Genetic analysis on Tuoba Xianbei remains excavated from Qilang Mountain Cemetery in Qahar Right Wing Middle Banner of Inner Mongolia

FEBS Letters 580 (2006) 6242–6246

Genetic analysis on Tuoba Xianbei remains excavated from Qilang Mountain Cemetery in Qahar Right Wing Middle Banner of Inner Mongolia Yu Changchuna,b, Xie Lib, Zhang Xiaoleia, Zhou Huia,c,*, Zhu Honga a

Ancient DNA Laboratory, Research Center for Chinese Frontier Archaeology, Jilin University, Changchun 130012, P.R. China b College of Life Science, Jilin Normal University, Siping 136000, P.R. China c College of Life Science, Jilin University, Changchun 130012, P.R. China Received 17 July 2006; revised 5 October 2006; accepted 12 October 2006 Available online 20 October 2006 Edited by Takashi Gojobori

Abstract Sixteen sequences of the hypervariable segment I (HVS-I, 16039–16398) in mtDNA control region from ancient Tuoba Xianbei remains excavated from Qilang Mountain Cemetery were analyzed. In which, 13 haplotypes were found by 25 polymorphic sites. The haplotype diversity and nucleotide diversity were 0.98 and 0.0189, respectively, and the mean of nucleotide number differences was 6.25. Haplogroup analysis indicates these remains mainly belong to haplogroup C (31.25%) and D (43.75%). According to the published data were considered, we can suggest that the Tuoba Xianbei presented a close genetic affinity to Oroqen, Outer Mongolian and Ewenki populations, especially Oroqen.  2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

three southward migrations, and finally founded the Northern Wei Dynasty and controlled the northern region of China (386–534 A.D.). By the time of the Tang Dynasty they had largely merged with Han populace. At present, the origin of Tuoba Xianbei ethnic group and its impacts on modern minorities of northern China are not yet clear. It will be very helpful for understanding in the origin, formation and development process of northern minorities of China by analyzing the data of mtDNA in Tuoba Xianbeis. In this study, we examined the sequences of HVS-I from 16 Tuoba Xianbei remains excavated in Qilang Mountain Cemetery that was indicated in Fig. 1, and compared the data with those of relevant populations.

Keywords: Qilang Mountain; Tuoba Xianbei; Mitochondrial DNA

2. Materials and methods

1. Introduction The PCR and ancient DNA technique have made it possible to identify the trace amount of DNA which still survived in archaeological remains [1–3]. Among the most informative genetic systems, mitochondrial DNA is the preferred target for recovering DNA from archaeological remains and characterizing the structure of a gene pool, which well-solved many important questions, such as human origin, migration and evolution, as well as population affinities and so on [4–9]. In ancient China, the Xianbei was another significant nomadic population succeeded by Xiongnu, which actually consisted of a federation of sizeable non-Han groups, and the Tuoba Xianbei was a very important sub-tribe in Xianbei Alliance. The Tuoba Xianbeis are said to be the northern-most branch of the Xianbei nomads, originally dwelled to the northeastern-most of all Xianbei tribles, in the region of the Great Xianbei Mountains, which is now the Erguna River valley of the northern section of the Great Xing’an Range. The language spoken by Tuoba Xianbei was almost the same with Mongolic. According to Chinese historical documents, from the 1st to the 3rd centuries, the Tuoba tribe had undergone

* Corresponding author. Fax: +86 431 8498031. E-mail address: [email protected] (Z. Hui).

2.1. Sampling The thighbone samples from 18 ancient Xianbei remains, provided by Dr. Wei-Jian (Inner Mongolia Institute of Archaeology), which were excavated from Qilang Mountain Cemetery in Qahar Right Wing Middle Banner of Inner Mongolia from 1995 to 1996. During this period, 20 graves were excavated, and out of which 18 remains were collected (numbered ZQ01–18). According to Wei et al. [10], these remains occupied between 4th and 5th centuries A.D., belong to Tuoba tribe of Xianbei alliance. 2.2. Contamination controls All stages of the work were carried out in term of aDNA-specific requirements [11]. First, before beginning our work, 2 decontamination methods, such as irradiating the samples 2–3 h with UV-light irradiation (254 nm) and removing the sample surface 3–4 mm depth with dentistry drill, were employed to remove the surface DNA deriving from other workers who had handled the samples before they reached our laboratory; secondly, the preparation of bone powders and the extraction and amplification of DNA were carried out in separate dedicated laboratories, routing sterilized by treatment with DNase away (Molecular Bioproduct, San Diego, CA), bleach and UV-light irradiation (254 nm), and the gloves, face masks and full-body suits must be used during all the handling processes; thirdly, the blankextractions, zero-controls and negative-controls must be carried out during the extraction and amplification of DNA, and fourthly, all results must be identical in two independent extractions and two independent amplifications, and compared with the sequences from the handlers. 2.3. DNA extraction and amplification The method of Kalmar et al. [12] was used for the bone powder preparation. The manual protocol of GENECLEAN Kit for ancient DNA (BIO101, USA) was used for DNA extraction. As for PCR amplification, the technique of Adcock et al. [13] was adopted, and

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Y. Changchun et al. / FEBS Letters 580 (2006) 6242–6246

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Fig. 1. The location of Qahar Right Wing Middle Banner Qilang Mountain Cemetery.

modified slightly. Four sets of primers for PCR were L16035–16055/ 16162–H16142, L16112–16131/H16237–16218, L16166–16185/16307– H16286 and L16261–16281/H16398–16379. The PCR conditions were described as follow: initial denaturizing at 94 C for 3 min, followed by 40 cycles of denaturizing at 94 C for 1 min, annealing at 55 C for 45 s, and extension at 72 C for 1 min, followed by the final extension 10 min. The quality of amplification was estimated on a 2% agarose gel, and the PCR products were purified using ‘QIAEX II’ gel extraction kit (QIAGen, Germany). 2.4. Cloning and sequencing The PCR products were cloned using TOPO TA Cloning kit (Invitrogen) according to the manufacture’s instruction, which can be used for assessing for damage, contaminations and jumping PCR [11]. Screening of white recombinant colonies was accomplished by PCR. The colonies were transferred into a 30 ll reactions mix with 67 mM Tris–HCl (pH 8.8), 2 mM MgCl2, 1 lM of each primer (M13 forward and reverse universal primers), 0.125 mM of each dNTP, and 0.75 U of Taq polymerase. After 5 min at 92 C, 30 cycles of PCR (30 s at 90 C, 1 min at 50 C, 1 min at 72 C) were carried out, the clones with an insert of the expected size were identified by agarose-gel electrophoresis. After purification of these PCR products with Microcon PCR diveces (Amicon), a volume of 1.5 ll was cycle-sequenced using the BigDye Terminater cycle sequencing kit (Applied Biosystems). Six clones of the same PCR products were sequenced, and finally, the sequences were analyzed on an Applied Biosystems DNA 377 sequencer. 2.5. RFLP analysis In order to confirm the haplogroup status of Tuoba Xianbei samples, three restriction sites (AluI 5176, DdeI 10394 and AluI 10397) in coding region were further detected. The primers for AluI 5176 were cited from Batista et al. [14], and for DdeI 10394 and AluI 10397 came from Kolman et al. [15]. 2.6. Data analysis The editing of sequences was performed by Clustal X1.83. The final information used for data analysis about each individual was the HVSI of 360 bp, nucleotide positions 16039–16398. Fst genetic distances, Genetic diversity, the mean number of pairwise difference were com-

puted by Arlequin 2000. The net genetic distances and phylogenetic tree were carried out using MEGA 3.0. To visualize the Fst distances among populations, the multidimensional scaling (MDS) analysis was performed by SPSS 12.0. For comparison, the published data from populations of Evenk [16], Inner Mongolian, Daur, Oroqen and Ewenki [17], Outer Mongolian [15], Buryat [18], Kazak [19], Uzbek [20], Turkey [21], Southern Han (Zhangjiang Han and Wuhan Han [22]), Northern Han (Qingdao Han, Fengcheng Han [22] and Xi’an Han [23]) were used.

3. Results and discussion 3.1. Sequence diversity and the mean number of pairwise differences Sixteen sequences of HVS-I (position 16039–16398) were obtained from 18 Tuoba Xianbei remains. Of these 18 subjects, we could not obtained PCR products from ZQ07. The sequence from ZQ01 was excluded, because it was ambiguous by the clone test. Among the 16 sequences, 13 different hyplotypes were identified by 25 polymorphic sites relative to the reference sequence [24], and thereinto, ZQ02 and ZQ10, ZQ08 and ZQ12, as well as ZQ16 and ZQ17 shared the same haplotypes. The haplotype diversity and the mean number of pairwise differences are 0.98 and 6.25. According to Pakendorf et al. [9], the gene diversity of the Tuoba Xoanbai (0.98) fell into those of the steppe populations (around 0.99) and the Siberian populations (0.93–0.95). 3.2. Nucleotide diversity Of these 25 mutation sites, 24 were transitions, only 1 was the transversion (16183 A fi C). The ratio of transition:transversion is 24:1, which agrees with other data obtained from humans [25]. Among the transitions, there were a high proportion of C M T substitutions compared to A M G, and the

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+ +

+ +

+ + + + +

G – – – – A – – – – – – – – – – –

T C C C C C C C – – – – – + + C C

+ + + + + + + + + + + +

+ + + + + + + + + + + +

3.3. Haplogroup analysis According to Wallace et al. [26], in Asia, the haplogroups include A, B, C, D, E, F and G, etc. Haplogroups A and B lack both the site DdeI 10394 and AluI 10397, whereas C and D contain these sites. Comparing haplogroup G and D, D lack AluI 5176 site, and G has it. For haplogroup A, its characteristic mutation sites are at positions 16362 (T to C), 16319 (G to A), 16290 (C to T) and 16223 (C to T), for B, at 16217 (T to C) and 16189 (T to C), for C, at 16327 (C to T), 16298 (T to C) and 16223 (C to T), and for D, at 16362 (T to C) and 16223 (C to T). Together with Kivisild et al. [27], Kong et al. [17] and Yao et al. [22], we can conclude that the samples ZQ02, 05, 09, 10, 15, 16 and 17 belong to D, ZQ03, 04, 08, 11 and 12 belong to C, ZQ13 and 14 belong to B, and ZQ06 belong to A, but the ZQ18 was not determined (Table 1). The haplogroup frequencies of the Tuoba Xianbei were 43.75% D, 31.25% C, 12.5% B, 6.25% A and 6.25% other. By comparing with the relevant populations, we found that the Tuoba Xianbei ethnic group presented a closer similarity to the Oroqen population (Table 2).

G – – – – – – – A A – – – – – A – Only the last three digits are given, e.g. site 093 means 16093.

G – – – – – A – A A – – – – C – – T – – – – – – – – – – – – – – – C CRS ZQ17 ZQ16 ZQ02 ZQ10 ZQ15 ZQ09 ZQ05 ZQ08 ZQ12 ZQ04 ZQ11 ZQ03 ZQ14 ZQ13 ZQ06 ZQ18

T C C C C C – – – – C C – – – – –

C – – – – – – – – – – – – – – T –

T – – – – – – – – – – – – C – – –

T – – – – – – – – – – – – – – – C

C – – – – – – – – – T – – – – – –

A – – – – – – – – – – – G – C – –

A – – – – C – – – – – – – C – – –

C – – – – – – T – – – – – – C – –

T – – – – C – – – – – – – C – – –

G – – – – – – A – – – – – – C – –

T – – – – – – – – – – – – C – – –

C – – – – – – – – – – – – T – – –

C T T T T T T T T T T T T – – T T

G – – – – – – A – – – – – – – – –

G – – – – – – A – – – – – – – – –

T – – – – – – – – – – C – – – – –

C – – T T – – – – – – – – – – T –

T – – – – – – – C C C C C – C – –

T C C – – – – C – – C – – – – – –

C – – – – – – – T T T T T – – – –

DdeI10394 AluI5176 3 1 9 2 8 8 2 7 4 2 5 5 2 2 3 2 1 8 2 1 7 2 1 3 1 8 9 1 8 4 1 8 3 1 7 5 1 7 4 1 7 2 1 3 6 1 2 9 1 2 6 1 0 4 0 9 3

Polymorphic nucleotide positions Mitotypes

Table 1 Variable sites of HSV-I in 16 Tuoba Xianbei individuals (position 16039–16398) and restriction sites

2 9 0

2 9 8

3 1 1

3 2 7

3 4 6

3 6 2

Restriction sites

AluI10397

D D D D D D D C C C C C B B A n.d.

Haplo-groups

A fi G only occurred one time at position 16175 in ZQ03 (Table 1). The nucleotide diversity is 0.0189, located between those of eastern Asian populations [19], which agreed with the conclusion of gene diversity.

3.4. Population comparison To compare with the populations published, the Fst genetic distances and the matrix of significant Fst P value (significance level = 0.05) were calculated and listed in Table 3. The Tuoba Xianbei presented the shortest Fst genetic distance to the Oroqen (0.017), Outer Mongolian (0.019) and Ewenki (0.020), the longest to the Turkeys (0.146), implying that there was a closer genetic affinity among the four populations. According to history documents, the forefathers of the Oroqen, Ewenki and Mongolian can be traced to the ancient ‘‘Shiwei’’ alliance, and the Shiwei and the Tuoba Xianbei all originated from ‘‘Eastern Hu’’ population [28,29], which indicated that our results agreed with history records. The genetic affinity among populations can be reflected well in the phylogenetic tree. An unrooted Neighbor-Joining tree of the 14 populations, based on the net average distance (the data not shown), was built using MEGA 3.0. The Xianbei population was located between the Oroqen and Outer Mongolian populations in the tree (Fig. 2). The MDS analysis can really reflects mtDNA similarities and dissimilarities among populations, and the Fst distances and net distances among the Xianbei and the populations compared can be interpreted well in MDS analysis plot (Fig. 3). In MDS map, the Xianbai population is situated very close to the Oroqen, Ewenki and Outer Mongolian populations, which supported the conclusion of the population tree. 4. Conclusion According to our results and the published data [9,19], we found that the Tuoba Xianbei bore the typical genetic characteristics from Northeastern Asia, and present some closer genetic affinities to the populations of Oroqen, Outer Mongolian and Ewenki, especially Oroqen. Since genetic informations in Tuoba Xianbei remains hold the key to study

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Table 2 The haplogroup distribution frequencies (%) between Tuoba Xianbei and 11 populations compared Haplogroups

Xib, N = 16

Dau, N = 45a

Oro, N = 44a

Ewn, N = 47a

Kor, N = 48a

IM, N = 48a

OM, N = 100b

Bur, N = 126c

Yak, N = 117c

SH, N = 72d

NH, N = 101d

Uzb, N = 58e

D C A B other

43.75 31.25 6.25 12.5 6.25

24.4 6.7 2.2 15.6 54.1

43.1 29.5 4.5 2.3 20.6

31.9 19.1 4.3 10.6 34.1

33.3 6.3 14.6 10.5 41.6

39.6 14 8.3 10.5 35.3

21 15.9 4 10 51

19.9 38.5 7.1 3.6 54.1

28.9 2.8 1.7 19.4 30.9

11.1 1.0 9.7 13.9 57

29.7 1.7 5.0

22.4

50.4

85.9

Population Tuoba Xianbei, Daur, Oroqen, Ewenki, Koran, Inner Mongolian, Outer Mongolian, Buryat, Yakut, Southern Han, Northern Han and Uzbek are abbreviated as Xib, Dau, Oro, Ewn, Kor, IM, OM, Bur, Yak, SH, NH and Uzb, respectively. a Data from Ref. [17]. b Data from Ref. [15]. c Data from Ref. [18]. d Data from Ref. [22]. e Data from Ref. [20].

Table 3 Population pairwise Fsts (below the diagonal) and the matrix of significant Fst P values (above the diagonal)

Xib Eve Bur OM IM Dau Oro Ewn Kor NH SH Kaz Uzb Tur

Xib

Eve

Bur

OM

IM

Dau

0.00000 0.05937 0.05942 0.01888 0.03000 0.04690 0.01744 0.01994 0.04151 0.02968 0.05461 0.05139 0.08808 0.14590

+ 0.00000 0.08885 0.04989 0.07758 0.07484 0.03679 0.05896 0.10005 0.08101 0.10773 0.09065 0.11408 0.16614

+ + 0.00000 0.02488 0.00582 0.02606 0.03291 0.03055 0.03233 0.01983 0.06481 0.04140 0.04992 0.11987

+ + + 0.00000 0.01025 0.00992 0.02706 0.01204 0.02282 0.01053 0.02893 0.00678 0.01618 0.05694

+ +

+ + +

+ 0.00000 0.01044 0.03138 0.02235 0.01027 0.00288 0.02288 0.02098 0.03277 0.09215

0.00000 0.03728 0.01210 0.01622 0.00531 0.02066 0.01704 0.03178 0.07753

Oro

Ewn

+ + + + + 0.00000 0.03192 0.03740 0.02950 0.05847 0.05159 0.08515 0.14585

+ + + + + 0.00000 0.03187 0.01795 0.03565 0.01970 0.03265 0.06970

Kor

NH

SH

Kaz

Uzb

Tur

+ + + + + + + + 0.00000 0.00755 0.02487 0.02715 0.05662 0.11704

+ + + +

+ + + + + + + + + + 0.00000 0.01770 0.03748 0.07151

+ + +

+ + + + + + + + + + +

+ + + + + + + + + + + +

+ + + 0.00000 0.01131 0.01299 0.02554 0.07197

+ + + + + + + 0.00000 0.00244 0.02870

0.00000 0.01195

0.00000

Populations Tuoba Xianbei, Evenk, Buryat, Outer Mongolian, Inner Mongolian, Daur, Oroqen, Ewenki, Korean, Northern Han, Southern Han, Kazakh, Uzbek and Turkey are abbreviated as Xib, Eve, Bur, OM, IM, Dau, Oro, Ewn, Kor, NH, SH, Kaz, Uzb and Tur, respectively.

Turkey

Uzbek

ZQ-Xianbei Oroqen

Kazakh

Ewenki Outer-Mongolian Evenks

Southern-Han

Inner-Mongolian

Northern-Han

Daur

Buryats

Korean 0 .0 0 0 5

Fig. 2. Unrooted neighbor-joining tree of Tuoba Xianbei group and 13 populations compared. The ZQ-Xianbei indicates the Tuoba Xianbei.

the origin of the nomadic populations from northern China, further mtDNA analyses on remains from other Xianbei cemeteries are necessary in order to have a complete understanding on Tuoba Xianbei genetic structure.

Acknowledgements: This study was supported by the National Science Fund for Fostering Talents in Basic Research (Grant Number, J0530184), Ministry of Education, China (the code, 00–07). We are grateful to Wei-jian who provided us the samples of Tuoba Xianbei remains.

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2

Evn

Tur

Dimension 2

1

Uzb

Ewn Kaz

0

Xib

OM Oro

Dau Bur

NH IM -1

Kor

SH

-4

-2

0

2

4

Dimension 1 Fig. 3. MDS plot of Tuoba Xianbei group and 13 populations compared. Populations Tuoba Xianbei, Evenk, Buryat, Outer Mongolian, Inner Mongolian, Daur, Oroqen, Ewenki, Korean, Northern Han, Southern Han, Kazakh, Uzbek and Turkey are abbreviated as Xib, Eve, Bur, OM, IM, Dau, Oro, Ewn, Kor, NH, SH, Kaz, Uzb and Tur, respectively.

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