Genetic polymorphism and forensic application of 23 autosomal STR loci in the Han population of Panjin City, Liaoning Province, Northeastern China

Abstract Background Short tandem repeats (STRs) are consecutive repetition of a repeat motif and widely used in forensic medicine and human genetics because of their high polymorphism. Subjects and methods In the current study, 23 autosomal STR loci were genotyped from 1263 unrelated healthy individuals living in Panjin City, Liaoning Province, Northeastern China using the VeriFilerTM Express PCR Amplification Kit. The population comparison was performed between the Panjin Han population and the other relevant groups to further explore the structure of Panjin Han and its relationship with the other groups. Results The results found 316 alleles across the 23 STRs and the corresponding allelic frequencies ranged from 0.5198 to 0.0004. Except for D3S1358, TPOX, TH01, and D3S1358, all STR loci were highly polymorphic (PIC > 0.7), with the Penta E locus having the highest degree of polymorphism (0.9147). For population comparison, the exact test of population differentiation found that no significant difference was observed between the Panjin Han and the other Han populations, except for Guangdong Han and Jiangxi Han. Conclusion The Panjin Han population showed significant differences with the other ethnic groups in China (Bouyei, Dong, Hui, Miao, Tibetan, and Uygur) and the foreign ethnic groups.


Introduction
Short tandem repeats (STRs), also known as microsatellites, are consecutive repetition of a repeat motif with three to six base nucleotides (Rubab et al. 2020). The mean mutation rate of autosomal STRs is 1.8 Â 10 À3 for paternal origin and 0.3 Â 10 À3 for maternal origin (Hamester et al. 2019). On account of their high polymorphism and strong discrimination ability, they can be widely used in forensic medicine and human genetics (Zhu et al. 2015;. Due to their high mutation rate, they can provide more information for exploring the population genetic structure and revealing the evolutionary relationships between different populations Guo 2017;He et al. 2017). In forensic medicine, STRs have high discrimination ability due to their variable repeat counts among different individuals. Thus, they can be utilised in personal identification and paternity testing (Adnan et al. 2016Zhan et al. 2018).
Panjin City, part of Liaoning Province, is located in Northeast China and the centre of the Liaohe Delta. Its land area is 4103 km 2 with a warm temperate continental semihumid monsoon climate. It is bordered by Anshan City in the east and northeast, Yingkou City in the southeast across the Daliao River, Jinzhou City in the west and northwest, and Liaodong Bay in the Bohai Sea in the south. Panjin had a population of over 1.44 million in 2019 (www.stats.gov.cn). Han is the most dominant ethnic group of Panjin city followed by other minority groups such as Manchu, Koreans, Mongolians, Hui, and Xibe.
In the current study, we used the VeriFiler TM Express PCR Amplification Kit (Lu et al. 2017) to explore the genetic characteristics of 1263 Han individuals from Panjin City, Northeastern China. Additionally, in order to understand the genetics and structural background of the Panjin Han population, we compared our population with other reference populations. Population comparisons including Reynold's genetic distance, neighbor-joining tree, and multi-dimensional scaling (MDS) analysis were carried out between the Panjin Han population and different ethnic groups to better understand the genetic background and structure of the Panjin Han population.

Ethical compliance
People's Republic of China, and in accordance with the standards of the Declaration of Helsinki (SYMC-20211015-15). All participants gave their informed consent either orally and with thumb prints (in case they could not write) or in writing after the study aims and procedures were carefully explained to them.

Study population
Blood samples were collected using the FTA cards from 1263 unrelated healthy individuals living in Panjin City, Liaoning Province, Northeastern China ( Figure 1).

DNA extraction, PCR amplification, and genotyping
Genomic DNA was extracted from FTA cards using Chelex-100 method (Walsh et al. 1991). A total of 23 autosomal STR loci (D3S1358, vWA, D16S539, CSF1PO,TPOX, D8S1179, D21S11, D18S51, Penta E, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, D6S1043, D10S1248, D1S1656, D12S391, D2S1338, and Penta D) were amplified simultaneously using the VeriFiler TM Express PCR Amplification Kit (ThermoFisher SCIENTIFIC, MA, USA). The 23 autosomal STR loci satisfied the requirements of the Chinese National autosomal DNA database as well as expanded Combined DNA Index System (Green et al. 2021). Multiplex amplification was conducted on a ProFlex TM PCR thermal cycler (ThermoFisher SCIENTIFIC), following the manufacturer's recommendations. Separation and detection were performed using the Applied Biosystems TM 3500 Genetic Analyser (ThermoFisher SCIENTIFIC). Internal controls (H 2 O as a negative control and 9947 A DNA as a positive control) were genotyped along with each batch of samples to ensure that the results were reproducible and accurate. The raw data was analysed using GeneMapper ID-X v1.2 software (ThermoFisher SCIENTIFIC).

Results and discussions
3.1. Allele frequencies and forensic parameters of the 23 autosomal STR loci

Hardy-Weinberg equilibrium (HWE)
Initially nineteen loci were in HWE, with D8S1197, TH01, D12S391, and Penta D not in HWE (p < 0.05). However, when  a sequential Bonferroni's correction was applied, all 23 loci were found to be in Hardly-Weinberg equilibrium (Table 1).

Linkage equilibrium (LE)
Linkage disequilibrium (LD) indicates the association between qualitative random variables corresponding to alleles at different STRs. Measuring the levels of linkage disequilibrium is important for gene mapping and it helps in the understanding of genome structure. Exact tests for LE between 253 pairs showed that the values of only 23 pairs were below 0.05 and thus displaying LD (Supplementary Table S2). When sequential Bonferroni's correction was applied, there were no pairs of loci displaying LD.

Population comparison with the other populations
In order to study the genetic structure of the Panjin Han population and its relationship with the other populations, the Reynold's genetic distance was calculated based on the allele frequencies across the 15 autosomal loci shared by the 21 included compared populations (Tie et al. 2006;Zuniga et al. 2006;Yan et al. 2007;Montelius et al. 2008;Wu et al. 2008;Gomes et al. 2009;Deng et al. 2011;Yoo et al. 2011;Chen et al. 2012;Tong et al. 2013;Zhang et al. 2015;Zhang 2015aZhang , 2015bXiao et al. 2016;Hongdan et al. 2017;Li et al. 2017;Xu, Feng, et al. 2017;He et al. 2018). The results showed that the Panjin Han had the nearest genetic distance with Liaoning Han (0.000289), followed by Heilongjiang Han ( Table 2). The exact test of population differentiation found that no significant difference was observed between the Panjin Han and the other Han populations, except for Guangdong Han (p < 0.0001) and Jiangxi Han (p ¼ 0.0190). Moreover, the Panjin Han population was significantly different to the other ethnic groups in China (Bouyei, Dong, Hui, Miao, Tibetan, and Uygur) and the foreign ethnic groups (Japanese, Korean, African, American, and European). The phylogenetic neighbor-joining (N-J) tree was generated to reflect the historical and geographical background of the compared populations ( Figure 2). The N-J tree exhibited significant differences between the Han population and the other minority groups. We also observed variations between Han populations from north to south, which was intricately sub-structured and clustered into three subgroups (northern Han, central Han and southern Han) in the previous study (Xu et al. 2009). Clearly, the Panjin Han population belongs to the northern Han subgroup, which also includes Heilongjiang, Jilin, Liaoning, Henan, and Hubei Han populations. These northern Han populations were also distributed in the close clusters in our N-J tree. In addition, the analysis of population differentiation found that The Panjin Han had major differences with the other Chinese minority ethnic populations and the foreign ethnic groups, which was also simultaneously mirrored in the N-J tree. The MDS plot was generated using the Reynold's genetic distance among the 22 compared populations (Figure 3). The Panjin Han and other Han groups were gathered together. The Chinese ethnic minorities, Korean and Japanese populations were scattered around the periphery. Additionally, the distribution of African, American, and European populations was more discrete. The location of the compared populations in the MDS plot was consistent with the results in the N-J tree. Geographically, Panjin city is located in Liaoning Province. The Liaoning Han integrated gradually with natives, such as Manchu, following geographical migration ). Consequently, the Liaoning Han population has its own unique genetic characteristics that are different from Han populations from other provinces. Similarly, the Panjin Han population belongs to the northern Han with its own unique structure characteristics.

Conclusions
In summary, 23 autosomal STR loci were genotyped in 1263 Panjin Han individuals using the VeriFiler TM Express PCR Amplification Kit. Population comparison was performed between the Panjin Han population and the other relevant groups to further explore the structure of Panjin Han and its relationship with the other groups. The results showed that the Panjin Han belong to the subgroup of northern Han and have major differences with the other Chinese minority ethnic populations and the foreign ethnic groups.

Author contributions
Hongbo Wang and Jun Yao developed the idea. Hongbo Wang analysed the results and wrote the manuscript. Bao-jie Wang and Cairui Xin conducted the experiment. All authors reviewed the manuscript.

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

Funding
The author(s) reported there is no funding associated with the work featured in this article.