WO2015007241A1

WO2015007241A1 - Molecular marker

Info

Publication number: WO2015007241A1
Application number: PCT/CN2014/082547
Authority: WO
Inventors: Feng Qin; Shengxue LIU; Xianglan WANG; Hongwei Wang
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2013-07-18
Filing date: 2014-07-18
Publication date: 2015-01-22
Anticipated expiration: 2016-01-18
Also published as: CN104293900A; CN104293900B

Abstract

Disclosed are methods and compositions for identifying, selecting and/or producing drought tolerant maize plants with beneficial haplotypes. Also disclosed are isolated nucleic acid sequences for regulating gene expression.

Description

MOLECULAR MARKER

FIELD OF THE INVENTION

The invention relates to methods and compositions for identifying, selecting and/or producing drought tolerant maize plants or germplasms and novel chromosomal segments and novel plants produced by such methods.

BACKGROUND OF THE INVENTION

Introduction

Maize (Zea mays L.) is one of the most planted crops world-wide and has tremendous value for providing food, forage, and other industrial products. Its productivity is frequently hampered by water scarcity and therefore, improved drought tolerance is an important goal in many breeding programs. Considerable research has been conducted to better understand the genetic and molecular basis for drought tolerance in plants with the idea that this research will provide information that will greatly increase the efficiency of traditional breeding programs to select for drought tolerance through the use of molecular markers. Alternatively, this research can be used to identify specific genes that can be used to improve drought tolerance in maize and other crop species using transformation technologies.

Abiotic stress research in Arabidopsis has revealed two major signaling pathways, one ABA-dependent and one ABA-independent,that control stress-inducible gene expression, "ABA" here referring to abscisic acid. DREBs/CBFs (Dehydration Responsive Element Binding proteins/C-repeat Binding Factors, hereafter referred as DREBs) are thought to be the major transcription factors (TFs) that control stress-inducible gene expression in the ABA- independent pathway [1]. DREB TFs, belonging to the APETALA2/Ethylene-Responsive Factor (AP2/ERF) superfamily of TFs, are able to bind a Dehydration Responsive Element (DRE, core motif: A/GCCGAC, also known as a C-repeat and low-temperature-responsive element [2]-[4], in the promoter region of many drought and/or cold stress-inducible genes. They were first identified using a yeast one-hybrid system to screening for the trans-factors of the DRE element identified in a set of drought and cold-inducible gene promoters [5], [6]. There are two groups of DREB genes in the Arabidopsis genome {DREB Is and DREB2s) that are composed of six and eight members, respectively [7]. Ectopic or selective expression of DREB1A/CBF3 can significantly enhance plant tolerance to multiple abiotic stresses, including drought, freezing and high salinity [6], [8]. Over-production of a constitutive active form of DREB2A (DREB2A-CA) protein conferred significant both drought and heat tolerance in transgenic plants [9], [10]. Thus, distinct from DREB 1, post-translational modification of the DREB2A protein was demonstrated to finely modulate its abundance and activity [11].

In plants, the DREB gene family consists of multiple genes. Studies in species such as rice, tomato, soybean, wheat, barley and maize, suggest that DREB genes play a central role in plant stress response [15], [16]. Although DREB genes are primarily involved in the regulation of water-stress-related gene expression, other functions have been noted for specific DREB genes. For example, DREB1D/CBF4 plays a role in plant drought stress tolerance which is in contrast to the homologous DREB1A/CBF3 gene that functions in cold response [12]. DREB1C/CBF2 has been characterized as a negative, but not a positive, regulator of plant cold stress response by tightly controlling DREBJA/CBF3 and DREBIB/CBFI expression [13]. DREB2C has been reported to play a role in heat rather than drought tolerance [14]. Thus, it is not possible to predict what effect any particular DREB gene or allelic form of such gene will have in a plant, when selected for by breeding or introduced ectopically. The functional divergence of different DREB genes has proven to be an attractive and challenging topic of research.

Overexpression of the Arabidopsis DREB2A gene does not result in a notable drought tolerant phenotype in transgenics, which is most likely a result of the instability of the ectopic expressed protein in plant cells [9], [11]. In maize, two DREB genes {ZmDREBlA and ZmDREB2A) belonging to the DREB1 and DREB2 subgroups, respectively, were cloned and demonstrated to be upregulated in response to plant water stress [17], [18]. However, previous reports indicate that transgenic plants constitutively overexpressing DREB2A-CA or ZmDREB2.1/2 A gene exhibited a dwarf phenotype in addition to enhanced drought tolerance [9], [18] which impacts negatively on yield. It was also found that, distinct from Arabidopsis DREB2A, ZmDREB2A gene expression in response to abiotic stress was regulated via an alternative splicing mechanism and that the expressed protein could directly activate downstream gene expression [18]. Similar findings in rice, wheat and barley, indicate the presence of a mechanism that finely modulates the activity of stress-inducible TF genes and suggest that the molecular mechanism is different in monocot and dicot plants [19]— [21].

There is a need for identifying genes in maize that enhance drought resistance can lead to more efficient crop production as this allows the identification, selection and production of maize plants with enhanced drought resistance, The present invention is aimed at addressing this need.

SUMMARY OF THE INVENTION

The invention is thus aimed at providing methods for identifying desirable drought resistance related alleles at particular chromosomal locations ("loci") in maize plants and selecting plants having such beneficial alleles at such loci for producing plants with enhanced drought resistance.

This invention describes five polymorphic loci (S P-503, S P-260, InDel-185, InDel- 154 and S P-150; collectively termed haplotype) in the 5'-UTR of the maize DREB gene

ZmDREB2. 7that confer resistance to drought in maize.

Detection of the presence or absence of particular alleles at each of these 5' polymorphic loci allows identifying and selecting a maize plant with a higher resistance to drought. This invention thus provides a method for molecular marker assisted selective breeding of maize.

The desirable alleles at these loci described hereincan be selected for as part of a breeding program in order to generate plants that carry desirable traits. An exemplary embodiment of a method for generating such plants includes the transfer by chromosomal recombination and introgression of nucleic acid sequences from plants that have desirable genetic information into plants that do not by crossing the plants.

Desirable loci can be introgressed, for example into commercially available plant varieties, using marker-assisted selection (MAS) or marker-assisted breeding (MAB). MAS and MAB 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 loci that encode the desired traits as described herein. Such identification and selection can be based on selection of informative markers that are associated with desired traits.

In a first aspect, the invention relates to a method for identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1.

In another aspect, the invention relates to a primer pair comprising SEQ ID NO. 3 and SEQ ID NO. 4. In another aspect, the invention relates to a primer pair comprising SEQ ID NO. 5 and SEQ ID NO. 6.

In another aspect, the invention relates to kit for identifying and/or selecting a maize plant with enhanced drought resistance comprising a primer pair comprising SEQ ID NO. 3 and SEQ ID NO. 4 and/or a primer pair comprising SEQ ID NO. 5 and SEQ ID NO. 6.

In another aspect, the invention relates to a method for identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype said method comprising a. obtaining a first maize plant that comprises within its genome a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1; b. crossing said first maize plant to a second maize plant; c. evaluating the progeny plants and d. identifying and/or selecting a maize plant with enhanced drought resistance that comprises said haplotype.

In another aspect, the invention relates to a method for producing a hybrid maize plant with enhanced drought resistance the method comprising a. providing a first plant with a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position b. providing a second plant that does not have a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1; c. crossing the first plant with the second plant to produce an Fl generation; d. identifying one or more members of the Fl generation that comprises the desired phenotype comprising said haplotype.

In another aspect, the invention relates to a recombined DNA segment comprising a 5' UTR ZmDREB2.7 allele from maize which comprises a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1.

In another aspect, the invention relates to an isolated nucleic sequence comprising or consisting of:

1) a ZmDREB2.7 promoter sequence which comprises nucleotides at positions 296 to 798 of SEQ ID NO. 1 (SEQ ID NO. 19);

2) a ZmDREB2.7comprising promoter sequence positions 1 to 798 of SEQ ID NO. 1 (SEQ ID NO. 20);

3) a ZmDREB2. 7 promoter sequence as defined in SEQ ID NO. 22;

4) nucleic acid sequences which exhibit at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% homology with the nucleic acid sequences defined in 1) or 2);

5) nucleic acid sequences which hybridize to the nucleic acid sequences defined in 1), 2), 3) or 4) under strict conditions. _{4 i 4 4 4 4}

in anomer aspect, t e invention re ates to vector comprising iso aieu nucieic sequence as above.

In another aspect, the invention relates to host cell comprising a vector an isolated nucleic sequence as above. In another aspect, the invention relates to use of a vector or an isolated nucleic sequence as above in conferring drought resistance.

In another aspect, the invention relates to method for conferring drought resistance to a plant comprising introducing and expressing in said plant a vector or an isolated nucleic sequence as above. In another aspect, the invention relates to a method for increasing drought resistance of a plant compared to a control plant comprising the steps of

a. targeted mutagenesis of a plant population to introduce the following mutations in the 5' UTR of ZmDREB2.7: a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1 and

b. identifying and selecting plants which comprise the desired mutations.

The invention is further described in the following non-limiting figures. BRIEF DESCRIPTION OF THE FIGURES Figure 1(A)-(E). The favorable allele of ZmDREB2.7 improves maize drought tolerance.

(A) Haplotypes of ZmDREB2. 7 in lines CIMBL70, 91, 92, CML118, Shen5003 and B73 (as reference genome) maize genotypes. The site of the start codon (ATG) was designated as SNP-503, SNP-260, SNP-150, InDel-185 and InDel-154 are the five DNA polymorphisms significantly associated with maize drought tolerance and are located in the 5'- UTR of ZmDREB2.7. The 20-bp InDel upstream of the ATG is in complete LD with the five polymorphisms in the four drought tolerant varieties. The location of PCR primers used for genotyping the InDel polymorphism of ZmDREB2. 7 in drought tolerant (CIMBL70, 91, 92, and CML118) and drought sensitive (Shen5003) inbred lines are indicated by arrows. The four significant nonsynonymous polymorphisms in the coding region of InDell41, S P142, SNP436 and S P661 are also shown.

(B) Phenotypic response of CEVIBL70, 91, 92, CML118 and Shen5003 to drought stress. The upper panel is a photo of plants growing under favorable water conditions while the lower panel plants re-watered for 6 days after the drought stress treatment was terminated.

(C) The survival rate of CEVIBL70, 91, 92, CML118 and Shen5003 plants exposed to moderate and severe drought stress. Data represent the mean of triplicates (t-test, *p,0.05, **p,0.01).

(D) Relative level of ZmDREB2.7 expression in CIMBL70, 91, 92, CML118 and Shen5003 grown under normal and drought stress conditions. The drought-stress treatment reflected a decrease in RLWC from 98% (unstressed) to 70%> (moderate drought), and 58%> (severe drought). Data represent the mean of three biological replicates (t-test, **p,0.01). From left to right for each line: normal growth, moderate drought, severe drought.

(E) The effect of the ZmDREB2.7 favorable allele on drought tolerance in four F2 segregating populations of maize. In each population, three distinct genotypes for ZmDREB2.7 were identified by DNA amplification: homozygous for the favorable allele, homozygous for the sensitive allele, and heterozygous for both alleles. The survival rate of the different genotypes was assessed and compared in the four populations. N indicates the number of F2 individuals tested in each population (t-test, *p,0.05, **p,0.01). From left to right for each line: homozygous tolerant, heterozygous, homozygous sensitive.

Figure 2. PCR amplification of molecular marker InDel-21.

The molecular marker InDel-21 was amplified in different maize inbred lines. Here electrophoresis channels 1-5 are maize inbred lines Shen5003, CIMBL70, CEVIBL91, CIMBL92 and CML118respectively.

Figure 3.PCR amplification of molecular marker InDel-21. PCR amplification of the InDel-21 molecular marker in each individual plant of different segregated groups. Here, from topto bottom, the four images are respectively the results for segregated group CEVIBL70 X Shen5003, CEVIBL91 X Shen5003, CEVIBL92 X Shen5003 and CML118 X Shen5003; electrophoresis channel PI is in each case the parent maize inbred line Shen5003, electrophoresis channel P2 being respectively parent-CIMBL70, CIMBL91, CEVIBL92 and CML118, the remaining electrophoresis channels being different individual plants of each segregated groups.

Figure 4. Correlation analysis between the ZmDREB2.7 genetic variation and droughtresistance of maize. The x axis represents the 2.1kb gene segment, including the ZmDREB2. 7 gene ATG upstream 600bp and downstream 1486bp, A within the initiation codon (ATG) being recorded as the y axis being "-logio (P value)". The 5 pronounced polymorphics of the promoter region and the 4 non-synonymous of the coding region are exchanged, a solid line is adopted for both to connect coordinates and for the linkage disequilibrium graph. "*" indicates a strong linkage (with a linkage disequilibrium value r²> 0.8).

Figure 5(A)-(C). Relative expression level and survival rate of the ZmDREB2. 7 gene.

Figures A, B and C are the results during normal growth after drought treatment at the seedling stage (RLWC=98%), medium drought (RLWC=70%) and extreme drought (RLWC=58%). Survival rate and relative expression levels are shown.

Figure 6. Allele frequencies of the two haplotypes within different subgroups.

Thesequences corresponding tothe 5'-UTR of ZmDREB2.7 from 105 randomly selected inbredlines were analyzed. Division of the population into subpopulations(MIXED, NSS, SS, and TST). TST = tropical or subtropical varieties NSS= temperate varieties; SS =B73 derivatives and MIXE= -varieties with no clear identity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature.

As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or

RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences. Thus, according to the various aspects of the invention, genomic DNA, cDNA or coding DNA may be used. In one embodiment, the nucleic acid is cDNA or coding DNA. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

The various aspects of the invention include aspects that do not involve the generation of transgenic plants by recombinant methods. However, other aspects of the invention as described herein involve the generation of a transgenic plants by recombinant methods.

For the purposes of the invention, "transgenic", "transgene" or "recombinant" means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either

(a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or

(b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or

(c) a) and b)

are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette - for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above - becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815 both incorporated by reference.

A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the different embodiments of the invention are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. According to the invention, the transgene is stably integrated into the plant and the plant is preferably homozygous for the transgene. Thus, any off spring or harvestable material derived from said plant is also preferably homozygous for the transgene.

Specific genetic loci correlating with particular phenotypes can be mapped in a plant's genome by the process of linkage mapping, where the strength of association between a genetic marker locus and the locus determining a phenotypic trait of interest is a function of the physical proximity (the genetic "linkage") on the chromosome of the marker locus and the trait locus. This allows the plant breeder to rapidly select and identify plants with the desired phenotype by detecting markers that show a statistically significant probability of co- segregation with a desired phenotype. Genetic markers that are within a gene that confers the desired trait, or indeed are based on the actual polymorphism that causes the desired trait, are in effect 100% linked and therefore 100% accurate in their predictive or diagnostic power. The invention is an example of such a 100% linked, directly causative marker. The invention is therefore directed to methods for identifying and selecting maize plants with enhanced resistance to drought through the analysis of the genotype by assessing the presence of markers. According to the invention, the identification of a haplotype that is associated with drought resistance in maize allows selection for resistance based solely on the genetic composition of the progeny. The invention thus provides methods for Marker-assisted selection (MAS) to identify/select a maize plant with enhanced drought resistance and which has the haplotype described herein. MASis a process by which phenotypes are selected based on marker genotypes. This is useful in Marker-assisted breeding (MAB) and the invention also involves methods for MAB as described herein.

The invention also provides a haplotype connected with drought resistance of maize and its molecular marker. A "haplotype" as used herein is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term "haplotype", "marker haplotypes" or "marker alleles" can refer to polymorphisms at a particular locus, such as a single marker locus, or polymorphisms at multiple loci along a chromosomal segment.

The term "maize" as used herein refers to a plant of the Zea mays L. ssp. mays and is also known as "corn". The term "maize plant" includes: whole maize plants, maize germplasm, maize plant cells, maize plant protoplast, maize plant cell or maize tissue cultures from which maize plants can be regenerated, maize plant calli, and maize plant cells that are intact in maize plants or parts of maize plants, such as maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips, and the like. The maize can be an inbred line, or a maize hybrid such as a maize single cross hybrid.

In general, MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with a phenotype, such as resistance to drought. Such markers are presumed to map near a gene or genes that give the plant its drought resistance phenotype, and are considered indicators for the desired trait, or markers. Plants are tested for the presence of a desired allele in the marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny. Thus, plants with enhanced resistance to drought can be selected for by detecting one or more marker alleles, and in addition, progeny plants derived from those plants can also be selected. Hence, a plant containing a desired genotype in a given chromosomal region (i.e. a genotype associated with enhanced resistance to drought) is obtained and then crossed to another plant. The progeny of such a cross can then be evaluated genotypically using one or more markers and the progeny plants with the same genotype in a given chromosomal region are then selected as having enhanced resistance to drought.

The inventors have analysed drought resistant maize lines and shownthat: (1) three S Ps and two InDels, upstream of the start codon of ZmDREB2. 7 are significantly associated with phenotypic variation in drought tolerance (Figure 5A); (2) consistent with the TF function of ZmDREB2. 7, a rapid induction of ZmDREB2.7 gene expression in response to a moderate drought stress is important in conferring plant drought-stress tolerance (Figure 5B); (3) the favorable allele of ZmDREB2.7 can effectively enhance plant drought tolerance in four distinct genetic backgrounds compared to the inferior allele (Figure IE).

The function of DREB TFs is to bind DRE sequences present in the promoter region of many stress-inducible genes and transactivate gene expression, the gene products of which may protect plants from stress impairment [1]. Thus, an early and quick response to an environmental stress signal is important for the proper function of a TF gene. This can be accomplished either by a rapid induction of gene expression in response to an environmental stimulus or by quick modulation of transactivation activity of the protein coded by the TF. As shown herein, genetic polymorphisms in the 5'-UTR of ZmDREB2.7 are associated with variation in maize drought tolerance.

Furthermore, differences in ZmDREB2.7gene expression in response to moderate drought stress, but not severe drought or normal growth conditions, were correlated with plant survival among different maize varieties. Thus, induction of ZmDREB2. 7 expression in early drought stress was important for plant survival in stress, which coincided with its function as a TF to activate downstream stress-responsive gene expression. The quicker induction of ZmDREB2. 7gene expression in the drought tolerant genotype of described herein compared to the sensitive genotypewas consistently observed (Figure ID). The inventors further analyzed the ZmDREB2.7 gene expression data in approximately seventy maize inbred lines based on tolerant or sensitive genotypes of ZmDREB2. 7, under well-watered, early and late drought stress conditions. The results demonstrated that, on average, the materials carrying the tolerant allele of ZmDREB2.7 had a significantly higher expression level than those carrying the sensitive allele in response to early drought stress.

The inventors have thus shown that the polymorphisms in the 5'-UTR oiZmDREB2.7 contribute to drought stress tolerance of maize seedlings, specifically early drought stress, and that the five polymorphisms in the gene promoter region are the functional variations responsible for the observed variations ingene expression and plant drought tolerance. Thefavourableallele of ZmDREB2. 7which shows these 5 polymorphismsis thus a valuable genetic resource for improving maize drought tolerance for example as a genetic marker in marker assisted breeding.

As explained herein, the presence or absence of the favourable allele that confers drought resistance can be detected using markers. As further explained herein, the marker can be a molecular marker. Markers can thus be used in the methods of the inventionto identify the favorable genotype.

A "marker" is a nucleotide sequence or encoded product thereof (e.g., a protein) used as a point of reference. A marker associated with enhanced drought resistance is a marker whose presence or absence can be used to predict whether and/or to what extend a plant will display a drought tolerant phenotype.

A marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay, e.g. many disease resistance traits, or, occurs at a late stage in plant development, e.g. kernel characteristics. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. The ideal situation is to have a marker in the gene itself, so that recombination cannot occur between the marker and the gene. For markers to be useful at detecting recombinations, they need to detect differences, or polymorphisms, within the population being monitored.

A "polymorphism" is a variation in the DNA that is too common to be due merely to new mutation. A polymorphism must have a frequency of at least 1% in a population. A polymorphism can be a single nucleotide polymorphism, or S P, or an insertion/deletion polymorphism, also referred to herein as an "InDel".

For molecular markers, differences are detected at the DNA level due to polynucleotide sequence differences (e.g. SSRs, RFLPs, FLPs, and SNPs). The genomic variability can be of any origin, for example, insertions, deletions, duplications, repetitive elements, point mutations, recombination events, or the presence and sequence of transposable elements. Molecular markers can be derived from genomic or expressed nucleic acids (e.g., ESTs) and can also refer to nucleic acids used as probes or primer pairs capable of amplifying sequence fragments via the use of PCR-based methods. The polymorphisms are not limited to single nucleotide polymorphisms (SNPs), but also include InDels, CAPS, SSRs, and VNTRs (variable number of tandem repeats).

Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well-established in the art. These include, e.g., DNA sequencing, PCR- based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Well established methods are also known for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).

A "molecular marker probe" as used hereinis a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Nucleic acids are "complementary" when they specifically hybridize in solution, e.g., according to Watson-Crick base pairing rules. Some of the markers described herein are also referred to as hybridization markers when located on an InDel region, such as the non-collinear region described herein. This is because the insertion region is, by definition, a polymorphism vis-a-vis a plant without the insertion. Thus, the marker need only indicate whether the InDel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g. SNP technology is used in the examples provided herein.

SNP markers detect single base pair nucleotide substitutions. SNPs can be assayed at a high level of throughput. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, mini sequencing and coded spheres. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode® (Qiagen), Invader® (Third Wave Technologies) and Invader Plus®, Snapshot® (Applied Biosystems), Taqman®( Applied Biosystems), KASP and Beadarrays® (Illumina).

A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene.

According to the invention and as further described below, markers identified herein can used in MAS to select maize plants with enhanced resistance to drought. In one aspect, the invention relates to a method for identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype comprisingpolymorphic loci XI, X2, X3, X4 and X5. The haplotype described above confers drought resistance. It is a combination of 5 polymorphic loci, XI, X2, X3, X4 and X5 which either show a SNP or a InDel. The 5 polymorphic loci X1-X5 correspond to position 296 with reference to SEQ ID NO. 1, position 539 with reference to SEQ ID NO. 1, the position between positions 614 and 615 with reference to SEQ ID NO. 1, position 646 with reference to SEQ ID NO. 1 and position 649 with reference to SEQ ID NO. 1 respectively. All of these positions are located in the5'UTR of ZmDREB2. 7 (SEQ ID NO. 1). Thus, the invention relates to a method for identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1.

The position of the 5 polymorphic loci can also be expressed with reference to the A nucleotide in the ATG start codon in SEQ ID NO. 1 as: SNP-503, SNP-260, InDel-185, InDel- 154 and SNP-150. The site of the ATG start codon is designated as +1. Thus, SNP-503 represents a single nucleotide polymorphism at the nucleotide at position 503upstream from the A in the (ATG) of the initiation codon; InDel-185 represents an insertion or deletion of one or a number of nucleotides downstream from the 185^th position upstream from the A in the (ATG) of the initiation codon (see Fig. 1 A).

The position of the primer or the sequence is thus also shown herein with reference to the A nucleotide in the initiation codon of the (ATG) of the ZmDREB2.7 gene of the maize inbred line B73 genomic DNA of SEQ ID No. 1 which is labelled as "+Γ.

SEQ ID NO. 1 is the nucleotide sequence of ZmDREB2.7 of the inbred maize line B73 which has been sequenced (Schnable et al, Science Vol. 326 no. 5956 pp. 1112-1115, 2009). The chromosomal region where ZmDREB2.7 is located is chromosome 1, bin 1.07. The accession number is GRMZM2G028386. The term ZmDREB2.7used hereinthus refers to the ZmDREB2. 7 gene in the B73 inbred line of maize having accession number GRMZM2G028386 and as shown in SEQ ID No. 1 or a functional variant thereof. Preferably, the term refers to the ZmDREB2.7 gene as shown in SEQ ID No. 1.

According to the various aspects of the invention, the term "functional variant of a nucleic acid sequence" as used herein, for example with reference to SEQ ID NO: 1, refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant ZmDREB2. 7 sequence, for example confers drought resistance when expressed in a transgenic plant. A functional variant also comprises a variant of the gene of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting protein, for example in non- conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non- conserved residues, to the wild type sequences as shown herein and is biologically active.

Thus, it is understood, as those skilled in the art will appreciate, that the aspects of the invention, including the methods and uses, encompass not only a ZmDREB2. 7 nucleic acid or protein sequence as described herein, for example a nucleic acid sequence comprising or consisting or SEQ ID NO: 1, a polypeptide comprising or consisting or SEQ ID NO: 2, but also functional variants of a ZmDREB2.7, that do not affect the biological activity and function of the resulting protein. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do however not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Generally, variants of ZmDREB2.7 have at least 75% 70%, 71%, 72%, 73%, 74%, 75%,

76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the amino acid represented by SEQ ID NO: lor 2. The genomic sequence of the ZmDREB2.7 gene within the DNA of the B73 maize inbred line genome is shown in SEQ ID NO. l .The full-length cDNA sequence is located at positions 701-2284 of the 5' UTR of SEQ ID NO. 1. The protein encoded by positions 799-1878 is shown in SEQ ID NO. 2. The A nucleotide of the ATG initiation codon starts is at position 799 of the 5' UTR of SEQ ID NO . 1.

The inventors have shown that there are two haplotypes shared by these five polymorphic loci in different maize inbred lines; thus there are three genotypes.

The aforementioned five polymorphic loci, XI, X2, X3, X4 and X5 correspond to the genotypes A,B and C respectively as shown below, depending on whether the genotype is homozygous or heterozygous for the polymorphism:

A) A/A, C/C, no nucleotide insertion/no nucleotide insertion, G/G and G/G (homozygous haplotype A) ;

B) C/C, T/T, CAC insertion/CAC insertion, deletion of 1 nucleotide/deletion of 1 nucleobase and C/C (homozygous haplotype B); C) A/C, C/T, no nucleotide insertion/CAC insertion, G/l nucleotide deletion and G/C (heterozygous haplotypes A and B).

Homozygous haplotype A shows enhanced drought resistance compared to a control maize plant which does not have the favourable haplotype and is drought sensitive. The control plant is preferablya drought sensitive line carrying haplotype B, including an inbred line. In one embodiment, said control is a drought sensitive line selected from table 1. In one embodiment, said control is Shen5003.

Homozygous haplotype B is drought sensitive. Heterozygous haplotype A/B (genotype C) is more drought resistant than haplotype B, but less drought resistant than haplotype A.

When referring to the detection of a haplotype, all aspects of the invention, including the methods of the invention relate to detecting haplotypepresent in a homozygous or heterozygous state (haplotype A or B) that is detecting genotypes A, B or C. In one embodiment, the methods of the invention relate to detecting haplotype A or detecting haplotype A/B.

In other words, the methods of the invention relate to detecting whether a maize plant is drought resistant or drought sensitive by detecting whether the plant shows the desired haplotype and whether the haplotype of the invention is present in a homozygous or heterozygous state. If the plant has haplotype A, it is drought resistant. If it has haplotype B, it is drought sensitive. If the plant has haplotype A/B, it shows more drought resistance than haplotype B, but less than haplotype A.

As explained above, markers can be used in a method of the invention to detect the presence or absence of the haplotype A, B or C as set out above. Thus, the invention also provides markers associated with enhanced drought resistance.

As shown in the examples, the ZmDREB2. 7 gene of the 105 maize inbred lines shown in table 1, including the sequence upstream from the ATG, was amplified. It was found that the homozygous haplotype A maize inbred line exhibited a deletion of the 20bp DNA segment downstream of the nucleotide in the 21^st position upstream from the initiation codon ATG in the ZmDREB2.7 gene. The homozygous haplotype B maize inbred line on the other hand exhibited an insertion of the 20bp DNA segment. Therefore, this polymorphic locus was named InDel-21 (SEQ ID NO. 21). The polymorphism of this locus can be used as a molecular marker to detect the condition of the 5' polymorphic loci in maize.

Thus, in one embodiment of the methods described herein, the method therefore comprises the detection of a molecular marker wherein said molecular is a deletion of the residues at positions 779to798with reference to SEQ ID NO. 1 (InDel-21). This position is located in theposition of the 5' terminal of SEQ ID NO. 1 immediately upstream of the ATG start codon.The residues 779-798 are gcacgaagctagtagtccag (InDel-21, SEQ ID NO. 21).

Whether or not the sequence as shown in SEQ ID NO. 7 is present can be detected by methods known in the art and described elsewhere herein, such as PCR or PCR-RFLP. If the sequence is present,i.e. there is no deletion, then the genotype is drought sensitive genotype B. If the sequence is not present, then the genotype is drought resistant genotype A. If the genotype is heterozygous, then two bands will be detected.

Thus, if the size of the PCR product is 46bp, then the genotype of the maize being sequenced is A; if the size of the PCR product is 66bp, then the genotype of the maize being sequenced is B; if the size of the PCR product is 46bp and 66bp, then the genotype of the maize being sequenced is C. This is shown in Fig. 2 and 3. Thus, plants that are homozygous or heterozygous for the 5 polymorphisms can be identified.

A PCR primer pair that can be used in the detection of the absence or presence of a deletion of the residues at positions 779to 798with reference to SEQ ID NO. 1, that is the presence or absence of SEQ ID NO. 21, is as follows:

Forward primer: 5' tcgccatcagtcgccata 3' SEQ ID NO. 3

Reverse primer: 5' gcggcggcacccgatccat 3' SEQ ID NO. 4

Other suitable primers can be designed by a person skilled in the art.

Thus, the invention relates to a method for identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1 wherein detecting comprises determining the presence of a deletion of the residues at positions 779to 798with reference to SEQ ID NO. 1. In another embodiment, the marker lies in the presence of the five polymorphism as described above.

The presence or absence of the polymorphism can be detected by methods known in the art, such as PCR amplification followed by sequencing, such as SNP mini-sequencing.

According to the invention, a primer pair used to detect the polymorphism can be as follows:

Forward primer: 5' gtccgtcagtccgtccttg 3' SEQ ID NO. 5

Reverse primer: 5' ggaaatggaatcggagtttgac 3' SEQ ID NO. 6

Other suitable primers can be designed by a person skilled in the art.

Thus, the invention relates to a method for identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1 wherein detecting comprises PCR amplification using SEQ ID NO. 5 and 6.

Other methods that can be used to detect SNPs are known in the art and described herein. These include, but are not limited to fluorescent detection of SNP-specific hybridization probes on PCR products such as Taqman® or Molecular Beacons. Other strategies such as Sequenom homogeneous Mass Extend (hME) and iPLEX genotyping systems involve MALDI-TOF mass spectrophotometry of SNP-specific PCR primer extension products.

In one embodiment, Kompetitive Allele Specific PCR (KASP) genotyping is used. This requires the presence of 1) a purified DNA sample, 2) two allele-specific forward primers, and 3) a common reverse primer. KASP is a SNP genotyping system FRET (Fluorescent Resonance Energy Transfer). FRET allows for the detection of SNP's without the need for a separation step. Coupled with the power of competitive allele specific PCR, the KASP is a well described system for determination of SNP or insertion / deletion genotypes.

In another aspect, the invention relates to an isolated and purified genetic marker associated with a drought yield trait in maize wherein the isolated and purified genetic marker permits identification of a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1.

In one embodiment, said marker is capable of detecting the presence or absence of the 5' polymorphic loci describe above. In one embodiment, said marker is capable of detecting the presence or absence of SEQ ID NO. 21 in ZmDREB2.7.

In one embodiment, said marker comprises a primer pair for the amplification of the region comprising the 5 polymorphic loci or for amplification of the region comprising the -21 InDel. In one embodiment, said marker comprises a nucleotide sequence of an amplification product or an informative fragment thereof from a nucleic acid sample isolated from a maize plant, wherein the amplification product is produced by amplifying a maize nucleic acid using a pair of oligonucleotide primers selected from among SEQ ID NOs: 3 and 4 or 5 and 6.

The invention also relates to a DNA marker that is linked to the maize drought resistance locus and can be amplified in a PCR reaction comprising a pair of

PCR oligonucleotide primers selected from the group consisting of:

a. primer pair 1 represented by a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4,

b. primer pair 2 represented by a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6,

or by any other marker that is statistically correlated and thus genetically linked to the drought resistance trait. The invention also relates to a composition comprising an amplification primer pair capable of amplifying a maize nucleic acid to generate a maize marker amplicon, wherein the maize marker amplicon corresponds to SEQ ID NO 21. In another aspect, the invention relates to an isolated nucleotide sequence selected from

SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.

In another aspect, the invention relates to a primer pair for use in detecting the presence of the presence or absence of the residues at positions 779 to 798with reference to SEQ ID NO. 1. In another aspect, the invention relates to a pair of primers wherein said first primer has nucleotide sequence SEQ ID NO. 3 and the second primer has nucleotide sequence SEQ ID NO. 4.

In another aspect, the invention relates to a primer pair for use in detecting the presence or absence of polymorphisms at position 296 of SEQ ID NO. 1 position 539 of SEQ ID NO. 1, positions 614 and 615 of SEQ ID NO. 1, position 646 of SEQ ID NO. 1, and position 649 of SEQ ID NO. 1. In another aspect, the invention relates to a pair of primers wherein said first primer has nucleotide sequence SEQ ID NO. 5 and the second primer has nucleotide sequence SEQ ID NO. 6.

In another aspect, the invention relates to a kit for the detection of a maize drought resistant haplotype comprising a primer pair wherein said first primer has nucleotide sequence SEQ ID NO. 3 and the second primer has nucleotide sequence SEQ ID NO. 4 and/or pair of primers wherein said first primer has nucleotide sequence SEQ ID NO. 5 and the second primer has nucleotide sequence SEQ ID NO. 6.

In accordance with another aspect of the invention the invention, novel varieties may be created by crossing plants of the invention followed by generations of selection as desired and inbreeding for development of uniform lines. New varieties may also be created by crossing with any second plant. In selecting such a second plant to cross for the purpose of developing novel lines, it may be desired to choose those plants which either themselves exhibit one or more selected desirable characteristics or which exhibit the desired characteristic(s) when in hybrid combination. Once initial crosses have been made, inbreeding and selection take place to produce new varieties. For development of a uniform line, often five or more generations of selfing and selection are typically involved. Uniform lines of new varieties may also be developed by way of doubled-haploids. This technique allows the creation of true breeding lines without the need for multiple generations of selfing and selection. In this manner true breeding lines can be produced in as little as one generation. Haploid embryos may be produced from microspores, pollen, anther cultures, or ovary cultures. The haploid embryos may then be doubled autonomously, or by chemical treatments (e.g. colchicine treatment). Alternatively, haploid embryos may be grown into haploid plants and treated to induce chromosome doubling. In either case, fertile homozygous plants are obtained. In accordance with the invention, any of such techniques may be used in connection with a plant of the present invention and progeny thereof to achieve a homozygous line.

Backcrossing can also be used to improve an inbred plant. Backcrossing transfers a specific desirable trait, such as elevated glucoraphanin, from one inbred or non-inbred source to a variety that lacks that trait. This can be accomplished, for example, by first crossing a parent (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate locus or loci for the trait in question. The progeny of this cross are then mated back to the recurrent parent (A) followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. After five or more backcross generations with selection for the desired trait, the progeny are heterozygous for loci controlling the characteristic being transferred, but are like the first parent for most or almost all other loci. The last backcross generation would be selfed to give pure breeding progeny for the trait being transferred. The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original variety. To accomplish this, a single locus of the recurrent variety is modified or substituted with the desired locus from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological constitution of the original variety. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross; one of the major purposes is to add some commercially desirable trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele may also be transferred. It may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.

Maize varieties can also be developed from more than two parents. The technique, known as modified backcrossing, uses different recurrent parents during the backcrossing. Modified backcrossing may be used to replace the original recurrent parent with a variety having certain more desirable characteristics or multiple parents may be used to obtain different desirable characteristics from each. In another aspect, the invention relates to a method for introgressing the haplotype described herein into a genetic background that lacks said haplotype. In some embodiments, the methods comprise crossing a donor comprising said allele with a recurrent parent that lacks said allele and repeatedly backcrossing progeny comprising said allele with the recurrent parent, wherein said progeny are identified by detecting, in their genomes, the presence of the haplotype described herein and which is associated with enhanced drought resistance.

Thus, one aspect of the current invention concerns methods for crossing a plant comprising the haplotype described herein with a second plant and the seeds and plants produced by such methods. These methods can be used for production and propagation of cultivated maize plants displaying desired drought resistance. The methods also can be used to produce hybrid maize seeds and the plants grown therefrom. Hybrid seeds are produced by crossing such lines with a second maize parent line. The hybrids may be heterozygous or homozygous for the introgression. The term "introgression" refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. Offspring comprising the desired allele can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background. For example, the chromosome 2 locus described herein may be introgressed into a recurrent parent that is not resistant or only partially drought resistant to. The recurrent parent line with the introgressed gene or locus then has enhanced drought resistance. The process of "introgressing" is often referred to as "backcrossing" when the process is repeated two or more times.

In another aspect, the invention thus relates to method for producing a hybrid maize plant with enhanced drought resistance the method comprising

a. providing a first plant with a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1;

b. providing a second plant that does not have a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1;

c. crossing the first plant with the second plant to produce an Fl generation and d. identifying one or more members of the Fl generation that comprises the desired phenotype comprising said haplotype.

Also within the scope of the invention is a plant orplant cell produced by such method. In another aspect, the invention thus relates to identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype comprising a. obtaining a first maize plant that comprises within its genome a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1.

b. crossing said first maize plant to a second maize plant;

c. evaluating the progeny plants and d. identifying and/or selecting a maize plant with enhanced drought resistance that comprises said haplotype.

In the methods above, detection can be carried out using the methods and primers described elsewhere herein.

A plant or plant part, for example a seed, obtained or obtainable by a method described above is also within the scope of the invention.

The invention also relates to a method of increasing the frequency of a drought tolerance phenotype in a population of maize plants comprising:

a. providing a first population of maize plants;

b. detecting the presence of a genetic marker that is associated with a drought tolerance trait;

c. selecting one or more corn plants exhibiting the drought tolerance genotype from the first population of v plants; and

d. producing an offspring population from the one or more selected v plants such that the drought tolerance phenotype occurs more frequently in the offspring population as compared to the first population.

The genetic marker is as described above. Thus, the marker can be the -21 InDel sequence as described above or the presence of the 5' polymorphic loci. Primer pairs SEQ ID NO. 3 and SEQ ID NO. 4 or SEQ ID NO. 5 and SEQ ID NO. 6 can be used in the detection.

The invention also relates to a recombined DNA segment comprising a 5' UTR ZmDREB2. 7 allele from maize which comprises a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1.

In one embodiment, the DNA segment is further defined as comprised within a cell. In another embodiment, the DNA segment is further defined as comprised within a seed. In yet another embodiment, the DNA segment is further defined as comprised within a plant. According to the invention, a maize plant that has the favourable haplotype has enhanced drought tolerance if the survival rates are at least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold higher than those of the control plant after exposure to drought and/or after exposure to drought and re- watering. Also according to the invention, a maize plant that has the favourable haplotype has enhanced drought tolerance if the rosette radius is at least 10, 20, 30, 40, 50% larger than that of the control plant after exposure to drought and/or after exposure to drought and re-watering. The plant may be deprived of water for 10-30, for example 20 days and then re-watered. Also according to the invention, a maize plant that has the favourable haplotype has enhanced drought tolerance if stomatal conductance (Gst) and transpiration are lower than in the control plant, for example at least 10, 20, 30, 40, 50% lower.

A maize plant that has the favourable haplotype also shows in creased yield when exposed to drought compared to a plant that does not have the favourable haplotype. The terms "increase", "improve" or "enhance" are interchangeable. Yield for example is increased by at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40% or 50% or more in comparison to a control plant. The term "yield" in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters. The term "yield" of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant. Thus, according to the invention, yield comprises one or more of and can be measured by assessing one or more of: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased number of seed capsules/pods, increased seed size, increased growth or increased branching, for example inflorescences with more branches. Preferably, yield comprises an increased number of seed capsules/pods and/or increased branching. Yield is increased relative to control plants.

In one embodiment, the drought stress is moderate or severe stress. Preferably, said stress is moderate drought stress. A plant according to the invention also shows reduced growth/yield penalties under moderate stress compared to a control plant. In other words, an improve in yield under moderate stress conditions can be observed. As explained herein, induction of ZmDREB2.7 expression in early drought stress is important for plant survival in stress, which coincided with its function as a TF to activate downstream stress-responsive gene expression. The quicker induction of expression in the drought tolerant genotype of the plants described herein compared to the sensitive genotype of was consistently observed.

For example, in plant research, drought tolerance is assessed predominantly under quite severe conditions in which plant survival is scored after a prolonged period of soil drying. However, in temperate climates, limited water availability rarely causes plant death, but restricts biomass and seed yield. Moderate water stress, that is suboptimal availability of water for growth can occur during intermittent intervals of days or weeks between irrigation events and may limit leaf growth, light interception, photosynthesis and hence yield potential. Leaf growth inhibition by water stress is particularly undesirable during early establishment. There is a need for methods for making plants with increased yield under moderate stress conditions. In other words, whilst plant research in making stress tolerant plants is often directed at identifying plants that show increased stress tolerance under severe conditions that will lead to death of a wild type plant, these plants do not perform well under moderate stress conditions and often show growth reduction which leads to unnecessary yield loss. Thus, in one embodiment of the methods of the invention, yield is improved under moderate stress conditions. The transgenic plants according to the various aspects of the invention show enhanced tolerance to these types of stresses compared to a control plant and are able to mitigate any loss in yield/growth. The tolerance can therefore be measured as an increase in yield as shown in the examples. The terms moderate or mild stress/stress conditions are used interchangeably and refer to non-severe stress. In other words, moderate stress, unlike severe stress, does not lead to plant death. Under moderate, that is non-lethal, stress conditions, wild type plants are able to survive, but show a decrease in growth and seed production and prolonged moderate stress can also result in developmental arrest. The decrease can be at least 5%-50% or more. Tolerance to severe stress is measured as a percentage of survival, whereas moderate stress does not affect survival, but growth rates. The precise conditions that define moderate stress vary from plant to plant and also between climate zones, but ultimately, these moderate conditions do not cause the plant to die.

Generally speaking, moderate drought stress is defined by a water potential of between and -2 Mpa. In one embodiment, the maize relative leaf water content (RLWC) at 95-100% is well- watered or favourablegrowth condition; RLWC at around 70-65% is moderate drought stress; RLWC at around 58-55%) is severe drought stress.

Drought tolerance can be measured using methods known in the art, for example assessing survival of the transgenic plant compared to a control plant, through leaf water potentials or by determining turgor pressure, rosette radius, water loss in leaves, growth or yield. Drought resistance can also be measured by assessing stomatal conductance (Gst) and transpiration in whole plants under basal conditions.

In another aspect, the invention relates to isolated nucleic sequences and their application in the regulation of drought resistance in maize. In one embodiment, the isolated nucleic acid sequences comprise or consist of:

1) a ZmDREB2. 7 promoter sequence which comprises nucleotides at positions 296 to 798 position of SEQ ID NO. 1 (SEQ ID NO. 19);

2) a ZmDREB2. 7 promoter sequence positions 1 to 798 of SEQ ID NO. 1 (SEQ ID NO. 20);

3) nucleic acid sequences which exhibit at least 70%, at least 75%, at least 80%>, at least 85%>, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% homology with the nucleic acid sequences defined in 1) or 2);

4) nucleic acid sequences which hybridize to the nucleic acid sequences defined in 1), 2) or 3) under strict conditions.

The invention also relates to a nucleic acid construct comprising a sequence as shown above in l)-4). In another aspect, the invention relates to a vector comprising a nucleic acid construct comprising a sequence as shown above in l)-4). Said sequence is preferably operably linked to a second nucleic acid sequence to direct expression of said nucleic acid sequence in a drought regulated manner. The second nucleic acid sequence can be selected from another DREB nucleic acid sequence. In one embodiment, for example a DREB sequence from another monocot plant. In one embodiment, for example a DREB sequence from another maize. However, a skilled person will appreciate that any nucleic acid sequence of interest which plays a role in drought resistance can be operably linked to the promoter sequence to direct expression for a beneficial drought response. In one embodiment, expression of the nucleic acid construct is in a monocot plant.

In another aspect, the invention relates to a host cell comprising a vector as described herein. The host cell can be selected from a plant cell or a bacterial cell, for example Agrobacterium. The invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described above.

In another aspect, the invention relates to the use of a nucleic acid construct or vector comprising a sequence as shown above in l)-4) in conferring drought regulated gene expression thus conferring resistance to a plant. In one embodiment, nucleic acid construct or vector confers early drought response.

In another aspect, the invention relates to a method for conferring or increasing drought resistance of a plant said method comprising introducing and expressing in said plant a nucleic acid construct comprising a sequence as shown above in l)-4) operably linked to a second nucleic acid sequence to direct expression of said nucleic acid sequence in a drought regulated manner.

In another aspect, the invention relates to plants obtained or obtainable by said method.

In another aspect, the invention relates to a plant comprising a nucleic acid construct wherein said construct comprises

3) a ZmDREB2. 7 promoter sequence as defined in SEQ ID NO. 22;

4) nucleic acid sequences which exhibit at least 70%, at least 75%, at least 80%, at least

85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% homology with the nucleic acid sequences defined in 1) or 2); 5) nucleic acid sequences which hybridize to the nucleic acid sequences defined in 1), 2), 3) or 4) under strict conditions. In one embodiment, said plant is a monocot plant, for example maize. In another embodiment, said plant is a dicot plant. In one embodiment, said plant is a crop plant, such as rape/canola, corn, sugar cane, palm trees, jatropha, soybeans, sorghum, sunflowers, cottonseed, Panicum virgatum (switchgrass), linseed, wheat, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials

(e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).

The nucleic acid or vector described above is used to generate transgenic plants using transformation methods known in the art. Thus, according to the various aspects of the invention, a nucleic acid comprising a ZmDREB2. 7 nucleic acid or a functional variant thereof is introduced into a plant and expressed as a transgene. The nucleic acid sequence is introduced into said plant through a process called transformation. The term "introduction" or "transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art. The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plants is now a routine technique in many species. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.

To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above. Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or Tl) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion). In another aspect, the invention relates to a method for increasing drought resistance of a plant by targeted genome editing to modify the ZmDREB2.7 promoter sequence to introduce the 5'UTR polymorphisms described herein.

Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulategenome editing through homologous recombination (HR)-mediated recombination events. To achieve effective genome editing via introduction of site-specificDNA DSBs, four major classes of customizable DNA bindingproteins can be used:meganucleasesderived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcriptionfactors, transcriptionactivator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guidedDNA endonuclease Cas9 from the type II bacterial adaptive immunesystem CRISPR (clustered regularly interspaced short palindromic repeats). Meganuclease, ZF, and TALE proteins all recognize specificDNA sequences through protein-DNA interactions. Althoughmeganucleases integrate its nuclease and DNA-bindingdomains, ZF and TALE proteins consist of individual modulestargeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinationsand attached to the nucleasedomain of Fokl to directnucleolytic activity toward specific genomic loci.

Upon delivery into host cells via the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters and activate transcription. Their targeting specificity is determined by a central domain of tandem, 33-35 amino acid repeats. This is followed by a single truncated repeat of 20 amino acids. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats.

These repeats only differ from each other by two adjacent amino acids, their repeat- variable di-residue (RVD). The RVD that determines which single nucleotide the TAL effector will recognize: one RVD corresponds to one nucleotide, with the four most common RVDs each preferentially associating with one of the four bases. Naturally occurring recognition sites are uniformly preceded by a T that is required for TAL effector activity. TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create a TAL effector nucleases (TALEN) which makes targeted DNA double-strand breaks (DSBs) in vivo for genome editing. The use of this technology in genome editing is well described in the art, for example in US8, 440,431, US 8,440, 432 and US8,450,471. The Golden Gate cloning method to assemble multiple DNA fragments can be used.

Another genome editing method that can be used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example in US8, 697,359 and references cited herein. In short, CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non- coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (I-III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). The Type II CRISPRis one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA: tracrRNA complex directs Cas9 to the target DNA via Wastson-Crick base- pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.

Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRIPSR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.

The single guide RNA (sgRNA) is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3.

Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the art.

In another aspect, the invention thus relates to a method for increasing drought resistance of a maize plant compared to a control plant comprising the steps of

c. targeted mutagenesis of a plant population to introduce the following mutations in the 5' UTR of ZmDREB2.7: a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1

d. identifying and selecting plants which comprise the desired mutations. Optionally, other modifications that act as a marker, such as -21 InDel can also be introduced so facilitate identification of plants with the desired genotype.

Plants can be identified by the methods described herein, for example using primer pairs SEQ ID NO. 5 and SEQ ID NO. 6 and/or, if -21 InDel is introduced, SEQ ID NO. 3 and SEQ ID NO. 4. Phenotypic tests to assess drought resistance can also be carried out. Furthermore, expression analysis to assess early induction of gene expression, can also be carried out.

The control plant is any plant that does not have the polymorphism described above. Plants obtained or obtainable by this method are also within the scope of the invention. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification, including reference to sequence database identifiers, are incorporated herein by reference in their entirety. Unless otherwise specified, when reference to sequence database identifiers is made, the version number is 1.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. The invention is further described in the following non-limiting examples.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

Examples

Example 1. Association Analysis of Natural Variation in ZmDREB Genes with Maize Drought Tolerance

Previous research reported that ZmDREB 1.1/1 A and 2.7/2_^4are transcription factors that play an important role in theregulation of maize drought-stress response [17,18]. In order tofurther investigate whether the natural variation in any of thegenes encoding ZmDREBl and

2 TFs was associated with thediversity in drought tolerance of maize varieties, an associationanalysis was conducted for each of the ZmDREB genes. Recently,525, 105 high- quality maize S P markers (minor allele frequency(MAF)>0.05) were identified from transcriptomic sequencing of amaize natural diversity panel consisting of 368 inbred lines fromtropical and temperate regions [22,23]. These markers were thenutilized to characterize the presence of genetic polymorphisms ineach of these 18 ZmDREB genes. Among the ZmDREB genes, 14were found to be polymorphic with 17 SNPs on average identified in each gene. The polymorphic information was currently absentfor four genes, due to above 60% missing rate in the genotypingdata. ZmDREB2.1/2A was found to be the most polymorphic, with 42 SNPs in this natural diversity panel. The drought stresstolerance of each variety was also investigated. The survival rate of seedlings under severe drought conditions was scored. Statistically ,the inbred lines from tropical regions exhibited higher survivalrates in comparison to those from temperate regions or B73derivatives. These data supported thehypothesis that varieties existing within the area of origination maypossess better and wider resistance than those in cultivated regions. Three kinds of statistical models were applied to identify significant genotypic and phenotypic associations. Specifically, a generallinear model (GLM), principle component analysis (PCA), and amixed linear model (MLM) were used in the associations. PCAwas applied to correct for spurious associations caused bypopulation structure. MLM incorporated both PCA and a Kinshipmatrix (to correct for the effect of cryptic relatedness) and wasconsidered to be effective for controlling false positives in theassociation analysis. The analysis detected significantassociations in the genetic variation in ZmDREB2.7and ZmDREB 2.3/ABI4 under different models. However, ZmDREB2.7was the gene that was the most significantly associated(21ogP= 3.07) with drought tolerance in this natural variation panel.

A natural variation group composed of 105 maize inbred lines was cultivated in a transplantation pool (6.0 x 1.4 x 0.22 metres, length x width x depth) using 5 tons of loam mixed with 0.25 tons of mature chicken manure to act as a transplantation base, in two separate pools. Each pool was separated into 105 small zones, with 9 seedlings being planted in each small zone. Watering was stopped once seedling age reached three true leaves, commencing drought treatment; this was continued for 7 days after the relative soil water content reached zero, then six days after watering was recommenced the survival rate was calculated. The drought data used for the purposes of statistical analysis was in all cases a mean value from individually repeated experiments. The results are as shown in table 1.

Table 1. Drought resistance survey of a natural variation group composed of 105 maize inbred lines Sequence Maize SubSurvival Drought Genotype inbred line group rate (%) resistance

1 CIMBL55 TST 100 Drought resistant Homozygous haplotype A

2 CIMBL70 TST 95 Drought resistant Homozygous haplotype A

3 CIMBL19 TST 90 Drought resistant Homozygous haplotype A

4 CML69 TST 81.25 Drought resistant Homozygous haplotype A

5 CML170 TST 80 Drought resistant Homozygous haplotype A

6 CIMBL92 TST 77.78 Drought resistant Homozygous haplotype A

7 GEMS 51 ss 77.78 Drought resistant Homozygous haplotype A

8 CIMBL2 TST 76.39 Drought resistant Homozygous haplotype A

9 CIMBL91 TST 74.44 Drought resistant Homozygous haplotype A

10 CIMBL123 TST 73.33 Drought resistant Homozygous haplotype A

11 Ry732 NSS 73.21 Drought resistant Homozygous haplotype A

12 CIMBL42 TST 72.22 Drought resistant Homozygous haplotype A

13 268 MIXED 70.83 Drought resistant Homozygous haplotype A

14 CML118 TST 68.75 Drought resistant Homozygous haplotype A

15 CIMBL115 TST 66.67 Drought resistant Homozygous haplotype A

16 CIMBL22 TST 66.67 Drought resistant Homozygous haplotype A

17 R08 NSS 63.75 Drought resistant Homozygous haplotype A

18 GEMS41 NSS 61.11 Drought resistant Homozygous haplotype A

19 CIMBL68 TST 58.89 Drought resistant Homozygous haplotype A

20 GEMS 59 NSS 58.57 Drought resistant Homozygous haplotype A

21 CIMBLl lO TST 57.5 Drought resistant Homozygous haplotype A

22 GEMS 13 ss 55.56 Drought resistant Homozygous haplotype A

23 CIMBLl MIXED 55.56 Drought resistant Homozygous haplotype A

24 CIMBL32 TST 52.78 Drought resistant Homozygous haplotype A

25 CIMBL33 TST 51.14 Drought resistant Homozygous haplotype A

26 CML298 TST 50.69 Drought resistant Homozygous haplotype A

27 CIMBL45 TST 50 Drought resistant Homozygous haplotype A

28 CML361 TST 49.21 Drought resistant Homozygous haplotype A

29 CIMBL95 MIXED 44.44 Drought resistant Homozygous haplotype A

30 CIMBL80 TST 41.25 Drought resistant Homozygous haplotype A

31 CIMBLl 27 TST 40.91 Drought resistant Homozygous haplotype A

32 CIMBL28 TST 38.89 Intermediate type Homozygous haplotype A JH96C MIXED 38.89 Intermediate type Homozygous haplotype A

CML171 TST 37.22 Intermediate type Homozygous haplotype A

CML172 TST 27.78 Intermediate type Homozygous haplotype A

CIMBL96 TST 27.78 Intermediate type Homozygous haplotype A

Syl l28 NSS 27.78 Intermediate type Homozygous haplotype A

CIMBL62 TST 26.79 Intermediate type Homozygous haplotype A

835a NSS 25.4 Intermediate type Homozygous haplotype A

CIMBL12 TST 25 Intermediate type Homozygous haplotype A

By4960 NSS 23.61 Intermediate type Homozygous haplotype A

05WN230 MIXED 21.11 Intermediate type Homozygous haplotype A

GEMS30 MIXED 18.06 Intermediate type Homozygous haplotype A

CIMBL56 TST 13.64 Intermediate type Homozygous haplotype A

CIMBL150 TST 11.11 Intermediate type Homozygous haplotype A

U8112 ss 11.11 Intermediate type Homozygous haplotype A

GEMS28 ss 11.11 Intermediate type Homozygous haplotype A

CIMBLIO MIXED 11.11 Intermediate type Homozygous haplotype A

M0113 NSS 10 Intermediate type Homozygous haplotype A

05W002 MIXED 10 Intermediate type Homozygous haplotype A

CML162 TST 7.14 Drought sensitive Homozygous haplotype A

CIMBL23 TST 5.56 Drought sensitive Homozygous haplotype A

CML304 TST 5.56 Drought sensitive Homozygous haplotype A

Ji63 NSS 5.56 Drought sensitive Homozygous haplotype A

CIMBL142 MIXED 5.56 Drought sensitive Homozygous haplotype A

GEMS32 MIXED 5 Drought sensitive Homozygous haplotype A

FCD0602 SS 4.55 Drought sensitive Homozygous haplotype B

TY6 TST 0 Drought sensitive Homozygous haplotype B

CIMBL18 TST 0 Drought sensitive Homozygous haplotype A

CIMBL69 TST 0 Drought sensitive Homozygous haplotype A

CML191 TST 0 Drought sensitive Homozygous haplotype A

CML480 TST 0 Drought sensitive Homozygous haplotype A

8902 ss 0 Drought sensitive Homozygous haplotype B

Dan360 ss 0 Drought sensitive Homozygous haplotype B

J4112 ss 0 Drought sensitive Homozygous haplotype B

Liao5114 ss 0 Drought sensitive Homozygous haplotype B Xun971 ss 0 Drought sensitive Homozygous haplotype B

Ye748 ss 0 Drought sensitive Homozygous haplotype B

Ye8001 ss 0 Drought sensitive Homozygous haplotype B

Zheng32 ss 0 Drought sensitive Homozygous haplotype B

Zheng35 ss 0 Drought sensitive Homozygous haplotype B

B73 ss 0 Drought sensitive Homozygous haplotype A

4F1 NSS 0 Drought sensitive Homozygous haplotype B

Dan3130 NSS 0 Drought sensitive Homozygous haplotype B

GEMS 58 NSS 0 Drought sensitive Homozygous haplotype B

GEMS60 NSS 0 Drought sensitive Homozygous haplotype A

JH59 NSS 0 Drought sensitive Homozygous haplotype B

Ji846 NSS 0 Drought sensitive Homozygous haplotype B

K14 NSS 0 Drought sensitive Homozygous haplotype B

K22 NSS 0 Drought sensitive Homozygous haplotype B

M97 NSS 0 Drought sensitive Homozygous haplotype B

M153 NSS 0 Drought sensitive Homozygous haplotype B

Mol7 NSS 0 Drought sensitive Homozygous haplotype A

Shen5003 NSS 0 Drought sensitive Homozygous haplotype B

Ye52106 NSS 0 Drought sensitive Homozygous haplotype B

ZaC546 NSS 0 Drought sensitive Homozygous haplotype B

Zhi41 NSS 0 Drought sensitive Homozygous haplotype B

Dan4245 NSS 0 Drought sensitive Homozygous haplotype A

Ry684 NSS 0 Drought sensitive Homozygous haplotype A

Ry737 NSS 0 Drought sensitive Homozygous haplotype B

Syl039 NSS 0 Drought sensitive Homozygous haplotype B

Sy3073 NSS 0 Drought sensitive Homozygous haplotype A

W138 NSS 0 Drought sensitive Homozygous haplotype A

Yu374 NSS 0 Drought sensitive Homozygous haplotype A

Zheng653 NSS 0 Drought sensitive Homozygous haplotype A

CIMBL137 MIXED 0 Drought sensitive Homozygous haplotype B

Qi205 MIXED 0 Drought sensitive Homozygous haplotype B

TY1 MIXED 0 Drought sensitive Homozygous haplotype B

TY2 MIXED 0 Drought sensitive Homozygous haplotype B

TY3 MIXED 0 Drought sensitive Homozygous haplotype B 101 TY5 MIXED 0 Drought sensitive Homozygous haplotype B

102 TY11 MIXED 0 Drought sensitive Homozygous haplotype B

103 7381 MIXED 0 Drought sensitive Homozygous haplotype A

104 CIMBL141 MIXED 0 Drought sensitive Homozygous haplotype A

105 WH413 MIXED 0 Drought sensitive Homozygous haplotype A

In table 1 the separation into the subgroups MIXED, NSS, SS and TST is based on the following paper: Yang X, Gao S, Xu S, Zhang Z, Prasanna B, et al. (2011) Characterization of a global germplasm collection and its potential utilization for analysis of complex quantitative traits in maize. Molecular Breeding 28: 511-526, where TST = tropical or subtropical varieties,

NSS = temperate varieties, SS = B73 derivatives and MIXED = varieties with no clear identity.

The academic paper that made the 105 maize inbred lines shown in table lpublic is: Yang X, Gao S, Xu S, Zhang Z, Prasanna B, et al. (2011) Characterization of a global germplasm collection and its potential utilization for analysis of complex quantitative traits in maize.

Molecular Breeding 28: 511-526 and Li H, Peng Z, Yang X, Wang W, Fu J, et al. (2013) Genome-wide association study dissects the genetic architecture of oil biosynthesis in maize kernels. Nat Genet 45: 43-50. Publicly available from the Institute of Botany of the Chinese Academy of Sciences.

According to the survival rates in table 1, drought resistance is divided into three types: drought resistant inbred lines, survival rate >40%; drought sensitive inbred lines, survival rate between <10%; intermediate type with a survival rate > 10 % and < 40%. Polymorphic loci statistics

In order to fully identify the DNA polymorphism present in theZmDREB2.7 gene, it was re-sequenced in 105 maize inbred lines. The 2.1kb genome segment of the non-translated 5' and 3' terminals of the coding sequence of the ZmDREB2. 7 gene of 105 maize inbred lines indicated in table 1 was divided into three sections for sequencing (for details of primers see table 2), then the results were compared using MEGA 5.0 (http://megasoftware.net). Based on the results of these comparisons of nucleotide polymorphism, 124 polymorphic loci were obtained, of which 102 were SNPs and 22 were InDels (as shown in table 4), the lowest allele frequency (MAF) being >0.05.

Table 2. Genetic codes of primers used in sequencing Name Sequence (5 '-3') Initiation position in primer 5' terminal

First section forward GTCCGTCAGTCCGTCCTTG SEQ -702

primer Fl ID NO. 5

First section reverse GGAAATGGAATCGGAGTTTGAC +654

primer Rl SEQ ID NO. 6

Second section forward CGAGGACTGCATTGCTAGCA -71

primer F2 SEQ ID NO. 7

Second section reverse TCAAAGAGGGACGACGAGC +1080

primer R2 SEQ ID NO. 8

Third section forward ACCACCAGACGATGTTCCA +722

primer F3 SEQ ID NO. 9

Third section reverse CCACGAATAACTAGGAGAAATA +1313

primer R3 SEQ ID NO. 10

Correlation analysis regarding polymorphic loci and drought resistant haplotype The association of each polymorphism with droughttolerance was analyzed again using the MLM model and thepairwise linkage disequilibrium (LD) of these polymorphisms wascalculated.

Using tassel 3.0 software, a mixed linear method was adopted, with attention being paid to kinship coefficients and population structure, examining the positive correlations between 124 polymorphic loci and drought resistance haplotype, computing lineage disequilibrium values for all the polymorphic loci pair by pair, finally using Sigmaplot 10.0 to generate the graph.

The results are as seen in figure 4. The results in figure 4 demonstrate that the -logio (P value) of the five newly determined loci upstream from the initiation codon (ATG) (SNP-503, -260, - 150 and InDel-185, -154) was 2.8, which is greater than that of other loci in the initiation zone; this confirms that these 5 loci are clearly linked with the drought resistance haplotype variation and were absolutely linked with the 105 material samples. The 3 non-synonymous substituted

SNPs (SNP142, 436, 661) and one 3bp InDell41 (non-synonymous substitution resulting in a change in amino acid) had clear correlations with haplotype variation. Synonymous substituted SNP408 (synonymous substitution notresulting in changes to amino acid) was the most pronounced loci, and exhibited strong lineage with InDell41. The two non-synonymous substituted SNPs (SNP142 and SNP661) exhibited strong lineage with the five pronounced loci in the initiation region. Pronounced correlation between level of expression of the ZmDREB2.7 gene and survival rate

In order to establish the contribution played by gene expression and protein activity in terms of drought resistance, analysis was carried out into 73 maize inbred lines: CIMBL55, CIMBL70, CML69, CIMBL92, CIMBL91, CIMBL42, CML118, CIMBL115, CIMBL22,

GEMS41, CIMBL68, CIMBLl, CML298, CIMBL95, SW92E114, CIMBL127, GEMS37, CIMBL28, CML171, CIMBL96, Syl l28, CIMBLl 2, By4960, GEMS30, CIMBLIO, U8112, GEMS28, MO 113, CML162, CML304, CIMBL23, CIMBLl 33, Ji63, GEMS 14, FCD0602, CIMBL69, GEMS60, CIMBLl 8, CIMBL27, CIMBLl 1, M153, Shen5003, D863F, K14, CIMBL137, Dan3130, JH59, Xun971, Zheng35, TY5, Ye8001, 8902, K22, Zheng32, Zhi41,

J4112, TY2, Tie7922, TY1, Dan360, TY6, TY3, Mol7, Ye478, CML189, Ye52106, CML166, GEMS44, CIMBLl 09, Syl039, By4839, Sy3073 and CIMBLl 43 (paper: Yang X, Gao S, Xu S, Zhang Z, Prasanna B, et al. (2011) Characterization of a global germplasm collection and its potential utilization for analysis of complex quantitative traits in maize. Molecular Breeding 28: 511-526 (publicly available from the Institute of Botany of the Chinese Academy of Sciences.)

Maize inbred lines listed herein are available from various stock collections, see for example http://www.maizego.org/Resources.html.

The results of correlation studies into the connection of mRNA levels of the ZmDREB2. 7 gene and survival rates under the three conditions ofnormal growth, slight and severe drought, were under slight drought (relative leaf water content RLWC= 70%) conditions, the level of expression of the ZmDREB2. 7 gene and survival rate exhibited pronounced correlations (P value of 7.14E-09); under normal growth (relative leaf water content RLWC = 98%) and extreme drought (relative leaf water content RLWC = 58%) conditions,there was no pronounced correlation with survival rate (P value being respectively 7.07E-01 and 3.14E-02).

The results indicate that where early expression of the ZmDREB2. 7 gene is induced, and this is not background or slowed expression, this is a major factor affecting the survival of maize where afflicted by drought. The methods and results of the correlation studies relating to relative expression levels of the ZmDREB2. 7 gene in the above mentioned 73 sample materials and survival rate are as follows:

Maize seeds were sterilised for 10 minutes using 1 %o (v/v) Topsin-M (Rotam Crop Sciences Ltd.), then washed three times in de-ionised water, then finally they were placed on a culture dish on top of filter paper then allowed to germinate at 28°C for three days, then the seeds that had germinated were transferred to nutrient soil. At seedling age of 3 leaves they were treated by cessation of water, leaves were removed at relative leaf water content (RLWC) 98%, 70%) and 58%> from not less than three seedlings, then total RNA separated out using the TRIZOL (Biotopped) method, this was followed by using the DNAse I (Takara) method to eliminate contaminants from the genome, then concentration was measured using a NanodroplOOO (Thermo Scientific Product, USA), and 5μg taken uniformly and run on 0.8%> agarose gel. Then ^g of total RNA was selected and recombinant M-MLV reverse transcriptase used, with ^g of Oligo (dT) 23 (Promega) used as primer, to carry out synthesis of the cDNAs. Then florescent real-time quantitative PCR was used, to analyse the ZmDREB2. 7 gene relative expression levelsin the 73 samples of genetic material. PCR amplification of the cDNA of the ZmDREB2.7 gene was carried out using the F4 and R4 specific primers, the ZmUbi-2 (GenBank: AFW66445.1) gene being used as the reference, with FU and RU as primers. Florescent real-time quantitative PCR was carried out using an Applied Biosystems Step One Real-Time PCR System (ABI, USA) florescent real-time quantitative PCR machine, each parallel experiment being set to repeat 3 times. Reporting was based on the methods of Livak KJ and Schmittgen TD (2001), i.e. 2^{" Δ Δ σΓ} calculation of relative expression levels.

A A C_T= ( C_T •Target ^T- ZmTJbi-2 ^ Time x - ( Or•Target ^T- ZmTJbi-2 ^ Time 0

Time x referring to any point in time,Time 0 referring to 1 multiple of target gene expression after ZmUbi-2 correction.

The sequences of the above mentioned primers are as follows: F4: 5 ' -TATGATGATGATGC ACTCC-3 ' SEQ ID NO. 11

R4: 5 ' -GAGTTGGAAATGGAATCG-3 ' SEQ ID NO. 12

FU: 5 ' -TGGTTGTGGCTTCGTTGGTT-3 ' SEQ ID NO.13

RU: 5'- GCTGCAGAAGAGTTTTGGGTACA -3' SEQ ID NO. 14

SPSS 12.0 software was then used to carry out correlation analysis of the survival rate and ZmDREB2. 7 gene expression levels of the 73 sample materials obtained, the results of which are as shown in figure 5(A)-(C).

Example 2. Protein transcription activation activity tests Protein transcription activation activity tests indicate that four non-synonymous mutations (S P142, 436, 661 and one 3bp InDell41) have no pronounced effect on protein activity, this being due to differences in expression of ZmDREB2.7, rather than due to differences in transcription activity, therefore making it a natural mutation that is a major factor affecting the drought resistance of maize. It is due to this that the polymorphism of the initiation region (the

SNP-503, -260, -150 and InDel-185, -154) of ZmDREB2.7 results in functional differences in terms of drought resistance in maize during the seedling stage. The detailed analytical methods and results being as follows: Genome DNA of the drought resistant maize inbred lines CIMBL70, CEVIBL91,

CIMBL92 and CML118 and the drought sensitive maizeinbred line Shen5003 and the control maize inbred line B73 were used separately as templates and the F5 and R5 primers used to carry out PCR amplification; the results of amplification were then subjected to PCR amplification separately using the F6 and R5 primers (the sequences of the primer as shown in table 5), the results of that amplification were then cloned separately on to the vector pBlueScript SK (Qin F, Kakimoto M, Sakuma Y,Maruyama K, Osakabe Y, et al. (2007) Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L. Plant J 50: 54-69. Publicly available from the Institute of Botany of the Chinese Academy of Sciences) between EcoK I and Sma I yielding an intermediate vector, then the intermediate vector was subjected to EcoK I and BamH I enzyme digestion; the ZmDREB2.7 gene segment was recovered, and linked to the vector pGBKT7(Zhang M, Fu Z, Tian D, Liu E, Dai J, et al. (2011) Investigation of the interaction between CREB-binding protein and STAT4/STAT6. Mol Biol Rep 38: 4805-4811. Publicly available from the Institute of Botany of the Chinese Academy of Sciences) between the EcoK I and BamH I enzyme digestion loci, then yeast strain AH109 was converted separately, in order to convert the empty vector pGBKT7 as a control, separately yielding recombinant yeast strains ZmDREB2.7-CIMBL70, ZmDREB2.7-CEVIBL91 , ZmDREB2.7-CEVIBL92, ZmDREB2.7-CML118, ZmDREB2.7- Shen5003, ZmDREB2.7-B73 and pGBKT7-Control, these 7 recombinant yeast strains were then cultured using SD/-T liquidnutrient medium (pGBKT7 vector contains tryptophan nutritional marker genes) then this was diluted to OD₆oo = 0.1, then ultrapure water was used to dilute these separately to a factor of 10°, 10¹, 10² and 10³, then dropout media were used, either

SD culture base which lacked tryptophan (SD/-T), SD culture base which lacked tryptophan and histidine (SD/-T-H) or SD culture base which lacked tryptophan, histidine and adenine

(SD/-T-H-A), culturing continuing for 3 days, increasing the aminotriazole (3-AT) concentration on the SD/-T-H-A template in steps (respectively 5, 10, 20, 30, 40 and 50mM, creating a filter pressure (yeast strain AH109 contains histidine and adenine synthesis reporter genes and carries the target vector pGBKT7 strain, making it capable of autonomously synthesising histidine and adenine; because the SD culture base lacks tryptophan, histidine and adenine (SD/-T-H-A), this guarantees that only the target vector pGBKT7 strain which it contains survives; 3-AT is a suppressor of histidine synthesis, increasing the 3-AT concentration has the actual effect of obstructing the synthesis of histidine by the yeast strain AH109; only if there are genes that exhibit high transcriptional activity present is it possible to activate the reporter genes in the AH109 yeast strain to synthesise histidine, thus guaranteeing the survival of the strain) culturing continuing for 6 days; results as shown in figure 6.

Table 3. Primers used in cloning

Example 3. Analysis of allelic frequency of haplotypes in different subgroups

Analysis of the frequency of occurrence of haplotypes A and B of the 105 samples of genetic material shown in table 1 was carried out, the results of which are shown in table 1 and figure 1(A)-(E). The results indicate that, the frequency of the type A haplotype genotype of the tropical sub varieties was highest, which is consistent with the fact that the survival rate of tropical sub varieties was higher than that of other sub varieties. This indicates that haplotype A is a valuable genetic resource in terms of enhancing drought resistance in maize.

In order to compare the genetic effect of different ZmDREB2.7alleles on drought tolerance in maize, four drought-tolerant,inbred lines (CIMBL70, 91, 92 and CML118 were selected andcrossed with a drought-sensitive variety (Shen5003) resulting infour segregating F2 populations. All four drought-tolerant lines have the same ZmDREB2.7 allelic sequence in the 59-UTR at fivesignificant loci, while Shen5003 has the opposite allele at all fiveloci. Thus, the ZmDREB2. 7 allele in the tolerant inbred lines wasconsidered to be the favorable/tolerant allele and the allele inShen5003 was inferior/sensitive. The DNA polymorphisms of the five varieties at the significant loci are shown in Figure 1 A. Additionally, a 20-bp InDel, located 21 -bp upstream of the startcodon, was found in the four drought-tolerant inbred lines. Although this polymorphism was not as significantly associated with drought tolerance in the 105 varieties as was the five locilocated in the 59-UTR, a pair of primers, surrounding the 20-bp InDel, was designed to distinguish the presence of the favourable ZmDREB2.7 allele by PCR, due to their close physical linkage (Figure 1A). A comparison of the level of drought tolerance in thefive parental materials is shown in Figures IB. When drought stress was applied to the plants, about 33.3% Shen5003 plantssurvived, while survival rate of the CEVIBL70, 91, 92 andCML118 was 100%, 88.1%, 65.5% and 100%, respectively (Figure 1C). Expression of ZmDREB2. 7 was significantly inducedin the four tolerant genotypes in response to a moderate droughtstress (RLWC = 70%)) whereas, it was not significantly upregulatedat all in the sensitive genotype (Figure ID).

More than 400 individual F2 plants in each of the four F2 segregating populations were genotyped for the presence of thefavorable/tolerant allele of ZmDREB2. 7 by PCRs. As expected, aMendelian inheritance pattern was observed for the ZmDREB2. 7favorable/tolerant allele in each of the four segregating populations. The segregation rate of homozygous tolerant, heterozygoustolerant/sensitive, and homozygous sensitive plants was approximatelyl :2: l . The survival rates of plantscarrying the three different assortments of ZmDREB2.7 alleles werethen compared after being subjected to a drought stress. As shownin Figure IE, plants that were homozygous for the favorable/tolerant allele of ZmDREB2.7 were more tolerant to drought stressthan plants that were homozygous for the inferior/sensitive allele.

Table 4. Parent genotype and survival statistical results after drought treatment

_> .

of Shen5003, ** indicates a significant difference P<0.01 when compared to the results of Shen5003. Here, the genotype results have all been confirmed by sequencing. Table 5. Genotypes of drought resistant and drought sensitive hybrid segregated groups and survival statistics after drought treatment

Plants that were heterozygous for the favorable and inferior allelesexhibited a level of drought tolerance that was intermediatebetween the plants that were homozygous favorable or homozygousinferior. Co-segregation of the ZmDREB2. 7 tolerant allelewith improved drought tolerance suggested the linkage of this locus with the trait in segregation populations. In maize, linkageanalyses using bi-parental crosses also reported QTLs (quantitativetrait loci) for drought tolerance within the chromosomal region(Chr. 1, bin 1.07) where the ZmDREB2.7 gene is located.

Collectively, these data further support the premise that natural variation in ZmDREB2.7 contributes to enhanced droughttolerance in different maize varieties. Importantly, the tolerant/favorable allele of ZmDREB 2.7represents a promising genetic resource for the development of drought-tolerant maize cultivarsusing traditional breeding approaches or genetic engineering.

Molecular marker InDel-21 and its specific primer was used to carry out typing analysis and drought resistance analysis of different maize, relying on the drought resistant maize inbred lines CIMBL70 (table 1, 2), CIMBL91 (table 1, 9), CIMBL92 (table 1, 6) and CML118 (table 1, 14) to act as the male parents, with drought sensitive maize inbred lines Shen5003 (table 1, 84) as the mother plant, these then being hybridized, then inbred for 1 generation, yielding four segregated groups CIMBL70 X Shen5003, CIMBL91 X Shen5003, CIMBL92 X Shen5003 and CML118 X Shen5003.

Taking the maize genome DNA as the template, PCR amplification was carried out using the primers designed in step 1, if the size of the amplified result was 46bp, then the maize being sequenced was homozygous haplotype A maize; if the size of the amplified result was 66bp, then the maize being sequenced was homozygous haplotype B maize; if the size of the amplified result was 46bp and 66bp, then the maize being sequenced was heterozygous haplotypes A and B.

The results shown in tables 4 and 5 demonstrate that: resistance to drought by maize was respectively from high to low: homozygous haplotype A maize > heterozygous haplotypes A and B maize > homozygous haplotypes B maize. In maize breeding, the homozygous haplotype A maize which exhibited higher resistance to drought should be bred. The results also demonstrate that there is a close physical linkage between the molecular marker InDel-21 and haplotype represented by the five polymorphic loci in the initiation codon region, therefore it is possible to indirectly select the haplotype represented by these five pronounced loci from the PCR resultant banding. References

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Sequence listing

SEQ ID NO. 1 ZmDREB2. 7 gene Zea mays

atgcatgcct actgtcacac aaaaaataca gtatattatt actgatggag aaaacaagga

aggatgggcc ggatggggat ggccgagggc aaagccgtcc gtcagtccgt ccttgttgcg

tgtgcgtgca tacggagacc ggagtcagcg gtatgatgca ggcaagcaga ccattgcaca

cgcagataca gatcccagcc gagcgtccag ctgccaagcc atgcatgtgg ctcgcggatc

ggcgcagtcc atggatagat ggagatggat ccatccatgg atagatcata gatagataga

taggcagccc atggccgtgg ctgcatctgc gggctgggcg ggctgcatca gcgtgacgcc

gtgacctcac cctggttcgg tcgccccccg gccgccacgt ggcccagcgg ccatgacgtg

gaccccacag gggcttccat gtgtcaagcc ccgctggccc ccaccacttc gtgtcacccg

cctccttcac ttggcgtgcc gcacccccac gcgtggcccc acgcccaggc cccgcatccc tacacggagg cgtcatgcag tgccatgcgc cggcttcccc cctgccccct ccgtccgccc gccttcattc cgcacaccac cgaaaactgg tgcccggcct gcagtgcagt gcaagccatg ccagctgcct atatatacca ggccagggag cgggagcctc acacacagtc acagcacacg cagccaccga ggactgcatt gctagcatcc atcgccatca gtcgccatat cgatctgcgc acgaagctag tagtccagat ggatcgggtg ccgccgccgg tctccatgca ggtggctgcg atgcagcgac atcagcagca gcagcagttc gtccaccacc tgcagcaggt ccaccagcaa ggtacgcagc acgagcaacc gccgccaccg caccagaacg gcagcagcag cagcggcagg accggcggcg gccgcaagtg ctgcccgctg cggcggtcgc gcaaggggtg catgaagggc aagggcgggc cggacaacca gcagtgcccc ttccgcggcg tccggcagcg cacctggggc aagtgggtgg ccgagatccg cgagcccaac cgcggcgcgc gcctctggct cggcaccttc ggcagcgcgc tcgaggccgc gcgcgcctac gacgccgcgg ccaggacgct ctacggcgac tgcgctcgcc taaacctgca gctagtgcct ccgtcggcgg ctgcggcagc cgccggagga ggaggaccgg cggtcgtcgc gtctccgtcc cctgacaccg tggctggccc tgctgctgct gctggtggtg gtggacacaa ctgccatcac cagtacctgc agcagcagca cgccatggcg gcgcctatga tgatgatgca ctcctcctgc tgctccgccg acgggtcgtc gtcaaactcc gattccattt ccaactcctg ctcgtcaccg gtgaccacgg cggcctcgcc agcctacagc caccaccaga cgatgttcca gacacctgca ctgcagccgt catgcggcgc aatgacgatg gcggccgctg cgccgcatgt gcagggcttc cacgtcggcg acgacgacac taccaccgcg atggcgatgc accgtcatca gcagatgatg cgcgagctgg cggaggcgcc tctgcaccag gaggcagacg acttcgagga cttcgtgacg cggctgccca aggcggagga cttcggcctg cagggcttcc aggaggtggc ccccgaggtg ttcgacgacg ccgccggcat ctgggaccac gcggccgcct gggagccccc caccatgatg atcgactctg gcgcccagcc ccagcagcag ctcgtcgtcc ctctttgact cgctcgtcga tgacgccgcg cgccctgcac cagctactgc ttcgttccca gctgcatcga actggccggt gtacgtggcg gagtgatacg acgcgcgcgc tatgcatgac accactgcac aggtggttct tgcatgtgtt gcttacgcct cgagacgtac gtacataata ccagtatgta tgtaccggat ggttactctg atatgactgt atttctccta gttattcgtg ggtttcattt ggataatgtt tcaggttttg taaaatatat actttagtag

tagtggtgtc ttaaatatat gctcctagct atatatctag tctctgtgtg gtatatgcat ggccgctagt tagcttgtac aatattacca tatatagata tattaatttc gcttttacta aata

SEQ ID NO. 2 ZmDREB2.7 protein Zea mays

Met Asp Arg Val Pro Pro Pro Val Ser Met Gin Val Ala Ala Met Gin

Arg His Gin Gin Gin Gin Gin Phe Val His His Leu Gin Gin Val His

Gin Gin Gly Thr Gin His Glu Gin Pro Pro Pro Pro His Gin Asn Gly Ser Ser Ser Ser Gly Arg Thr Gly Gly Gly Arg Lys Cys Cys Pro Leu

Arg Arg Ser Arg Lys Gly Cys Met Lys Gly Lys Gly Gly Pro Asp Asn

Gin Gin Cys Pro Phe Arg Gly Val Arg Gin Arg Thr Tip Gly Lys Trp

Val Ala Glu He Arg Glu Pro Asn Arg Gly Ala Arg Leu Trp Leu Gly

Thr Phe Gly Ser Ala Leu Glu Ala Ala Arg Ala Tyr Asp Ala Ala Ala

Arg Thr Leu Tyr Gly Asp Cys Ala Arg Leu Asn Leu Gin Leu Val Pro

Pro Ser Ala Ala Ala Ala Ala Ala Gly Gly Gly Gly Pro Ala Val Val

Ala Ser Pro Ser Pro Asp Thr Val Ala Gly Pro Ala Ala Ala Ala Gly

Gly Gly Gly His Asn Cys His His Gin Tyr Leu Gin Gin Gin His Ala

Met Ala Ala Pro Met Met Met Met His Ser Ser Cys Cys Ser Ala Asp

Gly Ser Ser Ser Asn Ser Asp Ser He Ser Asn Ser Cys Ser Ser Pro

Val Thr Thr Ala Ala Ser Pro Ala Tyr Ser His His Gin Thr Met Phe

Gin Thr Pro Ala Leu Gin Pro Ser Cys Gly Ala Met Thr Met Ala Ala

Ala Ala Pro His Val Gin Gly Phe His Val Gly Asp Asp Asp Thr Thr

Thr Ala Met Ala Met His Arg His Gin Gin Met Met Arg Glu Leu Ala

Glu Ala Pro Leu His Gin Glu Ala Asp Asp Phe Glu Asp Phe Val Thr

Arg Leu Pro Lys Ala Glu Asp Phe Gly Leu Gin Gly Phe Gin Glu Val

Ala Pro Glu Val Phe Asp Asp Ala Ala Gly lie Trp Asp His Ala Ala

Ala Trp Glu Pro Pro Thr Met Met He Asp Ser Gly Ala Gin Pro Gin

Gin Gin Leu Val Val Pro Leu

SEQ ID NO. \9ZmDREB2. 7 promoter sequence which comprises nucleotides at positionso 798 position of SEQ ID NO. 1 tagataggcagccc atggccgtgg ctgcatctgc gggctgggcg ggctgcatca gcgtgacgcc gtgacctcac cctggttcgg tcgccccccg gccgccacgt ggcccagcgg ccatgacgtg gaccccacag gggcttccat gtgtcaagcc ccgctggccc ccaccacttc gtgtcacccg cctccttcac ttggcgtgcc gcacccccac gcgtggcccc acgcccaggc cccgcatccc tacacggagg cgtcatgcag tgccatgcgc cggcttcccc cctgccccct ccgtccgccc gccttcattc cgcacaccac cgaaaactgg tgcccggcct gcagtgcagt gcaagccatg ccagctgcct atatatacca ggccagggag cgggagcctc acacacagtc acagcacacg cagccaccga ggactgcatt gctagcatcc atcgccatca gtcgccatat cgatctgcgc acgaagctag tagtccag

SEQ ID NO. 20ZmDREB2. 7 promoter sequence positions 1 to 798 of SEQ ID NO. 1 atgcatgcct actgtcacac aaaaaataca gtatattatt actgatggag aaaacaagga aggatgggcc ggatggggat ggccgagggc aaagccgtcc gtcagtccgt ccttgttgcg tgtgcgtgca tacggagacc ggagtcagcg gtatgatgca ggcaagcaga ccattgcaca cgcagataca gatcccagcc gagcgtccag ctgccaagcc atgcatgtgg ctcgcggatc ggcgcagtcc atggatagat ggagatggat ccatccatgg atagatcata gatagataga taggcagccc atggccgtgg ctgcatctgc gggctgggcg ggctgcatca gcgtgacgcc gtgacctcac cctggttcgg tcgccccccg gccgccacgt ggcccagcgg ccatgacgtg gaccccacag gggcttccat gtgtcaagcc ccgctggccc ccaccacttc gtgtcacccg cctccttcac ttggcgtgcc gcacccccac gcgtggcccc acgcccaggc cccgcatccc tacacggagg cgtcatgcag tgccatgcgc cggcttcccc cctgccccct ccgtccgccc gccttcattc cgcacaccac cgaaaactgg tgcccggcct gcagtgcagt gcaagccatg ccagctgcct atatatacca ggccagggag cgggagcctc acacacagtc acagcacacg cagccaccga ggactgcatt gctagcatcc atcgccatca gtcgccatat cgatctgcgc acgaagctag tagtccag

SEQ ID NO. 22ZmDREB2.7 promoter sequence from the drought tolerant inbred line CIMBL70.

GGCTCGCGGA TCGGCGCAGT CCATGGATAG ATGGAGATGG ATCCATCCAT GGATAGATCA

TAGATAGATA GATAGGCAGC CCATGGCCGT GGCTGCATCT GCGGGCTGGG CGGGCTGCAT

CAGCGTGACG CCGTGACCTC ACCCTGGTTC GGTCGCCCCC CGGCCGCCAC GTGGCCCAGC

GGCCACGACG TGGACCCCAC AGGGGCTTCC ATGTGTCAAG CCCCGCTGGC CCCCACCACT

TCGTGTCACC CGCCTCCTTC ACTTGGCGTG CCGCACCCCC ACGCGTGGCC CCACGCCCAG

GCCCCGCCTC CCTACACGGA GGCGTCATGC AGTGCCATGC GCCGGCTTCC CCCCTGCCCC

CTCCGTCCGC CCGCCTTCAT TCAGCTTCCG GCTTCCGCTG TTCCGCACAC CACCGAAAAC

TGGTGCACGG CCTGCAGTGC AGTGCATGCC ATGCCAGCTG CCTATATATA CCAGGCCAGG

GAGCGGGAGC CTCACACACA GTCACAGACT CACAGCACAC GCAGCCACCG AGGACTGCAT

TGCTAGCATC GTCCATCGCC ATCAGTCGCC ATATCTCGAT CTGC

Claims

CLAIMS What is claimed is:

1. A method for identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1.

2. A method according to claim 1 wherein identifying the haplotype comprises using PCR.

3. A method according to claim 2 wherein said PCR comprises using a primer pair comprising SEQ ID NO. 3 and SEQ ID NO. 4 and/or a primer pair comprising SEQ ID NO. 5 and SEQ ID NO. 6.

4. A primer pair comprising SEQ ID NO. 3 and SEQ ID NO. 4.

5. A primer pair comprising SEQ ID NO. 5 and SEQ ID NO. 6.

6. A kit for identifying and/or selecting a maize plant with enhanced drought resistance comprising a primer pair comprising SEQ ID NO. 3 and SEQ ID NO. 4 and/or a primer pair comprising SEQ ID NO. 5 and SEQ ID NO. 6.

7. A method for identifying and/or selecting a maize plant with enhanced drought resistance comprising detecting in the maize plant a haplotype said method comprising

a. obtaining a first maize plant that comprises within its genome a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1 ;

b. crossing said first maize plant to a second maize plant;

c. evaluating the progeny plants and

d. identifying and/or selecting a maize plant with enhanced drought resistance that comprises said haplotype.

8. A method according to claim 7 wherein identifying the haplotype comprises using PCR.

9. A method according to claim 7 wherein said PCR comprises using a primer pair comprising SEQ ID NO. 3 and SEQ ID NO. 4 or a primer pair comprising SEQ ID NO. 5 and SEQ ID NO. 6.

10. A method for producing a hybrid maize plant with enhanced drought resistance the method comprising

c. crossing the first plant with the second plant to produce an Fl generation;

d. identifying one or more membersof the Fl generation that comprises the desired phenotype comprising said haplotype.

11. A maize plant identified and/or selected by a method according to any of claims 1-10.

12. A recombined DNA segment comprising a 5' UTR ZmDREB2. 7 allele from maize which comprises a haplotype comprising a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1, a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1.

13. The DNA segment of claim 12 further defined as comprised within a cell.

14. The DNA segment of claim 12 further defined as comprised within a seed.

15. The DNA segment of claim 12 further defined as comprised within a plant.

16. An isolated nucleic sequence comprising or consisting of:

1) a ZmDREB2.7 promoter sequence which comprises nucleotides at positions 296 to 798 position of SEQ ID NO. 1 (SEQ ID NO. 19);

2) a ZmDREB2.7 promoter sequence positions 1 to 798 of SEQ ID NO. 1 (SEQ ID NO.

20);

3) a ZmDREB2. 7 promoter sequence positions as defined in SEQ ID NO. 22; 4) nucleic acid sequences which exhibit at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% and at least 99% homology with the nucleic acid sequences defined in 1) or 2);

5) nucleic acid sequences which hybridize to the nucleic acid sequences defined in 1), 2), 3) or 4) under strict conditions.

17. A vector comprising an isolated nucleic sequence according to claim 16.

18. A host cell comprising a vector according to claim 17 or an isolated nucleic sequence according to claim 16.

19. The use of a vector according to claim 17 or an isolated nucleic sequence according to claim 16 in conferring drought resistance.

20. A method for conferring drought resistance to a plant comprising introducing and expressing in said plant a vector according to claim 17 or an isolated nucleic sequence according to claim 16.

21. A method for increasing drought resistance of a maize plant compared to a control plant comprising the steps of

a. targeted mutagenesis of a plant population to introduce the following mutations in the 5' UTR of ZmDREB2. 7: a A nucleotide at the position that corresponds to position 296 of SEQ ID NO. 1, a C nucleotide at the position that corresponds to position 539 of SEQ ID NO. 1, the absence of a deletion between positions that correspond to positions 614 and 615 of SEQ ID NO. 1, a G nucleotide at the position that corresponds to position 646 of SEQ ID NO. 1 and a nucleotide G at the position that corresponds to position 649 of SEQ ID NO. 1

b. identifying and selecting plants which comprise the desired mutations.

22. A plant obtained or obtainable by the method of claim 21.