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A-Maize-ing Maize is one of our oldest and most important crops, having been domesticated approximately 9000 years ago in central Mexico. Schnable et al. (p. 1112 ; see the cover) present the results of sequencing the B73 inbred maize line. The findings elucidate how maize became diploid after an ancestral doubling of its chromosomes and reveals transposable element movement and activity and recombination. Vielle-Calzada et al. (p. 1078 ) have sequenced the Palomero Toluqueño ( Palomero ) landrace, a highland popcorn from Mexico, which, when compared to the B73 line, reveals multiple loci impacted by domestication. Swanson-Wagner et al. (p. 1118 ) exploit possession of the genome to analyze expression differences occurring between lines. The identification of single nucleotide polymorphisms and copy number variations among lines was used by Gore et al. (p. 1115 ) to generate a Haplotype map of maize. While chromosomal diversity in maize is high, it is likely that recombination is the major force affecting the levels of heterozygosity in maize. The availability of the maize genome will help to guide future agricultural and biofuel applications (see the Perspective by Feuillet and Eversole ).

In response to the correspondence by Vladimir V. Kapitonov and Jerzy Jurka (A universal classification of eukaryotic transposable elements implemented in Repbase. Nature Rev. Genet. 9 (2008), doi:10.1038/nrg2165-c1

Our knowledge of the structure and composition of genomes is rapidly progressing in pace with their sequencing. The emerging data show that a significant portion of eukaryotic genomes is composed of transposable elements (TEs). Given the abundance and diversity of TEs and the speed at which large quantities of sequence data are emerging, identification and annotation of TEs presents a significant challenge. Here we propose the first unified hierarchical classification system, designed on the basis of the transposition mechanism, sequence similarities and structural relationships, that can be easily applied by non-experts. The system and nomenclature is kept up to date at the WikiPoson web site.

Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%–4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.

Rice (Oryza sativa L.) is the most important human food crop in the world. The agronomic importance of rice, its shared evolutionary history with major cereal crops, and small genome size have led to the generation of a high-quality finished genome sequence by the International Rice Genome Sequencing Project (2005). The highly accurate and public IRGSP sequence now serves as a unifying research platform for a complete functional characterization of the rice genome. Such an analysis will investigate the rice transcriptome, proteome, and metabolome, with the goal of understanding the biological function of all 35,000 to 40,000 rice genes and applying that information to improve rice production and quality. This comprehensive analysis will utilize a variety of techniques and resources from expression and genome tiling arrays to collections of tagged mutant populations developed in elite cultivars grown around the world. Comparative genomics between the cereal genomes and within the genus Oryza will also play a critical role in our understanding of the rice genome (Ahn et al. 1993; Ahn and Tanksley 1993; Bennetzen and Ma 2003; Han and Xue 2003; Huang and Kochert 1994; Jena et al. 1994; Ma and Bennetzen 2004). By comparing genome organization, genes, and intergenic regions between cereal species, one can identify regions of the genome that are highly conserved or rapidly evolving. Such regions are expected to yield key insights into genome evolution, speciation, and domestication. The study of conserved noncoding sequences (CNSs) between cereal genomes will also increase our ability to understand and isolate regulatory elements required for precise developmental and temporal gene expression (Kaplinsky et al. 2002). The genus Oryza is composed of two cultivated (O. sativa and O. glaberrima) and 21 wild species (Khush 1997; Vaughan et al. 2003). Based on recent phylogenetic data, Ge et al. (1999) proposed that Porteresia coarctata should be included in the genus as the 24th Oryza species. Cultivated rice is classified as an AA genome diploid and has 6 wild AA genome relatives. The remaining 15 wild species are classified into 9 other genome types that include both diploid and tetraploid species. Figure 15.1 shows a proposed phylogenetic tree of the genus Oryza as described by Ge et al. (1999) based on the analysis of two nuclear genes and one chloroplast gene. The wild rice species offer a largely untapped resource of agriculturally important genes that have the potential to solve many of the problems in rice production that we face today, such as yield, drought, and salt tolerance as well as disease and insect resistance. To better understand the wild species of rice and take advantage of the IRGSP genome sequence, we have embarked on an ambitious comparative genomics program entitled the Oryza Map Alignment Project (OMAP). The long-term objective of OMAP is to create a genome-level closed experimental system for the genus Oryza that can be used as a research platform to study evolution, development, genome organization, polyploidy, domestication, gene regulatory networks, and crop improvement. The specific objectives of OMAP are to (1) construct deep-coverage large-insert bacterial artificial chromosome (BAC) libraries from 11 wild and 1 cultivated African Oryza species (O. glaberrima); (2) fingerprint and end-sequence clones from all 12 BAC libraries; (3) construct physical maps for all 12 Oryza species and align them to the IRGSP genome sequence; and (4) perform a detailed reconstruction of rice chromosomes 1, 3, and 10 across all 12 Oryza species (Wing et al. 2005). This chapter presents our current progress for OMAP and some early glimpses into the results we are finding.

The homeologous Orp1 and Orp2 regions of maize and the orthologous regions in sorghum and rice were compared by generating sequence data for >486 kb of genomic DNA. At least three genic rearrangements differentiate the maize Orp1 and Orp2 segments, including an insertion of a single gene and two deletions that removed one gene each, while no genic rearrangements were detected in the maize Orp2 region relative to sorghum. Extended comparison of the orthologous Orp regions of sorghum and japonica rice uncovered numerous genic rearrangements and the presence of a transposon-rich region in rice. Only 11 of 27 genes (40%) are arranged in the same order and orientation between sorghum and rice. Of the 8 genes that are uniquely present in the sorghum region, 4 were found to have single-copy homologs in both rice and Arabidopsis, but none of these genes are located near each other, indicating frequent gene movement. Further comparison of the Orp segments from two rice subspecies, japonica and indica, revealed that the transposon-rich region is both an ancient and current hotspot for retrotransposon accumulation and genic rearrangement. We also identify unequal gene conversion as a mechanism for maize retrotransposon rearrangement.

Orthologous regions in barley, rice, sorghum, and wheat were studied by bacterial artificial chromosome sequence analysis. General microcolinearity was observed for the four shared genes in this region. However, three genic rearrangements were observed. First, the rice region contains a cluster of 48 predicted small nucleolar RNA genes, but the comparable region from sorghum contains no homologous loci. Second, gene 2 was inverted in the barley lineage by an apparent unequal recombination after the ancestors of barley and wheat diverged, 11-15 million years ago (mya). Third, gene 4 underwent direct tandem duplication in a common ancestor of barley and wheat 29-41 mya. All four of the shared genes show the same synonymous substitution rate, but nonsynonymous substitution rates show significant variations between genes 4a and 4b, suggesting that gene 4b was largely released from the strong purifying selection that acts on gene 4a in both barley and wheat. Intergenic retrotransposon blocks, many of them organized as nested insertions, mostly account for the lower gene density of the barley and wheat regions. All but two of the retrotransposons were found in the regions between genes, while all but 2 of the 51 inverted repeat transposable elements were found as insertions in genic regions and outside the retrotransposon blocks.

Orthologous adh regions of the sorghum and maize genomes were sequenced and analyzed. Nine known or candidate genes, including adh1 , were found in a 225-kilobase (kb) maize sequence. In a 78-kb space of sorghum, the nine homologues of the maize genes were identified in a colinear order, plus five additional genes. The major fraction of DNA in maize, occupying 166 kb (74%), is represented by 22 long terminal repeat (LTR) retrotransposons. About 6% of the sequence belongs to 33 miniature inverted-repeat transposable elements (MITEs), remnants of DNA transposons, 4 simple sequence repeats, and low-copy-number DNAs of unknown origin. In contrast, no LTR retroelements were detected in the orthologous sorghum region. The unconserved sorghum DNA is composed of 20 putative MITEs, transposon-like elements, 5 simple sequence repeats, and low-copy-number DNAs of unknown origin. No MITEs were discovered in the 166 kb of DNA occupied by the maize LTR retrotransposons. In both species, MITEs were found in the space between genes and inside introns, indicating specific insertion and/or retention for these elements. Two adjacent sorghum genes, including one gene missing in maize, had colinear homologues on Arabidopsis chromosome IV, suggesting two rearrangements in the sorghum and three in the maize genome in comparison to a four-gene region of Arabidopsis . Hence, multiple small rearrangements may be present even in largely colinear genomic regions. These studies revealed a much higher degree of diversity at a microstructural level than predicted by genetic mapping studies for closely related grass species, as well as for comparisons of monocots and dicots.

Previously, we have demonstrated microcolinearity of gene composition and orientation in sh2/a1-homologous regions of the rice, sorghum, and maize genomes. However, the sh2 and a1 homologues are only about 20 kb apart in both rice and sorghum, while they are separated by about 140 kb in maize. In order to further define sequence organization and conservation in sh2/a1-homologous regions, we have completely sequenced a 42,446-bp segment of sorghum DNA. Four genes were identified: a homologue of sh2, two homologues of a1, and a putative transcriptional regulatory gene. A solo long terminal repeat of the retroelement Leviathan was detected between the two a1 homologues, and eight miniature inverted repeat transposable elements were found in this region. Comparison of the sorghum sequence with the sequence of the homologous segment from rice indicated that only the identified genes were evolutionarily conserved between these two species, which have evolved independently for over 50 million years. The introns of the a1 homologues have evolved faster than the introns of the sh2 homologue. The a1 tandem duplication appears to be an ancient event that may have preceded the ancestral divergence of maize, sorghum, and rice.

Comparative and Functional Genomics Comp Funct Genom 2002; 3: 165–166. Published online 21 March 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / cfg.164 Conference Review Frequent genic rearrangements in two regions of grass genomes identified by comparative sequence analysis Wusirika Ramakrishna 1 *, Jianxin Ma 1 , Phillip SanMiguel 2 , John Emberton 1 , Jorge Dubcovsky 3 , Bryan A Shiloff 4 , Zeyu Jiang 4 , Nils Rostoks 5 , Carlos S Busso 3 , Matthew Ogden 1 , Eric Linton 6 , Andris Kleinhofs 5 , Katrien M Devos 7 , Joachim Messing 6 and Jeffrey L Bennetzen 1 Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA Purdue University Genomics Core, S004 WSLR, Purdue University, West Lafayette, IN 47907, USA Department of Agronomy & Range Science, University of California, Davis, 95616 CA, USA National Center for Genome Resources, Santa Fe, NM 87505, USA Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA PGIR-Waksman Institute, Rutgers University, Piscataway, NJ 08855, USA John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK * Correspondence to: Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA. E-mail: wusirika@purdue.edu Grass genomes show extensive colinearity based on comparative genetic maps, although some large chromosomal rearrangements mark particular lineages. Small rearrangements involving one or a few genes would be missed by these comparative maps. Hence, we have undertaken sequence com- parisons between BACs (bacterial artificial chromo- somes) that contain 80–200 kb genomic segments of several grass species. Rp1 is a complex disease resistance locus in maize that provides race-specific resistance to the leaf rust disease caused by the fungus Puccinia sorghi. Sequence analysis of maize and sorghum Rp1 BACs revealed two Rp1 homologues and twelve other gene-homologous sequences, of which at least ten genes were truncated in one maize segment and eight gene-homologous segments were found in a second maize segment, of which two were Rp1-related and the other six were truncated. The truncated gene segments may have arisen by break repair, probably through homologous or illegitimate recombination. A 43 kb region with an Rp1 homologue, six truncated genes and three Opie retrotransposons was duplicated on two maize BACs. Estimation of divergence times for the Rp1 Copyright # 2002 John Wiley & Sons, Ltd. homologues, the three Opie elements and the inter- vening regions between duplicated regions are consistent with the duplication having occurred within the last 200 000 years. The Retrotransposons Opie-B, Opie-C and Opie-D were inserted before this duplication. In sorghum, the sequenced region includes a cluster of five Rp1 homologues, of which two are truncated with N-terminal deletions. Of the other three Rp1 homologues, a stop codon is present in one and a retrotransposon is inserted in another. The Rp1-homologous region in sorghum has several genes that are either duplicated, inverted, or both. Five duplicated genes other than the Rp1 homologues are present. Physical mapping revealed the presence of eleven Rp1 homologues that mapped to about 400 kb in B73 maize. Six Rp1 homologues mapped to about 50 kb in sorghum inbred BTr623 to a region on linkage group H that is colinear with maize chromosome 10. The Waxy1 gene encodes UDP-glucose starch glycosyl transferase, an enzyme that converts amy- lose to amylopectin. Wx1 is in syntenic locations in all of these grass species, although the maize Wx1 is within a paracentric inversion that places it near centromeric heterochromatin. Sequence analysis of

Background Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. Results A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the Sal I MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. Conclusion MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely understood at this time, MSLL technology flags both approximate boundaries and methylated genes that deserve additional investigation. MSLL and HMPR sequences provide a valuable resource for maize genome annotation, and are a uniquely valuable complement to any plant genome sequencing project. In order to make these results fully accessible to the community, a web display was developed that shows the alignment of MSLL, HMPR, and other gene-rich sequences to the BACs; this display is continually updated with the latest ESTs and BAC sequences.

Mycoplasma haemofelis is a mycoplasmal pathogen (hemoplasma) that attaches to the host's erythrocytes. Distributed worldwide, it has a significant impact on the health of cats causing acute disease and, despite treatment, establishing chronic infection. It might also have a role as a zoonotic agent, especially in immunocompromised patients. Whole genome sequencing and analyses of M. haemofelis strain Ohio2 was undertaken as a step toward understanding its survival and persistence. Metabolic pathways are reduced, relying on the host to supply many of the nutrients and metabolites needed for survival. M. haemofelis must import glucose for ATP generation and ribose derivates for RNA/DNA synthesis. Hypoxanthine, adenine, guanine, uracil and CMP are scavenged from the environment to support purine and pyrimidine synthesis. In addition, nicotinamide, amino acids and any vitamins needed for growth, must be acquired from its environment. The core proteome of M. haemofelis contains an abundance of paralogous gene families, corresponding to 70.6% of all the CDSs. This "paralog pool" is a rich source of different antigenic epitopes that can be varied to elude the host's immune system and establish chronic infection. M. haemofelis also appears to be capable of phase variation, which is particularly relevant to the cyclic bacteremia and persistence, characteristics of the infection in the cat. The data generated herein should be of great use for understanding the mechanisms of M. haemofelis infection. Further, it will provide new insights into its pathogenicity and clues needed to formulate media to support the in vitro cultivation of M. haemofelis.