Genetic diversity of 15 STR loci in Yunnan Va ethnic minority and the phylogenetic relationships with 26 other populations

Abstract Background The Va (also called “Wa”) people are an ethnic minority living mainly in the southwest of Yunnan Province Aim This study was conducted to obtain the genetic information and forensic statistical parameters of 15 autosomal short tandem repeat (STR) loci included in the AmpFlSTR®Identifiler™ kit (Applied Biosystems, Foster City, CA) in the Yunnan Va population, with a view to enriching the genetic databases of the Chinese Va population. Subjects and methods A total of 508 unrelated Chinese Va individuals were genotyped with this 15 STR kit, the genetic polymorphisms and associated forensic parameters were calculated. The genetic relationships between Chinese Va and 26 other Chinese populations were also evaluated. Results All of the STR loci reached the Hardy–Weinberg equilibrium after Bonferroni correction. A total of 159 alleles were observed with allele frequencies ranging from 0.000984 to 0.606299. The combined discrimination power (CDP) and the cumulative probability of excluding (CPE) of the 15 STR loci were 0.999 999 999 999 999 988 126 and 0.999 995 734, respectively. Our results indicated that the geographically adjacent or ethnically close populations showed a higher genetic affinity. Conclusions The results of this study will enrich the forensic databases of the Chinese Va population and could be applied in forensic analysis.


Introduction
The Va (also called "Wa") people are an ethnic minority and live predominately in the southwest of Yunnan Province. Ximeng and Cangyuan counties are the main places where the Va people live in compact communities, others are found scattered in the Lancang, Menglian, Shuangjiang, Gengma, Zhenkang counties and the Xishuangbanna Dai Autonomous Prefecture. In the areas where the Va people live, there are also Hans, Yis, Dais, Hanis, Lahus, Jingpos, Blangs, De'angs and Lisus. It is the 26th largest ethnic minority and according to the 2010 census has a population of 429,709, 98.8% of which were distributed in Yunnan. Historical records show that the forebears of today's Vas, Blangs and De'angs came under the rule of the Han Dynasty, the ancestors of the Bulang, Deang and Va ethnic groups were closely related with ancient Bai-Pu people (Cang 1997). The Va language belongs to the Palaung-Va language group of the Austroasiatic Language family. Before the founding of the People's Republic of China in 1949, except for some parts of the area where an alphabetic script was used, the Va people had no written language, and they kept records and accounted or passed messages with material objects, or by engraving bamboo strips. An alphabetic script was created for the Va people in 1957. The location of the studied population sample in this paper is shown in Supplementary  Figure S1.
To establish a database of the Va ethnic minority population from Yunnan Province, we used the AmpFlSTR V R Identifiler TM PCR Amplification Kit (Applied Biosystems, Foster City, CA) that includes 15 autosomal short tandem repeat (STR) loci, namely D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA. In this study, we presented the allele frequencies and the statistical forensic parameters of 15 STR loci. In addition, the pairwise genetic distances with other Chinese populations were compared.

Study population
Blood stains on the FTA card from 508 (421 males and 87 females) unrelated healthy Va individuals were collected from Pu'er city in Yunnan Province, after informed consent, and this study was approved by the ethics committee of

DNA typing
Genomic DNA was extracted with the Chelex-100 method and amplified with the AmpFlSTR V R Identifiler TM (Applied Biosystems) PCR Amplification kit on GeneAmp PCR system 9700 (Thermo Fisher) according to the manufacturer's recommendations (Walsh et al. 1991). 25 lL reaction volume was used for each sample, which contains 10.5 lL PCR reaction mix, 5.5 lL Primer set, 0.5 lL Taq Gold DNA polymerase, 7.5 lL ddH2O, and 1.0 lL template DNA. PCR conditions had the following steps: pre-denatured 95 C for 11 min, followed by 28 cycles of 94 C for 1 min, 59 C for 1 min, 72 C for 1 min, a final extension hold at 60 C for 60 min and a final soak at 4 C. Amplified products were genotyped on a 3130xl Genetic Analyser (Applied Biosystems). The electrophoretic sampling mixture included 1.0 lL amplified product, 8.7 lL Hi-Di formamide and 0.3 lL Gene ScanTM 500 LIZ Size Standard. Raw data analysis for STR was performed with the GeneMapper ID v3.2 software (Applied Biosystems).

Quality control
In all, 9947A and ddH 2 O were supplied in the kit and used as positive and negative controls, respectively. The typing and nomenclature assignments of DNA were based on the ISFG recommendations (Bar et al. 1997) and followed the guidelines for the publication of population data (Carracedo et al. 2014;Gusmao et al. 2017). Our lab has also been accredited with the China National Accreditation Service for Conformity Assessment (CNAS).

Statistical analysis
The distribution of allele frequencies, Hardy-Weinberg Equilibrium (HWE) and the statistical forensic parameters, including matching probability (MP), power of discrimination (PD), polymorphism information content (PIC), probability of exclusion (PE), typical paternity index (TPI), homozygosity, and heterozygosity were calculated using the modified PowerStats software (Zhao et al. 2003). The exact test of population differentiation was performed between the Yunnan Va and the other 26 Chinese populations using Arlequin v3.5 software (Excoffier and Lischer 2010). Pairwise genetic distances between populations were calculated using Phylip3.69 package (Retief 2000), and the results were applied to construct a neighbor-joining (NJ) phylogenetic tree with the MEGA 6.0 software (Tamura et al. 2013).

Results and discussion
Allele frequencies and forensic statistical parameters A total of 15 STR markers were genotyped in Va samples and their allele frequencies, along with a number of forensic statistical parameters of interest, are provided in Table 1. The observed heterozygosity of all the 15 markers was greater than 0.5 in the Va ethnic group. The lowest heterozygosity was 0.5650 (TPOX) and the highest was 0.8602 (FGA). The PIC ranged from 0.5288 (TPOX) to 0.8713 (D2S1338). Based on the heterozygosity and PIC, the FGA and D2S1338 are the most informative loci in the Va ethnic group. The PD value ranged from 0.7730 (TPOX) to 0.9680 (D2S1338), and the PE ranged from 0.2509 (TPOX) to 0.7151 (FGA). The combined power of discrimination (CPD) in the 15 STR loci was higher than 0.999 999 999 999 999 988 126, and the cumulative probability of exclusion (CPE) was 0.999 995 734. No statistically significant departure from the HWE was observed at any locus after the Bonferroni correction (p value > .05/ 15 % .0033) ( Table 1). A total of 159 alleles and 554 genotypes were identified with the corresponding allele frequencies varying from 0.000984 to 0.606299, and the number of alleles were observed at 15 STR loci ranging from 6 (TPOX) to 18 (FGA). The off-ladder alleles 19 and 23 were observed in two individuals at the CSF1PO, off-ladder allele 14 was observed at the TPOX, and off-ladder alleles 15 and 16 were observed at the FGA. Rare alleles 8.3 and 10.3 were observed at the TH01, and 20.2-24.2 were observed at the FGA, Therefore, these 15 loci exhibited high or medium polymorphism and can be used as genetic markers in human identity testing and paternity testing in the Va minority population in Yunnan, China.

Interpopulation comparison
The exact test of population differentiation was performed between the Va of the present study and 26 other Chinese populations (including 4 Han ethnic and 22 ethnic minority).
The p values at the same loci are shown in Supplementary  Table S1. After Bonferroni correction (p value < .05/ 286 % .000175, 286 was the number of tests), significant statistical differences were found between the present population and Yunnan Nu, Yunnan Derung, Xinjiang Kazakh, Xinjiang Uighur and Yunnan Nakhi populations at all compared STR loci, followed by Nantong Han, Guangdong Guangxi Gelao, Guangdong Guangxi Jing, Yunnan Hani and Yunnan Miao at 10 loci, however, there was no statistically significant difference between the studied population and the Yunnan Bai and Yunnan Dai at 8 STR loci.

Phylogenetic analysis
Genetic distances between the populations ranged from 0.001060 to 0.254626 as shown in Supplementary Table S2.
The largest genetic distances were observed between the Yunnan Va and Yunnan Derung (0.251508), followed by the Yunnan Nu (0.188323), whereas the shortest genetic distances were found between the Yunnan Va and Yunnan Han (0.019459), followed by the Guizhou Bouyei (0.020712). Furthermore, the population relationships on the basis of pairwise Fst were performed with Mega 6.0 and are shown by the NJ phylogenetic tree (see Figure 1). Six obvious clusters were observed in the NJ tree, including Nu and Derung from Yunnan, Kazakh and Uighur from Xinjiang, Hani from Suzhou and Yunnan, Lisu and Yi from Yunnan, Zhuang from Yunnan and Guangdong Guangxi, and Miao from Guizhou and Yunnan. In the NJ tree we observed that the Yunnan Va first clustered with the Yunnan Yi and Yunnan Lisu, and then grouped with the Yunnan Han population, one partial reason may be that the sample locations of these four studied groups are relatively closer to each other than the other groups. Kazakhs and Uighurs are Muslim groups, so these two populations grouped together with each other, which was consistent with previous reports (Yao et al. 2016).
between the two groups. Interestingly, the Yunnan Va were genetically closer to the Guizhou Bouyei than other populations except for Yunnan Han; a possible explanation for this phenomenon could be that ethnic fusions were common in these adjacent areas. Our results indicate that most populations that were geographically adjacent, with similar genetic backgrounds or were ethnically close, showed a higher genetic affinity. The population distribution patterns were consistent with previous studies (Zou et al. 2017;Li et al. 2017).
To further reveal the detailed genetic relationships of Chinese ethnic minorities, more STR loci, population data, and even more populations should be included in the analysis.

Conclusions
In the current study, we reported the allele frequencies and forensic statistical parameters of the AmpFlSTR V R Identifiler TM STR loci in the Chinese Va population from Yunnan province, which could serve as useful tools in forensic application and enrich the forensic database of Chinese populations.