Increasing calling accuracy, coverage, and read-depth in sequence data by the use of haplotype blocks.

High-throughput genotyping of large numbers of lines remains a key challenge in plant genetics, requiring geneticists and breeders to find a balance between data quality and the number of genotyped lines under a variety of different existing genotyping technologies when resources are limited. In this work, we are proposing a new imputation pipeline ("HBimpute") that can be used to generate high-quality genomic data from low read-depth whole-genome-sequence data. The key idea of the pipeline is the use of haplotype blocks from the software HaploBlocker to identify locally similar lines and subsequently use the reads of all locally similar lines in the variant calling for a specific line. The effectiveness of the pipeline is showcased on a dataset of 321 doubled haploid lines of a European maize landrace, which were sequenced at 0.5X read-depth. The overall imputing error rates are cut in half compared to state-of-the-art software like BEAGLE and STITCH, while the average read-depth is increased to 83X, thus enabling the calling of copy number variation. The usefulness of the obtained imputed data panel is further evaluated by comparing the performance of sequence data in common breeding applications to that of genomic data generated with a genotyping array. For both genome-wide association studies and genomic prediction, results are on par or even slightly better than results obtained with high-density array data (600k). In particular for genomic prediction, we observe slightly higher data quality for the sequence data compared to the 600k array in the form of higher prediction accuracies. This occurred specifically when reducing the data panel to the set of overlapping markers between sequence and array, indicating that sequencing data can benefit from the same marker ascertainment as used in the array process to increase the quality and usability of genomic data.

Genetic analysis reveals long-standing population differentiation and high diversity in the rust pathogen Melampsora lini.

A priority for research on infectious disease is to understand how epidemiological and evolutionary processes interact to influence pathogen population dynamics and disease outcomes. However, little is understood about how population adaptation changes across time, how sexual vs. asexual reproduction contribute to the spread of pathogens in wild populations and how diversity measured with neutral and selectively important markers correlates across years. Here, we report results from a long-term study of epidemiological and genetic dynamics within several natural populations of the Linum marginale-Melampsora lini plant-pathogen interaction. Using pathogen isolates collected from three populations of wild flax (L. marginale) spanning 16 annual epidemics, we probe links between pathogen population dynamics, phenotypic variation for infectivity and genomic polymorphism. Pathogen genotyping was performed using 1567 genome-wide SNP loci and sequence data from two infectivity loci (AvrP123, AvrP4). Pathogen isolates were phenotyped for infectivity using a differential set. Patterns of epidemic development were assessed by conducting surveys of infection prevalence in one population (Kiandra) annually. Bayesian clustering analyses revealed host population and ecotype as key predictors of pathogen genetic structure. Despite strong fluctuations in pathogen population size and severe annual bottlenecks, analysis of molecular variance revealed that pathogen population differentiation was relatively stable over time. Annually, varying levels of clonal spread (0-44.8%) contributed to epidemics. However, within populations, temporal genetic composition was dynamic with rapid turnover of pathogen genotypes, despite the dominance of only four infectivity phenotypes across the entire study period. Furthermore, in the presence of strong fluctuations in population size and migration, spatial selection may maintain pathogen populations that, despite being phenotypically stable, are genetically highly dynamic.

Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini.

BACKGROUND: Rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field. RESULTS: To facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. CONCLUSIONS: The M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.

Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines.

Although pioneered by human geneticists as a potential solution to the challenging problem of finding the genetic basis of common human diseases, genome-wide association (GWA) studies have, owing to advances in genotyping and sequencing technology, become an obvious general approach for studying the genetics of natural variation and traits of agricultural importance. They are particularly useful when inbred lines are available, because once these lines have been genotyped they can be phenotyped multiple times, making it possible (as well as extremely cost effective) to study many different traits in many different environments, while replicating the phenotypic measurements to reduce environmental noise. Here we demonstrate the power of this approach by carrying out a GWA study of 107 phenotypes in Arabidopsis thaliana, a widely distributed, predominantly self-fertilizing model plant known to harbour considerable genetic variation for many adaptively important traits. Our results are dramatically different from those of human GWA studies, in that we identify many common alleles of major effect, but they are also, in many cases, harder to interpret because confounding by complex genetics and population structure make it difficult to distinguish true associations from false. However, a-priori candidates are significantly over-represented among these associations as well, making many of them excellent candidates for follow-up experiments. Our study demonstrates the feasibility of GWA studies in A. thaliana and suggests that the approach will be appropriate for many other organisms.

Host ecotype generates evolutionary and epidemiological divergence across a pathogen metapopulation.

The extent and speed at which pathogens adapt to host resistance varies considerably. This presents a challenge for predicting when--and where--pathogen evolution may occur. While gene flow and spatially heterogeneous environments are recognized to be critical for the evolutionary potential of pathogen populations, we lack an understanding of how the two jointly shape coevolutionary trajectories between hosts and pathogens. The rust pathogen Melampsora lini infects two ecotypes of its host plant Linum marginale that occur in close proximity yet in distinct populations and habitats. In this study, we found that within-population epidemics were different between the two habitats. We then tested for pathogen local adaptation at host population and ecotype level in a reciprocal inoculation study. Even after controlling for the effect of spatial structure on infection outcome, we found strong evidence of pathogen adaptation at the host ecotype level. Moreover, sequence analysis of two pathogen infectivity loci revealed strong genetic differentiation by host ecotype but not by distance. Hence, environmental variation can be a key determinant of pathogen population genetic structure and coevolutionary dynamics and can generate strong asymmetry in infection risks through space.

Genome-wide survey of Arabidopsis natural variation in downy mildew resistance using combined association and linkage mapping.

The model plant Arabidopsis thaliana exhibits extensive natural variation in resistance to parasites. Immunity is often conferred by resistance (R) genes that permit recognition of specific races of a disease. The number of such R genes and their distribution are poorly understood. In this study, we investigated the basis for resistance to the downy mildew agent Hyaloperonospora arabidopsidis ex parasitica (Hpa) in a global sample of A. thaliana. We implemented a combined genome-wide mapping of resistance using populations of recombinant inbred lines and a collection of wild A. thaliana accessions. We tested the interaction between 96 host genotypes collected worldwide and five strains of Hpa. Then, a fraction of the species-wide resistance was genetically dissected using six recently constructed populations of recombinant inbred lines. We found that resistance is usually governed by single dominant R genes that are concentrated in four genomic regions only. We show that association genetics of resistance to diseases such as downy mildew enables increased mapping resolution from quantitative trait loci interval to candidate gene level. Association patterns in quantitative trait loci intervals indicate that the pool of A. thaliana resistance sources against the tested Hpa isolates may be predominantly confined to six RPP (Resistance to Hpa) loci isolated in previous studies. Our results suggest that combining association and linkage mapping could accelerate resistance gene discovery in plants.

Rapid genetic change underpins antagonistic coevolution in a natural host-pathogen metapopulation.

Antagonistic coevolution is a critical force driving the evolution of diversity, yet the selective processes underpinning reciprocal adaptive changes in nature are not well understood. Local adaptation studies demonstrate partner impacts on fitness and adaptive change, but do not directly expose genetic processes predicted by theory. Specifically, we have little knowledge of the relative importance of fluctuating selection vs. arms-race dynamics in maintaining polymorphism in natural systems where metapopulation processes predominate. We conducted cross-year epidemiological, infection and genetic studies of multiple wild host and pathogen populations in the Linum-Melampsora association. We observed asynchronous phenotypic fluctuations in resistance and infectivity among demes. Importantly, changes in allelic frequencies at pathogen infectivity loci, and in host recognition of these genetic variants, correlated with disease prevalence during natural epidemics. These data strongly support reciprocal coevolution maintaining balanced resistance and infectivity polymorphisms, and highlight the importance of characterising spatial and temporal dynamics in antagonistic interactions.

A conceptual framework for the evolution of ecological specialisation.

Ecology Letters (2011) 14: 841-851 ABSTRACT: Ecological specialisation concerns all species and underlies many major ecological and evolutionary patterns. Yet its status as a unifying concept is not always appreciated because of its similarity to concepts of the niche, the many levels of biological phenomena to which it applies, and the complexity of the mechanisms influencing it. The evolution of specialisation requires the coupling of constraints on adaptive evolution with covariation of genotype and environmental performance. This covariation itself depends upon organismal properties such as dispersal behaviour and life history and complexity in the environment stemming from factors such as species interactions and spatio-temporal heterogeneity in resources. Here, we develop a view on specialisation that integrates across the range of biological phenomena with the goal of developing a more predictive conceptual framework that specifically accounts for the importance of biotic complexity and coevolutionary events.

DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling.

In Arabidopsis, the flagellin-derived peptide flg22 elevates antibacterial resistance [1] and inhibits growth [2] upon perception via the leucine-rich repeat receptor-like kinase Flagellin-Sensitive 2 (FLS2) [3]. DELLA proteins are plant growth repressors whose degradation is promoted by the phytohormone gibberellin [4]. Here, we show that DELLA stabilization contributes to flg22-induced growth inhibition. In addition, we show that DELLAs promote susceptibility to virulent biotrophs and resistance to necrotrophs, partly by altering the relative strength of salicylic acid and jasmonic acid (JA) signaling. A quadruple-DELLA mutant (which lacks four out of the five Arabidopsis DELLA proteins [5]) was partially insensitive to gene induction by Methyl-Jasmonate (MeJA), whereas the constitutively active dominant DELLA mutant gai[6] was sensitized for JA-responsive gene induction, implicating DELLAs in JA-signaling and/or perception. Accordingly, the elevated resistance of gai to the necrotrophic fungus Alternaria brassicicola and susceptibility to the hemibiotroph Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000) was attenuated in the JA-insensitive coi1-16 mutant [7]. These findings suggest an explanation for why the necrotrophic fungus Gibberella fujikuroi, causal agent of the foolish-seedling disease of rice, makes gibberellin.

European maize genomes highlight intraspecies variation in repeat and gene content.

The diversity of maize (Zea mays) is the backbone of modern heterotic patterns and hybrid breeding. Historically, US farmers exploited this variability to establish today's highly productive Corn Belt inbred lines from blends of dent and flint germplasm pools. Here, we report de novo genome sequences of four European flint lines assembled to pseudomolecules with scaffold N50 ranging from 6.1 to 10.4 Mb. Comparative analyses with two US Corn Belt lines explains the pronounced differences between both germplasms. While overall syntenic order and consolidated gene annotations reveal only moderate pangenomic differences, whole-genome alignments delineating the core and dispensable genome, and the analysis of heterochromatic knobs and orthologous long terminal repeat retrotransposons unveil the dynamics of the maize genome. The high-quality genome sequences of the flint pool complement the maize pangenome and provide an important tool to study maize improvement at a genome scale and to enhance modern hybrid breeding.

Flax rust infection transcriptomics reveals a transcriptional profile that may be indicative for rust Avr genes.

Secreted effectors of fungal pathogens are essential elements for disease development. However, lack of sequence conservation among identified effectors has long been a problem for predicting effector complements in fungi. Here we have explored the expression characteristics of avirulence (Avr) genes and candidate effectors of the flax rust fungus, Melampsora lini. We performed transcriptome sequencing and real-time quantitative PCR (qPCR) on RNA extracted from ungerminated spores, germinated spores, isolated haustoria and flax seedlings inoculated with M. lini isolate CH5 during plant infection. Genes encoding two categories of M. lini proteins, namely Avr proteins and plant cell wall degrading enzymes (CWDEs), were investigated in detail. Analysis of the expression profiles of 623 genes encoding predicted secreted proteins in the M. lini transcriptome shows that the six known Avr genes (i.e. AvrM (avrM), AvrM14, AvrL2, AvrL567, AvrP123 (AvrP) and AvrP4) fall within a group of 64 similarly expressed genes that are induced in planta and show a peak of expression early in infection with a subsequent decline towards sporulation. Other genes within this group include two paralogues of AvrL2, an AvrL567 virulence allele, and a number of genes encoding putative effector proteins. By contrast, M. lini genes encoding CWDEs fall into different expression clusters with their distribution often unrelated to their catalytic activity or substrate targets. These results suggest that synthesis of M. lini Avr proteins may be regulated in a coordinated fashion and that the expression profiling-based analysis has significant predictive power for the identification of candidate Avr genes.

Marker development for the genetic study of natural variation in Arabidopsis thaliana.

We report AtPRIMER, an application that automates the discovery of new polymorphic markers between ecotypes of Arabidopsis thaliana. On specifying two ecotypes and the genomic region of interest, the script retrieves all corresponding single nucleotide polymorphisms (SNPs) and generates CAPS and/or dCAPS PCR primer sequences. We show that AtPRIMER accurately found specific polymorphic markers for our linkage mapping project. AtPRIMER will therefore be useful for efficient marker development with high density and specificity.

Comparative genomics of Australian isolates of the wheat stem rust pathogen Puccinia graminis f. sp. tritici reveals extensive polymorphism in candidate effector genes.

The wheat stem rust fungus Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. In this study, a draft genome was built for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly were performed resulting in a 92 Mbp reference genome for Pgt isolate 21-0. Approximately 13 Mbp of de novo assembled sequence in this genome is not present in the p7a reference assembly. This novel sequence is not specific to 21-0 as it is also present in three other Pgt rust isolates of independent origin. The new reference genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ∼10% of the genes each in germinated spores and haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 520 were classified as haustorial secreted proteins (HSPs). Comparison of 21-0 with two presumed clonal field derivatives of this lineage (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 25 HSP effector candidates. Some of these mutations could explain their novel virulence phenotypes.

The genome sequence and effector complement of the flax rust pathogen Melampsora lini.

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.

Population processes at multiple spatial scales maintain diversity and adaptation in the Linum marginale--Melampsora lini association.

Host-pathogen coevolution is a major driver of species diversity, with an essential role in the generation and maintenance of genetic variation in host resistance and pathogen infectivity. Little is known about how resistance and infectivity are structured across multiple geographic scales and what eco-evolutionary processes drive these patterns. Across southern Australia, the wild flax Linum marginale is frequently attacked by its rust fungus Melampsora lini. Here, we compare the genetic and phenotypic structure of resistance and infectivity among population pairs from two regions where environmental differences associate with specific life histories and mating systems. We find that both host and pathogen populations are genetically distinct between these regions. The region with outcrossing hosts and pathogens that go through asexual cycles followed by sexual reproduction showed greater diversity of resistance and infectivity phenotypes, higher levels of resistance and less clumped within-population spatial distribution of resistance. However, in the region where asexual pathogens infect selfing hosts, pathogens were more infective and better adapted to sympatric hosts. Our findings largely agree with expectations based on the distinctly different host mating systems in the two regions, with a likely advantage for hosts undergoing recombination. For the pathogen in this system, sexual reproduction may primarily be a survival mechanism in the region where it is observed. While it appears to potentially have adverse effects on local adaptation in the short term, it may be necessary for longer-term coevolution with outcrossing hosts.

Co-evolutionary interactions between host resistance and pathogen effector genes in flax rust disease.

Plant-pathogen co-evolutionary selection processes are continuous, complex and occur across many spatial and temporal scales. Comprehensive studies of the flax-flax rust pathosystem have led to the postulation of the gene-for-gene model, a genetic paradigm describing recognition events between host disease resistance proteins and pathogen effector proteins. The identification of directly interacting fungal effector proteins and plant disease resistance proteins in this pathosystem has facilitated the study of both the physical nature of these interactions and the evolutionary forces that have resulted in a molecular arms race between these organisms. The flax-flax rust pathosystem has also been detailed on the scale of interacting populations, and the integration of molecular- and population-scale datasets represents a unique opportunity to further our understanding of many poorly understood facets of host-pathogen dynamics. In this article, we discuss recent developments and insights in the flax-flax rust pathosystem and their implications for both long-term co-evolutionary dynamics in natural settings, as well as short-term co-evolutionary dynamics in agro-ecosystems.

The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana.

The downy mildew (Hyaloperonospora parasitica) effector proteins ATR1 and ATR13 trigger RPP1-Nd/WsB- and RPP13-Nd-dependent resistance, respectively, in Arabidopsis thaliana. To better understand the functions of these effectors during compatible and incompatible interactions of H. parasitica isolates on Arabidopsis accessions, we developed a novel delivery system using Pseudomonas syringae type III secretion via fusions of ATRs to the N terminus of the P. syringae effector protein, AvrRPS4. ATR1 and ATR13 both triggered the hypersensitive response (HR) and resistance to bacterial pathogens in Arabidopsis carrying RPP1-Nd/WsB or RPP13-Nd, respectively, when delivered from P. syringae pv tomato (Pst) DC3000. In addition, multiple alleles of ATR1 and ATR13 confer enhanced virulence to Pst DC3000 on susceptible Arabidopsis accessions. We conclude that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. Two ATR13 alleles suppressed bacterial PAMP (for Pathogen-Associated Molecular Patterns)-triggered callose deposition in susceptible Arabidopsis when delivered by DC3000 DeltaCEL mutants. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMP-triggered reactive oxygen species production in addition to callose deposition. Intriguingly, although Wassilewskija (Ws-0) is highly susceptible to H. parasitica isolate Emco5, ATR13Emco5 when delivered by Pst DC3000 triggered localized immunity, including HR, on Ws-0. We suggest that an additional H. parasitica Emco5 effector might suppress ATR13-triggered immunity.