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International audience; Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G -> A (C -> T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP.

Repetitive sequences, primarily transposable elements form an indispensable part of eukaryotic genomes. However, little is known about how these sequences originate, evolve and function in context of a genome. In an attempt to address this question, we performed a comparative analysis of repetitive DNA sequences in the genus Oryza, representing ~15 million years of evolution. Both Class I and Class II transposable elements, through their expansion, loss and movement in the genome, were found to influence genome size variation in this genus. We identified 38 LTRretrotransposon families that are present in 1,500 or more copies throughout Oryza, and many are preferentially amplified in specific lineages. The data presented here, besides furthering our understanding of genome organization in the genus Oryza, will aid in the assembly, annotation and analysis of genomic data, as part of the future genome sequencing projects of O. sativa wild relatives.

Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3-gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo-LTRs to full-length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3-associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement.

Long terminal repeat (LTR) retrotransposons constitute a significant portion of most eukaryote genomes and can dramatically change genome size and organization. Although LTR retrotransposon content variation is well documented, the dynamics of genomic flux caused by their activity are poorly understood on an evolutionary time scale. This is primarily because of the lack of an experimental system composed of closely related species whose divergence times are within the limits of the ability to detect ancestrally related retrotransposons. The genus Oryza, with 24 species, ten genome types, different ploidy levels and over threefold genome size variation, constitutes an ideal experimental system to explore genus-level transposon dynamics. Here we present data on the discovery and characterization of an LTR retrotransposon family named RWG in the genus Oryza. Comparative analysis of transposon content (approximately 20 to 27,000 copies) and transpositional history of this family across the genus revealed a broad spectrum of independent and lineage-specific changes that have implications for the evolution of genome size and organization. In particular, we provide evidence that the basal GG genome of Oryza (O. granulata) has expanded by nearly 25% by a burst of the RWG lineage Gran3 subsequent to speciation. Finally we describe the recent evolutionary origin of Dasheng, a large retrotransposon derivative of the RWG family, specifically found in the A, B and C genome lineages of Oryza.

A new technology was developed to assist gene-enrichment sequencing of any complex plant genome, employing maize as the test organism. Hypomethylated partial restriction (HMPR) libraries were constructed by using independent partial digestions with methylation-sensitive restriction enzymes HpaII (5′-CCGG-3′) and HpyCH4IV (5′-ACGT-3′). Fragments of 1-4 kb were purified and cloned, followed by sequence analysis of >2000 clones from 10 separate libraries. Organellar clones comprised ∼10% of each library but were useful in showing that no chimeric clones were generated and that digestion efficiencies were 10%-25% in different libraries. Four separate HMPR libraries, analyzed in detail, exhibited very similar degrees of gene enrichment and repeat depletion. Known gene homologies were found in ∼25% of the HMPR clones, compared with 70% for unfiltered maize sequences) and in providing an unmatched enrichment for the “unknown” sequences that contain promoters, introns, and other gene-adjacent regions.

The wild species of the genus Oryza offer enormous potential to make a significant impact on agricultural productivity of the cultivated rice species Oryza sativa and Oryza glaberrima. To unlock the genetic potential of wild rice we have initiated a project entitled the 'Oryza Map Alignment Project' (OMAP) with the ultimate goal of constructing and aligning BAC/STC based physical maps of 11 wild and one cultivated rice species to the International Rice Genome Sequencing Project's finished reference genome--O. sativa ssp. japonica c. v. Nipponbare. The 11 wild rice species comprise nine different genome types and include six diploid genomes (AA, BB, CC, EE, FF and GG) and four tetrapliod genomes (BBCC, CCDD, HHKK and HHJJ) with broad geographical distribution and ecological adaptation. In this paper we describe our strategy to construct robust physical maps of all 12 rice species with an emphasis on the AA diploid O. nivara--thought to be the progenitor of modern cultivated rice.

Little is known about the physical makeup of heterochromatin in the soybean (Glycine max L. Merr.) genome. Using DNA sequencing and molecular cytogenetics, an initial analysis of the repetitive fraction of the soybean genome is presented. BAC 076J21, derived from linkage group L, has sequences conserved in the pericentromeric heterochromatin of all 20 chromosomes. FISH analysis of this BAC and three subclones on pachytene chromosomes revealed relatively strict partitioning of the heterochromatic and euchromatic regions. Sequence analysis showed that this BAC consists primarily of repetitive sequences such as a 102-bp tandem repeat with sequence identity to a previously characterized ∼120-bp repeat (STR120). Fragments of Calypso-like retroelements, a recently inserted SIRE1 element, and a SIRE1 solo LTR were present within this BAC. Some of these sequences are methylated and are not conserved outside of G. max and G. soja, a close relative of soybean, except for STR102, which hybridized to a restriction fragment from G. latifolia. These data present a picture of the repetitive fraction of the soybean genome that is highly concentrated in the pericentromeric regions, consisting of rapidly evolving tandem repeats with interspersed retroelements.

Higher eukaryotic genomes, including those from plants, contain large amounts of repetitive DNA that complicate genome analysis. We have developed a technique based on DNA renaturation which normalizes repetitive DNA, and thereby allows a more efficient outcome for full genome shotgun sequencing. The data indicate that sequencing the unrenatured outcome of a Cot experiment, otherwise known as High-Cot DNA, enriches genic sequences by more than fourfold in maize, from 5% for a random library to more than 20% for a High-Cot library. Using this approach, we predict that gene discovery would be greater than 95% and that the number of sequencing runs required to sequence the full gene space in maize would be at least fourfold lower than that required for full-genome shotgun sequencing.

The finding in animal species of complexes homologous to the products of six Saccharomyces cerevisiae genes, origin of replication recognition complex (ORC), has suggested that ORC-related mechanisms have been conserved in all eukaryotes. In plants, however, the only cloned putative homologs of ORC subunits are the Arabidopsis ORC2 and the rice ORC1. Homologs of other subunits of plant origin have not been cloned and characterized. A striking observation was the absence from the Arabidopsis genome of an obvious candidate gene-homolog of ORC4. This fact raised compelling questions of whether plants, in general, and Arabidopsis, in particular, may have lost the ORC4 gene, whether ORC-homologous subunits function within a complex in plants, whether an ORC complex may form and function without an ORC4 subunit, whether a functional (but not sequence) protein homolog may have taken up the role of ORC4 in Arabidopsis, and whether lack of ORC4 is a plant feature, in general. Here, we report the first cloned and molecularly characterized five genes coding for the maize putative homologs of ORC subunits ZmORC1, ZmORC2, ZmORC3, ZmORC4 and ZmORC5. Their expression profiles in tissues with different cell-dividing activities are compatible with a role in DNA replication. Based on the potential of ORC-homologous maize proteins to bind each other in yeast, we propose a model for their possible assembly within a maize ORC. The isolation and molecular characterization of an ORC4-homologous gene from maize argues that, in its evolution, Arabidopsis may have lost the homologous ORC4 gene.

Genes from the SNF2 family play important roles in transcriptional regulation, maintenance of chromosome integrity and DNA repair. This study describes the molecular cloning and characterization of cereal genes from this family. The predicted proteins exhibit a novel C-terminal domain that defines a new subfamily designated SNF2P that includes human and mouse proteins. Comparison between genomic and cDNA sequences showed that cereal Snf2P genes consisted of 17 exons, including one only 8 bp long. Two barley alleles differed by the presence of a 7.7-kb non-LTR retrotransposon in intron 6. An alternative annotation of the orthologous Arabidopsis gene would improve its similarity with the other members of the subfamily. Intron 2 was not spliced out in approximately half of the rice Snf2P mRNAs present in leaves, resulting in a premature stop codon. Transcripts from the barley and wheat Snf2P genes were found in apexes, leaves, sheaths, roots and spikes. The Snf2P genes exist as single copies on wheat chromosome arm 5AmL and in the colinear regions on barley chromosome arm 4HL and rice chromosome 3. High-density genetic mapping and RT-PCR suggest that Snf2P is not a candidate gene for the tightly linked vernalization gene Vrn2. Fil: Yan, L.. University of California; Estados Unidos Fil: Echenique, Carmen Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; Argentina Fil: Busso, C.. Purdue University; Estados Unidos Fil: SanMiguel, P.. Purdue University; Estados Unidos Fil: Ramakrishna, W.. Purdue University; Estados Unidos Fil: Bennetzen, J.. Purdue University; Estados Unidos Fil: Harrington, S.. Cornell University; Estados Unidos Fil: Dubcovsky, J.. University of California; Estados Unidos

Rp1 is a complex disease resistance locus in maize that is exceptional in both allelic variability and meiotic instability. Genomic sequence analysis of three maize BACs from the Rp1 region of the B73 inbred line revealed 4 Rp1 homologs and 18 other gene-homologous sequences, of which at least 16 are truncated. Thirteen of the truncated genes are found in three clusters, suggesting that they arose from multiple illegitimate break repairs at the same sites or from complex repairs of each of these sites with multiple unlinked DNA templates. A 43-kb region that contains an Rp1 homolog, six truncated genes, and three Opie retrotransposons was found to be duplicated in this region. This duplication is relatively recent, occurring after the insertion of the three Opie elements. The breakpoints of the duplication are outside of any genes or identified repeat sequence, suggesting a duplication mechanism that did not involve unequal recombination. A physical map and partial sequencing of the Rp1 complex indicate the presence of 15 Rp1 homologs in regions of approximately 250 and 300 kb in the B73 inbred line. Comparison of fully sequenced Rp1-homologous sequences in the region demonstrates a history of unequal recombination and diversifying selection within the Leu-rich repeat 2 region, resulting in chimeric gene structures.

Sequencing of a contiguous 215-kb interval of Triticum monococcum showed the presence of five genes in the same order as in previously sequenced colinear barley and rice BACs. Gene 2 was in the same orienta- tion in wheat and rice but inverted in barley. Gene densi- ty in this region was 1 gene per 43 kb and the ratio of physical to genetic distance was estimated to be 2,700 kb cM -1 . Twenty more-or-less intact retrotransposons were found in the intergenic regions, covering at least 70% of the sequenced region. The insertion times of 11 retro- transposons were less than 5 million years ago and were consistent with their nested structure. Five new families of retroelements and the first full-length elements for two additional retrotransposon families were discovered in this region. Significantly higher values of GC content were observed for Triticeae BACs compared with rice BACs. Relative enrichment or depletion of certain dinu- cleotides was observed in the comparison of introns, ex- ons and retrotransposons. A higher proportion of transi- tions in CG and CNG sites that are targets for cytosine methylation was observed in retrotransposons (76%) than in introns (37%). These results showed that the wheat genome is a complex mixture of different se- quence elements, but with general patterns of content and interspersion that are similar to those seen in maize and barley.

Colinearity of a large region from barley (Hordeum vulgare) chromosome 5H and rice (Oryza sativa) chromosome 3 has been demonstrated by mapping of several common restriction fragment-length polymorphism clones on both regions. One of these clones, WG644, was hybridized to rice and barley bacterial artificial chromosome (BAC) libraries to select homologous clones. One BAC from each species with the largest overlapping segment was selected by fingerprinting and blot hybridization with three additional restriction fragment-length polymorphism clones. The complete barley BAC 635P2 and a 50-kb segment of the rice BAC 36I5 were completely sequenced. A comparison of the rice and barley DNA sequences revealed the presence of four conserved regions, containing four predicted genes. The four genes are in the same orientation in rice, but the second gene is in inverted orientation in barley. The fourth gene is duplicated in tandem in barley but not in rice. Comparison of the homeologous barley and rice sequences assisted the gene identification process and helped determine individual gene structures. General gene structure (exon number, size, and location) was largely conserved between rice and barley and to a lesser extent with homologous genes in Arabidopsis. Colinearity of these four genes is not conserved in Arabidopsis compared with the two grass species. Extensive similarity was not found between the rice and barley sequences other than within the exons of the structural genes, and short stretches of homology in the promoters and 3′ untranslated regions. The larger distances between the first three genes in barley compared with rice are explained by the insertion of different transposable retroelements.

Estimates of the size and copy number of the retrotransposons in a 240 kb region flanking theadh1gene of maize suggest that 33–62% of the maize genome is composed of the high copy-number retrotransposons found in this region. An additional 16% of the maize genome is estimated to be composed of middle and low copy-number retrotransposons. The sorghum genome, which is more than three-fold smaller than the maize genome, does not have any detected copies of the maize retrotransposons in a region orthologous to that of maizeadh1. Thus, it appears that retrotransposons have increased the size of the maize genome two- to five-fold since the divergence of maize and sorghum from a common ancestor about 16 million years ago.

Large regions of genomic colinearity have been demonstrated among grass species by recombinational mapping, but the degree of chromosomal conservation at the sub-centimorgan level has not been extensively investigated. We cloned the rice and sorghum genes homologous to the sh2 locus of maize on bacterial artificial chromosomes (BACs), and observed that a homologue of the maize a1 gene was also present on each of these BACs. In sorghum, we found a direct duplication of a1 homologues separated by about 10 kb. In maize, sh2 and a1 are approximately 140 kb apart and transcribed in the same direction, with sh2 upstream of a1 . In rice and sorghum, this arrangement is fully conserved. However, the sh2 and a1 homologues are separated by about 19 kb in both rice and sorghum. We found low-copy-number and repetitive DNAs between the sh2 and a1 homologues of sorghum and rice. The sh2 and a1 homologues cross-hybridized, but the repetitive DNA and most low-copy-number sequences between these genes did not. These results indicate that maize, sorghum, and rice have conserved gene order and composition in the sh2 - a1 region, but have acquired extensive qualitative and quantitative differences in the sequences between these genes.

We provide evidence for the location of matrix attachment sites along a contiguous region of 280 kb on maize chromosome 1. We define nine potential loops that vary in length from 6 kb to > 75 kb. The distribution of the different classes of DNA within this continuum with respect to the predicted structural loops reveals an interesting correlation: the long stretches of mixed classes of highly repetitive DNAs are often segregated into topologically sequestered units, whereas low-copy-number DNAs (including the alcohol dehydrogenase1 [adh1] gene) are positioned in separate loops. Contrary to expectations, several classes of highly repeated elements with representatives in this region were found to be transcribed, and some of these exhibited tissue-specific patterns of expression.

Mycoplasma haemocanis is a hemotrophic mycoplasma (hemoplasma), blood pathogen that may cause acute disease in immunosuppressed or splenectomized dogs. The genome of the strain Illinois, isolated from blood of a naturally infected dog, has been entirely sequenced and annotated to gain a better understanding of the biology of M. haemocanis. Its single circular chromosome has 919 992 bp and a low G + C content (35%), representing a typical mycoplasmal genome. A gene-by-gene comparison against its feline counterpart, M. haemofelis, reveals a very similar composition and architecture with most of the genes having conserved synteny extending over their entire chromosomes and differing only by a small set of unique protein coding sequences. As in M. haemofelis, M. haemocanis metabolic pathways are reduced and apparently rely heavily on the nutrients afforded by its host environment. The presence of a major percentage of its genome dedicated to paralogous genes (63.7%) suggests that this bacterium might use antigenic variation as a mechanism to evade the host’s immune system as also observed in M. haemofelis genome. Phylogenomic comparisons based on average nucleotide identity (ANI) and tetranucleotide signature suggest that these two pathogens are different species of mycoplasmas, with M. haemocanis infecting dogs and M. haemofelis infecting cats.

Mycoplasma wenyonii is a hemotrophic mycoplasma that causes acute and chronic infections in cattle. Here, we announce the first complete genome sequence of this organism. The genome is a single circular chromosome with 650,228 bp and G+C% of 33.9. Analyses of M. wenyonii genome will provide insights into its biology.

Mycoplasma haemocanis is a blood pathogen that may cause acute disease in immunosuppressed or splenectomized dogs. The genome of the strain Illinois is a single circular chromosome with 919,992 bp and a GC content of 35%. Analyses of the M. haemocanis genome will provide insights into its biology and in vitro cultivation requirements.

We have characterized the copy number, organization, and genomic modification of DNA sequences within and flanking several maize genes. We found that highly repetitive DNA sequences were tightly linked to most of these genes. The highly repetitive sequences were not found within the coding regions but could be found within 6 kb either 3′ or 5′ to the structural genes. These highly repetitive regions were each composed of unique combinations of different short repetitive sequences. Highly repetitive DNA blocks were not interrupted by any detected single copy DNA. The 13 classes of highly repetitive DNA identified were found to vary little between diverse Zea isolates. The level of DNA methylation in and near these genes was determined by scoring the digestibility of 63 recognition/cleavage sites with restriction enzymes that were sensitive to 5-methylation of cytosines in the sequences 5′-CG-3′ and 5′-CNG-3′. All but four of these sites were digestible in chromosomal DNA. The four undigested sites were localized to extragenic DNA within or near highly repetitive DNA, while the other 59 sites were in low copy number DNAs. Pulsed field gel analysis indicated that the majority of cytosine modified tracts range from 20 to 200 kb in size. Single copy sequences hybridized to the unmodified domains, while highly repetitive sequences hybridized to the modified regions. Middle repetitive sequences were found in both domains.Key words: genome organization, interspersed repetitive DNA, DNA modification.

Since the first fungal genome was sequenced in 1996, sequencing technologies have advanced dramatically. In recent years, it has become possible to cost-effectively generate vast amounts of DNA sequence data using a number of cell- and electrophoresis-free sequencing technologies, commonly known as "next" or "second" generation. In this chapter, we present a brief overview of next-generation sequencers that are commercially available now. Their potential applications in fungal genomics studies are discussed.

Background Extensive DNA rearrangement of genic colinearity, as revealed by comparison of orthologous genomic regions, has been shown to be a general concept describing evolutionary dynamics of plant genomes. However, the nature, timing, lineages and adaptation of local genomic rearrangement in closely related species (e.g., within a genus) and haplotype variation of genomic rearrangement within populations have not been well documented. Results We previously identified a hotspot for genic rearrangement and transposon accumulation in the Orp region of Asian rice (Oryza sativa, AA) by comparison with its orthologous region in sorghum. Here, we report the comparative analysis of this region with its orthologous regions in the wild progenitor species (O. nivara, AA) of Asian rice and African rice (O. glaberrima) using the BB genome Oryza species (O. punctata) as an outgroup, and investigation of transposon insertion sites and a segmental inversion event in the AA genomes at the population level. We found that Orp region was primarily and recently expanded in the Asian rice species O. sativa and O. nivara. LTR-retrotransposons shared by the three AA-genomic regions have been fixed in all the 94 varieties that represent different populations of the AA-genome species/subspecies, indicating their adaptive role in genome differentiation. However, LTR-retrotransposons unique to either O. nivara or O. sativa regions exhibited dramatic haplotype variation regarding their presence or absence between or within populations/subpopulations. Conclusions The LTR-retrotransposon insertion hotspot in the Orp region was formed recently, independently and concurrently in different AA-genome species, and that the genic rearrangements detected in different species appear to be differentially triggered by transposable elements. This region is located near the end of the short arm of chromosome 8 and contains a high proportion of LTR-retrotransposons similar to observed in the centromeric region of this same chromosome, and thus may represent a genomic region that has recently switched from euchromatic to heterochromatic states. The haplotype variation of LTR-retrotransposon insertions within this region reveals substantial admixture among various subpopulations as established by molecular markers at the whole genome level, and can be used to develop retrotransposon junction markers for simple and rapid classification of O. sativa germplasm.

Since the first fungal genome was sequenced in 1996, sequencing technologies have advanced dramatically. In recent years, it has become possible to cost-effectively generate vast amounts of DNA sequence data using a number of cell- and electrophoresis-free sequencing technologies, commonly known as "next" or "second" generation. In this chapter, we present a brief overview of next-generation sequencers that are commercially available now. Their potential applications in fungal genomics studies are discussed.

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 ).

Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3-gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo-LTRs to full-length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3-associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement.

Recent comprehensive sequence analysis of the maize genome now permits detailed discovery and description of all transposable elements (TEs) in this complex nuclear environment. Reiteratively optimized structural and homology criteria were used in the computer-assisted search for retroelements, TEs that transpose by reverse transcription of an RNA intermediate, with the final results verified by manual inspection. Retroelements were found to occupy the majority (>75%) of the nuclear genome in maize inbred B73. Unprecedented genetic diversity was discovered in the long terminal repeat (LTR) retrotransposon class of retroelements, with >400 families (>350 newly discovered) contributing >31,000 intact elements. The two other classes of retroelements, SINEs (four families) and LINEs (at least 30 families), were observed to contribute 1,991 and ∼35,000 copies, respectively, or a combined ∼1% of the B73 nuclear genome. With regard to fully intact elements, median copy numbers for all retroelement families in maize was 2 because >250 LTR retrotransposon families contained only one or two intact members that could be detected in the B73 draft sequence. The majority, perhaps all, of the investigated retroelement families exhibited non-random dispersal across the maize genome, with LINEs, SINEs, and many low-copy-number LTR retrotransposons exhibiting a bias for accumulation in gene-rich regions. In contrast, most (but not all) medium- and high-copy-number LTR retrotransposons were found to preferentially accumulate in gene-poor regions like pericentromeric heterochromatin, while a few high-copy-number families exhibited the opposite bias. Regions of the genome with the highest LTR retrotransposon density contained the lowest LTR retrotransposon diversity. These results indicate that the maize genome provides a great number of different niches for the survival and procreation of a great variety of retroelements that have evolved to differentially occupy and exploit this genomic diversity., Author Summary Although TEs are a major component of all studied plant genomes, and are the most significant contributors to genome structure and evolution in almost all eukaryotes that have been investigated, their properties and reasons for existence are not well understood in any eukaryotic genome. In order to begin a comprehensive study of TE contributions to the structure, function, and evolution of both genes and genomes, we first identified all of the TEs in maize and then investigated whether there were non-random patterns in their dispersal. We used homology and TE structure criteria in an effort to discover all of the retroelements in the recently sequenced genome from maize inbred B73. We found that the retroelements are incredibly diverse in maize, with many hundreds of families that show different insertion and/or retention specificities across the maize chromosomes. Most of these element families are present in low copy numbers and had been missed by previous searches that relied on a high-copy-number criterion. Different element families exhibited very different biases for accumulation across the chromosomes, indicating that they can detect and utilize many different chromatin environments.

Bacteriophage PhiV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the PhiV10 genome is 39 104 bp long and contains 55 predicted genes. PhiV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage epsilon15 and a prophage from E. coli APEC O1. The attachment site of PhiV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. PhiV10 encodes an O-acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block PhiV10 superinfection, indicating that the O157 antigen is most likely the PhiV10 receptor.

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.

Retrotransposons, transposable elements related to animal retroviruses, are found in all eukaryotes investigated and make up the majority of many plant genomes. Their ubiquity points to their importance, especially in their contribution to the large-scale structure of complex genomes. The nature and frequency of retro-element appearance, activation and amplification are poorly understood in all higher eukaryotes. Here we employ a novel approach to determine the insertion dates for 17 of 23 retrotransposons found near the maize adh1 gene, and two others from unlinked sites in the maize genome, by comparison of long terminal repeat (LTR) divergences with the sequence divergence between adh1 in maize and sorghum. All retrotransposons examined have inserted within the last six million years, most in the last three million years. The structure of the adh1 region appears to be standard relative to the other gene-containing regions of the maize genome, thus suggesting that retrotransposon insertions have increased the size of the maize genome from approximately 1200 Mb to 2400 Mb in the last three million years. Furthermore, the results indicate an increased mutation rate in retrotransposons compared with genes.

Milo disease in sorghum is caused by isolates of the soil-borne fungus Periconia circinata that produce PC-toxin. Susceptibility to milo disease is conditioned by a single, semi-dominant gene, termed Pc. The susceptible allele (Pc) converts to a resistant form (pc) spontaneously at a gametic frequency of 10(-3) to 10(-4). A high-density genetic map was constructed around the Pc locus using DNA markers, allowing the Pc gene to be delimited to a 0.9 cM region on the short arm of sorghum chromosome 9. Physically, the Pc-region was covered by a single BAC clone. Sequence analysis of this BAC revealed twelve gene candidates. Several of the predicted genes in the region are homologous to disease resistance loci, including one NBS-LRR resistance gene analogue that is present in multiple tandem copies. Analysis of pc isolines derived from Pc/Pc sorghum suggests that one or more members of this NBS-LRR gene family are the Pc genes that condition susceptibility.

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.

Maize is a staple crop of the grass family and also an excellent model for plant genetics. Owing to the large size and repetitiveness of its genome, we previously investigated two approaches to accelerate gene discovery and genome analysis in maize: methylation filtration and high C(0)t selection. These techniques allow the construction of gene-enriched genomic libraries by minimizing repeat sequences due to either their methylation status or their copy number, yielding a 7-fold enrichment in genic sequences relative to a random genomic library. Approximately 900,000 gene-enriched reads from maize were generated and clustered into Assembled Zea mays (AZM) sequences. Here we report the current AZM release, which consists of approximately 298 Mb representing 243,807 sequence assemblies and singletons. In order to provide a repository of publicly available maize genomic sequences, we have created the TIGR Maize Database (http://maize.tigr.org). In this resource, we have assembled and annotated the AZMs and used available sequenced markers to anchor AZMs to maize chromosomes. We have constructed a maize repeat database and generated draft sequence assemblies of 287 maize bacterial artificial chromosome (BAC) clone sequences, which we annotated along with 172 additional publicly available BAC clones. All sequences, assemblies and annotations are available at the project website via web interfaces and FTP downloads.

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.

Plants with a winter growth habit flower earlier when exposed for several weeks to cold temperatures, a process called vernalization. We report here the positional cloning of the wheat vernalization gene VRN2, a dominant repressor of flowering that is down-regulated by vernalization. Loss of function of VRN2, whether by natural mutations or deletions, resulted in spring lines, which do not require vernalization to flower. Reduction of the RNA level of VRN2 by RNA interference accelerated the flowering time of transgenic winter-wheat plants by more than a month. Fil: Yan, Liuling. University of California; Estados Unidos Fil: Loukoianov, Artem. University of California; Estados Unidos Fil: Blechl, Ann. United States Department of Agriculture; Estados Unidos Fil: Tranquilli, Gabriela Edith. University of California; Estados Unidos Fil: Ramakrishna, Wusirika. Michigan Technological University; Estados Unidos Fil: San Miguel, Phillip. Purdue University; Estados Unidos Fil: Bennetzen, Jeffrey L.. University of Georgia; Estados Unidos Fil: Echenique, Carmen Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina Fil: Dubcovsky, Jorge. University of California; Estados Unidos

Approximately 80% of the maize genome comprises highly repetitive sequences interspersed with single-copy, gene-rich sequences, and standard genome sequencing strategies are not readily adaptable to this type of genome. Methodologies that enrich for genic sequences might more rapidly generate useful results from complex genomes. Equivalent numbers of clones from maize selected by techniques called methylation filtering and High C 0 t selection were sequenced to generate ∼200,000 reads (approximately 132 megabases), which were assembled into contigs. Combination of the two techniques resulted in a sixfold reduction in the effective genome size and a fourfold increase in the gene identification rate in comparison to a nonenriched library.

A maize cDNA clone was isolated by virtue of its intense hybridization to total maize genomic DNA, indicating homology to highly repetitive sequences. Genomic homologues were identified and subcloned from an adh1-bearing maize yeast artificial chromosome (YAC). Sequencing revealed that the expressed sequence was part of a Ty3-gypsy-type retrotransposon. We discovered and sequenced two complete retrotransposons of this family, and named them Cinful elements because they are members of a family of maize retrotransposons including Zeon-1 and the first plant transposable element sequenced, the solo long terminal repeat (LTR) called Cin1. All are defective, as Cinful-1 and Cinful-2 elements lack gag and Zeon-1 lacks pol homology. Despite the apparent lack of an intact "autonomous" element, the Cinful family has expanded to a copy number of about 18 000, representing just under 9% of the maize genome. Both point mutations and major rearrangements, including possible gene acquisition, differentiate members of the Cinful family. Cinful family members were found to have an unusual feature that we also observed in two other Ty3-class retrotransposons of teosinte and tobacco: related tandem repeats that separate their internal domains with a gag- or pol-containing homology from a 3' segment of unknown function. The conserved and variable features identified provide insights into the origin, mutational history, and functional components of this major constituent of the maize genome.Key words: Cin1, genome evolution, sequence divergence, Ty3-gypsy elements, Zeon-1.

The sequences of large insert clones containing genomic DNA that is orthologous to the maize adh1 region were obtained for sorghum, rice, and the adh1 -homoeologous region of maize, a remnant of the tetraploid history of the Zea lineage. By using all four genomes, it was possible to describe the nature, timing, and lineages of most of the genic rearrangements that have differentiated this chromosome segment over the last 60 million years. The rice genome has been the most stable, sharing 11 orthologous genes with sorghum and exhibiting only one tandem duplication of a gene in this region. The lineage that gave rise to sorghum and maize acquired a two-gene insertion (containing the adh locus), whereas sorghum received two additional gene insertions after its divergence from a common ancestor with maize. The two homoeologous regions of maize have been particularly unstable, with complete or partial deletion of three genes from one segment and four genes from the other segment. As a result, the region now contains only one duplicated locus compared with the eight original loci that were present in each diploid progenitor. Deletion of these maize genes did not remove both copies of any locus. This study suggests that grass genomes are generally unstable in local genome organization and gene content, but that some lineages are much more unstable than others. Maize, probably because of its polyploid origin, has exhibited extensive gene loss so that it is now approaching a diploid state.

A 268-kb chromosomal segment containing sorghum (Sorghum bicolor) genes that are orthologous to the maize (Zea mays) Rp1 disease resistance (R) gene complex was sequenced. A region of approximately 27 kb in sorghum was found to contain five Rp1 homologs, but most have structures indicating that they are not functional. In contrast, maize inbred B73 has 15 Rp1 homologs in two nearby clusters of 250 and 300 kb. As at maize Rp1, the cluster of R gene homologs is interrupted by the presence of several genes that appear to have no resistance role, but these genes were different from the ones found within the maize Rp1complex. More than 200 kb of DNA downstream from the sorghumRp1-orthologous R gene cluster was sequenced and found to contain many duplicated and/or truncated genes. None of the duplications currently exist as simple tandem events, suggesting that numerous rearrangements were required to generate the current genomic structure. Four truncated genes were observed, including one gene that appears to have both 5′ and 3′ deletions. The maize Rp1region is also unusually enriched in truncated genes. Hence, the orthologous maize and sorghum regions share numerous structural features, but all involve events that occurred independently in each species. The data suggest that complex R gene clusters are unusually prone to frequent internal and adjacent chromosomal rearrangements of several types.

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.

Barley (Hordeum vulgare L.) is one of the most important large-genome cereals with extensive genetic resources available in the public sector. Studies of genome organization in barley have been limited primarily to genetic markers and sparse sequence data. Here we report sequence analysis of 417.5 kb DNA from four BAC clones from different genomic locations. Sequences were analyzed with respect to gene content, the arrangement of repetitive sequences and the relationship of gene density to recombination frequencies. Gene densities ranged from 1 gene per 12 kb to 1 gene per 103 kb with an average of 1 gene per 21 kb. In general, genes were organized into islands separated by large blocks of nested retrotransposons. Single genes in apparent isolation were also found. Genes occupied 11% of the total sequence, LTR retrotransposons and other repeated elements accounted for 51.9% and the remaining 37.1% could not be annotated.

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.

For the most part, studies of grass genome structure have been limited to the generation of whole-genome genetic maps or the fine structure and sequence analysis of single genes or gene clusters. We have investigated large contiguous segments of the genomes of maize, sorghum, and rice, primarily focusing on intergenic spaces. Our data indicate that much (>50%) of the maize genome is composed of interspersed repetitive DNAs, primarily nested retrotransposons that insert between genes. These retroelements are less abundant in smaller genome plants, including rice and sorghum. Although 5- to 200-kb blocks of methylated, presumably heterochromatic, retrotransposons flank most maize genes, rice and sorghum genes are often adjacent. Similar genes are commonly found in the same relative chromosomal locations and orientations in each of these three species, although there are numerous exceptions to this collinearity (i.e., rearrangements) that can be detected at the levels of both the recombinational map and cloned DNA. Evolutionarily conserved sequences are largely confined to genes and their regulatory elements. Our results indicate that a knowledge of grass genome structure will be a useful tool for gene discovery and isolation, but the general rules and biological significance of grass genome organization remain to be determined. Moreover, the nature and frequency of exceptions to the general patterns of grass genome structure and collinearity are still largely unknown and will require extensive further investigation.