2005 Annual Report 4d.Progress report. This report serves to document research conducted under a Non-funded Cooperative Agreement between ARS and Cold Spring Harbor Laboratory. Additional details of this research can be found in the report of the parent project 1907-21000-014-00D Comparative Plant Genomics. This works focuses on the developing resources to understand cereal genome evolution, by focusing on detail synteny analysis of rice chromosome 1 and homeologous chromosomes in maize and sorghum. In the last year, a preliminary website and database were established for the project (http://ware.cshl.edu/yia). We are using public datasets to establish points of contact between rice chromosome 1 and corresponding regions of maize and sorghum. Comparative maps were built using the rice pseudochromosome as a common reference sequence and the genetic and physical maps of maize and sorghum. This is an ongoing effort that progresses with the availability of new datasets. Our current comparative maps use the latest rice assembly from TIGR (release. 3)(http://www.tigr.org/tdb/e2k1/osa1/). Updated maize maps include the Oct 2004 release of the MMP FPC physical map (http://www.genome.arizona.edu/fpc/maize/) and the 2004 IBM2 Neighbors genetic map (http://www.maizegdb.org/). Sorghum maps comprise the 2004 release of the TAMU FPC physical map, and the genetic maps of Klein 2004 (http://sorgblast2.tamu.edu/) and Paterson 2003 (Bowers et al., 2003 Genetics 165:367). All comparative maps are available through the Gramene website (http://www.gramene.org/cmap/index.html). We are working with collaborators Davis and Klein laboratory to fill gaps in the physical maps of maize and sorghum within targeted regions and to anchor physical maps to genetic maps. We identified likely orthologues between rice/sorghum and rice/maize by comparing the rice assembly to all available expressed and genomic sequences. Results are posted in the Gramene Genome Browser. These sequences provide a resource for identifying BAC clones via PCR amplification of pooled libraries. Because the position of the presumptive rice orthologue is known, BAC identification can be targeted to find homeologous regions in maize and sorghum. We are currently identifying gaps in the comparative maps as targets for BAC identification. Potential genetic markers were identified as simple sequence repeats (SSR) in maize and sorghum sequences. Many of these could be anchored to FPC physical maps by virtue of overgo hybridization, presence within a BAC end, or anchoring to Rice. However, many FPC contigs lacked anchorage or orientation within genetic maps. The SSR sequences were communicated to our collaborators for polymorphism screening and genetic mapping. The Davis laboratory screened the maize markers against parental lines of eight mapping populations. Of the 244 markers tested, over half showed a polymorphism in at least one mapping population. Work is underway to choose robust markers corresponding to contigs of interest for targeted genetic mapping using the IBM lines and/or one of the other mapping populations. In sorghum, 54 putative SSR markers have been tested, and an additional 12 are in progress, in the laboratory of Klein. Genotype assays were successfully developed on 32 of the markers tested, and all of these have been mapped to chromosomes using the BTx623 x IS3620C population. Our strategy for building long-range, medium-resolution, comparative maps is to hybridize rice overgo probes (anchored to known positions in chromosome. 1)to maize and sorghum BACs, whose physical positions are known based on BAC fingerprinting. In collaboration with Dr. Patricia Klein we constructed a minimal tiling path of sorghum BAC clones corresponding to collinear regions of rice chromosome 1. These clones were hybridized to rice overgo probes in collaboration with Dr. Scott Jackson. To date, 288 probes have been tested, of which 16 probes positively hybridized with 66 BAC clones. Due to rapid progress being made with the maize FPC map, we have deferred building a maize minimal tiling path until after the next map update. However, our collaborator has included the ZMMBBc filter set, constituting 7x coverage of the maize genome, in overgo hybridizations. This has resulted in the identification of 138 BAC clones by 16 out of the 288 probes tested. Overgo hybridization data are displayed in browsable form at the project website (http://ware.cshl.edu/yia). Comparative maps revealed nearly continuous coverage of both the maize and sorghum physical maps over a region spanning 35-40 Mb on rice chromosome 1. To analyze micro-colinearity over this region we utilized a "BAC skim" strategy. The corresponding sorghum region was divided into 14 bins and 3-4 BAC clones were selected from each bin. The maize region corresponded to two FPC contigs and, in collaboration with the McCombie’s group, 30-40 BAC clones spanning each contig were selected. Each pool of BAC clones was sequenced to 2x coverage. Comparison of sequence reads to rice revealed selective alignment to the 35-40 Mb region of chromosome 1. Maize reads aligned to 17% of rice gene models in this region whereas sorghum reads aligned to 48% of gene models. High-resolution micro-colinearity is being examined using completely sequenced BAC clones. We aligned these BAC sequences (486 in all) to rice chromosome 1 and posted these data for viewing at the Project Website (http://ware.cshl.edu/yia). We are currently using these alignments and the skim data to select a sorghum BAC for complete sequencing. Our strategy is to choose a BAC that covers a gene-rich region in rice and corresponds to a completely sequenced region in maize. The resulting data set will provide a high-resolution 3-way micro-colinearity map over a ~150 kb region. This project supported one of three teaching fellows that participated in a curriculum development program at the CSHL Dolan DNA Learning Center in August 2004. This two-week program focused on concepts of gene annotation and colinearity of genes in cereals. In addition to interacting directly with Dr. Doreen Ware, the teaching fellows became familiar with the programs at the learning center and provided feedback on curriculum development.