A sensitive and specific PCR-based discrimination of split red vetch and lentil seeds

摘要

A PCR-based marker technique was developed to discriminate between morphologically similar split seed of vetch (Vicia sativa) and lentil (Lens culinaris subsp. culinaris). Sequence tagged microsatellite site (STMS) markers were more discriminatory than markers produced from the nontranscribed spacer (NTS) region of the 5S ribosomal RNA gene. A sequence characterized amplified region (SCAR) marker, developed from the 5S rRNA NTS region, was sensitive when resolved on agarose. However, the fluorescent-labeled 5S rRNA SCAR marker was unable to discriminate between vetch and lentil, probably because of the low copy number of the marker, and was not visualized on agarose. An STMS primer-pair (PSMPSAD123), developed from field pea, was able to discriminate split red cotyledon vetch from split red cotyledon lentil because it produced specific markers at 563 bp for lentil and 353 and 474 bp for vetch. The vetch-specific STMS marker was conserved among all species of the Vicia genus used in this study and was sensitive enough to discriminate both on agarose gels and on polyacrylamide gel-based fluorescent systems. The fluorescent-tagged STMS analysis revealed peaks for vetch and lentil at the expected sizes in admixtures of milled vetch and lentil seeds, and it was sensitive enough to detect one vetch seed in 1999 lentil seeds. The development of PCR-based tests for detecting the level of vetch seed contamination in lentil export seed may provide a method for quality assurance of export lentil seed.