Varying degrees in the conservation of gene order between species (synteny) have been observed. The first data reported for plants concerned the conservation of gene order between the tomato and potato genomes (Tanksley et aI., 1992). Subsequently, the comparative genetic analysis of cereal genomes yielded very exciting results.
These studies have compared the genomes of different species of cereals such as rice and wheat (Kurata et aI., 1994), wheat and rye (Devos et aI., 1993a) barley and wheat (Devos et aI., 1993b) maize and sorghum (Melake Berhan et al., 1993), maize and rice (Ahn and Tanksley 1993) or barley and rice (Saghai-Maroof et aI., 1996).
The conservation of gene order as indicated by molecular markers was remarkable, even in the case of species with large differences in genome size (such as in maize and sorghum or wheat and rice). This suggests that the difference in genome size is not due to a different number of genes but rather to a different amount of highly repetitive DNA with no known function. Recently, Van Deynze et al. (1995a,b) reported comparative mapping data between wheat, rice, maize and oat. In the database
"Grain genes" (http://wheat.pw.usda.gov/) consensus maps of several species belonging to the Triticeae are available. These data show that relative positions on homoeologous or orthologous (see Box 1) chromosome segments are conserved for agronomically important traits including resistance to leaf and stem rusts (Van Deynze et aI., 1995a).
For example, the close linkage between loci containing seed storage proteins (avenins in oat, glutenins and gliadins in wheat and hordeins in barley) and resistance genes (stem rust Pg9 and crown rust PcX in oat; stem rust Sr33, leaf rust LrlO/Lr21 and powdery mildew Pm3 in wheat; powdery mildew Mia in barley) seem to be very well conserved on the short arm of homoeologous group 1 chromosomes (Fig. 6). Thus, resistance genes in different species seem to be closely related and possibly origin from the same ancestral gene.
AS 1HS
Hor2 Avn B2IB4
Glil
Horl
Glu3
, Gli3
Oat Wheat Barley
Figure 6. Schematic representation of the synteny between the end of the homologs of group 1 chromosomes in oat, wheat and barley. Loci containing genes coding for seed storage proteins (avenins (Avn), glutenins and gliadins (Glu, Gli) and hordeins (Hor) are conserved in this region as well as genes encoding resistance genes for rust (Sr, Lr, Pg, PcX) and powdery mildew (Pm, Mia) diseases.
These findings open new possibilities for gene isolation strategies using positional cloning techniques, especially in the case of those plants for which huge genome size and high amount of repetitive sequences represent a barrier in chromosome walking strategies.
The conservation of gene order and composition of loci containing resistance genes will allow to use species with small genomes (rice, sorghum) and species for which tools such as YAC (yeast artificial chromosomes) and BAC (bacterial artificial chromosomes) libraries have been developed (maize, rice, barley) as genetic model plants.
This should greatly help to locate more precisely and eventually isolate genes even if the locus was originally identified in species with large or even polyploid genomes.
Fine scale analysis demonstrated a conserved gene organization along a small segment (1-2 cM genetically, 1 Mbp physically) of a rice chromosome if compared to the syntenic region in barley (Dunford et aI., 1995). The synteny between rice and barley was used by Kilian et al. (1995) to saturate the region of the barley RpgJ resistance gene in order to facilitate the map-based cloning of this gene.
The recent cloning of resistance genes against bacterial, fungal and viral diseases from different plant species showed that there are common features among sequences and conserved motifs (see chapter 4 and Jones and Jones, 1997). This opens up very exciting possibilities of gene isolation through homology-based cloning. So far, this approach has been used to successfully isolate candidate disease resistance genes in potato (Leister et at., 1996) and soybean (Kanazin et at., 1996) and is being tried for many other crops. In both cases the authors designed degenerate primers from two regions conserved among the nucleotide binding sites of the N resistance gene from tobacco, the RPS2 gene from Arabidopsis and the £6 gene from flax. They were able to amplify several classes of 500 bp DNA fragments differing in their nucleotide sequences but all containing the internal kinase 2/3 domains found in those resistance genes (see
116 B. Keller, C. Feuillet & M Messmer
Chapter 4). These fragments were subsequently used as probes for genetic mapping.
In both species, some of the isolated sequences mapped close to known disease resistance genes. Further work using transformation is necessary to verify whether some of the cloned sequences are part of race-specific resistance genes. This example shows that homology-based strategies and the conserved gene order in the genomes of related species can be used to isolate resistance gene candidates in species for which map-based cloning strategies are very difficult to develop. However, as a note of caution it should be remembered that not all characterised resistance genes belong to the class of proteins mentioned above. For genes encoding Serffhr protein kinases, such as the Pto gene which is responsible for resistance in tomato against Pseudomonas syringae (Martin et ai., 1993b), the homology-based approach is more difficult as there is a large number of kinases in eucaryotic genomes. Nevertheless it was possible by this approach to identify a new class of receptor-like kinase segregating with the leaf rust Lr 10 resistance locus in wheat (Feuillet et ai., 1997). There is no doubt that in the next years a number of disease resistance genes will be isolated by using conservation in gene order and/or homology-based cloning.
IV. Genetic Markers as Tools for the Characterization of Monogenic Resistance Genes
A. THE DEVELOPMENT OF GENETIC MARKERS FOR MONOGENIC
RESISTANCES
A genetic marker is broadly defined as a polymorphism between two plant lines which is inherited in a simple Mendelian way in the progeny of a cross between these lines. Several types of polymorphisms such as plant morphology, isozymes and DNA sequences have been used as genetic markers. The methods used to detect polymorphisms have evolved and improved in the last decades following the development of techniques in molecular biology.
In order to combine genes in a single individual (in the case of resistance genes this is called "pyramiding" resistance genes), it is necessary to determine the genotypes of plants and not just the phenotypes. The combination of complex resistances (quantitative resistances, see below) or the pyramiding of resistance genes is very difficult to achieve with classical breeding methods. By speeding up the process of selection, genetic markers represent an indispensible tool for plant improvement in general and for resistance breeding in particular. In addition, the development of high- density linkage maps composed of molecular markers is a prerequesite for resistance gene isolation through map-based cloning strategies as described in chapter 4.
Several excellent reviews on the application of genetic markers in plant breeding have been published (Paterson et aI., 1991; Winter and Kahl, 1995; Mohan et aI., 1997).
Here, we want to focus on the development of markers for resistance genes and the contribution of the different marker technologies for mapping, detection and finally isolation of disease resistance genes in crop plants.
B. THE IDENTIFICATION OF GENETIC MARKERS FOR RESISTANCE GENES