Nitrogen deficiency tolerance conferred by introgression of a QTL derived from wild emmer into bread wheat.

KEY MESSAGE: Genetic dissection of a QTL from wild emmer wheat, QGpc.huj.uh-5B.2, introgressed into bread wheat, identified candidate genes associated with tolerance to nitrogen deficiency, and potentially useful for improving nitrogen-use efficiency. Nitrogen (N) is an important macronutrient critical to wheat growth and development; its deficiency is one of the main factors causing reductions in grain yield and quality. N availability is significantly affected by drought or flooding, that are dependent on additional factors including soil type or duration and severity of stress. In a previous study, we identified a high grain protein content QTL (QGpc.huj.uh-5B.2) derived from the 5B chromosome of wild emmer wheat, that showed a higher proportion of explained variation under water-stress conditions. We hypothesized that this QTL is associated with tolerance to N deficiency as a possible mechanism underlying the higher effect under stress. To validate this hypothesis, we introgressed the QTL into the elite bread wheat var. Ruta, and showed that under N-deficient field conditions the introgression IL99 had a 33% increase in GPC (p < 0.05) compared to the recipient parent. Furthermore, evaluation of IL99 response to severe N deficiency (10% N) for 14 days, applied using a semi-hydroponic system under controlled conditions, confirmed its tolerance to N deficiency. Fine-mapping of the QTL resulted in 26 homozygous near-isogenic lines (BC4F5) segregating to N-deficiency tolerance. The QTL was delimited from - 28.28 to - 1.29 Mb and included 13 candidate genes, most associated with N-stress response, N transport, and abiotic stress responses. These genes may improve N-use efficiency under severely N-deficient environments. Our study demonstrates the importance of WEW as a source of novel candidate genes for sustainable improvement in tolerance to N deficiency in wheat.

A membrane associated tandem kinase from wild emmer wheat confers broad-spectrum resistance to powdery mildew.

Crop wild relatives offer natural variations of disease resistance for crop improvement. Here, we report the isolation of broad-spectrum powdery mildew resistance gene Pm36, originated from wild emmer wheat, that encodes a tandem kinase with a transmembrane domain (WTK7-TM) through the combination of map-based cloning, PacBio SMRT long-read genome sequencing, mutagenesis, and transformation. Mutagenesis assay reveals that the two kinase domains and the transmembrane domain of WTK7-TM are critical for the powdery mildew resistance function. Consistently, in vitro phosphorylation assay shows that two kinase domains are indispensable for the kinase activity of WTK7-TM. Haplotype analysis uncovers that Pm36 is an orphan gene only present in a few wild emmer wheat, indicating its single ancient origin and potential contribution to the current wheat gene pool. Overall, our findings not only provide a powdery mildew resistance gene with great potential in wheat breeding but also sheds light into the mechanism underlying broad-spectrum resistance.

Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69.

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

Dosage differences in 12-OXOPHYTODIENOATE REDUCTASE genes modulate wheat root growth.

Wheat, an essential crop for global food security, is well adapted to a wide variety of soils. However, the gene networks shaping different root architectures remain poorly understood. We report here that dosage differences in a cluster of monocot-specific 12-OXOPHYTODIENOATE REDUCTASE genes from subfamily III (OPRIII) modulate key differences in wheat root architecture, which are associated with grain yield under water-limited conditions. Wheat plants with loss-of-function mutations in OPRIII show longer seminal roots, whereas increased OPRIII dosage or transgenic over-expression result in reduced seminal root growth, precocious development of lateral roots and increased jasmonic acid (JA and JA-Ile). Pharmacological inhibition of JA-biosynthesis abolishes root length differences, consistent with a JA-mediated mechanism. Transcriptome analyses of transgenic and wild-type lines show significant enriched JA-biosynthetic and reactive oxygen species (ROS) pathways, which parallel changes in ROS distribution. OPRIII genes provide a useful entry point to engineer root architecture in wheat and other cereals.

Intracellular Reactive Oxygen Species-Aided Localized Cell Death Contributing to Immune Responses Against Wheat Powdery Mildew Pathogen.

Reactive oxygen species (ROS)- and hypersensitive response (HR)-mediated cell death have long been known to play critical roles in plant immunity to pathogens. Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a destructive wheat pathogen. Here, we report a quantitative analysis of the proportion of infected cells with local apoplastic ROS (apoROS) versus intracellular ROS (intraROS) accumulation in various wheat accessions that carry different disease resistance genes (R genes) at a series of time points postinfection. The proportion of apoROS accumulation was 70 to 80% of the infected wheat cells detected in both compatible and incompatible host-pathogen interactions. However, intensive intraROS accumulation followed by localized cell death responses was detected in 11 to 15% of the infected wheat cells, mainly in wheat lines that carried nucleotide-binding leucine-rich repeat R genes (e.g., Pm3F, Pm41, TdPm60, MIIW72, and Pm69). The lines that carry unconventional R genes, Pm24 (Wheat Tandem Kinase 3) and pm42 (a recessive R gene), showed fewer intraROS responses, whereas 11% of Pm24 line-infected epidermis cells still showed HR cell death, suggesting that different resistance pathways are activated there. Here, we also demonstrated that ROS could not act as a strong systemic signal for inducing high resistance to Bgt in wheat, although it induced the expression of pathogenesis-related genes. These results provide new insights into the contribution of intraROS and localized cell death to immune responses against wheat powdery mildew.

Genome-Wide Association Study in Bread Wheat Identifies Genomic Regions Associated with Grain Yield and Quality under Contrasting Water Availability.

The objectives of this study were to identify genetic loci in the bread wheat genome that would influence yield stability and quality under water stress, and to identify accessions that can be recommended for cultivation in dry and hot regions. We performed a genome-wide association study (GWAS) using a panel of 232 wheat accessions spanning diverse ecogeographic regions. Plants were evaluated in the Israeli Northern Negev, under two environments: water-limited (D; 250 mm) and well-watered (W; 450 mm) conditions; they were genotyped with ~71,500 SNPs derived from exome capture sequencing. Of the 14 phenotypic traits evaluated, 12 had significantly lower values under D compared to W conditions, while the values for two traits were higher under D. High heritability (H2 = 0.5-0.9) was observed for grain yield, spike weight, number of grains per spike, peduncle length, and plant height. Days to heading and grain yield could be partitioned based on accession origins. GWAS identified 154 marker-trait associations (MTAs) for yield and quality-related traits, 82 under D and 72 under W, and identified potential candidate genes. We identified 24 accessions showing high and/or stable yields under D conditions that can be recommended for cultivation in regions under the threat of global climate change.

Discovery of stripe rust resistance with incomplete dominance in wild emmer wheat using bulked segregant analysis sequencing.

Durable crop disease resistance is an essential component of global food security. Continuous pathogen evolution leads to a breakdown of resistance and there is a pressing need to characterize new resistance genes for use in plant breeding. Here we identified an accession of wild emmer wheat (Triticum turgidum ssp. dicoccoides), PI 487260, that is highly resistant to multiple stripe rust isolates. Genetic analysis revealed resistance was conferred by a single, incompletely dominant gene designated as Yr84. Through bulked segregant analysis sequencing (BSA-Seq) we identified a 52.7 Mb resistance-associated interval on chromosome 1BS. Detected variants were used to design genetic markers for recombinant screening, further refining the interval of Yr84 to a 2.3-3.3 Mb in tetraploid wheat genomes. This interval contains 34 candidate genes encoding for protein domains involved in disease resistance responses. Furthermore, KASP markers closely-linked to Yr84 were developed to facilitate marker-assisted selection for rust resistance breeding.

Capturing Wheat Phenotypes at the Genome Level.

Recent technological advances in next-generation sequencing (NGS) technologies have dramatically reduced the cost of DNA sequencing, allowing species with large and complex genomes to be sequenced. Although bread wheat (Triticum aestivum L.) is one of the world's most important food crops, efficient exploitation of molecular marker-assisted breeding approaches has lagged behind that achieved in other crop species, due to its large polyploid genome. However, an international public-private effort spanning 9 years reported over 65% draft genome of bread wheat in 2014, and finally, after more than a decade culminated in the release of a gold-standard, fully annotated reference wheat-genome assembly in 2018. Shortly thereafter, in 2020, the genome of assemblies of additional 15 global wheat accessions was released. As a result, wheat has now entered into the pan-genomic era, where basic resources can be efficiently exploited. Wheat genotyping with a few hundred markers has been replaced by genotyping arrays, capable of characterizing hundreds of wheat lines, using thousands of markers, providing fast, relatively inexpensive, and reliable data for exploitation in wheat breeding. These advances have opened up new opportunities for marker-assisted selection (MAS) and genomic selection (GS) in wheat. Herein, we review the advances and perspectives in wheat genetics and genomics, with a focus on key traits, including grain yield, yield-related traits, end-use quality, and resistance to biotic and abiotic stresses. We also focus on reported candidate genes cloned and linked to traits of interest. Furthermore, we report on the improvement in the aforementioned quantitative traits, through the use of (i) clustered regularly interspaced short-palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene-editing and (ii) positional cloning methods, and of genomic selection. Finally, we examine the utilization of genomics for the next-generation wheat breeding, providing a practical example of using in silico bioinformatics tools that are based on the wheat reference-genome sequence.

Functional characterization of powdery mildew resistance gene MlIW172, a new Pm60 allele and its allelic variation in wild emmer wheat.

Wild emmer wheat (Triticum dicoccoides, WEW) is an immediate progenitor of both the cultivated tetraploid and hexaploid wheats and it harbors rich genetic diversity against powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt). A powdery mildew resistance gene MlIW172 originated from WEW accession IW172 (G-797-M) is fine mapped in a 0.048 centimorgan (cM) genetic interval on 7AL, corresponding to a genomic region spanning 233 kb, 1 Mb and 800 kb in Chinese Spring, WEW Zavitan, and T. urartu G1812, respectively. MlIW172 encodes a typical NLR protein NLRIW172 and physically locates in an NBS-LRR gene cluster. NLRIW172 is subsequently identified as a new allele of Pm60, and its function is validated by EMS mutagenesis and transgenic complementation. Haplotype analysis of the Pm60 alleles reveals diversifications in sequence variation in the locus and presence and absence variations (PAV) in WEW populations. Four common single nucleotide variations (SNV) are detected between the Pm60 alleles from WEW and T. urartu, indicative of speciation divergence between the two different wheat progenitors. The newly identified Pm60 alleles and haplotypes in WEW are anticipated to be valuable for breeding powdery mildew resistance wheat cultivars via marker-assisted selection.

Erratum: Rybnikov et al. Selection for Plastic, Pathogen-Inducible Recombination in a Red Queen Model with Diploid Antagonists. Pathogens 2021, 10, 898.

In the original article, there was a mistake published in Figure 3 [...].

Selection for Plastic, Pathogen-Inducible Recombination in a Red Queen Model with Diploid Antagonists.

Antagonistic interactions and co-evolution between a host and its parasite are known to cause oscillations in the population genetic structure of both species (Red Queen dynamics). Potentially, such oscillations may select for increased sex and recombination in the host, although theoretical models suggest that this happens under rather restricted values of selection intensity, epistasis, and other parameters. Here, we explore a model in which the diploid parasite succeeds to infect the diploid host only if their phenotypes at the interaction-mediating loci match. Whenever regular oscillations emerge in this system, we test whether plastic, pathogen-inducible recombination in the host can be favored over the optimal constant recombination. Two forms of the host recombination dependence on the parasite pressure were considered: either proportionally to the risk of infection (prevention strategy) or upon the fact of infection (remediation strategy). We show that both forms of plastic recombination can be favored, although relatively infrequently (up to 11% of all regimes with regular oscillations, and up to 20% of regimes with obligate parasitism). This happens under either strong overall selection and high recombination rate in the host, or weak overall selection and low recombination rate in the host. In the latter case, the system's dynamics are considerably more complex. The prevention strategy is favored more often than the remediation one. It is noteworthy that plastic recombination can be favored even when any constant recombination is rejected, making plasticity an evolutionary mechanism for the rescue of host recombination.

Tandem Protein Kinases Emerge as New Regulators of Plant Immunity.

Plant-pathogen interactions result in disease development in a susceptible host. Plants actively resist pathogens via a complex immune system comprising both surface-localized receptors that sense the extracellular space as well as intracellular receptors recognizing pathogen effectors. To date, the majority of cloned resistance genes encode intracellular nucleotide-binding leucine-rich repeat receptor proteins. Recent discoveries have revealed tandem kinase proteins (TKPs) as another important family of intracellular proteins involved in plant immune responses. Five TKP genes-barley Rpg1 and wheat WTK1 (Yr15), WTK2 (Sr60), WTK3 (Pm24), and WTK4-protect against devastating fungal diseases. Moreover, a large diversity and numerous putative TKPs exist across the plant kingdom. This review explores our current knowledge of TKPs and serves as a basis for future studies that aim to develop and exploit a deeper understanding of innate plant immunity receptor proteins.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

TdPm60 identified in wild emmer wheat is an ortholog of Pm60 and constitutes a strong candidate for PmG16 powdery mildew resistance.

KEY MESSAGE: We identified TdPm60 alleles from wild emmer wheat (WEW), an ortholog of Pm60 from T. urartu, which constitutes a strong candidate for PmG16 mildew resistance. Deployment of PmG16 in Israeli modern bread wheat cultivar Ruta improved the resistance to several local Bgt isolates. Wild emmer wheat (WEW), the tetraploid progenitor of durum and bread wheat, is a valuable genetic resource for resistance to powdery mildew fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). PmG16 gene, derived from WEW, confers high resistance to most tested Bgt isolates. We mapped PmG16 to a 1.4-cM interval between the flanking markers uhw386 and uhw390 on Chromosome 7AL. Based on gene annotation of WEW reference genome Zavitan_V1, 34 predicted genes were identified within the ~ 3.48-Mb target region. Six genes were annotated as associated with disease resistance, of which TRIDC7AG077150.1 was found to be highly similar to Pm60, previously cloned from Triticum urartu, and resides in the same syntenic region. The functional molecular marker (FMM) for Pm60 (M-Pm60-S1) co-segregated with PmG16, suggesting the Pm60 ortholog from WEW (designated here as TdPm60) as a strong candidate for PmG16. Sequence alignment identified only eight SNPs that differentiate between TdPm60 and TuPm60. Furthermore, TdPm60 was found to be present also in the WEW donor lines of the powdery mildew resistance genes MlIW172 and MlIW72, mapped to the same region of Chromosome 7AL as PmG16, suggesting that TdPm60 constitutes a candidate also for these genes. Furthermore, screening of additional 230 WEW accessions with Pm60 specific markers revealed 58 resistant accessions from the Southern Levant that harbored TdPm60, while none of the susceptible accessions showed the presence of this gene. Deployment of PmG16 in Israeli modern bread wheat cultivar Ruta conferred resistance against several local Bgt isolates.

Genomic Architecture of Phenotypic Plasticity in Response to Water Stress in Tetraploid Wheat.

Phenotypic plasticity is one of the main mechanisms of adaptation to abiotic stresses via changes in critical developmental stages. Altering flowering phenology is a key evolutionary strategy of plant adaptation to abiotic stresses, to achieve the maximum possible reproduction. The current study is the first to apply the linear regression residuals as drought plasticity scores while considering the variation in flowering phenology and traits under non-stress conditions. We characterized the genomic architecture of 17 complex traits and their drought plasticity scores for quantitative trait loci (QTL) mapping, using a mapping population derived from a cross between durum wheat (Triticum turgidum ssp. durum) and wild emmer wheat (T. turgidum ssp. dicoccoides). We identified 79 QTLs affected observed traits and their plasticity scores, of which 33 reflected plasticity in response to water stress and exhibited epistatic interactions and/or pleiotropy between the observed and plasticity traits. Vrn-B3 (TaTF1) residing within an interval of a major drought-escape QTL was proposed as a candidate gene. The favorable alleles for most of the plasticity QTLs were contributed by wild emmer wheat, demonstrating its high potential for wheat improvement. Our study presents a new approach for the quantification of plant adaptation to various stresses and provides new insights into the genetic basis of wheat complex traits under water-deficit stress.

Structural rearrangements in wheat (1BS)-rye (1RS) recombinant chromosomes affect gene dosage and root length.

Good understanding of the genes controlling root development is required to engineer root systems better adapted to different soil types. In wheat (Triticum aestivum L.), the 1RS.1BL wheat-rye (Secale cereale L.) translocation has been associated with improved drought tolerance and a large root system. However, an isogenic line carrying an interstitial segment from wheat chromosome arm 1BS in the distal region of the 1RS arm (1RSRW ) showed reduced grain yield and shorter roots both in the field and in hydroponic cultures relative to isogenic lines with the complete 1RS arm. In this study, we used exome capture to characterize 1RSRW and its parental lines T-9 and 1B+40. We show that 1RSRW has a 7.0 Mb duplicated 1RS region and a 4.8 Mb 1BS insertion colinear with the 1RS duplication, resulting in triplicated genes. Lines homozygous for 1RSRW have short seminal roots, while lines heterozygous for this chromosome have roots of intermediate length. By contrast, near-isogenic lines carrying only the 1BS distal region or the 1RS-1BS duplication have long seminal roots similar to 1RS, suggesting a limited effect of the 1BS genes. These results suggest that the dosage of duplicated 1RS genes is critical for seminal root length. An induced deletion encompassing 38 orthologous wheat and rye duplicated genes restored root length and confirmed the importance of gene dosage in the short-root phenotype. We explored the expression profiles and functional annotation of these genes and discuss their potential as candidate genes for the regulation of seminal root length in wheat.

Introgression of the Powdery Mildew Resistance Genes Pm60 and Pm60b from Triticum urartu to Common Wheat Using Durum as a 'Bridge'.

Powdery mildew, caused by the fungus Blumeria graminis f. sp. tritici (Bgt), has limited wheat yields in many major wheat-production areas across the world. Introducing resistance genes from wild relatives into cultivated wheat can enrich the genetic resources for disease resistance breeding. The powdery mildew resistance gene Pm60 was first identified in diploid wild wheat Triticum urartu (T. urartu). In this study, we used durum as a 'bridge' approach to transfer Pm60 and Pm60b into hexaploid common wheat. Synthetic hexaploid wheat (SHW, AABBAuAu), developed by crossing T. urartu (AuAu) with durum (AABB), was used for crossing and backcrossing with common wheat. The Pm60 alleles were tracked by molecular markers and the resistance to powdery mildew. From BC1F1 backcross populations, eight recombinant types were identified based on five Pm60-flanking markers, which indicated different sizes of the introgressed chromosome segments from T. urartu. Moreover, we have selected two resistance-harboring introgression lines with high self-fertility, which could be easily used in wheat breeding system. Our results showed that the durum was an excellent 'bridge' for introducing the target gene from diploid T. urartu into the hexaploid cultivated wheat. Moreover, these introgression lines could be deployed in wheat resistance breeding programs, together with the assistance of the molecular markers for Pm60 alleles.