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

Jaemin Cho, Taewook Jung, Jungin Kim, Seokbo Song, Jeeyeon Ko, Koansik Woo, Jaesaeng Lee, Myeongeun Choe, Inseok Oh

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. All Korean waxy-grain sorghum used in this study contained the wx (a) allele, and one wx (a) allele-containing individual was also heterozygous for the wx (c) allele. No individuals possessed the wx (b) allele. The genotyping results were confirmed by iodine staining and amylose content analysis. Based on the sequence of the wx (a) allele, three different types of primers (wx (a) 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. 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.