Natl. Acad. Sci. USA ol.95,pp.11763-11768.sep Genetic relationship of populations in China J.Y.CHUab, W.HUANG b. c, S.O. KUANG, J M. WANG, JJ Xud, Z T. CHUa, Z.O. YANG, K.O. LIN, P Lre M. Wu, Z C GENG, CC. TANB, R F Dud, AND L JINg.h, i e of medical I of Medical sci Kunming, Peoples Republic of China; Rui-Jin Hospital, Shanghai Second Medical aInstitute of Genetics, Chinese Academy of Sciences, Beijing, People's Republic of China; Department of versity, Harbin, People's Republic of China; ' Institute of Cancer Research, Chinese Academy of Medical Sciences, Beijing. People's China;"Institute of Genetics, Fudan University, Shanghai, People's Republic of China; and Human Genetics Center, University of Texas-Houston, Contributed by Jiazhen Tan, June 26. 1998 ABSTRACT Despite the fact that the continuity of mor- Microsatellites have been widely used to study the genetic phology of fossil specimens of modern humans found in China relationship among human populations from different conti- has repeatedly challenged the Out-of-Africa hypothesis, Chi- nents(8-10). Simulation results indicated that microsatellite nese populations are underrepresented in genetic studies. Genetic profiles of 28 populations sampled in China sup shin generally provide a more reliable phylogenetic relation- among closely related populations than among distantly ported the distinction between southern and northern popu related ones(15)and therefore have been considered as ideal lations, while the latter are biphyletic. Linguistic boundaries markers to study closely related populations. However, closely are often transgressed across language families studied, re- related populations tend to live in the same geographical area flecting substantial gene flow between populations. Neverthe- and gene flow between neighboring populations can be sub- less, genetic evidence does not support an independent origin stantial, which may result in major changes in the original gene of Homo sapiens in China. The phylogeny also suggested that frequencies(16). In turn, the reliability of phylogeny infer it is more likely that ancestors of the populations currently ences in the presence of genetic admixture can be profoundly residing in East Asia entered from Southeast Asia compromised(17). Nevertheless, the ease of typing of micro- satellite alleles and the availability of large numbers of such The majority of China consists of the Han people(93.3%), and highly informative loci across the human genome made them 55 official minority nationalities(6.7%), most of which have the markers of choice in this study their own languages, are found predominantly in the periph eral regions. The number of living languages listed for China MATERIALS AND METHODS is 205 (1). Despite the fact that extensive variations among Han Chinese populations and minority populations in China have Twenty-eight populations speaking the languages that belong been observed (2-7). such populations are usually underre to six language families and currently residing in China were resented in genetic studies of worldwide populations(8-10 studied(see Table 1). The locations of those populations are The significance of an extensive study of Chinese populations indicatedin Fig. 2 and Table 1. Samples were collected through is twofold. First. the distinction between northern and south a coordinated effort of several institutes participating in the ern Chinese populations(Han and minority alike)has been Chinese Human Genome Diversity Project. Samples of four observed in the analyses of genetic markers (2-4)as well as Taiwanese Aborigine populations were kindly provided by m somatometric and nonmetric features (5-7). Most authors Hsu attributed such distinction simply to the presence of geo- DNA samples were extracted either directly from n ly graphic barriers(2-7). While it is true that a geographic barrier cytes or from immortalized cell lines. Some primers were maintains genetic difference if there is any, but it is irrelevant purchased from Perkin-Elmer Applied Biosystems division to a more interesting question: whether southern and northern populations are descendents of the same population or, alter Jolla, CA). Selected microsatellite loci were co-amplified in a single 5-Fl PCR. TaqStart antibody(CLONTECH) was used sources. Furthermore,the understanding of the origin of the to provide a hot-start mechanism. Following the PCR, al-ul populations in East Asia may shed light on the peopling of iberia and America. Second. the human fossil remains re 6% polyacrylamide sequencing gel. Electrophoresis was con- covered in China have also attracted attention. The regional ducted using an aBI373A sequencer configured with the B well as temporal continuity of fossil records from Homo erectus filter wheel during collection of fluorescence signal. genescan to Homo sapiens in this region(11-13) repeatedly challenged (Perkin-Elmer, Foster City, CA) was used to collect data the Out-of-Africa hypothesis, which suggests a complete re track lanes, measure fragment sizes, and to check the internal lacement of local populations by modern humans originatin size standard. Genotypes were called by Genotyper(Perkin- in Africa. The validity of this analysis(13) has been questioned Elmer. Foster cit ning method was used (14). Genetic evidence became necessary to verify such claims. convert raw data to allele frequency distribution. A systematic genetic study of Chinese populations using con- Phylogenies presented in Fig. I were constructed report y genetic markers therefore was conducted.This the neighbor-joining method (18). Genetic distance pr by Cavalli-Sforza and Edwards was used to estimate rticipating in the Chinese Human Genome Diversity Projec distance between populations(19). A population was selected (CHGDP) for phylogeny analysis only when the allele frequency distri butions of the population for all microsatellite loci were ary on this art dicate this fac bJ.Y. C. and W H. contributed equally to this work. To whom reprint requests may be addressed at: Human Genetics e 1998 by The National Academy of Sciences 0027-8424/98/9511763-6$2.00/0 Center, University of Texas, P.O. Box 20334, Houston, TX 77225 PnaSisavailableonlineatwww.pnas.org. c-mail: Ijin@@utsph.sph uth. tmc. edu 11763Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11763–11768, September 1998 Evolution Genetic relationship of populations in China J. Y. CHUa,b, W. HUANGb,c, S. Q. KUANGc , J. M. WANGc , J. J. XUd, Z. T. CHUa , Z. Q. YANGa , K. Q. LINa , P. LI e , M. WUf , Z. C. GENGg , C. C. TANg , R. F. DUd, AND L. JINg,h,i aInstitute of Medical Biology, Chinese Academy of Medical Sciences, Kunming, People’s Republic of China; c Rui-Jin Hospital, Shanghai Second Medical University, Shanghai, People’s Republic of China; dInstitute of Genetics, Chinese Academy of Sciences, Beijing, People’s Republic of China; eDepartment of Biology, Harbin Medical University, Harbin, People’s Republic of China; f Institute of Cancer Research, Chinese Academy of Medical Sciences, Beijing, People’s Republic of China; gInstitute of Genetics, Fudan University, Shanghai, People’s Republic of China; and hHuman Genetics Center, University of Texas-Houston, Houston, TX 77225 Contributed by Jiazhen Tan, June 26, 1998 ABSTRACT Despite the fact that the continuity of mor￾phology of fossil specimens of modern humans found in China has repeatedly challenged the Out-of-Africa hypothesis, Chi￾nese populations are underrepresented in genetic studies. Genetic profiles of 28 populations sampled in China sup￾ported the distinction between southern and northern popu￾lations, while the latter are biphyletic. Linguistic boundaries are often transgressed across language families studied, re￾flecting substantial gene flow between populations. Neverthe￾less, genetic evidence does not support an independent origin of Homo sapiens in China. The phylogeny also suggested that it is more likely that ancestors of the populations currently residing in East Asia entered from Southeast Asia. The majority of China consists of the Han people (93.3%), and 55 official minority nationalities (6.7%), most of which have their own languages, are found predominantly in the periph￾eral regions. The number of living languages listed for China is 205 (1). Despite the fact that extensive variations among Han Chinese populations and minority populations in China have been observed (2–7), such populations are usually underrep￾resented in genetic studies of worldwide populations (8–10). The significance of an extensive study of Chinese populations is twofold. First, the distinction between northern and south￾ern Chinese populations (Han and minority alike) has been observed in the analyses of genetic markers (2–4) as well as somatometric and nonmetric features (5–7). Most authors attributed such distinction simply to the presence of geo￾graphic barriers (2–7). While it is true that a geographic barrier maintains genetic difference if there is any, but it is irrelevant to a more interesting question: whether southern and northern populations are descendents of the same population or, alter￾natively, populations that arrived in China from different sources. Furthermore, the understanding of the origin of the populations in East Asia may shed light on the peopling of Siberia and America. Second, the human fossil remains re￾covered in China have also attracted attention. The regional as well as temporal continuity of fossil records from Homo erectus to Homo sapiens in this region (11–13) repeatedly challenged the Out-of-Africa hypothesis, which suggests a complete re￾placement of local populations by modern humans originating in Africa. The validity of this analysis (13) has been questioned (14). Genetic evidence became necessary to verify such claims. A systematic genetic study of Chinese populations using con￾temporary genetic markers therefore was conducted. This report reflects a collaborative effort made by several institutes participating in the Chinese Human Genome Diversity Project (CHGDP). Microsatellites have been widely used to study the genetic relationship among human populations from different conti￾nents (8–10). Simulation results indicated that microsatellite loci generally provide a more reliable phylogenetic relation￾ship among closely related populations than among distantly related ones (15) and therefore have been considered as ideal markers to study closely related populations. However, closely related populations tend to live in the same geographical area and gene flow between neighboring populations can be sub￾stantial, which may result in major changes in the original gene frequencies (16). In turn, the reliability of phylogeny infer￾ences in the presence of genetic admixture can be profoundly compromised (17). Nevertheless, the ease of typing of micro￾satellite alleles and the availability of large numbers of such highly informative loci across the human genome made them the markers of choice in this study. MATERIALS AND METHODS Twenty-eight populations speaking the languages that belong to six language families and currently residing in China were studied (see Table 1). The locations of those populations are indicated in Fig. 2 and Table 1. Samples were collected through a coordinated effort of several institutes participating in the Chinese Human Genome Diversity Project. Samples of four Taiwanese Aborigine populations were kindly provided by M. Hsu (Academia Sinica, Taiwan). DNA samples were extracted either directly from lympho￾cytes or from immortalized cell lines. Some primers were purchased from Perkin–Elmer Applied Biosystems Division and some were kindly provided by Sequana Therapeutics (La Jolla, CA). Selected microsatellite loci were co-amplified in a single 5-ml PCR. TaqStart antibody (CLONTECH) was used to provide a hot-start mechanism. Following the PCR, a 1-ml aliquot of PCR product was loaded on a standard denaturing 6% polyacrylamide sequencing gel. Electrophoresis was con￾ducted using an ABI373A sequencer configured with the B filter wheel during collection of fluorescence signal. GeneScan (Perkin–Elmer, Foster City, CA) was used to collect data, track lanes, measure fragment sizes, and to check the internal size standard. Genotypes were called by Genotyper (Perkin– Elmer, Foster City, CA). A binning method was used to convert raw data to allele frequency distribution. Phylogenies presented in Fig. 1 were constructed by using the neighbor-joining method (18). Genetic distance proposed by Cavalli-Sforza and Edwards was used to estimate genetic distance between populations (19). A population was selected for phylogeny analysis only when the allele frequency distri￾butions of the population for all microsatellite loci were The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y9511763-6$2.00y0 PNAS is available online at www.pnas.org. A Commentary on this article begins on page 11501. bJ.Y.C. and W.H. contributed equally to this work. i To whom reprint requests may be addressed at: Human Genetics Center, University of Texas, P.O. Box 20334, Houston, TX 77225. e-mail: ljin@utsph.sph.uth.tmc.edu. 11763