Genetic Analysis on Tuoba Xianbei Remains Excavated from Qilang Mountain Cemetery in Qahar Right Wing Middle Banner of Inner Mongolia
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector 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 three southward migrations, and finally founded the Northern Abstract Sixteen sequences of the hypervariable segment I (HVS-I, 16039–16398) in mtDNA control region from ancient Wei Dynasty and controlled the northern region of China Tuoba Xianbei remains excavated from Qilang Mountain (386–534 A.D.). By the time of the Tang Dynasty they had Cemetery were analyzed. In which, 13 haplotypes were found largely merged with Han populace. by 25 polymorphic sites. The haplotype diversity and nucleotide At present, the origin of Tuoba Xianbei ethnic group and its diversity were 0.98 and 0.0189, respectively, and the mean of impacts on modern minorities of northern China are not yet nucleotide number differences was 6.25. Haplogroup analysis clear. It will be very helpful for understanding in the origin, indicates these remains mainly belong to haplogroup C formation and development process of northern minorities of (31.25%) and D (43.75%). According to the published data were China by analyzing the data of mtDNA in Tuoba Xianbeis. considered, we can suggest that the Tuoba Xianbei presented a In this study, we examined the sequences of HVS-I from 16 close genetic affinity to Oroqen, Outer Mongolian and Ewenki Tuoba Xianbei remains excavated in Qilang Mountain Ceme- populations, especially Oroqen. Ó 2006 Federation of European Biochemical Societies. Published tery that was indicated in Fig. 1, and compared the data with by Elsevier B.V. All rights reserved. those of relevant populations. Keywords: Qilang Mountain; Tuoba Xianbei; Mitochondrial DNA 2. Materials and methods 2.1. Sampling The thighbone samples from 18 ancient Xianbei remains, provided by Dr. Wei-Jian (Inner Mongolia Institute of Archaeology), which 1. Introduction were excavated from Qilang Mountain Cemetery in Qahar Right Wing Middle Banner of Inner Mongolia from 1995 to 1996. During this The PCR and ancient DNA technique have made it possible period, 20 graves were excavated, and out of which 18 remains were to identify the trace amount of DNA which still survived in collected (numbered ZQ01–18). According to Wei et al. [10], these remains occupied between 4th and 5th centuries A.D., belong to archaeological remains [1–3]. Among the most informa- Tuoba tribe of Xianbei alliance. tive genetic systems, mitochondrial DNA is the preferred target for recovering DNA from archaeological remains and 2.2. Contamination controls characterizing the structure of a gene pool, which well-solved All stages of the work were carried out in term of aDNA-specific many important questions, such as human origin, migration requirements [11]. First, before beginning our work, 2 decontamina- and evolution, as well as population affinities and so on [4–9]. tion methods, such as irradiating the samples 2–3 h with UV-light irra- diation (254 nm) and removing the sample surface 3–4 mm depth with In ancient China, the Xianbei was another significant noma- dentistry drill, were employed to remove the surface DNA deriving dic population succeeded by Xiongnu, which actually con- from other workers who had handled the samples before they reached sisted of a federation of sizeable non-Han groups, and the our laboratory; secondly, the preparation of bone powders and the Tuoba Xianbei was a very important sub-tribe in Xianbei Alli- extraction and amplification of DNA were carried out in separate ance. The Tuoba Xianbeis are said to be the northern-most dedicated laboratories, routing sterilized by treatment with DNase away (Molecular Bioproduct, San Diego, CA), bleach and UV-light branch of the Xianbei nomads, originally dwelled to the north- irradiation (254 nm), and the gloves, face masks and full-body suits eastern-most of all Xianbei tribles, in the region of the Great must be used during all the handling processes; thirdly, the blank- Xianbei Mountains, which is now the Erguna River valley of extractions, zero-controls and negative-controls must be carried out the northern section of the Great Xing’an Range. The lan- during the extraction and amplification of DNA, and fourthly, all results must be identical in two independent extractions and two guage spoken by Tuoba Xianbei was almost the same with independent amplifications, and compared with the sequences from Mongolic. According to Chinese historical documents, from the handlers. the 1st to the 3rd centuries, the Tuoba tribe had undergone 2.3. DNA extraction and amplification The method of Kalmar et al. [12] was used for the bone powder preparation. The manual protocol of GENECLEANÒ Kit for ancient *Corresponding author. Fax: +86 431 8498031. DNA (BIO101, USA) was used for DNA extraction. As for PCR E-mail address: [email protected] (Z. Hui). amplification, the technique of Adcock et al. [13] was adopted, and 0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.10.030 Y. Changchun et al. / FEBS Letters 580 (2006) 6242–6246 6243 Fig. 1. The location of Qahar Right Wing Middle Banner Qilang Mountain Cemetery. modified slightly. Four sets of primers for PCR were L16035–16055/ puted by Arlequin 2000. The net genetic distances and phylogenetic 16162–H16142, L16112–16131/H16237–16218, L16166–16185/16307– tree were carried out using MEGA 3.0. To visualize the Fst distances H16286 and L16261–16281/H16398–16379. The PCR conditions were among populations, the multidimensional scaling (MDS) analysis was described as follow: initial denaturizing at 94 °C for 3 min, followed by performed by SPSS 12.0. For comparison, the published data from 40 cycles of denaturizing at 94 °C for 1 min, annealing at 55 °C for populations of Evenk [16], Inner Mongolian, Daur, Oroqen and Ewen- 45 s, and extension at 72 °C for 1 min, followed by the final extension ki [17], Outer Mongolian [15], Buryat [18], Kazak [19], Uzbek [20], 10 min. The quality of amplification was estimated on a 2% agarose Turkey [21], Southern Han (Zhangjiang Han and Wuhan Han [22]), gel, and the PCR products were purified using ‘QIAEX II’ gel extrac- Northern Han (Qingdao Han, Fengcheng Han [22] and Xi’an Han tion kit (QIAGen, Germany). [23]) were used. 2.4. Cloning and sequencing The PCR products were cloned using TOPO TA Cloning kit 3. Results and discussion (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 3.1. Sequence diversity and the mean number of pairwise PCR. The colonies were transferred into a 30 ll reactions mix with differences 67 mM Tris–HCl (pH 8.8), 2 mM MgCl2,1lM of each primer (M13 Sixteen sequences of HVS-I (position 16039–16398) were forward and reverse universal primers), 0.125 mM of each dNTP, obtained from 18 Tuoba Xianbei remains. Of these 18 subjects, 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, we could not obtained PCR products from ZQ07. The the clones with an insert of the expected size were identified by aga- sequence from ZQ01 was excluded, because it was ambiguous rose-gel electrophoresis. After purification of these PCR products with by the clone test. Among the 16 sequences, 13 different hyplo- Microcon PCR diveces (Amicon), a volume of 1.5 ll was cycle-se- types were identified by 25 polymorphic sites relative to the ref- quenced using the BigDye Terminater cycle sequencing kit (Applied erence sequence [24], and thereinto, ZQ02 and ZQ10, ZQ08 Biosystems). Six clones of the same PCR products were sequenced, and finally, the sequences were analyzed on an Applied Biosystems and ZQ12, as well as ZQ16 and ZQ17 shared the same haplo- DNA 377 sequencer. types. The haplotype diversity and the mean number of pair- wise differences are 0.98 and 6.25. According to Pakendorf 2.5. RFLP analysis et al. [9], the gene diversity of the Tuoba Xoanbai (0.98) fell In order to confirm the haplogroup status of Tuoba Xianbei sam- into those of the steppe populations (around 0.99) and the ples, three restriction sites (AluI 5176, DdeI 10394 and AluI 10397) Siberian populations (0.93–0.95). 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]. 3.2. Nucleotide diversity Of these 25 mutation sites, 24 were transitions, only 1 was 2.6. Data analysis the transversion (16183 A fi C). The ratio of transition:trans- The editing of sequences was performed by Clustal X1.83. The final information used for data analysis about each individual was the HVS- version is 24:1, which agrees with other data obtained from I of 360 bp, nucleotide positions 16039–16398. Fst genetic distances, humans [25]. Among the transitions, there were a high propor- Genetic diversity, the mean number of pairwise difference were com- tion of C M T substitutions compared to A M G, and the 6244 Y.