Development of a rapid one-step PCR protocol to distinguish between alfalfa (Medicago sativa) and sweet clover (Melilotus spp.) seeds

Distinguishing seeds of alfalfa (Medicago sativa) and sweet cover (Melilotus spp.) from one another is difficult because of similarities in the colour, shape, size and weight of the seeds. Existing methods to distinguish these seeds are usually time-consuming and inaccurate. In our study, a polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) method was developed to distinguish alfalfa and sweet clover seeds from one another. Based on differences in the DNA sequences of plastid matK spacer regions, a pair of primers was designed and used with simple PCR and PAGE techniques to identify 45 alfalfa and 34 sweet clover (8 white and 26 yellow) accessions that were collected from all over the world. Our results with this pair of primers were clearly different between the two species, supporting the reliability, sensitivity and simplicity of this method.

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