WO2016128898A1 - Riz semi-nain tolérant à la sécheresse et procédés et matériaux connexes - Google Patents

Riz semi-nain tolérant à la sécheresse et procédés et matériaux connexes Download PDF

Info

Publication number
WO2016128898A1
WO2016128898A1 PCT/IB2016/050686 IB2016050686W WO2016128898A1 WO 2016128898 A1 WO2016128898 A1 WO 2016128898A1 IB 2016050686 W IB2016050686 W IB 2016050686W WO 2016128898 A1 WO2016128898 A1 WO 2016128898A1
Authority
WO
WIPO (PCT)
Prior art keywords
qdty
rice
drought
dwarf
allele
Prior art date
Application number
PCT/IB2016/050686
Other languages
English (en)
Inventor
Nagendra Kumar SINGH
Arvind Kumar
Prashant VIKRAM
Shalabh DIXIT
Ajay Kohli
Original Assignee
International Rice Research Institute
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by International Rice Research Institute filed Critical International Rice Research Institute
Publication of WO2016128898A1 publication Critical patent/WO2016128898A1/fr

Links

Classifications

    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1225—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold or salt resistance
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • 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
    • 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
    • A01H6/4636—Oryza sp. [rice]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • GR varieties such as IR64, Swarna, MTU1010, Samba Mahsuri, BR11, Sabitri and TDK1 have become highly popular among farmers and are grown on large acreages in rain-fed areas due to their higher yield potential and preferred grain quality traits.
  • Rain-fed rice-growing areas comprise 45% of the total cultivated rice-growing area in the world, where drought in mild, moderate, or severe forms occur in most years.
  • Eastern India and adjoining areas of Nepal occupy a large drought-affected area with an estimate of around 17 million ha.
  • widespread severe drought in much of Asia not only resulted in agricultural production losses of hundreds of millions of dollars, but also pushed millions of people into poverty.
  • Production loss from major crop failures covering 2 million hectares is estimated at US$326 million, resulting in a 3.9% decline in the 2004 agricultural gross domestic product (GDP). More than half of the rural population of Thailand relies on farm income for their owns.
  • GDP agricultural gross domestic product
  • Describe herein are methods and materials useful for improving drought tolerance of sdl semi- dwarf rice varieties.
  • the present disclosure provides methods for breaking genetic linkages between loci for drought tolerance and un-desirable traits, including tall plant height, lodging, early maturity period duration, and low-yield under well-watered conditions.
  • the present disclosure further provides methods for improving drought tolerance in sdl semi-dwarf rice varieties involving marker-assisted selection (MAS) and backcrossing.
  • MAS marker-assisted selection
  • a method of producing a semi-dwarf drought tolerant rice plant comprising: a) providing a drought tolerant donor rice plant; b) transferring a nucleic acid from the donor plant to one or more recipient semi-dwarf drought susceptible rice plants, wherein the one or more recipient plants comprise a semi-dwarf allele of sdl comprising a Y342* mutation, and wherein the transfer results in the introduction of genomic material comprising a drought tolerant allele of qDTY; ⁇ from the donor rice plant in a corresponding genomic region of the one or more recipient rice plants; and c) identifying and selecting from the one or more recipient rice plants at least one rice plant retaining its original sdl allele, and comprises within its genome the drought tolerant allele of qDTY] , wherein the drought tolerant allele of qDTY] ⁇ is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: nksd
  • the method of producing a semi-dwarf drought tolerant rice plant further comprises: providing at least one additional drought tolerant donor rice plant; transferring a nucleic acid from the at least one additional donor plant to one or more recipient rice plants identified and selected in step c), wherein the transfer results in the introduction of genomic material comprising a drought tolerant allele of at least one of qDTY 3 j, qDTY 32 , and qDTY 62 from the at least one donor rice plant in a corresponding region of the one or more recipient rice plants identified and selected in step c); and identifying and selecting from the one or more recipient rice plants of step e) at least one rice plant retaining its original sdl allele, and which comprises within its genome at least one drought tolerant allele of a QTL selected from the group consisting of: qDTY 3 qDTY 3 2 ; and qDTY 62 , wherein the drought tolerant allele of qDTY 3 ⁇ is indicated by a genomic region on chromosome 3
  • the one or more recipient rice plants identified and selected in step f) are further selected for: high yield under irrigated conditions wherein the one or more recipient plants comprises qDTY 3 ⁇ medium maturation and high yield under irrigated conditions wherein the one or more recipient plants comprises qDTY 3 2 ; and semi-dwarf plant height and high yield under irrigated conditions wherein the one or more recipient plants comprises qDTY 62 .
  • the semi-dwarf allele(s) of sdl is indicated by a 383 bp deletion (set forth in SEQ ID NO: 32) spanning a first intron and second exon of sdl (SEQ ID NO: 31).
  • the deletion may be detected using primers flanking the deletion.
  • the primers comprise Sdl -forward (5 '-C ACGC ACGGGTTCTTCC AGGTG- 3 ') (SEQ ID NO: 33) and Sdl -reverse (5'- AGGAGAATAGGAGATGGTTTACC-3 ') (SEQ ID NO: 34).
  • the at least one marker may be detected in DNA isolated from the one or more recipient rice plants.
  • the transfer of the nucleic acid may be performed by a transgenic method, by crossing, by protoplast fusion, by a double haploid technique, or by embryo rescue.
  • the transfer of the nucleic acid may be performed by crossing the drought resistant donor plant with a semi-dwarf drought susceptible rice plant to produce progeny plants comprising the semi-dwarf sdl allele, and the drought tolerant allele of qDTYj j as an introgression, and wherein the identifying and selecting step is performed on one or more progeny plants.
  • the identifying and selecting step is performed by detecting the at least one marker in DNA isolated from the one or more progeny plants.
  • the identifying and selecting step may further comprise subjecting the at least one selected rice plant to a bioassay for measuring drought tolerance, and further selecting at least one rice plant that is drought tolerant.
  • the method further comprises a step of selfing the at least one selected rice plant.
  • the method may further comprise a step of selecting at least one rice plant resulting from the selfing step that maintains the semi-dwarf sdl allele and is homozygous for the drought tolerant qDTY allele.
  • the present inventors have also produced semi-dwarf drought tolerant rice plant by performing a method described herein.
  • the present disclosure also provides a drought tolerant rice plant, or part thereof, comprising the semi-dwarf allele of sdl comprising the Y342* mutation, and the drought tolerant allele of qDTYu, wherein the drought tolerant allele of qDTY ⁇ is not in its natural genetic background.
  • a method of producing a semi-dwarf drought tolerant inbred rice plant comprising: a) producing a semi-dwarf drought tolerant rice plant according to any one of the methods described herein; b) crossing the semi-dwarf drought tolerant rice plant with itself to yield progeny rice seed; growing the progeny rice seed to yield additional semi-dwarf drought tolerant rice plants; and repeating the crossing and growing steps from 0 to 7 times to generate a semi- dwarf drought tolerant inbred rice plant.
  • Step c) may further comprise the steps of identifying and selecting inbred rice plants that are homozygous for the drought tolerant allele of qDTYu.
  • the method may further comprise selecting inbred rice plants that exhibit semi-dwarf plant height and drought tolerance.
  • the present inventors have also produced semi-dwarf drought tolerant inbred rice plants using the method described herein.
  • a semi-dwarf drought tolerant inbred rice plant obtained or obtainable by the method described herein is also provided.
  • a hybrid rice plant or a part thereof, that exhibits semi-dwarf plant height and drought tolerance is provided, wherein the hybrid rice plant is obtained or obtainable by crossing an inbred rice plant obtained or obtainable by a method described herein with a rice plant that exhibits commercially desirable characteristics.
  • a plant part as described herein may be a seed.
  • a seed of a plant produced by a method described herein is provided.
  • the donor rice plant may be selected from the group consisting of: N22; Dagaddeshi; Apo; Vandana; and Black Gora.
  • the at least one additional donor rice plant may be selected from the group consisting of: IR55419-04; Apo; Vandana; RD7; IR74371-46-1-1 ; IR743-70-1-1 ; Dular; AdaySel; Black Gora; Brown Gora; Sathi 34- 36; Basmati 334; Basmati 370, IR77298-5-6-18, Moroberekan, and N22.
  • the recipient rice plant may be selected from the group consisting of: IR8; Jaya; IR36; IR64; Swarna; MTU1010; Sambha Mahsuri; BR1; RD1 ; Kalamkatit; TDK1 ; PSBRC80; NSICRC222; Dee-Geo-Woo-Gen, IRRI119, Purbachi (Chinese 1), Sabitri, and RD25.
  • the present disclosure provides a semi-dwarf drought tolerant rice plant, or part thereof e.g., grain, comprising: a) a semi-dwarf allele of sdl comprising a Y342* mutation; and b) a drought tolerant allele of qDTYu, wherein qDTYlj ⁇ is not in its natural genetic background, and wherein the drought tolerant allele of qDTYu is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: nksdtyl_l_34; nkstdyl_l_38; RM11943; RM431 ; RM12023; Rml2091 ; and RM12146.
  • the semi-dwarf drought tolerant rice plant described herein also comprises drought tolerant allele of at least one of qDTY 3 i , qDTY 3 2 , and qDTY 62 , wherein the drought tolerant allele of qDTY 3 ⁇ is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM520; RM416; and RM 16030, the drought tolerant allele of qDTY 3 2 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM7332;
  • the drought tolerant allele of qDTY 62 is indicated by a genomic region on chromosome 6 comprising at least one marker selected from the group consisting of: RM121 ; RM3; RM541 ; and RM275.
  • the semi-dwarf allele of sdl is indicated by a 383 bp deletion (SEQ ID NO: 32) spanning a first intron and second exon of sdl (SEQ ID NO: 31).
  • the disclosure provides a method for identifying a rice plant having a semi-dwarf drought tolerant phenotype, the method comprising: a) extracting genomic DNA from a rice plant; b) detecting in the rice plant a semi-dwarf allele of sdl comprising a Y342* mutation; c) detecting in the rice plant a drought tolerant allele of qDTYu, wherein the drought tolerant allele of qDTYu is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: RM11943; RM431 ; RM12023; Rml2091 ; and RM12146; and d) identifying the rice plant as having a semi-dwarf drought tolerant phenotype if the semi-dwarf allele of sdl and at least one marker linked to the drought tolerant allele of qDTYu are detected.
  • the present disclosure provides a method of producing a rice plant part, preferably grain, the method comprising: a) growing a semi-dwarf drought tolerant rice plant as described herein or a population of said rice plants; and b) harvesting the rice plant part(s) from the rice plant or rice plants.
  • the method may further comprise the steps of processing and/or packaging the rice plant part for sale.
  • the present disclosure provides a method of producing a product from rice or a processed rice material, the method comprising: a) obtaining grain of a semi-dwarf drought tolerant rice plant as described herein; and b) processing the grain to produce the product or material.
  • a product as described herein will thus comprise a processed rice material comprising: a) a semi-dwarf allele of sdl comprising a Y342* mutation; and b) a drought tolerant allele of qDTYu, wherein qDTYl u is not in its natural genetic background, and wherein the drought tolerant allele of qDTY L1 is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: nksdtyl_l_34; nkstdyl_l_38; RM11943; RM431 ; RM12023; Rml2091; and RM12146.
  • the processed rice material in the product described herein also comprises drought tolerant allele of at least one of qDTY 3 , qDTY 32 , and qDTY 62 , wherein the drought tolerant allele of qDTY 3 i is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM520; RM416; and RM16030, the drought tolerant allele of qDTY 3 2 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM7332; RM523; and RM545, and the drought tolerant allele of qDTY 62 is indicated by a genomic region on chromosome 6 comprising at least one marker selected from the group consisting of: RM121 ; RM3; RM541 ; and RM275.
  • the drought tolerant allele of qDTY 3 i is indicated by a genomic region on chromosome 3 comprising at least one marker
  • the semi-dwarf allele of sdl is indicated by a 383 bp deletion (SEQ ID NO: 32) spanning a first intron and second exon of sdl (SEQ ID NO: 31).
  • the method may comprise the further step of c) packaging the product or processed rice material for commercial sale.
  • the method of processing employed may vary depending on the product or material being produced.
  • the processing of the rice grain may comprise one or more steps selected from the group consisting of cleaning the grain, purifying the grain, milling the grain, grading the grain, weighing the grain, steaming or parboiling the grain and mixing the grain or a processed product thereof with one or more other components.
  • Other process steps for processing rice grain for the production of food and/or beverage products or ingredients thereof will be known to a person of skill in the art and are contemplated for use in the method described herein.
  • the present disclosure provides a processed rice material or a product comprising same, comprising: a) a semi-dwarf allele of sdl comprising a Y342* mutation; and b) a drought tolerant allele of qDTYu, wherein qDTYl; ⁇ is not in its natural genetic background, and wherein the drought tolerant allele of qDTYu is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: nksdtyl_l_34; nkstdyl_l_38; RM11943; RM431 ; RM12023; Rml2091 ; and RM12146.
  • the processed rice material or product comprising same further comprises a drought tolerant allele of at least one of qDTY 3 j, qDTY 3 2 , and qDTY 62 , wherein the drought tolerant allele of qDTY 3 ⁇ is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM520; RM416; and RM 16030, the drought tolerant allele of qDTY 3 2 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM7332;
  • the drought tolerant allele of qDTY 62 is indicated by a genomic region on chromosome 6 comprising at least one marker selected from the group consisting of: RM121 ; RM3; RM541 ; and RM275.
  • the semi-dwarf allele of sdl is indicated by a 383 bp deletion (set forth in SEQ ID NO: 32) spanning a first intron and second exon of sdl (SEQ ID NO: 31).
  • the processed rice material or product comprising same is produced from grain of a semi-dwarf drought tolerant rice plant as described herein.
  • the processed rice material or product comprising same is packaged for sale.
  • the processed rice material or product comprising same is a food ingredient, beverage ingredient, a food product or a beverage product.
  • examples of such products include, but are not limited to:
  • a food ingredient or beverage ingredient selected from the group consisting of rice wholemeal, rice flour, rice bran, rice starch, rice malt, rice vinegar, rice syrup, rice oil e.g., rice bran oil, and rice bran wax;
  • a food product may be selected from the group consisting of: leavened or unleavened breads, pasta, noodles, edible rice paper, animal fodder, breakfast cereals, snack foods, cakes, dumplings, puffed rice, pastries, confectionary and foods containing a rice flour-based sauce, or
  • a beverage product selected from the group consisting of rice milk and beverages containing ethanol produced from rice e.g., rice wine or sake.
  • the processed rice material or product comprising same is non-edible for humans.
  • materials and products which are non-edible for humans and which are produced from the hulls or husks of rice grain include, but are not limited to: fuel, bedding, incubation material, livestock feeds, concrete blocks, tiles, fiberboard, ceramics, cement, filters, charcoal briquettes, and products comprising rice bran wax e.g., cosmetics, shoe creams and polishing compounds.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • FIG. 1 Improved resolution of qDTY] ./ QTL in three different N22 derived RIL mapping populations (N22/ Swarna, N22/ IR64 and N22/ MTU1010) showing location of qDTY u distal to the sdl gene. Positions of marker are shown on the chromosome bar in Mbp.
  • FIG. 2 Development of N22/Swarna BIL population with fixed sdl allele for semi-dwarf height and synchronized plant height (PH) and number of days to 50% flowering (DTF) (similar to Swarna) and minimized background effects.
  • FIG. 3 Map representing positions of the SequnomMassARRAYcustom SNP assay markers used for fine mapping of qDTY; ./ QTL region.
  • FIG. 4 Recombination break point analysis of semi-dwarf recombinant BILs possessing different combination of alleles in the qDTY; / region and its effect on grain yield under drought.
  • Figure presents a total of 7 classes. Among these 4 (class A, B, C and D) are situated above and 3 (class E, F and G) belowsdl gene. Number of individuals in class A, B, C, D, E, F and G were 3, 2, 2, 6, 2, 3 and 2 respectively.
  • FIG. 5 Graphical genotyping results presenting four groups of random genotypes; Group 1. Pre- or GR drought tolerant varieties having tall sdl allele; Group 2.GR varieties having dwarf allele of sdl with drought sensitive alleles in the qDTYu region; Group 3. Indonesian landraces of East Java (rain-fed environment) with tallness allele of the sdl gene but have low yield potential; Group 4.GR varieties which are semi-dwarf but preferred in the rain-fed areas due to high yield potential.
  • FIG. 6 The relationship of plant height to known or novel sdl or WT haplotype and to the presence of the N22 qDTYu haplotype is presented. From a total of 954 genotypes, 657 had data available for height. Numbers are percentages in relation to their WT or sd classification. Forty three percent WT, tall genotypes are associated with the N22 haplotype and 51%, semi-dwarf genotypes are not so associated, thus confirming the hypothesis that selection for sdl also selected for drought susceptibility alleles. Additional sources of dwarfism and drought tolerance are the most likely explanations for the discrepancies noted in this classification.
  • FIGS. 7A-7F Physiological characterization of four selected qDTYl. l BILs.
  • FIG. 7A Flowering time across all field physiological studies.
  • FIG 7B Apparent leaf area in the drought stress treatment of the greenhouse lysimeter experiment.
  • FIG. 7C Shoot images at 49 days after planting from the greenhouse lysimeter experiment.
  • FIG. 7D Normalized difference vegetation index (NDVI) in the 2014DS field drought stress treatment (in which stress was initiated at 60 days after sowing (DAS).
  • DAS Normalized difference vegetation index
  • FIG. 7E Maximum root depth at harvest (54 days after planting) in the greenhouse lysimeter experiment.
  • FIG. 7F percent of root length at depth (below 30 cm) in the soil cores across all field studies. (RS; reproductive stage drought stress, NS; non-stress control).
  • FIG. 8 Neighbor-joining tree based on C.S. Chord (Cavalli-Sforza and Edwards, 1967) for eleven qDTY regions. Clusters 1-5 show classification of 132rice genotypes based on the allelic diversity.
  • FIG. 9 Allelic frequency patterns at 11 different DJF QTLs across traditional varieties, drought tolerant donors and modern high yielding GR varieties.
  • Clusters 1 -5 correspond to the clusters identified in the diversity analysis ( Figure 9).
  • FIG. 10 Representative gel picture showing allelic difference between tall and semi-dwarf allele among parents (N 22, Swarna, IR 64, and MTU1010) and the recombinants.
  • FIGS. 11A-11F FIG. 11 A) Apparent leaf area in the well-watered control treatment of the greenhouse lysimeter experiment.
  • FIG. 1 IB Normalized difference vegetation index (NDVI) in the 2013DS field drought stress treatment.
  • FIG. 11C Stem:leaf ratio in the 2014DS field well-watered control treatment.
  • FIG. 1 ID Stem:leaf ratio in the 2014DS field drought stress treatment.
  • FIG. 1 IE Maximum root depth at the end of the study in the well-watered control treatment of the greenhouse lysimeter experiment.
  • FIG. 1 IF Water uptake rates in the drought stress treatment of the greenhouse lysimeter experiment.
  • FIGS. 12A-12C Class analysis with marker loci within (FIG. 12A) qDTY 62 (RM3, RM541 and RM275), (FIG. 12B) qDTY 3J (RM520, RM416 and RM16030) and (FIG. 12C) qDTY 32 (RM7332, RM523 and RM545), in IR55419-04/2* TDK1, Apo/3*Swarna and Vandana/ Way Rarem
  • qDTY 62 (RM3, RM541 and RM275): I, IR55419-04 allele; T, TDK1 allele qDTY 3 (RM520, RM416 and RM16030): S, Swarna allele; A, Apo allele qDTY 32 (RM7332, RM523 and RM545): V, Vandana allele; R, Way Rarem allele.
  • FIG. 13 Soil water potential at a depth of 30 cm as measured by tensiometers in the drought stress treatments of the field physiology studies.
  • omics/networks-based attempts to produce drought tolerant landrace rice varieties have been unsuccessful. Describe herein are methods and materials useful for improving drought tolerance of sdl semi- dwarf rice varieties.
  • the present disclosure provides methods for breaking genetic linkages between loci for drought tolerance and desirable traits, including semi-dwarf plant height, low or non-lodging, medium maturity period duration, and high yield under well watered conditions.
  • the present disclosure further provides methods for improving drought tolerance in sdl semi-dwarf rice varieties involving marker assisted selection and backcrossing.
  • yield is intended to describe the amount of grain produced by a plant or a group, or crop, of plants of the disclosure. Yield can be measured in several ways, e.g. tonnes per nectar (t/ha or t ha or average grain yield per plant in grams.
  • phenotypic trait or similar is intended to refer to a distinct variant of an observable characteristic, e.g., yield under drought conditions, of a plant that may be inherited by a plant e.g., through breeding, or may be artificially incorporated into a plant e.g., by processes such as those involving transfer of genetic material with recombinant technologies.
  • the term "introgression”, “introgressed”, “introgress” or similar refers to the movement of one or more genes, or a group of genes, from one plant variety into the gene complex of another as a result of backcrossing i.e., crossing of interspecific hybrid with one of its parents.
  • backcrossing i.e., crossing of interspecific hybrid with one of its parents.
  • the process usually involves selfing or backcrossing to the recurrent parent to provide for an increasingly homozygous plant having essentially the characteristics of the recurrent parent in addition to the introgressed gene or trait.
  • backcross refers to a process in which the plant resulting from a cross between two parental lines is (repeatedly) crossed with one of its parental lines, wherein the parental line used in the backcross is referred to as the recurrent parent. Repeated backcrossing results in replacement of genome fragments of the donor parent with those of the recurrent.
  • the offspring of a backcross is designated “BCx” or “BCx population”, where "x” stands for the number of backcrosses.
  • a "transgenic plant cell” shall be understood to mean a plant cell that has been transformed with stably-integrated, non-natural, recombinant DNA, e.g.
  • a plant cell of this invention can be an originally-transformed or nucleases -modified plant cell that exists as a microorganism or as a progeny plant cell that is regenerated into differentiated tissue, e.g. into a transgenic plant with stably-integrated, non-natural recombinant DNA, or seed or pollen derived from a progeny transgenic plant.
  • ZFNs zinc- finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISP clustered regulator interspaced short palindromic repeat
  • a plant cell of this invention can be an originally-transformed or nucleases -modified plant cell that exists as a microorganism or as a progeny plant cell that is regenerated into differentiated tissue, e.g. into a transgenic plant with stably-integrated, non-natural recombinant DNA, or seed or pollen derived from a progeny transgenic plant.
  • transgenic plant or similar shall be understood to mean a plant whose genome has been altered by the stable integration of recombinant DNA.
  • a transgenic plant may include a plant regenerated from an originally-transformed plant cell and/or progeny transgenic plants from later generations or crosses of a transformed plant.
  • recombinant shall be understood to mean the product of artificial genetic recombination. Accordingly, in the context of "recombinant DNA”, the term shall not encompass DNA naturally-occurring within a cell that is the product of a natural recombination event. However, if such DNA is isolated and expressed using recombinant means, the expression construct comprising the isolated DNA may be recombinant, as may be the resulting RNA transcript and/or translated protein. Similarly, recombinant DNA shall be understood to encompass DNA which has been genetically engineered and/or constructed outside of a cell, including DNA containing naturally occurring DNA or cDNA or synthetic DNA.
  • percent identity describes the extent to which the sequences of DNA or protein segments are similar or invariant throughout a window of alignment of sequences, for example nucleotide sequences or amino acid sequences. Percent identity is calculated over the aligned length, preferably using a local alignment algorithm, such as BLASTn, BLASTp, BLASTx, tBLASTn and/or tBLASTx.
  • promoter shall be understood to describe a regulatory DNA element for initializing transcription.
  • a "promoter that is functional in a plant cell” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell, e.g. is it well known that Agrobacterium promoters are functional in plant cells.
  • plant promoters include promoters obtained from plants, plant viruses and bacteria, such as Agrobacterium and Bradyrhizobium bacteria.
  • operably-linked or “operable linkage” or similar shall be understood to mean that a coding nucleic acid sequence is linked to, or in association with, a regulatory sequence, e.g., a promoter, in a manner which facilitates expression of the coding sequence. Regulatory sequences include promoters, enhancers, and other expression control elements that are art -recognized and are selected to direct expression of the coding sequence.
  • the term “expressed” shall be understood to mean produced, e.g. a protein is expressed in a plant cell when its cognate DNA is transcribed to mRNA that is translated to the protein. Accordingly, an expression product shall include a transcription product i.e., mRNA, and/or a translation product i.e., protein.
  • Recombinant DNA constructs in accordance with the present disclosure may be assembled using methods well known to persons of ordinary skill in the art, and typically comprise one or more promoters operably-linked to a coding DNA sequence, the expression of which provides for ant enhanced agronomic trait or manifests as a new or different phenotype.
  • Other construct components may include additional regulatory elements, such as 5' leaders and introns for enhancing transcription, 3' untranslated regions (such as polyadenylation signals and sites), DNA for transit or signal peptides.
  • promoters that are active in plant cells have been described in the literature and will be known to a skilled person. These include, but are not limited to, promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens and the CaMV35S promoters from the cauliflower mosaic virus as disclosed in U.S. Pat. Nos. 5,164,316 and 5,322,938.
  • Useful promoters derived from plant genes are found in U.S. Pat. No: 5,641,876 which discloses a rice actin promoter, U.S. Pat.
  • the promoters may be altered to contain multiple "enhancer sequences" to assist in elevating gene expression.
  • enhancers are known in the art.
  • the expression of the selected protein may be enhanced.
  • These enhancers often are found 5' to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted upstream (5') or downstream (3') to the coding sequence.
  • these 5' enhancing elements are introns.
  • Particularly useful as enhancers are the 5' introns of the rice actin 1 ⁇ See e.g., U.S. Pat. No.
  • Quantitative trait locus shall be understood to encompass polymorphic genetic loci with at least two alleles that reflect differential expression of a continuously distributed phenotypic trait (quantitative trait).
  • association with refers to, for example, a nucleic acid and a phenotypic trait that are in linkage disequilibrium, i.e., the nucleic acid and the trait are found together in progeny plants more often than if the nucleic acid and phenotype segregated independently.
  • alleles or loci or DNA polymorphisms that associate at a frequency higher than expected for independent alleles or markers, such that they appear as a haplotype may be "associated” i.e., in linkage disequilibrium or LD.
  • the variant at one locus may be predictive of the variant at the other locus on an individual chromosome.
  • marker refers to a genetic locus (a "marker focus") used as a point of reference when identifying genetically-linked loci, such as a quantitative trait locus (QTL).
  • the term may also refer to nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes or primers.
  • the primers may be complementary to sequences upstream or downstream of the marker sequences.
  • the term can also refer to amplification products associated with the marker.
  • the term can also refer to alleles associated with the markers. Allelic variation associated with a phenotype allows use of the marker to distinguish germplasm on the basis of the sequence.
  • interval refers to a continuous linear span of chromosomal DNA with termini defined by and including molecular markers.
  • crossed or “cross” or similar as used herein refers to the fusion of gametes via pollination to produce progeny (i.e., cells, seeds or plants).
  • progeny i.e., cells, seeds or plants.
  • the term encompasses both sexual crosses (the pollination of one plant by another) and selling (self-pollination, i.e., when the pollen and ovule are from the same plant or from genetically identical plants).
  • stringent hybridization conditions refers to conditions under which a probe or nucleic acid will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Thijssen (Thijssen, 1993).
  • stringent conditions are selected to be about 5-10° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions are often: 50% formamide, 5xSSC, and 1% SDS, incubating at 42° C, or, 5xSSC, 1% SDS, incubating at 65° C, with wash in 0.2xSSC, and 0.1% SDS at 65° C.
  • a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. Additional guidelines for determining hybridization parameters are provided in numerous references, e.g. Current Protocols in Molecular Biology, eds. Ausubel, et al. 1995).
  • allele refers to any one of the different forms of a gene or DNA sequence at a single locus i.e., chromosomal location including a coding sequence, non-coding sequence or regulatory sequence.
  • breeding refers to any process that generates a progeny individual. Breeding can be sexual or asexual, or any combination thereof. Exemplary non-limiting types of breeding include crossings and selfings and combinations thereof.
  • selfing refers to the process of self-fertilization wherein an individual is pollinated or fertilized with its own pollen. Repeated selfing eventually results in homozygous offspring.
  • crossing refers to the fertilization of female plants (or gametes) by male plants (or gametes).
  • gamete refers to the haploid reproductive cell (egg or sperm) produced in plants by mitosis from a gametophyte and involved in sexual reproduction, during which two gametes of opposite sex fuse to form a diploid zygote.
  • the term generally includes reference to a pollen (including the sperm cell) and an ovule (including the ovum).
  • Crossing therefore generally refers to the fertilization of ovules of one individual with pollen from another individual.
  • recipient rice plant or “recipient plant” is used herein to indicate a rice plant that is to receive DNA obtained from a donor rice plant e.g., that comprises a QTL for drought tolerance.
  • Said recipient rice plant may or may not already comprise one or more QTLs for drought tolerance, in which case the term indicates a plant that is to receive an additional QTL.
  • donor tomato plant as used herein will be understood to mean the rice plant which provides at least one genetic element associated with drought tolerance.
  • homozygous refers to a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes.
  • heterozygous means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.
  • recombination refers to the exchange of genetic material between two homologous chromosomes during meiosis.
  • DNA that is originally present on a specific location within the chromosome, e.g. linked to a gene/locus is exchanged for DNA from another plant (i.e. maternal for paternal or vice versa).
  • another plant i.e. maternal for paternal or vice versa.
  • two flanking crossover or recombination events will usually be required.
  • this exchange has taken place on both sides of a gene/locus.
  • the term "genotype” refers to the genetic constitution of a cell or organism.
  • An individual's "genotype for a set of genetic markers” includes the specific alleles, for one or more genetic marker loci, present in the individual's haplotype.
  • a genotype can relate to a single locus or to multiple loci, whether the loci are related or unrelated and/or are linked or unlinked.
  • an individual's genotype relates to one or more genes that are related in that the one or more of the genes are involved in the expression of a phenotype of interest (e.g. a quantitative trait as defined herein).
  • a genotype comprises a summary of one or more alleles present within an individual at one or more genetic loci of a quantitative trait.
  • a genotype is expressed in terms of a haplotype
  • progeny means one or more genetic descendants or offspring.
  • population means a genetically heterogeneous collection of plants sharing a common genetic derivation.
  • hybrid shall be understood to mean any offspring of a cross between two genetically unlike individuals, more preferably the term refers to the cross between two (elite or inbred) breeding lines which will not reproduce true to the parent from seed.
  • Co-segregation occurs when an allele for a trait and the allele(s) for the markers segregate and are transmitted together because they are physically close together on the same chromosome (reduced recombination between them because of their physical proximity) resulting in a non-random association of their alleles.
  • Co-segregation also refers to the presence of two or more traits within a single plant of which at least one is known to be genetic and which cannot be readily explained by chance.
  • linkage refers to the tendency of alleles at different loci on the same chromosome to segregate together more often than would be expected by chance if their transmission were independent, in some embodiments as a consequence of their physical proximity. Linkage is measured by percent recombination between loci (centimorgan, cM).
  • Locus is understood within the scope of the invention to refer to a region on a chromosome, which comprises a gene or any other genetic element or factor contributing to a trait.
  • plant part may include, but is not limited to seeds, protoplasts, leaves, stems, roots, root tips, anthers, pistils, grain, embryo, pollen ovules, flower, shoot, tissue, petiole, cells, and meristematic cells.
  • natural genetic background is used herein to indicate the original genetic background of a QTL. Such a background may for instance be the genome of a wild accession of rice.
  • TDK1 is a popular tall rice variety harboring the tall allele of sdl but is susceptible to drought.
  • determining whether the sdl gene and qDTY L1 are linked required four variables (plant height (PH), grain yield (GY) under drought, presence of the sdl gene, and markers for qDTYu) to segregate. Re-mapping of qDTY L1 in three N22-derived original RIL populations (Vikram et al. 2011) using three additional SNP markers indicated qDTYu to be located downstream to the sdl gene ( Figure 1, Table 1).
  • qDTYj ⁇ BILs slightly earlier flowering time, a plastic shoot biomass response to drought, and the ability to increase root length at depth - act concertedly to confer higher yield under drought.
  • the dynamic shoot mass response may be a growth regulation response to conserve water.
  • NAM no apical meristem protein
  • the SNAC1 gene in rice is a typical example of this family reported to increase spikelet fertility and seed setting rate under severe drought stress (Hub et al. 2006).
  • Another gene of this complex, OsNACIO is reported to be responsible for root-specific expression imparting drought tolerance and enhancing grain yield under drought (Jing et al. 2010).
  • One of the serine/threonine protein kinases in this fine mapped region (LOC_Os01g66860) has been reported to show differential response under drought stress in cultivar N22 (Gorantla et al. 2007).
  • a drought responsive zinc finger protein factor has been isolated from rice cultivar N22 (Soren et al. 2011).
  • Other examples of zinc finger proteins related to drought tolerance in rice are ZFP252, ZatlO/STZ, and WRKY genes (Xu et al. 2008; Xiao et al. 2009; Wu et al. 2009).
  • STZ was reported to increase spikelet fertility and grain yield under drought (Xiao et al. 2009).
  • Further work with candidate genes within the qDTYj ⁇ region in terms of differential expression analysis and/or transgenic validation is necessary to pinpoint the gene(s) responsible for the increased drought tolerance conferred by qDTYn (Lenka et al. 2011 ; Vikram et al. 2011).
  • OsMADS21 - MADS-box family gene with MIKCc type-box OsMADS21 - MADS-box family gene with MIKCc type-box.
  • retrotransposon protein putative, gene 38587950 38588740 LOC_Os01g66460 Ty3-gypsy subclass, expressed retrotransposon protein, putative. gene 38589984 38591752 LOC_Os01g66470 Ty3-gypsy subclass, expressed gene 38594450 38594743 LOC_Os01g66480 expressed protein
  • retrotransposon protein putative, gene 38785135 38786060 LOC _Os01g66790 unclassified, expressed gene 38788306 38788417 LOC _Os01g66800 expressed protein
  • inactive receptor kinase Atlg27190 gene 38795480 38795636 LOC _Os01g66820 precursor, putative, expressed pectinacetylesterase domain gene 38803740 38803908 LOC _Os01g66830 containing protein, expressed pectinacetylesterase domain gene 38810316 38810451 LOC _Os01g66840 containing protein, expressed pectinacetylesterase domain gene 38815050 38815179 LOC .
  • _Os01g66850 containing protein, expressed transposon protein, putative, CACTA, En/Spm sub-class, gene 38838619 38840001 LOC .
  • auxin-responsive protein putative
  • a drought tolerant allele of qDTYu is indicated by at least one marker associated with the QTL, selected from a group consisting of: nksdtyl_l_34;
  • nkstdyl_l_38 RM11943; RM431 ; RM12023; RM12091 ; and RM12146.
  • markers disclosed herein indicate linked inheritance of genetic regions or the absence of observed recombination within such genetic regions. Therefore, it is noted that the markers listed herein indicate the chromosomal region where a QTL of the invention is located in the genome of the specified rice varieties and that those markers do not necessarily define the boundaries or the structure of that QTL. Thus, the part of a QTL that comprises the essential yield-improving nucleic acid sequence(s) may be considerably smaller than that indicated by the contiguous markers listed for a particular QTL. Such a part is herein referred to as a "yield- improving part" of a QTL.
  • a yield-improving part of a QTL need not necessarily comprise any of the listed markers.
  • other markers may be used to indicate the various QTLs, provided that such markers are genetically linked to the QTLs.
  • Table 4 High yielding drought tolerant recombinant lines with qDTY u , qDTY 62 , qDTY3.1, and qDTY 3 2
  • a yield-improving part of a QTL for drought tolerance in rice may be identified by using a molecular marker technique, for instance, with one or more of the markers for a QTL disclosed herein as being linked to said QTL, preferably in combination with a yield bioassay.
  • Rice plants that do not comprise a yield-improving part of a QTL of the present invention have a relatively lower yield.
  • the markers provided by the present invention may be used for detecting the presence of one or more QTLs of the invention in a rice plant suspected of being drought tolerant, and may therefore be used in methods involving marker-assisted breeding and selection of drought tolerant rice plants.
  • detecting the presence of a QTL of the invention is performed with at least one of the markers for a QTL described herein as being linked to the QTL.
  • the present invention therefore relates in another aspect to a method for detecting the presence of a QTL for improved yield under drought stress, comprising detecting the presence of a nucleic acid sequence of the QTL in a rice plant suspected of being tolerant of drought, wherein the presence of the nucleic acid sequence may be detected by the use of the said markers.
  • the nucleic acid sequence of a QTL of the present invention may be determined by methods known to the skilled person. For instance, a nucleic acid sequence comprising a QTL or a yield- improving part thereof may be isolated from a donor plant by fragmenting the genome of the plant and selecting those fragments harboring one or more markers indicative of the QTL. Subsequently, or alternatively, the marker sequences (or parts thereof) indicative of the QTL may be used as PCR amplification primers, in order to amplify a nucleic acid sequence comprising said QTL from a genomic nucleic acid sample or a genome fragment obtained from said plant. The amplified sequence may then be purified in order to obtain the isolated QTL. The nucleotide sequence of the QTL, and/or of any additional markers comprised therein, may then be obtained by standard sequencing methods.
  • the present invention therefore also relates to an isolated nucleic acid (preferably DNA) sequence that comprises a QTL of the present invention, or a drought tolerance-conferring part thereof.
  • an isolated nucleic acid (preferably DNA) sequence that comprises a QTL of the present invention, or a drought tolerance-conferring part thereof.
  • the markers that pinpoint the various QTLs described herein may be used for the identification, isolation and purification of one or more genes from rice that encode for drought tolerance.
  • the nucleotide sequence of a QTL of the present invention may, for instance, also be resolved by determining the nucleotide sequence of one or more markers associated with the QTL and designing internal primers for the marker sequences that may then be used to further determine the sequence of the QTL outside of the marker sequences.
  • the nucleotide sequence of the markers disclosed herein may be obtained by isolating the markers from the electrophoresis gel used in the determination of the presence of the markers in the genome of a subject plant, and determining the nucleotide sequence of the markers by, for instance, dideoxy chain terminating methods, which are well known in the art.
  • the method may also comprise the steps of providing a oligonucleotide or nucleic acid capable of hybridizing under stringent hybridization conditions to a nucleic acid sequence of a marker linked to the QTL, preferably selected from the markers disclosed herein as being linked to said QTL, contacting the oligonucleotide or nucleic acid with a genomic nucleic acid of a rice plant suspected of being drought tolerant, and determining the presence of specific hybridization of the oligonucleotide or nucleic acid to the genomic nucleic acid.
  • the method is performed on a nucleic acid sample obtained from the rice plant suspected of being drought tolerant, although in situ hybridization methods may also be employed.
  • the skilled person may, once the nucleotide sequence of the QTL has been determined, design specific hybridization probes or oligonucleotides capable of hybridizing under stringent hybridization conditions to the nucleic acid sequence of said QTL and may use such hybridization probes in methods for detecting the presence of a QTL of the invention in a rice plant suspected of possessing relatively higher yield during drought stress.
  • the mutation fully associated with semi-dwarf phenotype, and is characterized by a 383 bp deletion (set forth in SEQ ID NO: 32) spanning the first intron and second exon of the Sdl gene.
  • amplification product size using primers flanking the deletion were 348 bp and 731 bp for the dwarfing and tall alleles, respectively (primers: Sdl -Forward: 5'- CACGCACGGGTTCTTCC AGGTG-3 ' (SEQ ID NO: 33); and 5 i-Reverse:5'- AGGAGAATAGGAGATGGTTTACC- 3'(SEQ ID NO: 34)). Detection of this deletion by PCR thus identifies a dwarfing sdl allele.
  • the allelic identity of sdl is determined alongside identifying the presence of a QTL allele associated with drought tolerance in a rice plant.
  • a nucleic acid (preferably DNA) sequence comprising at least one QTL of the present invention (qDTY; A , qDTY 3 j, qDTY 3 2 , and qDTY 62 ) or a yield-improving part thereof, may be used for the production of a rice plant with improved drought tolerance.
  • the invention provides for the use of a QTL of the present invention or yield- improving parts thereof, for producing a rice plant with improved drought tolerance, which use involves the introduction of a nucleic acid sequence comprising said QTL in a rice plant having relatively low drought tolerance.
  • the nucleic acid sequence comprising the QTL is introduced in a semi-dwarf rice plant comprising a dwarfing allele of sdl.
  • said nucleic acid sequence may be derived from a suitable donor rice plant.
  • Suitable donor rice plants capable of providing a nucleic acid sequence comprising at least one of the herein described QTLs, or yield-improving parts thereof include but are not limited to N22, Dagaddeshi, Apo, IR55419-04, RD7, IR74371-46-1-1, IR743-70-1, Dular, AdaySel, Black Gora, Brown Gora, Sathi 34-36, Basmati 334, Basmati 370, Vandana, IR77298-5-6-18, and Moroberekan.
  • Other related rice varieties that exhibit drought tolerance and comprise one or more genes that encode for improved yield under drought stress may also be utilized as donor plants as the present invention describes how this material may be identified.
  • a suitable recipient rice plant is a rice plant that comprises a dwarfing allele of sdl and a drought susceptible QTL allele, including but not limited to IR8, Jaya, IR36, IR64, Swarna, MTU1010, Sambha Mahsuri, BR1, RD1, Kalamkatit, TDK1, PSBRC80, NSICR C 222, Dee-Geo-Woo-Gen, IRRI119, Purbachi (Chinese 1), Sabitri, and RD25.
  • the nucleic acid sequence may be transferred by crossing a donor rice plant with a semi-dwarf recipient rice plant (i.e. by introgression), by transformation, by protoplast fusion, by a doubled haploid technique, by embryo rescue, or by any other nucleic acid transfer system, optionally followed by selection of progeny plants comprising a dwarfing sdl allele and the QTL, and exhibiting both semi-dwarf plant height and drought tolerance.
  • a nucleic acid sequence comprising a QTL for drought tolerance according to the present invention, or a yield- improving part thereof may be isolated from said donor plant by using methods known in the art and the thus isolated nucleic acid sequence may be transferred to the recipient plant by transgenic methods, for instance by means of a vector, in a gamete, or in any other suitable transfer element, such as a ballistic particle coated with the nucleic acid sequence.
  • Plant transformation generally involves the construction of an expression vector that will function in plant cells.
  • a vector comprises a nucleic acid sequence that comprises a QTL for drought tolerance of the present invention, or a yield-improving part thereof, which vector may comprise one or more genes under control of, or operatively linked to, a regulatory element such as a promoter.
  • the expression vector may contain one or more such operably linked gene/regulatory element combinations, provided that at least one of the genes contained in the combinations encodes for drought tolerance.
  • the vector(s) may be in the form of a plasmid, and can be used alone or in combination with other plasmids to provide transgenic plants that have improved drought tolerance, using transformation methods known in the art, such as the Agrobacterium transformation system.
  • Expression vectors may include at least one marker gene, operably linked to a regulatory element (such as a promoter) that allows transformed cells containing the marker to be either recovered by negative selection (by inhibiting the growth of cells that do not contain the selectable marker gene), or by positive selection (by screening for the product encoded by the marker gene).
  • selectable marker genes for plant transformation include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide, or genes that encode an altered target which is insensitive to the inhibitor.
  • positive selection methods are known in the art, such as mannose selection.
  • marker-less transformation can be used to obtain plants without mentioned marker genes, the techniques for which are known in the art.
  • A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria that genetically transform plant cells.
  • rhizogenes carry genes responsible for genetic transformation of the plant.
  • Methods of introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant cells with Agrobacterium tumefaciens. Descriptions of Agrobacterium vectors systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber and Crosby, 1993 and Moloney et al., 1989. See also, U.S. Pat. No. 5,591,616. General descriptions of plant expression vectors and reporter genes and transformation protocols and descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer can be found in Gruber and Crosby, 1993.
  • Another method for introducing an expression vector into a plant is based on microprojectile- mediated transformation wherein DNA is carried on the surface of microprojectiles.
  • the expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes.
  • Another method for introducing DNA to plants is via the sonication of target cells.
  • liposome or spheroplast fusion has been used to introduce expression vectors into plants.
  • Direct uptake of DNA into protoplasts using CaCl 2 precipitation, polyvinyl alcohol, or poly-L-ornithine may also be used. Electroporation of protoplasts and whole cells and tissues has also been described.
  • selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.
  • the markers described herein may also be used for that purpose.
  • Zinc-finger nucleases ZFNs
  • transcription activator-like effector nucleases TALENs
  • CRISPR clustered regularly interspaced short palindromic repeat
  • the chimeric nucleases of ZFNs and TALENs are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain.
  • ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations.
  • any one of these technologies may be used to modify the genome of a rice plant.
  • Such modification may include modification, insertion, or deletion of a QTL or one or more individual genes associated with drought tolerance.
  • semi-dwarf rice plant comprising a dwarfing allele of the sdl gene may be modified from the drought susceptible qDTYu allele to the drought tolerant N22 qDTY u allele.
  • protoplast fusion can be used for the transfer of nucleic acids from a donor plant to a recipient plant.
  • Protoplast fusion is an induced or spontaneous union, such as a somatic hybridization, between two or more protoplasts (cells of which the cell walls are removed by enzymatic treatment) to produce a single bi- or multi-nucleate cell.
  • the fused cell which may even be obtained with plant species that cannot be interbred in nature, is tissue cultured into a hybrid plant exhibiting the desirable combination of traits. More specifically, a first protoplast can be obtained from a rice plant or other plant line that exhibits semi-dwarf plant height (and low or no lodging and fertilizer responsive) and drought tolerance.
  • a protoplast from rice N22 can be used.
  • a second protoplast can be obtained from a semi- dwarf rice comprising the sdl dwarfing allele along with other commercially desirable characteristics, such as, but not limited to low or no lodging, semi-dwarf plant height, medium maturity period duration, responsive to fertilizer, disease resistance, insect resistance, weed resistance, etc.
  • the protoplasts are then fused using traditional protoplast fusion procedures, which are known in the art.
  • embryo rescue may be employed in the transfer of a nucleic acid comprising one or more QTLs of the present invention from a donor plant to a recipient plant.
  • Embryo rescue can be used as a procedure to isolate embryo's from crosses wherein plants fail to produce viable seed. In this process, the fertilized ovary or immature seed of a plant is tissue cultured to create new plants (Pierik, 1999).
  • the present invention also relates to a method of producing a semi-dwarf drought resistant rice plant comprising the steps of performing a method for detecting the presence of at least one QTL associated with drought resistance in a donor rice plant according to the invention as described herein, and transferring one or more nucleic acid sequences comprising the at least one QTL thus detected, or a yield-improving part thereof, from the donor plant to a drought susceptible rice plant comprising a dwarfing sdl allele.
  • the transfer of said nucleic acid sequence may be performed by any of the methods described herein.
  • a preferred embodiment comprises transfer of a nucleic acid sequence by introgression from a drought tolerant rice plant to a semi-dwarf drought susceptible rice plant comprising a dwarfing sdl allele by crossing the plants.
  • This transfer may thus suitably be accomplished by using traditional breeding techniques.
  • QTLs are preferably introgressed into commercial semi-dwarf rice varieties using marker-assisted breeding (MAS).
  • Marker- assisted breeding or marker-assisted selection involves the use of one or more of the molecular markers for the identification and selection of those progeny plants that contain one or more of the genes that encode for the desired trait. In the present instance, such identification and selection is based on dwarfing sdl alleles and QTLs of the present invention or markers associated therewith.
  • MAS can also be used to develop near-isogenic lines (NIL) harboring the QTL of interest, allowing a more detailed study of each QTL effect and is also an effective method for development of backcross inbred line (BIL) populations (see, e.g., Nesbitt et al., 2001 ; van Berloo et al., 2001).
  • NIL near-isogenic lines
  • BIL backcross inbred line
  • Rice plants developed according to this preferred embodiment can advantageously derive a majority of their traits from the recipient plant, such as semi-dwarf plant height, low or no lodging, medium maturity period duration, and response to fertilizer, and derive drought tolerance from the donor plant.
  • a donor rice plant comprising a nucleic acid sequence encoding for drought tolerance is crossed with a drought susceptible rice plant comprising a dwarfing sdl allele that preferably exhibits commercially desirable characteristics, such as, but not limited to, low or no lodging, semi-dwarf plant height, medium maturity period duration, responsive to fertilizer, disease resistance, insect resistance, weed resistance, etc.
  • the resulting plant population is then self-pollinated and set seeds. The plants grown from the resulting seeds are then screened for semi-dwarf plant height and drought tolerance.
  • the population can be screened drought tolerance in a number of different ways.
  • the population can be screened by field evaluation over several seasons. Yield may be determined by weight of grain per hectare (e.g., t ha "1 , kg ha _1 ), average grain weight per plant, or any other method known in the art.
  • plants are further screened and selected for lodging characteristics, maturity period duration, and responsiveness to fertilizer. Preferably, selected plants in these embodiments exhibit low or no lodging, a medium maturity period duration, and are responsive to fertilizer.
  • Another aspect of the present invention relates to a semi-dwarf drought tolerant rice plant, or part thereof, comprising within its genome the dwarfing sdl allele and at least one QTL, or a yield- improving part thereof, associated with drought, wherein the QTL or the yield improving part thereof is not in its natural genetic background.
  • the QTL is qDTY] .
  • the at least one QTL is one or more of qDTY] , qDTY 3 , qDTY 3 2 , and qDTY 62 .
  • the rice plants having improved yield under drought stress of the present invention can be of any genetic type such as inbred, hybrid, haploid, dihaploid, parthenocarp, or transgenic. Further, the plants of the present invention may be heterozygous or homozygous for the drought tolerance trait. Preferably, the rice plants are homozygous.
  • the QTLs of the present invention, as well as those QTLs obtainable by a method of the invention, as well as yield-improving parts thereof may be transferred to any plant in order to provide for a plant having improved drought tolerance, the methods and plants of the invention are preferably related to rice (Oryza sativa).
  • Inbred semi-dwarf drought tolerant rice lines can be developed using the techniques of recurrent selection and backcrossing, selfing and/or dihaploids, or any other technique used to make parental lines.
  • drought tolerance can be introgressed into a target recipient semi-dwarf plant (which is called the recurrent parent) by crossing the recurrent parent with a first donor plant (which is different from the recurrent parent and referred to herein as the "nonrecurrent parent").
  • the recurrent parent is a plant that has relatively low yield under drought stress and possesses commercially desirable characteristics, such as, but not limited to low or no lodging, semi-dwarf plant height, medium maturity period duration, responsive to fertilizer, disease resistance, insect resistance, weed resistance, etc.
  • the non-recurrent parent comprises a nucleic acid sequence that encodes for drought tolerance.
  • the non-recurrent parent can be any plant variety or inbred line that is cross-fertile with the recurrent parent.
  • the progeny resulting from a cross between the recurrent parent and non-recurrent parent are backcrossed to the recurrent parent.
  • the resulting plant population is then screened.
  • the population can be screened in a number of different ways.
  • Plants that exhibit drought comprise the requisite nucleic acid sequences encoding for semi-dwarf plant height (sdl) and drought tolerance (e.g., qDTY; / ), and possess chosen commercially desirable characteristics, are then selected and selfed and further selected for a number of generations in order to allow for the rice plant to become increasingly inbred. This process of continued selfing and selection can be performed for two to five or more generations.
  • the result of such breeding and selection is the production of lines that are genetically homogenous for the genes associated with semi-dwarf plant height and drought tolerance, as well as other genes associated with traits of commercial interest.
  • MAS can be performed using one or more of the herein described molecular markers, hybridization probes or nucleic acids to identify those progeny that comprise a nucleic acid sequence encoding for semi-dwarf plant height and drought tolerance.
  • MAS can be used to confirm results obtained from quantitative bioassays.
  • the process is repeated.
  • the process of backcrossing to the recurrent parent and selecting for semi-dwarf plant height and drought tolerance is repeated for approximately five or more generations.
  • the progeny resulting from this process are heterozygous for the one or more qDTY QTLS encoding drought tolerance.
  • the last backcross generation is then selfed in order to provide for homozygous pure breeding progeny for semi-dwarf plant height and drought tolerance.
  • the semi-dwarf drought tolerant rice lines described herein can be used in additional crossings to create drought tolerant plants.
  • a first inbred semi-dwarf drought tolerant rice plant of the invention can be crossed with a second inbred rice plant possessing commercially desirable traits such as, but not limited to, low or no lodging, semi-dwarf plant height, medium maturity period duration, responsive to fertilizer, disease resistance, insect resistance, weed resistance, etc.
  • This second inbred rice line may or may not have relatively improved drought tolerance.
  • qDTY u MAS and MABC are described herein. MAS and MABC may be further applied to qDTY 3 1 , qDTY 3 2 , and qDTY 62 .
  • a primary motivation for development of molecular markers in crop species is the potential for increased efficiency in plant breeding through marker assisted selection (MAS) and marker assisted backcrossing (MABC).
  • Genetic marker alleles or alternatively, identified QTL alleles, are used to identify plants that contain a desired genotype at one or more loci and that are expected to transfer the desired genotype, along with a desired phenotype to their progeny.
  • Genetic marker alleles can be used to identify plants that contain a desired genotype at one locus or at several unlinked or linked loci (e.g., a haplotype) and that would be expected to transfer the desired genotype, along with a desired phenotype to their progeny.
  • the present invention provides the means to identify rice plants that are able to improve drought resistance by identifying plants having a specified quantitative trait locus, e.g., qDTY; , qDTY 3 qDTY 3 2 , and qDTY 62 , and homologous or linked markers. Similarly, by identifying plants having poor yield under drought stress, such low-yielding plants can be identified and, e.g., eliminated from subsequent crosses.
  • a specified quantitative trait locus e.g., qDTY; , qDTY 3 qDTY 3 2 , and qDTY 62 , and homologous or linked markers.
  • a desired phenotype e.g., semi-dwarf plant height or drought tolerance
  • polymorphic chromosomal locus e.g., a marker locus, gene, or QTL are determined to segregate together, it is possible to use those polymorphic loci to select for alleles corresponding to the desired phenotype; a process called marker-assisted selection (MAS).
  • MAS marker-assisted selection
  • a nucleic acid corresponding to the marker nucleic acid is detected in a biological sample (e.g., chromosomal DNA) from a plant to be selected.
  • This detection can take the form of hybridization of a probe nucleic acid to a marker, e.g., using allele-specific hybridization, Southern analysis, northern analysis, in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker, or the like.
  • a marker e.g., using allele-specific hybridization, Southern analysis, northern analysis, in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker, or the like.
  • a variety of procedures for detecting markers are described herein. After the presence (or absence) of a particular marker and/or marker allele in the biological sample is verified, the plant may be selected, i.e., used to make progeny plants by selective breeding.
  • Rice breeders combine modern irrigated rice varieties with desirable traits to develop improved rice varieties. Screening a large number of plants for drought tolerance can be expensive, time consuming and unreliable.
  • Use of the polymorphic loci described herein, and genetically-linked markers for drought tolerance and semi-dwarf plant height is an effective method for selecting varieties capable of fertility restoration in breeding programs. For example, one advantage of marker- assisted selection over field evaluations for drought resistance is that MAS can be done at any time of year regardless of the growing season. Moreover, environmental effects are irrelevant to marker- assisted selection.
  • Backcross breeding is the process of crossing a progeny back to one of its parents. Backcrossing is usually done for the purpose of introgressing one or a few loci from a donor parent into an otherwise desirable genetic background from the recurrent parent. The more cycles of backcrossing that are done, the greater the genetic contribution of the recurrent parent to the resulting variety. This is often necessary because donor parent plants may be otherwise undesirable. In contrast, varieties which are the result of intensive breeding programs may have excellent yield under irrigated conditions, semi-dwarf plant height, and low or no lodging, but are deficient in one desired trait such as drought tolerance. Backcrossing can be done to select for or against a trait.
  • molecular markers useful in MAS and MABC.
  • molecular markers can include restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), single nucleotide polymorphisms (SNP), and simple sequence repeats (SSR).
  • Simple sequence repeats (SSR) or microsatellites are regions of DNA where one to a few bases are tandemly repeated for few to hundreds of times. For example, a di- nucleotide repeat would resemble CACACACA and a trinucleotide repeat would resemble ATGATGATGATG.
  • Simple sequence repeats are thought to be generated due to slippage mediated errors during DNA replication, repair and recombination. Over time, these repeated sequences vary in length between one cultivar and another.
  • An example of allelic variation in SSRs would be: allele A being GAGAGAGA (4 repeats of the GA sequence) and allele B being GAGAGAGAGAGA (6 repeats of the GA sequence).
  • SSRs occur in a coding region, their survival depends on their impact on structure and function of the encoded protein. Since repeat tracks are prone to DNA-slippage mediated expansions/deletions, their occurrences in coding regions are limited by non-perturbation of the reading frame and tolerance of expanding amino acid stretches in the encoded proteins.
  • tri-nucleotide repeats or multiples thereof are more common in coding regions.
  • a single nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide - A, T, C or G - differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments from two individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case, there are two alleles: C and T.
  • Markers corresponding to genetic polymorphisms between members of a population can be detected by numerous well established methods (e.g., restriction fragment length polymorphisms, isozyme markers, allele specific hybridization (ASH), amplified variable sequences of the plant genome, self-sustained sequence replication, simple sequence repeat (SSR), single nucleotide polymorphism (SNP) or amplified fragment length polymorphisms (AFLP)).
  • restriction fragment length polymorphisms e.g., isozyme markers, allele specific hybridization (ASH), amplified variable sequences of the plant genome, self-sustained sequence replication, simple sequence repeat (SSR), single nucleotide polymorphism (SNP) or amplified fragment length polymorphisms (AFLP)
  • SSR simple sequence repeat
  • SNP single nucleotide polymorphism
  • AFLP amplified fragment length polymorphisms
  • hybridization formats include but are not limited to, solution phase, solid phase, mixed phase or in situ hybridization assays.
  • Markers which are restriction fragment length polymorphisms are detected by hybridizing a probe (which is typically a sub- fragment or a synthetic oligonucleotide corresponding to a sub-fragment of the nucleic acid to be detected) to restriction digested genomic DNA.
  • the restriction enzyme is selected to provide restriction fragments of at least two alternative (or polymorphic) lengths in different individuals and will often vary from line to line. Determining a (one or more) restriction enzyme that produces informative fragments for each cross is a simple procedure, well known in the art.
  • nucleic acid probes to the marker loci can be cloned and/or synthesized.
  • Detectable labels suitable for use with nucleic acid probes include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes and colorimetric labels.
  • Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents and enzymes. Labeling markers is readily achieved such as by the use of labeled PCR primers to marker loci.
  • the hybridized probe is then detected using, most typically, autoradiography or other similar detection technique (e.g., fluorography, liquid scintillation counter, etc.). Examples of specific hybridization protocols are widely available in the art.
  • Amplified variable sequences refer to amplified sequences of the plant genome which exhibit high nucleic acid residue variability between members of the same species. All organisms have variable genomic sequences and each organism (with the exception of a clone) has a different set of variable sequences. Once identified, the presence of specific variable sequence can be used to predict phenotypic traits.
  • DNA from the plant serves as a template for amplification with primers that flank a variable sequence of DNA (e.g., sdl). The variable sequence is amplified and then sequenced.
  • RNA polymerase mediated techniques e.g., NASBA
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • NASBA RNA polymerase mediated techniques
  • Oligonucleotides for use as primers are typically synthesized chemically according to the solid phase phosphoramidite triester method, or can simply be ordered commercially.
  • self-sustained sequence replication can be used to identify genetic markers.
  • Self- sustained sequence replication refers to a method of nucleic acid amplification using target nucleic acid sequences which are replicated exponentially in vitro under substantially isothermal conditions by using three enzymatic activities involved in retroviral replication: (1) reverse transcriptase, (2) Rnase H and (3) a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target.
  • AFLP amplified fragment length polymorphisms
  • ASH allele-specific hybridization
  • SNP single nucleotide polymorphisms
  • SSR simple sequence repeats
  • isozyme markers Methods of using the different types of molecular markers are known to those skilled in the art.
  • the qDTY u , qDTY 3 qDTY 3 2 , and qDTY 62 QTLs and sdl gene (or homologs thereof) in the genome of a plant exhibiting preferred phenotypic traits is determined by any method listed above, e.g., SNP, SSR, RFLP, AFLP, etc. If the nucleic acids from the plant are positive for one or more desired genetic markers, the plant can be selfed to create a true breeding line with the same genotype or it can be crossed with a plant with the same marker or with other desired characteristics to create a sexually crossed hybrid generation.
  • a drought tolerant donor rice plant is provided, and a nucleic acid from the donor plant is transferred to one or more recipient semi-dwarf drought susceptible rice plants.
  • the donor rice plant preferably comprises a drought tolerance-associated QTL (qDTY), such as qDTYu, qDTY 3 i , qDTY 3 2 , and qDTY 62 .
  • qDTY drought tolerance-associated QTL
  • the donor rice plant may be a rice variety including but not limited to N22, Dagaddeshi, Apo, IR55419-04, Vandana, RD7, IR74371-46-1-1, IR743-70-1-1, Dular, AdaySel, Black Gora, Brown Gora, Sathi 34-36, Basmati 334, Basmati 370, IR77298-5-6-18, and Moroberekan.
  • the QTL is qDTY ! h and the rice variety is one of N22, Dagaddeshi, Apo, Vandana, or Black Gora..
  • the QTL is any one or more of the QTLs qDTY !
  • the QTLs comprise qDTY a, and one or more of qDTY 3 , qDTY 3 2 , and qDTY 62 .
  • a preferred donor plant for qDTY 3 is a rice plant of variety Apo or IR55419-04
  • a preferred donor plant for qDTY 3 2 is a rice plant of variety Vandana, IR77298-5-6-18, Moroberekan, or N22
  • a preferred donor plant for qDTY 62 is a rice plant of variety IR55419-04, although other drought tolerant varieties comprising these QTLs may also be used.
  • the recipient rice plant comprises two or more of qDTY , qDTY 3 , qDTY 3 2 , and qDTY 62
  • the QTLs may be transferred to the donor plant one at a time, or two or more at a time.
  • donor plant genomes are stabilized (e.g., by backcrossing and/or selfing) following the transfer of each QTL.
  • the recipient semi-dwarf drought susceptible plant preferably has a plant height of less than 110 cm in lowland and less than 90 cm in upland, and comprises a dwarfing sdl allele.
  • a dwarfing allele is indicated by the mutation Y342*.
  • the mutation leads to a termination codon in the third exon of sdl -encoded gibberellin-20 oxidase, and is fully associated with the semi-dwarf phenotype. It is characterized by a 383 bp deletion (set forth in SEQ ID NO: 32) spanning the first intron and second exon of the Sdl gene (SEQ ID NO: 31). In certain embodiments, this deletion is used to identify a dwarfing sdl allele.
  • the deletion may be detected by any method known in the art.
  • the deletion is detected by PCR amplification utilizing primers flanking the deletion.
  • PCR amplification product size using primers flanking the deletion are 348 bp and 731 bp for the dwarfing and tall alleles, respectively (primers: Sdl -Forward: 5'- CACGCACGGGTTCTTCCAGGTG-3 ' (SEQ ID NO: 33); and 5 i-Reverse:5'- AGGAGAATAGGAGATGGTTTACC- 3'(SEQ ID NO: 34)). Detection of this deletion by PCR thus identifies a dwarfing sdl allele.
  • the one or more recipient rice plants are rice plants of a variety selected from IR8, Jaya, IR36, IR64, Swarna, MTU1010, Sambha Mahsuri, BR1, RD1, Kalamkatit, TDK1, PSBRC80, NSICRC222, Dee-Geo-Woo-Gen, IRRI119, Purbachi (Chinese 1), Sabitri, and RD25.
  • a rice plant from any variety wherein the rice plant exhibits semi-dwarf plant height and comprises the dwarfing sdl allele may be utilized as a recipient rice plant.
  • the one or more recipient rice plants in addition to comprising the dwarfing sdl allele and exhibiting semi-dwarf plant height, further exhibit high yields during irrigation (more than 5.0 tha low or no lodging, medium maturity period of 110-130 days, and responsiveness to fertilizer.
  • the transfer of the nucleic acid from the donor plant to the one or more recipient plants results in the introduction of genomic material comprising a drought tolerant allele of a QTL (e.g., qDTYu, qDTY 3 j, qDTY 3 2 , and qDTY 62 ) from the donor rice plant in a corresponding genomic region of the one or more recipient rice plants.
  • the transfer can be as a result of crossing (introgression), a transgenic method, by protoplast fusion, by a double haploid technique, or by embryo rescue.
  • the transfer is a result of crossing the donor plant with the one or more recipient plants.
  • the progeny produced by such a cross preferably retain the dwarfing sdl allele of the one or more recipient parent plants.
  • At least one rice plant retaining its original dwarfing sdl allele and a drought tolerant allele of one or more of qDTYu, qDTY 3 , qDTY 3 2 , and qDTY 62 is identified and selected.
  • DNA from progeny plants resulting from crossing donor and recipient plants is used for identifying and selecting.
  • a drought tolerant allele of qDTY ⁇ is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: nksdtyl_l_34; nkstdyl_l_38; RM11943; RM431 ; RM12023; Rml2091 ; and RM12146.
  • Drought tolerant alleles of qDTY 3 1 , qDTY 3 2 , and qDTY 62 are similarly indicated by genetic markers: the drought tolerant allele of qDTY 3 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM520; RM416; and RM16030, the drought tolerant allele of qDTY 32 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM7332; RM523; and RM545, and the drought tolerant allele of qDTY 62 is indicated by a genomic region on chromosome 6 comprising at least one marker selected from the group consisting of: RM121 ; RM3; RM541 ; and RM275.
  • rice plants comprising the dwarfing allele of sdl and the drought tolerant allele of qDTYj ⁇ are selected.
  • Rice plants identified and selected as semi-dwarf drought tolerant rice plants by means of genetic markers may be further selected for high yield under irrigated conditions, medium maturation periods, semi-dwarf plant height, low or no lodging, and responsiveness to fertilizer in field tests or bioassays.
  • Plant parts produced by a method described herein, or derived from a plant produced by a method described herein.
  • Plant parts may include, but are not limited to seeds, protoplasts, leaves, stems, roots, root tips, anthers, pistils, grain, embryo, pollen ovules, flower, shoot, tissue, petiole, cells, and meristematic cells.
  • a selected semi-dwarf drought tolerant rice plants described above is selfed or backcrossed to produce an inbred rice line.
  • the inbred rice line comprises the dwarf sdl allele and at least one drought tolerance-associated QTL (e.g., qDTYu, qDTY 3 j, qDTY 3 2 , or qDTY 62 ).
  • the inbred rice line is homozygous for these alleles.
  • a semi-dwarf drought tolerant rice plant comprises a semi- dwarf allele of sdl comprising a Y342* mutation and a drought tolerant allele of qDTYu, wherein qDTYl u is not in its natural genetic background.
  • the drought tolerant allele of qDTYu is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: nksdtyl_l_34; nkstdyl_l_38; RM11943; RM431 ; RM12023; Rml2091 ; and
  • the rice plant further comprises one or more of qDTY 3 , qDTY 3 2 , and qDTY 62 , wherein drought tolerant alleles of qDTY 3 , qDTY 3 2 , and qDTY 62 are indicated by genetic markers: the drought tolerant allele of qDTY 3 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM520; RM416; and RM16030, the drought tolerant allele of qDTY 3 2 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM7332; RM523; and RM545, and the drought tolerant allele of qDTY 62 is indicated by a genomic region on chromosome 6 comprising at least one marker selected from the group consisting of: RM121 ; RM3; RM541 ; and RM275.
  • Plant parts may include, but are not limited to seeds, protoplasts, leaves, stems, roots, root tips, anthers, pistils, grain, embryo, pollen ovules, flower, shoot, tissue, petiole, cells, and meristematic cells.
  • Also provided herein are methods of producing a product from rice or a processed rice material the method comprising: a) obtaining grain of a semi-dwarf drought tolerant rice plant as described herein; and b) processing the grain to produce the product or material.
  • a product from rice as described herein will thus comprise a processed rice material comprising: a) a semi-dwarf allele of sdl comprising a Y342* mutation; and b) a drought tolerant allele of qDTY !
  • qDTYlj ⁇ is not in its natural genetic background
  • the drought tolerant allele of qDTYi ⁇ is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: nksdtyl_l_34; nkstdyl_l_38; RM11943; RM431; RM12023; Rml2091 ; and RM12146.
  • the processed rice material in the product described herein may also comprise a drought tolerant allele of at least one of qDTY 3 j, qDTY 32 , and qDTY 62 , wherein the drought tolerant allele of qDTY 3 ⁇ is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM520; RM416; and RM16030, the drought tolerant allele of qDTY 3 2 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM7332; RM523; and RM545, and the drought tolerant allele of qDTY 62 is indicated by a genomic region on chromosome 6 comprising at least one marker selected from the group consisting of: RM121 ; RM3; RM541 ; and RM275.
  • the drought tolerant allele of qDTY 3 ⁇ is indicated by a genomic region on chromosome 3 comprising
  • the semi-dwarf allele of sdl is indicated by a 383 bp deletion (set forth in SEQ ID NO: 32) spanning a first intron and second exon of sdl (SEQ ID NO: 31).
  • the method described herein may comprise the further step of c) packaging the product or processed rice material for commercial sale.
  • processing of the rice grain in accordance with the method described herein may comprise one or more steps selected from the group consisting of cleaning the grain, purifying the grain, milling the grain, grading the grain, weighing the grain, steaming or parboiling the grain and mixing the grain or a processed product thereof with one or more other components.
  • Other process steps for processing rice grain for the production of food and/or beverage products or ingredients thereof will be known to a person of skill in the art and are contemplated for use in the method described herein.
  • a processed rice material or a product comprising same, comprising: a) a semi-dwarf allele of sdl comprising a Y342* mutation; and b) a drought tolerant allele of qDTY] , wherein qDTYlj ⁇ is not in its natural genetic background, and wherein the drought tolerant allele of qDTY a is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: nksdtyl_l_34; nkstdyl_l_38; RM11943; RM431 ; RM12023;
  • the processed rice material or product comprising same further comprises a drought tolerant allele of at least one of qDTY 3 , qDTY 3 2 , and qDTY 62 , wherein the drought tolerant allele of qDTY 3 i is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM520; RM416; and RM16030, the drought tolerant allele of qDTY 3 2 is indicated by a genomic region on chromosome 3 comprising at least one marker selected from the group consisting of: RM7332; RM523; and RM545, and the drought tolerant allele of qDTY 62 is indicated by a genomic region on chromosome 6 comprising at least one marker selected from the group consisting of: RM121 ; RM3; RM541 ; and RM275.
  • the semi-dwarf allele of sdl is indicated by a genomic region on chromosome 3 comprising at least one
  • the processed rice material or product comprising same is produced from grain of a semi-dwarf drought tolerant rice plant as described herein.
  • the processed rice material or product comprising same is packaged for sale.
  • the processed rice material or product comprising same is a food ingredient, beverage ingredient, a food product or a beverage product.
  • examples of such products include, but are not limited to:
  • a food ingredient or beverage ingredient selected from the group consisting of rice wholemeal, rice flour, rice bran, rice starch, rice malt, rice vinegar, rice syrup, rice oil e.g., rice bran oil, and rice bran wax;
  • a food product may be selected from the group consisting of: leavened or unleavened breads, pasta, noodles, edible rice paper, animal fodder, breakfast cereals, snack foods, cakes, dumplings, puffed rice, pastries, confectionary and foods containing a rice flour-based sauce, or
  • a beverage product selected from the group consisting of rice milk and beverages containing ethanol produced from rice e.g., rice wine or sake.
  • the processed rice material or product comprising same is non-edible for humans.
  • materials and products which are non-edible for humans and which are produced from the hulls or husks of rice grain include, but are not limited to: fuel, bedding, incubation material, livestock feeds, concrete blocks, tiles, fiberboard, ceramics, cement, filters, charcoal briquettes, and products comprising rice bran wax e.g., cosmetics, shoe creams and polishing compounds.
  • BIO 1628-33740 2450-33350 17790 ⁇ 5988 15490 ⁇ 4539 698-19050 845-21650 10760 ⁇ 7801 9347+2459
  • the effect of the sdl gene was analyzed in a set of 123 rice genotypes (Table 8) including landraces, traditional varieties, and cultivated varieties for rainfed as well as irrigated ecosystems in South and Southeast Asia. These lines were genotyped with a panel of 29 SNP markers underlying qDTYu with the Sequenom platform (described below). Gel based analysis with sdl gene-specific marker was also carried out. SNP markers showing spurious calls were omitted and a total of 22 markers (21 SNPs +sdl) were used for analysis. This set of diverse genotypes was screened for GY under RS at IRRI in DS2012 and DS2013 following the protocol described by Venuprasad et al. (2007).
  • RIL/ BIL populations were used to understand the linkage of qDTY 62 , qDTY3.1, qDTY 3 2 and tall plant height, reduced yield under irrigated conditions, and very early maturity, respectively.
  • Genotypic data available for these populations (Venuprasad et al. 2009, Vikram et al. 2011, and Dixit et al. 2014) was used for understanding the linkages, and identification of recombinants free from linkage drag within these populations ( Figure 12).
  • allelic diversity present for 11 qDTY loci a set of 123 diverse genotypes including short and tall plant types of traditional drought tolerant varieties, traditional drought-susceptible varieties, and modern rice varieties cultivated in rainfed and irrigated rice ecosystems was used.
  • Phenotyping under drought stress and irrigated conditions Phenotyping under drought stress and irrigated conditions.
  • N22/Swarna BIL population was screened under lowland reproductive stage drought stress (RS) and irrigated non-stress (NS) conditions in the 2012 dry season (DS) and 2013DS whereas qDTY] ⁇ homozygote plants (N22/Swarna, N22/IR64 and N22/MTU1010) were screened in 2012DS at IRRI, Philippines.
  • RS lowland reproductive stage drought stress
  • NS irrigated non-stress
  • the Apo/Swarna BCi-derived population was screened in 2006DS and 2007DS under RS and NS conditions, respectively; Vandana/Way Rarem lines were screened under upland RS and NS conditions in 2005DS and 2006DS; and the IR55419-04/TDK1 population was screened in 201 IDS and 2012DS under lowland RS and NS conditions, respectively.
  • the RS and NS experiments were laid out in an alpha lattice design with two replications. Seeds were sown in a nursery and transplanted after 21 -days. Single seedlings per hill were transplanted in 5-m single-row plots with row spacing of 0.2 m and 0.2 m between the hills in each row.
  • the four qDTY, ,-BILs and parents were also characterized in the field during the 2012 wet season (WS) (June 2012-October 2012), 2013DS (December 2012-April 2013), 2013WS (June 2013- October 2013), and 2014DS (December 2013-ApriI 2014) under transplanted lowland conditions including NS and RS treatments with four replicates per treatment.
  • Experimental plots were maintained flooded until 62, 75, 58, and 60 days after sowing (DAS) in 2013WS, 2013DS, 2013WS, and 2014DS, respectively, after which irrigation in the drought stress treatment was stopped and rain was excluded using an automatic rainout shelter.
  • DAS sowing
  • SSR genotyping of the N22 derived RILs and BILs was carried out for qDTY; A .
  • DNA of the populations was extracted from freeze-dried leaf samples that were cut in Eppendorf tubes and ground with a GENO grinder. Extraction was carried out by the modified CTAB method (Murray and Thomson 1980). DNA samples were stored in 2-mL deep-well plates (Axygen Scientific, California, USA). DNA samples were quantified on 0.8% agarose gel and concentration adjusted to
  • PCR amplification was carried with & ⁇ 5- iL reaction mixture having 50 ng DNA, 1 x PCR buffer, 100 ⁇ dNTPs, 250 ⁇ primers, and 1 unit Taq polymerase enzyme.
  • 8% non-denaturing polyacrilamide gels PAGE were used (Sambrook et al. 1989). The set of 123 random genotypes was genotyped with 65 SSR markers across 11 DTY loci.
  • the dwarfing allele of the rice plant height gene Sdl characterized by a 383 bp deletion (set forth in SEQ ID NO: 32) spanning the first intron and second exon of the Sdl gene (SEQ ID NO: 31), was assayed by PCR amplification using primers flanking this deletion.
  • the primers used were: Sdl -Forward: 5'-CACGCACGG GTTCTTCCAGGTG-3 ' (SEQ ID NO: 33)and Sdl-Reverse:5'-AGG AGAATA GGA GAT GGT TTA CC- 3'(SEQ ID NO: 34).
  • thermocycling program consisted of initial denaturation at 94°C for 5 min, followed by 45 cycles of amplification at 94°C for 30s, 55°C for 30s and 72°C for 1 min and a final extension at 72°C for 3 min. PCR product was visualized by electrophoresis in 2% Agarose Gel.
  • a 29-plex Sequenom SNP assay was designed and validated for fine mapping of the qDTYj ./ QTL located between 36.04-40.70Mb region on the long arm of rice chromosome 1 ( Figure 3, Table 9 and Table 10).
  • the primers were procured from IDT (Heverlee, Belgium).
  • the iPLEX GOLD SNP genotyping was performed as per the manufacturer's protocols and the genotype calls were analyzed using SequenomTyper 4.0 software.
  • the SNP calls in ACGT format generated for all the samples were converted to A, B and H format for easy visualization of the recombinant break points. In cases of heterozygote calls (due to incomplete fixing of the inbred lines), a manual inspection of peak intensity was carried to cross-check the validity of the calls.
  • Data was analyzed using graphical genotyping of the qDTYu region of the SNP calls from recombinant lines to identify the
  • Phenotypic means of entries were estimated using the following linear mixed model for the analysis of variance:
  • MT-MIM Multiple trait multiple interval mapping
  • q is the number of QTLs being fitted simultaneously
  • t is the number of analyzed traits
  • p is the number of non-genetic fixed factors
  • a is the additive effect
  • d is the dominant effect
  • X refers to the non-genetic fixed effect
  • B is the incidence matrix that links observation of the data with fixed effects
  • E is random error
  • i l-d.
  • N22/Swarna, 57 from N22/IR64 and 56 from N22/MTU1010) from the three populations was carried out. Segregation for the sdl gene was analyzed using a functional marker as described above, (iii) The semi-dwarf N22/Swarna RIL population was genotyped with SSR markers of qDTYu (RM11943, RM431, RM12023, RM12091 and RM12146), SNP markers of qDTYu, as well as with the sdl gene- based indel marker. This population was homozygous for the dwarf sdl allele. The N22/Swarna RIL population was also tested for genetic background.
  • Genotyping of 123 random genotypes was conducted with genome-wide SNP markers, SNP markers within the qDTY; ⁇ region and an sdl gene-based functional marker.
  • a genome-wide SNP panel was used for determination of population structure.
  • a SequenomMassARRAY multiplex assay was designed with 72 SNPs including two wells of 36 plexiPLEX gold chemistry. These 72 SNPs represented 72 conserved single copy rice genes and these SNPs represented 6 genes /chromosome, two genes each for all the telomeric and centromeric regions (Singh et al. 2010). All SNPs were used for the population structure analysis. Population structure was determined by STRUCTURE v.2.3.3 Software (Pritchard et al. 2000).
  • the genetic diversity analysis on the set of diverse varieties was conducted using the software Power Marker V3.25 (Liu and Muse 2005). Allele frequency was obtained using the bootstrap method with bootstrap number 10,000 and confidence interval 0.950. The frequency-based distance was obtained by the C.S. Chord method (Cavalli-Sforza and Edwards 1967). The neighbor-joining tree was constructed according to frequency-based distances obtained from PowerMarker using the software MEGA 5.2 (Tamura et al. 2011).
  • Recombinant classes were identified within qDTY 3 qDTY 3 2 ⁇ nd qDTY 62 based on the SSR marker data available for the three QTL.
  • Class means calculated based on the mean data of different recombinant classes were used to estimate the class effect on the respective phenotypic traits.
  • N22/MTU1010 populations were idl024366- idl024499, idl024499 -RM431 and idl024366- idl024499.
  • Fine mapping of the sdl/qDTYj ⁇ locus were idl024366- idl024499, idl024499 -RM431 and idl024366- idl024499.
  • SequnomMassARRAYSNP assay ( Figure 3, Table 9 and Table 10); comparison of mean yield under drought among different recombinant classes, namely A, B, and C that possessed the N22 allele at nksdtyl_l_30 with class D that lacked nksdtyl_l_30 but possessed the N22 allele at adjacent marker nksdtyl_l_34 and further down towards telomere showed that the difference of GY under RS between group (1) with N22 allele below sdl gene i.e. classes A+B+C+D and group (2) with Swarna allele below sdl gene i.e.
  • Example III Analysis of sdl.qDTYu region in traditional GR, and post-GR varieties.
  • Vandana and Sathi34-36 are derivatives from landraces Rajbhog, Kalakeri and Sathi, respectively.
  • This group has the tall allele of sdl along with the partial/full region of qDTYu.
  • Group 2 is composed of the GR varieties with the semi-dwarf allele of sdl and Swarna type qDTYu region.
  • Group 3 comprised Indonesian landraces of east Java, which have the tall allele of the sdl gene but with drought sensitive allele of qDTYu (all three varieties have Swarna allele at marker locus-nksl_l_30); these landraces are tall but do not flower under drought stress condition.
  • Group 4 represents GR varieties which had neither the sdl tall allele nor N22 allele of qDTYu. These are semi-dwarf and provide higher yield in the rainfed areas.
  • the performance of the genotypes in Group 4 under drought is neither due to sdl nor due to qDTYu.
  • the sdl profiles of genotypes in Groups 3 and 4 indicates that the sdl gene is not responsible for high grain yield under drought. Though not absolutely, comparative analysis of all four groups presented in Figure 5 indicates the responsiveness of qDTYu and non-responsiveness of sdl to drought tolerance. Table 12. Results of the association analysis in random rice genotypes for GY and yield related traits (HI, PHT, DTF, under reproductive stage stress (RS) and non-stress (NS) conditions.
  • Example IV Analysis of the qDTYu and sdl using 3K Rice genome sequence data.
  • LOC_Os01g66100 L266F
  • Speilmeyer et al., 2002 were represented by only 1% of the genotypes.
  • qDTYu homozygous lines were developed with the dwarf allele of the sdl gene from this BIL population. Plants with double cross-over events on both sides of the sdl gene were selected.
  • Four semi-dwarf qDTYu homozygous lines (IR91659:41-95-5-B, IR91659:41-95-6-B and IR91659:54-36-9-B) with plant height similar to Swarna under irrigated conditions showed a significant yield advantage over Swarna in the severe field drought experiment (Table 4).
  • Example V Physiological and molecular basis of the drought tolerance effect of qDTY L1 .
  • qDTYu BILs showed a dynamic response to the timing and severity of drought stress.
  • qDTY BIL IR91659:41-95-B showed higher apparent leaf area than Swarna early in the drought stress treatment (Figure 7B), but leaf area was similar among genotypes in the well-watered control ( Figure 11 A).
  • the height and plant type of the qDTYu BILs was similar to Swarna ( Figure 7C).
  • qDTYu BILs showed lower normalized difference vegetation index (NDVI) than Swarna when the stress treatment was initiated early (2014DS; Fig.
  • BILs IR91659:41-95-B showed a higher proportion of root length at depth across field seasons and treatments (Figure 7F), except in the seasons when the drought stress was very severe (2012WS and 2014DS).
  • the water uptake rates of qDTYu BILs in the drought treatment of the greenhouse lysimeter study were closely related to leaf area patterns across the study ( Figure 1 IF).
  • Example VI Linkage of other DTY QTLs with traits lost during GR era.
  • qDTY 62 Three other major effect qDTY also showed tight linkage with traits selected against during the GR.
  • Recombinant classes identified from a mapping population showed association of RM275 with tall plant height (Figure 12A). Two out of the four recombinant classes had the IR55419-04 allele at RM275. Both of these classes showed an increase in plant height compared to those with the TDK1 (drought-susceptible parent) allele at this locus. A difference of up to 4 cm under non-stress and up to 6 cm under stress was observed for mean PH of lines with IR55419-04 and TDK1 allele at this marker locus.
  • Swarna ILs with qDTY 3 ⁇ showed on par yield with Swarna under irrigated conditions and a significant yield advantage over Swarna under severe drought stress.
  • lines with qDTY 3 2 with later DTF than Vandana showed similar yield under drought as that of drought tolerant cultivar Vandana, the donor of qDTY 32 QTL (Table 12).
  • Example VII Proportion of qDTY alleles in traditional and GR varieties.
  • Cluster 1 was represented by well-known drought tolerant donors used in several drought studies; N22, Dular, AdaySel, Black Gora, Brown Gora, Sathi 34-36, basmati lines- Basmati 334 and Basmati 370, and IR55419-04.
  • Cluster 2 mostly included early GR varieties and was represented by IR8, Rasi, BR11, Ratna, RD15, Mahsuri and Sarjoo 50.
  • Cluster 3, included IR8, BR1, Jaya, RD1, Kalamkatit.
  • Cluster 4 included the presently cultivated semi-dwarf varieties developed around and after 1980: IR36, IR64, TDK1, PSBRC80, NSICRC222, Swarna, and Sambha Mahsuri classified in the present study as late GR varieties.
  • Cluster 5 included the drought tolerant varieties developed during GR era: Apo, RD7, IR74371 -46-1-1, and IR74371-70-1-1. The only exception in this group is Way Rarem, the cultivar which in itself is drought susceptible but has contributed the alleles for qDTY 12 1 .
  • the proportion of qDTY tolerant, sensitive, and other unknown alleles at 65 SSR marker loci at the 11 qDTY QTLs showed highest percentage of donor alleles in traditional upland drought tolerant varieties (Cluster 1 , 40%) followed by recently developed drought tolerant varieties (Cluster 5, 32%).
  • the proportion of drought tolerant alleles reduced to 21% in Cluster 2, 18% in Cluster 3, and 16% in Cluster 4 varieties (Figure 9).
  • dwarf line IR 91659-41-95-14-B showed similar yield to that of Swarna under control non- stress and a yield advantage of 0.8 to 1.6 t ha 1 under reproductive stage drought stress.
  • Table 15 Effect of dwarf line IR91659-41-95-14-1-B with qDTYj / under control non-stress and reproductive stage drought stress at Hyderabad, India
  • Example IX Production and evaluation of drought tolerant semi-dwarf lines introgressed with qDTY L1
  • the inventors also produced a drought tolerant semi-dwarf line with qDTYu by marker assisted backcross (MAB) breeding under environmental conditions which differ to those at the IRRI site in Los Banos, Philippines (where the drought tolerant QTL was identified).
  • MAB marker assisted backcross
  • qDTYu was introgressed in Swarna following MAB breeding. Lines were assessed for the presence of qDTY L1 and Sdl and those lines possessing qDTY L1 together with Sdl (Swarna) were evaluated at the IRRI South Asia Hub, Vietnamese, India under control non-stress (CONT) and reproductive stage drought stage (REPST). As observed in mapping population, the effect of qDTY L1 was consistent in the MAB derived lines developed and evaluated in both India and the Philippines. The introgressed lines with qDTYu showed yield advantage of 0.8 to 1.8 t ha 1 under reproductive stage drought stress while maintaining yield similar to Swarna when grown under control non-stress conditions (Table 16). Table 16: Effect of lines introgressed with qDTYj ⁇ following MAB breeding in Swrarna under control non-stress and reproductive stage drought stress at Hyderabad, India in different seasons.
  • Example X Production and evaluation of drought tolerant semi-dwarf lines introgressed with qDTY l lt qDTY 2 .i and qDTY 3 1
  • the inventors introgressed qDTY; l qDTY 2 1 , and qDTY 3 ⁇ into a Swarna subl variety following MAB breeding to assess the relationship between subl gene imparting tolerance to submergence and QTLs for grain yield under drought.
  • the MAB lines were assessed for the presence of qDTYu qDTY 2 1 , qDTY 3 j and subland those lines possessing the full complement of loci were evaluated at the IRRl site in Los Banos, Philippines and the IRRl South Asia Hub in Hyderabad, India under control non-stress (CONT) and reproductive stage drought stage (REPST), as well as under submergence conditions at the National Rice Research Project (NRRP) site, RARS, nepalganj, Nepal.
  • CONT non-stress
  • REPST reproductive stage drought stage

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Physiology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés et des matériaux utiles pour améliorer la tolérance à la sécheresse des variétés de riz semi-nain sd1. En particulier, la présente invention concerne des procédés permettant de rompre des liaisons génétiques entre locus pour une tolérance à la sécheresse et des caractéristiques non souhaitables, comprenant une hauteur de plante élevée, une verse, une durée de période de maturité très précoce, et un faible rendement dans des conditions bien arrosées. La présente invention concerne en outre des procédés permettant d'améliorer la tolérance à la sécheresse de variétés de riz semi-nain sd1 impliquant la sélection assistée par marqueurs et le rétro-croisement. L'invention concerne également des variétés de riz semi-nain tolérant à la sécheresse et des parties de celles-ci, y compris des grains, et des produits de riz dérivés de celles-ci.
PCT/IB2016/050686 2015-02-10 2016-02-10 Riz semi-nain tolérant à la sécheresse et procédés et matériaux connexes WO2016128898A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562114430P 2015-02-10 2015-02-10
US62/114,430 2015-02-10

Publications (1)

Publication Number Publication Date
WO2016128898A1 true WO2016128898A1 (fr) 2016-08-18

Family

ID=56615509

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2016/050686 WO2016128898A1 (fr) 2015-02-10 2016-02-10 Riz semi-nain tolérant à la sécheresse et procédés et matériaux connexes

Country Status (1)

Country Link
WO (1) WO2016128898A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148544A (zh) * 2016-08-29 2016-11-23 广西壮族自治区农业科学院水稻研究所 一种水稻3号染色体上与结实率相连锁的ssr引物对及其应用
CN111436364A (zh) * 2020-03-25 2020-07-24 上海天谷生物科技股份有限公司 一种节水抗旱稻三系不育系培育方法
CN111848765A (zh) * 2020-07-22 2020-10-30 中国水稻研究所 水稻基因OsFBK4及其突变体与应用
CN112364302A (zh) * 2020-11-11 2021-02-12 中国科学院东北地理与农业生态研究所 融合属性分级信息的生态系统属性组分组成结构描述方法
CN112649392A (zh) * 2020-12-15 2021-04-13 中国农业大学 一种快速鉴定小麦节水抗旱性的方法
CN113862387A (zh) * 2021-08-27 2021-12-31 上海市农业生物基因中心 水稻耐旱性调控基因OsNAC6的分子标记及其应用
CN115851824A (zh) * 2022-08-03 2023-03-28 贵州大学 一种降低大白果糯株高、提高产量并缩短生育期的方法以及sd1基因核心启动子和应用
CN117347282A (zh) * 2023-08-22 2024-01-05 中南大学 星基气溶胶光学厚度反演方法、装置及系统和存储介质
CN118006822A (zh) * 2024-01-24 2024-05-10 上海市农业生物基因中心 节水抗旱稻育种芯片（wdr6k）的探针组合、检测体系及应用

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CASELLA, L. ET AL.: "TILLING in European Rice: Hunting Mutations for Crop Improvement", CROP SCIENCE, vol. 53, 2013, pages 2550 - 62 *
GHIMIRE, K.H. ET AL.: "Identification and mapping of a QTL (qDTY1.1) with a consistent effect on grain yield under drought", FIELD CROPS RESEARCH., vol. 131, 2012, pages 88 - 96 *
KUMAR, A. ET AL.: "Breeding high-yielding drought-tolerant rice: genetic variations and conventional and molecular approaches", JOURNAL OF EXPERIMENTAL BOTANY., vol. 65, no. 21, 2014, pages 6265 - 78, Retrieved from the Internet <URL:http://jxb.oxfordjournals.org/content/65/21/6265.full.pdf+html?sid=c15f79b4-915f-47e3-acb3-8cfa1e6df30a> [retrieved on 20160323] *
MONNA, L. ET AL.: "Positional Cloning of Rice Semidwarfing Gene , sd1: Rice ''Green Revolution Gene '' Encodes a Mutant Enzyme Involved in Gibberellin Synthesis", DNA RESEARCH., vol. 9, 2002, pages 11 - 17 *
SINGH, R. ET AL.: "From QTL to variety-harnessing the benefits of QTLs for drought, flood and salt tolerance in mega rice varieties of India through a multi-institutional network", PLANT SCIENCE, vol. 242, 2016, pages 278 - 287, [retrieved on 20150820] *
VENUPRASAD, R. ET AL.: "A large-effect QTL for rice grain yield under upland drought stress on chromosome 1", MOLECULAR BREEDING., vol. 30, 2012, pages 535 - 47 *
VIKRAM, P. ET AL.: "Drought susceptibility of modern rice varieties: an effect of linkage of drought tolerance with undesirable traits", NATURE SCIENTIFIC REPORTS., vol. 5, 13 October 2015 (2015-10-13), Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602206/pdf/srep14799.pdf> [retrieved on 20160323] *
VIKRAM, P. ET AL.: "Genetic Diversity Analysis Reveals Importance of Green Revolution Gene (sdl Locus) for Drought Tolerance in Rice", AGRICULTURAL RESEARCH., vol. 5, no. 1, March 2016 (2016-03-01), pages 1 - 12, [retrieved on 20160123] *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148544A (zh) * 2016-08-29 2016-11-23 广西壮族自治区农业科学院水稻研究所 一种水稻3号染色体上与结实率相连锁的ssr引物对及其应用
CN111436364A (zh) * 2020-03-25 2020-07-24 上海天谷生物科技股份有限公司 一种节水抗旱稻三系不育系培育方法
CN111436364B (zh) * 2020-03-25 2023-08-18 上海天谷生物科技股份有限公司 一种节水抗旱稻三系不育系培育方法
CN111848765B (zh) * 2020-07-22 2021-10-08 中国水稻研究所 水稻基因OsFBK4及其突变体与应用
CN111848765A (zh) * 2020-07-22 2020-10-30 中国水稻研究所 水稻基因OsFBK4及其突变体与应用
CN112364302A (zh) * 2020-11-11 2021-02-12 中国科学院东北地理与农业生态研究所 融合属性分级信息的生态系统属性组分组成结构描述方法
CN112364302B (zh) * 2020-11-11 2023-05-02 中国科学院东北地理与农业生态研究所 融合属性分级信息的生态系统属性组分组成结构描述方法
CN112649392A (zh) * 2020-12-15 2021-04-13 中国农业大学 一种快速鉴定小麦节水抗旱性的方法
CN113862387A (zh) * 2021-08-27 2021-12-31 上海市农业生物基因中心 水稻耐旱性调控基因OsNAC6的分子标记及其应用
CN113862387B (zh) * 2021-08-27 2023-10-24 上海市农业生物基因中心 水稻耐旱性调控基因OsNAC6的分子标记及其应用
CN115851824A (zh) * 2022-08-03 2023-03-28 贵州大学 一种降低大白果糯株高、提高产量并缩短生育期的方法以及sd1基因核心启动子和应用
CN115851824B (zh) * 2022-08-03 2024-03-08 贵州大学 一种降低大白果糯株高、提高产量并缩短生育期的方法以及sd1基因核心启动子和应用
CN117347282A (zh) * 2023-08-22 2024-01-05 中南大学 星基气溶胶光学厚度反演方法、装置及系统和存储介质
CN117347282B (zh) * 2023-08-22 2024-05-28 中南大学 星基气溶胶光学厚度反演方法、装置及系统和存储介质
CN118006822A (zh) * 2024-01-24 2024-05-10 上海市农业生物基因中心 节水抗旱稻育种芯片（wdr6k）的探针组合、检测体系及应用

Similar Documents

Publication Publication Date Title
US11206776B2 (en) Markers associated with soybean rust resistance and methods of use therefor
WO2016128898A1 (fr) Riz semi-nain tolérant à la sécheresse et procédés et matériaux connexes
AU2008263879B2 (en) Improved pepper plant
US11032986B2 (en) Methods of creating drought tolerant corn plants using markers linked to cold shock domain-containing proteins and compositions thereof
US20150376638A1 (en) Breeding methods for enhanced grain yield and related materials and methods
EP2514304A1 (fr) Plante cucurbita résistante aux potyvirus
Liu et al. TaTPP‐7A positively feedback regulates grain filling and wheat grain yield through T6P‐SnRK1 signalling pathway and sugar–ABA interaction
ES2749964T3 (es) Planta resistente a un patógeno
EP2887796A2 (fr) Melon résistant à de multiples virus
EP2417261A1 (fr) Marqueurs associés à la résistance du soja à la rouille et leurs procédés d&#39;utilisation
US10036034B2 (en) Sequences involved in plant yield and methods of using
WO2010098670A1 (fr) Plants de concombre résistants au fusarium
WO2015103136A1 (fr) Plants de riz à texture peu crayeuse et matériaux et méthodes associés
Zhang et al. Loss of function of OsMADS3 via the insertion of a novel retrotransposon leads to recessive male sterility in rice (Oryza sativa)
WO2015087282A1 (fr) Plantes tolérantes à la germination anaérobie et matériels et procédés associés
KR102587723B1 (ko) 다량 개화 수박
WO2010025103A1 (fr) Compositions et procédés pour des marqueurs d’analyse étroitement liés au locus de résistance b s2 du poivre
CA2674243A1 (fr) Marqueurs genetiques de la resistance a l&#39;orobanche chez le tournesol
AU2014318042A1 (en) Molecular markers for blackleg resistance gene Rlm4 in Brassica napus and methods of using the same
US20230309480A1 (en) Methods of increasing outcrossing rates in gramineae
EP4434328A2 (fr) Nouveaux loci génétiques associés à la résistance à la pourriture de l&#39;épi du maïs
Sun et al. Quantitative trait loci (QTLs) for potassium chlorate resistance and low temperature tolerance in seedling stage in rice (Oryza sativa L.)
EP2473020B1 (fr) Plants de tomate démontrant une tolérance à la lumière en continu
Soriano et al. Mapping and tagging of simply inhereted traits
水多陽子 et al. Analysis of a pair of genes, DOPPELGANGER 1 (DPL1) and DOPPELGANGER 2 (DPL2) responsible for reproductive isolation between two rice subspecies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16748807

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16748807

Country of ref document: EP

Kind code of ref document: A1