ABSTRACT
Horizontal transfer (HT) refers to the exchange of genetic material between divergent species by mechanisms other than reproduction. In recent years, several studies have demonstrated HTs in eukaryotes, particularly in the context of parasitic relationships and in model species. However, very little is known about HT in natural ecosystems, especially those involving non-parasitic wild species, and the nature of the ecological relationships that promote these HTs. In this work, we conducted a pilot study investigating HTs by sequencing the genomes of 17 wild non-model species from a natural ecosystem, the Massane forest, located in southern France. To this end, we developed a new computational pipeline called INTERCHANGE that is able to characterize HTs at the whole genome level without prior annotation and directly in the raw sequencing reads. Using this pipeline, we identified 12 HT events, half of which occurred between lianas and trees. We found that mainly low copy number LTR-retrotransposons from the Copia superfamily were transferred between these wild plant species, especially those of the Ivana and Ale lineages. This study revealed a possible new route for HTs between non-parasitic plants and provides new insights into the genomic characteristics of horizontally transferred DNA in plant genomes.
Subject(s)
Ecosystem , Genome, Plant , Pilot Projects , Genome, Plant/genetics , Genomics , Retroelements , Phylogeny , Evolution, Molecular , Gene Transfer, Horizontal/geneticsABSTRACT
Abundant extrachromosomal circular DNA (eccDNA) is associated with transposable element (TE) activity. However, how the eccDNA compartment is controlled by epigenetic regulations and what is its impact on the genome is understudied. Here, using long reads, we sequence both the eccDNA compartment and the genome of Arabidopsis thaliana mutant plants affected in DNA methylation and post-transcriptional gene silencing. We detect a high load of TE-derived eccDNA with truncated and chimeric forms. On the genomic side, on top of truncated and full length TE neo-insertions, we detect complex structural variations (SVs) notably at a disease resistance cluster being a natural hotspot of SV. Finally, we serendipitously identify large tandem duplications in hypomethylated plants, suggesting that SVs could have been overlooked in epigenetic mutants. We propose that a high eccDNA load may alter DNA repair pathways leading to genome instability and the accumulation of SVs, at least in plants.
Subject(s)
Arabidopsis , Humans , Arabidopsis/genetics , DNA Transposable Elements/genetics , Genomic Instability/genetics , RNA Interference , DNA, CircularABSTRACT
The keystone of ribosome biogenesis is the transcription of 45S rDNA. The Arabidopsis thaliana genome contains hundreds of 45S rDNA units; however, they are not all transcribed. Notably, 45S rDNA units contain insertions/deletions revealing the existence of heterogeneous rRNA genes and, likely, heterogeneous ribosomes for rRNAs. In order to obtain an overall picture of 45S rDNA diversity sustaining the synthesis of rRNAs and, subsequently, of ribosomes in natura, we took advantage of 320 new occurrences of Arabidopsis thaliana as a metapopulation named At66, sampled from 0 to 1900 m of altitude in the eastern Pyrenees in France. We found that the 45S rDNA copy number is very dynamic in natura and identified new genotypes for both 5' and 3' External Transcribed Spacers (ETS). Interestingly, the highest 5'ETS genotype diversity is found in altitude while the highest 3'ETS genotype diversity is found at sea level. Structural analysis of 45S rDNA also shows conservation in natura of specific 5'ETS and 3'ETS sequences/features required to control rDNA expression and the processing of rRNAs. In conclusion, At66 is a worthwhile natural laboratory, and unraveled 45S rDNA diversity represents an interesting starting material to select subsets for rDNA transcription and alter the rRNA composition of ribosomes both intra- and inter-site.
ABSTRACT
Delphinium montanum DC. 1815, is an endangered larkspur endemic to the Eastern Pyrenees. For biogeographic and conservation purpose, a hybrid assembly approach based on long- and short-read genomic data allowed us to successfully assemble whole plastid genome of Delphinium montanum. The complete plastome is 154,185 bp in length, consisting of a pair of inverted repeats (IRs) of 26,559 bp, a large single-copy (LSC) region and a small single-copy region (SSC) of 84,746 and 16,320 bp, respectively. It was found to contain 136 genes, including 84 protein-coding genes, 44 trRNA genes and 8 rRNA genes. The overall GC content of the plastid genome is 38.3%. Phylogenetic inference supports the polyphyly of the Delphinium genus.
ABSTRACT
Extrachromosomal circular DNA (eccDNA) has been observed in different species for decades, and more and more evidence shows that this specific type of DNA molecules may play an important role in rapid adaptation. Therefore, characterizing the full landscape of eccDNA has become critical, and there are several protocols for enriching eccDNAs and performing short-read or long-read sequencing. However, there is currently no available bioinformatic tool to identify eccDNAs from Nanopore reads. More importantly, the current tools based on Illumina short reads lack an efficient standardized pipeline notably to identify eccDNA originating from repeated loci and cannot be applied to very large genomes. Here, we introduce a comprehensive tool to solve both of these two issues. Applying ecc_finder to eccDNA-seq data (either mobilome-seq, Circle-Seq and CIDER-seq) from Arabidopsis, human, and wheat (with genome sizes ranging from 120Mb to 17 Gb), we document the improvement of computational time, sensitivity, and accuracy and demonstrate ecc_finder wide applicability and functionality.
ABSTRACT
Active transposable elements (TEs) generate insertion polymorphisms that can be detected through genome resequencing strategies. However, these techniques may have limitations for organisms with large genomes or for somatic insertions. Here, we present a method that takes advantage of the extrachromosomal circular DNA (eccDNA) forms of actively transposing TEs in order to detect and characterize active TEs in any plant or animal tissue. Mobilome-seq consists in selectively amplifying and sequencing eccDNAs. It relies on linear digestion of genomic DNA followed by rolling circle amplification of circular DNA. Both active DNA transposons and retrotransposons can be identified using this technique.
Subject(s)
DNA Transposable Elements , DNA, Circular/isolation & purification , Animals , DNA, Plant/genetics , Nucleic Acid Amplification Techniques , Plants/genetics , Sequence Analysis, DNAABSTRACT
Rapid plant genome evolution is crucial to adapt to environmental changes. Chromosomal rearrangements and gene copy number variation (CNV) are two important tools for genome evolution and sources for the creation of new genes. However, their emergence takes many generations. In this study, we show that in Arabidopsis thaliana, a significant loss of ribosomal RNA (rRNA) genes with a past history of a mutation for the chromatin assembly factor 1 (CAF1) complex causes rapid changes in the genome structure. Using long-read sequencing and microscopic approaches, we have identified up to 15 independent large tandem duplications in direct orientation (TDDOs) ranging from 60 kb to 1.44 Mb. Our data suggest that these TDDOs appeared within a few generations, leading to the duplication of hundreds of genes. By subsequently focusing on a line only containing 20% of rRNA gene copies (20rDNA line), we investigated the impact of TDDOs on 3D genome organization, gene expression, and cytosine methylation. We found that duplicated genes often accumulate more transcripts. Among them, several are involved in plant-pathogen response, which could explain why the 20rDNA line is hyper-resistant to both bacterial and nematode infections. Finally, we show that the TDDOs create gene fusions and/or truncations and discuss their potential implications for the evolution of plant genomes.
Subject(s)
Arabidopsis/genetics , Disease Resistance/genetics , Gene Duplication , Gene Expression Regulation, Plant , Genes, rRNA , Gene Expression , Genes, Plant , Genome, Plant , Genomic InstabilityABSTRACT
In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1, a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice (Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.
Subject(s)
DNA-Directed RNA Polymerases/genetics , Oryza/genetics , Plant Proteins/genetics , RNA Interference , DNA Methylation , DNA-Directed RNA Polymerases/metabolism , Gene Knockdown Techniques , Oryza/metabolism , Plant Proteins/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolismABSTRACT
Global, segmental, and gene duplication-related processes are driving genome size and complexity in plants. Despite their evolutionary potentials, those processes can also have adverse effects on genome regulation, thus implying the existence of specialized corrective mechanisms. Here, we report that an N6-methyladenosine (m6A)-assisted polyadenylation (m-ASP) pathway ensures transcriptome integrity in Arabidopsis thaliana Efficient m-ASP pathway activity requires the m6A methyltransferase-associated factor FIP37 and CPSF30L, an m6A reader corresponding to an YT512-B Homology Domain-containing protein (YTHDC)-type domain containing isoform of the 30-kD subunit of cleavage and polyadenylation specificity factor. Targets of the m-ASP pathway are enriched in recently rearranged gene pairs, displayed an atypical chromatin signature, and showed transcriptional readthrough and mRNA chimera formation in FIP37- and CPSF30L-deficient plants. Furthermore, we showed that the m-ASP pathway can also restrict the formation of chimeric gene/transposable-element transcript, suggesting a possible implication of this pathway in the control of transposable elements at specific locus. Taken together, our results point to selective recognition of 3'-UTR m6A as a safeguard mechanism ensuring transcriptome integrity at rearranged genomic loci in plants.
Subject(s)
Adenosine/analogs & derivatives , Gene Expression Regulation, Plant , Plants/genetics , Plants/metabolism , Signal Transduction , Transcriptome , Adenosine/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Gene Expression Profiling , Genetic Loci , Mutation , Polyadenylation , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, GeneticABSTRACT
The recent release of genomic sequences for 3000 rice varieties provides access to the genetic diversity at species level for this crop. We take advantage of this resource to unravel some features of the retrotranspositional landscape of rice. We develop software TRACKPOSON specifically for the detection of transposable elements insertion polymorphisms (TIPs) from large datasets. We apply this tool to 32 families of retrotransposons and identify more than 50,000 TIPs in the 3000 rice genomes. Most polymorphisms are found at very low frequency, suggesting that they may have occurred recently in agro. A genome-wide association study shows that these activations in rice may be triggered by external stimuli, rather than by the alteration of genetic factors involved in transposable element silencing pathways. Finally, the TIPs dataset is used to trace the origin of rice domestication. Our results suggest that rice originated from three distinct domestication events.
Subject(s)
Domestication , Evolution, Molecular , Genetic Variation , Oryza/genetics , Retroelements/genetics , Datasets as Topic , Genetic Association Studies , Genome, Plant/genetics , Genomics/methods , PhylogenyABSTRACT
Ophrys aveyronensis is an orchid with disjunct geographic distribution. For biogeographic and conservation purpose, we sequenced its complete plastome using Illumina data. The complete plastome is 146,816 bp in length, consisting of a pair of inverted repeats (IRs) of 25,006 bp, a large single-copy (LSC) region and a small single-copy region (SSC) of 80,495 and 16,309 bp, respectively. It was found to contain 133 genes, including 86 protein-coding genes, up to 39 trRNA genes and 8 rRNA genes. The overall GC content of the plastid genome is 36.9%. Phylogenetic inference confirms that O. aveyronensis is very close to O. sphegodes.
ABSTRACT
BACKGROUND: Transposables elements (TEs) contribute to both structural and functional dynamics of most eukaryotic genomes. Because of their propensity to densely populate plant and animal genomes, the precise estimation of the impact of transposition on genomic diversity has been considered as one of the main challenges of today's genomics. The recent development of NGS (next generation sequencing) technologies has open new perspectives in population genomics by providing new methods for high throughput detection of Transposable Elements-associated Structural Variants (TEASV). However, these have relied on Illumina platform that generates short reads (up to 350 nucleotides). This limitation in size of sequence reads can cause high false discovery rate (FDR) and therefore limit the power of detection of TEASVs, especially in the case of large, complex genomes. The newest sequencing technologies, such as Oxford Nanopore Technologies (ONT) can generate kilobases-long reads thus representing a promising tool for TEASV detection in plant and animals. RESULTS: We present the results of a pilot experiment for TEASV detection on the model plant species Arabidopsis thaliana using ONT sequencing and show that it can be used efficiently to detect TE movements. We generated a ~0.8X genome coverage of a met1-derived epigenetic recombinant inbred line (epiRIL) using a MinIon device with R7 chemistry. We were able to detect nine new copies of the LTR-retrotransposon Evadé (EVD). We also evidenced the activity of the DNA transposon CACTA, CAC1. CONCLUSIONS: Even at a low sequence coverage (0.8X), ONT sequencing allowed us to reliably detect several TE insertions in Arabidopsis thaliana genome. The long read length allowed a precise and un-ambiguous mapping of the structural variations caused by the activity of TEs. This suggests that the trade-off between read length and genome coverage for TEASV detection may be in favor of the former. Should the technology be further improved both in terms of lower error rate and operation costs, it could be efficiently used in diversity studies at population level.
Subject(s)
Arabidopsis/genetics , DNA Transposable Elements/genetics , High-Throughput Nucleotide Sequencing , Sequence Analysis, DNA , Retroelements/genetics , Terminal Repeat Sequences/geneticsABSTRACT
Retrotransposons are mobile genetic elements abundant in plant and animal genomes. While efficiently silenced by the epigenetic machinery, they can be reactivated upon stress or during development. Their level of transcription not reflecting their transposition ability, it is thus difficult to evaluate their contribution to the active mobilome. Here we applied a simple methodology based on the high throughput sequencing of extrachromosomal circular DNA (eccDNA) forms of active retrotransposons to characterize the repertoire of mobile retrotransposons in plants. This method successfully identified known active retrotransposons in both Arabidopsis and rice material where the epigenome is destabilized. When applying mobilome-seq to developmental stages in wild type rice, we identified PopRice as a highly active retrotransposon producing eccDNA forms in the wild type endosperm. The mobilome-seq strategy opens new routes for the characterization of a yet unexplored fraction of plant genomes.
Subject(s)
DNA, Circular/genetics , Interspersed Repetitive Sequences/genetics , Plants/genetics , Retroelements/genetics , Arabidopsis/genetics , DNA, Circular/chemistry , DNA, Plant/chemistry , DNA, Plant/genetics , Endosperm/genetics , Flowers/genetics , Gene Expression Regulation, Plant , Genome, Plant/genetics , High-Throughput Nucleotide Sequencing , Models, Genetic , Mutagenesis, Insertional , Oryza/genetics , Phylogeny , Plant Leaves/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Iron toxicity is a root related abiotic stress, occurring frequently in flooded soils. It can affect the yield of rice in lowland production systems. This toxicity is associated with high concentrations of reduced iron (Fe(2+)) in the soil solution. Although the first interface of the element is in the roots, the consequences of an excessive uptake can be observed in several rice tissues. In an original attempt to find both genes and transposable elements involved in the response to an iron toxicity stress, we used a microarray approach to study the transcriptional responses of rice leaves of cv. Nipponbare (Oryza sativa L. ssp. japonica) to iron excess in nutrient solution. RESULTS: A large number of genes were significantly up- or down-regulated in leaves under the treatment. We analyzed the gene ontology and metabolic pathways of genes involved in the response to this stress and the cis-regulatory elements (CREs) present in the promoter region of up-regulated genes. The majority of genes act in the pathways of lipid metabolic process, carbohydrate metabolism, biosynthesis of secondary metabolites and plant hormones. We also found genes involved in iron acquisition and mobilization, transport of cations and regulatory mechanisms for iron responses, and in oxidative stress and reactive oxygen species detoxification. Promoter regions of 27% of genes up-regulated present at least one significant occurrence of an ABA-responsive CRE. Furthermore, and for the first time, we were able to show that iron stress triggers the up-regulation of many LTR-retrotransposons. We have established a complete inventory of transposable elements transcriptionally activated under iron excess and the CREs which are present in their LTRs. CONCLUSION: The short-term response of Nipponbare seedlings to iron excess, includes activation of genes involved in iron homeostasis, in particular transporters, transcription factors and ROS detoxification in the leaves, but also many transposable elements. Our data led to the identification of CREs which are associated with both genes and LTR-retrotransposons up-regulated under iron excess. Our results strengthen the idea that LTR-retrotransposons participate in the transcriptional response to stress and could thus confer an adaptive advantage for the plant.
ABSTRACT
Cooperation involving Plant Growth-Promoting Rhizobacteria results in improvements of plant growth and health. While pathogenic and symbiotic interactions are known to induce transcriptional changes for genes related to plant defense and development, little is known about the impact of phytostimulating rhizobacteria on plant gene expression. This study aims at identifying genes significantly regulated in rice roots upon Azospirillum inoculation, considering possible favored interaction between a strain and its original host cultivar. Genome-wide analyzes of Oryza sativa japonica cultivars Cigalon and Nipponbare were performed, by using microarrays, seven days post-inoculation with Azospirillum lipoferum 4B (isolated from Cigalon) or Azospirillum sp. B510 (isolated from Nipponbare) and compared to the respective non-inoculated condition. A total of 7384 genes were significantly regulated, which represent about 16% of total rice genes. A set of 34 genes is regulated by both Azospirillum strains in both cultivars, including a gene orthologous to PR10 of Brachypodium, and these could represent plant markers of Azospirillum-rice interactions. The results highlight a strain-dependent response of rice, with 83% of the differentially expressed genes being classified as combination-specific. Whatever the combination, most of the differentially expressed genes are involved in primary metabolism, transport, regulation of transcription and protein fate. When considering genes involved in response to stress and plant defense, it appears that strain B510, a strain displaying endophytic properties, leads to the repression of a wider set of genes than strain 4B. Individual genotypic variations could be the most important driving force of rice roots gene expression upon Azospirillum inoculation. Strain-dependent transcriptional changes observed for genes related to auxin and ethylene signaling highlight the complexity of hormone signaling networks in the Azospirillum-rice cooperation.
ABSTRACT
Vertical, transgenerational transmission of genetic material occurs through reproduction of living organisms. In addition to vertical inheritance, horizontal gene transfer between reproductively isolated species has recently been shown to be an important, if not dominant, mechanism in the evolution of prokaryotic genomes. In contrast, only a few horizontal transfer (HT) events have been characterized so far in eukaryotes and mainly concern transposable elements (TEs). Whether these are frequent and have a significant impact on genome evolution remains largely unknown. We performed a computational search for highly conserved LTR retrotransposons among 40 sequenced eukaryotic genomes representing the major plant families. We found that 26 genomes (65%) harbor at least one case of horizontal TE transfer (HTT). These transfers concern species as distantly related as palm and grapevine, tomato and bean, or poplar and peach. In total, we identified 32 cases of HTTs, which could translate into more than 2 million among the 13,551 monocot and dicot genera. Moreover, we show that these TEs have remained functional after their transfer, occasionally causing a transpositional burst. This suggests that plants can frequently exchange genetic material through horizontal transfers and that this mechanism may be important in TE-driven genome evolution.
Subject(s)
DNA Transposable Elements/genetics , Gene Transfer, Horizontal , Genome, Plant , Magnoliopsida/genetics , Retroelements/genetics , Species SpecificityABSTRACT
Transposable elements (TEs) are mobile entities that densely populate most eukaryotic genomes and contribute to both their structural and functional dynamics. However, most TE-related sequences in both plant and animal genomes correspond to inactive, degenerated elements, due to the combined effect of silencing pathways and elimination through deletions. One of the major difficulties in fully characterizing the molecular basis of genetic diversity of a given species lies in establishing its genome-wide transpositional activity. Here, we provide an extensive survey of the transpositional landscape of a plant genome using a deep sequencing strategy. This was achieved through paired-end mapping of a fourfold coverage of the genome of rice mutant line derived from an in vitro callus culture using Illumina technology. Our study shows that at least 13 TE families are active in this genotype, causing 34 new insertions. This next-generation sequencing-based strategy provides new opportunities to quantify the impact of TEs on the genome dynamics of the species.
Subject(s)
DNA Transposable Elements , Genome, Plant , Mutagenesis, Insertional , Oryza/genetics , Chromosome Mapping , Chromosomes, Plant , DNA, Plant/genetics , Genetic Variation , Sequence Analysis, DNAABSTRACT
The leucine-rich repeat class of receptor-like kinase (LRR-RLKs) encoding genes represents the largest family of putative receptor genes in the Arabidopsis thaliana genome. However, very little is known about the range of biological process that they control. We present in this paper the functional characterization of RLK7 that has all the structural features of a receptor-like kinase of the plant-specific LRR type. To this end, we identified and characterized three independent T-DNA insertion mutants, constructed lines carrying truncated versions of this putative receptor, one lacking the cytoplasmic kinase domain (RLK7Δkin) and the other one lacking 14 LRR repeats (RLK7ΔLRR) and generated RLK7 overexpressing lines. We thus provide evidences that RLK7 is involved in the control of germination speed and the tolerance to oxidant stress. First, consistent with the expression kinetics of the RLK7 gene in the seeds, we found that all three mutants showed a delay in germination, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines displayed a phenotype of more precocious germination. Second, a non-hypothesis driven proteomic approach revealed that in the seedlings of the three T-DNA insertion lines, four enzymes directly or indirectly involved in reactive oxygen species detoxification, were significantly less abundant. Consistent with this finding, the three mutants were less tolerant than the wild type to a hydrogen peroxide treatment, whereas the overexpressors, RLK7Δkin and RLK7ΔLRR lines presented the opposite phenotype.
Subject(s)
Adaptation, Physiological , Arabidopsis Proteins/physiology , Arabidopsis/physiology , Germination , Leucine/metabolism , Oxidative Stress , Arabidopsis/enzymology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Hydrogen Peroxide/metabolism , In Situ Hybridization , Mutation , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
An inverse genetic approach was used to gain insight into the role of AP2/ERF-type transcription factors genes during plant development in Arabidopsis thaliana. Here we show that the expression pattern of AtERF38, which is, among the organs tested, more intensively expressed in mature siliques and floral stems, is closely associated with tissues that undergo secondary cell wall modifications. Firstly, public microarray data sets analysis indicates that AtERF38 is coregulated with several genes involved in secondary wall thickening. Secondly, this was experimentally confirmed in different types of cells expressing a Pro(AtERF38)::GUS fusion: histochemical analysis revealed strong and specific GUS activity in outer integument cells of mature seeds, endodermal cells of the roots in the primary developmental stage and some sclerified cells of mature inflorescence stems. All of these cells are known or shown here to be characterized by a reinforced wall. The latter, which have not been well characterized to date in Arabidopsis and may be suberized, could benefit of the use of AtERF38 as a specific marker. We were not able to detect any phenotype in an insertion line in which ectopic expression of AtERF38 is caused by the insertion of a T-DNA in its promoter. Nevertheless, AtERF28 may be considered as a candidate regulator of secondary wall metabolism in particular cell types that are not reinforced by the typical deposition of lignin and cellulose, but that have at least in common accumulation of suberin-like lipid polyesters in their walls.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Plant Roots/metabolism , Plant Stems/metabolism , Seeds/metabolism , Transcription Factors/genetics , Arabidopsis/embryology , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Base Sequence , DNA Primers , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolismABSTRACT
Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.