About 1 million expressed sequence tag (EST) sequences comprising 125.3 Mb nucleotides were accreted from 51 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including abiotic stresses and pathogen challenges in common wheat (Triticum aestivum). Expressed sequence tags were assembled with stringent parameters after processing with inbuild scripts, resulting in 37,138 contigs and 215,199 singlets. In the assembled sequences, 10.6% presented no matches with existing sequences in public databases. Functional characterization of wheat unigenes by gene ontology annotation, mining transcription factors, full-length cDNA, and miRNA targeting sites were carried out. A bioinformatics strategy was developed to discover single-nucleotide polymorphisms (SNPs) within our large EST resource and reported the SNPs between and within (homoeologous) cultivars. Digital gene expression was performed to find the tissue-specific gene expression, and correspondence analysis was executed to identify common and specific gene expression by selecting four biotic stress-related libraries. The assembly and associated information cater a framework for future investigation in functional genomics.

Plasma membrane-associated and intracellular proteins and protein complexes play a pivotal role in pathogen recognition and disease resistance signaling in plants and animals. The two predominant protein families perceiving plant pathogens are receptor-like kinases and nucleotide binding-leucine-rich repeat receptors (NLR), which often confer race-specific resistance. Leaf rust is one of the most prevalent and most devastating wheat diseases. Here, we clone the race-specific leaf rust resistance gene Lr14a from hexaploid wheat. The cloning of Lr14a is aided by the recently published genome assembly of ArinaLrFor, an Lr14a-containing wheat line. Lr14a encodes a membrane-localized protein containing twelve ankyrin (ANK) repeats and structural similarities to Ca2+-permeable non-selective cation channels. Transcriptome analyses reveal an induction of genes associated with calcium ion binding in the presence of Lr14a. Haplotype analyses indicate that Lr14a-containing chromosome segments were introgressed multiple times into the bread wheat gene pool, but we find no variation in the Lr14a coding sequence itself. Our work demonstrates the involvement of an ANK-transmembrane (TM)-like type of gene family in race-specific disease resistance in wheat. This forms the basis to explore ANK-TM-like genes in disease resistance breeding.

The cloning of agriculturally important genes is often complicated by haplotype variation across crop cultivars. Access to pan-genome information greatly facilitates the assessment of structural variations and rapid candidate gene identification. Here, we identified the red glume 1 (Rg-B1) gene using association genetics and haplotype analyses in ten reference grade wheat genomes. Glume color is an important trait to characterize wheat cultivars. Red glumes are frequent among Central European spelt, a dominant wheat subspecies in Europe before the 20th century. We used genotyping-by-sequencing to characterize a global diversity panel of 267 spelt accessions, which provided evidence for two independent introductions of spelt into Europe. A single region at the Rg-B1 locus on chromosome 1BS was associated with glume color in the diversity panel. Haplotype comparisons across ten high-quality wheat genomes revealed a MYB transcription factor as candidate gene. We found extensive haplotype variation across the ten cultivars, with a particular group of MYB alleles that was conserved in red glume wheat cultivars. Genetic mapping and transient infiltration experiments allowed us to validate this particular MYB transcription factor variants. Our study demonstrates the value of multiple high-quality genomes to rapidly resolve copy number and haplotype variations in regions controlling agriculturally important traits.

Implementation of genomic tools is desirable to increase the efficiency of apple breeding. Recently, the multi-environment apple reference population (apple REFPOP) proved useful for rediscovering loci, estimating genomic predictive ability, and studying genotype by environment interactions (G × E). So far, only two phenological traits were investigated using the apple REFPOP, although the population may be valuable when dissecting genetic architecture and reporting predictive abilities for additional key traits in apple breeding. Here we show contrasting genetic architecture and genomic predictive abilities for 30 quantitative traits across up to six European locations using the apple REFPOP. A total of 59 stable and 277 location-specific associations were found using GWAS, 69.2% of which are novel when compared with 41 reviewed publications. Average genomic predictive abilities of 0.18-0.88 were estimated using main-effect univariate, main-effect multivariate, multi-environment univariate, and multi-environment multivariate models. The G × E accounted for up to 24% of the phenotypic variability. This most comprehensive genomic study in apple in terms of trait-environment combinations provided knowledge of trait biology and prediction models that can be readily applied for marker-assisted or genomic selection, thus facilitating increased breeding efficiency.

Introgressions of chromosomal segments from related species into wheat are important sources of resistance against fungal diseases. The durability and effectiveness of introgressed resistance genes upon agricultural deployment is highly variable-a phenomenon that remains poorly understood, as the corresponding fungal avirulence genes are largely unknown. Until its breakdown, the Pm17 resistance gene introgressed from rye to wheat provided broad resistance against powdery mildew (Blumeria graminis). Here, we used quantitative trait locus (QTL) mapping to identify the corresponding wheat mildew avirulence effector AvrPm17. It is encoded by two paralogous genes that exhibit signatures of reoccurring gene conversion events and are members of a mildew sublineage specific effector cluster. Extensive haplovariant mining in wheat mildew and related sublineages identified several ancient virulent AvrPm17 variants that were present as standing genetic variation in wheat powdery mildew prior to the Pm17 introgression, thereby paving the way for the rapid breakdown of the Pm17 resistance. QTL mapping in mildew identified a second genetic component likely corresponding to an additional resistance gene present on the 1AL.1RS translocation carrying Pm17. This gene remained previously undetected due to suppressed recombination within the introgressed rye chromosomal segment. We conclude that the initial effectiveness of 1AL.1RS was based on simultaneous introgression of two genetically linked resistance genes. Our results demonstrate the relevance of pathogen-based genetic approaches to disentangling complex resistance loci in wheat. We propose that identification and monitoring of avirulence gene diversity in pathogen populations become an integral part of introgression breeding to ensure effective and durable resistance in wheat.

Breeding for resistant crops is a sustainable way to control disease and relies on the introduction of novel resistance genes. Here, we tested three strategies on how to use transgenes from wheat to achieve durable resistance against fungal pathogens in the field. First, we tested the highly effective, overexpressed single transgene Pm3e in the background of spring wheat cultivar Bobwhite in a long-term field trial over many years. Together with previous results, this revealed that transgenic wheat line Pm3e#2 conferred complete powdery mildew resistance during a total of nine field seasons without a negative impact on yield. Furthermore, overexpressed Pm3e provided resistance to powdery mildew isolates from our worldwide collection when crossed into the elite wheat cultivar Fiorina. Second, we pyramided the four overexpressed transgenes Pm3a, Pm3b, Pm3d, and Pm3f in the background of cultivar Bobwhite and showed that the pyramided line Pm3a,b,d,f was completely resistant to powdery mildew in five field seasons. Third, we performed field trials with three barley lines expressing adult plant resistance gene Lr34 from wheat during three field seasons. Line GLP8 expressed Lr34 under control of the pathogen-inducible Hv-Ger4c promoter and provided partial barley powdery mildew and leaf rust resistance in the field with small, negative effects on yield components which might need compensatory breeding. Overall, our study demonstrates and discusses three successful strategies for achieving fungal disease resistance of wheat and barley in the field using transgenes from wheat. These strategies might confer long-term resistance if applied in a sustainable way.

The combined effects of enhanced total transgene expression level and allele-specificity combination in transgenic allele-pyramided Pm3 wheat lines result in improved powdery mildew field resistance without negative pleiotropic effects. Allelic Pm3 resistance genes of wheat confer race-specific resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) and encode nucleotide-binding domain, leucine-rich repeat (NLR) receptors. Transgenic wheat lines overexpressing alleles Pm3a, b, c, d, f, and g have previously been generated by transformation of cultivar Bobwhite and tested in field trials, revealing varying degrees of powdery mildew resistance conferred by the transgenes. Here, we tested four transgenic lines each carrying two pyramided Pm3 alleles, which were generated by crossbreeding of lines transformed with single Pm3 alleles. All four allele-pyramided lines showed strongly improved powdery mildew resistance in the field compared to their parental lines. The improved resistance results from the two effects of enhanced total transgene expression levels and allele-specificity combinations. In contrast to leaf segment tests on greenhouse-grown seedlings, no allelic suppression was observed in the field. Plant development and yield scores of the pyramided lines were similar to the mean scores of the corresponding parental lines, and thus, the allele pyramiding did not cause any negative effects. On the contrary, in pyramided line, Pm3b × Pm3f normal plant development was restored compared to the delayed development and reduced seed set of parental line Pm3f. Allele-specific RT qPCR revealed additive transgene expression levels of the two Pm3 alleles in the pyramided lines. A positive correlation between total transgene expression level and powdery mildew field resistance was observed. In summary, allele pyramiding of Pm3 transgenes proved to be successful in enhancing powdery mildew field resistance.

During the past decade, Sanger sequencing has been used to completely sequence hundreds of microbial and a few higher eukaryote genomes. In recent years, a number of alternative technologies became available, among them adaptations of the pyrosequencing procedure (i.e. "454 sequencing"), promising an approximately 100-fold increase in throughput over Sanger technology--an advancement which is needed to make large and complex genomes more amenable to full genome sequencing at affordable costs. Although several studies have demonstrated its potential usefulness for sequencing small and compact microbial genomes, it was unclear how the new technology would perform in large and highly repetitive genomes such as those of wheat or barley.

There is a large diversity of genetically defined resistance genes in bread wheat against the powdery mildew pathogen Blumeria graminis (B. g.) f. sp. tritici. Many confer race-specific resistance to this pathogen, but until now only the mildew avirulence gene AvrPm3a2/f2 that is recognized by Pm3a/f was known molecularly. We performed map-based cloning and genome-wide association studies to isolate a candidate for the mildew avirulence gene AvrPm2. We then used transient expression assays in Nicotiana benthamiana to demonstrate specific and strong recognition of AvrPm2 by Pm2. The virulent AvrPm2 allele arose from a conserved 12 kb deletion, while there is no protein sequence diversity in the gene pool of avirulent B. g. tritici isolates. We found one polymorphic AvrPm2 allele in B. g. triticale and one orthologue in B. g. secalis and both are recognized by Pm2. AvrPm2 belongs to a small gene family encoding structurally conserved RNase-like effectors, including Avra13 from B. g. hordei, the cognate Avr of the barley resistance gene Mla13. These results demonstrate the conservation of functional avirulence genes in two cereal powdery mildews specialized on different hosts, thus providing a possible explanation for successful introgression of resistance genes from rye or other grass relatives to wheat.

Iron and zinc deficiencies negatively impact human health worldwide. We developed wheat lines that meet or exceed recommended dietary target levels for iron and zinc in the grains. These lines represent useful germplasm for breeding new wheat varieties that can reduce iron and zinc deficiency-associated health burdens in the affected populations. Micronutrient deficiencies, including iron and zinc deficiencies, have negative impacts on human health globally. Iron-deficiency; anemia affects nearly two billion people worldwide and is the cause of reduced cognitive development, fatigue and overall low productivity. Similarly, zinc deficiency causes stunted growth, decreased immunity and increased risk of respiratory infections. Biofortification of staple crops is a sustainable and effective approach to reduce the burden of health problems associated with micronutrient deficiencies. Here, we developed wheat lines expressing rice NICOTIANAMINE SYNTHASE 2 (OsNAS2) and bean FERRITIN (PvFERRITIN) as single genes as well as in combination. NAS catalyzes the biosynthesis of nicotianamine (NA), which is a precursor of the iron chelator deoxymugeneic acid (DMA) required for long distance iron translocation. FERRITIN is important for iron storage in plants because it can store up to 4500 iron ions. We obtained significant increases of iron and zinc content in wheat grains of plants expressing either OsNAS2 or PvFERRTIN, or both genes. In particular, wheat lines expressing OsNAS2 greatly surpass the HarvestPlus recommended target level of 30 % dietary estimated average requirement (EAR) for iron, and 40 % of EAR for zinc, with lines containing 93.1 µg/g of iron and 140.6 µg/g of zinc in the grains. These wheat lines with dietary significant levels of iron and zinc represent useful germplasm for breeding new wheat varieties that can reduce micronutrient deficiencies in affected populations.

Routinely, fungal-fungal interactions (FFI) are studied on agar surfaces. However, this format restricts high-resolution dynamic imaging. To gain experimental access to FFI at the hyphal level in real-time, we developed a microfluidic platform, a FFI device. This device utilises microchannel geometry to enhance the visibility of hyphal growth and provides control channels to allow comparisons between localised and systemic effects. We demonstrate its function by investigating the FFI between the biological control agent (BCA) Clonostachys rosea and the plant pathogen Fusarium graminearum. Microscope image analyses confirm the inhibitory effect of the necrotrophic BCA and we show that a loss of fluorescence in parasitised hyphae of GFP-tagged F. graminearum coincides with the detection of GFP in mycelium of C. rosea. The versatility of our device to operate under both water-saturated and nutrient-rich as well as dry and nutrient-deficient conditions, coupled with its spatio-temporal output, opens new opportunities to study relationships between fungi.

Worldwide wheat production is under constant threat by fast-evolving fungal pathogens. In the last decades, wheat breeding for disease resistance heavily relied on the introgression of chromosomal segments from related species as genetic sources of new resistance. The Pm8 resistance gene against the powdery mildew disease has been introgressed from rye into wheat as part of a large 1BL.1RS chromosomal translocation encompassing multiple disease resistance genes and yield components. Due to its high agronomic value, this translocation has seen continuous global use since the 1960s on large growth areas, even after Pm8 resistance was overcome by the powdery mildew pathogen. The long-term use of Pm8 at a global scale provided the unique opportunity to study the consequences of such extensive resistance gene application on pathogen evolution.

A major QTL for angular leaf spot resistance in the common bean accession G5686 was fine-mapped to a region containing 36 candidate genes. Markers have been developed for marker-assisted selection. Common bean (Phaseolus vulgaris L.) is an important grain legume and an essential protein source for human nutrition in developing countries. Angular leaf spot (ALS) caused by the pathogen Pseudocercospora griseola (Sacc.) Crous and U. Braun is responsible for severe yield losses of up to 80%. Breeding for resistant cultivars is the most ecological and economical means to control ALS and is particularly important for yield stability in low-input agriculture. Here, we report on a fine-mapping approach of a major quantitative trait locus (QTL) ALS4.1(GS, UC) for ALS resistance in a mapping population derived from the resistant genotype G5686 and the susceptible cultivar Sprite. 180 F3 individuals of the mapping population were evaluated for ALS resistance and genotyped with 22 markers distributed over 11 genome regions colocating with previously reported QTL for ALS resistance. Multiple QTL analysis identified three QTL regions, including one major QTL on chromosome Pv04 at 43.7 Mbp explaining over 75% of the observed variation for ALS resistance. Additional evaluation of 153 F4, 89 BC1F2 and 139 F4/F5/BC1F3 descendants with markers in the region of the major QTL delimited the region to 418 kbp harboring 36 candidate genes. Among these, 11 serine/threonine protein kinases arranged in a repetitive array constitute promising candidate genes for controlling ALS resistance. Single nucleotide polymorphism markers cosegregating with the major QTL for ALS resistance have been developed and constitute the basis for marker-assisted introgression of ALS resistance into advanced breeding germplasm of common bean.

In the last hundred years, the development of improved wheat cultivars has led to the replacement of landraces and traditional varieties by modern cultivars. This has resulted in a decline in the genetic diversity of agriculturally used wheat. However, the diversity lost in the elite material is somewhat preserved in crop gene banks. Therefore, the gene bank accessions provide the basis for genetic improvement of crops for specific traits and and represent rich sources of novel allelic variation.

Using Roche/454 technology, we sequenced the chloroplast genomes of 12 Triticeae species, including bread wheat, barley and rye, as well as the diploid progenitors and relatives of bread wheat Triticum urartu, Aegilops speltoides and Ae. tauschii. Two wild tetraploid taxa, Ae. cylindrica and Ae. geniculata, were also included. Additionally, we incorporated wild Einkorn wheat Triticum boeoticum and its domesticated form T. monococcum and two Hordeum spontaneum (wild barley) genotypes. Chloroplast genomes were used for overall sequence comparison, phylogenetic analysis and dating of divergence times. We estimate that barley diverged from rye and wheat approximately 8-9 million years ago (MYA). The genome donors of hexaploid wheat diverged between 2.1-2.9 MYA, while rye diverged from Triticum aestivum approximately 3-4 MYA, more recently than previously estimated. Interestingly, the A genome taxa T. boeoticum and T. urartu were estimated to have diverged approximately 570,000 years ago. As these two have a reproductive barrier, the divergence time estimate also provides an upper limit for the time required for the formation of a species boundary between the two. Furthermore, we conclusively show that the chloroplast genome of hexaploid wheat was contributed by the B genome donor and that this unknown species diverged from Ae. speltoides about 980,000 years ago. Additionally, sequence alignments identified a translocation of a chloroplast segment to the nuclear genome which is specific to the rye/wheat lineage. We propose the presented phylogeny and divergence time estimates as a reference framework for future studies on Triticeae.

Crop breeding for resistance to pathogens largely relies on genes encoding receptors that confer race-specific immunity. Here, we report the identification of the wheat Pm4 race-specific resistance gene to powdery mildew. Pm4 encodes a putative chimeric protein of a serine/threonine kinase and multiple C2 domains and transmembrane regions, a unique domain architecture among known resistance proteins. Pm4 undergoes constitutive alternative splicing, generating two isoforms with different protein domain topologies that are both essential for resistance function. Both isoforms interact and localize to the endoplasmatic reticulum when co-expressed. Pm4 reveals additional diversity of immune receptor architecture to be explored for breeding and suggests an endoplasmatic reticulum-based molecular mechanism of Pm4-mediated race-specific resistance.

Identification of causal mutations in barley and wheat is hampered by their large genomes and suppressed recombination. To overcome these obstacles, we have developed MutChromSeq, a complexity reduction approach based on flow sorting and sequencing of mutant chromosomes, to identify induced mutations by comparison to parental chromosomes. We apply MutChromSeq to six mutants each of the barley Eceriferum-q gene and the wheat Pm2 genes. This approach unambiguously identified single candidate genes that were verified by Sanger sequencing of additional mutants. MutChromSeq enables reference-free forward genetics in barley and wheat, thus opening up their pan-genomes to functional genomics.

In plant and animal breeding, genomic prediction models are established to select new lines based on genomic data, without the need for laborious phenotyping. Prediction models can be trained on recent or historic phenotypic data and increasingly available genotypic data. This enables the adoption of genomic selection also in under-used legume crops such as common bean. Beans are an important staple food in the tropics and mainly grown by smallholders under limiting environmental conditions such as drought or low soil fertility. Therefore, genotype-by-environment interactions (G × E) are an important consideration when developing new bean varieties. However, G × E are often not considered in genomic prediction models nor are these models implemented in current bean breeding programs. Here we show the prediction abilities of four agronomic traits in common bean under various environmental stresses based on twelve field trials. The dataset includes 481 elite breeding lines characterized by 5,820 SNP markers. Prediction abilities over all twelve trials ranged between 0.6 and 0.8 for yield and days to maturity, respectively, predicting new lines into new seasons. In all four evaluated traits, the prediction abilities reached about 50-80% of the maximum accuracies given by phenotypic correlations and heritability. Predictions under drought and low phosphorus stress were up to 10 and 20% improved when G × E were included in the model, respectively. Our results demonstrate the potential of genomic selection to increase the genetic gain in common bean breeding. Prediction abilities improved when more phenotypic data was available and G × E could be accounted for. Furthermore, the developed models allowed us to predict genotypic performance under different environmental stresses. This will be a key factor in the development of common bean varieties adapted to future challenging conditions.

Grass powdery mildew (Blumeria graminis, Ascomycota) is a major pathogen of cereal crops and has become a model organism for obligate biotrophic fungal pathogens of plants. The sequenced genomes of two formae speciales (ff.spp.), B.g. hordei and B.g. tritici (pathogens of barley and wheat), were found to be enriched in candidate effector genes (CEGs). Similar to other filamentous pathogens, CEGs in B. graminis are under positive selection. Additionally, effectors are more likely to have presence-absence polymorphisms than other genes among different strains.

Blumeria graminis f. sp. tritici (B.g. tritici) is the causal agent of the wheat powdery mildew disease. The highly fragmented B.g. tritici genome available so far has prevented a systematic analysis of effector genes that are known to be involved in host adaptation. To study the diversity and evolution of effector genes we produced a chromosome-scale assembly of the B.g. tritici genome. The genome assembly and annotation was achieved by combining long-read sequencing with high-density genetic mapping, bacterial artificial chromosome fingerprinting and transcriptomics. We found that the 166.6 Mb B.g. tritici genome encodes 844 candidate effector genes, over 40% more than previously reported. Candidate effector genes have characteristic local genomic organization such as gene clustering and enrichment for recombination-active regions and certain transposable element families. A large group of 412 candidate effector genes shows high plasticity in terms of copy number variation in a global set of 36 isolates and of transcription levels. Our data suggest that copy number variation and transcriptional flexibility are the main drivers for adaptation in B.g. tritici. The high repeat content may play a role in providing a genomic environment that allows rapid evolution of effector genes with selection as the driving force.