Genome-Wide Association Mapping of Grain Micronutrients Concentration in Aegilops tauschii

Bread wheat is an important and the most consumed cereal worldwide. However, people with predominantly cereal-based diets are increasingly affected by micronutrient deficiencies, suggesting the need for biofortified wheat varieties. The limited genetic diversity in hexaploid wheat warrants exploring the wider variation present in wheat wild relatives, among these Aegilops tauschii, the wild progenitor of the bread wheat D genome. In this study, a panel of 167 Ae. tauschii accessions was phenotyped for grain Fe, Zn, Cu, and Mn concentrations for 3 years and was found to have wide variation for these micronutrients. Comparisons between the two genetic subpopulations of Ae. tauschii revealed that lineage 2 had higher mean values for Fe and Cu concentration than lineage 1. To identify potentially new genetic sources for improving grain micronutrient concentration, we performed a genome-wide association study (GWAS) on 114 non-redundant Ae. tauschii accessions using 5,249 genotyping-by-sequencing (GBS) markers. Best linear unbiased predictor (BLUP) values were calculated for all traits across the three growing seasons. A total of 19 SNP marker trait associations (MTAs) were detected for all traits after applying Bonferroni corrected threshold of -log10(P-value) ≥ 4.68. These MTAs were found on all seven chromosomes. For grain Fe, Zn, Cu, and Mn concentrations, five, four, three, and seven significant associations were detected, respectively. The associations were linked to the genes encoding transcription factor regulators, transporters, and phytosiderophore synthesis. The results demonstrate the utility of GWAS for understanding the genetic architecture of micronutrient accumulation in Ae. tauschii, and further efforts to validate these loci will aid in using them to diversify the D-genome of hexaploid wheat

Diversity, detection and exploitation: linking soil fungi and plant disease

Plant-associated fungi are incredibly diverse, comprising over a million species of mycorrhiza, endophytes, saprophytes and pathogens worldwide. This diverse fungal community is highly important for plant health. Many fungi are effective biocontrol agents that can kill or suppress fungal pathogens, with pathogen biocontrol found for both individual microorganisms and plant-associated fungal consortia. Meanwhile, increased plant community diversity aboveground corresponds to an increase in below-ground fungal community diversity, which contributes in turn to improved rhizosphere soil health and pathogen suppression. In this review, we discuss the role of fungal diversity in soil health and plant disease suppression and the various mechanisms by which mycorrhizal and endophytic fungi combat plant pathogenic fungi. We also discuss the array of diagnostic tools, both well-established and newly developed, which are revolutionising fungal pathogen detection and rhizosphere community analysis

Mutagenesis of Puccinia graminis f. sp. tritici and selection of gain-of-virulence mutants

Wheat stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance (Sr) genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each Sr gene must be confirmed after incorporation into an Sr-gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single Avr effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned Pgt Avrs. To accelerate Avr gene cloning, we outline a procedure to develop a mutant population of Pgt spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given Avr gene. We inoculated the mutant library onto plants containing Sr43, Sr44, or Sr45 and obtained 9, 4, and 14 mutants with virulence toward Sr43, Sr44, or Sr45, respectively. However, only mutants identified on Sr43 and Sr45 maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward Sr43. These also maintained their virulence profile on the stem rust international differential set containing 20 Sr genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance (R) genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several R genes without the need for multiple rounds of spore multiplication and mutagenesis

The wheat Sr22, Sr33, Sr35 and Sr45 genes confer resistance against stem rust in barley

In the last 20 years, stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), has re-emerged as a major threat to wheat and barley production in Africa and Europe. In contrast to wheat with 60 designated stem rust (Sr) resistance genes, barley’s genetic variation for stem rust resistance is very narrow with only ten resistance genes genetically identified. Of these, only one complex locus consisting of three genes is effective against TTKSK, a widely virulent Pgt race of the Ug99 tribe which emerged in Uganda in 1999 and has since spread to much of East Africa and parts of the Middle East. The objective of this study was to assess the functionality, in barley, of cloned wheat Sr genes effective against race TTKSK. Sr22, Sr33, Sr35 and Sr45 were transformed into barley cv. Golden Promise using Agrobacterium-mediated transformation. All four genes were found to confer effective stem rust resistance. The barley transgenics remained susceptible to the barley leaf rust pathogen Puccinia hordei, indicating that the resistance conferred by these wheat Sr genes was specific for Pgt. Furthermore, these transgenic plants did not display significant adverse agronomic effects in the absence of disease. Cloned Sr genes from wheat are therefore a potential source of resistance against wheat stem rust in barley

Resistance gene cloning from a wild crop relative by sequence capture and association genetics

Disease resistance (R) genes from wild relatives could be used to engineer broad-spectrum resistance in domesticated crops. We combined association genetics with R gene enrichment sequencing (AgRenSeq) to exploit pan-genome variation in wild diploid wheat and rapidly clone four stem rust resistance genes. AgRenSeq enables R gene cloning in any crop that has a diverse germplasm panel

A wheat kinase and immune receptor form host-specificity barriers against the blast fungus

Since emerging in Brazil in 1985, wheat blast has spread throughout South America and recently appeared in Bangladesh and Zambia. Here we show that two wheat resistance genes, Rwt3 and Rwt4, acting as host-specificity barriers against non-Triticum blast pathotypes encode a nucleotide-binding leucine-rich repeat immune receptor and a tandem kinase, respectively. Molecular isolation of these genes will enable study of the molecular interaction between pathogen effector and host resistance genes

Genome-Wide Association Study of Grain Architecture in Wild Wheat Aegilops tauschii

Aegilops tauschii, the D-genome progenitor of Triticum aestivum, encompasses huge diversity for various traits of potential economic importance such as yield, biotic and abiotic stress tolerance, quality and nutrition. In the present study, variation for grain size in Ae. tauschii germplasm was studied and its genetic basis dissected using genome-wide association study (GWAS). Grain length, width, and weight evaluated in 177 Ae. tauschii accessions over 3 years showed near normal distribution with 1.74-, 1.75-, and 2.82-fold variation, respectively. These lines were genetically characterized using genotyping-by-sequencing (GBS) protocol that produced 11,489 single nucleotide polymorphic (SNP) markers. Genetic diversity analysis revealed the presence of two distinct subgroups (designated as lineage 1 and 2) in Ae. tauschii. Based on GBS markers, the genetic similarity was calculated between the accessions and GWAS was conducted using 114 non-redundant accessions and 5,249 SNP markers. A total of 17 SNPs associated with grain size traits distributed over all the seven chromosomes were revealed with 6D, 5D, and 2D harboring most significant marker–trait associations. Some of the chromosomal regions such as 6D_66.4–71.1 cM, 1D_143.5–156.7 cM, and 2D_89.9–92.5 cM had associations with multiple traits. Candidate genes associated with cell division and differentiation were identified for some of the associated SNP markers. Further efforts to validate these loci will help to understand their role in determining grain size and allelic diversity in current germplasm and its effect on grain size upon transfer to bread wheat background

Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement

Aegilops tauschii, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 Ae. tauschii accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of Ae. tauschii geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through k-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse Ae. tauschii genomes. Exploiting the genomic diversity of the Ae. tauschii ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding

Identification of specificity‐defining amino acids of the wheat immune receptor Pm2 and powdery mildew effector AvrPm2

Plant nucleotide‐binding leucine‐rich repeat receptors (NLRs) act as intracellular sensors for pathogen‐derived effector proteins and trigger an immune response, frequently resulting in the hypersensitive cell death response (HR) of the infected host cell. The wheat (Triticum aestivum) NLR Pm2 confers resistance against the fungal pathogen Blumeria graminis f. sp. tritici (Bgt) if the isolate contains the specific RNase‐like effector AvrPm2. We identified and isolated seven new Pm2 alleles (Pm2e–i) in the wheat D‐genome ancestor Aegilops tauschii and two new natural AvrPm2 haplotypes from Bgt. Upon transient co‐expression in Nicotiana benthamiana, we observed a variant‐specific HR of the Pm2 variants Pm2a and Pm2i towards AvrPm2 or its homolog from the AvrPm2 effector family, BgtE‐5843, respectively. Through the introduction of naturally occurring non‐synonymous single nucleotide polymorphisms and structure‐guided mutations, we identified single amino acids in both the wheat NLR Pm2 and the fungal effector proteins AvrPm2 and BgtE‐5843 responsible for the variant‐specific HR of the Pm2 variants. Exchanging these amino acids led to a modified HR of the Pm2–AvrPm2 interaction and allowed the identification of the effector head epitope, a 20‐amino‐acid long unit of AvrPm2 involved in the HR. Swapping of the AvrPm2 head epitope to the non‐HR‐triggering AvrPm2 family member BgtE‐5846 led to gain of a HR by Pm2a. Our study presents a molecular approach to identify crucial effector surface structures involved in the HR and demonstrates that natural and induced diversity in an immune receptor and its corresponding effectors can provide the basis for understanding and modifying NLR–effector specificity