AU2014210372A1 - Herbicide tolerant barley - Google Patents

Abstract

Herbicide resistant barley plants and compositions and methods for producing herbicide resistant barley hybrids, in particular barley plants with mutated acetyl-CoA carboxylase (ACCase) genes that are resistant to ACCase inhibiting herbicides, such as barley varieties BOZO and HT107.

Description

WO 2014/113846 PCT/AU2014/000060 1 HERBICIDE TOLERANT BARLEY Field of the Invention 5 The present invention provides for compositions and methods for producing commercial barley plants that are resistant to herbicides. In particular, the present invention provides for barley plants, plant tissue and plant seed that contain acetyl-CoA carboxylase (ACC) genes and proteins that confer resistance to inhibition by herbicides that normally inhibit the activity of the ACC protein. Background Barley (Hordeum vulgare) is a major cereal grain and is a member of the grass family. Barley is used in malt production, brewing and for the production of cereal foods for 15 human and animal consumption and is generally ranked in the top five cereal crops globally in terms of quantity produced and area under cultivation. The control of weeds is a significant factor in the economic production of barley. The use of chemical herbicides to control weeds is popular and more desirable in most 20 circumstances compared to mechanical cultivation, which is generally more expensive and can result in damage to soil structure and erosion. Of particular interest to farmers is the use of herbicides with greater potency, broad weed spectrum effectiveness and rapid soil degradation. One such class of broad spectrum herbicides, are those compounds that inhibit the activity of the acetyl-CoA carboxylase 25 (ACC) enzyme in a plant. These are known as 'Group A' or 'Group 1' herbicides and include compounds within the following classes; aryloxyphenoxypropionates (FOP); cyclohexanediones (DIM): and phenylpyrazolins. While FOP and DIM herbicides are generally the first line of choice for removing grass weeds in broadleaf dicott) crops, rost of them cannot be used selectively in cereals 30 including barley. Therefore, grass weeds are sprayed with non-selective herbicides such as glyphosate before seeding barley but grass weeds that emerge later in the crop cannot be controlled selectively at present. There is a need in the art for improved barley varieties and methods for improving the production of barley 35 The present invention overcomes some or all of the shortcomings of the prior art Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 2 Summary of the Invention The present invention provides for barley plants and barley hybrids and methods for producing barley hybrids that are resistant to herbicides. In particular, the present 5 invention provides for barley plants and hybrids, plant tissue and plant seed that contain acetyl-CoA carboxylase (ACC) genes and proteins that confer resistance to inhibition by herbicides that normally inhibit the activity of the ACC protein. Cultivated commercial barley is susceptible to many ACC inhibiting herbicides that target monocot or grassy weed species. However, as described herein a barley genotype was 10 developed that exhibits tolerance to ACC inhibiting herbicides, Genetic analysis has identified genetic differences within germplasm that results in a ACC herbicide resistant phenotype. in one embodiment, the present invention provides for one or more barley plants whose germplasm comprises a mutation that renders the plant tolerant to ACC herbicides. 15 Moreover, in further embodiments the invention relates to the offspring (e.g., Fl, F2, F3, etc.) of a cross of said plant wherein the germplasm of said offspring has the same mutation as the parent plant. Therefore, embodiments of the present invention provide for barley hybrids whose germplasm contains a mutation, such that the phenotype of the Plants is ACC herbicide resistant. In some embodiments, said offspring (e.g., FI, F2, F3, 20 etc.) are the result of a cross between elite barley lines, at least one of which contains a germplasm comprising a mutation that renders the plant tolerant to AGC herbicides. In a first aspect, the invention is a barley plant, wherein said barley plant confers resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant. 25 Preferably, the barley plant is a commercial cultivar. Preferably, the acetyl-CoA carboxylase inhibiting herbicide is selected from the group consisting of; an aryloxyphenoxypropionate herbicide; and a cyclohexanedione herbicide. More preferably, the aryloxyphenoxypropionate herbicide is selected from the group consisting of; clodinafop-propargyl; diclofop-methyl; quizalofop-p-ethyl; fenoxyprop-p. 30 ethyl; haloxyfop; fluazifop; and propaquizafop. More preferably. the cyclohexanedione herbicide is selected from the group consisting of; clethodim; tralkoxydim; sethoxydim; tepraloxydim: and butroxydim. Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 3 In one preferred embodiment, the barley plant comprises an acetyl-CoA carboxylase gene, wherein said gene corresponds to the amino acid substitution selected from the group consisting of: lie 1 ,Leu; lie reuVal; and lie 1 933 Leu and lie 20 ,Val. For example, the Isoleucine at position 1933 (see sequence listing for sequence references) has been 5 substituted with a Leucine at the same position. Preferably, the barley plant comprises an acetyl-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain: and wherein the carboxyl transferase domain comprises anyone of the amino acids or 10 polypeptides selected from the group consisting of: e 13sLeu; 19 s 3 Leu and 20 1Val: SEQ ID No 1; 15 8 SEQ ID No 2; 4 SEQ ID No IandSEQlDNo2; SEQ ID No 3; SEQ ID No 4: and a SEQ ID No 8. 20 Preferably, the barley plant comprises an acetyl-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain and wherein the polynucleotide that corresponds to a carboxyi transferase domain comprises anyone of the nucleic acids and polynucleotides selected from the group consisting of; * sarTTA; 25 + + 5?.

7 TTA and 7GT: " SEQ ID No 5 Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 4 SEQ ID No 6; SEQ ID No 5 and SEQ ID No 6; and SEQ ID No 7. In one embodiment, the polynucleotide is introduced into the germpiasm of the barley 5 plant by hybridisation. Preferably, the polynucleotide is introduced into the germplasm of the barley plant by introgression. Methods for these techniques are widely available in the art. See also Principles of Plant Breeding, 2nd ed By R, W. Allard. John Wiley & Sons, New York, 1999. Alternatively, the polynucleotide is introduced into the germplasm of the barley plant by recombinant DNA engineering techniques. Methods for these techniques 10 are widely available in the art. See also In Vitro Plant Breeding S. Thirugnanakumar, K. Manivannan, M. Prakash, R. Narasimman and Y. Anitha. Vasline, Agrobios, 2009 In another preferred embodiment, the barley plant is not genetically modified. In a second aspect, the invention is a seed of a barley plant as described herein, 15 In a third aspect, the invention is a germplasm of a barley plant as described herein. In a fourth aspect, the invention is a method of producing a barley hybrid with increased resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant, said method comprising; selecting a first barley plant: 20 selecting a second barley plant; crossing the first barley plant with the second barley plant to produce hybrid offspring; selecting a hybrid from said hybrid offspring with increased resistance to inhibition by one or more acetyl-CoA carboxylase inhibitinq herbicides thereby to produce 25 the hybrid: and wherein the second barley plant is a barley plant as described herein. In a fifth aspect, the invention is a method of producing a barley hybrid with increased resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant, said method comprising; 30 selecting a first barley plant; isolating cells from the first barley plant; Rectifled Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 5 incorporating a foreign polynucleotide into the carboxyl transferase domain of the genome of the said cells of the first barley plant using recombinant DNA engineering techniques; growing offspring from the said cells: 5 selecting offspring with increased resistance to inhibition by one or more acetyk CoA carboxylase nhibiting herbicides thereby to produce the barley hybrid and wherein the foreign polynucleotide is selected from polynucleotides that correspond to a carboxyl transferase domain of a second barley plant; wherein the second barley plant is a barley plant as described herein; and 10 wherein the foreign polynucleotide is selected from the group consisting of; (a) reyTTA; (b) 62GTT, (c) Fa;'TTA and 6277 GTT; (d) SEQ ID No 5 15 (e) SEQ ID No 6; (f) SEQ ID No 5 and SEQ ID No 6; and (g) SEQ ID No 7. Preferably, in the fourth and fifth aspects described above, the first barley plant is selected from the group consisting of AC Metcalfe, Bass, Baudin, Buloke, CDC Copeland, 20 CDC Reserve, Commander, Fairview, Fathom, Flagship, Fleet, Gairdner, Henley, Hindmarsh, Keel, Navigator, Oxford, Propino, Scope, Sebastian, Skipper, SouthernStar, Quench, Vlamingh, VT Admiral, Westminster, W14593 and Wimmera. Preferably, in the fourth and fifth aspects described above, the second barley plant is selected from the group consisting of; BOZO: and HT017. 25 In a sixth aspect, the invention is a barley hybrid produced by the methods herein described. Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 6 in one emodiment, the hybrid comprises an acety-CoA carboxylase gene, wherein said gene corresponds to the amino acid substitution selected forn the group consisting of: Ile- 0 3 3 Leu; lie 2 MAlVal; and lie G 3 Leu and lie 20 ,aVal. Preferably, the hybrid comprises a acetyl-CoA carboxylase gene, further comprising a 5 polynucleotide that corresponds to a carboxyl transferase domain; and wherein the carboxyl transferase domain comprises anyone of the amino acids or polypeptides selected from the group consisting of, a aIsnLeu;

S

20 saVal; 10 * 1 ssLeu and 20 IVal; SEQ ID No 1: SEQ ID No 2: * SEQ ID No 1 and SEQ ID No 2; SEQ ID No 3: 15 * SEQ ID No 4: and * SEQ ID No. 8. Preferably, the hybrid comprises an acetyl-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain; and wherein the polynucleotide that corresponds to a carboxyl transferase domain comprises 20 nucleic acids selected from the group consisting of; E * qTA; e GT; * s, 7 TTA and 6277 GTT; SEQ ID No 5; 25 SEQIDNo6; Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 7 " SEQ ID No 5 and SEQ ID No 6; and " SEQ ID No T Preferably, the barley hybrid is a commercial cultivar. That is, it is adapted for us in the commercial production of barley. 5 In a seventh aspect, the invention is a seed of a barley hybrid as herein described. In an eighth aspect, the invention is germplasm of a barley hybrid as herein described. In a ninth aspect, the invention is a method of producing barley, the method comprising; (1) selecting a barley plant as described herein; (2) growing the said barley plant; 10 (3) harvesting barley from the barley plant; and wherein the barley plant has increased resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant. In a tenth aspect, the invention is a method of producing barley, the method comprising; 15 (1) selecting a barley hybrid as described herein; (2) growing the said barley hybrid; (3) harvesting barley from the barley hybrid: and wherein the hybrid has increased resistance to inhibition by one or more acetyi-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a 20 barley hybrid. Preferably, the methods of the ninth and tenth aspects of the invention improve weed control compared to weed control normally observed for producing barley. Preferably, the methods of the ninth and tenth aspects of the invention decrease contamination with weed species compared to contamination normally observed for 25 producing barley. In an eleventh aspect, the invention provides a method of controlling weeds in the vicinity of a barley plant as described herein, comprising: Rectiffed Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 8 (a) providing one or more acetyl-CoA carboxylase inhibiting herbicides (b) applying said one or more acetyl-CoA carboxylase inhibiting herbicides to a field comprising the barley plant as described herein; and (c) controlling weeds in the vicinity of said barley plant such that weed growth is 5 adversely affected by the application of said one or more herbicides and growth of said barley plant is not adversely affected. In a twelfth aspect; the invention provides a method of controlling weeds in the vicinity of a barley hybrid as described herein, comprising: (a) providing one or more acetyl-CoA carboxylase inhibiting herbicides 10 (b) applying said one or more acetyi-CoA carboxylase inhibiting herbicides to a field comprising the barley hybrid of as described herein: and (c) controlling weeds in the vicinity of said barley hybrid such that weed growth is adversely affected by the application of said one or more herbicides and growth of said barley hybrid is not adversely affected. 15 Preferably, in the methods of the eleventh and twelfth aspects of the invention, the acetyl CoA carboxylase inhibiting herbicide is selected from the group consisting of; an aryloxyphenoxypropionate herbicide and a cyclohexanedione herbicide. Preferably. the aryloxyphenoxypropionate herbicide is selected from the group consisting of; clodinafop propargyI; diclofop-methy; fenoxyprop-p-ethyl; quizalofop-p-ethy; haloxyfop; fluazifop; 20 and propaquizafop. Preferably, the cyclohexanedione herbicide is selected from the group consisting of; clethodim; sethoxydim; tepraloxydim; tepraloxydim; tralkoxydim; and butroxydim. Preferably, the phenylpyrazolin herbicide is pinoxaden Preferably, the one or more acetyi-CoA carboxylase inhibiting herbicides is applied at a 25 rate selected from the group consisting of: between 5ml/ha and 1000ml!ha: between 10mliha and 500mliha; between 50mliha and 400ml/ha; between 100mi/ha and 400ml/ha; between 200milha and 400ml/ha. In a thirteenth aspect, the invention is a barley plant or barley hybrid as described herein, where said barley plant or said barley hybrid confers resistance to inhibition by one or 30 more acetyl-CoA carboxylase inhibiting herbicides at levels that would normally rnhibit the growth of a barley plant, wherein said levels are selected from the group consisting of the following herbicide application rates: between 5milha and 1000mliha; between 10mi/ha Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 9 and 500mi/ha; between 50ml/ha and 400ml/ha; between 100lI/ha and 400mi/ha; between 200ml/ha and 400mliha; below 1000ml/ha: below 500mi/ha; below 400ml/ha; below 300mi/ha; below 200mlfha; below 100ml/ha; below 50ml/ha; below 40mliha: below 30mliha; below 20iI/ha; and below 10milha. 5 In one preferred embodiment of the invention, the carboxyl transferase domain of the barley plant or hybrid as described herein does not have 100% sequence homology to the following amino acid sequence: ,33cCys G/u Asn Leou His Gy Serne 3 In one preferred embodiment of the invention, the barley plant or barley hybrid as described herein is sensitive to acetolactate synthase enzyme inhibitor herbicides, 10 Preferably, the barley plant is sensitive to acetolactate synthase enzyme inhibitors herbicides selected from the group consisting of: imidazolines and sulfonylureas. Other aspects and advantages of the invention will become apparent to those skilled in the art from a review of the ensuing description. Brief Description of the Drawings 15 Figure 1: This figure presents the graphical representation of the effects of four ACCase inhibiting FOP herbicides on the grain yield of barley mutant 6OZO and cultivar Fleet as discussed in Example 2. Treatments with the same letter above the bar are not statistically significant based on the least significant difference at P=0.05. Figure 2: This figure presents the photographical data of the effects of four ACCase 20 inhibiting FOP herbicides on the grain yield of BOZO and cultivar Fleet as discussed in Example 2. BOZO is shown to the left of each photo and cultivar Fleet is shown in the right of each photo. Herbicide was applied perpendicular to sowing direction (left to right). A - 5h of August 2011 (10 DAA), B - 19 of August 2011 (23 DAA), C - 1 of September 2011 (37 DAA), and D - 111 of November 2011 (108 DAA). 25 Figure 3 This figure presents the graphical representation of the effects of three ACCase inhibiting DIM herbicides on the grain yield of barley mutant BOZO and cultivar Fleet as discussed in Example 2. Treatments with the same letter above the bar are not statistically significant based on the least significant difference at P=0.05. Figure 4: This figure presents HT017 three weeks after treatment with Fusilade in 30 comparison to sensitive genotypes. Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 10 Detailed Description of the invention General Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be 5 understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features, The present invention is not to be limited in scope by the specific embodiments described 10 herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein. The invention described herein may include one or more ranges of values (e.g. size, concentration etc). A range of values will be understood to include all values within the 15 range, including the values defining the range, and values adjacent to the range that lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range. The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby 20 incorporated by reference. Inclusion does not constitute an admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates. The disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein does not constitute an 25 admission that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates. Throughout this specification, unless the context requires otherwise, the word "comprise" or variations, such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer, or group of integers, but not the exclusion of any other 30 integers or group of integers. It is also noted that in this disclosure, and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised', "comprising" and the like can have the meaning attributed to it in US Patent law; e.g., they can mean "includes". "included", "including", and the like. Rectifled Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 11 The term "barley plant refers to the species Hordeum vulqare in its total genetic variation and its many varieties and to related species within the Hordeum genus. For example, the term includes the following varieties; AC Metcalfe, Bass, Baudin, Buloke, CDC Copeland, CDC Reserve, Commander, Fairview, Fathom, Flagship, Fleet, Gairdner, Henley, 5 Hindmarsh, Keel, Navigator, Oxford, Propino, Scope, Sebastian, Skipper, SouthernStar, Quench, Vlarningh, VT Admiral. Westminster, W14593 and Wimmera. The terms "increased resistance to herbicides", "herbicide resistant barley" and "resistance to inhibition by one or more acelyl-CoA carboxylase inhibiting herbicides" mean that the plant is not as adversely affected by the herbicide as its naturally occurring 10 counterpart or wild type. The term includes tolerance to herbicides and reduced sensitivity. Preferably, the herbicide does not kill the herbicide resistant barley. In one embodiment, the herbicide only kills a small percentage of herbicide resistant barley plants in a crop selected from a group of percentage ranges consisting of: less than 1% of the barley crop: less than 2%, less than 5%; less than 10%: less than 20%; less than 15 30%; less than 40%; and less than 50%. Preferably, the herbicide does not reduce the growth rate of the herbicide resistant barley. In one embodiment, the herbicide reduces the growth rate of the herbicide resistant barley by a percentage range selected from the group consisting of: less than a 1% reduction in growth rate; less than 2%: less than 5%: less than 10%; less than 20%: less than 30%; less than 40%; and less than 50%, 20 Preferably, the herbicide does not reduce the yield rate of the herbicide resistant barley. In one embodiment, the herbicide only reduces the yield rate of the herbicide resistant barley crop by a percentage range selected from the group consisting of: less than 1% of the herbicide resistant barley crop; less than 2%; less than 5%; less than 10%; less than 20%; less than 30%; less than 40%; and less than 50%. 25 The term "acetyl-CoA carboxylase inhibiting herbicides" are those herbicides that inhibit acety-CoA carboxylase (ACC) enzyme in a plant. These are known as 'Group A' or 'Group 1 herbicides and include compounds within the following classes; aryloxyphenoxypropionates (FOP); cyclohexanediones (DIM); and phenylpyrazolins. The term carboxyll transferase domain" refers to the domain on a polypeptide or protein 30 which carries out the reaction of transcarboxylation from biotin to an acceptor moleculeL There are two recognised types of carboxyl transferase. One of them uses acyl-CoA and the other uses 2-oxo acid as the acceptor molecule of carbon dioxide. The invention further provides a seed of the non-naturally occurring barley plant which has increased resistance to herbicides, Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 12 While not wishing to be bound by theory, it appears that the increased herbicide resistance developed by the present inventors results from changes to the physiology and/or biochemistry of the plant and thus interferes with the normal action of the herbicide. This may involve altered absorption and/or translocation of the herbicide or 5 altered ability of metabolic enzymes to bind the herbicide. Thus the invention extends to the altered elements in the plant that confer herbicide resistance including altered genes) and/or proteins) including enzymes, which genes and proteins are altered compared to their counterparts in herbicide sensitive barley plants. Preferably said altered genes and/or proteins are in isolated or substantially purified form. 10 The present invention will now be described with reference to the following non-limiting Examples. The description of the Examples is in no way limiting on the preceding paragraphs of this specification, but is provided for exemplification of the methods and compositions of the invention. Examples 15 It will be apparent to persons skilled in the materials and biological arts that numerous enhancements and modifications can be made to the above described processes without departing from the basic inventive concepts, All such modifications and enhancements are considered to be within the scope of the present invention, the nature of which is to be determined from the foregoing description and the appended claims. Furthermore, the 20 following Examples are provided for illustrative purposes only, and are not intended to limit the scope of the processes or compositions of the invention, EXAMPLE I The inventors developed a barley variety, named 'BOZO', whose phenotype is a reduced sensitivity to ACC herbicides, Genetic analysis has identified genetic differences within 25 the germplasm of BOZO (compared barley plants that are ordinarily sensitive to ACC herbicides) that results in an ACC herbicide resistant phenotype. The genotype of BOZO was compared to three different barley varieties. Plant materials consisted of four different barley lines Flagship, Alexis, BOZO and Clipper. Genomic DNA was extracted from a single cotyledon of 6-day old seedlings and 30 used as the template for polymerase chain reactions (PCR). Three sets of PCR primers were designed based on the published sequence of acetyl-CoA carboxylase mRNA from black grass Alopecurus myosuroides (GenBank: AJ310767) to amplify approximately 7.5 kb of carboxyl-transferase (CT) domain encompassing the regions known to be involved in sensitivity to ACCase herbicides (De'lye C and Michel S 2005 "Universal" primers for Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 13 PCR-sequencing of grass chioroplastic acetyl-CoA carboxylase domains involved in resistance to herbicides, Weed Res 45: 323-330). PCP amplification was carried out using standard conditions. All primer sets yielded single amplification products of expected sizes which were then purified and sequenced in both 5' and 3'directions using 5 either the forward or reverse primers as used for the initial POR. Sequencing reactions were carried out at the Australian Genome Research Facility, Waite Campus, University of Adelaide. SA. Sequences were then compiled and aligned against the black grass sequence AJ310767 using BioEdit sequence alignment editor (http://www.mbio.ncsu.edu/bioedit/bioeditt html) and assessed for polymorphisms, 10 A single nucleotide polymorphism (SNP) was identified in BOZO compared to the reference barley varieties Alexis, Flagship and Clipper, The SNP is at base position 5497 transitioning from A to T which results in an amino acid change from isoleucine to leucine (Tablel). Table 1. Summary of sequencing results showing the codons of interest and the 15 sequence found at that position in each of the four varieties. ---- ene 5497 6235| 626f 6277 | 3---------- - 6418 6442 (AJ310767) ATA TGG CTT ATT GATAGCAAG TGC GGT Alexi s AA TGG cTT-A GATAGCAAG T i dC, Bozo TA TGG CA AGCAAG Tid G d 4- TTT OTTG A Clippe A---- ---------- OTT AX j. T-c C Flagship ATA TGu CT "AOA The region of DNA in BOZO known in other plant varieties to be involved in sensitivity to ACCase herbicides in the carboxyl transferase domain, was sequenced and is presented 20 in SEQ ID 7. EXAMPLE 2 The extent of BOZO's resistance to ACCase herbicides was characterized in a field trial. The aim of the trial was to investigate tolerance of BOZO to four ACCase-inhibiting FOP herbicides, three ACCase-inhibiting DiM herbicides and one ALS-inhibiting imidazolinone 25 herbicide under field conditions. Methods Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 14 A field trial was conducted at Roseworthy Campus in the lower north region of the South Australia in 2011. The Roseworthy site has a Red-brown earth soil type and a Mediterranean type climate with an average annual rainfall of 440mm, The site was previously cropped to field peas and had a low weed pressure and good soil fertility 5 The trial was established in a replicated split plot design with BOZO barley and Fleet cullivar as the main plots and various herbicide treatments as split plots. Each plot was 1.5m wide and 5m long and each treatment was replicated 3 times. Barley strips were sown with a no-till plot seeder on the 7* of June 201 1. All herbicide treatments were applied on the 26t of July at 49 days after sowing when barley was at mid fillering 10 (approximately Z22-Z25). Herbicide treatments were applied with a spray boom delivering 10iOLha spray volume with surfactants used according to herbicide labels, Various herbicide treatments used in this study are shown in Table 1. Measurements recorded included crop establishment, normalised difference vegetation index (NDVI), crop canopy height at maturity, grain yield, grain size and visual observations of crop damage from the 15 treatments. Table 2. Herbicide treatments investigated in BOZO barley tolerance trial. LeoparO (quizalofop 99.5gL) 150 300, & 600 aUha Activator I ( 02 I% wv VePrdictTM (haloxyfop 529/L) 37.5 5 & 150 moLla Upitake T" (0.5 %6 wvv Fusiiade7"(laio 2gL 40 2 60mn Correct" (poauzfp10/) 10 0,&40mh atn*(.5 % Wv Aramo T M (tepraloxy'dim 200g/Lj 125 250, & 376 mha atn (%w) FactorTroxydtm 250gkg) 90, 1 & 360I'll /ha Serargefi(1% wv Intervix' (maramox 33g/L, 250, e7Q, & 1 m/ha Supercharge T (1 % wi) fmazapyir 1P/L)EA Results 20 Cornmercial cultivar Fleet produced about 10% higher grain yield than BOZO barley mutant in the absence of any herbicide treatment. However, application of ACCase inhibiting FOP herbicides resulted in severe phytotoxicity to Fleet but little damage to BOZO mutant barley. Application of quizalofop did not cause any detectable reduction in BOZO mutant barley yield even at 2x rate (Figure 1), in contrast, even %x caused 95% 25 reduction in grain yield of Fleet and a complete kill of this cultivar at 1x and 2x dose of this herbicide (Figure 2). Haloxyfop showed even greater activity against Fleet with complete crop mortality at % x rate. BOZO mutant barley showed reduction in yield at %xY and 1x rate but there was 28% reduction at the 2x rate of haloxyfop. The differences in herbicide tolerance between Fleet and OZO were also apparent for fluazifop and propaquizafop 30 (Figure 1). Rectifled Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 15 There were significant differences between Fleet and BOZO mutant barley in tolerance to three ACCase inhibiting DIM herbicides (Figure 3). Fleet showed 85-94% yield reduction even when treated with %x dose of DIM herbicides. In contrast, BOZ0 mutant barley line showed no yield reduction at %x and 1x dose of three DIM herbicides (Figure 3). 5 However, there was statistically significant yield reduction even in 30ZO mutant barley at the 2x rate of these herbicides, The results clearly show that tolerance to FOP herbicides also extends to DIM herbicides. The results from this study also showed that 60ZO and Fleet are both highly sensitive to the mixture of imazamox and imazapyr (Intervix m). These are imidazolines which are 10 ALS (acetolactate synthase enzyme) inhibitors (and are Group B or 2 herbicides). This result indicates that it would be feasible to control the volunteer plants of the BOZO line with this herbicide treatment and it would not pose any persistent weed threat to the cropping system. Summary 15 Controlled environment and field studies were undertaken with BOZO barley in 2010 and 2011 to determine its response to different ACCase inhibiting herbicides. These studies have clearly shown high level of tolerance to quizalofop and other ACCase inhibiting herbicides. As expected barley cultivars Flagship and Fleet used in these studies were killed by these herbicides. Supporting studies undertaken in the field in 2011 have shown 20 that it is feasible to selectively control barley grass (Hordeun gfaucurn) and brome grass (Bromus rigidus) growing in BOZO barley. Studies have also shown that BOZO barley can be controlled with imidazolinone herbicides as well as glyphosate. These herbicides will prevent 60ZO barley becoming a weed in following crops. EXAMPLE 3 25 A range of additional experiments have been conducted to further characterise BOZO and support the results presented above. A summary of these controlled environment and field based tests follows. 201 Pro ram BOZ Pot Experiment I 2010): Quizalofop at 0. 0.25, 0.5, 1.0, and 2.0 x recommended 30 labei rate (30g/ha) across BOZO mutant barley, Flagship barley cultivar and barley grass (Hordeum glaucum). Visual observations were taken. BOZO Pot Experiment 2 (20101: The objective was to confirm tolerance to quizalofop in 60Z0 mutant barley and investigate tolerance to other herbicides. Herbicides included Rectiffed Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 16 were quizaiofop, haloxyfop (ACCase-inhibiting FOP) and butroxydim (ACCase-inhibiting DIM) at rates 0, 0.25, 0.5. 1.0 & 2.0 x recommended label rate. Both BOZO mutant barley and FLAGSHIP barley cultivar were screened. Visual observations were made and plant biomass measured. S BZRAC field study (2010): The objective was to investigate tolerance of B0ZO mutant barley to quizalofop, haloxyfop and butroxydim under field conditions at Roseworthy SA. Rates included 0, 0.5 and 1 x recommended label rate and visual observations made. 2011 Program 10 B0Z0 Pot Experiment3 (2011): The objective was to investigate herbicide tolerance of BOZO mutant barley to a wide range of herbicides. Herbicides included quizalofop, haloxyfop (ACCase-inhibiting FOP), clethodim, tepraloxydim, butroxydim (ACCase inhibiting DIM), iodosulfuron, mesosulfuron (ALS-inhibiting sulfonylureas), imazamox /imazapyr (ALS-inhibiting imidazolinones), and glyphosate (membrane disrupters 15 glycines). Rates applied were 0, 0.25, 0.5, 1.0 & 2.0 x recommended label rates and both BOZO mutant barley and FLEET barley cultivar were screened, Visual observations and plant biomass measurements were made. BOZO Pot Experiment 4 (201 1: The objective was to investigate herbicide tolerance of BOZO mutant barley to quizalofop, haloxyfop, fluazifop, propaquizafop (ACCase 20 inhibiting FOP), and sethoxydim (ACCase-inhibiting DIM) herbicides. Rates applied were 0, 0.25, 0 5, 1.0 & 2.0 x field rates and both BOZO mutant barley and Fleet barley cultivar were screened. Visual observations and plant biomass measurements were made. BOZO field experiment RAC-D8 (2011) The objective was to investigate herbicide tolerance of BOZO mutant barley and barley grass (Hordeurm glaucum) control. This field 25 site at Roseworthy SA had low fertility and treatments included quizalofop, haloxyfop, fluazifop, (ACCase-inhibiting FOP), ciethodirn, tepraloxydim, butroxydim (ACCase inhibiting DIM) herbicides at 0, 0.5, 1.0, and 2.0 x recommended label rates. Visual assessment of crop damage, barley grass control, NDVI, crop canopy height at maturity, grain yield, and grain size was measured. 30 BOZO field experiment RAC-S2 (2011): reported above. B02 field experiment COOK2LAINS 2011: The objective was to investigate herbicide tolerance of BOZO mutant barley in a low rainfall environment and brome grass (Bromus rigidus) control. This field site had low fertility and herbicide treatments included Rectifled Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 17 quizalofop, haloxyfop. fluazifop, (ACCase-inhibiting FOP), clethodirn, tepraloxydim, butroxydim (ACCase-inhibiting DIM) herbicides at 0, 0.5: 1.0, and 2,0 x field rates. Visual observations of crop damage, brome grass control, NDVI, crop canopy height at maturity, grain yield, and grain size was measured. 5 BOZ field experiment MNTARG (2011: The objective was to investigate herbicide tolerance of BOZO mutant barley in a high rainfall environment. Treatments investigated included quizalofop, haloxyfop, fluazifop, (ACCase-inhibiting FOP), ciethodim, tepraloxydim, butroxydim (ACCase-inhibiting DIM) herbicides at 0, 0.5, 1.0, and 2 0 x field rates. Visual observations of crop damage, NDVI, crop canopy height at maturity, grain 10 yield, and grain size was measured, BOZO field experiment BUCKLEBOO (2011): The objective was to investigate herbicide tolerance of BOZO mutant barley in a low rainfall environment as well as barley grass (Hordeum g/aucun) control. Herbicide treatments were applied at 1 & 2 x recommended label rates and included quizalofop (ACCase-inhibiting FOP), clethodim 15 (ACCase-inhibiting DIM), and imazamox /imazapyr (ALS-inhibiting imidazolinones). Crop damage was assessed based on visual symptoms and panicle density was used to determine barley grass control. EXAMPLE 4 The inventors developed a barley variety, named 'HT01 7, whose phenotype is a reduced 20 sensitivity to ACC herbicides. Seed of the variety Flagship was mutagenised with 30mM ethyl methanesulfonate (EMS) and the Mo and M generations were grown and multiplied as unselected bulk populations. The M2 generation was sown at the Charlick Experimental Farm (Strathalbyn, South Australia) on 2 0" May 2009 at a density of 140 plants m- 2 . The area sown was 2,5 hectares equating to approximately 40 million 25 individual plants. The herbicide Fusilade (fluazifop) was applied at a rate of 5OOg / ha at the early tillering stage of plant development. At stem elongation surviving barley plants were transplanted to pots, grown through to maturity and grain was harvested from individual plants. Seed from the surviving plants was sown in pois in May 2010 and treated with 5OOg / ha Fusilade at the 3 leaf stage of development. Assessment of 30 survival was conducted three weeks after spraying and ~99% of plants were found to be sensitive to the herbicide but the line HT017 was confirmed as tolerant to fluazifop. Figure 4 presents HT017 three weeks after treatment with fluazifop in comparison to sensitive genotypes, Genetic analysis has identified genetic differences within the HT017 barley compared 35 barley plants that are ordinarily sensitive to ACC herbicides that results in an ACC Rectiffed Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 18 herbicide resistant phenotype. The genotype of HTOI 7 was compared to three different barley varieties using the same methods as described above under Example I and a single mutation corresponding to the amino acid substitution listed in Table 3 was observed. 5 A single nucleotide polymorphism (SNP) was identified in HT107 compared to the reference barley varieties BOZO, Alexis. Flagship and Clipper. The SNP is at base position 56277 transitioning from A to G which results in an amino acid change from isoleucine to valine (Table 3). Table 3 Summary of sequencing results showing the codons of interest and the 10 sequence found at that position in each of the five varieties, including HT107. R en 5497 6235 665 V 7736 6 648 5-42 (A J310767) ATA TGG CTT ATT GATAGCAAG TGC GGT A lexis A------ ---- OTT--------------- - A- -- A--- c Sozo TTA "FG - TT ATT GATAGOXAG 0k C Clipper ATA MG 5TT ATT AAG - - 1 - 6 Fagship ATA G 5 AT A aTkA- GGC GGATA iOTT T ATCAAG -C G G EXAMPLE 5 15 The tolerance of HT017 to Fusilade (fluazifop) was verified in a pot experiment. HT017 and the intolerant control barley HT066 were sown on 23" June 2010 with three replicates per treatment. Two treatments comprising 50Og/ha Fusilidate and 750g/ha Fusilade were applied on 23* July 2010 and one treatment remained unsprayed. The trial was assessed on 1 8* August 2010 and all HT066 plants were destroyed at both 20 application rates. All HT017 plants survived at both application rates. EXAMPLE 6 The mutant barley HTO1 7 was tested for tolerance against a range of Group A herbicides. Three replicate pots were sown for each herbicide treatment of the tolerant line HT017 and also the control barley HT066. Seed was sown 17" August 2010 and plants were Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 19 treated at the three leaf stage of plant development with the maKimum label rate of Fusilade (fluazifop), Select (clethodim), Factor (butroxydim), Verdict (haloxyfop), Targa (quizalofop) or Sertin (sethoxydim). All plants of the control barley HT066 were destroyed by the treatment and the ACCase-inhibiting herbicide tolerance of HT017 was confirmed. 5 Rectified Sheet (Rule 91) ISA/AU

Claims (27)

1. A barley plant, wherein said barley plant confers resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant.

2. A barley plant of claim 1, wherein the barley plant is a commercial cultivar.

3. A barley plant of claims 1 or 2, wherein the acetyi-CoA carboxylase inhibiting herbicide is selected from the group consisting of; a aryloxyphenoxypropionate herbicide and a cyclohexanedione herbicide.

4. A barley plant of claim 3, wherein the aryloxyphenoxypropionate herbicide is selected from the group consisting of; clodinafop-propargyl; diclofop-methy, fenoxyprop-p-ethyl: quizalofop-p-ethyl; haloxyfop fluazifop; and propaquizafopL

5. A barley plant of claim 3, wherein the cyclohexanedione herbicide is selected from the group consisting of; clethodim; sethoxydim; tepraloxydin: tralkoxydim; and butroxydim.

6. A barley plant of anyone of claims 1 to 5, wherein the barley plant comprises a acetyl-CoA carboxylase gene, wherein said gene corresponds to the amino acid substitution selected from the group consisting of: lle 1 m 3 Leu; lie 2 9 3SVal; and lie 1 , 3 ,Leu and lie 2 oeVal,

7. A barley plant of anyone of claims 1 to 5, wherein the barley plant comprises an acetyl-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain; and wherein the carboxyl transferase domain comprises anyone of the amino acids or polypeptides selected from the group consisting of; * 23Leu and 2 osVal; SEQ ID No 1; 6 SEQ ID No 2; Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 21 4 SEQ ID No Iand SEQ ID No2; SEQ ID No 3; 4 SEQ ID No 4; and 4 SEQ ID No 8.

8. A barley plant of anyone of claims I to 5, wherein the barley plant comprises an acetyl-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain; and wherein the polynucleotide that corresponds to a carboxyl transferase domain comprises anyone of the nucleic acids and polynucleotides selected from the group consisting of; 4 r,-TTA; * rrm7GTT; 4 , 4 WTTA and 62 mGTT: 4 SEQ ID No 5 4 SEQ ID No 6; * SEQ iD No 5 and SEQ ID No 6, and 4 SEQ iD No 7.

9. A barley plant of anyone of claims I to 8, wherein the barley plant is not genetically modified.

10. A seed of a barley plant of anyone of claims 1 to 9.

11 A germplasm of a barley plant of anyone of claims I to 9.

12. A method of producing a barley hybrid with increased resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant, said method comprising; selecting a first barley plant; selecting a second barley plant; Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 22 crossing the first barley plant with the second barley plant to produce hybrid offspring; selecting a hybrid from said hybrid offspring with increased resistance to inhibition by one or more acetyi-CoA carboxylase inhibiting herbicideacetyl-CoA carboxylase inhibiting herbicides thereby to produce the hybrid; and wherein the second barley plant is a barley plant of anyone of claims 1 to 9.

13. A method according to claim 12, wherein the first barley plant is selected from the group consisting of AC Metcalfe, Bass, Baudin, Buloke, CDC Copeland. CDC Reserve, Commander, Fairview, Fathom, Flagship, Fleet, Gairdner, Henley. Hindmarsh, Keel, Navigator, Oxford, Propino, Scope, Sebastian, Skipper, SouthernStar, Quench, Viamingh, VT Admiral, Westminster, W14593 and Wimmera.

14. A method according to claims 12 or 13, wherein the second barley plant is selected from the group consisting of, BOZO; and HTO 17.

15. A barley hybrid produced from the method of anyone of claims 12 to 14.

16. A barley hybrid of claim 15, wherein the hybrid comprises a acetyi-CoA carboxylase gene, wherein said gene corresponds to the amino acid substitution selected from the group consisting of: le 1 3 3 Leu; ile 2 s, 3 Val; and lle 3Leu and lie

17. A barley hybrid of claim 15, wherein the hybrid comprises a acetyi-CoA carboxylase gene, further comprising a polynucleotide that corresponds to a carboxyl transferase domain; and wherein the carboxyl transferase domain comprises anyone of the amino acids or polypeptides selected from the group consisting of; * 20 sVal; 33 m3Leu and 2 3 Val; SEQ ID No 1; SEQ ID No 2; Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 23 " SEQ iD No I and SEQ ID No 2: " SEQ ID No 3; " SEQ ID No 4; and Ib SEQ ID No 8.

18. A barley hybrid of claim 15, wherein the hybrid comprises a acetyl-CoA carboxylase gene further comprising a polynucleotide that corresponds to a carboxyl transferase domain; and wherein the polynucleotide that corresponds to a carboxyl transferase domain comprises nucleic acids selected from the group consisting of; K 4WTTA e 2 7mGTT: 54 q 7 TTA and smeGTT; K SEQ ID No 5 K SEQ ID No 6; K SEQ iD No 5 and SEQ ID No 6; and K SEQ ID No 7.

19. A barley hybrid according to anyone of claims 15 to 18, wherein the barley is a commercial cultivar.

20. A seed of a barley hybrid according to anyone of claims 15 to 19.

21. Germplasm of a barley hybrid according to anyone of claims 15 to 19.

22, A method of producing barley, the method comprising; (1) selecting a barley plant of claims 1 to 9; (2) growing the said barley plant; (3) harvesting barley from the barley plant and Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 24 wherein the barley plant has increased resistance to inhibition by one or more acetyl-CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley plant.

23. A method of producing barley, the method comprising; (1) selecting a barley hybrid according to anyone of claims 15 to 19; (2) growing the said barley hybrid; (3) harvesting barley from the barley hybrid; and wherein the hybrid has increased resistance to inhibition by one or more acetyl CoA carboxylase inhibiting herbicides at levels that would normally inhibit the growth of a barley hybrid,

24. A method of producing barley according to claims 22 or 23, wherein the method improves weed control compared to weed control normally observed for producing barley.

25. A method of producing barley according to claims 22 or 23, wherein the method decreases contamination with weed species compared to contamination normally observed for producing barley.

26. A method of controlling weeds in the vicinity of a barley plant of claims 1 to 9. comprising: (a) providing one or more acetyl-CoA carboxylase inhibiting herbicides (b) applying said one or more acetyl-CoA carboxylase inhibiting herbicides to a field comprising the barley plant of claims 1 to 9; and (c) controlling weeds in the vicinity of said barley plant such that weed growth is adversely affected by the application of said one or more herbicides and growth of said barley plant is not adversely affected.

27. A method of controlling weeds in the vicinity of a barley hybrid according to anyone of claims 15 to 19, comprising: (a) providing one or more acetyi-CoA carboxylase inhibiting herbicides (b) applying said one or more acetyl~CoA carboxylase inhibiting herbicides to a field comprising the barley hybrid according to anyone of clairns 15 to 19; and Rectified Sheet (Rule 91) ISA/AU WO 2014/113846 PCT/AU2014/000060 25 (c) controlling weeds in the vicinity of said barley hybrid such that weed growth is adversely affected by the application of said one or more herbicides and growth of said barley hybrid is not adversely affected. Rectified Sheet (Rule 91) ISA/AU