Mitochondrial DNA Variations in Russian and Belorussian Populations

OLGA BELYAEVA,1 MARINA BERMISHEVA,2 ANDREY KHRUNIN,1 PETR SLOMINSKY,1 NATALIA BEBYAKOVA,3 ELZA KHUSNUTDINOVA,2 ALEXEI MIKULICH,4 AND

SVETLANA LIMBORSKA1

Abstract

 The sequence of the first hypervariable segment (HVS-I) of mi­tochondrial DNA (mtDNA) was determined in 251 individuals from three eastern Slavonic populations, two Russian and one Belorussian. Within HVS-I, 78 polymorphic positions were revealed. Within-population diversity of HVS-I varies slightly among three samples; its estimates do not differ strongly from those for European populations. Haplotype diversity for three populations calculated in this study is 0.949; mean pairwise differences esti­mate is 3.59. To assign mtDNA sequences to major phylogenetic clusters, haplogroup-specific restriction polymorphisms were selectively typed in most samples. The haplogroup distribution in the total Eastern Slavonic sam­ple is similar to that reported for the European sample. However, the separate consideration of three Slavonic samples reveals the complicated structure of the mitochondrial gene pool in the Eastern European area. Data of this study support the proposed model of the origin of modern Eastern Slavs, which im­plies the admixture of ancient Slavonic tribes with pre-Slavonic populations of Eastern Europe. These data should contribute to general studies of mito-chondrial DNA variations in Europe.


European mitochondrial DNA (mtDNA) diversity, despite its comparatively low level, is widely employed in studies of origin and evolution of maternal lineages. Detailed studies were performed to describe mtDNA variations in separate popu­lations as well as to reveal general tendencies in the process of the colonization of Europe, which contributed to the modern European mitochondrial gene pool for­mation. The identification of phylogenetic mtDNA clades, or haplogroups, and analysis of their distribution were successfully used to reveal the continent-wide mitochondrial variation pattern and to trace prehistoric migrations during the col­onization of Europe (Richards et al. 1998; Macaulay et al. 1999; Simoni et al.





institute of Molecular Genetics of RAS, Moscow, Russia.

2Institute of Biochemistry and Genetics, Ufa Science Center of RAS, Ufa, Russia.

3Arkhangelsk State Medical Academy, Arkhangelsk, Russia.

4Institute of Ethnography and Anthropology, Minsk, Republic of Belarus.

Human Biology, October 2003, v. 75, no. 5, pp. 647-660.

Copyright © 2003 Wayne State University Press, Detroit, Michigan 48201-1309

KEY WORDS: MITOCHONDRIAL DNA, POLYMORPHISM, HAPLOGROUP, EASTERN SLAVONIC POPULATIONS



2000; Torroni et al. 2001; Richards et al. 2000). Recently, studies in several pop­ulations revealed that particular haplogroups could also be associated with longevity and diseases (Torroni et al. 1997; De Benedictis et al. 1999; Rose et al. 2001). Slavonic and, in particular, Eastern Slavonic populations, which inhabit a large part of the Eurasian territory, were significantly underrepresented in mtDNA diversity studies. Recently, data on coding and control region mtDNA variations and haplogroup distribution in several Russian, one Ukrainian, and one Polish samples were reported (Orekhov et al. 1999; Maliarchuk and Derenko 2000; Malyarchuk et al. 2002) and implicated to the question of origin of the Eastern Slavs (Malyarchuk and Derenko 2001). Here we present data on mtDNA polymorphism in three other native population samples belonging to two Eastern Slavonic peoples, Russians and Belorussians.

Materials and Methods 

Populations Studied. Population samples were collected after obtaining in­formed consent according to the following criteria: all individuals belong to the native population of the regions studied (at least three female generations living in the region), they are maternally unrelated, and they are of Slavonic origin (Rus­sian or Belorussian, depending on the sample). The Belorussian sample (92 indi­viduals) was collected in Bobruisk, located in the central part of the Republic of Belarus. Two Russian samples represent a native rural population of two geo­graphically distant regions of the Russian Plain. The first one (76 individuals) was collected in the settlement of Oshevensk in the Arkhangelsk region of northern Russia. This settlement is situated in the Kargopol district, in the southernmost part of the Arkhangelsk region, which was colonized during the very early period of the formation of the Russian State. Its isolated rural populations are supposed to be little affected by recent migrations and are very interesting with respect to genetic studies. The second sample (83 individuals) was collected in Bashkiria, Arkhangelsky district, but includes only ethnically Russian individuals. The Rus­sian population of the Ural region, to which Bashkiria belongs, was formed most­ly by migrants from southern areas of the country, and is supposed to differ ge­netically from the northern population.



Hypervariable Segment I Polymorphism Analysis. DNA was isolated from peripheral blood according to the standard phenol-chloroform extraction proto­col. The first hypervariable segment (HVS-I) fragment of the mitochondrial con­trol region was amplified by polymerase chain reaction (PCR) using primers con L2 (5'-CAC CAT TAG CAC CCA AAG CT 3') and con H2-B (5'TGA TTT CAC GGA GGA TGG TG-3') (Richards et al. 1996). Amplicons were sequenced in both directions by use of the Sanger dideoxy-chain-termination method and cy­cle-sequencing protocol with [y-33P]-labeled amplification primers. PCR and cy­cle sequencing reactions were performed in an MJR ptc-100 thermal cycler (MJ Research, MA, USA). Sequences were determined between positions 16040 and 16365 (Anderson et al. 1981). Positions different from the Cambridge reference sequence (CRS, Anderson et al. 1981) were identified. Simple haplotype diversi­ty, h, estimated by the method of Nei (Nei 1987), and mean pairwise differences were determined using DnaSP version 3.53 software (Rozas et al. 1999). The standard error of mean pairwise differences was estimated from 200 bootstrap replications of the primary data set. 



Restriction Fragment Length Polymorphism Analysis and Haplogroup As­signment. In some cases, haplogroup assignment of mtDNA sequences could be performed on the basis of diagnostic HVS-I motifs (Macaulay et al. 1999; Richards et al. 2000). For those lineages that do not have a distinctive HVS-I mo­tif, or in which this motif is ambiguous, additional coding-region restriction frag­ment length polymorphism (RFLP) markers were typed to confirm or exclude the affiliation with a particular haplogroup. In this study, selective RFLP polymor­phisms were analyzed in most individual samples. To determine the RFLP status of mitochondrial sequences, restriction endonuclease analysis was performed on mtDNA fragments amplified as described by Torroni et al. (1996, 1997). The 7025AluI site was tested to reveal haplogroup H sequences (-7025AM). Those +7025AluI samples lacking the 14766MseI site were identified as belonging to the HV cluster; from them, sequences bearing 16298C substitution on the HV background were identified as pre-V. In +7025AluI, +14766MseI sequences, the 12308HinfI site was tested to identify haplogroup U (+12308HinfI). Within hap-logroup U, sequences were assigned to subclusters on the basis of HVS-I diag­nostic motifs, as was done within haplogroups J and T. In cases of ambiguous se­quence motif, affiliation with haplogroup T was determined by +13366BamHI. 10394DdeI, 10397AluI sites were typed in all 16223T samples. The +10394DdeI, +10397AluI status was used as a marker of haplogroup M; +14465AccI was used as a marker of haplogroup X; and +10028AluI, +16389BamHI was used as a marker of haplogroup /. Haplogroups Wy C, and N were identified on the basis of HVS-I sequence (Macaulay et al. 1999). Lineages that could not be assigned us­ing HVS-I sequence motif and the restriction markers mentioned above were named "Other." The comparison of haplogroup distribution among samples was performed using POPGENE software version 1.32 (Yeh et al. 1997).

Results and Discussion 

HVS-I Sequence Variability. We performed the sequence analysis of the first hypervariable segment of the mitochondrial D-loop in three different Slavonic samples, two Russian and one Belorussian. Mitochondrial DNA of 251 individu­als was analyzed, including 92 Belorussian individuals from Bobruisk, 76 and 83 Russian individuals from Arkhangelsk region (Oshevensk) and Bashkiria, respec-tively.[5] 

5Sequence variants corresponding to HVSI haplotypes determined in this study were deposited in the Gen-Bank™ under accession numbers AY005336-AY005390 (Belorussian sample), AY005827-AY005866 (Russians, Oshevensk) and AF292943-AF292943 (Russians, Bashkiria). 

The total number of HVS-I polymorphic sites revealed in the three popula­tions is 78 (data on sequence variations and their distribution are presented in the Table 1). Most of them are transitions. Of the total number of substitutions in Russians (Bashkiria), 5 (11.1%) are transversions, versus 2 (4.4%) transversions in Russians (Oshevensk) and 3 (5.4%) transversions in Belorussians.

For HVS-I variations, the simple haplotype diversity was determined as a measure of within-population genetic diversity. It shows relatively high values, with the estimate 0.95 for three samples in total and the highest value for the northern Russian population (Table 2). Mean pairwise difference estimates, rela­tively low, range between 3.33 and 3.80, also reaching the highest value in Rus­sians (Oshevensk). Thus, all three Slavonic samples show high estimates of hap-lotype diversity combined with low values of mean pairwise differences, which is typical for European mitochondrial DNA diversity described elsewhere. The Rus­sian (Oshevensk) sample shows slightly higher haplotype diversity and number of mean pairwise differences than the two other samples.

The percentage of unique HVS-I haplotypes has the lowest value (68.3%) in Russians (Oshevensk) and the highest (84.6%) in Russians (Bashkiria). These estimates show that the female population of Oshevensk represents a more re­stricted pool than those of the two other Slavonic samples. The percentage of unique haplotypes estimated for the three populations in total (77.3%) is close to the estimate of approximately 77% for the European population (Richards et al. 1996). This finding suggests that treating Russians as one total population, as has been done in earlier studies, could mask significant heterogeneity and differences among local groups.

HVS-I and RFLP Combined Data. In addition to the sequence analysis, ma­jor haplogroup-defining RFLP markers were typed selectively in those samples that could not be assigned to any cluster on the basis of a diagnostic HVS-I motif. The HVS-I and RFLP typing revealed 139 different lineages (Table 1).

A relatively high number of lineages in Russians from Bashkiria ("Other," 15.7%; Table 3) was not assigned to any group. HVS-I and determined RFLP markers (+7025AluI, +14766MseI, -13366BamHI, -12308HinfI, and also -10397AluI, -10394DdeI for 16223T-sequences) imply that most probably these lineages belong to pre-HV or other clusters encompassed by R (Macaulay et al. 1999), excluding HV, U, and JT. At the least, the status of the 00073 position should be determined in assigning these sequences, but 00073 typing was not per­formed in this study.

The most abundant HVS-I type in all populations was CRS. Additional RFLP typing performed on these sequences revealed the heterogeneity of this
group. Interestingly, a relatively high proportion of CRS sequences belongs to haplogroup U (+12308HinfI). In Belorussian and Russian (Bashkiria) popula­tions it constitutes 33.3% and 36.8% of CRS lineages, correspondingly. Data from earlier studies reported that within Europe CRS lineages were assigned pre­dominantly to haplogroup H (Richards et al. 2000). Haplogroup H constitutes the majority of all three samples analyzed in this study, a finding similar to those for other European and Middle Eastern groups (Richards et al. 1998, 2000), as well as to those for previously reported Slavonic samples (Orekhov et al. 1999; Mal-yarchuk and Derenko 2001; Malyarchuk et al. 2002). Two 16223T substitutions were observed among haplogroup H sequences, one in each Russian sample. Se­quences bearing 16223T substitutions on the haplogroup H background were de­scribed also in another Slavonic sample, Ukrainians (Malyarchuk and Derenko 2001). As mentioned by the authors of this study, this substitution is rare among European sequences not belonging to I, W, or X groups. In addition, five more 16223T sequences observed in Russians (Bashkria) were assigned to "Other" and do not belong to M, I, W, or X clusters. A high representation of 16240C se­quences could be mentioned as another peculiarity of haplogroup H in the north­ern Russian population. This rare substitution, also reported by Helgason et al. (2001) in a Scottish sequence, was observed in seven maternally unrelated indi­viduals, five of them bearing the single 16240C transversion, and in two se­quences representing probable derivatives. 

The distribution of haplogroup pre-V in our samples could be of some in­terest, because in studies of Torroni et al. (1998, 2001) a postglacial recoloniza-tion of Europe and population expansion from southwestern to northeastern Eu­rope was inferred from the haplogroup V distribution. Pre-V lineages have equally significant frequencies (5.4% and 5.3%) in Belorussians and Russians (Osheven­sk), while in Russians (Bashkiria) this cluster is represented by a single individual sequence. This observation is consistent with the higher frequency of V in north­ern European populations.

Significant differences were noted in the representation of the HV mono-phyletic cluster. While more than half of individual lineages in Belorussians and Russians (Oshevensk) belong to this cluster, the population of Russians (Bashkiria) demonstrates the decreased level of 32.5%.

Haplogroup U sequences are widely distributed in the Eastern Slavonic samples described here. Their frequency in both Russian samples is higher than in the Russian sample reported by Malyarchuk and Derenko (2001) (28.6% and 26.3%, against 14.0%), though closer to the frequency in the Russian sample from the Malyarchuk et al. (2002) study (20%). In Russians (Bashkiria) hap-logroup U has higher diversity than in two other samples, with a greater number of different subclusters. As mentioned above, one unusual feature of haplogroup U in the two Eastern Slavonic populations consists of a high content of CRS line­ages. Also unlike findings for earlier reported Slavonic samples, haplogroup K has a significant frequency (7.9%) in the northern Russian population, but is rare in Belorussians and Russians (Bashkiria). U5, the most ancient European subcluster of U, is well represented in all three samples with frequencies close to the European average (Richards et al. 2000). The presence of the U5b1 subcluster in the northern Russian population should also be noted. U5b1 sequences in Rus­sians were also reported by Malyarchuk et al. (2002). This subcluster was de­scribed as specific for the Saami population (Lahermo et al. 1996). Its presence in the Russian (Oshevensk) sample seems to reflect an admixture of a Finno-Ugric component, but it is unclear how old this admixture could be. All individuals in­cluded in our sample were characterized as ethnically Russian, and inhabited the area where the sample was collected for at least three maternal generations. Cur­rently, due to geographical and sociological peculiarities, the Russian population of the Oshevensk settlement can be considered an isolate. The southern part of the Arkhangelsk region, where the Oshevensk settlement is situated, does not have immediate contact with Saami populations. So, a recent admixture seems to be less probable than an earlier admixture during the peopling of northern areas by Slavonic groups.

Haplogroup J shows a significant diversity in our Slavonic samples and in­cludes five different subclusters, in contrast to Russian samples reported earlier, where J sequences were represented mostly by 16069T-16126C types. Hap­logroup T is also diversified and occurs at a significant level in Belorussians and Russians (Oshevensk), but is less frequent in Russians (Bashkiria).

European-specific haplogroups I, W, and X are rare, with the exception of Russians (Bashkiria), where I contains four individual sequences.
In comparison to frequencies of cluster M in Belorussians and the northern Russian population, the frequency of cluster M in Russians (Bashkiria) is notably but not dramatically increased (five sequences). Although we collected samples from individuals who are ethnically Russian for at least three generations, we cannot exclude the possibility of some admixture with neighboring Asian popula­tions characterized by high frequencies of the cluster M. In the meantime, those undetermined "Other" sequences, which contain the 16223T motif (six individu­als), were tested for 10397AluI and 10394DdeI status. Because they lack restric­tion sites at these positions, however, this portion of the "Others" group could not contribute to the cluster M in Russians (Bashkiria). Thus, this population differs remarkably in the distribution of haplogroups within cluster R (Macaulay et al. 1999). Therefore, only the recent admixture, to which Russians (Bashkiria) are more exposed geographically, cannot easily explain the differences between this population and two others.

Conclusions. As follows from the above discussion, three eastern Slav sam­ples considered in total demonstrate mtDNA variations that are very close to vari­ations found in the European population as a whole. MtDNA haplotypes are sim­ilar to those found in Western and Central European populations. Nevertheless, the comparison of Slavonic samples of different ethnic and geographic origins re­veals the complicated structure of the mitochondrial gene pool in this area. This structure could reflect traces of female admixture between Slavonic and pre-Slavonic groups—in particular, Finno-Ugric tribes—during a colonization of northern Eastern Europe by Slavs. In this sense our data are in agreement with those from previous studies of Slavonic mtDNA (Malyarchuk and Derenko 2001) and a hybridization theory of the origin of Eastern Slavs (Alekseeva 1973), which imply their central European origin and subsequent admixture and assimilation of pre-Slavonic populations of Eastern Europe. This study also revealed no or low Mongoloid admixture in the mitochondrial gene pool of Eastern Slavs. However, the analysis of maternally inherited mtDNA could not effectively reveal the influ­ence of Mongoloid migrations, since they included mostly male individuals. Hap-logroup distribution in Belorussians and northern Russians has more similarity to that in northern European populations than in eastern Russian populations. The Russian (Bashkiria) population differs from the two other samples in the repre­sentation of several clusters, namely, HV, V, K, T. Besides the local admixture and assimilation of pre-Slavonic groups, this difference could support an existing opinion that Russian migrants of different geographic origin were involved in the processes of colonizing the northern and eastern parts of the Russian Plain. More detailed studies of Eastern European mtDNA variations, complemented by analy­sis of Y-chromosome loci, will allow revelation of some tendencies, which could reflect the main aspects of European gene pool formation.

Acknowledgments This work was partially supported by the Russian Art and Scientif­ic Foundation and the Russian Basic Research Foundation. 

Received 21 January 2003; revision received 28 April 2003.

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