The Development Of Single Nucleotide Polymorphism Markers Based On Expressed Sequence Tags In Maize

A single-nucleotide polymorphism (SNP) is a DNA sequence variation caused by nucleotides substitution, deletion or insertion. Compared with the commonly used molecular makers such as restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR), SNP is recognized as the third generation of molecular makers for its high density coverage, stable heredity, high correlation with phenotype, easy detection and automatical analysis, and so on. Due to the inadequate density of SSR markers, and the inadequate polymorphism of SNP markers developed based on sequence difference between only two genotypes, these two kinds of DNA molecular markers do not meet the current demand of fine mapping for maize genes. In this study, expressed sequence tags (EST) of maize (Zea mays) with different genetic background were downloaded from public databases, and used for the development of SNP molecular markers, with the help of different bioinformatical softwares and self-developed Perl scripts. High resolution melt (HRM) was used to evaluate the predicted SNP makers.Based on Windows XP and Linux operating systems, software such as Blast, cross_match, Phrap, ePrimer3, e-PCR and self-developed Perl scripts were used to construct an analysis system by bioinformatics approach. Moreover, using Bioperl modules, the scripts written with Perl language make automatization of the whole analysis process come true. The data of EST could be analyzed promptly and effectively. The analysis system includes Clustering EST sequences, clipping vectors, detecting low quality and repeated sequences, aligning EST sequences, mining SNP sites, designing and filtrating PCR primers, mapping SNP molecular marker on chromosome and calculating polymorphism information content (PIC).On the basis of the Cluster and alignment among 2018530 pieces of EST sequences, 80363 SNP loci were found out throughout the genome. According to the flanking conserved sequences beyond these SNP loci,12388 pairs of PCR primers were designed to amplify sequences involving 34721 SNP loci, and provided to be used as SNP molecular markers, among which 6008 contain only one SNP loci.12117 of these makers can be mapped on the ten maize chromosomes based on physical loci on sequenced genome of B73. Among of them, the density of SNP makers on chromosome 1 is much higher than that on others. For 12762 of these SNP sites, the polymorphism information content (PIC) is more than 0.4, showing their high polymorphism. Moreover, On the basis of Microsoft Windows XP operating system, Adobe Dreamweaver was used to establish a static platform of public database (http://www.sicau.edu.cn/web/yms/snp/snp.html) on Maize Research Institute of Sichuan Agricultural University web page, at which all the relative information about these SNP molecular markers is available.In order to validate the predicted SNP makers, nine of them were randomly picked out. High resolution melt curve is different according to the base of nucleotide fragment. The technology of HRM was used to evaluate the selected makers through typing the Real-Time PCR amplification, in which saturated fluorescent dye was added beforehand. Forty-six maize inbred lines were used in HRM typing. The fluorescence differential curve shown:polymorphism of nine SNP makers could be detected in the detectable inbred lines. Among of them, P5 and P6 had two genotypes, the rest had three genotypes. The melt curve shown:in those could be amplified, the melt curve was a specific peak, no non-specific amplification and primer dimmer were detected in the products. Because of primer designing and filtrating were based on inbred line 'B73' during the development of SNP markers, fluorescent signal of amplification may be related to the different base of primer binding region between B73 and other inbred lines. More or less, every maker had the phenomena that can't be amplified in certain maize inbred lines. Following high resolution melt, there is no fluorescent signal. Among of them, P4 had the best result which only couldn't be amplified in inbred line Dan340; P1 had the worst result which couldn't be amplified in fourteen inbred lines.In conclusion, a total of 12388 SNP markers were developed in this study.6008 markers of them contain single SNP loci, and all these markers showed high polymorphism, which can be used for association analysis, fine mapping and molecular marker-assisted selection (MAS) in maize breeding.