Forensic characterisation and polymorphism analysis of 16 X-chromosomal STRs in the Jining Han population in Eastern China

Abstract Background X-chromosomal short tandem repeats (X-STRs) are a useful supplementary approach to analysing autosomal markers in forensics and kinship studies; such markers are not well-characterised in many populations. Aim To investigate population genetic polymorphism and forensic characterisation of 16 X-STRs in the Jining Han population, and analyse genetic relationships with other Chinese populations. Subjects and methods Allele frequencies for 16 X-STR loci were obtained from a sample set of 527 unrelated individuals from the Jining Han population. Population genetic analyses of Jining Han and another 10 reference populations were conducted using phylogenetic tree, principal component analysis and multidimensional scaling. Results We detected 149 alleles, with frequencies ranging from 0.0013 to 0.8242. The combined powers of discrimination in males and females were 0.999999997194774 and 0.999999999999995, respectively. The combined mean exclusion change (MEC)Krüger, MECKishida, MECDesmarais, and MECDesmarais Duos values were 0.999974632649096, 0.999999976997582, 0.999999977013201, and 0.999993755768423, respectively. We detected relatively high genetic homogeneity in populations with similar ethnic or geographic origins, and a close relationship between the Jining Han and Beijing Han populations. Conclusions The present findings indicate that the 16 X-STR loci examined are highly polymorphic in the Han population of Jining, providing useful information for forensic science and population genetics studies.


Introduction
Short tandem repeats (STR) are the main genetic markers used in forensic DNA typing.Autosomal and Y-chromosome STR genetic markers are widely used in individual identification and kinship identification, and many commercial kits have been developed.Relatively little research has focused on X chromosome STR (X-STR) genetic markers in forensic science.However, X-STR markers are of interest to forensic scientists owing to their unique pattern of inheritance.The genetic characteristics of X chromosome genetic markers differ from those of the autosomal and Y chromosomes.The alleles of the two X chromosomes of the mother can be randomly passed to their children; fathers can only pass X chromosome genes to daughters, and the X chromosomes of daughters must come from grandmothers (Diegoli 2015).Owing to the specificity of their inheritance mode, X-STR loci are useful for personal identification, especially the identification of complex genetic relationships involving X chromosome inheritance.
Previous studies have shown that the Goldeneye 17X Kit (Peoplespot, Beijing, China) can be used for applications in forensic genetics, particularly for investigating paternity and genealogy (Sun et al. 2013).However, to date, the STR loci included in the Goldeneye 17X Kit have not been evaluated in the Jining Han population.In this study, we determined the forensic parameters and allele frequencies of the 16 X-STR loci included in the Goldeneye 17X Kit (DX6795, DXS9902, DXS8378, HPRTB, GATA165B12, DXS7132, DXS7424, DXS6807, DXS6803, GATA172D05, DXS6800, DXS10134, GATA31E08, DXS10159, DXS6789, and DXS6810) in the Jining Han population.This investigation of the applicability of the Goldeneye 17X Kit is expected to provide a basis for expanding population genetics data and analysing genetic relationships between the Jining Han and other Chinese populations (Sun et al. 2019;Yang et al. 2019;Chen et al. 2020;Tao et al. 2020;Zhang et al. 2021;Liu, Yao et al. 2022;Liu, Yuan et al. 2022;Qing et al. 2022).

DNA extraction and PCR
Peripheral blood samples of 527 unrelated, healthy individuals randomly chosen from the Jining Han population were collected with informed consent.Samples were obtained and used after obtaining the approval of the Medical Ethics Committee of Jining Medical University (Approval No. JNMC-2021-YX-008).FTA cards were used to save as samples.DNA was extracted from blood samples according to the Chelex-100 protocol, as described previously (Walsh et al. 2013).Multiplex PCR was performed using the Goldeneye 17X Kit (Peoplespot, China) and the ProFlex™ PCR System (Thermo Fisher Scientific, Waltham, MA, USA).

Genotyping and quality control
Amplification products were separated by capillary electrophoresis on an Applied Biosystems® 3500 Genetic Analyser (Thermo Fisher Scientific, Waltham, MA, USA).Allele designations were determined by the comparison of sample PCR fragments with allelic ladders provided with the Goldeneye 17X kit.All steps were conducted using laboratory internal control standards and kit controls.Raw data were analysed using GeneMapper ID-X v.1.6(Thermo Fisher Scientific, Waltham, MA, USA) for genotype assignment.All experiments were conducted at the Forensic Science Centre of Jining Medical University, an accredited laboratory (ISO 17025), in accordance with quality control standards (Dang et al. 2020).

Data analysis
A modified Powerstats spreadsheet was used to calculate forensic parameters, including allele frequencies for the 16 X-STR loci.Exact tests of Hardy-Weinberg equilibrium (HWE) for each locus were performed using Genepop version 4.2 (http://genepop.curtin.edu.au/).The polymorphism information content (PIC), gene diversity (GD), power of discrimination in males (PDM), power of discrimination in females (PDF), and mean exclusion chance (MEC) in trios and duos (MEC Krüger , MEC Kishida , MEC Desmarais , and MEC Desmarais Duos ) were estimated using StatsX (Statistics for X-STR) version 2.0 (Lang et al. 2019).A principal component analysis (PCA) was implemented in SPSS version 21.0.Phylip 3.695 was used to analyse the genetic distances among the Jining Han and other populations.F ST and p-values were estimated for 16 X-STR loci to evaluate differentiation between Jining Han populations and other groups using Arlequin 3.5.A phylogenetic tree was reconstructed using Mega 7.0 with the neighbor-joining method.Furthermore, a two multidimensional scaling (MDS) plot was built using SPSS version 21.0.Values of p < 0.05 were considered significant.populations, indicating close genetic relationships among these populations.A MDS plot (Figure 3) showed that the Tibetan, Kazakh, and Yunnan Miao groups were isolated, while the Jining Han population clustered together with other populations, except the Yunnan Han.These results are consistent with those of the PCA and demonstrate that Chinese Han populations from different administrative divisions are closely related.
The genetic distances between the Jining Han and 10 other ethnic groups are presented in Table S7.In addition,  we constructed phylogenetic tree of these populations using the neighbor-joining method (Figure 4).In the phylogenetic tree based on genetic distances, all 11 ethnic groups were divided into two main clusters corresponding to ethnic origins.The Jining Han population exhibited the greatest distance from the Tibetan population (R ST = 5.9437).We detected the shortest distances between the Jining Han and the Beijing Han population (R ST = 0.0017).The relationships based on genetic distances were similar to those based on the PCA and phylogenetic analysis.
In conclusion, the 16 X-STR loci had abundant polymorphisms in the Han population in Jining City and can be used in forensic and genetic research.Furthermore, we found that populations with the same ethnic origin or a close geographical origin exhibit relatively high genetic relatedness.This study expands the available data for X chromosome genetic polymorphisms in the Chinese Han population.Finally, interested researchers can contact the corresponding author to obtain data.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Figure 2 .
Figure 2. Principal component analyses (PCA) based on 16 overlapping X-sTR loci between the Jining Han (Red) and 10 reference Chinese populations (Black).

Figure 3 .
Figure 3. multidimensional scaling (mDs) plot based on pairwise F sT values among Jining Han and other populations.

Figure 4 .
Figure 4. Phylogenetic analysis of the relationships among the Jining Han population and 10 reference populations.The phylogenetic tree was constructed using the neighbor-joining method based on 16 overlapping sTR loci using mega 7.0.