Mol Gen Genet (1996) 251:52-59 © Springer-Verlag 1996 Susan W. Gorman. Dawn Banasiak Cecelia FaMey - Sheila McCormick A 610 kb YAC clone harbors 7 cM of tomato (tycopersicon esculentum) DNA that includes the male sterile 14 gene and a hotspot for recombination Received: 6 July 1995 / Accepted: 8 November 1995 Abstract Pollen development requires both sporo- phytic and gametophytic gene expression. We are using a map-based cloning technique to isolate sporophytic genes which, when mutant, cause pollen abortion and a male sterile (ms) phenotype in tomato (Lycopersicon esculentum). We have genetically characterized one ms locus (ms14) using RFLP analysis and identified flank- ing markers. High-resolution genomic physical map- ping indicates that the ms14 locus is located m a ~300 kb region. We have identified a YAC clone with an insert size of ,,-610 kb that contains the msl4- linked markers, reflects the organization of the physical map and therefore most probably contains the msl4 gene. In addition, we present evidence that the relation- ship between physical and genetic distance in this chromosomal region changes abruptly from ,-~ 105-140 kb/cM to less than 24kb/cM, and suggest that the TG393-TG104 region is a hotspot for recombi- nation. Key words Positional (map-based) cloning " Pulsed field electrophoresis • Physical mapping. RFLP mapping • Pollen development Introduction Pollen development in higher plants is a complex pro- cess that requires the coordination of gene expression in both the sporophytic and gametophytic tissues of the anther (reviewed in McCormick 1993). Mutations in male sterile (ms) loci result in aborted pollen and gener- ally require the homozygous recessive condition in Communicated by R. Hagemann S. W. Gorman • D. Banasiak • C. Fairley • S. McCormick (E~]) Plant Gene Expression Center, USDA/ARS and University of California-Berkeley, 800 Buchanan St., Albany, CA 94710, USA order to exhibit the phenotype. This suggests that these genes act in the sporophytic tissues of the anther but affect the developing microspores (gametophytes). Male sterile genes might be expressed before or during meiosis in the sporophytic pollen mother cell or in the tapetum or other sporophytic cells of the anther. We hope to develop an understanding of the sporophytic contribution to pollen development by studying the products of male sterile loci. Current biochemical and molecular information do not provide a clear picture of the mechanism(s) of nuclearly encoded male sterility. Although three genes have been isolated that produce a male sterile pheno- type when mutant, their gene products do not provide obvious clues to the roles that these genes play during pollen development. For example, Moffatt and Somer- ville (1988) isolated an adenine phosphoribosyl trans- ferase (aprt-) Arabidopsis mutant, but how a lesion in a nucleic acid salvage pathway disrupts pollen develop- ment is unclear. The ms2 gene from Arabidopsis (Aarts et al. 1993) contains a small region homologous to a wheat mitochrondrial ORF, but no function has been ascribed to this mitochondrial region or to the gene product of the ms2 gene. In maize, a male sterile gene designated Ms45 (M. Albertsen, personal communica- tion) was cloned (Albertsen et al. 1993) that showed 33% homology over 256 amino acids with strictosidine synthase. Although the action of strictosidine synthase in the indole alkaloid pathway is understood, it is not clear whether or how indole sythesis might be involved in pollen development. These examples serve to illus- trate the complexities of male gametophyte develop- ment and, therefore, it will probably be necessary to isolate and characterize a large number of male sterile gene products before we can develop a comprehensive understanding of the sporophytic contribution(s) to pollen development. In tomato (Lycopersicon escuIen- turn), more than 40 spontaneous ms loci have been identified (summarized in Kaul 1988). We have targeted for isolation one of these male sterile genes, ms14; in