263 315iCH1E(1)
189 295263 309iC 315iCH1E(1)
209263 315iCH1W(1)
261263 309iC 315iCH1E(1)
263263 315iCH1E(1)
311257 263 315iCH1W(1)
183AC 189 362257 263 309iCCC 315iCH1W(1)
183AC 189 193iC 235 260263 315iCH1W(1)
183AC 189 288263 309iCH1E(1)
CRS73 263 315iCH1W(1)
CRS143 263 309iC 315iCH1W(1)
CRS195 257 263 309iCC 315iCH1E(1)
CRS263 315iCH1E(2)
CRS263 309iC 315iCH1E(1)
134 16273 263 309iC 315iCH1aE(1)
16273 263 309iC 315iCH1aE(1) W(1)
16273 263 315iCH1aE(1)
16273 263 309iCC 315iCCH1aE(1)
80 189 193iC 356151 263 309iC 315iCH1bE(1)
188 189 356 362263 315iCH1bE(1)
189 356263 315iCH1bW(1)
189 356151 263 309iCC 315iCH1bE(1)
189 356 362263 315iCH1bE(1)
183AC 189 356 362263 309iCH1bE(1)
183AC 189 193iC 356263 309iC 315iCH1bE(1)
CRS263 309iC 315iCH2W(1)
CRS249dA 263 309iCC 315iCH2E(1)
CRS315iCH2E(1) W(1)
35473 263 309iC 315iCH2aE(1)
157 192263 315iCH4W(1)
311146 200 263 309iCC 315iCH4E(1)
CRS73 263 315iCH4W(1)
CRS263 315iCH4E(1)
304146 150 195 198 263 309iC 315iCH5W(1)
304146 263 315iCH5W(1)
304146 195 263 315iCH5E(1)
114 172 304 311146 263 315iCH5aW(1)
218 304263 309iC 315iCH5aE(1)
304152 263 309iC 315iCH5aE(1)
304263 309iC 315iCH5aE(2) W(1)
304263 315iCH5aE(1) W(1)
304 343263 309iC 315iCH5aE(1)
362152 239 263 309iC 315iCH6E(1)
362239 263 309iC 315iCH6E(2) W(2)
362239 263 309iCC 315iCH6E(1)
362 400239 263 315iCH6E(1)
362152 239 263 315iCH6E(1)
265263 309iCC 315iCH7E(1)
CRS263 315iCH7E(1)
CRS263 309iC 315iCCH7E(1)
CRS263 309iC 315iCH7E(1)
278 293 311195 263 309iC 315iCH11aE(1)
293 311195 263 315iCH11aE(1) W(1)
214 217 33573 151 152 195 246 263 309iC 315iCHV2E(1)
214 217 33573 151 152 195 246 263 279 309iC 315iCHV2E(1)
172 300 311146 263 309iC 315iCCHV3W(1)
311263 309iC 315iCHV3E(1)
311 354263 315iCHV3E(1)
113AC 172 311263 309iCC 315iCCHV3W(1)
129 145 223 39173 146 152 199 204 207 250 263 309iCIE(1)
129 172 223 311 39173 199 203 204 250 263 315iCI1E(2) W(1)
129 172 223 311 39173 199 203 204 250 263 309iC 315iCI1E(1)
129 172 223 311 39173 189 199 203 204 250 263 315iCI1W(1)
129 172 223 311 39173 199 204 250 263 315iCI1E(1)
129 223 311 368 39173 152 199 204 250 263 309iC 315iCI1E(1)
129 223 311 39173 189 193 199 204 250 263 309iCI1E(1)
86 129 223 39173 152 199 204 207 239 250 263 315iCI3E(1)
69 126 145 222 235 261 27173 263 295 309iCJ1aE(2)
69 126 145 222 235 261 27173 263 295 309iC 315iCJ1aE(1)
69 126 145 222 235 261 271 29073 263 295 309iC 315iCJ1aE(1)
69 126 145 172 222 26173 242 263 295 309iC 315iCJ1b1W(1)
69 126 26173 263 295 309iCJ1cE(1)
69 92 12673 185 228 263 295 309iC 315iCJ1cW(1)
69 126 172 18973 143 185 228 263 295 315iCJ1cW(1)
69 126 153 39073 150 185 195 228 263 295 315iCJ1cW(1)
69 126 147 209 242 31173 185 188 228 263 295 309iC 315iCCJ1cW(1)
69 126 189 193iC 24573 185 228 263 295 309iC 315iCCJ1cE(1)
69 12673 185 195 228 263 295 309iC 315iCJ1cE(1)
69 12673 185 188 228 263 295 309iC 315iCJ1cE(1)
69 12673 146 185 188 222 228 263 295 315iCJ1cE(1)
69 12673 146 185 188 228 263 295 309iC 315iCJ1cW(1)
69 12673 185 188 228 263 295 315iCJ1cE(1)
69 126 145 231 26173 150 152 195 215 263 295 315iCJ2aW(1)
69 126 145 231 26173 150 152 195 215 263 295 311 315iC 319J2aW(1)
224 31173 263 315iCKE(1)
224 31173 146 195 263 309iC 315iCKE(1)
224 31173 263 309iC 315iCKE(1)
51 223 278 294 309 39073 143 146 152 195 263 309iC 315iCL2aE(1)
172 189 192 218 223 278 294 309 39073 143 146 152 195 263 309iC 315iCL2aE(1)
129 185 189 193dC 223 249 31173 195 200 263 309iC 315iCM1b1aE(1)
129 223 230 233 304 34473 199 263 309iC 315iCM35bE(1)
145 176CG 223 39073 152 263 315iCN1bW(1)
126 294 29673 263 309iC 315iCCTW(1)
126 294 29673 263 315iCTW(1)
126 294 29673 263 309iC 315iCTW(1)
126 172 186 189 294 298 39973 263 315.CTW(1)
126 274 294 30473 263 315.CTW(1)
126 181 189 193iC 294 29673 152 263 309iC 315iCTW(1)
126 294 296 36273 263 309iC 315iCTW(1)
126 182AC 183AC 189 193iC 294 296 29873 150 195 263 315iCTW(1)
126 182AC 183AC 189 294 29673 195 263 315iCTW(1)
126 294 296 30473 263 315iCTE(1)
126 140 189 294 296 31173 195 263 309iC 315iCTE(1)
126 172 294 30473 195 263 309iC 315iCTE(1)
126 294 304 318AT73 152 263 309iCC 315iCTE(1)
126 234 294 296 30473 146 263 309iC 315iCTE(1)
126 292 294 296 30473 146 263 315iCTE(1)
126 153 207AC 29473 150 263 309iC 315iCTE(1)
126 294 296 30473 263 315iCTE(1)
126 294 30473 152 263 309iC 315iCTE(1)
126 163 189 221 243 294 31173 263 315iCT1W(1)
93 126 163 186 189 29473 263 309iC 315iCT1E(1)
126 163 186 189 29473 152 195 263 309iC 315iCT1E(2)
129 183AC 189 247 249 28873 146 150 152 195 263 285 309iC 315iC 385U1W(1)
51 129GC 182AC 183AC 189 291 300 36273 152 217 263 309dC 315iC 340U2W(1)
51 129GC 183AC 189 193iC 36273 152 204 263 309dC 315iC 340U2E(1)
35673 263 309iC 315iCU4W(1)
261 35673 195 263 315iCU4E(1)
35673 146 152 195 263 309iCC 315iCU4E(1)
179 35673 195 263 309iC 315iCU4W(1)
179 35673 195 263 315iCU4E(1)
134 35673 152 195 263 296 315iCU4aE(1)
35673 146 195 263 310U4aW(1)
189 35673 195 263 310U4aE(1)
35673 195 263 310U4aE(1)
CRS73 195 263 310U4aE(2)
256 270 320 39973 195 263 309iC 315iCU5aW(1)
256 270 39973 263 309iCU5aW(1)
192 234 256 27073 263 309iC 315iCU5aE(1)
256 270 39973 263 309iC 315iCU5aE(1)
93 183AC 189 193iC 27073 150 152 263 315iCU5bW(1)
27073 150 263 315iCU5bW(1)
174 192 31173 150 263 315iCU5bW(1)
192 27073 150 204 207 235 263 309iCC 315iCU5bW(1)
144 189 27073 150 263 309iCC 315iCU5bE(1)
189 27073 150 152 263 315iCU5bE(1)
147 183AC 189 27073 150 263 309iCC 315iCU5bE(1)
189 193iC 27073 150 152 263 309iC 315iCU5bE(1)
183AC 189 193iC 27073 150 152 263 315iCU5bE(1)
93 189 27073 150 263 315iCU5bE(1)
144 189 27073 150 263 315iCU5bE(1)
129 189 194iC 27073 150 152 263 285 309iC 315iCU5bE(1)
318AT73 152 263 309iCU7E(1)
29872 263 309iC 315iCHV0E(2) W(1)
29872 152 263 315iCHV0E(1)
29872 195 228 263 309iC 315iCHV0E(1)
29872 263 309iCC 315iCHV0E(3)
223 29273 189 194 195 199 204 207 263 315iCWW(1)
193 223 29273 119 152 189 195 204 207 263 315iCWW(1)
192 223 292 32573 189 194 195 204 207 263 309iC 315iCWW(1)
29273 143 189 195 204 207 263 315iCWE(1)
223 292 295 32473 189 195 204 207 263 315iCWW(1)
223 29273 189 195 204 207 263 309iC 315iCWW(1)
189 223 27873 150 153 195 225 226 263 309iC 315iCXW(1)
93 184CA 189 223 27873 153 195 225 226 263 315iCXE(1)
189 192 223 278 39973 143 195 225 226 235 263 309iC 315iCXE(1)
Table 2.  Haplogroup distributions (no. of individuals and % values in parentheses) in Slovaks in comparison with Czech population
HGSlovaksCzechs
West region (n = 70)East region (n = 137)In total (n = 207)(n = 179)
  1. Data for Czechs are from Malyarchuk et al. (2006b).

H*7 (10.0)18 (13.1)25 (12.1)26 (14.5)
H1*8 (11.4)13 (9.5)21 (10.1)16 (8.9)
H1a1 (1.4)4 (2.9)5 (2.4)5 (2.8)
H1b1 (1.4)6 (4.4)7 (3.4)8 (4.5)
H2*2 (2.9)2 (1.5)4 (1.9)6 (3.4)
H2a101 (0.7)1 (0.5)3 (1.7)
H42 (2.9)2 (1.5)4 (1.9)1 (0.6)
H5*2 (2.9)1 (0.7)3 (1.4)4 (2.2)
H5a3 (4.3)6 (4.4)9 (4.3)2 (1.1)
H62 (2.9)6 (4.4)8 (3.9)3 (1.7)
H704 (2.9)4 (1.9)2 (1.1)
H11a1 (1.4)2 (1.5)3 (1.4)3 (1.7)
HV01 (1.4)7 (5.1)8 (3.9)4 (2.2)
HV202 (1.5)2 (1.0)0
HV32 (2.9)2 (1.5)4 (1.9)3 (1.7)
I2 (2.9)8 (5.8)10 (4.8)5 (2.8)
J1a04 (2.9)4 (1.9)5 (2.8)
J1b11 (1.4)01 (0.5)2 (1.1)
J1c5 (7.1)7 (5.1)12 (5.8)12 (6.7)
J2a2 (2.9)02 (1.0)2 (1.1)
N1a0001 (0.6)
N1b1 (1.4)01 (0.5)2 (1.1)
N9a0001 (0.6)
K03 (2.2)3 (1.4)7 (3.9)
M1b01 (0.7)1 (0.5)0
M35b01 (0.7)1 (0.5)0
T*9 (12.9)9 (6.6)18 (8.7)17 (9.5)
T11 (1.4)3 (2.2)4 (1.9)5 (2.8)
U11 (1.4)01 (0.5)0
U21 (1.4)1 (0.7)2 (1.0)1 (0.6)
U30004 (2.2)
U4*2 (2.0)3 (2.2)5 (2.4)2 (1.1)
U4a1 (1.4)5 (3.6)6 (2.9)3 (1.7)
U5a2 (2.9)2 (1.5)4 (1.9)9 (5.0)
U5b4 (5.7)8 (5.8)12 (5.8)6 (3.4)
U701 (0.7)1 (0.5)0
U8a0001 (0.6)
W5 (7.1)1 (0.7)6 (2.9)1 (0.6)
X1 (1.4)2 (1.5)3 (1.4)3 (1.7)
A0001 (0.6)
D0001 (0.6)
M70002 (1.1)
L2a02 (1.5)2 (1.0)0
image

Figure 1. The phylogenetic tree of haplogroups M5a and M35 based on complete mitochondrial genome sequences. The tree is rooted in macrohaplogroup M. Numbers along links refer to substitutions scored relative to the rCRS (Andrews et al. 1999). Transversions are further specified, recurrent mutations are underlined. A plus sign (+) denotes an insertion. Four additional complete sequences were taken from the literature (Kivisild et al. 2006; Malyarchuk et al. 2006a; Sun et al. 2006) and designated by TK, BM, and CS, respectively, followed by “#” and the original sample code. For sample TK#As32 only coding region information was available.

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Previously, using complete mtDNA sequencing approach we found that another frequent Roma lineage with motif: 16129–16223-16291–16298 belongs to Indian-specific haplogroup M5 (Malyarchuk et al. 2006a). However, due to the progress of mitochondrial genome sequencing in Indian populations (Kivisild et al. 2006; Sun et al. 2006), it is possible to define the exact phylogenetic position of these Romani mtDNAs more precisely. We present here the completely sequenced Roma-specific M5 haplotype revealed in the Russian gene pool (individual Rus44 from study by Malyarchuk & Derenko, 2001) and compare it with the published M5-mtDNAs of Roma (Polish individual PL173; Malyarchuk et al. 2006a) and Indian/Asian origin (individuals R61 and As32 from studies by Sun et al. (2006) and Kivisild et al. (2006), respectively). As a result, we are able to considerably improve the M5 phylogenetic tree reconstructed by Sun et al. (2006). The results presented in Fig. 1 show that M5a1 trunk is defined by transitions at 4916 and 15287, and the Roma-specific branch of this subgroup designated as M5a1b is determined by transitions at four positions (1303, 3954, 6461 and 9833). It is worthwhile to note that one of the M5a1a samples (R61) was taken from South India (Andhra Pradesh), while another sample (As32) was of unknown Asian ancestry. Thus, our findings point to a possible Indian origin of both Roma-specific haplogroups, M5a1b and M35b.

Previously, we have found that the Polish Roma population is characterized by high incidence (18.8%) of haplogroup J1* lineage, defined by HVS I motif 16069–16126-16145–16222-16235–16261-16271 (Malyarchuk et al. 2006a). This and a similar haplotype, lacking only the 16271 transition, are very rare in European Roma populations, being found only in the Spanish, Bulgarian and Hungarian Roma (Gresham et al. 2001; Egyed et al. 2007). Among Europeans, such haplotypes have been revealed only in French (0.5%; Dubut et al. 2004), Hungarian (0.5%; Egyed et al. 2007) and Czech (about 3%; Vanecek et al. 2004; Malyarchuk et al. 2006b) populations. In the present study, we have found that 2.9% of individuals from eastern Slovakia are characterized by exactly the same J1*-haplotype. Taking into account its similarity with J1-haplotypes revealed in Southwestern Pakistani populations (Quintana-Murci et al. 2004) and thus assuming that this haplotype might have been characteristic of the ancestral Romani population (Malyarchuk et al. 2006a), we completely sequenced two J1*-samples revealed in Slovakia (Fig. 2). Comparison with published data (pooled in MitoMap mtDNA tree (Ruiz-Pesini et al. 2007)) demonstrated that these J1*-haplotypes belong to a new subhaplogroup J1a defined by transition at 8460. This subhaplogroup appears to be a sister clade to subhaplogroup J1b. Therefore, the J1a contribution to the Roma, and through them to some European gene pools, can be caused by gene flow from Indo-Pakistani region. One of the haplogroup J Slovak sample (Slv174; 16069–16126–16261–73–263–295) was characterized by uncertain HVS II motif, due to the lack of any subhaplogroup-diagnostic mutation according to classification developed in Palanichamy et al. (2004) and Carelli et al. (2006). To determine its phylogenetic status, we completely sequenced this sample and found that it belongs to subhaplogroup J1c being accompanied by transition at 14798, despite the lack of diagnostic mutation at position 228. It is unclear however, whether this sample should occupy the ancestral node for J1c-phylogeny or it is the result of back-mutation at position 228 (Fig. 2).

image

Figure 2. The phylogenetic tree of subhaplogroup J1 based on complete mitochondrial genome sequences. The tree is rooted in haplogroup J. Numbers along links refer to substitutions scored relative to the rCRS (Andrews et al. 1999). Recurrent mutations are underlined. For subhaplogroups J1b1 and J1b2 only diagnostic mutations are shown according to classification (Palanichamy et al. 2004).

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As in other European populations, the most frequent haplogroup in Slovaks is haplogroup H that encompasses 45.4%. Similar frequencies of this haplogroup (44%) have been previously revealed in Czech population (Malyarchuk et al. 2006b). Among the Slovaks, twelve subhaplogroups (H*, H1*, H1a, H1b, H2*, H2a1, H4, H5*, H5a, H6, H7, and H11a) were found using an RFLP approach described by Loogväli et al. (2004). Table 2 shows haplogroups frequency distribution in western and eastern Slovaks, in comparison with the Czechs. Some differences in between-population distribution of H-subgroups can be revealed (for instance, the lower frequency of H1b, H6 and H7 or higher frequency of H5* in western Slovaks), however, they are statistically insignificant. Between-population comparisons for distribution of all mtDNA haplogroups and subhaplogroups (Table 2) demonstrate the absence of statistical differences between Slovak and Czech populations (P > 0.35 in all cases). When comparisons on the level of individual haplogroups were performed, the only haplogroup W was revealed more frequently in western Slovaks than in eastern Slovaks and Czechs (P = 0.018 and 0.004, respectively). Chi-square analysis of mtDNA haplogroup frequencies in Slovaks in comparison with Slavonic-speaking populations, such as Czechs, Poles, Bosnians, Slovenians and Russians (according to data by Malyarchuk et al. (2006b)) has also revealed the population homogeneity (with P > 0.15 in all comparisons).

A similar conclusion (namely a lack of between-population differences in Slovaks and Czechs) also follows from an analysis of molecular variance (AMOVA) performed on the level of HVS I sequences. For such analysis, different European populations – Slavonic-speaking (Slovaks, Czechs, Slovenians, Bosnians, Poles, Russians), German-speaking (West Germans, South Germans, Austrians) and Finno-Ugric-speaking (Finns, Karelians, Estonians) – were used. Analysis of between-population differentiation based on FST-distances revealed that only 0.16% of variation was due to differences among populations (P = 0.013). Significant pairwise FST-differences (P < 0.05) were found mainly between some Finno-Ugric populations (Finns and Karelians) and Slavonic and German populations (Table 3). The MDS analysis performed on the basis of pairwise FST values revealed that Slovak populations do not cluster together. Western Slovaks are located together with the Czechs and Austrians (in accordance with their geographic proximity), whereas eastern Slovaks are placed close to Slovenians (Fig. 3).

Table 3.  Between-population differences based on pairwise FST-distances inferred from mtDNA HVS I variation data
PopulationCZEPOLSLOBOSRUSAUSSGEWGEESTFINKARSLV (W)
  1. Abbreviations for the populations are as follows: SLV (W) – western Slovaks, SLV (E) – eastern Slovaks, CZE – Czechs, POL – Poles, SLO – Slovenians, BOS – Bosnians, RUS – Russians, AUS – Austrians, SGE – South Germans, WGE – West Germans, EST – Estonians, FIN – Finns, KAR – Karelians. *significant differences (p < 0.05).

POL0.0013 
SLO0.00310.0009 
BOS0.00320.00060.0003 
RUS−0.0028−0.00080.0019−0.0011 
AUS−0.00310.00100.0015−0.0007−0.0046 
SGE0.00070.00010.0004−0.0012−0.0014−0.0008 
WGE0.0010−0.0024−0.00100.0021−0.00260.00030.0005 
EST−0.0007−0.00310.0017−0.0016−0.0006−0.001−0.0007−0.0082 
FIN0.0085*0.00380.0076*0.00170.0087*0.0082*0.00430.0026−0.0018 
KAR0.0078*0.0052*0.0089*0.00340.00560.0087*0.0062*−0.0009−0.00630.0010 
SLV (W)−0.0038−0.00060.00410.0044−0.0024−0.00230.00120.0009−0.00300.00580.0094* 
SLV (E)0.00080.00230.0067*0.00210.00050.00250.00170.0036−0.00240.00500.00400.0007
image

Figure 3. Multidimensional scaling plot of FST distances between Slovaks and the surrounding European populations based on mtDNA HVS I variation data (stress value 0.001). Populations designated as in Table 3.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Although the mtDNA analysis appears to be an effective tool in the reconstruction of population history, such studies of European populations do not allow researchers to identify any specific features clearly distinguishing the Slavs from neighboring populations (Malyarchuk & Derenko 2001; Malyarchuk et al. 2002). This means that questions such as the reconstruction of proto-Slavonic migrations and the search for the Slav homeland may not be answered by use of mtDNA markers. Meanwhile, Y-chromosome studies have shown that two genetically distant groups of the Slavs – Southern and Western/Eastern ones – can be recognized by means of SNP and/or STR analyses (Pericic et al. 2005; Rebala et al. 2007). Results of recent Y-STR analysis in different Slavonic populations, including Slovaks (Rebala et al. 2007), suggest that the expansion of medieval Slavs started from their putative homeland in the basin of the middle Dnieper (Ukraine). However, this conclusion seems to be uncertain because the distribution pattern of insignificant P-values for population pairwise FST-distances (Table 1 of Rebala et al. (2007)) indicates that Y-STR genetic homogeneity in Slavs extends from Slovakia, Ukraine and central Belarus to western parts of Russia, thus suggesting that Slavonic expansion might also have started in Central Europe. In any case, additional studies based on STR-SNP diversity analysis of Y-chromosome and complete mtDNA sequencing, in combination with autosomal loci variation, are required to solve the key problems of the genetic history of the Slavs.

One of the important questions is the nature and extent of non-European admixture in gene pools of Slavs. It is worth noting that gene pools of Slovaks and Czechs are characterized by a common component represented by the Roma-specific mtDNA lineages. The Roma (also known as Gypsies), who are believed to be of Indian origin, nowadays represent a large population spread over all of Europe, with their highest concentrations in southeastern Europe and the Iberian Peninsula (Kalaydjieva et al. 2001). It is suggested that by the 13th century the Roma had entered the Balkans and some groups moved slowly through the territories inhabited by the Slavs (Demeter et al. 2000). The first migration of small groups of the Roma from Hungary throughout Bohemia to the north and west of Europe took place at the beginning of the 15th century (Demeter et al. 2000). According to the latest Census in 2001 the Roma form the minority group, representing about 3.5% of the total Slovak population. However, not all Roma people may have claimed themselves as Roma, so the real number is unclear. Genetic studies have shown that the Roma populations share a common genetic history, as evidenced by mtDNA and Y-chromosomal markers as well as by several disease loci (Gresham et al. 2001; Kalaydjieva et al. 2001; Morar et al. 2004; Malyarchuk et al. 2006a). However, despite the obvious evidence that some Roma founder mtDNA lineages belong to haplogroups widespread across South Asia, there is no detailed phylogenetic characterization of such mtDNAs. In the present study, we extend considerably our knowledge about the phylogenetic position of Roma-specific lineages revealed in gene pools of different Slavonic groups (such as Slovaks, Czechs, Poles, and Russians), by using complete mtDNA sequencing approach. As a result, we have found that some Roma-specific M-lineages belong to the Indian haplogroups M5a1 and M35. Moreover, a novel subhaplogroup J1a, which is present with a marked frequency in gene pools of the Roma as well as Slovaks and Czechs, has been described. Further reconstruction of the phylogeographic structure of subhaplogroup J1a, requires new complete mtDNA data from India and Southwest Asia.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This research received support from the Program of Basic Research of Russian Academy of Sciences “Biodiversity and gene pools dynamics”. B.A.M. was supported in part by the Russian Science Support Foundation. We are grateful to Dr. Tomasz Grzybowski for help in this study.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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