Phylogenetic characteristics and population genetic diversity of 15autosomal STR loci in Tujia population of Guizhou of China

Gao Hongyan ¹,Luo Li ¹,Long Fei ²,Luo Jia ²,Wu Jian ¹,Zhang Hao ¹,Chen Pengyu ¹

(1.Center of Forensic Expertise,Affiliated Hospital of Zunyi Medical University,Zunyi Guizhou 563099,China; 2.Criminal Technology Department,Zunyi Public Security Bureau,Zunyi Guizhou 563003,China)

[Abstract ]Objective To investigate the genetic polymorphisms of 15 STR loci in the Tujia ethnic minority of Guizhou province,and explore the genetic relationship with other populations at home and abroad.Methods Fifteen STR loci (CSF1PO,D3S1358,D5S818,D7S820,D8S1179,D13S317,D16S539,D18S51,D21S11,FGA,TH01,TPOX,vWA,D2S1338 and D19S433) were amplified in 353 unrelated healthy Tujia population residing in northern Guizhou.Modified-Powerstates and Arlequin were used to calculate the allele frequencies and forensic parameters.Phylip,Mega and SPSS software were applied to structure population genetic relation.Results No evidence of deviation from the Hardy-Weinberg equilibrium (HWE) was identified.A total of 392 alleles was founded and the allelic frequencies spanned from 0.0014 (D7S820) to 0.5227 (TPOX).The cumulative power of discrimination (CDP) and the cumulative probability of exclusion(CPE) of 15 STR loci were 0.99999999999999999541338655 and 0.999998251,respectively.Genetic distance,N-J tree and MDS revealed that the farthest genetic distance was between Guizhou Tujia and Argentina population,whereas the nearest was among Guizhou Tujia and Hubei Tujia.Conclusion These 15 STR loci have high polymorphisms,which could be utilized as a powerful tool for forensic paternity test and personal identification in Guizhou Tujia population.The population comparison demonstrates that Guizhou Tujia had a close genetic relationship with adjacent geographic Hubei Tujia and Han populations,whereas Guizhou Tujia was obviously different from Argentina population.

[Key words ]forensic science; short tandem repeat; genetic polymorphisms; Guizhou Tujia population

Short tandem repeat is a kind of variable number of tandem repeat (VNTR),which was first described as a valid tool in forensic science and medicine in the early 1990s^[1].STR is also a short DNA fragment with a simple repeat of 3～7 base pairs in length.Nowadays,STR has become an indispensable application value in forensic science on account of high polymorphism,multiplex amplification and labouring saving^[2].Guizhou province is located in the southwestern hinterland of China,bording Chongqing,Sichuan,Yunnan,Guangxi and Hunan province.Guizhou is also a multi-ethnic province,where the majority is Han population(62%),and the followings are Miao(12%),Bouyei(8%),Dong(5%),Tujia(4%) and any other ethnic groups(7%) (https://en.wikipedia.org/wiki/Guizhou) .Tujia is the 8th largest ethnic minority in China,reside in the Wuling Mountains,straddling the common borders of Hunan,Hubei,Guizhou and Chongqing province.populations genetic data of most populations in Guizhou Province have been reported,while that of Tujia ethnic minority is lack.In our study,we investigated 353 Tujia residents from Yinjiang and Yanhe,the most populous areas of Tujia in Guizhou,so as to establish the autosomal STR database of Guizhou Tujia population for forensic application in personal identification and parentage testing,and relationship exploring between Guizhou Tujia and other 24 populations^[3-25].

秦明月刚走出卢局长办公室，手机就响了，还是边峰打来的，他想想还是接了。边峰说：“秦队，你别不够意思啊，我们一起喝喝茶如何？”

1Materials and methods

1.1 Materials Under the international ethical guidelines,we randomly obtained the blood from 353 Tujia healthy individuals residing in northern Guizhou whose three generations were unrelated,all the samples were restored by EDTA anticoagulant tube under -20℃ until use.All the individuals signed informed consent.

1.2 PCR amplification and genotyping Genomic DNA was extracted from Chelex-100 described by Walsh et al^[26].15 STR loci and an Amelogenin locus were amplified by using the AmpFlSTR Identifiler Plus PCR Amplification Kit (Applied Biosystems,Foster City,CA,USA) following the manufacturer's advice in a 10μl volume system,including Master Mix 4μl,DNA polymerase 0.2μl,a mixture of upstream and downstream primers 2μl,ddH₂O 2.8μl and the template DNA samples for 1μl.PCR was performed by GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems,Foster City,CA,USA).PCR cycling was employed as 95 °C for 3 min,followed by 30 cycles of 94 °C for 30 s,60 °C for 1 min,72 °C for 1 min,and a fnal extension at 60 °C for 12 min and 4 °C for preservation.PCR products were detached through capillary gel electrophoresis by ABI3130 genetic analyzer (Applied Biosystems,Foster City,CA,USA).Under the electrophoresis,allelic genotypes were identified using GeneScan Analysis V3.7^[27] (Applied Biosystems,Foster City,CA,USA) and Genetyper V3.7 software^[27] (Applied Biosystems,Foster City,CA,USA).A positive control and negative control standards were went through all the experiments.

1.3 Statistical analysis The allele frequencies,matching probability (MP),power of discrimination (PD),polymorphism information content (PIC),power of exclusion (PE),paternity Index (PI) were calculated using Modified-Powerstates.The probability of Hardy Weinberg equilibrium (HWE),linkage disequilibrium (LD),heterozygosity (Ho) were computed by Arlequin V3.1^[28].The Nei’s genetic distances were calculated by Phylip V3.695 software (http:// evolution.genetics.washington.edu/ phylip.html.) and visualized as the neighboring joining tree(N-J tree) in Mega V7.0 software^[29].Moreover,multidimensional scaling (MDS) of Guizhou Tujia and other reference populations were plotted using SPSS V17.0 software^[30].

2.1 The allelic frequency and corresponding forensic parameters were performed in Table 1.A total of 392 alleles were founded in the 15 loci,and the allelic frequencies spanned from 0.0014 to 0.5227.All of the 15 loci were in agreement with the HWE and LD after Bonferroni correction.The highest MP was observed at TPOX (0.2205) and the lowest was at FGA (0.0283).The lowest PD was detected at TPOX (0.7795),while the highest was at FGA (0.9717).The PIC results came to the same thing,all of the 15 loci reached above 0.6 other than TPOX (0.5395).The maximum He was observed at D2S1338 (0.8924) and the minimum at TPOX (0.6232),PE discovered the same results.The CDP and the CPE of 15 STR loci were 0.99999999999999999541338655 and 0.999998251,respectively.

第一，题本编制。结合等值 IRT题目的难度参数估计数值，对难易排序合理的试题题本进行编制。而在题本当中的每页都涵盖多个难度接近亦或是相同的试题，试题来源都是实际测验，同样也可以是题库中没有被运用的试题。

2Results

1.4 Reference populations To explore the pollution genetic structure between the Guizhou Tujia and neighbouring populations,a total of 24 populations mainly living in Guizhou and neighbouring Provinces including 11 Hans and 10 minorities,as well as 3 foreign populations were selected for comparison.These 24 comparison groups belong to 3 different languages families:①Sino-Tibetan language family(Guangxi Zhuang,Guizhou Bouyei,Guizhou Dong,Guizhou Miao,Guangdong Han,Yunnan Han,Yunnan Yi,Guizhou Han,Hubei Tujia,Guizhou Tujia,Sichuan Han,Chongqing Han,Hunan Han,Hubei Han,Shanghai Han,Liaoning Han,Tianjin Han,Beijing Han,Tibetan); ②Altaic language family(Korean,Liaoning Manchu,Xinjiang Kazakh,Xinjiang Uyghur); ③Indo-European family(Argentina and Equatorial Guinea).

Tab 1The distribution of allele frequencies ,forensic efficiency and statistical parameters on 15STR loci in Guizhou Tujia population (n =353).

Tab 1(continued )

MP:matching probability; PD:Power of Discrimination; PIC:Polymorphism Information Content; PE:Power of Exclusion; PI:Paternity Index; Ho:observed Heterozygosity; p -HWE:probability for Hardy Weinberg equilibrium (HWE).

2.3 In the MDS plot on the basis of all populations (Figure 2A),we also observed that the Chinese came together,while Argentina and Equatorial Guinea population were far away from Chinese.In the Sino-Tibetan languages MDS plot (Figure 2B),we discovered that Yunnan Yi and Tibet Tibetan were at the upper right corner; Guizhou Dong,Guizhou Bouyei and Guizhou Zhuang were at the upper left corner; Guizhou Miao was at the left bottom; the remaining populations were in the middle of plot.

试验采用随机区组设计，3次重复，小区面积50 m2。栽培密度为133 395株/hm2，株行距均为25 cm×30 cm。每小区随机抽取10株进行株高、叶片数、分蘖数的测量统计。测产时采用三点法，每小区随机抽取3个点，每个点选取1 m2进行测产。试验具体设置如表1所示。

Tab 2The Nei 's genetic distance between the Guizhou Tujia and other 24reference populations

Fig 1phylogenetic tree on the basis of neighbor joining algorithm among 25populations

2.2 We used the neighbour joining tree to explore genetic relationships between Guizhou Tujia and other populations (Table 2 and Figure 1).The neighbour joining tree was on the basis of the Nei’s distances,demonstrating that the remoter genetic distance was between Guizhou Tujia,Argentina population(0.1124) and Equatorial Guinea population (0.1939),whereas the nearest was among Guizhou Tujia,Guizhou Han (0.0023),Sichuan Han (0.0025),Hunan Han (0.0029),Hubei Han (0.0031),Yunnan Han (0.0033),Hubei Tujia (0.0033) and Chongqing Han (0.0035).The N-J tree was clustered in two groups:The Argentina and Equatorial Guinea grouped in one branch,and the other 23 comparisons clustered together in another branch.Guizhou Tujia gathered with Hubei Tujia first and then got together with the rest of populations.

Fig 2Multidimensional Scaling analysis based on 15overlapped STR loci of Guizhou Tujia and other 24reference populations

3Discussion

Our results showed all of the 15 loci were in agreement with the HWE,this means that the population sample collection meets the statistical requirements,the population is representative as well as the genotypes are reliable^[24].All of the 15 loci were in accord with the linkage equilibrium after Bonferroni correction,this demonstrates that loci are independent of each other,and the product law can be used to calculate the forensic parameters^[10].A total of 392 alleles were founded in the 15 loci,and the allelic frequencies spanned from 0.0014 to 0.5227.The DP and PE reflected the practical application value of genetic markers in forensic personal identification and paternity test,respectively.It is generally believed that the loci have a better value of forensic application when DP>0.7 and PE>0.5^[14].The lowest PD was detected at TPOX (0.7795),while the highest was at FGA (0.9717).The PE results came to all of the 15 loci reached above 0.5 other than CSF1PO (0.4918),D3S1358 (0.4637),TH01 (0.3574) and TPOX (0.3197).According to the product law,the CDP and the CPE of 15 STR loci were 0.99999999999999999541338655 and 0.999998251,respectively.Therefore,these 15 STR loci have high polymorphism and have the potential to employ in paternity test and personal identification in Guizhou Tujia population.

A comprehensive population genetic structure among Guizhou Tujia and other 24 populations was revealed using N-J tree and MDS via 15 STR loci according to their ethic,linguistic family and geographic characteristics.Except Argentina and Equatorial Guinea populations,all others are homogenous groups as revealed in this study.Guizhou Tujia keeps the genetic affinity with ethnically different Han populations from geographically-neighboring positions,including Guizhou Han,Sichuan Han,Hunan Han,Hubei Han,Yunnan Han,Hubei Tujia and Chongqing Han.Whereas relatively distant to other Hans from other regions of China,such as Shanghai Han,Liaoning Han,Tianjin Han,Beijing Han etc.These results demonstrated that Guizhou Tujia have a more wide range of gene exchange with surrounding Han populations.Furthermore,remarkable hereditary differences among the compared Chinese minority groups were screened based on diverse methods and datasets.The closer genetic relationship was displayed between Guizhou Tujia and Hubei Tujia other than other ethnic minorities.From both the N-J tree and MDS plot,Guizhou Tujia clustered closely with the same ethnic population Hubei Tujia,while showed genetic difference with other ethnic minority groups from Southwest China (Guizhou Dong,Guizhou Miao,Guizhou Bouyei,Yunnan Yi and Guangxi Zhuang) as well as from other remote regions of China (Tibet Tibetan,Liaoning Manchu,Xinjiang Kazakh,Xinjiang Uyghur).Obvious geographical and linguistical cluster pattern can be discovered among these comparisons that Guizhou Tujia grouped with the same Tibeto-Burman language and adjacent Han populations,and far away from Liaoning Manchu,Xinjiang Kazakh,Xinjiang Uyghur,which belongs to Altaic language family and geographically distant.

In conclusion,our study revealed that these 15 STR loci are genetically polymorphic and discriminating in Guizhou Tujia population and could be used as a powerful tool in paternity test and personal identification.The population comparison demonstrated that Guizhou Tujia had a closer genetic relationship with adjacent geography such as Hubei Tujia who belong to the same ethnicity,as well as Han majority from adjacent regions such as Sichuan Han,whereas was obviously different from linguistically distinct populations from other countries such as Argentina.

[References ]

[1]Edwards A,Civitello A,Hammond H A,et al.DNA typing and genetic mapping with trimeric and tetrameric tandem repeats[J].American Journal of Human Genetics ,1991,49 (4):746-756.

[2]Butler J M.Genetics and genomics of core short tandem repeat loci used in human identity testing[J].Journal of Forensic Sciences ,2006,51 (2):253-265.

[3]Chen P,Wang B,He G,et al.Population genetic analyses of 20 autosomal STR loci in Chinese Han population from Tianjin in Northern China[J].Forensic Science International Genetics,2017,27:184-185.

[4]Yang M,Ren Z,Ji J,et al.Population genetic data and mutations of 22 autosomal STR loci in Guizhou Han population[J].Forensic Science International Genetics,2017,29:29-30.

[5]Yao J,Wang B J.Genetic variation of 25 Y-chromosomal and 15 autosomal STR loci in the han chinese population of liaoning province,northeast China[J].Plos One 2016,11 (8):0160415.

[6]Guo F,Li J,Wei T,et al.Genetic variation of 17 autosomal STR loci in the zhuang ethnic minority from Guangxi zhuang autonomous region in the south of China[J].Forensic Science International Genetics,2017,28:51-52.

[7]Zhang L.Population data for 15 autosomal STR loci in the bouyei ethnic minority from Guizhou province,outhwest China[J].Forensic Science International Genetics ,2015,17:108-109.

[8]Zhang L.Population data for 15 autosomal STR loci in the Dong ethnic minority from Guizhou Province,Southwest China[J].Forensic Science International Genetics,2015,16:237-238.

[9]Zhang L,Zhao Y,Guo F,et al.Population data for 15 autosomal STR loci in the Miao ethnic minority from Guizhou Province,Southwest China[J].Forensic Science International Genetics,2015,16:3-4.

[10]Zhang X,Hu L,Du L,et al.Population data for 20 autosomal STR loci in the Yi ethnic minority from Yunnan Province,Southwest China[J].Forensic Science International Genetics,2017,28:43-44.

[11]Jie Z,Yuanyan Z,Gang S.Genetic polymorphisms of fifteen short tandem repeats in Tujia ethnic group of Hubei province[J].Chinese Journal of Medical Genetics,2009,26 (6):701-704.

[12]Li Z,Zhang J,Zhang H,et al.Genetic polymorphisms in 18 autosomal STR loci in the Tibetan population living in Tibet Chamdo,Southwest China[J].International journal of legal medicine 2018; 132 (3):733-734.

[13]Jin X,Guo Y,Wei Y,et al.Phylogenic analysis and forensic genetic characterization of Chinese Uyghur group via autosomal multi STR markers[J].Oncotarget,2017,8 (43):73837-73845.

[14]Feng C,Wang X,Wang X,et al.Genetic polymorphisms,forensic efficiency and phylogenetic analysis of 15 autosomal STR loci in the Kazak population of Ili kazak autonomous prefecture,northwestern China[J].Annals of Human Biology,2018,45 (2):160-165.

[15]Xie J,Shao C,Zhou Y,et al.Genetic distribution on 20 STR loci from the Han population in Shanghai,China[J].Forensic Science International Genetics,2014,9:30-31.

[16]Hong S B,Kim S H,Kim K C,et al.Korean population genetic data and concordance for the PowerPlex(R) ESX 17,AmpFlSTR Identifiler(R),and PowerPlex(R) 16 systems[J].Forensic Science International Genetics,2013,7 (3):47- 51.

[17]Liu J,Guo L,Qi R,et al.Allele frequencies of 19 autosomal STR loci in Manchu population of China with phylogenetic structure among worldwide populations[J].Gene,2013,529 (2):282-287.

[18]Parolin M L,Real L E,Martinazzo L B,et al.Population genetic analyses of the powerplex(R) fusion kit in a cosmopolitan sample of chubut province (patagonia argentina) [J].Forensic Science International Genetics,2015,19:221-222.

[19]Alves C,Gusmao L,Lopez-Parra A M,et al.STR allelic frequencies for an African population sample (Equatorial Guinea) using AmpFlSTR Identifiler and Powerplex 16 kits[J].Forensic Science International Genetics,2005,148 (2-3):239-242.

[20]Ruan X,Wang W N,Yang Y R,et al.Genetic variability and phylogenetic analysis of 39 short tandem repeat loci in Beijing Han population[J].Hereditas,2015,37 (7):683-691.

[21]Bai R,Zhao L,Sun J,et al.Genetic variation of 18 STR loci in the Changsha Han population from Hunan Province,South Central China[J].Forensic Science International Genetics,2017,31:43-45.

[22]Xiao C,Zhang W,Wei T,et al.Population data of 21 autosomal STR loci in Chinese Han population from Hubei province in Central China[J].Forensic Science International Genetics,2016,20：13-14.

[23]Tong D,Chen Y,Ou X,et al.Polymorphism analysis and evaluation of 19 STR loci in the Han population of Southern China[J].Annals of Human Biology,2013,40 (2):191-196.

[24]Zou X,Li Y,Li P,et al.Genetic polymorphisms for 19 autosomal STR loci of Chongqing Han ethnicity and hylogenetic structure exploration among 28 Chinese populations[J].International Journal of Legal Medicine,2017,131 (6):539- 1542.

[25]He G,Li Y,Wang Z,et al.Genetic diversity of 21 autosomal STR loci in the Han population from Sichuan province,Southwest China[J].Forensic Science International Genetics,2017,31:e33-35.

[26]Walsh P S,Metzger D A,Higuchi R.Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material[J].BioTechniques,1991,10 (4):506-513.

[27]AmpF STR Identifiler Plus PCR Amplification Kit [EB/OL].[2019-04-13].http://www.appliedbiosystems.com

[28]Laurent Excoffier,Laval G,Schneider S.Arlequin ver 3.1-An integrated software package for population genetics data analysis[J].Evol Bioinform Online,2007,1:47-50.

[29]Kumar S,Stecher G,Tamura K.MEGA7:Molecular evolutionary genetics analysis version 7.0 for bigger datasets[J].Mol Biol Evol,2016,11(7):1870-1874.

[30]Hansen J.Using SPSS for windows and macintosh analyzing and understanding data[J].The American Statistician,2005,59(1):113.

贵州土家族 15个常染色体 STR基因座遗传多态性及群体遗传关系分析

高红艳¹，罗 丽¹，龙 飞²，罗 佳²，吴 建¹，张 昊¹，陈鹏宇¹

(1.遵义医科大学附属医院 司法鉴定中心，贵州 遵义 563099； 2.遵义市公安局 刑事技术处，贵州 遵义 563003)

[摘 要] 目的 通过调查法医学中常规应用的15个STR基因座在贵州土家族人群中的遗传多态性，评估其在该人群中的法医学应用价值，比较与国内外其他群体的遗传关系。方法 应用Identifiler Plus复合STR试剂盒对贵州地区353例健康土家族无关个体进行15个STR基因座(CSF1PO、D3S1358、D5S818、D7S820、D8S1179、D13S317、D16S539、D18S51、D21S11、FGA、TH01、TPOX、vWA、D2S1338、D19S433)的分型，采用Modified-Powerstates、Arlequin计算等位基因频率和法医遗传学参数，采用Phylip、Mega、SPSS软件构建群体遗传关系。结果 15个STR基因座均符合Hardy-Weinberg平衡，共发现392个等位基因，等位基因频率范围为0.0014(D7S820)～ 0.5227(TPOX)。15个STR基因座的累积个人识别率(CDP)和累积非父排除率(CPE)分别为0.99999999999999999541338655和0.999998251。遗传距离、系统发生树(N-J tree)、多维尺度分析(MDS)显示贵州土家族与湖北土家族遗传关系最近，与阿根廷群体遗传关系最远。结论 15个STR基因座具有高度的遗传多态性和鉴别能力，适用于贵州土家族人群的法医学个人识别和亲子鉴定。贵州土家族与同民族同语系的湖北土家族遗传关系最近，与贵州汉族、四川汉族、重庆汉族地理位置毗邻的群体遗传关系较近，而与属于不同语系地理位置较远的阿根廷群体距离最远。

[关键词] 法医学；短串联重复(STR)；遗传多态性；贵州土家族

[中图法分类号] DF795.2

[文献标志码] A

[文章编号] 1000-2715(2019)05-0488-07

[基金项目] 遵义医学院附属医院博士启动基金资助项目(NO:201501)。

[通信作者] 陈鹏宇，男，博士，副教授，研究方向：法医物证学，E-mail:pychenfs@163.com。

[收稿2019-04-14;修回2019-06-17]

(编辑：谭秀荣)

标签：法医学论文;  短串联重复(STR)论文;  遗传多态性论文;  贵州土家族论文;  遵义医科大学附属医院司法鉴定中心论文;  遵义市公安局刑事技术处论文;