KASP Assay for G2373A SNP Genotyping

A KASP assay targeting the G2373A SNP was developed (K1-K2/K3; Online Resource 1 and 2) and validated in the three T4-2235 plants. Briefly, two allele specific reverse primers were designed for the G2373A SNP incorporating either the G or A polymorphism at the 3′ end (primer K2 for the wildtype (G) and K3 for the mutant (A) allele). The common primer was developed to discriminate against the B genome by utilising a 3 bp indel present in the A genome (primer K1). Allele specific primers were synthesised with FAM and HEX tails and reactions and cycling conditions were as described before (Trick et al. 2012 (link)). Fluorescent end-point genotyping was carried out using a Tecan Safire plate reader (Tecan Group AG, Mannerdorf, Switzerland).

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Simmonds J., Scott P., Brinton J., Mestre T.C., Bush M., del Blanco A., Dubcovsky J, & Uauy C. (2016). A splice acceptor site mutation in TaGW2-A1 increases thousand grain weight in tetraploid and hexaploid wheat through wider and longer grains. TAG. Theoretical and Applied Genetics. Theoretische Und Angewandte Genetik, 129, 1099-1112.

Publication 2016

Safire plate reader

Allele Assay Genome Indel Plants Polymorphism Primers Tails

Corresponding Organization :

Other organizations : Norwich Research Park, John Innes Centre, University of California, Davis

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Variable analysis

independent variables

G2373A SNP

dependent variables

Fluorescent end-point genotyping

control variables

Utilising a 3 bp indel present in the A genome to discriminate against the B genome
Reactions and cycling conditions as described in Trick et al. 2012

controls

Positive control: Wildtype (G) allele (primer K2)
Negative control: Mutant (A) allele (primer K3)

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