A "gene-island" sequencing strategy has been developed that expedites the targeted acquisition of orthologous gene sequences from related species for comparative genome analysis. A 152-kb bacterial artificial chromosome (BAC) clone from sorghum (Sorghum bicolor) encoding phytochrome A (PHYA) was fully sequenced, revealing 16 open reading frames with a gene density similar to many regions of the rice (Oryza sativa) genome. The sequences of genes in the orthologous region of the maize (Zea mays) and rice genomes were obtained using the gene-island sequencing method. BAC clones containing the orthologous maize and rice PHYA genes were identified, sheared, subcloned, and probed with the sorghum PHYAcontaining BAC DNA. Sequence analysis revealed that approximately 75% of the cross-hybridizing subclones contained sequences orthologous to those within the sorghum PHYA BAC and less than 25% contained repetitive and/or BAC vector DNA sequences. The complete sequence of four genes, including up to 1 kb of their promoter regions, was identified in the maize PHYA BAC. Nine orthologous gene sequences were identified in the rice PHYA BAC. Sequence comparison of the orthologous sorghum and maize genes aided in the identification of exons and conserved regulatory sequences flanking each open reading frame. Within genomic regions where micro-colinearity of genes is absolutely conserved, gene-island sequencing is a particularly useful tool for comparative analysis of genomes between related species.Many species within the grass family Poaceae provide staple grain and forage supplies for humans and animals and thus are of great economic and humanitarian importance. Members of the grass family are found in wide ranging areas of the world, demonstrating adaptability to diverse environmental conditions. Although genome sizes can vary greatly in the grasses (e.g. 420 Mb and 16,000 Mb for rice [Oryza sativa] and hexaploid wheat [Triticum aestivum], respectively [Arumuganathan and Earle, 1991]), recombinational mapping studies using common DNA markers indicate that gene order is generally conserved within long physical intervals between family members (Hulbert et al., 1990; Ahn and Tanksley, 1993;Van Deynze et al., 1998;Goff et al., 2002). This information has been used to construct comparative genetic maps among many grass species (for review, see Gale, 1997, 2000;Gale and Devos, 1998). The large variation in genome size observed in the grass family is attributable in part to differences in ploidy and to variation in the abundance of repetitive elements, primarily retrotransposons, located between low copy number genic regions in the genome (SanMiguel et al., 1996). In maize (Zea mays), retrotransposons are estimated to make up 50% to 80% of the genome (SanMiguel and Bennetzen, 1998). The repetitive sequences have little apparent sequence conservation among species (Hulbert et al., 1990; Bennetzen et al., 1994;Chen et al., 1998).Because of their widespread economic importance and genetic resources, rice and maize have been focal poin...