Conversion of AFLP bands into high-throughput DNA markers
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O R I GI N A L P A P E R
K. Meksem á E. Ruben á D. Hyten K. Triwitayakorn á D. A. Lightfoot
Conversion of AFLP bands into high-throughput DNA markers Received: 18 May 2000 / Accepted: 21 November 2000 / Published online: 17 January 2001 Ó Springer-Verlag 2001
Abstract The conversion of AFLP bands into polymorphic sequence-tagged-site (STS) markers is necessary for high-throughput genotype scoring. Technical hurdles that must be overcome arise from genome complexity (particularly sequence duplication), from the lowmolecular-weight nature of the AFLP bands and from the location of the polymorphism within the AFLP band. We generated six STS markers from ten AFLP bands (four AFLPs were from co-dominant pairs of bands) in soybean (Glycine max). The markers were all linked to one of two loci, rhg1 on linkage group G and Rhg4 on linkage group A2, that confer resistance to the soybean cyst nematode (Heterodera glycines I.). When the polymorphic AFLP band sequence contained a duplicated sequence or could not be converted to a locus-speci®c STS marker, direct sequencing of BAC clones anchored to a physical map generated locus-speci®c ¯anking sequences at the polymorphic locus. When the polymorphism was adjacent to the restriction site used in the AFLP analysis, single primer extension was performed to reconstruct the polymorphism. The six converted AFLP markers represented 996 bp of sequence from alleles of each of two cultivars and identi®ed eight insertions or deletions, two microsatellites and eight single-nucleotide polymorphisms (SNPs). The polymorphic sequences were used to design a non-electrophoretic, ¯uorometric assay (based on the TaqMan technology) and/or develop electrophoretic STS markers for high-throughput genotype determination during marker-assisted breeding for resistance to cyst nematode. We conclude that the converted AFLP markers contained polymorphism at a Communicated by R. Hagemann K. Meksem (&) á E. Ruben á D. Hyten K. Triwitayakorn á D. A. Lightfoot Department of Plant Soil and General Agriculture (Room 176), Center for Excellence in Soybean Research, Teaching and Outreach, Southern Illinois University at Carbondale, Carbondale, IL 62901-4415, USA E-mail: [email protected] Fax: +1-618-4537457
10- to 20-fold higher frequency than expected for adapted soybean cultivars and that the eciency of AFLP band conversion to STS can be improved using BAC libraries and physical maps. The method provides an ecient tool for SNP and STS discovery suitable for marker-assisted breeding and genomics. Key words Ampli®ed fragment length polymorphism (AFLP) á Soybean á Positional cloning á Disease resistance á Soybean cyst nematode
Introduction The use of ampli®ed fragment length polymorphism (AFLP) markers for genetic map construction in plants has accelerated genome analysis and genetic improvement. AFLP markers have been used to create genetic maps of both small and large genomes (Alonso-Blanco et al. 1998; Han et al. 1999), including those of many cultivated crop species (Becker et al. 1995; Gerats et al. 1995;
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