title: Rapid genetic mapping of ESTs using SNP pyrosequencing and indel analysis.
abstract: We describe an effective systematic approach to genetic mapping of cDNA clones, including those obtained from EST sequencing. The EST of interest is first partially sequenced from the 3'-end. PCR primers which bracket the 3'-UTR segment of the cDNA are designed. The corresponding gene segment is amplified from the parents of the mapping population, using primers equipped with 3'- and 5'-extensions to facilitate direct sequencing of PCR products. Comparison of the sequences obtained from the mapping parents frequently reveals single nucleotide polymorphisms or insertion / deletion polymorphisms, which can then be genotyped in a mapping population. The genotyping of SNPs is performed by pyrosequencing, a sequencing-by-synthesis method that has been used successfully in SNP diagnostics. SNP analysis of up to 96 samples, a number required to produce meaningful genetic segregation data, can be rapidly accomplished in parallel. The parental genotype of three loci, stearoyl-ACP desaturase, nucleoside-diphosphate kinase and sucrose synthetase-1 were determined by conventional sequencing, and the polymorphism so identified were scored by the pyrosequencing of 94 individuals of a maize recombinant-inbred population. These loci were successfully placed onto chromosomes 3, 7 and 9 respectively. This method is generally applicable to most plant species, which show sufficient sequence diversity in the 3'-UTR region of genes.
authors.affiliation: DuPont Crop Genetics, Molecular Genetics Group, 1, Innovation Way, Newark, DE 19711, USA. Ada.S.Ching@USA.dupont.com
authors.forename: Ada
authors.initials: A
authors.lastname: Ching
authors.forename: Antoni
authors.initials: A
authors.lastname: Rafalski
mesh_terms: D020413:3' Untranslated Regions; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D020224:Expressed Sequence Tags; D010944:Plants / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods; D003313:Zea mays / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant