Characterization of T-DNA insertion sites in Arabidopsis thaliana and the implications for saturation mutagenesis

A key component of a sound functional genomics infrastructure is the availability of a knockout mutant for every gene in the genome. A fruitful approach to systematically knockingout genes in the plant Arabidopsis thaliana has been the use of transferred-DNA (T-DNA) from Agrobacterium tumefaciens as an insertional mutagen. One of the assumptions underlying the use of T-DNA as a mutagen is that the insertion of these DNA elements into the Arabidopsis genome occurs at randomly selected locations. We have directly investigated the distribution of T-DNA insertions sites in populations of transformed Arabidopsis using two different approaches. To begin with, we utilized a polymerase chain reaction (PCR) procedure to systematically catalog the precise locations of all the T-DNA elements inserted within a 65 kb segment of chromosome IV. Of the 47 T-DNA insertions identified, 30% were found within the coding regions of genes. We also documented the insertion of T-DNA elements within the centromeric region of chromosome IV. In addition to these targeted T-DNA screens, we also mapped the genomic locations of 583 randomly chosen T-DNA elements by sequencing the genomic DNA flanking the insertion sites from individual T-DNA-transformed lines. 35% of these randomly chosen T-DNA insertions were located within the coding regions of genes. For comparison, coding sequences account for 44% of the Arabidopsis genome. Our results demonstrate that there is a small bias towards recovering T-DNA insertions within intergenic regions. However, this bias does not limit the utility of T-DNA as an effective insertional mutagen for use in reverse-genetic strategies.     

GABI-Kat SimpleSearch: a flanking sequence tag (FST) database for the identification of T-DNA insertion mutants in Arabidopsis thaliana

SUMMARY: GABI-Kat SimpleSearch is a database of flanking sequence tags (FSTs) of T-DNA mutagenized Arabidopsis thaliana lines that were generated by the GABI-Kat project. Sequences flanking the T-DNA insertion sites were aligned to the A.thaliana genome sequence, annotated with information about the FST, the insertion site and the line from which the FST was derived. A web interface permits text-based as well as sequence-based searches for relevant insertions. GABI-Kat SimpleSearch aims to help biologists to quickly find T-DNA insertion mutants for their research. AVAILABILITY: http://www.mpiz-koeln.mpg.de/GABI-Kat/     

Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR

Thermal asymmetric interlaced (TAIL-) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T-DNA insertions in Arabidopsis thaliana is described. Insertion-specific products were amplified from 183 of 190 tested T-DNA insertion lines. Reconstruction experiments indicate that the technique can recover single-copy sequences from genomes as complex as common wheat (1.5 × 10¹⁰ bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T-DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T-DNA insertions confer hygromycin resistance, can be used for fine-scale mapping of linked phenotypic loci.     

An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics

The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.     

High-Throughput Analysis of T-DNA Location and Structure Using Sequence Capture

Agrobacterium-mediated transformation of plants with T-DNA is used both to introduce transgenes and for mutagenesis. Conventional approaches used to identify the genomic location and the structure of the inserted T-DNA are laborious and high-throughput methods using next-generation sequencing are being developed to address these problems. Here, we present a cost-effective approach that uses sequence capture targeted to the T-DNA borders to select genomic DNA fragments containing T-DNA—genome junctions, followed by Illumina sequencing to determine the location and junction structure of T-DNA insertions. Multiple probes can be mixed so that transgenic lines transformed with different T-DNA types can be processed simultaneously, using a simple, index-based pooling approach. We also developed a simple bioinformatic tool to find sequence read pairs that span the junction between the genome and T-DNA or any foreign DNA. We analyzed 29 transgenic lines of Arabidopsis thaliana, each containing inserts from 4 different T-DNA vectors. We determined the location of T-DNA insertions in 22 lines, 4 of which carried multiple insertion sites. Additionally, our analysis uncovered a high frequency of unconventional and complex T-DNA insertions, highlighting the needs for high-throughput methods for T-DNA localization and structural characterization. Transgene insertion events have to be fully characterized prior to use as commercial products. Our method greatly facilitates the first step of this characterization of transgenic plants by providing an efficient screen for the selection of promising lines.     

Distribution and characterization of over 1000 T-DNA tags in rice genome

We generated T-DNA insertions throughout the rice genome for saturation mutagenesis. More than 1,000 flanking sequences were mapped on 12 rice chromosomes. Our results showed that T-DNA tags were not randomly spread on rice chromosomes and were preferentially inserted in gene-rich regions. Few insertions (2.4%) were found in repetitive regions. T-DNA insertions in genic (58.1%) and intergenic regions (41.9%) showed a good correlation with the predicted size distribution of these sequences in the rice genome. Whereas, obvious biases were found for the insertions in the 5'- and 3'-regulatory regions outside the coding regions both at 500-bp size and in introns rather than in exons. Such distribution patterns and biases for T-DNA integration in rice are similar to that of the previous report in Arabidopsis, which may result from T-DNA integration mechanism itself. Rice will require approximately the same number of T-DNA insertions for saturation mutagenesis as will Arabidopsis. A database of the T-DNA insertion sites in rice is publicly available at our web site (http://www.genomics.zju.edu.cn/ricetdna).     

Chromosomal translocations are a common phenomenon in Arabidopsis thaliana T-DNA insertion lines

Ordered collections of Arabidopsis thaliana lines containing mapped T-DNA insertions have become an important resource for plant scientists performing genetic studies. Previous reports have indicated that T-DNA insertion lines can have chromosomal translocations associated with the T-DNA insertion site, but the prevalence of these rearrangements has not been well documented. To determine the frequency with which translocations are present in a widely-used collection of T-DNA insertion lines, we analyzed 64 independent lines from the Salk T-DNA mutant collection. Chromosomal translocations were detected in 12 of the 64 lines surveyed (19%). Two assays were used to screen the T-DNA lines for translocations: pollen viability and genome-wide genetic mapping. Although the measurement of pollen viability is an indirect screen for the presence of a translocation, all 11 of the T-DNA lines showing an abnormal pollen phenotype were found to contain a translocation when analyzed using genetic mapping. A normal pollen phenotype does not, however, guarantee the absence of a translocation. We observed one T-DNA line with normal pollen that nevertheless had a translocation based on genetic mapping results. One additional phenomenon that we observed through our genetic mapping experiments was that the T-DNA junctions on the 5'- and 3'-sides of a targeted gene can genetically separate from each other in some cases. Two of the lines in our survey displayed this 'T-DNA borders separate' phenomenon. Experimental procedures for efficiently screening T-DNA lines for the presence of chromosomal abnormalities are presented and discussed.     

Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing 13 804 T-DNA flanking sequences from an enhancer-trap mutant library

We isolated 13 804 T-DNA flanking sequence tags (FSTs) from a T-DNA insertion library of rice. A comprehensive analysis of the 13 804 FSTs revealed a number of features demonstrating a highly non-random distribution of the T-DNA insertions in the rice genome: T-DNA insertions were biased towards large chromosomes, not only in the absolute number of insertions but also in the relative density; within chromosomes the insertions occurred more densely in the distal ends, and less densely in the centromeric regions; the distribution of the T-DNA insertions was highly correlated with that of full-length cDNAs, but the correlations were highly heterogeneous among the chromosomes; T-DNA insertions strongly disfavored transposable element (TE)-related sequences, but favored genic sequences with a strong bias toward the 5' upstream and 3' downstream regions of the genes; T-DNA insertions preferentially occurred among the various classes of functional genes, such that the numbers of insertions were in excess in certain functional categories but were deficient in other categories. The analysis of DNA sequence compositions around the T-DNA insertion sites also revealed several prominent features, including an elevated bendability from -200 to 200 bp relative to the insertion sites, an inverse relationship between the GC and TA skews, and reversed GC and TA skews in sequences upstream and downstream of the insertion sites, with both GC and TA skews equal to zero at the insertion sites. It was estimated that 365 380 insertions are needed to saturate the genome with P = 0.95, and that the 45 441 FSTs that have been isolated so far by various groups tagged 14 287 of the 42 653 non-TE related genes.     

Insertional mutagenesis of genes required for seed development in Arabidopsis thaliana.

The purpose of this project was to identify large numbers of Arabidopsis genes with essential functions during seed development. More than 120,000 T-DNA insertion lines were generated following Agrobacterium-mediated transformation. Transgenic plants were screened for defective seeds and putative mutants were subjected to detailed analysis in subsequent generations. Plasmid rescue and TAIL-PCR were used to recover plant sequences flanking insertion sites in tagged mutants. More than 4200 mutants with a wide range of seed phenotypes were identified. Over 1700 of these mutants were analyzed in detail. The 350 tagged embryo-defective (emb) mutants identified to date represent a significant advance toward saturation mutagenesis of EMB genes in Arabidopsis. Plant sequences adjacent to T-DNA borders in mutants with confirmed insertion sites were used to map genome locations and establish tentative identities for 167 EMB genes with diverse biological functions. The frequency of duplicate mutant alleles recovered is consistent with a relatively small number of essential (EMB) genes with nonredundant functions during seed development. Other functions critical to seed development in Arabidopsis may be protected from deleterious mutations by extensive genome duplications.     

Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics.

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.     

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

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.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

Extensive Phenotypic Variation among Allelic T-DNA Inserts in Arabidopsis thaliana

T-DNA insertion mutants are a tool used widely in Arabidopsis thaliana to disrupt gene function. We phenotyped multiple homozygous T-DNA A. thaliana mutants at each of two loci (AT1G11060 and AT4G00210). We measured life history traits, including germination, size at reproduction and fruit production. Allelic T-DNA lines differed for most traits at AT1G11060 but not at AT4G00210. However, insertions in exons differed from other insertion positions in AT4G00210 but not in AT1G11060. We found evidence for additional insertions in approximately half of the lines, but found few phenotypic consequences. In general, our results suggest that a cautious interpretation of T-DNA phenotypes is warranted.     

ATIDB: Arabidopsis thaliana insertion database

Insertional mutagenesis techniques, including transposon- and T-DNA-mediated mutagenesis, are key resources for systematic identification of gene function in the model plant species Arabidopsis thaliana. We have developed a database (http://atidb.cshl.org/) for archiving, searching and analyzing insertional mutagenesis lines. Flanking sequences from approximately 10 500 insertion lines (including transposon and T-DNA insertions) from several tagging programs in Arabidopsis were mapped to the genome sequence through our annotation system before being entered into the database. The database front end provides World Wide Web searching and analyzing interfaces for genome researchers and other biologists. Users can search the database to identify insertions in a particular gene or perform genome-wide analysis to study the distribution and preference of insertions. Tools integrated with the database include a graphical genome browser, a protein search function, a graphical representation of the insertion distribution and a Blast search function. The database is based on open source components and is available under an open source license.     

Creation of a collection of morphological insertional mutants of Arabidopsis thaliana

A collection of transgenic Arabidopsis thaliana plants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12-18% of the total number of A. thaliana genes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.