JP2019141045A - Sorghum plants with increased herbicide resistance comprising mutated polynucleotide encoding mutant large subunit of acetohydroxy acid synthase protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a herbicide which exhibits improved resistance to a herbicide, for example, a herbicide targeting an AHAS enzyme, particularly imidazolinone and a sulfonylurea, as compared with a wild-type sorghum plant. SOLUTION: A sorghum plant containing at least one polynucleotide encoding a polypeptide having an alanine-to-threonine substitution at position 93 of the sorghum AHAS protein large subunit in its genome. The sorghum plant may contain, in its genome, one, two or three or more copies of the polynucleotide encoding the mutated large subunit of sorghum AHAS or the sorghum AHAS polypeptide of the invention. In this context, the sorghum plant may be tolerant of any herbicide capable of inhibiting AHAS enzyme activity. [Selection diagram] None

Description

Sorghum (sorghum species) is a genus of plants containing about 20 grasses. It is native to the tropical and subtropical regions of East Africa and is grown everywhere on the continent for cereals for human and animal consumption, and is used as feed and for the production of alcoholic beverages. Since it is gluten free, sorghum is preferred for individuals suffering from celiac disease.

Sorghum is more tolerant of drought and excess soil moisture content than many cereals. It can grow properly under various soil and weather conditions. Similarly, it responds favorably to irrigation and requires a minimum of 250 mm during its life cycle,
The optimal irrigation range is 400-550mm.

In order to achieve rapid and uniform germination and thereby good crop transplantation, the soil must have an appropriate moisture content at the time of planting. The maximum water demand begins 30 days after germination and continues until the grain is filled, the most important stages are those of conical inflorescence formation and flowering, at which point the lack of water results in a decrease in yield Because.

In addition, sorghum has the ability to remain dormant during drought and resumes growth under favorable periods, but these stress situations can affect performance.

Sorghum requires high temperatures for normal development and, as a result, is more sensitive to low temperatures than other crops. Germination requires a soil temperature of at least 18 ° C; actual active growth of the plant is not achieved until a temperature of 15 ° C is reached, with an optimum temperature of about 32 ° C.

Acetohydroxyacid synthase (AHAS) is the first enzyme that catalyzes the synthesis of branched amino acids valine, leucine and isoleucine. AHAS is sulfonylurea,
It is the target of several herbicides such as imidazolinone, triazolopyrimidine and pyrimidyloxybenzoic acid. The former two herbicide families are widely used in modern agriculture because of their low toxicity and high effectiveness against weeds.

The imidazolinone family of herbicides include imazetapyr, imazaquin, and imazapill.

Some sulfonylureas present in the market are metsulfuron-methyl, chlorosulfuron, nicosulfuron, synosulfuron, imidasulfuron, halosulfuron, rimsulfuron, trisulfuron-methyl, and tribenuron-methyl.

However, plants that are resistant to imidazolinone and / or sulfonylurea family of herbicides; for example, Zea mays, Arabidopsis thalian
a, Brassica napus, Glycine max, Nicotiana t
There are species such as abacum, and Oryza sativa (Sebastian et al. (1989) Crop Sci., 29: 1403-1408; Swanson et al., 1
989 Theor. Appl. Genet. 78: 525-530; Newhouse
e et al. (1991) Theor. Appl. Genet. 83: 65-70; Sath
asivan et al. (1991) Plant Physiol. 97: 1044-1050
Page; Mourand et al. (1993) J. MoI. Heredity 84: 91-96; US Pat. No. 5,545,822). AHA imparting resistance to imidazolinone type herbicides
Point mutations in the large subunit of S have been described in sunflower (WO2
007/0118920).

Plants that are resistant to imidazolinone or sulfonylurea type herbicides have also been detected. These plants naturally acquire resistance and are used for crop breeding, resulting in herbicide-tolerant varieties. Analysis of resistant plants determined point mutations in the sunflower AHAS protein that resulted in the substitution of the amino acid Ala by Val (White).
(2003) Weed Sci. 51: 845-853).

Further, U.S. Pat. Nos. 4,761,373; 5,331,107; 5,304,
No. 732; No. 6,211,438; No. 6,211,439; and No. 6,222,1
No. 00 discloses plants that are resistant to imidazolinone herbicides. All of these plants generally describe the use of altered AHAS genes to elicit herbicide resistance in plants, and specifically disclose certain imidazolinone resistant corn lines. US Pat. No. 5,731,180 and US Pat. No. 5,767,361 describe isolated genes having a single amino acid substitution in the wild-type monocot AHAS amino acid sequence resulting in imidazolinone-specific tolerance. I am considering.

Patent documents WO 2006/007373 and WO 2006/060634 disclose mutations that confer resistance to imidazolinone type herbicides in wheat plants.

Publication "Amino acids conferring herbicide resistance in tobacco acetohydroxy acid synthase"
Describe various point mutations in AHAS that confer herbicide tolerance (GM C
rops 1: 2, 62-67; Feb. 16, 2010).

Patent document US20110011563 relates to a herbicide-tolerant sorghum plant achieved by changing the acetyl CoA carboxylase gene. Also disclosed are a sorghum plant that is resistant to the herbicide dinitroaniline (US201100205686) and a sorghum plant that is resistant to the herbicide acetolactate synthase (WO2008 / 073800).

The sorghum plant of the present invention has a herbicide such as AHAS compared to a wild type sorghum plant.
It shows improved resistance to enzyme-targeting herbicides, particularly imidazolinones and sulfonylureas. In particular, the sorghum plant of the present invention (Sorghum bicolor)
It contains at least one polynucleotide in its genome. The polynucleotide is
Encodes a large subunit of AHAS having an alanine to threonine substitution at position 93 or equivalent position of the large subunit of sorghum AHAS, said plant being selected from the imidazolinone group compared to wild-type sorghum plants There is an increased resistance to one or more herbicides such as herbicides. Sorghum plants contain sorghum AHA in their genome.
It may contain 1, 2, 3 or more copies of a polynucleotide encoding a mutant large subunit of S or a sorghum AHAS polypeptide of the invention. In this context, the sorghum plant may be tolerant to any herbicide capable of inhibiting AHAS enzyme activity, ie, the sorghum plant is not limited to imazetapil,
Imidazolinone-type herbicides such as imazapyr and imazapic or, but are not limited to, chlorosulfuron, methylsulfuron methyl, sulfometuron methyl, chlorimuron ethyl, thiofensulfuron methyl, tribenuron methyl, Bensulfuron methyl, nicosulfuron, etameturflon methyl, rimsulfuron, triflusulfuron methyl, trisulfuron, primsulfuron methyl, synosulfuron, amidosulfuron,
It may be tolerant to sulfonylurea herbicides such as fluzasulfuron, imazosulfuron, pyrazosulfuron ethyl, and halosulfuron.

In a preferred embodiment, the sorghum plant of the present invention that is resistant to herbicides belonging to the group of imidazolinone or sulfonylurea is a sorghum line designated VT11-11331-BK, whose seeds are under the Budapest Treaty. And deposited with the NCIMB collection under the accession number NCIMB 41870 on October 12, 2011. The mutated VT11-11331-BK plant, part thereof and seed thereof have the nucleotide sequence set forth in SEQ ID NO: 1 encoding in its genome an AHAS large subunit polypeptide having the sequence set forth in SEQ ID NO: 2. Mutant AHAS comprising a polynucleotide
Contains genes. The amino acid sequence of SEQ ID NO: 2 corresponding to the large subunit of AHAS is
One amino acid differs from the wild-type amino acid sequence of the large subunit of sorghum AHAS (SEQ ID NO: 3), the difference comprising a substitution of alanine to threonine at position 93, or equivalent position, of the large subunit of sorghum AHAS .

The herbicide-tolerant sorghum plant genetic resources of the present invention are useful for the introduction of tolerance traits by gene transfer into other sorghum varieties. The sorghum plant of the present invention comprises progeny and seed of a plant comprising a polynucleotide encoding an AHAS large subunit and having a substitution of alanine to threonine at position 93 or equivalent position of said large subunit of sorghum AHAS, Said plants show increased resistance to one or more imidazolinones and / or sulfonylurea herbicides compared to wild type sorghum plants.

In a preferred embodiment, the herbicide-tolerant sorghum plant comprises a resistance trait of NCIMB41870, the plant described in NCIMB41870, the progeny of NCIMB41870 plant,
It may be a mutant of the NCIMB 41870 plant and a descendant of the NCIMB 41870 mutant. The plant may be transgenic or non-transgenic and may belong to any plant species suitable for agricultural use or other uses such as houseplants.

Further provided is a sorghum seed comprising in its genome at least one polynucleotide encoding a polypeptide having an alanine to threonine substitution at position 93 of the sorghum AHAS protein large subunit. This seed germinates a plant with increased resistance to one or more herbicides of the imidazolinone group compared to a wild-type sorghum plant,
Generate. In a preferred embodiment, the seed is a seed deposited as NCIMB 41870.

A method for identifying a plant that is resistant to a herbicide in a group of imidazolinones or sulfonylureas, comprising:
a) providing a nucleic acid sample derived from a sorghum plant;
b) amplifying a region corresponding to the AHAS gene from a sorghum plant present in the nucleic acid sample;
c) identifying a sorghum plant that is resistant to the imidazolinone herbicide based on the presence of at least one mutation in the amplified nucleic acid sample that confers resistance to the imidazolinone herbicide. Further provided is the above method. In a preferred embodiment, NCIMB 418
Plants containing at least one mutation in the AHAS, such as a mutation in 70, are selected;
Said at least one mutation in the AHAS gene encodes an AHAS polypeptide or large subunit containing an Ala93Thr substitution compared to the wild-type sorghum AHAS amino acid sequence. The plant may be a monocotyledonous plant or a dicotyledonous plant. Preferably, the plant is an agricultural target plant such as sorghum, rice, corn, soybean, wheat, oats, barley, rye, flax, cotton, sugarcane, sunflower and the like.
The method of identification may use a specific point mutation SNP marker.

The present invention is a method for controlling agricultural plants, for example weeds in the vicinity of a sorghum plant, wherein the sorghum plant is a herbicide such as a herbicide derived from the group of imidazolinones and / or sulfonylureas. The method is provided which is resistant to. In a preferred embodiment, the herbicide is imidazolinone. The imidazolinone herbicide may be imazetapil, imazapic, or imazapill. The plant is NCIMB 41870.
Or may be a derivative, mutant, or progeny plant thereof.

The present invention includes, but is not limited to, the following nucleotide sequences:
The sequence set forth in SEQ ID NO: 1,
A nucleotide sequence encoding the polypeptide of SEQ ID NO: 2,
A polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 and encoding said polypeptide exhibiting herbicide-tolerant AHAS activity;
-A nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1, encoding a polypeptide comprising a large subunit of AHAS;
The nucleotide sequence exhibiting herbicide-tolerant AHAS activity;
Or a polypeptide comprising one or more of its complementary sequences. Preferably, said polynucleotide encodes a polypeptide of an AHAS large unit comprising an Ala93Thr substitution.

The present invention also provides at least 95% of the following nucleotide sequence: SEQ ID NO: 1, nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2, SEQ ID NO: 2 amino acid sequence
A nucleotide sequence encoding said polypeptide exhibiting herbicide-tolerant AHAS activity; a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1 Also provided are expression cassettes comprising at least one polynucleotide encoding a polypeptide comprising a large subunit of AHAS and having said nucleotide sequence exhibiting herbicide-tolerant AHAS activity, or a complementary sequence thereof. Preferably, the polynucleotide is an AHA containing an Ala93Thr substitution.
Encodes a large unit polypeptide of S; said sequence being operably linked to a nucleotide sequence for expression, eg, one or more promoters, enhancers or other known regulatory sequences. The promoter may be a promoter for expression in plants, plant tissues, chloroplasts, animals, bacteria, fungi or yeast cells.

The present invention provides the following nucleotide sequence: a) the nucleotide sequence set forth in SEQ ID NO: 1, b)
A nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2, c) a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein said polypeptide exhibits herbicide-tolerant AHAS activity D) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1, encoding a polypeptide comprising a large subunit of AHAS and exhibiting herbicide-resistant AHAS activity Provided is a transformation vector comprising at least one polynucleotide having one of the nucleotide sequences, further comprising an operably linked sequence that induces expression of said nucleotide sequence and a selectable marker. Vectors can be used to transform bacteria, fungi, yeast, plant cells or animal cells, each adapted for a particular case.

The present invention relates to a nucleotide sequence as set forth in SEQ ID NO: 1, incorporated into its genome; b
C) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2; c) a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the polypeptide exhibits herbicide-tolerant AHAS activity D) a nucleotide sequence encoding; d) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1, encoding a polypeptide comprising a large subunit of AHAS and exhibiting herbicide-tolerant AHAS activity E) comprising at least one promoter operably linked to a polynucleotide selected from a nucleotide sequence perfectly complementary to one of the nucleotide sequences of a) to (d); Induces gene and expression of said nucleotide sequence At least one operatively linked to that nucleotide sequence
A transformed plant further comprising one promoter is provided. A promoter is a promoter that induces polypeptide expression in plants, such as plant tissue or chloroplasts. The transformed plant may be a monocotyledonous plant such as sorghum, corn, rice or wheat; or a dicotyledonous plant such as sunflower, Arabidopsis, tobacco or rape. Transformed plants are resistant to herbicides (imidazolinones and sulfonylureas) when equal amounts of the herbicide are applied to both compared to the same wild type plant.

The present invention also provides a method for obtaining a herbicide-tolerant plant or a plant with increased resistance to a herbicide, comprising i) transforming a plant cell with an expression cassette comprising a polynucleotide, ii) a plant cell Regenerating and obtaining a herbicide-tolerant plant,
The polynucleotide comprises the following nucleotide sequence: a) a nucleotide sequence set forth in SEQ ID NO: 1, b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2, c) at least 95% of the amino acid sequence of SEQ ID NO: 2 A polypeptide having sequence identity, the nucleotide sequence encoding said polypeptide exhibiting herbicide-tolerant AHAS activity;
d) a nucleotide sequence having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1, wherein said nucleotide sequence encodes a polypeptide comprising a large subunit of AHAS and exhibits herbicide-tolerant AHAS activity; and e Also provided is said method, having at least one of the nucleotide sequences perfectly complementary to one of the nucleotide sequences a) to (d). The DNA expression cassette comprises at least one promoter for inducing expression of the polypeptide in plants, such as plant tissues or chloroplasts.
The transformed plant may be a monocotyledonous plant, such as sorghum, corn, rice, or wheat; or a dicotyledonous plant, such as sunflower, Arabidopsis, tobacco, soybean or rape. Transformed plants are more resistant to herbicides (imidazolinones and sulfonylureas) when compared to wild type plants when equal amounts of the herbicide are applied to both. The plant contains a large subunit of AHAS that is resistant to herbicides.

FIG. 5 shows dose response curves of imidazolinone resistant mutant sorghum system (ADV-IMI-R) and original self-pollinated sorghum system 80237 or wild type system. The herbicides assayed were imazetapil, imazapyr and imazapic with application rates of 0 (control), 1X, 2X, 3X and 4X. Herbicidal effect was measured as a percentage of dry matter DM in air tissue compared to untreated controls; each value is an average of three assays. FIG. 5 shows dose response curves of imidazolinone resistant mutant sorghum system (ADV-IMI-R) and original self-pollinated sorghum system 80237 or wild type system. The herbicides assayed were imazetapil, imazapyr and imazapic with application rates of 0 (control), 1X, 2X, 3X and 4X. Herbicidal effect was measured as a percentage of dry matter DM in air tissue compared to untreated controls; each value is an average of three assays. FIG. 5 shows dose response curves of imidazolinone resistant mutant sorghum system (ADV-IMI-R) and original self-pollinated sorghum system 80237 or wild type system. The herbicides assayed were imazetapil, imazapyr and imazapic with application rates of 0 (control), 1X, 2X, 3X and 4X. Herbicidal effect was measured as a percentage of dry matter DM in air tissue compared to untreated controls; each value is an average of three assays. FIG. 5 shows an alignment of a mutated ADV-IMI-R AHAS nucleotide sequence with the original endogomic sorghum line 80237 or wild type line. The nucleotide G to A substitution is shown at position +277, which distinguishes both sequences. Different primers used to amplify overlapping amplicon sequences are underlined. FIG. 5 shows an alignment of a mutated ADV-IMI-R AHAS nucleotide sequence with the original endogomic sorghum line 80237 or wild type line. The nucleotide G to A substitution is shown at position +277, which distinguishes both sequences. Different primers used to amplify overlapping amplicon sequences are underlined. FIG. 4 shows a genotyping assay for SNP G / A at codon 93 of the sorghum AHAS gene. Specific SNP-SbAHAS markers were analyzed in the genotype of 177 F2 progeny plants derived from ADV-IMI-RxF2 90523. Three clusters are clearly defined: dots corresponding to homozygous plants of allelic mutants (A / A) (black circles), triangles corresponding to heterozygous plants (A / G) (black triangles) and wild Identified as a homozygous rhombus (black rhombus) of type allele (G / G). Matching the nucleotide sequence of the sorghum AHAS gene (Accession No. GM6633363.1) with the sorghum genome sequence (Sorghum bicolor) deposited in the Phytosome nucleotide sequence database (http://www.phytosome.net/search.php) It is a figure which shows the result obtained by this. This matching indicates that the AHAS GM6633363.1 sequence shows a highly significant homology (value e = 0) to the AHAS sequence located on chromosome 4 of the sorghum genome, which indicates that the gene of interest It is present in the group. S. It is a figure which shows the connection map of bicolor chromosome 4. FIG. The distance between adjacent markers is given in centimorgans (cM). The chromosomal location of resistance to imazetapil referring to 7 SSRs in chromosome 4 of the genotype of SNP-SbAHAS and 177 F2 plants is shown. This map was built using the JoinMap software using the default parameters of LOD = 3 and the maximum Kosambi distance of 50 cM.

To obtain herbicide-tolerant plants, endogomic sorghum (Sorghum bico)
lor) line 80237 plants were treated with an aqueous solution of ethyl methanesulfonic acid (EMS). Treated seeds were planted and left for open pollination. A total of 546 M2 plants were obtained by selecting 273 M1 plants and planting the two seeds of each plant on the nursery.
Pollen from one plant in each pair was collected and used to pollinate the other plants in the pair. M3 seeds obtained from each of 273 pollinated M2 plants were harvested. M3 offspring were planted between a total of 273 bushes. 10 to 50 plants from each M3 furrow
0 ml La a. i. / Ha imazetapill was sprayed. 68 plants from the furrows showed normal growth and absence of symptoms after treatment with herbicides, were considered resistant to the herbicides and were identified as VT09-9754. A lineage of resistant plants from the furrow was identified and named 80237EMS2-192 (hereinafter referred to as ADV-IMI-R). Herbicide-tolerant M7 mutant plants and seeds (named VT11-11331-BK) selected from the original ADV-IMI-R mutant were obtained and under the terms of the Budapest Treaty, 20
Deposited with the NCIMB collection under the accession number NCIMB41870 on October 12, 2011.

The present invention is not limited to sorghum plants mutated with EMS. Other mutation methods,
For example, sorghum plants obtained by methods such as irradiation and chemical mutagens are also within the scope of the present invention. Herbicide-tolerant mutant plants can also be obtained using selective pressure on cells cultured with herbicides and the process of selection of resistant cells to produce herbicide-tolerant plants. For details on mutation and breeding methods, see "Principles of Cul."
tivar Development "Fehr, 1993, Macmillan Pu
can be found in the Blissing Company, which is hereby incorporated by reference.

Subsequently, the effect of herbicide spraying on the imidazolinone-tolerant plant mutant ADV-IMI-R (original mutation) was compared to the response of the endogomic sorghum line 80237 (Advanta-owned elite line). To that end, plants were treated on the field with three herbicides belonging to the imidazolinone family: imazetapyr, imazapyr, and imazapic. Four different application rates were assayed: 1X, 2X, 3X, for each herbicide
And 4X. Table 1 shows the recommended field application rate (1X) for each herbicide.

Ten days after spraying, all plants were assayed for dry matter (DM) in aerial tissue. The result is shown in FIG.

The response of the ADV-IMI-R mutant of the present invention was compared to that of the endogomic line 80237. Table 2 shows the effect of different application rates of imazetapil, imazapyr, and imazapic, expressed as a percentage of dry matter compared to the untreated control (DM% control). The disclosed value is the average of 3 experiments.

These results indicate that the ADV-IMI-R mutants of the present invention are resistant to the three tested imidazolinone herbicides even at 4X application rates. Conversely, the original endogomic line 80237 is clearly sensitive to all herbicides, even when the recommended application rate (1X) is applied.

As can be seen, the herbicide-tolerant sorghum plants of the present invention are imazetapill, imazapic,
Or it is resistant to imidazolinone herbicides such as imazapyr.

The imidazolinone-tolerant sorghum plants can be sprayed with an amount that is four times the recommended amount for use of any of the known imidazolinone herbicide groups. The resistant plant is, for example, NCIMB41
It may have a tolerance trait as observed in plants derived from 870 seeds, or it may contain alanine at position 93 or equivalent position of the sorghum AHAS protein in the plant, offspring, or genome thereof derived therefrom. In other plants comprising at least one polynucleotide encoding a polypeptide having a threonine to threonine substitution and increased resistance to one or more herbicides from the imidazolinone group compared to wild-type sorghum plants There may be.

Furthermore, one skilled in the art will recognize that such amino acid positions may vary depending on whether an amino acid is added or removed from, for example, the N-terminus of the amino acid sequence. Let's go. “Equivalent position” is intended to mean a position that is within the same conserved region as the exemplified amino acid positions.

In the present invention, the terms “tolerance” to herbicides and “tolerance” to herbicides have the same meaning and equivalent ranges when used in connection with herbicides such as imidazolinones or sulfonylureas.

The present invention provides plants, plant cells, and plant tissues that are resistant to an effective amount of herbicide.
By “effective amount” is meant an amount of herbicide that can inhibit the growth of wild-type plants, plant cells or tissues, but does not produce a severe effect on resistant plants, plant cells or plant tissues. An effective amount of herbicide is the recommended amount to eliminate weeds. A wild type plant, cell or tissue is one that does not exhibit a herbicide tolerance trait of a herbicide belonging to the group of, for example, imidazolinone or sulfonylurea. The term “plant” encompasses a plant or plant part at any stage, which may be a seed, leaf, flower, stem, tissue or organ known to a knowledgeable botanist.

To detect mutations, the mutant sorghum AHAS gene of the present invention encoding a polypeptide having acetohydroxy acid synthase activity, wherein the AHAS polypeptide or large subunit is herbicide resistant, was sequenced.

Comparison of the AHAS gene DNA sequence from the mutant plant with the AHAS gene DNA sequence from the wild-type sorghum plant identified a point mutation in which the G nucleotide at nucleotide position +277 was changed to A. The original sorghum 80237 system (wild type) is identified from the ADV-IMI-R mutant of the present invention. When deduced AHAS amino acid sequences from mutant and wild type systems, nucleotide substitutions (SEQ ID NO: 1
GCG by ACG of ACG) encodes a mutant polypeptide at codon 93, exhibits acetohydroxy acid synthase activity, and corresponds to the large subunit of the sorghum AHAS protein of the mutant ADV-IMI-R system (SEQ ID NO: It was observed to result in the substitution of alanine in 2) to threonine (Ala93Thr).

A plant comprising a polynucleotide encoding a polypeptide having a substitution of alanine to threonine (Ala93Thr) at position 93 of said polypeptide or at an equivalent position of another AHAS enzyme belonging to a different species, plant part, seed, progeny thereof, Transgenic plants and the like are also within the scope of the present invention.

Polynucleotides or polypeptides having at least 85%, 95%, or 98% sequence identity are also within the scope of the invention. The percent sequence identity is determined by the alignment of two amino acid sequences or two nucleotide sequences. The percentage of alignment between two sequences can be expressed, for example, by the following formula:
(Amount of the same position / total amount of overlapping positions) × 100
Can be used to calculate.

The percentage of alignment between two sequences can be determined using various mathematical algorithms, such as Altschul et al. (1990) J. MoI. Mol. Biol. 215: 403 pages NBLA
Determined using algorithms included in ST and XBLAST programs. BLA
When using ST, Gapped BLAST, and PSI-Blast programs:
Default parameters of corresponding programs (eg, XBLAST and NBLAST) can be used. http: // www. ncbi. nlm. nih. See gov. Another preferred non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The alignment can also be performed by manual inspection.

The present invention includes polynucleotides and proteins that confer herbicide resistance. When referring to a herbicide-tolerant polynucleotide, it is understood to mean a polynucleotide encoding a herbicide-tolerant AHAS protein. Herbicide-tolerant AHAS proteins may be natural or mutant, and they confer resistance to herbicides belonging to the group of imidazolinones or sulfonylureas.

Inheritance of resistance to imidazolinones, such as imazetapyr, was evaluated in isolated F2 sorghum populations generated by crossing an imazetapyr sensitive system with an ADV-IMI-R mutant. The F2 plants thus obtained were sprayed with imazetapil (Pivot®, BASF) at an application rate of 3 ×, 20 days at stage V6 after germination, and phenotypic symptoms were evaluated 10 days after treatment. The results are provided in Table 3.

Results classify F2 plant populations based on visual symptoms with phenotypic scoring that allows individuals to be grouped into three phenotypic categories: intact plants, yellowish plants and dead plants Show that you can.

Sequencing has shown that the herbicide-tolerant sorghum mutants of the present invention have point mutations in the polypeptide having acetohydroxy acid synthase activity or the large subunit of AHAS protein. The nucleotide sequence set forth in SEQ ID NO: 1 indicates that nucleotide G is changed to A at nucleotide position +277 (corresponding to codon 93), which is the induced ADV-IMI-R mutation of the present invention. It identifies the original sorghum endogamic system 80237 (sensitive to herbicides) from the body. This molecular marker was named SNP-SbAHAS.

SNP-S for detecting plants, plant parts or seeds that may be herbicide-resistant
To determine the usefulness of bAHAS markers, the herbicide-resistant ADV-IMI-R mutant (VT09-9754-48-6-BK selection) and the herbicide-sensitive endogamic system 90
F2 population generated by crossing with 523 (herein ADV-IMI-R
177 plants derived from (designated x90523 F2 population) were tested. FIG. 3 shows by way of example the allelic discrimination of several plants from the F2 population. The two tested controls were grouped into expected clusters (homozygous resistant and homozygous sensitive ADV-IMI-R mutant and wild type 90523, respectively). These data allow the molecular marker SNP-SbAHAS to identify the three allelic combinations at nucleotide position +277 of a polynucleotide encoding a polypeptide having acetohydroxy acid synthase activity or the large subunit of the sorghum AHAS protein. It is confirmed that it is useful.

One skilled in the art will identify herbicide-tolerant plants, for example, herbicides belonging to the group of imidazolinones and / or sulfonylureas, eg, imidazolepyr, imazapic, etc. in view of the disclosure of SNP-SbAHAS markers and their use Can do. In addition to the methods described herein, the identification method may be any other known method, for example,
ASA (Soleimani et al. (2003) Plant Mol Biol Rep 2
1: 281-288), PAMSA (Gaudet et al. (2007) Plant Mol.
Biol Rep 25: 1-9), SSCP (Germano and Klein (
1999) Theor Appl Gene 99: 37-49) or TaqMa
n <(R)> (Jones et al. (2008) Best Management Scie
nce 64: 12-15).

The correlation between the resistance phenotype and the number of mutant alleles was analyzed using the SNP-SbAHAS marker. For this purpose, the yellowing and death phenotypes of individual plants derived from F2 progeny (ADV-IMI-Rx90523) were tested after spraying with imazetapill. At the same time, the genotype of the SNP-SbAHAS marker was tested. The results are shown in Table 4. The correlation (measured as a phenotypic assessment) between the molecular marker SNP-SbAHAS and resistance to imazetapy was determined using the chi-square independent test. This test showed that induced mutations in the AHAS gene (specific SNP-SbAHA) that follow phenotype and genotype co-segregation
Probability p = 1.948e- ⁴⁶ was obtained which showed a highly significant correlation between resistance to imazetapil and genotyped using the S marker).

In addition, genetic mapping of herbicide tolerance and specific SNP-SbAHAS markers was performed. Based on the sorghum AHAS sequence disclosed in GenBank (Accession No. GM6633363.1, SEQ ID NO: 4), a BLAST analysis was performed to determine the Sorghum bic
The DNA sequence of the olor and the Phytosome database (http: //www.p
hytosome. net / search. php). The results shown in FIG. 4 show that the AHAS sequence associated with resistance to herbicides belonging to the imidazolinone group is Sorg
It is located in chromosome 4 of the hum bicolor genome. This information-based fingerprinting (genotyping) procedure was performed for the mutant ADV-IMI-R system and the Endogammic 90253 system of the present invention using various SSR type DNA molecular markers. Seven polymorphic SSRs located on chromosome 4 were selected for genotyping (Mace, ES et al. A consensus genetic map of s
orghum that integrals multiple component
t map and high-throughput Diversity Arr
ay Technology (DArT) markers, BMC Plant Bi
olology, 2009, 9:13; Srinivas, G et al. Exploration
and mapping of microsatellite markers f
rom subtracted drought stress ESTs in So
rghum bicolor (L.), Moench. Theor. Appl. Gene
t. 2009, 118: 703-717; Ramu, P et al., In silico ma.
pping of important genes and markers ava
available in the public domain for efficien
t songhum breathing, Mol Breeding 2010, 26:
409-418; http: // www. lbk. ars. usda. gov / psg
d / sorghum / 2009 Sorghum SEAMs_LBKARS. xls).

Using the results of this procedure, it was possible to genetically map both the SNP-SbAHAS marker and the resistance phenotype, which indicated that the resistance to imidazolinone and the specific SNP-SbAHAS marker are chromosome 4 of Sorghum bicolor. 5 flanking the previously identified SSR and is located in chromosome 4 (FIG. 5).
).

The polynucleotide of the present invention (SEQ ID NO: 1) can be introduced into a plant to obtain a transgenic plant. There are a variety of methods for introducing polynucleotides into plants, particularly plants for agricultural purposes, such as corn, soybeans, sorghum, wheat, sugarcane, flax, sunflower or other plants; vegetables, trees, or foliage plants. Methods for introducing polynucleotides to obtain transgenic plants are well known in the art, and recombinant methods can also be used.

For example, there are various methods for plant transformation using vectors (An, G. et al. (
1986) Plant Pysiol. 81: 301-305; Fry, J. et al. Et al (1
987) Plant Cell Rep. 6: 321-325; Block, M.M. (
1988) Theor. Appl Genet. , 76: 767-774; Hinch
ee et al. (1990) Stadler. Genet. Symp. , 203212.203 ~
212; Barcelo et al. (1994) Plant J. et al. 5: 583-592; B
Ecker et al. (1994) Plant J. et al. , 5: 299-307; Borkowski
a et al. (1994) Acta Physiol Plant. 16: 225-230;
Christou et al. (1992) Trends Biotechnol. , 10: 239
˜246; D'Halluin et al. (1992) Bio / Technol. , 10:30
9-314; Dhir et al. (1992) Plant Physiol. , 99: 81-8
8; Casas et al. (1993) Proc. Nat. Acad Sci. USA 90:
11212-11216; Dong, J. et al. A. And Mchuchen, A .; (199
3) Plant Sci. 91: 139-148; Golovkin et al. (1993).
Plant Sci. 90: 41-52; Guo Chin Sci. Bull.
38: 2072-2078; Asano et al. (1994) Plant Cell Re
p. , 13; Christou, P .; (1994) Agro. Food. Ind. Hi
Tech. 5: 17-27; Eapen et al. (1994) Plant Cell Re
p. 13: 582-586; Hartman et al. (1994) Bio-Technol.
ogy 12: 919923; Christou, P .; (1993) In Vitro
Cell. Dev. Biol. -Plant; 29P: 119-124; Davie
s et al. (1993) Plant Cell Rep. , 12: 180-183; Rita
la et al. (1994) Plant. Mol. Biol. 24: 317-325; and Wan, Y. et al. C. And Lemaux, P.A. G. (1994) Plant Physio
l. 104: 3748).

Several methods and genetic engineering techniques for transforming plants are described in Sambrook.
(1989) Molecular cloning, A laboratory m
annual, Cold Spring Harbor Press, Plainview
, N. Y. Ausubel FM et al., Current Protocols in M
Olecular Biology, John Wiley & Sons, New Y
ork, N.M. Y. It is described in.

Vectors are described in US Pat. No. 4,945,050 and Casas et al., Proc. Nat
l. Sci. , USA, 90: 11212, 1993, and the like.

For example, plants transformed with the polynucleotides of the invention express herbicide-resistant AHAS proteins and exhibit resistance traits to herbicides such as imidazolinones and / or sulfonylureas. “Herbicide-tolerant AHAS protein” refers to an AHAS activity that is higher than that of wild-type AHAS activity when the herbicide is in the presence of a herbicide that inhibits AHAS activity at a concentration that inhibits AHAS activity of the wild-type AHAS protein It means a protein showing

The polynucleotide sequences of the present invention can be used to produce resistant AHA in a subject, eg, a plant to be agriculturally targeted.
It can be contained in an expression cassette for expressing S protein. This cassette includes, for example, a regulatory sequence operably linked to at least one promoter.
Promoters are well known in the art and can be, for example, constitutive, inducible, tissue specific, chloroplast specific promoters, all of which are functional in plants. The cassette further includes a transcription termination sequence known in the art. The cassette may contain various regulatory sequences to improve the expression efficiency of the herbicide-tolerant AHAS polynucleotide, such as enhancers such as introns, viral sequences; or bound or not bound to said polynucleotide. Other polynucleotide sequences of interest that are different from the polynucleotides of the invention; or leader sequences may be included.

Promoters include, but are not limited to, constitutive, inducible, tissue or organ specific promoters such as 35S promoter, wound or chemical inducible promoter, heat shock promoter, tetracycline inducible promoter and the like (
Chao et al., 1999, Plant Physiol. 120: 979; US Pat. No. 5,187,267, US Pat. No. 5,057,422).

Suitable transcription terminators that may be useful in plants are also known to those skilled in the art, for example, Odell et al., 1985, Nature 313: 810; Sanfaco
n et al., Genes Dev. , 5: 141, 1990; Munroe et al., 1990 G
ene 91: 151; Rosenberg et al., 1987, Gene 56: 125; Joshi et al., Nucleic Acid Res. 15: 9627, 1987.

“Operably linked” is intended to refer to a functional linkage between a promoter and a second sequence. The promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. The term “operably linked” means that the nucleic acid sequences to be linked are continuous and, if necessary to join two protein coding regions, are continuous and in the same reading frame. Means. The cassette may be a plurality of expression cassettes.

The polynucleotides of the invention can be linked to other polynucleotides in the form of polynucleotide fusions or separately. Other polynucleotides may encode, for example, herbicide tolerance, insect tolerance or other plant parts well known to those skilled in the art.

In order to improve expression in plants, the polynucleotides of the invention can be modified, for example, by containing codons that are favorable to the plant, by deleting sequences such as introns or harmful sequences. Also within the scope of the invention are polynucleotides that are mutated, eg, resistant AHAS polypeptides of the invention that exhibit deletions or insertions, or other mutations that differ from those described herein. “Mutant polynucleotide” means a polynucleotide having a permanent change in its nucleotide sequence.

The polynucleotides of the invention can be altered by substitutions, deletions, truncations, and insertions. Methods for such operations are generally known in the art. Methods for mutagenesis and nucleotide sequence changes are well known in the art (Kunkel (
1985) Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) Methods in Enzymol. 154: 36
7-382; Walker and Gaastra (1983) Techniques.
in Molecular Biology (MacMillan Publishing)
ng Company, New York) and references cited therein). Dayhoff et al. (1978) Atlas of Protein, which is incorporated herein by reference for amino acid substitutions that do not affect the biological activity of the protein of interest.
Sequence and Structure (Natl. Biomed. Res. F)
ound. , Washington, D.C. C. ) Can be found.

The DNA containing the AHAS polynucleotide of the present invention can be introduced into various plants as part of a vector. The selection of a suitable vector depends on the method intended to be used for transformation and the plant to be transformed. The person skilled in the art is familiar with various vectors, for example Ti and / or Ri. T-DNA is also used as a flanking region for incorporation of Ti or Ri vectors (WO84 / 02913, He
rrera-Estrella et al., 1983, Nature 303: 209; Hor
sch et al., 1984, Science 223: 496; Fraley et al., 1983,
Proc. Natl. Acad. Sci, USA 80: 4803).

Vectors include expression cassettes and other nucleotide sequences such as selectable markers such as markers that confer resistance to antibiotics or herbicides and polynucleotides of interest in plants, such as the AHAS large subunit polypeptide of SEQ ID NO: 2. A sequence for inducing expression of may be included.

A vector comprising a polynucleotide encoding a resistant AHAS polypeptide of the invention comprises
It may be useful to obtain transgenic plants that are resistant to imidazolinone and sulfonylurea type herbicides. The transformed plant may be any type of plant, such as a dicotyledonous or monocotyledonous plant, including but not limited to, sorghum (Sorghum bicolor, Sorghum vulgare), corn (Zea mays), Brassica species (eg, B. napus, B. rapa,
B. juncea), alfalfa (Medicago sativa), rice (Or
yza sativa), rye (Secale cereal), millet (Penn)
isetum glaucum), (Panicum milaceum), (Set
aria italica), (Eleusine coracana), sunflower (H
elianthus annuus), wheat (Triticum aestivum),
T.A. Turbidum ssp. durum), soybean (Glycine max), tobacco (Nicotiana tabacum), peanut (Arachis hypo)
gaea), cotton (Gossypium barbadense, Gossypium)
hirsutum), sweet potato (Ipomoea battus), cassava (M
anihot esculenta, coffee (Coffea spp.), coconut (Cocos nucifera), safflower (Carthamus tinctor)
ius), pineapple (Ananas comosus), citrus tree (Citrus)
spp. ), Cocoa (Theobroma cacao), tea (Camellia s)
inensis), banana (Musa spp.), avocado (Persea amer)
icana), olives (Olea europaea), cashew (Anacardi)
um occidentale), almond (Prunus amygdalus),
Sugar beet (Beta vulgaris), potato (Solanum tube)
erosum), sugar cane (Saccharum spp.), vegetables, foliage plants, and plants for agriculture such as trees. Preferably, the plant of the present invention is sorghum.

The present invention is better illustrated by the following examples, which should not be construed as limiting its scope. On the contrary, other embodiments, modifications and equivalents of the present invention are possible after reading this specification, and those skilled in the art will depart from the spirit of the invention and / or the appended claims. It should be clearly understood that recommendations can be made without.

[Mutagenesis of the endogomic sorghum line 80237 and selection of the imidazolinone resistant mutant 80237EMS2-192]
32,000 pre-germinated sorghum seeds derived from the endogomic line 80237 were soaked in an aqueous solution of 0.05% v / v ethylmethane sulfonic acid (EMS) for 16 hours. Treated seeds on 21st December 2007, Experimental Stadio
n of ADVANTA SEEDS in VENDA TURTO, Prov
ince of Santa Fe, Argentina plot 1 (33 ° 41 '
47 "S; 61 ° 58'45" W) and left them for open pollination.

A total of 273 M1 plants were selected, and two seeds from each plant were transferred to the Experimental Station of Advanced Seed on May 23, 2008.
s in Oran, Province of Salta, Argentina (22
The plant was planted on a nursery bed at 49 ° 37 ″ S; 64 ° 20′14 ″ W) to obtain a total of 546 M2 plants. Pollen was collected from one plant in each pair and used to pollinate the other plant in the pair. M3 seeds obtained from each of 273 pollinated M2 plants were harvested.
A total of 273 furrow M3 descendants on December 16, 2008 Vendo Turto
50 plants derived from each M3 ridge between 100 and 23 December 2008
ml L a. i. / Ha imazetapill was sprayed. 68 plants originating from the intercostals showed normal growth and absence of symptoms after treatment with the herbicide, were considered to be resistant to the herbicide and were identified as VT09-9754. A lineage of resistant plants derived from the furrows was identified and named 80237EMS2-192 (hereinafter referred to as ADV-IMI-R).

  Table 5 shows the history of generations.

[Analysis of response rate to treatment with imidazolinone in resistant ADV-IMI-R mutants compared to the original sorghum endogamic line 80237]
Sorghum 80237 (Advanta's own elite) and the imidazolinone-resistant ADV-IMI-R mutant (original mutation) of the present invention, on the field, three herbicides in the imidazolinone group: imazetapill, imazapill , And Imazapic were sprayed. Four different application rates were assayed for each herbicide: 1X, 2X, 3X and 4X (
1X is the recommended application rate) (see Table 1 above).

A field experiment was carried out in Venedo Turto and planting was carried out on 1 December 2010. The herbicide was sprayed 20 days after germination (stage V6). All treatments were compared to their corresponding untreated controls. The experimental design consisted of plots divided into 3 replicates (main plot: treatment with herbicide; secondary plot: system).

Ten days after spraying, all plants were assayed for dry matter (DM) in aerial tissue. Herbicide response was assessed as the DM percentage in the corresponding untreated control aerial tissue.

Thus, a system that is sensitive to herbicide i for each application rate j,
DM _ij % control = (DM _ij * 100) / expressed as DM control. The DM _ij % control is the average of 3 experiments for each treatment from each system.

[Sequencing of the sorghum AHAS gene encoding the mutant polypeptide of the present invention having acetohydroxy acid synthase activity and resistance to imidazolinone]
Sequencing studies were performed on leaf tissue derived from the ADV-IMI-R mutant (VT11-11331-BK selection) and the wild type endogomic line 80237. Genomic DNA
Was isolated and resuspended in water at a final concentration of 100 ng / μl. For sequencing of the acetohydroxy acid synthase (AHAS) gene from ADV-IMI-R mutant and sorghum line 80237, based on the sorghum AHAS sequence disclosed in GenBank under the accession number GM6633363.1 Specific primers for amplification by polymerase chain reaction (PCR) were designed. 5 overlapping DNs representing the complete AHAS coding sequence
Primers were designed to generate the A segment (amplicon). The designed primer sequences are as follows.

The PCR mix was in a final volume of 25 μl and the following components: 1 × reaction buffer (Invi
trogen), 0.2 mM dNTP (GE Healthcare), 2.5 mM
MgCl ₂ (Invitrogen), 0.2 μM of each primer, 0.5 μl Pla
It had tinum Taq (5 U / μl) (Invitrogen) and 100 ng genomic DNA. PCR reaction was performed using GeneAmp PCR System 9700.
Performed in a thermocycler (Perkin-Elmer), amplification conditions were as follows: initial denaturation at 94 ° C for 1 minute, then 94 ° C for 45 seconds, 57 ° C for 45 seconds, and 72 ° C for 70 minutes. 35 cycle steps per second, as well as a final extension step at 72 ° C. for 10 minutes.

Both are ADV-IMI-R products as well as SbAH for the original sorghum line 80237
No amplification product was obtained when AS-F1 and SbAHAS-R1OLD1-R2 primers were used.

To overcome this problem, two new primers were designed to generate a sixth amplicon:

  A sixth amplicon was obtained using the same PCR conditions as above.

2 μl of each DNA product resulting from amplification by PCR was examined by agarose gel electrophoresis, and the molecular weight marker Low DNA Mass Ladder (Invitrolog
en) and fragment size and DNA concentration estimates were analyzed. The remaining PCR products were purified from Wizard® SV gel (Promega) and PCR Clean-
Purification using Up System (Promega). Purified DNA is converted to Big
Dye (registered trademark) Terminator v3.1 Cycle Sequencin
Sequenced using g System (Applied Biosystems) according to manufacturer's instructions.

A sequencing file of acetohydroxy acid synthase obtained for each amplicon
CAP3 Sequence Assembly Program (http: // pb
il. univ-lyon1. fr / cap3. php). The resulting DNA sequence of the acetohydroxy acid synthase gene is designated as Clustal W version 2.1.
Alignment was performed using the program (http://www.clustal.org).

[Inheritance of Imazetapil Resistance in an Isolated F2 Sorghum Population Generated by Crossing an Imazetapill Sensitive System with a Resistant ADV-IMI-R Mutant]
Sorghum 90523 (Advanta's own elite system, sensitive to imidazolinone) and imidazolinone resistant ADV-IMI-R mutant (VT09-9754-48-6)
Targeted mating was performed with (BK selection). The resulting F1 plant was self-pollinated and the F2 seed was harvested. F2 seeds were planted in the field of Venado Turerto in October 2010. F2 plants (177 plants) with an application rate of 3x (application rate recommended by the manufacturer for commercial use is 1X, which is equal to 100 ml ai / ha), imazetapill ( Pivot (R), BASF). Before spraying, ADV-IMI
Leaf tissue was collected from each of 177 F2 plants derived from both the -R mutant (VT09-9754-48-6-BK selection) and the wild-type line 90523 to isolate genomic DNA and genotype Resuspended in water at a final concentration of 100 ng / μl for use in decision analysis.

The herbicide was sprayed 20 days after germination (stage V6). The plants are evaluated 10 days after spraying,
The herbicidal effects were classified into three categories according to the symptoms assessed visually according to the following phenotypic scoring:
−1 = no damage −2 = yellowing -3 = dead

[Development of DNA marker specific for SNP-SbAHAS]
AH replacing nucleotide G at nucleotide position +277 (codon 93) with A
A single point mutation in the AS gene distinguishes the original endogomic sorghum line 80237 (herbicide sensitivity) from the induced ADV-IMI-R mutant. Using the following group of double-labeled primers and probes, molecular markers (herein SNP-SbAH
Designed AS):

AB 7500 Thermocycler (Applied Biosystems, Fost
er City, CA, US) was performed by real-time PCR allele discrimination assay. 12.5 μl of 2X Perfect
a® qPCR Supermix (Quanta Biosciences,
Gaithersburg, MD, US), 0.08 μM primers (forward and reverse), 0.4 μM probes (1 and 2), 10 μl genomic DNA and 25 μl final volume containing DNase-free water to make the final volume It was prepared with. Amplification conditions were one initial denaturation cycle at 95 ° C. for 10 min followed by 50 cycles of denaturation at 92 ° C. for 15 seconds and hybridization / extension at 60 ° C. for 1 min. The results of the allele discrimination assay are expressed as AB Sequence Detection System.
em (SDS) 7500 1.4 software program (Applied Biosy)
analysis after amplification using stems, Foster City, CA, US).

[Correlation between herbicide tolerance and AHAS mutation in ADV-IMI-R mutant]
Individual F2 progeny plants obtained by crossing ADV-IMI-Rx90523 were phenotyped (no damage, yellowing and death) after nebulization with imazetapil (using the method described in Example 4) and then in AHAS Molecular marker SN specific for the induced mutation of
Genotyping was performed using P-SbAHAS (using the method described in Example 5).

[Gene mapping of herbicide tolerance and specific SNP-SbAHAS markers]
Herbicide tolerance and specific SNP-SbAHAS markers were gene mapped. Ge
Based on the sorghum AHAS sequence disclosed in nbank (Accession No. GM6633363.1), a BLAST analysis was performed, which was performed on the Phytosome database (http:
// www. phytosome. net / search. Sor deposited at php)
Matched to the genomic DNA sequence of ghum bicolor.

The genotyping (fingerprinting) of the mutant ADV-IMI-R system and the endogomic system 90253 was performed using a group of SSR type molecular markers. Sorghu
Seven polymorphic SSRs located on chromosome 4 of the m biocolor genome were selected for genotyping (Mace, ES et al. A consensus genetic map).
of sorghum that integrates multiple comp
onent maps and high-throughput Diversity
Array Technology (DArT) markers, BMC Plan
t Biology, 2009, 9:13; Srinivas, G et al., Explora
tion and mapping of microsatellite marke
rs from subtracted drought stress ESTs i
n Sorghum bicolor (L.), Moench. Theor. Appl.
Genet. 2009, 118: 703-717; Ramu, P et al., In silic.
o mapping of important genes and markers
available in the public domain for effi
cent sorghum breeding, Mol Breeding 2010
26: 409-418; http: // www. lbk. ars. usda. gov
/ Psgd / sorghum / 2009SorghumSEAMs_LBKARS. xl
s).

  The seven selected polymorphic SSRs were as follows:

The resulting PCR fragments derived from each of the tested SSRs were converted into automated sequencing equipment AB
Resolved by capillary electrophoresis using I3130x1 (Applied Biosystems). These seven SSRs were genotyped by ADV-IMI-Rx90.
This was done in 177 individuals of F2 progeny obtained from 523 crosses. In addition, these same 177 F2 progeny plants were identified as SNPs specific for the introduced mutation in the AHAS gene.
-Analyzed for the presence of SbAHAS markers. The results were categorized according to the phenotypic rating at the time of imazetapill application for F2 progeny plants and the computer program JoinMap (
Van Ooijen, JW and Voorips, RE, JoinMap 3.0 S
ofware for the calculation of genetic l
Inkage maps, Plant Research International
2001, Wageningen, The Netherlands).

Claims (60)

A sorghum plant comprising at least one polynucleotide in the genome encoding a polypeptide having an alanine to threonine substitution at position 93 of the large subunit of the sorghum AHAS protein, wherein the sorghum plant comprises at least one polynucleotide compared to a wild type sorghum plant. Such a plant having increased resistance to one or more herbicides. The sorghum plant of claim 1, wherein the herbicide is selected from the group consisting of imidazolinones and sulfonylureas. The sorghum plant according to claim 2, wherein the imidazolinone herbicide is selected from the group consisting of imazetapill, imazapill, and imazapic. The sorghum plant of claim 1, wherein the at least one polynucleotide comprises SEQ ID NO: 1.   The sorghum plant of claim 1, wherein the encoded polypeptide comprises SEQ ID NO: 2. The sorghum plant according to claim 1, comprising a mutation in the AHAS gene according to NCIMB41870. The sorghum plant of claim 1, wherein the sorghum plant comprises an increased resistance trait to one or more herbicides of the imidazolinone group similar to the resistance described for NCIMB41870. 8. A sorghum plant according to claim 6 or 7, wherein the sorghum plant having increased resistance to one or more herbicides is useful for introducing a resistance trait by introgression into another sorghum plant.   The sorghum plant according to claim 1, wherein the plant is transgenic.   The sorghum plant of claim 1, wherein the plant is non-transgenic. A herbicide-tolerant sorghum plant comprising a resistance trait of NCIMB41870, wherein the sorghum plant is selected from the group consisting of a plant such as NCIMB41870, a progeny of NCIMB41870 plant, a NCIMB41870 mutant plant and a NCIMB41870 mutant progeny.   The sorghum plant according to claim 11, wherein the herbicide belongs to the imidazolinone group.   12. A sorghum plant according to claim 11, wherein the plant is transgenic.   12. A sorghum plant according to claim 11, wherein the plant is non-transgenic. A sorghum seed comprising in the genome at least one polynucleotide encoding a polypeptide having an alanine to threonine substitution at position 93 of the large subunit of the sorghum AHAS protein. 16. The seed of claim 15, wherein the seed produces a plant with increased tolerance to one or more herbicides in the imidazolinone group as compared to a wild type sorghum plant. 16. The seed of claim 15, wherein the seed comprises a mutation in the AHAS gene shown in deposited seed NCIMB 41870.   The seed of the plant of any one of Claim 1 to 10.   The seed of the plant according to any one of claims 11 to 14. a) preparing a nucleic acid sample derived from a sorghum plant;
b) amplifying a region corresponding to the AHAS gene present in the nucleic acid sample derived from the sorghum plant;
c) identifying herbicide-tolerant plants based on the presence of at least one said mutation in the amplified nucleic acid sample that is a mutation that confers resistance to herbicides from the imidazolinone group. A method for identifying herbicide-tolerant plants. 21. The method of claim 20, wherein the nucleic acid sample comprises at least one mutation, such as a mutation present in MCIMB 41870. 21. The method of claim 20, wherein the at least one mutation comprises an Ala93Thr substitution in the encoded polypeptide, and the polypeptide has acetohydroxy acid synthase activity.   21. The method of claim 20, wherein the nucleic acid sample comprises SEQ ID NO: 1.   23. The method of claim 22, wherein the polypeptide comprises SEQ ID NO: 2.   21. The method of claim 20, wherein the plant is a monocotyledonous plant.   21. The method of claim 20, wherein the plant is a dicotyledonous plant.   21. The method of claim 20, wherein the plant is a transgenic plant.   21. The method of claim 20, wherein the plant is a non-transgenic plant. 21. The herbicide is selected from the group of imidazolinones and sulfonylureas.
The method described in 1. A method for controlling weeds in close proximity to a crop plant, comprising applying an effective amount of a herbicide in the imidazolinone group or sulfonylurea group to the weeds and crop plants. 32. The method of claim 30, wherein the imidazolinone herbicide is selected from the group consisting of imazetapill, imazapic, and imazapill. The crop plant is
a) a sorghum plant comprising in its genome at least one polynucleotide encoding a polypeptide having an alanine to threonine substitution at position 93 of the sorghum AHAS protein, wherein imidazolinone or Said plant having increased resistance to one or more herbicides in the group of sulfonylureas,
b) a sorghum plant comprising a mutation in the AHAS gene, such as in NCIMB 41870,
c) a sorghum plant comprising an increased resistance trait to one or more herbicides in the group of imidazolinones or sulfonylureas, such as the resistance exhibited by NCIMB 41870,
31. The method of claim 30, wherein d) is selected from the group consisting of plants, progeny, derivatives or mutants of sorghum plants a) to c). a) the nucleotide sequence set forth in SEQ ID NO: 1,
b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2,
c) a nucleotide sequence encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 and having herbicide-tolerant AHAS activity;
d) having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1;
A nucleotide sequence encoding a polypeptide comprising a large subunit of AHAS and exhibiting herbicide-tolerant AHAS activity;
e) a polynucleotide comprising a nucleotide sequence selected from the group consisting of nucleotide sequences perfectly complementary to one of the nucleotide sequences a) to (d). 34. The polynucleotide of claim 33, wherein the polynucleotide encodes a polypeptide comprising an Ala93Thr substitution in the large subunit of AHAS. 34. An expression cassette comprising the nucleotide sequence of claim 33 operably linked to an expression cassette. Further comprising at least one promoter that directs expression of the polynucleotide;
36. The expression cassette of claim 35. 37. An expression cassette according to claim 36, wherein the promoter is selected from the group comprising promoters for expression in plants, bacteria, fungi, animal cells and protozoa.   36. A cell transformed with the expression cassette of claim 35. The cells are selected from the group consisting of bacteria, fungi, yeast, plant cells, and animal cells;
40. The cell of claim 38. a) the nucleotide sequence set forth in SEQ ID NO: 1,
b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2,
c) a nucleotide sequence encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 and having herbicide-tolerant AHAS activity;
d) having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1;
A nucleotide sequence encoding a polypeptide comprising a large subunit of AHAS and exhibiting herbicide-tolerant AHAS activity;
e) having a polynucleotide comprising a nucleotide sequence selected from the group consisting of nucleotide sequences perfectly complementary to one of the nucleotide sequences a) to (d), wherein the selectable gene induces expression of said nucleotide sequence A transformation vector further comprising at least one promoter operably linked to a nucleotide sequence capable of. 41. Use of a vector according to claim 40 for transforming a cell selected from the group comprising bacteria, fungi, yeast, plant cells and animal cells. a) the nucleotide sequence set forth in SEQ ID NO: 1,
b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2,
c) a nucleotide sequence encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 and having herbicide-tolerant AHAS activity;
d) having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1;
A nucleotide sequence encoding a polypeptide comprising a large subunit of AHAS and exhibiting herbicide-tolerant AHAS activity;
e) at least one promoter integrated in the genome of the plant operably linked to a polynucleotide selected from the group comprising a nucleotide sequence perfectly complementary to one of the nucleotide sequences a) to (d) A transformed plant, wherein the vector further comprises a selectable gene, at least one promoter operably linked to a nucleotide sequence capable of inducing expression of said nucleotide sequence. 43. The plant of claim 42, wherein the promoter is selected from the group consisting of a tissue specific promoter and a chloroplast specific promoter.   43. The plant of claim 42, wherein the plant is a monocotyledonous plant.   43. The plant of claim 42, wherein the plant is a dicotyledonous plant. The plant is selected from the group consisting of sorghum, corn, rice, and wheat;
45. A plant according to claim 44. 46. The plant of claim 45, wherein the plant is selected from the group consisting of soybean and rape. 43. The plant of claim 42, wherein the plant comprises increased herbicide tolerance compared to a non-transformed plant. 43. The plant of claim 42, wherein the herbicide is selected from the group consisting of imidazolinones and sulfonylureas. i) a) the nucleotide sequence set forth in SEQ ID NO: 1
b) a nucleotide sequence encoding the polypeptide set forth in SEQ ID NO: 2,
c) a nucleotide sequence encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 and having herbicide-tolerant AHAS activity;
d) a nucleotide sequence encoding a polypeptide having at least 85% identity to the nucleotide sequence set forth in SEQ ID NO: 1, comprising a large subunit of AHAS and exhibiting herbicide-tolerant AHAS activity; and e) nucleotide sequence a ) -D), an expression cassette or vector comprising a polynucleotide having a nucleotide sequence selected from the group of nucleotide sequences perfectly complementary to one of said expression cassettes, said expression cassette being operably linked to said polynucleotide Transforming a plant cell with the expression cassette or vector further comprising at least one promoter, wherein the promoter induces expression of the polynucleotide;
ii) regenerating said plant cells to obtain herbicide resistant plants, a method for obtaining herbicide resistant plants or plants with increased herbicide tolerance.   51. The method of claim 50, wherein the promoter comprises a plant tissue specific promoter.   51. The method of claim 50, wherein the promoter comprises a chloroplast specific promoter. 51. The method of claim 50, wherein the herbicide is selected from the group consisting of imidazolinones and sulfonylureas. 54. The method of claim 53, wherein the imidazolinone herbicide is selected from the group comprising imazetapill, imazapic, and imazapill.   51. The method of claim 50, wherein the plant is a monocotyledonous plant.   51. The method of claim 50, wherein the plant is a dicotyledonous plant. 56. The method of claim 55, wherein the plant is selected from the group consisting of sorghum, corn, rice and wheat. 57. The method of claim 56, wherein the plant is selected from the group consisting of soybean and rape. 51. The method of claim 50, wherein the plant comprises increased herbicide tolerance compared to a non-transformed plant. 51. The method of claim 50, wherein the plant comprises a large subunit of AHAS and exhibits herbicide-tolerant AHAS activity.

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