Genetic variation and forensic characteristic analysis of 25 STRs of a novel fluorescence co-amplification system in Chinese Southern Shaanxi Han population

We analyzed the genetic polymorphisms of 15 autosomal and 10 Y-chromosomal STR loci in 214 individuals of Han population from Southern Shaanxi of China and studied the genetic relationships between Southern Shaanxi Han and other populations. We observed a total of 150 alleles at 15 autosomal STR loci with the corresponding allelic frequencies ranging from 0.0023 to 0.5210, and the combined power of discrimination and exclusion for the 15 autosomal STR loci were 0.99999999999999998866 and 0.999998491, respectively. For the 10 Y-STR loci, totally 100 different haplotypes were obtained, of which 94 were unique. The discriminatory capacity and haplotype diversity values of the 10 Y-STR loci were 0.9259 and 0.998269, respectively. The results demonstrated high genetic diversities of the 25 STR loci in the population for forensic applications. We constructed neighborjoining tree and conducted principal component analysis based on 15 autosomal STR loci and conducted multidimensional scaling analysis and constructed neighbor-joining tree based on 10 Y-STR loci. The results of population genetic analyses based on both autosomal and Y-chromosome STRs indicated that the studied Southern Shaanxi Han population had relatively closer genetic relationship with Eastern Han population, and distant relationships with Croatian, Serbian and Moroccan populations.


INTRODUCTION
Short tandem repeats (STRs) are widely distributed in human genome and play a significant role in forensic DNA analysis. Autosomal STRs have been the most common genetic markers in forensic applications and could be used to solve most of the personal identification and paternity testing cases [1,2]. However, Y-chromosomal STRs www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 33), pp: 55443-55452 Research Paper www.impactjournals.com/oncotarget (Y-STRs) would be more helpful in some special cases, such as mixed stain detection for sexual-assault cases, the paternal migration history tracing [3], and so on, because STRs on the non-recombining region of Y-chromosome do not participate in meiotic recombination and are unaltered when inherited from father to son [4][5][6]. At present, autosomal STRs or Y-STRs are often used alone in forensic applications. However, when autosomal STRs and Y-STRs were co-amplification in a single fluorescence multiplex system, they would be useful in both personal identification and paternity testing cases, for those Y-STRs could be helpful in the determination of gender and the reconstruction of paternal lineage [7,8].
Southern Shaanxi region is the south part of Shaanxi province, covering the area from the south of Qinling Mountains to the north of Ba Mountain with the Han River flowing through from west to east; Southern Shaanxi consists of three cities: Hanzhong, Ankang and Shangluo, from west to east where mainly reside the Han population [9,10]. To further understand the genetic background of Southern Shaanxi Han population and provide population genetic data for forensic identification, we firstly studied 15 autosomal STR loci and 10 Y-STR loci together in Chinese Han population from Southern Shaanxi region, calculated the forensic parameters, and collected previously published population data with overlapping loci of both autosomal STRs and Y-STRs, to discuss the genetic relationships between the studied population and other populations.

The analysis of allelic frequencies and forensic parameters for the 15 autosomal STRs
According to the results of Hardy-Weinberg equilibrium (HWE) tests (presented in Table 1), all the autosomal STR loci showed no deviations from HWE (p>0.05). Tests of linkage disequilibrium (LD) were performed for all pairs of autosomal STR loci and the results were shown in Table 2. No LD was observed at a significance level of 0.0033 (α = 0.05/15) after Bonferroni correction, which indicated these autosomal STR loci were relatively independent. As summarized in Table 3, there were totally 150 alleles found at the 15 autosomal STR loci in the Southern Shaanxi Han population. As summarized in Table 1, the observed heterozygosity (Ho) in the studied population ranged from 0.6075 (TPOX locus) to 0.8411 (D8S1179 and D18S51 loci). The loci D18S51 and TPOX showed the highest and lowest expected heterozygosity (He), respectively. The power of discrimination (PD) and power of exclusion (PE) ranged from 0.7944 (TPOX locus) to 0.9623 (D2S1338 locus); and 0.2999 (TPOX locus) to 0.6774 (D8S1179 and D18S51 loci), respectively. The combined PD and PE were 0.99999999999999998866 and 0.999998491, respectively. All the 15 autosomal STR loci were found to be highly polymorphic in Southern Shaanxi Han population and the high value, which indicated their large potentiality for forensic individual identification.

Principal component analysis based on the 15 overlapping autosomal STRs
The principal component analysis (PCA) was performed among the Southern Shaanxi Han population and other 19 populations using the allelic frequencies of the 15 overlapping autosomal STR loci. The PCA result was shown in Figure 1. The first and second components accounted for 44.00 and 12.73% of the total variance, respectively; and the cumulative contribution of them was 56.73%, which was over half of the total variance. According to Figure 1, Southern Shaanxi Han was located in left part, close to Eastern Han, Liaoning Han and Jilin Han population. Moroccan, Croatian and Serbian gathered in the right edge of the plot, which were relatively far away from Southern Shaanxi Han.

Phylogenetic analysis based on the 15 overlapping autosomal STRs
The neighbor-joining tree (NJ tree) of the Southern Shaanxi Han and other 19 populations based on allelic frequencies of 15 overlapping autosomal STR loci was shown in Figure 2. In the NJ tree, the Southern Shaanxi Han population was also observed to be close with the

Phylogenetic analysis based on the 10 Y-STRs
As shown in Figure 4, the genetic relationships of the studied Southern Shaanxi Han and other 19 populations at the 10 overlapping Y-STRs were analyzed by the phylogenetic tree. In the NJ tree, the Southern Shaanxi Han population was also clustered close with the Eastern Han, Liaoning Han, Korean, Shandong Han and Xibe populations. Moreover, Croatian, Serbian, Moroccan and Yi populations were located furthest to Southern Shaanxi Han population. The phylogenetic result was similar to MDS result.

Sample collection and DNA extraction
We collected 214 blood samples (108 males and 106 females) from unrelated healthy Han individuals living in the south of Shaanxi province (Southern Shaanxi), China. All the samples were collected according to the criteria for selection as follows: their ancestors within three generations should be unrelated individuals and members of Han ethnic group, with no migration. All volunteers without diseases signed informed consents. The present study followed the human rights and the ethical principle of Xinjiang Medical University and approved by institutional ethics committee, Xinjiang medical university, China. Genomic DNA was extracted using the Chelex-100 method as described by Walsh et al [47].
Electrophoresis was performed using an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Fragment sizing was supported using the AGCU Marker SIZ-500 (AGCU ScienTech Incorporation, Wuxi, Jiangsu, China) internal size standard and allelic ladder as basis for comparison. Alleles were identified using the GeneMapper® ID V3.2 (Applied Biosystems, Foster City, CA, USA). The 9948 cell-line (Promega, Madison, WI, USA) DNA was also genotyped using the reagent as control.

Statistical analysis
Fifteen autosomal STRs were statistically analyzed as follows. We calculated the allelic frequencies and tested the Hardy-Weinberg equilibrium using the modified powerstat v1.2 spreadsheet [48]. The linkage disequilibrium of autosomal STRs was analyzed by Genepop v4.0.10 (http://genepop.curtin.edu.au/). The pairwise Fst and p values between the studied Han population and reference populations at 15 overlapping autosomal STR loci were estimated by the program ARLEQUIN v3.1 (http://cmpg.unibe.ch/software/ arlequin3). Principal component analysis was performed with MVSP 3.1 (http://www.kovcomp.com) based on allelic frequencies of 15 overlapping autosomal STR loci, which was used to explore the extent of correlation genetic relationships. Neighbor-joining tree based on allelic frequencies of 15 overlapping autosomal STR loci were calculated using the Phylip-3.69 Software (http:// evolution.gs.washington.edu/phylip.html). Based on the 10 overlapping Y-STR loci, another NJ tree was obtained by Phylip-3.69 Software (http://evolution.gs.washington. edu/phylip.html). Multidimensional scaling analysis of Y-STR based on Rst values was constructed by statistical software SPSS version 13.0 (SPSS Inc., Chicago, IL). Gene diversity and haplotype diversity were calculated using Nei's formula [49].

CONCLUSION
In summary, the results demonstrated high genetic diversities of the 15 autosomal STR loci and 10 Y-STR loci in the Southern Shaanxi Han population and the studied polymorphic markers were suitable for forensic DNA cases, which could offer a new tool for forensic investigation. In this study, 15 autosome and 10 Y chromosome STR loci were amplified by the multi-color fluorescence technique in a single PCR reaction, and genotyping profile of both autosomal STR and Y chromosome STR loci could be obtained simultaneously. This made it possible for forensic geneticists to get the information of personal identification, gender determination and pedigree investigation in one step, which is a time, labor and cost saving strategy. The results of population differentiation, principal component analysis, multidimensional scaling analysis and phylogenic analysis indicated the Southern Shaanxi Han population had closer genetic relationship with the Eastern Han population and more distant relationships with populations from other races. The present data which combined autosomal STR loci and Y-STR loci will be useful for the enrichment of Chinese genetic information resources and provide valuable forensic data for forensic DNA cases.