FONZIE: An optimized pipeline for minisatellite marker discovery and primer design from large sequence data sets

<p>Abstract</p> <p>Background</p> <p>Micro-and minisatellites are among the most powerful genetic markers known to date. They have been used as tools for a large number of applications ranging from gene mapping to phylogenetic studies and isolate typing. However, identifying micro-and minisatellite markers on large sequence data sets is often a laborious process.</p> <p>Results</p> <p>FONZIE was designed to successively 1) perform a search for markers via the external software Tandem Repeat Finder, 2) exclude user-defined specific genomic regions, 3) screen for the size and the percent matches of each relevant marker found by Tandem Repeat Finder, 4) evaluate marker specificity (i.e., occurrence of the marker as a single copy in the genome) using BLAST2.0, 5) design minisatellite primer pairs via the external software Primer3, and 6) check the specificity of each final PCR product by BLAST. A final file returns to users all the results required to amplify markers. A biological validation of the approach was performed using the whole genome sequence of the phytopathogenic fungus <it>Leptosphaeria maculans</it>, showing that more than 90% of the minisatellite primer pairs generated by the pipeline amplified a PCR product, 44.8% of which showed agarose-gel resolvable polymorphism between isolates. Segregation analyses confirmed that the polymorphic minisatellites corresponded to single-locus markers.</p> <p>Conclusion</p> <p>FONZIE is a stand-alone and user-friendly application developed to minimize tedious manual operations, reduce errors, and speed up the search for efficient minisatellite and microsatellite markers departing from whole-genome sequence data. This pipeline facilitates the integration of data and provides a set of specific primer sequences for PCR amplification of single-locus markers. FONZIE is freely downloadable at: <url>http://www.versailles-grignon.inra.fr/bioger/equipes/leptosphaeria_maculans/outils_d_analyses/fonzie</url></p

Comprehensive annotation of the Parastagonospora nodorum reference genome using next-generation genomics, transcriptomics and proteogenomics

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch (SNB), is an economically important pathogen of wheat (Triticum spp.), and a model for the study of necrotrophic pathology and genome evolution. The reference P. nodorum strain SN15 was the first Dothideomycete with a published genome sequence, and has been used as the basis for comparison within and between species. Here we present an updated reference genome assembly with corrections of SNP and indel errors in the underlying genome assembly from deep resequencing data as well as extensive manual annotation of gene models using transcriptomic and proteomic sources of evidence (https://github.com/robsyme/Parastagonospora_nodorum_SN15). The updated assembly and annotation includes 8,366 genes with modified protein sequence and 866 new genes. This study shows the benefits of using a wide variety of experimental methods allied to expert curation to generate a reliable set of gene models

Regulation of proteinaceous effector expression in phytopathogenic fungi

Effectors are molecules used by microbial pathogens to facilitate infection via effector-triggered susceptibility or tissue necrosis in their host. Much research has been focussed on the identification and elucidating the function of fungal effectors during plant pathogenesis. By comparison, knowledge of how phytopathogenic fungi regulate the expression of effector genes has been lagging. Several recent studies have illustrated the role of various transcription factors, chromosome-based control, effector epistasis, and mobilisation of endosomes within the fungal hyphae in regulating effector expression and virulence on the host plant. Improved knowledge of effector regulation is likely to assist in improving novel crop protection strategies

The genomic determinants of adaptive evolution in a fungal pathogen

Antagonistic host-pathogen co-evolution is a determining factor in the outcome of infection and shapes genetic diversity at the population level of both partners. While the molecular function of an increasing number of genes involved in pathogenicity is being uncovered, little is known about the molecular bases and genomic impact of hst-pathogen coevolution and rapid adaptation. Here, we apply a population genomic approach to infer genome-wide patterns of selection among thirteen isolates of the fungal pathogen Zymoseptoria tritici. Using whole genome alignments, we characterize intragenic polymorphism, and we apply different test statistics based on the distribution of non-synonymous and synonymous polymorphisms (pN/pS) and substitutions (dN/dS) to (1) characterise the selection regime acting on each gene, (2) estimate rates of adaptation and (3) identify targets of selection. We correlate our estimates with different genome variables to identify the main determinants of past and ongoing adaptive evolution, as well as purifying and balancing selection. We report a negative relationship between pN/pS and fine-scale recombination rate and a strong positive correlation between the rate of adaptive non-synonymous substitutions (ωa) and recombination rate. This result suggests a pervasive role of Hill-Robertson interference even in a species with an exceptionally high recombination rate (60 cM/Mb). Moreover, we report that the genome-wide fraction of adaptive non-synonymous substitutions (α) is ~ 44%, however in genes encoding determinants of pathogenicity we find a mean value of alpha ~ 68% demonstrating a considerably faster rate of adaptive evolution in this class of genes. We identify 787 candidate genes under balancing selection with an enrichment of genes involved in secondary metabolism and host infection, but not predicted effectors. This suggests that different classes of pathogenicity-related genes evolve according to distinct selection regimes. Overall our study shows that sexual recombination is a main driver of genome evolution in this pathogen

RNA-seq-Based gene annotation and comparative genomics of four fungal grass pathogens in the genus Zymoseptoria identify novel orphan genes and species-specific invasions of transposable elements

The fungal pathogen Zymoseptoria tritici is a prominent pathogen of wheat. The reference genome of the isolate IPO323 is one of the best-assembled eukaryotic genomes and encodes more than 10,000 predicted genes. However, a large proportion of the previously annotated gene models are incomplete with either no start or no stop codons. The availability of RNA-seq data allows better predictions of gene structure. We here used two different RNA-seq datasets, de novo transcriptome assemblies, homology-based comparisons, and trained ab initio gene callers to generate a new gene annotation of Z. tritici IPO323. The annotation pipeline was also applied to re-sequenced genomes of three closely related species of Z. tritici: Z. pseudotritici, Z. ardabiliae, and Z. brevis. Comparative analyses of the predicted gene models in the four Zymoseptoria species revealed sets of species-specific orphan genes enriched with putative pathogenicity-related genes encoding small secreted proteins that may play essential roles in virulence and host specificity. De novo repeat identification allowed us to show that few families of transposable elements are shared between Zymoseptoria species while we observe many species-specific invasions and expansions. The annotation data presented here provide a high quality resource for future studies of Z. tritici and its sister species as it provides detailed insights into gene and genome evolution of fungal plant pathogens