Plant & Animal Genome VI Conference

P79

GENERATION OF CODOMINANT, PCR-BASED MARKERS FROM AFLP BANDS

USDA-ARS and Department of Horticulture, 1575 Linden Drive, University of Wisconsin-Madison, Madison, WI 53706 USA

The amplified fragment length polymorphism (AFLP) is a powerful marker class, allowing rapid and simultaneous evaluation of 50-70+ potentially polymorphic sites. Well adapted to linkage mapping and diversity assessment, AFLPs are primarily dominant in nature. That fact and relatively high cost and technological difficulty limit use of AFLPs for marker aided selection and other locus-specific applications. In carrot the Y2 locus conditions carotene accumulation in the root xylem core. We identified AFLP fragments linked to the dominant Y2 allele and pursued the conversion of those AFLP fragments to forms useful for plant breeding, characterization of wild populations, and gene cloning. We refer to these converted marker forms as breeder-friendly markers (BFMs). BFMs are codominant, PCR-based markers that are comparatively inexpensive, rapid, and simple, requiring no radioactivity. The short length of AFLPs (~60 bp to 500 bp) precludes development of longer, more specific primers (a SCAR-like approach). Instead, inverse PCR (iPCR) primers were designed from sequence information from cloned AFLP fragments and used to generate DNA sequence information outside and immediately adjacent to the AFLP fragment. Comparison of sequences associated with Y2 vs. y2 revealed differences allowing BFM design. For example, PCR primers flanking indels amplified allele specific bands that distinguish homozygotes and heterozygotes. Another approach that should be more generally applicable is conversion of AFLPs to cleaved amplified polymorphic sequences (CAPS), using restriction site polymorphisms flanking the AFLP fragments. Conversion of AFLPs to breeder-friendly forms greatly expands the usefulness of this marker class.