遗 传 学 报 Acta Genetica Sinica, December 2006, 33 (12)：1060–1072 ISSN 0379-4172 Y-chromosome Genotyping and Genetic Structure of Zhuang Populations CHEN Jing1,2, LI Hui2,3,①, QIN Zhen-Dong2, LIU Wen-Hong2, LIN Wei-Xiong4, YIN Rui-Xing5, JIN Li2, PAN Shang-Ling1,① Department of Pathophysiology, Guangxi Medical University, Nanning 530021, China; MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai 200433, China; Department of Genetics, School of Medicine, Yale University, New Haven CT 06520-8005, USA; Medical Research Center of Guangxi Medical University, Nanning 530021, China; Institute of Cardiovascular Diseases, the First Affiliated Hospital, Guangxi Medical University, Nanning 530021, China Abstract: Zhuang, the largest ethnic minority population in China, is one of the descendant groups of the ancient Bai-Yue Linguistically, Zhuang languages are grouped into northern and southern dialects To characterize its genetic structure, 13 East Asian-specific Y-chromosome biallelic markers and Y-chromosome short tandem repeat (STR) markers were used to infer the haplogroups of Zhuang populations Our results showed that O*, O2a, and O1 are the predominant haplogroups in Zhuang Frequency distribution and principal component analysis showed that Zhuang was closely related to groups of Bai-Yue origin and therefore was likely to be the descendant of Bai-Yue The results of principal component analysis and hierarchical clustering analysis contradicted the linguistically derived north-south division Interestingly, a west-east clinal trend of haplotype frequency changes was observed, which was supported by AMOVA analysis that showed that between-population variance of east-west division was larger than that of north-south division O* network suggested that the Hongshuihe branch was the center of Zhuang Our study suggests that there are three major components in Zhuang The O* and O2a constituted the original component; later, O1 was brought into Zhuang, especially eastern Zhuang; and finally, northern Han population brought O3 into the Zhuang populations Key words: Y chromosome; Zhuang; internal genetic structure The nonrecombining portion of the human Y-chromosome (NRY), which is paternally inherited and does not undergo recombination during cell division, is prone to form population-specific polymorphisms In addition, single nucleotide polymorphisms (SNP) on the Y-chromosome, which has a lower probability of recurrent mutations and higher reliability, are more group- and area-specific and can record more accurately human historical migrations and evolutionary events, a quality because of which they are rapidly being accepted as one of the most effec- tive markers for studying human evolution and origin[1,2] Using the techniques of denaturing highperformance liquid chromatography (DHPLC) and single-stranded conformation polymorphism (SSCP), Underhill et al.[1,2] have investigated several Y-chromosome biallelic markers from populations worldwide in the past and constructed 131 Y-haplogroups and mapped human evolution genealogy In Asia, on the basis of 19 East Asian-specific polymorphic markers on the Y-chromosome, Su et al.[3,4] established 17 Y-haplogroups, of which were specific to Received: 2006-02-23; Accepted: 2006-04-07 This work was supported by the Priority Project of the National Natural Science Foundation of China (No 39993420), the Science Foundation of Guangxi Province (No.GSN0339041, GSY0542044) and the Genographic Project of National Geography ① Corresponding authors LI Hui, E-mail:LiHui.Fudan@gmail.com,Tel:+86-21-6564 2419; PAN Shang-Ling, E-mail: s.pan@gxmu.net.cn ,Tel: +86-771-535 8292 CHEN Jing et al.: Y-chromosome Genotyping and Genetic Structure of Zhuang Populations the East Asian population By clearly tracing the paternal migration route in East Asia and the Pacific Region, Su et al found that the South-Asian group had more Y-haplogroups compared with the NorthAsian group, indicating that the East Asians originated from the south On the basis of the data of Su et al., Li et al.[5,6] studied dozens of ethnic groups in southern China using these Y-SNP markers and explained their origin, migration, mixture, and evolution, thereby adding important genetic information and evidence to the origin of these groups With a population of more than 16 million, Zhuang is China’s largest minority population, with 94% of its population living in the Guangxi autonomous region The Zhuang language belongs to the Kam-Tai linguistic family, the Tai-Kadai sublinguistic family, and the Tai-Sek branch[7], which can be subclassified into Southern and Northern dialects bounded by Yongjiang River (for details on the distribution of the Zhuang branches, see Fig 1) It should be noted that the Bouyei ethnic group in the Guizhou Province actually belongs to the same popu- 1061 lation as Zhuang, as shown by their language and culture, and the so-called Shui Hu in the Yunnan Province, which is completely different from Shui people in Guizhou, is in fact Bouyei Therefore, all of these ethnic groups are considered Zhuang academically Unfortunately, the Zhuang population does not have its own written script and has to use Han characters to record events historically, and these records might be incomplete On the basis of the few available historical records, Zhuang can be traced back to the ‘Luo-Yue’ and ‘Xi’Ou’ groups, 2000 years ago[7] However, the origin of Zhuang might be more complicated than expected because Zhuang might have experienced complex evolvement and migration and to a great extent, may have a close relationship with the origin of Thai and Lao in southeast Asia At the same time, Zhuang might be mixed with other surrounding ethnic groups, especially with Han Therefore, many questions emerge: what is the exact origin of Zhuang? Is there any genetic evidence to support their historical migration events? Is it reasonable to classify Zhuang into South and North groups just Fig Distribution of Zhuang branches North Zhuang dialect group: Guibei, Liujiang, Hongshuihe, Yongbei, Youjiang, Bouyei, Qiubei, Nhang, Tai-Mène South Zhuang dialect group: Yongnan, Tày, Man-Caolan, Nung, YanGuang, WenMa, E (Wuse), Tsün-Lao 1062 遗传学报 based on dialects without any genetic indication? Can the genetic nature of Zhuang provide any information for the study of the origin and migration of the surrounding populations? Furthermore, when the early east-Asian population moved northward via the Guangxi region, were there any original genetic materials retained in the native Zhuangs? Very few published reports are available about Zhuang’s paternal genetic structure, and most of these have mainly focused on some of its special branches In this study, the Y-haplogroups were typed for eight branches of the Zhuang population with 13 biallelic markers and Y-chromosome short tandem repeats (STR), and for every haplogroup, the frequency was calculated, the principal component analysis was carried out, and the heredity framework was drawn The authors of this study hope to determine Zhuang’s inherited structure at a genetic level and to provide genetic data for further studies on Zhuang’s linguistics, origin, transformation, and admixing with other ethnic groups Acta Genetica Sinica Vol.33 No.12 2006 Table Sample distribution of eight branches of Zhuang from Guangxi province Branches Abbreviation Size Yongbei YB 23 Youjiang YJ Guibian/Bouyei GB Hongshuihe HSH 39 Guibei GN 21 Yongnan YN 19 Zuojiang/Tày ZJ 15 Dejing/Nung DJ Total 129 SNPs without length changes, i.e., substitution or transversion, PCR-RFLP (Restriction Fragment Length Polymorphism) was used[4,6,8 10] The Y-hap- logroup of every subject was determined by the integrated analysis of the Y-SNP typing results of the two typing methods For PCR, a primers mix was used, which contained M175, 0.04 μL; M121, 0.03 μL; M134, 0.03 μL; M117, 0.06 μL; M111, 0.08 μL; and M15, 0.02 1 Materials and Methods Sample collection and DNA extraction A total of 129 blood samples were collected from eight representative Zhuang-living areas in the Guangxi Province, each representing eight Zhuang branches DNA was extracted from white blood cells using the traditional phenol-chloroform method[8] Table shows the details on the different branches and the sample sizes All individuals who were Zhuang for more than three generations and were unrelated healthy males were asked to sign informed consent at the time of recruitment Y-chromosome biallelic typing Two strategies were introduced to type Y-SNP For SNPs with length variation, i.e., deletion or insertion, fluorescence PCR (primer information shown in Table 2) was used, the obtained product was electrophoresed on a 3100 genetic analyzer (ABI company, USA) to determine the individual’s genotype For μL Each PCR reaction (volume μL) included KodDash polymerase (TOYOBA) 0.5 U, 0.26 μL Primer Mix, 10 ng of genomic DNA, and buffer The PCR conditions were as follows: 30 cycles of 98℃ for 10 s, 55℃ for s, 74℃ for s, and a final extension at 4℃ To test the degree of variation among different branches, Y-STR was typed using the same method that was used for typing Y-SNP Information on Y-STR primers is also shown in Table The volume of each PCR system was μL, containing KodDash polymerase, 0.05 μL; 10× buffer, 0.5 μL; dNTP (2.5 mmol/L each), 0.4 μL; genomic DNA, 10 ng; mixture of primers, 0.4 μL Usually, PCR reaction was carried out in two panels Panel included primer pairs DYS389, 0.05 μL × (forward and reverse); DYS390, 0.07 μL × 2; DYS391, 0.08 μL × 2; with a total volume of 0.4 μL Panel included DYS388, 0.05 μL × 2; DYS392, 0.07 μL × 2; DYS393, 0.02 μL × 2; DYS19, 0.06 μL × CHEN Jing et al.: Y-chromosome Genotyping and Genetic Structure of Zhuang Populations Table 1063 Y-Chromosome SNP, STR fluorescence primers Locus M121 M117 M175 M134 M15 M111 DYS388 DYS389 DYS390 DYS391 DYS392 DYS393 DYS394 Primer sequence (5′→3′) F:ACAAAGACCTGGACAGATTAC R:CCCTTAAAAACAGCATGATA F:GTACGAAGAAAATCAAGGCTATTA R:TTGGGTAGAAAAACTGCAAGTAG F:TTGAGCAAGAAAAATAGTACCCA R:TTCAGTTAGCCTTGATTGACTGT F:AGAATCATCAAACCCAGAAGG R:TCTTTGGCTTCTCTTTGAACAG F:ACAAATCCTGAACAATCGC R:GTCTGGGAAGAGTAGAGAAAAG F:TAACATAAACAGTATGCCAAA R:TGCCCTAAAGTTAATACCAG F:GTGAGTTAGCCGTTTAGCGA R:CAGATCGCAACCACTGCG F:CCAACTCTCATCTGTATTATCTATG R:TCTTATCTCCACCCACCAGA F:TATATTTTACACATTTTTGGGCC R:TGACAGTAAAATGAACACATTGC F:CTATTCATTCAATCATACACCCA R:GATTCTTTGTGGTGGGTCTG F:TCATTAATCTAGCTTTTAAAAACAA R:AGACCCAGTTGATGCAATGT F:GTGGTCTTCTACTTGTGTCAATAC R:AACTCAAGTCCAAAAAATGAGG F:CTACTGAGTTTCTGTTATAGT R:ATGGCATGTAGTGAGGACA Fluorescence Product size (bp) Wide type Mutant type FAM 123 118 FAM 317 313 FAM 226 221 NED 232 231 FAM 151 142 HEX 197 195 FAM FAM NED HEX NED HEX HEX F: forward; R:reverse Statistical analysis The Y-haplogroup of every individual was defined based on the experimental results of the authors of this study and the NRY haplogroup tree of East-Asia shown in reference [11] In practice, to ensure that an effective genetic sample size has been obtained, for every branch, other published data such as those on Bouyeis, Zhuangs, Suis in Yunnan[12], Bouyeis in Guizhou [13], and unpublished data (Fudan University, data not shown) such as those on Tianlin Zhuangs, Shangsi Zhuangs, Wuse Zhuangs, and Man-Caolan Zhuangs in Guangxi were added to the relevant Zhuang branches Hierarchical clustering analysis was carried out to show the genetic distance (affinity) among Guangxi Zhuang branches, using SPSS13.0 by calculating the frequency of different haplogroups in every branch The phylogeny relationship among Zhuang branches was carried out by combining analysis of hierarchical clustering, principal component analysis, and association analysis to determine the association between phylogeny and Y-chromosome haplogroups; this was then observed using the gradient distribution chart which was drawn using Surfer7.0 software for every principal component according to its geographic distribution, in which principal component value served as the height values Variances among and within populations resulting from Y-haplotype frequencies of different branches were calculated using AMOVA (Analysis of MOlecular VAriance framework) in Arlequin1.1 software to further elucidate the phylogeny of different Zhuang branches Finally, Zhuang’s genetic framework was drawn using the network 11.0 software with the same Y-STR haplogroups of different branches to show the detailed difference and association among Zhuang branches 1064 遗传学报 Results Distribution of NRY haplogroups in Zhuang branches The haplogroup frequencies of different Zhuang branches stemming from the typing results of Y-SNP were calculated As shown in Table 3, Zhuang’s Y-haplogroups mainly cluster around O*, O1, O2a, and O3, the four most common haplogroups in East Asia Haplogroup O* is the most frequent, followed by O2a and O1, showing that Zhuang is a typical southern group in East Asia and possesses more ancient Y-haplogroups Interestingly, O3, O3e, and O3e1, the characteristic haplogroups for East Asian northern group were also frequently found in Zhuangs, showing a common gene communication between the two populations 2 Principal component analysis Principal component analysis was carried out using SPSS 13.0 software, and the principal component dot plot of Y-SNP frequencies was drawn (Fig 2) according to Y-SNP typing results of different branches, as obtained by the authors of this study, and additional data of Yunnan Bouyeis, Zhuangs, Suis, Guangxi Tianlin, Shuangsi, Wuse, Man-Caolan Zhuangs, and Guizhou Bouyeis In this analysis, Tianlin was merged with the Youjiang branch, Shangsi was merged with the Zuojiang branch, and Table Acta Genetica Sinica Vol.33 No.12 2006 Bouyei with the Guibian branch The result of principal component analysis showed that the cumulative contribution of principal component (pc1) and component (pc2) accounted for 82.5% of the total difference It is obvious from Fig that 12 Zhuang branches and other correlated groups mainly gathered into two bigger groups Hongshuihe, Guibei, Yongbei, Yongnan, and ManCaolan were located in the upper part of the principal components plot and constituted the first group, whereas Guibian, Zuojiang, Youjiang branch, Yunnan Zhuang, and Yunnan Bouyei clustered in the lower part of the chart, and constituted the second group Yunnan Shui Hu seemed to be isolated from all branches, but was somewhat closer to the second group on pc2 As shown in Fig 2, it is the pc2 that separated these branches into two groups Considering the geographic location of each branch, it was observed that the difference between the two groups was, genetically, an east–west rather than a southnorth profile, contradicting the traditional south-north grouping of Zhuangs by linguistic factors Using the values of pc1 and pc2 as height values, the principal component gradient distribution diagram was drawn in the same manner as drawing relief map with contours according to the geographic location of each Zhuang branch (Fig 3) In the relief map of pc1, it was observed that the peak value was near the Guangxi-Vietnam border, which gradually changed Y-SNP haplogroup frequencies of branches of Zhuang Branches Size C D D1a F* YB 23 8.70 YJ Tianlin 22 GB HSH 39 2.56 GN 21 4.76 YN 19 ZJ 15 Shangsi 15 13.33 DJ 100.00 4.35 K* O O1 4.35 21.704 17.39 40.00 20.00 5.13 4.76 5.26 6.67 O2a* O2a1 O3 8.70 21.74 8.70 O3a O3e* O3e1 4.35 4.35 20.00 20.00 13.64 4.55 25.00 25.00 25.00 5.13 23.08 10.26 5.13 7.69 20.51 5.13 4.76 38.10 4.76 4.76 4.76 14.29 14.29 5.26 15.79 10.53 10.53 10.53 31.58 33.33 72.73 4.55 25.00 40.00 20.00 4.55 60.00 20.00 6.67 5.26 5.26 15.38 CHEN Jing et al.: Y-chromosome Genotyping and Genetic Structure of Zhuang Populations Fig 1065 The principal components plot of Y-SNP frequencies of Zhuang populations Fig Geographic map of Y-SNP principal components of Zhuang populations X coordinate: longitude; Y coordinate: latitude The geographic map of Y-SNP principal components of Zhuang branches was drawn using principal component value as contour, wherein, the lower the value, the darker of the color For pc1 (left), the peak value was near the Guangxi-Vietnam border, which gradually changed north-eastward and north-westward The peak value of pc2 that classifies Zhuang branches into two main groups appeared in the east Hongshuihe basin (right), exhibiting an east-to-west gradient It is quite clear that contour lines run along rivers, indicating that native Zhuangs migrated upstream along rivers in Guangxi in early times 1066 遗传学报 Acta Genetica Sinica Vol.33 No.12 2006 served Theoretically, the more positive correlated study, it was observed that pc1 was positively associated with O2a (r = 0.69, p = 0.02) and that the values of pcl were all positive Therefore, the meaning of pc1 underscores the positive correlation of Zhuang branches and Yunnan Shui Hu with O2a, showing that these ethnic groups are all typical southern groups of East-Asian population, a conclusion that is consistent with the historical records of Zhuang’s Baiyue origin For pc2, the number of positively correlated haplogroups was almost the same as that of the negative ones, with an opposite trend being found between O* and O2a: haplogroup O* was positively but O2a was negatively associated with pc2 Both pc2 and haplogroup O* were clearly related to longitude, implying that haplogroup O* was the main component of pc2, i.e., it is O* that separates the Zhuang branches into two main groups With the westward movement of haplogroup O*, O2a retreated in the same direction This process agrees with the lower frequency of O2a in east Zhuang branches No significant correlation was found between other haplogroups and the two principal components haplogroup a principal component has, the more dis- north-eastward and north-westward This might be a clue to the spreading of the East Asian population in ancient Guangxi when it first entered East Asia Because pc2 classifies Zhuang branches into two main groups, its significance might be more definite The peak value of pc2 appeared in the east Hongshuihe basin, exhibiting an east-to-west gradient In addition, a higher value of pc2 was seen in northwest Guangxi bordering the Yunnan Province This might be influenced by data of the Yunnan Bouyeis Furthermore, from Fig it is quite clear that contour lines run along rivers, indicating that native Zhuangs migrated upstream along rivers in Guangxi in early times Correlation analysis To understand the meaning of each principal component, correlation analysis was carried out to seek the origin of each principal component Each principal component was sorted out after calculating the correlation coefficient for every haplogroup such that the positively and negatively correlated haplogroups of the principal components could be ob- tinct is its genetic structure, and the more practical meaning it has For more detail, see Figs and Apparently, the structure of pc2 is clearer than that of pc1, after comparing the correlation coefficient values of the two principal components There were many contradictions in the variables among positively correlated haplogroups in pc1, whereas in pc2, most positively associated variables fell within the positive correlation area Moreover, pc2 was markedly positively correlated with longitude, showing that pc2 was more significant than pc1 Further analysis of pc1 showed that the number of positive correlated haplogroups, despite their weak correlations, was bigger than that of the negative groups This is attributed mainly to the difference between O2a and O3e, where O2a was a southern aboriginal haplogroup in the East Asian population and O3e was probably a northern haplogroup In this Hierarchical clustering analysis To further elucidate the relationship among Guangxi Zhuang branches, hierarchical clustering analysis was carried out with average linkage (between groups) in SPSS 13.0 software and the results are shown in Fig 6, in which Mien and Yi are the foreign groups It is clear that the center of all Zhuang branches emerges in the Hongshuihe area, evolving gradually toward Yongbei, Yongnan, and Guibian along the Hongshuihe river, then toward Guibei and Man-Caolan, and finally toward Zuojing, Youjiang, and Yunnan, providing strong evidence once again that the difference among Zhuang branches is fundamentally east-west and not south- north, as traditionally believed Intriguingly, Wuse Zhuangs living in Yongle, Rongshui, a northwest county in Guangxi, are genetically close to the remote Zuojiang Zhuangs who live in southwestern Guangxi, implying a special moving event in ancient times CHEN Jing et al.: Y-chromosome Genotyping and Genetic Structure of Zhuang Populations 1067 Fig Correlations among principal components, Y-SNP frequencies, longitude, and latitude of Zhuang branches Principal components were ranked after their correlation coefficient for relevant haplogroup were calculated to estimate the positively and negatively correlated haplogroups of the principal components Each correlation coefficient was marked with relevant color according to its value There were many contradictions in the variables among positively correlated haplogroups in pc1 (left), whereas in pc2, most positively associated variables fell within the positive correlation area (right) Fig Correlations and statistical significance among principal components, Y-SNP frequencies, longitude, and latitude of Zhuang branches r: correlation coefficient; P: probability value; long.: longitude; lat.: latitude Chromatism staff gauge shows different p values pc1 was positively associated with O2a, a typical south aboriginal haplogroup in East Asian population, underscoring the positive correlation of Zhuang branches and Yunnan Shui Hu with O2a For pc2, the number of positively correlated haplogroups was almost the same as that of the negative ones, with an opposite trend being observed between O* and O2a, in which haplogroup O* was positively but O2a was negatively associated with pc2 1068 遗传学报 Acta Genetica Sinica Vol.33 No.12 2006 within populations was quite the opposite, further suggesting that the classification of east and west Zhuang is more reasonable than that of the southnorth grouping if these groups need to be distinguished However, because the east-west difference is graded due to Zhuang population migration, it is very difficult to differentiate Zhuangs into two distinct groups genetically Fig Dendrogram of Y-SNP of Zhuang branches Yi and Mien, two non-Baiyue populations living in southwest China, were introduced to illustrate the cluster of Zhuang and its relationship with surrounding non-Baiyue groups This dendrogram showed that the ancestor of Zhuang gathered in Hongshuihe area first, then spread toward Yongjiang and Youjiang basin, and the surrounding area Genetically, Zhuang was much far away from Yi and Mien AMOVA analysis Linguists divide the Zhuangs into the southern group and northern group, according to their dialects If this classification has a genetic basis, the variance among populations should be greater, whereas the variance within populations should be smaller in the molecular variance framework analysis As suggested by the pc2 results, the AMOVA results were compared between the southern and northern groups and between the eastern and western groups (Table 4) However, no differences were observed, as once anticipated Instead, the variance among populations of the eastern-western groups was far greater than that of the northern-southern groups, whereas the variance Table Network analysis of Y-chromosome STR haplogroup of Guangxi Zhuang branches The combination of several short tandem repeats (STR) in NRY comprises another type of Y-chromosome haplogroup, which reflects minute genetic variances in different populations with the same Y-SNP This differs from Y-SNP haplogroup, which can only be roughly used in the classification of systematic phylogeny After sorting out different Zhuang branches on the basis of Y-SNP information, the genetic network was drawn using Network 11.0 with Y-STR data from individuals of all branches to show the relationship and differences between branches with the same Y-SNP haplogroups As shown in Fig 7, the information of STR network of haplogroup O* is more abundant due to its higher frequency in all Zhuang branches Nine individuals from Hongshuihe branch with haplogroup O* had Y-STRs, the highest Y-STRs frequency of all, three of which were shared by Guibian, Guibei, and Yongbei individuals In addition, Hongshuihe Zhuangs were closely related with individuals from other haplogroups, especially Yongbei and Guibei individuals, establishing its central position in all Results of AMOVA of northern-southern group and eastern-western group of Zhuang branches Eastern–western Northern-southern Eastern: HSH, YN, YB, CL, GN Northern: YB, YJ, HB, HSH, GB Western: ZJ, YJ, GB, E, DJ Southern: YN, ZJ, DJ, CL, E Among populations 17.851 3.461 Within populations 18.539 32.929 Groups CHEN Jing et al.: Y-chromosome Genotyping and Genetic Structure of Zhuang Populations 1069 Fig STR network of haplogroups O*, O1, O2a, and O3* of Zhuang branches In the network of haplogroups O* and O1, Hongshuihe branch had more STR haplogroup polymorphisms and was closely related with others, especially Yongbei and Guibei individuals, establishing its central position in all Zhuang branches In the O2a network, Zuojiang branch had more Y-STRs In the O3* network, no shared STR haplogroup was seen Zhuang branches Only one Guibei individual shared the same Y-STR haplogroups with that of Hongshuihe although Guibei branch also had STR haplogroups that were distributed at the edge of the O* haplogroup network More linkage was also found among Zuojiang, Youjiang, and Dejing branches, which, as a closed group, was more isolated from others Taken together, all these findings verify and agree with results from the principal component analysis and the above-mentioned clustering analysis Likewise, in the O1 network, Hongshuihe branch had more STR haplogroup polymorphisms than others and occupied the center of the framework However, it failed to maintain its leading position in O2a and gave way to Zuojiang branch as a result of fewer individuals carrying the O2a haplogroup There was no shared STR haplogroup or any remarkable difference among branches in the O3* network One possible explanation is that O3* is not the dominant and characteristic haplogroup in Zhuangs 1070 3 遗传学报 Discussion The origin of Zhuangs Historical records, linguistic studies, and ethnology investigations, all suggest that Zhuang might have been derived from Luoyue and Xi’ou division of ancient Baiyue In this study, O*, O2a, and O1, the characteristic Y-SNP haplogroups of Baiyues, were found to be frequently present in Zhuangs In addition, principal component analysis confirmed that Zhuangs clustered with Yuannan Shuihu and Bouyei, proven typical descendants of Baiyue Therefore, all these studies strongly indicate that Zhuang stemmed from the ancient Baiyue group, which is consistent with historical records and cultural anthropological results[10,12,14] Genetic structure of northern Zhuang dialect group and southern Zhuang dialect group Without a written language, Zhuang’s important historic events were less reliably documented and were passed to the next generation by limited oral folklores and by a few recent references written by Han Chinese Coupled with several large-scale population movement and chaos brought on by wars, it is hard to explore the development, differentiation, communication, and evolvement of Zhuang’s inner branches using historical records Therefore, Zhuangs can only be divided into a northern dialect group and a southern dialect group, according to their approximate difference in spoken languages No striking difference in the genetic structure of the two dialect groups was found, which was an unexpected result The principal component analyses showed that, either on pc1 or pc2, there was no clustering in both branches within the southern dialect group or branches belonging to the northern dialect group The geographic map and clustering analysis of principal component did not present a south-to-north gradual change and gathering In other words, the linguistic definition of south Zhuang and north Zhuang lacks relevant genetic supports and both Acta Genetica Sinica Vol.33 No.12 2006 groups are in fact a genetically homogeneous population Instead, Zhuangs exhibit, if any, a Hongshuihe-centered, east-to-west gradually diffusing process in their heredity structure Indeed, according to legend, the name of ‘Zhuang’ ethnic group stemmed from ‘Yangke river’, the ancient name of Hongshuihe river, and this may not be a coincidence Principal component analysis showed that Zhuang was divided into eastern and western subgroups according to their geographical distribution However, clustering analysis suggested that Guangxi Zhuangs gradually expanded westward from the Hongshuihe branch In the STR haplogroup network of Zhuang branches, especially in the network of the O* haplogroup, Hongshuihe branch was also in the center Zuojiang, Youjiang, and Dejing branches within the territory of the western subgroup were more close to each other in terms of genetic framework and were a little away from the eastern subgroup 3 Genetic levels of Zhuangs Table shows the Y-SNP haplogroup frequencies of Zhuang branches, from which it can be seen that two older haplogroups, O* and O2a, were more frequently present, suggesting that these two haplogroups might be the original components of Zhuangs and remained as heredity markers from early inhabitants when they entered Guangxi and spread in the east Asian continent In pc1 geographic map, multiple centers could be seen and expansions in the south-to-northeast and south-to-southwest directions were observed Furthermore, pc1 was proved in correlation analysis to be positively associated with haplogroup O2a, which might be more than 10 000 years old according to the STR age estimate[15], suggesting that the ancestors of Zhuangs had carried marker O2a when they first entered into Guangxi area More interestingly, haplogroup O* was estimated to be around 200 00 years old, suggesting that it might be another primary component It is possible that the early East Asian population went straight to Hongshuihe area first when they arrived in east Asian CHEN Jing et al.: Y-chromosome Genotyping and Genetic Structure of Zhuang Populations continent and then migrated and developed around in the region now called Guangxi Finally, the inner structure of O1 haplogroup showed that some components of Zhuang scattered from east to west This is within 100 00 years and is possibly caused by the mixture of Xi’Ou with LuoYue, becoming the second genetic level of Zhuangs Historically, several massive settlements of soldiers and officials into Guangxi by governmental policies and a couple of big southward movements of Zhuangs might have contributed to the mixture of Zhuang’s gene pool In the results of this study, O3, a characteristic haplogroup in north group in East Asian population, was also considerably higher in Zhuangs Unfortunately, either subgroups or center for haplogroup O3 was not obtained, indicating that this haplogroup is a foreign component and can be traced to the intermixing of northern Chinese and Guangxi locals, which is also consistent with 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mitochondrial dan in coding region in 16 Ethnic populations of Yunnan Acta Genetica Sinica, 2005, 32(2): 118-123 (in Chinese with an English abstract) [15] LI H On dividing of Baiyue people’s hereditary frame Study of Ethnics in Guangxi, 2002 70(4): 26-31 (in Chinese with an English abstract) 1072 遗传学报 Acta Genetica Sinica Vol.33 No.12 2006 壮族 Y 染色体分型及其内部遗传结构 陈 晶1,2，李 辉2,3，覃振东2，刘文泓2，林伟雄4，尹瑞兴5，金 力2，潘尚领1 广西医科大学病理生理教研室，南宁 530021; 复旦大学生命科学院教育部现代人类学重点实验室，上海 200433； 耶鲁大学医学院遗传系，新港，康奈狄格州 06520-8005； 广西医科大学医学实验中心，南宁 530021； 广西医科大学第一附属医院老年心血管病研究所，南宁 530021 摘 要: 壮族是中国最大的少数民族，与东南亚的泰老族群关系密切，在东亚人群的遗传结构研究中地位非常特殊。本研究 调查了壮族各个支系的 Y 染色体多样性，通过主成分分析、聚类分析和分子方差分析，揭示壮族的内部父系遗传结构。结 果发现，壮族的主要 Y 染色体单倍群为 O*, O2a, O1。传统的对壮族按方言分为南北二组的分类方法在遗传上并没有依据， 壮族支系体现出从东往西的梯度变化过程。这说明壮族的结构中有几个层次，最早的成分普遍出现在各个支系中，第二层 是由东部来的百越核心成分，第三层是北方来的汉族成分。壮族内部遗传结构的分析将有助于对东亚人群的南来起源的研 究。 关键词: Y-染色体；壮族；内部遗传结构 作者简介: 陈晶（1980-）女，硕士研究生，研究方向：分子人类学。E-mail:chenjing.gxmu@gmail.com ... 25 .00 25 .00 5 .13 23 .08 10 .26 5 .13 7.69 20. 51 5 .13 4.76 38 . 10 4.76 4.76 4.76 14 .29 14 .29 5.26 15 .79 10 .53 10 .53 10 .53 31. 58 33.33 72.73 4.55 25 .00 40. 00 20. 00 4.55 60. 00 20. 00 6.67 5.26 5.26 15 .38... 21 4.76 YN 19 ZJ 15 Shangsi 15 13 .33 DJ 10 0 .00 4.35 K* O O1 4.35 21. 704 17 .39 40. 00 20. 00 5 .13 4.76 5.26 6.67 O2a* O2a1 O3 8. 70 21. 74 8. 70 O3a O3e* O3e1 4.35 4.35 20. 00 20. 00 13 .64 4.55 25 .00 ... which contained M175, 0. 04 μL; M1 21, 0. 03 μL; M134, 0. 03 μL; M 117 , 0. 06 μL; M 111 , 0. 08 μL; and M15, 0. 02 1 Materials and Methods Sample collection and DNA extraction A total of 12 9 blood samples