Harbans Bariana

The Gene Sr33, an Ortholog of Barley Mla Genes, Encodes Resistance to Wheat Stem Rust Race Ug99

Resistance May Not Be Futile Recently, Ug99, a particularly devastating strain of wheat stem rust fungus, has emerged, which could potentially threaten food security. Now, two genes have been cloned that offer resistance to Ug99. Saintenac et al. (p. 783, published online 27 June) cloned Sr35 from Triticum monococcum, a diploid wheat species not often cultivated. Periyannan et al. (p. 786, published online 27 June) cloned Sr33 from Aegilops tauschii, a diploid wild grass that contributed to the hexaploid genome of cultivated wheat. The genes both encode proteins that show features typical of other disease resistance proteins and offer opportunities to slow the pace of Ug99 progression. Two resistance genes are identified that could protect wheat from a virulent fungus that can severely reduce crop yields. Wheat stem rust, caused by the fungus Puccinia graminis f. sp. tritici, afflicts bread wheat (Triticum aestivum). New virulent races collectively referred to as “Ug99” have emerged, which threaten global wheat production. The wheat gene Sr33, introgressed from the wild relative Aegilops tauschii into bread wheat, confers resistance to diverse stem rust races, including the Ug99 race group. We cloned Sr33, which encodes a coiled-coil, nucleotide-binding, leucine-rich repeat protein. Sr33 is orthologous to the barley (Hordeum vulgare) Mla mildew resistance genes that confer resistance to Blumeria graminis f. sp. hordei. The wheat Sr33 gene functions independently of RAR1, SGT1, and HSP90 chaperones. Haplotype analysis from diverse collections of Ae. tauschii placed the origin of Sr33 resistance near the southern coast of the Caspian Sea.

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.

Implementation of markers in Australian wheat breeding

Genetic associations of morphological, biochemical, and DNA markers with economically important traits can be used for indirect selection of the traits. Chromosomal linkage between pseudo-black chaff and the stem-rust resistance gene Sr2, and between the red glume gene (Rg1) and the stripe rust resistance gene Yr10, have been used in this way for many years. Similarly, linkages between disease resistance genes, such as Sr38, Lr37, and Yr17, have been used to achieve resistance to multiple diseases while selection is performed for resistance to one disease. Alleles at the Glu loci, assessed as protein differences, have been used as predictors of dough strength. More recently, DNA markers have been developed and used, especially to select for resistance to cereal cyst nematode, a trait which is difficult and expensive to assess with conventional bioassays. We found that the major use of DNA markers was for selection for traits of substantial economic importance, which were primarily determined by a single gene, and where the non-marker assay was expensive and unreliable. The other uses of markers were for pyramiding several genes influencing one trait, or for rapid backcrossing.

Mapping of durable adult plant and seedling resistances to stripe rust and stem rust diseases in wheat

Doubled haploid populations of CD87/Katepwa, Cranbrook/Halberd, and Sunco/Tasman were assessed for seedling response to stem rust and stripe rust. The CD87/Katepwa population was also screened as adult plants in the field against stripe rust. The respective parents differed in presence or absence of various stem rust and stripe rust resistance genes. At least 4 resistance loci controlled adult plant resistance to stripe rust in the CD87/Katepwa population, and based on quantitative trait loci mapping results, two of these were contributed by CD87. Pedigree information indicated that these regions correspond to durable adult plant stripe rust resistance genes Yr18 and Yr29. Yr29 was mapped to the distal region of chromosome 1BL. The third gene, contributed by Katepwa, YrKat, was located in chromosome arm 2DS. Sr30 mapped distal to markers abg3 and P36/M61-170 in chromosome arm 5DL. Genes Yr7 and Pbc (completely linked with durable stem rust resistance gene Sr2) showed close associations with markers in chromosome arms 2BL and 3BS, respectively. A distally located genomic region in chromosome 6AS also affected the expression of Pbc. The temperature-sensitive stripe rust resistance gene, YrCK, carried by Sunco showed monogenic inheritance and was located in chromosome arm 2DS. Several markers showed complete association with Triticum timopheevi derived stem rust resistance gene Sr36. Microsatellite markers stm773 and gwm271A were validated on a set of wheat genotypes and were found to be diagnostic for the detection of Sr36. TheSr36-linked Xstm773 allele showed better amplification than the Sr36-linked Xgwm271A allele. These markers could be used for marker assisted identification of Sr36 in breeding populations.

A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat

As there are numerous pathogen species that cause disease and limit yields of crops, such as wheat (Triticum aestivum), single genes that provide resistance to multiple pathogens are valuable in crop improvement. The mechanistic basis of multi-pathogen resistance is largely unknown. Here we use comparative genomics, mutagenesis and transformation to isolate the wheat Lr67 gene, which confers partial resistance to all three wheat rust pathogen species and powdery mildew. The Lr67 resistance gene encodes a predicted hexose transporter (LR67res) that differs from the susceptible form of the same protein (LR67sus) by two amino acids that are conserved in orthologous hexose transporters. Sugar uptake assays show that LR67sus, and related proteins encoded by homeoalleles, function as high-affinity glucose transporters. LR67res exerts a dominant-negative effect through heterodimerization with these functional transporters to reduce glucose uptake. Alterations in hexose transport in infected leaves may explain its ability to reduce the growth of multiple biotrophic pathogen species.

Breeding triple rust resistant wheat cultivars for Australia using conventional and marker-assisted selection technologies

Stem rust susceptibility of European wheats under Australian conditions posed a significant threat to wheat production for the early British settlers in Australia. The famous Australian wheat breeder, William Farrer, tackled the problem of stem rust susceptibility through breeding fast-maturing wheat cultivars. South-eastern Australia suffered a severe stem rust epidemic in 1973, which gave rise to a national approach to breeding for rust resistance. The National Wheat Rust Control Program was set up in 1975, modelled on the University of Sydney’s own rust resistance breeding program, at the University of Sydney Plant Breeding Institute, Castle Hill (now Cobbitty). Back-crossing of a range of sources of resistance provided genetically diverse germplasm for evaluation in various breeding programs. Current efforts are directed to building gene combinations through marker-assisted selection. Major genes for resistance to stem rust and leaf rust are being used in the back-crossing program of the ACRCP to create genetic diversity among Australian germplasm. Stripe rust and to a lesser extent leaf rust resistance in the Australian germplasm is largely based on combinations of adult plant resistance genes and our knowledge of their genomic locations has increased. Additional genes, other than Yr18/Lr34 and Yr29/Lr46, appeared to control adult plant resistance to both leaf rust and stripe rust. Two adult-plant stem rust resistance genes have also been identified. The development of selection technologies to achieve genotype-based selection of resistance gene combinations in the absence of bioassays has evolved in the last 5 years. Robust molecular markers are now available for several commercially important rust resistance genes. Marker-assisted selection for rust resistance is performed routinely in many wheat-breeding programs. Modified pedigree and limited back-cross methods have been used for breeding rust-resistant wheat cultivars in the University of Sydney wheat-breeding program. The single back-cross methodology has proved more successful in producing cultivars with combinations of adult plant resistance genes.

Validation of molecular markers for wheat breeding

Five sets of markers were assessed for their usefulness in breeding, two linked to wheat stem rust gene Sr2, several markers linked to a chromosome segment conferring Yr17/Lr37/Sr38 resistance, two reported markers for the linked genes Lr35 andSr39, one for Lr28, and one linked to flour colour. The gene for Sr2 confers adult plant resistance to stem rust (Puccinia graminis f.sp. tritici) and was originally transferred to bread wheat from the tetraploid emmer (‘Yaroslav’) to the cultivars Hope and H-44. The gene is located on the short arm of chromosome 3B and confers a durable adult plant resistance to stem rust usually expressed only in the field. The chromosome segment carrying the Lr37, Sr38, Yr17 resistance genes is located on 2AS and was originally introduced into wheat through an Aegilops ventricosa Triticum persicum cross, followed by a cross to the cultivar Marne (VPM1). The flour colour quantitative trait locus was originally described in a Yarralinka Schomburg cross and is located on chromosome 7A. The primers as originally developed required optimisation for more routine use in a breeding program.

A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes.

BackgroundBread wheat is an allopolyploid species with a large, highly repetitive genome. To investigate the impact of selection on variants distributed among homoeologous wheat genomes and to build a foundation for understanding genotype-phenotype relationships, we performed population-scale re-sequencing of a diverse panel of wheat lines.ResultsA sample of 62 diverse lines was re-sequenced using the whole exome capture and genotyping-by-sequencing approaches. We describe the allele frequency, functional significance, and chromosomal distribution of 1.57 million single nucleotide polymorphisms and 161,719 small indels. Our results suggest that duplicated homoeologous genes are under purifying selection. We find contrasting patterns of variation and inter-variant associations among wheat genomes; this, in addition to demographic factors, could be explained by differences in the effect of directional selection on duplicated homoeologs. Only a small fraction of the homoeologous regions harboring selected variants overlapped among the wheat genomes in any given wheat line. These selected regions are enriched for loci associated with agronomic traits detected in genome-wide association studies.ConclusionsEvidence suggests that directional selection in allopolyploids rarely acted on multiple parallel advantageous mutations across homoeologous regions, likely indicating that a fitness benefit could be obtained by a mutation at any one of the homoeologs. Additional advantageous variants in other homoelogs probably either contributed little benefit, or were unavailable in populations subjected to directional selection. We hypothesize that allopolyploidy may have increased the likelihood of beneficial allele recovery by broadening the set of possible selection targets.

Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture

Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5–15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.

The successful application of a marker-assisted wheat breeding strategy

A number of useful marker-trait associations have been reported for wheat. However the number of publications detailing the integrated and pragmatic use of molecular markers in wheat breeding is limited. A previous report by some of these authors showed how marker-assisted selection could increase the genetic gain and economic efficiency of a specific breeding strategy. Here, we present a practical validation of that study. The target of this breeding strategy was to produce wheat lines derived from an elite Australian cultivar ‘Stylet’, with superior dough properties and durable rust resistance donated from ‘Annuello’. Molecular markers were used to screen a BC1F1 population produced from a cross between the recurrent parent ‘Stylet’ and the donor parent ‘Annuello’ for the presence of rust resistance genes Lr34/Yr18 and Lr46/Yr29. Following this, marker-assisted selection was applied to haploid plants, prior to chromosome doubling with cochicine, for the rust resistance genes Lr24/Sr24, Lr34/Yr18, height reducing genes, and for the grain protein genes Glu-D1 and Glu-A3. In general, results from this study agreed with those of the simulation study. Genetic improvement for rust resistance was greatest when marker selection was applied on BC1F1 individuals. Introgression of both the Lr34/Yr18 and Lr46/Yr29 loci into the susceptible recurrent parent background resulted in substantial improvement in leaf rust and stripe rust resistance levels. Selection for favourable glutenin alleles significantly improved dough resistance and dough extensibility. Marker-assisted selection for improved grain yield, through the selection of recurrent parent genome using anonymous markers, only marginally improved grain yield at one of the five sites used for grain yield assessment. In summary, the integration of marker-assisted selection for specific target genes, particularly at the early stages of a breeding programme, is likely to substantially increase genetic improvement in wheat.