Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in Claviceps purpurea Sclerotia.

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea.

Mining Indole Alkaloid Synthesis Gene Clusters from Genomes of 53 Claviceps Strains Revealed Redundant Gene Copies and an Approximate Evolutionary Hourglass Model.

Ergot fungi (Claviceps spp.) are infamous for producing sclerotia containing a wide spectrum of ergot alkaloids (EA) toxic to humans and animals, making them nefarious villains in the agricultural and food industries, but also treasures for pharmaceuticals. In addition to three classes of EAs, several species also produce paspaline-derived indole diterpenes (IDT) that cause ataxia and staggers in livestock. Furthermore, two other types of alkaloids, i.e., loline (LOL) and peramine (PER), found in Epichloë spp., close relatives of Claviceps, have shown beneficial effects on host plants without evidence of toxicity to mammals. The gene clusters associated with the production of these alkaloids are known. We examined genomes of 53 strains of 19 Claviceps spp. to screen for these genes, aiming to understand the evolutionary patterns of these genes across the genus through phylogenetic and DNA polymorphism analyses. Our results showed (1) varied numbers of eas genes in C. sect. Claviceps and sect. Pusillae, none in sect. Citrinae, six idt/ltm genes in sect. Claviceps (except four in C. cyperi), zero to one partial (idtG) in sect. Pusillae, and four in sect. Citrinae, (2) two to three copies of dmaW, easE, easF, idt/ltmB, itd/ltmQ in sect. Claviceps, (3) frequent gene gains and losses, and (4) an evolutionary hourglass pattern in the intra-specific eas gene diversity and divergence in C. purpurea.

Sympatric divergence of the ergot fungus, Claviceps purpurea, populations infecting agricultural and nonagricultural grasses in North America.

The ergot diseases of agricultural and nonagricultural grasses are caused by the infection of Claviceps spp. (Hypocreales, Ascomycota) on florets, producing dark spur-like sclerotia on spikes that are toxic to humans and animals, leading to detrimental impacts on agriculture and economy due to the downgrading of cereal grains, import-export barriers, reduced yield, and ecological concerns. At least seven phylogenetic lineages (phylogenetic species) were identified within the premolecular concept of C. purpurea s.l. (sensu lato) in agricultural areas and vicinities in Canada and the Western United States. Claviceps purpurea s.s (sensu stricto) remained as the most prevalent species with a wide host range, including cereal crops, native, invasive, and weedy grasses. The knowledge on genetic diversity and distribution of C. purpurea s.s. in North America is lacking. The objective of the present study was to shed light on genetic differentiation and evolution of the natural populations of C. purpurea s.s. Multilocus DNA sequences of samples from Canada and the Western USA were analyzed using a phylogenetic network approach, and population demographic parameters were investigated. Results showed that three distinct genetically subdivided populations exist, and the subdivision is not correlated with geographic or host differentiations. Potential intrinsic mechanisms that might play roles in leading to the cessation of gene flows among the subpopulations, that is, mating and/or vegetative incompatibility, genomic adaptation, were discussed. The neutrality of two house-keeping genes that are widely used for DNA barcoding, that is, translation elongation factor 1-α (TEF1-α) and RNA polymerase II second largest subunit (RPB2), was challenged and discussed.

Four phylogenetic species of ergot from Canada and their characteristics in morphology, alkaloid production, and pathogenicity.

Four ergot species (Claviceps ripicola, C. quebecensis, C. perihumidiphila, and C. occidentalis) were recognized based on analyses of DNA sequences from multiple loci, including two housekeeping genes, RNA polymerase II second largest subunit (RPB2), and translation elongation factor 1-α (TEF1-α), and a single-copy ergot alkaloid synthesis gene (easE) encoding chanoclavine I synthase oxidoreductase. Morphological features, ergot alkaloid production, and pathogenicity on five common cereal crops of each species were evaluated and presented in taxonomic descriptions. A synoptic key was also provided for identification.

Genetic analysis of loose smut (Ustilago tritici) resistance in Sonop spring wheat.

BACKGROUND: The genetics of resistance to loose smut of wheat (Triticum aestivum L.) caused by the fungus Ustilago tritici (Pers.) Rostr. is not well understood. This study examines loose smut resistance in Sonop (TD-14), a South African spring wheat variety. A doubled haploid (DH) population of 163 lines derived from the cross Diamont/TD-14 was studied. The parents and progenies were inoculated with U. tritici races T2, T9, and T39 individually in growth facilities at Morden and Swift Current, Canada. Loose smut incidence (LSI) and partial loose smut resistance (PLSR) were assessed. RESULTS: A whole genome linkage map was developed consisting of 11,519 SNP loci found on 31 linkage groups spanning 2845 cM. A new major resistance gene Ut11 was located to the distal end of chromosome arm 7BS. Ut11 conferred resistance to U. tritici race T2, but not races T9 and T39. Quantitative trait locus (QTL) mapping identified four QTL controlling LSI in the Diamont/TD-14 DH population on chromosomes 3B, 4B, 5B, and 7B (at Ut11) with TD-14 contributing the resistance alleles at three of these loci. The major QTL QUt.mrc-5B was effective against all three races and explained up to 81% of the phenotypic variation. The only QTL identified for PLSR coincided with the LSI QTL QUt.mrc-5B indicating that this locus affected both loose smut incidence and partial smutting of spikes. CONCLUSIONS: A race-specific resistance gene Ut11 and a broadly effective resistance QTL QUt.mrc-5B were the main loci controlling loose smut resistance in the differential line TD-14 (cultivar Sonop). This study provides insight into the genetics of loose smut resistance in spring wheat Sonop and the single nucleotide polymorphism (SNP) markers linked to the resistance gene Ut11 and QTL QUt.mrc-5B will be useful for selecting loose smut resistance in breeding programs.

Mapping of the Oat Crown Rust Resistance Gene Pc39 Relative to Single Nucleotide Polymorphism Markers.

Crown rust, caused by Puccinia coronata f. sp. avenae Eriks. (Pca), is among the most important oat diseases resulting in significant yield losses in many growing regions. A gene-for-gene interaction is well established in this pathosystem and has been exploited by oat breeders to control crown rust. Pc39 is a seedling crown rust resistance gene that has been widely deployed in North American oat breeding. DNA markers are desired to accurately predict the specific Pc genes present in breeding germplasm. The objectives of the study were as follows: (i) to map Pc39 in two recombinant inbred line (RIL) populations (AC Assiniboia/MN841801 and AC Medallion/MN841801) and (ii) to identify single nucleotide polymorphism (SNP) markers for postulation of Pc39 in oat germplasm. Pc39 was mapped to a linkage group consisting of 16 SNP markers, which placed the gene on linkage group Mrg11 (chromosome 1C) of the oat consensus map. Pc39 cosegregated with SNP marker GMI_ES01_c12570_390 in the AC Assiniboia/MN841801 RIL population and was flanked by the SNP markers avgbs_126086.1.41 and GMI_ES15_c276_702, with genetic distances of 1.7 and 0.3 cM, respectively. In the AC Medallion/MN841801 RIL population, similar results were obtained but the genetic distances of the flanking markers were 0.4 and 0.4 cM, respectively. Kompetitive Allele-Specific PCR assays were successfully designed for Pc39-linked SNP loci. Two SNP loci defined a haplotype that accurately predicted Pc39 status in a diverse panel of oat germplasm and will be useful for marker-assisted selection in oat breeding.

Genetic and transcriptional dissection of resistance to Claviceps purpurea in the durum wheat cultivar Greenshank.

KEY MESSAGE: Four QTL for ergot resistance (causal pathogen Claviceps purpurea) have been identified in the durum wheat cultivar Greenshank. Claviceps purpurea is a pathogen of grasses that infects flowers, replacing the seed with an ergot sclerotium. Ergot presents a significant problem to rye, barley and wheat, in particular hybrid seed production systems. In addition, there is evidence that the highly toxic alkaloids that accumulate within sclerotia can cross-contaminate otherwise healthy grain. Host resistance to C. purpurea is rare, few resistance loci having been identified. In this study, four ergot resistance loci are located on chromosomes 1B, 2A, 5A and 5B in the durum wheat cv. Greenshank. Ergot resistance was assessed through analysis of phenotypes associated with C. purpurea infection, namely the number of inoculated flowers that produced sclerotia, or resulted in ovary death but no sclerotia, the levels of honeydew produced, total sclerotia weight and average sclerotia weight and size per spike. Ergot testing was undertaken in Canada and the UK. A major effect QTL, QCp.aafc.DH-2A, was detected in both the Canadian and UK experiments and had a significant effect on honeydew production levels. QCp.aafc.DH-5B had the biggest influence on total sclerotia weight per spike. QCp.aafc.DH-1B was only detected in the Canadian experiments and QCp.aafc.DH-5A in the UK experiment. An RNASeq analysis, undertaken to identify wheat differentially expressed genes associated with different combinations of the four ergot resistance QTL, revealed a disproportionate number of DEGs locating to the QCp.aafc.DH-1B, QCp.aafc.DH-2A and QCp.aafc.DH-5B QTL intervals.

Chromosomal location of the crown rust resistance gene Pc98 in cultivated oat (Avena sativa L.).

KEY MESSAGE: SNP loci linked to the crown rust resistance gene Pc98 were identified by linkage analysis and KASP assays were developed for marker-assisted selection in breeding programs. Crown rust is among the most damaging diseases of oat and is caused by Puccinia coronata var. avenae f. sp. avenae (Urban and Marková) (Pca). Host resistance is the preferred method to prevent crown rust epidemics. Pc98 is a race-specific, seedling crown rust resistance gene obtained from the wild oat Avena sterilis accession CAV 1979 that is effective at all growth stages of oat. Virulence to Pc98 has been very low in the Pca populations that have been tested. The objectives of this study were to develop SNP markers linked to Pc98 for use in marker-assisted selection and to locate Pc98 on the oat consensus map. The Pc98 gene was mapped using F2:3 populations developed from the crosses Pc98/Bingo and Pc98/Kasztan, where Pc98 is a single-gene line carrying Pc98. Both populations were evaluated in seedling inoculation experiments. Pc98 was mapped relative to Kompetitive Allele-Specific PCR SNP markers in both populations, placing Pc98 on the Mrg20 linkage group of the consensus map. Pc98 was bracketed by two SNP markers GMI_ES22_c3052_382_kom399 and GMI_ES14_lrc18344_662_kom398 in the Pc98/Bingo mapping population with genetic distances of 0.9 cM and 0.3 cM, respectively. Pc98 co-segregated with four SNP markers in the Pc98/Kasztan population, and the closest flanking markers were GMI_DS_LB_6017_kom367 and avgbs2_153634.1.59_kom410 with genetic distances of 0.7 cM and 0.3 cM, respectively. Two SNP loci defined a haplotype that accurately predicted Pc98 status in a diverse group of oat germplasm, which will be valuable for marker-assisted selection of Pc98 in breeding of new oat cultivars.

Mapping of the stem rust resistance gene Pg13 in cultivated oat.

KEY MESSAGE: The widely deployed, oat stem rust resistance gene Pg13 was mapped by linkage analysis and association mapping, and KASP markers were developed for marker-assisted selection in breeding programs. Pg13 is one of the most extensively deployed stem rust resistance genes in North American oat cultivars. Identification of markers tightly linked to this gene will be useful for routine marker-assisted selection, identification of gene pyramids, and retention of the gene in backcrosses and three-way crosses. To this end, high-density linkage maps were constructed in four bi-parental mapping populations using SNP markers identified from 6K oat Infinium iSelect and genotyping-by-sequencing platforms. Additionally, genome-wide associations were identified using two sets of association panels consisting of diverse elite oat lines in one set and landrace accessions in the other. The results showed that Pg13 was located at approximately 67.7 cM on linkage group Mrg18 of the consensus genetic map. The gene co-segregated with the 7C-17A translocation breakpoint and with crown rust resistance gene Pc91. Co-segregating markers with the best prediction accuracy were identified at 67.7-68.5 cM on Mrg18. KASP assays were developed for linked SNP loci for use in oat breeding.

Phylogeny of Canadian ergot fungi and a detection assay by real-time polymerase chain reaction.

The ergot disease of cereals has become increasingly important in agricultural areas of Canada since 1999. Generally, this disease is considered to be caused by Claviceps purpurea, but the taxonomy of Claviceps from these areas has not been well studied. The objectives of this study were (i) to determine the phylogenetic lineages (phylogenetic species) present in agricultural areas of Canada and (ii) to develop a molecular assay that can separate the lineages on crops from other lineages. Genetic diversity of Claviceps collected from agriculture areas in Canada were investigated using multilocus sequence typing. The loci sequenced include nuc rDNA internal transcribed spacer (ITS1-5.8S-ITS2 = ITS), partial fragments of translation elongation factor 1-α (TEF1), RNA polymerase II second largest subunit (RPB2), ß-tubulin (tubB), and two ergot alkaloid synthesis genes (easA, easE). Based on individual locus and concatenated alignments, phylogenetic analyses revealed seven lineages within the premolecular concept of C. purpurea, of which five corresponded with undescribed species (G2b and G4-7). Although lineages G2-7 had narrow host ranges, lineage G1 (= C. purpurea s.s.) had a broad host range that overlapped with other lineages. A molecular diagnostic quantitative polymerase chain reaction (qPCR) assay was developed and validated with 185 samples from a wide range of host plants and geographic origins, including 10 phylogenetic species in C. sect. Claviceps, 8 in C. sect. Pusillae, 1 in C. sect. Citrinae, and 1-2 species from Alternaria, Fusarium, and Penicillium. The assay can detect lineage G1 at a concentration of 7.5 pg/µL and distinguish it from other Claviceps species and lineages. This facilitates disease management by detecting the inocula from nonagriculture host plants.

Mapping Oat Crown Rust Resistance Gene Pc45 Confirms Association with PcKM.

Molecular mapping of crown rust resistance genes is important to effectively utilize these genes and improve breeding efficiency through marker-assisted selection. Pc45 is a major race-specific crown rust resistance gene initially identified in the wild hexaploid oat Avena sterilis in the early 1970s. This gene was transferred to cultivated oat (Avena sativa) and has been used as a differential for identification of crown rust races since 1974. Previous research identified an association between virulence to Pc45 and PcKM, a crown rust resistance gene in the varieties 'Kame' and 'Morton'. This study was undertaken to reveal the relationship between Pc45 and PcKMPc45 was studied in the crosses 'AC Morgan'/Pc45 and 'Kasztan'/Pc45, where Pc45 is the differential line carrying Pc45 F2 progenies and F2:3 families of both populations were inoculated with the crown rust isolate CR258 (race NTGG) and single gene segregation ratios were observed. SNP markers for PcKM were tested on these populations and linkage maps were generated. In addition, 17 newly developed SNP markers identified from genotyping-by-sequencing (GBS) data were mapped in these two populations, plus another three populations segregating for Pc45 or PcKMPc45 and PcKM mapped to the same location of Mrg08 (chromosome 12D) of the oat chromosome-anchored consensus map. These results strongly suggest that Pc45 and PcKM are the same resistance gene, but allelism (i.e., functionally different alleles of the same gene) or tight linkage (i.e., two tightly linked genes) cannot be ruled out based on the present data.

Nine draft genome sequences of Claviceps purpurea s.lat., including C. arundinis, C. humidiphila, and C. cf. spartinae, pseudomolecules for the pitch canker pathogen Fusarium circinatum, draft genome of Davidsoniella eucalypti, Grosmannia galeiformis, Quambalaria eucalypti, and Teratosphaeria destructans.

This genome announcement includes draft genomes from Claviceps purpurea s.lat., including C. arundinis, C. humidiphila and C. cf. spartinae. The draft genomes of Davidsoniella eucalypti, Quambalaria eucalypti and Teratosphaeria destructans, all three important eucalyptus pathogens, are presented. The insect associate Grosmannia galeiformis is also described. The pine pathogen genome of Fusarium circinatum has been assembled into pseudomolecules, based on additional sequence data and by harnessing the known synteny within the Fusarium fujikuroi species complex. This new assembly of the F. circinatum genome provides 12 pseudomolecules that correspond to the haploid chromosome number of F. circinatum. These are comparable to other chromosomal assemblies within the FFSC and will enable more robust genomic comparisons within this species complex.

Genetic analysis and molecular mapping of a seedling crown rust resistance gene in oat.

KEY MESSAGE: Genetic analysis and genome mapping of a major seedling oat crown rust resistance gene, designated PcKM, are described. The chromosomal location of the PcKM gene was identified and linked markers were validated. Crown rust (Puccinia coronata Corda f. sp. avenae Eriks) is the most important foliar disease of oats and can cause considerable yield loss in the absence of appropriate management practices. Utilization of novel resistant genes is the most effective, economic and environmentally sound approach to control the disease. Crown rust resistance present in the cultivar 'Morton' was evaluated in a population developed from the cross OT3019 × 'Morton' to elucidate the genetic basis of resistance. Crown rust reaction evaluated in field nurseries and greenhouse tests demonstrated that resistance provided by 'Morton' was controlled by a single gene, temporarily designated as PcKM. The gene was initially linked to a random amplified polymorphic DNA band and subsequently converted into a sequence characterized amplified region (SCAR) marker. Genotyping with the PcKM SCAR on the 'Kanota' × 'Ogle' population, used to create the first oat chromosome-anchored linkage map, placed the PcKM gene on chromosome 12D. Consensus map markers present in the same region as the PcKM SCAR were tested on the OT3019 × 'Morton' population and two additional phenotyped populations segregating for PcKM to identify other markers useful for marker-assisted selection. Three markers were perfectly linked to the PcKM phenotype from which TaqMan and KBioscience competitive allele-specific PCR assays were developed and validated on a set of 25 oat lines. The assays correctly identified PcKM carriers. The markers developed in this study will facilitate fine mapping of the PcKM gene and simplify selection for this crown rust resistance.

A major quantitative trait locus conferring adult plant partial resistance to crown rust in oat.

BACKGROUND: Crown rust, caused by Puccinia coronata f. sp. avenae, is the most important disease of oat worldwide. Adult plant resistance (APR), based upon partial resistance, has proven to be a durable rust management strategy in other cereal rust pathosystems. The crown rust APR in the oat line MN841801 has been effective for more than 30 years. The genetic basis of this APR was studied under field conditions in three recombinant inbred line (RIL) populations: 1) AC Assiniboia/MN841801, 2) AC Medallion/MN841801, and 3) Makuru/MN841801. The populations were evaluated for crown rust resistance with the crown rust isolate CR251 (race BRBB) in multiple environments. The 6 K oat and 90 K wheat Illumina Infinium single nucleotide polymorphism (SNP) arrays were used for genotyping the AC Assiniboia/MN841801 population. KASP assays were designed for selected SNPs and genotyped on the other two populations. RESULTS: This study reports a high density genetic linkage map constructed with oat and wheat SNP markers in the AC Assiniboia/MN841801 RIL population. Most wheat SNPs were monomorphic in the oat population. However the polymorphic wheat SNPs could be scored accurately and integrated well into the linkage map. A major quantitative trait locus (QTL) on oat chromosome 14D, designated QPc.crc-14D, explained up to 76% of the APR phenotypic variance. This QTL is flanked by two SNP markers, GMI_GBS_90753 and GMI_ES14_c1439_83. QPc.crc-14D was validated in the populations AC Medallion/MN841801 and Makuru/MN841801. CONCLUSIONS: We report the first APR QTL in oat with a large and consistent effect. QPc.crc-14D was statistically significant in all environments tested in each of the three oat populations. QPc.crc-14D is a suitable candidate for use in marker-assisted breeding and also an excellent target for map-based cloning. This is also the first study to use the 90 K wheat Infinium SNP array on oat for marker development and comparative mapping. The Infinium SNP array is a useful tool for saturating oat maps with markers. Synteny with wheat suggests that QPc.crc-14D is orthologous with the stripe rust APR gene Yr16 in wheat.