U.S. patent application number 14/540478 was filed with the patent office on 2015-03-19 for herbicide resistant barley.
The applicant listed for this patent is AGRICULTURE VICTORIA SERVICES PTY LTD. Invention is credited to Michael Materne, David Moody, Christopher Pittock.
Application Number | 20150075070 14/540478 |
Document ID | / |
Family ID | 41264332 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150075070 |
Kind Code |
A1 |
Materne; Michael ; et
al. |
March 19, 2015 |
Herbicide Resistant Barley
Abstract
The application discloses non-naturally occurring barley plants
with increased resistance to herbicides, particularly imidazolinone
herbicides. Also disclosed are the barley seeds per se, an isolated
nucleic acid incorporating the nucleic acid sequence conferring
imidazolinone resistance, a method of inhibiting weed growth in the
vicinity of a barley plant, an a method of growing a barley crop.
Seeds have been deposited at NCIMB.
Inventors: |
Materne; Michael; (Horsham,
AU) ; Pittock; Christopher; (Horsham, AU) ;
Moody; David; (Horsham, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AGRICULTURE VICTORIA SERVICES PTY LTD |
ATTWOOD |
|
AU |
|
|
Family ID: |
41264332 |
Appl. No.: |
14/540478 |
Filed: |
November 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12991172 |
Jan 28, 2011 |
8889951 |
|
|
PCT/AU09/00558 |
May 6, 2009 |
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14540478 |
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Current U.S.
Class: |
47/58.1R ;
504/130; 800/300 |
Current CPC
Class: |
A01H 1/06 20130101; A01H
5/10 20130101; A01G 22/00 20180201; A01N 43/50 20130101 |
Class at
Publication: |
47/58.1R ;
800/300; 504/130 |
International
Class: |
A01H 5/10 20060101
A01H005/10; A01G 1/00 20060101 A01G001/00; A01N 43/50 20060101
A01N043/50 |
Foreign Application Data
Date |
Code |
Application Number |
May 6, 2008 |
AU |
2008902205 |
Claims
1. A non-naturally occurring barley plant with increased resistance
to imidazolinone herbicides compared to a wild type Buloke barley
plant and deposited under NCIMB Deposit accession number NCIMB
41547, NCIMB 41550 or NCIMB 41551.
2. The non-naturally occurring barley plant of claim 1, deposited
as NCIMB Deposit accession number NCIMB 41547.
3. The non-naturally occurring barley plant of claim 1, deposited
as NCIMB Deposit accession number NCIMB 41550.
4. The non-naturally occurring barley plant of claim 1, deposited
as NCIMB Deposit accession number NCIMB 41551.
5. A seed of the plant of claim 1, said seed having increased
resistance to imidazolinone herbicides compared to seed of a wild
type Buloke barley plant.
6. A barley plant, or a descendent of a barley plant, grown or
otherwise derived from the seed of claim 5.
7. A method of inhibiting weed growth in the vicinity of a barley
plant comprising growing the barley plant of claim 1 and applying
imidazolinone herbicide to said barley plant and its vicinity
sufficient to inhibit weed growth.
8. The method of claim 7 wherein the imidazolinone herbicide
comprises at least two constituents selected from the group
consisting of imazamox, imazapyr, and imazapic.
9. The method of claim 8 wherein the imidazolinone herbicide is
applied after plant emergence.
10. The method of claim 9 wherein the imidazolinone herbicide used
is a combination of imazamox and imazapyr applied post emergence at
a rate of 12.375 g per hectare and 5.625 g per hectare
respectively; 24.75 and 11.25 g per hectare respectively; 41.25 and
18.75 g per hectare respectively or 49.5 and 22.5 g per hectare
respectively.
11. The method of claim 9 wherein the imidazolinone herbicide used
is a combination of imazamox and imazapyr applied at a rate of
26.25 and 8.75 g per hectare respectively or 42 and 14 g per
hectare respectively.
12. A method of growing a barley crop comprising the steps of:
sowing the seed of claim 5; and cultivating said seed and a plant
resulting therefrom to produce a barley crop.
13. The method of claim 12 further comprising a step of applying an
imidazolinone herbicide to said crop to inhibit growth of weeds in
the crop.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to grain plants having
herbicide resistance or tolerance. More specifically the present
invention relates to herbicide resistant barley plants.
BACKGROUND OF THE INVENTION
[0002] Food production, particularly grain production is of
increasing importance worldwide due to growing populations and
other pressures such as reduction in arable land, climate change
and the increasing use of crops for biofuel production.
[0003] Herbicides play an important role in modern agriculture by
maximizing grain production, pasture production, quality and
profitability. Herbicides allow undesirable plants to be killed or
suppressed and thereby reduce competition with desirable plants
such as crop plants for nutrients, water and the like.
[0004] Imidazolinone and sulphonylurea herbicides inhibit the
growth and development of susceptible plants, including a wide
range of weeds, by inhibiting the plant enzyme Acetohydroxy acid
synthase (AHAS), also known as acetolactate synthase (ALS). AHAS
synthesizes branch chain amino acids, a key function in plants.
Importantly, imidazolinone and sulphonylurea herbicides also have
relatively low toxicity to animals, including humans.
[0005] Imidazolinones include the following compounds imazethapyr,
imazaquin, imazapyr, imazapic, imazamox and the like.
[0006] Commercially available imidazolinone herbicides include ON
DUTY.RTM. (imazapic+imazapyr) INTERVIX.RTM. (imazamox+imazapyr)
SPINNAKER.RTM. (imazethapyr), RAPTOR.RTM. (imazamox) and FLAME.RTM.
(imazapic).
[0007] Although naturally occurring resistance to herbicides has
been described in some crops many important food crops are
susceptible to herbicides including imidazolinone.
[0008] Barley (Hordeum vulgare) is an important grain crop
internationally because it is used in malt production, brewing and
for human and animal feed. It has the advantage of being more salt
tolerant than wheat and can be grown successfully on poorer quality
land and/or in drier environments.
[0009] Weed control is a major issue in barley production. Good
chemical weed control is desirable to maximize production of the
crop and limit the need for mechanical cultivation and resultant
damage to soil structure and erosion. In particular it would be
desirable to utilize a greater range of low toxicity herbicides to
control the full spectrum of a variety of weeds in a barley
cropping system. For example, as most barley is resistant to
sulphonylureas, this herbicide can be applied post emergence to
growing barley crops, however, imidazolinones cannot be applied
post emergence because barley is susceptible to this class of
herbicides. Further, the crop cannot even be sown into soil
containing imidazolinones because of its susceptibility to this
herbicide.
[0010] The above references to and descriptions of prior proposals
or products are not intended to be, and are not to be construed as,
statements or admissions of common general knowledge in the
art.
SUMMARY OF THE INVENTION
[0011] The invention provides a non-naturally occurring barley
plant which has increased resistance to herbicides compared with
the wild type barley.
[0012] The invention also provides a seed of the non-naturally
occurring barley plant. Further the invention provides a method of
inhibiting weed growth in the vicinity of a barley plant comprising
growing a barley plant with increased resistance to a herbicide
compared to a wild type barley plant and applying under suitable
conditions imidazolinone to said barley plant with increased
resistance and its vicinity sufficient to inhibit weed growth.
[0013] The invention also relates to a method of growing a barley
crop comprising sowing the seeds of the invention and cultivating
the seeds and resultant plants under suitable conditions to produce
a crop.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows a sequence alignment for the AHAS gene
localised around base 1742 (indicated by a box) of the following
imidazolinone resistant mutant lines:
VBHT 0805 (SEQ ID NO: 8)
VBHT 0806 (SEQ ID NO: 9)
VBHT 0802 (SEQ ID NO: 10)
[0015] and the following wild type comparators:
AF059600 (SEQ ID NO: 5)
Buloke (SEQ ID NO: 6)
Hindmarsh (SEQ ID NO: 7).
[0016] FIG. 2 shows amino acid sequence alignment of the AHAS gene
for six plant species localised around mutant DNA nucleotide base
1742 identified in lines EMS05*06HI005 and EMS05*06HI006
(H.vulgare_AHAS_mutant) and the resulting alteration of a Serine
(S) to an Asparagine (N):
Nicotiana tabacum AHAS/1-24 (SEQ ID NO: 11) Brassica napus
AHAS1/1-24 (SEQ ID NO: 12) Brassica napus AHAS2/1-24 (SEQ ID NO:
13) Hordeum vulgare AHAS/1-24 (SEQ ID NO: 14) Hordeum vulgare AHAS
mutant/1-24 (SEQ ID NO: 15) Oryza saliva AHAS/1-24 (SEQ ID NO: 16)
Triticum aestivum AHAS/1-24 (SEQ ID NO: 17) Zea mays AHAS/1-24 (SEQ
ID NO: 18).
[0017] FIG. 3 shows alignment of resequenced AHAS gene for VBHT
0805, VBHT 0806, VBHT 0802 and VBHT 0810 compared to the wild type
reference sequences (AHAS.sub.--H.vulgare_AF059600 and Buloke):
VBHT 0805 (SEQ ID NO: 1)
VBHT 0806 (SEQ ID NO: 2)
VBHT 0802 (SEQ ID NO: 19)
[0018] AHAS H. vulgare AF059600 (SEQ ID NO: 20)
VBHT 0810 (SEQ ID NO: 21)
Buloke (SEQ ID NO: 22)
[0019] FIG. 4 shows the imidazolinone resistance of the mutated
lines VBHT0802, VBHT0805, VBHT0806 and VBHT0810 compared to the
wild type lines Buloke and Hindmarsh in experiments at Horsham,
Victoria, Australia in 2007 (Table 3), and 2008 (Table 4) and at
Marinna, NSW, Australia in 2008 (Table 5). The experiments are
described in Example 5.
[0020] FIG. 5 shows in Table 6 yield (t/ha) of imidazolinone
resistant mutant lines compared with Buloke at 9 sites in
Australia. The experiments are described in Example 6.
[0021] FIG. 6 shows in Table 7 morphological and DNA comparison of
imidazolinone resistant mutant barley lines with parent cultivar
Buloke. The experiments are described in Example 7.
[0022] FIG. 7 shows in Table 8 malting quality for VBHT0805
compared to Buloke. The experiments are described in Example
10.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention provides a non-naturally occurring barley
plant which has increased resistance to herbicides compared to wild
type barley, particularly resistance to imidazolinone herbicides.
Progeny of the barley plants and descendants having the increased
herbicide resistance trait or characteristic are also included in
the invention. The invention also includes hybrids of the barley
plants of the invention.
[0024] The term "non-naturally occurring" refers to a plant which
does not occur in nature and is the result of human intervention.
Preferably the plants are produced by mutagenesis, more preferably
chemically induced mutagenesis.
[0025] The term "barley plant" refers to the species Hordeum
vulgare in its total genetic variation and its many varieties such
as Buloke and Hindmarsh, and to related species within the Hordeum
genus.
[0026] The terms "increased resistance to herbicides" and
"herbicide resistant barley" mean that the plant is not as
adversely affected by the herbicide as its naturally occurring
counterpart or wild type. The term includes tolerance to
herbicides.
[0027] The invention further provides a seed of the non-naturally
occurring barley plant which has increased resistance to
herbicides, particularly imidazolinone herbicides.
[0028] 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 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
gene(s) and/or protein(s) including enzymes, which genes and
proteins are altered compared to their counterparts in herbicide
sensitive barley plants.
[0029] Preferably said altered genes and/or proteins are in
isolated or substantially purified form.
[0030] The plants and seeds of the invention include plants and
seeds that are mutations, recombinant variants and genetically
engineered derivatives or derivatives derived by other means such
as conventional breeding, which retain the altered elements
described above. The invention also extends to plant parts other
than seeds of the herbicide resistant barley, including plant
cells, plant tissues and plant organs whether produced by plant
tissue culture or otherwise.
[0031] In a preferred embodiment the seeds of the invention are
VBHT 0802, VBHT 0803, VBHT 0805, VBHT 0806, VBHT 0807, VBHT 0809 or
VBHT0810 as described herein or are plants derived from these
seeds. The invention also provides seeds or plants which are
mutants, derivatives and variants thereof having increased
imidazolinone resistance compared to wild type barley plants.
[0032] Samples of the following seeds were deposited in accordance
with the Budapest Treaty on 18 Apr. 2008 at NCIMB Ltd of Ferguson
Building, Craibstone Estate, Buckburn, Aberdeen, Scotland and
accorded accession numbers as shown:
TABLE-US-00001 TABLE 1 Deposits Designation Accession No. VBHT 0802
NCIMB 41547 VBHT 0803 NCIMB 41548 VBHT 0805 NCIMB 41549 VBHT 0806
NCIMB 41550 VBHT 0809 NCIMB 41551
[0033] The invention also provides a method of inhibiting weed
growth in the vicinity of a barley plant comprising growing a
barley plant with increased resistance to a herbicide compared to a
wild type barley plant and applying under suitable conditions
imidazolinone to said barley plant and vicinity sufficient to
inhibit weed growth.
[0034] The invention also relates to a method of inhibiting weed
growth in a barley crop said method comprising growing a herbicide
resistant barley crop and applying a imidazolinone herbicide to
said crop under suitable conditions to inhibit weed growth.
[0035] The imidazolinone may be used as a combination of imazamox
and imazapyr at a rate of 12.375 g per hectare and 5.625 g per
hectare respectively; 24.75 and 11.25 g per hectare respectively;
41.25 and 18.75 g per hectare respectively or 49.5 and 22.5 g per
hectare respectively. Alternatively, the imidazolinone may be used
as a combination of imazapic and imazapyr at a rate of 26.25 and
8.75 g per hectare respectively or 42 and 14 g per hectare
respectively.
[0036] The invention also relates to a method of growing a barley
crop comprising sowing the seed of the invention and cultivating
the seed and resultant plant under suitable conditions to produce a
crop.
[0037] Preferably the herbicide resistant barley used for the crop
is produced by mutagenesis, more preferably chemical
mutagenesis.
[0038] Still more preferably the herbicide resistant crop is, or is
derived from, NCIMB 41547, NCIMB 41548, NCIMB 41549, NCIMB 41550 or
NCIMB 41551 or has the herbicide resistant characteristics
thereof.
[0039] The following non limiting examples describe the
invention.
EXAMPLE 1
Production of Mutants
[0040] 33.3kg (approximately 812,195 seeds) of the barley variety
Buloke was soaked in 0.25% Ethyl methane sulfonate (EMS) and dried
using an air blower. The parent Buloke is a high yielding variety
which produces grain of malting quality. Surprisingly Buloke is
suited to areas where brome grass is a problem weed. In selecting
this strain, the inventors hoped to develop an imidazolinone
resistant barley variety which could be grown under conditions
where brome grass and other weeds may be controlled with
imidazolinone herbicides.
EXAMPLE 2
M1 Generation
[0041] Mutated barley seed from Example 1 was sown in a plot of
0.5ha at Horsham, Victoria, Australia in 2006. Management was
consistent with those recommended for barley in the region. At
maturity the plot was harvested by an experimental plot harvester.
300g samples were taken from each full seed bin and bulked together
to form a representative smaller composite (150 kg) and remaining
seed was bulked together as a composite sample (600 kg).
EXAMPLE 3
M2 Generation
[0042] 200 kg of mutated barley seed from example 2 (approx. 4.88
million seeds) was sown on 2 ha of land at Horsham, Victoria,
Australia in 2007. Emerged plants were sprayed with ON DUTY.RTM. at
a rate of 80 g/ha (active ingredient. Imazapic 42 g+Imazapyr 14 g)
and 20 surviving plants were harvested individually by hand and
retained.
EXAMPLE 4
Preliminary Evaluation Of M2 Selections For Resistance To
Imidazolinone Herbicides.
[0043] The ten plants with the largest dry matter and seed yield
from Example 3 were identified and designated BULOKE-EMS05*06HI001,
BULOKE-EMS05*06HI002, BULOKE-EMS05*06HI003, BULOKE-EMS05*06HI004,
BULOKE-EMS05*06HI005, BULOKE-EMS05*06HI006, BULOKE-EMS05*06HI007,
BULOKE-EMS05*061HI008, BULOKE-EMS05*06HI009, BULOKE-EMS05*06HI010.
30 seeds from each selection were sown in 10 pots (3 seeds per pot)
in a glasshouse. Half of the pots for each selection were sprayed
with ON DUTY.RTM. at a rate of 50 g/ha (active ingredient. Imazapic
26.25 g+Imazapyr 8.75 g) and each selection classified for
resistance to ON DUTY.RTM. based on the reaction of progenies.
Plants were deemed resistant if they appeared unaffected by ON
DUTY.RTM. (no visual symptoms) or susceptible if they were killed
by ON DUTY.RTM., [0044] The following Homozygous susceptible
(escapes in field screening) selections were identified and
discontinued from further evaluation: BULOKE-EMS05*06HI001,
BULOKE-EMS05*06HI004, BULOKE-EMS05*06HI008. [0045] The following
Homozygous resistant selections were identified and resistant
plants bulked together for evaluation: BULOKE-EMS05*06HI002,
BULOKE-EMS05*06HI005, BULOKE-EMS05*06HI006, BULOKE-EMS05*06HI009,
BULOKE-EMS05*06HI010. [0046] Although homozygous for resistance to
ON DUTY, BULOKE-EMS05*06HI009 was segregating for genes that
affected plant type and single plants were harvested separately for
further evaluation. [0047] Heterozygous susceptible/resistant
selections were identified and individual resistant plants were
harvested for further evaluation: BULOKE-EMS05*06HI003,
BULOKE-EMS05*06HI007 A homozygous herbicide resistant line
representing each original resistant M2 plant selection was
selected for submission to NCIMB and further evaluation. These
lines were given shorter, new names to improve communications as
shown in Table 2:
TABLE-US-00002 [0047] TABLE 2 Original and new names for homozygous
herbicide resistant lines Name Original name VBHT0802
BULOKE-EMS05*06HI002 VBHT0803 BULOKE-EMS05*06HI003-06GI003 VBHT0805
BULOKE-EMS05*06HI005 VBHT0806 BULOKE-EMS05*06HI006 VBHT0807
BULOKE-EMS05*06HI007-06GI001 VBHT0809 BULOKE-EMS05*06HI009-06G082
VBHT0810 BULOKE-EMS05*06HI010
EXAMPLE 5
Field Evaluation For Tolerance To Intervix.RTM.
a) Selection Of Lines With Homozygous Resistance To Imidazolinone
Herbicides
[0048] Seed from single plants selected in the glasshouse (Example
4) were sown in 5 m paired rows at Horsham, Victoria, Australia, in
2007 and sprayed with 0.75 L/ha of Intervix.RTM. (active ingredient
Imazamox 33 grams/kg+Imazapyr 15 grams/kg) post emergence and rows
that were visually unaffected by the herbicide were harvested for
further evaluation (data not presented). Homozygous
imidazolinone_resistant lines VBHT0803 and VBHT0806 were identified
and submitted to NCIMB.
[0049] b) Evaluation Of Resistance To Imidazolinone Herbicides
The imidazolinone resistance of the mutated lines VBHT0805,
VBHT0806, and VBHT0810 were compared to the wild type lines Buloke
and Hindmarsh in experiments at Horsham, Victoria, Australia in
2007 and 2008 and Mariana, NSW, Australia in 2008. At Horsham the
experiments were sown in a split plot design with 2 replications in
2007 and 3 replications in 2008, and at Marinna a factorial design
was used with 3 replications. At Horsham genotypes (Selections)
were allocated as subplots and herbicide treatment as mainplots
Herbicide treatments were: [0050] Horsham 2007--no applied
Intervix.RTM. and Intervix.RTM. applied post emergence at 0.75 L/ha
(active ingredient24.75 g imazamox+11.25 g imazapyr) [0051] Horsham
2008--no applied Intervix.RTM. and Intervix.RTM. applied post
emergence at 0.375 L/ha (active ingredient 12.375 g imazamox+5.625
g imazapyr), 0.75 L/ha (active ingredient 24.75 g imazamox+11.25 g
imazapyr), 1.5 L/ha (active ingredient 49.5 g imazamox+22.5 g
imazapyr). [0052] Marinna 2008--no applied Intervix.RTM. and
Intervix.RTM. applied post emergence at 0.375 L/ha (active
ingredient 12.375 g imazamox+5.625 g imazapyr), 0.75 L/ha (active
ingredient 24.75 g imazamox+11.25 g imazapyr), 1.25 L/ha (active
ingredient 41.25 g imazamox+18.75 g imazapyr). Management of all
experiments was consistent with established commercial practices
except the application of Intervix.RTM. herbicide. Plots were
harvested using a small plot harvester, and seed weighed, converted
to yield in t/ha and analyzed using REML. A 500 gram sample from
replication 1, no applied Intervix.RTM. was evaluated for malting
quality using established methods (see Example 10). Intervix
applied at all rates significantly reduced the grain yield of the
varieties Buloke and Hindmarsh compared to the control treatment
(no applied Intervix) in all three experiments (Tables 3, 4 and 5).
In both cases all plants in the Intervix.RTM. treated plots turned
yellow, ceased to grow and died. In contrast, there was no
significant difference in the grain yield of VBHT0805, VBHT0806 or
VBHT0810 when treated with Intervix.RTM. at any rate compared to
the control treatment (no applied Intervix). Furthermore, no visual
symptoms of damage could be observed in the Intervix treated plots
of VBHT0805, VBHT0806 or VBHT0810. VBHT0805, VBHT0806 or VBHT0810
are all derived from Buloke that was treated with EMS and clearly
exhibit improved resistance to Intervix.RTM. at rates from 0.375
L/ha (active ingredient 12.375 g imazamox+5.625 g imazapyr) to 1.5
L/ha (active ingredient 49.5 g imazamox+22.5 g imazapyr) compared
to the wild type parent Buloke and the cultivar Hindmarsh.
EXAMPLE 6
Field Evaluation To Compare The Yield Of Imidazolinone Resistant
Mutant Barley Lines With Buloke.
[0053] The yield (t/ha) of imidazolinone resistant mutant lines
VBHT0805, VBHT0806 or VBHT0810 were compared with Buloke at 9 sites
in Australia. Paskeville and Callington were located in the state
of South Australia, Forbes, Temora and Junee Reefs in New South
Wales, Dimboola, Elmore and Mt Mercer in Victoria, and Northam
early and late sown in Western Australia. VBHT0805 and VBHT0802 had
the highest yields across all sites and averaged 4% higher than
Buloke indicating that the mutation process had not cause
deleterious mutations that significantly affect grain yield in
Australia. Alternatively VBHT0806 was 4% lower yielding than
Buloke. These results are shown in Table 6.
EXAMPLE 7
Morphological And DNA Comparison Of Imidazolinone Resistant Mutant
Barley Lines With Buloke
[0054] The morphological characteristics and DNA of imidazolinone
resistant mutant barley lines VBHT0802, VBHT0803, VBHT0805,
VBHT0806, VBHT0807, VBHT0809 and VBHT0810 were compared with the
wild type Buloke. Table 7 describes the distinctive morphological
and DNA characteristics identified. VBHT0803 differs from Buloke in
having purple awns and is shorter than Buloke but not a dwarf.
VBHT0809 has purple awns and is a dwarf plant with a prostrate
vegetative growth habit. VBHT0805, VBHT0807 and VBHT0810 have
identical haplotypes suggestive of being derivatives of a single
mutational event. VBHT0802, VBHT0803, VBHT0806 and VBHT0809 all
have unique haplotypes that is likely to be due to the
heterogeneity within the original Buloke sample used for the
mutagenesis and indicates that each represents a unique mutational
event for resistance to imidazolinone herbicides. The haplotypes of
VBHT0805, VBHT0807 and VBHT0810 differed from the Buloke reference
sample (VB0105*12) at 41 of 1424 single polynucleotide polymorphism
(SNP) loci.
EXAMPLE 8
DNA Sequencing Of The AHAS Gene Of Imidazolinone Resistant Mutant
Barley Lines.
[0055] a. Overview:
[0056] Mutant lines of barley (Hordeum vulgare L.) screened for
resistance to the herbicide imidazolinone and identified as
containing resistance determinants as per the previous examples
were tested to confirm and characterise the mutant lines, the
suspected mutant alleles of the acetohydroxyacid synthase (AHAS)
gene, and resequenced to identify DNA base changes. The DNA
sequences were collated and compared to wild type sequences and
known mutations.
b. Experimental Approaches and Results:
[0057] The plant material consisted of the wild type genotypes,
Buloke along with 4 mutant lines derived from Buloke numbered
VBHT0802 (NCIMB 41547), VBHT0805 (NCIMB 41549), VBHT0806 (NCIMB
41550) and VBHT0810 (not deposited). A collection of publicly
available reference sequences of the AHAS gene was assembled from
Genbank. The sequences were obtained from a range of
monocotyledonous species, (including barley) and were used for PCR
primer design to conserved regions of the gene to ensure optimal
primer performance. A total of 20 primers were designed in forward
and reverse orientation to enable maximal coverage of the available
gene sequence and provide multiple options for primer pairing to
mitigate the possibility of poor primer performance.
[0058] PCR amplification using standard conditions was tested on
all supplied DNA templates. Amplification performance was low, with
all possible primer combinations and resulted in the production of
multiple products as resolved on an agarose gel. Nested PCR was
employed to reduce amplification product complexity and to increase
reaction specificity. Using the nested PCR strategy a region of the
AHAS gene was uniquely amplified and was subjected to direct
sequencing using BigDye 3.1 resolved on an Abi 3730xl. The
sequences were then aligned using Sequencher v3.7 and assessed for
polymorphism.
[0059] Several variant mutated bases were identified. At base
coordinate 1431 line VBHT0810 has a transition from G to A.
However, this change is unlikely to be causal for the observed
phenotype of herbicide resistance as this specific nucleotide is
the third base in the triplet codon and codes for the same amino
acid and is therefore synonymous.
[0060] In lines VBHT0805 (NCIMB 41549) and VBHT0806 (NCIMB 41550)
at base coordinate 1742 G has been mutated to A, also a transition.
This causes alteration of the resulting amino acid from serine to
asparagine (FIG. 1).
[0061] Using cross-species comparative analysis, the serine amino
acid that has been mutated in lines VBHT0805 (NCIMB 4159) and
VBHT0806 (NCIMB 41550) has been identified as being highly
conserved in other species (FIG. 2).
[0062] Comparison of this serine to asparagine specific change to
known causal mutations that confer plant herbicide resistance,
conforms to previously identified and characterised changes (Li et
al 2008; Jander et al. 2004; Sathasivan et al. 1990).
c. Conclusions:
[0063] Mutant lines VBHT0805 (NCIMB 41549) and VBHT0806 (NCIMB
41550) are likely to express herbicide resistance due to alteration
of coordinate 1742 changing from a G to an A, which alters the
amino acid from a serine to an asparagine. This mutation is
referred to as SER653. The imidazolinone herbicide resistance shown
in the barley of the present invention is surprisingly high
compared to levels found in other species. This was unexpected and
while not wishing to be bound by theory it may be possibly due to
the high copy number of the AHAS gene in barley.
[0064] The mutation(s) causing herbicide resistance in lines
VBHT0802, VBHT0803, VBHT0807, VBHT0809 and VBHT0810 are currently
unknown. Mutations are likely to be in the unsequenced part of the
AHAS gene and may have alternate mechanisms for resistance.
VBHT0802 has a different genetic control for resistance than
VBHT0805 and VBHT0806 and does not have the mutation SER653.
EXAMPLE 9
Further Sequencing Of VBHT0802, VBHT0803, VBHT0805, VBHT0806,
VBHT0807, VBHT0809 And VBHT0810
[0065] Further work is being carried out using standard sequencing
techniques similar to those of Example 8 to characterise the AHAS
gene and other possibly relevant genes involved in conferring
imidazolinone resistance in VBHT0802 (NCIMB 41547), VBHT0803 (NCIMB
41548), VBHT0805 (NCIMB 41549), VBHT0806 (NCIMB 41550), VBHT0807
(not deposited), VBHT0809 (NCIMB 41551) and VBHT0810 (not
deposited).
[0066] Even though the SER653 mutation has been identified in
VBHT0805 (NCIMB 41549) and VBHT0806 (NCIMB 41550), these are
included in the further sequencing since it is believed there may
be another mutation in the part of the gene that was not
sequenced.
EXAMPLE 10
Suitability For Malt Production.
[0067] Buloke is currently recognised as a malting cultivar in
Australia. Tests for suitability for malt production were carried
out in accordance with the standard procedures of the European
Brewing Congress. The results shown in Table 8 demonstrate that
VBHT0805 (NCIMB 41549) is equivalent to Buloke in its suitability
for malting.
EXAMPLE 11
Production Of Herbicide Resistant Derivates Of VBHT0805 Via
Backcrossing With Hindmarsh.
[0068] The imidazolinone resistance from VBHT0805 has been
transferred through backcrossing to derivatives of the high
yielding dwarf barley variety Hindmarsh that is susceptible to
imidazolinone herbicides. Lines from these crosses have been
identified that retain the characteristics of Hindmarsh, including
dwarf growth habit, but also have resistance to imidazolinone
herbicides that is comparable to VBHT0805. Hindmarsh is genetically
different and unrelated to Buloke, the original line used to
develop VBHT0805, and these findings indicate that the
imidazolinone resistance discovered and outlined in the above
examples can be transferred to progeny and are thus derivatives of
VBHT0805.
REFERENCES
[0069] Li D., Barclaya I., Jose K., Stefanova K., Appels R. 2008. A
mutation at the A1a122 position of acetohydroxyacid synthase (AHAS)
located on chromosome 6D of wheat; improved resistance to
imidazolinone and a faster assay for marker selection. Mol
Breeding. Published Online early. Jander G., Baerson S., Hudak J.,
Gonzalez K., Gruys K., Last R. 2003 Ethylmethanesulfonate
Saturation Mutagenesis in Arabidopsis to Determine Frequency of
Herbicide Resistance. Plant Physiologyl 31 p139-16.
[0070] Sathasivan K., Haughn G., Murai N. 1990 Nucleotide sequence
of a mutant acetolactate synthase gone from an
imidazolinone-resistant Arabidopsis thaliana var. Columbia. Nucleic
Acids Research 18 2188.
Sequence CWU 1
1
511075DNAHordeum vulgare VBHT 0805 1gtatgttggt ggcggctgcg
ctgcatctgg cgaggagttg cgccgctttg ttgagctcac 60tggaattcca gttacaacta
ctctgatggg ccttggcaac ttccccagtg acgacccact 120gtcactgcgc
atgcttggga tgcatggtac cgtgtatgca aattatgcag tagataaggc
180tgacctgttg cttgcatttg gtgtgcggtt tgatgatcgc gtgactggga
aaattgaggc 240ttttgcaagc aggtccaaga ttgtgcacat tgacattgat
ccagctgaga ttggcaagaa 300caagcagcca catgtctcca tttgtgcaga
tgttaagctt gctttacagg ggttgaatgg 360tctattaagt ggcagcaaag
cacaacaggg tctagatttt ggtccatggc acaaggagtt 420ggatcagcag
aagagggagt ttcctctagg atacaagact tttggtgagg caatcccacc
480gcagtatgct atccaggtac tggatgagct gacaaaaggg gaggcgatta
ttgccacagg 540tgttgggcag catcagatgt gggcggctca gtattacact
tacaagcggc cacgtcagtg 600gctgtcttcg tctggtttgg gggcaatggg
atttgggttg ccagctgcag ctggcgcttc 660tgtggccaac ccaggtgtca
cagttgttga cattgatggg gatggtagtt tcctcatgaa 720cattcaggag
ttggcgttga tccgtattga gaacctccca gtgaaggtga tgatattgaa
780caaccagcac ctgggaatgg tggtgcagtg ggaggatagg ttttacaagg
ccaaccgggc 840gcacacatac cttggcaacc cagaaaatga gagtgagata
tatccagatt ttgtgacgat 900tgctaaagga ttcaatgttc cggcagttcg
tgtgacaaag aagagtgaag tcagtgcagc 960tatcaagaag atgcttgaga
ccccagggcc gtacctgctg gatatcattg tcccgcatca 1020ggagcacgtg
ctgcctatga tcccaaacgg tggtgctttc aaggacatga tcatg
107521143DNAHordeum vulgare VBHT 0806 2caccatctac tgaatcgctt
gagcaggtcc tgcgcctggt tggcgaggca cggcgcccga 60ttctgtatgt tggtggcggc
tgcgctgcat ctggcgagga gttgcgccgc tttgttgagc 120tcactggaat
tccagttaca actactctga tgggccttgg caacttcccc agtgacgacc
180cactgtcact gcgcatgctt gggatgcatg gtaccgtgta tgcaaattat
gcagtagata 240aggctgacct gttgcttgca tttggtgtgc ggtttgatga
tcgcgtgact gggaaaattg 300aggcttttgc aagcaggtcc aagattgtgc
acattgacat tgatccagct gagattggca 360agaacaagca gccacatgtc
tccatttgtg cagatgttaa gcttgcttta caggggttga 420atggtctatt
aagtggcagc aaagcacaac agggtctaga ttttggtcca tggcacaagg
480agttggatca gcagaagagg gagtttcctc taggatacaa gacttttggt
gaggcaatcc 540caccgcagta tgctatccag gtactggatg agctgacaaa
aggggaggcg attattgcca 600caggtgttgg gcagcatcag atgtgggcgg
ctcagtatta cacttacaag cggccacgtc 660agtggctgtc ttcgtctggt
ttgggggcaa tgggatttgg gttgccagct gcagctggcg 720cttctgtggc
caacccaggt gtcacagttg ttgacattga tggggatggt agtttcctca
780tgaacattca ggagttggcg ttgatccgta ttgagaacct cccagtgaag
gtgatgatat 840tgaacaacca gcacctggga atggtggtgc agtgggagga
taggttttac aaggccaacc 900gggcgcacac ataccttggc aacccagaaa
atgagagtga gatatatcca gattttgtga 960cgattgctaa aggattcaat
gttccggcag ttcgtgtgac aaagaagagt gaagtcagtg 1020cagctatcaa
gaagatgctt gagaccccag ggccgtacct gctggatatc attgtcccgc
1080atcaggagca cgtgctgcct atgatcccaa acggtggtgc tttcaaggac
atgatcatgg 1140agg 114331108DNAHordeum vulgare VBHT 0802
3ccatctactg aatcgcttga gcaggtcctg cgcctggttg gcgaggcacg gcgcccgatt
60ctgtatgttg gtggcggctg cgctgcatct ggcgaggagt tgcgccgctt tgttgagctc
120actggaattc cagttacaac tactctgatg ggccttggca acttccccag
tgacgaccca 180ctgtcactgc gcatgcttgg gatgcatggt accgtgtatg
caaattatgc agtagataag 240gctgacctgt tgcttgcatt tggtgtgcgg
tttgatgatc gcgtgactgg gaaaattgag 300gcttttgcaa gcaggtccaa
gattgtgcac attgacattg atccasctga gattggcaag 360aacaarcagc
cacatgtctc catttgtgca gatgttaagc ttgctttaca ggggttgaat
420ggtctattaa gtggcagcaa agcacaacag ggtctagatt ttggtccatg
gcacaaggag 480ttggatcagc agaagaggga gtttcctcta ggatacaaga
cttttggtga ggcaatccca 540ccgcagtatg ctatccaggt actggatgag
ctgacaaaag gggaggcgat tattgccaca 600ggtgttgggc agcatcagat
gtgggcggct cagtattaca cttacaagcg gccacgtcag 660tggctgtctt
cgtctggttt gggggcaatg ggatttgggt tgccagctgc agctggcgct
720tctgtggcca acccaggtgt cacagttgtt gacattgatg gggatggtag
tttcctcatg 780aacattcagg agttggcgtt gatccgtatt gagaacctcc
cagtgaaggt gatgatattg 840aacaaccagc acctgggaat ggtggtgcag
tgggaggata ggttttacaa ggccaaccgg 900gcgcacacat accttggcaa
cccagaaaat gagagtgaga tatatccaga ttttgtgacg 960attgctaaag
gattcaatgt tccggcagtt cgtgtgacaa agaagagtga agtcagtgca
1020gctatcaaga agatgcttga gaccccaggg ccgtacctgc tggatatcat
tgtcccgcat 1080caggagcacg tgctgcctat gatcccag 11084834DNAHordeum
vulgare VBHT 0810 4ctgaatcgct tgagcaggtc ctgcgcctgg ttggcgaggc
acggcgcccg attctgtatg 60ttggtggcgg ctgcgctgca tctggcgagg agttgcgccg
ctttgttgag ctcactggaa 120ttccagttac aactactctg atgggccttg
gcaacttccc cagtgacgac ccactgtcac 180tgcgcatgct tgggatgcat
ggtaccgtgt atgcaaatta tgcagtagat aaggctgacc 240tgttgcttgc
atttggtgtg cggtttgatg atcgcgtgac tgggaaaatt gaggcttttg
300caagcaggtc caagattgtg cacattgaca ttgatccagc tgagattggc
aagaacaagc 360agccacatgt ctccatttgt gcagatgtta agcttgcttt
acaggggttg aatggtctat 420taagtggcag caaagcacaa cagggtctag
attttggtcc atggcacaag gagttggatc 480agcagaagag ggagtttcct
ctaggataca agacttttgg tgaggcaatc ccaccgcagt 540atgctatcca
ggtactggat gagctgacaa aaggggaggc gattattgcc acaggtgttg
600ggcagcatca gatgtgggcg gctcagtatt acacttacaa gcggccacgt
cagtggctgt 660cttcgtctgg tttgggggca atgggatttg ggttgccagc
tgcagctggc gcttctgtgg 720ccaacccagg tgtcacagtt gttgacattg
atggggatgg tagtttcctc atgaacattc 780aggagttggc attgatccgt
attgagaacc tcccagtgaa ggtgatgata ttga 834524PRTHordeum vulgare 5His
Val Leu Pro Met Ile Pro Asn Gly Gly Ala Phe Lys Asp Met Ile 1 5 10
15 Met Glu Gly Asp Gly Arg Thr Ser 20
* * * * *