Young, intact and nested retrotransposons are abundant in the onion and asparagus genomes.

BACKGROUND AND AIMS: Although monocotyledonous plants comprise one of the two major groups of angiosperms and include >65 000 species, comprehensive genome analysis has been focused mainly on the Poaceae (grass) family. Due to this bias, most of the conclusions that have been drawn for monocot genome evolution are based on grasses. It is not known whether these conclusions apply to many other monocots. METHODS: To extend our understanding of genome evolution in the monocots, Asparagales genomic sequence data were acquired and the structural properties of asparagus and onion genomes were analysed. Specifically, several available onion and asparagus bacterial artificial chromosomes (BACs) with contig sizes >35 kb were annotated and analysed, with a particular focus on the characterization of long terminal repeat (LTR) retrotransposons. KEY RESULTS: The results reveal that LTR retrotransposons are the major components of the onion and garden asparagus genomes. These elements are mostly intact (i.e. with two LTRs), have mainly inserted within the past 6 million years and are piled up into nested structures. Analysis of shotgun genomic sequence data and the observation of two copies for some transposable elements (TEs) in annotated BACs indicates that some families have become particularly abundant, as high as 4-5 % (asparagus) or 3-4 % (onion) of the genome for the most abundant families, as also seen in large grass genomes such as wheat and maize. CONCLUSIONS: Although previous annotations of contiguous genomic sequences have suggested that LTR retrotransposons were highly fragmented in these two Asparagales genomes, the results presented here show that this was largely due to the methodology used. In contrast, this current work indicates an ensemble of genomic features similar to those observed in the Poaceae.

The dynamics of LTR retrotransposon accumulation across 25 million years of panicoid grass evolution.

Sample sequence analysis was employed to investigate the repetitive DNAs that were most responsible for the evolved variation in genome content across seven panicoid grasses with >5-fold variation in genome size and different histories of polyploidy. In all cases, the most abundant repeats were LTR retrotransposons, but the particular families that had become dominant were found to be different in the Pennisetum, Saccharum, Sorghum and Zea lineages. One element family, Huck, has been very active in all of the studied species over the last few million years. This suggests the transmittal of an active or quiescent autonomous set of Huck elements to this lineage at the founding of the panicoids. Similarly, independent recent activity of Ji and Opie elements in Zea and of Leviathan elements in Sorghum and Saccharum species suggests that members of these families with exceptional activation potential were present in the genome(s) of the founders of these lineages. In a detailed analysis of the Zea lineage, the combined action of several families of LTR retrotransposons were observed to have approximately doubled the genome size of Zea luxurians relative to Zea mays and Zea diploperennis in just the last few million years. One of the LTR retrotransposon amplification bursts in Zea may have been initiated by polyploidy, but the great majority of transposable element activations are not. Instead, the results suggest random activation of a few or many LTR retrotransposons families in particular lineages over evolutionary time, with some families especially prone to future activation and hyper-amplification.

Targeting xa13, a recessive gene for bacterial blight resistance in rice.

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide. Thirty bacterial blight resistance (R) genes (21 dominant genes and 9 recessive genes) in rice have been identified. They are the main sources for the genetic improvement of rice for resistance to Xoo. However, little is known about the recessive R genes. To clone and characterize the recessive R genes, we fine-mapped xa13, a fully recessive gene for Xoo resistance, to a DNA fragment of 14.8 kb using the map-based cloning strategy and a series of sequence-based molecular markers. Sequence analysis of this fragment indicated that this region contains only two apparently intact candidate genes (an extensin-like gene and a homologue of nodulin MtN3) and the 5' end of a predicted hypothetical gene. These results will greatly facilitate the isolation and characterization of xa13. Four PCR-based markers, E6a, SR6, ST9 and SR11 that were tightly linked to the xa13 locus, were also developed. These markers will be useful tools for the marker-assisted selection of xa13 in breeding programs.

Isolation and characterization of genomic and transcribed retrotransposon sequences from sorghum.

Reverse transcriptase sequences from both major classes of retrotransposons were amplified from sorghum genomic DNA, leaf mRNA and callus protoplast mRNA. Sequence analysis of clones derived from genomic DNA demonstrated the presence of a wide variety of copia-like and gypsy-like elements. Twenty-four families of copia-like elements were found, of which at least thirteen were expressed in callus protoplasts. Two families (containing forty-eight subfamilies) of gypsy-like elements were discovered, both closely related to Huck of maize. At least twenty-seven of these subfamilies were expressed in callus protoplasts. Most of these elements were expressed at high levels in protoplasts derived from embryogenic callus, but expression of only a few was detected (at low levels) in leaves. Sequence divergence within individual families was quite high, and all relatedness profiles were consistent with vertical transmission of these elements. These data indicate that sorghum contains a large number and diversity of retrotransposons, and that some may be useful as transposon tagging systems in callus protoplasts.

Enrichment of gene-coding sequences in maize by genome filtration.

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 C0t selection were sequenced to generate approximately 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.

Cereal genes similar to Snf2 define a new subfamily that includes human and mouse genes.

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 5A(m)L 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.

[Construction of genetic map in sorghum and fine mapping of the germination stimulant production gene response to Striga asiatica].

Sorghum is the fifth important crop in the world. It is also the major food resource in African countries. Striga asiatica is a parasitism weed on sorghum and some other important crops. In this report, two sorghum lines with the difference in response to Striga asiatica, SRN39 (lower Germination Stimulant-GermStim production) and Shanguihong (high GermStim production), were selected as the parents for the construction of a recombinant inbred (RI) population. Ninety-four RI lines were collected for the molecular analysis and GermStim production evaluation. A genetic map was constructed with 251 molecular markers that distributed on 10 different linkage groups. The map covers sorghum genome of 1,779 cm with an average map distance of 7.1 cm between linked markers. It is one of the complete sorghum molecular map in the world. Co-segregation analysis indicated that the germination stimulant gene (GermStim) was located on linkage group J, which was at a distance of 13 cm from the closed marker. Further RAPD analysis between two parents and two DNA pools different in the amount of germination stimulant production, several polymorphic DNA fragments were identified and cloned. Mapping results showed two of them flanked with the GermStim gene at a distance of 1.6 cm and 2.1 cm respectively.

Target sites for SINE integration in Brassica genomes display nuclear matrix binding activity.

Short interspersed nuclear elements (SINEs) are ubiquitous components of complex animal and plant genomes. SINEs are believed to be important players in eukaryotic genome evolution. Studies on SINE integration sites have revealed non-random integration without strict nucleotide sequence requirements for the integration target, suggesting that the targeted DNA might assume specific secondary structures or protein associations. Here, we report that S1 SINE elements in the genomes of Brassica show an interesting preference for matrix attachment regions (MARs). Ten cloned genomic regions were tested for their ability to bind the nuclear matrix both before and after a SINE integration event. Eight of the genomic regions targeted by S1 display strong affinity for the nuclear matrix, while two show weaker binding. The SINE S1 did not display any matrix-binding capacity on its own in either non-methylated or methylated forms. In vivo, an integrated S1 is methylated while the surrounding genomic regions may remain undermethylated or undergo methylation. However, tested genomic regions containing methylated S1, with or without methylated flanking genomic sequences, were found to vary in their ability to bind the matrix in vitro. These results suggest a possible molecular basis for a preferential targeting of SINEs to MARs and a possible impact of the integration events upon gene and genome function.

Comparative sequence analysis of colinear barley and rice bacterial artificial chromosomes.

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.

The many hues of plant heterochromatin.

Recent sequence and cytogenetic analyses of heterochromatin in Arabidopsis, together with other results from Arabidopsis and maize, indicate that plant heterochromatin can have very different origins, compositions and dynamics. Shared features that must determine and/or be a result of its unique biological properties are also revealed.

Transposable element contributions to plant gene and genome evolution.

Transposable elements were first discovered in plants because they can have tremendous effects on genome structure and gene function. Although only a few or no elements may be active within a genome at any time in any individual, the genomic alterations they cause can have major outcomes for a species. All major element types appear to be present in all plant species, but their quantitative and qualitative contributions are enormously variable even between closely related lineages. In some large-genome plants, mobile DNAs make up the majority of the nuclear genome. They can rearrange genomes and alter individual gene structure and regulation through any of the activities they promote: transposition, insertion, excision, chromosome breakage, and ectopic recombination. Many genes may have been assembled or amplified through the action of transposable elements, and it is likely that most plant genes contain legacies of multiple transposable element insertions into promoters. Because chromosomal rearrangements can lead to speciating infertility in heterozygous progeny, transposable elements may be responsible for the rate at which such incompatibility is generated in separated populations. For these reasons, understanding plant gene and genome evolution is only possible if we comprehend the contributions of transposable elements.

Structural domains and matrix attachment regions along colinear chromosomal segments of maize and sorghum.

Although a gene's location can greatly influence its expression, genome sequencing has shown that orthologous genes may exist in very different environments in the genomes of closely related species. Four genes in the maize alcohol dehydrogenase (adh1) region represent solitary genes dispersed among large repetitive blocks, whereas the orthologous genes in sorghum are located in a different setting surrounded by low-copy-number DNAs. A specific class of DNA sequences, matrix attachment regions (MARs), was found to be in comparable positions in the two species, often flanking individual genes. If these MARs define structural domains, then the orthologous genes in maize and sorghum should experience similar chromatin environments. In addition, MARs were divided into two groups, based on the competitive affinity of their association with the matrix. The "durable" MARs retained matrix associations at the highest concentrations of competitor DNA. Most of the durable MARs mapped outside genes, defining the borders of putative chromatin loops. The "unstable" MARs lost their association with the matrix under similar competitor conditions and mapped mainly within introns. These results suggest that MARs possess both domain-defining and regulatory roles. Miniature inverted repeat transposable elements (MITEs) often were found on the same fragments as the MARs. Our studies showed that many MITEs can bind to isolated nuclear matrices, suggesting that MITEs may function as MARs in vivo.

Colinearity and its exceptions in orthologous adh regions of maize and sorghum.

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.

Plant genomics takes root, branches out.

For the past nine years, an international consortium of researchers have collaborated on a project to provide a full set of genomics tools for the model plant species Arabidopsis thaliana. Among the goals of this project were the complete sequence of the Arabidopsis genome, which may be completed in the year 2000, four years ahead of schedule. Arabidopsis was an appropriate choice as the first target of plant genomics because of its excellent genetics, outstanding research community and small genome size. Until very recently, it appeared that comprehensive high-throughput plant genomics in the public sector would largely begin and end with Arabidopsis. Over the past two years, this situation has changed completely.

Plant retrotransposons.

Retrotransposons are mobile genetic elements that transpose through reverse transcription of an RNA intermediate. Retrotransposons are ubiquitous in plants and play a major role in plant gene and genome evolution. In many cases, retrotransposons comprise over 50% of nuclear DNA content, a situation that can arise in just a few million years. Plant retrotransposons are structurally and functionally similar to the retrotransposons and retroviruses that are found in other eukaryotic organisms. However, there are important differences in the genomic organization of retrotransposons in plants compared to some other eukaryotes, including their often-high copy numbers, their extensively heterogeneous populations, and their chromosomal dispersion patterns. Recent studies are providing valuable insights into the mechanisms involved in regulating the expression and transposition of retrotransposons. This review describes the structure, genomic organization, expression, regulation, and evolution of retrotransposons, and discusses both their contributions to plant genome evolution and their use as genetic tools in plant biology.