Genetic polymorphism and forensic efficiency of 21 autosomal STR loci from Shandong Han population in Northern China

Abstract Background Highly polymorphic autosomal STR loci are useful for understanding population structure better and for forensic application, however the non-CODIS STR loci in the Han population of Shandong, located in Northern China, are not well-characterised. Aim To investigate population genetic polymorphism and forensic efficiency of 21 autosomal STR loci from the Shandong Han population in Northern China and reveal the genetic relationships with other populations both at home and abroad. Subjects and methods In this study, population genetic data of 21 autosomal STR loci included in the Goldeneye DNA ID 22NC Kit that includes four CODIS loci and 17 non-CODIS loci were determined for 523 unrelated Han individuals in Shandong. Results Significant deviations from Hardy–Weinberg equilibrium were not observed. A total of 233 alleles were detected with allele frequencies ranging from 0.0010 to 0.3728. The combined power of discrimination was 0.99999999999999999999999990011134, and the combined power of exclusion was 0.99999999788131. Furthermore, in an analysis of population differentiation Nei’s standard genetic distance and multidimensional scaling analysis, which were conducted based on the overlapping 15 STR loci, revealed that the Shandong Han population was most closely related to populations in close geographic proximity. Conclusions This study demonstrated that the 21 autosomal STR loci included in the GoldeneyeTM DNA ID 22NC system are highly polymorphic and suitable for forensic identification and paternity testing in the Shandong Han population. Additionally, the present results enrich the population genetic database.


Introduction
short tandem repeats (stRs), as the most important genetic markers, have been widely used in paternity testing and individual identification (Butler 2007;Ziętkiewicz et al. 2012).Many commercial stR multiplex amplification kits targeting the new combined DNa index system (cODis) stR core loci proposed by cODis core loci Working Group are suitable for applications in forensics (hares 2012, 2015;lian et al. 2016;he et al. 2018;Zhang et al. 2019).however, in some cases, including duo parentage analyses in which samples from the father or mother are lacking, complex kinship cases, and cases with stR mutations, a larger number of stR loci is needed for accurate identification.so additional stR loci independent of those in the current cODis system are needed as supplementary tools.some population data for various non-cODis systems have been reported (shen et al. 2013; song et al. 2014; li et al. 2018).however, the shandong han population has not been evaluated using the commercially available Goldeneye™ DNa iD 22Nc Kit (Peoplespot inc., Beijing, china), which simultaneously amplifies 21 autosomal stR loci, including four cODis loci (D1s1656, D2s441, D10s1248, and D3s1358) and 17 non-cODis loci (D4s2366, D6s477, D22Gata198B05, D15s659, D8s1132, D3s3045, D14s608, D17s1290, D3s1744, D18s535, D13s325, D7s1517, D10s1435, D11s2368, D19s253, D7s3048, and D5s2500).We performed the first investigation of allele frequencies and forensic parameters of 21 autosomal stR loci among 523 unrelated healthy chinese han individuals living in shandong, Northern china, using the Goldeneye™ DNa iD 22Nc Kit.Our results provide basic stR population data for forensic assays in the region and phylogenetic analyses.

DNA preparation and PCR
after obtaining written informed consent from all participants and ethical approval by the Medical ethics committee of Jining Medical University (NO.JNMc-2021-YX-008), according to the Declaration of helsinki, bloodstain samples were collected from 523 unrelated healthy individuals (240 males and 283 females) from the shandong han population of Northern china.Fta cards were used to save samples.Without DNa extraction, DNa was directly amplified using the commercially available Goldeneye™ DNa iD 22Nc Kit on Geneamp PcR 9700 thermal cycler (thermo Fisher scientific, Waltham, Ma, Usa) according to the manufacturer's instructions.the conditions were an initial denaturation at 95 °c for 2 minutes, 28 cycles of 94 °c for 5 seconds, 60 °c for 90 seconds, 62 °c for 60 seconds, and a final extension at 60 °c for 5 minutes.

Genotyping and quality control
PcR products were separated using an aBi 3500 Genetic analyser (thermo Fisher scientific) by capillary electrophoresis.allele identification was performed using GeneMapper iD-X version 1.5 (thermo Fisher scientific).all analyses included a positive control from the 9,948 DNa samples as well as a negative control without DNa.all experiments were conducted at the Forensic science centre of Jining Medical University, an accredited laboratory (isO 17025), in accordance with quality control guidelines.the laboratory has been accredited by the china National accreditation service for conformity assessment (cNas l9338).

Data analysis
allele frequencies and forensic parameters, including the observed heterozygosity (h O ), expected heterozygosity (h e ), matching probability (MP), power of discrimination (PD), power of exclusion (Pe), polymorphism information content (Pic), and typical paternity index (tPi), were evaluated using Powerstats version 1.2 (Promega, Madison, Wi, Usa) (tereba 1999).hardy-Weinberg equilibrium (hWe) at each locus, and linkage disequilibrium (lD) for all pairwise stR loci, were evaluated using arlequin version 3.5 (excoffier and lischer 2010).interpopulation differentiation based on the allele frequency distributions of the studied loci between shandong han and other reference populations was analysed by analysis of molecular variance (aMOVa) implemented in arlequin version 3.5.Nei's standard genetic distance was computed using Phylip3.69. a multidimensional scaling analysis (MDs) was performed using sPss.

Results and discussion
the genotype data of 21 autosomal stR loci included in the Goldeneye™ DNa iD 22Nc Kit in shandong han are listed in supplementary table s1.allele frequencies and forensic parameters for 21 autosomal stR loci are shown in supplementary table s2. in total 233 alleles were observed with frequencies ranging from 0.0010 to 0.3728.all 21 autosomal stR loci were highly polymorphic, with unique allele numbers ranging from 7 (D3s1358) to 15 (D17s1290, D7s1517, and D1s1656).although D13s325 exhibited a deviation from hWe (p = 0.0182), no statistically significant deviations from hWe were observed after Bonferroni correction (p > 0.05/21).the observed heterozygosity and homozygosity values ranged from 0.7304-0.8795and 0.1205-0.2696,respectively.the MP, PD, Pic, Pe, and tPi values were 0.0307-0.1262,0.8738-0.9693,0.6752-0.8580,0.4768-0.7538,and 1.8546-4.1508,respectively.lD was not detected between pairs of stR loci, as shown in supplementary table s3, which is related to the distribution of stR loci on chromosomes.in general, there is no linkage relationship among loci when they are either located on different chromosomes or on the same chromosome but with a large physical distance; indicating that the 21 autosomal stR loci were independent of each other and the principle of multiplication could be utilised to calculate the combined discrimination power (cDP) and combined probability of exclusion (cPe).the cDP and cPe using all 21 stR loci were 0.99999999999999999 999999990011134 and 0.99999999788131, respectively, which demonstrated the extremely good efficiency in forensic application of individual identifications and paternity testing.
allele frequency distributions of the 17 non-cODis stR loci in the studied population were compared with 11 previously published population datasets from china and other countries.Owing to the limited stR loci in different multiplex amplification stR kits, only a portion of the 17 non-cODis stR loci were used for comparisons.the p-values of the interpopulation difference comparisons between the studied population and reference populations are shown in supplementary table s4.significant differences were observed at 1/7 loci in comparison with a han population in central china, 2/17 loci in a han population in hainan, 5/17 loci in a li population in hainan, 1/17 loci in a Uygur population in Xinjiang, and 1/7 loci in a Japanese population after Bonferroni correction.however,however, no statistically significant differences were detected in comparisons between the study population and the han in southern china, the hui in Xinjiang, the tibetans, and the Mongolians at 17 stR loci; the han from Northern china at 15 stR loci; or the Koreans at five stR loci.this suggested that the genetic differences between han people living in different regions of china are lower than they are to some ethnic minorities, which is consistent with the existing research (Xie et al. 2015;liu et al. 2016).Nei's genetic distances among the nine chinese populations were analysed based on 15 shared stR loci (D4s2366, D6s477, D22Gata198B05, D15s659, D8s1132, D3s3045, D14s608, D17s1290, D18s535, D13s325, D10s1435, D11s2368, D19s253, D7s3048, and D5s2500), as shown in table 1. the largest genetic distances were observed between shandong han and a li population (0.0508), followed by a Uygur population (0.0320), and the smallest distances were found between the shandong han and Northern han (0.0030), and between the shandong han and a Mongolian population (0.0054).a multidimensional scaling plot based on the genetic distance matrix is presented in Figure 1. the southern han and Northern han from different administrative regions fall into different quadrants in the MDs plots.the shandong han population was placed in the lower right quadrant and kept relatively close with the Northern han population.these results demonstrated that genetic relatedness was explained by geographic distance and ethnic origin.
in conclusion, genetic polymorphisms of 21 autosomal stR loci included in the Goldeneye™ DNa iD 22Nc Kit were evaluated for the first time in the shandong han population.the 21 autosomal stR loci were highly polymorphic.accordingly, the kit can be used as a complement to current commercial kits targeting cODis core loci for forensic identification and relatedness tests, especially in the region, and for phylogenetic analyses.additionally, our data provide valuable information for other population genetic and diversity studies.

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

Funding
this work was supported by the shandong Provincial Natural science Foundation [ZR2019P h039]; the project of shandong educational science planning [2020Zc255]; shandong undergraduate university teaching reform research project [M2020069]; Research Fund for academician lin he New Medicine [JYhl2018Ms13].

Figure 1 .
Figure 1.multidimensional scaling plots displaying the genetic relationships between shandong Han and eight reference populations.The stress value and squared correlation (RsQ) value were 0.09598 and 0.98288, respectively.

Table 1 .
nei's standard genetic distances between our studied population and eight previously published Chinese populations.