Deletion mutation as a means of isolating avirulence genes in flax rust

Summary The interaction between flax rust,Melampsora lini, and its host, flax,Linum usitatissimum, has been extensively studied, and certain genetic features make the system an appropriate choice to utilize in isolating genes conferring avirulence in rust. A mutant that was selected for virulence on Lx plants was isolated, after treatment with gamma rays, from a strain that is genotypicallyA-L5,A-L6,A-L7,A-Lx/A-L5,A-L6,a-L7,a-Lx. These four specificities are tightly linked. Breeding tests showed that this mutant was genotypicallyA-L5,A-L6,a-L7,a-Lx/a-L5,a-L6,a-L7,a-Lx and, when made homozygous for the mutant chromosome, was virulent onL5,L6,L7, andLx. This result excludes somatic recombination as a source of the mutation and indicates deletion as a likely cause. A 250 bp genomic sequence from a strain of rust homozygous for these four linked avirulence genes (A-L5,A-L6,A-L7,A-Lx) was isolated, using a method that allows the differential cloning of the specific DNA sequences located within a deletion in the mutant genome. This clone hybridized to two EcoRI bands in genomic DNA from the strain homozygous for the four linked avirulence genes and from the strain homozygousA-L5 andA-L6 and heterozygousA-L7 andA-Lx, but showed no homology to DNA from the strain carrying the putative chromosomal deletion. The correlation between the genetically characterized deletion mutation and the isolation of a sequence from within a region of chromosome missing from this strain of rust suggests that this 250 bp tract may be part of, or closely linked to, the defined set of avirulence genes.     

Development of an efficient two-element transposon tagging system in Arabidopsis thaliana

Summary Modified Ac and Ds elements, in combination with dominant markers (to facilitate monitoring of excision, reinsertion and segregation of the elements) were introduced into Arabidopsis thaliana ecotype Landsberg erecta. The frequencies of somatic and germinal transactivation of the Ds elements were monitored using a streptomycin resistance assay. Transactivation was significantly higher from a stable Ac (sAc) carrying a 537 by deletion of the CpG-rich 5 untranslated leader of the transposase mRNA than from a wild-type sAc. However, substitution of the central 1.77 kb of the transposase open reading frame (ORF) with a hygromycin resistance marker did not alter the excision frequency of a Ds element. -Glucuronidase (GUS) or iaaH markers were linked to the transposase source to allow the identification of plants in which the transposase source had segregated away from the transposed Ds element, eliminating the possibility of somatic or germinal re-activation. Segregation of the excision marker, Ds and sAc was monitored in the progeny of plants showing germinal excision of Ds. 29% of the plants inheriting the excision marker carried a transposed Ds element.     

Ds transposition mediated by transient transposase expression in Heiracium aurantiacum

The feasibility of using transient transposase expression to mobilize Ds elements for gene tagging in Hieracium aurantiacum was evaluated. A T-DNA construct carrying the Ac transposase gene and either a visible marker gene (uidA) or the conditionally-lethal marker gene (codA) was transferred to H. aurantiacum leaf discs (previously transformed with a Ds element) by co-cultivation with Agrobacterium tumefaciens. Shoots were regenerated directly from the co-cultivated leaf discs under selection for antibiotic resistance resulting from Ds excision. Most regenerants carried unique transposition events. Of 84 regenerated plants, twenty one (25%) did not express the marker gene and the DNA coding sequence of the transposase could not be detected in seven (8.3%). Potential advantages of this method over conventional gene-tagging methods are: rapid recovery of individual transposition events; regenerated plants are isogenic; and the transient nature of transposase expression should facilitate the stabilisation of the transposed element.     

Improved flax regeneration from hypocotyls using thidiazuron as a cytokinin source

Summary The effects of thidiazuron, benzyladenine and zeatin were tested with respect to bud regeneration of different flax explants from hypocotyls, cotyledons and apices of two fibre varieties (Ariane, Viking) and one linseed variety (Antarès). These three cytokinins were tested either alone or in combination with naphthalene acetic acid, indole acetic acid or 2,4-dichlorophenoxyacetic acid.Hypocotyls were the most responsive explants. Thidiazuron was significantly the most effective followed by benzyladenine, and then zeatin, in inducing organogenesis from hypocotyl segments. The optimal thidiazuron concentration for bud regeneration from hypocotyls was 0.1–0.3 M in combination with 0.01 M of naphthalene acetic acid. Six days after plating, shoot initials began to appear on hypocotyl sections compared with ten to fifteen days when using benzyladenine or zeatin.     

Recombination among genes at the L group in flax conferring resistance to rust

Summary Fourteen of the known genes conferring resistance to rust in flax occur in the L group, and recombinational analysis has been used to study their fine structure. Three important features were observed. (a) Similar to the findings of Shepherd and Mayo, only susceptible recombinants were detected among the testcross progeny of 11 of the 15 heterozygotes involving pairs of L genes. Some of these recombinants showed variation in the degree of their susceptibility and appeared to be unstable in nature. (b) A new class of recombinants exhibiting a modified type of resistance was recovered. They occurred rarely but consistently, with frequencies similar to that of susceptible recombinants. (c) Rare resistant plants occurred among the progeny of susceptible recombinants. In each case, the specificity of the resistant plant corresponded to only one of the parental types. The relative roles of seed contamination, mutation, recombination and the transposition of genetic elements are discussed to account for these features.     

Development of Ac and Ds transposon tagging lines for gene isolation in diploid potato

For the development of an efficient transposon tagging strategy it is important to generate populations of plants containing unique independent transposon insertions that will mutate genes of interest. To develop such a transposon system in diploid potato the behavior of the autonomous maize transposable element Ac and the mobile Ds element was studied. A GBSS (Waxy) excision assay developed for Ac was used to monitor excision in somatic starch-forming tissue like tubers and pollen. Excision of Ac results in production of amylose starch that stains blue with iodine. The frequency and patterns of blue staining starch granules on tuber slices enabled the identification of transformants with different Ac activity. After excision the GBSS complementation was usually not complete, probably due to the segment of DNA flanking Ac that is left behind in the GBSS gene. Molecular and phenotypic analyses of 40 primary transformants classified into 4 phenotypic classes revealed reproducible patterns. A very high percentage (32.5%) of the primary transformants clearly showed early excision in the first transformed cell as displayed both by the analysis of the GBSS excision marker gene as well as DNA blot analyses. Genotypes useful for tagging strategies were used for crosses and the frequency of independent germinal transpositions was assessed. In crosses to Ds genotypes, excision of Ds was revealed that correlated to the activity of the Ac genotype. A line displaying Ac amplification to multiple copies conferred a high frequency of independent Ds transpositions. The genotypes described here are useful in somatic insertion mutagenesis aimed at the isolation of tagged mutations in diploid potato.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3 Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

Construction of an inducible transposon,INAc, to develop a gene tagging system in higher plants

An inducible transposable element, termed INAc (inducible Activator), was constructed for development of a gene tagging system in higher plants. The advantage of such an inducible element is that, unlike the native transposon, its excision can be induced at any time during plant development and the resulting mutants are stable after removal of the inducer. A fusion of the SA inducible promoter (PR-1a) with the Ac transposase gene was inserted together with a hygromycin resistance gene between ca. 400 bp sequences from each end of the maize Ac element, yielding INAc. The INAc element was introduced into tobacco and tomato plants. A high frequency of spontaneous transposition was apparent in primary transformed tomato calli but not in tobacco calli. Treatment of tobacco plants with salicylic acid induced transposition of INAc in both somatic and germinal tissue, with germinal transposition events being revealed by characterization of the progeny of transformed plants whose flowers were exposed to SA. The INAc element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species.     

Transgenic tomato lines containing Ds elements at defined genomic positions as tools for targeted transposon tagging

We have introduced a genetically marked Dissociation transposable element (Ds ^HPT ) into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. Probes for the flanking regions of the T-DNA and transposed Ds ^HPT elements were obtained with the inverse polymerase chain reaction (IPCR) technique and used in RFLP linkage analyses. The RFLP map location of 11 T-DNAs carrying Ds ^HPT was determined. The T-DNAs are distributed on 7 of the 12 tomato chromosomes. To explore the feasibility of gene tagging strategies in tomato using Ds ^HPT , we examined the genomic distribution of Ds ^HPT receptor sites relative to the location of two different, but very closely linked, T-DNA insertion sites. After crosses with plants expressing Ac transposase, the hygromycin phosphotransferase (HPT) marker on the Ds element and the excision markers -glucuronidase (GUS) and Basta resistance (BAR) facilitated the identification of plants bearing germinally transposed Ds ^HPT elements. RFLP mapping of 21 transposed Ds ^HPT elements originating from the two different T-DNA insertions revealed distinct patterns of reintegration sites.     

Mapping Ds insertions in barley using a sequence-based approach

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgare subsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M.-A. GrandbastienThe first three authors contributed equally to this work     

Gelling agent influences the detrimental effect of kanamycin on adventitious budding in flax

Flax (Linum usitatissimum L.) hypocotyls were cultivated on regeneration media containing various concentrations of kanamycin (an aminoglycoside antibiotic commonly used to select transgenic plant material) solidified with three different gelling agents: gellan gum, agar and a mixture of both. The inhibitory effect of kanamycin on bud regeneration was analyzed. A significant interaction was observed between the nature of the gelling agent and the kanamycin concentration. The antibiotic concentration needed to strongly inhibit bud production varied greatly with the nature of the gelling agent. Gellan gum lowered the inhibitory effect of kanamycin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

Maize Ac/Ds transposon system leads to highly efficient germline transmission of transgenes in medaka (Oryzias latipes)

As the medaka is a popular fish model in genetics, developmental biology and toxicology, the development of an efficient transgenic medaka technique is important for a variety of biological experiments. Here we demonstrated that the maize transposon system, Ac/Ds, greatly improved the transgenesis of microinjected DNA. Using the Ac/Ds system, two types of stable transgenic medaka lines, Tg(hsp70:gfp) and Tg(cyp1a1:gfp), were established with germline transmission rates of 83.3% (10/12) and 100.0% (4/4) from GFP-expressing founders, respectively. The percentages of transgenic progeny ranged between 3.1% and 100.0% in F1 from different transgenic founders. Interestingly, multiple insertions were found from transgenic founders and the cloned insertion sites confirmed the transposition mediated by Ac transposase. In addition, we demonstrated the inducible GFP expression in both GFP transgenic medaka lines. In Tg(hsp70:gfp) whose gfp gene was under the control of a heat shock inducible medaka hsp70 promoter, GFP expression was induced ubiquitously after heat shock. In Tg(cyp1a1:gfp), the gfp gene was driven by medaka cyp1a1 promoter that could be activated by various xenobiotic chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); indeed, GFP expression was found to be induced in the liver, intestine and kidney by TCDD. Our data presented here demonstrated the highly efficient transgenesis with the aid of the maize Ac/Ds transposon system.     

Botanical analysis of a bundle of flax (Linum usitatissimum L.) from an early medieval site in northern Poland; a contribution to the history of flax cultivation and its field weeds

A bundle of flax (Linum usitatissimum L.) radiocarbon dated to 1210±70 uncal B.P. (830±90 cal A.D.) was analysed for its macrofossil content. Apart from stems, capsules and seeds of flax., a large number of diaspores (fruits and seeds) from other plants was identified. Field weeds were the most numerous taxa present, among them three flax field weeds,Spergula maxima, Camelina alyssum andCuscuta cpilinum. Development of the specific flax weed community is discussed. Indicator values are used to characterize the edaphic conditions of this early medieval flax field. The field weeds spectrum also suggests that this flax was sown as a summer crop after an earlier crop of millet.     

Antisense transgenesis of tobacco with a flax pectin methylesterase affects pollen ornamentation

Summary. Antisense transgenesis of tobacco (Nicotiana tabacum) with a partial flax (Linum usitatissimum L.) pectin methylesterase (Lupme3) cDNA sequence yielded plants with altered pollen content. Moreover, the characteristically sculptured cell wall surrounding the pollen grains was modified in transgenic tobacco plants: the wavy ornamentation was dramatically reduced, suggesting the involvement of the demethylation of pectin in the pollen cell wall-specific structure. Germination of pollen was decreased and the pollen tube surface aspect was also different in transgenic plants.Correspondence and reprints: Laboratoire de Biotechnologies et Physiologie Végétales, Faculté des Sciences, Université de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex, France.