The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2.

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.

Mycoplasma haemocanis--the canine hemoplasma and its feline counterpart in the genomic era.

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.

Complete genome sequence of Mycoplasma wenyonii strain Massachusetts.

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.

Complete genome sequence of Mycoplasma haemocanis strain Illinois.

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.

Genome of Mycoplasma haemofelis, unraveling its strategies for survival and persistence.

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.

Exceptional lability of a genomic complex in rice and its close relatives revealed by interspecific and intraspecific comparison and population analysis.

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.

Exceptional diversity, non-random distribution, and rapid evolution of retroelements in the B73 maize genome.

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 approximately 35,000 copies, respectively, or a combined approximately 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.

Evolutionary dynamics of an ancient retrotransposon family provides insights into evolution of genome size in the genus Oryza.

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 complex history of rearrangement in an orthologous region of the maize, sorghum, and rice genomes.

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.

High-Cot sequence analysis of the maize genome.

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.

Methylation-spanning linker libraries link gene-rich regions and identify epigenetic boundaries in Zea mays.

Complex cereal genomes are largely composed of small gene-rich regions intermixed with 5 kb to 200 kb blocks of repetitive DNA. The repetitive DNA blocks are usually 5-methylated at 5'-CG-3' and 5'-CNG-3' cytosines in most or all adult tissues, while the genes are generally unmethylated at these sites. We have developed methylation-spanning linker library (MSLL) technology as a tool to span large methylated DNA blocks and thereby link unmethylated genic regions. MSLL clones contain insertions of large fragments that are size fractionated over gels after complete digestion of total genomic DNA with restriction enzymes that are sensitive to the 5-methylation of cytosines in 5'-CG-3' and 5'-CNG-3' sequences. Our data indicate that the end sequences of maize MSLL clones are greatly depleted in repetitive DNAs and enriched in genes relative to total genomic DNA. Combined with other gene-enrichment approaches, MSLL technology can efficiently generate fully-linked contiguous sequences in complex genomes that are resistant to shotgun sequencing.

Genomic sequencing reveals gene content, genomic organization, and recombination relationships in barley.

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.

Transposable elements, genes and recombination in a 215-kb contig from wheat chromosome 5A(m).

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 orientation in wheat and rice but inverted in barley. Gene density 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 retrotransposons were less than 5 million years ago and were consistent with their nested structure. Five new families of retro-elements 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 dinucleotides was observed in the comparison of introns, exons and retrotransposons. A higher proportion of transitions 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 sequence elements, but with general patterns of content and interspersion that are similar to those seen in maize and barley.