- Email: [email protected]
Genetic analysis of 24 Y-STR loci in the Miao ethnic minority from Yunnan Province, southwestern China
Accepted Manuscript Title: Genetic analysis of 24 Y-STR loci in the Miao ethnic minority from Yunnan Province, southwestern China Author: Xiufeng Zhang PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Author: Tao Gu PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Author: Jinyong Yao PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Author: Canming Yang PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Author: Lei Du PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Authors: Jing bo Pang, Min Rao, Aiting Nie, Liping Hu, Shengjie Nie PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Received date: Revised date: Accepted date:
28-10-2016 2-2-2017 11-2-2017
Please cite this article as: Jing bo Pang, Min Rao, Aiting Nie, Liping Hu, Shengjie Nie, Genetic analysis of 24 Y-STR loci in the Miao ethnic minority from Yunnan Province, southwestern China, Forensic Science International: Genetics http://dx.doi.org/10.1016/j.fsigen.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title：Genetic analysis of 24 Y-STR loci in the Miao ethnic minority from Yunnan Province, southwestern China Xiufeng Zhang1†, Tao Gu2†, Jinyong Yao3, Canming Yang4, Lei Du5, Jing bo Pang1, Min Rao1, Aiting Nie1, Liping Hu 1*, Shengjie Nie 1* 1 School of Forensic Medicine, Kunming Medical University, Yunnan Province, People’s Republic of China 2 Center for Disease Prevention and Control of Kunming, Yunnan Province, People’s Republic of China 3 Honghe Public Security Bureau, Yunnan Province, People’s Republic of China 4 Eryuan Public Security Bureau, Yunnan Province, People’s Republic of China 5 Chenggong Public Security Bureau, Yunnan Province, People’s Republic of China † Xiufeng Zhang and Tao Gu contributed equally to this work. Email address:[email protected]; [email protected]
* Corresponding author: All correspondence should be addressed to: Dr. Shengjie Nie and Liping Hu. School of Forensic Medicine, Kunming Medical University, Yunnan Province, People’s Republic of China, 650500; Tel.: 0871-65922837; Fax: 0871-65922837; Email address: [email protected]; [email protected]
Abstract In the present study, 24 Y-chromosomal short tandem repeat (Y-STR) loci were analyzed in 252 unrelated Miao male individuals from Pingbian county, Honghe Hani and Yi Autonomous Prefecture, Yunnan province, southwestern China. The gene diversity of the 24 Y-STR loci in the studied group ranged from 0.2683 (DYS391) to 0.9312 (DYS527a/b). According to haplotypic analysis of the 24 Y-STR loci, 214 different haplotypes were obtained, 186 of which were unique. The overall haplotype diversity and discrimination capacity were calculated to be 0.9983 and 0.8492, respectively. In addition, three different triplications were observed at the DYS527a/b
marker, and 1 intermediate allele and six single off-ladder alleles were observed at four markers. We analyzed interpopulation differentiations by making comparisons between the Yunnan Miao ethnic minority and 18 other ethnic groups. The results obtained using pairwise genetic distances, multidimensional scaling plot, and neighbor-joining tree at the same set of 17 Y-filer loci indicated that Yunnan Miao had a closer genetic relationship with Yunnan Han and Hunan Miao individuals. The present results may provide useful information for paternal lineages in forensic cases and can also increase our understanding of the genetic relationship between Miao individuals and other groups. Keywords: Genetic distribution; Y-STR; Miao ethnic minority; Haplotypic structure The Miao ethnic group in China is primarily distributed across Guizhou, Hunan, Yunnan, and Sichuan provinces and the Guangxi Zhuang Autonomous Region. It is the 4th largest ethnic minority and had a population of 9,426,007 at the 2010 census. By the end of 2012, there were one hundred and fifty-six thousand (155630) people living in Pingbian county, Honghe Hani and Yi Autonomous Prefecture, Yunnan province. Pingbian county in Yunnan province is one of the five Miao Autonomous Counties in China. In addition to the Han ethnicity, there are 16 ethnic minorities living in Pingbian, such as Miao, Yi, Zhuang and Yao. This population totals one hundred thousand (103298), accounting for 66.3% of the total population in Pingbian. The language spoken by Miao people belongs to the Miao-Yao branch of the Chinese –Tibetan language family. Due to extended co-existence with the Han and other ethnic minorities, Miao people also speak the Han, Dong and Zhuang languages. They have been writing their own Miao language based on Latin since 1956[1]. The Miao opts for monogamy. Marriage in the same paternal lineage is restricted, while intermarriage among first cousins on the maternal side is a taboo. The sampling location of Yunan Miao in this paper is indicated in Figure S1. To establish a database of the Miao ethnic minority population residing in Yunnan province, China, we used an AGCU Y24（AGCU ScienTech Inc., China）kit, which includes 24 Y-chromosomal short tandem repeat (Y-STR) loci, namely, DYS391, DYS389Ⅰ, DYS439, DYS389Ⅱ, DYS438, DYS449, DYS456, DYS458, DYS437, DYS635, DYS448, DYS527a/b, Y-GATA-H4, DYS447, DYS19, DYS392, DYS522, DYS393, DYS388, DYS390, DYS385a/b and DYS444. In this study, we presented allele frequencies and haplotype distribution of 24 Y-STR loci in the Miao group and compared pairwise genetic distances with other populations. A total of 252 blood samples from Miao ethnic individuals were collected from Pingbian county, Honghe Hani and Yi Autonomous Prefecture, Yunnan province, Southwest China, after informed consent. DNA extracted with Chelex-100 was amplified in a GeneAmp PCR system 9700 (Applied Biosystems, USA) using an AGCU Y24（AGCU ScienTech Inc., China）PCR Amplification kit, separated by capillary electrophoresis on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, USA), and subsequently analyzed by GeneMapper v.3.2 software (Applied Biosystems, Foster City, CA)[2]. DNA typing and assignment of
nomenclature were based on the ISFG recommendations[3, 4]. Control DNA (9948) and ddH2O were used as positive and negative controls, respectively. Allelic frequencies were estimated by direct gene counting. Single-marker gene diversity (GD) was calculated according to Nei with the formula GD= n(1- Σpi2)/(n-1), where n and pi denote the total number of the samples and the relative frequency of the i-th allele, respectively[5]. Haplotype diversity was calculated similarly to GD. The discrimination capacity (DC) was defined as the ratio between the number of different haplotypes and the total number of haplotypes. To compare our data with other published data, genetic distances (Rst) were measured by analysis of molecular variance (AMOVA) and visualized using two multidimensional scaling (MDS) plots via the YHRD database (http://www.yhrd.org/Analyse/AMOVA). A neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 6.0 software to illustrate the relationships between populations [6]. The allele frequency and locus diversity of the 24 Y-STR loci in the Miao group are shown in Table S2. In this study, 199 different alleles were observed at all loci, and corresponding allelic frequencies ranged from 0.0040 to 0.8452. Twenty-one Y-STR loci had a high level of genetic polymorphisms, with the GD values exceeding 0.5, markers DYS527a/b (GD=0.9312) and DYS385a/b (GD=0.9213) on the one hand, and DYS391 (0.2683) and DYS438 (0.3164) on the other marked the extremes of the GD distribution (see Table S2). The haplotype distribution is presented in Table S3. A total of 214 different haplotypes were found in 252 male individuals, of which 186 (86.92﹪) were unique, 22 were observed twice, 4 were shared by 3 individuals, 1 was shared by 4 individuals, and 1 was shared by 6 individuals. The overall haplotype diversity was calculated to be 0.9983 with a discrimination capacity of 0.8492. In addition, three different triplications (19,20,22; 18,19,22 and 22,23,24) were observed in seven Miao samples at the DYS527a/b marker. One intermediate allele (20.2) was observed at the DYS527a/b marker; this allele was also observed in another ethnic group (Hani ethnic living in Honghe) (unpublished data). Six single off-ladder alleles (allele 14 at DYS448, allele 12, 12.1 at DYS458 and 13, 14, 17 at DYS447) were observed in this study （see Table S1）. All non-standard alleles are highlighted in Table S1 and were confirmed by retyping. The results indicated that the 24 Y-STR kit had a high power of discrimination and could be used for paternal lineage identification for the Miao ethnic group in Yunnan province. To achieve extensive illustration of the genetic relationship, Pairwise Rst and P value were calculated using AMOVA on the same 17 Y-STR loci between Yunnan Miao and 18 other populations data previously submitted to Y-STR haplotype reference database (YHRD, http://www.yhrd.org) (Table S4), namely, Liaoning, China [Northern Han] (YP000797), Yunnan, China [Han][7], Xinjiang, China [Kazakh][8], Liaoning, China [Manchu][9], Inner Mongolia, China [Mongolian][10], Fuzhou,China [She][11], Qinghai, China [Tibetan][12], Youyang Chongqing, China[Tujia][13], Xinjiang, China [Uygur][8], Yunnan, China [Bai] (YP000902), Xishuangbanna, China [Dai] (YP000903), Hunan, China [Dong] (YP001039), Hunan, China [Miao][14], Liaoning, China [Xibe][15], Guangxi, China [Zhuang][16], South Korea [Korean][17], Central Thailand, Thailand [Thai][18] and Vietnam [Kinh][19],
with the statistical significance determined by a permutation test (10,000 replicates). The pairwise Rst and associated P values among 19 populations are shown in Table S4. Significant genetic differences were observed between all groups (P values <0.0026, after Bonferroni’s correction) except Yunnan, China [Han] (P value=0.0200), and Hunan, China [Miao] (P value =0.0043). Based on Rst values, the Yunnan, China [Han] (Rst = 0.0266), and Hunan, China [Miao] (Rst = 0.0336), seemed genetically closer to Yunnan, China [Miao], than other populations. The MDS plot (Figure S2) and NJ phylogenetic tree (Figure S3) built from the RST distance matrices visually indicated this relationship. As the language spoken by Yunnan Miao and Hunan Miao people belong to the Miao-Yao branch of the Chinese–Tibetan language family, they have similar genetic backgrounds. Additionally, Yunnan Han and Yunnan Miao inhabit contiguous geographic regions. Therefore, the close proximity may have led to intermarriage, so they have a greater opportunity for gene exchange. The potential gene flow would help explain the close relationship between these groups. Our laboratory has participated in and passed the YHRD quality control; the population accession number is YA004179 (2016-06-02) (Yunnan, China [Miao]). This manuscript follows the new guidelines for publication of population data requested by the journal[20]. Acknowledgments: We would like to thank the donors who contributed samples for this study. This work was supported by grants from the Natural Science Foundation of China (31660310), the Science and Technology Planning Projects of Yunnan Province (2013FZ066, 2014FZ005) and the Yunnan Educational Science Foundation program (2014Y180). Conflict of Interest: All authors declare that they have no conflicts of interest.
References： 1.
P. Lewis: Ethnologue, Languages Of The World. 16th ed., edition: SIL International,Dallas; 2009.
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P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, Biotechniques. 10 (1991) 506-513.
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W. Bar, B. Brinkmann, B. Budowle, A. Carracedo, P. Gill, P. Lincoln, W. Mayr, B. Olaisen, DNA recommendations. Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. International Society for Forensic Haemogenetics, Int. J. Legal. Med. 110 (1997) 175-176.
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P.J. Lincoln, DNA recommendations--further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems, Forensic. Sci. Int. 87 (1997) 181-184.
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M. Nei: Molecular Evolutionary Genetics. Columbia University Press, New York,; 1987.
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K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular Evolutionary Genetics Analysis version 6.0, Mol Biol Evol. 30 (2013) 2725-2729.
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Y.M. Yang, T. Gu, Y.B. Zeng, C.J. Xiao, B.F Chen, L. Shi, B.Y. Xu, Q. Jing, Q.Y Zhuang, W. Zhang, et al, Genetic polymorphism of 11 Y-chromosomal STR loci in Yunnan Han Chinese, Forensic. Sci. Int. Genet. 4 (2010) e67-69.
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W. Shan, A. Ablimit, W. Zhou, F. Zhang, Z. Ma, X. Zheng, Genetic polymorphism of 17 Y chromosomal STRs in Kazakh and Uighur populations from Xinjiang, China, Int. J. Legal. Med. 128 (2014) 743-744.
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J. He, F. Guo, Population genetics of 17 Y-STR loci in Chinese Manchu population from Liaoning Province, Northeast China, Forensic. Sci. Int. Genet. 7 (2013) e84-85.
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X. Fu, Y. Fu, Y. Liu, J. Guo, Y. Liu, Y. Guo, J. Yan, J. Cai, J. Liu, L. Zha, Genetic polymorphisms of 26 Y-STR loci in the Mongolian minority from Horqin district, China, Int. J. Legal. Med. 130 (2016) 941-946.
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R. Bai, Y. Liu, X. Lv, M. Shi, S. Ma, Genetic polymorphisms of 17 Y chromosomal STRs in She and Manchu ethnic populations from China, Forensic. Sci. Int. Genet. 22 (2016) e12-14.
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B. Zhu, Y. Wu, C. Shen, T. Yang, Y. Deng, X. Xun, Y. Tian, J. Yan, T. Li, Genetic analysis of 17 Y-chromosomal STRs haplotypes of Chinese Tibetan ethnic group residing in Qinghai province of China, Forensic. Sci. Int. 175 (2008) 238-243.
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Y.R. Yang, Y.T. Jing, G.D. Zhang, X.D. Fang, J.W. Yan, Genetic analysis of 17 Y-chromosomal STR loci of Chinese Tujia ethnic group residing in Youyang Region of Southern China, Leg. Med. (Tokyo). 16 (2014) 173-175.
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W.H. Liu, H. Liu, J Yang, the genetic polymorphism of 26 Y-STR loci in Miao population from Hunan province, Journal of Forensic Medicine. 31 (2015) 307-308.
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F. Guo, L. Zhang, X. Jiang, Population genetics of 17 Y-STR loci in Xibe ethnic minority from Liaoning Province, Northeast China, Forensic. Sci. Int. Genet. 16 (2015) 86-87.
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H. Luo, F. Song, L. Zhang, Y. Hou, Genetic polymorphism of 23 Y-STR loci in the Zhuang minority population in Guangxi of China, Int. J. Legal. Med. 129 (2015) 737-738.
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S.H. Kim, N.Y. Kim, S.B. Hong, N.S. Cho, J.J. Kim, M.S. Han, W. Kim, Genetic polymorphisms of 16 Y chromosomal STR loci in Korean population, Forensic. Sci. Int. Genet. 2 (2008) e9-10.
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T. Siriboonpiputtana, U. Jomsawat, T. Rinthachai, J. Thanakitgosate, J. Shotivaranon, N. Limsuwanachot, P. Polyorat, B. Rerkamnuaychoke, Y-chromosomal STR haplotypes in Central Thai population, Forensic. Sci. Int. Genet. 4 (2010) e71-72.
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F. Miranda-Barros, C. Romanini, L.A. Perez, N.D. Nhu, T.D. Phan, E.F. Carvalho, C. Vullo, L. Gusmao, Y Chromosome STR haplotypes in different ethnic groups of Vietnam, Forensic. Sci. Int. Genet. 22 (2016) e18-20.
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A. Carracedo, J.M. Butler, L. Gusmao, A. Linacre, W. Parson, L. Roewer, P.M. Schneider, Update of the guidelines for the publication of genetic population data, Forensic. Sci. Int. Genet. 10 (2014) A1-2.
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Author: Tao Gu PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Author: Jinyong Yao PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Author: Canming Yang PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Author: Lei Du PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Authors: Jing bo Pang, Min Rao, Aiting Nie, Liping Hu, Shengjie Nie PII: DOI: Reference:
S1872-4973(17)30035-2 http://dx.doi.org/doi:10.1016/j.fsigen.2017.02.006 FSIGEN 1661
To appear in:
Forensic Science International: Genetics
Received date: Revised date: Accepted date:
28-10-2016 2-2-2017 11-2-2017
Please cite this article as: Jing bo Pang, Min Rao, Aiting Nie, Liping Hu, Shengjie Nie, Genetic analysis of 24 Y-STR loci in the Miao ethnic minority from Yunnan Province, southwestern China, Forensic Science International: Genetics http://dx.doi.org/10.1016/j.fsigen.2017.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title：Genetic analysis of 24 Y-STR loci in the Miao ethnic minority from Yunnan Province, southwestern China Xiufeng Zhang1†, Tao Gu2†, Jinyong Yao3, Canming Yang4, Lei Du5, Jing bo Pang1, Min Rao1, Aiting Nie1, Liping Hu 1*, Shengjie Nie 1* 1 School of Forensic Medicine, Kunming Medical University, Yunnan Province, People’s Republic of China 2 Center for Disease Prevention and Control of Kunming, Yunnan Province, People’s Republic of China 3 Honghe Public Security Bureau, Yunnan Province, People’s Republic of China 4 Eryuan Public Security Bureau, Yunnan Province, People’s Republic of China 5 Chenggong Public Security Bureau, Yunnan Province, People’s Republic of China † Xiufeng Zhang and Tao Gu contributed equally to this work. Email address:[email protected]; [email protected]
* Corresponding author: All correspondence should be addressed to: Dr. Shengjie Nie and Liping Hu. School of Forensic Medicine, Kunming Medical University, Yunnan Province, People’s Republic of China, 650500; Tel.: 0871-65922837; Fax: 0871-65922837; Email address: [email protected]; [email protected]
Abstract In the present study, 24 Y-chromosomal short tandem repeat (Y-STR) loci were analyzed in 252 unrelated Miao male individuals from Pingbian county, Honghe Hani and Yi Autonomous Prefecture, Yunnan province, southwestern China. The gene diversity of the 24 Y-STR loci in the studied group ranged from 0.2683 (DYS391) to 0.9312 (DYS527a/b). According to haplotypic analysis of the 24 Y-STR loci, 214 different haplotypes were obtained, 186 of which were unique. The overall haplotype diversity and discrimination capacity were calculated to be 0.9983 and 0.8492, respectively. In addition, three different triplications were observed at the DYS527a/b
marker, and 1 intermediate allele and six single off-ladder alleles were observed at four markers. We analyzed interpopulation differentiations by making comparisons between the Yunnan Miao ethnic minority and 18 other ethnic groups. The results obtained using pairwise genetic distances, multidimensional scaling plot, and neighbor-joining tree at the same set of 17 Y-filer loci indicated that Yunnan Miao had a closer genetic relationship with Yunnan Han and Hunan Miao individuals. The present results may provide useful information for paternal lineages in forensic cases and can also increase our understanding of the genetic relationship between Miao individuals and other groups. Keywords: Genetic distribution; Y-STR; Miao ethnic minority; Haplotypic structure The Miao ethnic group in China is primarily distributed across Guizhou, Hunan, Yunnan, and Sichuan provinces and the Guangxi Zhuang Autonomous Region. It is the 4th largest ethnic minority and had a population of 9,426,007 at the 2010 census. By the end of 2012, there were one hundred and fifty-six thousand (155630) people living in Pingbian county, Honghe Hani and Yi Autonomous Prefecture, Yunnan province. Pingbian county in Yunnan province is one of the five Miao Autonomous Counties in China. In addition to the Han ethnicity, there are 16 ethnic minorities living in Pingbian, such as Miao, Yi, Zhuang and Yao. This population totals one hundred thousand (103298), accounting for 66.3% of the total population in Pingbian. The language spoken by Miao people belongs to the Miao-Yao branch of the Chinese –Tibetan language family. Due to extended co-existence with the Han and other ethnic minorities, Miao people also speak the Han, Dong and Zhuang languages. They have been writing their own Miao language based on Latin since 1956[1]. The Miao opts for monogamy. Marriage in the same paternal lineage is restricted, while intermarriage among first cousins on the maternal side is a taboo. The sampling location of Yunan Miao in this paper is indicated in Figure S1. To establish a database of the Miao ethnic minority population residing in Yunnan province, China, we used an AGCU Y24（AGCU ScienTech Inc., China）kit, which includes 24 Y-chromosomal short tandem repeat (Y-STR) loci, namely, DYS391, DYS389Ⅰ, DYS439, DYS389Ⅱ, DYS438, DYS449, DYS456, DYS458, DYS437, DYS635, DYS448, DYS527a/b, Y-GATA-H4, DYS447, DYS19, DYS392, DYS522, DYS393, DYS388, DYS390, DYS385a/b and DYS444. In this study, we presented allele frequencies and haplotype distribution of 24 Y-STR loci in the Miao group and compared pairwise genetic distances with other populations. A total of 252 blood samples from Miao ethnic individuals were collected from Pingbian county, Honghe Hani and Yi Autonomous Prefecture, Yunnan province, Southwest China, after informed consent. DNA extracted with Chelex-100 was amplified in a GeneAmp PCR system 9700 (Applied Biosystems, USA) using an AGCU Y24（AGCU ScienTech Inc., China）PCR Amplification kit, separated by capillary electrophoresis on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, USA), and subsequently analyzed by GeneMapper v.3.2 software (Applied Biosystems, Foster City, CA)[2]. DNA typing and assignment of
nomenclature were based on the ISFG recommendations[3, 4]. Control DNA (9948) and ddH2O were used as positive and negative controls, respectively. Allelic frequencies were estimated by direct gene counting. Single-marker gene diversity (GD) was calculated according to Nei with the formula GD= n(1- Σpi2)/(n-1), where n and pi denote the total number of the samples and the relative frequency of the i-th allele, respectively[5]. Haplotype diversity was calculated similarly to GD. The discrimination capacity (DC) was defined as the ratio between the number of different haplotypes and the total number of haplotypes. To compare our data with other published data, genetic distances (Rst) were measured by analysis of molecular variance (AMOVA) and visualized using two multidimensional scaling (MDS) plots via the YHRD database (http://www.yhrd.org/Analyse/AMOVA). A neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 6.0 software to illustrate the relationships between populations [6]. The allele frequency and locus diversity of the 24 Y-STR loci in the Miao group are shown in Table S2. In this study, 199 different alleles were observed at all loci, and corresponding allelic frequencies ranged from 0.0040 to 0.8452. Twenty-one Y-STR loci had a high level of genetic polymorphisms, with the GD values exceeding 0.5, markers DYS527a/b (GD=0.9312) and DYS385a/b (GD=0.9213) on the one hand, and DYS391 (0.2683) and DYS438 (0.3164) on the other marked the extremes of the GD distribution (see Table S2). The haplotype distribution is presented in Table S3. A total of 214 different haplotypes were found in 252 male individuals, of which 186 (86.92﹪) were unique, 22 were observed twice, 4 were shared by 3 individuals, 1 was shared by 4 individuals, and 1 was shared by 6 individuals. The overall haplotype diversity was calculated to be 0.9983 with a discrimination capacity of 0.8492. In addition, three different triplications (19,20,22; 18,19,22 and 22,23,24) were observed in seven Miao samples at the DYS527a/b marker. One intermediate allele (20.2) was observed at the DYS527a/b marker; this allele was also observed in another ethnic group (Hani ethnic living in Honghe) (unpublished data). Six single off-ladder alleles (allele 14 at DYS448, allele 12, 12.1 at DYS458 and 13, 14, 17 at DYS447) were observed in this study （see Table S1）. All non-standard alleles are highlighted in Table S1 and were confirmed by retyping. The results indicated that the 24 Y-STR kit had a high power of discrimination and could be used for paternal lineage identification for the Miao ethnic group in Yunnan province. To achieve extensive illustration of the genetic relationship, Pairwise Rst and P value were calculated using AMOVA on the same 17 Y-STR loci between Yunnan Miao and 18 other populations data previously submitted to Y-STR haplotype reference database (YHRD, http://www.yhrd.org) (Table S4), namely, Liaoning, China [Northern Han] (YP000797), Yunnan, China [Han][7], Xinjiang, China [Kazakh][8], Liaoning, China [Manchu][9], Inner Mongolia, China [Mongolian][10], Fuzhou,China [She][11], Qinghai, China [Tibetan][12], Youyang Chongqing, China[Tujia][13], Xinjiang, China [Uygur][8], Yunnan, China [Bai] (YP000902), Xishuangbanna, China [Dai] (YP000903), Hunan, China [Dong] (YP001039), Hunan, China [Miao][14], Liaoning, China [Xibe][15], Guangxi, China [Zhuang][16], South Korea [Korean][17], Central Thailand, Thailand [Thai][18] and Vietnam [Kinh][19],
with the statistical significance determined by a permutation test (10,000 replicates). The pairwise Rst and associated P values among 19 populations are shown in Table S4. Significant genetic differences were observed between all groups (P values <0.0026, after Bonferroni’s correction) except Yunnan, China [Han] (P value=0.0200), and Hunan, China [Miao] (P value =0.0043). Based on Rst values, the Yunnan, China [Han] (Rst = 0.0266), and Hunan, China [Miao] (Rst = 0.0336), seemed genetically closer to Yunnan, China [Miao], than other populations. The MDS plot (Figure S2) and NJ phylogenetic tree (Figure S3) built from the RST distance matrices visually indicated this relationship. As the language spoken by Yunnan Miao and Hunan Miao people belong to the Miao-Yao branch of the Chinese–Tibetan language family, they have similar genetic backgrounds. Additionally, Yunnan Han and Yunnan Miao inhabit contiguous geographic regions. Therefore, the close proximity may have led to intermarriage, so they have a greater opportunity for gene exchange. The potential gene flow would help explain the close relationship between these groups. Our laboratory has participated in and passed the YHRD quality control; the population accession number is YA004179 (2016-06-02) (Yunnan, China [Miao]). This manuscript follows the new guidelines for publication of population data requested by the journal[20]. Acknowledgments: We would like to thank the donors who contributed samples for this study. This work was supported by grants from the Natural Science Foundation of China (31660310), the Science and Technology Planning Projects of Yunnan Province (2013FZ066, 2014FZ005) and the Yunnan Educational Science Foundation program (2014Y180). Conflict of Interest: All authors declare that they have no conflicts of interest.
References： 1.
P. Lewis: Ethnologue, Languages Of The World. 16th ed., edition: SIL International,Dallas; 2009.
2.
P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, Biotechniques. 10 (1991) 506-513.
3.
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