The maize transposable element Ac/Ds was chosen to construct a two-component gene trap system for barley. Two versions of Ac expressing either wild type transposase (TPase) or an N-terminally truncated transposase (TPase_103–807 32) under control of the native Ac promoter were used (Figure 1a). Both TPase-expressing elements were immobilized by removal of the five terminal bases from the 5' terminal inverted repeat (TIR) sufficient to abolish Ac transposition 33. The non-autonomous Ds element named GTDsB carries the uidA reporter gene encoding β-glucuronidase (GUS) 31. The reporter gene is preceded by engineered intron and triple splice acceptor sequences upstream of the ATG codon (Figure 1b). Each of the three constructs was stably transformed into barley cultivar Golden Promise by particle bombardment. To verify the integration of intact copies and estimate the transgene copy number, in order to select parental lines for crosses, we subjected independent lines, seven carrying Ac and 34 harbouring GTDsB, to DNA gel blot analysis. Eleven GTDsB lines with low (one to three), medium (four to seven) and high (up to 12) copy number and four TPase lines were selected as starter lines (Table 1). Two TPase lines express wild type TPase and two the truncated TPase_103–807 protein. The number of integrated Ac TPase copies was between one and four in the different lines. The expression of a functional TPase was confirmed with plants from all four TPase lines (C.K. Friedrich, personal communication) using a transient assay for TPase activity 34. From crosses of the four TPase lines with each of the 11 GTDsB lines we obtained F₁ progeny for 30 different combinations.

DNA gel blot analysis was employed to study the transposition of GTDsB in the gene trap lines. In these experiments the occurrence of a new GTDsB-hybridizing DNA fragment in comparison to the corresponding GTDsB parental line was chosen as a criterion to indicate transposition of GTDsB. We performed analysis of GTDsB excision and reinsertion events in 79 F₁ plants originating from 29 independent crosses. Nine plants (11%) derived from six independent crosses showed novel hybridizing bands that were not present in the parental GTDsB lines (Table 1).

For a second set of experiments we rescued progeny harbouring both TPase and GTDsB constructs from 28 selfed F₁ plants (F₂ parent, Table 1), each derived from an independent cross of different parental lines. A total of 191 F₂ plants, including an average of five siblings per independent cross, with the exception of lines GT39 and GT80 with one and 54 plants each, were analyzed. New GTDsB-hybridizing bands were detected in 79 F₂ plants (41%) representing 21 of the 28 F₁ gene trap lines (75%). Examples of DNA hybridization patterns are shown in Figure 2. Unique hybridization patterns, suggesting independent transposition events, were found in 49 F₂ plants (26%). Independent transposition events can be due to either transposition in independent cells of the F₁ plant, which subsequently were transmitted to progeny, or to somatic transposition in the F₂ seedling (for example see Figure 2b, plants GT80/10 and GT80/13). In contrast, early transposition of GTDsB in the F₁ generation may result in all progeny inheriting the same insertion (for example see Figure 2a, GT82/1–3). Additionally a transposition event having occurred in one of the barley tillers during the development of the F₁ plant may lead to several but not all F₂ siblings showing the same new GTDsB insertion (for example see Figure 2a, GT54/3–5). Transposition of GTDsB occurred in progeny of crosses with each of the 11 GTDsB parental lines (Table 1), evidencing that every independent parental GTDsB line carries transposition competent constructs.

The gene trap F₂ population was also screened for visible phenotypic abnormalities. In 21 of the 191 (11%) gene trap F₂ plants deviations from barley wild type phenotype were observed (Table 1). These included reduced fertility (4/21), aberrant leaf pigmentation (4/21) and stunted growth (9/21) (data not shown). Interestingly, in two plants, GT29/6 and GT39/6, showing asymmetric internodes leading to bending stems and stunted growth with shortened ears respectively, the phenotypic deviation coincided with an independent transposition event. A possible connection between the transposition event and the conspicuous phenotype must be examined in further experiments.

To prove the persistence of GTDsB transpositional activity in later generations, we subjected a total of 252 F₄ plants originating from the F₂ plants GT54/8, GT29/6, GT49/7 and GT41/3 to DNA gel blot analysis. In five of the 67 GT54/8 progeny (7.5%), 22 of the 64 GT29/6 progeny (34.4%), 10 of the 59 GT49/7 progeny (17%) and three of the 64 GT41/3 progeny (4.8%) new GTDsB-hybridizing DNA fragments were detected. These transposition events must have occurred either in the F₂ and F₃ generation or in somatic tissue of the F₄ plants.

We employed TAIL-PCR 35 to obtain DNA sequences flanking transposed GTDsB constructs from gene trap F₂ plants. In total, 32 genomic sequences ranging from 111 to 678 bp were isolated and compared to publicly available databases using BLAST searches. We considered Expectation (E) values below 1e-6 to assign a putative identity to a flanking sequence. As evidenced by similarity to expressed sequence tags (ESTs) from members of the Triticeae and maize, 19 of the 32 GTDsB insertions (59%) are located in transcribed genomic regions (Table 2). Moreover, 15 GTDsB flanking sequences (47%) show high identity to ESTs from barley (Table 2). Assuming an average sequence length of about 400 bp, the 419 146 non-overlapping barley ESTs in the databases 21 represent approximately 168 Mb of total sequence, or about 3% of the 4873 Mb barley genome 16. Consequently, a random fragment of barley DNA would have on average a 3% chance of being homologous to a barley EST in the database. Considering that more than 80% of the barley genomic sequences are intergenic heterochromatin 36 and therefore not expressed, the frequent identity of the flanking genomic sequences to barley ESTs clearly indicates a preference for GTDsB insertion into coding regions.

The expression of the GUS reporter gene was assayed by histochemical GUS staining in 141 F₂ plants, comprising 1 to 8 progeny of the 28 individual F₁ gene trap lines (Table 3). Staining for GUS expression was performed in leaves, nodes, immature florets, pollinated florets, immature grains and seedlings covering several stages of barley development. The leaves, nodes and immature florets were collected from developmental stage 49 defined following the Zadoks code system for growth staging in barley 37. Pollinated florets, immature grains and seedlings represent developmental stages 65–69, 83–85 and 10 respectively. For the majority of the 141 F₂ plants multiple explants were examined (one leaf, two nodes, 8 pollinated florets, 20 immature florets, 8 immature grains and 8 seedlings), resulting in a total of 5134 analysed explants. This experimental approach was chosen to enable the distinction of somatic from heritable events. We assume that, if only one explant of a certain organ type shows GUS expression, it may be considered as a somatic event. In contrast, if the majority or all explants of the same organ type from a single plant exhibit an equal GUS expression pattern, the event may be transmitted to progeny. Due to the two-element approach, these inheritable events can be stabilized by segregation of the TPase construct and are amenable to further analysis.

Expression of the GUS reporter could be detected in 51% (72/141) of the analysed F₂ plants (Table 3). Moreover, GUS expression was found in 24 of the 28 F₁ lines and in progeny of all GTDsB parental lines used. Examples of GUS expression in various organs are shown in Figure 3. In immature florets and in pollinated florets GUS activity was primarily detected in the palea and lemma (for examples see Figure 3a and 3b). In addition, in three cases GUS activity appeared in the stigma and pistil (data not shown). In all samples the GUS signals in culm nodes corresponded to the example shown in Figure 3d. The seedlings displayed GUS expression primarily in the scutellum (for example see Figure 3e). In five cases GUS activity could be observed in the roots (data not shown). In the majority of GUS positive seeds the expression was localized in the endosperm (for example see Figure 3c and 3f). However, in three cases GUS signals were observed in the pericarp (data not shown).

Out of the 72 GUS positive plants 45 showed GUS expression restricted to one or two explants, even when occurring in several organs, indicating that the majority of the events is due to somatic transposition of GTDsB. In such cases only a limited portion of somatic tissue carries the new GTDsB insertion and can consequently be expected to express GUS. In contrast, in 14 F₂ plants GUS expression was detected in the same tissue in at least 50% of the explants (Table 4) denoting candidates for heritable events. In eight of these candidates GUS expression was detected in scutellar tissue of seedlings (GT54/5-GT15/4) or in the endosperm of immature grains (GT63/4). Analysis of progeny will confirm the heritability of these gene trap events enabling identification of GTDsB integration sites.

The GUS staining frequency ranged between 3% and 26% in individual organs (Table 5). As expected, the highest frequencies of 26% and 24% were observed in grains and seedlings representing mostly tissues of the F₃ generation. As a consequence of transposition in F₃ tissues early events of the F₃ generation can be detected in addition to events that occurred in the preceding generations.

The expression of GUS depends on the transcriptional fusion between the reporter open reading frame and upstream gene sequences following the insertion of GTDsB into a transcription unit. Consequently, correct and efficient splicing of the gene trap construct by the host spliceosome is crucial and has been already shown for GTDsB in transient experiments 31. We aimed to demonstrate that splicing of stably integrated GTDsB constructs in the gene trap barley lines is accomplished just as accurately. For these experiments the gene trap line GT35 was chosen, since the same GUS expression pattern (Figure 3a) was found in 100% of the pollinated florets in all progeny tested, indicating an inheritable gene trap event (Table 4). Additionally, RNA gel blot analysis confirmed the occurrence of uidA-specific transcripts exceeding the size of the original uidA transcript by 1.1 and 0.4 kb, thus indicating the presence of transcriptional fusions encoding GUS in the florets of gene trap line GT35 (data not shown). We used 5'-RACE (rapid amplification of cDNA ends 38), to isolate spliced transcripts encoding GUS. Out of 17 isolated 5' sequences, in 14 the splice site A1 and in three A2 has been properly used to generate the reporter gene transcript. These findings are consistent with previous studies of GTDsB splice products in transiently transformed barley tissue, revealing that the splice acceptor sites A2 and A3 were utilized with almost equal frequencies but eleven times less frequent than A1 31.

To generate cwAc (clipped wing Ac) containing an immobilized Ac element expressing wild type TPase under the regulatory control of the native Ac promoter, five base pairs of the 5' Ac end in pJAc 62 have been deleted according to Healy et al. 33. For cwAcΔ102, expressing a transposase shortened by 102 amino acids at the N-terminus (TPase_103–807, 32), the Ac element was immobilized in the same manner. The Ds based gene trap construct GTDsB carries a promoterless uidA gene preceded by a triple splice acceptor site 31.

Immature embryos of barley (Hordeum vulgare L.; cv. Golden Promise) were transformed via particle bombardment either with GTDsB, cwAc or cwAcΔ102. To facilitate the selection of transgenic plants a synthetic pat gene (Peter Eckes, unpublished) encoding a phosphinotricin acetyltransferase that confers resistance to glufosinate-type herbicides was co-transformed. Transformation and regeneration of transgenic barley plants was performed as described by Scholz et al. 26. Barley plants were grown in the greenhouse at 16–18°C day/12–13°C night temperatures with a 16 h photoperiod.

To generate the gene trap lines, plants carrying GTDsB constructs were crossed with plants expressing either wild type transposase (TPase) or an N-terminal truncated transposase (TPase_103–807). F₁ plants containing TPase as well as GTDsB constructs were selected by PCR. Individual F₁ plants, also referred to as gene trap lines, were named GT followed by a unique number. For the F₂ generation, progeny of 28 selfed F₁ plants containing both constructs were selected in the same way. Likewise, the F₃ and F₄ generation was produced by two further rounds of selfing and PCR selection.

Genomic DNA was extracted from several leaf tips per plant as described by Palotta et al. 63. To determine the copy number of GTDsB and TPase constructs, DNA from GTDsB and TPase plants was digested with BamHI, MunI or XbaI. The integration of intact GTDsB elements was proved by digestion with EcoRI and HindIII, which release a 3.5 kb fragment from the pGTDsB transformation vector containing the complete GTDsB cassette. The integration of intact TPase constructs was confirmed by digestion with BamHI and PacI. For analysis of transposition events the genomic DNA of F₁, F₂ and F₄ gene trap plants was digested with BglII, BcuI or BamHI, all cutting only once in the GTDsB element. Digested DNA was subjected to DNA gel blot analysis as described by Scholz et al. 26. For the detection of GTDsB elements a 637 bp uidA fragment was digoxigenin labeled by PCR. The TPase specific probe was prepared by labelling a 734 bp Ac fragment with digoxigenin.

DNA regions flanking GTDsB inserts in gene trap lines were amplified by TAIL-PCR as described 3564. 10 ng of genomic DNA were used as template DNA. The specific primers for the GTDsB 5' end were: R-GUS-A (5'-CCCACAGGCCGTCGAGTTT-3'), R-GUS-1 (5'-CACGGGTTGGGGTTTCTACA-3') and 3pAH2-2.1 (5'-CCGTATTTATCCCGTTCGTTTTCGTTA-3'). The following arbitrary primers were used: AD1 (5'-NGTCGASWGANAWGAA-3'), AD2 (5'-GTNCGASWCANAWGTT-3'), AD3 (5'-WGTGNAGWANCANAGA-3'), AD4 (5'-NTCGASTWTSGWGTT-3'); AD5 (5'-NGTASASWGTNAWCAA-3'), AD6 (5'-TGWGNAGWANCASAGA-3'), AD7 (5'-AGWGNAGWANCAWAGG-3'), AD8 (5'-CAWCGICNGAIASGAA-3') and AD9 (5'-TCSTICGNACITWGGA-3'). Specific tertiary PCR fragments were purified from agarose gels with Recochips (TaKaRa, Shiga, Japan) and sequenced (DNA-Cloning-Service, Hamburg, Germany). The BLAST algorithm 65 was used to compare each sequence to the publicly available databases GenBank, EMBL/EBI and DDBJ.

Plant material for GUS staining was collected from gene trap F₂ plants considering the Zadoks code system for growth staging in barley 37. Two nodes, one leaf and 20 florets were collected from one tiller at growth stage 49. Nodes were divided, leafs dissected and florets cut from the spike. To obtain the pollinated florets eight spikelets were collected from one ear at growth stage 65–69. The awns of all florets were cut off, while sterile lateral spikelets were partially kept. At growth stage 83–85, eight grains were collected from one ear and divided after the removal of the lemma and palea. For seedling analysis, eight embryos were isolated from one ear and germinated for five days on sterile plates. Expression of GUS in barley tissues was assayed essentially as described by Jefferson 66 and Dai et al. 67. Materials, with the exception of grains, were pre-treated with 90% acetone for 2 h at 20°C. Grains were pre-treated with 70% ethanol for 2 min. Explants were washed twice in 50 mM sodium phosphate (pH 7) transferred into microtiter wells containing GUS staining solution (50 mM sodium phosphate pH 7, 10 mM EDTA, 0,05% Triton-X 100, 100 μg/ml Chloramphenicol, 1 mM X-Gluc), vacuum infiltrated for 2 min and incubated at 37°C for 24 h. Tissues were cleared by several changes of 96% ethanol and stored in 75% ethanol. The samples were observed under a light microscope (SZX9, Olympus, Japan) and images captured using a CCD camera (ColorView, Olympus, Japan) and appropriate software (analySIS, Soft Imaging System GmbH, Münster, Germany).

Total RNA was extracted as described by Chomczynski and Sacchi 68 from sterile lateral spikelets of gene trap line GT35. 1 μg was used as a template in a reverse transcription reaction (Thermoscript Reverse Transcriptase, Invitrogen, Karlsruhe, Germany) with a gene specific primer (GSP) binding to the uidA coding region in GTDsB (R-GUS-D, 5'-CGCTGGCCTGCCCAACCTTT-3'). After RNA degradation with RNase H (Invitrogen, Karlsruhe, Germany) a homopolymeric tailing reaction with Terminal Deoxynucleotidyl Transferase (MBI Fermentas, St.Leon-Rot, Germany) and dGTP was carried out. The tailed cDNA was used as a template in a PCR with a tail binding Primer (CB3, 5'-CCCCCCCCTCCCCCCC-3', H. Schmidt, unpublished) and a nested GSP (R-GUS-B, 5'-CGCGCTTTCCCACCAACGCT-3'). 1 μl PCR product was subjected to a second PCR with CB3 and a third GSP (R-GUS-A, 5'-CCCACAGGCCGTCGAGTTT-3'). Specific DNA fragments were extracted from agarose gels and subcloned for analysis.