Forensic characterization of 15 autosomal STRs in four populations from Xinjiang, China, and genetic relationships with neighboring populations.

The Xinjiang Uyghur Autonomous Region of China (XUARC) harbors 47 ethnic groups including the Manchu (MCH: 0.11%), Mongols (MGL: 0.81%), Kyrgyz (KGZ: 0.86%) and Uzbek (UZK: 0.066%). To establish DNA databases for these populations, allele frequency distributions for 15 autosomal short tandem repeat (STR) loci were determined using the AmpFlSTR Identifiler PCR amplification kit. There was no evidence of departures from Hardy-Weinberg equilibrium (HWE) in any of the four populations and minimal departure from linkage equilibrium (LE) for a very small number of pairwise combinations of loci. The probabilities of identity for the different populations ranged from 1 in 1.51 × 1017 (MCH) to 1 in 9.94 × 1018 (MGL), the combined powers of discrimination ranged from 0.99999999999999999824 (UZK) to 0.9999999999999999848 (MCH) and the combined probabilities of paternal exclusion ranged from 0.9999979323 (UZK) to 0.9999994839 (MCH). Genetic distances, a phylogenetic tree and principal component analysis (PCA) revealed that the MCH, KGZ and UZK are genetically closer to the Han population of Liaoning and the Mongol population of Mongolia while the MGL are closer to Han, Japanese, Korean, Malaysian, Hong Kong Han and Russians living in China.

Introduction

Xinjiang is a multi-ethnic region and has played an important role in connecting eastern Eurasia and western Eurasia. It was crossed by the famous Silk Road, which linked trade between East Asia, Central Asia, and Europe[1]. Many ethnic groups, including the Manchu (MCH), Mongols (MGL), Kirgiz (KGZ) and Uzbek (UZK) have lived there for hundreds of years[2].

The Manchu founded two Chinese Dynasties on the country’s inner plains: the Jin Dynasty, founded by the Nvzhen people, and the Qing Dynasty, founded by Huang Taijin in 1635. The history of the Manchu can be traced back 6000–7000 years ago (6–7 kya). Although the Manchu people can be found in all over China[3], they represent only 0.11% of the Xinjiang population[4].

The Mongols came from the area around the east bank of the ancient Wangjian River (present-day Eerguna River) in Inner Mongolia. “Mengwu” is the earliest Chinese name for “Mongolia”. It first appeared in the Tang dynasty (618–907). “Mongol” was initially the name for one of the Mongolian tribes. At the beginning of the 13th century, the Mongolian tribe headed by Genghis Khan unified the other tribes in the region and gradually formed a new ethnic community. Therefore, “Mongolia” became the name for a nationality instead of a tribe[5]. As well as Mongolia, Mongols currently live mainly in the Inner Mongolia Autonomous Region and some prefectures of Xinjiang Uygur Autonomous Region like Bayingolin (South East) and Bortala (North West). They represent 0.81% of the Xinjiang population[4].

The Kyrgyz (or Kirgiz) live mainly in the southwest of Xinjiang, especially in the Kezhilesu Kyrgyz autonomous state. They have a long history and have been known in China by many names. In the Han dynasty, they were called “Gekun” or “Jiankun”. Later they were called “Qigu” in the Jin dynasty; “Jiankun”, “Jikasi” or “Qiliqisi” in the Tang and Song dynasty; and “Jirjisi” or “Qirjisi” in the Yuan and Ming periods. All these names were based on “Kyrgyz”, which has had different Chinese translation at different times. The etymology of “Kyrgyz” is thought to be “40 tribes” or “40 girls”[2]. While the Kyrgyz are primarily located in Kyrgyzstan, they represent only 0.86% of the Xinjiang population[4].

The name “Uzbek” first originated with Uzbek Khan, a local ruler in the Mongol Empire in the 14th century. The Uzbeks are an ancient Iranian people that intermingled with nomadic Mongol and Turkic tribes that invaded Central Asia between the 11th and 15th centuries. The Uzbeks that live in China live mostly in Xinjiang near the border with Russia and the former Soviet Central Asian republics. Uzbeks have been trading in western China for centuries. In the 16th century, they began to settle in cities in Xinjiang. Most Uzbeks in China still live in the cities and are engaged in trading or business[1]. They represent 0.066% of the Xinjiang population[4].

Short tandem repeat (STR) loci, also referred to as microsatellites or simple sequence repeats (SSRs), are DNA sequences that contain a repeat motif of 2–6 bp and are characterized by a high level of relatively stable polymorphisms, a dense, uniform chromosomal distribution as well as short sequence lengths, which facilitates detection and analysis by PCR and sequencing[6,7]. All these features render STRs as powerful genetic markers for inter-population studies[8] and for the reconstruction of recent human evolutionary history[9]. In view of their high level of variability, autosomal STRs have been the most common genetic markers used in forensic applications, including personal identification and paternity testing[10]. Most forensic laboratories use commercially available kits for multiple STR genotyping[11].

There have been previous studies of STR genotypes in the Uighur[12] and Kazak[13] populations of Xinjiang but the Manchu, Mongol, Kyrgyz and Uzbek populations remain uncharacterised. In the present study, the 15autosomal STRs in the AmpFLSTR Identifiler kit (Applied Biosystems, Foster City, CA, USA) were examined in the MCH, MGL, KGZ and UZK minorities of the Xinjiang Uyghur Autonomous Region (XUAR).

Results and Discussion

Forensic parameters

The distribution of allele frequencies and forensic statistical parameters in the four Xinjiang ethnic minorities are available from the authors upon request. Totals of 152, 165, 153 and 168 unique alleles were found in the Manchu, Mongol, Kyrgyz and Uzbek populations, respectively. The combined powers of discrimination (CPDs) for the 15 STR loci were 0.999 999 999 999 999 984 833, 0.999 999 999 999 999 990 057, 0.999 999 999 999 999 996 333 and 0.999 999 999 999 999 998 244, respectively. The combined powers of exclusion (CPE) for the 15 STR loci were 0.999 999 416, 0.999 999 483, 0.999 997 932 and 0.999 998 973, respectively. The probabilities of identity for the different populations were 1/1.51 × 1017, 1/1.75 × 1018, 1/3.66 × 1018 and to 1/9.94 × 1018, respectively. D2S1338 had the highest heterozygosities and powers of discrimination (PDs) in all four populations. FGA was the most polymorphic locus in the Mongol (20 unique alleles) and Uzbek (19 unique alleles) populations, respectively. D18S51 was most polymorphic in the Manchu population (18 unique alleles) while D18S51, D21S11 and FGA all had 15 unique alleles in the Kyrgyz population. Informativeness can be quantitatively measured by the polymorphism information content. Theoretically, PIC values can range from 0 to 1. At a PIC of 0, the marker has only one allele. At a PIC of 1, the marker would have an infinite number of alleles. A PIC value of greater than 0.7 is considered to be highly informative. Clearly, markers with greater numbers of alleles tend to have higher PIC values and thus are more informative[14]. The Manchu and Mongol populations have four loci with PIC < 0.7 while the Uzbek and Kyrgyz populations have only two loci with PIC < 0.7. Therefore, most loci exhibited a high informativeness, showing the potential of the Identifiler panel for differentiation of individuals and for paternity testing for the four ethnic minority populations in the Xinjiang Uyghur Autonomous Region of China.

Hardy-Weinberg equilibrium (HWE)

All of the loci were in Hardy-Weinberg Equilibrium (HWE) in the Kyrgyz population (p > 0.05), while one STR locus was out of HWE for Manchu (D7S820), two loci for Mongol (CSF1PO, D19S433) and four loci for Uzbek (D18S51, D2S1338, D7S820 and FGA). However, when a sequential Bonferroni correction[15] was applied to mitigate against the so-called “multiple comparison problem” (where for a significant p-value of 0.5, 5% of tests are likely to be significant by chance), no loci in any of the four populations were found to be out of HWE.

Linkage equilibrium (LE)

Linkage disequilibrium (LD) can be caused by association between adjacent alleles co-inherited from single, ancestral chromosomes but may also be a result of selection, random genetic drift, the rate of mutation or recombination, nonrandom mating, founder effects, sampling effects, recent admixture, and population substructure[16]. Exact tests for linkage equilibrium (LE) showed that the p-values of 50 pairwise combinations of STR loci (11 in Mongolia and Manchu, 13 in Kyrgyz and 15 in Uzbek) were below 0.05 and thus displaying LD. After a sequential Bonferroni correction[15], only five pairs were out of LE. These were TH01/D8S1179 and D18S51/D13S317 in the Manchu population, vWA/D21S11 and D2S1338/D19S433 in the Uzbek population and FGA/D13S317 in the Kyrgyz population. All pairwise combinations of loci were in LE in the Mongol population. Therefore, of the 105 pairwise LE tests in each population, a maximum of two were out of LE in any population. Application of the “product rule” for calculation of random match probabilities across multiple loci is fully justified in the Mongol population and is unlikely to produce significant errors in the other three populations.

Cluster analysis with STRUCTURE

STRUCTURE analysis of the four populations from Xinjiang provided no evidence of population structure for any repetition at any value of K. That is, each repetition yielded ancestry proportions for each individual that were approximately equally distributed between each ancestral cluster and were no different between the four populations. STRs for forensic identity testing, such as those included in the Identifiler panel, are selected for high heterozygosity and minimal allele frequency differences between populations and so they generally make poor ancestry informative markers (AIMs) which require large allele frequency differences between populations. Further, pairwise FST between the four populations were generally < 0.03 except for Mongols at D5S818, D13S317, D16S539, D18S51, D19S433, FGA, TPOX and vWA. While we may have expected Mongols to exhibit some differentiation from the other three populations, it is not surprising that Manchus, Kyrgyz and Uzbeks are not differentiated by the STRs in the Identifiler panel using STRUCTURE.

Comparison with other populations

An AMOVA was utilized for comparison between the four populations in this study and previously published population studies employing the same 15 STR loci. Genetic distances (FST) and associated p-values for each locus are available from the authors upon request.. The largest genetic distances in the Manchu, Mongol, Kyrgyz and Uzbeck populations were observed at vWA, D19S433, FGA and TPOX, respectively, while the lowest distances were observed at D8S1179 in the Manchu population and at CSF1PO in the Mongol, Kyrgyz and Uzbek populations. Genetic distances between populations based on Nei’s formula[17] are available from the authors upon request. These were used to construct a neighbor-joining tree of the four populations from Xinjiang and the other populations (Fig. 1). The Manchu and Kyrgyz are most closely related and they share a most recent common ancestor with the Uzbeks and a second most recent common ancestor with Mongols (from Mongolia) and ethnic Han from Liaoning province. Mongols from Xinjiang were most closely related to Russians in China, Hui from Qinghai, Manchu from Liaoning and Salar from Qinghai.

Figure 1

Neighbour-joining tree of the Manchu, Mongol, Kyrgyz and Uzbek populations from Xinjiang in relation to other regional populations.

PCA was applied to normalized allele frequencies at the 15 STR loci in the Manchu, Mongol, Kyrgyz and Uzbek populations of Xinjiang (Fig. 2A), in other populations from Xinjiang (Uyghurs and Kazakhs: Fig. 2B) in other populations from China (Fig. 2C) and in other populations from neighboring countries (Fig. 2D). In Fig. 2A, the Manchu and Kyrgyz are clustered in the lower right quadrant closer to each other than to Uzbeks. Mongols appear in the upper right quadrant, away from Manchu, Kyrgyz and Uzbeks. These proximities are consistent with the phylogenetic relationships observed in the neighbor-joining tree (Fig. 1). In Fig. 2B, the Manchu, Kyrgyz, Uzbeks and Kazakhs are clustered in the lower right quadrant while the Uyghurs and Mongols are clustered in the upper right. In Fig. 2C, the Manchu, Kyrgyz, Uzbek, Kazakh and Mongols of East Mongolia (China) are clustered in the lower right quadrant while the Miao, Dong, Bouyei, Mongols from Xinjiang, Hui from Qinghai, Dongxiang from Qinghai, Salar from Qinghai, Russians in China, Han and Manchu of Liaoning are clustered in the upper right. Finally, in Fig. 2D, the Manchu, Kyrgyz, Uzbek and Kazakhs cluster with the Mongols from Mongolia, away from other populations. At all resolutions, PCA supports the genetic proximity of Manchu, Kyrgyz, Uzbek and Kazakhs in Xinjiang while Mongols in Xinjiang display greater genetic distance from these populations as well as from other Mongols in Mongolia and China. This interpretation is also consistent with Fig. 1.

Figure 2

(A) Principal component analysis (PCA) based on the 15 autosomal STR loci of the four populations from Xinjiang in this study. (B) Principal component analysis (PCA) based on the 15 autosomal STR loci of the four populations from Xinjiang in this study and two other Xinjiang populations from previous studies (Uyghur and Kazakhs). (C) Principal component analysis (PCA) based on the 15 autosomal STR loci of the four populations from Xinjiang in this study and other Chinese populations from previous studies. (D) Principal component analysis (PCA) based on the 15 autosomal STR loci of the four populations from Xinjiang in this study and other populations from neighboring countries.

Concluding remarks

In this study, forensic characterization of 15 autosomal STR loci in the Manchu, Mongol, Kyrgyz and Uzbek minority populations of Xinjiang was performed. The AmpFlSTR Identifiler panel was found to be appropriate for forensic identity testing and paternity testing in these populations with a high power of discrimination, no significant departures from HWE at any loci and minimal departure from LE for a very small number of pairwise combinations of loci. Population genetic analyses indicated that the Manchu, Kyrgyz and Uzbek were closely related while the Mongols of Xinjiang had a closer genetic relationship with Russians in China, Hui from Qinghai, Manchu from Liaoning and Salar from Qinghai. Surprisingly, Mongols from Mongolia and China were more closely related to Manchu, Kyrgyz and Uzbek than to Mongols in Xinjiang, perhaps suggesting an ancient divergence when Mongols originally migrated to present day Xinjiang.

Materials and Methods

Samples and DNA extraction

Blood samples were collected from a total of 1842 unrelated healthy individuals from the XUAR (1157 males, 685 females), including 306 Manchu (208 males, 98 females), 507 Mongols (male: 275, female: 232), 550 Kyrgyz (329 males, 221 females) and 479 Uzbek (345 males, 134 females). All participants gave their informed consent either orally and with thumb print (in case they could not write) or in writing after the study aims and procedures were carefully explained to them in their own language. The study was approved by the ethical review board of the China Medical University, Shenyang Liaoning Province, People’s Republic of China and in accordance with the standards of the Declaration of Helsinki. All blood samples were stored at −20 °C before DNA extraction. Genomic DNA was extracted from blood stains using the TIANamp Blood Spots DNA Kit (TIANGEN BIOTECH BEIJING CO., LTD) according to the manufacturer’s instructions and the concentration of DNA was quantified by absorption at 260 nm using an ultraviolet spectrophotometer (UV-2800AH, UNICO).

PCR amplification

PCR co-amplification of fifteen autosomal STR loci (D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, CSF1PO, D16S539, D7S820, D13S317, D2S1338, D19S433, and D5S818) were performed in a fluorescence-based multiplex reaction using the AmpFLSTR Identifiler kit (Applied Biosystems, Foster City, CA, USA). From 1 to 2 ng of the target DNA was amplified according to the manufacturer’s recommended protocol. Thermal cycling was conducted under the following conditions: 95 °C for 11 min; 28 cycles of 94 °C for 60 s, 59 °C for 60 s, 72 °C for 60 s; and a final extension of 60 °C for 45 min. All loci were amplified in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, Foster City, CA).

Genotyping

Amplified products were analyzed with reference to ABI GeneScan 500 LIZ internal size standard (Life Technologies) and AmpFlSTR Identifiler Allelic Ladder using an ABI 3130xl genetic analyzer (Applied Biosystems, Foster City, CA) according to the AmpFLSTR Identifiler standard protocol. Analysis of data obtained from the genetic analyzer was performed using GeneMapper software v3.5.

Quality control

Negative (autoclaved deionized H2O) and positive (AmpFlSTR Control DNA 9947 A) controls were employed for DNA extraction, DNA quantitation, PCR amplification and capillary electrophoresis. All negative controls displayed an absence of amplified product while positive controls were consistent with known genotypes.

Statistical analysis

Allelic frequencies and important forensic parameters, such as match probability (MP), power of discrimination (PD), power of exclusion (PE) and polymorphism information content (PIC) were calculated using PowerStats V1.2[18]. Observed heterozygosity (Ho), expected heterozygosity (He), pairwise FST and exact tests for Hardy–Weinberg equilibrium (HWE) and linkage equilibrium (LE) between pairwise combinations of loci were performed using Arlequin v3.5 based on a likelihood ratio test for unknown gametic phase[19]. Empirical distributions were obtained from 10,000 permutations. Principal components analysis was performed with MVSP 3.1 (http://www.kovcomp.com) based on allelic frequencies of the 15 autosomal STR loci. Nei’s standard genetic distances between currently studied and previously published populations (Russian[20], Saraki Pakistan[21], Korean[22], Punjabi[23], Indian[24], Morocco[25], Eastern Turkey[26], Hong Kong[27], Japanese[28], Interior Sindh (unpublished), Hungarian[29], South Iran[30], Azerbaijan[31], Turkish Cypriot[32], Afghanistan[33], Bangladesh[34], Malaysia[35], Kadazan Malaysia[36], Sindh Pakistan[37], Iraq[38], Pashtuns Afghanistan[39], Tajik Afghanistan[39], Uzbek Afghanistan[39], Turkmen Afghanistan[39], Mongols of Mongolia[40], Hazara Afghanistan[39], Kuala Lumpur Malaysia[41], Miao[42], East Mongolia of China[43], Dong[44], Bouyei[45], Han Liaoning[46], Manchu Liaoning[47], Hui Qinghai[48], Uyghur China[49], Russian in China[50], Dongxiang Qinghai[51], Salar Qinghai[51], Kazakh China[13]) were generated using the Phylip 3.69 package[52] and visualized with Mega7 software[53].

Cluster analysis using STRUCTURE

STRUCTURE (version 2.2)[54] was used to determine if there was any population structure within and between the Manchu, Mongol, Kyrgyz and Uzbek populations from Xinjiang. Raw genotypes are available from the authors upon request. The Admixture model with correlated allele frequencies was employed without prior population information (USEPOPINFO = 0). The number of inferred clusters (K) was varied from 2 to 10 with 10 repetitions of each K value and a total of 10,000 burnins and 10,000 Markov chain Monte Carlo (MCMC) simulations for each repetition.