Genetic diversity and phylogenetic characteristics of 38 Y-STR Loci in Lisu population from Kunming,Yunnan Province,Southwest China

Abstract

Y-Chromosomal short tandem repeat polymorphism (Y-STR) plays an important role in forensic practice due to its unique patrilineal inheritance pattern. Y-STRs of male individuals from different regions differ in terms of relevant forensic parameters and phylogenetic relationships. Although a large number of scholars have reported studies on the Y-STRs of different groups in different regions, there are still insufficient data on the Y-STR population genetics of the Lisu population in Kunming. To enrich the relevant research, a total of 801 healthy male participants from the Lisu ethnic group in Kunming, Yunnan Province, Southwest China, were included in this study. The DeepTyper Y41 Human DNA Typing Kit was used to co-amplify the 38 Y-STR loci. Haplotypes and associated forensic parameters were analyzed. Multidimensional scaling and neighbor-joining (N-J) phylogenetic tree were performed to complete the phylogenetic analyses based on the genetic distance (Rst) values in the Y-STR Haplotype Reference Database. 656 haplotypes were observed among the 801 male individuals. The value of haplotype diversity, haplotype matching probability and discrimination capacity was 0.9991, 0.0022 and 0.8190, respectively. Phylogenetic analyses showed that the Kunming Lisu population was closer to the Yunnan Han, the Liangshan Yi and the Yunnan Dai in phylogenetic relationship. Overall, this study enriches the research on population genetics on Y-STRs in Chinese ethnic minorities. The data can be utilized for population genetics and forensic research, specifically in the construction of genealogical databases.

Keywords

Genetic diversity phylogenetic characteristics Y-STR Lisu population Kunming

Introduction

Accounting for around 0.3% of the total DNA in the human genome differs between people, making us unique individuals, this difference makes it possible to distinguish between different individuals.^1,2 These variable regions of the human genome provide the capability to use DNA information for human identity verification purposes.³ They mainly exist in nuclear chromosome DNA, including not only autosomal chromosomes, but also sex chromosomes that determine the gender of an individual such as Y-chromosome and X-chromosome.¹ Nowadays, the Y chromosome, as a male-specific chromosome, has now become a very useful tool for studying human origins, migration, evolution, genetics, anthropology, history, genealogy, as well as forensic science fields due to its paternal inheritance and stable characteristics.⁴ The human Y chromosome plays an irreplaceable role not only in paternity testing, kinship analysis, familial searching, and male relative differentiation towards male individual identification, but also paternal lineage identification and inferring paternal bio-geographic ancestry in cases such as sexual assault, missing person and disaster victim identification, etc.⁵ Among them, the most extensive application of human Y-chromosome in forensic investigations is the determination of male suspects in sexual assault cases, including rape and gang rape, especially in some cases where autosomal DNA analysis fails to provide information, such as high levels of female DNA in the presence of minor amounts of male DNA.⁶ Currently, the Y chromosome short tandem repeats (Y-STRs) are the most commonly used genetic markers on the Y-chromosome. The haplotypes and frequencies of the Y-STRs vary among populations of different races, ethnicities, and regions. Therefore, it is necessary to establish different Y-STRs databases that covering various ethnic regions.

The Lisu ethnic group mainly resides in Nujiang Lisu Autonomous Prefecture and Diqing Tibetan Autonomous Prefecture in Yunnan Province.⁷ The rest are scattered in other regions of Yunnan, China, northeastern India, and the border area between Thailand and Myanmar. According to China's seventh national population census in 2020, the Lisu ethnic group has a population of 762,996, ranking 19th among China's ethnic minorities.⁸ To date, only one publication focused on the genetic diversity (GD) of Y chromosome short tandem repeat (Y-STR) among the Lisu ethnic minority in Chuxiong Yi Autonomous Prefecture.⁹ The forensic-related Y-STR data in Kunming Lisu population remains unclear, and there is a lack of research on the origin and migration of the Lisu in Kunming through Y-STR. In this study, we analyzed the relevant forensic parameters and phylogenetic relationships of Kunming Lisu population with other reported populations, which could be helpful in forensic fields such as male individual tracking, genealogical databases, as well as in the establishment of Y-STR reference databases, and improving some references for the origin of the Lisu in Kunming.

Materials and methods

Sample preparation

After informed consent, the blood stain samples were collected using DNA blood collection card from 801 unrelated healthy male Lisu individuals who are residents in Luquan Yi and Miao Autonomous County, Kunming City, Yunnan Province, Southwest China, for at least three generations and are confirmed as Lisu ethnic group which determined by registered residence file, identity information and pedigree documents. This study has been approved by the Ethics Committee of Kunming Medical University (No. KMMU2023MEC052) and complies with the ethical standards of the Helsinki Declaration.

DNA extraction, amplification and typing

Genomic DNA was extracted with the Chelex-100, which is the most commonly used method of DNA extraction in forensic science.¹⁰ The DeepTyper Y41 Human DNA Typing Kit (Guangzhou Koalson Biotechnology Co, Ltd, China) was used to amplify 38 Y-STR and 3 Y-Indel loci.¹¹ The total volume of PCR reaction system is 10 μL for each sample, including 2 μL of PCR Master Mix, 2 μL of Primer Mix, 5 μL of Nuclease-Free Water and 1 μL of DNA template. 9948 is a positive control. DNA fragments were amplified in a GeneAmp^® PCR System 9700 thermal cycler (Thermo Fisher Scientific, USA) according to the manufacturer's recommendations of the typing kit. The PCR procedures were as follows: pre-denaturation at 95 °C for 5 min, followed by 28 cycles of amplification (94 °C for 10 s, 62 °C for 90 s, 70 °C for 30 s), final extension hold at 60 °C for 15 min. PCR products were separated by capillary electrophoresis on an Applied Biosystems^® 3500 Genetic Analyzer (Thermo Fisher Scientific, USA). Genotyping was conducted using the GeneMapper™ ID-X v1.5 software (Thermo Fisher Scientific, USA). DNA typing and nomenclature assignment followed the recommendations of the International Society of Forensic Genetics (ISFG).^12,13 Moreover, the participating laboratory in this study has been accredited by the China National Accreditation Service for Conformity Assessment (CNAS).

Statistical analysis

The haplotype frequencies and allele frequencies of the Y-STRs were calculated through direct counting. Briefly, the haplotype frequency is calculated by dividing the number of haplotypes by the number of samples; whereas the allele frequency is calculated by dividing the number of allele occurrences by the sample size. The haplotype diversity (HD) were calculated using the formula: HD = n(1 − ∑Pi²)/(n − 1), where Pi represents the relative frequency of the allele, and N represents the sample size. The haplotype match probability (HMP) was calculated through the formulas: HMP = ∑Pi² (Pi is the frequency of the haplotype).¹⁴ The value of GD and discrimination capacity (DC) were calculated using the online tool of STR Forensic Analysis (STRAF).¹⁵

Pairwise genetic distances (Rst) between the Kunming Lisu and other populations were obtained by selecting 27 loci for analysis of molecular variance (AMOVA) using the Y chromosome haplotype reference database (YHRD) online tool (www.yhrd.org).¹⁶ Sixteen Chinese ethnic groups and three other foreign ethnic groups were selected for the study of phylogenetic relationships with the Kunming Lisu. Next, we used a multidimensional scaling (MDS) plot based on the Rst values in this database to visualise the pairwise genetic distances. The references of the related populations also were downloaded from the website. The heatmap of pairwise Rst values was performed by the heatmap package of R Software v. 3.3. On the basis of the pairwise Rst matrix, the neighbor-joining (N-J) phylogenetic tree was constructed using MEGA v6.0 software to further verify the genetic relationships between 20 populations.^17,18

Results

Relevant forensic parameters

Among 38 Y-STR loci in 801 male samples, a total of 656 different haplotypes were identified, of which 584 (89.02%) were unique, and 9 haplotypes were repeated 5 times or more. The HD value for the 656 haplotypes was 0.9991. The HMP and DC values were 0.0022 and 0.8190, respectively. Supplementary Table S1 shows the results for complete haplotypes, haplotype frequencies, HMP, HD, and DC. The GD values of 38 Y-STR loci ranged from 0.0369 (DYS645) to 0.9412 (DYS385), as shown in Figure 1. A total of 361 different alleles were found at all loci, with the highest allele frequency at locus 8 of DYS645. The allele frequencies and the detailed GD values of 38 Y-STR loci are presented in Supplementary Table S2 and S3, respectively.

Figure 1.

Genetic diversity (GD) of 38 Y-STR loci of Kunming Lisu population. These GD values were calculated using the online tool of STR Forensic Analysis (STRAF).

Population comparison study

AMOVA

All populations, which selected for phylogenetic relationships, and relevant references are shown in Supplementary Table S4. The pairwise Rst matrix and corresponding p values among all 20 populations are shown in Supplementary Table S5, and the heatmap of pairwise Rst values for 20 populations are shown in Figure 2. The Kunming Lisu had the smallest Rst value with the Yunnan Han (Rst = 0.0337), followed by the Yunnan Dai (Rst = 0.0369) and Liangshan Yi (Rst = 0.0442), while the Aba Tibetans (Rst = 0.3274) had the largest RST value with the Kunming Lisu, followed by London British Indians, Pakistanis, Bangladeshis and Sri Lankans (Rst = 0.1898).

Figure 2.

Heatmap of the pairwise genetic distances (Rst) values among the Kunming Lisu population and other reference populations. The Rst values between different populations were calculated via analysis of molecular variance (AMOVA) via the online tool of the YHRD database (www.yhrd.org). The heatmap was performed by the heatmap package of R software v. 3.3.

MDS plot of Kunming Lisu and other populations

The MDS plot (Figure 3) shows that the Yunnan Lisu, Yunnan Han, Yunnan Dai and Liangshan Yi were in the second quadrant, and the Yunnan Lisu ethnic group gets together with these three populations. Three Tibetans (Aba, Liangshan and Lhasa) were clustered separately in the first quadrant, away from the Kunming Lisu. Chinese Hans from different regions (Fujian, Yunnan and Shaanxi) gather together and distribute at the junction of the second and third quadrants.

Figure 3.

MDS plot between the Kunming Lisu and other populations. The MDS plot was generated directly from the online YHRD database on the basis of Rst values.

N-J phylogenetic tree of Kunming Lisu and other populations

The N-J phylogenetic tree (Figure 4) shows similar phylogenetic relationships to the MDS plot. All 20 populations are divided into two branches: the Tibetan group forms one branch, and the remaining 17 populations form another branch. The Kunming Lisu and Yunnan Dai are grouped close to each other, followed by the Liangshan Yi. The Kunming Lisu is far from the three Tibetan groups in the N-J phylogenetic tree.

Figure 4.

Neighbor-joining (N-J) phylogenetic tree based on the Rst values among 20 populations. The N-J phylogenetic tree was constructed using MEGA v6.0 software.

Discussion

The level of HD, DC and HMP values may reflect the level of genetic polymorphism in this population at the selected locus.¹⁹ Compared to Y-STR studies of other populations, such as Chuxiong Lisu (423 samples, 23 loci), Guizhou Miao (455samples, 36 loci), Jieyang Han (238 samples, 23 loci), and Central Kazakhstan population (112 samples, 27 loci), this study used a larger number of samples and Y-STR loci (801 samples, 39 loci) for a comprehensive GD study.⁹^20–22 The results of the relevant forensic parameters, such as HD and DC values, showed that the Kunming Lisu people have higher GD. At the same time, the smaller HMP values indicate that the likelihood of the two Kunming Lisu individuals having the same haplotype is reduced.

For the GD values of individual loci, except for DYS645, the other 37 Y-STR loci with high GD values are suitable for Y-STR typing in Kunming Lisu population. Previous Y-STR studies in other ethnic groups such as Hunan Dong, Guizhou Buyei, and Yunnan Hui have also found low GD values for the DYS645 locus.^23–25 This suggests that the locus DYS645 is poorly polymorphic in some Chinese populations, including the Kunming Lisu, and that there is a need to find a better polymorphic locus alternative. Meanwhile, higher GD values (＞0.8) were found for the Y-STR loci (DYS385, DYS449, DYS518, DYS527, DYS570, and DYF387S1) in this study these loci can help distinguish between close and distant male relatives in Kunming Lisu population in this study.

AMOVA is a statistical method for assessing the genetic structure and differentiation of populations. It estimates the evolutionary distance between populations from haplotypes (or genotypes), with the smaller the RST value approaching, the smaller the distance between populations.²⁶ This analysis is widely used in Y-STR studies and is the most commonly used method to study genetic distances between different populations.²⁷ In this study, we found that the Kunming Lisu had the smallest Rst values with the Yunnan Han, Yunnan Dai and Liangshan Yi groups, and the Kunming Lisu and these three groups were also close to each other in the MDS plot and N-J phylogenetic tree. These results show that the Kunming Lisu are genetically closer to these three groups, which is consistent with and in line with a large number of studies on the origin and migration of the Lisu. The Lisu had close ties with the Yi and Naxi ethnic groups in terms of ethnic origin, and even in the Ming Dynasty, the Lisu people were still regarded as a branch of the Yi ethnic group.²⁸ This may be the reason for the close relationship between Kunming Lisu and Liangshan Yi. During the Yuan and Ming dynasties, with the large-scale migration of the Lisu ancestors, some of them went south along the Jinsha River and entered places such as Luquan and Dayao.²⁹ At present, the Lisu ethnic group lives together with Han, Bai, Yi, Naxi and other ethnic groups, forming characteristics of large dispersion and small settlements.³⁰ This may explain why Kunming Lisu was closely related to Yunnan Han and Liangshan Yi. Similarly, Chinese Tibetans in different regions focus together due to the same linguistic origin and geographical proximity. Previous studies have also found that Tibetans from different regions cluster together, which reveals genetic homogeneity among Chinese Tibetans.³¹

In this study, we investigated the GD and phylogenetic relationships of the Kunming Lisu by analysing the Y-STR. However, this was done by analysing only Y-STR, and further analysis of other genetic markers was lacking. With the increasing popularity of high-throughput sequencing technology and the completion of human Y-chromosome sequencing, the use of high-throughput sequencing to detect a variety of genetic markers will provide us with more information about the origin, migration and evolution of the Lisu, which will be of great value for forensic applications.³²

Conclusions

In conclusion, this study reported for the first time that 38 Y-STR loci were highly GD in male individuals of the Kunming Lisu ethnic group, and these data may provide useful information for forensic practice in these areas. Meanwhile, this study assessed the phylogenetic relationships between the Lisu and 19 reference groups, revealing the close affinities of these Kunming Lisu with the Yunnan Han and Liangshan Yi. These findings will not only enrich Y-STR data of ethnic minority populations in China, but also be applicable to population genetics and forensic DNA analysis.

Supplemental Material

sj-pdf-1-msl-10.1177_00258024251399257 - Supplemental material for Genetic diversity and phylogenetic characteristics of 38 Y-STR Loci in Lisu population from Kunming, Yunnan Province, Southwest China

Supplemental material, sj-pdf-1-msl-10.1177_00258024251399257 for Genetic diversity and phylogenetic characteristics of 38 Y-STR Loci in Lisu population from Kunming, Yunnan Province, Southwest China by Xinjian Zheng, Wei Wei, Yue Wang, Chongchong Xu, Shixu Wang, Jiahui Peng, Chaojie Xiang, Yujie Fan and Shurong Zhong in Medicine, Science and the Law

Footnotes

Acknowledgements

We would like to thank all the participants who provided the sample for this study.

ORCID iDs

Xinjian Zheng

Shurong Zhong

Ethical considerations

The experimental protocol was established according to the ethical guidelines of the Helsinki Declaration and was approved by the Human Ethics Committee of Kunming Medical University (No. KMMU2023MEC052). Written informed consent was obtained from individual or guardian participants.

Consent to participate

Prior to blood sample collection, we received written informed consent from the participants after informing them about the purpose, procedures, potential risks and benefits of this study. We also respected any decisions made by participants during the blood sample and necessary personal information collection process. The personnel involved in this study received medical ethics training, including protection of personal privacy. We also train our study staff on privacy and sign confidentiality agreements to protect the security of participants’ personal information.

Consent for publication

In informed consent, participants are informed and agree that the data involved in this study may be made public in various ways, including but not limited to academic papers, databases, academic forums, academic conferences, etc. All authors have consented to the publication of the results of this study in Medicine, Science and Law, and all or part of this manuscript has not been published elsewhere.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (82260273, 81660232 and 81000577).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data availability

The data that support the findings of this study are available from the corresponding author [Shurong Zhong], upon reasonable request.

Supplemental material

Supplemental material for this article is available online.

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