Genome-based development of 15 microsatellite markers in fluorescent multiplexes for parentage testing in captive tigers.

As one of the most endangered species, tiger (Panthera tigris) inbreeding has become an urgent issue to address. Using a microsatellite (short tandem repeat, STR) identification system, paternity testing may be helpful to avoid inbreeding in captive breeding programs. In this study, we developed a genome-based identification system named tiger pedigree identification multiplex system (TPI-plex). By analyzing the entire tiger genome, 139,967 STR loci were identified and 12.76% of these displayed three to six alleles among three re-sequenced individual tiger genomes. A total of 204 candidate STRs were identified and screened with a reference population containing 31 unrelated captive tigers. Of these, 15 loci were chosen for inclusion in the multiplex panel. The mean allele number and mean expected heterozygosity (He) were 7.3333 and 0.7789, respectively. The cumulative probability of exclusion (CPE) and total probability of discrimination power (TDP) reached 0.999999472 and 0.999999999999995, respectively. The results showed that the TPI-plex system can be applied in routine pedigree identification for captive tigers. We also added a sex identification marker named TAMEL into the TPI-plex for sex determination.

Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

Mutation rate analysis at 19 autosomal microsatellites.

Previous studies have demonstrated that a large sample size is needed to reliably estimate population- and locus-specific microsatellite mutation rates. Therefore, we conducted a long-term collaboration study and performed a comprehensive analysis on the mutation characteristics of 19 autosomal short tandem repeat (STR) loci. The STR loci located on 15 of 22 autosomal chromosomes were analyzed in a total of 21,106 samples (11,468 parent-child meioses) in a Chinese population. This provided 217,892 allele transfers at 19 STR loci. An overall mutation rate of 1.20 × 10(-3) (95% CI, 1.06-1.36 × 10(-3) ) was observed in the populations across 18 of 19 STR loci, except for the TH01 locus with no mutation found. Most STR mutations (97.7%) were single-step mutations, and only a few mutations (2.30%) comprised two and multiple steps. Interestingly, approximately 93% of mutation events occur in the male germline. The mutation ratios increased with the paternal age at child birth (r = 0.99, p<0.05), but not maternal age. Last, with the combination analysis of the data from the southern Chinese population, we drew a picture of 19 STR mutations in China. In conclusion, the data from this study will provide useful information in parentage testing, kinship analysis, and population genetics.

[Mutation of suspected autosomal STR loci: 227 cases retrospective analysis].

OBJECTIVE: To study the suspected autosomal STR loci mutation cases. METHODS: A total of 227 suspected autosomal STR loci mutation cases were selected from Center of Forensic Sciences, Beijing Genomics Institute. The allelic mutation cases were screened and the number of mutation of each STR loci was statistically analyzed. The CPI value was calculated in order to study the characteristics and rules of the mutations. RESULTS: In the 227 suspected mutation cases, 3 cases were excluded paternity, and 228 mutations were observed at 18 STR loci in the rest of the cases. The average number of STR mutation loci was 1-2. The maximum of mutation step was 4. After using 20A amplification kit, the CPI values in 3 non-parentage cases were all less than 10(4). After using 20A and 10G amplification kits, the CPI values were all larger than 10(4) in all standard parents-child triplet cases and in 99.45% of diad cases. CONCLUSION: The allelic mutation of STR loci is relatively common in forensic cases. By increasing the number of the required STR loci and supplementing the samples of the triplet, the identification errors could be decreased to a great extent when suspected autosomal STR loci mutation occurs.

Genetic polymorphism analysis of 15 STR loci in Chinese Hui ethnic group residing in Qinghai province of China.

In the present study, we investigated the diversity distributions of allelic frequencies of 15 short tandem repeats (STRs) loci in a sample of Chinese Hui ethnic group in the Ningxia Hui Autonomous Region. The allelic frequencies of the 15 STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) were obtained from 2975 unrelated healthy Hui individuals. The STR genotyping data of all the samples were generated by DNA extraction, multiple amplification, GeneScan and genotype analysis. The genetic distances among different populations were calculated by using Nei's method and a phylogenetic tree was constructed based on the allelic frequencies of the same 15 STR loci using the neighbor-joining method. A total of 185 alleles were observed in the Hui population, with the corresponding allelic frequencies ranging from 0.0002 to 0.5322. Chi-Square tests showed that all STR loci were in Hardy-Weinberg equilibrium. The forensic statistical parameters of all the loci showed high values. The population data in this study were compared with the previously published population data from other ethnics or areas. The Hui population showed significant differences from the Minnan Han, Uigur, Ewenki, Yi, Tibetan, Maonan and Malay ethnic minority groups in some loci, and from the South Morocco population and the Moroccan population in all the loci. Our results are valuable for human individual identification and paternity testing in the Chinese Hui population and are expected to enrich the genetic information resources of Chinese populations.

[Detection of ABO genotype genetic polymorphism by multiplex-PCR based sequencing and application in forensic medicine].

Multiplex PCR-direct sequencing method was established to detect 9 different SNPs in exon 6 and exon 7 of ABO genes and could identify at least 28 different ABO genotypes. Population study was carried out in a sample of 80 unrelated Chinese Tibetan minority individual dwelled in Qinghai Province. The method was also applied to forensic cases. A variety of degeneration forensic samples, including blood stain, hair root, swab, bone and mixed stain were successfully identified by this efficient method and in conformance with serological typing. There were no significant deviations from Hardy-Weinberg equilibrium in ABO genotypes of Tibetan population. The heterozygosity, polymorphic information content, discrimination power, paternity of exclusion, and probability of genetic identity were 0.675, 0.672, 0.874, 0.391, and 0.126 respectively. The gene frequency of ABO was O>B>A. The multiplex PCR-directed sequencing method can accurately and reliably detect ABO genotypes in many kinds of samples, and it improves personal identification efficiency. The ABO genotype is high variance in Qinghai Tibetan minority group, and it can be applied in forensic medicine and population genetic study.

Genetic polymorphism of mitochondrial DNA HVS-I and HVS-II of Chinese Tu ethnic minority group.

We analyzed the two hypervariable segments HVS-I and HVS-II of 108 Chinese Tu ethnic minority group samples for forensic and population genetics purposes. Comparing with Anderson sequence, 79 polymorphic loci in HVS-I and 40 in HVS-II were found in Chinese Tu ethnic minority group mtDNA sequences, and 90 and 64 haplotypes were then defined. Haplotype diversity and the mean pairwise differences were 0.9903+/-0.0013 and 5.7785 in HVS-I, and 0.9777+/-0.0013 and 3.5819 in HVS-II, respectively. By analyzing the hypervariable domain from nucleotide 1,6180 to 1,6193 in HVS-I, we defined some new types of sequence variations. We also compared the relationship between Tu population and other populations using mtDNA HVS-I sequences. According to Rst genetic distances, the phylogenetic tree showed that the Tu population, the Xi'an Han population, the Chinese Korean, and the Mongol ethnic group were in a clade. This indicated a close genetic relationship between them. There were far relations between the Tu population and other Chinese southern Han populations, Siberian, European, African, and other foreign populations. The results suggest that Tu population has a multi-origin and has also merged with other local populations.

[Sequence polymorphism of mtDNA control region in Chinese Qinghai Tibetan ethnic group and Han population].

OBJECT: To study sequence polymorphism of mtDNA control region in Chinese Qinghai Tibetan group and Han population. METHODS: Venous blood samples from 69 unrelated Qinghai Tibetans and Han individuals were collected and their mtDNA control region sequences were analyzed. Polymorphism indicators were calculated. The genetic distances based on Fst and Rst among eleven groups from different districts include the Qinghai Tibetan and Han population were elucidated using Nei's method. Phylogenetic tree was constructed. RESULTS: There were 56 polymorphic loci and 59 loci found in the mtDNA control region of Tibetan group and Han population, respectively. It was indicated by the Rst distance that there was a far distance between Qinghai Tibetan and the other populations (P<0.05), and the distance was much closer between Qinghai Han and Xi'an Han, Mongolian, Changsha Han populations (P>0.05). CONCLUSION: There is unique genetic polymorphism of mtDNA control region both in Qinghai Tibetan and Han population. These findings may be useful in forensic identification, population genetic and migration studies.

Population genetic analysis of 15 STR loci of Chinese Tu ethnic minority group.

We studied and established a DNA database of 15 euchromosome STRs (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) in a population sample of 151 unrelated individuals of Tu ethnic minority group. Allelic frequencies and statistical parameters of Tu population were calculated. Totally 136 alleles were observed, with the corresponding allelic frequencies ranging from 0.0033 to 0.5359. Chi-square test showed that all STR loci agreed with Hardy-Weinberg equilibrium. Our study population data were compared with the previously publishing population data of other ethnic groups or areas. Our results of present study were valuable for human identification and paternity tests in Chinese Tu population.

Genetic analysis of 15 STR loci in Chinese Han population from West China.

Allele frequencies for 15 short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA) were obtained from 7,636 unrelated individuals of Chinese Han population living in Qinghai and Chongqing, China. Totally 206 alleles were observed, with the corresponding allele frequencies ranging from 0.0001-0.4982. Chi-square test showed that all of the STR loci agreed with the Hardy-Weinberg equilibrium. We also compared our data with previously published population data of other ethnics or areas. The results are valuable for human identification and paternity testing in Chinese Han population.

Genetic analysis of 15 STR loci on Chinese Tibetan in Qinghai Province.

We report allele frequencies and statistical parameters of 15 short tandem repeats (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) determined in 850 unrelated individuals of Chinese Tibetan, an ethnic group residing in Qinghai Province, China. We observed 155 alleles with allele frequencies ranging from 0.0006 to 0.5682. The distribution of these observed genotypes were not significantly different from the expected distribution according to Hardy-Weinberg equilibrium. The forensic parameters from the data showed high values. In conclusion, the 15 STR loci are useful for forensic analysis, paternity tests for Tibetans in the region, and population genetic studies.

Genetic polymorphisms of 15 STR loci of Chinese Dongxiang and Salar ethnic minority living in Qinghai Province of China.

Fifteen autosomal STRs loci were analyzed from two samples of 178 healthy unrelated autochthonous individuals of Chinese Dongxiang and Salar ethnic minority groups using a multiplex PCR system. Allele frequencies distribution and statistical parameters for all STR loci, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, were determined by the AmpFISTR Identifiler Kit. The observed genotype frequencies and expected of genotype frequencies were evaluated by chi(2)-test and the Fisher exact tests. chi(2)-test showed that the agreement with Hardy-Weinberg equilibrium (p>0.05) was for all studied STR loci of two populations. The data in the present study can be used greatly for routine forensic application in the region, and enrich Chinese ethnical genetic informational resources.