2. Guangzhou Forensic Science Institute, Guangzhou510030, China
2. 广州市刑事科学技术研究所， 广东 广州 510030
The PowerPlex®21 System is a widely used commercial kit for short-tandem repeat (STR) genotyping[1-3] , which yields reliable results with a robust design . Developed by Promega Corporation in 2012, this system has been shown to have a good inhibitor tolerance, a rapid PCR protocol and a good compatibility with direct amplification of FTA card punches and pretreated swabs . This system contained 17 combined DNA index system (CODIS) loci (the original set including D3S1358, D13S317, D16S539, D18S51, CSF1PO, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179 and FGA with an additional set including D1S1656, D2S1338, D12S391 and D19S433) and D6S1043, Penta E and Penta D, which contains more loci than other widely used kits such as PowerPlex 16 System (Promega, Madison, WI, USA), AmpFℓ STR Identifier System (Applied Biosystems, Foster City, CA, USA), AmpFℓ STR Sinofiler System and GoldeneyeTM DNA ID system 20A kit (Goldeneye Ltd, Beijing, China).
So far in China little population-based genetic data of the PowerPlex®21 System have been available, especially in terms of the locus D1S1656. In this study, we carried out a population investigation using this kit among the Chinese Han population living in Guangdong Province of South China. The data we obtained may enrich the international DNA database that can be used as a reference for human identification in forensic studies and genetic diversity studies.SUBJECTS AND METHODS Population
Bloodstains and mouth swabs of 2367 unrelated Chinese Han individuals living in Guangdong Province in South China were collected from the Center of Forensic Science, Southern Medical University (Guangzhou, China). The native birthplaces of these individuals were mostly Guangdong Province (1744 individuals), and a small portion of them were born in nearby provinces and regions including Guangxi Zhuang Autonomous Region (71 individuals), Hunan Province (196 individuals), Sichuan Province (175 individuals), Jiangxi Province (83 individuals) and Hubei Province (97 individuals). Informed consent to participating in this study was obtained from all the individuals.DNA Extraction and PCR amplification
Genomic DNA was extracted according to the Chelex-100 method and proteinase K protocol . The extracted DNA was amplified using the PowerPlex®21 System (Promega) in the GeneAmp 9700 PCR System (Applied Biosystems) following the manufacturer's protocol.Genotyping and quality control
The PCR products were detected by capillary electrophoresis in ABI 3130xl Genetic Analyzer (Applied Biosystems) using CC5 ILS 500 size standard and reference allelic ladder provided along with the PowerPlex®21 System. Data analysis and genotyping were performed automatically using GeneMapper ID v3.2 software (ABI Company, Forster City, USA). The internal controls (negative control and the 2800 M DNA positive control) were genotyped along with each batch of samples to ensure the reproducibility and accuracy of the results.Data analysis
The allelic frequencies, matching probability, power of discrimination, power of exclusion, polymorphism information content and typical paternity index were calculated using the PowerStats V12.xls software (http://www.promega.com/geneticidtools/). The P values of exact test of Hardy-Weinberg's equilibrium, expected heterozygosity and observed heterozygosity were calculated using Genepop software (http://genepop.curtin.edu.au/). The allele frequencies in this southern Chinese Han population were compared with the published data of other populations using Fst pairwise distance by Arlequin v3.5 software . A phylogenetic tree showing the inter-population relationshipwas constructed with the neighbor-joining method and based on Fst distances with 1000 bootstrap replications by software package POPTREE2 according to the allele frequency data of 15 shared STR loci (D3S1358, D13S317, D16S539, D18S51, D2S1338, CSF1PO, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D19S433 and FGA) from this present study and 13 reported populations. A principal component analysis (PCA) plot was drawn with Past 3.11 software based on allelic frequencies of 15 STRs.RESULTS
Tab. 1 shows the statistical parameters of the 20 STR loci in this Han population. In this population, the expected heterozygosity ranged from 0.6040 (TPOX) to 0.9120 (Penta E), and the observed heterozygosity ranged from 0.6054 (TPOX) to 0.9071 (Penta E); the and 0.9852 (Penta E), and the combined power of discrimination for the 20 STR loci was over 0.999 999 999 999 999 999 999 999 999 999. The power of exclusionvaried between 0.2974 (TPOX) and 0.8099 (PentaE), and the combined probability of excluding paternity for the 20 loci was over 0.999 999 999 999 999 999 999 999 999 999. The polymorphism information content varied between 0.5428 (TPOX) and 0.9053 (Penta E). Among all the studied loci, no significant deviations from Hardy-Weinberg expectations were observed after Bonferroni correction except for the locus D5S818 (P＜0.0025).
The differences between this Chinese Han population and other populations are shown in Tab. 2 and Tab. 3. Fst distance was used to compare this population and 13 other reported populations in relation to the allele frequencies of 15 autosomal STR loci. The neighbor-joining phylogenetic tree generated is shown in Fig. 1. The Northern Italian and Argentinian populations were clustered by one branch, and the Asian populations were clustered by another branch. Seven Chinese ethnic minority groups containing Hui (Qinghai), Miao (Guizhou), Dong (Guizhou), Miao (Guangxi), Hui (Guangxi), Yi (Guangxi) and Yi (Yunnan) were clustered by one small branch, and Tu (Qinghai) was far away from the other ethnic minority groups. The southern Chinese Han population in this study was near Yi (Yunnan) group.
Principal component analysis of allele frequencies in this Han population under study and 13 reported populations was performed. As shown in Fig. 2, the Argentinian and northern Italian populations were on the left upper quadrant, and 6 Chinese ethnic minority populations were up on the right. The Japanese, Philippine and Korean populations were on the left lower quadrant. The southern Chinese Han population under study and Yi (Yunnan) population were on the right lower quadrant.
In this study, we evaluated the performance of PowerPlex®21 System and presented allele frequencies and forensically relevant statistical parameters of 20 STR loci in a Chinese Han population in South China.
A STR locus can be considered highly polymorphic when its PD value is over 0.80 or/and its PE value is over 0.50 . The results of this study showed that most of the examined loci were highly polymorphic. The combined discrimination power and the probability of excluding paternity of the 20 STR loci were both over 0.999 999 999 999 999 999 999 999 999 999, suggesting that PowerPlex®21 System is suitable for forensic personal identification and paternity testing.
We found that after Bonferroni correction (P＜0.05/ 20 [0.0025] ), the P value of exact test for Hardy-Weinberg equilibrium was less than 0.0025 (P= 0.0013) for the locus D5S818. This divergence from Hardy-Weinberg equilibrium may be due to an excess of homozygotes. In contrast, the discrepancies reported in this study can be easily identified by using two commercial kits from different manufacturers. We previously showed the existence of a silent allele of D5S818, caused by mutations at primer-binding sites, in 6 members of 3 paternity cases . The primers for D5S818 in this system need to be optimized to better adapt to southern Chinese Han population.
In this southern Chinese Han population, we found significant differences from Yi (Guangxi) group at 8 STR loci from, from Hui (Guangxi) group at 3 STR loci, from Miao (Guangxi) group at 3 STR loci, from Dong (Guizhou) group at 5 STR loci, from Miao (Guizhou) group at 10 STR loci, from Tujia (Qinghai) group at 2 STR loci (vWA and D7S820), from Yi (Yunnan) group at 2 STR loci (D3S1358 and D21S11), from Hui (Qinghai) group  at 10 STR loci, from Philippine group at 9 STR loci, and from Korean group at 8 STR loci. Significant differences were found in this Chinese Han population at all the loci except for D18S51 and D5S818 from the Japanese population  and at all these 20 STR loci from the northern Italian population  . Only two STR loci (D16S539 and CSF1PO) showed no significant differences between this southern Chinese Han population and the Argentinian population  . The southern Chinese Han population showed significant differences at the locus D1S1656 from 5 foreign populations and also from the Han populations in Beijing (P＜0.0001) , Zhejiang Province (P＜0.0001) and Fujian Province of China (P＜0.0001) . These results indicate that D1S1656 hasa highly ethnic diversity and is suitable for Chinese population.
The neighbor-joining phylogenetic tree showed the clustering of all the Asian populations as one group and of the northern Italian and Argentinian populations as another. The southern Chinese Han population showed the nearest affinities to Yi (Yunnan) population. The cluster branch of the Chinese populations showed the nearest affinity between Guangxi and Guizhou minority populations, while the Tu (Qinghai) population branched away from the other Chinese populations. The phylogenetic tree showed that the populations were clustered basically consistent with their distributions on the continental plate. The result of principal component analysis was in agreement with the phylogenetic tree and indicated a clear pattern of regional distribution.
In conclusion, our results demonstrate that these 20 STR loci can provide highly informative polymorphic data for paternity testing, individual identification and genetic population studies. PowerPlex®21 System can serve as an efficient tool in forensic science and in anthropology of southern Chinese Han population.
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