| Summary: | Aroma and amylose content are the key determinants of grain quality and commercial value in rice (Oryza sativa L.). Here, we report a novel agarose-based multiplex polymerase chain reaction (PCR)-assay for the unambiguous identification of genes controlling aroma (fgr) and amylose content (Wx) traits in rice. The multiplex assay was developed following the validation of two previously reported gene-specific primers, namely, the fgr-SNP and Wx-SSR. Twenty aromatic and non-aromatic rice genotypes with a wide range of variation in amylose content were used in this study. Optimization of primer concentration and critical parameters for thermal cycling, inclusive of cycle number and annealing temperature, were performed to obtain optimal results for the multiplex amplification. The resulting amplification products displayed well-differentiated allelic variants of both fgr and Wx genes on a 3% MetaPhor agarose gel, at the cost of less than $0.15 per sample. MetaPhor agarose gel electrophoresis (MAGE) was effectively employed as a genotyping method which offers an alternative to polyacrylamide gel electrophoresis (PAGE); a system, commonly used in rice research, that can be technically challenging and time-consuming. Without the need of expensive probes or specialized equipment, this newly developed multiplex assay is suitable for researchers, whose studies are dependent on mass genotyping, and in molecular laboratories with limited resources.
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