# Patent

## Title
Rice resistant to HPPD and accase inhibiting herbicides

## Abstract

Rice is described that is tolerant/resistant to a plurality of herbicides, for example, ACCase and HPPD inhibitors. Use of the rice for weed control and methods of producing tolerant/resistant rice are also described.

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/869,608, filed Aug. 23, 2013, and is a Continuation-in-Part of copending U.S. patent application Ser. No. 13/975,034, filed Aug. 23, 2013, which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61/692,861 filed Aug. 24, 2012; this application is also a Continuation-in-Part of copending U.S. patent application Ser. No. 13/554,675, filed Jul. 20, 2012, which claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Nos. 61/510,585, filed Jul. 22, 2011, and 61/541,832, filed Sep. 30, 2011. The disclosures set forth in the referenced applications are incorporated herein by reference in their entireties.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 13, 2014, is named 706191_SEQ_US.txt and is 79 KB in size.
BACKGROUND
Mutant rice is disclosed that is (1) resistant/tolerant to both HPPD and ACCase inhibiting herbicides; or (2) resistant/tolerant only to 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibiting herbicides. Methods of weed control are disclosed using rice with these herbicide resistant/tolerant crops in fields. Methods to produce herbicide resistant/tolerant rice are also disclosed.
Value of Rice Crops
Rice is an ancient agricultural crop and today is one of the principal food crops of the world. There are two cultivated species of rice: Oryza sativa L., the Asian rice, and Oryza glaberrima Steud., the African rice. The Asian species constitutes virtually all of the worlds cultivated rice and is the species grown in the United States. Three major rice producing regions exist in the United States: the Mississippi Delta (Arkansas, Mississippi, northeast Louisiana, southeast Missouri), the Gulf Coast (southwest Louisiana, southeast Texas), and the Central Valley of California. Other countries, in particular in South America and the East, are major rice producers.
Rice is one of the few crops that can be grown in a shallow flood as it has a unique structure allowing gas exchange through the stems between the roots and the atmosphere. Growth in a shallow flood results in the best yields and is the reason that rice is usually gown in heavy clay soils, or soils with an impermeable hard pan layer just below the soil surface. These soil types are usually either not suitable for other crops or at best, the crops yield poorly.
The constant improvement of rice is imperative to provide necessary nutrition for a growing world population. A large portion of the world population consumes rice as their primary source of nutrition and crops must thrive in various environmental conditions including competing with weeds and attacks by unfavorable agents. Rice improvement is carried out through conventional breeding practices and also by recombinant genetic techniques. Though appearing straightforward to those outside this discipline, crop improvement requires keen scientific and artistic skill and results are generally unpredictable.
Although specific breeding objectives vary somewhat in the different rice producing regions of the world, increasing yield is a primary objective in all programs.
Plant breeding begins with the analysis and definition of strengths and weaknesses of cultivars in existence, followed by the establishment of program goals, to improve areas of weakness to produce new cultivars. Specific breeding objectives include combining in a single cultivar an improved combination of desirable traits from the parental sources. Desirable traits may be introduced due to spontaneous or induced mutations. Desirable traits include higher yield, resistance to environmental stress, diseases and insects, better stems and roots, tolerance to low temperatures, better agronomic characteristics, nutritional value and grain quality.
For example, the breeder initially selects and crosses two or more parental lines, followed by selection for desired traits among the many new genetic combinations. The breeder can theoretically generate billions of new and different genetic combinations via crossing. Breeding by using crossing and selfing, does not imply direct control at the cellular level. However, that type of control may be achieved in part using recombinant genetic techniques.
Pedigree breeding is used commonly for the improvement of self-pollinating crops such as rice. For example, two parents which possess favorable, complementary traits are crossed to produce an F1 generation. One or both parents may themselves represent an F1 from a previous cross. Subsequently a segregating population is produced, by growing the seeds resulting from selfing one or several F1s if the two parents are pure lines, or by directly growing the seed resulting from the initial cross if at least one of the parents is an F1. Selection of the best individual genomes may begin in the first segregating population or F2; then, beginning in the F3, the best individuals in the best families are selected. “Best” is defined according to the goals of a particular breeding program e.g., to increase yield, resist diseases. Overall a multifactorial approach is used to define “best” because of genetic interactions. A desirable gene in one genetic background may differ in a different background. In addition, introduction of the gene may disrupt other favorable genetic characteristics. Replicated testing of families can begin in the F4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F6 and F7), the best lines or mixtures of phenotypically similar lines are tested for potential release as new parental lines.
Backcross breeding has been used to transfer genes for a highly heritable trait into a desirable homozygous cultivar or inbred line which is the recurrent parent. The source of the trait to be transferred is called the donor parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent. After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The process is used to recover all of the beneficial characteristics of the recurrent parent with the addition of the new trait provided by the donor parent.
Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for at least three or more years. The best lines are candidates for new commercial varieties or parents of hybrids; those still deficient in a few traits may be used as parents to produce new populations for further selection.
These processes, which lead to the final step of marketing and distribution, usually take from 8 to 12 years from the time the first cross is made and may rely on the development of improved breeding lines as precursors. Therefore, development of new cultivars is not only a time-consuming process, but requires precise forward planning, efficient use of resources, and a minimum of changes in direction. The results include novel genetic combinations not found in nature.
Some improvement of rice through breeding may be restricted to the natural genetic variation in rice and hybridizing species, such as wild rice. The introduction of new variation in a breeding program is usually through the crossing program as described, such as pedigree or backcross breeding. However, occasionally natural mutations are found that result in the introduction of new traits such as disease resistance or height changes. Breeders have also developed new traits by inducing mutations (small changes in the DNA sequence) into a rice genome. Some of these mutations or combination of genes are not found in nature. Commonly, EMS or sodium azide plus MNU are used as mutagenic agents. These chemicals randomly induce single base changes in DNA, usually of G and C changed to A and T. Overall effects are unpredictable. Most of these changes have no effect on the crop as they fall either outside the gene coding regions or don't change the amino acid sequence of the gene product. However, some produce new traits or incorporate new DNA changes into previous lines.
The breeder has no direct control of mutation sites in the DNA sequence. The identification of useful changes is due to the random possibility that an effective mutation will be induced and that the breeder will recognize the phenotypic effects of the change and will be able to select rice having that mutation. Seeds are treated with the mutagenic chemical and immediately planted to grow and produce M2 seed. The M2 seed will carry numerous new variations; therefore, no two experiments will produce the same combinations. Among these variations new traits previously not existing in rice and unavailable for selection by a plant breeder may be found and used for rice improvement.
To find new traits the breeder must use efficient and strategic selection strategies as the process is completely random and has an extremely low frequency of useful new combinations. Among thousands of induced new genetic variants there may be only one with a desirable new trait. An optimal selection system will screen through thousands of new variants and allow detection of a few or even a single plant that might carry a new trait. After identifying or finding a possible new trait the breeder must develop a new cultivar by pedigree or backcross breeding and extensive testing to verify the new trait and cultivar exhibits stable and heritable value to rice producers.
Using recombinant genetic techniques, nucleic acid molecules with mutations that encode improved characteristics in rice, may be introduced into rice with commercially suitable genomes. After a mutation is identified by whatever course, it may be transferred into rice by recombinant techniques.
Applications of Herbicide Resistance Patents in Rice
Weeds and other competitors for resources in crop fields compete for resources and greatly reduce the yield and quality of the crop. Weeds have been controlled in crops through the application of selective herbicides that kill the weeds, but do not harm the crop. Usually selectivity of the herbicides is based on biochemical variations or differences between the crop and the weeds. Some herbicides are non-selective, meaning they kill all or almost all plants. Non-selective or broad spectrum herbicides can be used in crops if new genes are inserted that express specific proteins that convey tolerance or resistance to the herbicide. Resistance to herbicides has also been achieved in crops through genetic mutations that alter proteins and biochemical processes. These mutations may arise in nature, but mostly they have been induced in crops or in vitro in tissue cultures or by inducing mutations in vivo. Unfortunately in some instances, especially with repeated use of a particular herbicide, weeds have developed resistance through the unintended selection of natural mutations that provide resistance. When weeds become resistant to a particular herbicide, that herbicide is no longer useful for weed control. The development of resistance in weeds is best delayed through alternating the use of different modes of action to control weeds, interrupting development of resistant weeds.
Rice production is plagued by broad leaf plants and a particularly hard to control weed called red rice. One difficulty arises because red rice is so genetically similar to cultivated rice (they occasionally cross pollinate) that there are no selective herbicides available that target red rice, yet do not harm the cultivated rice. Control is currently provided in commercial rice production through the development of mutations found in rice that render rice resistant to broad spectrum herbicides e.g. imidazolinone and sulfonylurea herbicides. Rice resistant to herbicides that inhibit other deleterious plants, such as broad leaf plants, are needed.
Finding new mutations in rice that makes it resistant to a variety of herbicides, and to combinations of herbicides with alternative modes of action, would greatly benefit rice production. Obtaining and incorporating genes for herbicide resistance into rice genomes with additional favorable characteristics and alternative resistances is challenging, unpredictable, time consuming and expensive, but necessary to meet the world's increasing food needs.
SUMMARY
Described and disclosed herein are novel and distinctive rice lines with unique resistances to herbicides in particular HPPD and ACCase inhibiting herbicides and combinations thereof. For example, a mutant rice line designated ML0831266-03093 is disclosed that is resistant/tolerant to HPPD inhibiting herbicides (ATCC deposit PTA-13620). The HPPD inhibiting herbicides include mesotrione, benzobicyclon, and combinations thereof. An embodiment of a mutant rice line designated ML0831265-01493 (ATCC deposit PTA-12933, mutation G2096S) is resistant/tolerant to ACCase inhibitors.
Embodiments of rice resistant to both HPPD and ACCase inhibitors, include rice designated PL121448M2-80048 (ATCC deposit PTA-121362) and PL 1214418M2-73009 (ATCC deposit PTA-121398).
A method to control weeds in a rice field, wherein the rice in the field includes plants resistant to a plurality of herbicides, includes:

    
    
        a. using herbicide resistant/tolerant rice in the field; and
        b. contacting the rice field with a plurality of herbicides, for example, one of which is an HPPD inhibiting herbicide, another an ACCase inhibitor.
    
    


Rice lines either singly or multiply resistant/tolerant extend the useful life of several herbicides due to being able to rotate the kinds of herbicides applied in grower's fields thus slowing the development of weed resistance. Several methods are possible to deploy these resistances into hybrids or varieties for weed control, as well as options for hybrid seed production. The rice lines described herein represent new methods for weed control in rice and can be deployed in any of many possible strategies to control weeds and provide for long-term use of these and other weed control methods. In particular, mutant rice tolerant to HPPD inhibiting herbicides and to both HPPD and ACCase inhibitors are disclosed.
Rice production for good yields requires specific weed control practices. Some herbicides are applied at the time of planting and others are applied before a permanent flood is applied, few weeds can grow in a full flood.
Through developing sources of resistance to multiple herbicides, more options are available for weed control in rice. The rice lines claimed provide the ability to use herbicides with a new mode of action for weed control. The ability to use an HPPD inhibiting herbicide in combination with an ACCase inhibitor, represents a mode of action not previously reported in rice. The use of these rice lines including combining lines with resistance to herbicide with other modes of action provides new options for weed control in grower's fields thus slowing the development of weed resistance. Several methods are possible to deploy this resistance in hybrids for weed control as well as options for hybrid seed production.
Cells derived from herbicide resistant seeds, plants grown from such seeds and cells derived from such plants, progeny of plants grown from such seed and cells derived from such progeny are within the scope of this disclosure. The growth of plants produced from deposited seeds, and progeny of such plants will typically be resistant/tolerant to HPPD inhibiting and ACCase inhibiting herbicides at levels of herbicide that would normally inhibit the growth of a corresponding wild-type plant.
A method for controlling growth of weeds in the vicinity of herbicide resistant/tolerant rice plants is also within the scope of the disclosure. One example of such methods is applying one or more herbicides to the fields of rice plants at levels of herbicide that would normally inhibit the growth of a rice plant. For example, at least one herbicide inhibits HPPD activity. A plurality includes, for example, HPPD and ACCase inhibitors. Surprisingly, some mixtures of herbicide increased the activity of all components. Such methods may be practiced with any herbicide that inhibits HPPD and/or ACCase activity and any resistant rice mutation, e.g., the embodiments disclosed herein.
Unexpectedly, using a mixture of HPPD and ACCase inhibiting herbicides, provided better results than when each herbicide was applied separately.
A method for growing herbicide resistant/tolerant rice plants includes (a) planting resistant rice seeds; (b) allowing the rice seeds to sprout; (c) applying one or more herbicides to the rice sprouts at levels of herbicide that would normally inhibit the growth of a rice plant. For example, at least one of the herbicides inhibits HPPD, other herbicides include ACCase inhibitors.
Methods of producing herbicide-tolerant rice plants may also use a transgenes or plurality of transgenes. One embodiment of such a method is transforming a cell of a rice plant with transgenes, wherein the transgenes encode an HPPD and an ACCase enzyme that confers tolerance in resulting rice plants to one or more herbicides. Any suitable cell may be used in the practice of these methods, for example, the cell may be in the form of a callus.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a graphical representation of a trait tolerance trial; the trial was planted, mesotrione was applied at the indicated rates about 1 month later (3-4 leaf stage), and a second application was applied on the two indicated treatments about 1 week later; the rice was evaluated for the percent of injury or damage as compared to unsprayed rice twenty-one days after the first herbicide application; line ML0831266-03093 is the HPPD resistant line and R0146 and P1003 are control lines with no induced mutation; the line PL1214418M2-73009 was selected out of a cross between the HPPD tolerant line ML0831266-03093 (parent is P1003) and the ACCase resistant line ML0831265-01493 (parent is R0146). The newly developed line PL1214418M2-73009 shows equivalent tolerance as the original HPPD tolerant line ML0831266-03093.
FIG. 2 is a graphical representation of a trait tolerance trial; the trial rice were planted, quizalofop was applied at the indicated rates about 5 weeks later (initiating tillering); the rice was evaluated for the percent of injury or damage as compared to unsprayed rice four weeks after herbicide application; line ML0831265-01493 is the ACCase resistant line and R0146 and P1003 are control lines; the line PL1214418M2-73009 was selected out of a cross between the HPPD tolerant line ML0831266-03093 (parent is P1003) and the ACCase tolerant line ML0831265-01493 (parent is R0146). The newly developed line PL1214418M2-73009 which has combined resistance shows equivalent or better as the original ACCase resistant line ML0831265-01493.
FIG. 3 (A, B) are photographs of line ML0831266-03093 that in addition to resistance to HPPD herbicides also has enhanced resistance to ACCase herbicides. Enhanced resistance is demonstrated by plants from the HPPD resistant line ML0831266-03093 (A) surviving an application of quizalofop (77 gm ai/ha) whereas the wild-type parent line P1003 (B) does not survive. Photographs taken four weeks after herbicide application.
FIG. 4 is a graphical representation of results of a trait tolerance trial wherein mesotrione was applied at the indicated rates about 34 days after planting. (initiating tillering) the rice was evaluated for the percent of injury or damage as compared to unsprayed rice four weeks after herbicide application; the HPPD tolerant line ML0831266-03093 and the line with combined HPPD and ACCase tolerance PL1214418M2-73009 show a similar response indicating the full HPPD tolerance was recovered in the new line; the line PL1214418M2-73001 was selected to only carry the HPPD tolerant mutation from the HPPD line ML0831266-03093 and the line PL1214418M2-73013 was selected to only carry the HPPD non-induced tolerance from the HPPD line ML0831266-03093; P1003 is the non-mutant parent line for the HPPD tolerant line ML0831266-03093; note that over 420 gmai/ha, even the lines with genetic resistance may be injured.
FIG. 5 illustrates grass weed control by the ACCase inhibitor quizalofop; control is shown by the prevalent grass weed (dead plants) being killed by quizalofot (77 gm ai/ha) while the resistant line ML0831265-01493 (live plants) were not injured.
FIG. 6 (A, B) are photographs of results of trait tolerance trials; weed control by mesotrione herbicide applied pre-planting was evaluated; the plots were planted with a hybrid of the HPPD tolerant line ML0831266-03093; just before planting, the plot on the right (B) received an application of mesotrione at 210 gmai/ha; the plot on the left (A) had no herbicide applied either pre-plant or post-emergence; pictures were taken four weeks after planting showing differences in weed appearance.
FIG. 7 (A, B) are photographs of rice growth versus stunted growth in a trait tolerance trial; both plots have rice line P1003 (carries some non-induced tolerance to HPPD herbicides); the herbicide treatments were applied at the initiation of tillering; the photographs were taken four weeks after herbicide application; only the ACCase herbicide fluazifop was applied to the plot on the left (A) (210 gmai/ha); at this rate fluazifop was not as active, no killing of the rice plants was observed; the right plot (B) was sprayed with a tank mixture of the ACCase herbicide fluazifop (210 gmai/ha) and the HPPD herbicide mesotrione (210 gmai/ha); combining herbicides improves activity.
FIG. 8 is a graphical representation of results of a trait tolerance trial quizalofop was applied at the indicated rates about 30 days after planting (3-4 leaf stage), and a second application was applied on the two indicated treatments. The rice was evaluated for the percent of injury or damage as compared to unsprayed rice twenty-one days after the first herbicide application; line ML0831265-01493 is the ACCase tolerant line with the G2096S mutation; line ML0831265-02283 is also tolerant to ACCase herbicides however the tolerance is not from a mutation in the ACCase coding gene, R0146 is the parent line for both of the ACCase tolerant lines; P1003 is a control line.
FIG. 9 shows results of a trait mapping experiment; an F2 population was derived from a cross between a cytoplasmic male sterile line A0109 and the ACCase tolerant line ML0831265-0228; the F2 individuals in the population were genotyped, sprayed with quizalofop (116 gmai/ha) twenty-six days after planting, and evaluated for tolerance to quizalofop nineteen days after herbicide application; using QTL mapping software, a major QTL for tolerance was identified on chromosome one (indicated by the arrow).
FIG. 10 is a scattergram showing results of a mutation mapping experiment; an F2 population was derived from a cross between the ACCase tolerant line ML0831265-02283 and the parent line R0146; the F2 population was genotyped, sprayed with quizalofop (116 gmai/ha), and evaluated for resistance to quizalofop; only mutations including ACCase resistant mutations will be segregating in the population; the SNP index is a measure of the proportion of sequencing reads that carry a variation from the non-mutant line R0146; a score of one indicates that all sequencing reads had the variation; 19 variations (mutations) had an index of one and grouped together on chromosome one (circled) indicating the probable location of the tolerance causing mutations. (SEQ ID NOs: 208-226)
FIG. 11 shows graphical results of HPPD trait tolerance trials; mesotrione was applied at the 3-4 leaf stage of rice (ML0831266-03093; P1003 and R0146) and evaluated twenty-one days after application for response to the herbicide; the rice was evaluated for the percent of injury or damage [see DEFINITIONS] as compared to unsprayed rice; the last two treatments included a sequential application two weeks after the first application.
FIG. 12 shows graphical results of HPPD trait tolerance trial; a pre-plant application of mesotrione was applied (ML0831266-03093; P1003 and R0146) at 210 gmai/ha followed by post-emergent mesotrione applications at the 3-4 leaf stage at the indicated rates; the rice was evaluated for the percent of injury or damage as compared to unsprayed rice twenty-one days after the post-emergent application.
FIG. 13 graphically represents results of trait mapping experiments; an F2 population was derived from a cross between the HPPD tolerant line ML0831266-03093 and the ACCase tolerant line ML0831265-01493; the population was genotyped, sprayed with mesotrione at 105 gmai/ha, and evaluated for tolerance to mesotrione; plants inheriting either the non-induced tolerance or the mutation tolerance from parent line ML0831266-03093 were expected to live; using QTL mapping software, a major QTL for tolerance was identified on chromosome two (indicated by the bold arrow). (X axis=chromosome number in the genome; Y axis=score, correlation with phenotype (resistance)).
FIG. 14 graphically represents results of trait mapping experiments; an F2 population was derived from a cross between the HPPD tolerant line ML0831266-03093 and the ACCase tolerant line ML0831265-01493; the population was genotyped, sprayed with mesotrione at 105 gmai/ha, evaluated for tolerance to mesotrione, and sprayed again with mesotrione at 630 gmai/ha, and evaluated for tolerance to mesotrione; only plants inheriting the mutation for tolerance from parent line ML0831266-03093 were expected to live; using QTL mapping software a major QTL for tolerance was identified on chromosome one (indicated by the arrow) (X axis—chromosome number in the genome; Y-axis=score, correlation with phenotype).
FIG. 15 shows results of mutation mapping experiments; an F2 population was derived from a cross between the HPPD tolerant line ML0831266-03093 and the parent line P1003; the population was genotyped, sprayed with mesotrione at 840 gmai/ha and evaluated for tolerance to mesotrione; only mutations including the HPPD tolerant mutation segregate in the population; the SNP index is a measure of the proportion of sequencing reads that carry a variation from the line P1003; a score of one indicates that all sequencing reads had the variation; ten variations (mutations) had an index of one and grouped together on chromosome one (circled) indicating the probable location of the tolerance causing mutation. (SEQ ID NOS: 227-236)
FIGS. 16 (A, B) are a tabular representation of segregants identified in a F4 population; the F4 population was derived from F2 selections carrying both HPPD and ACCase tolerance from a cross between the HPPD tolerant line ML0831266-03093 and the ACCase tolerant line ML0831265-01493; each row represents a different line and each column is a molecular marker within the QTL for the HPPD tolerant mutation on chromosome 1.(B) is a continuation of (A) black lines (boundaries) represent results of the genotype of the HPPD tolerant line ML0831266-03093 (20) and the genotype of the ACCase tolerant line ML0831265-01493 (10); observing the tolerance level of these lines to the HPPD herbicide mesotrione allows the tolerance mutation to be mapped to a specific gene.
FIG. 17 is a tabular representation of segregants identified in a F4 population; the F4 population was derived from F2 selections carrying both HPPD and ACCase tolerance from a cross between the HPPD tolerant line ML0831266-03093 and the ACCase tolerant line ML0831265-01493; each row represents a different line and each column is a molecular marker within the QTL for the HPPD non-induced tolerance on chromosome two; black lines (boundaries) represent results of the genotype of the HPPD tolerant line ML0831266-03093 (20) and the unshaded represents the genotype of the ACCase tolerant line ML0831265-01493 (10); observing the tolerance level of these lines to the HPPD herbicide mesotrione allows the resistance/tolerance to be mapped to a specific gene.
FIG. 18 (A, B, C) are photographs of controls used in the mesotrione herbicide bioassay after spraying one application of mesotrione at 105 gai/ha followed by a second application of 630 gai/ha; (A) the mutant line ML0831266-03093 shows little damage from the spray application, while (B) the unmutated type P1003 is severely injured (damaged) and (C) the other type of rice, R0146, used to make ACCase mutant line ML0831265-01493 is completely killed.
FIG. 19 (A, B) illustrates rate response of the mutant line ML0831266-03093, the unmutated original (parental) line P1003, and a different type of rice R0146; mesotrione was applied across all plots immediately following planting at a rate of 210 gai/ha; the response rates were applied at the 2-3 leaf stage; response was recorded twenty-one days following the foliar application. (A)=initial results and (B)=subsequent results.
FIG. 20 shows a DNA sequence for the carboxyl transferease coding region in the ACCase coding gene; a single nucleotide change (box) that encodes a mutation from G2096S is identified in the mutant line ML0831265-0149 which is designated as 09PM72399. (SEQ ID NO: 202) [NIPPONBARE (SEQ ID NO: 200) is a control; R0146 (SEQ ID NO: 201) is the original line treated with a mitogen to produce a mutation population.
FIG. 21 shows comparison of protein sequences for the carboxyl transferase region of the ACCase gene; the line with code 09PM72399 (SEQ ID NO: 204) is the line ML0831265-0149; this line shows a change of a single amino acid (box) at position 2096, relative to Black-Grass; R0146 (SEQ ID NO: 204) is the original line treated with a mutagen to produce a mutation population. [NIPPONBARE is SEQ ID NO: 203]
FIG. 22 illustrates a response of different germplasms (genotypes) of rice with different genetic backgrounds to mesotrione applied at the 2-3 leaf stage; wherein different rates of mesotrione are applied.
FIG. 23 is a photograph showing phenotypic differences (resistance) of an F2 population derived from the cross of ML0831266-03093 with tolerance to mesotrione and ML0831265-01493 with tolerance to ACCase herbicides; mesotrione was applied with one application of 105 gai/ha followed by a second application of 630 gai/ha; individuals (circled) are selected as inheriting the mesotrione tolerance, whereas the plants that did not inherit the tolerance are dead or severely injured.
FIG. 24 is a flow chart of a type of cross used in some embodiments disclosed herein.



DETAILED DESCRIPTION
Mutation Population and Establishment
A mutation breeding program was initiated to develop proprietary herbicide resistant/tolerant lines. A permanent mutant population was created by exposing approximately 10,000 seeds (estimated by the average weight of a kernel) of three rice lines including P1003, R0146, and P1062 to both mutagens sodium azide (AZ) and methyl-nitrosourea (MNU). The treated seeds were planted. Individual plants were harvested creating 8,281 potentially mutation lines. The lines have been maintained as a permanent mutant population for trait screening.
Development of Tolerance to HPPD and ACCase Inhibiting Herbicides by Combining the Tolerances in Lines ML0831266-03093 and ML0831265-01493
Herbicides that target the HPPD enzyme, primarily control broad leaf weeds. However trials in rice show prevalent control of grass weeds in rice, including red weedy rice especially with mesotrione herbicide.
On the other hand, the primary weed target of ACCase herbicides is monocot plants including rice, grass weeds, and red rice. However, some ACCase herbicides have lower activity on rice. This weakness is likely transferred to red rice as the plants are very closely related. Combining the HPPD tolerance and the ACCase tolerance into a single rice line allows a broad spectrum weed control strategy for rice. The HPPD herbicide controls broad leaf weeds and enhances the effect of ACCase herbicides for control of monocot weeds including red rice.
Combining the HPPD tolerance with the ACCase tolerance into a single rice line was initiated with the HPPD tolerance mapping project by crossing the HPPD tolerant line ML0831266-03093 to the ACCase tolerant line ML0831265-01493. In mapping the F2 population plants were selected for HPPD tolerance by applying mesotrione first at a low rate (105 gmai/ha) followed by a high rate (630 gmai/ha). In this process molecular markers were also developed allowing future selection of HPPD tolerance by either markers or herbicide tolerance screening or both.
After identifying plants that were tolerant to the HPPD herbicide mesotrione, they were also tested with the ACCase tolerance functional marker for the G2096S mutation in the ACCase donor parent line ML0831265-01493. Information to develop ACCase G2096S markers are in FIGS. 20, 21.
After this process, a set of 25 F2 plants with the ACCase mutation to herbicide resistance, and the HPPD genetic herbicide resistance on chromosome 1 and chromosome 2, in at least the heterozygous condition, were identified. The plants were transplanted to another field for harvesting at maturity. Out of the 25 plants, eight were homozygous for the ACCase mutation and one plant was homozygous for the ACCase mutation, the HPPD tolerance mutation, and the non-induced tolerance gene. The 25 plants were bagged at flowering and the seed harvested at maturity from each plant individually.
An early maturing group of plants was harvested as early as possible and the seeds planted in the greenhouse to help quickly advance to the F4 generation. Selections on the F3 plants were made by molecular markers flanking the HPPD tolerance mutation and native tolerance the ACCase functional mutation. Homozygous plants for all the selected genomic regions were advanced to the F5 generation. The F5 seed was confirmed to carry tolerance to ACCase herbicides and the HPPD herbicide mesotrione. Among the F5 lines PL1214418M2-80048 was selected due to a high seed yield and being homozygous for the ACCase tolerance mutation at position G2096S, the HPPD tolerance mutation, and the HPPD tolerance native gene. Seed from the line PL1214418M2-80048 was deposited at the ATCC and given a deposit number PTA-121362 (see Table 8).
A second line was developed by planting F3 seed in rows. The plants were sprayed with the HPPD herbicide mesotrione and selected for little or no injury as compared to unsprayed controls. Leaf tissue was also collected and the plants were tested for inheritance of the ACCase tolerance mutation G2096S, the HPPD mutation tolerance, and the HPPD non-induced tolerance. Plants homozygous for all three tolerance genes or QTLs were identified and harvested. The F4 seed (PL1214418M2-73009) was bulked together from plants carrying all three tolerance genes or QTLs and used for testing or as a new donor line for tolerance to both ACCase and HPPD herbicides. Seed of the source PL1214418M2-73009 was deposited at the ATCC and given a deposit number PTA-121398 (see Table 8).
Tolerance of New Lines Combining HPPD and ACCase is Equivalent and Selectable in Breeding Populations
The seed source PL1214418M2-73009 was developed from a cross between the HPPD resistant line ML0831266-03093 and the ACCase resistant line ML0831265-01493 and was sufficient to allow testing to verify equivalent tolerance to HPPD and ACCase inhibitors in new lines. Two trials were conducted to measure recovery of tolerance to both ACCase and HPPD herbicides in the new line PL1214418M2-73009. Recovery of tolerance in the line combining the two traits will illustrate that the traits are heritable and can be used to produce new varieties and hybrids carrying herbicide resistance. These trials are important as often times it is difficult to recover complex QTLs for quantitative traits or in some cases a traits response is dependent upon the genetic background. In the first trail the lines resistance to mesotrione (HPPD herbicide) was evaluated by planting line PL1214418M2-73009, the HPPD resistant line ML0831266-03093 and wild-type rice line P1003 and R0146 in plots (5 feet×10 feet). Mesotrione was applied at 0.5×, 1×, 2×, and 4× multiples of the labeled application rate (210 gmai/ha). Two additional treatments were included with a 1× and 2× rate followed by a second application 14 days afterward with the same rates. Full recovery of the HPPD resistance from line ML0831266-03093 was achieved in the line PL1214418M2-73009 as it and the original trait line had the same response to the herbicide applications (FIG. 1). These results show that the HPPD resistant trait can be bred and selected to develop commercial products.
Another trial was conducted to confirm recovery of the ACCase inhibitor resistance from the G2096S mutation as in the line ML0831265-01493. In this trial the new line PL1214418-73009 with combined HPPD and ACCase tolerance was planted in a row along with other various lines including the original donor line ML0831265-01493 (planted in a plot), ML0831266-03093, P1003, R0146 parent line for ACCase tolerance. The lines were all tested with the ACCase herbicides fluazifop at 0.5×, 1×, 2×, and 4× multiples of the label application rate (210 gmai/ha) and quizalofop at 0.5×, 1×, 2×, and 4× multiples of the label application rate (77 gmai/ha). In these trials the three new lines that inherited the ACCase tolerance all showed equivalent tolerance to the ACCase herbicides as did the donor line ML0831265-01493 (FIG. 2).
The tolerance to HPPD herbicides is more complex than the ACCase tolerance because it requires two genes that are different from the gene targeted by the herbicide. In spite of this greater complexity, the equivalent tolerance was recovered through selection of both the native tolerance gene and the mutation tolerance. The ACCase parent line ML0831265-01493 in this cross was highly sensitive to the HPPD herbicide mesotrione and thus was not expected to contribute any towards HPPD tolerance. Resistance/tolerance to HPPD herbicides is mostly likely caused by these two genes alone as they were the focus of the selection process, and the new line PL1214418M2-73009 shows equivalent resistance. These results show that the resistance for both ACCase and HPPD inhibitors is inherited and can be bred into any rice for commercial development of both HPPD and ACCase inhibitor resistance in rice.
Identification of Unexpected Increased Tolerance to ACCase Herbicides by the HPPD Tolerance Mutation
During the process of showing equivalent resistance of the ACCase tolerance in the new line PL1214418M2-73009, the HPPD tolerant line ML0831266-03093 was also evaluated for response to ACCase tolerance by application of the ACCase herbicides fluazifop at 0.5×, 1×, 2×, and 4× multiples of the label application rate (210 gmai/ha) and quizalofop at 0.5×, 1×, 2×, and 4× multiples of the label application rate (77 gmai/ha). In the field trials the resistant HPPD line ML0831266-03093 was planted in a row adjacent to the parent line P1003. During observations it became clear that the line ML0831266-03093 had more resistance to the ACCase inhibiting herbicides than did line P1003 (FIG. 3). These results suggest that the HPPD tolerance mutation has activity against ACCase inhibiting herbicides in addition to HPPD inhibiting herbicides. By combining the HPPD tolerance with the ACCase tolerance the newly developed line PL1214418M2-73009, or any other new line and other derived lines because resistance is heritable and can be bred into lines e.g. progeny may carry a higher tolerance to ACCase inhibiting herbicides than lines developed from only the ACCase inhibitor resistant line ML0831265-01493.
Identification of the Tolerance Contribution from the HPPD Tolerance Mutation and the Non-Induced Tolerance Gene from P1003
During the breeding process to develop new lines (PL1214418M2-80048 and PL1214418M2-73009) with resistance/tolerance to HPPD and ACCase herbicides, two other lines were also investigated to determine the contribution of the HPPD tolerance mutation and the HPPD tolerance native gene. The line PL1214418M2-73001 carries ACCase tolerance and only the HPPD tolerance, whereas mutation PL1214418M2-73013 carries ACCase tolerance and only the HPPD native tolerance gene. These selections allow the estimation of the tolerance effect of each of the two genes required for tolerance to HPPD herbicides. The tolerance effect of each gene was measured by growing the lines in single rows including the newly developed line PL1214418M2-73009 that carries both the HPPD tolerance from the mutation and the non-induced tolerance, the HPPD tolerant line ML0831266-0309, and the non-induced parent line P1003. The field plots were sprayed at the 4 leaf stage with the HPPD herbicide mesotrione at 0.5×, 1×, 2×, and 4× multiples of the labeled application rate of 210 gmai/ha.
The plots were evaluated 4 weeks after the herbicide was applied. The results showed that the native tolerance gene alone (PL1214418M2-73013) gave tolerance levels similar to the parent line P1003 (FIG. 4). This result would be expected if the native tolerance gene located on chromosome 2 located within the QTL flanking markers is the only causative source of tolerance in the non-mutant line P1003. The line PL1214418M2-73001 that carries only the HPPD mutation located on chromosome 1 within the QTL flanking markers, shows intermediate tolerance between the non-mutant line P1003 and the HPPD mutant line ML0831266-0309. This result shows that the HPPD mutation provides not only enhanced tolerance but also a greater level of tolerance than the native tolerance gene. In addition it also suggests that the HPPD mutation functions independently of the HPPD native tolerance gene. The two genes also appear to function in an additive manner as only by combining the two in the new line PL1214418M2-73009 does the tolerance level become equivalent to the original mutant line ML0831266-0309.
Controlling Weeds and Red Rice in Rice Crops with ACCase Inhibitors and Mesotrione Herbicides (HPPD Inhibitors)
The herbicide activity or ability to control non-mutant rice, such as line R0146 and P1003, is a good predictor of how well the herbicides will control red rice or wild weedy rice in a rice crop. Red rice and wild weedy rice are very similar to rice, even with the ability to cross with rice. This similarity is the reason these weeds are so difficult to control in a rice crop. The mutant lines (ML0831265-01493, ML0831265-02283, ML0831266-03093, PL1214418M2-80048, and PL1214418M2-73009) disclosed offer a new weed control strategy for red rice, wild weedy rice, and other weeds common in rice crops. These lines give rice tolerance to herbicides that will normally kill or cause yield reducing injury to the rice crop.
While testing the tolerant lines, the parent lines were also tested to serve as controls and as an indication of commercial potential as a red rice/wild weedy rice control strategy. These trials showed that select treatments of the herbicides applied alone or in various combinations and application timings offer a new weed control strategy in rice crops.
Rice is tolerant to certain ACCase inhibitor herbicides, for example cyhalofop is registered for use in rice. However other ACCase herbicides kill or severely injure rice to varying degrees. After testing, several of these other herbicides including fluazifop and quizalofop were found to offer good control of common grass weeds, such as barnyard grass, in rice (FIG. 5). Control of common weeds in rice was also achieved with mesotrione alone, especially when applied pre-plant or at higher rates (2× the labeled rate of 210 gmai/ha) (FIG. 6). The applied rates of both types of herbicides giving the weed control are well within the tolerance level of the respective ACCase and HPPD tolerant lines including the combined lines carrying tolerance to both HPPD and ACCase herbicides.
Development of the HPPD and ACCase tolerance into single lines (PL1214418M2-80048 and PL1214418M2-73009) gives the opportunity for an additional weed control strategy involving applications of ACCase and HPPD herbicides in a tank mix or individually at different times. The very effective pre-plant application of the HPPD herbicide mesotrione can now be followed with ACCase herbicides applied alone or in combination with HPPD herbicides. This strategy provides full spectrum weed control in a rice crop by broad leaf weed control provided by the HPPD herbicide, and grass weed control by the ACCase herbicide. In addition the control of grasses and red rice/weedy rice by ACCase herbicides is greatly enhanced by the activity provided by the HPPD inhibiting herbicide. This strategy is anticipated as being especially effective for control of red rice when an ACCase inhibiting herbicides are used that have lower activity on rice (FIG. 7).
This particular weed control system is highly useful in rice crops due to some weeds, including red rice, developing tolerance to currently used herbicides. Use of this weed control strategy allows rotation of different modes of action herbicides in rice crops. By rotating different modes of herbicide action the development of resistant weeds is slowed or prevented allowing for longer term use of all available weed control methods.
Tolerance/Resistance to ACCase Inhibitors
1. Validation of the Mutant Line ML0831265-02283 for Tolerance to ACCase Herbicides
After screening a large mutant population, the line ML0831265-02283 also survived application of the ACCase herbicide quizalofop. The line was increased to obtain sufficient seed for larger trials to evaluate its tolerance to ACCase herbicides. The tolerance to ACCase herbicides in line ML0831265-02283 was validated by planting in the field plots (5 feet by 10 feet) of the line, the non-mutant parent line R0146, a second non-mutant line P1003, and the ACCase tolerant line ML0831265-01493. The ACCase herbicide quizalofop was applied at the four leaf stage at 0.5×, 1×, 2×, and 4× multiples of the labeled rate (77 gmai/ha). Twenty one days after the herbicide was applied the plots were evaluated for percent injury caused to the rice based on control plots that had no herbicide application (FIG. 8). The data confirms the tolerance of line ML0831265-02283 and it may even carry more tolerance than line ML0831265-01493 as shown by less injury at the 2× and 4× rates of quizalofop.
2. Identification of the Causal Mutation for Tolerance to ACCase Herbicides in Line ML0831265-02283
Often tolerance to ACCase herbicides is derived from a mutation in the carboxyl transferase region of the ACCase gene, as is the case in tolerant line ML0831265-01493 (mutation at G2096S). However after sequencing the carboxyl transferase region of the ACCase gene in line ML0831265-02283 no mutation was found. This result indicates that the tolerance in line ML0831265-02283 is derived from a non-target site process.
Finding the causal mutation for tolerance in line ML0831265-02283 involved linkage mapping and mutation mapping as (“mut mapping”) described for finding the causal mutation and native tolerance for HPPD tolerance in line ML0831266-0309. (Wright et al., 2011)
Linkage mapping to find the chromosomal region or QTL causing the tolerance in line ML0831265-02283 requires a population segregating for the trait. This population was made by crossing the tolerant line with the male sterile cytoplasm line A0109. The F1 collected from this cross was grown and allowed to self-pollinate to make a F2 population. The F2 population will segregate for ACCase tolerance. Eight hundred F2 seeds were planted and leaf tissue was collected from the seedlings to allow genotyping of each plant. When the seedlings where three weeks old the whole F2 population was sprayed with quizalfop (116 gmai/ha). The seedlings were evaluated for tolerance nineteen days after the herbicide application. Standard QTL mapping software was used to analyze the genotypes of each F2 individual and the associated tolerance response to identify molecular markers linked to the herbicide tolerance. After this analysis a genomic region (QTL) was identified for the tolerance on chromosome one (FIG. 9). Linked markers flanking the QTL and markers inside the QTL flanking the peak of the QTL were identified as being suitable to select the herbicide tolerance derived from line ML0831265-02283 (TABLE 5). Approximately 250 genes are between the flanking markers.
The mutation mapping strategy to find the causal mutation was employed in the same manner as used to find the QTL for HPPD mutation tolerance. A mutation mapping population was created to find the causal tolerance mutation through genomic sequencing by next-generation sequencing. The mutant line ML0831265-02283 was crossed back to the original non-mutant parent R0146. The F1 progeny of the cross were selfed to produce a F2 population that is segregating for the tolerance causing mutation. Only mutations are segregating in this population because the mutations are the only genomic difference between ML0831265-02283 and R0146.
The F2 population was planted as individuals, and leaf tissue was collected and DNA extracted from each individual to use for genotyping after the population was phenotyped. The ACCase herbicide quizalofop was applied to the F2 population at the 3-4 leaf stage and a concentration of 116 gmai/ha. Individuals that survived the herbicide application were scored as tolerant and those that died were scored as susceptible.
The DNA derived from a set of twenty surviving F2 individuals and twenty that were killed was each respectively bulked together and sequenced along with both the mutant line ML0831265-02283 and the non-mutant parent line R0146. Mapping the causal mutation was based on an index accessing the frequency of all mutations in the bulk representing the surviving individuals. The index was derived from the proportion of sequencing reads that carried a variation different from the non-mutant parent line R0146. The more sequencing reads with the variation the closer the index was to one and if all sequencing reads had the variation the index equaled one.
The analysis of these results showed two groups including 19 mutations of eleven mutations on chromosome one with an index score of one (FIG. 10). This result confirms the QTL linkage mapping results as the mutations identified here all are located within the QTL region identified on chromosome one by linkage mapping. Molecular markers (SNP) were made for each of the mutations (TABLE 6). These markers are used in fine mapping to find the causal mutation and for breeding the ACCase tolerance derived from line ML0831265-02283 into commercial rice lines.
Tolerance/Resistance to HPPD Inhibitors: Herbicide Screening
Mesotrione (Callisto®), is an herbicide that inhibits the plant enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD). Callisto® is a postemergent and preemergent herbicide used to control annual broadleaf weeds in corn and certain other crops. The herbicide only damages some rice at lower rates but kills other types of rice. All rice appears to be at least damaged by higher herbicide rates. Finding resistance to Callisto® herbicide in rice results in a new mode of action for controlling broadleaf weeds and some grasses, in rice.
Resistance to mesotrione herbicide was found by screening the permanent mutant population. All lines in the permanent mutant population were planted into a dry seed bed. Within twelve hours after planting mesotrione (Callisto®) was applied at a rate of 255.1 gm ai per acre. The field was immediately flushed with water and kept moist through periodic flushing. The seedlings grew, many were bleached white and all lines derived from R0146 died whereas plants lived from 21 lines derived from the P1003 and 2 derived from P1062 mutation populations. The HPPD gene was sequenced, and no genetic mutation was identified causing any amino acid substitutions (SEQ ID NOs: 1, 2, 3).
Validating the Mutant Line ML0831266-03093 for Tolerance to HPPD Inhibiting Herbicides
After the initial screening of the “mutation population,” the lines with no damage were selected and tested in additional experiments using different rates of the herbicide. In particular, a rate response experiment was conducted in which two different rates of mesotrione were applied pre-emergence, plus an additional foliar application was also applied. This experiment differentiated one mutant line as having superior resistance (less injury) to the mesotrione herbicide as compared to the control (FIG. 19, Table 1). Progeny of this designated ML0831266-03093 are maintained as a new line carrying resistance to mesotrione herbicide. Seeds are deposited under ATCC PTA-13620. The line (ML0831266-03093) is a source of resistance that is backcrossed into proprietary rice lines or used directly in breeding to develop new proprietary rice lines. The developed lines are a source of herbicide resistance for use in development of new hybrids that offer an alternative mode of action to control weeds in rice. Affording this opportunity to growers is of great value both in providing high yields and in extending the useful life of available weed control technologies. These herbicide resistant traits can be tracked through the simple application of herbicides to growing plants.
The mutant line ML0831266-03093 was found to carry tolerance to mesotrione (a common HPPD inhibiting herbicide) through screening the line with different rates of the herbicide. The tolerance level of ML0831266-03093 was found to be much greater than the original non-mutant (native) line P1003.
The original line P1003, carries natural tolerance to mesotrione. This non-induced resistance of the original line sometimes masked the resistance of the mutant line making the enhanced resistance of the mutant line ML0831266-03093 not obvious. Finding the value of the mutant line ML0831266-03093 was only achieved with careful testing over two years, in different locations, and using different rates and timings of herbicide application. The high tolerance of the mutant line ML0831266-03093 is now apparent and documented through a rate response trial measuring the response of line ML0831266-03093 and non-mutant control lines to different rates of mesotrione (FIG. 11).
The validating trials included testing applications of mesotrione applied just before planting (pre-plant applications), after planting at various stages of rice growth (post applications), and combinations of both pre-plant and post applications. The discovered tolerance to HPPD inhibiting chemicals was apparent for both pre-plant and post-emergent applications (FIG. 12). The mutant line ML0831266-03093 shows tolerance greater than natural rice has to HPPD herbicides in all application regimes.
Further validation of the trait involved testing the mutant line ML0831266-03093 in the presence of common rice weeds. The mutant line was completely tolerant to the applied rates of mesotrione whereas the prevalent weed population was well controlled by the herbicide (FIG. 6). This level of grass weed control was completely unexpected as mesotrione is labeled for controlling broadleaf weeds in monocot crops. This result indicates that mesotrione and other HPPD inhibiting herbicides in combination with line ML0831266-03093 and derived lines, represent a new weed control system in rice. The high activity of the mesotrione on grass weeds and certain types of rice indicates that the system could be used to control red rice in a rice crop.
Mesotrione and other HPPD inhibiting herbicides target the HPPD gene. An increase in herbicide tolerance could be achieved through a mutation in the HPPD gene. A mutation within the gene sequence can alter the enzyme structure sufficiently to prevent it from being inhibited by the herbicide, but still allow it to carry-out its normal physiological function. Assuming this as a plausible tolerance mechanism, the HPPD gene was sequenced by Sanger sequencing in both the mutant line ML0831266-03093 and the original line P1003. Surprisingly, no mutation was found in the HPPD gene (SEQ ID NO: 1). The herbicide tolerance in line ML0831266-03093 appears to be derived from a non-target site process.
Two different methods were used to find the tolerance causing mutation. The first method involved using a QTL mapping strategy only employing a unique phenotyping process to find both the tolerance causing mutation and the gene causing the natural tolerance. The second method involved sequencing the entire genome of F2 plants derived from a cross to the non-mutant parent. [P1003]
1. QTL Mapping to Find the Causal Mutation and the Natural Tolerance Causing Gene
The mutant line ML0831266-03093 contains high tolerance to mesotrione and possibly other HPPD inhibiting herbicides, due to both a new mutation and a native tolerance gene present in the original non-mutant line P1003. The mesotrione tolerant line ML0831266-03093 was crossed to another mutant line ML0831265-01493. This second mutant line ML0831265-01493 lacks the native tolerance gene and is highly susceptible to mesotrione. However line ML0831265-01493 does have tolerance to ACCase herbicides due to a mutation in the ACCase gene that changes amino acid 2096 from glycine to serine. This mutation alters the enzyme making it unaffected by certain ACCase herbicides, however it still retains its normal physiological function. The mutation site for change the amino acid 2096 most commonly arises in weeds as a change to alanine rather than the only rarely found serine change. (FIGS. 20, 21) A molecular marker was developed based on the sequencing information, to test for inheritance of the mutation G2096S.
The F1 progeny from the cross of line ML0831266-03093 to ML0831265-01493 was selfed to produce a large population of F2 individuals. Each F2 individual was genotype with a set of 192 SNP markers (Table 3) that were polymorphic between the parents, to fully cover the genome with molecular markers.
Next in a QTL mapping strategy was to spray the herbicide on the F2 individuals and observe those that survive. However, this strategy could introduce complications due to both the native tolerance gene and the tolerance mutation segregating. A different strategy was used in which first mesotrione was applied at a low rate (105 gmai/ha). At this rate all plants inheriting the tolerance causing mutation, the native tolerance, or both, survived, whereas plants inheriting the corresponding genomic regions from the line ML0831265-01493 died as they are highly sensitive to the herbicide. QTL analysis based on this phenotyping method identified one QTL located on chromosome 2 (FIG. 13). After phenotyping the F2 plants with a low application rate of mesotrione a second high rate (630 gmai/ha) was applied, and the plants were again phenotyped. After analysis, a second QTL was found on chromosome 1 representing the higher tolerance achieved from the tolerance mutation (FIG. 14). With this strategy the two genes were resolved, one of which is the mutation (chromosome 1) and the other the native tolerance (chromosome 2).
Based on the QTL positions and linked markers a set of markers was identified that flank the mutation and native tolerance QTLs (TABLE 4). These markers define the region containing the HPPD tolerance causing genes. In addition this set of markers can be used for breeding purposes to develop new lines carrying tolerance to HPPD herbicides. The use of these markers allows selection without having to apply the herbicide to breeding populations.
2. Mutation Mapping to Find the Tolerance Causal Mutation
A mutation mapping population was created to find the tolerance causal mutation through genomic sequencing by next-generation sequencing. The mutant line ML0831266-03093 was crossed back to the original non-mutant parent P1003. The F1 progeny of the cross was selfed to produce a F2 population that will be segregating for the tolerance causing mutation. Only mutations will be segregating in this population because the mutations are the only genomic difference between ML0831266-03093 and P1003.
The F2 population was planted as individuals and leaf tissue collected and DNA extracted from each individual to use for genotyping after the population was phenotyped. In this method all of the population will carry the native tolerance gene rendering the population tolerant to a certain level to mesotrione herbicide. To differentiate the native tolerance from the tolerance causal mutation mesotrione was applied to the population with a high rate (840 gmai/ha) so that all individuals without the tolerance causal mutation died.
The DNA derived from a set of twenty surviving F2 individuals and twenty that were killed was each respectively bulked together and sequenced along with both the mutant line ML0831266-03093 and the non-mutant parent line P1003. Mapping the causal mutation was based on an index accessing the frequency of all mutations in the bulk representing the surviving individuals. The index was derived from the proportion of sequencing reads that carried a variation different from the non-mutant parent line P1003. The more sequencing reads with the variation the closer the index was to one and if all sequencing reads had the variation the index equaled one.
A single mutation causing the high tolerance to mesotrione was predicted. Instead the data showed a peak of mutations carrying a score of 1 introducing another level of difficulty in finding the causal mutation. The result did confirm that the QTL on chromosome 1 found through linkage mapping is the genomic location of the tolerance casual mutation (FIG. 15). Within the peak of mutations we found seven mutations with an index of one, none of which are an obvious cause for tolerance to mesotrione herbicide. (TABLE 7) Markers were developed for the mutations to facilitate finding the casual resistance mutation(s).
A set of lines was identified with recombination points evenly distributed within the identified QTLs and mutations (FIG. 16, FIG. 17). These lines were recovered in a homozygous condition for each recombination allowing phenotyping for herbicide tolerance on multiple individuals (full plots). Analysis of these lines allowed the tolerance mutation and native gene to be narrowed to a small region of the chromosome.
Through the described strategy the specific genomic regions containing the tolerance causal mutation and the native tolerance gene are now known and useful for developing commercial products. The commercial products are useful in rice production as they survive application of mesotrione herbicide at rates that will control prevalent weeds including red rice without harming the rice crop. The specific genomic location allows the use of molecular markers on the flanking regions of each QTL to select for the HPPD tolerant trait in the development of commercial products.
Genetic mapping of the three groups of F2 individuals including the set of individuals sprayed with mesotrione at only 105 gm ai/ha, the set followed by a sequential application of 630 gm ai/ha, and the final group sprayed with 420 gm ai/ha shows two genes controlling resistance to mesotrione. In the population sprayed with 105 gm ai/ha a single QTL found on chromosome 2 with strong linkage to SNP marker BG-id2004662 acted in a mostly dominate manner. This marker and QTL identifies the inherent tolerance in line P1003. The marker is useful for breeding and selection of new mesotrione tolerant lines. The discovery of this QTL facilitates commercial development of new rice varieties with a new method for controlling weeds through the use of mesotrione herbicide. The finding of the linked marker BG-id2004662 is a novel finding and selection strategy for breeding and selecting the tolerance to mesotrione and other herbicides derived from line P1003.
In the two groups of F2 individuals sprayed with the higher rates of mesotrione (420 and 630 gm ai/ha) a second QTL was found with strong linkage to SNP marker WG-id1002788. This QTL is the demonstrated genetic position of the causal mutation for high tolerance to mesotrione. The combined tolerance of the QTL developed through mutation breeding on chromosome 1 and the QTL discovered in line P1003 provides a novel tolerance to mesotrione and combined with the linked molecular markers facilitates quick and efficient breeding of new rice varieties (FIG. 24).
Genetic mapping for the genomic location for resistance in line ML0831266-03093 is carried out by common QTL mapping strategies. The resistant line ML0831266-03093 may be crossed with a highly sensitive line and the resulting F1 seeds grown and plants selfed to produce a F2 mapping population segregating for the resistance trait. Finding trait linked markers is done by genotyping each F2 plant, spraying the plants with an appropriate concentration of the herbicide, and associating the molecular genotypes with the phenotype of each individual. This process will identify a genomic region between markers for the causal mutation for resistance.
Identifying the mutation causing the herbicide resistance is possible through a variety of processes. The mutant line and the original non-mutant line sequences are prepared and compared to identify mutations within the region of the QTL found through common trait mapping methods. Then markers are developed to the sequence differences, they are testing on a phenotyped segregating population such as the one used for QTL identification or make a new similar population. In another method, next generation sequencing is used to sequence and compare a bulk of individuals that are resistant to either a bulk of susceptible individuals or to the original non-mutant line. In this method the causal mutation is found through the resistant bulk having the highest portion of the mutation or sequence difference as compared to the susceptible bulk or non-mutant original line.
A recent publication for these methods includes Akira Abe, et al.; Nature Biotechnology 30, 174-178 (2012) doi:10.1038/nbt.2095, Published online 22 Jan. 2012.
EXAMPLES
Example 1
Production of Hybrid Rice Resistant to One HPPD Inhibiting Herbicide
The HPPD inhibiting herbicide resistance provided by ML0831266-03093 is deployed individually into hybrids through either the male or female parent resulting in the hybrid seed being resistant to the herbicide. If the resistance is deployed in only the male parent, then in addition to its use for weed control, the herbicide when applied to hybrid seed kills contaminating female selfed seed. On the other hand if the resistance is deployed only through the female parent, growers may eliminate contaminating male selfed seed.
Growers may alternate the type of resistance they purchase and apply in their fields to reduce the chance that weeds develop resistance to the herbicide. The HPPD inhibiting herbicide, though primarily for control of broad leaf weeds, also allows for some enhanced control of red rice. At higher rates it will kill certain types of rice. If resistance arose in red rice from cross pollination, it could still be controlled with a different herbicide class in the next season.
Example 2
Production of Hybrid Rice with High Level of Resistant to HPPD Inhibiting Herbicides
The HPPD inhibiting herbicide resistance provided by ML0831266-03093 is deployed into both the male and female parents of a hybrid. The resulting hybrid seed may carry resistance to mesotrione and other HPPD inhibiting herbicides. Resistance provided in this manner is stronger and offers better weed control through the possibility of being able to apply higher rates of herbicide.
Example 3
Production of Hybrid Rice Resistant to Multiple Herbicides
The herbicide resistance for at least 2 herbicides is deployed in a single hybrid through making both the female and male parent resistant to both herbicides. Deployment in this manner results in hybrid seed being homozygous for both resistances. By providing resistance in homozygous condition in the hybrid for both herbicide classes the hybrid seed shows maximum level of resistance. In addition by deploying both resistances together, the grower has the option to select either herbicide to apply in a given season, alternatively, both herbicides could be applied within the same season. The ability to rotate herbicides provides the opportunity to extend the life of the herbicides through delaying the development of weed resistance. This method also allows for the use of both herbicide classes for weed control during hybrid seed production.
Example 4
Production of Hybrid Rice Resistant to Mesotrione and at Least One Other Herbicide Class
1. Resistance to mesotrione and at least one other herbicide class is deployed in a single hybrid by using a male parent that carries resistance to the mesotrione (or the other herbicide) and a female that carries the other resistance. The method allows the grower to make a single purchase but to be able to choose which herbicide to apply. A single class of herbicide may be used in any one season and rotated between seasons, or alternatively both herbicides could be applied within a single season. In addition, deployment by this method, elements contaminating selfed seed of both parents in the hybrid seed through application of both herbicides, or one type or the other, are eliminated through application of only one herbicide.
2. In another method of deployment the mesotrione resistance and any other herbicide resistance is deployed through making a hybrid with a male parent that carries both resistances. The grower then has the option to choose which herbicide class to apply or to apply both within a single season. In addition, through the application of either herbicide contaminating selfed female seed would be eliminated. Alternatively both herbicide class resistances are provided in the female parent, giving the grower the same options for weed control.
3. Another embodiment is to deploy the mesotrione resistance to both parents, and another herbicide resistance into only one parent, such as the male parent. The hybrid seed are then homozygous for the mesotrione resistance but not the other. A scheme like this is used to make an early application with the herbicide put into only the male parent, providing weed control and elimination of contaminating female selfs. Later in the season mesotrione may be applied or another HPPD inhibitor herbicide. The useful life of both herbicides is extended through limiting or eliminating the development of weed resistance. In another application this method allows the use of mesotrione or other HPPD inhibitor herbicide to control weeds in seed production fields, allowing for cleaner seed.
4. Alternatively a different herbicide could be deployed in both of the hybrid parents and the mesotrione/HPPD inhibitor is deployed in only the male parent.
5. Other embodiments for deploying herbicide resistant lines include other traits such as resistance to other classes of herbicides, or other traits of importance.
Example 5
Seed Production
The herbicide resistance is also used for seed production. As an example, if it is deployed into the female parent, making it resistant, the herbicide is applied to the seed production field to kill the male plants before setting seed so that a seed production field is harvested as a bulk. In addition the purity of the seed may also be verified through deploying two herbicide resistances with only one in each parent. Selfed seed is detected and eliminated by applying herbicide put into the other parent.
Example 6
Control of Broadleaf Weeds and Limited Control of Grasses
The resistance when deployed in a hybrid, by any combination, provides resistance to mesotrione or other HPPD inhibiting herbicides. This deployment results in a new mode of action in rice to control broadleaf weeds with some limited control of grasses such as red rice. Further options or broad spectrum control of weeds is provided by deployment in the same hybrid another resistance to herbicides providing grass weed control, such as ACCase inhibiting herbicides. Through deployment with other modes of action development of weed resistance is more likely to be prevented through the use of multiple modes of action.
Example 7
Selection of Herbicide Resistant Rice Using a Herbicide Bioassay
Selection of material inheriting mesotrione tolerance is accomplished by a simple herbicide bioassay. A high rate of mesotrione (at least 420 gm ai/ha) is applied allowing differentiation of heterozygous individuals from homozygous individuals and the tolerance level of the mutation line from the inherent tolerance level in the background of some types of rice. In one example a rate of 105 gm ai/ha is applied followed three weeks later by a second application of 630 gm ai/ha. In another example a rate of 420 gm ai/ha is applied in a single application. Yet another example entails applying the herbicide at a rate of 630 gm ai/ha. Herbicide applications are done at the three to four leaf stage of seedling growth. The ideal situation is to have also planted near or within the plants to be selected a set of plants from the original mutant mesotrione tolerant donor line ML0831266-03093, a row of plants of the wild-type of the mutation line, P1003, and a row of the line involved as the other parent in the cross. These control lines allow easy differentiation for inheritance of the tolerance provided by the ML0831266-03093 mutant line through comparison of the response in the plants to be selected with the control lines. Only plants that live and are relatively healthy will have inherited and be homozygous for the tolerance level provided by the ML0831266-03093 mutant line (FIG. 18).
Example 8
Production of Rice Resistant to Both HPPD and ACCase Inhibiting Herbicides
The mutant line ML0831266-03093 that is tolerant to mesotrione and likely other herbicides including HPPD inhibitors, was crossed with mutant line ML0831265-01493 having tolerance to ACCase herbicides and more specifically “fop” type of ACCase herbicides. In one example the ML0831266-03093 plants are the female parent and pollination is by a plant from line ML0831265-01493. In another embodiment the parents are reversed so that ML0831266-03093 serves as the pollinating parent. The resulting F1 seed are harvested having inherited both mesotrione and ACCase herbicide tolerance. The F1 individual carries tolerance to both herbicides at a partially dominant level so they show some tolerance but not to the same level as the tolerant parent lines.
The F1 seeds are planted and the resulting plants are allowed to self-pollinate to produce F2 seed making a population segregating for tolerance to both mesotrione and ACCase herbicides. This population is screened by an herbicide bioassay to identify individuals that have inherited tolerance from the original mutant line ML0831266-03093 and are homozygous for the resistance. A high rate of mesotrione is applied allowing differentiation of heterozygous individuals from homozygous individuals and the tolerance level of the mutation line from the tolerance level in the background of some types of rice including the original line used for mutation to create the ML0831266-03093 line, which was P1003 (FIG. 3).
In one example a rate of 105 gm ai/ha is applied followed three weeks later by a second application of 630 gm ai/ha. In another example a rate of 420 gm ai/ha is applied in a single application. Yet another example entails applying the herbicide at a rate of 630 gm ai/ha. Herbicide applications are done at the three to four leaf stage of seedling growth. The ideal situation is to have also planted near or within the F2 population a set of plants from the original mutant mesotrione donor line ML0831266-03093, a row of plants of the wild-type of the mutation line, P1003, and a row of the line involved as the other parent ML0831265-01493. These control lines will allow easy differentiation for inheritance of the tolerance provided by the ML0831266-03093 mutant line through comparison of the response in the F2 plants to these control lines. Only plants that live and are relatively healthy will have inherited and be homozygous for the tolerance level provided by the ML0831266-03093 mutant line.
Example 9
A Co-Dominant Marker Assay to Select and Develop ACCase and HPPD Tolerant Rice Lines
A simple co-dominant marker assay is available to select for inheritance to ACCase herbicides derived from line ML0831265-01493. The marker is developed as a single nucleotide polymorphic marker and detects the causal mutation at position G2096S (blackgrass number) for ACCase tolerance in line ML0831265-01493. All of the surviving plants following the mesotrione bioassay as employed in Example 8 are sampled for tissue collection, the DNA is extracted by known methods and the samples are tested with the SNP assay. A subset of the surviving plants are then also identified as carrying homozygous tolerance to ACCase herbicides through marker assisted selection.
Individuals with tolerance to both mesotrione and ACCase herbicides are selfed to produce F3 families and further selected for other important agronomic characters. The F3 lines are selfed and purified to derive a new line or variety with dual resistance to mesotrione and ACCase herbicides. Such lines are highly valuable as the use of both herbicides provides more complete and broad-spectrum weed control.
In another embodiment the individuals with tolerance to both mesotrione and ACCase are used as trait donors in a backcross (BC) breeding program. After selecting one individual or a few individuals they are used either as the pollinating parent or the female parent. Another more elite and desirable line serves as the recurrent parent to which the traits are transferred.
Following the first cross the F1 plants are crossed again to the recurrent parent. The resulting backcross seed from this cross and ongoing crosses to the recurrent parent are tested with either markers or through herbicide bioassays for inheritance of the herbicide tolerance or a combination of markers and bioassays. In the best situation markers for the functional mutations are used. Alternatively an herbicide bioassay for mesotrione is applied to the BC seed or possibly the BC seed is progeny tested to verify inheritance of the tolerance. Furthermore an herbicide bioassay is used to identify individuals that also inherited tolerance to ACCase herbicides. This process is repeated until the recurrent parent genome is recovered along with the two new traits for tolerance to mesotrione and ACCase herbicides. After the last backcross individuals are selfed to recover the dual herbicide tolerances in a homozygous resistant level in at least one plant.
In yet another embodiment the individuals with resistance to both mesotrione and ACCase herbicides are crossed to a third line and subsequently selfed or even crossed with other lines. The resulting new lines and germplasm is tested and evaluated for other agronomic important traits. Finally new varieties or male and female lines are developed with tolerance to both mesotrione or other HPPD herbicides and ACCase herbicides a combination novel to rice.
Example 10
Mutant Rice ML0831266-03093
The mutant line ML0831266-03093 is demonstrated to carry a high tolerance level to mesotrione herbicide beyond the tolerance found naturally in some rice types including the original mutation treated line P1003. The mutant line is planted in rows or alternatively whole plots are planted and rows of the unmutated line (P1003) and other types of rice or whole plots are planted. Mesotrione is applied pre-emergence or alternatively it is applied post-emergence at the three to four leaf stage of the rice plants. Various rates of mesotrione are applied pre-emergence, pre-emergence followed by post-emergent, or post-emergent with a single or sequential application. Post-emergent applications are applied at the 3-4 leaf stage of the rice.
With low rates (105 gm ai/ha) of mesotrione applied both the mutant line as well as the original unmutated line survive. However, other types of rice, such as the mutant line with ACCase tolerance ML0831265-01493 and the associated unmutated line R0146 are killed at these rates of mesotrione. Applying mesotrione herbicide at higher rates clearly shows new and novel tolerance level as only the mutant line ML0831266-03093 survives while the original unmutated line P1003 and all other tested lines are killed or severally injured. The higher tolerance to mesotrione makes the line ML0831266-03093 of commercial value as both the tolerance can be controlled or bred into new varieties and it is of a high enough level to allow commercial weed control in rice with the application of mesotrione herbicide and possibly other HPPD inhibiting herbicides (FIG. 19A initial results, FIG. 19B new results after 1 year, and FIG. 22-trial results on various rice lines).
Example 11
Chromosomal Locations of Mutations Related to HPPD Inhibiting Resistance
The mesotrione tolerant line ML0831266-03093 is tolerant to rates of 420 gm ai/ha and even a dual application of mesotrione first at a rate of 105 gm ai/ha followed three weeks later by an application of 630 gm ai/ha. The line ML0831266-03093 with this high level of tolerance is crossed with a line highly sensitive to mesotrione one being line ML0831265-01493, which has tolerance to ACCase herbicides. The cross is made and the resulting F1 seeds are harvested, planted, and allowed to self to produce a F2 population. The F2 population is grown and tissue is collected from individual plants. Each F2 plant and parental lines are tested with a set of 192 SNP markers identified as being polymorphic between the two mutant lines ML0831266-03093 and ML0831265-01493. The set of polymorphic markers was identified as evenly spaced across the rice genome after testing both parental lines with a set of 796 SNP markers. All 192 markers including two found with linkage to target traits were selected from the 44 k SNP set described by Zhao et al. 2011.
Seedlings of size 3 to 4 leaves are sprayed with mesotrione herbicide. In one set of 89 plants mesotrione is first applied at 105 gm ai/ha killing 23 plants. Both the mutant line control ML0831266-03093 and the unmutated line P1003 survived the herbicide application while the unmutated line R0146 was killed. The surviving plants including some additional plants making a total of 95 are then sprayed with another treatment of mesotrione at a rate of 630 gm ai/ha killing or injuring 67 while 28 survived. (FIG. 23) The surviving ratio of plants to those killed or injured fits a one quarter ratio with a chi squared value of 1.03, well within the expected for a single recessive gene. Another set of F2 individuals of size 78 individuals are sprayed with a single application of mesotrione herbicide at a rate of 420 gm ai/ha resulting in 26 plants surviving also fitting in the expected one quarter ratio for a single recessive gene with a chi square value of 2.88 (Table 2).
Example 12
Crosses Between Mutant Lines Resistant to Different Herbicides
The cross between mutant line ML0831266-03093 (ATCC PTA-13620) carrying tolerance to mesotrione and possibly other HPPD inhibiting herbicides with mutant line ML0831265-01493 with (ATCC PTA-12933) tolerance to ACCase herbicides produced F1 seed inheriting both herbicide tolerances. Following selfing of the F1 plants F2, individuals are selected either through herbicide bioassays or alternatively with molecular markers. In the case of ACCase a functional molecular marker is described such that the mutation at position G2096S (based on the black grass numbering system) is selected. Furthermore using either markers linked to the QTLs on chromosome 1 and chromosome 2 or herbicide bioassays recovery of tolerance to mesotrione and ACCase including other HPPD herbicides or other herbicides.
Individuals selected for tolerance to mesotrione including other HPPD herbicides and possibly other herbicides may be used in a backcross conversion program or in breeding to develop new varieties and hybrids with a commercial level of tolerance to mesotrione and other herbicides. Selection with either bioassays or the chromosome 1 and chromosome 2 QTLs leads to the recovery of the inherent tolerance in P1003 along with the mutant tolerance for development of a novel variety or hybrid with herbicide tolerance and representing new weed control options in rice. The tolerance level of the mutant line is superior to other lines and allows for various commercial application methods.
The individual plants with tolerance to both herbicides are used in breeding to develop new varieties and hybrids with tolerance to both ACCase inhibiting herbicides, mesotrione, other HPPD inhibiting herbicides, and other herbicides. The new varieties and hybrids are commercial products. The commercial products are used commercially for rice production. In the production process both ACCase and mesotrione or other herbicides may be applied to the rice crop to control weeds. In one example mesotrione or other herbicides are applied preplant to control germinating weeds and provide residual weed control. Following germination of the rice crop ACCase herbicides are applied for controlling grass weeds. In another example both mesotrione or the equivalent is applied preplant and a second application is made post emergent along with an ACCase herbicide with one or two applications. In another example both mesotrione and an ACCase herbicide or other herbicides including other HPPD herbicides are applied post emergent with one or two applications. In this manner a new and novel strategy is implemented to provide full spectrum weed control in rice. In addition these herbicides have new not previously used in rice modes of action. This strategy therefore has commercial application not only for weed control but as a method to extend the useful life of this strategy and others through the application of multiple modes of action for weed control.
Example 13
Identification of the Causal Mutation for Tolerance to Mesotrione and Other HPPD Herbicides
The mutant line ML0831266-03093 is crossed back to the original line, P1003, used for induction of mutations. The F1 seed is grown and the plants selfed to produce an F2 population segregating for tolerance to mesotrione. Each F2 plant is labeled and a leaf sample is collected for DNA extraction. The F2 plants are then sprayed with mesotrione herbicide at the 3-4 leaf stage at a rate of 630 gmai/ha. Among the surviving set of the least injured twenty are identified and used for DNA extraction to represent individuals that inherited the mesotrione tolerance and presumably the causal mutation for tolerance. Out of the plants killed by the herbicide application a set of twenty is also identified for DNA extraction to represent individuals that have the wild-type allele at the causal tolerance locus.
The leaf tissue from the identified individuals is used for DNA extraction and DNA is combined from each set to make a bulk of individuals carrying the tolerance and a bulk lacking the tolerance. In addition DNA is also extracted from leaf tissue derived from the original line, P1003, used for mutation treatment. These samples are used in next generations sequencing at 30× coverage and compared to the rice reference sequence, NIPPONBARE, to the original line used for mutation treatment, P1003, and to each other. Using these comparisons it is possible to identify among the group with tolerance a single mutation being present across all individuals and thus highly likely to be the causal mutation for tolerance to mesotrione.
The genome regions suspected to carry the causal mutation are sequenced by Sanger sequencing technology in both the mutant line ML0831266-03093 and the original non-mutant line P1003. Following the identification of real SNP markers or some other suitable marker is developed and the whole F2 phenotyped segregating population is tested to identify linkage of the suspected causal mutation to the phenotype.
Other characterizations and processes are also applicable to verify the function of the causal mutation. For example the gene containing the mutation is identified through comparison to the published full rice sequence and related databases. Furthermore the gene product or enzyme can be isolated and characterized to describe its normal function and function with the new mutation especially in relation to its function in the presence of mesotrione.
Example 14
Double Mutant Resistant to HPPD and ACCase Herbicides
A simple co-dominant marker assay is available to select for inheritance to ACCase herbicides derived from line ML0831265-01493. The marker is developed as a single nucleotide polymorphic marker and detects the causal mutation at position G2096S (blackgrass number) for ACCase tolerance in line ML0831265-01493. All of the surviving plants following the mesotrione bioassay as employed in example 11 are sampled for tissue collection, the DNA is extracted by known methods and the samples are tested with the SNP assay. A subset of the surviving plants are then also identified as carrying homozygous tolerance to ACCase herbicides through marker assisted selection.
Individuals with tolerance to both mesotrione and ACCase herbicides were selfed to produce F3 families and further selected for other important agronomic characters. The F3 lines were selfed and purified to derive a new line or variety with dual resistance to mesotrione and ACCase herbicides. Such lines are highly valuable as the use of both herbicides provides more complete and broad-spectrum weed control. For example line PL1214418M2-73009 (ATCC deposit PTA-121398) and PL1214418M2-80048 (ATCC deposit PTA-121362) contains tolerance to both HPPD inhibiting herbicide mesotrione and the ACCase inhibiting herbicide fluazifop (see Table 8). Other related lines have also been developed and are highly useful for use as a new weed control system in rice employing both ACCase and HPPD types of herbicide.
In another embodiment the individuals with tolerance to both mesotrione and ACCase are used as trait donors in a backcross breeding program. After selecting one individual or a few individuals they will be used either as the pollinating parent or the female parent. Another more elite and desirable line serves as the recurrent parent to which the traits are transferred. Following the first cross the F1 plants are crossed again to the recurrent parent. The resulting backcross seed from this cross and ongoing crosses to the recurrent parent are tested with either markers or through herbicide bioassays for inheritance of the herbicide tolerance or a combination of markers and bioassays. In the best situation markers for the functional mutations are used. Alternatively an herbicide bioassay for mesotrione is applied to the BC seed or possibly the BC seed is progeny tested to verify inheritance of the tolerance. Furthermore an herbicide bioassay is used to identify individuals that also inherited tolerance to ACCase herbicides. This process is repeated until the recurrent parent genome is recovered along with the two new traits for tolerance to mesotrione and ACCase herbicides. After the last backcross individuals are selfed to recover the dual herbicide tolerances in a homozygous resistant level in at least one plant.
In yet another embodiment the individuals with resistance to both mesotrione and ACCase herbicides are crossed to a third line and subsequently selfed or even crossed with other lines. The resulting new lines and germplasm is tested and evaluated for other agronomic important traits. Finally new varieties or male and female lines are developed with tolerance to both mesotrione or other HPPD herbicides and ACCase herbicides a combination novel to rice.
Example 15
Full Spectrum Weed Control in Rice Based on Dual Resistance to Both ACCase and HPPD Herbicides
In the production process both ACCase and mesotrione or other herbicides may be applied to the rice crop to control weeds. In one example mesotrione or other herbicides are applied preplant to control germinating weeds and provide residual weed control. Following germination of the rice crop ACCase herbicides are applied for controlling grass weeds. In another example both mesotrione is applied preplant and a second application is made post emergent along with an ACCase herbicide with one or two applications. In another example both mesotrione and an ACCase herbicide or other herbicides including other HPPD herbicides are applied post emergent with one or two application. In this manner a new and novel strategy is implemented to provide full spectrum weed control in rice. In addition these herbicides have not previously been used in rice modes of action. This strategy therefore has commercial application not only for weed control but as a method to extend the useful life of this strategy and others through the application of multiple modes of action for weed control.
Seed Deposits Under Budapest Treaty
Seed deposits by Ricetec AKTIENGESELLSCHAFT were made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110, United States of America. The dates of deposit and the ATCC Accession Numbers are: ML0831266-03093 (PTA-13620, Mar. 19, 2013); ML0831265-01493 (PTA-12933, May 31, 2012); ML0831265-02283 (PTA-13619, Mar. 19, 2013); PL1214418M2-73009 (PTA-121398, Jul. 18, 2014); PL1214418M2-80048 (PTA-121632, Jun. 30, 2014) (see also Table 8). All restrictions will be removed upon granting of a patent, and the deposits are intended to meet all of the requirements of 37 C.F.R. §§1.801-1.809, and satisfy the Budapest Treaty requirements. The deposit will be maintained in the depository for a period of thirty years, or five years after the last request, or for the enforceable life of the patent, whichever is longer, and will be replaced as necessary during that period.
DEFINITIONS
In the description and tables which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:
Acetyl-Coenzyme. A carboxylase (ACCase; EC 6.4.1.2) enzymes synthesize malonyl-CoA as the start of the de novo fatty acid synthesis pathway in plant chloroplasts. ACCase in grass chloroplasts is a multifunctional, nuclear-genome-encoded, very large, single polypeptide, transported into the plastid via an N-terminal transit peptide. The active form in grass chloroplasts is a homodimeric protein.
ACCase enzymes in grasses are inhibited by three classes of herbicidal active ingredients. The two most prevalent classes are aryloxyphenoxypropanoates (“FOPs”) and cyclohexanediones (“DIMs”). In addition to these two classes, a third class phenylpyrazolines (“DENs”) has been described.
Certain mutations in the carboxyl transferase region of the ACCase enzyme results in grasses becoming resistant to ACCase herbicides. In the weed Black-Grass at least five mutations have been described which provide resistance to FOP or DIM class of ACCase herbicides. Some mutations rendering ACCase enzymes resistant to these herbicides may be associated with decreased fitness.
Allele. Allele is any one of many alternative forms of a gene, all of which generally relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
Backcrossing. Process of crossing a hybrid progeny to one of the parents, for example, a first generation hybrid F1 with one of the parental genotypes of the F1 hybrid.
Blend. Physically mixing rice seeds of a rice hybrid with seeds of one, two, three, four or more of another rice hybrid, rice variety or rice inbred to produce a crop containing the characteristics of all of the rice seeds and plants in this blend.
Cell. Cell as used herein includes a plant cell, whether isolated, in tissue culture or incorporated in a plant or plant part.
Cultivar. Variety or strain persisting under cultivation.
Embryo. The embryo is the small plant contained within a mature seed.
Essentially all the physiological and morphological characteristics. A plant having essentially all the physiological and morphological characteristics of the hybrid or cultivar, except for the characteristics derived from the converted gene.
Grain Yield. Weight of grain harvested from a given area. Grain yield could also be determined indirectly by multiplying the number of panicles per area, by the number of grains per panicle, and by grain weight.
Injury to Plant. Is defined by comparing a test plant to controls and finding the test plant is not same height; an abnormal color, e.g. yellow not green; a usual leaf shape, curled, fewer tillers.
Locus. A locus is a position on a chromosome occupied by a DNA sequence; it confers one or more traits such as, for example, male sterility, herbicide tolerance, insect resistance, disease resistance, waxy starch, modified fatty acid metabolism, modified phytic acid metabolism, modified carbohydrate metabolism and modified protein metabolism. The trait may be, for example, conferred by a naturally occurring gene introduced into the genome of the variety by backcrossing, a natural or induced mutation, or a transgene introduced through genetic transformation techniques. A locus may comprise one or more alleles integrated at a single chromosomal location.
Induced. As used herein, the term induced means genetic resistance appeared after treatment with mutagen.
Non-induced. As used herein, the term non-induced means genetic resistance not known to be induced; is at different location in the genome, than induced resistance.
Plant. As used herein, the term “plant” includes reference to an immature or mature whole plant, including a plant from which seed or grain or anthers have been removed. Seed or embryo that will produce the plant is also considered to be the plant.
Plant Part. As used herein, the term “plant part” (or a rice plant, or a part thereof) includes protoplasts, leaves, stems, roots, root tips, anthers, seed, grain, embryo, pollen, ovules, cotyledon, hypocotyl, glumes, panicles, flower, shoot, tissue, cells, meristematic cells and the like.
Quantitative Trait Loci (QTL). Genetic loci that controls to some degree numerically measurable traits that are usually continuously distributed.
Regeneration. Regeneration refers to the development of a plant from tissue culture.
Resistance/Resistant1. The inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type resistance may be naturally occurring or induced by such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis. 1 Weed Science Society of America, Weed Technology, vol. 12, issue 4 (October-December, 1998, p. 789)
Single Gene Converted (Conversion). Single gene converted (conversion) includes plants developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of an inbred are recovered, while retaining a single gene transferred into the inbred via crossing and backcrossing. The term can also refer to the introduction of a single gene through genetic engineering techniques known in the art.
Tolerance/Tolerant. The inherent ability of a species to survive and reproduce after herbicide treatment implies that there was no selection or generic manipulation to make the plant tolerant.
Resistance/tolerance are used somewhat interchangeably herein; for a specific rice plant genotype information is provided on the herbicide applied, the strength of the herbicide, and the response of the plant.










Sequence listing of HPPD gene and protein















LOC_Os02g07160 sequence information


Genomic sequence length: 2760 nucleotides


CDS length: 1341 nucleotides


Protein length: 446 amino acids


Putative Function: glyoxalase family protein, putative, expressed


 


Genomic Sequence


>LOC_Os02g07160 (SEQ ID NO: 1)


ACGCCGCCACTGTCATCCACTCCCCCACACCCCACGACGCGCCACGCCACGCCGCGCCGC


GCCGCGCCATGCCTCCCACTCCCACCCCCACCGCCACCACCGGCGCCGTCTCGGCCGCTG


CGGCGGCGGGGGAGAACGCGGGGTTCCGCCTCGTCGGGCACCGCCGCTTCGTCCGCGCCA


ACCCGCGGAGCGACCGGTTCCAGGCGCTCGCGTTCCACCACGTCGAGCTCTGGTGCGCCG


ACGCCGCGTCCGCCGCGGGCCGGTTCGCCTTCGCCCTGGGCGCGCCGCTCGCCGCCAGGT


CCGACCTCTCCACGGGGAACTCCGCGCACGCCTCCCTCCTCCTCCGCTCCGCCTCCGTCG


CGTTCCTCTTCACCGCCCCCTACGGCGGCGACCACGGCGTCGGCGCGGACGCGGCCACCA


CCGCCTCCATCCCTTCCTTCTCCCCAGGCGCCGCGCGGAGGTTCGCCGCGGACCACGGCC


TCGCGGTGCACGCCGTGGCGCTGCGCGTCGCCGACGCGGCCGACGCCTTCCGCGCCAGCG


TCGCGGCCGGTGCGCGCCCGGCGTTCCAGCCCGCCGACCTCGGCGGTGGCTTCGGCCTCG


CGGAGGTGGAGCTCTACGGCGACGTCGTGCTCCGCTTCGTCAGCCACCCGGACGGCGCCG


ACGCGCCCTTCCTCCCGGGTTTCGAGGGCGTCAGCAACCCGGGCGCCGTGGACTACGGCC


TCCGCCGGTTCGACCACGTCGTCGGCAACGTGCCGGAGCTCGCTCCGGTAGCCGCGTACA


TCTCCGGGTTCACCGGGTTCCACGAGTTCGCCGAGTTCACCGCCGAGGACGTGGGCACCG


CCGAGAGCGGCCTCAACTCGGTGGTGCTCGCCAACAACGCGGAGACCGTGCTGCTGCCGC


TCAACGAGCCGGTGCACGGCACCAAGCGGCGGAGCCAGATACAGACGTACCTGGACCACC


ACGGCGGCCCGGGGGTGCAGCACATCGCGCTGGCCAGCGACGACGTGCTCGGGACGCTGA


GGGAGATGCGGGCGCGCTCCGCCATGGGCGGCTTCGAGTTCTTGGCGCCGCCGCCGCCCA


ACTACTACGACGGCGTGCGGCGGCGCGCCGGGGACGTGCTCTCGGAGGAGCAGATCAACG


AGTGCCAGGAGCTCGGGGTGCTCGTGGACAGGGATGACCAGGGGGTGTTGCTCCAGATCT


TCACCAAGCCAGTAGGAGACAGGTAAAATCCTCACCTCTTTCATGATGAAAATGGCTTAT


GAATTCAGATTTGCAGTTATTTGTTGGCACATAGCATCGATTAGGCGCAGAAAGGTGTCA


AGCATTATGAAATTAATCCAGAATGCTTGAATAATACAGTATAATATATGATAGTGAGCT


CTGTGATACTCCATGGATACTCTTTATGTGTCTCCATGAATCCATGATGCGCCTTTCTGA


AGATTGTGACACTAGAAAGGGAATAAAGCTGAATGTGCATAGGAAAAAAATGAAAAGCCA


ATGTGTGTCTGTTTATGCCTTCTTGCAAGCATATCCCAGTTCCTTTTTGCCGGCATGTTG


TAATGCAGATAGCCAGCCACATATAGCTACTTAATTAGTGAGTACTCCCTCTCACAATGT


AAGTCATTCTAGTATTTTCCACATTCATATTGATGCTAATCTATCTAGATTCATTAGCAT


CAATATGAATATGGGAAATACTAGAATGACTTACATTGTGAAACGGAGGAAGTATTACTT


ACTACATCTAAGGTCCATGGATTCCTTTTTTTACAAAAGAAAGAAAGAATCTTATGGCAA


CTCCATCAGCATAAACCAGCAATGCTGCTGGGAACAACTTAAACTTTAGGTTCAGGAGGT


TGTAATTGTCTTTAAGCTTAATAGTCTGATTCAGTCAGTATTCTAATTTCTGCTGCATCT


TTGCTATTGTTATTTCCTCTCTGTGACTCCAAATCTAACTGGATCAGCTATTTCACTCAG


GCCAACCTTTTTCTTGGAGATGATACAAAGGATTGGGTGCATGGAGAAGGATGAGAGTGG


GCAGGAGTACCAGAAGGGCGGCTGCGGCGGGTTTGGGAAGGGCAACTTCTCGGAGCTGTT


CAAGTCCATTGAGGAGTATGAGAAATCCCTTGAAGCCAAGCAAGCCCCTACAGTTCAAGG


ATCCTAGGTAGGAACTGGAGGCCTGGAGCAACAGATGTAACCAGTGTATTTGTATTATGG


AGCAGAAGAAAAAAGATGTGCTTTCACTGCTTTGTGATATGTGTCATGCAAGTTGATGTT


GTAATTTGTGGAAGCTGAAGACAAATGATGGTACAATCACTGTAATAGATAATAGACATG


GATCACATACAAGAATGTAACCTAGTGTTGGCATTGCTGCTGTACAATCTTGCTTGGAAA


TAAAATAATAATCAACCTGGAGAAAGAATGTAACCTACTGTTGGCATTGCTGATGTACAA


TCTTGCTTGGAAATAAAATAAGAATCAACCAAGAGAATCTGTCCTTGTGATGCTTGTGAT


CTTCTGGTGTCTTTTTATTTAACAGAATGTAGTGGTCCTCTGCTGCCTCCAACCGTCCAG


GGTAAAAGTGTAAACCGTGGGCTGAGTTACAGCGAATTGCAGTTAGCAATCTGCAAGAGA


CAGGGGATGAACAGAGTAAGGTCAATAGTTCAGTGTATGACATGATCATCTTGTTTCGTG


GCCTTAAATGGCAAGAAAATGGGCTTGTCAGATCTCAAAGAACTCCTATATGTTAAAAGG


 


CDS (SEQ ID NO: 2)


>LOC_Os02g07160.1 (SEQ ID NO: 2)


ATGCCTCCCACTCCCACCCCCACCGCCACCACCGGCGCCGTCTCGGCCGCTGCGGCGGCG


GGGGAGAACGCGGGGTTCCGCCTCGTCGGGCACCGCCGCTTCGTCCGCGCCAACCCGCGG


AGCGACCGGTTCCAGGCGCTCGCGTTCCACCACGTCGAGCTCTGGTGCGCCGACGCCGCG


TCCGCCGCGGGCCGGTTCGCCTTCGCCCTGGGCGCGCCGCTCGCCGCCAGGTCCGACCTC


TCCACGGGGAACTCCGCGCACGCCTCCCTCCTCCTCCGCTCCGCCTCCGTCGCGTTCCTC


TTCACCGCCCCCTACGGCGGCGACCACGGCGTCGGCGCGGACGCGGCCACCACCGCCTCC


ATCCCTTCCTTCTCCCCAGGCGCCGCGCGGAGGTTCGCCGCGGACCACGGCCTCGCGGTG


CACGCCGTGGCGCTGCGCGTCGCCGACGCGGCCGACGCCTTCCGCGCCAGCGTCGCGGCC


GGTGCGCGCCCGGCGTTCCAGCCCGCCGACCTCGGCGGTGGCTTCGGCCTCGCGGAGGTG


GAGCTCTACGGCGACGTCGTGCTCCGCTTCGTCAGCCACCCGGACGGCGCCGACGCGCCC


TTCCTCCCGGGTTTCGAGGGCGTCAGCAACCCGGGCGCCGTGGACTACGGCCTCCGCCGG


TTCGACCACGTCGTCGGCAACGTGCCGGAGCTCGCTCCGGTAGCCGCGTACATCTCCGGG


TTCACCGGGTTCCACGAGTTCGCCGAGTTCACCGCCGAGGACGTGGGCACCGCCGAGAGC


GGCCTCAACTCGGTGGTGCTCGCCAACAACGCGGAGACCGTGCTGCTGCCGCTCAACGAG


CCGGTGCACGGCACCAAGCGGCGGAGCCAGATACAGACGTACCTGGACCACCACGGCGGC


CCGGGGGTGCAGCACATCGCGCTGGCCAGCGACGACGTGCTCGGGACGCTGAGGGAGATG


CGGGCGCGCTCCGCCATGGGCGGCTTCGAGTTCTTGGCGCCGCCGCCGCCCAACTACTAC


GACGGCGTGCGGCGGCGCGCCGGGGACGTGCTCTCGGAGGAGCAGATCAACGAGTGCCAG


GAGCTCGGGGTGCTCGTGGACAGGGATGACCAGGGGGTGTTGCTCCAGATCTTCACCAAG


CCAGTAGGAGACAGGCCAACCTTTTTCTTGGAGATGATACAAAGGATTGGGTGCATGGAG


AAGGATGAGAGTGGGCAGGAGTACCAGAAGGGCGGCTGCGGCGGGTTTGGGAAGGGCAAC


TTCTCGGAGCTGTTCAAGTCCATTGAGGAGTATGAGAAATCCCTTGAAGCCAAGCAAGCC


CCTACAGTTCAAGGATCCTAG


 


Protein (SEQ ID NO: 3)


>LOC_Os02g07160.1


MPPTPTPTATTGAVSAAAAAGENAGFRLVGHRRFVRANPRSDRFQALAFHHVELWCADAA


SAAGRFAFALGAPLAARSDLSTGNSAHASLLLRSASVAFLFTAPYGGDHGVGADAATTAS


IPSFSPGAARRFAADHGLAVHAVALRVADAADAFRASVAAGARPAFQPADLGGGFGLAEV


ELYGDVVLRFVSHPDGADAPFLPGFEGVSNPGAVDYGLRRFDHVVGNVPELAPVAAYISG


FTGFHEFAEFTAEDVGTAESGLNSVVLANNAETVLLPLNEPVHGTKRRSQIQTYLDHHGG


PGVQHIALASDDVLGTLREMRARSAMGGFEFLAPPPPNYYDGVRRRAGDVLSEEQINECQ


ELGVLVDRDDQGVLLQIFTKPVGDRPTFFLEMIQRIGCMEKDESGQEYQKGGCGGFGKGN


FSELFKSIEEYEKSLEAKQAPTVQGS*
















TABLE 1







Phenotypic characteristics of a mutant rice line showing resistance


to mesotrione, with comparison to the original unmutated parent line.
















Days to










50%
Plant
Plant

Sheath





Designation
Heading
Height
Type
Pubesence
Color
Awns
TKW, g
yield/plant


















Unmutated
85
96.5
erect
glaborous
purple
None
23.15
N/A


parent line




inside










sheath





ML0831266-
82
94
erect
glaborous
purple
None
24.55
4.5


03093F2




inside










sheath
















TABLE 2







Numbers of tolerant plants after three different herbicide


bioassays applied to an F2 individuals derived from a cross of ML0831266-03093


with tolerance to mesotrione and ML0831265-01493 with tolerance to ACCase


herbicides.













One





application of





mesotrione at





105 gai/ha



Single
Single
followed by



application of
application of
second



mesotrione at
mesotrione at
application of



105 gai/ha
420 gai/ha
630 gai/ha













Parameters
Tolerant
Susceptible
Tolerant
Susceptible
Tolerant
Susceptible
















Observed (o)
64
23
26
52
28
67


Expected (e)
21.75
65.25
19.5
58.5
23.75
71.25


Deviation (o − e)
42.25
−42.25
6.5
−6.5
4.25
−4.25


Deviation (o − e)2
1785.06
1785.06
42.25
42.25
18.06
18.06


(o − e)2/e
82
27.35
2.166
0.722
0.76
0.253










Chi-Square Value
109.35
2.88
1.013


Degrees of
1
1
1


freedom (df)





Probability value

0.08919242
0.31393809


Critical Chi-
3.84
3.84
3.84


Square value at





p = 0.05
















TABLE 3







Markers used in QTL analysis











SEQ ID



MARKER
NO:














WG-id11001864
8



WG-id11002275
9



WG-id11003701
10



WG-id11007323
11



WG-wd10001341
12



WG-wd11001701
13



WG-wd2002275
14



WG-wd7000143
15



BG-id10001133
16



BG-id10003147
17



BG-id10004614
18



BG-id1001716
19



BG-id1012406
20



BG-id1015060
21



BG-id1020809
22



BG-id1026723
23



BG-id11000280
24



BG-id11000643
25



BG-id11001000
26



BG-id11003263
27



BG-id11005541
28



BG-id11011578
29



BG-id12001413
30



BG-id12003453
31



BG-id12006669
32



BG-id12010130
33



BG-id2000100
34



BG-id2001406
35



BG-id2002159
36



BG-id2004662
37



BG-id2006793
38



BG-id2008132
39



BG-id2009032
40



BG-id2010498
41



BG-id2012278
42



BG-id2013398
43



BG-id3002278
44



BG-id3006415
45



BG-id3007343
46



BG-id3008063
47



BG-id3008702
48



BG-id3011050
49



BG-id3011406
50



BG-id3015075
51



BG-id4001244
52



BG-id4002084
53



BG-id4004010
54



BG-id4010543
55



BG-id4012206
56



BG-id5003430
57



BG-id5004121
58



BG-id5011704
59



BG-id5014703
60



BG-id6007975
61



BG-id6011524
62



BG-id6016683
63



BG-id6016941
64



BG-id7006069
65



BG-id8000032
66



BG-id8004971
67



BG-id8006271
68



BG-id9003596
69



BG-ud11001609
70



BG-ud7000168
71



BG-ud7000468
72



BG-ud7001467
73



BG-ud9000404
74



BG-ud9000939
75



BG-wd12000096
76



BG-wd5002107
77



BG-wd7000537
78



BG-wd8000300
79



WG-id10000057
80



WG-id1000027
81



WG-id10000678
82



WG-id10005716
83



WG-id10006397
84



WG-id10006890
85



WG-id10007362
86



WG-id1000987
87



WG-id1002788
88



WG-id1003490
89



WG-id1004858
90



WG-id1005915
91



WG-id1006413
92



WG-id1007758
93



WG-id1008433
94



WG-id1011077
95



WG-id1013249
96



WG-id1015747
97



WG-id1019114
98



WG-id1022207
99



WG-id1023338
100



WG-id12004473
101



WG-id12005677
102



WG-id12007189
103



WG-id12008113
104



WG-id12009381
105



WG-id2000711
106



WG-id2003035
107



WG-id2003988
108



WG-id2005453
109



WG-id2005879
110



WG-id2007502
111



WG-id2011561
112



WG-id2011986
113



WG-id2014452
114



WG-id2015344
115



WG-id2016104
116



WG-id3000020
117



WG-id3003557
118



WG-id3003855
119



WG-id3004338
120



WG-id3005216
121



WG-id3005783
122



WG-id3009997
123



WG-id3010769
124



WG-id3013945
125



WG-id3016222
126



WG-id3017628
127



WG-id3018382
128



WG-id4000023
129



WG-id4001471
130



WG-id4002895
131



WG-id4004798
132



WG-id4005527
133



WG-id4005882
134



WG-id4006725
135



WG-id4007645
136



WG-id4008100
137



WG-id4008430
138



WG-id4008947
139



WG-id4009312
140



WG-id4009705
141



WG-id4011039
142



WG-id4011619
143



WG-id4011820
144



WG-id5001055
145



WG-id5002055
146



WG-id5002453
147



WG-id5002782
148



WG-id5004697
149



WG-id5006824
150



WG-id5007247
151



WG-id5007583
152



WG-id5008807
153



WG-id5009334
154



WG-id5010535
155



WG-id6000075
156



WG-id6001960
157



WG-id6002888
158



WG-id6003335
159



WG-id6004012
160



WG-id6004657
161



WG-id6005348
162



WG-id6007016
163



WG-id6010853
164



WG-id6012703
165



WG-id6014165
166



WG-id6016119
167



WG-id7000480
168



WG-id7001929
169



WG-id7002851
170



WG-id7003936
171



WG-id7004491
172



WG-id8000555
173



WG-id8001575
174



WG-id8002235
175



WG-id8005634
176



WG-id8006703
177



WG-id8007014
178



WG-id8007344
179



WG-id8007751
180



WG-id9000056
181



WG-id9002563
182



WG-id9002755
183



WG-id9005502
184



WG-id9006187
185



WG-id9006850
186



WG-id9007344
187



WG-ud1001267
188



WG-ud7000348
189



WG-ud7001018
190



WG-ud7002024
191



WG-id1028225
192



WG-id11000006
193



WG-id11007850
194



WG-id11008114
195



WG-id11009132
196



WG-id12002544
197



WG-id11006215
198



WG-id11002912
199

















TABLE 4 







SNP markers













Chromo-



SEQ ID


ID
some
upstream sequence
Allele
downstream sequence
NO:





WG-
1
ATGCCCACGGCGGCGGCGGCGGAGGA
C/T
GTCGGAAATGCCTGCCACGGGCTGTTCC
4


id1002788

GGAGGAGGAGGAGGAGCTAAGGAGCG

CGCAGGTATTGAGAAATGAGCGCTGAG





GCGCGGTACGTCGCCGGTGCTGTTCTGC

TTCCTGACGCGTTTAAATCCACTGATTA





TTTGTAGCCGCTGCTGTCCT

GCTGAGTTCCCTTCCAA



 


WG-
1
GAGAGTGGAGGAGGAGGACGAGTGGA
A/G
CAAGCAAAGGAAGCAAGCAAAAGAAA
5


id1003490

GGTGGAGGTGGCGCGCGGCTGCGCGG

AAAAGCCCGGGAATTTACCTGGCGGGA





TGCGCTTCTTTTTTTTTTCTTTTTTTGTTC

ATGCCCTACTTGGCAGCGCCGCCCGTCT





CCGCCGCAACCAAAGGAG

CTCTCCACAAACGCCCTGC



 


BG-
2
TTCTATCTCAAGGCGGCAATAGAATCAT
C/T
GCCAAATGTCTGATGAATTGCTCTTGCT
6


id2004662

AGATGCTAGAGTCCAGAAGAAGGCCAA

CTGATGTTGAGCCCGATGAAGTTGTTAG





AGACTTGAAATTTTCAGTTGAGAATGAG

CTGCTGAGGACATGATCGGTACCACCTA





CAATCCAAGGTGATGCT

TATTGACAACCCTGAT



 


WG-
2
TCAGTGTTCACGGACCCTACATGGAGTT
A/G
TATCAATCGATCAATCACCAATCGGATG
7


id2003988

CTCCTAAGTTCAACTACAAGAGACATAG

GTACCAAATCCAAAACAACAGTTGGGG





CCCATAGGGTAATGCCCTCACTTTCCAG

AAAACTGATCCTACCAACCCAGCTCAAC





CTCTTTAACTATGGAG

TAATTTTGCAGTGCTAC
















TABLE 5 







Unknown ACCase mutation-QTL start and end SNP marker sequences

















SEQ



Chromo-



ID


ID
some
Upstream sequence
Allele
Downstream sequence
NO:





id1019752
1
Ataaagatgaggtgtttgatgaattaaaggccgca
g/t
acagcaactgatgcagctgctgcgaaagcccata
206




gggttgaagaggccatgtagctttacagatatttc

ttaggcgccagcttcatccagatgtctgttccca





cagtgaaaatgctttgcttcttgaatttga

ggacaagaatacttctggtcatgaactttttg



 


id1025754
1
tcacatgatctgcaactgtcaacagtcttaccgga
t/c
gcatgatgtgtctatactctatactgcaaagatg
207




attggattctgaaggtggatactccacctgtccac

aatactaacaagtttttcttgggcttaaaagaag





cataagtccttatttgtcagaggttacaat

aaaaactaggaacagcctcactagtttgctag
















TABLE 6 







List of mutations identified in Quizalofop mutant. These mutations were identified using


MutMap method (whole genome sequencing)

















SEQ ID


ID
Chromosome
Upstream sequence
Allele
Downstream sequence
NO:





Chr1QMM32785958
1
tgtttcaagtttggttgctgaaaaacgtacggacaaa
g/a
cttcggtggtttgccaagaacatgaaacctggggataa
208




ccatgaactaacttgctattttgctccacatgggttt

ggatttttttcgactttgctaatgctggtcgtctggat





gtctcgaagcaaccaggaattgatca

taggaacagaacaggtattgctac



 


Chr1QMM33147227
1
agtctaaatgggctgcactttgattgggctgggttca
a/g
gggtaaacggtgtgcggacgtgagacgagaaaagcatg
209




tatgagattaggggaaaaaacacgaacattccagtaa

agagaaaacgatctgtgtgcatgcatagggctggacga





aatggggagtgtgaactgtgaagaag

aaagctcgtgactcgttagctcgc



 


Chr1QMM33201871
1
tagggcttctgatagccctccatctgtccgtcctttg
t/c
tcttcaactcgattggaacagcaggctccgtgtatgtg
210




cccgtttgcttcttggcctaaaccaccgaaaaggtgg

taactatggctgtgtttagatctaaagtttagattcaa





gtccgttttgctggacgcctggaata

agtatagatttaaacttcagtcat



 


Chr1QMM34374172
1
tgctgattctcaggctgattctacttggttggtagaa
a/g
gagaaaactcgctacactttataggaaatgaactactc
211




aatctacttatccaggaacaagcgtagggtaactttt

ttctaccgacggatatcctaaaggctatcctctggtac





cctttttttctcagctatgtgaaaag

ctcggccttacgctaactcaaaac



 


Chr1QMM34482064
1
cgatcccagggaggttgtggaagtgcttctcatgacc
a/g
acctgaaggctggtgccatcatgttggccagtaatctg
212




ttcgaggcactgcggcgtaggcatcttcaattggatg

agggctaacattgggaagaggattggagctgtccctgg





agacacaagagactagcaaacgtgca

agttgaagtaggggatattttcta



 


Chr1QMM34680949
1
aattccaatttcatcccatttgtcccattccctcctg
a/g
ccctccatcagctgaaaaattaccagaaacaaaatatt
213




attactttgccaagaaaaataagcctgtggagaattc

catctggaaatctgggaaattttcagaacagctcacag





atcagatgcaggaatagtgccagaag

gtgatgggcagtcaggcagcaaac



 


Chr1QMM34976760
1
gttttggttgctattaatcgattgagcaagtagggga
t/c
cgattacaccgttgtgttcgtaataattaaatctttac
214




aatattcctatcatctatgcttcaaataaagttttct

aacaagatctcacatgattatattttgatgaaaaatca





cttaaattactcatccgatttacaat

caaattacttttatgatatgtcta



 


Chr1QMM35498447
1
gtcccgcctggtgacgatttccatgggcattgcgccg
a/g
agtacacgcgaagactgtaggtagaggtgcttttcccg
215




actgactgtgtcggcagcatgcatcgtctcgggcgtt

cgaaaagtggcagtagcggcggttggacagtaaccttc





caacgtgtggaggggacgacgtataa

gtgtatggttgtgtgttcatctca



 


Chr1QMM35779866
1
tgcttgtgcgttcactgttcagagaagctggttatcc
a/g
tcacccagcacaattgactggctgagtgttgcattaag
216




tccctgataagaacagccgggaggtcagtgtgctatg

caaatctggaccggatttgagggaattttctcgcgcag





gttttgtttagttctggaatgatcca

tggagatctatgataaatctcgta



 


Chr1QMM36160202
1
ctgcttcgccaacggcctcgaggcgaggctggcaggt
a/g
cgtaccagctttatttggcagcttgcccatttaagaag
217




actggtagtcaaatttacaagaactacacgataactc

atctctcattactttgccaatcaaacaattttgaatgc





ggcttccatgcactgatgtgctgaaa

tgtggagaaggccaagaaagttca



 


Chr1QMM36386713
1
tcacattctggttgttgagggtccactgataccttta
a/g
cacagcagcagtacatgcaatgcaagatgcccagatga
218




cctgttgcagtttattgttttaaataatccaatcaaa

tgagaataaaggcaatggcaaaaatataagtgctagtt





cttttgtttgagcttattgctgaata

ctatttcaaaataacaaacagaca



 


Chr1QMM36447011
1
tatgatgatgcttattatagcctaaggtatgtacttt
a/g
ccactctacttatgagaagccgggccaccacattcata
219




taagatttagttcgaagtaatgcccatctggcaagtt

cattacagccagaaacaacaaatccaggaagttaatac





aattccagcattaacgtgttctaaaa

gtgattaagaatgcatcaaacaag



 


Chr1QMM36747244
1
tcgatatgttgggttttttctctttactagtagcatg
a/g
ctccgaggacgaggaggaggacgactaatttggcagct
220




ccatctagtgtgcatcttacgtagtggaatattatcg

cagctcacctgcacggctgcactgtgctgtgcccggtg





ggcacccaatattcggctcgcacaaa

ggcgaagccatttcacccgcgggc



 


Chr1QMM28257622
1
tctgaaaacgcatggccgaaataagatgcaagaacac
a/g
ctccgaggacgaggaggaggacgactaatttggcagct
221




ctgcaaaataatctcaggatcagtccaggtacgcatt

tgctgcttgtaattctctgaatttctgaatgaaagaaa





ctctactgttatctactgaaccagag

agaaaagaaaagaagcgaaactgg



 


Chr1QMM29278751
1
cttaaattctcatgttttattcccgttgcaacgaacg
a/g
gtgtggcgcctacgtgatcgttggtttgtttcgcttgt
222




gtcatttcttttagtgtccataaatagctataagagg

ttgggcatacagctatgagaactttgttgggggcccat





catcgatcatcgcagcaagccgactg

actacttatcatcatgtgtctaat



 


Chr1QMM29340100
1
gaactaaacacacccggaatgtgatggatccgaatct
a/g
aaaaacataggaataagaatcctatgtgaattggtact
223




gctgtagttgatactgtgaatgtaacttgtaggcctc

gttcatccctttgatttgtaggaattgaacaaaggaaa





atttgattttctagaaaaaaatggag

agcatggggaaaaaaatcctatga



 


Chr1QMM29875869
1
agcgccacgccgcggccagcgccgtggtgttctccgg
a/g
atccggcggctccacagggccgtcggcaacgcggtcgt
224




gtggcatcgcttgagctacatcaccaccgatggccac

cgacgacaagtacttggtcttcgggaccggctccaccc





ttaaagtccgttgagctcgatcgcca

acctgatcaacgcgctggtgtacg



 


Chr1QMM30173016
1
acgggcggtaccagctacctgtcacagacatgtgggc
a/g
tccacgggcgcaccgagcgtccaccgccccccgcgata
225




ccagcttaacgctaacgcgctgatggccccacatggc

tccgggggtttaggcgatatttagccacgagaggggga





agcgacctgccccctctgtccctccg

ggggagagtaggagaccgacgctt



 


Chr1QMM30198493
1
agaaagacttgctttagctttattgtttcttttccat
a/g
cacttcaaaagggatgaaggaaagaaggctgtcatatc
226




attctatattcttgaaaaggactgcaaagctcttcta

aattactcaatcatgaccagatcatcgatctgatgcag





gtatatgcaattagctgctttggaag

ttaaaaatttcattaattttgcca
















TABLE 7 







List of mutations identified in Mesotrione mutant. Mutations were


identified through MutMap method (whole genome sequencing).

















SEQ ID


ID
Chromosome
Upstream sequence
Allele
Downstream sequence
NO:















Chr1CMM3349975
1
atcgatgtaattagtgatgtcaatcaatggtccaga
a/g
Tacagcatattccaaggaatctgtgtgcttcctat
227




tggcatttggagtcttcgagccttatctttagtgtc

caggttttgctggaatggaaaattgtgctggctca





acttgcattttcagttccaaatgaacatctggaaga

gttttgctgttccccctacttttcaataaatcagg





aggcttggatatctttggaatttcattgataggatg

ttcaccttcttttcgcagaccagttgaattttgta





aaaatctcttgccgcatgaagcctgacggagtttcc

tattctctttacaagcagatgctgaagagtggcct





gttagccaaagtggcatcag

aataaattatttggtgaaacctcta



 


Chr1CMM3568176
1
Taaccttatagtgggacaaggactgaaaagcagttc
a/g
aaatagattttctacaagcacatgatcattggtgg
228




ctcttgctttaacccagagagggtcaacatttttct

gcactgcccacagataaaactagctagctctcggc





ccctgtaagttccaatgctccacaatatttgtatca

agtcttacccatgggaaacagtaggatctatcgaa





agtgttttgaggttccaggatatgcttcagaatcca

aaatcaagcagctgctttgatataactttccaggc





ttgctccccatctcaatagaatgcataagctgaaaa

agtatagaaatctggcaaagagattagcagctcac





ggcaagaaaaagtggaataa

ctcagaagtgaaatttggagggcaa



 


Chr1CMM4052710
1
ccaagcggagacatcgcttccatgagcaattcacgg
a/c
tgcagcagtcagctcatcagttcttgtaagactca
229




tttttcactagatctctcacc

ctgatcaacacagattgtgcagccactaagttact





ctttctctgatccattctggaccagcctctaatgcg

tatactgcattgctatggtgataatttaagggaat





tagagcgccaggcgctgcccgatgatggaagcacag

gccctggtaaaagattcaagtggatgcgggaaatt





ataggtatgttatcttgcactttgagcagctgtgca

catgaaactgaaagaactcaatattgactcttaca





tgaagaccgtcagcttcgtttgggtgggcaat

tcagcattttcaagcctaagcagag



 


Chr1CMM4203161
1
tttaaatcaaatcttaaaaatataaatcataaataa
t/c
tttataagtatggagggagtatccatttcacatat
230




ctatcaagttgttgagtttaaaaatataaaaattat

acttatggtcttgtttacatcccaacaaattttag





ataaatatatttgtcttgaaaaatactttcataaaa

ccaaaaacatcacatcaaatatttagccacatgta





gtatacatatatcactttttaataaatatttttata

taggacattaaatataaaaaaacaattacacagtt





aaaacaagaagtcaaagttatgttttagagaccgcg

tgcatgtaaattgcgagacgaatcttttgagccta





tctctgttctaaacgacttc

attacgccattatttgacaatgtgg



 


Chr1CMM4388604
1
cggaactatgactaactcctctccgtaagcttcttt
t/c
ttccagtgttactcaaaatctagctactggaaaca
231




gtaatatgtattgctgctgtacttggtctcattatc

ataccaatattatagaacaaacagctgatgttatc





tccttacagatatatatacattttttgcagggtata

acaaaaatagacaatagtatgagttcaccacggat





tccacttcatcttctccgtgacattgagactagggt

gagaataacagaaaggaatggtataagggacaaca





ttggctcctggccgtggagtcagagagtcagtgtaa

ccaccccatcattccatcaacatttgcaactcttt





agctgacggagaatatgcac

gagtctaatggcgaaggcgtacata



 


Chr1CMM4425961
1
caactactgacaacaagtgccatgtctaaatttctg
t/c
caacttttaaaaatgtgggaacaatcaaaccatat
232




aacatgcacacaacacacaaatgatgaatatggtga

gcttgagatatacccacaaagccatcggcggccgc





aaccgcaattagcattagaaagttttaactctagaa

ttacagcagagtacaccctcatcttgcacgcctcc





atcaatttccaagttgtaatccccatactcccaacc

gaagaagcaggagccgtgatcaacacgagcatctt





cagaagggaaaaaaaacaactccaaaaccc

gtcctgctccaccccacacagtgacgagtctaggg





cacgacgaaggcgcgctcctaaacc





 


Chr1CMM4454432
1
cttcctgtcgtgagtgactgggtggtgggctcaatc
t/c
tagtgctgttctgaacctcttgcgcatacattaac
233




ggcctggcccgatacgatgcaagcgcgtggctgggc

atgttttatctaatctaataaacatgattaaattt





aggagatcggacggtgctgattgttggggcgacgtg

agcgtttgcttttacagtagtagaaatatgaaatt





gccgcgtgggcgaatgaatagtgaacagtaccgacg

gaacaatggttagtctgaggaatcataagcctatg





tgaggtttataggattttatatgactaggggtgaac

atctagctggagtcttctccggtttaagctaccaa





gttggatagaagggaatgtg

ttgaaacatattaattgatgcctga



 


Chr1CMM4496531
1
aatgtgctcaacttcatatatatgtgtgttgagcac
a/g
aatattcaactttactcaatgttttgtttaacaag
234




atagctcatatataagatcaatggttagatcaatgg

ttccttttggtcacttgccaatttttctagatcat





tttttgggttagatcaatggttgagcacatagctca

acagtacaatctattgatcacaattcacattgaat





tatatatgtgtgctcaacggctcacacacaacttca

aactaggtcaagccattctgtacatgcccatgcat





tatatatgtacccaaaaaagcactattagatcaatg

gaacttactgtactaatattatcttagattaattt





gttataattgtttcaccacg

atcctgaaacttatagtcatatgtg



 


Chr1CMM3714792_CA-C
1
gcatgaaagctgagaccatcaccaggttgatcgttg
 c/ca
aaaaaaaagtaacaggtagcagactttcaactaac
235




ttgctgctattataagatgccaaaatcggcaaatcg

ctggcatgaaagcttggattgtcactggtttgatt





gtcattcactcaaggattggacacaaga

gttgctgctgctgttatgaaagctgtata



 


Chr1CMM3931650_A-T
1
acgttgcttaggtagcaccttgatttaatcaaatgc
t/a
ttgtacaggagtgtactacatccacatacaatgaa
236




tagctagttgatgccaggtggcacactgcggacgga

cagtagtagtagcagcagctatatactccagttgc





tttgtttgtcagtttccctgcattacac

ctagtcgtacacaaagtataattaatcaca




















TABLE 8







TRAIT





RESISTANCE/TOLERANCE
ATCC


LINE
SOURCE DESIGNATION
TO INHIBITORS
DEPOSIT







P1003

HPPD (NON-INDUCED)



R0146

HPPD SENSITIVE


ML0831266-

HPPD
PTA-13620


03093

(INDUCED + NON-
Mar. 19, 2013




INDUCED)


ML0831265-
09PM72399
ACCASE
PTA-12933


01493

G2096S MUTATION
May 31, 2012


ML0831265-

ACCASE
PTA-13619


02283

(UNKNOWN MUTATION)
Mar. 19, 2013


PL1214418M2-

ACCASE MUTATION
PTA-121398


73009

HPPD (INDUCED + NON-
Jul. 18, 2014




INDUCED)


PL1214418M2-

ACCASE MUTATION
PTA-121362


80048

HPPD (INDUCED + NON-
Jun. 30, 2014




INDUCED)


PL1214418M2-

ACCASE-G2096S


73001

HPPD INDUCED


PL1214418M2-

ACCASE-G2096S


73013

HPPD (NON-INDUCED)
















TABLE 9







Agronomic characteristics of two lines carrying both HPPD and ACCase resistance/tolerance.















Days to









50%
Plant


Sheath




Designation
Heading
Height
Plant Type
Pubesence
Color
Awns
yield/plant





PL1214418M2-
range 56-94
range 57-80 cm
erect to
variation of
variation
None
13.39 gm


80048


intermediate
glaborous
between








and smooth
green and









purple




PL1214418M2-
85
range 84-108 cm
erect to
variation of
variation
None
NA


73009


intermediate
glaborous
between








and smooth
green and









purple









PUBLICATIONS
All publications cited in this application are herein incorporated by reference

    Akira, Abe et al., Genome sequencing reveals agronomically important loci in rice using MutMap Nature Biotechnology 30, 174-178 (2012), Published online 22 Jan. 2012.
    Wright, Mark H. et al., Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa. Nat. Comm 2:467|DOI: 10.1038/ncomms1467, Published Online 13 Sep. 2011. The marker information can be accessed from The Rice Diversity Home Page and downloading the file “44K GWAS Data” (http://www.ricediversity.org/index.cfm).
    Zhao, Keyan et al. (2011). Genome-wide association mapping reveals a rich genetic architecture of complex traits in Oryza sativa. Nat Comm 2:467|DOI: 10.1038/ncomms1467, Published Online 13 Sep. 2011

## Claims

1. A mutant rice plant resistant to both HPPD and ACCase inhibiting herbicides, wherein said mutant rice plant is produced from seeds deposited in the ATCC under Accession numbers PTA-121398 or PTA-121362.

2. The mutant rice plant of claim 1, wherein the HPPD inhibiting herbicides are selected from the group consisting of mesotrione, benzobicyclon, and combinations thereof.

3. The mutant rice plant of claim 1, wherein the HPPD inhibiting herbicides are selected from the group consisting of topramezone, tembotrione, isoxaflutole, and combinations thereof.

4. An F1 progeny rice plant of the mutant rice plant of claim 1, or a part thereof, wherein the F1 progeny rice plant is resistant to both HPPD and ACCase inhibiting herbicides.

5. A tissue culture produced from protoplasts or cells from the mutant rice plant of claim 1, wherein said cells or protoplasts of the tissue culture are produced from a plant part selected from the group consisting of leaves, pollen, embryos, cotyledon, hypocotyl, meristematic cells, roots, root tips, pistils, anthers, flowers, stems, glumes, and panicles.

6. A rice plant regenerated from the tissue culture of claim 5, wherein the plant has all the morphological and physiological characteristics of the mutant rice plant of claim 1.

7. A method for controlling weeds in a rice field, the method comprising:
(a) having the mutant rice plant of claim 1, or the F1 progeny rice plant of claim 5 in the field, wherein the mutant rice plant or the F1 progeny rice plant is resistant to a plurality of herbicides; and
(b) contacting the rice field with at least one of the plurality of the herbicides to which the mutant rice plant is resistant at levels known to kill weeds wherein the plurality of herbicides comprise herbicides selected from the group consisting of ACCase and HPPD inhibitors.

8. The method of claim 7, wherein the mutant rice plant or the F1 progeny rice plant is resistant to both ACCase and HPPD inhibitors.

9. The method of claim 7, wherein the ACCase inhibiting herbicides are selected from the group consisting of aryloxyphenoxy propionate, fluazifop and quizalofop.

10. A method to develop a rice plant resistant to a plurality of herbicides comprising HPPD and ACCase inhibitors, the method comprising:
(a) introgressing genetic material derived from ATCC deposited lines PTA-13620 and PTA-12933 into rice lines through breeding, tissue culture or transformation, wherein said genetic material causes resistance to HPPD and ACCase inhibitors;
(b) selecting progeny of the introgressed lines that demonstrate the plurality of resistance,: and
(c) producing inbred or hybrid plants from the progeny.

11. The method of claim 10, further comprising application of mesotrione at a rate of at least 420-630 gm ai/ha and quizalofop at a rate of at least 154 gm ai/ha and up to 210 gm ai/ha, to the rice plant resistant to the plurality of herbicides comprising HPPD and ACCase inhibitors.

12. A method of isolating a DNA molecule with a mutation associated with tolerance to mesotrione or other HPPD inhibiting herbicides, said method comprising isolating a DNA sequence from seeds deposited in the ATCC under Accession number PTA-13620, wherein the mutation is linked to, or located between, SNP markers with nucleic acid sequences selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5, the mutation causing tolerance to mesotrione or other HPPD inhibiting herbicides.

13. A recombinant nucleic acid comprising the mutant DNA molecule of claim 12.

14. A mutant rice plant resistant to both HPPD and ACCase inhibiting herbicides, said mutant rice comprising the mutant DNA molecule of claim 12 and an ACCase tolerance mutation G2096S.