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ABSTRACT: Four Waxy haplotypes, previously identified as each having a different combination of three single nucleotide polymorphisms (SNPs)
in the Waxy gene, were highly correlated with apparent amylose content and pasting properties, which are important grain quality traits
for predicting cooked rice (Oryza sativa L.) texture and processing properties (Chen et al. in J Cereal Sci 47:536–545, 2008a; Chen et al. in J Cereal Sci 48:781–788, 2008b). Three allele-specific PCR markers were developed to genotype the three aforementioned functional SNPs in a single PCR amplification.
Each marker contained two allele-specific primers and one common primer. For each marker, the two allele-specific primers
differed by one base at the 3′-end to provide discrimination of SNP alleles, and were labeled with unique fluorescence probes.
An additional mismatched base, the third base from the 3′-end, was inserted in some allele-specific primers to increase selectivity.
The amplification step of the PCR thermal cycling program was initially set for 20× touch-down cycles with the annealing temperature
of the first cycle approximately 6°C above the thermal melting temperature of all three primers at a touch-down rate of −0.3°C
per cycle, and followed by 25× regular thermal cycles with the annealing temperature at their thermal melting temperature.
The allelic genotypes for each SNP were distinguished from each other by both their differential primer-allele fluorescences
and their amplification product lengths. The simplicity of these assays makes it easy to utilize these markers as part of
a marker-assisted selection strategy in rice breeding programs selecting for these important grain quality traits.
Molecular Breeding 26(3):513-523. · 3.25 Impact Factor