Genetic mapping using the Diversity Arrays Technology (DArT)

224 Pages·2007·10.22 MB·English

by  Wittenberg, Alexander

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

Alexander H.J. Wittenberg
Promotor:
Prof. Dr. R.G.F. Visser
Hoogleraar in de Plantenveredeling
Wageningen Universiteit

Co-promotoren:
Dr. Ir. H.J. Schouten, Senior Onderzoeker, Plant Research International, Wageningen
Universiteit en Researchcentrum.
Dr. T.A.J. van der Lee, Onderzoeker, Plant Research International, Wageningen Universiteit
en Researchcentrum.

Promotiecommissie:
Prof. Dr. A.G.M. Gerats (Radboud Universiteit Nijmegen)
Dr. Ir. G.H.J. Kema (Wageningen Universiteit)
Dr. J.N.A.M. Rouppe van der Voort (Keygene N.V., Wageningen)
Prof. Dr. W.J. Stiekema (Wageningen Universiteit)

Dit onderzoek is uitgevoerd binnen de onderzoeksschool ‘Experimental Plant Sciences’.
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

Alexander H.J. Wittenberg

Proefschrift
ter verkrijging van de graad van doctor
op gezag van de rector magnificus
van Wageningen Universiteit
Prof. dr. M.J. Kropff
in het openbaar te verdedigen
op vrijdag 16 maart 2007
des namiddags te 13.30 uur in de Aula
CIP-DATA Koninklijke Bibliotheek, Den Haag

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

Alexander H.J. Wittenberg (2007)

PhD thesis, Wageningen University, The Netherlands
With summaries in English and Dutch

ISBN 90-8504-629-7
Contents

Chapter 1 General introduction and outline of thesis 7

Chapter 2 Genome-wide profiling using Diversity Arrays Technology (DArT);
theoretical considerations and practical protocols 25

Chapter 3 Validation of the high-throughput marker technology DArT using the
model plant Arabidopsis thaliana 59

Chapter 4 High-density maps of the fungus Mycosphaerella graminicola, using
Diversity Arrays Technology (DArT) reveal frequent loss of
chromosomes 81

Chapter 5 Quantitative trait loci determine specificity to bread and durum wheat
cultivars in the fungal wheat pathogen Mycosphaerella graminicola 109

Chapter 6 Whole-genome alignment of DArT and SSR markers positioned on
genetic linkage maps of Mycosphaerella graminicola 129

Chapter 7 General discussion 151

Summary 167

Samenvatting 170

Nawoord 173

Curriculum Vitae 176

Education certificate of the EPS graduate school 177

Appendices
Appendix I DArT protocol 181
Appendix II Genetic linkage maps of Mycosphaerella graminicola 189
Appendix III Alignment genetic maps with physical map 208
Appendix IV Colour figures 215

Chapter 1

General introduction and outline of thesis
Chapter 1
Genetic diversity

Genetic diversity is generally defined as the amount of genotypic (on the DNA level)
variability present in a group of individuals. Genetic diversity gives species the ability to
adapt to changing environments, including new pests and diseases and new climatic
conditions, such as global warming (Peters and Lovejoy 1992; Parmesan and Yohe 2003).
Knowledge about diversity present in germplasm collections around the world and genetic
relationships among breeding materials is therefore essential in crop improvement strategies.
Agriculture began thousands of years ago when farmers began to raise wild plants and
animals for food. They learned to select for individuals with desirable traits. This process of
domestication resulted in a wide range of varieties that helped them to survive under difficult
conditions. In contrast, modern agriculture has evolved into the practice of raising
monocultures of crops, in which most of the gene variants are the same in every individual of
a particular variety or breed. These monocultures are highly productive but their reduced
genetic variability leaves them with a diminished capacity to deal with new diseases, pests,
and other changes in environmental conditions. A good example is the Irish potato famine
(1845-1849), that in part could be attributed to the fact that there were only a few different
genetic strains of potatoes grown, making it easier for the oomycete Phytophthora infestans to
infect and kill the majority of the crop. The pseudo-fungus is after 150 years still causing
major problems for many farmers (Fry and Goodwin 1997).

A number of methods have been developed over the past decades for the analysis of genetic
diversity in germplasm accessions, breeding lines and populations. These methods have relied
mainly on the availability of genetic markers (Avise 2004). A genetic marker represents
variation at a particular site on the genome, which is inherited in a Mendelian way, is easy to
assay and can be followed over generations. Genetic markers can be ordered on the
chromosomes and in this way the entire genome of an individual can be visualized on a
genetic linkage map.
For the construction of genetic linkage maps three types of genetic markers can be employed:
morphological (plant traits), biochemical (proteins and isozymes) and molecular (DNA-
based) markers. Morphological and biochemical markers have enabled the mapping of several
important qualitative traits on the first genetic linkage maps in animal (Sturtevant 1913), plant
(MacArthur 1934; Emerson 1935) and fungal (Lindegren 1936) genomes. These markers are
however limited in number and are therefore less efficient for the identification of complex
quantitative traits (e.g. yield and quality). In addition morphological markers are influenced
by the environment, are development stage specific and can have pleiotropic effects.
Molecular markers are numerous in number and do provide the capacity for complete genome
coverage. These markers enable the identification and genetic localization of the contributing
genetic factors as quantitative trait loci (QTLs) and their utilization in crop improvement.
The development and availability of new high-throughput cost effective marker systems has
important implications for the future of marker assisted selection (MAS) and breeding
strategies in general. Genome-wide information in the form of graphical genotypes, marker
8
Introduction
information and known location of key loci for both desirable and undesirable alleles is
available for some of the major crop species. The challenge for the coming years will be how
we can design optimal breeding strategies that make use and integrate all of this information
(Peleman and van der Voort 2003). These strategies for “whole genome breeding” are
currently underway in a number of crops and will have major impacts on modern agriculture.
A brief overview of the most commonly used molecular markers types is presented below.

Types of molecular markers

Molecular markers reflect heritable differences (e.g. polymorphisms) in homologous DNA
sequences among individuals. These differences may be due to single nucleotide
polymorphisms (SNPs), insertions or deletions (INDELs) or rearrangements (translocations or
inversions). The methods of detection of polymorphism involve the use of 1) restriction
endonuclease, 2) nucleic acid hybridization or 3) DNA sequence amplification. A large
number of reviews have been published on molecular markers and their application in crop
improvement (Jain 2002; Lörz and Wenzel 2005). The decision which marker system is the
most appropriate to use will depend on the species, the objective of the marker work and on
the resources available. Here the most widely used molecular marker technologies will be
described.

1) Restriction Fragment Length Polymorphism (RFLPs)
RFLPs are differences in restriction fragment lengths caused by SNPs or INDELs that create
or destroy restriction endonuclease recognition sites. Both the basis and techniques for RFLPs
(Botstein 1980) in plant genome mapping have been extensively reviewed (Tanksley et al.
1989). RFLPs are assayed by hybridizing labelled (c)DNA probes to a Southern blot
(Southern 1975) of genomic DNA digested with various restriction enzymes. Marker alleles
are identified by size differences of the restriction fragments to which these probes hybridize.
The RFLP marker technology allowed the construction of the first whole-genome linkage
maps in plants (Bernatzky and Tanksley 1986; Helentjaris et al. 1986) and initiated the rapid
developments in the field of comparative genomics (Gale and Devos 1998; Paterson et al.
2000).
Some advantages of the use of RFLPs are that, if single-copy, most markers can be scored co-
dominant, are locus specific and high-throughput PCR-based markers can easily be developed
from the probe sequences. Some limitations to the use of RFLPs are:
• Development of RFLP probe sets and markers is labor intensive and the multi-step
protocol is time-consuming.
• Analysis requires large amounts (1-10 µg/gel lane) of high-quality DNA.
• RFLPs are difficult to automate/multiplex and therefore have a low genotyping
throughput.
• RFLP probes must be physically maintained and thus are difficult to share between
laboratories.
9
Chapter 1
2) Random Amplified Polymorphic DNAs (RAPDs)
RAPD markers are defined as DNA polymorphisms produced by “rearrangements or
deletions at or between oligonucleotide primer binding sites in the genome” (Welsh and
McClelland 1990; Williams et al. 1990). The method simultaneously amplifies DNA
fragments with a single random-sequence primer (usually 10-base oligomers) in a low-
stringency PCR (35-45°C). These fragments are separated on conventional agarose gels and
RAPDs are identified by the presence or absence of a particular fragment (i.e. band). RAPD
markers can be converted into simple and robust PCR markers termed Sequence
Characterized Amplified Regions (SCARs) by developing site specific primer pairs from
cloned RAPD fragments. DNA Amplification Fingerprinting (DAF) is a modified approach of
the RAPD technique. It employs one or more primers as short as five nucleotides in length to
produce complex banding patterns that are resolved by polyarcylamide gel electrophoresis.
The major advantage of the use of RAPDs is the use of universal primers (Tingey et al. 1994).
Other advantages are the small amount of DNA required (5-25 ng/individual) and the relative
low start-up costs (Waugh and Powell 1992). The major limitations to the use of RAPDs are:
• The reproducibility of RAPD assays across laboratories is generally low (Perez et al.
1998).
• Most RAPD markers are dominant, although some can be converted into locus-
specific co-dominant markers (Davis et al. 2005).
• The homology of fragments across genotypes cannot be ascertained without
sequencing.

3) Amplified Fragment Length Polymorphism (AFLP)
Amplified Fragment Length Polymorphism (AFLP™) is a successful, PCR-based multi-locus
fingerprinting technique that efficiently identifies DNA polymorphisms without prior
sequence information (Vos et al. 1995; Mueller and Wolfenbarger 1999). The polymorphisms
are scored by differences in restriction fragment lengths caused by SNPs or INDELs in or
adjacent to the endonuclease restriction sites. AFLP assays are performed by selectively
amplifying a subset of genomic restriction fragments using PCR. The selectivity is achieved
by using selective nucleotides that are added to the 3’ ends of the PCR primers that anneal to
the adapters ligated to the restriction sites. Only restriction fragments in which the nucleotides
flanking the restriction site match the selective nucleotides will be amplified. The subset of
amplified fragments is then separated with gel electrophoresis to generate the fingerprints.
The development of the AFLP method has had a large impact on genomics as it was the first
method that cost-effectively enabled the identification and typing of a large number of
markers throughout the genome using a simple and robust protocol (Blears et al. 1998). A
major improvement has been made by switching from radioactive to fluorescent dye-labeled
primers for the detection of fragments in gel-based or capillary DNA sequencers (Schwarz et
al. 2000). The success of the technology mainly can be contributed to the high multiplex ratio
and genotyping throughput, the high reproducibility, the low amount of DNA required (200-
10