WO2016128898A1 - Semi-dwarf drought tolerant rice and related methods and materials - Google Patents

Abstract

Describe herein are methods and materials useful for improving drought tolerance of sd1 semi- dwarf rice varieties. In particular, the present disclosure provides methods for breaking genetic linkages between loci for drought tolerance and un-desirable traits, including tall plant height, lodging, very early maturity period duration, and low yield under well watered conditions. The present disclosure further provides methods for improving drought tolerance in sd1 semi-dwarf rice varieties involving marker assisted selection and backcrossing. Also described are semi-dwarf drought tolerant rice varieties and parts thereof, including grain, and rice products derived therefrom.

Description

SEMI- DWARF DROUGHT TOLERANT RICE AND RELATED METHODS AND MATERIALS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to United States Provisional Application Number 62/114,430, filed on 10 February 2015, the entire disclosure of which is expressly incorporated herein by reference for all purposes.

BACKGROUND OF THE INVENTION

The introduction of semi dwarf genes (sdl and Rhtl) in rice and wheat during the Green Revolution (GR) helped achieve the food security by developing semi-dwarf, lodging tolerant, and fertilizer responsive rice and wheat varieties. This was one of the most significant achievements in the history of agricultural research. Rice breeding since the GR era has continued to incorporate the semi- dwarf sdl allele, and several rice mega-varieties have been developed for irrigated ecosystem. GR rice varieties show substantial yield grain mainly in the irrigated areas. However, some of the 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.

However, these and other GR varieties are highly sensitive to drought stress. Rejection of undesired traits, such as tall plant height, reduced yield under irrigated conditions, and early days to flowering, during breeding for semi-dwarf and high yielding rice varieties during and since the GR era might have led to the unintentional loss of genes or quantitative trait loci (QTLs) associated with drought tolerance.

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. For example, eastern India and adjoining areas of Nepal occupy a large drought-affected area with an estimate of around 17 million ha. In 2004, 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 livelihoods. In 2004, the normally lush tropical southern Chinese island of Hainan suffered its worst drought in 50 years, with 12 million hectares of farmland affected. Vietnam's eight central highland provinces suffered their worst drought in 28 years, affecting around 1 million people and causing an estimated $80 million worth of crop losses. Coping with recurrent drought is part of life for millions of Asia's rural poor.

In addition to rain-fed areas, in recent years, many of the irrigated areas have also experienced drought as a result of water shortages from receding water table, as well as increased competition for water reserves from other industries and human consumption.

The 1 % annual genetic gain of yield in rice is far below the 2% increase required to meet the increasing demands for rice in coming decades. Given the large area under rain-fed rice cultivation and the increasing incidence of drought stress in a range of cultivation systems, improving the drought tolerance of GR varieties would significantly help to fill this gap, providing for sustainable rice production and help ensure food security, particularly for those regions where rice is a staple part of the diet, such as Asia and Africa.

SUMMARY OF THE INVENTION

Describe herein are methods and materials useful for improving drought tolerance of sdl semi- dwarf rice varieties. In particular, 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.

In one aspect, a method of producing a semi-dwarf drought tolerant rice plant is provided, the method 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; _Λ 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] _Λ 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. In particular embodiments, the method further comprises selecting from the rice plants identified and selected in step c) one or more rice plants exhibiting semi-dwarf plant height and drought tolerance.

In one embodiment, 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 qDTY₃ j, qDTY₃₂, and qDTY₆₂ 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: qDTY₃ qDTY_{3 2}; and qDTY₆₂, wherein the drought tolerant allele of qDTY_{3 Λ} 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 qDTY₆₂ 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. In certain embodiments, 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 qDTY₃ \ 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 qDTY₆₂.

In one embodiment, 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. In certain embodiments, 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).

In one embodiment, the at least one marker may be detected in DNA isolated from the one or more recipient rice plants.

In one embodiment, 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.

In another embodiment, 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.

In one embodiment, 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.

In yet another embodiment, 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. Thus, 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 _Λ is not in its natural genetic background.

In another aspect, a method of producing a semi-dwarf drought tolerant inbred rice plant is provided, said method 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. Thus, in another aspect, a semi-dwarf drought tolerant inbred rice plant obtained or obtainable by the method described herein is also provided.

In yet another aspect, 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.

It will be appreciated that a plant part as described herein may be a seed. Thus, in another aspect, a seed of a plant produced by a method described herein is provided.

In accordance with any of the methods described herein, the donor rice plant may be selected from the group consisting of: N22; Dagaddeshi; Apo; Vandana; and Black Gora.. Similarly, 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.

In accordance with any of the methods described herein, 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.

In another aspect, 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 _Λ 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. In one example, 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 qDTY₆₂, wherein the drought tolerant allele of qDTY_{3 Λ} 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;

RM523; and RM545, and the drought tolerant allele of qDTY₆₂ 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. In one example, 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).

In yet another aspect, 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.

In another aspect, 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.

In a further aspect, 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. In one example, the processed rice material in the product described herein also comprises drought tolerant allele of at least one of qDTY₃ , qDTY₃₂, and qDTY₆₂, 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 qDTY₆₂ 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. In one example, 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.

A skilled person will appreciate that the method of processing employed may vary depending on the product or material being produced. However, 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.

In yet another aspect, 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; _Λ 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. In one example, the processed rice material or product comprising same further comprises a drought tolerant allele of at least one of qDTY₃ j, qDTY_{3 2}, and qDTY₆₂, wherein the drought tolerant allele of qDTY_{3 Λ} 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;

RM523; and RM545, and the drought tolerant allele of qDTY₆₂ 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. In one example, 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).

Preferably, the processed rice material or product comprising same is produced from grain of a semi-dwarf drought tolerant rice plant as described herein. In one example, the processed rice material or product comprising same is packaged for sale.

According to any example described herein, 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:

(i) 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;

(ii) 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

(iii) a beverage product selected from the group consisting of rice milk and beverages containing ethanol produced from rice e.g., rice wine or sake.

In another example, the processed rice material or product comprising same is non-edible for humans. Examples of 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.

Also provided is the use of grain from a semi-dwarf drought tolerant rice plant as described herein, or part thereof, as animal feed or food, or to produce feed for animal consumption or food for human consumption.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, 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.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the Patent Office upon request and payment of the necessary fee.

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). 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) qDTY₆₂ (RM3, RM541 and RM275), (FIG. 12B) qDTY_3J (RM520, RM416 and RM16030) and (FIG. 12C) qDTY₃₂ (RM7332, RM523 and RM545), in IR55419-04/2* TDK1, Apo/3*Swarna and Vandana/ Way Rarem

populations, respectively. Bar graphs show different degree of effects of recombinant classes on GY under non-stress conditions, DTF and PHT for the three QTLs respectively. qDTY₆₂ (RM3, RM541 and RM275): I, IR55419-04 allele; T, TDK1 allele qDTY₃ (RM520, RM416 and RM16030): S, Swarna allele; A, Apo allele qDTY₃₂ (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.

DETAILED DESCRIPTION

Throughout this disclosure, various publications, patents and published patent specifications are referenced. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

With the advent of the GR era, the traditional rice cultivars commonly grown in the rainfed rice growing areas of the world were replaced by semi-dwarf, non-lodging and fertilizer-responsive modern rice varieties. Semi-dwarf cultivars became popular as they produce much higher yields without any lodging under well managed irrigated conditions. The popular semi-dwarf GR varieties such as IR64, Swarna, Sambha Mahsuri, Sabitri and many others have been cultivated for decades but they are highly susceptible to drought in comparison to their traditional counterparts. This is likely to be the result of the loss of many valuable drought tolerance and other abiotic stresses loci tightly linked to traits rejected during the development of high yielding GR rice varieties.

Drought adversely affected two billion people in the last century. Expected climato- demography changes predict exacerbated drought scenarios. Rice is the major food and livelihood crop for poor inhabits of many drought prone areas. Drought tolerant rice varieties may therefore alleviate poverty and hunger. Despite this being the case, breeding lines, QTLs, genes or

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. In particular, 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.

General Definitions

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, feature, composition of matter, group of steps or group of features or compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, features, compositions of matter, groups of steps or groups of features or compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this

specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in quantitative genetics, molecular genetics, plant breeding, agronomy etc).

Unless otherwise indicated, the recombinant DNA, recombinant protein and cell culture techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, ohn Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present).

For other techniques and terms referred to herein, reference is made to Allard, R. W. Principles of Plant Breeding, 2nd Edition, Wiley New York, 1999, and specifically to the Glossary therein.

Throughout this specification, unless the context requires otherwise, the word

"comprise", or variations such as "comprises" or "comprising", is understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Specific definitions

The term "yield", "yielding" or similar as used herein 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.

As used herein, the term "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.

As used herein, 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. In plant breeding, 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.

The term "backcross", "backcrossing" or similar 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.As used herein, 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. by Agrobacterium- mediated transformation, bombardment using microparticles coated with recombinant DNA, transfection, viral transduction or other method, or by programmable site-specific nucleases, e.g. zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regulator interspaced short palindromic repeat (CRISP)/Cas-based RNA-guided DNA endonucleases, or other nuclease . 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.

As used herein, the term a "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.

The term "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.

The term "percent identity" as used herein 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.

As used herein, the term "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. Thus, plant promoters include promoters obtained from plants, plant viruses and bacteria, such as Agrobacterium and Bradyrhizobium bacteria.

As used herein, the term "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. As used herein, 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.

Numerous 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. NO: 7,151,204 which discloses a maize chloroplast aldolase promoter and a maize aldolase (FDA) promoter, and US Patent Application Publication 2003/0131377 Al which discloses a maize nicotianamine synthase promoter. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant nucleic acids of the present invention to provide for expression of desired genes in transgenic plant cells.

Furthermore, the promoters may be altered to contain multiple "enhancer sequences" to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence within a construct of the disclosure, 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. In some instances, 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. 5,641,876) and rice actin 2 genes, the maize alcohol dehydrogenase gene intron, the maize heat shock protein 70 gene intron (U.S. Pat. No. 5,593,874) and the maize shrunken 1 gene. See also US Patent Application Publication 2002/0192813 Al which discloses 5', 3' and intron elements useful in the design of effective plant expression vectors.

The term "quantitative trait locus", "quantitative trait loci" or "QTL" as used herein 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).

The term "associated with" or "associated" or similar as used herein 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. Similarly, 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. When variants of two genetic loci are in strong linkage disequilibrium, the variant at one locus may be predictive of the variant at the other locus on an individual chromosome.

The term "marker" or "molecular marker" or "genetic 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.

The term "interval" as used herein refers to a continuous linear span of chromosomal DNA with termini defined by and including molecular markers.

The term "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). 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).

The phrase "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).

Generally, 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. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes

complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). 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. For PCR, 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).

The term "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.

As used herein, the term "breeding" or similar refer 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.

The term "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.

The term "crossing" as used herein refers to the fertilization of female plants (or gametes) by male plants (or gametes). The term "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.

The term "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.

The term "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.

As used herein, the term "homozygous" refers to a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes. Conversely, the term

"heterozygous" means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.

The term "recombination" or "recombine" refers to the exchange of genetic material between two homologous chromosomes during meiosis. During a recombination event in a plant, 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). In order to exchange only the required material, and maintain the valuable original information on the chromosome as much as possible, two flanking crossover or recombination events will usually be required. In a double recombinant this exchange has taken place on both sides of a gene/locus.

As used herein, 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. As is known in the art, 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. In some embodiments, 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). Thus, in some embodiments a genotype comprises a summary of one or more alleles present within an individual at one or more genetic loci of a quantitative trait. In some embodiments, a genotype is expressed in terms of a haplotype

As used herein, the term "progeny" means one or more genetic descendants or offspring.

As used herein, the term "population" means a genetically heterogeneous collection of plants sharing a common genetic derivation.

As used herein, the term "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.

The term "segregate", as used herein, refers to the separation of paired alleles during meiosis so that members of each pair of alleles appear in different gametes.

The term "co-segregation" as used herein 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.

As used herein, the term "linkage" or similar 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.

As used herein, the term "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.

The term "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. General Description

Several drought grain yield QTLs have been identified at IRRI during the past few years (Bernier et al. 2007; Venuprasad et al. 2009; Vikram et al. 2011 ; Ghimire et al. 2012; Mishra et al. 2013; Yadaw et al. 2013; Swamy et al. 2013). Some of these are reported to be associated with undesirable traits such as tall PH {qDTYu and qDTY₆₂), reduced yield under irrigated condition (qDTY₃ ) and very early DTF (qDTY_{3 2})- In all these four cases, the characteristic traits of landraces such as tall plant height, low yield potential and earliness are closely associated with DTY QTLs. qDTYu (Vikram et al. 2011; Ghimire et al. 2012) is co-located on rice chromosome 1 with the sdl gene responsible for the GR. In addition to its effect on grain yield under drought, the region encompassing qDTY_L1 and sdl also showed an effect on increased plant height. The studies described herein examined whether the increase in grain yield under drought reported for qDTY_L1 is due to close genetic linkage of qDTY_L1 with sdl, or due to a pleiotropic effect of the sdl tallness allele itself. It has also been observed that there several tall rice varieties highly susceptible to drought. For example, TDK1 is a popular tall rice variety harboring the tall allele of sdl but is susceptible to drought. As described herein, 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).

In a BC₃F₄ population of 160 lines with similar plant height, days to flowering, fixed sdl allele, and a close genetic background, the position of qDTY_L1 was validated to be distal to the sdl locus. Re-mapping of qDTY_L1 in RILs as well as RIL recombinants showed the QTL location to be between sdl and RM431 (~500Kb). The recombination breakpoint analysis targeted high density SNP assay on 160 recombinants also confirmed qDTY_L1 region below sdlgene, between markers nksdtyl_l_34- nksdtyl_l_38 (Figure 4).

Association analysis on a random set of 123 varieties also revealed that the sdl gene has a significant association with PH but not with GY under RS (Table 1). Multi trait-multiple interval mapping (MT-MIM) analysis also showed no significant association of GY with PH under drought stress (Table 2). A similar approach has been followed by others to test pleiotropic effects of other co- located QTLs (Kumawat et al. 2012). Table 1. qDTYj _Λ interval and effect in the three N22 derived RIL populations for GY under RS

F- Additive effect

Population Marker interval value (%) R² Season idl024366- idl024499 41 31 19.1 2009DS

RM12091-

RM12146 10 9 6 2010DS idl024366-

N22/Swarna idl024499 42 25 35.4 Combined

idl024499-RM431 89 30 8.8 2009DS

RM12091-

RM12146 10 9 5.1 2010DS

N22/IR64 idl024499-RM431 85 26 9.1 Combined

idl024366- idl024499 41 19 11.7 2009DS idl024366- idl024499 17 11 8.1 2010DS idl024366-

N22/MTU1010 idl024499 49 18 14.3 Combined

idl024366- idl024499 36 29 18.8 2009DS idl024366- idl024499 15 22 9.1 2010DS

RIL idl024366-

Recombinants idl024499 41 27 21.2 Combined

Re- mapping/fine mapping, association analysis, as well as statistical analysis indicated that qDTYi i is located distal to the sdl gene and is tightly linked thereto. Further, the effect on increase in grain yield under drought contributed by this region is shown herein to be due to qDTYi , whereas the sdl gene did not contribute to drought tolerance. Therefore, due to tight linkage between the tall allele of sdl and the drought tolerant allele of qDTYi _.i, the transfer of semi-dwarf allele of sdl led to the loss of the drought tolerant qDTYi allele in the semi-dwarf GR varieties during their development, leading to their increased susceptibility to drought.

Table 2. Pleiotropy effect of grain yield QTL qDTYi on yield component traits using QGene software (combined analysis over two years).

Population Environment DTF PHT BIO HI

NS 0.7(1.65) 0.9(2.42) 8.4(2.93) 0.5(1.86)

N22/ IR64 RS 1.8(2.12) 2.1(2.26) 6.7(2.96) 0.9(1.92)

NS 1.6(1.84) 2.4(2.47) 3.7(3.21) 1.5(1.69)

N22/ Swarna

RS 1.4(2.01) 1.6(2.41) 3.7(2.73) 1.8(2.39)

Values in the bracket are LOD threshold values for significance. DTF= days to 50% flowering, PHT= plant height, BIO= biomass, HI= harvest index

That breeding for the semi-dwarf trait led to introgression of drought susceptibility was also revealed from the analysis of the qDTY _Λ and sdl using the IK Rice genome SNP annotation from the 3K sequence data. This was noticed only recently, when GR varieties showed high reduction in yield due to increased occurrence of drought in recent years.

Physiological mechanisms linked to the presence of qDTY; _Λ further demonstrated that drought tolerance by this QTL is independent of plant height. The mechanisms observed in the qDTYj _Λ 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.

Of the candidate genes (Table 3) identified within the qDTY] _Λ region, NAM (no apical meristem protein) is one of the components of the drought responsive NAC gene complex. 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 _Λ 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).

Table 3. List of the candidate genes at distal end of sdl gene and tightly linked with RM431 (38.4 - 39.0 Mb)

S.No. feature start end Locus name Putative function

No apical meristem protein,

1 gene 38401207 38401533 LOC _Os01j ?66120 putative, expressed

armadillo/beta-catenin repeat family

2 gene 38409201 38409365 LOC _Os01j ?66130 protein, putative, expressed

plus -3 domain containing protein,

3 gene 38424266 38424621 LOC _Os01j ?66140 putative, expressed

4 gene 38432869 38433670 LOC _Os01j ?66150 expressed protein

pentatricopeptide, putative,

5 gene 38435460 38437074 LOC _Os01j ?66160 expressed

SNARE associated Golgi protein,

6 gene 38439863 38440460 LOC _Os01j Φ6Π0 putative, expressed

7 gene 38443627 38443810 LOC _Os01j ?66180 cytochrome c, putative, expressed

8 gene 38450202 38450501 LOC _Os01j ?66190 expressed protein

9 gene 38460094 38460564 LOC _Os01j ?66200 expressed protein

retrotransposon protein, putative,

10 gene 38465531 38465598 LOC _Os01j ?66210 Ty3-gypsy subclass gene 38474878 38475218 LOC_Os01g66230 csAtPR5, putative, expressed

mitochondrion protein, putative, gene 38475617 38475831 LOC_Os01g66240 expressed

S-locus-like receptor protein kinase, gene 38481235 38484007 LOC_Os01g66250 putative, expressed

gene 38484468 38485604 LOC_Os01g66260 expressed protein

AP2 domain containing protein, gene 38489525 38490169 LOC_Os01g66270 expressed

transcriptional regulator, putative. gene 38493839 38494134 LOC_Os01g66280 expressed

OsMADS21 - MADS-box family gene with MIKCc type-box.

gene 38500880 38501056 LOC_Os01g66290 expressed

KH domain containing protein, gene 38509123 38509734 LOC_Os01g66300 putative, expressed

gene 38516417 38516517 LOC_Os01g66310 expressed protein

gene 38519987 38520259 LOC_Os01g66320 expressed protein

ATP-dependent Clp protease ATP- binding subunit clpX, putative. gene 38525642 38526409 LOC_Os01g66330 expressed

gene 38527961 38528185 LOC_Os01g66340 expressed protein

DUF647 domain containing protein, gene 38534823 38535329 LOC_Os01g66350 putative, expressed

2-C-methyl-D-erythritol 4- phosphate cytidylyltransferase, gene 38541818 38542109 LOC_Os01g66360 putative, expressed

gene 38557970 38558381 LOC_Os01g66379 expressed protein

gene 38559137 38559341 LOC_Os01g66400 expressed protein

gene 38561851 38561924 LOC_Os01g66410 expressed protein

PHD finger protein, putative, gene 38570487 38570861 LOC_Os01g66420 expressed

gene 38574115 38574132 LOC_Os01g66440 expressed protein

retrotransposon protein, putative, gene 38582407 38582574 LOC_Os01g66450 unclassified, expressed

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

no apical meristem protein, gene 38611435 38612105 LOC_Os01g66490 putative, expressed

phosphoribosylformylglycinamidine gene 38623771 38623836 LOC_Os01g66500 synthase, putative, expressed

MLO domain containing protein. gene 38627746 38628127 LOC_Os01g66510 putative, expressed

serine/threonine-protein kinase gene 38635322 38635651 LOC_Os01g66520 RIO-like, putative, expressed gene 38637331 38638234 LOC_Os01g66530 ARGOS, putative, expressed gene 38645466 38645708 LOC_Os01g66544 expressed protein

signal recognition particle 72 kDa gene 38650518 38650715 LOC_Os01g66560 protein, putative, expressed

ZOS 1 -19 - C2H2 zinc finger gene 38655236 38656447 LOC_Os01g66570 protein, expressed

RNA polymerase III RPC4 domain gene 38661257 38661310 LOC_Os01g66580 containing protein, expressed gene 38676217 38676530 LOC_Os01g66590 DUF260 domain containing protein, putative, expressed

gene 38687370 38688097 LOC_ Os01g66600 rhodanese-like, putative, expressed serine/threonine-protein kinase receptor precursor, putative, gene 38692318 38692621 LOC_ Os01g66610 expressed

glucosyltransferase, putative, gene 38696091 38696135 LOC_ Os01g66620 expressed

S -domain receptor-like protein gene 38699042 38699146 LOC_ Os01g66630 kinase, putative, expressed

S-domain receptor-like protein gene 38704701 38704872 LOC_ Os01g66640 kinase, putative, expressed gene 3871661 1 38716756 LOC_ Os01g66650 expressed protein

gene 38718491 38719023 LOC_ Os01g66660 expressed protein

gene 38723347 38724165 LOC_ Os01g66670 expressed protein

S-domain receptor-like protein gene 38729814 38729926 LOC_ Os01g66680 kinase, putative, expressed gene 38736544 38737856 LOC_ Os01g66690 ZIP4/SP022, putative, expressed beta-hexosaminidase precursor, gene 38745734 38745918 LOC_ Os01g66700 putative, expressed

polygalacturonase, putative. gene 38749035 38749487 LOC_ Os01g66710 expressed

NADP-dependent oxidoreductase. gene 3875261 1 38752935 LOC _Os01g66720 putative, expressed

exosome complex exonuclease gene 38759291 38759513 LOC _Os01g66730 RRP40, putative, expressed

inactive receptor kinase Atlg27190 gene 38759757 38759950 LOC _Os01g66740 precursor, putative, expressed retrotransposon protein, putative, gene 38767579 38768280 LOC _Os01g66750 unclassified, expressed

inactive receptor kinase At2g26730 gene 38770126 38770260 LOC _Os01g66760 precursor, putative, expressed retrotransposon protein, putative, gene 38778041 38778137 LOC _Os01g66780 unclassified

retrotransposon protein, putative, gene 38785135 38786060 LOC _Os01g66790 unclassified, expressed gene 38788306 38788417 LOC _Os01g66800 expressed protein

gene 38790403 38790510 LOC _Os01g66810 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_. _Os01g66870 expressed

BTBZ1 - Bric-a-Brac, Tramtrack, and Broad Complex BTB domain with TAZ zinc finger and Calmodulin-binding domains, gene 38843725 38843862 LOC_. _Os01g66890 expressed

gene 38854092 38854277 LOC_. _Os01g66900 expressed protein

gene 38859999 38860214 LOC_. _Os01g66910 expressed protein

Ser/Thr protein phosphatase family gene 38864020 38864322 LOC_. _Os01g66920 protein, putative, expressed kinase, pfkB family, putative,

74 gene 38887107 38887723 LOC _Os01j ?66940 expressed

75 gene 38888805 38889245 LOC _Os01j ?66950 expressed protein

selenoprotein precursor, putative,

76 gene 38891151 38891518 LOC _Os01j ?66960 expressed

zinc finger, C3HC4 type domain

77 gene 38897220 38897714 LOC _Os01j ?66970 containing protein, expressed

78 gene 38902608 38902752 LOC _Os01j Φ6980 expressed protein

79 gene 38909394 38909598 LOC _Os01j ?66990 expressed protein

membrane-associated 30 kDa

protein, chloroplast precursor,

80 gene 38915255 38915492 LOC _Os01j φΊΟΟΟ putative, expressed

81 gene 38916092 38916366 LOC _Os01j φΊΟΙΟ expressed protein

auxin-responsive protein, putative,

82 gene 38924789 38925118 LOC _Os01j ?67030 expressed

OsRhmbd5 - Putative Rhomboid

83 gene 38928602 38930270 LOC _Os01j ?67040 homologue, expressed

calreticulin precursor protein,

84 gene 38934590 38934770 LOC _Os01j ?67054 putative, expressed

85 gene 38938902 38939459 LOC _Os01j ?67070 expressed protein

86 gene 38945620 38945638 LOC _Os01j ?67080 expressed protein

IQ calmodulin-binding motif

domain containing protein,

87 gene 38946700 38946868 LOC _Os01j ?67090 expressed

In addition to linkage between sdl and qDTY , the association of other drought GY QTLs qDTY₆₂, qDTY₃ j, and qDTY_{3 2} are described herein. The positive alleles of qDTY₆₂, qDTY₃ j, and qDTY_{3 2}were responsible for tall plant height, low yield under irrigated conditions and early maturity respectively (Venuprasad et al. 2009; Vikram et al. 2011 ; Dixit et al. 2012). Tall plant height, lower yield under irrigated conditions, and early DTF leading to early maturity were typically associated drought tolerance. Indeed, most of the known drought tolerant donors are intermediate/tall (more than 110-130 cm in lowland, 90-125 cm in upland) to tall (more than 130 cm in lowland, more than 125 cm in upland) in height, have low yield potential (less than 3.0 tha^"1) and mature early (80-100 days) compared with the GR varieties that are semi-dwarf in height (less than 110 cm in lowland and less than 90 cm in upland), have higher yield potential (more than 5.0 tha ^_1) and possess medium maturity period duration (110-130 days). The study described herein indicates that the linkages of QTLs for grain yield under drought (qDTY) with tall plant height, lower yield potential, and early maturity are the reason for traditional drought tolerant varieties having tall height, lower yield potential and early maturity characteristics.

An allelic analysis with 11 drought GY QTLs showed a genetic shift in modern varieties with respect to these loci (Figure 8). A sharp decrease in the frequency of drought tolerant alleles in GR varieties as compared to the traditional drought tolerant varieties/drought tolerant donors was observed (Figure 9). The loss of drought tolerant alleles continued as breeding for semi-dwarf varieties proceeded with time from IR8 in the 1960s to Jaya in 1970s to IR 36, IR 64, Swarna and Samba Mahsuri in 1980s and beyond (Figure 9). This trend resulted from replacement of drought tolerant alleles (alleles conferring adaptation to adverse conditions) with alleles adapted to irrigated ecosystems (favorable conditions) through intercrossing among the GR varieties, and is the approach by which breeding has progressed globally from 1966 until now. Linkage of drought tolerant alleles with unfavorable traits/traits rejected for in the GR period (tall plant height, lower yield potential, and very early maturity) is the key factor for the loss of the drought tolerant alleles during selection for semi-dwarf height, high yield potential, and medium maturity period duration during the GR era.

As shown and described herein, new drought-tolerant semi-dwarf lines have been developed through the successful breakage of linkages between loci for drought tolerance and desirable traits. Semi-dwarf drought tolerant genotypes homozygous for a drought tolerant qDTY] _Λ allele, and grain yield similar to Swarna under favorable conditions, were successfully developed (Table 4).

Genotypes with qDTY₃ j, qDTY_{3 2} and qDTY₆₂ QTLs without unfavorable linkages of traits were also identified. In all four cases, the semi-dwarf plants with significant yield advantage under drought over the recipient parents were developed without any compromise for yield potential under favorable conditions. Bringing back drought tolerant alleles in semi-dwarf cultivars through systematic marker assisted introgression programs is described herein.

In certain embodiments described herein, 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.

Because the nucleic acid sequence of a QTL that is responsible for conferring drought tolerance may only be a fraction of an entire QTL herein identified, 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. As a result, a yield-improving part of a QTL need not necessarily comprise any of the listed markers. Also, 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, qDTY₆₂, qDTY3.1, and qDTY_{3 2}

Stress Non stress

QTL Genotype

DTF PHT GY DTF PHT GY

IR 91659:41-95-B 90 72 3079 94 84 4530

IR 91659:54-36-B 92 68 3299 96 82 5027

A: qDTY_u IR 91659:41-95-B 86 69 3123 96 81 4487

Swarna 102 63 561 96 84 4312

LSD^{0 05} 3 6 771 3 5 1246

IR 90266-B-491-1 83 92 1750 74 109 5750

IR 90266-B-155-1 86 96 1637 73 113 5740

B: qDTY₆₂ IR 90266-B-53-1 84 102 1947 70 116 5672

TDK1 99 73 173 74 111 5985

LSD^{0 05} 4 10 714 8 14 1578

IR81896-B-B-309 98 82 1928 92 138 5174

IR81896-B-B-481 98 76 929 95 127 5592

C: qDTY_{3 1} IR81896-B-B-305 102 85 808 94 130 6717

Swarna 54 0 103 82 4121

LSD^{0 05} 7 24 664 4 4 873

IR 79971-B-102-B 79 84 920 83 110 3547

IR 79971-B-421-B 84 79 756 84 110 3156

D: qDTY_{3 2} IR 79971-B-86-B 68 76 847 73 101 3311

Vandana 64 73 617 63 94 2180

LSD^{0 05} 5 10 241 3 12 782

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. Preferably, 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. Thus, 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. For instance, 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.

In embodiments of such methods for detecting the presence of a QTL in a rice plant, 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. Preferably, 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. Alternatively, and in a more preferred embodiment, 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.

Also provided herein are methods for determining the allelic identity of the Green Revolution semi-dwarf gene (sdl; LOC_Os01g66100). Described herein is a mutation (Y342*) of the sdl gene that leads to a termination codon in the third exon of sdl -encoded gibberellin-20 oxidase. 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. PCR

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. In certain embodiments, the allelic identity of sdl is determined alongside identifying the presence of a QTL allele associated with drought tolerance in a rice plant.

Production of Semi-Dwarf Drought Tolerant Rice Plants by Transgenic Methods.

According to one aspect of the present invention, a nucleic acid (preferably DNA) sequence comprising at least one QTL of the present invention (qDTY; _A, qDTY₃ j, qDTY_{3 2}, and qDTY₆₂) or a yield-improving part thereof, may be used for the production of a rice plant with improved drought tolerance. In this aspect, 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. In particular embodiments the nucleic acid sequence comprising the QTL is introduced in a semi-dwarf rice plant comprising a dwarfing allele of sdl. As stated, 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, IR743-70-1-1, 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.

Once identified in a suitable donor rice plant, the nucleic acid sequence that comprises a QTL for drought tolerance according to the present invention, or a yield-improving part thereof, may be transferred to a suitable recipient plant by any method available. In certain embodiments, 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_C222, Dee-Geo-Woo-Gen, IRRI119, Purbachi (Chinese 1), Sabitri, and RD25.

For instance, 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. For transgenic methods of transfer, 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. In the present invention, such 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). Many commonly used selectable marker genes for plant transformation are known in the art, and 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. Several positive selection methods are known in the art, such as mannose selection. Alternatively, marker-less transformation can be used to obtain plants without mentioned marker genes, the techniques for which are known in the art.

One method for introducing an expression vector into a plant is based on the natural transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria that genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.

rhizogenes, respectively, 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.

General methods of culturing plant tissues are provided, for example, by Miki et al., 1993 and by Phillips, et al., 1988. A reference handbook for molecular cloning techniques and suitable expression vectors is Sambrook and Russell (2001).

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. Alternatively, liposome or spheroplast fusion has been used to introduce expression vectors into plants. Direct uptake of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, or poly-L-ornithine may also be used. Electroporation of protoplasts and whole cells and tissues has also been described.

Following transformation of target tissues, expression of the above 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.

Production of Semi-Dwarf Drought Resistant Rice Plants by Programmable Site-Specific Nucleases.

Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases comprise a powerful class of tools useful in genome engineering. 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.

Site-specific nucleases induce DNA double-strand breaks that stimulate non-homologous end joining and homology directed repair at targeted genomic loci. A thorough review of the ZFN, TALEN, and CRISPR/Cas-based RNA-guided DNA endonuclease is available (Gaj et al., 2013). Further discussion of ZNFs may be found in US Patents 8,106,255, 8,399,218, and 8,592,645. Further discussion of TALENs may be found in US Patent 8,697,853. Further discussion of CRISPR/Cas- based RNA-guided DNA endonucleases may be found in US Patent 8,697,359, and in J.D. Sander & J.K. Juong (2014).

In certain aspects, any one of these technologies (ZFNs, TALENs, and CRISPR/Cas-based RNA guided DNA endonucleases) 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. For example, 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.

Production of Semi-Dwarf Drought Resistant Rice Plants by Non-Transgenic Methods.

In an alternative embodiment for producing a semi-dwarf drought tolerant rice plant, 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. For example, 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.

Alternatively, 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). 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.

As discussed above, traditional breeding techniques can be used to introgress a nucleic acid sequence encoding for drought tolerance into a recipient semi-dwarf drought susceptible rice plant. In one method, which is referred to as pedigree breeding, 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. For example, 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. In certain embodiments, 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.

A Semi-Dwarf Drought Tolerant Rice Plant, or a Part Thereof, Obtainable by a Method of the Invention is Also an Aspect of the Present Invention.

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. In particular embodiments, the QTL is qDTY] . In yet other embodiments, the at least one QTL is one or more of qDTY] , qDTY₃ , qDTY_{3 2}, and qDTY₆₂.

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. Although 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. In a method of selection and backcrossing, 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. Instead of using phenotypic pathology screens of bioassays, 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.

Alternatively, MAS can be used to confirm results obtained from quantitative bioassays. Once the appropriate selections are made, 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. For example, 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.

Marker Assisted Selection and Backcrossing.

qDTY_u MAS and MABC are described herein. MAS and MABC may be further applied to qDTY_{3 1}, qDTY_{3 2}, and qDTY₆₂.

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; , qDTY₃ qDTY_{3 2}, and qDTY₆₂, 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.

After a desired phenotype, e.g., semi-dwarf plant height or drought tolerance, and a

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). In brief, 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 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.

Another use of MAS in plant breeding is to assist the recovery of the recurrent parent genotype by backcross breeding. 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.

There are many kinds of molecular markers useful in MAS and MABC. For example, 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). When 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. Among all possible SSRs, tri-nucleotide repeats or multiples thereof are more common in coding regions.

A single nucleotide polymorphism (SNP) 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)).

The majority of genetic markers rely on one or more properties of nucleic acids for their detection. For example, some techniques for detecting genetic markers utilize hybridization of a probe nucleic acid to nucleic acids corresponding to the genetic marker. 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 (RFLP), 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. After separation by length in an appropriate matrix (e.g., agarose) and transfer to a membrane (e.g., nitrocellulose, nylon), the labeled probe is hybridized under conditions which result in equilibrium binding of the probe to the target followed by removal of excess probe by washing. 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. Preferably, 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.

In vitro amplification techniques are known in the art. Examples of techniques sufficient to direct persons of skill through such in vitro methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), Ο,β-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA), are readily found in the art. One of skill will appreciate that essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase.

Oligonucleotides for use as primers, e.g., in amplification reactions and for use as nucleic acid sequence probes, are typically synthesized chemically according to the solid phase phosphoramidite triester method, or can simply be ordered commercially.

Alternatively, 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.

As mentioned above, there are many different types of molecular markers, including amplified fragment length polymorphisms (AFLP), allele-specific hybridization (ASH), single nucleotide polymorphisms (SNP), simple sequence repeats (SSR), and isozyme markers. Methods of using the different types of molecular markers are known to those skilled in the art.

The qDTY_u, qDTY₃ qDTY_{3 2}, and qDTY₆₂ 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.

In particular embodiments described herein, methods for producing a semi-dwarf drought tolerant rice plant are described. 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 qDTY₆₂. 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. In particular embodiments, the QTL is qDTY_{! h} and the rice variety is one of N22, Dagaddeshi, Apo, Vandana, or Black Gora.. In other embodiments, the QTL is any one or more of the QTLs qDTY_! , qDTY_{3 1}, qDTY_{3 2}, and qDTY₆₂. In yet other embodiments, the QTLs comprise qDTY a, and one or more of qDTY₃ , qDTY_{3 2}, and qDTY₆₂. In particular embodiments a preferred donor plant for qDTY₃ 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, and a preferred donor plant for qDTY₆₂ is a rice plant of variety IR55419-04, although other drought tolerant varieties comprising these QTLs may also be used. Wherein the recipient rice plant comprises two or more of qDTY , qDTY₃ , qDTY_{3 2}, and qDTY₆₂, the QTLs (nucleic acids) may be transferred to the donor plant one at a time, or two or more at a time. Preferably, 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. In certain embodiments, 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. In particular embodiments, 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. In particular embodiments, 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. However, 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. In certain embodiments, 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, qDTY₃ j, qDTY_{3 2}, and qDTY₆₂) 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. In a preferred embodiment, 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.

Following the transfer of the nucleic acid from the donor plant to the one or more recipient plants, at least one rice plant retaining its original dwarfing sdl allele and a drought tolerant allele of one or more of qDTYu, qDTY₃ , qDTY_{3 2}, and qDTY₆₂ is identified and selected. Preferably, DNA from progeny plants resulting from crossing donor and recipient plants is used for identifying and selecting. A drought tolerant allele of qDTY _Λ 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 qDTY₆₂ are similarly indicated by genetic markers: the drought tolerant allele of qDTY₃ 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 qDTY₃₂ 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 qDTY₆₂ 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. In a preferred embodiment, rice plants comprising the dwarfing allele of sdl and the drought tolerant allele of qDTYj _Λ are selected.

A number of the above-mentioned markers and the primer sequences for their identification are known in the art e.g., as described in IRGSP: The map-based sequence of the nee genome. Nature 2005, 436:793-800 and as available at http://archive.gramene.org/markers/microsat/all-ssr.html. However, for convenience, primer sequences for the above-mentioned markers characterizing qDTYu, qDTY₃ j, qDTY_{3 2}, and qDTY₆₂ are provided in Tables 5 and 6 below.

Table 5. Details of markers for identifying drought tolerance alleles of qDTYj _Λ QTL region.

Table 6. Details of markers for identifying drought tolerance alleles of qDTYu, qDTY₃ , qDTY_{3 2}, and qDTY₆₂ QTL region.

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.

Also provided herein are plants, or 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.

Also provided herein are methods for producing a semi-dwarf drought tolerant inbred rice plant. To produce such a plant, a selected semi-dwarf drought tolerant rice plants described above is selfed or backcrossed to produce an inbred rice line. Preferably, the inbred rice line comprises the dwarf sdl allele and at least one drought tolerance-associated QTL (e.g., qDTYu, qDTY₃ j, qDTY_{3 2}, or qDTY₆₂). Even more preferably, the inbred rice line is homozygous for these alleles.

In another aspect, a semi-dwarf drought tolerant rice plant, or a part thereof, 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

RM12146. In particular embodiments, the rice plant further comprises one or more of qDTY₃ , qDTY_{3 2}, and qDTY₆₂, wherein drought tolerant alleles of qDTY₃ , qDTY_{3 2}, and qDTY₆₂ are indicated by genetic markers: the drought tolerant allele of qDTY₃ 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 qDTY₆₂ 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 described herein are methods for identifying a rice plant as having a semi-dwarf drought tolerant phenotype. These methods comprise of extracting genomic DNA from a rice plant, detecting in the rice plant a semi-dwarf allele of sdl comprising a Y342* mutation using methods and markers described herein, detecting in the rice plant a drought tolerant allele of qDTYu, qDTY₃ , qDTY_{3 2}, qDTY₆₂ or a combination thereof utilizing the markers disclosed and described herein, and 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 a drought tolerant qDTY allele are detected.

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_! , wherein qDTYlj _Λ is not in its natural genetic background, and wherein the drought tolerant allele of qDTYi _Λ 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. In certain embodiments, the processed rice material in the product described herein may also comprise a drought tolerant allele of at least one of qDTY₃ j, qDTY₃₂, and qDTY₆₂, wherein the drought tolerant allele of qDTY_{3 Λ} 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 qDTY₆₂ 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. In one example, 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.

A skilled person will appreciate that the method of processing employed may vary depending on the product or material being produced. However, 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.

Also provided herein is 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 _Λ 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;

Rml2091 ; and RM12146. In one example, the processed rice material or product comprising same further comprises a drought tolerant allele of at least one of qDTY₃ , qDTY_{3 2}, and qDTY₆₂, 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 qDTY₆₂ 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. In one example, 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).

Preferably, the processed rice material or product comprising same is produced from grain of a semi-dwarf drought tolerant rice plant as described herein. In one example, the processed rice material or product comprising same is packaged for sale.

According to any example described herein, 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:

(i) 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;

(ii) 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

(iii) a beverage product selected from the group consisting of rice milk and beverages containing ethanol produced from rice e.g., rice wine or sake.

In another example, the processed rice material or product comprising same is non-edible for humans. Examples of 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.

Also provided is the use of grain from a semi-dwarf drought tolerant rice plant as described herein, or part thereof, as animal feed or food, or to produce feed for animal consumption or food for human consumption.

Examples

The following examples serve to explain the present disclosure in more detail. These examples should not be construed as being exhaustive or exclusive as to the scope of the present disclosure. Example I. Materials and Methods

Plant material and genotyping.

Seven sets of plant materials were used:

1) The previously identified qDTY; _/region in three populations with 292 lines (N22/Swarna), 289 lines (N22/IR64), and 362 lines (N22/MTU1010) were genotyped with SNP markers underlying qDTYj i using the Fluidigm SNP genotyping platform (K-Biosciences Ltd) for improved resolution of the map of the qDTY_u/sdl region. RIL recombinants from the three populations were also genotyped with SNP markers flanking qDTYj _Λ and with an sdl gene specific marker (Figure 10).

2) An N22/4*Swarna backcross population was used for the qDTYj lsdl linkage and fine mapping study. To develop this population, a total of 3000 BC₃Fj plants were grown and plants with comparable semi-dwarf plant height to Swarna were selected. Of these, 217 semi-dwarf plants were further genotyped with qDTY_u markers RM11943, RM431, RM12023, RM12091 and RM12146. Only two plants were identified in which qDTYu was segregating. These two plants were analyzed with the sdl gene based marker for confirming the semi-dwarf allele and selfed for generation advance, and the resulting 180 BC₃F_2:3 N22/Swarna plants were analyzed with the qDTYu markers. Among 180 BC₃F₃ N22/Swarna plants, there were 20 heterozygote recombinants for flanking markers for the sdl gene as well as qDTYu. These heterozygotes were selfed to produce -1200 BC₃F₄ plants which were then genotyped to select 160 recombinants based on the two flanking markers of qDTYu. In this BC F₄ N22/Swarna population, DTF, PH and the sdl allele were similar in all selected lines. Background QTL effects were also neutralized in this population (Figure 2). Another QTL, qDTY_{3 2} identified in N22/Swarna population in a previous study by Vikram et al. (2011) was also fixed. Along with qDTY_{3 2}, there were five introgressed regions (RM246, RM278, RM335, RM434 and RM551) in the recombinant lines. N22 alleles for these markers were fixed in the recombinant lines and the population was segregating for only one (RM411) of the 120 markers used to test the background effect, and this one marker did not show significance for any of the tested drought -related traits. The fine mapping study was conducted on these 160 recombinants (BC₃F_4:5 and BC₃F_4:6). Phenotypic variation for GY and related traits in the recombinant BIL population is presented in Table 7.

Table 7. Phenotypic variation for GY related traits under RS and NS in the dwarf N22/Swarna BIL population

NON STRESS STRESS

RANGE MEAN ± SD RANGE MEAN ± SD

DS2012 DS2013 DS2012 DS2013 DS2012 DS2013 DS2012 DS2013

DTF 59-127 62-99 96±7 88±6 70-119 62-105 100±6 92±6

PHT 64-122 71-112 94±6 84±6 52-88 52-99 72±5 67±6

BIO 1628-33740 2450-33350 17790±5988 15490±4539 698-19050 845-21650 10760±7801 9347+2459

GY 557-11590 368-10970 5448±2160 5341±1530 0-6869 71-4238 1872±696 1735+624

HI 0.043-0.50 0.105-0.499 0.296±0.078 0.401±0.06 0.001-0.44 0.001-0.50 0.21+0.13 0.21+0.09

3) Four qDTYu near isogenic lines (NILs) IR 91659-41-95-B, IR 91659-41-36-B, IR 91659-41- 39-B, IR 91659-41-59-B along with parents N22 and Swarna were characterized in lysimeter as well as in field studies from 2012-2014 to understand the physiological mechanism of drought tolerance conferred by qDTYj

4) 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).

5) 1000 rice genotypes that were recently sequenced by IRRI under the 3K Rice Genome project was used to study the linkage pattern between sdl allele and qDTYu N22 allele.

6) RIL/ BIL populations were used to understand the linkage of qDTY₆₂, qDTY3.1, qDTY_{3 2} and tall plant height, reduced yield under irrigated conditions, and very early maturity, respectively. A BQF₃ derived population developed from the cross of IR55419-04/ TDK1 (365 lines), a BQF₄ derived population developed by the cross Apo/Swarna (490 lines), and a F₃ derived population developed from the cross Vandana/Way Rarem (242 lines) was used to understand the linkage in the qDTY₆₂, qDTY₃ j, and qDTY_{3 2} regions, 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).

7) In order to understand the 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.

The 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] _Λ homozygote plants (N22/Swarna, N22/IR64 and N22/MTU1010) were screened in 2012DS at IRRI, Philippines. 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. In the NS treatment, 5 cm of standing water were maintained after transplanting throughout the crop season, and then drained before harvesting. Standard IRRI crop management practices for drought screening of lowland rice were followed (Kumar et al. 2008; Venuprasad et al. 2007). Water was drained 30 days after transplanting (DAT) from the RS treatment for the stress imposition. Stress was imposed until severe leaf rolling was observed in at least 70% of the lines, at which time the field was re-watered then drained again after 24 hours to initiate a second stress cycle (Venuprasad et al. 2007). Phenotypic data of screening of the N22-derived RIL populations was reported previously (Vikram et al. 2011). Observations of the number of days to 50% flowering (DTF), plant height (PH), biomass (BIO), grain yield (GY), and harvest index (HI) were measured according to previously described protocols (Venuprasad et al. 2009; Bernier et al. 2007).

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. The drought stress experiments were re-watered periodically as needed to sustain the plants until harvest. Soil water potential was monitored with one tensiometer per replicate that was installed at a depth of 30 cm in each RS experiment. The drought stress across physiology study field seasons was most severe in 2012WS and 2014DS (Figure 13). Table 8. List of 123 genotypes/varieties used for the qDTYl. l/ sdl association with GY under RS.

S.N. Genotype/variety S.N. Genotype/variety S.N. Genotype/variety S.N. Genotype/variety S.N. Genotype/variety

1 AI JIAO NAN TE 26 CR 1014 51 JIRASAIL 76 MTU1010 101 RTS 12

2 ANNADA 27 DAVAO 52 JOYNA 77 MUEY NONG (WANG DIN) 102 RTS 14

3 APO 28 DEE-GEO-WOO-GEN 53 KAJALSAIL 78 N22 103 RTS 5

4 BALAM 29 DHAGAD DESHI 54 KALAKERI 79 NAN TE HAO 104 SAFRI 17

5 BASHFUL 30 DHALASAITA 55 KALAMKATI 80 NAZIRSAIL 105 SAMBA MAHSURI

6 BASMATI 334 31 DHOLI BORO 56 KALIJIRA 81 NIAW 106 SARJOO 50

7 BASMATI 370 32 DINORADO 57 KAO TAH KONG 82 NSIC RC 192 107 SARJU 49

8 BENGAWAN 33 DURGABHOG 58 KASALATH 83 NSIC RC 222 108 SATHI 34-36

9 BETONAN 34 GIE 57 59 KATARIBHOG 84 NSICRC 9 109 SMAGUING

10 BHADOIA 233 35 GS 529;DULAR 60 KHAO DAWK MALI 105 85 PANKAJ (IR 5-114-3-1) 110 SINAMPAGA SELECTION

11 BINNATOHA 36 HOING 61 KHAO GAEW 86 PELITA 1 1 111 SOM CAU 70 A

12 BLACK GORA 37 IR 1561-228-3-3 62 KHAO HAWM 87 PELITA 12 112 SWARNA

13

BPI RI 10 38 IR 36 63 KHAO TAH OO 88 PSBRC 68 113 T 1

14 BR 1 39 IR 42 64 KHAO TAH HAENG 89 PSBRC 80 114 T 136

15 BR 11 40 IR 64 65 KHAO TAH PIENG 90 PSBRC 82 115 TADUKAN

16 BR 2 41 IR 8 66 KU 113-1 91 PURBACHI (CHINESE 1) 116 TAICHUNG 65

17 BR 21 42 IR74371-46-1-1 67 LALSAR 92 RASI 117 TAICHUNG NATIVE 1

18 BROWN GORA 43 IR74371-70-1-1 68 LATISAIL 93 RATNA 118 TKM 6

19 CHAMPA TONG 54 44 IRRI 119 69 LEUANG YAI NONG 94 RD 1 119 UPLRI 4

20 CHAU 45 IRRI 123 70 MACAN BINUNDOK 95 RD 15 120 UPLRI 7

21 CHIEM CHANH 46 IRRI 141 71 MAGAWK DONG 269-7-7 (9) 96 RD 2 121 VANDANA

22 CHINIGURA 47 JAGANATH 72 MAHSURI 97 RD 25 122 WAGWAG

23 CIKAPUNDUNG 48 JAYA 73 MAKMUR 98 RD 6 123 ZHENSHAN 2

24 CO 18 49 JC 148 74 MARIN AH 99 RD 7

25 CO 25 50 JHONA 349 75 MOHINI SAIL 100 RD 9

Four BILs and parents N22 and Swarna were grown in a greenhouse lysimeter experiment from May to July 2013 in large iysimeters (95 cm tali x 20 cm diameter), as described by Kijoji et al. (2013). Five replicates of two treatments (NS, RS) were included in a completely randomized design. The soil in the Iysimeters was maintained flooded until 20 days after planting, after which the iysimeters in the drought stress treatment were drained. Water uptake rates were determined by weighing, at which time images of the shoot were acquired to measure apparent leaf area in Image! until plants were harvested at 60 days after sowing. Maximum root depth was determined as the distance from the base of the plant to the deepest root in the lysimeter.

Shoot growth was monitored by NDVI (normalized difference vegetation index) measured around mid-day using a GreenSeeker Handheld Sensor (NTech Industries, CA, USA) that was carried through the field (2013DS) or mounted about 1 m above the soil from a rack that rolled along the tracks of the rainout shelter (20I4DS). Aboveground biomass partitioning was monitored throughout the season in 2014DS, in which two hills per plot were harvested at each sampling, the leaf blades were separated from the sheaths and culms (the stems), and dry weight was determined. Root samples were taken at 128, 119, 1 13, and 101 DAS following rewatering of the drought stress treatment in 2013WS, 2013DS, 2013WS, and 2014DS, respectively using a 4-cm-diameter core sampler

(fabricated at IRRI, Los Banos, Philippines) to a depth of 60 em (divided into 15-cm segments) and root length determined according to Henry et al. (201 1). 'Percent deep roots' was calculated as the root length below the soil depth of 30 cm as a percent of the total root length in the soil core.

Genotyping with SSR markers of DTY QTLs.

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

approximately 25 ng iL . For SSR analysis PCR amplification was carried with & \5- iL reaction mixture having 50 ng DNA, 1 x PCR buffer, 100 μΜ dNTPs, 250 μΜ primers, and 1 unit Taq polymerase enzyme. To resolve the PCR products, 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.

Genotyping for the alleles of the Sdl gene.

Apart from phenotypic measurements for the presence of sdl, 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). Product size for the dwarfing allele and the tallness alleles were 348 and 731 bp, respectively. This region of the Sdl gene is difficult to amplify consistently due to high GC content, thus the PCR reaction was optimized for consistent amplification with minor modifications to the protocol of Masouleh et al. (2009). For a 20 μΐ PCR reaction: 2μ1 of lOx Enhancer buffer from Invtrogen (50mM ), 1.2 μΐ of MgCl₂ (50mM ), 3.0 μΐ of primers (5pM each), 1 μΐ of dNTPs (2.5 mM each), 6 μΐ of 2X Enhancer, 0.2 μΐ of Platinum ® Taq polymerase (5U/ μΐ) and 2.0 μΐ (20ng/ μΐ) template was used. The 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.

SequonomMassARRAY assay for fine mapping ofqDTY .

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). A total of 46 SNPs located in the 36.36Mb to 40.06 Mb region on chromosome 1 and polymorphic between parents N22 and Swarna (cf. OryzaSNP database;

http://oryzasnp.piar:tbioiogy.ir:SH.edu/) were taken for assay design using the Sequenom MALDI-TOF Mass ARRAY system (http://www.sequenom.com). The multiplex assay was designed using MassARRAY Assay design 4.0 software and the reaction for a single tube multiplex containing 29 assays was optimized. The assay also included the sdl deletion locus with three possible SNP alleles, in addition to 28 SNPs located in 28 different genes in the QTL region (Figure 3). After

standardization of the primer concentrations for MALDI-TOF and validation of the assays in test samples, DNA samples of backcross inbred lines showing recombination between qDTYj _Λ and QTL flanking markers in a large N22/Swarna fine mapping population and 46 random varieties were analyzed. All SNP calls worked well, except for one designed for the sdl del where only one of the alleles (T), representing wild type (tall) allele was called due to high GC content of the amplicon resulting in poor amplification. Therefore, a PCR assay was optimized for the separate genotyping of the Sdl alleles. Table 9. Details of SNPs for custom MALDI assay (for the qDTY QTL region).

Table 10. Details of SNPs for custom MALDI assay (for the qDTY_u QTL region).

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

recombination break points and find association with the phenotyping data.

Statistical analysis.

Statistical analysis was conducted using CROPSTAT v.4.2.3 (available at www.irri.org).

Phenotypic means of entries were estimated using the following linear mixed model for the analysis of variance:

Pi_jk = M + R; + B_j (R;) + L_k + e_ijk [equation 1]

where P _k is the measurement recorded on a plot, M is the mean over all plots, and R, B, L, and e are replications, blocks, lines, and error, respectively. For estimating the entry means, replications and blocks within replicates were considered random whereas entries were fixed. While estimating the entry means across years, season effects were also taken as random.

Statistical analyses for the physiology experiments were performed in R v. 2.15.2 (R

Foundation for Statistical Computing, 2012) by ANOVA (aov script) for genotype and replication; and LSD mean comparison was used as the post -hoc test. For measurements conducted on multiple dates, a repeated measures analysis was conducted with the mixed model ASREML using Wald's test in R, with 'genotype' and 'days after sowing' as fixed variables and 'replicate' as a random variable.

Multiple trait analysis.

Multiple trait multiple interval mapping (MT-MIM) analysis in the N22/IR64 and N22/Swarna RIL population was used to test the pleiotropic effect of qDTY on associated traits as described by Kumuwat et al (2012). This MT-MIM analysis was performed with QGene software version 4.3.10 with 1000 permutations at significance level p < 0.01 (Joehanes and Nelson 2008). Combined mean phenotypic data of two years was used for the analysis. The model used for MT-MIM was

X B 4- E [equation 2]

··: · · I :>:.t. !l X t · where 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, and i=l-d.

QTL analysis using additional markers in the qDTYu region.

Genetic map distances between markers were estimated according to their position on the physical map in reference to rice variety Nipponbare. One mega base pair (Mb) was assumed to be equivalent to -3.92 cM (IRGSP 2005). The QTL analysis in different populations was performed using Inclusive Composite Interval Mapping (ICIM) software (Li et al. 2007). Results were also confirmed with the QTL Network software v2.1 following a mixed model-based composite interval mapping method described by Yang et al. (2008). Candidate intervals were first selected, then significance of association of intervals with the trait of interest was analyzed, and additive effect explained by significantly associated interval was estimated. The significance level for the determination of the candidate intervals, detection of putative QTLs, and their additive effects was P < 0.01. The F- value threshold for significance of the QTL was determined using 1000 permutation tests. The window size and walk speed used for the genome scan were 10 cM and 1 cM, respectively.

Mapping of qDTYu for improving resolution.

A three-way approach was followed for the fine-mapping of qDTYu through N22-derived RILs, RIL recombinants and N22/4*Swarna BIL populations, (i) In order to identify the break point events between sdl and qDTYu, three SNP markers were added in and around sdl. One SNP was between RM11943 and sdl (idl024167) and two SNPs were between sdl and RM431 (idl024366 and idl024499). These markers were based on a panel of 44,000 SNPs published by Zhao et al. (2011). Genotyping was carried out using the Fluidigm SNP genotyping platform by K-Biosciences Ltd. QTL analysis was conducted on the phenotypic data from two field seasons (described above), (ii) These additional SNPs were used to test recombination between sdl and qDTYu. Recombinant lines among RILs between markers RM11943 and RM431 flanking both the sdl gene and qDTYu locus were identified. Tight linkage between sdl and qDTYu reduced the probability of double recombination in this interval; therefore only the recombinant lines between the flanking markers were analyzed to find lines with break points between the sdl and qDTYu. Further, due to a low number of recombinant lines in the individual populations, a pooled analysis of the 161 recombinant lines (48 from

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. A randomly selected set of 48 lines from this population was analyzed with 120 polymorphic markers between N22 and Swarna evenly spread throughout the twelve chromosomes. Among these polymorphic markers at six marker loci (RM246, RM278, RM335, RM411, RM434 and RM551), the N22-derived qDTYu allele was homozygous. In order to test whether these markers were correlated with GY under RS, these six markers were run on N22/Swarna RIL population (292 lines) originally used for mapping of qDTYu. Association analysis in random varieties.

Genotyping of 123 random genotypes was conducted with genome-wide SNP markers, SNP markers within the qDTY; _Λ 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). A burn in period of 100000 with 10000 MCMC was followed. The qDTYu specific SNP assay (Figure 3) was used for marker trait associations. Association of qDTY; _Λ SNP markers as well as the sdl gene with drought-related traits was analyzed using population structure file, kinship matrix, genotypic and drought related phenotypic data of 123 rice genotypes (Table 11) through TASSEL (Bradbury et al. 2007) using the mixed linear model (MLM).

Table 11. Fst values of the genotypes grouping 123 genotypes in to four different sub populations.

Genotype Fstl Fst2 Fst3 Fst4

AIJIAONANTE 0.013 0.114 0.008 0.865

ANNADA 0.003 0.018 0.015 0.965

Apo 0.005 0.19 0.01 0.795

BALAM 0.01 0.169 0.709 0.112

BASHFUL 0.008 0.548 0.02 0.424

BASMATI334 0.917 0.024 0.019 0.04

BASMATI370 0.856 0.022 0.065 0.057

BENGAWAN 0.004 0.971 0.007 0.018

BETONAN 0.013 0.91 0.031 0.046

BHADOIA233 0.011 0.012 0.966 0.011

BINNATOHA 0.004 0.011 0.975 0.01

Blackgora 0.003 0.004 0.989 0.004

BPIRI10 0.013 0.324 0.008 0.656

BR1 0.005 0.007 0.982 0.006

BR11 0.005 0.59 0.016 0.389

BR2 0.005 0.06 0.891 0.044

BR21 0.006 0.278 0.008 0.707

Browngora 0.003 0.003 0.99 0.004

CHAMP ATONG54 0.006 0.965 0.005 0.023

CHAU 0.02 0.056 0.006 0.918

CHIEMCHANH 0.011 0.017 0.009 0.963

CHINIGURA 0.008 0.92 0.015 0.057

CIKAPUNDUNG 0.004 0.974 0.005 0.017

C018 0.006 0.008 0.977 0.009 C025 0.005 0.008 0..978 0.008

CR1014 0 .008 0.782 0. .037 0.173

DAVAO 0 .011 0.655 0. .212 0.122

DEE-GEO-WOO-GEN 0 .003 0.085 0. .007 0.905

Dhagaddeshi 0 .008 0.313 0. .303 0.376

DHALASAITA 0 .014 0.043 0. .923 0.021

DHOLIBORO 0 .006 0.005 0. .984 0.005

DINORADO 0 .988 0.004 0. .004 0.003

DURGABHOG 0 .979 0.008 0. .006 0.007

GIE57 0 .007 0.319 0. .009 0.665

DULAR 0 .004 0.01 0. .977 0.009

HOING 0 .004 0.942 0. .005 0.049

IR156122833 0 .004 0.631 0. .009 0.356

IR36 0 .011 0.011 0. .967 0.011

IR42 0 .004 0.062 0. .791 0.143

IR64 0 .007 0.064 0. .012 0.917

IR8 0 .004 0.104 0. .007 0.885

IR743714611 0 .037 0.235 0. .013 0.714

IR743717011 0 .005 0.081 0. .007 0.908

IRRI119 0 .003 0.056 0. .006 0.935

IRRI123 0 .005 0.352 0. .008 0.634

IRRI141 0 .004 0.318 0. .005 0.673

JAGANATH 0 .006 0.647 0. .068 0.279

JAYA 0 .004 0.519 0. .041 0.436

JC148 0 .003 0.009 0. .979 0.009

JHONA349 0 .012 0.452 0. .164 0.372

JIRASAIL 0 .009 0.314 0. .156 0.522

JOYNA 0 .011 0.676 0. .011 0.302

KAJALSAIL 0.03 0.352 0. .549 0.068

Kalakeri 0 .003 0.004 0. .987 0.005

KALAMKATI 0 .004 0.076 0. .018 0.902

KALIJIRA 0 .972 0.009 0. .009 0.01

KAOTAHKONG 0 .008 0.929 0. .009 0.054

KASALATH 0 .004 0.048 0. .625 0.322

KATARIBHOG 0 .003 0.122 0. .011 0.863

KH AOD A WKM ALI 105 0 .006 0.967 0. .007 0.02

KHAOGAEW 0 .006 0.87 0. .024 0.099

KHAOHAWM 0 .005 0.323 0. .019 0.653

KHAOTAHOO 0 .014 0.884 0. .004 0.097

KHAOTAHHAENG 0 .015 0.801 0. .034 0.149

KHAOTAHPIENG 0 .006 0.942 0. .006 0.046

KU1131 0 .006 0.915 0. .008 0.072

LALSAR 0 .015 0.455 0. .111 0.419

LATISAIL 0 .003 0.936 0. .007 0.053

LEUANGYAINONG 0 .008 0.966 0. .005 0.021

MACANBINUNDOK 0 .005 0.468 0. .006 0.521 MAGAWKDONG26977(9) 0.987 0.004 0.004 0.005

MAHSURI 0.008 0.894 0.019 0.079

MAKMUR 0.003 0.971 0.004 0.022

MARIN AH 0.003 0.934 0.013 0.05

MOHINISAIL 0.006 0.501 0.051 0.442

MTU1010 0.005 0.064 0.006 0.924

MUEYNONG(WANGDIN) 0.525 0.114 0.171 0.19

N22 0.003 0.004 0.987 0.005

NAN THE AO 0.004 0.177 0.014 0.805

NAZIRSAIL 0.004 0.966 0.005 0.025

NIAW 0.007 0.64 0.067 0.285

NSICRcl92 0.007 0.263 0.009 0.72

NSICRc222 0.009 0.112 0.011 0.868

NSICRC9 0.004 0.242 0.013 0.741

PANKAJ(IR511431) 0.004 0.957 0.005 0.034

PELITAI1 0.004 0.969 0.005 0.022

PELITAI2 0.005 0.966 0.007 0.023

PSBRC68 0.003 0.047 0.006 0.944

PSBRC80 0.023 0.102 0.006 0.869

PSBRc82 0.036 0.674 0.046 0.243

PURBACHI(CHINESEl) 0.003 0.124 0.012 0.861

RASI 0.009 0.59 0.006 0.395

RATNA 0.003 0.045 0.005 0.947

RD1 0.003 0.093 0.006 0.897

RD15 0.005 0.669 0.019 0.306

RD2 0.003 0.774 0.012 0.21

RD25 0.003 0.54 0.006 0.451

RD6 0.006 0.686 0.017 0.291

RD7 0.005 0.852 0.01 0.133

RD9 0.023 0.131 0.137 0.709

RTS 12 0.006 0.071 0.007 0.917

RTS 14 0.013 0.829 0.037 0.121

RTS5 0.023 0.378 0.011 0.588

SAFRI17 0.019 0.495 0.013 0.473

Sambamahsuri 0.005 0.029 0.019 0.947

SARJOO50 0.007 0.04 0.011 0.942

SARJOO 49 0.005 0.7 0.007 0.288

SATHI-34-36 0.005 0.032 0.94 0.023

SMAGUING 0.983 0.004 0.005 0.008

SINAMPAGASELECTION 0.988 0.004 0.005 0.003

SOMCAU70A 0.006 0.241 0.016 0.737

SWARNA 0.006 0.553 0.013 0.428

Tl 0.01 0.011 0.947 0.032

T136 0.008 0.06 0.893 0.039

TADUKAN 0.007 0.063 0.008 0.922

TAICHUNG65 0.005 0.007 0.981 0.007 T AICHUNGN ATI VE 1 0.004 0.007 0.982 0.007

TKM6 0.092 0.675 0.009 0.223

UPLRI4 0.006 0.204 0.009 0.781

UPLRi7 0.007 0.585 0.004 0.405

Vandana 0.089 0.805 0.074 0.033

WAGWAG 0.201 0.631 0.009 0.16

ZHENSHAN2 0.02 0.097 0.017 0.866

Genetic diversity analysis of varietal set.

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).

QTL class analysis.

Recombinant classes were identified within qDTY₃ qDTY_{3 2}^nd qDTY₆₂based 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.

Example II. Analysis of pleiotropy vs. tight linkage between sdl and qDTY ₁

The question whether the drought tolerance was due to a pleiotropic effect of the sdl gene or there was a tight genetic linkage between the qDTYi _A and sdl genes was addressed through two different approaches: (i) statistical analysis of multiple traits for linkage vs. pleiotropy by saturation mapping in the RIL mapping populations used for the identification of qDTYi .i, and (ii) fine mapping of qDTYi locus using region-specific SNP markers in a large backcross inbred line (BIL) population. a) Multiple trait analysis and re-mapping of the sdl/qDTY Ί in N22-derived RIL populations.

Multiple trait analysis in N22/IR64 and N22/Swarna RIL populations did not show any significant pleiotropic effects of qDTYi on days to flowering (DTF), plant height (PH) and harvest index (HI) under reproductive-stage drought stress (RS) (Table 2). A re-mapping of the qDTYi QTL interval (RM 11943 - RM431) in the three N22 derived RIL populations (N22/Swarna, N22/IR64, and N22/MTU1010) using three addition SNP markers in the region revealed that qDTYi J was located distal to sdl gene (Figure 1 , Table 1). The QTL intervals in N22/Swarna, N22/IR64, and

N22/MTU1010 populations were idl024366- idl024499, idl024499 -RM431 and idl024366- idl024499. Further analysis of 161 recombinant inbred lines (RILs; 48 from N22/Swarna, 57 from N22/IR64 and 56 from N22/ MTU1010) using sdl gene based marker and its flanks (Figure 10) revealed that the qDTY _Λ position to be below the sdl gene between interval idl024366- idl024499 in combined analysis over both years (Table 1). b) Fine mapping of the sdl/qDTYj _Λ locus.

In a recombination breakpoint analysis of the 160 N22/2* Swarna BIL population with fixed DTF and sdl alleles that segregated for markers in the qDTY] _Λ interval using a 29-plex

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. classes E+F+G was 970 Kg/ha confirming presence of qDTY_L1 gene below sdl. Results of fine mapping suggested QTL location between nksdty_l_34 - nksdty_l_38.

(Figure 4).

Example III. Analysis of sdl.qDTYu region in traditional GR, and post-GR varieties.

Association analysis on a set of random varieties revealed significant association of markers within qDTYu with DTF, PH, grain yield (GY) and harvest index (HI). The sdl gene was found to be significantly associated with PH but not with GY under RS (Table 12). Four different varietal groups were identified among 123 random genotypes originating from South and Southeast Asia, that were genotyped for sdl and qDTYj _/markers and compared for phenotypic variation for GY and related traits among medium- and late-duration groups (Figure 5; Tables 13 and 14). Group 1 represents drought tolerant genotypes which are either pre-GR varieties or landrace derivatives such as N22, Vandana and Sathi34-36. N22, 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.

Trait Year Environment Locus Site Significance R²

DTF 2012 NS nksdtyl_] _46 39880000 * 0.06271

GY 2012 NS nksdtyl_] _44 39700000 ** 0.09045

HI 2012 NS nksdtyl_] _20 37900000 ** 0.08222

PHT 2012 NS nksdtyl_] _25 38180000 * 0.05196

DTF 2012 S nksdtyl_] _46 39880000 * 0.05987

PHT 2012 s sdl 38330000 * 0.06308

DTF 2013 NS nksdtyl_] _34 38840000 * 0.0458

HI 2013 NS nksdtyl_] _47 39930000 * 0.07348

HT 2013 NS nksdtyl_] _46 39880000 * 0.06903

GY 2013 S nksdtyl_] _34 38840000 ** 0.05674

DTF Combined NS nksdtyl_] _46 39880000 * 0.06938

DTF Combined NS nksdtyl_] _40 39250000 * 0.05867

HI Combined NS nksdtyl_] _25 38180000 * 0.06236

PHT Combined NS nksdtyl_] _46 39880000 * 0.06003

GY Combined S nksdtyl_] _38 39080000 * 0.05159

PHT Combined s sdl 38330000 * 0.07501

S: Stress; NS: non stress; Combined: combined analysis over two years

*: Significant at 5 %; **: Significant at 1 %

R²: Phenotypic variance explained by the marker

Table 13. Phenotypic variation for GY related traits under RS and NS in random varieties of medium duration

NON STRESS STRESS

TRAIT RANGE MEAN ± SD RANGE MEAN ± SD

DS2012 DS2013 DS2012 DS2013 DS2012 DS2013 DS2012 DS2013

DTF 58 - 105 61 - 109 72 ± 9 81 ± 10 51 - 91 63 - 103 71 ± 9 78 ± 8

PHT 68 - 156 68 - 140 111 ± 18 100 ± 18 67 - 134 49 - 117 99 ± 15 85 ± 15

BIO 2835 - 30850 3650 - 43500 13280 ± 5430 12430 ± 4611 2650 - 33400 550 - 49000 9063 ± 4045 7052 ± 6394

GY 702 - 9278 372 - 10850 3880 ± 1794 3078 ± 1372 363 - 5296 0.1 - 2807 2534 ± 1108 1178 ± 707

HI 0.12 - 0.50 0.04 - 0.49 0.344 ± 0.07 0.36 ± 0.09 0.026 - 0.50 0.00001 - 0.50 0.313 ± 0.09 0.39 ± 0.04

Table 14. Phenotypic variation for GY related traits under RS and NS in random varieties of late duration.

NON STRESS STRESS

TRAIT RANGE MEAN ± SD RANGE MEAN ± SD

DS2012 DS2013 DS2012 DS2013 DS2012 DS2013 DS2012 DS2013

DTF 62 - 112 60 - 108 83 ± 12 76 ± 13 61 - 123 62 - 109 88 ± 14 86 ± 12

PHT 72 - 164 76 - 169 126 ± 19 119 ± 20 72 - 157 65 - 146 113 ± 18 103 ± 15

BIO 5204 - 40550 1946 - 17460 15180 ± 6905 7707 ± 3198 2300 - 38000 2100 - 16000 12130 ± 5052 3761 ± 1797

GY 439 - 8120 1319 - 8957 3206 ± 1863 4372 ± 2006 14 - 5763 0 - 4711 2156 ± 1222 1379 ± 1271

HI 0.02 - 0.50 0.07 - 0.49 0.234 ± 0.11 0.35 ± 0.09 0.004 - 0.49 0.001 - 0.50 0.21 ± 0.11 0.18 ± 0.14 Example IV. Analysis of the qDTYu and sdl using 3K Rice genome sequence data.

A linkage between the Sdl allele and drought tolerance in the tall landraces, most of which are drought tolerant, is further supported through SNP analysis of the recently available 1000 genome SNP annotation of the 3K Rice Genome Project (3K RGP, 2014). Relation of the culm length to the type of cultivar revealed that advanced breeding lines or released varieties that were part of GR comprised 62% of the genotypes with 50 to 90 cm culm length. In contrast, 90% of the genotypes with >90 cm culm lengths were landraces. Semi-dwarf plants arising from one of the two well-known sdl alleles (LOC_Os01g66100; L266F; Speilmeyer et al., 2002) were represented by only 1% of the genotypes. However, 100% association was found between dwarf genotypes (<50 cm) and a novel mutation (Y342*) leading to a termination codon in the third exon of LOC_Os01g66100 encoded gibberellin-20 oxidase. Also, a haplotype with three mutations in intron 2 was significantly related to semi-dwarfism. The one known and two novel alleles of the sdl, fully associated with semi-dwarf phenotype, were not linked to the putatively drought tolerant N22 haplotype of the 58 downstream genes spanning qDTYu. Contrarily, in 43% of the tall genotypes (WT gibberellin-20 oxidase;

LOC_Os01g66100), the haplotype of the downstream 58 genes was significantly similar to the N22 haplotype, the source of drought tolerance (Figure 6). These results reinforced the hypothesis that sdl was linked to drought sensitive alleles and that selecting for sdl had selected for drought sensitivity. The drought sensitive alleles would not be identified as a QTL in a cross.

Finally, after analyzing qDTYu - sdl linkage and fine mapping, 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.

In detailed physiological studies on four drought tolerant recombinant BILs in the Swarna genetic background to understand the mechanism of drought tolerance conferred by the qDTYu ^trie four recombinant BILs and Swarna showed a varying level of flowering time difference under well watered (NS) and drought stress (RS) conditions, with significantly shorter flowering time in the qDTY] i BILs than in Swarna in 2013WS and 2014DS under drought stress and in 2013DS and 2014DS in the well-watered control (Figure 7A). Drought stress was imposed early enough around 30 days after transplanting to minimize the effect of early DTF on drought stress. Swarna did not flower in the drought stress treatment of 2013DS. The above ground shoot mass of qDTYu BILs showed a dynamic response to the timing and severity of drought stress. In a greenhouse lysimeter study, 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).In the field, qDTYu BILs showed lower normalized difference vegetation index (NDVI) than Swarna when the stress treatment was initiated early (2014DS; Fig. 7D), and qDTYu BILs showed higher NDVI than Swarna early in the drought stress treatment when the stress treatment was initiated late (2013DS; Figure 1 IB). Starting from reproductive stage, the qDTYu BILs showed a greater allocation to stem mass as a proportion of the above ground biomass in the field under drought and in the well-watered control (Figure 1 lC-1 ID) in 2014DS. The allocation to deep root growth was higher in the qDTYu BILs than in Swarna. The qDTYu BILs showed higher maximum root depth in the drought stress treatment of the greenhouse lysimeter study (Figure 7E) but not in the well-watered control (Figure 1 IE). 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.

Three other major effect qDTY also showed tight linkage with traits selected against during the GR. qDTY₆₂ with tall plant height, qDTY₃ with reduced grain yield under irrigated conditions, and qDTY 3 2 with very early days to flowering. Presence of the qDTY₆₂ donor (IR55419-04) allele at three marker loci (RM3, RM541 and RM275) within qDTY₆₂ led to increased yield under drought.

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. For qDTY 3 i, three marker loci (RM520, RM416 and RM16030) were associated with increased yield under drought in an Apo/Swarna population (Venuprasad et al. 2009). However, class analysis based on the three marker loci within this QTL showed that the presence of drought tolerant Apo allele at RM16030 led to a decline in yield under irrigated conditions (Figure 12B). For qDTY_{3 2}, presence of the drought tolerant qDTY_{3 2} allele from Vandana at three marker loci RM7332, RM523 and RM545 results in reduced DTF and days to maturity (DM) (Figure 12C). While this earliness was advantageous under severe drought stress, DTF less than 80 days reduces yield potential under irrigated condition, and this has been the main reason that most of the high yielding GR varieties were bred to mature after more than 110 days leading to rejection of this QTL during development of GR varieties. Recombinant lines with drought tolerance alleles of the qDTY₆₂, qDTY₃ and qDTY_{3 2} QTLs with desired PH, GY and DTF, were identified. Semi-dwarf plants with qDTY₆₂ high yielding plants with qDTY₃ j and medium duration high yielding lines with qDTY_{3 2} were identified from a large back cross inbred populations (Table 12). The semi-dwarf lines with qDTY₆₂ with plant height similar to TDK 1 under irrigated conditions showed higher yield than TDK 1 under severe drought stress.

Swarna ILs with qDTY_{3 Λ} showed on par yield with Swarna under irrigated conditions and a significant yield advantage over Swarna under severe drought stress. Similarly, 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 qDTY₃₂ QTL (Table 12).

Example VII. Proportion of qDTY alleles in traditional and GR varieties.

Genetic diversity analysis for qDTY alleles clearly separated the drought tolerant lines from drought susceptible lines and grouped into different clusters (Figure 8). The diversity analysis also separated the traditional drought tolerant donors (Cluster 1) with recently developed drought tolerant varieties (Cluster 5) and further separated GR varieties in to three distinct groups: early GR varieties represented by IR8 (Cluster 2), mid GR varieties represented by Jaya (Cluster 3) and late GR varieties represented by presently cultivated varieties IR36, IR64, Swarna, MTU1010, and Sambha Mahsuri (Cluster 4) (Figure 9). 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%). Among the GR varieties, the proportion of drought tolerant alleles reduced to 21% in Cluster 2, 18% in Cluster 3, and 16% in Cluster 4 varieties (Figure 9).

Interestingly, the frequency of the recipient allele (contributed by drought-susceptible GR varieties) increased from 34% in traditional donor varieties to 45% and 49% in early GR varieties (Clusters 2 and 3) and to 58% in recent GR varieties (Cluster 4). In recently-developed drought tolerant varieties, the frequency of recipient allele frequency decreased to 41% (Figure 9). Example VIII. Validation of drought tolerant semi-dwarf varieties with qDTY ₁

To evaluate performance of the dwarf line IR 91659-41-95-14-B carrying qDTY_L1 under environmental conditions which differ to those at the IRRI site in Los Banos, Philippines (where the drought tolerant QTL was identified), a growing trial was carried out at the South Asia Hub IRRI site in Hyderabad, India. The growing trial compared the performance of the dwarf line IR 91659-41-95- 14-B carrying qDTYu relative Swarna, the corresponding recipient dwarf line, under control non- stress (CONT) and reproductive stage drought stress (REPST).

Similar to the performance of the mapping population used to break linkage between Sdl and qDTYu, 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. These result validated the earlier finding on effect of qDTYu to enhance yield under drought (Table 15) and demonstrate the effect of qDTYu in diverse growing environments.

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

CONT- Control non-stress; REPST- Reproductive stage drought stress; DS- Dry season, WS- Wet season

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).

Briefly, 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, Hyderabad, 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 _Λ following MAB breeding in Swrarna under control non-stress and reproductive stage drought stress at Hyderabad, India in different seasons.

CONT- Control non-stress; REPST- Reproductive stage drought stress; DS- Dry season, WS- Wet season

Example X. Production and evaluation of drought tolerant semi-dwarf lines introgressed with qDTY_{l lt} qDTY₂.i and qDTY_{3 1}

The inventors introgressed qDTY; _l qDTY_{2 1}, and qDTY_{3 Λ} 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}, qDTY₃ 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.

The subl and qDTY QTLs combined well and the MAB lines with drought QTLs in Swarna subl background provided yields similar to Swarna subl under control non-stress (CONT) and 17 days submergence (SUB), and an yield advantage of 0.9 to 1.0 t ha ¹ under drought (Table 17).

Table 17: Performance of drought tolerant lines qDTYj _Λ qDTY_{2 1}, qDTY₃ j and subl gene

CONT- Control non-stress; REPST- Reproductive stage drought stress; SUB- submergence screening; DS- Dry season, WS- Wet season

While the invention has been described with reference to various and preferred embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Other variables may also be modified without departing from the essential scope of the invention.

Therefore, it is intended that the invention not be limited to a particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all

embodiments falling within the scope of the claims.

Claims

1. 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] _Λ 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; _A, wherein the drought tolerant allele of qDTY; _Λ 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.

2. The method of claim 1 , further comprising selecting from the rice plants identified and selected in step c) one or more rice plants exhibiting semi-dwarf plant height and drought tolerance.

3. The method of claim 1, further comprising:

d) providing at least one additional drought tolerant donor rice plant;

e) 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 qDTY₃ j, qDTY_{3 2}, and qDTY₆₂ 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 f) 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 comprises within its genome at least one drought tolerant allele of a QTL selected from the group consisting of: qDTY₃ \ qDTY_{3 2}; and qDTY₆₂, wherein the drought tolerant allele of qDTY₃ 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 qDTY₆₂ 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.

4. The method of claim 3, wherein 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 qDTY₃ 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 qDTY₆₂.

5. The method of claim 1, wherein the semi-dwarf allele of sdl is indicated by a 383 bp deletion spanning a first intron and second exon of sdl.

6. The method of claim 5, wherein the 383 bp deletion is detected using primers flanking the

deletion.

7. The method of claim 6, wherein the primers are:

Sdl -forward (5 '-C ACGC ACGGGTTCTTCC AGGTG- 3 ') (SEQ ID NO: 33); and

Sdl -reverse (5'-AGGAGAATAGGAGATGGTTTACC-3') (SEQ ID NO: 34).

8. The method according to any one of claims 1 to 7, wherein the at least one marker is detected in DNA isolated from the one or more recipient rice plants.

9. The method according to any one of claims 1 to 8, wherein the transfer of the nucleic acid is performed by a transgenic method, by crossing, by protoplast fusion, by a double haploid technique, or by embryo rescue.

10. The method according to any one of claims 1 to 9, wherein the transfer of the nucleic acid is 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 qDTY] _Λ as an introgression, and wherein the identifying and selecting step is performed on one or more progeny plants.

11. The method according to any one of claims 1 to 10, wherein the identifying and selecting step is performed by detecting the at least one marker in DNA isolated from the one or more progeny plants.

12. The method according to any one of claims 1 to 11, wherein the identifying and selecting step further comprises 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.

13. The method according to any one of claims 1 to 12, wherein the method further comprises a step of selfing the at least one selected rice plant.

14. The method according to any one of claims 1 to 13, wherein the method further comprises a step of selfing the at least one selected rice plant one or more times, and 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.

15. A drought tolerant rice plant, or part thereof, produced by the method according to any one of claims 1 to 14, wherein the plant or part thereof comprises the semi-dwarf allele of sdl comprising the Y342* mutation, and the drought tolerant allele of qDTY; _A, wherein the drought tolerant allele of qDTYu is not in its natural genetic background.

16. A method of producing a semi-dwarf drought tolerant inbred rice plant comprising:

a) producing a semi-dwarf drought tolerant rice plant according to the method of any one of claims 1 to 14;

b) crossing the semi-dwarf drought tolerant rice plant with itself to yield progeny rice seed; c) growing the progeny rice seed to yield additional semi-dwarf drought tolerant rice plants; and optionally

d) repeating the crossing and growing steps from 1 to 7 times to generate a semi-dwarf drought tolerant inbred rice plant.

17. The method of claim 16, wherein step c) further comprises the steps of identifying and selecting inbred rice plants that are homozygous for the drought tolerant allele of qDTYu.

18. The method of claim 16, wherein step c) further comprises the steps of identifying and selecting inbred rice plants that exhibit semi-dwarf plant height and drought tolerance.

19. A semi-dwarf drought tolerant inbred rice plant obtained or obtainable by the method of any one of claims 16 to 18.

20. A hybrid rice plant, or a part thereof, that exhibits semi-dwarf plant height and drought tolerance, wherein the hybrid rice plant is obtained or obtainable by crossing an inbred rice plant obtained or obtainable by the method of any one of claims 16 to 18 with a rice plant that exhibits commercially desirable characteristics.

21. A seed of a plant produced by any one of claims 15, 19 or 20.

22. The method of any one of claims 1 to 14 or 16 to 18, wherein the donor rice plant is selected from the group consisting of: N22; Dagaddeshi; Apo; Vandana; and Black Gora.

23. The method of any one of claims 1 to 14 or 16 to 18 or 22, wherein the recipient rice plant is selected from the group consisting of: IR8; Jaya; IR36; IR64; Swarna; MTU1010; Sambha Mahsuri; BRl ; RDl ; Kalamkatit; TDKl; PSBRC80; NSICR_C222; Dee-Geo-Woo-Gen; IRRI119; Purbachi (Chinese 1); Sabitri; and RD25.

24. The method of any one of claims 1 to 14 or 16 to 18 or 22 or 23, wherein the at least one

additional donor rice plant is 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.

25. A semi-dwarf drought tolerant rice plant, or part thereof, comprising:

a) a semi-dwarf allele of sdl comprising a Y342* mutation; and

b) a drought tolerant allele of qDTY] , wherein qDTY] _Λ is not in its natural genetic

background, and wherein the drought tolerant allele of qDTY; _Λ 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.

26. A method for identifying a rice plant as having a semi-dwarf drought tolerant phenotype,

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 qDTY] , wherein the drought

tolerant allele of qDTY] _Λ is indicated by a genomic region on chromosome 1 comprising at least one marker selected from the group consisting of: RM 11943; RM431 ; RM 12023; 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 qDTY] _Λ are detected.

27. The method of claim 26, wherein the semi-dwarf allele of sdl is indicated by a 383 bp deletion spanning a first intron and second exon of sdl.

28. The method of claim 27, wherein the 383 bp deletion is detected using primers flanking the deletion.

29. The method of claim 28, wherein the primers are:

Sdl -forward (5 '-C ACGC ACGGGTTCTTCC AGGTG- 3 ') ; and

Sdl -reverse (5'-AGGAGAATAGGAGATGGTTTACC-3').