Comparison of lactase persistence polymorphism in ancient and present-day Hungarian populations

Comparison of Lactase Persistence Polymorphism inAncient and Present-Day Hungarian Populations

Dora Nagy,1,2* Gyongyver Tomory,1 Bernadett Csanyi,1 Erika Bogacsi-Szabo,1 Agnes Czibula,1

Katalin Priskin,1 Olga Bede,2 Laszlo Bartosiewicz,3 C. Stephen Downes,4 and Istvan Rasko1

1Institute of Genetics, Biological Research Centre of Hungarian Academy of Sciences, Szeged H-6726, Hungary2Department of Pediatrics, University of Szeged, Szeged H-6725, Hungary3Archeological Institute of the Hungarian Academy of Sciences, Budapest H-1014, Hungary4School of Biomedical Sciences, University of Ulster, Coleraine BT521SA, Northern Ireland

KEY WORDS lactose intolerance; ancient DNA; 10th–11th century bones; mitochondrial DNA

ABSTRACT The prevalence of adult-type hypolacta-sia varies ethnically and geographically among popula-tions. A C/T–13910 single nucleotide polymorphism(SNP) upstream of the lactase gene is known to be asso-ciated with lactase non-persistence in Europeans. Theaim of this study was to determine the prevalence of lac-tase persistent and non-persistent genotypes in currentHungarian-speaking populations and in ancient bonesamples of classical conquerors and commoners from the10th–11th centuries from the Carpathian basin; 181present-day Hungarian, 65 present-day Sekler, and 23ancient samples were successfully genotyped for the C/T-13910 SNP by the dCAPS PCR-RFLP method. Additionalmitochondrial DNA testing was also carried out. In an-cient Hungarians, the T-13910 allele was present only in11% of the population, and exclusively in commoners of

European mitochondrial haplogroups who may havebeen of pre-Hungarian indigenous ancestry. This is de-spite animal domestication and dairy products havingbeen introduced into the Carpathian basin early in theNeolithic Age. This anomaly may be explained by theHungarian use of fermented milk products, their greaterconsumption of ruminant meat than milk, cultural dif-ferences, or by their having other lactase-regulatinggenetic polymorphisms than C/T-13910. The low preva-lence of lactase persistence provides additional informa-tion on the Asian origin of Hungarians. Present-dayHungarians have been assimilated with the surroundingEuropean populations, since they do not differ signifi-cantly from the neighboring populations in their posses-sion of mtDNA and C/T-13910 variants. Am J PhysAnthropol 145:262–269, 2011. VVC 2011 Wiley-Liss, Inc.

Lactose intolerance (adult-type hypolactasia, lactasenon-persistence) is a common trait worldwide, whichvaries between populations, both ethnically and geo-graphically. The activity of the lactase enzyme, whichfacilitates the digestion of milk-derived lactose,decreases after weaning in most humans, but persists insome; when it does not persist, lactose intoleranceresults. The prevalence of adult-type lactose intoleranceamong Caucasian populations in Europe varies between3 and 70%, in contrast with up to 100% among Asians(Sahi, 1994; Swallow, 2003). European lactase persist-ence is associated with a single nucleotide polymorphism(SNP), 13910 base pairs (bp) upstream of the lactasegene, in one of the introns of the MCM6 gene (Enattahet al., 2002), which appears to function also as a regula-tory region for lactase transcription (Olds and Sibley,2003; Troelsen et al., 2003). The CC213910 genotype isassociated with lactase non-persistent, CT213910 andTT213910 genotypes with lactase persistent phenotypes(Enattah et al., 2002).Several hypotheses have been proposed to explain the

geographic distribution of lactase persistence. The mostcommonly acknowledged view is the ‘‘gene-culture co-evolution/culture-historical’’ hypothesis, which supposesthat lactase persistence most likely originated as amutation in populations which used milk or milk prod-ucts from domesticated animals as an important sourceof adult nutrition (Simoons, 1970; Beja-Pereira et al.,2003). A strong positive selection is then supposed tohave occurred for the lactase persistent phenotype, fromthe ancestral lactase non-persistent phenotype, around

5,000–10,000 years ago (Bersaglieri et al., 2004). An al-ternative, the ‘‘reverse cause’’ hypothesis, is that somepopulations were pre-adapted for the use of milk as anadult food, having acquired a substantial frequency ofthe T213910 allele by random drift, before they took updairy farming (Burger et al., 2007).The population we are concerned with here, Hungar-

ian pastoralist nomads, after a period of migration frommuch further east, entered Europe as seven major tribesthat invaded the Carpathian basin from across the encir-cling mountains around 895 AD. The Hungarian lan-guage belongs to the Finno-Ugric branch of the Uralicfamily, a diverse group, living in Northern Europe andin the regions west and east of the Ural. During the

Additional Supporting Information may be found in the onlineversion of this article.

Grant sponsor: Hungarian National Research and DevelopmentPrograms; Grant numbers: OM-00050/2004.

*Correspondence to: Dora Nagy, Institute of Genetics, BiologicalResearch Centre of Hungarian Academy of Sciences, POB 521,Szeged H-6701, Hungary. E-mail: [email protected]

Received 31 August 2010; accepted 13 December 2010

DOI 10.1002/ajpa.21490Published online 1 March 2011 in Wiley Online Library

(wileyonlinelibrary.com).

VVC 2011 WILEY-LISS, INC.

AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 145:262–269 (2011)

migration they might have been in contact with Finno-Ugric populations, Kazars, Petchenegs, Bolgars, Savirs,Kabars, and Alans living in the northwestern and south-western regions of the Ural and in the Caucasus. TheCarpathian basin had been settled for thousands ofyears before the arrival of the Hungarians, by Dacians,Romans, Sarmatians, Goths, Huns, Avars, Slavs, andmany others: it is probable that on the eve of the Hun-garian conquest the majority of the indigenous popula-tion were Slavic (Tomory et al., 2007).The aim of this study was to evaluate the prevalence

of C/T213910 genotypes in remains from the Hungarianpopulation of the 10th–11th centuries, as compared withcurrent Hungarian-speaking populations. Random sam-ples were evaluated, and our results are analyzed rela-tive to other populations, which are believed to havebeen in contact with Hungarians during the migratoryperiod, and after the settlement of the Hungarians inthe Carpathian basin.

MATERIALS AND METHODS

Present-day samples

In this study, unrelated adults from different partsof Hungary and Transylvania were involved, 181 Hungar-ians and 65 Seklers (or Szekely, an outlying minorityHungarian-speaking population in eastern Transylvania,supposedly descendants of one of the seven invadingtribes). The individuals gave their informed consent to thestudy, which was approved by the local ethical committee.

DNA extraction from present-day samples. GenomicDNA was extracted from the root section of individualhairs or buccal smears, using the Chelex-based method(Walsh et al., 1991).

Mitochondrial DNA (mtDNA) testing of present-daysamples. The hypervariable region I (HVSI) of the con-trol region and, if necessary, HVSII and coding regionsof the mtDNA were analyzed either by sequencing or bythe PCR-RFLP method to elicit the mtDNA haplogroupof each sample. The method was identical to thatdescribed by Tomory et al. (2007). Polymorph positions ofthe mtDNA were identified using the revised CambridgeReference Sequence (Andrews et al., 1999). The sampleswere mtDNA haplogrouped based on mutational pat-terns summarized by Tomory et al. (2007). All present-day Sekler samples were mitochondrially haplogrouped.Seventy-one of the 181 present-day Hungarian sampleswere randomly selected for mtDNA testing.

C/T213910 genotyping of present-day samples. Thederived cleaved amplified polymorphic sequence method(dCAPS) was used to analyze the genotypes. A restrictionenzyme recognition site including the SNP was introducedinto the PCR product by the forward primer containing amismatch to template DNA, using the dCAPS Finder 2.0program (http://helix.wustl.edu/dcaps/dcaps.html). Theforward primer was: 50-GGCAATACAGATAAGATAATGGAG-30; reverse primer: 50-CCTATCCTCGTGGAATGCAGG-30; mismatching nucleotide underlined (Nagy et al.,2009). PCR amplification was carried out in 40 ll reac-tions containing 13 Ampli Taq Gold Buffer, 6 lM of eachprimer, 200 lM of each deoxynucleotide-triphosphate(dNTP), 2.5 mM MgCl2, 20 ng DNA extract, and 1 UAmpliTaq Gold Polymerase (Applied Biosystems, CA). Theamplification protocol: 6 min at 948C, 35 cycles of 938C

for 45 s, 548C for 45 s, 728C for 60 s, and final extension at728C for 5 min.Seven microliter of the 119 bp-modified PCR product

was subjected to NlaIV and HinfI (Fermentas, Ontario,Canada) restriction enzyme digestions, with 5 U restric-tion enzyme and 13 reaction buffer in a reaction volumeof 20 ll, to detect the C213910 and T213910 alleles. NlaIVenzyme cleavage resulted in two fragments (96 bp and23 bp) for the C allele, while HinfI digestion yielded a97 bp and a 22 bp fragment for the T allele. Theenzyme-cleaved PCR products were run on 8% nativepolyacrylamide gel and visualized after ethidium bro-mide staining by UV transillumination with the UVPBioImaging System (Upland, CA).

Ancient samples

Forty-two bone samples in an excellent state of biomo-lecular preservation, originating from burials in theperiod of the Hungarian conquest were included in theanalysis. The samples, provided by the ArcheologicalInstitute of the Hungarian Academy of Sciences, had beenexcavated in cemeteries from the 10th–11th centuriesfrom different regions of the Carpathian basin (TableS1a). Both the burial sites and the bones were archaeolog-ically and anthropomorphologically well-defined. Thegroups we have called classical conquerors and com-moners were distinguished on the basis of the grave find-ings. Classical Hungarian conquerors were those exca-vated from rich graves, containing a horse skull, harness,arrow- or spear-heads, mounted belts, braided ornamentsand earrings. Commoners were found in graves withimpoverished burial remains. All 42 ancient bone sampleshad previously yielded reproducible mtDNA [bone sam-ples were identical to those analyzed by Tomory et al.(2007) and to not yet published samples; Table S1a]. Proc-essing of ancient samples was carried out according to thestudy of Kalmar et al. (2000), Tomory et al. (2007), andCsanyi et al. (2008).

Bone powdering and DNA extraction from ancientsamples. The surface of the bones (femurs) was washedwith diluted bleach and distilled water and was treatedwith UV-C irradiation at 1 J/cm2 for 30 min. A 2 cm 3 3cm portion was cut from each bone epiphysis and thesurface of these portions was washed with bleach andremoved (at least 2–3 mm deep) with a UV-C treatedsterilized sand disk. The bone portion was then treatedat each side with UV-C light at 1 J/cm2 for 30 min andground into fine powder by using mineralogy mill(Retsch MM301; Haan, Germany) and then stored insterile tubes at 48C. Bone powder (1.3–1.5 g) was sus-pended in 10 ml EDTA and incubated overnight at 378Cwith continuous vertical rotation. The samples were cen-trifuged (2000g for 15 min), EDTA was removed and thesediment was resuspended in 10 ml EDTA everyday for3–5 days. The sediment was suspended in 1.8 ml ofextraction buffer, incubated overnight at 378C with con-tinuous vertical rotation, and centrifuged at 12,000 rpmfor 10 min. The supernatant, containing the DNA, isstored at 2208C.

DNA isolation from ancient samples. Standard isola-tion methods were used as described by Kalmar et al.(2000) and alternatively when needed, a modifiedmethod incorporating the DNeasy Tissue Kit (Qiagen,Valencia, CA) was used. In this modified method, DNAwas isolated from 350-ll bone extract, by treatment with

263LACTASE PERSISTENCE IN ANCIENT AND RECENT HUNGARIANS

American Journal of Physical Anthropology

350 ll 4 M NH4-acetate and 700 ll 96% EtOH at 2708Cfor 10 min. The mixture was transferred into DNeasyMini spin column and centrifuged at 6000g for 1 min.The column was washed twice and DNA was eluted in afinal volume of 40 ll (Tomory et al., 2007).

Mitochondrial DNA (mtDNA) testing of ancientsamples. All ancient samples were previously tested formtDNA haplogroups (Tomory et al., 2007; Table S1b).

C/T213910 genotyping of ancient samples. After thesuccessful and contamination-free mtDNA amplification,C/T213910 genotyping was carried out on the ancient sam-ples. The primers and restriction enzymes were identicalto those used in the reactions of the present-day samples.The standard amplification reaction of the ancient

samples contained 10 ll of bone isolation, 200 lM ofeach of the dNTPs, 25 pmol of each primer, 2.5 mMMgCl2, 13 Colorless GoTaq Flexi Buffer, and 1.25 UGoTaq Hot Start Polymerase (Promega, Wisconsin) in atotal reaction mixture volume of 50 ll. The amplificationprotocol was: 948C for 6 min, 10 cycles of 30 s at 938C, 40 sat 568C, 40 s at 728C, 40 cycles of 30 s at 938C, 40 s at548C, 40 s at 728C, and a final extension at 728C for 5 min.Twenty of the 42 ancient samples yielded DNA in the

PCR reaction. The remaining 22 samples were subjectedto a modified improved primer extension preamplifica-tion (mIPEP) method (Hanson et al., 2005; Csanyi et al.,2008) to enhance the efficiency of the amplification. Tenmicroliter aliquots of mIPEP products were used insubsequent C/T213910 genotyping. Three further ancientsamples were successfully typed (Table S1b).Ten microliter of the PCR product was subjected to

restriction enzyme digestions as described in connectionwith the present-day samples (Fig. S1).

Contamination prevention and authentication

To prevent any possible contamination with modernDNA, strict precautions were taken during each step ofthe ancient sample preparation, as described by Tomoryet al. (2007). The 11 persons, who participated in thesample processing or worked in the labs, were mtDNAtested and C/T213910 genotyped (Table S2). The numberof persons involved in the processing was minimized asmuch as possible in order to prevent contamination. Allsteps of sample processing (bone powdering, DNA extrac-tion, preamplification, amplification and post-PCR analy-sis) were carried out wearing appropriate protectiveclothing (gloves, face mask, hair net, glasses, and labora-tory coats) in separate rooms dedicated for ancient DNAwork and free of other molecular work. All workspacesand appliances were cleaned with bleach and subse-quently irradiated with 1 J/cm2 UV-C light for 2 h beforeuse. All solutions used were filtered and subsequentlyirradiated with UV-C light for 30 min. During all stepsUniversal Fit Filter Tips (Corning Incorporated, Lowell,MA) were used for pipetting. PCR and Eppendorf tubeswere sterilized before use by autoclaving.The surface of the bone samples was cleaned and

removed as described above in order to prevent possiblecontamination. Bones were powdered independently by atleast two researchers with different mtDNA haplogroupsand C/T213910 genotypes, at least two times each. In eachcase, two independent DNA extractions were carried out,and at least two successful mIPEP and/or PCR amplifica-tions were performed from each extract and/or mIPEPproduct to assess the reproducibility and authenticity of

the results. Those samples were accepted that had con-sistent results in all successful PCR reactions and had dif-ferent results from the haplogroup and genotype resultsof the researchers who analyzed the samples. Extraction,preamplification, amplification, and digestion blanks(with no bone powder, template DNA) were used as nega-tive controls in each reaction to screen for contaminantsentering the process at any stage. Positive controls (CC,CT, and TT213910) were also used in each digestion.Due to DNA degradation, primers were designed to

amplify short sequences of templates during mtDNAtesting (Tomory et al., 2007) and C/T213910 genotyping(119bp-long PCR product).To prove the authenticity of ancient human DNA fur-

ther, DNA was isolated from an ancient horse remain,excavated from one of the human burial sites, andamplified with both the horse-specific (forward: 50-CACCATACCCACCTGACATGCA-30 and reverse: 50-GCTGATTTCCCGCGGCTTGGTG-30) and the human-specificC/T213910 primers. Only the horse-specific primersresulted in amplification product.

Statistical analysis

The GraphPad Prism version 4.00 for Windows soft-ware (GraphPad Software, San Diego, CA) was used.The Fisher exact test was performed to compare the C/T213910 genotypes in the ancient and present-day popula-tions. Deviation from Hardy-Weinberg equilibrium wascalculated (Rodriguez et al., 2009) in present-day andancient Hungarian populations concerning the C/T213910

genotypes. A probability level P \ 0.05 was consideredto be statistically significant.

RESULTS

The genotyping of the C/T213910 autosomal SNP wassuccessful in 23 ancient bone samples (13 classical con-querors, nine commoners, and one not determined). TheC/T213910 genotype, and the mtDNA haplogroup resultsand the features of the bone samples are shown in TableS1a and S1b. The prevalence of the C/C213910, C/T213910,

and T/T213910 genotypes among the 23 ancient Hungar-ians was 87%, 4%, and 9% (Table 1); as compared with39%, 50%, and 11% among 181 present-day Hungarians;and 29%, 62%, and 9% among 65 present-day Seklers(Table 2). The allele frequencies associated with lactasepersistence (T213910) in the groups of ancient, present-day Hungarians and present-day Seklers were 10.9%,35.9%, and 40%, respectively. Although all 13 classicalconquerors had C/C213910 genotype, three of the com-moners displayed C/T213910 (11%) and T/T213910 geno-types (22%) (Table 1). The T213910 allele frequency was28% among the commoners.The additional mtDNA testing identified six major

mtDNA haplogroups (H, U, T, N1a, JT, X) among Hun-garian conquerors, six among commoners from the timeof the conquest (H, HV, M, R, T, U) and 13 (H, HV, I, J,K, JT, M, R, T, U, V, W, X) among present-day Hungar-ian-speaking populations. The three ancient sampleswith a lactase persistent genotype were all commonersand all displayed haplogroup H, which is the most com-mon in Europe (Richards et al., 1998). Two of these sam-ples showed TT213910 genotype. Although they both dis-played haplogroup H, their mutations in the HVSII andcoding regions of the mtDNA were not identical whichexcludes their maternal relationship. They were buried

264 D. NAGY ET AL.


in graves of different location and time which may fur-ther support that these individuals were not related toeach other (Table S1a and S1b). Haplogroup N1a (indica-tive of a Near Eastern, Asian origin—Haak et al., 2005)and haplogroup M (indicative of an Asian origin—Maca-Meyer et al., 2001) were present in 9% and 4% of theancient Hungarians, but absent or very rare in the pres-ent-day Hungarian and Sekler populations. The highprevalence of haplogroup U (23% of the ancient sam-ples), and especially the haplogroup U4, is characteristicof Finno-Ugric populations and populations in south-eastern Europe and western Siberia (Richards et al.,1998; Bermisheva et al., 2002) (Table S3).Significant difference was found in the C/T213910

genotypes and allele frequencies between the ancient

Hungarian conquerors and the present-day Hungarian-speaking populations. No significant difference wasfound between the present-day Hungarian-speaking pop-ulations and ancient Hungarian commoners (Table 3).Table S4 presents the frequencies of the T213910 allele,

digesters and non-digesters in present-day populations ofthe Uralic linguistic family and populations which accord-ing to historical accounts (Vekony, 2002) might well havebeen in contact with ancient Hungarians during theirwestward migratory period, and after the settlement ofthe Hungarians in the Carpathian basin. Present-dayHungarian-speaking populations exhibit a similar preva-lence of the T213910 allele to those in neighboring coun-tries, such as Austria, the Czech Republic, Slovenia, andGermany (Hogenauer et al., 2005; Gerbault et al., 2009;

TABLE 1. Distribution of mtDNA haplogroups and C/T213910 genotypes among ancient Hungarians

Number of classicalconquerors (%)

Number ofcommoners (%)

Total number ofancient Hungarians (%)

CC213910 CT213910 TT213910 Total CC213910 CT213910 TT213910 Total CC213910 CT213910 TT213910 Total

mtDNAhaplogroup

13 (100) 0 0 13 (100) 6 (67) 1 (11) 2 (22) 9 (100) 20 (87) 1 (4) 2 (9) 23 (100)

H 4 0 0 4 (31) 0 1 2 3 (33.5) 4 1 2 7 (31)U 3 0 0 3 (23)a 2 0 0 2 (22.5)b 5 0 0 5 (23)T 2 0 0 2 (15)c 1 0 0 1 (11)d 3 0 0 3 (13)N1a 2 0 0 2 (15) 0 0 0 0 2 0 0 2 (9)JT 1 0 0 1 (8) 0 0 0 0 1 0 0 1 (4)X 1 0 0 1 (8) 0 0 0 0 1 0 0 1 (4)HV 0 0 0 0 1 0 0 1 (11) 1 0 0 1 (4)R 0 0 0 0 1 0 0 1 (11) 1 0 0 1 (4)M 0 0 0 0 1 0 0 1 (11) 1 0 0 1 (4)J 0 0 0 0 0 0 0 0 1e 0 0 1 (4)

mtDNA, mitochondrial DNA. Further types were identified within haplogroup U:a U4 in two cases,b U3 and U5a1 in one case each; haplogroup T:c T3 in 1 case,d T2 in 1 case; haplogroup J:e J2 in 1 case, the social status of this sample was not classified.

TABLE 2. Distribution of mtDNA haplogroups and C/T213910 genotypes among present-day Hungarian-speaking populations

Number of present-day Hungarians (%) Number of present-day Seklers (%)

CC213910 CT213910 TT213910 Total CC213910 CT213910 TT213910 Total

71 (39) 90 (50) 20 (11) 181 (100)a 19 (29) 40 (62) 6 (9) 65 (100)a

mtDNA haplogroup 30 (42) 37 (52) 4 (6) 71 (100)b 19 (29) 40 (62) 6 (9) 65 (100)b

H 13 15 1 29 (41) 6 16 2 24 (36)U 0 6 0 6 (8)c 2 8 1 11 (16.5)d

T 3 2 0 5 (7)e 4 6 1 11 (16.5)f

J 5 3 0 8 (11)g 1 3 1 5 (8)h

K 4 3 0 7 (10) 3 4 0 7 (11)V 2 1 1 4 (6) 0 0 0 0HV 2 0 1 3 (4.2) 2 0 0 2 (3)W 0 3 0 3 (4.2) 0 1 1 2 (3)R 1 2 0 3 (4.2) 0 0 0 0X 0 2 0 2 (3) 1 0 0 1 (2)M 0 0 1 1 (1.4) 0 0 0 0JT 0 0 0 0 0 1 0 1 (2)I 0 0 0 0 0 1 0 1 (2)

mtDNA, mitochondrial DNA.a Number of all C/T213910 genotyped samples.b Number of all mtDNA tested and C/T213910 genotyped samples. Further types were identified within haplogroup U:c U5, U5a, and U5b in one case each and U4 in two cases,d U3, U4, and U5a1a in one case each and U5a1 in five cases; haplogroup T:e T1 and T3 in one case each and T2 in two cases,f T2, T2b, T3, and T5 in one case each and T1a in five cases; haplogroup J:g J1 and J1a in one case each andh J2 in one case.



GLAD). In contrast, the group of all ancient Hungariansdisplayed a significantly lower prevalence. Although theprevalence of the T213910 allele in the subgroup of ancientcommoners was similar to that of the present-dayHungarians, North-west Russians, Austrians, Slovaians,Chechs, and Germans; in the subgroup of classical con-querors it corresponded well with the prevalence of Ob-Ugric present-day populations, such as Khantys, or Maris,and certain Central-Asian and Turkish populations(Lember et al., 1995; Kozlov et al., 1998; Enattah et al.,2007; Sun et al., 2007; GLAD) (Table S4).Significant deviation from Hardy Weinberg Equilib-

rium was calculated in the group of ancient Hungarians(P \ 0.001) and in the subgroups of commoners (P \0.05). No significant deviation was found in the present-day Hungarian population (P[ 0.10).

DISCUSSION

We succeeded in genotyping the C/T213910 autosomalSNP in an unprecedented number of ancient bone sam-ples. It has been proposed (Burger et al., 2007) thatallele determination by PCR of ancient samples may bemisleading on account of the possibility of allele dropout:the ancient DNA may be so sparse that random distribu-tion of the alleles in the PCR reaction mixture mayresult in only one allele being present. If this happens, aheterozygote would be falsely reported as a homozygote.However, this effect would only strengthen our conclu-sions. Dropout might remove both alleles from the reac-tion mixture, so nothing would appear in our dCAPS-PCR results. The much more probable loss of one allelefrom homozygotes would not affect the results. Randomloss of one allele or the other from heterozygotes wouldreduce the proportion of apparent heterozygotes, andincrease apparent homozygotes, both CC and TT. Butour analysis detected no TT homozygotes in classicalconquerors, and unusually few in commoners. If alleledropout has occurred, that would not affect the firstresult, and would imply that the true level of TT homo-zygotes in commoners is even lower than our estimate.Beside this explanation we made efforts to reduce thepossible allele dropout. High-quality and -quantity bonepowder was used to optimize DNA extraction; preampli-fication method (mIPEP) was applied to increase thequantity of template DNA before PCR reaction; the num-ber of amplification cycles were increased to reach thedetection limit of the machine without strong artifacts;and amplification results were concluded from several,consistent PCR reactions as it is suggested in studies on

ancient DNA by Burger et al. (1999) and Hummel(2003).To interpret our results correctly, we favor a multidisci-

plinary approach, including data on genetic testing ofmtDNA and Y chromosomes, Hungarian history, and directand indirect evidence from archaeology and ethnography.The ability to digest lactose into adulthood is one of

the traits that developed in mankind under strong selec-tion pressure (Bersaglieri et al., 2004). The selectionforces could have been the nutritional benefit, thewater and electrolyte contents of the milk or theimproved calcium absorption after milk consumption.The ‘‘gene-culture co-evolution’’ hypothesis (Simoons,1970; McCracken, 1971; Holden et al., 2002) postulatesthat the selection force was the nutritional benefit inthose nomad pastoral populations which originally usedprocessed, lactose-low milk products (cheese, yoghurt,kumis, etc) for which the lactase persistent allele wasnot advantageous, but who subsequently had strongselective pressure for those who could also drink rawmilk. Such selection appears to have operated substan-tially later than the earliest domestication of ruminantsin the Middle East, around 8000 BC, and later than thefirst use of milk, as assessed via the d13C values of fattyacids in pottery vessels; that is, around 7000 BC in theNear East and south-eastern Europe (Evershed et al.,2008). Though only small-scale dairying existed between7000 and 4000 BC, it increased in the period 4000–3000BC (Craig et al., 2005). Beja-Pereira et al. (2003) haveargued that the ability for adults to consume milk co-evolved with the ability of dairy cattle to give high milkyields; and that adult lactose tolerance evolved in north-ern Neolithic Europe, where the relatively cool climateallows unfermented milk to be stored for a while.There is good evidence that Early Neolithic farmers

lacked adult lactose tolerance. Burger et al. (2007) con-cluded that the lactase persistent T213910 allele was notobserved in human remains from European Neolithicand Mesolithic sites (5840-2267 BC), whereas it was inone Mediaeval sample (AD 400-600). Population studies(Enattah et al., 2007) are consistent with the T213910 al-lele having arisen twice, once between 5 and 12 thou-sand years ago in the haplotype which is now dominantin Europe, and once 1400–3000 years ago in a regionnorth of the Caucasus and west of the Urals. Itan et al.(2009) used a demic computer simulation model toexplore the spread of European lactase persistence, andconcluded that it originated in central Europe, in aregion including modern Hungary, about 5500 BC. Themolecular evidence, then, is in favor of gene/culture co-evolution, not of the reverse-cause hypothesisHow do our results fit into this picture? Present-day

Hungarian-speaking populations and ancient commonersexhibit a similar prevalence of the T213910 allele to thosein neighboring countries, such as Austria, the CzechRepublic, the Ukraine, and Slovenia. In contrast, the an-cient classical conquerors displayed a significantly lowerprevalence, which corresponds well with those of pres-ent-day populations of the Uralic linguistic family, suchas the Khantys, Mansis and Maris, and certain Central-Asian and Turkish populations (Table S4). This is con-sistent with the original Hungarian invaders, havingroots far to the east of modern Hungary, being a minor-ity in the Carpathian basin, further diluted in the subse-quent turbulent history of that area. This is supportedby previous Hungarian studies on mitochondrial hap-logroups and Y chromosome (Tomory et al., 2007; Csanyi

TABLE 3. Comparison of the distribution of C/T213910

genotypes and allele frequencies in present-day and ancientHungarian-speaking populations

Present-dayHungarians

Present-daySeklers

Classicalconquerors

All ancient HungariansGenotype P\ 0.0001 P\ 0.0001 –Allele frequency P 5 0.0004 P 5 0.0002 –

Classical conquerorsGenotype P\ 0.0001 P\ 0.0001 –Allele frequency P\ 0.0001 P\ 0.0001 –

CommonersGenotype P 5 0.1617 P 5 0.0537 P 5 0.0545Allele frequency P 5 0.6169 P 5 0.4399 P 5 0.0079

P\ 0.05 is considered to be statistically significant.

266 D. NAGY ET AL.


et al., 2008), which showed the genetic assimilation ofthe present-day Hungarians with their geographicalneighbors, but also a significant Asian influence on thegenetics of the Hungarian conquerors, from their Sibe-rian origins and from the genetic effect of those popula-tions with whom the ancient Hungarians came intocontact during their westwards migrations, such as theKazars, Petchenegs, Bolgars, Savirs, and Iranians.Among the archaeological remains from the period of

the Hungarian conquest, the absence of the T213910 allelein the classical conquerors, but not in the commoners,might suggest a hierarchical difference in nutrition. Itmight also be due to some of the commoners beingdescendants of the pre-existing inhabitants of the Carpa-thian basin, who lived a settled life and had a differentdairy culture from that of the Hungarian conquerors.There is some evidence for a change in stockraising

practices at the time of the Hungarian conquest. Inarchaeological sites in the Carpathian basin from theSarmatian (1st–4th centuries AD), Slavic and Avar (5th–9th centuries AD), and Hungarian conquest period (9th–10th centuries AD) cattle remains dominated animalbone assemblages; but during the Hungarian conquest, asmall increase occurred in the number of horse bonesand a small decrease in the number of cattle bones. Thefragmentation of the bones indicated a butchering pro-cess; the change could therefore be due to differentialconsumption of horse and cattle meat (Batrosiewicz,2003). Nevertheless, milk products must have beenrenewable sources of food for the ancient Hungarian pas-toralists; mobile pastoralists collect milk from theirherds, including mares (Batrosiewicz, 2003; Outram etal., 2009).The absence of adult lactose tolerance in the ancient

Hungarians we have studied is compatible with theirmilking their herds, given milk fermentation (Myles etal., 2005; Ingram et al., 2009). While fresh milk was thebasic dairy food in Scandinavia, a situation in accordwith the cold climate and good sanitation, in south-east-ern Europe and in south-western Asia processed foodsprepared from soured milk were preferred. Milk was lessappetizing as a fresh warm beverage than as fermentedfoods (Kosikowski, 1981; Outram et al., 2009). Thelactose content of fresh milk is 4.42–5.15 g/g% incattle (Cerbulis et al., 1974; Miglior et al., 2006), 4.66–4.82 g/g%in the goat (Baldi et al., 2002; Contreras et al.,2009), 4.57–5.40 g/g% in the sheep (Fuertes et al., 1998;Addis et al., 2005), and 6.91–7.04 g/g% in the horse(Caroprese et al., 2007). Since the lactose content can bereduced by 50–60% by bacterial fermentation (Kilara etal., 1975), processed milk products have no or low lactosecontents (ranging 0–3.7 g/g%). Lactose malabsorbersreported fewer or no symptoms after consuming fer-mented milk products (Alm, 1982; Savaiano et al., 1987).Such processing of milk is of considerable antiquity, tojudge by the archaeological evidence. The presence ofdegraded milk fats (high abundances of C16:0 and C18:0

fatty acids) and lipid pyrolysis products (mid- and long-chain ketones) in some archaeological ceramics suggeststhat the dairy products were heated, perhaps as part oftheir processing (Craig et al., 2005; Evershed et al.,2008). This is supported by the fact that a high fre-quency of ruminant milk lipids has been detected fromsuch ceramics, whereas raw milk lipids (high abundan-ces of C4:0 to C12:0 fatty acids) are rapidly destroyed byburial (Evershed et al., 2008; Copley et al., 2003). Hun-garian pastoralists, averse to drinking fresh milk, would

have had little or no selection for lactose tolerance inearlier millennia.Alternatively, the low prevalence of European-type

lactase persistence in ancient Hungarians may be due totheir having one or more non-European polymorphismsin the lactase regulatory regions. Recent studies sum-marized by Ingram et al. (2007), Enattah et al. (2008),and by Itan et al. (2010), have revealed other alleles thatcan also produce adult lactose tolerance: G213907,G213915, and C214010 in East African populations, a com-pound G213915/C23712 allele in Saudi and other MiddleEastern populations. Some cases of undoubted lactosetolerance have not yet been identified with anyknown allele in the control regions sequenced (Itan etal., 2010); notably, lactose tolerant northern Chinesewhose closeness to the central Asian steppe may allowsome common ancestry with early Hungarians lack theT213910 allele, and must have some other unidentifiedallele instead (Sun et al., 2007). It is therefore possiblethat ancient Hungarians were lactose tolerant despitetheir lack of the characteristic European allele. In viewof the fairly high proportion of European Y chromosomeand mitochondrial haplogroups among Hungarian con-querors (Tomory et al., 2007; Csanyi et al., 2008), that isperhaps unlikely: and in view of our ignorance of thenon-European allele that might be involved, it is prema-ture to search for it in ancient Hungarian samples.In conclusion, in ancient Hungarians, the T213910 allele

was present only in 11% of the population, and exclusivelyin commoners of European mitochondrial haplogroupswho may have been of pre-Hungarian indigenous ances-try. This is despite animal domestication and dairy prod-ucts having been introduced into the Carpathian basinearly in the Neolithic Age. This anomaly may be explainedby the Hungarian use of fermented milk products, theirgreater consumption of ruminant meat than milk, cul-tural differences, or by their having other lactase-regulat-ing genetic polymorphisms than C/T213910. The low preva-lence of lactase persistence provides additional informa-tion on the Asian origin of Hungarians. Present-dayHungarians have been assimilated with the surroundingEuropean populations, since they do not differ signifi-cantly from the neighboring populations in their posses-sion of mtDNA and C/T213910 variants.

ACKNOWLEDGMENTS

The authors thank Mrs. Maria Rado and Mrs. Gabri-ella Leho†cz for their skilled technical assistance, andBalazs Mende and Peter Lango for the archaic samples.

LITERATURE CITED

Addis M, Cabiddu A, Pinna G, Decandia M, Piredda G, Pirisi A,Molle G. 2005. Milk and cheese fatty acid composition insheep fed Mediterranean forages with reference to conjugatedlinoleic acid cis-9, trans-11. J Dairy Sci 88:3443–3454.

Alm L. 1982. Effect of fermentation on lactose, glucose, and gal-actose content in milk and suitability of fermented milk prod-ucts for lactose intolerant individuals. J Dairy Sci 65:346–352.

Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turn-bull DM, Howell N. 1999. Reanalysis and revision of the Cam-bridge reference sequence for human mitochondrial DNA. NatGenet 23:147.

Anthoni SR, Rasinpera HA, Kotamies AJ, Komu HA, Pihlaja-maki HK, Kolho KL, Jarvela IE. 2007. Molecularly definedadult-type hypolactasia among working age people with refer-



ence to milk consumption and gastrointestinal symptoms.World J Gastroenterol 13:1230–1235.

Baldi A, Modina S, Cheli F, Gandolfi F, Pinotti L, Baraldi ScesiL, Fantuz F, Dell’Orto V. 2002. Bovine somatotropin adminis-tration to dairy goats in late lactation: effects on mammarygland function, composition and morphology. J Dairy Sci85:1093–1102.

Bartosiewicz L. 2003. A millennium of migrations: proto-historicmobile pastoralism in Hungary. Bull Fla Mus Nat Hist44:101–130.

Beja-Pereira A, Luikart G, England PR, Bradley DG, Jann OC,Bertorelle G, Chamberlain AT, Nunes TP, Metodiev S, Fer-rand N, Erhardt G. 2003. Gene-culture coevolution betweencattle milk protein genes and human lactase genes. NatGenet 35:311–313.

Bermisheva M, Tambets K, Villems R, Khusnutdinova E. 2002.Diversity of mitochondrial DNA haplotypes in ethnic popula-tions of the Volga-Ural region of Russia. Mol Biol (Mosk)36:990–1001.

Bersaglieri T, Sabeti PC, Patterson N, Vanderploeg T, SchaffnerSF, Drake JA, Rhodes M, Reich DE, Hirschhorn JN. 2004.Genetic signatures of strong recent positive selection at thelactase gene. Am J Hum Genet 74:1111–1120.

Burger J, Hummel S, Herrmann B, Henke W. 1999. DNA pres-ervation: a microsatellite-DNA study on ancient skeletalremains. Electrophoresis 20:1722–1728.

Burger J, Kirchner M, Bramanti B, Haak W, Thomas MG. 2007.Absence of the lactase-persistence-associated allele in earlyNeolithic Europeans. PNAS 104:3736–3741.

Caroprese M, Albenzio M, Marino R, Muscio A, Zezza T, Sevi A.2007. Behavior, milk yield, and milk composition of machineand hand-milked Murgese mares. J Dairy Sci 90:2773–2777.

Cerbulis J, Farrell HM Jr. 1974. Composition of milks of dairycattle. I. Protein, lactose, and fat contents and distribution ofprotein fraction. J Dairy Sci 58:817–827.

Contreras A, Paape MJ, Miller RH, Corrales JC, Luengo C, San-chez A. 2009. Effect of bromelain on milk yield, milk composi-tion and mammary health in dairy goats. Trop Anim HealthProd 41:493–498.

Copley MS, Berstan R, Dudd SN, Docherty G, Mukherjee AJ,Straker V, Payne S, Evershed RP. 2003. Direct chemical evi-dence for widespread dairying in prehistoric Britain. PNAS100:1524–1529.

Craig OE, Chapman J, Heron C, Willis LH, Bartosiewicz L, TaylorG, Whittle A, Collins A. 2005. Did the first farmers of centraland eastern Europe produce dairy foods? Antiquity 79:882–894.

Csanyi B, Bogacsi-Szabo E, Tomory G, Czibula A, Priskin K,Cso†sz A, Mende B, Lango P, Csete K, Zsolnai A, Conant EK,Downes CS, Rasko I. 2008. Y-chromosome analysis of ancientHungarian and two modern Hungarian-speaking populationsfrom the Carpathian Basin. Ann Hum Genet 72:519–534.

Czeizel A, Flatz G, Flatz SD. 1983. Prevalence of primary adultlactose malabsorption in Hungary. Hum Genet 64:398–401.

Enattah NS, Jensen TG, Nielsen M, Lewinski R, Kuokkanen M,Rasinpera H, El-Shanti H, Seo JK, Alifrangis M, Khalil IF,Natah A, Ali A, Natah S, Comas D, Mehdi SQ, Groop L,Vestergaard EM, Imtiaz F, Rashed MS, Meyer B, Troelsen J,Peltonen L. 2008. Independent introduction of two lactase-per-sistence alleles into human populations reflects different his-tory of adaptation to milk cultures. Am J Hum Genet 82:57–72.

Enattah NS, Sahi T, Savilahti E, Terwilliger JD, Peltonen L,Jarvela I. 2002. Identification of a variant associated withadult-type hypolactasia. Nat Genet 30:233–237.

Enattah NS, Trudeau A, Pimenoff V, Maiuri L, Auricchio S,Greco L, Rossi M, Lentze M, Seo JK, Rahgozar S, Khalil I,Alifrangis M, Natah S, Groop L, Shaat N, Kozlov A, Verschub-skaya G, Comas D, Bulayeva K, Mehdi SQ, Terwilliger JD,Sahi T, Savilahti E, Perola M, Sajantila A, Jarvela I, PeltonenL. 2007. Evidence of still-ongoing convergence evolution of thelactase persistence T213910 alleles in humans. Am J HumGenet 81:615–625.

Evershed RP, Payne S, Sherratt AG, Copley MS, Coolidge J,Urem-Kotsu D, Kotsakis K, Ozdogan M, Ozdogan EA, Nieu-wenhuyse O, Akkermans PMMG, Bailey D, Andeescu RR,

Campbell S, Farid S, Hodder I, Yalman N, Ozbas�aran M,Bicakci E, Garfinkel Y, Levy T, Burton MM. 2008. Earliestdate for milk use in the Near East and southeastern Europelinked to cattle herding. Nature 455:528–531.

Flatz G, Henze HJ, Palabiyikoglu E, Dagalp K, Turkkan T.1986. Distribution of the adult lactase phenotypes in Turkey.Trop Geogr Med 38:255–258.

Fuertes JA, Gonzalo C, Carriedo JA, San Primitivo F. 1998. Pa-rameters of test day milk yield and milk components for dairyewes. J Dairy Sci 81:1300–1307.

Gerbault P, Moret C, Currat M, Sanchez-Mazas A. 2009. Impact ofselection and demography on the diffusion of lactase persistence.PLoS One 4:3:6369. doi: 10.1371/journal.pone.0006369.

Global Lactase Persistence Association Database: GLAD website: http://www.ucl.ac.uk/mace-lab/GLAD/

Haak W, Forster P, Bramanti B, Matsumura S, Brandt G,Tanzer M, Villems R, Renfrew C, Gronenborn D, Alt KW, Bur-ger J. 2005. Ancient DNA from the first European farmers in7500-year-old Neolithic sites. Science 310:1016–1018.

Hanson EK, Ballantyne J. 2005. Whole genome amplificationstrategy for forensic genetic analysis using single or few cellequivalents of genomic DNA. Anal Biochem 346:246–257.

Helgason A, Hickey E, Goodacre S, Bosnes V, Stefansson K,Ward R, Sykes B. 2001. mtDNA and the islands of the NorthAtlantic: estimating the proportions of Norse and Gaelicancestry. Am J Hum Genet 68:723–737.

Holden C, Mace R. 2002. Pastoralism and the evolution of lac-tase persistence. In: Leonard WR, Crawford MH, editors. Thehuman biology of pastoral populations. Cambridge: Cam-bridge University Press. p 280–307.

Hogenauer C, Hammer HF, Mellitzer K, Renner W, Krejs GJ,Toplak H. 2005. Evaluation of a new DNA test compared withthe lactose hydrogen breath test for the diagnosis of lactasenon-persistence. Eur J Gastroenterol Hepatol 17:371–376.

Hummel S. 2003. DNA markers. In: Ancient DNA typing: meth-ods, strategies and applications. Berlin, Heidelberg: Springer-Verlag. p 19–41.

Ingram CJ, Elamin MF, Mulcare CA, Weale ME, Tarekegn A,Raga TO, Bekele E, Elamin FM, Thomas MG, Bradman N,Swallow DM. 2007. A novel polymorphism associated withlactose tolerance in Africa: multiple causes for lactase persist-ence? Hum Genet 120:779–788.

Ingram CJE, Mulcare CA, Itan Y, Thomas MG, Swallow DM.2009. Lactose digestion and the evolutionary genetics of lac-tase persistence. Hum Genet 124:579–591.

Itan Y, Jones BL, Ingram CJ, Swallow DM, Thomas MG. 2010.A worldwide correlation of lactase persistence phenotype andgenotypes. BMC Evol Biol 10:36.

Itan Y, Powell A, Beaumont MA, Burger J, Thomas MG. 2009.The origins of lactase persistence in Europe. PLoS ComputBiol 5:e1000491. doi: 10.1371/journal.pcbi.1000491.

Kalmar T, Bachrati CZ, Marcsik A, Rasko I. 2000. A simple andefficient method for PCR amplifiable DNA extraction from an-cient bones. Nucleic Acids Res 28:E67.

Khabarova Y, Torniainen S, Nurmi H, Jarvela I, Isokoski M,Mattila K. 2009. Prevalence of lactase persistent/non-persis-tent genotypes and milk consumption in a young populationin north-west Russia. World J Gastroenterol 15:1849–1853.

Kilara A, Shahani KM. 1975. Lactase activity of cultured andacidified dairy products. J Dairy Sci 59:2031–2035.

Kosikowski FW. 1981. Dairy foods of the world—evolution,expansion and innovation. J Dairy Sci 64:996–1007.

Kozlov AI, Balanovskaia EV, Nurbaev SD, Balanovskii OP.1998. Genogeographic primary hypolactasia in the Old Worldpopulations. Genetika 34:551–561.

Lember M, Tamm A, Piirsoo A, Suurmaa K, Kermes K, KermesR, Sahi T, Isokoski M. 1995. Lactose malabsorption in Khantsin western Siberia. Scand J Gastroenterol 30:225–227.

Lember M, Torniainen S, Kull M, Kallikorm R, Saadla P, RajasaluT, Komu H, Jarvela I. 2006. Lactase non-persistence and milkconsumption in Estonia. World J Gastroenterol 12:7329–7331.

Maca-Meyer N, Gonzalez AM, Larruga JM, Flores C, CabreraVM. 2001. Major genomic mitochondrial lineages delineateearly human expansions. BMC Genet 2:13.

268 D. NAGY ET AL.


McCracken RD. 1971. Lactase deficiency—example of dietaryevolution. Curr Anthropol 12:479.

Miglior F, Sewalem A, Jamrozik J, Lefebvre DM, Moore RK.2006. Analysis of milk urea nitrogen and lactose and theireffect on longevity in Canadian dairy cattle. J Dairy Sci89:4886–4894.

Myles S, Bouzekri N, Haverfield E, Cherkaoui M, Dugoujon J-M,Ward R. 2005. Genetic evidence in support of a shared Eura-sian-North African dairying origin. Hum Genet 117:34–42.

Nagy D, Bogacsi-Szabo E, Varkonyi A, Csanyi B, Czibula A,Bede O, Tari B, Rasko I. 2009. Prevalence of adult-type hypo-lactasia as diagnosed with genetic and lactose hydrogenbreath tests in Hungarians. Eur J Clin Nutr 63:909–912.

Olds LC, Sibley E. 2003. Lactase persistence DNA variantenhances lactase promoter activity in vitro: functional role asa cis regulatory element. Hum Mol Genet 12:2333–2340.

Outram AK, Stear NA, Bendrey R, Olsen S, Kasparov A, Zai-bert V, Thorpe N, Evershed RP. 2009. The earliest horse har-nessing and milking. Science 323:1332–1335.

Pakendorf B, Stoneking M. 2005. Mitochondrial DNA andhuman evolution. Annu Rev Genomics Hum Genet 6:165–183.

Pericic M, Barac Lauc L, Martinovic Klaric I, Janicijevic B,Rudan P. 2005. Review of Croatian genetic heritage asrevealed by mitochondrial DNA and Y chromosomal lineages.Croat Med J 46:502–513.

Richards MB, Macaulay VA, Bandelt HJ, Sykes BC. 1998. Phy-logeography of mitochondrial DNA in western Europe. AnnHum Genet 62:241–260.

Rodriguez S, Gaunt TR, Day INM. 2009. Hardy-Weinberg equi-librium testing of biological ascertainment for Mendelian ran-domization studies. Am J Epidemiol 169:505–514. DOI10.1093/aje/kwn359.

Sahi T. 1994. Genetics and epidemiology of adult-type hypolac-tasia. Scand J Gastroenterol Suppl 202:7–20.

Savaiano DA, Levitt MD. 1987. Milk intolerance and microbe-containing dairy foods. J Dairy Sci 70:397–406.

Semino O, Passarino G, Oefner PJ, Lin AA, Arbuzova S, BeckmanLE, De Benedictis G, Francalacci P, Kouvatsi A, Limborska S,Marcikiae M, Mika A, Mika B, Primorac D, Santachiara-Bener-ecetti AS, Cavalli-Sforza LL, Underhill PA. 2000. The geneticlegacy of Paleolithic Homo sapiens sapiens in extant Europeans:a Y chromosome perspective. Science 290:1155–1159.

Simoons FJ. 1970. Primary lactose intolerance and the milkinghabit: a problem in biological and cultural interrelations, II. Aculture historical hypothesis. Am J Dig Dis 45:489–497.

Sun HM, Qiao YD, Chen F, Xu LD, Bai J, Fu SB. 2007. The lac-tase gene -13910T allele cannot predict the lactase-persistencephenotype in north China. Asia Pac J Clin Nutr 16:598–601.

Swallow DM. 2003. Genetics of lactase persistence and lactoseintolerance. Annu Rev Genet 37:197–219.

Torroni A, Bandelt HJ, D’Urbano L, Lahermo P, Moral P, Sell-itto D, Rengo C, Forster P, Savontaus ML, Bonne-Tamir B,Scozzari R. 1998. mtDNA analysis reveals a major late Paleo-lithic population expansion from southwestern to northeast-ern Europe. Am J Hum Genet 62:1137–1152.

Tomory G, Csanyi B, Bogacsi-Szabo E, Kalmar T, Czibula A,Cso†sz A, Priskin K, Mende B, Lango P, Downes CS, Rasko I.2007. Comparison of maternal lineage and biogeographicanalysis of ancient and modern Hungarian populations. Am JPhys Anthropol 134:354–368.

Troelsen JT, Olsen J, Moller J, Sjostrom H. 2003. An upstreampolymorphism associated with lactase persistence hasincreased enhancer activity. Gastroenterology 125:1686–1694.

Vekony G. 2002. Magyar o†stortenet–Magyar honfoglalas. Buda-pest: Nap Kiado.

Walsh PS, Metzger DA, Higuchi R. 1991. Chelex 100 as a me-dium for simple extraction of DNA for PCR-based typing fromforensic material. Biotechniques 10:506–513.



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Comparison of lactase persistence polymorphism in ancient and present-day Hungarian populations