Genotypic identification using molecular methods

The IBEB differential system provides a macroscopic way to investigate the B.

lactucae and lettuce interaction phenotypically. For further understanding the mechanism, molecular methods are essential tools to investigate these interactions genotypically. There are several kinds of markers showing the potential for

development of B. lactucae race genotypic identification.

1.4.3.1 RFLP, AFLP and RADP markers

RFLP (restriction fragment length polymorphism), AFLP (amplified fragment length polymorphism) and microsatellite marker RAPD (randomly amplified polymorphic DNA) have been used in molecular studies and are invaluable for generating fingerprints of pathogen isolates (Dickinson, 2003). These markers have been used

for genetic mapping of B. lactucae (Hulbert et al., 1988; Sicard et al., 2003).

Faba bean genetic diversity studies using RADP and AFLP indicated some of the technical limitation of these techniques. RAPD markers have been used to study the

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genetic diversity within European and Mediterranean Faba bean germplasm (Zeid et

al., 2003). AFLP markers have been used to assess genetic diversity among elite

Faba bean inbred lines (Zeid et al., 2003). However, these two molecular marker

systems have some technical limitations. The RAPD technique has low laboratory-to-laboratory reproducibility, and the AFLP technology has a high cost. However ISSR techniques can overcome the above limitations and yield more polymorphisms than other molecular techniques (Terzopoulos and Bebeli, 2008).

1.4.3.2 Ribosomal Internal Transcribed Spacer (ITS) markers

Peagy et al. (2008) indicated that the internal transcribed spacer region has been

widely sequenced for DNA regions in fungi. It has typically been most useful for

molecular systematics at the species level, and even within species (e.g. to identify

geographic races). Variation among individuals rDNA repeats can sometimes be observed within both the ITS and IGS regions, due to the higher degree of variation of ITS regions than other regions of rDNA. ITS1+ITS4 primers have been frequently used and several taxon-specific primers have been described that allow selective amplification of fungal sequences specifically describing amplification of basidiomycete ITS sequences from mycorrhiza samples (Gardes and Bruns, 1993).

1.4.3.3 Inter Simple Sequence Repeat markers (ISSR)

ISSR (inter-simple sequence repeat) (Zietkiewicz et al., 1994) is a general term for a

genome region between microsatellite loci. The complementary sequences of two neighbouring microsatellites are used as PCR primers which are generated from a single-primer PCR reaction where the primer is designed from di- or trinucleotide

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al., 1998); the variable region between them gets amplified. The limited length of

amplification cycles during PCR prevents excessive replication of overly long contiguous DNA sequences, so the result will be a mix of a variety of amplified DNA strands which are generally short but vary much in length. So far, ISSR-PCR has been demonstrated to provide highly reproducible results and generate abundant

polymorphisms in a number of host pathogen interactions (Ajibade et al., 2000).

ISSR markers have been developed for identification of B. lactucae races (Wagner

and Idczak, 2004).

1.4.3.4 Simple Sequence Repeats (SSR) markers

Microsatellites or simple sequence repeats (SSRs) are tandem repeats of 2-8 base pairs (bp) that can vary extensively in the number of repeats. They are valuable as genetic markers because they are co-dominant, detect high levels of allelic diversity,

and are easily and economically assayed by PCR (McCouch et al., 1997).

Microsatellite DNA loci have become important sources of genetic information for a variety of purposes. Specific microsatellite repeats primers must be developed to amplify microsatellite loci by PCR (Glenn and Schable, 2005). These microsatellite regions of DNA are among the most variable in the genome, thus primer-binding

sites are not well conserved among distantly related species (Primmer et al., 1996).

Although microsatellite loci have now been developed for hundreds of species these loci have not been isolated from many additional species (Glenn and Schable, 2005).

Many different strategies for obtaining microsatellite DNA loci have been described. Cloning small genomic fragments and using radiolabelled oligonucleotide probes of

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microsatellite repeats to identify clones with microsatellites works well in organisms

with abundant microsatellite loci (Weber et al., 1991). It does not work well when

microsatellite repeats are less abundant. Therefore another two enrichment strategies

have been developed: (1) uracil-DNA selection (Ostrander et al., 1992) and (2)

hybridization capture (Armour et al., 1994; Kandpal et al., 1994; Kijas et al., 1994).

Hybridization capture allows selection prior to cloning, and therefore is faster and easier to achieve with multiple samples than uracil-DNA selection (Glenn and Schable, 2005).

1.4.3.5 Effector based markers

Oomycete cytoplasmic effectors have been discovered through their avirulence (Avr)

function, where they have the ability to trigger hypersensitive cell death in host

genotypes with corresponding disease resistance (R) genes (Morgan and Kamoun,

2007; Allen et al., 2004; Armstrong et al., 2005). Recent studies indicated that RxLR

effectors possess virulence function (Bos et al., 2006; Sohn et al., 2007; Dou et al.,

2008). Multiple candidate effectors from the oomycete pathogen Hyaloperonospora

arabidopsidis (Hpa) have recently been studied to ascertain if they can suppress the

host plant immunity (Fabro et al., 2011), which showed that Single Nucleotide

Polymorphisms (SNPs) in effector candidates between seven Hpa isolates (Cala2,

Emco5, Emoy2, Hind2, Maks9, Noco2, Waco9) were detected. These studies show a possibility that effectors candidates could be developed as markers to identify the variation between the oomycete isolates.

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HYPOTHESIS AND OBJECTIVE OF THE PROJECT

The overall aim of the project is to develop and use molecular methods to study

changes in population structure of B. lactucae on lettuce within agricultural

ecosystems. An important aspect was to develop molecular markers for epidemiological studies and compare these with existing methods for differentiating

B. lactucae races. Physiological methods were applied for phenotypic B. lactucae

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