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GrainGenes Reference Report: CAM-7-803

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Reference	CAM-7-803
Title	Rapid genetic mapping of ESTs using SNP pyrosequencing and indel analysis
Journal	Cellular and Molecular Biology Letters
Year	2002
Volume	7
Pages	803-810
Author	Ching A Rafalski A
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 3prime-end. PCR primers which bracket the 3prime-UTR segment of the cDNA are designed. The corresponding gene segment is amplified from the parents of the mapping population, using primers equipped with 3prime- and 5prime-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 3prime-UTR region of genes
External Databases	Pubmed: 12378241
Keyword	[ Hide all but 1 of 18 ] chromosome chromosome maps clone complementary dna diversity dna sequencing genetic mapping genetic polymorphism genotype inbred line locus maize mapping methodology molecular genetics polymerase chain reaction segregation sucrose

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