High throughput discovery and genotyping of single nucleotide polymorphisms in maize.

摘要

Single nucleotide polymorphisms (SNPs) are the most frequent DNA markers for genetic mapping and disease diagnostics in eukaryotic genomes. We developed DNA microarray-based approaches to score the inheritance of SNPs. The barcode oligonucleotide ligation assay (BOLA) exploits the fact that DNA ligase will join two oligonucleotides that are properly aligned by a guide DNA but will fail to do so if the 3′ end of one of the oligonucleotides is mispaired with the guide. Molecular barcodes (20 mer oligonucleotides) were used to target the ligation products to specific addresses on an oligonucleotide microarray slide. This method was tested with a set of maize SNPs. The genotypes of 17 inbred lines were used to build a dendrogram that appears to agree with the pedigrees of the inbred lines. We also developed a DNA microarray formatted single base extension (SBE) method for SNP genotyping. SBE primers were printed and immobilized on DNA microarray slides. DNA fragments flanking the SNP regions were amplified with polymerase chain reactions (PCR). The PCR products, either denatured by heating or digested to single strands with T7 gene 6 exonuclease, were hybridized with the SBE primers. The single base extension reaction was subsequently conducted on the surface of DNA microarray slides. With the single stranded PCR as template, DNA polymerase separately adds a single base of ddNTPs labeled with one of the four dyes, Texas Red®, tetramethylrhodamine, fluorescein, and eosin to the 3′ ends of the SBE primers. Our preliminary data showed that the DNA template prepared either way produced a high signal to noise ratio. Our preliminary results also suggested acceptable accuracy of this SBE method. We also studied the polymorphisms at the DNA sequence level between Illinois High Oil (IHO) and Illinois Low Oil (ILO) populations in maize. Forty-one percent (9 of 22) of the PCR products was polymorphic between IHO and ILO, but 48% (11 of 23) was polymorphic between B73 and Mo17, indicating that IHO and ILO were very different at the genomic sequence level although they were derived from the same open pollinated population.