|Title||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|
|Source||Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Henk Schouten; Theo van der Lee. - [S.l.] : S.n. - ISBN 9789085046295 - 222|
PRI Biodiversity and Breeding
Biointeracties and Plant Health
|Publication type||Dissertation, internally prepared|
|Keyword(s)||genetische kartering - technieken - nucleotidenvolgordes - genomen - arabidopsis thaliana - plantenziekteverwekkers - mycosphaerella - genetische merkers - plantenveredeling - genetic mapping - techniques - nucleotide sequences - genomes - arabidopsis thaliana - plant pathogens - mycosphaerella - genetic markers - plant breeding|
|Categories||Plant Breeding and Genetics (General)|
|Abstract||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 ecotypes
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.