Plant & Animal Genome VI Conference Plant & Animal Genome VI Conference
P86
AFLP, amplified fragment length polymorphism, is a powerful genotyping technique which requires limited sequence information for the exploitation of altered restriction endonuclease recognition sequences between individuals. Although AFLP bands can be genetically linked to a given trait or phenotype and are inherited in a Mendelian fashion, the dominant nature of the marker system (+ / null) represents a disadvantage associated with this technique for some applications. We proposed to take advantage of the rapid identification of polymorphisms detected with AFLP, and convert the bands to single, specific amplification products associated with a trait that could detect both alleles in an individual. The current study describes the identification and isolation of AFLP bands for assay of hybrid purity in Brassica oleracea. AFLP was used to identify both male- (pollen source) and female- (pollen recipient) specific bands which were subsequently isolated from the acrylimide gel and sequenced. PCR primers were developed from the AFLP band sequence. In some cases, a dominant (plus-minus) amplification was observed consistent with the plus-minus amplification of the individuals with the AFLP assay. However, in an effort to develop a co-dominant test, PCR primers were designed that would amplify both alleles corresponding to a given AFLP band. Isolation of products from each individual revealed DNA sequence differences that were exploitable either as restriction site polymorphisms or as primer-binding polymorphisms for the development of a co-dominant test. The identification of linked AFLP loci and subsequent conversion to allele-specific tests represents a useful tool for marker assisted selection, hybrid purity, and diagnostic testing.