KASP Marker for causal SNPs in PHS resistance gene TaPHS1
Contributed by Shubing Liu and Guihua Bai
Pre-harvest sprouting (PHS) in wheat (physiologically mature grains germinating in spikes before harvest) causes significant losses in grain yield and quality, particularly in the regions with prolonged wet weather during a harvest season. Direct annual losses caused by PHS approach US$ 1 billion worldwide (1).
Resistance to PHS in wheat is a complex trait that is affected by both genotype and environment (2). Quantitative trait loci (QTLs) for PHS resistance have been reported on most wheat chromosomes (2-7). A major QTL for PHS resistance was mapped on 3AS (8) and recently the gene, designated as TaPHS1, underlying this QTL was cloned using map-based cloning (9).
We identified two key mutations in the coding region of the gene that are responsible for the change in PHS susceptibility, One is a GT-to-AT transition at the 5’ donor splice-site of the intron 3, which extends the exon 3 into intron 3 and the other is an A-to-T transversion that generates a premature stop codon and result in a truncated protein in susceptible genotypes (106 amino acids). Two KASP assays were developed to diagnose these two mutations in TaPHS1.
Two KASP assays were designed: KASPTaPHS1-646 for the mis-splicing site and KASPTaPHS1-666 for the premature stop codon. PCR can be run in a regular PCR thermal cycler and PCR product can be read in a Real-Time PCR Cycler or a fluorescence scanner following manufacturer instruction (http://www.kbioscience.co.uk/reagents/KASP_manual.pdf). A pre-plate scan before PCR is required to remove background fluorescence.
KASP assay for KASPTaPHS1-646:
Forward primers: GGTGGAACAGATGCAACTAAAGG -FAM GGTGGAACAGATGCAACTAAAGA -HEX Reverse primer: GTGAGTGTTATATGAAACTAATGATCCATT
KASP assay for KASPTaPHS1-666:
Forward primers: GTGAGTGTTATATGAAACTAATGATCCATTT -FAM GTGAGTGTTATATGAAACTAATGATCCATTA -HEX Reverse primer: ACCGGGTGGAACAGATGCAACTAAA
The diagnostic value of the two SNP was evaluated in a set of 82 wheat PHS resistant and susceptible cultivars. In KASPTaPHS1-646, ‘G’ allele co-segregated with PHS resistance and “A” allele co-segregated with PHS susceptibility (Figure. 1). In KASPTaPHS1-666, ‘A’ allele present in the resistant accessions and ‘T’ allele present in the susceptible accessions.
Conditions presented here should be considered only as a starting point of the PCR optimization for individual laboratories.
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