ORIGINAL PAPER M. R. Ponce á P. Robles á J. L. Micol High-throughput genetic mapping in Arabidopsis thaliana Received: 24 September 1998 / Accepted: 9 December 1998 Abstract To facilitate rapid determination of the chro- mosomal location of novel mutations, we have improved current approaches to gene mapping using microsatellite length polymorphisms. The high-throughput linkage analysis method described here allows a novel gene to be tested for linkage against the whole genome of a multi- cellular eukaryote, Arabidopsis thaliana, in a single polyacrylamide gel. The procedure is based on the si- multaneous co-ampli®cation of 21 microsatellites in a single tube, using a multiplex PCR mix containing 21 primer pairs, each including one oligonucleotide labeled with one of three ¯uorescent dyes that have dierent emission wavelengths. The ampli®cation products, which range in number from 21 to 42, depending on the genotype of the individual being tested, are electropho- resed in a single lane on a polyacrylamide gel. The use of an automated fragment analyzer makes it possible to perform linkage analysis on a one gel-one gene basis using DNA samples from 19 F 2 individuals obtained from an outcross involving a mutant and a wild-type that is genetically polymorphic with respect to the eco- type in which the mutant was generated. Discrimination of the ampli®cation products is facilitated not only by labeling with dierent ¯uorochromes, but also by prior testing of dierent sequences for the ability to prime the ampli®cation of each microsatellite, in order to ensure that multiplex PCR yields compatible ampli®cation products of non-overlapping size. The method is par- ticularly useful in large-scale mutagenesis projects, as well as for routine mapping of single mutants, since it reveals the map position of a gene less than 24 h after the F 2 individuals to be analyzed have become available. The concepts employed here can easily be extended to other biological systems. Key words Simple sequence length polymorphism (SSLP) á Multiplex PCR á Fluorescence-based genetic mapping á Arabidopsis Introduction Among the procedures currently available for gene mapping by linkage analysis, one of the most widely preferred is the simple sequence length polymorphism (SSLP) method, which is based on the presence of highly polymorphic microsatellites in most, if not all, eukary- otic genomes. SSLPs were ®rst discovered (Litt and Luty 1989; Smeets et al. 1989; Tautz 1989; Weber and May 1989) and exploited for linkage analysis (Hearne et al. 1992) in mammals. More recently, mapping procedures based on SSLP have been introduced in plants, with the description of about 50 microsatellites in Arabidopsis thaliana (Bell and Ecker 1994; http://cbil.humgen. upenn.edu:80/atgc/SSLP_info/nga_sequences.html). Gene mapping by SSLP is performed in Arabidopsis on genomic DNA samples obtained from the F 2 progeny of an outcross between a mutant and a wild-type strain that is genetically polymorphic with respect to the eco- type in which the mutant was obtained. Linkage analysis is performed by ampli®cation of the polymorphic DNA regions, followed by determination of the PCR product sizes. For recessive alleles of a gene whose map position is unknown, DNAs from at least twenty F 2 individuals displaying the mutant phenotype are used as a template on a one microsatellite (one primer pair) ± one PCR ampli®cation basis. The usual approach consists of choosing ®ve microsatellites, one from each of the ®ve Arabidopsis chromosomes, which are then sequentially tested for linkage. If linkage is observed at the ®rst at- tempt, a minimum of 20 PCR ampli®cations is neces- sary, each ampli®cation requiring one lane in a Mol Gen Genet (1999) 261: 408±415 Ó Springer-Verlag 1999 Communicated by G. JuÈrgens M. R. Ponce á P. Robles á J. L. Micol (&) DivisioÂn de GeneÂtica, Universidad Miguel HernaÂndez Campus de San Juan, E-03550 Alicante, Spain e-mail: jlmicol@umh.es Tel.: +34-96-5919455; Fax: +34-96-5919434