Wolfgang Spielmeyer

A Putative ABC Transporter Confers Durable Resistance to Multiple Fungal Pathogens in Wheat

Agricultural crops benefit from resistance to pathogens that endures over years and generations of both pest and crop. Durable disease resistance, which may be partial or complete, can be controlled by several genes. Some of the most devastating fungal pathogens in wheat are leaf rust, stripe rust, and powdery mildew. The wheat gene Lr34 has supported resistance to these pathogens for more than 50 years. Lr34 is now shared by wheat cultivars around the world. Here, we show that the LR34 protein resembles adenosine triphosphate–binding cassette transporters of the pleiotropic drug resistance subfamily. Alleles of Lr34 conferring resistance or susceptibility differ by three genetic polymorphisms. The Lr34 gene, which functions in the adult plant, stimulates senescence-like processes in the flag leaf tips and edges.

Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene

The introduction of semidwarf rice (Oryza sativa L.) led to record yield increases throughout Asia in the 1960s. The major semidwarfing allele, sd-1, is still extensively used in modern rice cultivars. The phenotype of sd-1 is consistent with dwarfism that results from a deficiency in gibberellin (GA) plant growth hormones. We propose that the semidwarf (sd-1) phenotype is the result of a deficiency of active GAs in the elongating stem arising from a defective 20-oxidase GA biosynthetic enzyme. Sequence data from the rice genome was combined with previous mapping studies to locate a putative GA 20-oxidase gene (Os20ox2) at the predicted map location of sd-1 on chromosome 1. Two independent sd-1 alleles contained alterations within Os20ox2: a deletion of 280 bp within the coding region of Os20ox2 was predicted to encode a nonfunctional protein in an indica type semidwarf (Doongara), whereas a substitution in an amino acid residue (Leu-266) that is highly conserved among dioxygenases could explain loss of function of Os20ox2 in a japonica semidwarf (Calrose76). The quantification of GAs in elongating stems by GC-MS showed that the initial substrate of GA 20-oxidase activity (GA53) accumulated, whereas the content of the major product (GA20) and of bioactive GA1 was lower in semidwarf compared with tall lines. We propose that the Os20ox2 gene corresponds to the sd-1 locus.

Perfect markers for the Rht-B1b and Rht-D1b dwarfing genes in wheat

Abstract.PCR-based markers were developed to detect the point mutations responsible for the two major semi-dwarfing genes Rht-B1b (Rht1) and Rht-D1b (Rht2) in wheat. These markers were validated by testing 19 wheat varieties of known Rht genotype. They included Rht-B1b and Rht-D1b dwarfs, double-mutant varieties and tall wheats. These were correctly genotyped with the Rht-B1b and Rht-D1b-specific primers, as well as markers specific for the tall alleles Rht-B1a and Rht-D1a. Using a family of doubled-haploid lines segregating for Rht-B1b and Rht-D1b, the markers were mapped to the expected homoeologous regions of chromosomes 4B and 4D, respectively. Both markers were strongly correlated with a reduction in height, accounting for 23% (Rht-B1b) and 44% (Rht-D1b) of the phenotypic variance in the population. These markers will have utility in marker-assisted selection of the Rht-B1b and Rht-D1b genes in wheat breeding programs.

A Physical Map of the 1-Gigabase Bread Wheat Chromosome 3B

As the staple food for 35% of the worlds population, wheat is one of the most important crop species. To date, sequence-based tools to accelerate wheat improvement are lacking. As part of the international effort to sequence the 17–billion–base-pair hexaploid bread wheat genome (2n = 6x = 42 chromosomes), we constructed a bacterial artificial chromosome (BAC)–based integrated physical map of the largest chromosome, 3B, that alone is 995 megabases. A chromosome-specific BAC library was used to assemble 82% of the chromosome into 1036 contigs that were anchored with 1443 molecular markers, providing a major resource for genetic and genomic studies. This physical map establishes a template for the remaining wheat chromosomes and demonstrates the feasibility of constructing physical maps in large, complex, polyploid genomes with a chromosome-based approach.

Breeding for improved water productivity in temperate cereals: phenotyping, quantitative trait loci, markers and the selection environment.

Consistent gains in grain yield in dry environments have been made by empirical breeding although there is disturbing evidence that these gains may have slowed. There are few examples where an understanding of the physiology and the genetics of putative important drought-related traits has led to improved yields. Success will first depend on identifying the most important traits in the target regions. It will then depend on accurate and fast phenotyping, which, in turn, will lead to: (1) trait-based selection being immediately transferable into breeding operations and (2) being able to identify the underlying genes or the important genomic regions (quantitative trait loci), perhaps leading to efficient marker-based selection (MBS). Genetic complexity, extent of genotypeenvironment (GE) interaction and sampling cost per line will determine value of phenotyping over MBS methods. Here, we review traits of importance in dry environments and review whether molecular or phenotypic selection methods are likely to be the most effective in crop improvement programs and where the main bottlenecks to selection are. We also consider whether selection for these traits should be made in dry environments or environments where there is no soil water limitation. The development of lines/ populations for trait validation studies and for varietal development is also described. We firstly conclude that despite the spectacular improvements in molecular technologies, fast and accurate phenotyping remains the major bottleneck to enhancing yield gains in water-limited environments. Secondly, for most traits of importance in dry environments, selection is generally conducted most effectively in favourable moisture environments.

A Sodium Transporter (HKT7) Is a Candidate for Nax1, a Gene for Salt Tolerance in Durum Wheat

Durum wheat (Triticum turgidum subsp. durum) is more salt sensitive than bread wheat (Triticum aestivum). A novel source of Na+ exclusion conferring salt tolerance to durum wheat is present in the durum wheat Line 149 derived from Triticum monococcum C68-101, and a quantitative trait locus contributing to low Na+ concentration in leaf blades, Nax1, mapped to chromosome 2AL. In this study, we used the rice (Oryza sativa) genome sequence and data from the wheat expressed sequence tag deletion bin mapping project to identify markers and construct a high-resolution map of the Nax1 region. Genes on wheat chromosome 2AL and rice chromosome 4L had good overall colinearity, but there was an inversion of a chromosomal segment that includes the Nax1 locus. Two putative sodium transporter genes (TmHKT7) related to OsHKT7 were mapped to chromosome 2AL. One TmHKT7 member (TmHKT7-A1) was polymorphic between the salt-tolerant and -sensitive lines, and cosegregated with Nax1 in the high-resolution mapping family. The other TmHKT7 member (TmHKT7-A2) was located within the same bacterial artificial chromosome contig of approximately 145 kb as TmHKT7-A1. TmHKT7-A1 and -A2 showed 83% amino acid identity. TmHKT7-A2, but not TmHKT7-A1, was expressed in roots and leaf sheaths of the salt-tolerant durum wheat Line 149. The expression pattern of TmHKT7-A2 was consistent with the physiological role of Nax1 in reducing Na+ concentration in leaf blades by retaining Na+ in the sheaths. TmHKT7-A2 could control Na+ unloading from xylem in roots and sheaths.

Development of PCR markers for the selection of wheat stem rust resistance genes Sr24 and Sr26 in diverse wheat germplasm

The use of major resistance genes is the most cost-effective strategy for preventing stem rust epidemics in Australian wheat crops. The long-term success of this strategy is dependent on combining resistance genes that are effective against all predominant races of the pathogen, a task greatly assisted by the use of molecular markers linked to individual resistance genes. The wheat stem rust resistance genes Sr24 and Sr26 (derived from Agropyron elongatum) and SrR and Sr31 (derived from rye) are available in wheat as segments of alien chromosome translocated to wheat chromosomes. Each of these genes provides resistance to all races of wheat stem rust currently found in Australia .We have developed robust PCR markers for Sr24 and Sr26 (this study) and SrR and Sr31 (previously reported) that are applicable across a wide selection of Australian wheat germplasm. Wheat lines have recently become available in which the size of the alien segments containing Sr26, SrR and Sr31 has been reduced. Newly developed PCR-markers can be used to identify the presence of the shorter alien segment in all cases. Assuming that these genes have different gene-for-gene specificities and that the wheat industry will discourage the use of varieties carrying single genes only, the newly developed PCR markers will facilitate the incorporation of two or more of the genes Sr24, Sr26, SrR and Sr31 into wheat lines and have the potential to provide durable control to stem rust in Australia and elsewhere.

Comparative mapping of HKT genes in wheat, barley, and rice, key determinants of Na+ transport, and salt tolerance

Salt tolerance of plants depends on HKT transporters (High-affinity K(+) Transporter), which mediate Na(+)-specific transport or Na(+)-K(+) co-transport. Gene sequences closely related to rice HKT genes were isolated from hexaploid bread wheat (Triticum aestivum) or barley (Hordeum vulgare) for genomic DNA southern hybridization analysis. HKT gene sequences were mapped on chromosomal arms of wheat and barley using wheat chromosome substitution lines and barley-wheat chromosome addition lines. In addition, HKT gene members in the wild diploid wheat ancestors, T. monococcum (A(m) genome), T. urartu (A(u) genome), and Ae. tauschii (D(t) genome) were investigated. Variation in copy number for individual HKT gene members was observed between the barley, wheat, and rice genomes, and between the different wheat genomes. HKT2;1/2-like, HKT2;3/4-like, HKT1;1/2-like, HKT1;3-like, HKT1;4-like, and HKT1;5-like genes were mapped to the wheat-barley chromosome groups 7, 7, 2, 6, 2, and 4, respectively. Chromosomal regions containing HKT genes were syntenic between wheat and rice except for the chromosome regions containing the HKT1;5-like gene. Potential roles of HKT genes in Na(+) transport in rice, wheat, and barley are discussed. Determination of the chromosome locations of HKT genes provides a framework for future physiological and genetic studies investigating the relationships between HKT genes and salt tolerance in wheat and barley.

The effect of different height reducing genes on the early growth of wheat

Genes that reduce height without compromising seedling vigour or coleoptile length have great potential for wheat improvement. We therefore investigated the effects of various reduced height (Rht) genes on the early stages of plant development, using a combination of near isogenic, recombinant, mutant and wild type comparisons. Gibberellin (GA) insensitivity caused by Rht-B1b or Rht-D1b was associated with reduced leaf elongation rate and coleoptile length. Similar results were found for two other sources of dwarfing, Rht11 and Rht17. We found one class of Rht genes (e.g. Rht8) which had no effect on coleoptile length, leaf elongation rate or responsiveness to GA, indicating that these dwarfing genes may act later in wheat development to reduce height and increase harvest index, without affecting early growth. A third class of Rht genes was found in three durum backgrounds. These had reduced coleoptile lengths and leaf elongation rates, but had a greater response to GA than the corresponding tall varieties. We discuss these results in relation to the possible mechanisms underlying the reduction in height and the suitability of the different Rht genes for wheat improvement.

Strategies for efficient implementation of molecular markers in wheat breeding

Although molecular markers allow more accurate selection in early generations than conventional screens, large numbers can make selection impracticable while screening in later generations may provide little or no advantage over conventional selection techniques. Investigation of different crossing strategies and consideration of when to screen, what proportion to retain and the impacts of dominant vs. codominant marker expression revealed important choices in the design of marker-assisted selection programs that can produce large efficiency gains. Using F2 enrichment increased the frequency of selected alleles allowing large reductions in minimum population size for recovery of target genotypes (commonly around 90%) and/or selection at a greater number of loci. Increasing homozygosity by inbreeding from F2 to F2:3 also reduced population size by around 90% in some crosses with smaller incremental reductions in subsequent generations. Backcrossing was found to be a useful strategy to reduce population size compared with a biparental population where one parent contributed more target alleles than the other and was complementary to F2 enrichment and increasing homozygosity. Codominant markers removed the need for progeny testing reducing the number of individuals that had to be screened to identify a target genotype. However, although codominant markers allow target alleles to be fixed in early generations, minimum population sizes are often so large in F2 that it is not efficient to do so at this stage. Formulae and tables for calculating genotypic frequencies and minimum population sizes are provided to allow extension to different breeding systems, numbers of target loci, and probabilities of failure. Principles outlined are applicable to implementation of markers for both quantitative trait loci (QTL) and major genes.