SNP discovery by ecotilling using capillary electrophoresis

Eliott, F., Cordeiro, G., Bundock, P. C. and Henry, R. J. (2008). SNP discovery by ecotilling using capillary electrophoresis. In R. J. Henry (Ed.), Plant genotyping II: SNP technology (pp. 78-87) Wallingford, United Kingdom: CABI Publishing. doi:10.1079/9781845933821.0078

Author Eliott, F.
Cordeiro, G.
Bundock, P. C.
Henry, R. J.
Title of chapter SNP discovery by ecotilling using capillary electrophoresis
Title of book Plant genotyping II: SNP technology
Place of Publication Wallingford, United Kingdom
Publisher CABI Publishing
Publication Year 2008
Sub-type Research book chapter (original research)
DOI 10.1079/9781845933821.0078
ISBN 9781845933821
Editor R. J. Henry
Chapter number 5
Start page 78
End page 87
Total pages 10
Total chapters 15
Language eng
Abstract/Summary Ecotilling (derived from targeting-induced local lesions in genomes (TILLING)) is a high-throughput method of detecting naturally-occurring DNA polymorphisms in specific gene sequences. Sugarcane, a complex polyploid species, was utilized as a model to develop and test new protocols for high throughput ecotilling using capillary electrophoresis (CE) systems. Segregating single nucleotide polymorphisms (SNPs) were easily identified in the progeny of the sugarcane population using the ABI 3730 CE system. In the SPS gene family 1, a cluster of SNP peaks formed a distinctly different pattern in each of the parents Q165 and IJ76-514, and these two patterns clearly segregated in the progeny. In ecotilling or TILLING using gel-based systems, polymorphisms are typically localized to within ±10 bases. In the analyses using CE, SNPs were typically localized to within ±3 bases of that determined through sequence information. A higher level of precision in the assignment of fragment sizes based on the size standards was achieved with the sensitivity of a CE system compared with gel electrophoresis systems. In the two sugarcane genotypes, Q165 and IJ76-514, SNP loci in SPS gene family III, identified through ecotilling, corresponded precisely with SNPs identified in and through the respective alignment of the 58 and 86 sequences amplified with the same set of primers used in ecotilling. An ecotilling strategy utilizing the sensitivity of a CE system has been shown as a robust means for genetic mapping in sugarcane. In a number of instances, multiple single-dose SNPs belonging to homoeologous genes were detected and mapped to separate locations on the Q165 map. A summary of the optimized protocol for ecotilling using CE is also presented.
Keyword Induced point mutations
Resistance gene analogs
Functional genomics
Reverse genetics
Q-Index Code B1
Q-Index Status Provisional Code
Institutional Status Non-UQ

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Created: Fri, 21 Oct 2011, 10:45:13 EST by Professor Robert Henry on behalf of Qld Alliance for Agriculture and Food Innovation