Development of a waxy gene real-time PCR assay for the quantification of sorghum waxy grain in mixed cereal products

Abstract

Background: Waxy-grain sorghum is used in most of the commercial cereal products in Korea. Worldwide, three waxy mutant alleles have been identified in the sorghum germplasm, and DNA markers for these alleles have been developed to identify the waxy genotype. However, that detection method cannot be used to determine the proportion of waxy content in samples containing both waxy and non-waxy sorghum. This study developed an assay that can be used to detect and quantify the waxy content of mixed cereal samples. Results: All Korean waxy-grain sorghum used in this study contained the wxa allele, and one wxa allele-containing individual was also heterozygous for the wxc allele. No individuals possessed the wxb allele. The genotyping results were confirmed by iodine staining and amylose content analysis. Based on the sequence of the wxa allele, three different types of primers (wxa allele-specific, non-waxy allele-specific, and nonspecific) were designed for a quantitative real-time PCR (qPCR) assay; the primers were evaluated for qPCR using the following criteria: analytical specificity, sensitivity and repeatability. Use of this qPCR assay to analyze mixed cereal products demonstrated that it could accurately detect the waxy content of samples containing both waxy and non-waxy sorghum. Conclusions: We developed a qPCR assay to identify and quantify the waxy content of mixed waxy and non-waxy sorghum samples as well as mixtures of cereals including sorghum, rice and barley. The qPCR assay was highly specific; the allele-specific primers did not amplify PCR products from non-target templates. It was also highly sensitive, detecting a tiny amount (>0.5%) of waxy sorghum in the mixed samples; and it was simple and repeatable, implying the robust use of the assay.