CA2561992A1 - Polynucleotides and uses thereof - Google Patents

Abstract

The present invention relates to inter alia, polynucleotides and methods of use thereof and to plants containing the polynucleotides. More specifically, the invention relates to specific wheat events which events contain a nucleotide sequence encoding a trichothecene 3-O-acetyltransferase. Further, the invention relates to methods of identifying specific wheat events which events contain a nucleotide encoding a trichothecene 3-O-acetyltransferase. More specifically, the invention provides for the identification of a transferase-containing event, in particular, the Event 1, via a method which uses at least one of the following techniques: a polynucleotide amplification reaction; a polynucleotide hybridisation reaction; and the formation of a protein-antibody complex.

Description

POLYNUCLEOTIDES AND USES THEREOF
The present invention relates to inter alia, polynucleotides and methods of use thereof and, in particular, to wheat plants comprising said polynucleotides.
The invention also relates to methods of identifying specific wheat events which contain a gene capable of conferring Fusarium resistance on said wheat plants.
Numerous fungi are serious pests of economically important agricultural crops.
Further, crop contamination by fungal toxins is a major problem for agriculture throughout to the world. Mycotoxins are toxic fungal metabolites, often found in agricultural products, that are characterized by their ability to cause health problems for vertebrates.
Trichothecenes are sesquiterpene epoxide mycotoxins produced by species of Fusarium, Trichothecium, and Myrothecium that act as potent inhibitors of eukaryotic protein synthesis. Fusarium species that produce such trichothecenes include F.
acuminatum, F.
15 crookwellense, F. culmorum, F. equiseti, F. graminearum (Gibberella zeae), F. lateritium, F. poae, F. sambucinum (G. pulicaris), and F. sporotrichioides (Marasas, W.F.O., Nelson, P.E., and Toussoun, T.A. 1984).
As previously described (A. E. Desjardins and T. M Hohn, Mycotoxins in plant 20 pathogenesis. Mol.Plant-Microbe Interact. 10 (2).147-152, 1997), both acute and chronic mycotoxicoses in farm animals and in humans have been associated with consumption of wheat, rye, barley, oats, rice and maize contaminated with Fusarium species that produce trichothecene mycotoxins. Experiments with chemically pure trichotheeenes at low dosage levels have reproduced many of the features observed in mouldy-grain toxicoses in 25 animals, including anaemia and immunosuppression, haemorrhage, emesis and feed refusal. Historical and epidemiological data from human populations indicate an association between certain disease epidemics and consumption of grain infected with Fusariurn species that produce trichothecenes. In particular, outbreaks of a fatal disease known as alimentary toxic aleukia, which has occurred in Russia since the nineteenth 30 century, have been associated with consumption of over-wintered grains contaminated with Fusarium species that produce the trichothecene T-2 toxin. In Japan, outbreaks of a similar disease called akakabi-byo or red mould disease have been associated with grain infected with Fusariurn species that produce the trichothecene, deoxynivalenol (hereinafter "DON"). Trichothecenes were detected in the toxic grain samples responsible for recent human disease outbreaks in India and Japan. There exists, therefore, a need for agricultural methods for preventing and, crops having reduced levels of, mycotoxin contamination.
Further, trichothecene-producing Fusarium species are destructive pathogens and attack a wide range of plant species. The acute phytotoxicity of trichothecenes and their occurrence in plant tissues also suggest that these mycotoxins play a role in the pathogenesis of Fusarium on plants. This implies that mycotoxins play a role in disease l0 and, therefore, reducing their toxicity to the plant may also prevent or reduce disease in the plant. Further, reduction in disease levels may have the additional benefit of reducing mycotoxin contamination of the plant and particularly in grain where the plant is a cereal plant.
15 Various methods of controlling diseases in plants, such as corn ear rot, stock rot or wheat head blight, have been used with varying degrees of success. One method of controlling plant disease has been to apply an antimicrobial chemical to crops. This method has numerous, art-recognized problems. Alternatively, a more recent method involves the use of biological control organisms ("biocontrol") which are natural 20 competitors or inhibitors of the pest organism. However, it is difficult to apply biocontrol to large areas, and even more difficult to cause those living organisms to remain in the treated area for an extended period of time. More recently, techniques in recombinant DNA have provided the opportunity to insert into plant cells, cloned genes that express antimicrobial compounds. However, this technology has given rise to concerns about 25 eventual microbial resistance to well-known, naturally occurring antimicrobials. Thus, a continuing need exists to identify naturally occurring antimicrobial agents, such as proteins, which can be formed in plant cells directly by translation of a single gene.
A trichothecene 3-O-acetyltransferase that catalyses the acetylation of a number of 30 different Fusarium trichothecenes including DON at the C3 hydroxyl group has been identified in F. sporotrichioides (S. P. McCormick, N. J. Alexander, S. C.
Trapp, and T.
M. Hohn. Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusczrium sporotrichioicles. Applied.Environ.Mierobiol. 65 (12):5252-5256, 1999).

Acetylation of trichothecenes at the C3 hydroxyl group significantly reduces their toxicity in vertebrates and plants and results in the reaction product 3-acetyldeoxyvalenol (hereinafter "3ADON"), see Kimura et al. below.
The sequence of stnictural genes encoding trichothecene 3-O-acetyl transferases from Fusarium graminearum and Fusaria~m sporotrichioides, as well as sequences of other orthologs, has been published. See, e.g. Kimura et al., Biosci.
Biotechnol. Biochem., 62(5): 1033-1036 (1998), and Kimura et al., FEBS Letters, 435:163-168 (1998).
Further, it has been speculated that the gene from Fusarium sporotrichioides encoding a to trichothecene 3-O-acetyl transferase may be useful in developing plant varieties with increased resistance to Fusarium. See e.g. Hohn, T.M. et al. Molecular Genetics of Host-Specific Toxins in Plant Disease, 17-24 (1998) and Kimura et al. J. Biological Chemistry, 273(3):1654-1661 (1998).
I S Plants expressing these trichothecene 3-O-acetyltransferase genes have now been made and have been shown to be trichothecene and Fusarium resistant. The basic technology is described in International Patent Application Publication Number WO
00/60061.
20 The present invention provides, inter alia, a specific wheat event (referred to hereinafter as 'Event 1') and methods for the identification thereof. This specific event has been selected based on, inter alia, its agronomic performance and the fact that it is a single copy event. It is believed that the characteristics of this event are far superior to like transformants based upon, inter alia, the integration site of the transgene during the 25 transformation process. The present invention utilises a trichothecene 3-O-acetyltransferase gene isolated from F. sporotrichioides. It will be appreciated that the event of the invention can be defined by way of the unique nucleotide sequence which is formed when the construct used to create the event inserts in the wheat genome. In particular, the nucleotide sequence at the 5' and 3' event boundaries is distinctive of the 3o specific event.
Therefore, according to the present invention there is provided a polynucleotide which comprises a first region comprising the sequence depicted as SEQ ID NO:
1 and a further region which comprises the sequence depicted as SEQ ID NO: 2. It is noted that SEQ ID NO: 1 is the sequence of the 5' end of the event of the invention. In particular, nucleotides 1 to 1393 of SEQ ID NO: 1 are wheat genomic sequence, 1394 to 1563 are vector sequence and 1564 to 1788 are promoter sequence. In SEQ ID NO: 2, which is the sequence of the 3' end of the event of the invention, nucleotides 1 to 204 are terminator sequence, 205 to 426 are vector sequence and 427 to 2471 are wheat genomic sequence.
In a further embodiment said polynucleotide comprises a region which can be amplified by an amplification reaction which reaction uses the primers depicted as SEQ
1o ID NO: 5 and 6. These primers are located in the coding sequence of the trichothecene 3-O-acetyltransferase gene encoded by the event of the present invention (SEQ ID
NO: 5) and in the wheat genomic sequence 3' to the insertion site (SEQ ID NO: 6). In a still further embodiment said polynucleotide comprises a still further region which encodes a' 3-O-acetyltransferase from F. sporotrichioides. In a still further embodiment said polynucleotide comprises a region which provides for the maize polyubiquitin promoter operably linked to said 3-O-acetyltransferase.
In a further aspect of the invention there is provided a polynucleotide which comprises at least 18 contiguous nucleotides of the sequence depicted as SEQ
ID NO: 3.
2o Still further provided is a polynucleotide which comprises at least 20 contiguous nucleotides of the sequence depicted as SEQ ID NO: 3. Still further provided is a polynucleotide which comprises at least 25 contiguous nucleotides of the sequence depicted as SEQ ID NO: 3. Still further provided is a polynucleotide which comprises the sequence depicted as SEQ ID NO: 3.
Still further provided is a polynucleotide which comprises at least 35 contiguous nucleotides of the sequence depicted as nucleotides 1364 to 1423 of SEQ ID NO:
1. Still further provided is a polynucleotide comprising at least 40 contiguous nucleotides of the sequence depicted as nucleotides 1364 to 1423 of SEQ ~ NO: 1. Still further provided is 3o a polynucleotide comprising at least 50 contiguous nucleotides of the sequence depicted as nucleotides 1364 to 1423 of SEQ ID NO: 1. Still further provided is a polynucleotide comprising the sequence depicted as nucleotides 1364 to 1423 of SEQ ID NO: 1.

Still further provided is a polynucleotide comprising at least 70 contiguous nucleotides of the sequence depicted as nucleotides 1334 to 1453 of SEQ ID NO:
1. Still further provided is a polynucleotide comprising at least 90 contiguous nucleotides of the sequence depicted as nucleotides 1334 to 1453 of SEQ ID NO: 1. Still further provided is a polynucleotide comprising at least 100 contiguous nucleotides of the sequence depicted as nucleotides 1334 to 1453 of SEQ ID NO: 1. Still further provided is a polynucleotide comprising the sequence depicted as nucleotides 1334 to 1453 of SEQ ID NO: 1.
Still further provided is a polynucleotide comprising at least 110 contiguous to nucleotides of the sequence depicted as nucleotides 1304 to 1483 of SEQ ID
NO: 1. Still further provided is a polynucleotide comprising at least 130 contiguous nucleotides of the sequence depicted as nucleotides 1304 to 1483 of SEQ ID NO: 1. Still further provided is a polynucleotide comprising at least 150 contiguous nucleotides of the sequence depicted as nucleotides 1304 to 1483 of SEQ ID NO: 1. Still further provided is a polynucleotide comprising the sequence depicted as nucleotides 1304 to 1483 of SEQ ID NO: 1.
Still further provided is a polynucleotide comprising at least 160 contiguous nucleotides of the sequence depicted as nucleotides 1274 to 1513 of SEQ ID NO:
1. Still further provided is a polynucleotide comprising at least 200 contiguous nucleotides of the 2o sequence depicted as nucleotides 1274 to 1513 of SEQ ID NO: 1. Still further provided is a polynucleotide comprising at least 220 contiguous nucleotides of the sequence depicted as nucleotides 1274 to 1513 of SEQ )D NO: 1. Still further provided is a polynucleotide comprising the sequence depicted as nucleotides 1274 to 1513 of SEQ ID NO: 1.
Still further provided is a polynucleotide comprising at least 50, 100, 150, 200, 300, 400 or 500 contiguous nucleotides of SEQ ID NO: 1 said polynucleotide containing the nucleotide junction between nucleotides 1393 and 1394 of SEQ ID NO: 1.
Still further provided is a polynucleotide which comprises the sequence depicted as SEQ ID
NO: 1.
Still further provided is a sequence which is the complement of a sequence described above.

In a further aspect of the invention there is provided a polynucleotide which comprises at least 18 contiguous nucleotides of the sequence depicted as SEQ
ID NO: 4.
Still further provided is a polynucleotide which comprises at least 20 contiguous nucleotides of the sequence depicted as SEQ ID NO: 4. Still further provided is a polynucleotide which comprises at least 25 contiguous nucleotides of the sequence depicted as SEQ ID NO: 4. Still further provided is a polynucleotide which comprises the sequence depicted as SEQ ID NO: 4.
Still further provided is a polynucleotide which comprises at least 35 contiguous to nucleotides of the sequence depicted as nucleotides 397 to 456 of SEQ ID
NO: 2. Still further provided is a polynucleotide which comprises at least 40 contiguous nucleotides of the sequence depicted as nucleotides 397 to 456 of SEQ ID NO: 2. Still further provided is a polynucleotide which comprises at least 50 contiguous nucleotides of the sequence depicted as nucleotides 397 to 456 of SEQ ID NO: 2. Still further provided is a 15 polynucleotide which comprises the sequence depicted as nucleotides 397 to 456 of SEQ
ID NO: 2.
Still further provided is a polynucleotide which comprises at least 70 contiguous nucleotides of the sequence depicted as nucleotides 367 to 486 of SEQ ID NO:
2. Still 20 further provided is a polynucleotide which comprises at least 90 contiguous nucleotides of the sequence depicted as nucleotides 367 to 486 of SEQ ID NO: 2. Still further provided is a polynucleotide which comprises at least 100 contiguous nucleotides of the sequence depicted as nucleotides 367 to 486 of SEQ ID NO: 2. Still further provided is a polynucleotide which comprises the sequence depicted as nucleotides 367 to 486 of SEQ
25 ID NO: 2.
Still further provided is a polynucleotide which comprises at least 110 contiguous nucleotides of the sequence depicted as nucleotides 337 to 516 of SEQ ID NO:
2. Still further provided is a polynucleotide which comprises at least 130 contiguous nucleotides 30 of the sequence depicted as nucleotides 337 to 516 of SEQ ID NO: 2. Still further provided is a polynucleotide which comprises at least 150 contiguous nucleotides of the sequence depicted as nucleotides 337 to 516 of SEQ ID NO: 2. Still further provided is a _7_ polynucleotide which comprises the sequence depicted as nucleotides 337 to 5156 of SEQ
ID NO: 2.
Still further provided is a polynucleotide which comprises at least 50, 100, 150, 200, 300, 400 or 500 contiguous nucleotides of SEQ ID NO: 2, said polynueleotide containing the nucleotide junction between nucleotides 426 and 427 or SEQ ID
NO: 2.
Still further provided is a polynucleotide which comprises the sequence depicted as SEQ
ID NO: 2.
Still further provided is a sequence which is the complement of a sequence described above.
In a still further aspect there is provided a wheat plant which comprises a polynucleotide described above. In a still further embodiment there is provided a wheat seed which comprises the polynucleotide as described above. In a still further embodiment said wheat plant or seed comprises "Event 1".
In a still further aspect there is provided a wheat plant which comprises a polynueleotide which can be amplified during an amplification reaction which reaction uses a primer comprising the sequence depicted as SEQ ID NO: 5 and a primer comprising the sequence depicted as SEQ ID NO: 6.
In a still further aspect there is provided a wheat plant which comprises the polynucleotide depicted as SEQ ID NO: 7 or SEQ ID NO: 8.
The present invention further provides a method for detecting a plant which contains the polynucleotide depicted as SEQ ID NO: 1 or the polynucleotide depicted as SEQ ID NO: 2 said method comprising: (a) preparing a sample containing the genomic DNA of the plant to be tested; and (b) designing a pair of primers which are suitable for use in an amplification reaction to amplify a sequence selected from the group consisting of: (i) a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO: 3 and the complement thereof and (ii) a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO: 4 and the complement _g_ thereof; and (c) adding said pair of primers to said sample and the means for performing an amplification reaction; and (d) performing an amplification reaction; and (e) visualising the thus amplified sequence.
There are many amplification methods that may be used in accordance with the methods of the invention. The underlying principle, a known technique to those skilled in the art, is the polymerase chain reaction (PCR). The amplification product from a PCR
reaction may be visualised by staining with ethidium bromide and excitation with UV
light, typically after size separation using agarose gel electrophoresis. In a particular to embodiment of the invention variations of the PCR principle such as TaqManTM may be used. Such techniques involve labelling at least one of the primers involved in the amplification process with a fluorescent dye. When unbound, the primer adopts a conformation such that no fluorescence can be detected. However, when the primer is bound to a piece of DNA, the conformation changes and fluorescence can be detected. In this way, the amplification process can be monitored in real-time, the intensity of fluorescence corresponding directly to the level of amplification. Further embodiments of the present invention include, but are not limited to, RACE PCR.
The present invention further provides a method for detecting a plant which 2o contains the polynucleotide depicted as SEQ ID NO: 1 said method comprising: (a) preparing a sample containing the genomic DNA of the plant to be tested; and (b) designing a pair of primers which are suitable for use in an amplification reaction to amplify a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO: 3 and the complement thereof; and (c) adding said pair of primers to said sample and the means for performing an amplification reaction; and (d) performing an amplification reaction; and (e) visualising the thus amplified sequence.
The present invention further provides a method as described above wherein said primers are suitable for use in an amplification reaction to amplify a sequence comprising 3o at least 20 contiguous nucleotides of the sequence depicted as SEQ ID NO: 3 and the complement thereof. In a still further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 25 contiguous nucleotides of the sequence depicted as SEQ ID NO: 3 and the complement thereof. In a still further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising the sequence depicted as SEQ ID NO: 3 and the complement thereof.
The present invention still further provides a method as described above wherein the sequence to be amplified by said amplification reaction comprises a sequence containing the nucleotide junction of genomic sequence-transgene cassette insert (c-c) provided as nucleotides 1393/1394 of SEQ ID NO: 1. The person skilled in the art will appreciate that this junction can be used to characterise and thus identify the event and so it is well within the ambit of said skilled person to design and produce oligonucleotide primer sequences that are suitable for use in an amplification reaction to amplify the sequence which comprises the aforesaid junction. For example, the amplification product may comprise a small region of the genomic sequence, which genomic sequence is indicated as comprising nucleotides 1 to 1393 of SEQ ID NO: 1 and a larger region of the insert sequence, which insert sequence is indicated as the sequence comprising nucleotides 1394 to 1788 of SEQ ID NO: 1. Alternatively, the amplification product may comprise a small region of the insert sequence and a larger region of the genomic sequence. The person skilled in the art will also appreciate that the primer sequences suitable for use in an amplification reaction may be designed based on the genomic sequence which is 5' i.e.
upstream of nucleotide number 1 of SEQ ID NO: 1 and the insert or genomic sequence 2o which is 3' i.e. downstream of nucleotide number 1788 of SEQ ID NO: 1.
The present invention further provides a method for detecting a plant which contains the polynucleotide depicted as SEQ ID NO: 1 said method comprising:
(a) preparing a sample containing the genomic DNA of the plant to be tested; and (b) designing a pair of primers which are suitable for use in an amplification reaction to amplify a sequence comprising at least 35 contiguous nucleotides of the sequence depicted as nucleotides 1364 to 1423 of SEQ ID NO: 1 and the complement thereof; and (c) adding said pair of primers to said sample and the means for performing an amplification reaction; and (d) performing an amplification reaction; and (e) visualising the thus 3o amplified sequence. In a further embodiment said primers are suitable for use in an ampliFcation reaction to amplify a sequence comprising at least 40 contiguous nucleotides of the sequence depicted as nucleotides 1364 to 1423 of SEQ ID NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 50 contiguous nucleotides of the sequence depicted as nucleotides 1364 to 1423 of SEQ ID NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising the sequence depicted as nucleotides 1364 to 1423 of SEQ ID NO: 1.
In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 70 contiguous nucleotides of the sequence depicted as nucleotides 1334 to 1453 of SEQ ID NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a to sequence comprising at least 90 contiguous nucleotides of the sequence depicted as nucleotides 1334 to 1453 of SEQ ID NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 100 contiguous nucleotides of the sequence depicted as nucleotides 1334 to 1453 of SEQ ID
NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising the sequence depicted as nucleotides 1334 to 1453 of SEQ ID NO: 1.
In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 110 contiguous nucleotides of the sequence depicted as nucleotides 1304 to 1483 of SEQ ID NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 130 contiguous nucleotides of the sequence depicted as nucleotides 1304 to 1483 of SEQ ID NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 150 contiguous nucleotides of the sequence depicted as nucleotides 1304 to 1483 of SEQ ID NO:
1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising the sequence depicted as nucleotides 1304 to 1483 of SEQ
ID NO: 1.
3o In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 160 contiguous nucleotides of the sequence depicted as nucleotides 1274 to 1513 of SEQ ID NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 200 contiguous nucleotides of the sequence depicted as nucleotides 1274 to 1513 of SEQ ID NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 220 contiguous nucleotides of the sequence depicted as nucleotides 1274 to 1513 of SEQ ID
NO: 1. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising the sequence depicted as nucleotides 1274 to 1513 of SEQ ID NO: 1.
In a still further embodiment said primers are suitable for use in an amplification to reaction to amplify a sequence comprising at least 50, 100, 150, 200, 300, 400 or 500 contiguous nucleotides of SEQ ID NO: 1 said sequence containing the nucleotide junction between nucleotides 1393 and 1394 of SEQ ID NO: 1. The primers referred to above are suitable for use in an amplification reaction to amplify the sequences mentioned above and the complementary sequences thereof.
The present invention still further provides a sequence which is the amplification product of the method described above.
The present invention still further provides a sequence which is the complement of 2o a sequence described above.
The present invention still further provides a method as mentioned above wherein the thus amplified product comprises a sequence as described above.
The present invention still further provides a method as described above wherein said pair of primers comprise a forward primer which comprises a sequence which when read in the 5'-~3' direction is identical to a region of the sequence depicted as nucleotides 1 to 1393 of SEQ ID NO: 1 and the reverse primer comprises a sequence which when read in the 5'-~3' direction is identical to a region of the reverse complement of the sequence 3o depicted as nucleotides 1394 to 1788 of SEQ ID NO: 1. The person skilled in the art will recognise that a number of primers suitable for use in the methods of the invention may be created based on the sequences provided herein and the complementary sequences thereto.
In addition to this, as mentioned above, such primer sequences may be based on the sequence 5' and 3' (upstream and downstream) of the sequences depicted as SEQ
ID NO:
1 and it is well within the capability of the skilled person to identify such 5' and 3' sequence.
In a particular embodiment of the invention said pair of primers comprise the sequences depicted as SEQ ID NO: 5 and 6.
The present invention further provides a method for detecting a plant which contains the polynucleotide depicted as SEQ ID NO: 2 said method comprising:
(a) preparing a sample containing the genomic DNA of the plant to be tested; and (b) designing a pair of primers which are suitable for use in an amplification reaction to amplify a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO: 4 and the complement thereof; and (c) adding said pair of primers to said sample and the means for performing an amplification reaction; and (d) performing an amplification reaction; and (e) visualising the thus amplified sequence.
The present invention further provides a method as described above wherein said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 20 contiguous nucleotides of the sequence depicted as SEQ ID NO: 4 and the complement thereof. In a still further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 25 contiguous nucleotides of the sequence depicted as SEQ ID NO: 4 and the complement thereof. In a still further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising the sequence depicted as SEQ ID NO: 4 and the complement thereof.
The present invention still further provides a method as described above wherein the sequence to be amplified by said amplification reaction comprises a sequence containing the nucleotide junction of transgene cassette insert-genomic sequence (c-g) provided as nucleotides 426/427 of SEQ ID NO: 2. The person skilled in the art will appreciate that this junction can be used to characterise and thus identify the event and so it is well within the ambit of said skilled person to design and produce oligonucleotide primer sequences that are suitable for use in an amplification reaction to amplify the sequence which comprises the aforesaid junction. For example, the amplification product may comprise a small region of the genomic sequence, which genomic sequence is indicated as comprising nucleotides 427 to 2471 of SEQ ID NO: 2 and a larger region of the insert sequence, which insert sequence is indicated as the sequence comprising nucleotides 1 to 426 of SEQ ID NO: 2. Alternatively, the amplification product may comprise a small region of the insert sequence and a larger region of the genomic sequence. The person skilled in the art will also appreciate that the primer sequences suitable for use in an amplification reaction may be designed based on the insert or genomic sequence which is 5' i.e. upstream of nucleotide number 1 of SEQ ID
NO: 2 and the genomic sequence which is 3' i.e. downstream of the genomic sequence nucleotide 1o number 427 of SEQ ID NO: 2.
The present invention further provides a method for detecting a plant which contains the polynucleotide depicted as SEQ ID NO: 2 said method comprising:
(a) preparing a sample containing the genomic DNA of the plant to be tested; and (b) designing a pair of primers which are suitable for use in an amplification reaction to amplify a sequence comprising at least 35 contiguous nucleotides of the sequence depicted as nucleotides 397 to 456 of SEQ ID NO: 2 and the complement thereof; and (c) adding said pair of primers to said sample and the means for performing an amplification reaction; and (d) performing an amplification reaction; and (e) visualising the thus 2o amplified sequence. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 40 contiguous nucleotides of the sequence depicted as nucleotides 397 to 456 of SEQ ID NO: 2. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 50 contiguous nu:.leotides of the sequence depicted as nucleotides 397 to 456 of SEQ )D NO: 2. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising the sequence depicted as nucleotides 397 to 456 ofd SEQ ID NO: 2.
In a further embodiment said primers are suitable for use in an amplification 3o reaction to amplify a sequence comprising at least 70 contiguous nucleotides of the sequence depicted as nucleotides 367 to 486 of SEQ ID NO: 2. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 90 contiguous nucleotides of the sequence depicted as nucleotides 367 _ ly _ to 486 of SEQ ID NO: 2. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 100 contiguous nucleotides of the sequence depicted as nucleotides 367 to 486 of SEQ ID NO:
2. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising the sequence depicted as nucleotides 367 to 486 of SEQ
ID NO: 2.
In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 110 contiguous nucleotides of the to sequence depicted as nucleotides 337 to 516 o:f SEQ ID NO: 2. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 130 contiguous nucleotide, of the sequence depicted as nucleotides 337 to S 16 of SEQ ID NO: 2. In a further embodiment said primers are suitable for use in an amplification reaction to amplify a sequenc~° comprising at least 150 contiguous nucleotides of the sequence depicted as nucleotides 337 to 516 of SEQ ID NO:
2. In a further embodiment said primers are suitable fir use in an amplification reaction to amplify a sequence comprising the sequence depicted as nucleotides 337 to 516 of SEQ
ID NO: 2.
2o In a still further embodiment said primers are suitable for use in an amplification reaction to amplify a sequence comprising at least 50, 100, 150, 200, 300, 400 or 500 contiguous nucleotides of SEQ ID NO: 2 said sequence containing the nucleotide junction between nucleotides 426 and 427 of SEQ ID NO: 2. The primers referred to above are suitable for use in an amplification reaction to amplify the sequences mentioned above and the complementary sequences thereof.
The present invention still further provides a sequence which is the amplification product of the method described above.
3o The invention further provides a sequence which is the complement of a sequence described above.

The present invention still further provides a method as mentioned above wherein the thus amplified product comprises a sequence as described above.
The present invention still further provides a method as described above wherein said pair of primers comprise a forward primer which comprises a sequence which when read in the 5'-~3' direction is identical to a region of the sequence depicted as nucleotides 1 to 426 of SEQ ID NO: 2 and the reverse primer comprises a sequence which when read in the 5'~3' direction is identical to a region of the reverse complement of the sequence depicted as nucleotides 427 to 2471 of SEQ ID NO: 2. The person skilled in the art will 1 o recognise that a number of primers suitable for use in the methods of the invention may be created based on the sequences provided herein and the complementary sequences thereto.
In addition to this, as mentioned above, such primer sequences may be based on the sequence 5' and 3' (upstream and downstream) of the sequences depicted as SEQ
ID NO:
2 and it is well within the capability of the skilled person to identify such 5' and 3' t5 sequence.
The present invention still further provides a method for detecting a plant which contains the polynucleotide depicted as SEQ ID NO: 1 and/or the polynucleotide depicted as SEQ ID NO: 2 said method comprising: (a) preparing a sample containing the genomic 2o DNA of the plant to be tested; and (b) designing a probe which is capable of hybridising to a sequence selected from the group consisting of a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO: 3 and a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ
ID NO: 4;
and (c) adding at least one of the probes of step (b) to said sample under conditions which 25 allow said probe to hybridise with a complementary nucleic acid within said sample; and (d) removing substantially non-hybridised probe by washing; and (e) detecting the thus hybridised probe to identify if the sample contains said polynucleotide. In a particular embodiment of said method said probe comprises at least 20 contiguous nucleotides. In a still further embodiment of said method, said probe comprises at least 50, 100, 150, 200, 30 300, 400 or 500 contiguous nucleotides of SEQ ID NO: 1, said probe containing the nucleotide junction between nucleotides 393 and 394 of SEQ ID NO: 1 or at least 50, 100, 150, 200, 300, 400 or 500 contiguous nucleotides of SEQ ID NO: 2, said probe containing the nucleotide junction between nucleotides 426 and 427 of SEQ ID NO: 2. In a still further embodiment of the invention, said probe may comprise a fragment of a relevant polynucleotide described within this specification. In particular, said probe may comprise a polynucleotide sequence which is capable of hybridising with a sequence which characterises the event described in the present application. In a still further embodiment of said method, said washing takes place under high stringency conditions.
Said probe may be generated and labelled using techniques well known to the person skilled in the art.
The probe may be, for example, a PCR product or restriction digestion fragment.
to In a further embodiment, the probe as described herein may be tagged with a fluorescent, radioactive, enzymatic or other suitable label to enable hybridisation to be detected.
In a still further embodiment of the present invention there is provided a method of hybridising a probe to the complementary nucleic acid within the sample under stringent conditions and detecting whether the probe has hybridised. High stringency hybridisation 15 conditions are well known to the skilled person and comprise, for example:
hybridisation at a temperature of about 65°C in a solution containing 6 x SSC, 0.01%
SDS and 0.25%
skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2 x SSC and 0.1% SDS. The skilled person may alternatively select the following hybridisation conditions, viz., hybridisation at a temperature of between 60°C
20 and 65°C in 0.3 strength citrate buffered saline containing O.I% SDS
followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS.
The person skilled in the art may also select further hybridisation conditions that are equally understood to be "high stringency" conditions. Suitable techniques for detecting plant material derived from the event described herein based on the hybridisation principle 25 include, but are not limited to Southern Blots, Northern Blots and in-situ hybridisation.
The skilled person is familiar with these techniques. Typically, they involve incubating a probe with a sample, washing to remove unbound probe, and detecting whether the probe has hybridised. Said detection method is dependent on the type of tag attached to the probe - for example, a radioactively labelled probe can be detected by exposure to and 30 development of x-ray film. Alternatively, an enzymatically labelled probe may be detected by conversion of a substrate to effect a colour change.

In a still further aspect there is provided a method for identifying a plant comprising the event described herein said method comprising (a) preparing a sample containing the genomic DNA of the plant to be tested; (b) digesting said DNA
via a restriction enzyme; (c) separating the digested DNA fragments and transferring the thus separated fragments to a membrane; (d) probing the thus bound fragments with a probe, designed as described above, which probe has be labelled to allow its visualisation; (e) removing substantially non-hybridised probe; and (f) detecting the thus hybridised probe wherein said event can be characterised by said probe hybridising to fragments having a particular size.
In a further aspect there is provided a wheat event which is capable of being identified by a method according to the invention. In a particular embodiment said method is the one according to the preceding paragraph.
In a still further aspect of the invention there is provided a method for detecting a plant which contains a protein capable of being encoded by a polynucleotide depicted as SEQ ID NO: ?, said method comprising: (a) preparing a protein-extract of the plant to be tested; and (b) providing an antibody which is capable of binding to a trichothecene 3-O-acetyltransferase protein from F. sporotrichioides; (c) adding said antibody to said extract under conditions which allow said antibody to bind to said protein within said extract; and (d) detecting the thus bound antibody to identify if the extract contains said protein.
In a further aspect of the invention there is provided a method for detecting a plant which comprises a trichothecene 3-O-acetyltransferase from F. sporotrichioides said method comprising: (a) preparing a protein-extract of the plant to be tested;
and (b) providing an antibody which is capable of binding to a trichothecene 3-O-acetyltransferase protein from F. sporotrichioides (c) adding said antibody to said extract or said extract to said antibody under conditions which allow said antibody to bind to said transferase protein within said extract; and (d) detecting the thus bound antibody to identify if the extract contains said transferase protein. In a still further aspect there is provided a method for detecting a plant as described above which plant comprises Event I .

Suitable methods of detecting plant material derived from the event described herein which methods are based on said antibody binding include, but are not limited to Western Blots, Enzyme-Linked ImmunoSorbent Assays (ELISA) and SELDI mass spectrometry. The skilled person is familiar with these and further immunological techniques. Typical steps include incubating a sample with an antibody that binds to the said protein, washing to remove unbound antibody, and detecting whether the antibody has bound. Many such detection methods are based on enzymatic reactions - for example the antibody may be tagged with an enzyme such as horseradish peroxidase, and on application of a suitable substrate, a colour change detected. Suitable antibodies may be to monoclonal or polyclonal.
In another aspect of the invention there is provided a method of detecting plant material derived from an event described herein said method comprising obtaining a sample for analysis; making a protein extract of the sample; providing a test strip or dipstick designed to detect the presence of a said protein present within the sample;
incubating the test strip or dipstick with the sample; and detecting whether said protein is present.
In one embodiment, this method is an antibody-based detection method for the 2o events referred to herein and uses test strips or dipsticks. Typical steps include incubating a test strip or dipstick with a sample and observing the presence or absence of coloured bands on the test strip or dipstick. The coloured bands are indicative of the presence of a protein in the sample. Such test strip or dipstick tests are usually protein specific, and may be used for rapid testing of samples in the field.
In one embodiment, the dipstick utilises an antibody or antibodies, or fragment/fragments thereof, specific for the trichothecene 3-O-acetyltransferase from F.
sporotrichioides as encoded by nucleotides 3591 to 4970 of SEQ ID NO: 7.
Antibody fragments include, but are not limited to, Fab, modified Fab, diFab, Fab', F(ab')2 or FV
3o fragment, immunoglobulin light chain or heavy chain monomer, single chain FV (scFV) or nanobody. The antibody or fragment thereof may be monoclonal or polyclonal.
In a particular embodiment, the antibody is an antibody secreted by cell lines selected from the group consisting of DSM ACC 2679 and DSM ACC 2680 or an antibody which is capable of inhibiting the binding to the trichothecene 3-O-acetyltransferase of an antibody secreted by cell lines selected from the group consisting of DSM ACC 2679 and DSM ACC
2680.
It is noted that methods for producing both monoclonal and polyclonal antibodies and fragments thereof are well known in the art.
Suitable test strips or dipsticks and materials for their use are described in PCT
application WO 02/27322 and are, for example, lateral-flow immunostrips comprising a detection membrane of cellulose acetate, cellulose, nitrocellulose or nylon, supported on a plastic backing. Such an immunostrip may be produced using membranes and filters to through which a liquid sample is drawn by capillary action. The protein in the sample reacts with the antibodies contained in the immunostrip as it moves the length of the strip and is captured at a line that becomes visible. Suitable means of detection are, for example, colloidal gold and coloured latex beads.
is In a particular embodiment, a line of specific anti-trichothecene 3-O-acetyltransferase antibody, as described above, is sprayed on a test strip, which is suitably made from nitrocelluose supported on a plastic backing. A reagent control line of anti-mouse antibody is sprayed in parallel above the first antibody line. The membrane is flanked on the top by an absorption pad and on the bottom by a pad containing dried .>.o colloidal gold labelled anti-trichothecene 3-O-acetyltransferase antibody. In a preferred embodiment, the colloidal gold-labelled anti-trichothecene 3-O-acetyl transferase antibody is different from the first antibody sprayed as the test line. In a particular embodiment, the colloidal gold-labelled anti-trichothecene 3-O-acetyl transferase antibody is the antibody secreted by cell line DSM ACC 2679 and the antibody sprayed at the test line is the 2.5 antibody secreted by cell line DSM ACC 2680. A sample application pad flanks the colloidal gold pad. In use, the sample application pad is placed in a sample of extracted tissue or this sample is applied to the pad in another way, for example, by pipette. Any trichothecene 3-O-acetyltransferase protein contained within the sample flows up the strip and becomes bound by the colloidal gold labelled-anti-trichothecene 3-O-acetyltransferase 3o antibody. As it continues up the strip, the protein also becomes bound by the anti-trichothecene 3-O-acetyltransferase antibody at the test line. Excess gold conjugate is captured at the reagent control line. In a positive test, that is, if trichothecene 3-O-acetyltransferase is present in the sample, a double red line appears: the lower line indicates the presence of trichothecene 3-O-acetyltransferase while the upper line is the control line signalling a properly working device.
In a still further aspect of the invention there is provided a kit of parts which comprises a pair of primers as described above and instructions for performing the method as described above and means for performing an amplification reaction and optionally means for preparing the sample to be tested. In a still further embodiment there is provided a kit of parts which comprises an antibody as described above and instructions for performing the method as described above and means for performing the method as described above and optionally means for preparing the sample to be tested. In a still further embodiment of the present invention, said kit of parts may comprise DNA
amplification-detection technology such as PCR or TaqManTM. In a still further embodiment of the present invention, said kit of parts may comprise probe hybridisation-detection technology such as Southern Blots, Northern Blots or in-situ Hybridisation. In another embodiment of the present invention, said kit of parts may comprise antibody binding-detection technology such as Western Blots, ELISA's, SELDI mass spectrometry, test strips or dipsticks. In a further embodiment of the present invention, said kit of parts may comprise any combination of the aforementioned detection technologies. In a still further embodiment, said kit of parts may comprise in the form of instructions one or more of the methods described above.
In a still further aspect there is provided a wheat seed which comprises a polynucleotide as described herein. Further provided is a wheat plant or seed which is derivable from an event as described herein.
In a still further aspect the plant or seed according to the invention further comprises a polynucleotide which provides for a trait selected from the group consisting of: insect resistance and/or tolerance; fungal resistance and/or tolerance;
disease resistance and/or tolerance; improved resistance and/or tolerance to stress; a substance having pharmaceutical activity and/or any other desired agronomic trait.
In a still further aspect there is provided a plant or seed according to the invention which is used in a method of breeding. For example, the plants may be used to transfer the trait which provides for trichothecene/Fusarium resistance into a plant of the same genus but having a different background germplasm. Such breeding into a different germplasm may be desired if the plant is to be grown in under conditions where an alternative germplasm is favourable. Methods for breeding that can be used to transfer the trait into a different background germplasm are well known in the art. Of course, it is noted that, when using these breeding methods for transferring the trait into a different germplasm, the resultant wheat plant will still contain the unique nucleotides of the invention as the insertion site for the trait will not be changed.
In a still further aspect there is provided the use of a plant or seed according to the invention to generate explant material for use in a method of transformation of said explant with a further genetic trait. Once provided with the events that can be identified by the methods of the present invention it is well within the capabilities of the person skilled in the art to generate such explant material and use in further transformation procedures. Furthermore, once provided with the events that can be identified by the methods of the present invention it is well within the capabilities of the person skilled in the art to use said events in breeding methods as described herein.
In a still further aspect there is provided a polynucleotide comprising a sequence selected from the sequences depicted in the sequence listing as SEQ ID NOS: 1 to 8 inclusive.
In a still further aspect there is provided a polynucleotide consisting of a sequence selected from the sequences depicted in the sequence listing as SEQ ID NOS: 1 to 7 inclusive.
Throughout this specification the term "seed" may be interchanged with the term "grain".
Throughout this specification the term "containing" when used in the context "plant containing" or "nucleotide containing" may be interchanged with the term " »
comprising .

The invention will now be described by way of the following non-limiting examples with reference to the Sequence Listing of which:
SEQ ID NO: 1 = Event 1 5'~3' (coding strand) Genomic sequence-Insert. In particular, nucleotides 1 to 1393 are wheat genomic sequence, 1394 to 1563 are vector sequence and 1564 to 1788 are promoter sequence.
SEQ ID NO: 2 = Event 1 5'~3' (coding strand) Insert-Genomic sequence. In particular, nucleotides 1 to 204 are terminator sequence, 205 to 426 are vector sequence and 427 to 2471 are wheat genomic sequence.
t0 SEQ ID NO: 3 = Event 1 S'-~3' (coding strand) Genomic sequence-Insert junction. This sequence is situated at nucleotides 1379 to 1408 of SEQ ID NO: 1.
SEQ ID NO: 4 = Event 1 5'-~3' (coding strand) Insert-Genomic sequence junction. This sequence is situated at nucleotides 412 to 441 of SEQ ID NO: 2.
SEQ ID NO: 5 = Event 1 5'~3' (coding strand) insert primer. This sequence is situated at nucleotides 4819 to 4841 of SEQ ID NO: 7.
SEQ ID NO: 6 = Event 1 5'~3' (coding strand) genomic primer. This sequence is situated at nucleotides 5673 to 5693 of SEQ ID NO. 7.
SEQ ID NO: 7 = Event 1 5'~3' (coding strand) Genomic sequence- Insert-Genomic sequence.
SEQ ID NO: 8 = Event 1 5'~3' (coding strand) Genomic sequence- Insert-Genomic sequence.

_23_ E~CAMPLES
General molecular biology methods arG carried out according to Sdmbrook et al.
(1989) 'Molecular cloning: A laboratory Manual, 2nd Edition. Cold Spring Harbour Lab.
Press.
Pol~rnuc:lcotidc synthesis 'V'twious pc~lynucleotidcs based on the sequence information as described in the Sequence Listing can b~ synthesised chemically using standard techniques well known to the person skilled in the art. Those poiynuclaotides include the primer sequences depicted in the Sequence Listing.
Transformation of Wheat Transformation of ~uvhcz~t using a particle bombardment technique was carried out 1~ using a construct comprising the expression cassette contained within the sequence depicted as SEQ ~ NO: 7 (nucleotides 1565 to 5290). Tbis cassette comprises maize ubiquitin promoter-maize ubiquitin intron-tricbothecene 3-D-acetyltransferase from F.
sPorocrechivtdes-nos terminator. In addition, wheat plants were also bombarded with an ~cxprcssion cassette derived from plasmid pCiB9818, as described in '~?U'Cl 00/60061. This ?o ;piasmid is a 611 1 base pair circular plasmid and the cassette used comprises the maize ubiquitin promoter (including a portion of the chop and introit) opera'bly linked to the phosphate mann4b.e isomerase selectable marker, an inserted i'F.PC introit #9 and a termination sequence from the CaMV 35S gene.
2s rnitnature zygotic wheat emtbryos are isolated at 11 to 14 days post-anthesis (dpa) firm surFacc sterilised caryopses and are pre-incubated on 3MS3S medium (Murashige and Skoog salts, MS vitamin, 300mg/1 glutaminc, 1S0 mg/1 asparagine, 3 mg/1 2,4-D.
30g/1 sucrose, and 7811 phytagar, pH at 5.8) for 5 to 7 days in the dark ac 22°C. Those embryos displaying the required embryonic phenotype arc transferred to plasmolysis 3e medium (3MS as above wvith 15p~1 of maltose in place of. the sucrose) for 3 to 4 hours in the dark pre-hon7bttrdinent_ Bombardment is carried out twice using 650psi rupture disks to decelerate particles coated with approximately 1mg/2 shots of 0.31.iM geld (mated using 0.17-0.67pg DNA/2 shots). The shock wave is dissipated by a stainless steel mesh baffle positioned above the trugec plate. Post-bombardment, etrtbryos continue tc~ be incubated in plasmolytis mzdium until a total of approximately 24 hours plasmolysis haS
occurred.
Embryos are then incubated in callus initiation medium (3MS3S) far 4 weeks in the dark without subculturing after which the resultant calli are dissected into 2 to 5mm pieces.
Theca pieces of calli are incubated on regenerationlseleetion medium NG1M.SS
(108/1 of mannoso, 58/1 of sucrose, 5 mg/! GA3 and lmg/1 NAA) for 2 to 3 days in the dark and then in the light for 2 weeks, Whole callus pieces are transferred to shoot and root initiationlselection medium MS2S.5M (MS salts and MS vitamins with Sg/1 mannose and 20811 sucrose) for 3 to 4 weeks and then onto root initiationlselection medium 1/x MSOS I .SM+a.SNAA (~Talf strength of MS salts and MS vitamin, 15gl1 mannose and to O.Smg/t NAA) for a further 3 to 4 weeks. Resultant plants are moved to the greenhouse when they are large enough and have adequate roots, Plants era transplanted to soil in the greenhouse find grown to maturity.
Analysis of insertion Sites for Event 1.
IS al Sample Preparation & PCR
The tissue samples for PCR detection of specific events can be prepared by any of the routine plant DNA exUCactiOn protocols_ For example, 1(10 mg of wheat leaf material are harvested from chc plant to be analysed and processed according to Qiagen's DN&asy Plant Miniprcp Kit. rn a typical PCR reaction, approximately 1 pg of DNA is used and 2o the reactions are carried out using standard methods. The ieacii0n mixture.
in addition to the saxnpie DNA, may include a final concentration of lx Clontech Advantage 2T"~ Taq Polymerise Buffer, 0.25 rnM dNTP mixture, lp.M of each primer used and lx Clvntcch .Advantage 2T"'' Taq Polymerise eniyme.
?s The PCR conditions are set to, for example, a denaturing cycle of 94°C for 3 minutes, then 35 amplification cycles of 94°C for 15 seconds;
60°C for 30 seconds; and i'2°C fur 45 seconds, followed 'by a final extension of 72°C for S minutes. The PCR
product is visualized on a 1°lo agarose gel-by-gel electopharesis. A
positive result is indicated by a PCR product of the correct size.

Further confirmation of the identity of the PCR product, is obtained by segueneing using probes used to amplify the product.

R further detection method involves the use the PCR product as a hybridisation probo for Southern Blot detection. The genomic DNA from the wheat plant to be analysed can be digested with a reslxiction endnnuelease. By employing a standard Southern Hlc~t protocol with this DNA, the pCR prUduct will give a unique hybridisation signal when probed against the specific event.
b~Mappin of Insertion Sites far Event 1:
The position of the inserted DNA is determined using TAIL PC12 (thermal tn asymmetric interlaced PCIZ). which is able to recover the sequence fianl.ing the insertion at hoth the left and right border of the inserted DNA. A protocol for carrying out the TAT~L PCR reaction is detailed, inter alia, in Liu et al., 1995 (The Plant Journal, vol. 8(3), pages 457 to 463). Once deterrninad, knowlcd.gc of the flanking sequence and insertion sites can be wed to design primers for event specific identification. Such primers ax'e 1~ useful in, far example, breeding techniques in which the $vent 1 trait is transferred to a different background germplasm.
Cenomic-insert Junction Genomic sequence = nuclaotidea 1 to 1393 of SEQ ID NO: 1.
20 Insert sequence = nucleotides 1394 to 1788 of SEQ ID NO: 1.
Insert-Gcnamic Junction Insert s~qucncc = nucleotides 1 to 426 of SEQ ID NO: 2_ Genonve seyuenee = nucleotides 427 to 2,471 of SEQ ID N4: 2.
~;l Event sQeci Fic identification her PCIZ
Samplea of plant material are prepared using techniques well known in the art_ 1PC12 is performed on the samples using the following primers:
3o rdentitication of insartivn of terminator end of constnlet was using the primers ciepietc:d as SEQ ID 1~TOS: 5 and 6. Approximate size of 1'CR fragment yielded = 877 bp.
Possible PCR conditions a.re as listed above.

Results are visualised using gal electrophoresis in accordance Nrith protocols well known to the person skilled in the art.
hnmunostrip assays This example describes the use of Irnmunosirip assays to test for the presence of lrichotheeene 3-D-acetyltransferasc in a sample.
a) ,~oCedure for pre"parinR extract t 0 Two, approximately 1 cm=, discs of leaf tissue are cut from a leaf of the plant to be tcsccd. 0.4-0.5 m~ of exCraCdon buffer is added and the tissue extracted using a plastic stirrer until a green extract is farmed and the tissue is fibrous.
h) Pre-paration of Immunostrip Briefly, the lateral-flow immunostrip comprises a detection membrane of nitxocellulose (2.5 x l8exn) supported on a plastic backing (AristaTM brand plastic CiISSGttCS, Bethlehem, PA), in which a 1 mm line of specific mouse anti-trichothecena 3-D-sc:etyleransfcrasc monoclonal antibody is sprdyed. A reagent control line of donkey anti-mousy antibody is sprayed in p3rullcl above the first antibody line.
'7'he bottom end 2U portion of the strip of nitrocellulose is oYer-layered with a piece of treated polyester strip.
The polyester strip is first treated with a solution B (90.59b HSA, 0.5%
polyvinylalcohol and 0.1%n TrIIori X-10: 50tnM phosphate buffer pH ?.4) and the colloidal gold conjugated mouse anti trichothecene 3-D-acetyleransferase antibody. The polyester strip is allowed to dry and is then overlaycred with a sample application pad of cotton, which has been 2s pretreated with a solution C (0.136 Triton X-100 and O.1M T~orate buffer pH
8.5) and ailo~uvced to dry. Flanking the other end, or top cad, of the nitrocellulose strip Is another wotton pad co absorb the solution from the samples after it passes over the lost arttibvdy and control antibody areas on the nitrocellulose. The completed immunostrip may then be cut into smaller test strips.
3() c:) Asady far trichothecanc 3-U-acecvltransfcrase usingyimm"u,~a5ttips The assay is performed by placing the bottom of the immllnosttip into 40DEt1 of extracted tissue. After waiting approximately ~ to 10 minutes, the reSUlts appear. rf tricholhecene 3-O-acetyltransferase is present in lhc sample, a double red line appears: the lower line indicates the presence of ts~ichothecene 3-O-acctylrransfcrase while the upper lint is the Control line signalling a properly working device. If no trichothccenc 3-O-acetyltransfcrase is present, only a single red control line appears.
Fungal resistance assays Event 1 in a background germplasm of 9850055-03 (AgrlPro) vcras trialled against the same germplasrn with no triehotheeene 3-D-aeecyltransfcrase gcnc and against two commercial wheat varieties, AC Barrie and Alsen.
y0 Test and control plants were grown in the field at a number of ~:ites throughout the LIS and Canada. At each sits, 8 replicates each of Event 1 and the three control lines were planted, each replicate in a standard plot size of S' by 12' (approx.imately 1.5'm by 3.7m).
The seeding rate was approximately SOg/plot. Four of the replicates were artificially t5 inoculated with Fusarictrr~ grarreinearum spores, the other four were left for natural infestation.
For artificial inoculation, Fusurium ~rumtnearum cultures may be grown as described in WO 00160061. The spores wcrr applied to the plants in a spray with a z0 concentration of SO,OpO sporas/nzl. lOml of spore suspension was used per foot (approximately U.3m) and the spores were sprayed onto the wheat spikes in two directions using a 110° flat fan with the filler removed. Spraying txras performed in the late aftcrnoon/evcning co maximise disease. The spores were applied at anthesis, when the :majority of the plants have reached Zatloks scale QS 6.5 (plot is at 50°lo anThesis) and then 25 again, threw days after the first inoculation.
At harvest. 5' (approximately 1.5m) sections were harvested from the two centre rows of each plot, by hand, in order to obtain approximately 2008 of grain sample.
30 Disease was evaluated by counting the number of x'usarium damaged kernels (FD~j in the sample and by assaying for mycotoxin contamination. p'DK is a visual ttait and is used by grain traders to grade wheat gualiCy. For assaying mycotoxin Contamination, a SUg grain sample was ground to a fine powder in a mill and the -zs-concentration of DON determined using a commercially available test (e.g.
DONtest TAGS" mycotoxin testing syatern; Trilogy Analytical L.ahoratory, Tnc., Washington, MO).
The following table shows the results of the field trials and is the average data obttained. from the 10 locations where the trial was carried out.
'TABLE 1 Genotype % FDT~ pprn DON

9850055-03 + Event 7.2 12.3 9sso055-03 - l~~ent 9.6 17.7 AC 8arrie 8.2 13.0 Alsen 7.0 14.6 in The field trials were repeated and the 1501~T concentration was again analysed. The results are shown in Table 2. In this table, trials 1 to 5 are carried out using natural infestation and trials 6 tol4 using inoculation.
TAB 1.E 2 Trial 9850055-03 + 98S0055-03 - HventAC $arrie Alsen $Vent 1 I.

1 I.73 2.67 3.80 2.54 2 1.16 3.21 2_95 4.49 3 0.75 2.11 3.05 2.20 4 1.88 .4.63 4.36 x.65 0.40 1.47 I .54 2.17 6 1.73 3.56 5.38 3.29 7 4.09 9.42 8.85 8.94 8 0.39 1.39 1.92 1.44 9 1.03 2.46 4.22 4.91 0.54 4.32 4.86 8.97 11 2.42 5.26 6.41 8.22 12 0.56 1.63 1.43 1.88 13 3.34 8.62 7.$1 9.19 14 1.12 2.11 2.53 2.55 Mean 1.51 3.78 4.22 4.67 In both cases, it cRn be seen that the ppm DON is decreased in rrvhcat containing Event 1 when cc~mparcd to the same germplasm lacking the event and to the two commercial standards. Xn the first trial, it can be seen that the %FDK is reduced compared to the same ~enheat line without Event I and is the same as or lower than the %FDK in the two commercial standards Alsen and AC Harric.
Uthcr modifications of the present invention will be apparent to those skilled in the art without dcpartin~ from the Scope of the invention which i.s defined by the appended U claims. All publications oiled brrcin are hereby incorporated by reference in their entirety for a!I purposes to the same extent as if each individual publication were specifically and individually indicated to be so incorporated by reference.

SEQUENCE LISTING
<110> Syngenta Ltd Syngenta Participations AG
<120> Polynucleotides and Uses Thereof <130> Event 1 <160> 8 <17~)> PatentIn version 3.1 <210> 1 <21:1> 1788 <212> DNA
<21:3> Synthetic <401)>

tgtc~gagaagcatagaccaacataatgtcactagtatgcctctacttgactagctcatta60 atcaaagatgattatgtttcctaaccatagacatgtgttgtcatttgattaacgggatca120 catcattaggagaatggtgtgattgacatgacccattccgttagcctagcacttgatcgt180 ttac3tatattgctattgctttcttcatgacttatacatgttcctgtaactatgggattat240 gcaactcccgtttaccggaggaacactttgagtgctaccaaacgtcacaacgtaactggg300 tgata ggagtactacaggtgtctccaaaggtacatgttgggttggcgtatttcga360 ataaa gatt:aggttttgtcactccgattgtcagagaggtatctctgggccctctcggtaatgcac420 atcactataagccttgcaagcaatgtagctaatgagttagttacagaatgatgcattacg480 taac:gactaaagagacttgccggtaatgagttaaactaggtattagataccgacgattga540 atct:cgggcaagtaacataccgatgacaaagggaacaacgtatgttgttatgcggtttga600 ccgataaagatcttcgtagaatatgtaggagccaatatgagcatccaggttccgctattg660 gttattgatcgagaatagttctaggtcatgtctacatagttctcgaacccgtagggtccg720 cacc~cttaacgttatgatgacagttttattatgagtttatatattttgatcctttcgacc780 ttggttccgtaggagcaagtcagaaaggttgagaaacagaaccatctgatttgattcgtt840 cccaatagccatgagatgatcatcttagggtgatccttttgtcaacggatgctcctatta900 cactcgtagtctctgaaggatgagaacccactatgtagcatctacatcgataattcaagc960 att~~tatacgtcattagtccgattctttgtaggaactacccgtaataacgaacttgcaaa1020 atggatctgtttatcataaagagattcattgttcctgaccctgcttcaccttaattgtta1080 tttgaacaaaaagatcacaataaacttttggtaaaagttctatcttggtcggagtgggga1140 tag~~atttctcttctgcatgtctatggagttttgcaaaacccaaacacctcagagataga1200 tat;~gaggtaggaatttgtcgaacgaaccacactccttcgtagacgtcaggagtccattg1260 atgaaaaggggctggggaaagcttaaacccaagtcctacagtgatggatataagcgcaat1320 tga;~attcctggggagttatacatttgtgtattgataagaccgttcacaatttcttgaag1380 ctc<~atctcccccctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgcc1440 aggc3ttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactc1500 actatagggcgaattgggtaccgggccccccctcgaggtcgacggtatcgataagcttgc1560 atgcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatg1620 tctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagttta1680 tct<~tctttatacatatatctaaactttactctacgaataatataatctatagtactaca1740 ata<~tatcagtgttttagagaatcatataaatgaacagttagacatgg 1788 <210>

<21:L>

<212>
DNA

<21:3> hetic Synt <400>

cggt:cttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaa 60 catc3taatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattata 120 catt:taatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgc 180 ggtc3tcatctatgttactagatcgggagctccagcttttgttccctttagtgagggttaa 240 ttgc:gcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctca 300 caat=tccactcaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgag 360 tga~3ctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgt420 cgt~3ccgtttttttcgctttttttctcccatcccatcttggacgggcccagaagcttttc4S0 tcatgtttgaatggaggtcttccgcaaatgaatttccatataggaattatataatgtgcc540 tgc,agatattgattgaaagcttgtttcaattgaacgaggatgtgaacgagaaggcaacaa600 aag~3gagcttggagcaagaggagtcatttagtagttcacgaaaaaaaggcatagtagaga660 aag'tctgaggagcaggaaaatttggtcacgtagatcttctatttcgccggaattcgttga720 ttg~~tccgtactcatttacaatggaaaaactcctgttcgttgtaagatcactgaaggtca780 taa~atttggagagtttgcttttacacggagacgaagaccctatcgaacaaatagaggaaa840 ggg~3aaaaagtaaagtctaagcgacatatggatcaataaggaaatccaatttcggtaatc900 cga;~tcccatataggtcttttcttctctctcgaccagccctaggttggggataatgaatt960 tttc~gaatcttgttgggagatgcttcgctccgagtccgactgggacggaatgggggaccc1020 aggcccttcaatgcctccctccggatccactaactcggaagagctattcaaagcttttct1080 ggci=gaccgtgaggacgcctctgcatcccgggagcagaccgctcccccggaagctgtggt1140 cagl:caagagcggcatccgaatcccgctccaatgcctggagtggcagcggaagcagaaca1200 gaaccctccttcttttgattgggagggacgacctctctctctttgagaggaaagttctct1260 acaaaaggcccgcgcctgtcacgcccagattctggacacattgcgtgatattactctgaa1320 gaacggagaggagctccgtgaagaagactatcgcaccatgcttgatttcatgttgagccg1380 actc:cagtccgacgtcaatccccgggcgctgaaggagctcctgaacagactccaaaacgg1440 tcg<~gcacgatctcaaacttctagggcggcaaggagattcctcgacgaggactagtgaaa1500 cttt:ttttgagagagaaatttccagtcatatcaggaatcagtacaaagtagttgaccctt1560 acaagcacactgaaatgcaaacacaaaagatcatgaacacctagagtaccctaaaacaac1620 cataacacaagaagatctccggagccagtgtcatcatccatgaatcttgagagaagaccc1680 ctgc:tgcagcagaaggatctgcaaccagtcgcaaacaggtcatcatcttcgaccacagta1740 caattgccgccacgctgcttcctcctttcttgacaccagcgctgagagggcatggacatc1800 aatccaacacacctgcaacagccgtcgccatctttggctttgagtaccgcgaaaaatgtc1860 ccttccgaaaggaagagaactcgagtcattcgaccggtccagcaccgcggccgggccatc1920 cgcccgggcaaagcaggatctcttcaaaacttagagcatcaacaactagacttgcaaatc1980 cggcccctcaaacgatcgtgggcacgtccgtgtgcagtgaccggacacgtctcaaatttt2040 aacaggtgcatccggacatctcatattagattctcgaaractaaaataaacctacactac2100 gtcggtcacctagctactcgtcatcagagawgtcgacgatctctgtgcccagctctggca2160 gcatgggcggcagctgcagctccggctcctccgacggctccggctgctctgctccggcct2220 cttccgcctctatctccgcgtcgagttcggcgaagagcgcgtcggacgccgcctgctcct2280 gacagaggaacgcccggttttcctccacataggcctcatcctggatggcctgctgctcag2340 ccatctcctctgacggggcgacgctaaactccgtctccgcatgctccatgttgaaggcca2400 acagctgctgctctggcttctccacctccatcagagccatcttctcctcctcttccctts2460 gmttcttcact 2471 <210> 3 <211> 30 <212> DNA
<213> Synthetic <400> 3 agctcgatct cccccctggc gaaaggggga 30 <210> 4 <211> 30 <212> DNA
<213> Synthetic <400> 4 gaa;~cctgtc gtgccgtttt tttcgctttt 30 <21i7> 5 <211> 23 <212> DNA
<21:3> Synthetic <400> 5 gacctcgctt tgaacctttt gag 23 <210> 6 <211> 21 <212> DNA
<213> Synthetic <400> 6 cgagaaggca acaaaaggga g 21 <210>

<211>

<212>
DNA

<213>
Synthetic <400>

tgt~3gagaagcatagaccaacataatgtcactagtatgcctctacttgactagctcatta 60 atc~aaagatgattatgtttcctaaccatagacatgtgttgtcatttgattaacgggatca 120 catcattaggagaatggtgtgattgacatgacccattccgttagcctagcacttgatcgt 180 tta~~tatattgctattgctttcttcatgacttatacatgttcctgtaactatgggattat 240 gca;~ctcccgtttaccggaggaacactttgagtgctaccaaacgtcacaacgtaactggg 300 tgattataaaggagtactacaggtgtctccaaaggtacatgttgggttggcgtatttcga 360 gat'~aggttttgtcactccgattgtcagagaggtatctctgggccctctcggtaatgcac 420 atc~~ctataagccttgcaagcaatgtagctaatgagttagttacagaatgatgcattacg 480 taacgactaaagagacttgccggtaatgagttaaactaggtattagataccgacgattga 540 atctcgggcaagtaacataccgatgacaaagggaacaacgtatgttgttatgcggtttga 600 ccg<~taaagatcttcgtagaatatgtaggagccaatatgagcatccaggttccgctattg 660 gttattgatcgagaatagttctaggtcatgtctacatagttctcgaacccgtagggtccg 720 cacgcttaacgttatgatgacagttttattatgagtttatatattttgatcctttcgacc 780 ttggttccgtaggagcaagtcagaaaggttgagaaacagaaccatctgatttgattcgtt 840 cccaatagccatgagatgatcatcttagggtgatccttttgtcaacggatgctcctatta 900 cactcgtagtctctgaaggatgagaacccactatgtagcatctacatcgataattcaagc 960 attgtatacgtcattagtccgattctttgtaggaactacccgtaataacgaacttgcaaa1020 atggatctgtttatcataaagagattcattgttcctgaccctgcttcaccttaattgtta1080 tttgaacaaaaagatcacaataaacttttggtaaaagttctatcttggtcggagtgggga1140 tagcatttctcttctgcatgtctatggagttttgcaaaacccaaacacctcagagataga1200 tatagaggtaggaatttgtcgaacgaaccacactccttcgtagacgtcaggagtccattg1260 atgaaaaggggctggggaaagcttaaacccaagtcctacagtgatggatataagcgcaat1320 tgaaattcctggggagttatacatttgtgtattgataagaccgttcacaatttcttgaag1380 ctcgatctcccccctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgcc1440 agggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactc1500 actatagggcgaattgggtaccgggccccccctcgaggtcgacggtatcgataagcttgc1560 atgcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatg1620 tctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagttta1680 tctatctttatacatatatctaaactttactctacgaataatataatctatagtactaca1740 ataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaa1800 ttgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcct1860 ttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatt1920 tagggtttagggttaatggtttttatagactaatttttttagtacatctattttattcta1980 ttttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataattta2040 gatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaatta2100 aaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccg2160 tcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaag2220 cagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccg2280 ttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccg2340 gcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcc2400 caccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacacc2460 ctctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaa2520 tccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctc2580 taccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttc2640 atgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatg2700 cgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatc2760 ctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgtt2820 gcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcg2880 ggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtc2940 gttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatc3000 tgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcg3060 atctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttt3120 tgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcgg3180 agtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtg3240 tcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtat3300 acatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcat3360 atgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttg3420 atcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctg3480 ccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtt3540 tggtgttacttctgcaggtcgactctagaggatccagaattcgtgatcaaatggccgcaa3600 caagcagcacaagcagccagtcttttgacatagagctcgacatcatcggccagcaaccgc3660 ctcttctttcaatctacacccagatcagtctcgtttaccccgtctctgatccctcccagt3720 atcccaccatcgtcagcacccttgaggaaggcctaaaacgcctctctcaaaccttcccat3780 gggtcgcgggccaggtcaagaccgagggcatcagcgaaggaaacacaggaacttccaaga3840 tcattccatatgaggagacaccccgtcttgtggtgaaagacctccgtgatgattcctcag3900 cgccaacgatcgaggggttgagaaaggcgggtttccccttagagatgtttgacgagaacg3960 tcgtcgctccgaggaagacattagctatcggacctggcaatggccccaacgacccgaagc4020 ctgtgttgctattgcagctcaacttcattaagggcggactcattctcaccgtcaacggac4080 aacatggtgctatggacatgacaggacaagatgcaattattcgtcttctctccaaggcgt4140 gccgcaacgaatcattcaccgaggaggaaatctcggccatgaacctcgatcgcaagacgg4200 tagtccctctccttgaaaactacaaagttggtcctgagctagaccaccagatcgccaaac4260 ctgcgcctgctggcgacgctccacccgcaccggccaaggcaagctgggcgttcttttcat4320 tcactcccaaggccctctcggagctgaaagacgcagccacaaagactcttgacgcgtcgt4380 ccaagtttgtgtcaactgatgatgctctttcggcgtttatctggcaatcaacctcgcgcg4440 tacgtctcgcaagattggatgcttccacacctactgaattctgccgcgctgtcgacatgc4500 ggggcccaatgggcgtatcaagcacatacccaggccttcttcaaaacatgacctaccatg4560 actcgaccgtcgccgaaatcgccaacgaaccacttggcgcaacagcatcacgcctgcgct4620 cggaactcaacagtgatcgtttgcgcagacgaacacaagctttggcgacgtacatgcatg4680 gcctgcctgacaagtcgagcgtctccctgaccgccgatgcgaatccgtcaagcagcatca4740 tgctgagttcctgggccaaggtgggatgctgggagtatgactttgggtttggactgggta4800 agcctgagagtgtgagaagacctcgctttgaaccttttgagagtttgatgtactttatgc4860 ccaagaagcctgatggggagtttacggcgtccatttctctgagggatgaggatatggaga4920 gactaaaggcggatgaggagtggacaaagtacgcaaagtatattgggtagatagtttact4980 agactactgcaggatatcgtggatccactagttctagagcggccgcgaatttccgcgatc5040 gttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatga5100 ttatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatga5160 cgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcga5220 tagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgt5280 tactagatcgggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcg5340 taatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccactcaac5400 atacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcaca5460 ttaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccgttttttt5520 cgctttttttctcccatcccatcttggacgggcccagaagcttttctcatgtttgaatgg5580 aggtcttccgcaaatgaatttccatataggaattatataatgtgcctgcagatattgatt5640 gaaagcttgtttcaattgaacgaggatgtgaacgagaaggcaacaaaagggagcttggag5700 caagaggagtcatttagtagttcacgaaaaaaaggcatagtagagaaagtctgaggagca5760 ggaaaatttggtcacgtagatcttctatttcgccggaattcgttgattgctccgtactca5820 tttacaatggaaaaactcctgttcgttgtaagatcactgaaggtcataaatttggagagt5880 ttgcttttacacggagacgaagaccctatcgaacaaatagaggaaaggggaaaaagtaaa5940 gtctaagcgacatatggatcaataaggaaatccaatttcggtaatccgaatcccatatag6000 gtcttttcttctctctcgaccagccctaggttggggataatgaatttttcgaatcttgtt6060 gggagatgcttcgctccgagtccgactgggacggaatgggggacccaggcccttcaatgc6120 ctccctccggatccactaactcggaagagctattcaaagcttttctggctgaccgtgagg6180 acgcctctgcatcccgggagcagaccgctcccccggaagctgtggtcagtcaagagcggc6240 atccgaatcccgctccaatgcctggagtggcagcggaagcagaacagaaccctccttctt6300 ttgattgggagggacgacctctctctctttgagaggaaagttctctacaaaaggcccgcg6360 cctgtcacgcccagattctggacacattgcgtgatattactctgaagaacggagaggagc6420 tccgtgaagaagactatcgcaccatgcttgatttcatgttgagccgactccagtccgacg6480 tcaatccccgggcgctgaaggagctcctgaacagactccaaaacggtcgggcacgatctc6540 aaacttctagggcggcaaggagattcctcgacgaggactagtgaaactttttttgagaga6600 gaaatttccagtcatatcaggaatcagtacaaagtagttgacccttacaagcacactgaa6660 atgcaaacacaaaagatcatgaacacctagagtaccctaaaacaaccataacacaagaag6720 atctccggagccagtgtcatcatccatgaatcttgagagaagacccctgctgcagcagaa6780 ggatctgcaaccagtcgcaaacaggtcatcatcttcgaccacagtacaattgccgccacg6840 ctgcttcctcctttcttgacaccagcgctgagagggcatggacatcaatccaacacacct6900 gcaacagccgtcgccatctttggctttgagtaccgcgaaaaatgtcccttccgaaaggaa6960 gagaactcgagtcattcgaccggtccagcaccgcggccgggccatccgcccgggcaaagc7020 aggatctcttcaaaacttagagcatcaacaactagacttgcaaatccggcccctcaaacg7080 atcgtgggcacgtccgtgtgcagtgaccggacacgtctcaaattttaacaggtgcatccg7140 gacatctcatattagattctcgaaractaaaataaacctacactacgtcggtcacctagc7200 tactcgtcatcagagawgtcgacgatctctgtgcccagctctggcagcatgggcggcagc7260 tgcagctccggctcctccgacggctccggctgctctgctccggcctcttccgcctctatc7320 tccgcgtcgagttcggcgaagagcgcgtcggacgccgcctgctcctgacagaggaacgcc7380 cggttttcctccacataggcctcatcctggatggcctgctgctcagccatctcctctgac7440 ggggcgacgctaaactccgtctccgcatgctccatgttgaaggccaacagctgctgctct7500 ggcttctccacctccatcagagccatcttctcctcctcttcccttsgmttcttcact 7557 <210>

<211>

<212>
DNA

<213>
Synthetic <400>

tgtggagaagcatagaccaacataatgtcactagtatgcctctacttgactagctcatta 60 atcaaagatgattatgtttcctaaccatagacatgtgttgtcatttgattaacgggatca 120 catcattaggagaatggtgtgattgacatgacccattccgttagcctagcacttgatcgt 180 ttagtatattgctattgctttcttcatgacttatacatgttcctgtaactatgggattat 240 gcaactcccgtttaccggaggaacactttgagtgctaccaaacgtcacaacgtaactggg 300 tgattataaaggagtactacaggtgtctccaaaggtacatgttgggttggcgtatttcga 360 gattaggttttgtcactccgattgtcagagaggtatctctgggccctctcggtaatgcac 420 atcactataagccttgcaagcaatgtagctaatgagttagttacagaatgatgcattacg 480 taacgactaaagagacttgccggtaatgagttaaactaggtattagataccgacgattga 540 atctcgggcaagtaacataccgatgacaaagggaacaacgtatgttgttatgcggtttga600 ccgataaagatcttcgtagaatatgtaggagccaatatgagcatccaggttccgctattg660 gttattgatcgagaatagttctaggtcatgtctacatagttctcgaacccgtagggtccg720 cacgcttaacgttatgatgacagttttattatgagtttatatattttgatcctttcgacc780 ttggttccgtaggagcaagtcagaaaggttgagaaacagaaccatctgatttgattcgtt840 cccaatagccatgagatgatcatcttagggtgatccttttgtcaacggatgctcctatta900 cactcgtagtctctgaaggatgagaacccactatgtagcatctacatcgataattcaagc960 attgtatacgtcattagtccgattctttgtaggaactacccgtaataacgaacttgcaaa1020 atggatctgtttatcataaagagattcattgttcctgaccctgcttcaccttaattgtta1080 tttgaacaaaaagatcacaataaacttttggtaaaagttctatcttggtcggagtgggga1140 tagcatttctcttctgcatgtctatggagttttgcaaaacccaaacacctcagagataga1200 tatagaggtaggaatttgtcgaacgaaccacactccttcgtagacgtcaggagtccattg1260 atgaaaaggggctggggaaagcttaaacccaagtcctacagtgatggatataagcgcaat1320 tgaaattcctggggagttatacatttgtgtattgataagaccgttcacaatttcttgaag1380 ctcgatctcccccctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgcc1440 agggttttcccagtcacgacgttgtaaaacgacggccagtgarykcgcgtaatacgactc1500 actatagggcgaattgggtaccgggccccccctcgaggtcgacggtatcgataagcttgc1560 atgcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatg1620 tctaagttataaaaaattaccacatattttttttgtcacacttgtttgaagtgcagttta1680 tctatctttatacatatatytaaactttactctacgaataatataatctatagtactaca1740 ataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaa1800 ttgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcct1860 ttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatt1920 tagggtttag ggttaatggt ttttatagac taattttttt agtacatcta ttttattcta 1980 ttttagcctc taaattaaga aaactaaaac tctattttag tttttttatt taataattta 2040 gatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaatta2100 aaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccg2160 tcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaag2220 cagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccg2280 ttggacttgctccgctgtcggcatccagaaattgcgtggcggagcggcagacgtgagccg2340 gcacggcaggcggcctcctcctcctctcacggcacggcagctacgggggattcctttccc2400 accgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccc2460 tctttccccaacctcgtgttgttcggagcgcacacacacacaaccagatctcccccaaat2520 ccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctct2580 accttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttca2640 tgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggatgc2700 gacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcc2760 tgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttg2820 catagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgg2880 gtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcg2940 ttctagatcggagtagaattctgtttcaaactacctggtggatttattaattttggatct3000 gtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcga3060 tctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgcttttt3120 gttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcgga3180 gtagaatactgtttcaaactacctggtgtattta.ttaattttggaactgtatgtgtgtgt3240 catacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtata3300 catgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctattcata3360 tgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttga3420 tcttgatatacttggatgatggcatatgcagcagctatatgtggatttttttagccctgc3480 gactactgcaggatatcgtggatccactagttctagagcggccgcgaatttccgcgatcg5040 ttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgat5100 tatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgac5160 gttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgat5220 agaaaacaaaatatagctgcgcraacwaggataaattatcgcgcgcggtgtcatctatgt5280 tactagatcgggagctccagcttttgttccctttagtgagggttaattscgmgcttggca5340 gtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaa5400 catacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcac5460 attaattgcgttgcgctcactgcccgctttccagtcgggaracctgtcgtgccgtttttt5520 tcgctttttttctcccatcccatcttggacgggcccagaagcttttctcatgtttgaatg5580 gaggtcttccgcaaatgaatttccatataggaattatataatgtgcctgcagatattgat5640 tgaaagcttgtttcaattgaacgaggatgtgaacgagaaggcaacaaaagggagcttgga5700 gcaagaggagtcatttagtagttcacgaaaaaaggcatagtagagaaagtctgaggagca5760 ggaaaatttggtcacgtagatcttctatttcgccggaattcgttgattgctccgtactca5820 tttacaatggaaaaactcctgttcgttgtaagatcactgaaggtcataaatttggagagt5880 ttgcttttacacgggagacgaagaccctatcgaacaaatagaggaaaggggaaaaagtaa5940 agtctaagcgacatatggatcaataaggaaatccaatttcggtaatccgaatccccatat6000 aggtcttttcttcwywctcgacc 6023 cttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgttt3540 ggtgttacttctgcaggtcgactctagaggatccagaattcgtgatcaaatggccgcaac3600 aagcagcacaagcagccagtcttttgacatagagctcgacatcatcggccagcaaccgcc3660 tcttctttcaatctacacccagatcagtctcgtttaccccgtctctgatccctcccagta3720 tcccaccatcgtcagcacccttgaggaaggcctaaaacgcctctctcaaaccttcccatg3780 ggtcgcgggccaggtcaagaccgagggcatcagcgaaggaaacacaggaacttccaagat3840 cattccatatgaggagacaccccgtcttgtggtgaaagacctccgtgatgattcctcagc3900 gccaacgatcgaggggttgagaaaggcgggtttccccttagagatgtttgacgagaacgt3960 cgtcgctccgaggaagacattagctatcggacctggcaatggccccaacgacccgaagcc4020 tgtgttgctattgcagctcaacttcattaagggcggactcattctcaccgtcaacggaca4080 acatggtgctatggacatgacaggacaagatgcaattattcgtcttctctccaaggcgtg4140 ccgcaacgaatcattcaccgaggaggaaatctcggccatgaacctcgatcgcaagacggt4200 agtccctctccttgaaaactacaaagttggtcctgagctagaccaccagatcgccaaacc4260 tgcgcctgctggcgacgctccacccgcaccggccaaggcaagctgggcgttcttttcatt4320 cactcccaaggccctctcggagctgaaagacgcagccacaaagactcttgacgcgtcgtc4380 caagtttgtgtcaactgatgatgctctttcggcgtttatctggcaatcaacctcgcgcgt4440 acgtctcgcaagattggatgcttccacacctactgaattctgccgcgctgtcgacatgcg4500 gggcccaatgggcgtatcaagcacatacccaggccttcttcaaaacatgacctaccatga4560 ctcgaccgtcgccgaaatcgccaacgaaccacttggcgcaacagcatcacgcctgcgctc4620 ggaactcaacagtgatcgtttgcgcagacgaacacaagctttggcgacgtacatgcatgg4680 cctgcctgacaagtcgagcgtctccctgaccgccgatgcgaatccgtcaagcagcatcat4740 gctgagttcctgggccaaggtgggatgctgggagtatgactttgggtttggactgggtaa4800 gcctgagagtgtgagaagacctcgctttgaaccttttgagagtttgatgtactttatgcc4860 caagaagcctgatggggagtttacggcgtccatttctctgagggatgaggatatggagag4920 actaaaggcggatgaggagtggacaaagtacgcaaagtatattgggtagatagtttacta4980

Claims (17)

1. A polynucleotide which comprises a first region comprising the sequence depicted as SEQ ID NO: 1 and a further region which comprises the sequence depicted as SEQ ID NO: 2.

2. A polynucleotide which comprises:
a) at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO:

3;
b) at least 35 contiguous nucleotides of the sequence depicted as nucleotides 1364 to 1423 of SEQ ID NO: 1; or c) at least 50 nucleotides of the sequence depicted as SEQ ID NO: 1, said polynucleotide encompassing nucleotides 1393 and 1394 of SEQ ID NO: 1.

3. A polynucleotide which comprises:
a) at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO:

4;
b) at least 35 nucleotides of the sequence depicted as nucleotides 397 to 456 of SEQ ID NO: 2; or c) at least 50 nucleotides of the sequence depicted as SEQ ID NO: 2, said polynucleotide encompassing nucleotides 426 and 427 of SEQ ID NO: 2.

4. A wheat plant comprising a polynucleotide according to any one of claims 1 to 3.

5. Seed of the wheat plant according to claim 4 which comprises the polynucleotide according to any one of claims 1 to 3.

6. A method for detecting a plant which contains the polynucleotide depicted as SEQ
ID NO: 1 said method comprising:
a) preparing a sample containing the genomic DNA of the plant to be tested;
b) designing a pair of primers which are suitable for use in an amplification reaction to amplify a sequence comprising at least 18 contiguous -31-~

nucleotides of the sequence depicted as SEQ ID NO: 3 and the complement thereof;
c) adding said pair of primers to said sample and the means for performing an amplification reaction;
d) performing an amplification reaction; and e) visualising the thus amplified sequence.

7. A method for detecting a plant which contains the polynucleotide depicted as SEQ
ID NO: 2 said method comprising:
a) preparing a sample containing the genomic DNA of the plant to be tested;
b) designing a pair of primers which are suitable for use in an amplification reaction to amplify a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO: 4 and the complement thereof;
c) adding said pair of primers to said sample and the means for performing an~
amplification reaction;
d) performing an amplification reaction; and e) visualising the thus amplified sequence.

8. A method according to claim 6 or claim 7 wherein said sequence comprises at least 20 contiguous nucleotides.

9. A method for detecting a plant which contains the polynucleotide depicted as SEQ
ID NO: 1 and/or the polynucleotide depicted as SEQ ID NO: 2 said method comprising:
a) preparing a sample containing the genomic DNA of the plant to be tested;
b) designing a probe which is capable of hybridising to a sequence selected from the group consisting of a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO: 3 and a sequence comprising at least 18 contiguous nucleotides of the sequence depicted as SEQ ID NO: 4;

c) ~adding at least one of the probes of step (b) to said sample under conditions which allow said probe to hybridise with a complementary nucleic acid within said sample;~
d) ~removing substantially non-hybridised probe; and e) ~detecting the thus hybridised probe to identify if the sample contains said polynucleotide.

10. ~A method according to claim 9 wherein said sequence comprises at least 20 contiguous nucleotides.

11. ~A method according to claim 9 or claim 10 wherein said substantially non-hybridised probe is removed by rinsing said probe under high stringency conditions.

12. ~A method for detecting a plant which contains a protein capable of being encoded by the polynucleotide depicted as SEQ ID NO: 7 said method comprising:
a) ~preparing a protein-extract of the plant to be tested;
b) ~providing an antibody which is capable of binding to a trichothecene 3-O-acetyltransferase protein;
c) adding said antibody to said extract or said extract to said antibody under conditions which allow said antibody to bind to said protein within said extract; and d) ~detecting the thus bound antibody to identify if the extract contains said protein.

13. ~A kit of parts which comprises a pair of primers as defined in claim 6 or claim 7 and instructions for performing the method of claim 6 or claim 7 and means for performing an amplification reaction and optionally means for preparing the sample to be tested.

14. ~A kit of parts which comprises an antibody as defined in claim 12 and instructions for performing the method of claim I2 and means for performing the method according to claim 12 and optionally means for preparing the sample to be tested.

15. A dipstick for use in the method of claim 14 comprising an anti-trichothecene 3-O-acetyltransferase antibody.

16. A dipstick comprising a) a test line of specific anti-trichothecene 3-O-acetyltransferase antibody;
b) a reagent control line of anti-mouse antibody;
c) a pad containing dried colloidal gold labelled anti-trichothecene 3-O-acetyltransferase antibody; and d) a sample application pad.

17. ~A dipstick according to claim 16, wherein the anti-trichothecene 3- O-acetyltransferase antibody and the dried colloidal gold labelled anti-trichothecene 3-D-acetyltransferase antibody are independently selected from the group consisting of an antibody secreted by cell line DSM ACC 2679 and an antibody secreted by cell line DSM ACC 2680.