2006 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 1907-21000-023-00D "Comparative Genomic Analyses, Bioinformatics and Resource Development for Cereal Genomes." We are using public datasets to establish points of contact between genomes of rice, 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. 4)(http://www.tigr.org/tdb/e2k1/osa1/). Updated maize maps include the July 2005 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). The resulting comparative maps are available through the Gramene website (http://www.gramene.org/cmap/index.html). Using these data we have identified conserved syntenic blocks between rice and maize (Zhao, W. and Ware, D, In Preparation). These maps, also displayed in Gramene (http://www.gramene.org/Oryza_sativa/syntenyview?otherspecies=Zea_mays), reveal that rice chromosome 1 is orthologous to two maize chromosomes, 3 and 8, consistent with the well-documented tetraploidy event in the maize lineage. By contrast only chromosome 3 in sorghum (aka linkage group A) shows orthology to rice chromosome 1. For each of the maize or sorghum orthologues, conserved syntenic blocks are distributed over the entire length of the rice chromosome. Furthermore, only relatively small gaps exist between syntenic blocks in rice, with exception of the centromeric region. An early priority of this project was the construction of BAC-based comparative maps of maize and sorghum to rice chromosome 1. With the maturation of integrated genetic and physical maps by the Maize Mapping Project and our collaborator Dr. Patricia Klein this goal has been largely achieved. We nevertheless developed a strategy for genetically anchoring orphan FPC contigs using simple sequence repeat (SSR) markers. A total of 22,980 SSR’s were mined from maize and 11,914 mined from sorghum using EST, BAC-end, and other genome survey sequences. These data are posted on the project web-site (http://ware.cshl.org/cgi-bin/data_view?action=ssr_hits). Our collaborator, Dr. Georgia Davis, screened 244 maize SSR’s against parental lines of eight mapping populations. Over half (137) showed a polymorphism in at least one population. To further evaluate this approach, twenty-two were mapped using the B73 x Mo17 population. All but three markers mapped to the region predicted by the integrated genetic and physical map. In sorghum, 54 putative SSR markers have been tested in the laboratory of Dr. Patricia Klein. Genotype assays were successfully developed on 32 of the markers tested, and all of these have been mapped using the BTx623 x IS3620C population. To build long-range, high-resolution comparative maps we evaluated overgo hybridization technology in collaboration with Dr. Scott Jackson. This effort took advantage of the Oryza Map Alignment Project (OMAP), in which 1,723 probes were designed at 25-kb intervals along rice chromosome 1. This density corresponds to about one probe per three protein-coding genes. Probes were hybridized to physically-mapped maize and sorghum BAC clones. Dr. Patricia Klein provided ~3500 sorghum BAC clones from chromosome 3, giving ~10X coverage of that chromosome. For maize, the ZMMBBc filter set was used, constituting 7x coverage of the genome. In tests of 288 probes the hybridization success-rate was lower than we would have liked, just 6%. Based on alignment data, we attributed the low success rate to probe-to-target mismatches. The resulting data was sufficient to build a comparative map between rice chromosome 1 and sorghum chromosome 3, showing long-range colinearity and one major inversion, consistent with previously published maps. However, due to the low success rate, we have decided not to proceed with hybridization of the remaining overgo probes. This work is included in a manuscript published in BMC Genomics (Barbara L Hass-Jacobus, Montona Futrell-Griggs, Brian Abernathy, Rick Westerman, Jose-Luis Goicoechea, Joshua Stein, Patricia Klein, Bonnie Hurwitz, Bin Zhou, Fariborz Rakhshan, Abhijit Sanyal, Navdeep Gill, Jer-Young Lin, Jason G Walling, Mei Zhong Luo, Jetty Siva S Ammiraju, Dave Kudrna, Hye Ran Kim, Doreen Ware, Rod A Wing, Phillip San Miguel, and Scott A Jackson - Integration of hybridization-based markers (overgos) into physical maps for comparative and evolutionary explorations in the genus Oryza and in Sorghum). 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 clone skim sequencing strategy. The minimum tiling path within the corresponding region of sorghum consisted of 51 clones, and these were divided into 13 adjacent pools containing 3-4 BAC clones per pool. The maize region corresponded to two FPC contigs and a minimum tiling path across each contig (30-40 clones each) was pooled. Each pool of BAC clones was sequenced to 1-2x coverage. Skim sequences were aligned to the TIGR (Release. 3)rice assembly and gene model annotations. Significant alignments to non-transposon coding regions were used to build comparative maps. This analysis revealed that approximately 67% of sorghum genes within the skim-sequenced region were collinear with the targeted region of rice chromosome 1, whereas collinearity was observed for approximately 50% of maize genes. Non-collinear genes were distributed over the remaining 11 rice chromosomes. On chromosome 5, the corresponding rice genes were clustered within a 4 Mb region, and displayed collinearity across the skim sequence pools. This region was previously shown to be homologous with chromosome 1 due to an ancient whole-genome duplication event preceding the origin of grasses. Many, but not all, of duplicated genes were lost from one paralogous region or the other in the rice genome by a process called diploidization. We are investigating the hypothesis that continued and differential diploidization following the divergence of grass lineages contributed to some of the losses in colinearity observed in present-day species. The extent of collinearity within the targeted rice region was also examined. For this analysis we excluded TIGR gene models corresponding to transposable elements as well as hypothetical models that lack EST and other homology-based evidence. We also treated tandemly-duplicated genes as single loci, since distinguishing orthologues from paralogues would be difficult in the non-assembled skim data. Within the remaining set of gene models approximately 67% aligned to the sorghum skim sequence, whereas approximately 35% aligned to maize skim sequence. The lower rate of colinearity with maize is consistent with lineage-specific gene losses from maize chromosome 8. Such losses could have resulted from tetraploidization and subsequent diploidization in the maize progenitor. According to this hypothesis we would predict that many of the non-collinear rice loci are present in the homoeologous region of maize chromosome 3. To test this hypothesis we are planning to generate skim sequence data from this region. High-resolution micro-colinearity is being examined using completely sequenced BAC clones. Many finished or near-finished maize BACs are available through the SMTG consortium and from Dr. Richard McCombie’s lab. We aligned these sequences (486 in all) to rice chromosome 1 and posted these data for viewing at the Project Website (http://ware.cshl.edu/yia). Two sequenced BACs correspond to homeologous regions of chromosomes 3 and 8 (AC155539 and AC147015, respectively). Using the skim sequence data we selected and completely sequenced the corresponding BAC clone (SB_IBa82G24) from sorghum chromosome 3. The resulting 4-way map supports observations based on skim sequencing. Greater collinearity was observed between rice and sorghum than between either with individual maize chromosomal regions. Disruptions of collinearity could be attributed to scattered gene loss from the two maize homoeologues. When considered together these regions of maize chromosome 3 and 8 contain the full complement of genes that are in common between rice and sorghum.