B. THE IDENTIFICATION OF GENETIC MARKERS FOR RESISTANCE GENES
118 B. Keller, C. Feuillet & M Messmer
In the absence of NILs, bulk segregant analysis (Giovannoni et ai., 1991; Michelmore et al., 1991) can be used for the identification of genetic markers linked to a target region. In this approach, samples of a segregating population are pooled into different classes determined by their phenotype (resistant or susceptible). A marker present in only one pool but not in the other one represents a potential marker for the selected phenotypic trait. This approach has been used to characterize resistance loci such as the plr locus in lettuce (Kesseli et ai., 1993) and the cyst nematode Ccn-D 1 resistance gene in wheat (Eastwood et ai., 1994).
C. CLASSES OF GENETIC MARKERS
1. Morphological and biochemical markers
The first type of marker used were morphological markers for which a distinct morphological trait is determined by a single gene which is linked to the gene of interest. Such markers are principally very easy to work with and quick to score, but there are also serious limitations for their use. There are relatively few such markers in any given species and the expression of the morphological marker can be influenced by the environment. In addition, they are often expressed only at a specific growth stage or in a particular organ of the plant. Consequently, this type of marker only allows the development of reasonably good linkage maps in a few species (e.g. maize, barley, pea, tomato). Most of the morphological markers, which are often mutations, can not be used in breeding programs due to their strong and negative effects on the plant. A very limited number of morphological markers have been found which are linked to resistance genes and useful for practical breeding. Singh (1992, 1993) demonstrated a genetic linkage between leaf tip necrosis (Ltn, Fig. 7) and a number of resistance genes against leaf rust (Lr34), yellow rust (Yr18) and barley yellow dwarf virus (Bdv 1) in wheat.
Figure 7. Leaf tip necrosis as a morphological marker for the wheat leaf rust resistance gene Lr34. Flag leaves of near-isogenic wheat lines with Lr34 (left) and without Lr34 (right) are shown. Leaf tip necrosis can be observed in the line with Lr34.
The second type of markers are biochemical markers which are based on differences in charged amino acids of enzymes with little or no effect on the enyzmatic activity. Such biochemically distinct isozymes are the products of different alleles of the coding gene.
Maps based on isozyme markers have been established for several plant species and several isozymes have been found to be linked with disease resistance genes. They are used in many breeding programs to select efficiently for the presence of a particular resistance gene. A phosphoglucomutase is closely linked to the Mo gene conferring resistance to the bean yellow mosaic virus in pea (Weeden et aI., 1984) and an alcohol dehydrogenase isoform is a good marker for resistance to the pea enation mosaic virus (En) (Weeden and Provvidenti, 1987). A complete linkage was described between the endopeptidase isozyme Ep-Dlb and an eyespot resistance gene derived from Aegilops ventricosa (McMillin et aI., 1986) as well as between Ep-Dlc and the wheat leaf rust resistance gene Lr19 introgressed from Agropyron elongatum (Winzeler et aI., 1995).
These isozyme markers have been extensively used in improvement of crop resistance by breeding. However, similarly to morphological markers, there is only a small number of genetic loci which can be detected by these markers and their expression is often restricted to specific developmental stages or tissues (Table 2).
2. Molecular (DNA) Markers
The integration of DNA markers in genetic mapping projects substantially increased the number of markers available for agronomically important traits. The many advantages of DNA markers in comparison to morphological and isozyme markers (see Table 2) led to the intensive development and integration of those markers in breeding programs in recent years.
The first group of molecular markers developed were the RFLP (Restriction Fragment Length Polymorphism) markers which are based on the detection of polymorphic DNA fragments after restriction digests. Because RFLPs are mostly codominant markers (i.e. heterozygotes are easily identifiable because they combine the phenotype of the two homozygotes) and there is basically an almost unlimited number of loci, they represent very good and informative markers in linkage analysis and marker-assisted selection. The development of these markers led to a dramatic improvement in the construction of linkage maps and the elucidation of loci contributing to quantitative traits, so called quantitative trait loci (QTLs), in many crops. RFLP markers have been found to be linked with disease resistance genes in many species such as Arabidopsis, lettuce, flax, potato, tomato, maize, rice, wheat, barley etc.
Despite their usefulness, the application of RFLP in marker-assisted selection remains time-consuming and expensive because of the technical difficulties and the labor intensive methods.
The recent development of markers based on the polymerase chain reaction (peR) accelerated the development and use of molecular markers. The detection of peR markers is faster than with the more complex technique of RFLP markers. In addition, the important steps of the reaction and also of detection can be automated, saving costs and increasing the number of samples that can be analyzed. peR markers have been identified either by screening with arbitrary primers or with specific primers designed from known sequences.
Table 2. Comparison of the different genetic marker systems. The usefulness of a particular marker type for map-based cloning is estimated in terms of time to find very closely linked markers and the possibility to use them as tools for gene isolation. Morphological Isozyme RFLP RAPD Microsatellites AFLP Marker inheritance dominant codominant, codominant, dominant codominant codominant, multiple alleles multiple alleles multiple alleles dominant Developmental stage of detection mostly adult plant early stage all tissues at all tissue at all tissue at all tissue at tissue-specific early stages early stages early stages early stages expression (seeds) (seeds) (seeds) Number of loci limited limited -unlimited -unlimited -unlimited -unlimited Degree of polymorphism low intermediate intermediate high high very high Synteny, comparative mapping (yes) no yes no no ? Analysis easy in some cases fast, easy, time consuming, fast, simple, fast, simple, fast, (time, feasibility, cost, safety) cheap no automation, automation, automation, automation, expensive problems with expensive expensive reproducibility Application in breeding programs limited good limited limited good limited (?) Usefulness for map-based cloning impossible impossible very long (fast if PCR fast if PCR very fast screening of large screening of large insert libraries insert libraries can can be done) be done. Otherwise useless as probe for further cloning (repetitive)
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'"Arbitrary primers are used for RAPD (Random Amplified Polymorphic DNA) markers (Williams et aI., 1990). RAPD markers were the first PCR-based markers described. They are based on the amplification of DNA fragments between short (usually 10 bases) arbitrary oligonucleotide primers and allow the detection of polymorphisms resulting from insertions, deletions and single base changes that alter the binding sequence. RAPD markers may contain repetitive sequences within the amplified fragment and therefore may detect genomic sequences which are inaccessible to RFLP analysis. RAPDs have been used as markers for resistance genes in several species with different degree of success. In sugar beet (Uphoff and Wricke, 1992) and tomato (Klein-Lankhorst et aI., 1991), RAPD markers were found which are closely linked to nematode resistance genes or to a resistance gene against Pseudomonas in tomato (Martin et aI., 1991). In wheat, Schachermayr et al. (1994, 1995) detected RAPD markers linked to the leaf rust resistance genes Lr9 and Lr24.
RAPD markers have not become widely used in marker-assisted selection because they are difficult to reproduce between different laboratories and even between different PCR thermal cyclers. In addition, they are usually dominant markers and therefore do not distinguish between heterozygotes and homozygotes. In some cases, these disadvantages have been overcome by using the RAPD fragment as a probe to highlight an RFLP on Southern (genomic DNA) blots to obtain codominant markers (Eastwood et al., 1994; Martin et aI., 1993a). Another alternative to RAPD markers are the sequence- tagged-site (STS) or sequence characterized amplified region (SCAR) markers where PCR primers are designed from mapped, low copy number sequences (from RFLP probes or RAPD fragments). This allows the specific amplification and identification of a unique sequence at a known location in the genome. Such markers have been successfully developed among others in lettuce for Dm genes for resistance against downy mildew (Paran and Michelmore, 1993), in bean for the Are gene against anthracnose (Adam-Blondon et al., 1994), in wheat for leaf rust (Schachermayr et aI., 1994, 1995, 1997; Feuillet et al., 1995) and cereal cyst nematode (Williams et aI., 1996a) resistance genes and for powdery mildew resistance loci in cereals (Mohler and lahoor, 1996).
Very recently, Zabeau and Vos (1993) developed a new technique which combines the reliability of the RFLP method with the power of the PCR technique. The amplified fragment length polymorphism (AFLP, Vos et aI., 1995) technique leads to the amplification of a random subset of restriction fragments without prior knowledge of nucleotide sequence. AFLP has been adapted to study complex genomes such as those of higher plants. To date, only few reports concerning AFLP markers linked to disease resistance genes have been published. However, the work of Thomas et al. (1995a) on AFLP markers linked to the Cj9 resistance gene in tomato demonstrates the usefulness of AFLP analysis for positional cloning strategies. Indeed, the authors were able to detect AFLP markers located on the opposite sides of the resistance gene separated by only 15.5 kb. The development of the AFLP technology addresses one of the most limiting factors in map-based cloning strategies, i.e. the ability to rapidly test thousands of loci for polymorphisms and identify markers very closely linked to the resistance gene.
For the same reasons, AFLP will probably become the technique of choice to map genetic loci contributing to more complex, quantitative traits (QTLs, see below).
122 B. Keller, C. Feuillet & M Messmer
In Fig. 8, different types of molecular markers are shown for some disease resistance genes. It is difficult to assign a relative advantage for one type of molecular marker in comparison to the others. Powell et al. (1996) recently performed an interesting comparison of the RFLP, RAPD, SSR (simple sequence repeats or microsatellites) and AFLP systems and concluded that each type of marker has different and complementary properties. It is likely that the integration of all different type of markers in genetic linkage maps will be the solution for generating highly saturated maps, The combination of RFLPs and RAPDs on tomato, maize and rice linkage maps has already enabled the detection of a number of markers tightly linked to disease resistance genes. This has represented the primary step towards the cloning of some of these genes. Indeed, the first cloned plant disease resistance gene (HM 1 in maize) was isolated by a combination of transposon mutagenesis and RFLP mapping (Johal and Briggs, 1992). Similarly, two race-specific resistance genes were cloned through map-based cloning in tomato (Pto, Martin et aI., 1993b) and rice (Xa21, Song et aI., 1995), The integration of new classes of promising marker systems such as AFLP and SSR (microsatellites, ROder et al., 1995) should enable the cloning of other resistance genes from more complex genomes.
Better linkage maps are also necessary for QTL analysis and the identification of genes involved in genetically complex quantitative resistance. In addition, the generation of marker systems which are easier to handle and to automate opens up interesting opportunities for application in marker-assisted breeding.
D. IMPACT OF THE ISOLATION OF RACE-SPECIFIC RESISTANCE GENES