Genetic study in Aedes (Stegomyia) aegypti (Linnaeus, 1762) from Londrina (Paraná State, Brazil): an approach to population structure and pyrethroid resistance

Abstract Londrina is the fourth most populous city in southern Brazil. Its subtropical weather with rain in all seasons, as well as its high population density, make the city perfect for the Aedes aegypti (Linnaeus, 1762) life cycle. Over the last few years, Londrina presented high infestation indexes and was one of the cities with the most reported cases of dengue. Uncontrolled use of synthetic insecticides may influence the mosquito's genetic composition. In this paper, we studied mitochondrial DNA and kdr mutations in Aedes aegypti. The analysis of the ND4 gene in 330 specimens showed the presence of 27 haplotypes. The pyrethroid resistance alleles (kdr) evaluated are present in the collected populations, with a 50% frequency of the Val1016Ile and 48% of the Phe1534Cys mutations. Such analysis of the mutations in the populations collected at the State University of Londrina's campus - a microenvironment that differs from the rest of the city - showed frequencies of 57% and 62%, respectively. The low gene flow observed, Nm = 0.11 and Nm = 0.10, along with the elevated differentiation, Fst = 0.19 and Fst = 0.18, among populations suggest an influence of genetic drift. The strong presence of resistance alleles kdr in the city is evident, which demonstrates that even with the interruption of the use of pyrethroids by the National Dengue Control Program, resistance may be maintained due to domestic use. Thus, the results have shown the need for genetic monitoring, alongside other entomological surveillance monitoring tools, to create strategies of mosquito control.

Development of KASP marker for cytoplasmic male sterility in Nicotiana tabacum and utilization in trait introgression

Cytoplasmic male sterility (CMS) is an important trait for large-scale hybrid seed production which avoids manual emasculation and undesired horizontal spread of pollen.Rearrangements in mitochondrial genome in terms of deletions and insertions are frequent causes leading to CMS. Mitochondrial ATP synthase is a multisubunit molecular machine which is involved in synthesis of ATP. In this study, three mutations in ATPase subunit 6 were identified and their cosegregation with male sterility was established using tobacco male sterile hybrids and Nicotiana suvaolensis. A breeder friendly Kompetitive allele specific polymerase chain reaction (KASP) SNP marker was developed for high throughput and quick genotyping. Introgression of this trait into selected germplasm lines (n = 9) was achieved based on foreground for CMS and background selection for recurrent parent using KASP marker and 50K custom tobacco SNP array, respectively. Analysis of genotyping data from SNP array revealed the presence of 88–99% of recurrent parent genome in BC3F1 plants. The selected BC3F1 plants exhibit CMS and are indistinguishable from the fertile recurrent parent (germplasm) in terms of plant morphology.

Rapid detection of drug-resistant Mycobacterium tuberculosis directly from clinical specimens using allele-specific polymerase chain reaction assay

Background & objectives: Rapid detection of drug resistance in Mycobacterium tuberculosis (MTB) is essential for the efficient control of tuberculosis. Hence, in this study a nested-allele-specific (NAS) PCR, nested multiple allele-specific PCR (NMAS-PCR) and multiple allele-specific (MAS) PCR assays were evaluated that enabled detection of the most common mutations responsible for isoniazid (INH) and rifampicin (RIF) resistance in MTB isolates directly from clinical specimens. Methods: Six pairs of primers, mutated and wild type, were used for the six targets such as codon 516, 526 and 531 of rpoB, codon 315 of katG and C15-T substitution in the promoter region of mabA-inhA using allele-specific (AS) PCR assays (NAS-PCR, NMAS-PCR and MAS-PCR). The performance of AS PCR method was compared with phenotypic drug susceptibility testing (DST). Results: The usefulness of AS PCR assays was evaluated with 391 clinical specimens (251 Acid fast bacilli smear positive and MTB culture positive; 93 smear negative and MTB culture positive; 47 smear positive and MTB culture negative) and 344 MTB culture positive isolates. With culture-based phenotypic DST as a reference standard, the sensitivity and specificity of the NAS-PCR, NMAS-PCR and MAS-PCR assay for drug resistance-related genetic mutation detection were 98.6 and 97.8 per cent for INH, 97.5 and 97.9 per cent for RIF and 98.9 and 100 per cent for multidrug resistance (MDR). Interpretation & conclusions: The performance of AS PCR assays showed that those could be less expensive and technically executable methods for rapid detection of MDR-TB directly from clinical specimens.

Evaluation of High Resolution Melting Technique for Detection of JAK2-V617F Mutation in Formalin-Fixed Paraffin-Embedded Specimen from Myeloproliferative Neoplasm Cases

@#Introduction: Myeloproliferative neoplasm (MPN) is a group of myeloid disorders which leads to erythrocytosis, thrombocytosis and leucocytosis. MPN with BCR-ABL positive is chronic myeloid leukaemia (CML) while BCR-ABL negative MPN includes polycythaemia Vera (PV), essential thrombocytemia (ET) and primary myelofibrosis (PMF). One of the major criteria for diagnosis of BCR-ABL negative MPN is the presence of JAK2-V617F mutation which is positive in 95% of PV and around 60% of ET and MF. Beside peripheral blood specimen, formalin-fixed paraffin-embedded (FFPE) marrow specimen can be used for detection of this mutation. Unfortunately, FFPE produces low quality DNA that put a challenge for successful amplification of DNA. We aimed to evaluate the utility of High Resolution Melting (HRM) analysis for detection of JAK2-V617F mutation in FFPE specimen from MPN cases. Materials and Methods: This study is a descriptive crosssectional study. Forty FFPE marrow specimens were retrieved from the years 2014-2016. Bio-Rad Precision Melt Analysis software was used for analysis of HRM data. Allele-specific PCR was done for validation of results. Positive samples were subjected to Sanger sequencing. Results: JAK2-V617F mutation was positive in 13 out of 40 MPN cases. Level of agreement between HRM and AS-PCR was 97.5%. Conclusion: HRM is a rapid and powerful diagnostic assay which is suitable for detection of JAK2-V617F mutation in FFPE marrow specimen.

Identification of Cervi Cornu Pantotrichum and Cervi Cornu by Allele-specific PCR / 中国实验方剂学杂志

Objective: To obtain a rapid,efficiency and convenient polymerase Chain reaction(PCR) identification method for medicinal Cervi Cornu Pantotrichum,Cervi Cornu and its common adulterates. Method: Based on three single nucleotide polymorphisms (SNP) of Cytb gene DNA sequences among Cervus nippon,C. elaphus and its adulterants,a pair of species-specific primers (LR-238.F and LR-238.R) was designed,the reaction conditions were optimized,and the PCR method for identification was explored and verified in terms of tolerance and feasibility. Result: Through the established allele-specific PCR method,under the annealing temperature of 56℃ and cycle number of 35,250 bp of fragments were amplified from DNA templates of Cervi Cornu Pantotrichum,Cervi Cornu and its subspecies in origin animal samples as well as herbal medicines. All of the adulterants species of Przewalskium albirostris,Cervus eldi,Odocoileus hemionus,Dama dama,Alces alces,Elaphurus davidianus,Capreolus pygargus,Rusa unicolor and Rangifer tarandus were negative by the PCR assay. Conclusion: The identification primer is highly specific,and the allele-specific PCR identification method established in this paper can accurately identify the medicinal Cervi Cornu Pantotrichum and Cervi Cornu.

Identification of Fallopia multiflora by allele-specific polymerase chain reaction / 中草药

Objective :To establish a rapid molecular identification method for Fallopia multiflora and its adulterants. Methods: Based on psbA-trnH sequences of F. multiflora and its adulterants, the SNP site was searched and the specific primers were designed. The allele-specific PCR amplification of F. multiflora and its adulterants from different producing areas was carried out and the reaction system was optimized. Results: When the annealing temperature was raised to 48 ℃ with 30 cycle number, only the template DNA of F. multiflora could be amplified to obtain the specific 191 bp band whereas the diagnostic PCRs of the other adulterants were all negative. Conclusion: It’s simple and reliable to identify the authenticity of F. multifloa by allele loci specific PCR.

Allele-specific PCR to identification of Achyranthis Bidentatae Radix and Cyathulae Radix formula granules / 中国中药杂志

To establish a robust and accuracy molecular method to identify Achyranthis Bidentatae Radix and Cyathulae Radix formula granules. ITS sequences of Achyranthes bidentata and Cyathula officinalis were aligned, specific SNPs (single nucleotide polymorphisms) were excavated, specific primers were designed and allele-specific PCR method was established. The genomic DNA was successfully extracted from the herbal medicine and its formula granules by using an improved CTAB (cetyltrimethyl ammonium bromide) method and then performed PCR with the designed primers. The 187 bp specific band could be amplified only in the presentation of C. officinalis and its granules when use of C. officinalis specific primers, whereas the 162 bp band could be amplified only in the presentation of A. bidentata and its granules when use of A. bidentata specific primers. This method was also successfully applied in the identification of commercial formula granules.

Identification of uighur medicine Cichorium glandulosum and its adulterants by Allele-Specific PCR / 中草药

Objective To establish molecular identification method of Cichorium glandulosum and its adulterants Cichorium intybus by Allele-Specific PCR. Methods The samples of C. glandulosum and C. intybus were collected in different geographical areas. The DNA was extracted, and rbcL gene segments were amplified and sequenced directionally. The multiple sequences were aligned by using Clustal W. Specific primers were designed and amplified according to its variable sites, and PCR reaction system was optimized to determine detection limits and establish Allele-Specific PCR identification method. Results According to Allele-Specific PCR system established in this study for C. glandulosum, the optimization results was a total of 30 μL reaction system containing TaqDNA polymerase 0.25 μL, 10 × buffer 2.5 μL, dNTP 2.0 μL, primer 0.5 μL, template DNA 2 μL, and ddH2O 22.25 μL. The most suitable PCR amplification procedure is one cycle of predegeneration at 94 ℃ for 3 min; 32 cycles of denaturing at 94 ℃ for 30 s, annealing at the primer temperature 55 ℃ for 30 s and extending at 72 ℃ for 1 min, and extending at 72 ℃ for 7min. Through the detection of 20 medicinal materials of C. glandulosum and C. intybus, the result showed that 230 bp amplified band of target fragment was identified for C. glandulosum but no amplified band was observed for its adulterants. Conclusion In this study, we established and optimized the Allele-Specific PCR identification technology of C. glandulosum and its adulterants C. intybus, which can accurately, reliably, and effectively identify these two medicinal materials.

Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

Background: Cultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by theΔ12 fatty acid desaturase (FAD) encoded byAhFAD2AandAhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C→A:T) ofAhFAD2Aand an "A" insertion ofAhFAD2Bresulted in high-oleic acid phenotype. Detection ofAhFAD2mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detectAhFAD2genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detectionAhFAD2genotype of large number of breeding materials. Results: Here, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC4F2seeds was aabb. Conclusions: Due to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable fordeterminingAhFAD2genotype than other methods.

Typing of fragment length polymorphism marker with primer SNP-binding at the last second 3'end / 中国法医学杂志

Objective To successfully get the full PCR alleles of the Insertion/Deletion marker rs10644346 in which a SNP-binding exists at the 3'end region of the primer.Methods Based on the AS-PCR,a common forward primer and two reverse primers with allele-specific oligonucleotides at the last second 3'end instead of the terminus were tentatively designed for typing 150 unrelated individuals and 10 father-mother--child trios from Htnan province in South-central China.Simultaneously,9 samples were typed with all the above three primers (the two primer sets which consist of the common forward primer and one of the reverse primers).Results PCR amplicons were well detected in the 150 unrelated individuals after being typed with the three primers,and the amplified fragments of parental and filial generations among 10 father-mother-child trios conformed to Mendel's principles.Allele missing was found in the two-primer group.Conclusion The primers designed by locating the specific nucleotide at the last second 3'end rather than terminal position were demonstrated also effective in getting specific alleles if perfect mismatch and PCR conditions are guaranteed,and the design strategy can provide an optional reference to rescue markers of SNP-binding primers for forensic practice.

Establishment of allele-specific PCR for the detection of CYP3A5 and MDR-1 gene polymorphisms and its clinical application / 临床检验杂志

Objective To establish an allele-specific PCR method for the detection of cytochrome P-450 CYP3A5 (A6986G) and multiding resistance gene MDR-1 (C3435T) polymorphisms,and investigate the correlations of their polymorphisms with blood tacrolimus (Tac) concentration/dose (C/D) ratio in renal transplant recipients.Methods The allele-specific PCR primers were designed according to the polymorphism sites of CYP3A5 (A6986G) and MDR-1 (C3435T) genes.Then,their polymorphisms in the genomic DNA of peripheral blood samples from 72 renal transplant recipients were analyzed,and the results were validated by DNA sequencing.The blood Tac concentration was determined by the chemiluminescence microparticle immunoassay and the differences of concentration,dose and C/D ratio of blood Tac in renal transplant recipients with different genotypes were compared at 1 month after transplantation.Results The coincidence rate between the established allele-specific PCR and DNA sequencing was 100％.The frequencies of CYP3A5 * 1/* 1,* 1/* 3 and * 3/* 3 genotypes in 72 renal transplant recipients were 18.1％,31.9％ and 50.0％,respectively,and those of MDR-1 C/C,C/T and T/T genotypes were 27.8％,58.3％ and 13.9％,respectively.There were significant differences in blood Tac concentration (P =0.014) and Tac C/D ratio (P =0.019) between different CYP3A5 genotypes of renal transplant recipients.Further analysis found that the Tac C/D ratio of CYP3A5 * 3/* 3 genotype was significantly higher than that of CYP3A5 * 1/* 1 and * 1/* 3 genotypes (P ＜ 0.05).Conclusion The allele-specific PCR method for the detection of CYP3A5 and MDR-1 polymorphisms is successfully established and the polymorphism of CYP3A5 * 3 gene in renal transplant recipients is obviously correlated with the pharmacokinetics of Tac.

Modified allele-specific PCR improves HER2 Ile655Val detection by reducing genotyping errors

Background: A reliable method to detect gene polymorphisms must be established to eliminate genotyping errors due to false PCR amplification. In the previous study, we have developed AS-PCR (Allele Specific-Polymerase Chain Reaction) to detect HER2 Ile655Val gene polymorphism with good specificity and sensitivity, yet it produces some errors. This study is aimed to eliminate the source of genotyping errors mainly by betaine treatment and PCR program modification. Methods: Genotyping errors produced by AS-PCR was qualitatively and quantitatively evaluated using two genomic DNA that each contained AA genotype and GG genotype of HER2 Gene. Betaine treatment or PCR program modification was tested to eliminate the occurrence of genotyping errors during AS-PCR amplification. Results: The types of genotyping errors exhibited by HER2 Ile655Val AS-PCR are diverse, ranging from LDO (Locus Drop Out), preferential amplification to ADO (Allele Drop Out). The rate of genotyping errors was from 10% to 50% depending on the amount and ratio of DNA template and the annealing temperature of PCR. In the mixed DNA template model, the betaine treatment has shown to reduce ADO only in preferentially amplified GG genotype amplicon. Alternatively, reducing the template of the heterozygous DNA by half ( -0.5 ng of DNA template) for such case has effectively recovered the AS-PCR from ADO. Furthermore, increasing the denaturation temperature to 96 °C with an annealing time of 40 s at the first 10 cycles of AS-PCR has succeeded in eliminating severe preferential amplification of AA genotype amplicon by preventing the DNA template with GG genotype from forming into a G-quadruplex structure. The guideline offered in this study has been successfully applied for clinical samples of breast cancer that show preferential amplification. Conclusion: Betaine and the modifying AS-PCR program can reduce significantly genotyping errors making AS-PCR for HER2 Ile655Val detection more reliable to be used as a molecular tool for genotyping purpose (AU)

Molecular authentication of Jinyinhua formula granule by using allele-specific PCR / 中国中药杂志

Traditional authentication method is hard to identify herb's authenticity of traditional Chinese medicine(TCM) formula granules because they have lost all their morphological characteristics. In this study, a new allele-specific PCR method was established for identifying the authentication of Jinyinhua formula granule (made from Lonicerae Japonicae Flos) based on an SNP site in trnL-trnF fragment. Genomic DNA was successfully extracted from Lonicerae Japonicae Flos and its formula granules by using an improved spin column method and then PCR was performed with the designed primer. Approximately 110 bp specific bands was obtained only in the authentic Lonicerae Japonicae Flos and its formula granules, while no bands were found in fake mixed products. In addition, the PCR product sequence was proved from Lonicerae Japonicae Flos trnL-trnF sequence by using BLAST method. Therefore, DNA molecular authentication method could make up the limitations of character identification method and microscopic identification, and quickly identify herb's authenticity of TCM formula granules, with enormous potential for market supervision and quality control.

Design of an allele-specific PCR assay to genotype the rs12255372 SNP in a pilot study of association between common TCF7L2 polymorphisms and type 2 diabetes in Venezuelans

Objective The global burden of diabetes mellitus will impact strongly American countries in the coming decades. Type 2 diabetes mellitus (T2DM) is a multifactorial disease and the basis for its genetic susceptibility remains not fully understood. Different population studies have demonstrated that variants of the TCF7L2 gene are strongly associated with an increased risk of T2DM. Moreover, institutions or countries with limited budget to conduct genetic research need cost effective methods for detecting DNA variants. Subjects and methods We standardized a rapid and simple allele-specific PCR method for genotyping the rs12255372 single nucleotide polymorphism (SNP) in a pilot study exploring the association of three TCF7L2 polymorphisms (rs7903146, rs12255372 and DG10S478) with T2DM in 70 patients and 73 controls from Venezuela. Results The performance of the designed allele-specific PCR reaction for rs12255372 genotyping was reliable and accurate. Patients carrying the TCF7L2 rs7903146 T allele (CT + TT genotypes) and heterozygous CT genotype had a significantly higher risk for T2DM (OR = 2.9 and 2.3, respectively). Although rs12255372 and DG10S478 risk alleles predominated in T2DM group no statistical significance was found. Conclusions We developed a novel allele-specific PCR method for easier and rapid detection of rs12255372 polymorphism without the use of expensive instrumentation and reagents. Our study in a relatively small sample of the Venezuelan population replicated the association of the rs7903146 SNP with T2DM. Further studies with larger sample size and more biochemical data should be conducted to explore the genetic basis of T2DM susceptibility in Venezuela.

Comparison of Several Methods and Their Progresses on Molecular Authentication of Lonicera japonica Thunb / 世界科学技术-中医药现代化

Authentications of Chinese herbal medicine have a critical effect in Chinese clinical medicine. DNA molecular marker, as an important component for true or false authentication, is more and more widely used in iden-tification of Chinese medicinal materials. At the same time, many new methods for authentication of Chinese medici-nal materials are continuously emerging. But the systematically comparative analysis of these new methods is lack. The present study taking Lonicera japonica as an example, systematically compared principles, characteristics, ex-periment methods, detection time and the application scope of express sequence tag-simple sequence repeat (EST-SSR), polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific PCR (AS-PCR), DNA barcoding and loop-mediated isothermal amplification (LAMP), and put forward corresponding improve-ment opinions. This study can help to screen appropriate approach for rapid authentication of L. japonica and offer demonstrating to other Chinese herbal medicines.

Investigation of Correlation between Gene Polymorphism of Apolipoprotein E and Mycoplasma Pneumoniae Infection in Children / 现代检验医学杂志

Objective To study the relationship between gene polymorphism of apolipoprotein E (apolipoproteinE,ApoE)of peripheral blood and Mycoplasma pneumoniae infection in children.Methods Collected 236 cases serum of inpatient and outpatient screening in children with Mycoplasma pneumoniae infection and healthy children between March 2011 and March 2014 in the First Affiliated Hospital of Xi’an Medical University and Xi’an Children’s Hospital,at the age of 3~8 years old,divided into two groups:110 cases of control group and 126 cases of Mycoplasma pneumoniae infection in chil-dren.Used multiple allele-specific PCR (multi-AS PCR)to detect gene polymorphism of ApoE in each group.Results ApoE gene was polymorphic and 6 genotypes:3 homozygous (ε2/2,ε3/3,ε4/4)and 3 heterozygote (ε3/2,ε3/4,ε4/2).Theε3/2 had four bands,ε3/3,ε3/4 and 4/2 had three bands,ε2/2 andε4/4 had two bands.ε3/3 of ApoE genotype distribution in two groups was the most common,control group was 66.7%,infection group was 46.4%.Allele frequencies ofε3 and genotype frequencies ofε3/3 inMycoplasmapneumoniae infection of children were lower than those in control group (P<0.05).But allele frequencies ofε4 and genotype frequency ofε4/4 in Mycoplasma pneumoniae infection of children were increased, which were compared with those in control group (P<0.05).Conclusion There were an association between ApoE gene polymorphism and the incidence of Mycoplasma pneumoniae infection in children.Allelesε3 seems to be a protective factor and allelesε4 may contribute to the development of Mycoplasma pneumoniae infection of children.

Allele-specific PCR for a cost-effective & time-efficient diagnostic screening of spinal muscular atrophy.

Background & objectives: Genetic diagnosis of spinal muscular atrophy (SMA) is complicated by the presence of SMN2 gene as majority of SMA patients show absence or deletion of SMN1 gene. PCR may amplify both the genes non selectively in presence of high amount of DNA. We evaluated whether allelespecific PCR for diagnostic screening of SMA is reliable in the presence of high amount of genomic DNA, which is commonly used when performing diagnostic screening using restriction enzymes. Methods: A total of 126 blood DNA samples were tested in amounts ranging 80-200 ng, referred for the genetic diagnosis of SMA using both conventional PCR-RFLP and allele-specific PCR. Results: The results from both methods showed agreement. Further, allele-specific PCR was found to be a time-efficient and cost-effective method. Interpretation & conclusions: Our study demonstrated the accuracy of our allele-specific PCR and the results were comparable compatible with that of PCR-RFLP, indicating its practical application in SMA diagnostic screening.

Discrepancy in Genotyping of Apolipoprotein E between Allele-Specific PCR and Fluorescence Resonance Energy Transfer or Sequencing / 대한진단검사의학회지

The human apolipoprotein E (APOE) gene contains several single-nucleotide polymorphisms (SNPs) that are distributed across the gene. The genotype of the APOE gene has important implications as a risk factor for various diseases. We observed 2 cases in which the results of allele-specific PCR (AS-PCR) of the APOE gene were not consistent with those of fluorescence resonance energy transfer (FRET) or sequencing analysis. In these cases, genotyping by AS-PCR showed that patients were epsilon2 homozygotes, while sequencing analysis and FRET showed that they were epsilon2/epsilon3 heterozygotes. Herein, we describe the causes of the errors in genotyping and describe the significance of these errors.

A Novel Mutation of ADAR Gene Identified in a Chinese Pedigree with Dyschromatosis Symmetrical Hereditaria / 医学研究杂志

Objective To discover the mutation of ADAR gene in a pedigree with dyschromatosis symmetrical hereditaria(DSH). Methods We investigated this family and collected blood samples of the individuals in this family. Mutation screening was carried out by PCR and direct sequencing. The allele specific primer was designed for the mutation point, and allele-specific PCR was carried out on the patients, normal family members and 40 normal individuals. Results A single nucleotide deletion (c.1642 delC) was identified in exon3 of ADAR gene in the patients of this family. This mutation was not detected in the normal family members and in any of the control individuals. Conclusion This single nucleotide deletion was responsible for the disease in the family.

Haplogroup diversity of 8 biallelic markers on human Y chromosome in Wuhan Han population / 中国法医学杂志

Objective To screen polymorphic biallelic markers on human Y chromosome in Han population , calculations of their allele and haplogroup frequency distributions to provide data for forensic application and population evolution studies. Methods Genotyping of 8 biallelic markers on human Y chromosome (M9, M89, M111, Ml19, M122, M134, IMS-JST0033050 and SRY +465) were carried out in a sample of 160 unrelated Chinese male individuals living in Wuhan using fragment length discrepant allele specific PCR (FLDAS-PCR) and PAGE technique. Results Genetic polymorphism were identified for all 8 biallelic markers in Wuhan Han population. Gene Diversity (GD) ranges from 0.0126 to 0.4830. A total of 9 different haplogroups(Hg 1-9)were observed and the haplogroup diversity (HD) were 0.7776. Conclusion The haplogroups formed by 8 biallelic markers are highly polymorphic, and can be used in conjunction with Y-STRs in forensic medicine and population evolution studies.