Polymorphism Research And Application Of Seven X-STR Loci In Northern Han Chinese People

Objective: Short tandem repeats (STR) is the mainstream geneticmarkers in paternity test and forensic identification. Because of its' uniquegenetic method and structure characteristics, the X chromosome STR geneticmarkers has an incomparable advantage compare with autosomal STR or Ychromosome genetic markers in some special or complex kinship cases. Butthe forensic applications of X-STR is not mature, only one kit namedInvestigator Argus X-12kit which was produced by Qiagen company in2010,including12X-STR loci distributed among the four linkage groups, has justbeen introduced to the commercialization. However, there are differentfrequency distributions of these loci in many different populations, especiallyin Chinese northern Han population, some loci do not have highpolymorphism, and the forensic efficiency of the kit also needs to be furtherimproved. Therefore, this study aimmed to screen more X-STR loci to detectgenetic data of Northern China, thus select high polymorphism loci of Hanpeople, so as to provide basic data for development of the X chromosome STRkit.Methods:1Selection of the loci: In accordance with filter, login NCBI and UCSCwebsite to select the X-STR loci, the screen conditions as follows: no relevantforensic report; the loci are all tetranucleotide repeat units; the segmentslength of PCR products are distributed among100-400bp; there are nocomplementary sequences between different genetic loci primers especially3'end; and have similar amplification conditions. According to these, this articlescreen17loci to be a research target, employ PCR amplification andpolyacrylamide gel electrophoresis silver stain method, thus investigate40unrelated healthy individuals in HeBei Han people for genetic polymorphism purpose. Preliminary,7X-STR loci that thought to be polymorphism wereobtained, and then research the detail.2Construction of multiplx amplification systems: Aimming at these7polymorphism X-STR loci, the application of fluorescence STR profilingtechnology to construct two fluorescence multiplx amplification systems. Theprimers concentration, Mg2+concentration, annealing temperature and recyclenumber are optimized. Using AB3130gene analyzer to detect the PCRproducts, and the results are analysed by Genemapper3.2software, the nameof the allele based on the recommended naming principles of the internationalsociety of forensic genetics (ISFG). Each allele was sequenced for validatingand analyzing their repeat sequence structures.3Population genetic investigation: Among genetic investigation ofnorthern Han people, total364unrelated healthy individuals (200males and164females) was detected by the above two multiplx amplification systems.The allele frequency distribution of the7X-STR loci was calculated, andHardy-Weinberg equilibrium of all the loci were test by chi-square goodnessof fit test, while allele frequency and linkage disequilibrium analysis werecalculated using Arlequin software version3.1.4Evaluation of forensic application: Species specificity and repeatabilityof the polymorphism loci were studied using the fluorescence multiplxamplification systems of the7X-STR loci. And the application of all the lociin kinship cases were estimated.Results:1Construction of multiplx amplification systems: The primers of thepreliminary screening7X-STR loci, DXS1268, GATA83F09, GGAT4B02,GATA42D03, GATA22E12, GATA130D02and DXS2498were labeled withtwo fluorescences. Fluorescence FAM was labeled on the forward primers ofthe DXS1268, GATA83F09, GGAT4B02, GATA42D03, GATA22E12loci,while fluorescence HEX was labeled on the reverse primers of theGATA130D02and DXS2498loci. And among the two multiplx amplificationsystems, DXS1268, GGAT4B02, GATA42D03, GATA22E12loci were included in system1, as well as GATA83F09, GATA130D02, DXS2498lociwere included in system2. The peak of the allele profiling was sharp andsymmetry.2The results of population genetic investigation: The detection of the164unrelated female individual of northern Han people showed that,DXS1268was detected5alleles and11kinds of genetic typing; GATA42D03was detected6alleles and10kinds of genetic typing and GATA22E12wasdetected4alleles and10kinds of genetic profiling;8alleles and20kinds ofgenetic typing were tested in GATA83F09; while GGAT4B02wasinvestigated4alleles and9sorts of genetic typing; GATA133D02found5alleles and12kinds of genetic profiling; however, DXS2498was detected4alleles and6kinds of genetic typing. The detection of the200unrelated maleindividual showed that, DXS1268found six alleles and GATA42D03showedsix alleles, four alleles were found in GATA22E12locus and six alleles werediscovered in GATA83F09locus, while GGAT4B02was detected five alleles,meanwhile five and three alleles were detected in locus GATA133D02andlocus DXS2498. The Hardy-Weinberg equilibrium results showed that, therewere no significant differences in all the7X-STR loci except the DXS2498locus (P>0.05), therefore, the rest of the study would never involve DXS2498locus. During the364unrelated individuals of the northern Han people,chi-square test results showed that there were no significant differences inallele distribution between men and women (P>0.05), therefore, the allelefrequency of men and women can be combined for general forensic geneticsparameters statistics calculation. Among them, the observative heterozy-gosity (Ho) of loci DXS1268, GATA42D03, GATA22E12, GATA83F09,GGAT4B02and GATA130D02were as follows:0.6118,0.4809,0.6221,0.7129,0.3160,0.6044；the Polymorphism information content (PIC) were0.5362,0.4422,0.5610,0.6654,0.2940,0.5377, respectively; the womendiscrimination power (PDfemale) were0.7737,0.6918,0.7961,0.8701,0.5101,0.7769and the men discrimination power (PDmale) were0.6118,0.4809,0.6221,0.7129,0.3160,0.6044; the triplet mean exclusion chance (MECKishida) were0.5362,0.4421,0.5610,0.6654,0.2904,0.5365, and the duo meanexclusion chance (MECDesmarais Duo) were0.3922,0.2295,0.4141,0.5237,0.1792,0.3929. According to the results above mentioned, based on thestandard PIC>0.5, the4loci DXS1268, GATA22E12, GATA83F09,GATA130D02were screened as high polymorphism loci.3Evaluation of forensic application: The fluorescence multiplxamplification systems of the7X-STR loci aforesaided, there were no specifictyping found in common animals such as pig, rat, sheep, oxen and rabbit. Theresults illustrated that the systems have certain species specificity; for the10two-generation pedigree cases which the relationship between father anddaughter are verified, the daughters can all win their fathers' X chromosomesteady. The results demonstrate these7X-STR loci have preferable geneticstability.Conclusions: Population genetic data of the7X-STR loci was obtainedthrough the experiment, and the4high polymorphism loci DXS1268,GATA22E12, GATA83F09, GATA130D02were screened, which can providebasic data for the development of X-STR kit.