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
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F'igure 8. Different types of DNA markers:
(A) RFLP marker for the wheat leaf rust resistance gene Lr24. Southern blot hybridization pattern of HindIII digested genomic DNA from the resistant parent Lr24lArina. the susceptible parent Oberkulmer and a segregating F2 population. The arrowhead indicates the fragment which segregates with the Lr24 resistance gene.
(B) AFLP analysis of three spring wheat and two winter wheat varieties. Template DNAs were digested with MseI and EcoRI. The amplification step was performed with primers containing the 3'-variable extension CTC or CTG, respectively.
(C) RAPD marker for the wheat leaf rust resistance gene Lr9. Amplification of a polymorphic DNA fragment produced by the random primer OPR-IS on genDmic DNA. The arrowhead indicates the position of the polymorphic band segregating with the Lr9 resistance gene in a F2 progeny deriv~d from the cross between the susceptible parent Arina and the resistant near isogenic line Lr917* Arina.
(D) STS marker for the wheat leaf rust resistanc~ gene Lr9. Amplification of a unique and specific DNA fragment with the primers 11311+2 designed from a RAPD marker linked to the Lr9 resistance gene.
Amplification was performed on genomic DNA from the susceptible parent Arina and the resistant NIL Lr917* Arina as well as F2 plants derived from their cross.
(The phenotype of the F2 plants is indicated as follows: R= resistant, S=susceptible)
124 B. Keller, C. Feuillet & M Messmer
Durable resistance obtained by pyramiding several resistance genes by classical breeding is a very long and costly process. The use of molecular markers can improve the efficiency of the selection process (by tracking the resistance gene as well as the genetic background of the recurrent parent) but the cloning of the resistance genes themselves should also greatly help to achieve broad spectrum resistance. Indeed, DNA probes derived from the sequences of cloned resistance genes represent ideal markers to track the genes efficiently during the selection process. It is also possible that once several effective resistance genes are cloned, they can be transformed as a cassette to crop plants. These gene pyramids should confer more durable resistance and would be easy to follow in the breeding program. In addition, the understanding of resistance mechanisms should allow the creation of new resistance genes by manipulating domains responsible for specificity to obtain resistance against a broad range of pathogens (Bent, 1996). In many cases pyramiding strategies have not succeeded because of the negative effects the combined genes have in the genetic background of elite lines, often leading to loss of other agronomically useful traits. Thus, the introduction of single resistance gene against a broad spectrum of pathogen races should avoid these problems by limiting the number of steps in the selection process and the negative effect of associated genes.
V. Vertical and Horizontal Resistance
Race-specific resistance genes and the corresponding avirulence genes in the pathogens belong to the genetically most intensively studied systems. These monogenic resistances are also of great importance for resistance breeding in many crop plants and, as discussed above, there are considerable efforts to find markers for these genes and to clone them. However, it has to be emphasized that this type of resistance represents only one form of resistance present in the gene pool of plants. Race-specific resistance is also called vertical resistance in contrast to the genetically (and physiologically) different type of non race-specific resistance which is often referred to as horizontal or polygenic resistance as there are several genes involved (see Box 3). Other terms used for this type of resistance are quantitative or partial resistance. The latter is a particularly useful term as it is purely descriptive and does not imply any genetic knowledge which is usually not present. The genes involved in this type of resistance are often called minor genes (in contrast to the major genes in vertical resistance).
As this type of resistance is sometimes only expressed in the adult stage, such resistance is also described as adult plant resistance. There is a lot of discussion about the terminology of vertical and horizontal resistance. Here, we follow the terminology suggested by Robinson (1969). Horizontal resistance is characterized by the absence of genetic interactions between the host genotype and the pathogen genotype (in contrast to the race-specific genetic interaction in vertical resistance in gene-for-gene relationships). Oligogenicity (2-6 genes) is a characteristic property of horizontal resistance. If a resistance is based on several genes it is probably not race-specific (some exceptions are described below). A large part of the genetic understanding of such complex resistances comes from new studies using molecular markers to analyze
the role of different genomic regions in the inheritance of a characteristic polygenic disease resistance. The methods and the results of such studies as well as the application of these results for resistance breeding will now be described.
Box 3. Vertical and horizontal resistance.
The tenns vertical and horizontal resistance were used by van rler Plank (1963) to describe the graphical representation of the relationships between host varieties and different races of the pathogen. A variety has vertical resistance if it is resistant against some of the existing pathogen races, but not to others (the variety shown below would be resistant to races 2,5,6,8):
Resistance
Susceptibility
2 3 4 5 6 7 8 9 10 11 pathogen races
Horizontal resistance is a resistance that is evenly spread against all races of the pathogen, i.e. a raGe.flon-specific resistance:
Resistance
Susceptibility
2 3 4 5 6 7 8 9 10 11 pathogen races
VI. Quantitative Resistance
A. ASSESSMENT OF QUANTITATIVE RESISTANCE
In the case of qualitative or monogenic resistance fitting the gene-for-gene concept, host plants can be classified as resistant or s·usceptible. In contrast, quantitative resistance is characterized by a continuous distribution in the level of resistance. The degree of resistance is determined by the potential of the host plant to slow down the growth
126 B. Keller. C. Feuillet & M Messmer
and/or the multiplication of the pathogen (Parlevliet, 1979). The continuous distribution in the expression of quantitative resistance results from the interaction of the plant genotype, the pathogen population and environmental effects. The effect of a single gene compared to the variation caused by other loci and non-genetic factors is too small to cause a discontinuity in the phenotypic distribution. Thus, the presence or absence of an individual gene will not dramatically alter the phenotypic distribution, and is therefore called a minor gene, i.e. a gene having a "minor" effect (Falconer, 1981).
The more genes and environmental factors that are involved in disease development, the more the phenotypic expression of quantitative resistance will show a normal distribution (Fig. 9). The resistance of bread wheat against septoria nodorum leaf blotch caused by the fungal pathogen Stagonospora nodorum (Berk.) Castellani & Germano (syn. Septoria nodorum) is a typical quantitative trait. All wheat genotypes infected with this pathogen will show necrotic leaf tissue (Fig. 10), so there is no complete resistance. However, the percentage of necrotic leaf tissue measured after a given time varies considerably between different genotypes, i.e. they show different levels of resistance. Due to the complex interactions between the host and the pathogen, the level of resistance has to be defined in terms of developmental stage of the plant (seedling vs. adult plant), assessed traits (necrotic tissue vs. sporulation frequency), assessed organs (leaf vs. ear), pathogen population (isolate vs. mixture) and growing conditions (field vs. growth chamber). Therefore, quantitative resistance is not absolute but can only be assessed relative to other genotypes exposed to the same pathogen population and tested under the same conditions. For various host-pathogen systems quantitative resistance has been described in the literature e.g., maize/northern com leaf blight (Ullstrup, 1977), cereals/rust (Wilcoxon, 1981), barley/powdery mildew (J~rgensen,
1994), barley/Rhynchosporium secalis (Habgood, 1974), and potato/Phytophthora infestans (Wastie, 1991). Quantitative resistance often corresponds to partial resistance which is defined as reduced pathogen sporulation on a host with susceptible infection type (Parlevliet, 1979) and to horizontal resistance characterized by the absence of significant host-pathogen interaction (Van der Plank, 1968). In this chapter, the definition of quantitative resistance is based solely on the phenotypic distribution of the trait and does not imply anything about race specificity. Parlevliet (1979) gave a good overview of the different components of resistance that reduce the rate of epidemic development. He distinguished between factors which reduce the amount of disease present at the start of infection (infection frequency) and delay the epidemic, from factors which reduce the infection rate and slow down the epidemic development.
The major components of resistance which affect the reproduction rate of the pathogen are the reduction of infection frequency, lengthening of latent period and decrease of spore production. In situations with one reproductive cycle of the pathogen during disease development (e.g. smut in cereals) resistance is the sum of these components whereas in the case of a large number of reproductive cycles (e.g. rusts in cereals) the latent period is of major importance (Parlevliet, 1979). The correlation between the single components varies in different host/pathogen systems and at different developmental stages. Both factors delaying the epidemic and/or reducing the rate of epidemic development can be race-specific as well as non race-specific (Parlevliet, 1979) and can be under monogenic or polygenic control (Nelson, 1978; Parlevliet, 1979).
Box 4. Terminology of plant disease and disease resistance
infection: the entry of an organism or virus into a host and the establishment of a permanent or temporary parasitic relationship
resistance: the ability of an organism to withstand or oppose the operation of or to lessen or overcome the effects of an injurious or pathogenic factor
race non-specific: resistance to all races of a pathogen
race specific: resistance to some races of a pahtogen, but not to others
complete resistance: multiplication of the pathogen is totally prevented, no spore production
incomplete resistance: refers to all resistances that allow some spore production partial resistance: a form of incomplete resistance, in which spore production is reduced even though the host plants are susceptible to infection (susceptible infection type)
susceptibility: the inability of an organism to defend itself or to overcome the effects of invasion by a pathogenic organism or virus
tolerance: ability of the host to endure the presence and multiplication of the pathogen, can be expressed by less severe disease symptoms and/or limited yield reduction. Severity of symptoms and the amounts of damage to the host genotypes should be compared at equal amounts of the pathogen at the same stage of host development.
disease incidence: number of infected plants
disease severity: area or amount of plant tissue affected by disease
area under disease progress curve: disease severity measured several times during epidemic development
infection frequency: proportion of successful infections - spores that result in sporulating lesions
infection rate: multiplication rate of the pathogen on the host, increasse in disease severity spore production: number of spores produced per lesion, per affected area or per time period incubation period: time period between infection and first visible symptoms
latent period: time period from infection to spore production
infectious period: time period over which the diseased tissue sporulates
monocyclic: one reproductive cycle of the pathogen during reproductive cycle of the host polycyclic: large number of reproductive cycles of the pathogen during reproductive cycle of the host
B. BIOMETRIC APPROACH FOR QUANTITATIVE RESISTANCE TRAITS
Many efforts were made to investigate the genetic basis of quantitative resistance (for review see Geiger and Heun, 1989). Analogous to other quantitative traits such as yield or plant height, quantitative genetic theory can be applied to estimate genetic effects in controlled crosses (Falconer, 1981; Mather and Jinks, 1971). As described by Sprague (1983), quantitative genetics is an attempt to deal with the phenotypic expression
128 B. Keller, C. Feuillet & M Messmer
Relative frequency of wheat progenies
20 parent 8
~
15
10
5
o
10 14 18 22 26 30 34 38 42 46 50 54 58Percentage of necrotic leaf tissue averaged over two years
Figure 9. Phenotypic distribution of wheat progeny (homozygous, recombinant inbred lines) derived from a cross between two parents with different levels of resistance (indicated by the arrows) against septoria nodorum leaf blotch caused by the pathogen Stagonospora nodorum (Berk.) Castellani & German.
The damage caused by the disease was estimated as percentage of necrotic leaf tissue.
Figure 10. Symptoms of septoria nodorum leaf blotch in wheat caused by the pathogen Stagonospora nodorum (Berk.) Castellani & German. S. nodorum is a necrotrophic pathogen causing lens-shaped chlorotic and necrotic lesions on the leaves. The three flag leaves show different percentages of necrotic leaf tissue.
resulting from the joint contribution of all genes involved.' The phenotypic value is divided into the genotypic value (complex of all genes involved) and the deviation caused by environmental effects, genotype-environmental interaction and experimental error. Accordingly, the phenotypic variation of a population observed in replicated trials over several environments can be split into the amount of variation caused by the respective effects. The broad sense heritability (h2) is defined as the ratio between genotypic variance and phenotypic variance (Hallauer and Miranda Fo, 1981). Only the heritable part, i.e., genetic variance, can be exploited in breeding. In contrast to other quantitative traits, the expression of quantitative resistance not only depends on the genotype of the host and environmental effects but also on epidemiological factors like the genetic composition of the pathogen population, host-pathogen interaction and pathogen-environmental interaction which has an influence on the time of infection and infection pressure (Fig. 11). Since the control of the pathogen population in the field is difficult, these effects are often neglected in quantitative genetic studies and can increase the environmental effects as well as the genotype-environmental interaction.
Components of the phenotypic variance
Figure 11. Composition of the phenotypic variance observed for the level of quantitative resistance on the host plant after infection with a pathogen.
The genotypic value can be further divided into different genetic effects (Mather and Jinks, 1971): the additive effect is defined as the average effect of an allele substitution at a given locus (half the difference between homozygous parents). The dominance effect is caused by allelic interaction (i.e. the heterozygous genotype deviates from the mean of the homozygous parents) whereas epistatic effects are caused by the interaction of two or more loci. Thus, the genetic variance across all loci can be partitioned into additive, dominance and epistatic variance components (Fig.12). The ratio of additive to dominance variance is important for efficient breeding strategies. While for hybrid cultivars and vegetatively propagated cultivars all variance components can be used,
130 B. Keller, C. Feuillet & M Messmer
the breeder can only rely on additive effects for homozygous cultivars of self-pollinating species. For example, if there is a high proportion of dominance variance that covers the additive variance, selection in early generations, where there is still a high proportion of heterozygosity, is not very effective for inbred line development.
Components of the genetic variance of the host plant
Figure 12. Composition of genotypic variance of the host plant for the level of resistance after infection with the pathogen. The sectors of the circle indicate the contribution of each locus involved in the expression of quantitative resistance. Additive variance results from the additive effects of single alleles at different genetic loci. Dominance variance is due to the interaction of different alleles present at the same locus in heterozygous plants, whereas epistatic variance is due to the interaction of alleles between different loci.
The heritability of quantitative resistance in many host/pathogen systems is generally high, indicating that genetic effects are more important for the difference in resistance than environmental effects. Thus, the ranking of cultivars based on the expressed resistance level does not change dramatically under different growing conditions.
Biometric analysis of resistance against fungal diseases in small grain cereals (e.g.
leaf rust in wheat, powdery mildew in rye) revealed that the additive variance was the predominant source of genetic variation for resistance for both self-pollinating and cross-pollinating species against obligate and facultative pathogens (for review see Geiger and Heun, 1989). The cumulative dominance effects across all loci were of minor importance and heterosis could be observed for resistance or susceptibility depending on the crosses for the same host/pathogen system, e.g. rye/Erysiphe graminis and wheat/Stagonospora nodorum. Therefore, heterozygous plants are not necessarily more resistant than homozygous host plants. Transgressive segregation in both directions, i.e., some progeny which are more or less resistant than either parent (as shown in Fig. 9), was also observed for resistance in wheat against leaf rust, stem rust and powdery mildew indicating that both parents, the one with the high resistance level as well as the one with the low resistance level, contributed positive and negative alleles to the progeny.
It is speculated that only a limited range of biochemical processes is involved in disease resistance. As a consequence less genes might be responsible for the expression of resistance than for other agronomic traits such as stress tolerance or yield, which are the result of many different physiological processes and therefore truely polygenic. For many pathosystems two to ten effective factors were found to be responsible for the expression of quantitative resistance which seems to be a lower limit of the number of genes involved (Geiger and Heun, 1989).