Complex polyploid and hybrid species in an apomictic and sexual tropical forage grass group: genomic composition and evolution in Urochloa (Brachiaria) species.

BACKGROUND AND AIMS: Diploid and polyploid Urochloa (including Brachiaria, Panicum and Megathyrsus species) C4 tropical forage grasses originating from Africa are important for food security and the environment, often being planted in marginal lands worldwide. We aimed to characterize the nature of their genomes, the repetitive DNA and the genome composition of polyploids, leading to a model of the evolutionary pathways within the group including many apomictic species. METHODS: Some 362 forage grass accessions from international germplasm collections were studied, and ploidy was determined using an optimized flow cytometry method. Whole-genome survey sequencing and molecular cytogenetic analysis were used to identify chromosomes and genomes in Urochloa accessions belonging to the 'brizantha' and 'humidicola' agamic complexes and U. maxima. KEY RESULTS: Genome structures are complex and variable, with multiple ploidies and genome compositions within the species, and no clear geographical patterns. Sequence analysis of nine diploid and polyploid accessions enabled identification of abundant genome-specific repetitive DNA motifs. In situ hybridization with a combination of repetitive DNA and genomic DNA probes identified evolutionary divergence and allowed us to discriminate the different genomes present in polyploids. CONCLUSIONS: We suggest a new coherent nomenclature for the genomes present. We develop a model of evolution at the whole-genome level in diploid and polyploid accessions showing processes of grass evolution. We support the retention of narrow species concepts for Urochloa brizantha, U. decumbens and U. ruziziensis, and do not consider diploids and polyploids of single species as cytotypes. The results and model will be valuable in making rational choices of parents for new hybrids, assist in use of the germplasm for breeding and selection of Urochloa with improved sustainability and agronomic potential, and assist in measuring and conserving biodiversity in grasslands.

Genome-wide association study for deoxynivalenol production and aggressiveness in wheat and rye head blight by resequencing 92 isolates of Fusarium culmorum.

BACKGROUND: Fusarium culmorum is an important pathogen causing head blight of cereals in Europe. This disease is of worldwide importance leading to reduced yield, grain quality, and contamination by mycotoxins. These mycotoxins are harmful for livestock and humans; therefore, many countries have strict regulatory limits for raw materials and processed food. Extensive genetic diversity is described among field populations of F. culmorum isolates for aggressiveness and production of the trichothecene mycotoxin deoxynivalenol (DON). However, the causes for this quantitative variation are not clear, yet. We analyzed 92 isolates sampled from different field populations in Germany, Russia, and Syria together with an international collection for aggressiveness and DON production in replicated field experiments at two locations in two years with two hosts, wheat and rye. The 30x coverage whole-genome resequencing of all isolates resulted in the identification of 130,389 high quality single nucleotide polymorphisms (SNPs) that were used for the first genome-wide association study in this phytopathogenic fungus. RESULTS: In wheat, 20 and 27 SNPs were detected for aggressiveness and DON content, respectively, of which 10 overlapped. Additionally, two different SNPs were significantly associated with aggressiveness in rye that were among those SNPs being associated with DON production in wheat. Most of the SNPs explained only a small proportion of genotypic variance (pG), however, four SNPs were associated with major quantitative trait loci (QTLs) with pG ranging from 12 to 48%. The QTL with the highest pG was involved in DON production and associated with a SNP most probably located within the Tri4 gene. CONCLUSIONS: The diversity of 92 isolates of F. culmorum were captured using a heuristic approach. Key phenotypic traits, SNPs, and candidate genes underlying aggressiveness and DON production were identified. Clearly, many QTLs are responsible for aggressiveness and DON content in wheat, both traits following a quantitative inheritance. Several SNPs involved in DON metabolism, among them the Tri4 gene of the trichothecene pathway, were inferred as important source of variation in fungal aggressiveness. Using this information underlying the phenotypic variation will be of paramount importance in evaluating strategies for successful resistance breeding.

Flow Cytometry-Based Determination of Ploidy from Dried Leaf Specimens in Genomically Complex Collections of the Tropical Forage Grass Urochloa s. l.

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the 'brizantha' and 'humidicola' agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy.

A new genome allows the identification of genes associated with natural variation in aluminium tolerance in Brachiaria grasses.

Toxic concentrations of aluminium cations and low phosphorus availability are the main yield-limiting factors in acidic soils, which represent half of the potentially available arable land. Brachiaria grasses, which are commonly sown as forage in the tropics because of their resilience and low demand for nutrients, show greater tolerance to high concentrations of aluminium cations (Al3+) than most other grass crops. In this work, we explored the natural variation in tolerance to Al3+ between high and low tolerant Brachiaria species and characterized their transcriptional differences during stress. We identified three QTLs (quantitative trait loci) associated with root vigour during Al3+ stress in their hybrid progeny. By integrating these results with a new Brachiaria reference genome, we identified 30 genes putatively responsible for Al3+ tolerance in Brachiaria. We observed differential expression during stress of genes involved in RNA translation, response signalling, cell wall composition, and vesicle location homologous to aluminium-induced proteins involved in limiting uptake or localizing the toxin. However, there was limited regulation of malate transporters in Brachiaria, which suggests that exudation of organic acids and other external tolerance mechanisms, common in other grasses, might not be relevant in Brachiaria. The contrasting regulation of RNA translation and response signalling suggests that response timing is critical in high Al3+-tolerant Brachiaria.

Candidate Genes for Aggressiveness in a Natural Fusarium culmorum Population Greatly Differ between Wheat and Rye Head Blight.

Fusarium culmorum is one of the species causing Fusarium head blight (FHB) in cereals in Europe. We aimed to investigate the association between the nucleotide diversity of ten F. culmorum candidate genes and field ratings of aggressiveness in winter rye. A total of 100 F. culmorum isolates collected from natural infections were phenotyped for FHB at two locations and two years. Variance components for aggressiveness showed significant isolate and isolate-by-environment variance, as expected for quantitative host-pathogen interactions. Further analysis of the isolate-by-environment interaction revealed the dominant role of the isolate-by-year over isolate-by-location interaction. One single-nucleotide polymorphism (SNP) in the cutinase (CUT) gene was found to be significantly (p < 0.001) associated with aggressiveness and explained 16.05% of the genotypic variance of this trait in rye. The SNP was located 60 base pairs before the start codon, which suggests a role in transcriptional regulation. Compared to a previous study in winter wheat with the same nucleotide sequences, a larger variation of pathogen aggressiveness on rye was found and a different candidate gene was associated with pathogen aggressiveness. This is the first report on the association of field aggressiveness and a host-specific candidate gene codifying for a protein that belongs to the secretome in F. culmorum.

Candidate gene based association mapping in Fusarium culmorum for field quantitative pathogenicity and mycotoxin production in wheat.

BACKGROUND: Quantitative traits are common in nature, but quantitative pathogenicity has received only little attention in phytopathology. In this study, we used 100 Fusarium culmorum isolates collected from natural field environments to assess their variation for two quantitative traits, aggressiveness and deoxynivalenol (DON) production on wheat plants grown in four different field environments (location-year combinations). Seventeen Fusarium graminearum pathogenicity candidate genes were assessed for their effect on the aggressiveness and DON production of F. culmorum under field conditions. RESULTS: For both traits, genotypic variance among isolates was high and significant while the isolate-by-environment interaction was also significant, amounting to approximately half of the genotypic variance. Among the studied candidate genes, the mitogen-activated protein kinase (MAPK) HOG1 was found to be significantly associated with aggressiveness and deoxynivalenol (DON) production, explaining 10.29 and 6.05% of the genotypic variance, respectively. CONCLUSIONS: To the best of our knowledge, this is the first report of a protein kinase regulator explaining differences in field aggressiveness and mycotoxin production among individuals from natural populations of a plant pathogen.