Typing Of 26 Y Chromosome Biallelic Markers In Wuhan Han Population Using Snapshot Minisequencing

Background The development of human DNA genetic markers had gone through the following three stages. The first stage was established in the late 80s, known as restriction fragment length polymorphism (RFLP). The second stage was variable number tandem repeats (VNTR) based on PCR technology, including minisatellite and microsatellite DNA markers. The third stage was ballelic markers, which included single nucleotide polymorphisms (SNPS) and insertion/deletions (Indels). At present, ballelic marker systems have gradually become a research hotspot due to the advantages of widely distribution, genetic stability, low mutation rate and easily to analyze. Human Y chromosome is paternally inherited and insulated from meiotic recombination except for the pseudoautosomal region. Accordingly, Genetic markers located on the non-recombining portion of the Y chromosome are of increasing importance in tracing human evolution through male lineages as well as application in a variety of forensic situations including personal identification in sexual assault cases and paternity testing to reveal the relationship between a son and his putative father. Although Y-chromosome STRs are highly informative and therefore are the markers of first choice in forensic cases, binary polymorphisms, such as SNPs and indels, are promising markers for forensic analysis because of some special characteristics of their abundance, simplicity and low mutation rate.Objective The aim of the present study was to study the genetic polymorphism of 26 Y chromosome biallelic markers and to discuss its application value in forensic science and human evolution. Methods According to some references and Y-SNP database, we selected 26 Y chromosome biallelic markers which were polymorphic in East Asian populations. The primers both for PCR amplification and the minisequencing reaction were designed to have an annealing temperature around 60°C using the web-based software Primer 3, and minisequencing primer sequences upstream and downstream adjacent to the polymorphism site were selected that have the appropriate length and tm characteristics. The selected 26 loci were combined in three multiplex reactions according to their fragment size and interaction. First, the target fragments which contained the polymorphism sites were amplified using the specific primers. After cleaned up with shrimp alkaline phosphatase (SAP) and exonuclease I (Exo I), the single base extension reaction was performed using the allelic-specific single base extension primers. Then, 120 unrelated healthy male individuals in Wuhan Han population were typed by using the established multiplex detection system. In addition, we constructed phylogenetic network of Wuhan Han population based on the typing results of 26 Y chromosome biallelic markers with NETWORK 4.5.1.6 software.Results (1) Three fluorescent-multiplex PCR system for typing 26 Y chromosome biallelic markers were successfully established. For each multiplex detection system, the typing results showed that two different colours of product peaks denoted two different alleles of a binary locus, and the fragment sizes of alleles among different loci were different. (2) All of 26 Y chromosome biallelic markers were polymorphic in Wuhan Han population. Total 26 haplotypes were observed from 120 male unrelated individuals in Wuhan Han population, and the frequency range was 0.0083~0.1250. The haplotype diversity was estimated to be 0.9349. (3) The phylogenetic network of 26 Y chromosome biallelic markers in Wuhan Han population was successfully constructed.