2012 Annual Report 1a.Objectives (from AD-416): Evaluate carrot and cucumber genetic marker relationships to chromosomes. 1b.Approach (from AD-416): 1) Compare linkage map location to marker sequence for 15 genes each in carrot and cucumber. 2) Compare carrot and cucumber marker sequence to GenBank and other data bases. 3) Evaluate sequences for carrot in BAC library. 3.Progress Report: This is the final report for project 3655-21000-049-20S, terminating in August 2012. Marker genes in single-copy bacterial artificial chromosome (BACs) (24) for carrot and fosmid clones (76) for cucumber were localized on all chromosomes of each crop. Both carrot and cucumber are plant species with few cross-compatible related species. With the availability of numerous molecular markers combined with comparative fluorescence in situ hybridization (FISH) analysis of pachytene chromosomes, this project provided insights into the genetic differentiation between species to advance our understanding of chromosome evolution and the domestication history of these crops. Pachytene chromosome-based karyotypes were developed and several chromosome rearrangements were uncovered by FISH mapping of genetically anchored bacterial artificial chromosome clones in carrot and fosmid clones in cucumber. For carrot the chromosomal location of Recombinant deoxyribonucleic acid (rDNA) gene clusters was determined for the first time, and a satellite repeat from the centromeric regions common to all carrot chromosomes was discovered and used to demonstrate that heterochromatic domains were mainly confined to the pericentromeric regions of each chromosome. For cucumber, strategically selected markers from the genetic map and draft genome assembly were employed to screen for fosmid clones for use as probes in comparative fluorescence in situ hybridization analysis of pachytene chromosomes to investigate genetic differentiation between wild and cultivated species. These studies provide evidence of the evolutionary relationships between cultivated carrot and cucumber and their related species and reveal patterns of chromosome evolution. Chromosomes were associated with linkage groups and rearrangements noted for cucumber, relationships among taxa were clarified, and data submitted to GenBank. With this information, carrot and cucumber breeders and researchers can better predict the feasibility of incorporating new genes by crossing with related species and the genetic origin of these crops is better understood. This research relates to Objective 2, Utilize current biotechnology to discover and evaluate genetic variation and to map agriculturally important traits in Allium, Cucurbit, and Daucus germplasm, and to develop genetic and breeding stocks.