WO2013026085A1 - Disease resistant plant breeding method - Google Patents

Abstract

A method of plant breeding is provided which includes crossing one or more plants to produce a progeny plant, wherein at least one of the plants has been selected as having at least partial adult stage resistance to one or more plant diseases in response to exposure to a disease-causing organism or pathogen. The plant may be grown in a controlled environment which modulates plant growth and development, including factors such as (i) timing of exposure to the pathogen, (ii) photoperiod; (iii) temperature; (iv) humidity and/or (v) carbon dioxide. Identifying and selecting the plants as resistant to the plant disease and subsequent crossing occurs within a single generation, whereby multiple cycles occur within a year. Also provided is a method of identifying genetic markers associated with disease resistance. A preferred adult stage resistance is adult plant resistance. Preferred plants are crop plants such as cereals and legumes.

Description

TITLE

DISEASE RESISTANT PLANT BREEDING METHOD

FIELD

THIS INVENTION relates to plant breeding. More particularly, this invention relates to rapid selection and breeding of plant cultivars that display enhanced disease resistance.

BACKGROUND

Plant disease-causing organisms such as fungi, nematodes, bacteria and insects can cause extensive damage to commercial crops including cereals and legumes, although without limitation thereto.

In cereals, rusts are a particular problem. For example, stripe rust or yellow rust (YR) caused by the fungus Puccinia striiformis f. sp. tritici (Pst), is an important disease of wheat (Triticum aestivum L.). YR causes regular crop losses ranging from 0.1 to 5% with rare events accounting for much higher losses (Wellings 2011). Severe epidemics between 1984 and 1987 caused losses of up to 84% to wheat crops in southern New South Wales, Australia (Murray et al. 1994). Nevertheless, sustainable protection against rust pathogens can be achieved by deploying diverse resistance genes in new cultivars (Hong and Singh 1996). Other rusts include wheat stem rust (SR) and leaf rust (LR) which are also caused by Pucciniamycete fungi.

In the case of rusts and other plant pathogens, adult plant resistance (APR) is best expressed by at the adult plant stage of development and is conditioned by the additive and/or epistatic effects of multiple genes often conferring low levels of resistance (partial resistance) individually. Therefore, APR is thought to provide more durable resistance than seedling (i.e. major gene) resistance, which is often pathotype specific and may be overcome by mutations within the pathogen population (Mcintosh 1992; Hong and Singh 1996; Lagudah et al. 2006; Pretonus et al. 2007). However, selection for APR in breeding programs is typically conducted in field screening nurseries, where the expression of APR may be influenced by factors such as weather patterns, inoculum load and sequential infection, or by other plant characteristics such as maturity and the presence of other diseases. Furthermore, such breeding programmes can take many years (e.g. 10-20) to produce disease-resistant cultivars due to the fact that crossing and selection of resistant breeding plants requires the plants to reach maturity before disease resistance becomes apparent and selectable. For example, APR is typically assessed post-anthesis.

SUMMARY

The present invention recognizes the need to more efficiently develop, produce or breed disease-resistant plant cultivars by selective breeding. Accordingly, the present invention provides a controlled environment which modulates plant growth and development such that identification and selection of plants displaying enhanced disease resistance, particularly adult plant resistance (APR), before the plant reproduces or ceases being capable of reproduction (e.g. before the conclusion of anthesis) or is otherwise used for subsequent breeding. This is of particular relevance to plants genetically segregating for disease resistance, where identification of disease resistance, selection and breeding can occur in the same generation. This means that iterative cycles of identification, selection and breeding for disease resistance can occur in a relatively "compressed" timeframe, thereby decreasing the total time taken to develop new, disease-resistant plant varieties. In a preferred embodiment, the present invention provides use of a controlled environment which accelerates plant growth and development and/or facilitates the display or expression of disease resistance by the plant.

In one aspect, there is provided a method of plant breeding including the step of crossing one or more plants to produce one or more progeny plants, wherein at least one of the plants has been selected as at least partly resistant to one or more plant diseases in response to exposure of the plant to a disease-causing organism or pathogen.

Preferably, after exposure to the disease-causing organism or pathogen the plant is grown in a controlled environment which modulates plant growth and development and/or facilitates the expression of disease resistance.

In a preferred embodiment, after exposure to the disease-causing organism or pathogen the plant is grown in a controlled environment which accelerates plant growth and development and/or facilitates the expression of disease resistance.

Suitably, the progeny plant has or displays at least partial resistance to said disease. Preferably, the progeny plant has greater or enhanced resistance to said plant disease and/or said organism or pathogen compared to one or both of said one or more selected plants used for crossing.

Preferably, the method further includes identifying one or more at least partly disease-resistant plants as resistant after exposure of the one or more plants to the disease-causing organism or pathogen and growth of the one or more plants in a controlled environment which modulatesplant growth and development and/or facilitates the expression of disease resistance, thereby facilitating identification of the disease-resistant plants.

In a preferred embodiment, identifying one or more at least partly disease- resistant plants occurs after growth in a controlled environment which accelerates plant growth and development and or facilitates the expression of disease resistance.

Suitably, the controlled environment provides an artificial environment for plant growth and includes controllable factors such as timing of exposure to the disease-causing pathogen or organism, photoperiod, temperature, humidity and carbon dioxide (CO₂) levels. The controlled environment may be provided before and/or after exposure to the disease-causing pathogen. Suitably, the controlled environment accelerates plant growth and development and/or facilitates the expression of adult stage disease resistance, such as manifested by adult plant resistance (APR), compared to that which would occur under normal field conditions. Suitably, the adult stage disease resistance (e.g. APR) is different to that observed or manifested at seedling stages.

Suitably, identification and/or selection of the one or more plants occurs before the plant reproduces or ceases being capable of reproduction.

Preferably, the one or more plants have a single reproductive phase in its lifetime.

In a preferred embodiment, selection of, and crossing, the one or more plants occurs in the same generation.

Preferably, the one or more plants are genetically segregating for disease resistance.

In one embodiment, each of the one or more plants are Fi hybrids, F₂, F3 or F„ plants, wherein n is the number of generations since the initial cross. In an alternative embodiment, at least one of the one or more plants used for crossing having fixed or non-segregating genotypes, such as a parental plant (i.e a back-cross) or other plant.

Suitably, the one or more plants are crop plants. Preferably, the crop plant is a cereal or legume.

Suitably, the disease is caused by a disease-causing organism or pathogen of plants such as a fungus, worm, protozoan, virus or bacterium. In one particular embodiment, the disease is caused by a fungus, inclusive of Pucciniamycetes that infest wheat, barley and rye stems, leaves and grains, although without limitation thereto.

In one particular embodiment, plants resistant to a plurality of different diseases may be identified or selected for breeding. A non-limiting example is identification and selection of wheat plants resistant to leaf rust (LR), stem rust (SR) and stripe rust (YR).

In another aspect, the invention provides a progeny plant produced or bred by the method of the aforementioned aspect.

In yet another aspect, here is provided a method of identifying a genetic marker associated with plant disease resistance including the step of identifying one or more genetic markers in a genome of one or more plants that are associated with, or linked to, disease resistance after exposure of the one or more plants to a disease pathogen and growth of the one or more plants in a controlled environment which modulates plant growth and development.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES

Reference is made to the following Figures which assist in understanding non-limiting embodiments of the invention described in detail herein wherein:

Figure 1 shows seedling stripe rust responses of five wheat lines used as controls, when tested against Pst pt. 134 El 6 A+. Disease developed at constant 11°C; Figure 2 shows adult stripe rust responses on leaves of 'EGA Gregory' infected with Pst pt. 134 El 6 A+. Disease developed at post-inoculation temperature of 23/11°C (day/night) and 11°C constant. Disease response on leaf 3 is displayed for plants grown in a temperature controlled glasshouse (22 ± 2°C) under continuous light. The pair of leaves on the right demonstrates increased resistance when disease developed at 11 °C;

Figure 3 shows mean disease response and confidence intervals (95%) for wheat lines used as controls and assessed for APR to stripe rust while screening the F₂ populations in the field and under controlled environmental conditions. Confidence intervals were derived from separate analyses of experiments for each environment;

Figure 4 shows comparative responses to Pst pt. 134 El 6 A+ of wheat lines used as controls for pairs of leaves (leaf 3) from adult plants grown under controlled environmental conditions (i.e. constant light and temperature);

Figure 5 shows the distribution of disease responses of F₂ individuals from crosses 'Wyalkatchem' ^χ 'ST93' and Ή45' ^χ 'ST93' assessed for APR to stripe rust under controlled environmental conditions and in the field. The mean disease response for parents are indicated by arrows;

Figure 6 shows disease response for F₃ progeny from two crosses; 'Wyalkatchem' 'ST93' (red) and Ή45' * 'ST93' (black), assessed for APR to stripe rust in the field at Gatton in 2010, plotted against disease response of individual F₂ plants assessed for APR to stripe rust in the field at Warwick in 2009;

Figure 7 described the observed combinations of disease resistance and susceptibility to the three rust pathogens in wheat; stripe rust (YR), stem rust (SR) and leaf rust (LR), observed on the same leaf of an individual plant. L-R: 1) susceptible to YR and resistant LR and SR, 2) susceptible to LR and resistant to SR,

3) susceptible to SR and resistant to LR, and 4) resistant to both SR and LR;

Figure 8 demonstrates a full range in disease response to stem rust observed in the experiment: ranging from resistant (left) to very susceptible (right);

Figure 9 shows the distribution of disease response to the three rusts (stripe, stem and leaf rust) for an H45 derived population assessed under controlled environmental conditions; and Figure 10 shows the distribution of disease responses to the three rusts (stripe, stem and leaf rust) for a Wyalkatchem derived population assessed under controlled environmental conditions. DETAILED DESCRIPTION

The present invention has arisen, at least in part, from the discovery that relatively early display and assessment of adult stage disease resistance phenotypes before the reproductive phase allows cross-breeding within the same plant generation. In particular, this is facilitated by the use of controlled environmental conditions (CEC), incorporating variable inoculation timing, controlled light, temperature, humidity and/or carbon dioxide (CO₂) levels that may provide more uniform conditions for modulating the expression and assessment of adult stage resistance (e.g. APR) and/or other heritable modes of adult resistance, that may also include elements of seedling resistance or other genes that influence or modify expression of resistance genes. In some embodiments, use of an extended photoperiod accelerates plant development and so plants will attain the adult plant reproductive phase sooner, further assisting rapid assessment of disease resistance. Disease screening will thereby permit year-round assessment and breeding, which is expected to greatly shorten the typical time period required for breeding new plant cultivars with disease resistance. This provides rapid and accurate pheriotyping of fixed plant lines and an oppurtunity to enrich genetically segregating populations for resistance to diseases caused by plant pathogens during progress to development of inbred lines.

In one particular example relating to wheat rusts, identification of rust- resistant plants can be completed within only five weeks from time of sowing, and permits year-round phenotyping, assessment and selection (up to 10 consecutive cycles per year).

In one aspect, there is provided a method of plant breeding including the step of crossing one or more plants to produce one or more progeny plants, wherein at least one of the plants has been selected as resistant to one or more plant diseases after exposure to a disease-causing pathogen, preferably in a controlled environment which modulates plant growth and development and/or facilitates the expression of disease resistance in plants. In another aspect, the invention provides a progeny plant produced or bred by the method of the aforementioned aspect.

Suitably, the progeny plant has or displays at least partial resistance to said disease.

Preferably, the progeny plant has greater or enhanced resistance to said plant disease and/or said organism or pathogen compared to one or both of said one or more selected plants used for crossing.

Preferably, the method includes identifying one or more disease-resistant plants resistant to said plant disease after exposure of the one or more plants to the disease-causing pathogen, and preferably after growth of the one or more plants in a controlled environment which modulates plant growth and development and/or facilitates the expression of disease resistance in plants.

As used herein, plants suitably include any crop plant of agronomic importance that is susceptible to one or more disease-causing organisms or pathogens. Non-limiting examples include monocot crop plants of the Poaceae (formerly known as Gramineae) family which include grasses such as cereals and sugarcane; other monocot crop plants such as pineapple, banana and onion; legumes; fruit crops; cucurbits; root vegetables such as potatoes, carrots and beet; crop plants of the Brassicaceae family (e.g. cabbage, cauliflower, broccoli); crop plants of the Solanaceae family including Solarium spp such as potato, tomato, eggplant, Capsicum spp and Nicotania spp; and oil-producing crop plants such as sunflower, canola, rapeseed and field mustard.

In particular embodiments, the crop plants are cereals such as wheat, barley, maize, oats, sorghum, millet, rye and rice, although without limitation thereto.

In other particular embodiments, the crop plants are legumes such as soybean, alfalfa, clover, beans, lentils, garden pea, pulses (e.g. cowpea, pigeonpea, chickpea), lupins, mesquite, and peanuts, although without limitation thereto.

Plants may be annuals or perennials.

In certain embodiments, it may be advantageous that the plant has a single, relatively short and/or defined reproductive phase, an example of which would be a cereal such as wheat which typically dies after a single reproductive phase during its lifetime In other embodiments, the plant may have a longer reproductive phase that may be characterized by repeated, successive or continuous flowering.

By "resistanf or "resistance" in the context of disease-resistant plants is meant that the plant displays relatively reduced sensitivity or susceptibility to the detrimental effect(s) of a causative organism or pathogen, displays relatively reduced or ameliorated symptoms of the disease caused by the organism or pathogen, and/or possesses or expresses one or more genes that confer, underlie or contribute to at least partial resistance to the disease, compared to a plant that is more disease susceptible. Resistance may be manifested by relatively reduced or ameliorated levels of pathogen infestation, tissue damage (e.g stem, leaf or root damage, wilting, chlorosis, necrosis etc), compromised or suppressed growth and development and/or inhibition or suppression of metabolic processes (e.g nitrogen fixation, photosynthesis, carbon fixation etc), although without limitation thereto. Assessment of disease resistance may be performed by methods well known to persons skilled in the art and such as those described hereinafter in the Examples. Suitably, disease resistance is in a form that is optimally, preferentially or maximally expressed or displayed at an adult stage of plant development. Adult stage resistance is distinguishable from seedling resistance, which is manifested or displayed by plant seedlings and typically, although not exclusively, is controlled by a single gene that provides or underlies the resistance, although adult stage resistance may include elements of seedling resistance (such as expression of one or more genes associated with seedling resistance).

In a preferred embodiment, adult stage resistance is "adult plant resistance" (APR), which is any form of disease resistance which is optimally or preferentially manifested, expressed and/or examined at the adult plant stage of development rather than at seedling stage, typically conditioned by the additive and/or epistatic effects of each of multiple genes that may each confer low levels of resistance (e.g. partial resistance) individually. In this context, "adult plant resistance", also includes "slow rusting" or "partial resistance".

Disease-causing organisms and pathogens include fungi, worms, insects, bacteria, viruses and protozoa. Disease-causing pathogens may also include different pathotypes of the same pathogen. 3

Viruses may include those of the Potyviridae family, Bromoviridae family, Geminiviridae family inclusive of tomato yellow leaf curl virus and the Luteoviridae family inclusive of barley yellow dwarf virus, although without limitation thereto.

Fungi may include ascomycete and basidiomycte phytopathogens. The fungi may be biotrophic fungi or necrotrophic fungi. Non-limiting examples of fungi are Pucciniamycetes that infest wheat, barley and rye stems, leaves and grains. In particular embodiments relating to cereals such as wheat and barley, the pathogens are biotrophic fungi which are the causative agents of cereal rusts. These include fungi such as Puccinia striiformis f. sp. tritici (Pst) which causes yellow or stripe rust in wheat, Puccinia graminis which causes stem rust in wheat, Puccinia triticina which causes leaf rust in wheat and Puccinia hordeii which causes leaf rust in barley, although without limitation thereto. Another fungus is Pyrenophora such as Pyrenophora teres f. teres which is a necrotrophic fungus that causes net blotch in barley.

Other non-limiting examples of fungi include Fusarium spp. such as

Fusarium graminearum and Fusarium oxysporum, although without limitation thereto.

In particular embodiments relating to worms, the disease-causing organisms include nematodes such as Heterodera, Meloidogyne, Globodera and Belonolaimus spp. In plant species, most of economic damage is caused by the sedentary endoparasitic nematodes of the family Heteroderidae. This family includes the cyst nematodes (Heterodera and Globodera) and the root-knot nematodes (Meloidogyne) which infect thousands of plant species and cause severe losses in yield of many crop plants. Legumes such as soybean are particularly susceptible to Heterodera nematodes.

In other particular embodiments, nematodes may be pathogens of cereals, inclusive of cereal cyst nematode (also known as cereal root eelworm or oat cyst nematode) or pathogens of cereals and/or other crop plants such as root-lesion nematodes (Pratylenchus spp such as Pratylenchus thornei and Pratylenchus neglectus).

In certain embodiments, resistance to more than one disease-causing organism or pathogen (i.e. a plurality of disease-causing organisms or pathogens) may be identified and/or selected according to the invention. In this regard, it will also be appreciated that the plurality of pathogens may be of different types (e.g. virus, fungus) or of different species within a particular pathogen type (e.g. different fungus species and/or different virus species). A non-limiting example applicable to cereals such as wheat is the identification, selection and breeding of wheat plants resistant to leaf rust (LR), stem rust (SR) and stripe rust (YR).

A preferred feature of the invention is growth of the plant in a controlled environment which modulates plant growth and development. The controlled environment may be referred to herein as "controlled environment conditions", abbreviated to "CEC".

While in some embodiments it may be advantageous to accelerate plant growth and development to thereby accelerate identification, selection and/or crossing for disease resistance, in some alternative embodiments it may be advantageous to at least temporarily slow down or decelerate plant growth and development to facilitate identification, selection and/or crossing.

The controlled environment provides an artificial environment for plant growth and includes controllable factors such as timing of exposure to the pathogen, photoperiod (e.g. constant 24 hr light or diurnal 12 hr photoperiod), temperature (e.g. constant or variable such as diurnally variable), humidity and/or carbon dioxide (CO₂) levels. The controlled environment may be provided pre- and/or post- inoculation. Suitably, the controlled environment accelerates plant growth and development, such as manifested by APR or other forms of adult stage disease resistance, compared to that which would occur under normal field conditions.

It will be appreciated that suitably one or plurality of plants are initially exposed to the pathogen at an early stage in plant growth prior to identifying one or more disease-resistant plants. Timing of exposure to the disease-causing organism or pathogen (e.g. by inoculation) is generally an important, controllable factor in identifying adult stage disease resistance, such as APR.

Suitably, exposure to a disease-causing organism or pathogen such as by inoculation, will depend upon the plant type, the plant tissue affected by the pathogen and the pathogen itself, which can be determined by persons skilled in the art. This will determine the timing of exposure and the part or site of the plant (e.g. leaves, stem etc.) exposed to, or inoculated with, the organism or pathogen. 12 000203

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Suitably, inoculation of a plant with disease-causing organism or pathogen occurs before anthesis. This may occur at a seedling stage, late tillering or stem elongation stages of plant development. In embodiments relating to cereals such as wheat or barley under extended photoperiod conditions, this is typically between 2 and 6 weeks of age. In alternative embodiments under ambient light conditions, this may be between 5-10 weeks of age.

In embodiments relating to cereals, the timing of exposure to a disease- causing organism or pathogen may be expressed in terms of Zadoks growth stage (GS). Suitably, exposure occurs before anthesis, which is Zadoks stages 60-69.

For example, in embodiments relating to stripe rust in wheat, APR was observed in 3 -week-old plants (i.e. after sowing) following inoculation of leaves at the late tillering to stem elongation phase (Zadoks growth stages 21-31). In other embodiments, selection for APR to stripe rust, leaf rust and stem rust in wheat was possible through assessment of stripe rust on leaves 3-4, leaf rust on flag-1 and flag leaf (Zadoks growth stages 11-19), and stem rust on the elongated stem (Zadoks growth stages 30-39).

In embodiments relating to barley, moderate levels of APR (such as to net blotch) can be detected by inoculating barley plants grown for 3 weeks (21 days) under a continuous (constant) light photoperiod and controlled temperature (15°C) at the Zadoks growth stage 32 (i.e. second node can be detected). If inoculated at this growth stage, it would be possible to cross barley plants. However, wheat plants inoculated slightly earlier in development (i.e. at late tillering growth stages = Zadoks stages 21-22 and onwards) express APR to stripe rust (see Table 3). Thus, it may be preferable to inoculate barley plants several days earlier (e.g. 2.5 weeks, at the late tillering stage) rather than 3 weeks, to allow even more time for ease of crossing the selected plants. Net blotch in barley was assessed only 9 days post- inoculation, which saves some time compared to stripe rust in wheat, which was assessed 14 days post-inoculation.

Typically, provision of an appropriate temperature post-inoculation is advantageous for accelerating disease resistance and identification of disease- resistant plants. Temperature may be any temperature in the range 10°C-35°C.

The temperature may be a constant temperature or a variable temperature. A preferred variable temperature varies diumally. Constant temperature would typically be at about 11°C-32°C, 16°C-32°C, inclusive of 18°C-30°C, 20°C-28°C and 22°C-25°C.

Diurnal temperatures would typically vary from a minimum of about 10°C-20°C to a maximum of about 18°C-30°C.

Examples include about 8°C-22°C, 10°C-20°C, 12°C-18°C, 14°C-16°C or about 16°C-30°C, inclusive of the ranges referred to above.

In embodiments relating to stripe rust in wheat, a diurnal (i.e night/day) temperature range of about 11°C-22°C or 12°C-18°C was particularly advantageous. In other embodiments, a diurnally variable temperature of about 20°C-22°C (night- day) may assist development of stem rust and leaf rust.

In the context of the temperatures and temperature ranges described herein "abouP refers to the stated temperature or temperature range ±1 or 2°C.

Light may be provided at a constant level or may be varied, such as diurnally. A diurnal photoperiod would typically comprise no light overnight (e.g for about 12 hrs) and a low level of light during daylight hours to supplement natural light. In some embodiments, an extended photoperiod is utilized where light is provided for a period longer than would normally occur under normal, seasonal conditions in the field. Extended photoperiods would typically be greater than 12 hrs per 24 hr period and may include constant light for the duration of plant growth under CEC. Growth under extended photoperiod conditions may be particularly advantageous for inducing rust resistance in cereals.

It will be appreciated that use of extended light photoperiods assists accelerating plant development, thus identification of disease-resistant plants, selection and crossing can optimally be completed within 4-5 weeks. However, in an alternative less preferred embodiment identification of disease-resistant plants, selection and crossing of the selected plants could also be conducted in a regular glasshouse without the use of controlled lighting (i.e. using ambient light), but would take longer to do so (e.g. 8-9 weeks).

In other embodiments, humidity may be controlled to achieve controlled environment conditions. Typically, elevated humidity may be provided as part of controlled environment conditions. Elevated humidity may be at least 70%, 80%, 90%, 95% or up to 100% relative humidity. By way of example, it may be advantageous to maintain cereal plants in high humidity (e.g. up to 100% relative humidity) for a period after inoculation with rust pathogens to facilitate inoculation and growth of the pathogen.

In still further embodiments, the level of CO2 may be controlled. Typically, the level of CO2 in air is about 0.04% or 400 ppm by volume. The invention contemplates providing elevated CO2, preferably at a level 1.2-5.0 times that normally present in air, which may accelerate photosynthesis and thereby accelerate plant growth and induction of disease resistance. In particular embodiments, elevated C0₂ is preferably at a level 1.5, 1.8, 2.0, 2.2, 2.5, 3.0, 3.5, 4.0 or 4.5 times the level of CO2 normally present in air.

It will be appreciated that the identification of disease-resistant plants described herein is suitably for the purpose of subsequent plant breeding from the disease-resistant plants.

In addition, one or more additional plant culture factors or conditions can be used to modulate plant development. Non-limiting examples include pruning or physically removing parts of the plant, use of fertilizer treatments, particular watering regimes or the like. In a preferred embodiment, such treatments may be used to extend the reproductive phase of plants for ease of crossing the selected disease-resistant plants.

Suitably, selection of the plant(s) identified as disease-resistant occurs before the plant reproduces or ceases being capable of reproduction.

In this context, "capable of reproduction^'1'' means that the plant is in a period of its reproductive phase characterized by anthesis including the production of flowers or seeds that enable sexual reproduction. In some embodiments, a reproductive stem, or flowering portion thereof, may be pruned or otherwise physically removed and one or more new tillers used to produce reproductive material for subsequent breeding.

By identifying and selecting disease-resistant plants before the plant reproduces or ceases being capable of reproduction, the present invention allows both identification and selection of disease-resistant plants to occur within the same plant generation.

In a preferred embodiment, identification, selection and crossing of the one or more disease-resistant plants occurs in the same generation. Crossing one or more plants may include that the one or more plants is the the same plant or are different plants.

Suitably, the one or more plants are segregating with respect to one or more disease-resistance genes. Alternatively, at least one of the plants has a fixed or non- segregating genotype such as a parental plant (i.e a back-cross) or other plant.

For example, either or both of the first and second plants may be Fi hybrids, F2, F3 or F„ plants, wherein n is the number of generations since an initial cross. It will therefore be appreciated that the one or more plants may be the same plant (e.g. self-pollinating plants) or different plants (e.g cross-pollinating plants). It will also be apparent that in some embodiments where different plants are crossed (referred to herein as "first" and "second" plants), only the first or the second plant is segregating with respect to one or more disease-resistance genes. For example, it may be advantageous to back-cross a later generation, segregating plant (e.g. Fi, F2, F3 etc) with a "parental" plant having a fixed or stable genotype to ensure that a desired genetic trait of the parental plant is carried by the progeny of the backcross. In this regard, also contemplated is plant breeding where disease resistance and/or the desired genetic trait of the parental plant is provided and carried by a transgene, as is well understood on the art. By way of example only, desired genetic traits of the parental plant (either provided by endogenous genes or transgenes) could be flowering time, fruit ripening time, grain size, plant height, endosperm colour, milling ability, nitrogen utilization, fruit colour and/or water content, although without limitation thereto.

In certain embodiments, the method of plant breeding may be facilitated by marker-assisted selection (MAS) of plants for breeding. While phenotypic assessment of disease resistance may alone be sufficient to identify disease-resistant plants, analysis of genetic markers associated with disease resistance may further assist the identification and selection of disease-resistant plants. Appropriate genetic markers may be associated with, linked to, genes that underlie disease resistance. The genetic markers may be linked to, or associated with, endogenous genes or transgenes that underlie a particular trait.

In particular embodiments, genetic markers may be useful for determining or distinguishing between adult stage resistance (e.g APR) and seedling resistance in a plant. 12 000203

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By way of example only such genetic markers that assist MAS may be amplification fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), random amplification of polymorphic DNA (RAPD), simple sequence repeat (SSR), diversity array technology (DArT), short tandem repeat (STR), microsatellite and/or single nucleotide polymorphism (SNP) markers, although without limitation thereto.

In certain embodiments, this aspect may include the use of genetic markers that are linked to Quantitative Trait Loci (QTL). Many phenotypic traits (such as pathogen resistance) are not associated with, or linked to, one or a few "major" genes but multiple segregating genes that each have different and sometimes minor "quantitative" contributions to phenotypic variation.

For example, a single-step method for QTL marker-assisted plant breeding can be utilized. In such an approach, in the first few breeding cycles, markers linked to the trait of interest are identified by QTL mapping and this information in used in the same population. In this approach, pedigree structures are created from families that are created by crossing a number of parents (e.g three-way or four way crosses). Both phenotyping and genotyping is performed using molecular markers mapped the possible location of QTL of interest. This will identify genetic markers and alleles associated or linked to the desired trait. Once these favourable marker alleles are identified, the frequency of such alleles will be increased and response to marker assisted selection is estimated. Marker alleles associated with, or linked to pathogen resistance will be further used in subsequent cycles of plant selection and breeding according to the aforementioned aspects of the invention.

By way of example, MAS can be used in combination with phenotypic selection, using markers linked to genes such as Yrl8; a gene that confers APR to stripe rust in wheat and/or Rph20 a gene which confers APR to leaf rust (Puccinia hordei) in barley. A non-limiting example of analysis of the csLV34 marker linked to the Yrl8 gene associated with APR to rusts in wheat is provided hereinafter in the Examples.

It will also be appreciated that the invention may assist or facilitate the identification of genetic markers associated with or linked to disease resistance, particularly APR. The genetic markers may be hitherto unknown genetic markers or may be known genetic markers not yet associated with, or liked to, resistance to a particular disease.

Accordingly, there is provided a method of identifying a genetic marker associated with plant disease resistance including the step of identifying one or more genetic markers in a genome of a plant that are associated with disease resistance after exposure (e.g. by inoculation) of the plant to one or more disease pathogens and growth of the plant in a controlled environment which accelerates plant growth and development and or facilitates the expression of disease resistance.

Suitably, the controlled environment is as hereinbefore described.

One or more genetic markers may be identified by (i) obtaining a nucleic acid sample from a plant after growth under controlled environment conditions and; (ii) analysis of the nucleic acid sample to identify said one or more genetic markers.

By way of example new genetic markers identified according to this aspect may include AFLP, RFLP, RAPD, SSR, DArT, STR, microsatellite or single SNP markers, although without limitation thereto.

In certain embodiments, this aspect may include the identification of new genetic markers that are linked to, or associated with, Quantitative Trait Loci (QTL), as hereinbefore described.

In some embodiments, identification of genetic markers that are linked to QTLs may be achieved using bi-parental cross populations, a cross between two parents which have a contrasting phenotype for the trait of interest are developed. Commonly used populations are recombinant inbred lines, doubled haploids (DH), back-crosses and F₂ plants. Linkage between the phenotype and genetic markers is tested in bred populations in order to determine the position of the QTL.

It will be appreciated that the foregoing aspects of the invention provide rapid identification, selection and breeding of plants that display resistance to disease pathogens, thereby providing an opportunity to enrich segregating populations with pathogen resistance genes and, ultimately, rapid development of stable, plant cultivars. In some embodiments, identification and selection of disease-resistance plants can be completed within only a few weeks from time of sowing, thereby permitting multiple (e.g. at least two, three, four, five and up to ten), year-round iterative cycles of identification, selection and breeding. This may include identification, selection and breeding for resistance to a plurality of different disease pathogens.

In the particular context of cereals such as wheat and barley, breeding populations carry seedling resistance genes, which are considered less sustainable when deployed alone in cereal cultivars (as the pathogen can easily mutate to overcome the resistance). However, if these genes are combined, they may be as effective as APR. For example, the "triple rust screen" described herein facilitates the identification, selection and breeding of disease resistance gene combinations in plants displaying superior levels of resistance, which may be the effect of multiple APR genes or multiple seedling resistance genes and may also involve other regions of the genome that influence expression of resistance genes (i.e. genetic modifiers) or for example Sr2 in wheat, which enhances the effectiveness of other resistance genes.

So that preferred embodiments of the invention may be fully understood and put into practical effect, reference is made to the following non-limiting Examples.

EXAMPLES

The Examples described herein provide an illustration of the use of controlled environment conditions (CEC) to identify and select wheat plants resistant to stripe or yellow rust (Example 1) or a plurality of rusts including stripe (or yellow rust), leaf rust and stem rust (Example 2). It will be appreciated that these examples are illustrative of the invention and should not be read as limiting the invention to these particular plants, diseases or the controlled environment conditions (CEC) applicable thereto.

EXAMPLE 1

Establishment of controlled environment conditions using stripe rust in wheat INTRODUCTION

Stripe or yellow rust (YR) is a significant problem in wheat crops worldwide.

The deployment of adult plant resistance (APR) genes in wheat cultivars is considered to be a sustainable management strategy, as these genes often confer partial resistance which is non-race specific. Screening for APR typically involves assessment of adult plants in the field, where expression may be influenced by environmental factors. Here we report a high-throughput screening method for YR APR that can be used to assess breeding populations grown under controlled environmental conditions (CEC). Inoculation of 3-week-old wheat plants grown under constant light and temperature provided disease responses typical of adult plants. Two F₂ populations (Ή45' * 'ST93' and 'Wyalkatchem' 'ST93') segregating for APR were assessed under both CEC and field conditions. These populations showed similar variation in disease response and lines assessed in both environments attained similar rankings. Phenotypic screening using CEC and continuous light provides an opportunity to both phenotype fixed lines for APR and to perform selection in segregating populations to increase the frequency of alleles for APR, and so accelerate the development of new wheat cultivars with durable resistance. MATERIALS AND METHODS

Plant material

Initial work used five Australian spring wheat lines with known YR responses (Table 1) to examine the responses to YR and to investigate impacts of growth stage, temperature and their interactions under CEC. The Australian bread wheat line 'Ellison' contains the seedling resistance gene Yrl7 (Banana and Mcintosh 1993); however, recent mutations in the Australian YR population have led to virulence for this gene. Seedlings that possess Yrl7 typically express a hypersensitive disease response under higher temperatures i.e. 18-20°C, but under lower temperatures (12-15°C) most lines possessing Yrl7 show susceptible or near- susceptible reactions to 7r77-avirulent Pst pathotypes (Banana and Mcintosh 1994; Qamar et al. 2008). 'EGA Gregory' has very good resistance in the field, which is conferred by at least two minor genes for APR (one of which is YrlS) and a major gene, Yr33 (Zahravi et al. 2003). 'Janz' and 'Baxter' have weaker levels of APR, mostly due to the presence of Yrl8, and 'Janz' is known to carry additional genes for YR APR (Banana et al. 2010). 'H45' was selected as a susceptible standard because although it carries the seedling YR resistance gene Yr7, this gene is not effective against the Pst pathotype 134 El 6 A+ (WA pathotype) used in all experiments. In addition to these five controls ('Ellison', 'EGA Gregory', ' Janz', 'Baxter' and Ή45') four other lines were evaluated for YR APR under CEC and in the field. These included a YR APR donor selected from a 'Sunco' x 'Tasman' doubled haploid (DH) population (Banana et al. 2001) and referred to here as 'ST93', 'Wyalkatchem' (supplied by the Australian Winter Cereals Collection, Tamworth, NSW) and two lines from CIMMYT carrying YR APR (supplied by R. P. Singh) referred to here as ^♦RSY10' (BABAX/LR42/ BABAX*2/3/VIVITSI) and 'RSY5'

(KIRITATI//ATTILA*2/PASTOR).

Two F₂ populations segregating for YR APR were developed by crossing 'ST93' to both 'H45' and 'Wyalkatchem'. 'Wyalkatchem' is moderately susceptible to YR, and is genetically heterogeneous for the APR gene(s) Lr46/Yr29 that confer partial resistance (Loughman et al. 2008). 'ST93' carries four to six APR genes for YR resistance coupled with high levels of resistance to leaf rust (LR) and stem rust (SR) (Bariana et al. 2007). Each F₂ population comprised 800 to 1000 individuals.

Pathogen

Pst pathotype 134 E16 A+ was used in all experiments conducted under CEC and in the field. This pathotype is a foreign incursion (thought to be from the USA) and has caused widespread damage across wheat growing regions in Australia since 2002 (Wellings 2007). This pathotype has virulence for Yr7 (Ή45') and is avirulent for Yrl7 ('Ellison').

Growth stage and temperature experiments

The five lines (Table 1) were first assessed for response to YR in a repeated experiment conducted under CEC. This experiment combined three factors: 1) age at inoculation (two or three weeks after sowing), 2) photoperiod during plant development (constant 24 h light or diurnal 12 h day/night photoperiod), and 3) post- inoculation temperature (constant 11°C or 23/11°C day/night).

Each experiment consisted of eight 100-cell seedling trays (10 ^χ 10 cells, with dimensions of 30 x 35 cm) split across two sowing dates (one week apart) and the two photoperiod treatments. For each sowing, approximately 200 seeds of each line were imbibed in Petri dishes lined with filter papers (Whatman^® 90 mm) for 24 h and then placed in a refrigerator (4°C) for 48 h to promote synchronous germination. Petri dishes were then maintained at room temperature (approx. 24°C) for 24 h to initiate germination prior to transplanting into the seedling trays. Lines were arranged in a completely randomised design, with 20 replicate plants of each of the five lines per tray. Seedling trays were placed in a controlled temperature glasshouse (22 ± 2°C) fitted with low-pressure sodium lamps to supplement natural light levels and to extend the photoperiod to 24 h. Although the lamps provided constant light, the photosynthetically available radiation (PAR) varied according to a diurnal cycle and the natural light levels present during daylight hours. To manually impose a diurnal photoperiod (12 h day/night cycle) on half the trays within the same glasshouse, trays were moved into a blackout shelter each day at 6 pm for 12 h. All trays were positioned on the same bench in the glasshouse. The repeat experiment was conducted in an identical manner in the same glasshouse, two months after the first experiment. Just prior to inoculation, the developmental GS was recorded for each plant using the decimal code scoring system for wheat (Zadoks et al. 1974).

Disease screening

All plants were inoculated at the same time with a suspension of urediniospores (in light mineral oil at a rate of 0.005 g/ml), applied evenly using an air-brush. Trays were incubated at 10°C overnight in a dew chamber (100% relative humidity maintained with a fogger).

Post-inoculation, trays were moved to growth cabinets with more accurate temperature control (±1°C). The experiment was again divided adopting two temperature treatments: (i) constant 11°C, and (ii) 23/11°C (day/night). A diurnal (12 h) photoperiod was used for both growth chambers. Trays in the warm growth chamber (11/23°C) were assessed for disease 14 days post-inoculation, whereas plants in the cool growth chamber (11°C constant) were scored 21 days post- inoculation because disease development was slower at the low temperature. The disease response was scored on up to three separate leaves per plant using a modified 1-9 scale (1 = very susceptible and 9 = immune), where units of the scale reflect the amount of diseased tissue and infection type. Individual plants were also assigned an overall disease response, which was the average response observed for the two upper-most infected leaves. Screening populations for YR APR under CEC

Results from the temperature ^χ growth stage experiment (described above) were used to design a procedure to screen the two segregating populations for YR APR under CEC. F2 seed derived from the two susceptible x resistant (APR) crosses was divided into two equal portions, so that half could be sown in the glasshouse and the other half in the field. Approximately 400 grains from each cross ('Wyalkatchem' 'ST93' and Ή45' ^χ 'ST93') along with the parents and controls ('Ellison', 'ST93', 'RSY10', 'RSY5\ 'EGA Gregory', 'Janz', 'Baxter', 'Wyalkatchem' and Ή45') were germinated according to the procedure described above. Germinated grains (60 per control and parent plus 400 F2 grains per cross) were transplanted in 16 x 100-cell seedling trays, adopting a completely randomised design.

Plants were grown in a temperature-controlled glasshouse (22 ± 2°C), fitted with sodium vapour lamps to provide constant light (as described above). Trays were regularly rotated and re-positioned in the glasshouse in order to reduce any edge effects. All plants were inoculated three weeks after sowing with urediniospores suspended in light mineral oil using an air-brush (as described above). At the time of inoculation, plants ranged in Zadok's growth stage from 21 to 31 (tillering to stem elongation). Plants were incubated post-inoculation in a dew chamber overnight (10°C) and then placed in a temperature controlled growth cabinet at 23/11°C (day/night temperature) for two weeks for disease development. Individual plants were scored for APR using the same 1-9 scale and an average disease response was assigned to each plant (as described above). Field assessment

The remaining F_∑ seeds (approximately 400 per cross) along with seeds of the same set of nine lines and parents used as controls were germinated in seedlings trays and grown for two weeks before transplanting into a field experiment at the Hermitage Research Facility, Warwick, Queensland. Seedlings were transplanted into 2-m long, two-row plots with a 20-cm spacing within and between rows (i.e. 20 plants per plot). F2 plants of each cross and the controls were randomly allocated to plots, split between two planting bays. Each control was replicated three times. Disease spreader rows containing a mix of lines (Ή45', 'Petrie' and 'EGA Wylie') susceptible to YR and covering a range of maturities were planted between the two bays and around the perimeter of the plots. Inoculation of spreader rows was performed using an injection method, where pre-emergent leaves were injected with a water suspension of urediniospores. Multiple assessments in the field from the time of anthesis were made to confirm phenotypes for APR, particularly for F₂ individuals. The above 1-9 scale was used for scoring.

In 2010, F3 progeny derived from 202 F₂ plants (101 per cross) were assessed for YR APR in unreplicated hill plots (15-20 seeds per plot) in a disease screening nursery at the University of Queensland Research Station, Gatton, Queensland.

DNA analyses

Leaf tissue was sampled from five plants of the parents ('Wyalkatchem', Ή45' and 'ST93') plus standards 'Janz' and 'Westonia' that carry the b and a alleles at the csLV34 marker locus, respectively. Following assessment of the two F₂ populations under CEC, eight plants were selected from each phenotypic extreme (very susceptible and resistant phenotypes) for each cross, and disease-free leaf tissue (from the flag leaf) was sampled separately from each plant (i.e. total 32 plants). In the field trial, leaf tissue samples were collected from all F₂ plants prior to disease infection. After final disease readings, eight F₂ plants were selected per phenotypic class (i.e. plants scored as 1 , 2, 3...9 for YR resistance) for each cross.

Genomic DNA was extracted from the leaf samples using a modified CTAB technique (Rogers and Bendich 1985). F2 DNA samples were then pooled according to phenotypic classes (eight samples per phenotypic class per cross). Equal concentrations of DNA from each F₂ plant were pooled to ensure equal allelic contributions from each plant to each bulk. The marker csLV34 is a bi-allelic locus, where the resistance allele is detected through the absence of a 79 bp insertion (Lagudah et al. 2006) using recommended protocols. PCR products were resolved on 2% agarose gels. Pooled samples that amplified a 229 bp fragment (a allele) were recorded as negative (-) for Yrl8 and those amplifying a 150 bp (b allele) fragment were positive (+) for Yrl8 (Lagudah et al. 2006). Analysis of CEC and field experiments

Analysis of variance (ANOVA) of the disease response data from growth stage x temperature interaction experiments was performed using the GLM (General Linear Model) procedure in Minitab^® V.15 (Minitab Statistical Software 2005), with the three main factors (age at inoculation, photoperiod and post-inoculation temperature) and genotypes as fixed factors, and repeated experiments (A and B) were considered a random factor. Mean disease response was calculated for each line and least significant difference (LSD) was derived using the individual significance level (corrected for multiple comparisons) at 0.1%. Means and confidence intervals (95%) were calculated for the nine lines assessed in both the field and CEC environments. Confidence intervals for each environment were calculated based on separate analyses due to differences in experimental design. The concordance between disease responses observed for F₂ individual plants and disease responses for derived F₃ progeny was calculated using the cross-tabulation and chi-square procedure (Kendall's tau-b coefficient) in Minitab^® V.15.

RESULTS

Characterisation experiment

Four of the five lines were susceptible to YR when inoculated as seedlings

(Fig. 1). Expression in 'Ellison' (YrlT) was influenced by growth stage and post- inoculation temperature. 'Ellison' was moderately susceptible as seedlings (GS12) when disease developed at a constant temperature of 11°C (Table 2). However, when 'Ellison' was grown under constant light for three weeks and inoculated at the tillering stage (GS 14,21), expression of Yrl7 resulted in a hypersensitive response without sporulation (Table 2). Post-inoculation temperature had little effect on the mean disease response for the remaining four lines used as controls. In the case of 'EGA Gregory', sporulation appeared to be more restricted when disease developed at 11°C constant (Fig. 2); however, these differences were not statistically significant (Table 2).

APR responses were observed for the moderately resistant line 'EGA Gregory', and to a lesser extent in 'Janz\ When 'EGA Gregory' was inoculated at the seedling stage (2 -week-old plants grown under diurnal photoperiod), it showed a susceptible to moderately susceptible response exhibiting heavy sporulation with some light chlorosis. In contrast, older plants of 'EGA Gregory' (3-week-old plants grown under continuous photoperiod) showed a moderately resistant response with restricted sporulation coupled with extensive blotched areas of chlorosis and necrosis (Fig. 1). 'Janz' and 'Baxter' are known to possess weak APR (YrI8) and displayed a similar disease response to YR across the treatments. However, some light chlorosis was observed in the case of 'Janz'. Chlorosis was more pronounced when inoculation was performed on plants grown under constant light for three weeks followed by incubation at 11°C (Table 2). As expected, the very susceptible control, Ή45', showed no APR and had a very susceptible disease response in all treatments (Table 2).

Overall, APR was best expressed by 3-week-old plants grown under constant light. At this stage most plants had produced 2-3 tillers (Table 3) and were at the stem elongation phase prior to inoculation, but the first node was readily detectable in most lines only a few days post-inoculation. This treatment also successfully ranked the lines according to published resistance levels for Pst pathotype 134 El 6 A+ (Table 1) and provided the best differentiation between the lines, regardless of post-inoculation temperature (Table 2). However, the disease developed much faster in the warmer post-inoculation temperature regime, allowing reliable scoring of disease resistance to be achieved one week earlier.

In most cases the upper-most inoculated leaves showed increased susceptibility when compared to lower leaves on the same plant (data not presented). As leaves aged, they displayed higher resistance levels (particularly for lines with APR). This was clear when comparing the disease response of leaf 2 of 'EGA Gregory' inoculated at the 3-week old stage, in comparison to leaf 2 inoculated at the 2-week old stage. Consequently, it was easier to detect early expression of APR by taking into account the disease response on lower leaves.

Expression of APR in lines assessed across environments

The nine lines used as controls demonstrated similar levels of YR APR under both CEC and field environments (Fig. 3). 'Ellison' showed a hypersensitive disease response in both environments, consistent with expression of Yrl7 against the avirulent Pst pathotype 134 El 6 A+ (Fig. 4). The three controls with better levels of APR ('ST93\ 'RSY10' and 'RSY5') and 'EGA Gregory' (Yr33 plus APR genes) showed moderate to high levels of APR in both screening environments (Fig. 4). As adult plants, these lines displayed partial resistance to the pathogen including restricted sporulation and chlorosis around the infected leaf tissue (Fig. 4). 'RSY10' and 'RSY5' (uncharacterised APR genes) provided similar levels of resistance to 'EGA Gregory'. 'Janz' and 'Baxter' both displayed considerably lower levels of resistance, with 'Janz' being slightly more resistant in the CEC (Fig. 3). When assessed in the CEC, 'H45' and 'Wyalkatchem' (i.e. susceptible parents of the two F2 populations) showed a susceptible disease response, i.e. heavy sporulation and no chlorosis or necrosis (Fig. 4). However, when grown in the CEC under continuous light, most 'Wyalkatchem' plants showed a prolonged tillering phase, and several days after inoculation the 'Wyalkatchem' plants did not show any signs of stem elongation. This was probably due to a vernalisation requirement. Other experiments characterising APR in 'Wyalkatchem' under CEC (data not presented) showed that when plants were inoculated at the stem elongation growth stage, 'Wyalkatchem' displayed increased resistance, attaining a moderately susceptible to moderately resistant disease response. When assessed in the field, 'Wyalkatchem' also displayed a low level of APR (Fig. 4). Screening F₂ populations for YR APR

Similar levels of APR were identified in the F2 populations when assessed in CEC or field environments (Fig. 5). Both populations displayed a distribution skewed towards susceptibility; however, the 'Wyalkatchem' ^χ 'ST93' population distribution was less skewed when assessed in the field. This population showed higher levels of resistance when compared to the 'H45' * 'ST93' population (Fig. 5). At the time of assessment under CEC, some individuals in the 'Wyalkatchem' ^χ 'ST93' population were not at the stem elongation growth stage (GS30), displaying a prolonged vegetative phase with profuse tillering. These individuals were assessed for resistance but were not included in analysis of population distributions and DNA sampling. The vast majority of F2 plants were assessed for YR APR prior to anthesis.

Assessment of F3 progeny in the field The responses of F2 plants assessed for YR APR in the field at Warwick in 2009 were very similar to those of their F3 progenies assessed for disease resistance at the Gatton field site the following year (Fig. 6). The test of concordance showed that there was a strong positive association between the disease responses in the F₂ and F3 generations (P < 0.001). The Kendall's tau-b measure of concordance was high and positive (0.74), indicating that the ranking of disease response observed on single F₂ plants and their F3 progeny increased together (Fig. 6). This was consistent for both populations. The mean disease responses of F3 progeny mostly fell within one score (higher and/or lower) of the F₂ disease response. For example, an F₂ individual plant assigned a score of 5 in the field at Warwick, resulted in F3 progeny with probabilities of 25%, 44% and 28% for a disease response of 4, 5 and 6 respectively, when assessed in the following year at Gatton (Table 4).

The frequency of Yrl8 in phenotypic classes for APR

DNA samples from individual plants (including susceptible and resistant phenotypes) from the Ή45' 'ST93' population were tested with the csLV34 marker. This confirmed the b allele at csLV34 was fixed in this population. In the 'Wyalkatchem' x 'ST93' population, all phenotypic classes sampled (including susceptible classes) contained the b allele for Yrl8 in both field and CEC environments. The only phenotypic class having a high frequency of homozygosity for the b allele (YrlS) was the most resistant bulk. This was consistent across both CEC and field environments (i.e. 6 in the CEC and 7 in the field).

DISCUSSION

Expression of APR under CEC

The hypersensitive resistance displayed by 3-week-old plants of 'Ellison' when YR developed under a cool post-inoculation temperature (11°C) demonstrates that 3- week-old plants grown under constant light display a disease response typical of adult plants, rather than the seedling response. 'Ellison' contains the seedling resistance gene Yrl 7. Seedlings that possess this gene, typically express high levels of resistance at warmer temperatures (i.e. 15-20°C); however, at lower temperatures (12-15°C) most lines possessing Yrl7 are susceptible to Pst pathotype 134 E16 A+ (Banana and Mcintosh 1994; Qamar et al. 2008). A number of studies have reported a temperature * growth stage interaction for Yrl7, which may also be influenced by low light levels (Banana and Mcintosh 1994).

The first node was readily detected approximately two days post-inoculation in plants grown for three weeks under constant light, thus as reported by Principe et al. (1992) in barley, growth under continuous light largely eliminated maturity differences among cultivars. Therefore the disease responses observed are likely adult-plant responses. Since the timing of inoculation is critical, a single inoculation (monocyclic test) around stem elongation (GS30) under controlled conditions likely provides a better measure of APR expression than data from field experiments where fresh infection can occur every night (polycyclic test). The link between growth stage and disease response is highlighted by the increased resistance expressed by 'EGA Gregory' when plants grown for three weeks under constant light were inoculated. 'EGA Gregory' carries the major resistance gene Yr33, which typically expresses better in adult plants and/or at high temperatures. 'EGA Gregory' also has Yrl8, plus additional uncharacterised APR genes, conferring a high level of resistance to YR in the field. Repeated experiments conducted under CEC demonstrate clearly the change in expression of these genes.

Park and Rees (1987) reported expression of YR APR at the mid-tillering growth stages in the field. This finding aligns well with results from the current study, where expression of APR corresponded with the spike initiation phase for plants grown in CEC. In addition, as the leaves on plants matured they showed improved resistance to the pathogen. This is also consistent with previous research by Singh (1992), where resistant infection types were best recognised in older leaves. Singh (1992) suggested that the products of APR genes accumulates at a slow rate, thus newly developed leaves are more susceptible than older ones.

Comparing expression of APR across environments

The nine wheat lines assessed across environments (CEC and field) attained similar rankings indicating similar expression of APR genes in both environments. The resistance conferred by Yrl8 is reported to be stronger than Yr29 (Lillemo et al. 2007; Lillemo et al. 2008; Martinez et al. 2001); however, in the current study 'Wyalkatchem' (Yr29) showed similar resistance to lines carrying YrJ8 ('Janz' and 'Baxter') when assessed in the field. Despite displaying a prolonged vegetative phase in the CEC, when inoculated at stem elongation 'Wyalkatchem' plants showed weak to moderate resistance. In the experiments conducted under CEC 'Baxter' (Yrl8) showed little resistance (across the various treatments); however, 'Janz' (also with Yrl8) showed light chlorosis on the upper leaves of adult plants, that was likely due to the effect of Yrl8 plus additional APR gene(s) (Banana et al. 2010). 'EGA Gregory' showed higher levels of APR (scores 5-7), due to expression of Yr33 and other APR genes (including YrlS). This suggests that screening under CEC can be employed to identify lines with polygenic APR, as those lines known to carry more genes tended to show higher levels of APR in the CEC.

Generally, the Ή45' ^χ 'ST93' population displayed lower levels of resistance than the 'Wyalkatchem' χ 'ST93' population, particularly when assessed in the field. This was expected as 'H45' is more susceptible than 'Wyalkatchem'. 'Wyalkatchem' is heterogeneous for Yr29 on chromosome IB (William et al. 2003; 2006); however, the plants used for crossing in the current study contained the gene.

Testing the csLV34 marker for Yrl8 alleles in the bulked DNA samples from the 'Wyalkatchem' ^χ 'ST93' population indicated that even moderately susceptible phenotypic classes (2 and 3) in the field contained heterozygous alleles (ab). However, the most resistant phenotypic class observed in both CEC and the field were homozygous for the b allele at the csLV34 locus. This suggested that individuals in the population displaying high levels of resistance were likely to carry Yrl8; however, when deployed alone Yrl8 only provides a weak level of resistance as in the case of 'Baxter'. Lillemo et al. (2008) suggested that combining just two APR genes (YrJ8 and Yr29) can provide good levels of resistance in the field, but the number of genes required for moderate-high levels of APR will depend upon the genes involved. In the current study, F₂ individuals may contain various combinations of 4-6 minor APR genes from 'ST93' to attain moderate-high levels of resistance.

The association between disease response of the F₂ plants and their F₃ progeny assessed in the field at Gatton 2010 suggests that phenotypic selection for YR APR is effective in early segregating generations and could be used to increase the frequency of APR genes in later generations. The high probability of a similar disease response to the proceeding generation, may partly be due to the large number of genes (with minor effect) contributing APR in these segregating populations and/or the care taken in phenotyping the F₂ populations.

Application within breeding programs

In recent years Australian rust pathogens have overcome several seedling resistance genes, including Yrl7, Lr24, Lr37 and Sr38 (Park 2008). As the industry strives toward durable rust resistance achieved by pyramiding seedling genes and/or polygenic APR genes, we believe that screening for YR resistance in CEC will provide another tool to assist breeding programs achieve this goal.

APR to rust pathogens is a priority for most wheat breeding programs around the world. Of the three rust diseases of wheat, YR is the most sensitive to environmental conditions (Stubbs 1988). The temperature sensitivity of YR resistance in Australian wheat lines has been well documented (Park et al. 1992). The ability to accurately control temperature, light levels and disease pressure in CEC may eliminate much of the environmental noise associated with field screening. Different temperature regimes may also be adopted when screening under CEC to select and pyramid resistance genes that are effective across a wide range of temperatures. This may assist the development of lines adapted to more variable future environments resulting from climate change. Further, use of warmer conditions post-inoculation may correspond better with warming temperatures typical of mid to late spring in the field, which is often the time of year when APR is expressed and scored in the field.

Marker assisted selection (MAS) for rust resistance genes is performed routinely in many wheat-breeding programs (Banana et al. 2007) and there has been much progress toward development of diagnostic molecular markers for the APR genes, such as the perfect marker for Lr34/Yrl8 reported by Lagudah et al. (2006) and the CAP (cleaved amplified polymorphic sequence) marker for Lr46/Yr29 (E. S. Lagudah, personal communication). However, MAS for APR genes can only target known and well-characterised resistance genes, and even then the presence or absence of other APR genes may determine the phenotype observed. Thus, breeding for resistance still requires phenotypic screening. The CEC phenotypic screening described here can be applied to both fixed lines and segregating populations and can provide results within five weeks after sowing. When CEC screening is combined with MAS, selection for both known and unknown APR genes is possible. Selection can be performed at any time of the year and with up to four generations grown per year (Hickey et al. 2009), faster development of rust resistant lines is feasible. The proposed screening method using CEC provides an opportunity to enrich populations with minor genes for APR and to assist the development of wheat lines with durable rust resistance.

Table 1: Spring wheat lines used as stripe rust (YR) controls - disease ratings, known resistance genes and information on presence/absence of the csLV34 marker linked to the APR gene Lr34IYrl8

Known YR

YR disease rating for pt. 134 csLV34

Line resistance APR

E16 A+ " status *

genes *

Ellison Resistant (R) Yrl7 - None

EGA Very

Moderately resistant (MR) Yr33 + Gregory good

Moderately resistant / moderately

Janz Yrl8+ + Weak susceptible (MR/MS)

Very

Baxter Moderately susceptible (MS) Yrl8 +

weak

H45 Very susceptible (VS) Yr7 + ^b None

⁸ Adapted from Australian Cereal Rust Control Program, Rust Report 2009 Volume 7 #2.

b Lr34IYrl8 lacks function for resistance to stripe rust in Ή45' (Lagudah et al. 2009).

Table 2: Mean disease responses of lines grown for two or three weeks prior to inoculation, under constant light or diurnal photopenod treatments, and with two post-inoculation temperature treatments (11/23 °C or 11°C constant)

Post- Diurnal (12 h)

Constant light (24 h)

inoculation photoperiod temperature 2 weeks 3 weeks 2 weeks 3 weeks

Ellison 7.4 7.8 7.6 7.7

EGA

3.9 5.5 2.9 4.0 Gregory

11/23°C

Janz 2.6 3.3 2.5 3.0

Baxter 2.7 2.8 2.4 2.6

H45 1.6 1.7 1.8 1.9

Ellison 4.5 7.4 3.4 5.9

EGA

3.7 5.8 3.0 4.1 Gregory

11°C constant

Janz 2.8 3.6 2.3 3.1

Baxter 2.3 3.1 2.2 2.7

H45 1.8 1.6 1.9 1.7

LSD at 0.1% (adjusted for multiple comparisons) between genotypes within each treatment is 0.40 (scoring based on 1-9 scale, where 9 = immune). Table 3: Zadoks' growth stages (GS) for the five wheat lines used as controls, recorded at time of inoculation, when grown in 100-cell trays for two or three weeks under constant light (24 h) or a diurnal (12 h) photoperiod. All plants were grown at a constant temperature of 22°C

Two weeks old Three weeks old

Line Diurnal (12 h) Constant Diurnal (12 h) Constant light photoperiod light (24 h) photoperiod (24 h)

Ellison GS12 GS13 GS13 GS14,21

GS14,21 -

EGA Gregory GS13 GS13 GS14

14,22

GS14,21 -

Janz GS13 GS13 GS14

15,21

GS14.21 -

Baxter GS13 GS13 GS14

14,22

H45 GS13 GS13 GS14 GS15 - 15,21

GS12 = 2-leaf stage, GS13 = 3-leaf stage, GS14 = 4-leaf stage, GS14,21 = 4 leaves main stem and 1 tiller, GS14,22 = 4 leaves main stem and 2 tillers, GS15 = 5-leaf stage etc.

Table 4: Distribution (%) of disease responses in F3 progeny based on the disease response of individual F₂plants assessed for adult-plant resistance to stripe rust in the field

Percentage for disease response of F3 progeny

F₂ disease

Total number of response 1 2 3 4 5 6 7

plants

1 38 62 0 0 0 0 0 13

2 3 43 42 9 0 3 0 35

3 0 7 49 34 10 0 0 41

4 0 0 29 50 21 0 0 34

5 0 3 0 25 44 28 0 32

6 0 0 0 13 44 41 3 32

7 0 0 0 0 8 69 23 13

8 0 0 0 0 0 100 0 2

Total number of

6 27 45 46 40 34 4 202 plants

EXAMPLE 2

Triple Rust Screening of Wheat

INTRODUCTION

Meeting global food demands for the future largely depends on the success of developing new cereal cultivars adapted to an ever-changing environment. Prospects of climate change and an evolving spectrum of disease and pest organisms, requires more efficient crop improvement strategies. We report the simultaneous rapid assessment of adult plant resistance (APR) to rust pathogens in wheat grown under controlled environmental conditions (CEC). This method permits efficient high- throughput screening of large segregating populations for APR to all three rusts; i.e. leaf rust (LR), stem rust (SR) and stripe rust (YR). We assessed two segregating populations (approximately 1000 individuals each) using the newly developed screening method. Prior to inoculation, wheat populations were grown for three weeks under constant light and temperate conditions to accelerate plant development. Selection for APR to all three rusts was possible through assessment of YR on leaves 3-4, LR on flag-1 and flag leaf, and SR on the elongated stem. This rust screen can be completed within only five weeks from time of sowing, and permits year-round phenotyping, screening and selection (up to 10 consecutive cycles per year). The rapid screening method provides accurate phenotyping of fixed lines and an oppurtunity to enrich segregating populations with genes for APR to all three rusts during progress to development of inbred lines.

EXPERIMENTAL PROCEDURE

Two three-way cross populations segregating for APR to the three rust pathogens were developed. An Fi cross was made to combine genes for rust resistance present in elite breeding lines; UQ01484 and UQ01488. The Fi donor plants were then crossed to two Australian wheat cultivars, H45 and Wyalkatchem, producing two three-way crosses. H45 carries the seedling YR resistance gene Yr7, but this gene is not effective against the pathotypes currently found in Australia (H45 is very susceptible to the pathotype 134 E16 A+ used in this study). Wyalkatchem is moderately susceptible to YR, and is mixed for the APR gene(s) Lr46/Yr29 that confer partial resistance (Loughman et al. 2008). Trial experiments combining inoculation of all three rust pathogens in wheat were conducted in 2009 and results from the characterisation experiments performed for APR to YR in wheat (see Example l)and APR to leaf rust in barley (Hickey et al. 2011) were used to design the triple rust screening methodology, as applied in the current study. In addition H45 carries leaf rust gene Lrl3 and stem rust genes Sr9g, Sri 7 and Sr30, while Wyalkatchem carries leaf rust genes Lr3a and Lr20, and stem rust genes Sr2, Sr9b and Sri 5 (as published in the Australian Cereal Control Program circulars). The triple rust screen was developed to enable simultaneous selection of all three rusts, to facilitate the development of new lines resistant to the three rusts.

Approximately 1000 grains were obtained from over 40 single plants in the each of the two three-way crosses (Fl Donor χ H45 and Fl Donor x Wyalkatchem). There were a variable number of grains from each plant in the three-way cross. Families labelled H001 to H082 and W002 to W102 were derived from these individual three-way cross plants in the H45 and Wyalkatchem populations respectively.

Grains were imbibed in Petri dishes lined with filter papers (Whatman 90 mm) for 24 h and then placed in a refrigerator (4°C) for 48 h to promote synchronous germination. Petri dishes were then maintained at room temperature (approx. 24°C) for 24 h to initiate germination prior to transplanting into the seedling trays. Germinated grains were transplanted using tweezers into 100-cell seedling trays (10 x 10 cells, with dimensions of 30 35 cm), and then grown in a temperature controlled glasshouse under constant light.

From previous experience, when Wyalkatchem is grown under constant light and temperature in the glasshouse, it displays a delayed time to flowering (approximately 1 week later than most Australian spring wheat cultivars). Therefore, the Wyalkatchem derived population was germinated 7 days earlier to ensure that majority of plants were post-stem elongation at the time of inoculation, which is critical for early detection of APR to YR in wheat (as previously described herein).

Transplanting was conducted by family (i.e. three-way cross plants), with parental lines replicated 15-20 times throughout the design. A total of 12 trays were planted and placed in a controlled temperature glasshouse (maintained at 21 ± 1°C) fitted with low-pressure sodium lamps to supplement natural light levels and to extend the photoperiod to 24 h, thereby accelerating the development of wheat plants (as detailed in Hickey et al. 2009). Although the lamps provided constant light, the photosynthetically available radiation (PAR) varied according to a diurnal cycle and the natural light levels present during daylight hours. Trays were positioned across two benches in the glasshouse.

One week after transplanting, the 100-cell seedling trays were placed on top of similar sized open tray (with no cells) filled with potting media with added Osmocote slow release fertilizer. This provided additional resources for rapidly developing wheat plants and liquid fertilizer was applied regularly. Wheat plants derived from the H45 population were grown under constant light for three weeks, whereas plants derived from the Wyalkatchem population were grown for four weeks. Just prior to inoculation, most wheat plants ranged in Zadok's growth stage from 21-31 (tillering to stem elongation; Zadoks et al. 1974).

Inoculation of stripe rust (YR)

Each population was inoculated separately, within a period of 48 h. Inoculation of stripe rust pathotype 134 El 6 A+ (Wellings 2007) was performed with a suspension of urediniospores (in light mineral oil at a rate of 0.005 g ml) applied evenly using an air-brush. Trays were incubated at 10°C overnight in a dew chamber (maintained at 100% relative humidity). Leaves 3 and 4 were inoculated.

Post-inoculation, trays were returned to the temperature controlled glasshouse. To assist disease development, the lights were programmed to a 12 h diurnal photoperiod and only used during the day to supplement light. The glasshouse temperature was set to a lower temperature of 18/12°C (day/night) to favour YR development.

Inoculation of stem rust (SR) and leaf rust (LR)

Once early signs of YR infection could be detected (i.e. light flecking of the leaves, approximately five to six days post-inoculation of YR), plants were then inoculated with a mix of stem rust (pathotypes 343-1,2,3,5,6 and 34-127+Sr38) and leaf rust (pathotype 104-2,3,6,(7)). Both SR and LR urediniospores were mixed together in an oil suspension (as described above for YR) and applied evenly using an air-brush. Inoculum was applied evenly in all directions and rows of plants were separated gently to allow consistent infection within the canopy of the wheat plants, including the elongated stems. Trays were incubated at 22°C overnight in a dew chamber (as described above). The flag-1 (for most plants leaf 5) and flag leaf had been produced on almost all plants at the time of inoculation.

Post-inoculation, trays were returned to the temperature controlled glasshouse. The lights remained programmed to a 12 h diurnal photoperiod. The glasshouse temperature was then set to a wanner temperature of 22/20°C (day/night) to favour development of SR and LR.

Assessment

, Approximately 10-11 days after inoculation of stem rust and leaf rust, both populations were assessed for APR to the three rust diseases. Individual plants were assigned an overall disease response, which was the average response observed for the two upper-most infected leaves (in the case of YR and LR) or infection on elongated stems (in the case of SR) using a 0-9 scale (McNeal et al., 1971; where 0 = immune and 9 = very susceptible). Units of the scale reflect the amount of diseased tissue and infection type.

RESULTS

Disease responses typical of those observed under field conditions were observed on plants infected under the system described here. A full range of symptoms for APR to YR (i.e. moderately resistant to very susceptible) was observed on leaves 3 and 4. Leaf rust APR was detected on the upper leaves (flag-1 and flag leaves covering the full range of responses from resistant to susceptible. It was possible to identify differential responses to YR, LR and SR when infection occurred on the same leaf (Figure 7). Likewise for SR, disease responses from susceptible to resistant were observed on the elongated stems (Figure 8).

The mean disease responses observed on three of the parental lines (H45, Wyalkatchem and UQ01484; Table 5) were similar to those previously observed under field conditions when exposed to the same pathotypes. These two adapted parents (H45 and Wyalkatchem) are clearly susceptible to YR, but have useful levels of resistance to both LR and SR (Table 5). The YR donor (UQ01488) displays APR to YR, carries Lr42 which confers high levels of resistance to LR, but is susceptible to SR (Table 5). This triple rust screening method was able to clearly distinguish between the parental lines for resistance to each of the three rusts. The families derived from each of the single plants in the three-way crosses varied significantly for their response to the three rusts (Table 6). Some families displayed high levels of resistance to all three rusts (e.g. H008 and H024; Table 6). For YR, high levels of resistance were conferred by APR genes, whereas, high levels of resistance to SR and LR was conditioned by a combination of seedling genes and APR genes (e.g. Sr2 for stem rust and Lr34 for leaf rust). This screening method allows selection of plants with "good gene combinations" (i.e. polygenic resistance) for resistance to rust pathogens expressed at the adult-plant stage.

The expected range of reaction types to each rust were observed on individual plants in each population (Figures 9 and 10); however, relatively few plants were found that combined the highest levels of resistance to all three rusts. Nevertheless it was possible to select approximately 90 individual plants from each cross which displayed high levels of resistance to the three rusts.

The selected plants were then transplanted into 1.4L pots, and crossed to the adapted parent (i.e. H4S or Wyalkatchem) in the same plant generation. If crossing could not be completed in the same generation, plants in the next generation would again segregate, therefore requiring a larger number of heads to be pollinated so that favourable genes are not lost.

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

Table 5: Mean disease response of parental lines to stripe rust (YR), stem rust (SR) and leaf rust (LR), when assessed simultaneously for disease resistance under controlled environmental conditions at the adult plant stage.

Table 6: Mean disease response of families derived from three-way crosses involving H45 (families H001 to H082) and Wyalkatchem (W002 to W 102) when inoculated with stripe rust (YR), stem rust (SR) and leaf rust (LR) at the adult plant stage, and grown under controlled conditions.

YR SR LR

Family N

Mean SE Mean SE Mean SE

H001 31 5.65 0.27 3.94 0.45 4.32 0.45

H002 12 5.13 0.42 3.26 0.71 3.72 0.62

H003 12 7.29 0.37 2.23 0.62 1.58 0.13

H004 28 5.41 0.31 1.90 0.04 3.48 0.48

H005 27 6.87 0.21 2.08 0.20 1.85 0.22

H006 23 4.00 0.15 2.90 0.33 2.86 0.45

H008 28 3.75 0.17 1.78 0.05 1.89 0.24

H009 31 4.57 0.18 1.83 0.03 2.71 0.34

H010 16 6.66 0.31 3.58 0.40 2.76 0.47

H011 20 4.40 0.20 1.56 0.06 3.55 0.68

H014 26 4.21 0.18 1.47 0.08 1.85 0.31

H015 27 6.39 0.25 2.61 0.29 4.24 0.53

H016 30 6.58 0.24 2.02 0.03 3.33 0.40

H019 18 7.25 0.18 4.86 0.54 3.85 0.51

H024 40 3.63 0.14 1.42 0.06 1.44 0.08

H025 20 6.45 0.32 2.68 0.51 2.08 0.22

H026 40 6.86 0.18 1.99 0.03 4.74 0.43

H030 21 4.98 0.23 1.32 0.09 1.82 0.36

H038 10 5.85 0.50 1.97 0.05 4.61 0.80

H041 33 4.08 0.22 2.11 0.17 2.38 0.19

H042 28 4.68 0.20 1.58 0.06 2.00 0.17

H043 10 4.65 0.45 1.61 0.14 2.59 0.74

H044 19 4.55 0.35 2.56 0.29 3.91 0.63

H045 39 5.28 0.26 1.95 0.22 2.64 0.29

H047 41 4.94 0.22 2.99 0.28 2.16 0.18 YR SR LR

Family N

Mean SE Mean SE Mean SE

H048 15 6.37 0.35 2.99 0.49 2.79 0.41

H049 77 4.90 0.14 3.01 0.22 2.50 0.21

H051 26 5.69 0.23 3.21 0.40 3.31 0.40

H052 10 4.10 0.34 1.84 0.10 2.47 0.63

H057 40 4.14 0.14 1.84 0.10 2.92 0.31

H058 26 5.15 0.22 1.49 0.08 2.14 0.33

H059 25 4.98 0.36 1.93 0.09 2.26 0.31

H061 25 6.06 0.27 2.43 0.21 3.29 0.47

H062 28 5.93 0.27 2.53 0.38 3.37 0.36

H063 15 8.00 0.30 1.54 0.08 3.33 0.41

H065 41 7.34 0.12 1.97 0.03 2.60 0.30

H067 3 5.83 0.93 1.87 0.13 1.93 0.13

H069 23 4.54 0.22 1.48 0.08 2.18 0.43

H072 19 6.74 0.29 4.36 0.57 3.39 0.53

H074 19 6.61 0.31 4.20 0.65 3.02 0.46

H075 40 7.10 0.23 3.54 0.32 4.32 0.40

H077 14 5.64 0.41 2.16 0.37 2.83 0.49

H080 22 5.41 0.30 1.55 0.21 1.93 0.32

H082 6 3.67 0.21 4.73 1.18 3.86 1.07

W002 2 4.50 0.50 3.35 2.35 1.38 0.38

W003 69 3.62 0.09 3.11 0.29 2.15 0.21

W006 5 3.10 0.10 3.53 0.85 3.05 1.24

W010 39 4.71 0.16 1.94 0.26 1.67 0.17

W014 13 5.12 0.29 2.25 0.44 1.92 0.35

W021 8 3.25 0.16 2.29 0.25 3.25 1.04

W023 20 4.50 0.37 2.18 0.30 1.78 0.35

W028 22 5.59 0.32 2.04 0.39 1.73 0.34

W032 15 6.97 0.23 2.19 0.29 1.95 0.26

W034 46 6.28 0.19 2.28 0.18 2.97 0.34

W035 14 7.25 0.20 2.43 0.30 3.69 0.71

W036 30 4.02 0.24 1.59 0.08 2.09 0.36

W039 2 3.00 0.00 3.35 2.35 1.55 0.55

W043 20 4.80 0.30 1.89 0.09 3.66 0.59

W050 45 5.58 0.20 3.68 0.34 4.56 0.45

W052 35 4.43 0.15 1.84 0.20 2.54 0.35

W054 39 5.13 0.29 5.01 0.39 3.16 0.35

W057 50 3.47 0.10 3.64 0.31 3.81 0.42

W065 15 5.13 0.37 2.42 0.52 2.14 0.43

W066 30 6.90 0.25 2.38 0.23 2.73 0.38

W067 40 6.96 0.16 2.95 0.25 4.25 0.47

W068 29 4.53 0.24 4.98 0.36 4.06 0.52

W070 13 4.92 0.28 4.54 0.65 3.92 0.78

W071 31 4.97 0.26 2.17 0.26 1.83 0.21

W072 4 4.63 0.38 1.65 0.23 2.56 1.08

W077 7 6.93 0.60 3.77 0.65 4.00 1.06

W081 30 6.60 0.14 2.21 0.17 4.49 0.56 YR SR LR

Family N

Mean SE Mean SE Mean SE

W082 39 8.35 0.15 2.23 0.20 4.60 0.44

W083 40 4.75 0.23 2.29 0.26 3.72 0.46

W084 7 6.43 0.54 2.46 0.61 2.39 0.78

W086 11 4.41 0.37 3.82 0.58 3.20 0.64

W089 11 7.00 0.29 3.88 0.57 2.79 0.63

W090 20 5.28 0.35 2.64 0.38 2.99 0.50

W091 10 6.85 0.51 2.65 0.53 2.93 0.70

W092 22 5.32 0.33 1.53 0.22 2.06 0.46

W095 8 3.63 0.16 2.49 0.80 1.28 0.14

W096 20 4.38 0.20 1.62 0.10 1.86 0.35

W097 4 3.13 0.13 1.68 0.25 1.38 0.22

W098 6 4.92 0.49 1.29 0.19 1.38 0.17

W099 10 3.35 0.15 2.34 0.28 2.85 0.71

W102 21 4.71 0.19 1.35 0.08 1.55 0.15

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Claims

1. A method of plant breeding including the step of crossing one or more plants to produce one or more progeny plants, wherein at least one of the plants has been selected as having at least partial adult stage resistance to a plant disease in response to exposure of the plant to one or more disease-causing organism or pathogen.

2. The method of Claim 1, which includes the step of distinguishing or differentiating the adult stage resistance from seedling resistance.

3. The method of Claim 1 or Claim 2, wherein the adult stage resistance is adult plant resistance (APR).

4. The method of any one of Claims 1-3, wherein said at least one of the plants has been selected as having at least partial adult stage resistance to the plant disease after growth in a controlled environment which modulates plant growth and development and/or facilitates the expression of disease resistance.

S. The method of any preceding claim, which prior to selection and crossing includes identifying at least one of said one or more plants at an adult stage as having at least partial adult stage resistance to said plant disease.

6. The method of any preceding claim, wherein identifying and/or selecting said at least one of the one or more plants as at least partly resistant to said plant disease occurs at an adult stage before the or each plant reproduces or ceases being capable of reproduction.

7. The method of Claim 6, wherein identifying and selecting said at least one of the one or more plants as at least partly resistant to said plant disease occurs before conclusion of anthesis.

8. The method of any preceding claim, wherein identifying, selecting and crossing the one or more disease-resistant plants occurs within a single generation.

9. The method of Claim 8, wherein multiple cycles of identifying, selecting and crossing the one or more disease-resistant plants occur within a year.

10. The method of Claim 9, wherein 2-10 cycles of identifying, selecting and crossing the one or more disease-resistant plants occur within a year.

11. The method of any preceding claim, wherein the plant is a crop plant.

12. The method of Claim 11, wherein the crop plant is of the family Poaceae.

13. The method of Claim 12, wherein the crop plant is a cereal.

14. The method of Claim 13, wherein the cereal is wheat or barley.

15. The method of Claim 11, wherein the crop plant is a legume.

16. The method of any preceding claim, wherein the disease is caused by a fungus, worm, protozoan, virus or bacterium.

17. The method of Claim 16, wherein the fungus is a Pucciniamycete.

18. The method of Claim 17, wherein the Pucciniamycete causes leaf rust, stem rust or stripe rust in cereals.

19. The method of Claim 17, wherein the disease is net blotch in barley.

20. The method of Claim 16, wherein resistance is to multiple diseases caused by a plurality of different disease-causing organisms or pathogens.

21. The method of any one of Claims 4-20, wherein the controlled environment includes controllable factors including (i) timing of exposure to the disease- causing pathogen or organism, (ii) photoperiod; (iii) temperature; (iv) humidity and/or (v) carbon dioxide (C0₂) levels.

22. The method of Claim 21 , wherein the temperature is in the range 10-35°C.

23. The method of Claim 21 or Claim 22, wherein a constant temperature is maintained.

24. The method of Claim 23, wherein the constant temperature is 11°C or 22°C.

25. The method of Claim 21 or Claim 22, wherein a variable temperature is provided.

26. The method of Claim 25, wherein the temperature is diurnally variable.

27. The method of Claim 26, wherein the diurnally variable temperature varies from a temperature in the range 10-20°C to a temperature in the range 18-30°C.

28. The method of any one of Claims 21-27, wherein a constant photoperiod is maintained.

29. The method of any one of Claims 21-28, wherein a diurnally variable photoperiod is provided.

30. The method of any one of Claims 21-29, wherein humidity is elevated.

31. The method of Claim 30, wherein humidity is in the range 70% to 100% relative humidity.

32. The method of any one of Claims 21-31, wherein C0₂ is elevated.

33. The method of Claim 32, wherein C0₂ is elevated at a level 1.5 to 4.5 times the level of C0₂ normally present in air.

34. The method of any one of Claims 4-33, wherein the controlled environment accelerates plant growth and development and/or facilitates the expression of disease resistance, compared to that which would occur under normal field conditions.

35. The method of any one of Claims 4-34, wherein the method further includes one or more additional plant culture factors or conditions to modulate plant development.

36. The method of any preceding claim, wherein at least one of the one or more plants are segregating with respect to disease resistance.

37. The method of any preceding claim, which includes the use of one or more genetic markers for marker assisted breeding.

38. A method of identifying a genetic marker associated with plant disease resistance including the step of identifying one or more genetic markers in a genome of a plant that are associated with, or linked to, at least partial adult stage resistance in response to exposure of the plant to one or more disease-causing pathogens or organims and growth of the plant in a controlled environment which accelerates plant growth and development and/or facilitates the expression of disease resistance.

39. The method of Claim 38, wherein a genetic marker is identified by:

(i) obtaining a nucleic acid sample from the plant after growth in plant in the controlled environment; and;

(ii) analysis of the nucleic acid sample to identify said genetic marker.