A resource of mapped dissociation launch pads for targeted insertional mutagenesis in the Arabidopsis genome

We describe a new resource for targeted insertional mutagenesis in Arabidopsis using a maize (Zea mays) Activator/Dissociation (Ds) two-element system. The two components of the system, T-DNA vectors carrying a Ds launch pad and a stable Activator transposase source, were designed to simplify selection of transposition events and maximize their usefulness. Because Ds elements preferentially transpose to nearby genomic sites, they can be used in targeted mutagenesis of linked genes. To efficiently target all genes throughout the genome, we generated a large population of transgenic Arabidopsis plants containing the Ds launch pad construct, identified lines containing single Ds launch pad inserts, and mapped the positions of Ds launch pads in 89 lines. The integration sites of the Ds launch pads were relatively evenly distributed on all five chromosomes, except for a region of chromosomes 2 and 4 and the centromeric regions. This resource therefore provides access to the majority of the Arabidopsis genome for targeted tagging.     

Transposon tagging in diploid strawberry

Fragaria vesca was transformed with a transposon tagging construct harbouring amino terminally deleted maize transposase and EGFP (Ac element), NPTII, CaMV 35S promoter (P35S) driving transposase and mannopine synthase promoter (Pmas) driving EGFP (Ds element). Of 180 primary transgenics, 48 were potential launch pads, 72 were multiple insertions or chimaeras, and 60 exhibited somatic transposition. T(1) progeny of 32 putative launch pads were screened by multiplex PCR for transposition. Evidence of germ-line transposition occurred in 13 putative launch pads; however, the transposition frequency was too low in three for efficient recovery of transposants. The transposition frequency in the remaining launch pads ranged from 16% to 40%. After self-pollination of the T(0) launch pads, putative transposants in the T(1) generation were identified by multiplex PCR. Sequencing of hiTAIL-PCR products derived from nested primers within the Ds end sequences (either P35S at the left border or the inverted repeat at the right border) of T(1) plants revealed transposition of the Ds element to distant sites in the strawberry genome. From more than 2400 T(1) plants screened, 103 unique transposants have been identified, among which 17 were somatic transpositions observed in the T(0) generation. Ds insertion sites were dispersed among various gene elements [exons (15%), introns (23%), promoters (30%), 3' UTRs (17%) as well as intergenically (15%)]. Three-primer (one on either side of the Ds insertion and one within the Ds T-DNA) PCR could be used to identify homozygous T(2) transposon-tagged plants. The mutant collection has been catalogued in an on-line database.     

Ac Transposition from a T-DNA Can Generate Linked and Unlinked Clusters of Insertions in the Tomato Genome

We have investigated the distribution of transposed Acs in the tomato genome. Our approach has been to clone the regions flanking the T-DNAs and transposed Acs from two transgenic lines of tomato and place these sequences on the tomato restriction fragment length polymorphism (RFLP) map. The distribution of transposed Acs around the T-DNA and at locations unlinked to the T-DNA indicates that Ac transposes to linked and unlinked sites in tomato as it does in maize. The structure and terminal sequence of these cloned elements shows that Ac remains intact after transposition. We discuss these results and their bearing on gene tagging strategies using Ac and Ds.     

Distribution of Unlinked Transpositions of a Ds Element from a T-DNA Locus on Tomato Chromosome 4

In maize, receptor sites for unlinked transpositions of Activator (Ac) elements are not distributed randomly. To test whether the same is true in tomato, the receptor sites for a Dissociation (Ds) element derived from Ac, were mapped for 26 transpositions unlinked to a donor T-DNA locus on chromosome 4. Four independent transposed Dss mapped to sites on chromosome 4 genetically unlinked to the donor T-DNA, consistent with a preference for transposition to unlinked sites on the same chromosome as opposed to sites on other chromosomes. There was little preference among the nondonor chromosomes, except perhaps for chromosome 2, which carried seven transposed Dss, but these could not be proven to be independent. However, these data, when combined with those from other studies in tomato examining the distribution of transposed Acs or Dss among nondonor chromosomes, suggest there may be absolute preferences for transposition irrespective of the chromosomal location of the donor site. If true, transposition to nondonor chromosomes in tomato would differ from that in maize, where the preference seems to be determined by the spatial arrangement of chromosomes in the interphase nucleus. The tomato lines carrying Ds elements at known locations are available for targeted transposon tagging experiments.     

Assignment of 44 Ds Insertions to the Linkage Map of Arabidopsis

Transposon tagging is a useful tool for biological studies. Transposon insertions have been used to obtain new mutants which are extremely helpful in understanding gene function. These insertions immediately provide a means to isolate the corresponding genes. Transposon tagging has also been used to clone genes previously defined by point mutations. In addition, transposon insertions into cloned genes that lack mutations can be generated to facilitate functional analysis. The maize Ac/Ds transposon elements are known to transpose to local sites with high frequencies and have been shown to function in several dicots. To generate a collection of Ds elements for the purpose of targeted insertional mutagenesis of mapped genes in Arabidopsis, we have mapped 44 Ds insertions by simple sequence length polymorphism (SSLP). Because the Arabidopsis genome project is advancing rapidly, many genes will be discovered whose functions are unknown. The mapped 44 Ds insertions will be a useful resource for post-genome analysis of gene functions in Arabidopsis.     

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.     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。     

State II dissociation element formation following activator excision in maize

Active Activator (Ac) elements undergo mutations to become nonautonomous Dissociation (Ds) elements at a low frequency. To understand the mechanism of Ds formation, we have developed high-throughput genetic and molecular screens to identify these rare Ds derivatives generated from any Ac insertion in the maize genome. Using these methods we have identified 15 new Ds elements derived from Ac insertions at eight different loci. Approximately half of the Ds elements contain filler DNA inserted at the deletion junction that is derived from sequences within or adjacent to Ac. In contrast to previous reports, several of these Ds elements lack direct repeats flanking the deletion junctions and filler DNA in the donor Ac. To accommodate our findings and those of others, we propose a model of slip mispairing during error-prone repair synthesis to explain the formation of state II Ds elements in maize. We discuss the use of these lines and molecular techniques developed here to capture somatic Ds transposition events in two-component Ac/Ds tagging programs in maize.     

An inducible targeted tagging system for localized saturation mutagenesis in Arabidopsis

We describe a system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis (Arabidopsis thaliana). In this system, the Ac and Ds elements are carried within the same T-DNA and a heat shock-inducible transposase fusion is utilized to control the levels of transposase gene expression, generating transpositions that can be subsequently stabilized without requiring crossing or segregation. We have mapped 40 single-copy lines by thermal asymmetric interlaced-PCR, which can be used as potential launch pads for heat shock mutagenesis. Using a starter line selected for detailed analysis, the efficiency of tagging over a 50-kb region in the genome was examined. Hits were obtained in the targeted genes with multiple alleles for most genes, with approximately equal numbers of hits detected in genes on either side of the T-DNA. These results establish the feasibility of our approach for localized saturation mutagenesis in Arabidopsis. This system is very efficient and much less laborious as compared to conventional crossing schemes and may be generally applicable to other plant species for which large-scale T-DNA tagging is not currently feasible.     

T-DNA and transposon tagging in aspen

Abstract: We have investigated the somatic activity of the maize Activator (Ac) element in haploid and diploid aspen with the objective of developing an efficient transposon-based system for gene isolation in the model tree species Populus. It was shown that Ac is reinserted, frequently into or near coding regions in aspen, and therefore can be used for gene tagging studies. A number of phenotypic variants were also found following transformation of constructs harbouring the rolC gene. Comparative analyses of T-DNA flanking regions of variants and wild type lines indicate that T-DNA insertion has occurred in or near coding regions. However, the frequency of T-DNA insertion into genes is about one half of the frequency of Ac insertion hitting coding sequences. The results obtained give a proof-of-concept for transposon tagging in a tree system. Given the long generation cycles in tree species, gene tagging strategies are practical only to obtain dominant gain-of-function mutants that do not require selfing or test crossing. In order to obtain recessive loss-of-function mutants, we have regenerated haploid lines from immature pollen. These lines were successfully transformed with a construct containing the rolC transgene from Agrobacterium rhizogenes and Ac element from maize. The results indicate that Ac is also active in haploid aspen and hence can be used in general for gene tagging in trees.     

激活标签法构建拟南芥突变体库及其表型分析

以拟南芥(ArabMopsis thaliana)野生生态型(Columbia)植株为实验材料,以含有激活标记双元质粒pCB260的农杆菌进行转化,并以抗除草剂Basta为筛选标记,构建了拟南芥激活标签突变体库,所用pCB260双元质粒含有两个Ds位点、一个GFP标记基因与一个抗basta标记基因,可以方便高效地筛选转基因植物.目前经初步筛选获得了约10 000个独立转化株系(T1代),其中约50个株系具有明显的表型变化,包括花期改变、株型变异、叶形特异、育性降低、花发育异常、种子颜色变浅等.运用TAIL-PCR技术,成功获得了其中10个表型特异株系的T-DNA侧翼序列,分别分布于拟南芥基因组的5条染色体上.     

Distribution of T-DNA carrying a Ds element on rice chromosomes

Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agrobacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from independent transformants and classified into six main types on the basis of the origin of filler DNA between the right border of T-DNA and flanking sequence of rice genome. Type I sequences were the most common and showed canonical integration that T-DNA right border was followed by rice genome sequence with or without filler DNA of no more than 50 bp, while type II sequences displayed a vector-genome combination that T-DNA right border was followed by a vector fragment and then connected with rice genome sequence. The location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using BLAST analysis. The 340 Ds insertions at an average interval of 0.8 megabase (Mb) constructed a basic framework of Ds starter points on whole rice chromosomes. The frequency of T-DNA (Ds) inserted into the exons of predicted genes on chromosome one was 21%. Knowledge of T-DNA (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis.     

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).     

Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging efficiency. The 3' end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3' end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.     

Distribution of T-DNA carrying a Ds element on rice chromosomes

Rice is one of the most important crops in the world, and is widely studied as a model for cereal ge-nomics because of its small genome size (about 430 Mbp), and its colinearity at the sequence level with limited regions of other cereal genomes. In addition, there are a large number of rice databases document-ing molecular markers, genome sequences, EST se-quences and trait mutants[1—4]. Functional genomic studies of rice are increasing with the availability of the complete genome sequence. …     

Generation and flanking sequence analysis of a rice T-DNA tagged population

Insertional mutagenesis provides a rapid way to clone a mutated gene. Transfer DNA (T-DNA) of Agrobacterium tumefaciens has been proven to be a successful tool for gene discovery in Arabidopsis and rice (Oryza sativa L. ssp. japonica). Here, we report the generation of 5,200 independent T-DNA tagged rice lines. The T-DNA insertion pattern in the rice genome was investigated, and an initial database was constructed based on T-DNA flanking sequences amplified from randomly selected T-DNA tagged rice lines using Thermal Asymmetric Interlaced PCR (TAIL-PCR). Of 361 T-DNA flanking sequences, 92 showed long T-DNA integration (T-DNA together with non-T-DNA). Another 55 sequences showed complex integration of T-DNA into the rice genome. Besides direct integration, filler sequences and microhomology (one to several nucleotides of homology) were observed between the T-DNA right border and other portions of the vector pCAMBIA1301 in transgenic rice. Preferential insertion of T-DNA into protein-coding regions of the rice genome was detected. Insertion sites mapped onto rice chromosomes were scattered in the genome. Some phenotypic mutants were observed in the T₁ generation of the T-DNA tagged plants. Our mutant population will be useful for studying T-DNA integration patterns and for analyzing gene function in rice.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by D. Mackill