WO2013071366A1 - Drought tolerant plants produced by modification of the stay-green stgx locus - Google Patents
Drought tolerant plants produced by modification of the stay-green stgx locus Download PDFInfo
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- WO2013071366A1 WO2013071366A1 PCT/AU2012/001423 AU2012001423W WO2013071366A1 WO 2013071366 A1 WO2013071366 A1 WO 2013071366A1 AU 2012001423 W AU2012001423 W AU 2012001423W WO 2013071366 A1 WO2013071366 A1 WO 2013071366A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/10—Seeds
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
- A01H6/4666—Sorghum, e.g. sudangrass
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the present disclosure teaches the generation of drought tolerant plants.
- the present disclosure enables manipulation of a phenotypic characteristic referred to as "stay- green" to facilitate drought adaptation in plants by recombinant, mutagenic, breeding and/or selection methods.
- Plant management practice systems to increase crop yield and harvest efficiency in water-limited environments are also taught herein.
- Drought is the single most important constraint to cereal production worldwide.
- Sorghum is a repository of drought resistance mechanisms, which include C 4 photosynthesis, deep roots and thick leaf wax which enable growth in hot and dry environments. Drought tolerance makes sorghum especially important in dry regions such as sub-Saharan Africa, western-central India, north-eastern Australia, and the southern plains of the US. With increasing pressure on the availability of scarce water resources, the identification of traits associated with grain yield under drought conditions becomes more important.
- the drought adaptation mechanism identified in sorghum which results in the retention of green leaves for longer periods during grain filling under drought is known as 'stay-green' .
- grain yield is a function of transpiration (T). transpiration efficiency (TE), and harvest index (HI) [Passioura ( 1977) J Aust. Inst. Agric. Sci. 45: 1 17- 120].
- T transpiration
- TE transpiration efficiency
- HI harvest index
- grain yield is linked to post-anthesis T (Turner (2004) J. Exp. Bot. 55:2413-2425; Manschadi et al. (2006) Fund. Plant. Biol. 55:823- 837), because HI increases with the fraction of total crop T used after anthesis (Passioura, (1977) supra; Sadras and Connor (1991) Field Crops Res. 26:227-239; Hammer (2006) Agric. Sci. 79: 16-22).
- post-anthesis T is associated with reduced drought stress around anthesis, which can positively affect crop growth rate at anthesis of cereals and hence grain number (Andrade et al. (2002) Crop Sci. 42: 1 173- 1 179; Van Oosterom and Hammer (2008) Field Crops Res. 108:259-268). If the total amount of available water is limited, post-anthesis T can be increased by restricting pre-ahthesis T. This can be achieved by restricting canopy size, either genetically or through crop management. However, a smaller canopy will only reduce total T if its TE is not compromised. Significant genotypic differences in TE have been reported for sorghum (Hammer et al. (1997) Aust. J. Agric. Res.
- post-anthesis water use can be increased by increasing the total amount of water accessed by the crop, either through deeper rooting or reduced lower limit of water extraction (Manschadi et al. (2006) supra).
- stay-green trait affects a number of the above processes in sorghum.
- stay- green reduces water use during the pre-anthesis period by restricting canopy size (via reduced tillering and smaller leaves).
- stay-green improves water accessibility by increasing the root:shoot ratio. There is some experimental evidence for better water extraction in stay-green lines, although more research is required. These root responses could also be explained by enhanced auxin transport (Wang et al. (2009) Molecular Plant 2( ⁇ ):823-831 ).
- stay- green increases the greenness of leaves at anthesis, effectively increasing photosynthetic capacity, and, therefore, TE (providing that photosynthesis increases proportionately more than conductance). The increase in leaf greenness is an indirect affect of reduced leaf mass, i.e. nitrogen is concentrated in the leaf.
- QTLs Quantitative trait loci are used to identify genomic regions in sorghum associated with and/or which otherwise facilitate the stay-green phenotype.
- the QTLs identify stay-green (Stg) X wherein X is a numeral increasing from 1 which represents the region on a chromosome comprising loci associated with the stay-green phenotype.
- a region within StgX is referred to as StgXm wherein m is an alphabetical designation such as Stg3a and Stg3b.
- X is 1 and the region is Stg l on chromosome 3 between markers txp581 and txp38 of sorghum or its equivalent in another plant genome.
- X is 2 and the region is Stg2 on chromosome 3 between markers txp530 and txp31 of sorghum or its equivalent in another plant genome.
- X is 3 and the region is Stg3 on chromosome 2 between markers txp471 and txp l 79 of sorghum or its equivalent in another plant genome.
- Stg3 is divided into Stg3a (region between txp298 and sPb-2568) and Stg3b (region between sPb-2568 and txp l 79).
- X is 4 and the region is Stg4 on chromosome 5 between markers txp283 and tx l5 of sorghum or its equivalent in another plant genome.
- FIG. 68 provides a diagram of how many of the genes in Stgl , Stg2, Stg3a, Stg3b and Stg4 affect the stay-green phenotype.
- StgX comprise loci which encode proteins or regulatory agents such as microRNAs, the level of expression of which, facilitate the stay-green phenotype. Selection of a genetic locus or genetic region at StgX in a crop plant including elevating or reducing expression of an indigenous locus or loci is proposed to shift water use by the plant to the post-anthesis period or increase accessibility of water during crop growth or increase transpiration efficiency thereby increasing harvest index (HI) and grain yield under water-limited conditions. It is further proposed that StgX is part of a genetic and physiological network associated with drought adaptation. Polymorphic variants of loci within an StgX may also affect levels of expression.
- the present disclosure teaches the selection of plant breeding parents which express a particular polymorphism as well as introducing an StgX to a plant by any number of means including recombinant means or via standard breeding protocols. Mutagenesis of existing (i.e. indigenous) loci is also contemplated herein.
- Taught herein is a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, said method comprising modulating the level of expression of an existing or introduced StgX locus or loci in all or selected tissue in plant, the StgX corresponding to the location on a chromosome within a sorghum plant or its equivalent in another plant, which StgX encodes a product, the level of which, is associated with or facilitates a stay-green phenotype which phenotype includes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water-limited conditions, and wherein StgX is identified by fine structure mapping.
- a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into the plant or a parent of the plant an agent selected form the list consisting of: (i) a genetic agent comprising one or more loci, located in a region selected from Stgl on chromosome 3 between txp581 and txp38; Stg2 on chromosome 3 between txp530 and txp31 ; Stg3a (region between txp298 and sPb-2568); Stg3b (region between sPb-2568 and txpl 79); and Stg4 on chromosome 5 between txp283 and tx l 5 of sorghum or its equivalent in another plant, the level of expression of which, is associated with or facilitates a stay-green phenotype, which phenotype includes a shift in water use to the post-an
- This aspect encompasses using recombinant techniques to introduce one or more loci into a plant as well as using breeding protocols to select plants having a particular expression profile of the one or more loci. Mutagenesis followed by selection may also be used to alter expression profiles or patterns in indigenous loci.
- a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising modulating the level of expression of an existing or introduced StgX locus or loci in all or selected tissue in a plant, which locus or loci corresponding to a locus or loci located at Stgl , Stg2, Stg3 (including Stg3a and Stg3b) and/or Stg4 on a chromosome -within a sorghum plant or its equivalent in another plant which encodes a product, the level of which, is associated with or facilitates a stay-green phenotype, which phenotype includes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water-limited conditions, and wherein StgX is identified by fine structure mapping.
- a the level of expression of one or more loci in an StgX region facilitates inter alia a particular plant canopy architecture which enables a plant to become more water efficient.
- the locus or loci in StgX therefore, is/are referred to herein as a "drought adaptation gene(s)" or "drought adaption locus/loci". Examples of loci are listed in Tables 1A through 1C.
- a locus is selected from Table I B.
- the locus encodes a protein associated with auxin such as a pin-like inflorescence (PIN) protein.
- PIN proteins are auxin efflux carriers which contain transmembrane domains and are mainly localized in the plasma membranes.
- the locus encoding a PIN protein is represented herein as PIN.
- Other examples of a genetic loci are IPA-1 (Ideal Plant Architecture 1 ), WFP (Wealthy farmers Panicle), squamosa Spl (promoter binding protein-like) and CCD7/8.
- the present disclosure teaches introducing one or more stay-green loci into a plant or introducing a functional equivalent such as a cDNA or up-regulating or down regulating expression of an indigenous locus or loci. This includes recombinant techniques, breeding, hybridization and selection protocols and mutagenesis methods.
- SbPINn is used to describe a SbPIN protein produced in sorghum wherein n is a numeral defining the auxin efflux carrier component and n is 1 through 1 1 .
- Reference to “SbPINn” includes its homologs and orthologs in other plants. Examples of SbPINn loci are those which encode SbPIN4 and SbPIN2 and their equivalents in other plants.
- the level or location of expression of a PIN or level of expression of a PIN with a particular polymorphic variation is proposed herein to facilitate expression of the stay- green phenotype.
- the PIN may be introduced or .its level of expression altered by recombinant means, standard breeding protocols and mutagenesis methods.
- SbPIN4 corresponds to the OsPIN5
- SbPIN2 corresponds to OsPIN3a.
- the term “Os” refers to rice (refer to Table 1 A).
- the locus is IPA-1.
- the level of expression of IPA- 1 or level or location of expression of IPA-1 with a particular polymorphic variation is proposed herein to facilitate the stay-green phenotype
- the locus is WFP.
- the level or location of expression of WFP or level of expression of WFP with a particular polymorphic variation is proposed herein to facilitate the stay-green phenotype
- the locus is Spl.
- the level or location of expression of Spl or level of expression of Spl with a particular polymorphic variation is proposed herein to facilitate the level of the stay-green phenotype.
- the locus is CCD7/8.
- the level or location of expression of CCD7/8 or level of expression of CCD7/8 with a particular polymorphic variation is proposed herein to facilitate expression of the stay- green phenotype.
- the stay-green loci may be expressed in all plant tissue or in selected tissue. Differential expression may also be selected.
- SbPIN2 for Sorghum bicolor member of the auxin efflux carrier component 2 family
- SbPIN4 is at Stg l
- on chromosome 3 fine -mapped to a region between markers txp563 and txp442 are taught herein to be responsible for the stay- green trait in sorghum resulting in a range of phenotypes that confer drought adaptation via increased water use at anthesis (due to reduced tillering and smaller leaves), increased water accessibility (due to enhanced root:shoot ratio), increased transpiration efficiency under mild water deficit (due to higher leaf nitrogen concentration), increased biomass per leaf area under terminal water deficit (due to increased transpiration per leaf area) and increased grain yield, grain size and lodging resistance.
- Reference to the txp markers in sorghum extends to the equivalent markers in the genome of other plants.
- Another aspect taught herein is a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a one or more loci corresponding to SbPINl to 1 1 , IPA-1 , WFP, Spl and/or CCD7/8 or a functional equivalent thereof or an agent which modulates expression of an indigenous form of one or more of these loci wherein the level and/or location of expression of the one or more loci causes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- a "functional equivalent" of a locus includes a cDNA molecule or a homolog from another plant species. This aspect includes a recombinant approach to introduce a locus or a breeding protocol to introduce or select a locus with a particular expression profile.
- the present disclosure further teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA encoding a locus located in Stgl or a molecule which modulates expression of an indigenous locus. Examples are listed in Tables 1 A through 1 C, such as Table I B.
- the present disclosure further teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA encoding a locus located in Stg2 or a molecule which modulates expression of an indigenous locus. Examples are listed in Tables 1 A through 1 C, such as Table I B.
- the present disclosure further teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA encoding a locus located in Stg3a or a molecule which modulates expression of an indigenous locus. Examples are listed in Tables 1 A through 1 C, such as Table IB.
- the present disclosure further teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA encoding a locus located in Stg3b or a molecule which modulates expression of an indigenous locus. Examples are listed in Tables 1A through 1C, such as Table IB.
- the present disclosure further teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA encoding a locus located in Stg4 or a molecule which modulates expression of an indigenous locus. Examples are listed in Tables 1 A through 1 C, such as Table 1 B.
- the present disclosure further teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA encoding a PIN protein, or a molecule which modulates expression of an indigenous PIN locus.
- PINs are SbPIN 1 to 1 1 which include SbPIN4 and SbPIN2 and other SbPINs listed in Table 1 A as well as their equivalent in other plants.
- the present disclosure teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA which encodes IPA-1 or a functional homolog or ortholog thereof or an agent which modulates the level of expression of an indigenous IPA-1 to cause a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the present disclosure teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA which encodes WFP or a functional homolog or ortholog thereof or an agent which modulates the level of expression of an indigenous WFP to cause a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the present disclosure teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA which encodes Spl or a functional homolog or ortholog thereof or an agent which modulates the level of expression of an indigenous Spl to cause a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the present disclosure teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA which encodes CCD7/8 or a functional homoiog or ortholog thereof or an agent which modulates the level of expression of an indigenous CCD7/8 to cause a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the plants are modified or selected to change the level of expression of two or more of loci listed in Tables I B or 1 C.
- the plants are modified or selected to change the level of expression of two or more of PIN, IPA- 1 , WFP, Spl and/or CCD7/8 and/or two or more P v.
- Genetically modified plants and their progeny exhibiting the stay-green trait are also enabled herein as well as seeds, fruit and flowers and other reproductive or propagating material.
- Such "genetically modified plants” include plants modified by recombinant means as well as plants selected through breeding protocols and/or plants subjected to mutagenesis and selection.
- Genetic material is enabled herein which encodes a product which is associated with or facilitates a stay-green phenotype which phenotype includes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water- limited conditions, and encoded by a locus in the StgX region wherein X is a numeral corresponding to the location on the chromosome and wherein StgX is identified by line structure mapping is enabled thereon as in a functional equivalent of the StgX.
- the genetic material is useful for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species as well as for developing markers for selection of traits during breeding protocol.
- Genetic material contemplated herein includes cDNA, genomic DNA and germplasm encoding one or more of a locus listed in Tables 1 A through 1C, such as Table I B. [0049] Genetic material contemplated herein includes cDNA, genomic DNA and germplasm encoding one or more of a PIN, IPA-1 , WFP, Spl and/or CCD7/8. Reference to a "PIN" includes one or more PINs.
- the plant management system includes the generation of a drought adapted crop including cereal plants using the selection and expression of an StgX locus or a functional, equivalent thereof alone or in combination with the introduction of other useful traits such as grain size, root size, salt tolerance, herbicide resistance, pest resistance and the like.
- the plant management system comprises generation of drought adapted plants and agricultural procedures such as irrigation, nutrient requirements, crop density and geometry, weed control, insect control, soil aeration, reduced tillage, raised beds and the like.
- StgX locus in sorghum examples include SbPINl to 11 , IPA-1, WFP, Spl and CCD7/8 and their equivalents in other plants.
- loci examples include located in Stgl , Stg2, Stg3a, Stg3b and/or Stg4 (see Tables 1 A through 1C such as Table IB).
- a business model is also taught herein for improved economic returns on crop yield, the model comprising generating crop plants having a selected StgX trait or elevated or reduced StgX trait resulting in the crop plant having a shift in water use by the plant to the post-anthesis period thereby increasing HI and grain yield under water-limited conditions, obtaining seed from the generated crop plant and distributing the seed to grain producers to enhance yield and profit.
- the present disclosure further teaches markers for the stay-green phenotype for use in breeding programs for drought tolerant plants, the markers comprising a stay-green X (StgX) locus, wherein X is a numeral corresponding to the location on a chromosome within a sorghum plant or its equivalent in another plant which encodes a product which is associated with or facilitates a stay-green phenotype which phenotype includes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water-limited conditions.
- StgX stay-green X
- markers include Stg l on chromosome 3 between txp581 and txp38; Stg2 on chromosome 3 between txp530 and txp31 ; Stg3 is divided into Stg3a (region between txp298 and . sPb-2568) and Stg3b (region between sPb-2568 and txpl 79) and Stg4 on chromosome 5 between txp283 and txpl 5. Examples include those listed in Tables IB and 1 C. These markers are based on the sorghum genome but extend to the equivalents in" another plant genome.
- marker adjacent or proximal to the genomic locations given above may also be used to screen for particular progeny in a breeding program.
- a set of biomarkers is taught herein including Stgl on chromosome 3 between txp581 and txp38; Stg2 on chromosome 3 between txp530 and txp31 ; Stg3 is divided into Stg3a (region between txp298 and sPb-2568) and Stg3b (region between sPb- 2568 and tx l 79) and Stg4 on chromosome 5 between txp283 and txpl 5 on chromosome 5 of sorghum, or the equivalent in the genome of another plant.
- Such markers are useful in breeding protocols designed to generate plants exhibiting the stay-green phenotype. Examples of loci are listed in Tables 1 A through 1C. Particular examples are in Table 1 B.
- the locus is in Stgl selected from PINS, GID I L2, P45098A 1. indole-3-acetate and brassinosteroid insensitive.
- the locus is in Stg2 and is auxin efflux carrier component 3a (PIN3a).
- the locus is in Stg3a selected from leaf senescence protein-like (Sb02g023510), leaf senescence protein-like (Sb02g023520), RAMOS A 1 C2H2 zinc- finger transcription factor (Sb02g024410), putative auxin-independent growth promoter (Sb02g024540), similar to dehydration-responsive protein-like (Sb02g024670), similar to glucose transporter (Sb02g024690), WRKY transcription factor 76 (Sb02g024760), glutamine synthetase-like protein (Sb02g025100), senescence-associated protein DH (Sb02g025180), putative alanine aminotransferase (Sb02g025480), auxin-induced protein- like (Sb02g025610), auxin-induced protein-like (Sb02g025620), putative far-red impaired response protein (Sb02g025670), similar to
- the locus is in Stg3b selected from putative auxin-independent growth promoter (Sb02g027470), squamosa promoter-binding-like protein 1 7 (Sb02g028420), similar to Os09g0505400 (OsPIN9) protein (Sb02g029210), squamosa promoter-binding-like protein 17 (Sb02g029300) similar to auxin-induced protein-like (Sb02g029630).
- the locus is in Stg4 selected from brassinosteroid LRR receptor (Sb05g006842), brassinosteroid LRR receptor (Sb05g006860), putative far-red impaired response protein (Sb05g007130), cytochrome P450 84A 1 (Sb05g007210), gibberellin receptor GID1 L2 (Sb05g007270), gibberellin receptor GID1 L2 (Sb05g007290), sucrose- phosphate synthase (Sb05g007310), aquaporin SIPl -1 (Sb05g007520), gibberellin 20 oxidase 2 (Sb05g008460), OsIAA29 - auxin-responsive (Sb05g008510), OsIAA29 - auxin- responsive (Sb05g008512), protein gibberellin receptor GID1 L2 (Sb05g008610), similar to aminotransferase, putative (Sb05g006842
- HWLD high water, low density (least water stressed)
- PASM post-anthesis stem mass
- Table 1A provides information on PIN's from sorghum and rice. Table 1A
- Fine-mapped region region between txp563 and txp581 containing 60 annotated genes:
- Fine-mapped region region between txp512 and txp2 containing 15 annotated genes:
- Stg3a entire region (region between txp298 and sPb-2568 containing 520 annotated genes):
- Stg3b entire region (region between sPb-2568 and txp179 containing 291 annotated genes):
- FIG. 1 is a graphical representation showing the relation between culms per m 2 and green leaf area at anthesis in a range of NILs containing various Stg introgressions.
- Figure 3 is a graphical representation showing the histogram of predicted values for culms per plant in the Stgl fine-mapping population averaged over three seasons.
- FIG. 4 is a tabulated representation showing the marker data (BB/TT) for the Stgl fine-mapping population.
- BB designates both alleles are like the allele of the stay- green parent
- TT designates both alleles are like the allele of the senescent parent
- x designates no marker data available.
- Black and red font color for markers designate actual and inferred marker status, respectively.
- Green and brown shading for genotypes across the top designate a stay-green (low tillering) and senescent (high tillering) phenotype, respectively. Markers highlighted in pink indicate the likely location of a "low-til lering" " gene.
- Figure 5 is a graphical representation showing a histogram of culms per plant at 44 DAE for five genotypes grown under two water regimes.
- the genotypes comprise RTx7000 (recurrent parent), 6078-1 (donor parent), and three selections from the Stgl fine-mapping population.
- HWLD high water, low .density (10 plants/m 2 ).
- LWLD low water, low density (10 plants/m 2 ).
- Figure 6 is a tabulated representation showing marker data (BB/TT) for a subset of the Stgl fine-mapping population.
- BB designates both alleles are like the allele of the stay-green parent
- TT designates both alleles are like the allele of the senescent parent
- BT designates one allele is like the allele of the stay-green parent
- the other is like the allele of the senescent parent
- x designates no marker data available. Green and brown shading for genotypes across the top designate a stay-green (low tillering) and senescent (high tillering) phenotype, respectively. Markers highlighted in pink indicate the likely location of a "low-tillering" gene.
- Figure 7 is a graphical representation showing the phenotypic variation in the Stgl fine-mapping population for presence of T2.
- Figure 8 is a graphical representation showing the phenotypic variation in the Stgl fine-mapping population for presence of T3.
- Figure 9 is a tabulated representation showing a histogram of T2 presence for eight high-tillering and eight low-tillering recombinants from the Stgl fine-mapping population.
- Figure 10 is a tabulated representation showing marker data (BB/TT) for the Stgl fine-mapping population.
- BB designates both alleles are like the allele of the stay-green parent
- TT designates both alleles are like the allele of the senescent parent
- BT designates one allele is like the allele of the stay-green parent
- the other is like the allele of the senescent parent
- x designates no marker data available.
- Green and brown shading for genotypes across the top designate a stay-green (low-tillering) and senescent (high-tillering) phenotype, respectively.
- Figure 11 is a tabulated representation showing a histogram of total tiller number per plant for five high-tillering and three low-tillering recombinants from the Stg l fine- mapping population. A value of 2.5 was chosen as the arbitrary cut-off between high and low tillering.
- Figure 12 is a tabulated representation showing marker data (BB/TT) for the Stg l fine-mapping population.
- BB designates both alleles are like the allele of the stay-green parent
- TT designates both alleles are like the allele of the senescent parent
- BT designates one allele is like the allele of the stay-green parent
- the other is like the allele of the senescent parent
- x designates no marker data available.
- Black and red font color for markers designate actual and inferred marker status, respectively.
- Green and brown shading for genotypes across the top designate a stay-green (low-tillering) and senescent (high-tillering) phenotype, respectively. Markers highlighted in pink indicate the likely location of a "low-tillering" gene.
- Figures 13A through D are graphical representations showing the leaf size distribution of mainstem and tillers for RTx7000 and 6078- 1 (Stgl NIL) grown in lysimeters under low and high VPD conditions.
- Figure 15 is a tabulated representation showing marker data (BB/TT) for the Stg l fine-mapping population.
- BB designates both alleles are like the allele of the stay-green parent
- TT designates both alleles are like the allele of the senescent parent
- BT designates one allele is like the allele of the stay-green parent
- the other is like the allele of the senescent parent
- x designates no marker data available.
- Black and red font color for markers designate actual and inferred marker status, respectively.
- Green and brown shading for genotypes across the top designate a stay-green (small leal and senescent (large leaf) phenotype, respectively. Markers highlighted in pink indicate the likely location of a "small leafsize" gene.
- FIG. 16 is a graphical representation showing the leaf size distribution (LI -6) for the parents of the St l fine-mapping population grown in an igloo.
- Figure 17 is a graphical representation showing the leaf length distribution (L 1-6) for the parents of the Stgl fine-mapping population grown in an igloo.
- Figure 18 is a graphical representation showing the leaf width distribution (L I -6) for the parents of the Stgl fine-mapping population grown in an igloo.
- Figure 19 is a graphical representation showing the leaf size distribution (11- 1 1 ) for the parents of the Stg l fine-mapping population grown in an igloo.
- Figure 20 is a graphical representation showing the leaf length distribution (L l - 10) for the parents of the Stgl fine-mapping population grown in an igloo.
- Figure 21 is a tabulated representation showing a histogram of phenotypic variation for L10 length in a subset of the Stgl fine-mapping population grown in an igloo.
- Figure 22 is a tabulated representation showing marker data (BB/TT) for a subset of the Stgl fine-mapping population.
- BB designates both alleles are like the allele of the stay-green parent
- TT designates both alleles are like the allele of the senescent parent
- BT designates one allele is like the allele of the stay-green parent
- the other is like the allele of the senescent parent
- x designates no marker data available.
- Black and red font color for markers designate actual and inferred marker status, respectively.
- Green and brown shading for genotypes cross the top designate a stay-green (short leaf) and senescent (long leaf) phenotype, respectively.
- Figure 23 is a schematic representation showing the likely marker location (pink shading) of a gene conferring both "low-tillering" and "small-leaf size” phenotypes in the Stgl region.
- Figure 24 is a diagrammatic representation showing that increased water availability at anthesis is achieved via reduced water use due to two mechanisms (reduced tillering and smaller leaves) in plants containing the Stgl region.
- Figure 25 is a tabulated representation showing that canopy size is modulated by both constitutive and adaptive responses controlled by a gene(s) in the Stg 1 region.
- Figure 27 is a graphical representation showing the relation between the area of leaf 12 and the total green leaf area at anthesis for the two parents (6078-1 and RTx7000) and three recombinants from the Stgl fine-mapping population.
- Figure 28 is a graphical representation showing the relation between total green leaf area (cm 2 /m 2 ) and crop water use (mm) at anthesis for the two parents (6078- 1 and RTx7000) and three recombinants from the Stgl fine-mapping population.
- Figure 29 is a graphical representation showing the relation between green leaf area and water use (T) in four Stg QTL and the recurrent parent (RTx7000) in lysimetry studies under two levels of VPD.
- Figure 30 is a graphical representation showing a histogram of phenotypic variation for the "roo shoot ratio" at L6 in the Stgl fine-mapping population grown in an igloo.
- Figure 31 is a graphical representation showing the temporal pattern of cumulative crop water use for RTx7000 and Stgl grown under low-water and low-density (20 plants/m 2 ) conditions. The vertical lines marks anthesis.
- Figure 32 is a graphical representation showing the relation between the length (mm) and greenness (SPAD) of leaf 10 in the Stgl fine-mapping population grown in an igloo.
- Figure 33 is a graphical representation showing the relation between leaf greenness (SPAD) and leaf photosynthesis in a subset of lines from the Stgl fine-mapping population, including the parents.
- Figure 34 is a graphical representation showing the relation between leaf greenness (SPAD) and WUE (Licor) in a subset of lines from the Stg l fine-mapping population, including the parents.
- Figure 35 is a graphical representation showing the relation between leaf greenness (SPAD) and WUE (Licor) in four Stg Nils (Stgl , Stg2, Stg3 and Stg4) and the recurrent parent (RTx7000).
- Figure 36 is a graphical representation showing the relation between transpiration per leaf area and transpiration efficiency area in four Stg QTL and the recurrent parent (RTx7000) in lysimetry studies under two levels of VPD.
- Figure 37 is a graphical representation showing the relation between CWU (mm) before and after anthesis in a subset of lines from the Stgl fine-mapping population, including the parents, grown under high density (HD) and low density (HD) conditions.
- Figures 38A and B are graphical representations showing patterns of cumulative water use for Stgl and RTx7000 grown under LWHD and conditions.
- Figure 39 is a graphical representation showing the relation between CWU (mm) before and after anthesis in four Stg QTL and the recurrent parent (RTx7000) grown under low water (LW) and low density (LD) conditions.
- Figure 46 is a tabulated representation showing marker data (BB/TT) for RTx7000 (recurrent parent), 6078-1 (NIL containing complete Stgl region), 10709-5 (NIL containing lower 1 /3 of the Stgl region), 10604-5 (NIL containing upper 3/4 of the Stgl region), and 10568-2 (NIL containing upper 1/2 of the Stgl region).
- BB designates both alleles are like the allele of the stay-green parent
- TT designates both alleles are like the allele of the senescent parent
- BT designates one allele is like the allele of the stay-green parent
- the other is like the allele of the senescent parent
- x designates no marker data available. Black and red font color for markers designate actual and inferred marker status, respectively.
- PASM post-anthesis stem mass
- PAB post-anthesis biomass
- RTx7000 recurrent parent
- PASM post-anthesis stem mass
- RTx7000 recurrent parent
- PASM post-anthesis stem mass
- PAB post-anthesis biomass
- RTx7000 recurrent parent
- RWC relative water content
- FL-2 mid-grain filling
- RTx7000 recurrent parent
- LWP leaf water potential
- Figure 64A through C are graphical representations showing results from running a sorghum crop simulation model using the generic variety Buster with the usual 2 tillers/plant (HT) versus a Buster with only 1 tiller/plant (LT) in a well-watered (WW) and a terminally stressed (TS) virtual environment.
- Figure 64 A shows simulated leaf area index (LAI) over time (0- 120 days after sowing).
- Figure 64 B shows simulated extractable soil water (EWS) over time (0- 120 days after sowing).
- Figure 64 C shows simulated biomass and grain yield (kg/ha) over time (0-120 days after sowing).
- Figure 65 is a diagrammatical representation showing comparisons of PIN, SPL and CCD7/8 orthologs aligned with QTL for stay-green for the sorghum chromosomes 1 through 5.
- Figure 66 is a diagrammatical representation showing comparisons of PIN, SPL and CCD7/8 orthologs aligned with QTL for stay-green for the sorghum chromosomes 6 through 10.
- Figure 67A is a graphical representation of differential expression of SbPIN4 (Stgl candidate) under well-watered conditions. Under well-watered conditions, this gene is down-regulated in young root tips Tx642 and Stg l NIL compared to Tx7000.
- Figure 67B is a graphical representation of differential expression of SbPIN4 (Stgl candidate) under water-deficient conditions. Under water-deficient conditions, this gene is up-regulated in most tissues, but especially in expanding leaves of Tx642 and Stgl NIL compared to Tx7000.
- Figure 67C is a graphical representation of differential expression of SbPIN2 (Stg2 candidate) under well-watered conditions. Under well-watered conditions; this gene is slightly up-regulated in stem and root tissues of Tx642 and Stg l NIL compared to Tx7000.
- Figure 67D is a graphical representation of a differential expression of SbPlN2 (Stage 2 candidate) under water-deficient conditions. Under water-deficient conditions, this gene is up-regulated in most tissues of Tx642 and Stgl NIL compared to Tx7000.
- Figure 68 is a diagrammatic representation of the network of genes identified at Stgl , Stg2, Stg3a, Stg3b and Stg4 and the proposed affect on the stay-green phenotype.
- the present disclosure teaches loci associated with and which facilitate the stay- green phenotype in crop including cereal plants.
- the loci are referred generically as StgX wherein X is a numeral from 1 and above corresponding to a genetic locus or genetic loci region on a particular chromosome in a crop plant.
- a sub-region is referred to as StgXm where m is an alphabetical designation of a region within StgX.
- the level and/or location of expression of an StgX locus is taught herein to facilitate a physiological and genetic network which induces or promotes a shift in water use by the crop plant to the post- anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency thereby increasing harvest index (HI) and grain yield under water- limited conditions.
- “Expression” of an StgX includes up-regulating or down-regulating expression levels as well as selection of a polymorphic variant which is expressed at a higher level or which encodes a more active or efficient product.
- An example of a "physiological network” includes a plant canopy architecture which induces or promotes a shift in water use by the crop plant to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency thereby increasing harvest index (HI) and grain yield under water-limited conditions.
- the locus may itself confer this phenotype or a functional equivalent thereof such as a cDNA encoding a protein encoded by the locus.
- manipulation of the stay-green phenotype may be by recombinant engineering, breeding and selection as well as by chemical, radioactive or genetic mutagenesis followed by selection.
- X is 1 and the region is Stgl on, chromosome 3 between markers txp581 and txp38 of sorghum or its equivalent in another plant genome.
- X is 2 and the region is Stg2 on chromosome 3 between markers txp530 and txp31 of sorghum or its equivalent in another plant genome.
- X is 3 and the region is Stg3 is divided into Stg3a (region between txp298 and sPb-2568) and Stg3b (region between sPb-2568 and txpl 79) of sorghum or its equivalent in another plant genome.
- X is 4 and the region is Stg4 on chromosome 5 between markers txp583 and txp l S of sorghum or its equivalent in another plant genome. These markers or markers adjacent Or proximal thereto are also useful in breeding programs to' generate plants which exhibit the stay-green phenotype in sorghum or other plants.
- a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into the plant' or a parent of the plant an agent selected form the list consisting of: (i) a genetic agent comprising one or more loci, located in a region selected from Stgl on chromosome 3 between txp58 l and txp38; Stg2 on chromosome 3 between txp530 and txp31 ; Stg3a (region between txp298 and sPb-2568); Stg3b (region between sPb-2568 and txpl 79); and Stg4 on chromosome 5 between txp283 and txpl 5 of sorghum or its equivalent in another plant, the level of expression of which, is associated with or facilitates a stay-green phenotype.
- an agent selected form the list consisting of: (i) a genetic agent comprising one or more
- phenotype includes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water-limited conditions; and (if) an agent which up-regulates or down-regulates an indigenous form of the locus or loci.
- the StgX regions are defined as follows Stgl : Fine-mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes, Candidates in tail between txp580 and txp38 containing 178 annotated genes; Stg2: Fine-mapped region between txp512 and txp2 containing 15 annotated genes, Larger region between txp31 and txp530 containing 241 annotated genes; Stg3a: Entire region between txp298 and sPb-2568 containing 520 annotated genes; Stg3b: Entire region between sPb-2568 and txpl 79 containing 291 annotated genes; Stg4: Entire region defined by txp283 and txpl 5 containing 306 annotated genes. These markers or markers adjacent or proximal
- a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into the plant or a parent of the plant an agent selected form the list consisting of Stgl : Fine-mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes, Candidates in tail between txp580 and txp38 containing 178 annotated genes; Stg2: Fine-mapped region between txp512 and txp2 containing 15 annotated genes, Larger region between txp31 and txp530 containing 241 annotated genes; Stg3a: Entire region between txp298 and sPb-2568 containing 520 annotated genes; Stg3b: Entire region between sPb-2568 and txp!
- Stg4 Entire region defined by txp283 and txpl S containing 306 annotated genes; sorghum or its equivalent in another plant, the level of expression of which, is associated with or facilitates a stay-green phenotype, which phenotype includes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water- limited conditions; and an agent which up-regulates or down-regulates an indigenous form of the locus or loci.
- a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species comprising introducing into the plant or a parent of the plant an agent selected form the list consisting of a locus selected from Table I B of sorghum or its equivalent in another plant, the level of expression of which, is associated with or facilitates a stay-green phenotype, which phenotype includes a shift in water use to the post-a thesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water- limited conditions; and an agent which up-regulates or down-regulates an indigenous form of the locus or loci.
- a method for generating a genetically modified plant which uses water more efficiently than a ⁇ -genetically modified plant of the same species comprising introducing into the plant or a parent of the plant an agent selected form the list consisting of a locus selected from Table 1 A of sorghum or its equivalent in another plant, the level of expression of which, is associated with or facilitates a stay-green phenotype, which phenotype includes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water- limited conditions; and an agent which up-regulates or down-regulates an indigenous form of the locus or loci.
- the level of expression of StgX alone or in combination with the operation of a genetic or physiological network alters plant architecture including plant canopy architecture to enhance or otherwise promote efficient water use.
- the modified architecture is modified plant canopy architecture.
- progeny includes immediate progeny as well as distant relatives of the plant, as long as it stably expresses the StgX trait first introduced to an earlier parent.
- Reference to a "crop plant” includes a cereal plant.
- the crop plants enabled herein include sorghum, ' wheat, oats, maize, barley, rye and rice, abaca, alfalfa, almond, apple, asparagus, banana, bean-phaseolus, blackberry, broad bean, canola, cashew, cassava, chick pea, citrus, coconut, coffee, corn, cotton, fig, flax, grapes, groundnut, hemp, kenaf, lavender, mano, mushroom, olive, onion, pea, peanut, pear, pearl millet, potato, ramie, rapseed, ryegrass, soybean, strawberry, sugarbeet, sugarcane, sunflower, sweetpotato, taro, tea, tobacco, tomato, triticaie, truffle and yam.
- the drought tolerance mechanisms of sorghum are used to promote drought tolerance in sorghum as well as other crop plants.
- the genetically modified plant uses water more efficiently than a non-genetically modified plant of the same species.
- a "genetically modified plant" may be produced by recombinant DNA means, selected via a breeding protocol and/or selected following a mutagenesis procedure.
- drought tolerance includes drought escape, drought adaptation, drought resistance, reduced sensitivity to drought conditions, drought insensitive, enhanced water use efficiency as well as an ability to shift water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency, thereby increasing HI and grain yield under water-limited conditions.
- Plants exhibiting drought tolerance are described as “drought adapted plants” or “plants exhibiting reduced sensitivity to water-limited conditions”. It is taught herein that drought tolerance is induced, facilitated by. or otherwise associated with the stay-green phenotype.
- genetically modified in relation to a plant, includes an originally derived genetically modified plant as well as any progeny, immediate or distant which stably express the stay-green trait.
- the present disclosure teaches both classical breeding techniques to introduce the genetic agent, i.e. a stay-green locus or loci or a functional equivalent thereof such as cDNA or a genomic fragment or an agent which alters ⁇ expression of the locus or the protein encoded thereby as well as genetic engineering technology.
- genetic engineering means and “recombinant means”.
- Markers defining StgX can also be screened during breeding protocols to monitor transfer of particular genetic regions.
- a specific StgX or StgX region can be genetically inserted by recombinant means into a plant cell or plant callus and a plantlet regenerated.
- a "genetically modified" plant includes a parent or any progeny as well as any products of the plant such as grain, seed, propagating material, pollen and ova.
- Regions defining StgX in sorghum are located at Stgl on chromosome 3 between txp581 and txp38; Stg2 on chromosome 3 between txp530 and txp31 ; Stg3 is divided into Stg3a (region between txp298 and sPb-2568) and Stg3b (region between sPb- 2568 and txpl 79) and Stg4 on chromosome 5 between txp283 and txpl 5.
- regions defining Stg in sorghum are located at Stg l : Fine-mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes, Candidates in tail between txp580 and txp38 containing 178 annotated genes; Stg2: Fine-mapped region between txp512 and txp2 containing 15 annotated genes, Larger region between txp31 and txp530 containing 241 annotated genes; Stg3a: Entire region between txp298 and sPb-2568 containing 520 annotated genes; Stg3b: Entire region between sPb-2568 and txpl 79 containing 291 annotated genes; Stg4: Entire region defined by txp283 and txpl 5 containing 306 annotated genes.
- the present disclosure extends to equivalent regions or equivalent
- Reference to the "stay-green phenotype” includes characteristics selected from enhanced canopy architecture plasticity, reduced canopy size, enhanced biomass per unit leaf area at anthesis, higher transpiration efficiency, increased water use during grain filling, increased plant water status during grain filling, reduced pre:post anthesis biomass ratio, delayed senescence, increased grain yield, larger grain size, and reduced lodging.
- StgX includes QTLs at Stg 1 , 2, 3 (including Stg3a and Stg3b), 4, etc which represent a particular locus or group or region of loci associated with drought adaptation.
- StgX is Stgl located on chromosome 3 between markers txp581 and txp38 of sorghum.
- the StgX is Stg2 located on chromosome 3 between markers txp530 and txp31 of sorghum.
- the StgX is Stg3a (region between txp298 and sPb-2568) or Stg3b (region between sPb-2568 and txpl 79) of sorghum.
- the StgX is Stg4 located on chromosome 5 between markers txp283 and txpl S of sorghum.
- Stgl Fine- mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes, Candidates in tail between txp580 and txp38 containing 178 annotated genes; Stg2: Fine-mapped region between txp512 and txp2 containing 15 annotated genes.
- Stg3a Entire region between txp298 and sPb-2568 containing 520 annotated genes:
- Stg3b Entire region between sPb-2568 and txp l 79 containing 291 annotated genes;
- Stg4 Entire region defined by txp283 and txpl 5 containing 306 annotated genes.
- the StgX contemplated for use herein may be an isolated naturally occurring genetic element or a particular variation may be artificially induced or selected through classical or recombinant breeding practices.
- a particular polymorphic variant may result in high expression levels or a more stable expression product or a product which is more or less pleiotropic within a genetic or physiological network.
- Reference to an StgX includes a cDNA encoding a product as well as a genomic locus or region which may or may not include a promoter region, 5' and 3' untranslated regions, introns, exons and the like.
- a "cDNA" is an example of a functional equivalent of an StgX.
- the present disclosure further teaches markers for the stay ⁇ green phenotype for use in breeding programs for drought tolerant plants, the markers comprising a quantitative trait locus (QTL), stay-green X (StgX), wherein X is a numeral corresponding to the location on a chromosome within a sorghum plant or its equivalent in another plant which encodes a product which is associated with or facilitates a stay-green phenotype which phenotype includes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water-limited conditions.
- QTL quantitative trait locus
- StgX stay-green X
- markers include Stgl on chromosome 3 between txp581 and txp38; Stg2 on chromosome 3 between txp530 and txp31 ; Stg3 which is divided into Stg3a (region between txp298 and sPb-2568) and Stg3b (region between sPb-2568 and txpl 79) and Stg4 on chromosome 5 between txp283 and txpl 5. These markers are based on the sorghum genome but extend to the equivalents in another plant genome.
- markers include Stgl : Fine-mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes, Candidates in tail between txp580 and txp38 containing 178 annotated genes; Stg2: Fine-mapped region between txp512 and txp2 containing 15 annotated genes, Larger region between txp31 and txp530 containing 241 annotated genes; Stg3a: Entire region between txp298 and sPb-2568 containing 520 annotated genes; Stg3b: Entire region between sPb-2568 and txpl 79 containing 291 annotated genes; Stg4: Entire region defined by txp283 and txpl 5 containing 306 annotated genes. 101501 Examples of suitable markers include a locus listed in Tables 1 A through 1 C.
- markers include a locus listed in Table 1 B.
- a set of biomarkers is enabled herein including txp581 to txp38 on chromosome 3 of sorghum; txp530 to txp31 on chromosome 3 of sorghum; txp298 to sPb- 2568 and sPb-2568 to txpl 79 on chromosome 2 of sorghum; and txp283 to txp l 5 on chromosome 5 of sorghum, or the equivalent in the genome of another plant.
- a set of biomarkers is further enabled herein including Stgl : Fine-mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes, Candidates in tail between txp580 and txp38 containing 178 annotated genes; Stg2: Fine-mapped region between txp512 and txp2 containing 15 annotated genes, Larger region between txp31 and txp530 containing 241 annotated genes; Stg3a: Entire region between txp298 and sPb-2568 containing 520 annotated genes; Stg3b: Entire region between sPb-2568 and txpl 79 containing 291 annotated genes; Stg4: Entire region defined by txp283 and txpl 5.
- biomarkers containing 306 annotated genes.
- a set of biomarkers is further taught as listed in Table 1 B. Such markers are useful in breeding protocols designed to generate plants exhibiting the stay-green phenotype. Alternatively, markers adjacent or proximal to these regions may be used in the breeding protocol. [01531 The present disclosure teaches the use of genetic material corresponding to an StgX or genetic material which alters expression of an indigenous StgX locus or a genetic equivalent thereof to facilitate the stay-green phenotype.
- An "indigenous" locus means a locus present in a parent plant prior to breeding, recombinant intervention or mutagenesis. By “alters” includes “modulates”.
- the present disclosure enables plants genetically modified according to the methods taught herein as well as seeds, fruit, flowers and other reproductive or other propagating material.
- the present disclosure also teaches use of root stock and propagating stock. This is based on the premise that the seeds, fruit, flowers, reproductive and propagating material exhibit or can pass on the stay-green phenotype introduced into the ultimate parent(s).
- Reference to an "agent which up-regulates StgX” includes promoters, microRNAs, genes and chemical compounds which facilitate increased expression of StgX or increased activity of a StgX product.
- An agent may also be an intr n of a genomic StgX which is part of an natural genetic network to facilitate expression.
- An agent may also be a functional equivalent of a StgX (or QTL) such as a cDNA.
- the StgX encodes a locus selected from Stgl , Stg2, Stg3a, Stg3b and Stg4 as listed in Table IB (and Table 1A). The interaction of some of these loci in various networking pathways is shown in Figure 68.
- the StgX encodes a locus selected from Stg l , Stg2, Stg3a. Stg3b and Stg4 as listed in Table 1 B (and Table 1 C).
- the StgX encodes a ⁇ protein.
- a PIN protein produces an auxin gradient in cells and contains transmembrane domain and is mainly localized in the plasma membrane.
- PIN proteins are the rate limiting factors of auxin transport and provide vectorial direction for the auxin flows.
- an StgX encodes a PIN protein.
- Introduction of a StgX de novo in a plant or elevation of its expression or the expression of its homolog or ortholog facilitates exhibition of one or more features or sub-features associated with the stay-green phenotype.
- PIN proteins are efflux carriers of auxin which mediate polar auxin transport (PAT) from cell to cell as opposed to the transport of auxin through, the xylem (Rashotte et al. (2000) Plant Cell 13: 1683- 1697; Friml et al. (2003) Current Opinion in Plant Biology (5:7-12).
- the term 'PIN' is derived from the PIN-like inflorescence which develops in Arabidopsis when auxin transport is defective.
- a number of PIN proteins are known (see Forestan and Varotto (2009) Plant Physiology; and Wang et al. (2009) supra).
- the present disclosure teaches SbPINn, where n is a numeral from 1 through 1 1 (Table 1 A). However, the instant disclosure teaches equivalent or homolog PINs from other plants.
- SbPIN2 for Sorghum bicolor auxin efflux carrier component 2
- SbPIN4 is at Stgl
- on chromosome 3 fine -mapped to a region between markers txp563 and txp 442 are taught herein to be responsible for the stay-green trait in sorghum resulting in a range of phenotypes that confer drought adaptation via increased water use at anthesis (due to reduced tillering and smaller leaves), increased water accessibility (due to enhanced roo shoot ratio), increased transpiration efficiency under mild water deficit (due to higher leaf nitrogen concentration), increased biomass per leaf area under terminal water deficit (due to increased transpiration per leaf area) and increased grain yield, grain size and.
- SbPIN4 corresponds to the OsPIN5 and SbPIN2 corresponds to OsPlN3a.
- Os refers to rice (refer to Table 1 A).
- Another aspect taught herein is a method for generating a genetically modified plant which uses water more efficiently than a non-geneticall modified plant of the same species, the method comprising introducing into a plant or parent of the plant a one or more loci of a functional equivalent thereof or an agent which modulates expression of an indigenous one or more loci wherein the level of expression of the one or more loci causes a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the present disclosure further teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus selected from the list provided in Table I B or a molecule which modulates expression of an indigenous locus.
- the present disclosure further teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA encoding a PIN protein, or a molecule which modulates expression of an indigenous PIN locus.
- PINs include SbPIN4 and SbPIN2 and other SbPFNs listed in Table 1 A as well as their equivalent in other plants.
- sorghum SbPIN4 and SbPIN2 are major drought adaptation genes which have been fine-mapped in multiple studies to a region between markers txp536 and txp442 on chromosone3 (Stgl) and a region between makers txp512 and txp530 on chromosome 3 (Stg2). Differences in auxin signalling explain all of the multiple phenotypes observed in plants containing SbPlN4 or 2. Another gene is SbPIN5.
- Phenotypes exhibited by SbPFN4 and SbPIN2 plants are explained directly by changes in auxin efflux and include reduced tillering, smaller leaves (both length and width), reduced leaf mass and increased root:shoot ratio. Phenotypes exhibited by SbPIN4 and SbPIN2 plants can also be explained indirectly (or as emergent consequences of these direct effects) and include increased availability of water at anthesis, higher leaf N concentration at anthesis, increased transpiration and biomass per unit leaf area, higher transpiration efficiency, retention of green leaf area during grain filling, increased harvest index, higher grain yield, larger grain size and increased lodging resistance. Enabled herein is that SbPIN4 or 2 is operative alone or together across other major cereal and crop species to enhance drought adaptation in localities worldwide where water limits crop growth post-anthesis.
- the level of expression of an StgX such as Stgl, Stg2, Stg3a, Stg3b and/or Stg4 (as defined in Table I B), (SbPIN4) and/or Stg2 (SbPIN2) [see Table 1 A] in all or certain plant tissue confers or confer drought adaptation both directly, and indirectly, ultimately leading to higher grain yield, larger grain size, and lodging resistance under water-limited conditions.
- the level of expression of an StgX such as Stgl (SbPIN4) and/or Stg2 (SbPIN2) in all or certain plant tissue confers or confer drought adaptation both directly, and indirectly, ultimately leading to higher grain yield, larger grain size, and lodging resistance under water-limited conditions.
- This aspect extends to OsPINS which corresponds to SbPIN4 and OsPIN3a which corresponds to SbPIN2.
- Other PIN proteins taught herein include those listed in Table 1 A and their equivalents in other plants.
- StgX encodes a Spl (squamosa promoter binding protein-like) such as but not limited to Spl 14.
- the present disclosure teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA which encodes CCD7/8 or a functional homolog or ortholog thereof or an agent which modulates the level of expression of an indigenous CCD7/8 to cause a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the present disclosure teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA which encodes WFP or a functional homolog or ortholog thereof or an agent which modulates the level of expression of an indigenous WFP to cause a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the present disclosure teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA which encodes Spl or a functional homolog or ortholog thereof or an agent which modulates the level of expression of an indigenous Spl to cause a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the present disclosure teaches a method for generating a genetically modified plant which uses water more efficiently than a non-genetically modified plant of the same species, the method comprising introducing into a plant or parent of the plant a locus or cDNA which encodes CCD7/8 or a functional homolog or ortholog thereof or an agent which modulates the level of expression of an indigenous CCD7/8 to cause a shift in water use to the post-anthesis period or increased accessibility of water during crop growth or increased transpiration efficiency resulting in increased harvest index and grain yield under water limiting conditions.
- the plants are modified or selected to change the level of expression of two or more of SbPIN l to 1 1 , IPA- 1 , WFP, Spl and/or CCD7/8.
- two or more includes 2, 3, 4, 5, 6, 7, 8. 9. 10 and 1 1 .
- Increased water availability at anthesis is achieved via reduced water use due to two mechanisms (reduced tillering and smaller leaves) in plants containing the St l or Stg2 regions. Both mechanisms, individually, appear to reduce canopy size by about 9%, on average.
- the 'low-tillering' mechanism dominates in low density environments when tillering potential is high.
- the 'small-leaf mechanism dominates in high density environments when tillering potential is low. Combined, these two mechanisms provide crop plants with considerable plasticity to modify canopy architecture in response to the severity of water limitation.
- T/LA transpiration per unit leaf area
- TE transpiration efficiency
- StgX lines such as Stgl or Stg2 lines is also observed when water deficit is less severe.
- Increased TE via introgressing Stg l or Stg2 is proposed to be due to a) proportionally higher photosynthetic capacity compared with stomatal conductance, due to smaller, thinner and greener leaves, and/or b) a decrease in transpiration while maintaining biomass. Lysimetry studies indicate that both of these mechanisms contribute to higher TE in Stgl or Stg2 lines, with the reduction in transpiration the primary mechanism.
- Changes in transpiration per unit leaf area is proposed to be due to a) number of stomata, b) size of stomatal aperture, c) changes in the timing of stomatal opening and closing relative to VPD, and/or d) the number of hair base cells (which affects the boundary layer and hence T/LA).
- Introgressing Stgl for example, into RTx7000 modified leaf anatomy by increasing the number of bundle sheath cells surrounding the vascular bundle.
- Increased water use during grain filling is achieved via (i) increased water availability at anthesis and (ii) increased water accessibility (better water extraction and deeper or greater lateral spread) during grain filling.
- Crop water use (CWU) before anthesis was negatively correlated with CWU after anthesis in an artificial drought (rain-out shelter [ROS]) experiment.
- ROS rain-out shelter
- StgX such as Stgl or Stg2 confers drought adaptation by being associated with pre- and post-anthesis biomass production.
- the Stgl or Stg2 region for example, reduce the pre:post anthesis biomass ratio below a critical level, increasing grain yield and lodging resistance.
- the level and location of expression of StgX such as Stgl , Stg2, Stg3 (including Stg3a and Stg3b) and/or Stg4 (e.g. as defined in Table I B) facilitates one or more of the following phenotypes:
- each of the key StgX mechanisms maps to a defined region, suggesting that the action of a single gene has multiple pleiotrophic effects.
- the present invention further contemplates a business model to enhance economic returns from crop production.
- a business model is also taught herein for improved economic returns on crop yield, the model comprising generating crop plants having a selected StgX trait or elevated or reduced StgX trait resulting in the crop plant having a shift in water use by the plant to the post-anthesis period thereby increasing HI and grain yield under water-limited conditions, obtaining seed from the generated crop plant and distributing the seed to grain producers to enhance yield and profit.
- Taught herein is a plant management system to reduce crop reliance on water or to otherwise improve water use efficiency and to enhance grain or product yield.
- the plant management system includes the generation of a drought adapted crop including cereal plants using the selection and expression of an StgX locus or a functional equivalent thereof alone or in combination with the introduction of other useful traits such as grain size, root size, salt tolerance, herbicide resistance, pest resistance and the like.
- the plant management system comprises generation of drought adapted plants and agricultural procedures such as irrigation, nutrient requirements, crop density and geometry, weed control, insect control, soil aeration, reduced tillage, raised beds and the like.
- StgX examples include Stgl : Fine-mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes, Candidates in tail between txp580 and txp38 containing 178 annotated genes; Stg2: Fine-mapped region between txp512 and txp2 containing 15 annotated genes, Larger region between txp3 1 and txp530 containing 241 annotated genes; Stg3a: Entire region between txp298 and sPb-2568 containing 520 annotated genes; Stg3b: Entire region between sPb-2568 and txp !
- Stg4 Entire region defined by txp283 and txpl 5 containing 306 annotated genes. Examples of loci in these regions are listed in Table 1 B (and Table 1 C). Examples of a StgX locus include SbPINl to 1 1 , IPA-1 , WFP, Spl and CCD7/8 and their equivalents in other plants.
- the disclosure teaches a means to induce or enhance drought adaptation capacity in a plant by introducing do novo one or more features of the stay-green phenotype or elevating expression of an existing one or more StgX loci in a plant and/or selecting an StgX polymorphic variant with improved or enhanced expression or product activity.
- the manipulation of the stay-green phenotype may be done alone or as part of an integrated plant management system which may include further trait selection and/or improved agronomical techniques.
- the resulting crops use water more efficiently and have a higher yield of grain and increased grain size.
- the business model extends to collecting seed from drought adapted or enhanced crop plants for distribution to growers to ultimately increase grain yield.
- the present disclosure further teaches the use of a genetic agent selected from (i) a StgX locus; (ii) a functional equivalent of the StgX locus; and (iii) an agent which modulates expression of an indigenous StgX locus in the manufacture of a drought adapted plant.
- a "functional equivalent” includes a cDNA.
- StgX loci are identified encoding one or more of a locus listed in Table IB which, when expressed or up-regulated or down-regulated in all or selected tissues in a plant or when a particular polymorphic variant of any one or more is selected in breeding or by genetic engineering, promotes a stay-green phenotype.
- StgX loci are identified encoding one or more of SbPI l to 1 1 , IPA- I , WFP, SPL and/or CCD7/8 which, when expressed or up-regulated or down- regulated in all or selected tissues in a plant or when a particular polymorphic variant of any one or more is selected in breeding or by genetic engineering, promotes a stay-green phenotype.
- Genetically modified plants and their progeny exhibiting the stay-green trait are also enabled herein as well as seeds, fruit and flowers and other reproductive or propagating material.
- the locus is in Stgl selected from PIN5, GID 1 L2, P45098A1 , indole-3-acetate and brassinosteroid insensitive.
- the locus is in Stg2 and is auxin efflux carrier component 3a (PIN3a).
- the locus is in Stg3a selected from leaf senescence protein-like (Sb02g023510), leaf senescence prote n-like (Sb02g023520), RAMOSA1 C2H2 zinc- finger transcription factor (Sb02g024410), putative auxin-independent growth promoter (Sb02g024540), similar to dehydration-responsive protein-like (Sb02g024670), similar to glucose transporter (Sb02g024690), WRKY transcription factor 76 (Sb02g024760), glutamine synthetase-like protein (Sb02g025100), senescence-associated protein DH (Sb02g025180), putative alanine aminotransferase (Sb02g025480), auxin-induced proteinlike (Sb02g025610), auxin-induced proteinlike (Sb02g025610), aux
- the locus is in Stg3b selected from putative auxin-independent growth promoter (Sb02g027470), squamosa promoter-binding-like protein 17 (Sb02g028420), similar to Os09g0505400 (OsPIN9) protein (Sb02g029210), squamosa promoter-binding- ike protein 17 (Sb02g029300) similar to auxin-induced protein-like (Sb02g029630).
- the locus is in Stg4 selected from brassinosteroid LRR receptor (Sb05g006842), brassinosteroid LRR receptor (Sb05g006860), putative far-red impaired response protein (Sb05g007130), cytochrome P450 84A1 (Sb05g007210), gibberellin receptor GID1 L2 (Sb05g007270), gibberellin receptor GID1L2 (Sb05g007290), sucrose- phosphate synthase (Sb05g007310), aquaporin SIP 1-1 (Sb05g007520), gibberellin 20 oxidase 2 (Sb05g008460), OsIAA29 - auxin-responsive (Sb05g008510), OsIAA29 - auxin- responsive (Sb05g008512), protein gibberellin receptor GID1 L2 (Sb05g0086 0), similar to aminotransferase, putative (Sb05g006842
- Stgl A quantitative trait locus (QTL) referenced to as Stgl which is an example of an StgX has been identified which increases or enhances water use efficiency by sorghum plants, Stgl encodes a sorghum bicolor member of the auxin efflux carrier component 4 family, PIN4 (or SbPIN4).
- This major drought adaptation gene has been fine-mapped in multiple studies to a 152 gene block between markers txp563 and txp442. Changes in auxin efflux explains all of the multiple phenotypes observed in plants containing SbPIN4.
- the candidate gene (and promoter region) is sequenced in the two parents of the fine-mapping population (RTx7000 and Tx642) to identify a single nucleotide polymorphism.
- RNA expression profiling of the Stgl fine-mapping population is also conducted for a subset of lines, times and organs.
- Phenotypes exhibited by SbPIN4 plants that could be explained directly by enhanced auxin availability include reduced tillering, smaller leaves (both length and width), reduced leaf mass and increased root.shoot ratio.
- Phenotypes exhibited by SbPIN4 plants that could be explained indirectly (or as emergent consequences of these direct effects) include increased availability of water at anthesis, higher leaf N concentration at anthesis, increased transpiration and biomass per unit leaf area, reduced pre:post anthesis biomass ratio, higher transpiration efficiency, retention of green leaf area during grain filling, increased harvest index, higher grain yield, larger grain size and increased lodging resistance. It is proposed that SbPIN4 works across other major cereal and crop species to enhance drought adaptation in localities worldwide where water limits crop growth post- anthesis.
- Stgl (SbPIN4) confers drought adaptation both directly, and indirectly, ultimately leading to higher grain yield, larger grain size, and lodging resistance under water-limited conditions.
- StgX regions are defined as follows: Stgl : Fine-mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes, Candidates in tail between txp580 and txp38 containing 178 annotated genes; Stg2: Fine-mapped region between txp512 and txp2 containing 15 annotated genes, Larger region between txp31 and txp530 containing 241 annotated genes; Stg3a: Entire region between txp298 and sPb-2568 containing 520 annotated genes; Stg3b: Entire region between sPb-2568 and tx l 79 containing 291 annotated genes; Stg4: Entire region defined by txp283 and txpl 5 containing 306 annotated genes.
- Canopy size is further reduced (adaptive response) in a mild drought (-10%) and more severe drought ( ⁇ 15%).
- Low tillering is primarily a constitutive response.
- Small leaf size is both a constitutive and adaptive response.
- Increased water availability at anthesis is also achieved via increased water accessibility (better water extraction and deeper or greater lateral spread).
- T/LA transpiration per unit leaf area
- TE transpiration efficiency
- Increased TE via introgressing Stgl may be due to a) proportionally higher photosynthetic capacity compared with stomatal conductance, due to smaller, thinner and greener leaves, and/or b) a decrease in transpiration while maintaining biomass. Lysimetry studies indicate that both of these mechanisms contribute to higher TE in Stg l lines, with the reduction in transpiration the primary mechanism.
- Changes in transpiration per unit leaf area could be due to a) number of stomata, b) size of stomatal aperture, c) changes in the timing of stomatal opening and closing relative to VPD, and/or d) the number of hair base cells (which affects the boundary layer and hence T/LA).
- Introgressing Stgl into RTx7000 reduced the number of stomata and increased the number of hair base cells per unit leaf area in leaves 7 and 10; both mechanisms can conserve water by reducing T/LA.
- Introgressing Stgl into RTx7000 modified leaf anatomy by increasing the number of bundle sheath cells surrounding the vascular bundle.
- the increased number of cells in the bundle sheath might also contribute to increased photosynthetic assimilation and hence TE.
- Increased water use during grain filling is achieved via (i) increased water availability at anthesis and (ii) increased water accessibility (better water extraction and deeper or greater lateral spread) during grain filling. a) Increased wate availability at anthesis
- Crop water use (CWU) before anthesis was negatively correlated with CWU after anthesis in an ROS experiment.
- CWU Crop water use
- Stgl region confers drought adaptation via a link between pre- and post-anthesis biomass production. The Stg l region reduces the pre.post anthesis biomass ratio below a " critical level, increasing grain yield and lodging resistance.
- Delayed leaf senescence (stay-green), higher grain yield and lodging resistance are consequences of higher plant water status during grain filling (due to increased water use during grain filling).
- RTx7000 produced 41% more (P ⁇ 0.05) culms/m2 than B35 (14.07 vs. 10.00).
- Introgression of the Stgl region alone into RTx7000 (6078-1) reduced culms/m2 significantly (PO.05) compared with RTx7000 (9.40 vs. 14.07).
- additional introgressions of either Stg2 or Stg4 increased culm numbers to 10.49 (1 ,2 combination) and 10.74 (1 ,4 combination). Note that the three near- isolines containing no Stg regions (2212-3, 2235-1 1 and 6120-16) also exhibited high tillering equivalent to Tx7000.
- RTx7000 produced almost eight- fold more (P ⁇ 0.05) GLAAt than B35 (15460 vs. 1980). Introgression of the Stg l region alone into RTx7000 (6078-1 ) reduced GLAAt significantly (P ⁇ 0.05). compared with RTx7000 (3121 vs 15460). Compared with Stgl only, additional introgressions of either Stg2 or Stg4 increased GLAAt to 4187 (1 ,2 combination) and 4797 (1 ,4 combination). All lines containing Stgl (in any combination) were not significantly different (P ⁇ 0.05) in GLAAt from Stgl alone.
- Table 4 is a list of genes located between markers txp581 and txp563. Note that a strong candidate for low tillering (auxin efflux carrier component 5) is located in an 18- gene block between markers txp563 and txp441.
- a subset of the Stgl fine-mapping population was grown in the field at the Rain- Out Shelter (ROS) under high and low water conditions, with each water treatment split for high and low density. This created four water regimes with increasing levels of water deficit: HWLD (least stressed) ⁇ HWHD ⁇ LWLD ⁇ LWHD (most stressed). The number of culms per plant was measured at 44 days after emergence in each plot. Differences were most obvious in the Low Density (LD) treatment, since expression of tillering is maximized in this treatment. On average, RTxVOOO, 10568-2 and 10709-5 produced 27% more culms per plant than 6078-1 and 10604-5 under LWLD conditions (2.05 vs.
- ROS Rain- Out Shelter
- the total number of tillers was the sum of T2, T3 and T4, where T2 was the tiller emerging from the axil of leaf 2 (and so on for T3 and T4), including secondary tillers.
- Significant genotypic variation was observed for all of the traits relating to tillering in this study (Table 5), with heritabi lilies generally above 30.
- T4 tiller numbers also varied among genotypes. 6078-1 produced a T4 tiller in 3 ⁇ of 4 replicates, while RTx7000 produced a T4 in all 4 replicates. Hence the Stgl introgression essentially prevented the growth of T2 and T3 tillers in a RTx7000 background.
- a 'marker x trait' analysis identified a 7 cM region between txa3676 and txp442 containing about 60 genes that was significant (PO.Q5) for all of the key tillering traits except for the presence of T4 (Table 6), suggesting a tillering gene(s) is located in this block. Note that the top of this block is the same breakpoint (between txa3676 and txp536) as already identified by the field fine-mapping studies, validating the previous result with a more specific phenotype (T2) under controlled conditions.
- Table 6 is a summary of P-values for various tillering traits measured at the LI 1 " harvest. Significant differences (PO.05) are shaded in yellow while differences (P ⁇ 0.1 ) are shaded in green.
- Table 7 shows the presence of tillers (T1-T3) and total tiller number for eight high-tillering recombinants (brown shading) and eight low-tillering recombinants (green shading) from the Stgl fine-mapping population.
- Stepping up through the markers in Figure 10 gain-of-function (low tillering) is achieved in recombinant 10604-1 -157-5 at marker txp587. This would mean the low- tillering gene(s) resides in a block extending down to (but not including) txp446 and up to (but not including) txp581. Stepping down through the markers, gain-of-function (low tillering) is achieved in three recombinants (10604-1-195-5, 10604-1-56-7, 10604-1-477-4) at one of three markers: txp563, txa3676 or txa2986 (missing marker data prevents a more exact location).
- Table 8 shows the presence of tillers (T1-T4), including secondary tillers, and total tiller number for five high-tillering recombinants (brown shading) and three low- tillering recombinants (green shading) from the Stg l fine-mapping population.
- Stepping up through the markers in Figure 12 gain-of-function (low tillering) is achieved in recombinant 10604-1 -157-5 at marker txp587.
- Stgl confers two mechanisms for reducing canopy size: a) reduced tillering, and b) reduced leaf size. Combined, these two mechanisms provide a fair degree of plasticity for the plant to modify canopy architecture in response to environmental and/or management factors.
- leaf size distribution pattern was similar for 6078-1 and RTx7000 in the milder water deficit (LD), yet leaves were significantly smaller in 6078-1 (up to 18% smaller) under greater water deficit (HD), suggesting an adaptive response by Stgl plants to increasing water deficit.
- introgressing the Stgl region into RTx7000 reduced the size of the four largest leaves (L10-L13) by an average of 16.5% in the more severe water deficit (HD). Since there was little tillering in either genotype in this treatment, reduced leaf size in 6078-1 should have markedly decreased canopy size and hence crop water use (assuming similar transpiration per unit leaf area).
- 0261] Note that the leaf size reduction mechanism associated with Stgl appears to operate in both the presence (LD) and absence of tillering (HD), but appears to be best expressed under HD where uniculm and high water deficit conditions generally occur.
- Leaf area varied significantly (PO.001 ) among genotypes with a heritability approaching 60 for leaves 4 and 5. Introgressing the Stgl region into RTx7000 reduced the area of leaves 1-6 ( Figure 16). For example, L6 area was 22% higher in RTxTOOO (67.4 cm2) than 6078-1 (55.3 cm2), although this difference was not significant at the P ⁇ O.05 level. The area of L6 ranged from 47.8 cm2 to 93.9 cm2 (LSD [0.05] 21), with a heritability of 42.
- Table 9 is a summary of predicted means, P-value and heritability of leaf size traits measured at the Ll l harvest. Significant differences (PO.05) are shaded in yellow
- GLA green leaf area.
- DW dry weight.
- SLW_L9_L1 1 specific leaf weight.
- 16 txp581 new 150.3-151.9 0.03 0.1 1 0.60 0.87 0.22 0.19 0.01 0.91 0.49 0 91 0.52
- J0273J Leaf number and length were linearly correlated for the parents of the Stgl fine- mapping population ( Figure 20). Introgressing Stgl into RTx7000 resulted in a reduction in the length of leaves 8-10, with L10 being 7% shorter in 6078- 1 than RTx7000 (550 vs. 592 mm).
- Gain-of- function (short leaf) is achieved in recombinant 10604-1 -222- 1 between markers txa2986 and txp542. This would mean the small-leaf gene(s) resides in a block extending down to (but not including) txp58 Land up to (but not including) txp440. Hence the 'small leaf gene maps to the same region as the 'low tillering' gene.
- Reduced crop water use at anthesis can be caused by a) a smaller canopy size with equivalent transpiration per unit leaf area, b) an equivalent canopy size with lower transpiration per unit leaf area, or c) a smaller canopy size and lower transpiration per unit leaf area.
- T Transpiration
- LA leaf area
- T/LA transpiration per leaf area
- T Transpiration
- RTx7000 4898 vs 7082 cm2
- T/LA Transpiration
- RTx7000 5.15 v.v 4.70
- the net result was a 22% reduction in water use per plant (T) in Stgl compared with RTx7000 (25.6 v.v 32.7 1), primarily due to reduced canopy size.
- the increase in T/LA exhibited by Stgl may itself be a drought adaptation mechanism, cooling the leaf and enabling photosynthesis to continue.
- the plasticity in T/LA appears to be particularly important in the regulation of plant water status.
- Increased water availability at anthesis may also be achieved via increased water accessibility due to better water extraction and/or deeper or greater lateral spread of roots in plants containing the Stgl regio
- Root mass and root:shoot ratio were higher in Stg l than RTx7000 at the Leaf 6 stage. There was considerable transgressive segregation for these traits in the Stgl fine-mapping population. The relation between root mass and root:shoot ratio highlights the opportunity for further genetic advance in these traits.
- Root mass per leaf area ratio can be used as a drought adaptation index at the seedling stage since it integrates the capacity of the plant to access water (root mass) with the capacity of the plant to utilise water (leaf area). A higher index indicates a greater capacity to access water per unit leaf area. Stgl exhibited a higher root mass per leaf area ratio relative to RTx7000 due to both a higher root mass and a smaller leaf area.
- root harvest index (root.totalbio ratio) at the L6 stage mapped to txa3676 and txa2986, the same location as the Stgl candidate gene (Table ⁇ ).
- Table 1 1 is a summary of P- values for various leaf size (blue shading), tillering (brown shading) and biomass (grey shading) traits measured at the L6 and L I 1 harvests. Significant differences (P ⁇ 0.05) are shaded in yellow while differences (P ⁇ 0.1 ) are shaded in green.
- Table 12 shows mainstem, tiller and total biomass per leaf area for RTx7000 (recurrent parent) and a number of near-isogenic lines containing various Stgl introgressions grown under high and low water stress at Biloela, Queensland, Australia.
- Stgl and RTx7000 displayed equivalent B/LA under low water stress. However, T was -7% lower in Stgl , due to ⁇ 10% lower T/LA which, in turn, increased TE by -9% ( Figure 53). Therefore, Stgl maintained biomass but used less water compared with RTx7000.
- B/LA was ⁇ 6% higher in Stg l compared with RTx7000.
- B/LA was positively correlated with T/LA but not with TE.
- the higher .B LA displayed by Stgl was due to higher T/LA.
- B/LA was positively correlated with T/LA and negatively correlated with TE under high water stress.
- Stgl used -22% less water than RTx7000 during the pre-anthesis period. Therefore, Stgl would have significantly more water available to fill grain, despite lower biomass at anthesis.
- Increased TE via introgressing Stgl may be due to a) proportionally higher photosynthetic capacity compared with stomatal conductance, due to smaller, thinner and greener leaves, or b) a decrease in transpiration per leaf area while maintaining biomass per leaf area
- Greener leaves may increase photosynthetic capacity and therefore water use efficiency.
- photosynthesis increased with SPAD value until reaching a plateau at a SPAD of -48.5 ( Figure 33).
- the line (6078-1) with the highest SPAD value (51.6) exhibited a relatively low rate of photosynthesis (32.1 J/m2/d). This result is either a) anomalous, or b) indicates a real decline in photosynthesis at high SPAD values.
- Leaf greenness (SPAD) and WUE were positively correlated in a subset of the Stgl fine-mapping population ( Figure 34).
- Increased TE via introgressing Stgl may be due to a) proportionally higher photosynthetic capacity compared with stomatal conductance, due to smaller, thinner and greener leaves, or b) a decrease in transpiration per leaf area while maintaining biomass per leaf area
- Transpiration efficiency was negatively correlated with transpiration per leaf area (T/LA) under low and high VPD conditions ( Figure 36) in a set of Stg NILs, including the recurrent parent (RTx7000).
- the ranking of Stg NILs relative to RTx7000 interacted with VPD conditions. For example, T/LA in Stgl was lower relative to RTx7000 under high VPD conditions, yet higher than RTx7000 under low VPD conditions.
- Changes in transpiration per unit leaf area could be due to a) number of stomata, b) stomatal aperture, c) changes in the timing of stomatal opening and closing relative to VPD, and/or d) number of hair base cells (which affects the boundary layer and hence
- T/LA Introgressing Stg l into RTx7000 variously affected T/LA, depending on VPD conditions. Relative to RTx7000, Stgl increased T/LA by -9% under low VPD and decreased T/LA by -10% under high VPD.
- T/LA can be regulated by a) the number of stomata per unit leaf area, b) the size to the stomatal aperture, c) the timing of stomatal opening and closing, and/or d) the number of hair base cells (which affects the boundary layer and hence T/LA). Measurements of two of these four components (a and d) have been made.
- Increased water use during grain filling is achieved via (i) increased water availability at anthesis and (it) increased water accessibility (better water extraction and deeper or greater lateral spread) during grain filling a) Increased water availability at anthesis
- Crop water use (CWU) before anthesis was negatively correlated with CWU after anthesis in the ROS experiment ( Figure 37). For example, saving 20 mm of water before anthesis (165 vs 185 mm) enabled the utilization of an additional 20 mm after anthesis (80 mm vs 60 mm). So all of the water conserved before anthesis was util ized by the crop after anthesis. Overall, a 25% increase in water use after anthesis in this experiment resulted in a 25% increase in grain yield (400 vs 300 g/m2). This translated to 50 kg/ha of grain for every additional mm of water available. While this data supports the concept that increased water use during grain filling is achieved via increased water availability at anthesis, it does not tell us anything about increased water accessibility during grain filling. b) Increased water accessibility during grain filling
- Post-anthesis stem mass is a component of lodging resistance. Analysis of this component provides some understanding of how Stg introgressions affect lodging resistance. Reducing PPBR from >8 to ⁇ 4 resulted in a gradual increase in post-anthesis stem mass. However, further reducing PPBR below 4 resulted in a relatively sharp increase in post-anthesis stem mass. Introgressing Stg 1 into RTx7000 increased post-anthesis stem mass under both LD (marginal increase) and HD (significant increase) conditions.
- the pre:post anthesis biomass ratio also affected lodging resistance ( Figure 44).
- post anthesis stem mass PASM
- PPBR post anthesis stem mass
- a high pre:post anthesis biomass ratio increased the amount of stem reserves remobilized during grain filling, thus reducing stem biomass and increasing the likelihood of lodging.
- Stgl a significantly reduced the amount of stem reserves mobilized under LD (-5 vs 65 g/m2) and HD (-80 v.v 140 g/m2).
- Stgl significantly reduced the amount of stem reserves mobilized under HD (-80 vs 140 g/m2), but not LD.
- the extent of stem reserves mobilized was greater under HD than LD, reflecting the greater water deficit under HD.
- the difference in stem reserve mobilization between HD and LD was twofold in RTx7000 (about 140 vs 70 g/m2).
- pre-anthesis biomass varied by only -5% (from 522 to 552 g/m2) among genotypes, yet post-anthesis biomass varied almost twofold (from 173 to 313 g/m2).
- post-anthesis biomass varied almost twofold (from 173 to 313 g/m2).
- 10709-5 and RTx7000 both produced -550 g/m2 of pre- anthesis biomass, yet the Stg l recombinant (10709-5) produced ⁇ 60% more post-anthesis biomass (310 vs 130 g/m2).
- the two density treatments provide a continuum in the range of PPBR from ⁇ 2 to >5, although the slope of the regression was greater for LD than HD.
- reducing PPBR from ⁇ 6 to -3.5 resulted in a gradual increase in PAB from -130 g/m2 (RTx7000) to -180 g/m2 ( 10604-5).
- Further reducing PPBR below ⁇ 3 under LD resulted in a steeper increase in PAB, presumably because more water was available during grain filling when the PPBR ratio fell below three.
- the pre:post anthesis biomass ratio also affected lodging resistance.
- post anthesis stem mass PASM
- PASM post anthesis stem mass
- PPBR post anthesis stem mass
- a high pre:post anthesis biomass ratio increased the amount of stem reserves remobilized during grain filling, thus reducing stem biomass and increasing the likelihood of lodging.
- Stgl significantly reduced the amount of stem reserves mobilized under HD (-100 160 g/m2). The extent of stem reserves mobilized was greater under HD than LD, reflecting the greater water deficit under HD.
- LWP leaf water potential
- RWC relative water content
- the RWC of FL-2 was negatively correlated with the relative rate of leaf senescence at mid-grain filling under both high and low densities in a set of Stg NILs including the recurrent parent. Correlations for HD and LD were parallel, but offset by about 0.35 units of leaf senescence, i.e. for a given level of RWC, say 70, the relative rates of leaf senescence were 2.1 and 2.45 for LD and HD, respectively. Introgressing the Stg l region into RTx7000 increased RWC at mid-grain filling (FL-2) and decreased the relative rate of leaf senescence under both HD and LD, although the impact was greater under HD.
- Post-anthesis biomass is mainly comprised of a) post-anthesis stem mass (PASM), a measure of stem reserve mobilization and a component of lodging resistance, and b) grain yield.
- PASM post-anthesis stem mass
- Grain-growers require that both grain yield and lodging resistance be maximized, i.e. they do not want one at the expense of the other.
- Post-anthesis stem mass was highly linearly correlated with PAB under HD and LD conditions ( Figure 52). While Stgl had little impact on PASM under the milder drought (LD), the amount of dry mass translocated from the stem during grain filling was much less in Stgl compared with RTx7000 (85 vs. 339 g/m2) under the more severe drought (HD).
- PASM PA-100 vs. -50 g/m2
- LD -100 vs. -50 g/m2
- Stgl produced -28% more PAB than Stgla for equivalent stem reserve utilisation.
- Stgl a and to a lesser extent Stgl both increased PAB relative to RTx7000.
- Post-anthesis stem mass was highly linearly correlated with PAB under HD and LD conditions (Figure 56. Genetic variation in utilization of post-anthesis stem reserves ranged from -30- 1 10 g/m2 under LD, and from -100- 160 g/m2 under HD, reflecting the higher level of stress in the HD treatment. Under HD, the Stgl parent (6078- I ) and two of the Stg l recombinants (10604-5 and 10709-5) utilized significantly less stem reserves compared with RTx7000 (-100 vs.160 g/m2), yet produced more PAB than RTx7000 (-170 vs. 130 g/m2).
- RWC at mid-grain filling in FL-2 was positively correlated with grain yield under HD and LD.
- grain yield was higher under LD than HD for a given level of RWC.
- RWC and grain yield were higher in Stgl than RTx7000 under both crop densities. For example in Stgl under HD, a 26% increase in RWC was associated with a 58% increase in grain yield, relative to RTx7000.
- CWU during grain filling remained low (at a benchmark of ⁇ 85 and 95 mm for LD and HD, respectively) until the pre:post anthesis biomass ratio fell below ⁇ 3 and 2.5 for LD and HD, respectively ( Figure 58). Below this critical value, CWU during grain filling increased significantly for each incremental reduction in this ratio. Under both densities, Stgl introgressions reduced the PPBR sufficiently to significantly increase CWU relative to RTx7000.
- CWU during grain filling remained low (at a benchmark of ⁇ 58 mm for HD) until the pre:post anthesis biomass ratio fell below -3.5 ( Figure 62). Note that the PPBR did not drop below the critical threshold in any genotype under HD, hence CWU during grain filling remained relatively low for all genotypes in this treatment. However, as genotypes fell below this critical value in the LD treatment, CWU during grain filling increased significantly for each incremental reduction in this ratio. Only one Stgl recombinant (10709-5) increased CWU during grain filling relative to RTx7000.
- the score (S value: a measure of the similarity of the query to the sequence shown), E-value (the probability due to chance, that there is another alignment with a similarity greater than the given S score), %ID and length of sequence homology for each of the 1200 hits were collated.
- the relationship between the 4 measures was analyzed and the S score was selected as the main measure to assess likelihood of sequence similarity.
- 3 S score categories were identified (> 1000; >499 and ⁇ 1000; ⁇ 499) and a list of 1 1 sorghum genes with scores >499 (i.e. in the first 2 categories) was produced. See Table 13.
- SbPIN4 was generally (across all conditions) more highly expressed in roots and steins and less expressed in leaves, while SbPIN2 generally showed higher expression in leaves and stems and lower expression in roots).
- Tables IB and 1C provide examples of loci located at: Stgl : Fine-mapped region between txp563 and txp581 containing 60 annotated genes, Larger middle region between txp440 and txp580 containing 307 annotated genes.
- Stg2 Fine-mapped region between txp512 and txp2 containing 15 annotated genes, Larger region between txp31 and txp530 containing 241 annotated genes; Stg3a: Entire region between txp298 and sPb-2568 containing 520 annotated genes; Stg3b: Entire region between sPb-2568 and txpl79 containing 291 annotated genes; Stg4: Entire region defined by txp283 and txplS containing 306 annotated genes, How these various loci fit in various biochemical and physiology pathways is depicted in Figure 68.
- the locus is in Stgl selected from PIN5. GID1 L2, P45098A1 , indole-3-acetate and brassinosteroid insensitive.
- the locus is in Stg2 and is auxin efflux carrier component 3a (PIN3a),
- the locus is in Stg3a selected from leaf senescence protein-like (Sb02g023510), leaf senescence protein-like (Sb02g023520), RAMOSA l C2H2 zinc- finger transcription factor (Sb02g024410), putative auxin-independent growth promoter (Sb02g024540), similar to dehydration-responsive protein-like (Sb02g024670), similar to glucose transporter (Sb02g024690), WRKY transcription factor 76 ' (Sb02g024760), glutamine synthetase-like protein (Sb02g025100), senescence-associated protein DH (Sb02g025180), putative alanine aminotransferase (Sb02g025480), auxin-induced protein- like (Sb02g025610), auxin-induced protein-like (Sb02g025620), putative far-red impaired response protein (Sb02g025670),
- the locus is in Stg3b selected from putative auxin-independent growth promoter (Sb02g027470), squamosa promoter-binding-like protein 17 (Sb02g028420), similar to Os09g0505400 (OsPIN9) protein (Sb02g029210), squamosa promoter-binding-like protein 17 (Sb02g029300) similar to auxin-induced protein-like (Sb02g029630).
- the locus is in Stg4 selected from brassinosteroid LRR receptor (Sb05g006842), brassinosteroid LRR receptor (Sb05g006860), putative far-red impaired response protein (Sb05g007130), cytochrome P450 84A1 (Sb05g007210), gibberellin receptor GID1L2 (Sb05g007270), gibberellin receptor GID1 L2 (Sb05g007290), sucrose- phosphate synthase (Sb05g007310), aquaporin SIPl-1 (Sb05g007520), gibberellin 20 oxidase 2 (Sb05g008460), OsIAA29 - auxin-responsive (Sb05g008510), OsIAA29 - auxin- responsive (Sb05g008512), protein gibberellin receptor GID1 L2 (Sb05g008610), similar to aminotransferase, putative (Sb05g006842),
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EP12850261.4A EP2780921A4 (en) | 2011-11-16 | 2012-11-16 | Drought tolerant plants produced by modification of the stay-green stgx locus |
CA2855995A CA2855995A1 (en) | 2011-11-16 | 2012-11-16 | Drought tolerant plants produced by modification of the stay-green stgx locus |
CN201280065782.6A CN104520954A (en) | 2011-11-16 | 2012-11-16 | Drought tolerant plants produced by modification of the stay-green STGX locus |
UAA201406657A UA118334C2 (en) | 2011-11-16 | 2012-11-16 | Drought tolerant plants produced by modification of the stay-green stgx locus |
BR112014011943A BR112014011943A8 (en) | 2011-11-16 | 2012-11-16 | DROUGHT TOLERANT PLANTS PRODUCED BY MODIFICATION OF THE STGX LOCUUS BEING GREEN |
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US14/358,725 US10590431B2 (en) | 2011-11-16 | 2012-11-16 | Drought tolerant plants produced by modification of the stay-green STGX locus |
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CN104725495A (en) * | 2015-04-01 | 2015-06-24 | 山西省农业科学院棉花研究所 | Cotton GhWRKY51 transcription factor, and coding gene and application thereof |
CN104725496A (en) * | 2015-04-02 | 2015-06-24 | 江苏省农业科学院 | Gossypium aridum WRKY transcription factor GarWRKY9 for regulating blossoming of plant and application |
WO2020188504A1 (en) * | 2019-03-19 | 2020-09-24 | Universidad Nacional Del Litoral | A method to improve the agronomic characteristics of plants |
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US10767189B2 (en) * | 2015-08-18 | 2020-09-08 | Monsanto Technology Llc | Methods for producing cotton plants with enhanced drought tolerance and compositions thereof |
CN106399324A (en) * | 2016-08-30 | 2017-02-15 | 山东农业大学 | Apple auxin delivery vector gene MdPIN1 for regulating root growth, and application thereof |
CN110204600B (en) * | 2019-05-07 | 2022-06-14 | 扬州大学 | BnSPL14 gene, protein and application thereof in controlling cabbage type rape plant type |
CN110452911B (en) * | 2019-06-08 | 2022-05-24 | 吉林大学 | Corn ATP (adenosine triphosphate) binding cassette transporter protein E2 gene ZmABCE2 and application thereof |
CN114591971B (en) * | 2022-03-30 | 2022-12-20 | 西北农林科技大学 | Drought-resistant VvCCD7 gene of grape as well as amino acid sequence and application thereof |
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Cited By (4)
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CN104725495A (en) * | 2015-04-01 | 2015-06-24 | 山西省农业科学院棉花研究所 | Cotton GhWRKY51 transcription factor, and coding gene and application thereof |
CN104725495B (en) * | 2015-04-01 | 2018-07-24 | 山西省农业科学院棉花研究所 | Cotton GhWRKY51 transcription factors and its encoding gene and application |
CN104725496A (en) * | 2015-04-02 | 2015-06-24 | 江苏省农业科学院 | Gossypium aridum WRKY transcription factor GarWRKY9 for regulating blossoming of plant and application |
WO2020188504A1 (en) * | 2019-03-19 | 2020-09-24 | Universidad Nacional Del Litoral | A method to improve the agronomic characteristics of plants |
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