A universal molecular method for rapid identification of alfalfa and dodder seeds

There are numerous similarities between Medicago sativa seeds and those of Cuscuta spp. with respect to color, size, and shape. Traditionally, the evaluation of seed purity has been based primarily on morphological methods, which are mainly dependent on visual assessments. However, given that the morphological indices of similar forage seeds are often similar or overlapping, the consistency of replicated seed observations is unreliable, and the process is laborious and time-consuming. By way of resolving these problems, in this study, we investigated the utility of a molecular approach based on sequence differences between the chloroplast rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large) genes of alfalfa and parasitic dodder, using dodder-specific primers designed for PCR amplification of alfalfa and dodder DNA. In this approach, the purity of alfalfa seeds was assessed by observing whether fragments of dodder DNA were amplified. In addition, this method can also accurately amplify DNA from mixtures of dodder seeds in alfalfa when the proportions of dodder to alfalfa seed DNA and seed number were 1 ꞉ 10 000 and 1 ꞉ 1 000, respectively. Thus, given its reliability, rapidity, and sensitivity, this method can provide a convenient detection technology for customs quarantine, thereby enabling assessments of alfalfa seed purity. Moreover, it has considerable potential for extensive application in the seed production and breeding of forage crops.