Outstanding : the dispensable chromosomes of Mycosphaerella graminicola

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Genetic mapping using the Diversity Arrays Technology (DArT) : application and validation using the whole-genome sequences of Arabidopsis thaliana and the fungal wheat pathogen Mycosphaerella graminicola

Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds- to thousands of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. In contrast to many other gel-based marker technologies DArT markers can be sequenced readily. Chapter 1 gives an overview of the most widely used genetic marker methods and their limitations. In addition some background information is provided on the wheat infecting fungal pathogen Mycosphaerella graminicola , in which we applied DArT.In Chapter 2 the DArT procedure is described in more detail. All steps in the DArT protocol are explained and discussed. In addition some adaptations to the DArT procedure with respect to the complexity reduction and use of adapter sequences are demonstrated on the basis of experiments performed in M. graminicola . Finally, the specifically developed software (DArTsoft) and the application of DArT markers in the mapping process are described.Chapter 3demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana . Restriction fragments from a genomic representation of the ecotype Landsberg erecta (L er ) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypesColumbia(Col) and L er and of individuals from an F 2 population obtained from aCol× L er cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the L er markers and alignment with the availableColgenome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms.In Chapter 4DArT was used to construct two high-density genetic linkage maps of the haploid wheat pathogen M. graminicola . The isolate IPO323 that originates from a bread wheat field was crossed in planta with either another bread wheat isolate IPO94269 or the durum wheat isolate IPO95052. Host species specificity of the plant pathogenic fungus M. graminicola towards hexaploid bread wheat and tetraploid durum wheat is well-documented. Although M. graminicola has an important sexual cycle and in some parts of the world durum wheat and bread wheat are grown side by side, in nature most isolates are pathogenic to either bread wheat or durum wheat. The generation of progeny from the IPO323 x IPO95052 in planta cross was therefore unexpected as these parental isolates are avirulent to durum wheat and bread wheat, respectively. The segregation of markers was followed in the progenies of both crosses. In total 2078 markers (of which 1793 were DArT markers) could be mapped. The maps of the individual crosses showed absolute co-linearity to a constructed bridge map, with the exception of eight markers that appeared to reveal translocations between IPO95052 and IPO94269. Graphical genotyping enabled the identification of dispensable chromosomes in 15-20 % of the progenies, although these chromosomes were present in both parents. We demonstrate that this is due to aberrations (partly by non-disjunction) during meiosis. The loss of chromosomes did not seem to affect viability and pathogenicity. This is the first report on detailed meiotic events using high-density mapping in a haploid filamentous fungus. We show that DArT is a highly efficient and robust marker technology for genetic mapping. The DArT markers can be readily sequenced and are currently being deployed in the assembly of the 9X M. graminicola IPO323 genome sequence.In Chapter 5 the genetic linkage map of the IPO323 x IPO95052 cross was used to map several quantitative trait loci (QTL) for host species specificity in M. graminicola .Virulence of the progeny (n=163) was tested on four bread wheat cultivars and three durum wheat cultivars. All progeny isolates grew normally in vitro,but in contrast to crosses performed with two bread wheat isolates, nearly 50 % were unable to cause disease on any of the generally susceptible bread wheat and durum wheat cultivars. Interestingly, other progeny caused disease on either bread wheat cultivars or durum wheat cultivars or both wheat species. This proves that the observed host species specificity was overcome in a single generation. In addition, some progeny were able to cause disease on the bread wheat cv. Shafir even though both parents are avirulent to this cultivar. Detailed analyses allowed the identification of QTLs that determine specificity using the high-density genetic linkage map containing 1144 molecular markers, described in the previous chapter. In total, we identified nine QTLs on seven linkage groups. Remarkably, no specific locus was identified that explains the host species specificity to either durum or bread wheat. One locus with a very high LOD value had a major effect on specificity towards all tested durum wheat cultivars, but was previously identified as a locus involved in cultivar specificity. To our knowledge, this is the first comprehensive QTL analysis on virulence performed on a plant pathogenic fungus. We conclude that specificity in M. graminicola is controlled by many genes and that the long-accepted notion of species specificity among isolates of M. graminicola may in fact be a mix of genetically inherited factors, whereby the distinction between host species specificity and cultivar specificity is not clear-cut.Chapter 6shows the alignment of the DArT markers to the genome of M. graminicola that recently was sequenced by the US Department of Energy-Joint Genome Institute (DOE-JGI). Assembly (version 1.0) of the 9X shotgun sequence resulted in the identification of 129 scaffolds ranging in size from 99 kb to 6.36 Mbp and covering 41.9 Mbp. In this assembly a total of 36 telomeres were identified. Sequencing of DArT markers used for the construction of two high-density genetic linkage maps in M. graminicola provided us the opportunity to align the genetic maps with the physical (sequence) map. By performing BLAST and BLAT analysis of the sequences obtained, 1069 IPO323, 724 IPO95052 and 24 SSR markers could be positioned on the 22 largest scaffolds in the assembly allowing the most comprehensive alignment among sequenced fungal genomes. Comparison of the positions of these markers on the sequence map with the order of the markers on the genetic linkage maps resulted in a nearly complete co-linearity of the order of markers, covering >99 % of the current assembly. When a 2 cM confidence interval was applied, 99 % of the markers were co-linear with the genome sequence. The alignment provided evidence that scaffolds 7 and 17, 10 and 14 and also 12 and 22 belong to one chromosome and that scaffolds 4 and 9 were wrongly assembled in the first draft. Furthermore, we identified large differences in genetic versus physical distance in certain genomic regions demonstrating that recombination frequencies increase towards the telomeres. In addition, we identified significant differences in the recombination rates in the different crosses studied. The integration of the genetic maps with the physical map will: 1) assist in finishing the genome assembly; 2) facilitate map-based cloning strategies of genes involved in pathogenesis and 3) help to understand the processes that occur during meiosis and recombination.Finally, Chapter 7 gives a general discussion. The advantages and disadvantages of the DArT marker method as well as alternative array-based technologies are discussed. We conclude that DArT is a cost effective marker technology which generates the extremely high quality data needed for high-density mapping. In addition the ease in which sequences of the markers are obtained, allow large-scale alignments between genetic and physical maps. This makes DArT the method of choice especially for species for which large genetic variation exists, limited financial resources are available or for complex polyploid genomes that may not be amenable to the whole-genome sequence approach. In addition the results obtained using the high-density genetic linkage maps from M. graminicola and the biological implications for the fungal population in respect to adaptation to the host are discussed

Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana

Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. This paper demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana. Restriction fragments from a genomic representation of the ecotype Landsberg erecta (Ler) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypes Columbia (Col) and Ler and of individuals from an F2 population obtained from a Col × Ler cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the Ler markers and alignment with the available Col genome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphism

Transposons passively and actively contribute to evolution of the two-speed genome of a fungal pathogen

Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in "arms races" with their hosts. In many pathogens, genes mediating virulence cluster in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of these compartments, and the role of transposons therein, remains limited. Here, we show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen Verticillium dahliae. We show that highly variable lineage-specific (LS) regions evolved by genomic rearrangements that are mediated by erroneous double-strand repair, often utilizing transposons. We furthermore show that recent genetic duplications are enhanced in LS regions, against an older episode of duplication events. Finally, LS regions are enriched in active transposons, which contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of pathogen virulence

Transposons passively and actively contribute to evolution of the two-speed genome of a fungal pathogen

Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in "arms races" with their hosts. In many pathogens, genes mediating virulence cluster in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of these compartments, and the role of transposons therein, remains limited. Here, we show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen Verticillium dahliae. We show that highly variable lineage-specific (LS) regions evolved by genomic rearrangements that are mediated by erroneous double-strand repair, often utilizing transposons. We furthermore show that recent genetic duplications are enhanced in LS regions, against an older episode of duplication events. Finally, LS regions are enriched in active transposons, which contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of pathogen virulence.</p

Meiosis drives extraordinary genome plasticity in the haploid fungal plant pathogen Mycosphaerella graminicola

Meiosis in the haploid plant-pathogenic fungus Mycosphaerella graminicola results in eight ascospores due to a mitotic division following the two meiotic divisions. The transient diploid phase allows for recombination among homologous chromosomes. However, some chromosomes of M. graminicola lack homologs and do not pair during meiosis. Because these chromosomes are not present universally in the genome of the organism they can be considered to be dispensable. To analyze the meiotic transmission of unequal chromosome numbers, two segregating populations were generated by crossing genetically unrelated parent isolates originating from Algeria and The Netherlands that had pathogenicity towards durum or bread wheat, respectively. Detailed genetic analyses of these progenies using high-density mapping (1793 DArT, 258 AFLP and 25 SSR markers) and graphical genotyping revealed that M. graminicola has up to eight dispensable chromosomes, the highest number reported in filamentous fungi. These chromosomes vary from 0.39 to 0.77 Mb in size, and represent up to 38% of the chromosomal complement. Chromosome numbers among progeny isolates varied widely, with some progeny missing up to three chromosomes, while other strains were disomic for one or more chromosomes. Between 15¿20% of the progeny isolates lacked one or more chromosomes that were present in both parents. The two high-density maps showed no recombination of dispensable chromosomes and hence, their meiotic processing may require distributive disjunction, a phenomenon that is rarely observed in fungi. The maps also enabled the identification of individual twin isolates from a single ascus that shared the same missing or doubled chromosomes indicating that the chromosomal polymorphisms were mitotically stable and originated from nondisjunction during the second division and, less frequently, during the first division of fungal meiosis. High genome plasticity could be among the strategies enabling this versatile pathogen to quickly overcome adverse biotic and abiotic conditions in wheat field

Effector discovery in the fungal wheat pathogen Zymoseptoria tritici

Fungal plant pathogens, such as Zymoseptoria tritici (formerly known as Mycosphaerella graminicola), secrete repertoires of effectors to facilitate infection or trigger host defence mechanisms. The discovery and functional characterization of effectors provides valuable knowledge that can contribute to the design of new and effective disease management strategies. Here, we combined bioinformatics approaches with expression profiling during pathogenesis to identify candidate effectors of Z.¿tritici. In addition, a genetic approach was conducted to map quantitative trait loci (QTLs) carrying putative effectors, enabling the validation of both complementary strategies for effector discovery. In¿planta expression profiling revealed that candidate effectors were up-regulated in successive waves corresponding to consecutive stages of pathogenesis, contrary to candidates identified by QTL mapping that were, overall, expressed at low levels. Functional analyses of two top candidate effectors (SSP15 and SSP18) showed their dispensability for Z.¿tritici pathogenesis. These analyses reveal that generally adopted criteria, such as protein size, cysteine residues and expression during pathogenesis, may preclude an unbiased effector discovery. Indeed, genetic mapping of genomic regions involved in specificity render alternative effector candidates that do not match the aforementioned criteria, but should nevertheless be considered as promising new leads for effectors that are crucial for the Z.¿tritici–wheat pathosyste