湖南漢族人群21個(gè)STR基因座的遺傳多態(tài)性（英文）

鄒鷹，郭娟娟，李情鵬，左道宏，劉金山，郭亞東，閆杰，扎拉嘎白乙拉，蔡繼峰，蘭玲梅

（1.中南大學(xué)基礎(chǔ)醫(yī)學(xué)院法醫(yī)學(xué)系，湖南長沙　410013；2.中南大學(xué)湘雅醫(yī)學(xué)院，湖南長沙　410013；3.內(nèi)蒙古民族大學(xué)附屬醫(yī)院內(nèi)科，內(nèi)蒙古通遼　028000）

·綜述·

湖南漢族人群21個(gè)STR基因座的遺傳多態(tài)性（英文）

鄒鷹1，郭娟娟1，李情鵬2，左道宏2，劉金山3，郭亞東1，閆杰1，扎拉嘎白乙拉1，蔡繼峰1，蘭玲梅1

（1.中南大學(xué)基礎(chǔ)醫(yī)學(xué)院法醫(yī)學(xué)系，湖南長沙410013；2.中南大學(xué)湘雅醫(yī)學(xué)院，湖南長沙410013；3.內(nèi)蒙古民族大學(xué)附屬醫(yī)院內(nèi)科，內(nèi)蒙古通遼028000）

目的調(diào)查湖南地區(qū)漢族人群21個(gè)STR基因座（D3S1358、D13S317、D7S820、D16S539、Penta E、D2S441、TPOX、TH01、D2S1338、CSF1PO、Penta D、D10S1248、D19S433、vWA、D21S11、D18S51、D6S1043、D8S1179、D5S818、D12S391和FGA）的遺傳多態(tài)性。方法共采集560例湖南漢族健康無關(guān)個(gè)體血液樣本，使用Chelex-100法提取DNA，應(yīng)用AGCU EX22試劑盒及9700 PCR擴(kuò)增儀進(jìn)行復(fù)合擴(kuò)增，擴(kuò)增產(chǎn)物使用310遺傳分析儀進(jìn)行分離分析。結(jié)果共發(fā)現(xiàn)248個(gè)等位基因，等位基因頻率分布在0.001~0.518。除Penta E（P=0.023）外，其余基因座的基因型分布均符合Hardy-Weinberg平衡。21個(gè)基因座的累積個(gè)人識(shí)別率、累積非父排除率、累積匹配率分別為0.999 999 999 999 999 999 999 999 8、0.999 999 998和1.36×10-25。結(jié)論21個(gè)STR基因座在湖南漢族人群中呈高度多態(tài)性。本研究可為法醫(yī)學(xué)個(gè)人識(shí)別及親子鑒定提供有價(jià)值的數(shù)據(jù)及理論基礎(chǔ)。

法醫(yī)遺傳學(xué)；多態(tài)現(xiàn)象，遺傳；短串聯(lián)重復(fù)序列；湖南；漢族

Introduction

Han nationality is the main nationality with the largestpopulation，notonlyinChinabutalso worldwide.It is generally believed that the ancestors of Han nationality can be traced back to Yan Emperor and Yellow Emperor.The Han people also call themselves“Hua Xia Zi Sun”and“Tang people.”Because of the diversity of gene，region，languageandculture，quiteanumberofdifferent tribes and branches evolved in the formation process of Han nationality.Most historians argue thatthe core of Han nationality was“pre-Qin”and that a unified Han nationality was formed during the QinandHanDynasties.HunanProvince，also called“Xiang Chu”and“Xiao Xiang”，is located at the central south China.With a long history，the province has played an important role in Chinese culture and economy.A trace of human activities was discovered in the Paleolithic Period；100 000 years ago the activities of planting rice came into being.During the early of Western Han Dynasty，Changsha County was established and Hunan gradually became the culture center of the southeast of China.At present，Hunan Province is composed of 54 nationalities besides Han nationality，with a household register population of 65683722（in 2010）［1］.

Short tandem repeat（STR），which is occasionally referred to microsatellite sequence，is a kind of DNA sequence inherited by Mendel co-dominance，easy and quick to standardize and genotype，and with high polymorphism［2］.STR typing methods have become major detecting means in forensic DNA analysis，gene mapping and genetic linkage analysis of forensic science.In forensic routine casework，Power-Plex?16 System Kit（Promega，Madison，WI），AmpF?STR?Identifiler?Plus PCR Amplification Kit and AmpF?STR?Sinofiler?PCR Amplification Kit（Applied Biosystems，F(xiàn)oster City，CA）are commonly practiced in the world.For a higher level ofdiscriminatorypower，however，extendedloci may be required in single paternity testing［3］and the paternity testing observed STR mutations［4］.In the current study，AGCU EX22 STR kit［5］was used to evaluate the21STRloci（D3S1358，D13S317，D7S820，D16S539，Penta E，D2S441，TPOX，TH01，D2S1338，CSF1PO，Penta D，D10S1248，D19S433，vWA，D21S11，D18S51，D6S1043，D8S1179，D5S818，D12S391 and FGA）performance in population genetics research，human identification，and paternity testing in forensic sciences.

Materials and methods

Samples and DNA extraction

A total of 560 blood samples were collected from unrelated healthy individuals of the Han population，222malesand238females，inHunan Province.The human Genomic DNA was extracted from the blood samples with the method of Chelex-100 extraction.The current study was approved by the Ethics Committee of Central South University Xiangya School of Medicine（Code:201303147），with the written informed consents provided for the collection of the samples and subsequent analyses.

PCR amplification

AGCU EX22 STR kit was applied to the amplification reactions of the 21 autosomal STRs loci following the manufacturer’s instructions.The 10 μL reaction volumes contained 1.0 μL（0.5-2.0 ng）genomic DNA，2.0 μL EX22 Primers，4.0 μL AGCU PCR Reaction buffer，0.4 μL Taq and 0.6 μL ddH2O.Temperature cycling conditions for PCR reactions were as follows:2-min denaturation at 95℃，10 cycles for 30s at 94℃，1min at 60℃，1min at 72℃，20 cycles for 30 s at 90℃，1 min at 58℃，1min at 72℃，and 10-min elongation at 72℃.All amplifications were conducted on a GeneAmp PCR 9700 thermal cycler（Applied Biosystems，F(xiàn)oster City，CA，USA）.

DNA typing

A 310 Genetic Analyzer（Applied Biosystems，F(xiàn)oster City，CA，USA）was used to separate and detect the PCR products by capillary electrophoresis.A microliter PCR product or allelic ladder was mixed with 15 μL Hi-Di formamide（Applied Biosystems，F(xiàn)oster City，CA，USA）and 0.5 μL internal lane standard CC5 ILS 600（Promega，Madison，WI，USA），before denatured at 95℃for 3 min，immediately followed by 3-min chilling on ice.Gene-Mapper ID 3.2 software（Applied Biosystems，F(xiàn)oster City，CA，USA）was used to analyze STR alleles by comparison with kit allelic ladders.Standard reference in all experiments was genotyped control DNA from cell line 9947A（Promega，Madison，WI，USA）. Statistical analysis

ModifiedPowerStats v1.2softwarepackage（Promega，Madison，WI，USA）was used to calculateallelicfrequencies，polymorphicinformation content（PIC），matching probability（MP），discrimination power（DP），probability of paternity exclusion（PE），observed heterozygosity（Ho）and exact tests of the Hardy-Weinberg equilibrium of the 21 STR loci［6］；SHEsis software［7］，to calculate the value of D′and r2；and Arlequin v3.11 software［8］，to compare the data of Hunan Han popu-lation with the previously published data of other populations. A Neighbor-Joining（N-J）phylogenetic tree based on pairwise Nei’s genetic distance［9］was developed through the phylogeny inference package（Phylip）version 3.695［10］.

Results and discussion

The diversity distribution was made of allelic frequencies and forensic statistical parameters of 13 Combined DNA Index System（CODIS）loci and 8 non-Combined DNA Index System（non-CODIS）loci（Penta E，D2S441，D2S1338，Penta D，D10S1248，D19S433，D6S1043 and D12S391）（Table 1&Table 2）.The allelic frequencies of 248 alleles at 21 STR loci ranged from 0.001 to 0.518；the genotype frequencies of 910 genotypes，from 0.001 8 to 0.373 2；and the MP，DP，PIC，PE and Ho，from 0.015 to 0.226，0.774 to 0.985，0.554 to 0.909，0.360 to 0.773 and 0.654 to 0.889，respectively.The combined powerof discrimination，combined power of exclusion，and combined matching probability of the 21 STR loci were approximately 0.9999999999999999999999998，0.999999998，and 1.36×10-25，respectively.Observed genotype distributions for each locus showed no deviationsfromHardy-Weinbergequilibriumexcept Penta E（P=0.023）.The possible causes were the errors in technology and standard of DNA typing，or random sampling.The D′values ranged from 0.072 to 0.249，whereas the r2values，from 0.001 to 0.005.The value of D′and r2suggested that the alleles were not in linkage disequilibrium.

Theresultswerecomparedwithpreviously published population data at the same STR loci using Arlequin v3.11 software（Table 3）.No statistically significant differences（P＜0.05）were observed between the current population and Singapore Han［11］populations for all the 13 CODIS STR loci.Statistically significant differences were found between Hunan Han and Anhui Han［12］，Zhejiang Han［13］，Changzhou Han［14］，Shaanxi Han［15］，Gansu Tu［16］，Hong Kong Han［17］，Qinghai Dongxiang［18］，Chongqing Han［19］，Yunnan Yi［20］，Xinjiang Kazak［21］，Qinghai Hui［22］，Qinghai Salar［23］，Shandong Han［24］and Fujian She［25］populations for 2（15），2（15），2（15），3（15），3（15），4（15），4（15），5（14），6（13），12（19），11（15），12（15），14（15）and 19（20）STR loci，respectively（The number within parentheses is the number of common STR loci between two populations）.Our results indicated the previous finding［26］thatallelicfrequencydistributionsvariedinthe populationsfromdifferentethnicgroupsinthe same country and even from the same ethnic group who migrated to live in different regions and areas.

Table 1Allelic frequency distributions of 21 STR loci in Hunan Han population samples（n=560）

Continued

Table 2Genetic parameters of 21 loci in Hunan Han population samples（n=560）

Furthermore，an un-rooted phylogenetic tree was constructed by comparing the allelic frequencies for the 13 CODIS STR loci between Hunan Han population and the other 15 populations［11-25］（Fig.1）. The analysis showed that Fujian She，Yunnan Yi，Hong Kong Han，Singapore Han，Hunan Han and Zhejiang Han population，who reside in South China or Middle Asian regions，belonged to a cluster. Zhejiang Han population lives more closely to the Hunan Han population，and they have very close relationships.From the West China Gansu Tu，Qinghai Hui，Xinjiang Kazak，Qinghai Dongxiang，Qinghai Salar，Chongqing Han and Shaanxi Han population showed close genetic relationship，while Anhui Han and Shandong Han population from North China belongedtooneclade.Geneexchangebetween populations generally occurs in the same department or near the geographic area［27］.The geographical factors play an important role in the formation of genetic polymorphisms，and the heterogeneity level between STR loci in populations from distant geographical distribution is higher［28］.

Conclusion

Thedatademonstratethe21STRlociare highly discriminating and polymorphic in the Hunan Han population.The 13 CODIS core STRs and additional 8 new autosomal STRs can obtain more information for routine forensic casework.Additionally，the population data can improve forensic DNA database，enrich Chinese population genetic information resources and provide genetic evidence for the origin of the Hunan Han population.

Acknowledgements

This study was supported by the Natural Science Foundation for the Youth（No.81302621）and the Fundamental Research Funds for the Central Universities of Central South University（2015zzts276）.

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（Editor:LI Cheng-tao）

Genetic Polymorphisms of 21 STR Loci in Hunan Province-based Han Population

ZOU Ying1，GUO Juan-juan1，LI Qing-peng2，ZUO Dao-hong2，LIU Jin-shan3，GUO Ya-dong1，YAN Jie1，ZHA Lagabaiyila1，CAI Ji-feng1，LAN Ling-mei1
（1.Department of Forensic Science，School of Basic Medical Sciences，Central South University，Changsha 410013，China；2.Xiangya School of Medicine，Central South University，Changsha 410013，China；3.Department of Internal Medicine，Affiliated Hospital of Inner Mongolia University for Nationalities，Tongliao 028000，China）

Objective To investigate the genetic polymorphisms of 21 short tandem repeat（STR）loci（D3S1358，D13S317，D7S820，D16S539，Penta E，D2S441，TPOX，TH01，D2S1338，CSF1PO，Penta D，D10S1248，D19S433，vWA，D21S11，D18S51，D6S1043，D8S1179，D5S818，D12S391 and FGA）.Methods A total of 560 blood samples were collected from unrelated healthy individuals of Han population in Hunan Province.Chelex-100 extraction method was applied to the extraction of genomic DNA，and an AGCU EX22 Kit and 9700 STR amplification was used in amplification reactions.The products were separated and analyzed on 310 Genetic Analyzer.Results A total of 248 alleles were observed，the allelic frequencies ranging from 0.001 to 0.518.Observation of genotype distributions for each locus showed no deviations from Hardy-Weinberg equilibrium except Penta E（P=0.023）.The combined power of discrimination，combined power of exclusion，and combined matching probability of the 21 STR loci were approximately 0.999 999 999 999 999 999 999 999 8，0.999 999 998，and 1.36×10-25，respectively. Conclusion The 21 STR loci show high polymorphisms in the Han population，which can provide valuable data and a theoretical basis for forensic individual identification and paternity testing.

forensic genetics；polymorphism，genetic；short tandem repeat sequences；Hunan；Han nationality

LAN Ling-mei，M.S.，experimentalist，major in forensic genetics；E-mail:lanlingmei0731@163.com

（date:2015-07-20）

DF795.2

A

10.3969/j.issn.1004-5619.2016.05.010

1004-5619（2016）05-0356-07

Author:ZOU Ying，M.S.，experimentalist，major in forensic genetics；E-mail:zouhawk@163.com