Haplotypic polymorphisms and forensic applications in Chinese Hunan Han population based on a series of Y-STR loci: a perspective of paternal inheritance

Abstract Background Hunan, a multinational province in China, possesses more than fifty ethnic groups, such as the Han, Yao, Tujia, Miao, and so on. Aims To evaluate the forensic efficiency of the novel panel and investigate the genetic relationships between Hunan Han population and 12 other reference populations from China. Subjects and methods Haplotypic data of 153 unrelated males of Hunan Han population were investigated using the AGCU Y SUPP Plus amplification system containing 27 Y-chromosomal short tandem repeat (STR) loci. Forensic parameters were calculated to evaluate the application efficiency of this panel in Hunan Han population. Results Haplotype diversity, discrimination capability, and match probability values were 0.9999999977, 1.0000, and 0.0065, respectively. Pairwise fixation index values demonstrated that the minimal genetic differentiation (0.0073) was found between Hunan Han population and Hunan Yao group, while the maximal genetic differentiation (0.0651) was observed between Hunan Han and Guangxi Yao group from the perspective of the patrilineal DNA analysis. Conclusions The haplotype distributions of 27 Y-STR loci in Hunan Han population exhibited remarkable polymorphisms. Moreover, this panel has potential advantages for the forensic applications regarding family investigations, paternity testing of the paternal line, and population genetics.


Background
Hunan province (latitude 24 38 0 -30 08 0 , longitude 108 47 0 -114 15 0 ), called "Xiang" for short, is located in the middle reaches of the Yangtze River and on the edge of the Yunnan-Guizhou Plateau in China.Surrounded by mountains on its three sides, Hunan is a mountainous province.This peculiar topography acts as a natural obstacle that blocks population immigration, making this area popular for human genetic study for population geneticists (Statistics 2012).Hunan province possesses more than fifty ethnic groups like the Yao, Tujia, Miao, and so on.Most of the ethnic minorities reside in the western region of Hunan province, which encompasses a total population of more than 6.6 million.The Han is the most populous population in Hunan province, accounting for nearly 90% of the overall population of the province (https://www.hunan.gov.cn).
The commonly used Y-STR loci are located on the nonrecombination region of the Y chromosome and are inherited only by male individuals in the family.Due to their unique inheritance pattern (paternal inheritance), the Y-STR loci have been widely utilised in forensic practice and recognised by forensic practitioners as they yield tremendous superiorities in mixed stain, paternity testing, family investigation, and ancestry inference.
In this study, we investigated allele and haplotype polymorphisms of the 27 Y-STR loci in 153 males from the Hunan Han population as well as the population genetic relationships among Hunan Han and reference populations in the light of paternal inheritance.

Ethics statement
This research was conducted according to the ethical principle of medical research involving a human subject that the World Medical Association Declaration of Helsinki recommended previously and was approved by the Ethics Committee of Xi'an Jiaotong University Health Science Centre and the Southern Medical University (Approval number: 2019-1039).The research protocol conformed to the guidelines and requirements of the above two ethics committees.

Population sample and DNA amplification and genotyping
A total of 153 unrelated healthy male volunteers living in Hunan province were recruited for this research, and they declared that their families were Han people who had lived in Hunan province for at least three generations.Their blood samples were collected after the written informed consents were obtained.The GeneAmp PCR System 9700 Thermal Cyclers (Applied Biosystems, Foster City, USA) was applied to conduct the PCR amplification following the manufacturer's protocol recommended by the Y SUPP Plus.The multiplex PCR amplification reaction was conducted in 25 ll PCR volume, which consisted of about 1.0 mm 2 bloodstain sample, 10 ll of Reaction Mix, 5 ll of Y SUPP Plus Primer Mix, 1 ll of Taq polymerase, and 9 ll of sdH 2 O.For DNA pre-denaturation, the thermal cycling parameter was set at 95 C for 2 min; then it was set at 29 cycles of 94 C for 30 s, 60 C for 1 min, 72 C for 1 min; and finally it was set at 60 C for 15 min and stored at 4 C.The PCR products were separated on the Genetic Analyser ABI 3500xL (Applied Biosystems, Foster City, USA).The GeneMapper ID-X software (Applied Biosystems, Foster City, USA) was applied to analyse Y-STR genotypes.

Data management and statistical analysis
Allele and haplotype frequencies of 27 Y-STR loci and other forensic parameters, such as haplotype diversity (HD), genetic diversity (GD), discrimination capability (DC), and match probability (MP) were calculated in the Hunan Han population, according to the recommendations from the DNA Commission of the International Society of Forensic Genetics (ISFG) (Gusmão et al. 2017).To illustrate the genetic relationships between Hunan Han and its neighbouring populations, raw data from 12 populations from China were acquired (Liu et al. 2018;Feng et al. 2020;Zhou et al. 2021).The detailed information on the studied population and all reference populations are presented in Supplementary found that 52 deletions or duplicated events occurred.Due to the primer binding site variants, deletions of the target region, or the loss of locus, the deletions (null alleles) were observed at DYS389I, DYS389II, DYS437, DYS446, DYS447, DYS448 loci, and the duplications were observed at DYS385a/b, DYS527a/b, DYS557, and DYS460 loci (Nan et al. 2021).We can see that the duplications and deletions of Y-STR loci are frequently observed in Chinese populations.
In fact, the reason for the duplications of Y-STR loci could probably be understood from the Y chromosome sequence distributions.Previously published studies reported that most sequences of the Y chromosome appear more than once, often in palindromic patterns (Rozen et al. 2003;Skaletsky et al. 2003).Since few genes on the Y chromosome resulted in fewer restrictions on its maintenance of sequence context, insertion polymorphisms were likely to occur.Since there is no recombination with the other chromosomes, the nonrecombination region on the Y chromosome may accumulate these insertion events and therefore generate multiple copies and primer binding sites in original single-copy loci (Butler et al. 2005).Initially, Y-STR loci like DYS385a/b, DYS527a/b, and DYF404S1a/b might possess two identical primer binding sites and the same copies for the reasons mentioned above, producing one allele peak (peak height and peak area are larger than those of normal allele peak).Subsequently, one of the alleles might be mutated in a onestep mutation at a rate of $10 À3 per generation, resulting in two divergent allelic peaks (Dupuy et al. 2004).Similarly, the generations of tri-copy and tetra-copy were also highly possible due to cumulative effects (Park et al. 2019).
Twenty-five Y-STR loci showed high GD values higher than 0.5, revealing that those loci were genetically high polymorphisms in the Hunan Han population.We observed 153 haplotypes among 153 individuals at 27 Y-STR loci, all of which were unique.The HD, DC, and MP values were 0.9999999977, 1.0000, and 0.0065, respectively.The 27 Y-STR loci provided high polymorphisms in Hunan Han population, which were suitable for forensic application in this population and could serve as a useful tool for forensic investigation.
To investigate the genetic relationships between Hunan Han and other reference populations in China, the pairwise F ST values, along with corresponding p-values, among 13 populations were calculated, and are listed in Supplementary Table 3.The Han population in Hunan province was observed to possess significant differences with all reference populations (p < 0.05).Pairwise comparisons between the Hunan Han, Huan Yao and Tujia groups became no longer statistically significant differences after the Bonferroni correction (p ¼ 0.05/78 ¼ 0.00064).The minimal genetic differentiation (0.0073) was found between Hunan Han population and Hunan Yao group, while the largest genetic differentiation (0.0651) was observed between Hunan Han population and Guangxi Yao group.The MDS plot was conducted based on the pairwise F ST matrix by the R version 4.0.4,and the plot is shown in Supplementary Figure 1.The stress value of the MDS was 0.1025 in this study and the MDS plot showed that the populations from Hunan province were comparatively clustered compared with populations from Guangxi province.Gelao, Zhuang, Miao, and Yulin Han populations from Guangxi province were basically gathered in the upper left part of the MDS.The studied Hunan Han population clustered with Hunan Yao, Hunan Miao, Hunan Tujia, and Hunan Dong populations.These results indicated that Hunan Han population has close genetic relationships with Yao, Miao, Tujia, and Dong groups in Hunan province.The abovementioned results were basically in accordance with previously published studies (Feng et al. 2009;Guo et al. 2017).
It is not hard to see that the more detailed genetic relationships between the studied population and reference populations need further discussion and exploration due to the small sample size collected and the limited number of Y-STR loci used in this study.Besides, the inhabitation characteristics and national culture can also influence genetic exchange (Laland et al. 2010;Fan et al. 2016).As more population data of these 27 Y-STR loci are acquired, we should be able to reveal the genetic background of Hunan Han more comprehensively.

Comments
In conclusion, this research investigated the allele frequencies as well as forensic parameters of 27 Y-STR loci in Hunan Han population using the above panel.These 27 Y-STR loci exhibited remarkable polymorphisms and could serve as a useful tool to be applied in population genetics and forensic application in Hunan Han population.Population genetic investigation revealed that Hunan Han population had a close genetic relationship with Hunan Yao group.

Table 1 .
(Excoffier and Lischer 2010)warewas applied to calculate the pairwise F ST values and corresponding p-values(Excoffier and Lischer 2010).A multidimensional scaling plot (MDS) was constructed based on the pairwise F ST values by R software.
(Fu et al. 2021)2021).Hailun et al. investigated 1160 twogeneration families from the Chinese Han population and