# Patent

## Title
Method for obtaining a plant with a lasting resistance to a pathogen

## Abstract

Corresponding results were obtained with A method for obtaining a plant, in particular a cultivated lettuce plant (L. sativa), with a lasting resistance to a pathogen, in particular Bremia lactucae, comprising of providing one or more specific DNA markers linked to one or more resistance genes, determining the presence of one or more resistance genes in a plant using these DNA markers, subsequently crossing a first plant comprising one or more resistance genes with a second plant comprising one or more resistance genes, and selecting from the progeny a plant in which one or more resistance genes are present by using the DNA markers. The invention further relates to the plants obtained with this method, seeds and progeny of these plants, as well as progeny thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a division of U.S. patent application Ser. No. 11/146,392 filed Jun. 6, 2005, which is a division of U.S. patent application Ser. No. 09/959,037 filed Mar. 8, 2002, now U.S. Pat. No. 6,903,249, which in turn is a National Phase application of PCT Application Serial No. PCT/NL2000/000241 filed Apr. 13, 2000, each of which is incorporated herein by reference in its entirety. This application claims foreign priority to Netherlands Patent Application No. 101 1819 filed Apr. 16, 1999.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for obtaining a plant with a lasting resistance to a pathogen. The invention also relates to a plant in which two or more resistance genes to the pathogen are present, in addition to seeds and progeny of this plant, and progeny thereof.
The invention relates particularly to a method for obtaining a cultivated lettuce plant (L. sativa) with a lasting resistance to Bremia lactucae. The invention also relates to DNA-markers which are specifically linked to a resistance gene to Bremia lactucae. The invention further relates to a cultivated lettuce plant (L. sativa) in which two or more Dm-resistance genes are present, and to seeds and progeny of this plant, and progeny thereof.
2. Description of the Related Art
The disease which is caused by the fungus Bremia lactucae Regel is known as downy mildew. Downy mildew occurs worldwide and represents a great problem for both the yield and quality of cultivated lettuce. The fungus can infect the lettuce plant at any stage of growth, after which the first symptoms of downy mildew consist of the appearance of chlorotic yellow spots on the leaf surface. Within 24 to 48 hours a white fluffy fungus growth then becomes visible an the lower leaf surface as an indication of spore formation. During the infection the lesions become increasingly larger and more chlorotic until the leaves become completely brown.
Bremia lactucae is one of the so-called Oomycetes, a class of relatively primitive fungi. Other known fungi of this group are for instance Phytium and Phytophtora. The fungus B. lactucae contains different physiological species (“physios”) and is host-specific. Bremia lactucae is known as a very variable pathogen. New physios occur relatively frequently through mutation of the avirulence genes during the spore formation preceding the propagation of B. lactucae. 
Within the Lactucae genus, to which the cultivated lettuce (Lactuca sativa) belongs, there are different species which are resistant to Bremia lactucae Regel. The resistance is based in most cases on qualitative genes, known as Dm-resistance genes (Dm=Downy mildew). The resistance mechanism is known as a gene-for-gene working principle based on the specific interaction between products of the Dm-resistance gene and the pathogen-specific avirulence gene, which results in resistance of the lettuce plant (Michelmore et al., Plant Pathology 33, 301-315, 1984). This resistance mechanism has also been demonstrated for diverse other resistance genes in different other plant species (Michelmore et al., Genome Research, 8, 1113-1130, 1998).
A large number of Dm-resistance genes have already been identified which can bring about resistance to specific physics of Bremia lactucae Regel. Genetic research has shown that these Dm-resistance genes often occur clustered in groups on the same chromosome. Four such linking groups on different chromosomes in the genome of lettuce have been demonstrated which contain different Dm-resistance genes (Farrara et al., Plant Pathology 36, 499-514, 1987). Newly identified Dm-genes can often be classified into one of the known resistance linking groups.
A major problem however is that Bremia lactucae physics continue to occur which “break down” the resistance resulting from the known Dm-resistance genes in the present cultivated lettuce varieties. This implies that Bremia lactucae physios occur to which there is no resistance in present cultivated lettuce varieties. Resistance genes can however sometimes still found in old lettuce cultivars, but particularly in wild Lactucae species related to cultivated lettuce, such as for instance L. virosa and L. serriola. A number of broad-spectrum Dm-resistance genes have been identified with a resistance to all tested Bremia physios.
Dm-resistance genes from old lettuce cultivars or from wild lettuce species can be crossed into cultivated lettuce to once again obtain resistance. Crossed-in Dm-resistance genes have been demonstrated in conventional manner by means of an artificial Bremia lactucae disease test. For this purpose a number of leaf punches—(diameter 18-20 mm) or seedlings of the lettuce plant are inoculated with different physios of B. lactucae. After 10 to 14 days the degree of development and sporulation on the punches/seedlings is then examined. On the basis hereof it is possible to judge whether a tested lettuce plant or improved line is resistant or susceptible to the tested B. lactucae physios.
When it is known that two or more new Dm-resistance genes occur in different linking groups, these resistance genes can be brought together (‘stacked’) in a cultivated lettuce plant by crossing-in, thereby reducing the danger of the resistance being broken down. Stacking of a plurality of qualitative broad-spectrum Dm-resistance genes from different linking groups can however not be carried out with the conventional Bremia lactucae disease test because, when one qualitative Dm-resistance gene is present, total resistance is already detected in the disease test and the possible presence of a second broad-spectrum Dm-resistance gene will therefore not be detected. It is therefore not possible to select precisely those plants in which two or more qualitative broad-spectrum Dm-resistance genes are present and thus obtain plants with a lasting resistance to B. lactucae. 
It is therefore desirable for a method to be developed with which, after crossing of qualitative resistance genes into a plant, those plants can be identified and selected in which two or more resistance genes are present.
SUMMARY OF THE INVENTION
The general object of the present invention is therefore to provide a method for obtaining a plant with a lasting resistance to a pathogen. A particular objective of the present invention is to provide a method for obtaining a cultivated lettuce plant (L. sativa) with a lasting resistance to B. lactucae. 
The invention provides for this purpose a method for obtaining a plant with a lasting resistance to a pathogen, comprising of providing one or more specific DNA-markers linked to one or more resistance genes, determining the presence of one or more resistance genes in a plant using these DNA-markers, subsequently crossing a first plant comprising one or more resistance genes with a second plant comprising one or more resistance and selecting from the progeny a plant in which two or more resistance genes are present using the DNA-markers.
The present invention particularly provides a method for obtaining a cultivated lettuce plant (L. sativa) with a lasting resistance to Bremia lactucae, comprising of providing one or more specific DNA-markers linked to one or more Dm-resistance genes, determining the presence of one or more Dm-resistance genes in a cultivated lettuce plant and/or wild lettuce plant using these DNA-markers, subsequently crossing a cultivated lettuce plant comprising at least one or more Dm-resistance genes with another cultivated lettuce plant or a wild lettuce plant comprising at least one or more Dm-resistance genes, and selecting from the progeny thereof a cultivated lettuce—plant with two or more Dm-resistance genes using the DNA-markers.
With the method according to invention plants, particularly cultivated lettuce plants, can be obtained in simple manner which comprise two or more resistance genes, particularly two or more Dm-resistance genes, with a lasting resistance to a pathogen, particularly Bremia lactucae. The selection of plants in which two or more qualitative resistance genes are present can only be accomplished using molecular DNA-markers which can demonstrate the specific genes in the genome of the lettuce plant. With the conventional disease tests it is not possible to demonstrate the presence of two or more qualitative resistance genes in a cultivated lettuce plant. The method according to invention can also be used for quantitative resistance genes.
According to the invention the resistance genes are preferably qualitative resistance genes, and the resistance genes are preferably located in different linking groups.
In order to enable identification and selection of a plant with two or more resistance genes, use is made of specific molecular DNA-markers linked to the resistance genes. Use can be made herefor of different DNA-markers such as for instance RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphism), SCAR (sequence characterized amplified region) etc. The specific DNA-markers linked to the resistance genes are developed in accordance with per se known techniques (Paran et al., Genome 34, 1021-1027, 1991; Paran et al., TAG 85, 985-993, 1993). The application of such DNA-markers to stack different resistance genes in a plant, in particular to combine different broad-spectrum Dm-resistance genes in a lettuce plant (L. sativa), in order to obtain a plant, particularly a cultivated lettuce plant (L. sativa), with a lasting resistance to a pathogen, particularly Bremia lacticae, has however not previously been described.
According to the present invention DNA-markers have been found for four Dm-resistance genes, particularly qualitative broad-spectrum Dm-resistance genes from the Lactuca family. Using these DNA-markers it has been established that the four Dm-resistance genes are located in separate linking groups, whereby stacking of the Dm-resistance genes in cultivated lettuce (L. sativa) is possible.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a listing of the sequence of DNA marker A (SEQ ID NO: 1);
FIG. 2 is a listing of the sequence of DNA marker B (SEQ ID NO: 2);
FIG. 3 is a listing of the sequence of DNA marker C.sub.2 (SEQ ID NO: 3);
FIG. 4 is a listing of the sequence of DNA marker C.sub.3 (SEQ ID NO: 4);
FIG. 5 is a listing of the sequence of DNA marker C.sub.4 (SEQ ID NO: 5);
FIG. 6 is a listing of the sequence of DNA marker D.sub.2 (SEQ ID NO: 6);
FIG. 7 is a representation of marker analysis of marker A in 24 tested F2 individuals;
FIG. 8 is a representation of marker analysis of marker B in 24 tested F2 individuals;
FIG. 9 is a representation of marker analysis of marker C.sub.1 in 24 tested F2 individuals;
FIG. 10 is a representation of marker analysis of marker C.sub.2 in 24 tested F2 individuals;
FIG. 11 is a representation of marker analysis of marker C.sub.3 in 24 tested F2 individuals;
FIG. 12 is a representation of marker analysis of marker C.sub.4 in 24 tested F2 individuals;
FIG. 13 is a representation of marker analysis of marker D.sub.1 in 24 tested F2 individuals; and
FIG. 14 is a representation of marker analysis of marker D.sub.2 in 24 tested F2 individuals;



DETAILED DESCRIPTION OF THE INVENTION
There are different methods of demonstrating whether different resistance genes are present in the same or in different linking groups. The position of the DNA-markers can be determined by generating a so-called genetic map or by studying the dependent or independent segregation of the different DNA-markers in relation to each other. In the present invention it was determined by studying the segregation of the DNA-markers that the specific DNA-markers linked to the Dm-resistance genes segregate independently of each other and are therefore located in four different linking groups.
In the research leading up to the present invention the individuals susceptible and resistant to the same B. lactucae phenotype from a population of plants which segregate for B. lactucae—resistance were tested with commercially obtainable RAPD-primers (OPA-01 to OPAN-20, Operon Technologies, Alameda, USA; UBC 1 to 800, University of British Columbia, Vancouver, Canada). RAPD analysis is a per se known technique (Williams at al., Nucleic Acids Research, 18, 6531-6535, 1990) based on the use of primers with a random sequence for the purpose of amplifying random segments of the genomic DNA. Among the amplification products polymorphisms can then be demonstrated on an agarose gel and can be used as genetic markers.
1600 primers (from Operon technologies, and the University of British Columbia, UBC 1 to 800) were used for the study. The DNA of the plants was mixed with the primers in a suitable amplification mixture and subsequently amplified. The amplification products were analysed on an agarose gel for the presence of suitable DNA-markers.
The ‘candidate’ molecular DNA-markers found in the RAPD-analysis were tested on the individuals of the segregating population, whereafter it was possible to establish which of these DNA-markers were physically linked in suitable manner to the different studied qualitative Dm-resistance genes. In this way the following DNA-markers were identified: DNA-marker A (primer OPAF06, 451 bp); DNA-marker B (primer OPAM10, 555 bp); DNA-marker C1 (primer OPW16, 750 bp), DNA-marker C2 (primer OPL03, 276 bp), DNA-marker C3 (primer OPAE19, 675 bp) and DNA-marker C4 (primer UBC711, 1083 bp); and DNA-marker D1 (primer OPW04, 520 bp) and DNA-marker D2 (primer OPW19, 963 bp). The sequence of the markers A, B, C2, C3, C4 and D2 was then determined and are shown in FIGS. 1-6.
The DNA-markers found were subsequently used to select a cultivated lettuce plant with two or more Dm-resistance genes, after introgression of the resistance genes from wild lettuce species, such as for instance Lactuca virosa and L. serriola. The crossing into cultivated lettuce varieties of two or more resistance genes, particularly qualitative broad-spectrum Dm-resistance genes, from wild lettuce species, such as for instance L. virosa, has not been described previously.
If crossing of two lettuce plants is not successful via the normal methods, use can be made for crossing of the Dm-resistance genes into a cultivated lettuce plant of known cell-biological techniques such as embryo rescue (Maisonneuve, Agronomie 7, 313-319, 1987) or protoplast fusion (Maisonneuve et al., Euphytica 85, 281-285, 1995). In the present invention the different Dm-resistance genes were crossed in as described in Example 2.
Introduction of a new broad-spectrum Dm-resistance gene into one of the four known linking groups can result as a consequence of recombination processes in crossing-out of Dm-resistance genes already present in the linking group, or other resistance genes or horticultural traits with high value. In order to prevent this new qualitative resistance genes with a broad-spectrum Dm-resistance are preferably introgressed into each of the separate linking groups.
The wild lettuce plant used for the method according to invention can for instance be chosen from L. saligna, L. altaica, L. aculeata, L. homblei, L. indica, L. tenerrima, L. squarrosa, L. viminea, L. augustana, L. quercina, and L. cacadensis. However, other suitable wild lettuce plants can also be used according to the invention. The wild lettuce plant is preferably L. virosa or L. serriola, more preferably L. virosa. 
The method according to the invention is preferably used to stack qualitative resistance genes, such as Dm-resistance genes, in cultivated lettuce (L. sativa). This further includes for instance head lettuce varieties (L. sativa Lineaus capitata), such as iceberg lettuce, batavia lettuce and butterhead lettuce, varieties of leaf lettuce for picking (L. sativa Lineaus acephala), such as curly leaf lettuce and stem lettuce, cos lettuce (L. sativa Lineaus romana), leaf lettuce for cutting (L. sativa Lineaus secalina) and asparagus lettuce (L. sativa Lineaus angustana).
The method according to the invention for obtaining a plant with a lasting resistance to a pathogen, as described for cultivated lettuce, can be used in analogous manner for other cultivated crops or other plants, and other pathogens. As non-limitative examples are for instance mentioned obtaining a lasting resistance to determined nematodes, such as Meloidogyne javanica, M. arenaria, and M. incognita, or to Oidium lycopersici in tomato, and obtaining a lasting potyvirus resistance in paprika by crossing-in two or more pvr resistance genes (pvr=potyvirus resistance).
The present invention further provides DNA-markers which are specifically linked to a Dm-resistance gene, and which comprise a DNA-fragment with a sequence which is at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homologous to a sequence as shown in any of the FIGS. 1-6.
The invention further relates to a plant in which two or more resistance genes to a pathogen are present, generally, and particularly to a cultivated lettuce plant (L. sativa) in which two or more Dm-resistance genes are present, and to the seeds and progeny of the plant, particularly the cultivated lettuce plant, or the progeny thereof.
A lasting resistance is thus understood to mean in the present invention that there are present in a plant at least two or more resistance genes, for instance two or more broad-spectrum Dm-resistance genes, to a pathogen. The pathogen is for instance B. lactucae, but can also be any other organism capable of causing disease in plants, such as for instance fungi, viruses, nematodes, bacteria, (parasitic) insects etc.
In a particularly suitable embodiment of the method according to the invention a Dm-resistance gene is a qualitative, broad-spectrum Dm-resistance gene to the fungus Bremia lactucae. 
The invention is described in more detail with reference to the following non-limitative examples and figures, in which:\
FIGS. 1-6 show respectively the sequence of the DNA-markers A, B, C2, C3, C4, D2; and
FIGS. 7-14 show eight DNA-markers according to the invention in 24 tested F2-individuals. Marker A was identified with primer OPAF06 (451 bp); marker B was identified using primer OPAM10 (555 bp), marker C1 using primer OPW16 (750 bp), marker C2 using primer OPL03 (276 bp), marker C3 using primer OPAE19 (675 bp) and marker C4 using primer UBC711 (1083 bp; DNA-marker D1 was identified with primer OPW04 (520 bp), and marker D2 with primer OPW19 (963 bp).
EXAMPLES
Example 1
Marker Analysis in Lettuce F2 Populations which Split for a Bremia lactucae Regel Resistance Gene
The techniques used to provide fast and directed molecular DNA-markers closely associated with resistance genes to B. lactucae are per se known (Paran et al., Genome 34, 1021-1027, 1991; Paran et al., TAG85, 985-993, 1993; Williams et al., Nucleic Acids Research, 18, 6531-6535, 1990), and can be used in analogous manner for identification of DNA-markers in other crops.
From a population (see crossing scheme, example 2) of more than 300 plants which segregate for B. lactucae resistance the individuals susceptible and resistant to the same B. lactucae phenotype were pooled separately (5 plants per pool). These pools were examined using 1600 commercially obtained RAPD-primers (Operon Technologies, Alameda USA, OPA-01 to OPAN-20; and University or British Columbia UBC 1 to 800). The PCR mixture for the DNA-markers A, B, C1, C2, C3, C4, D1 and D2 was amplified under standard RAPD conditions.
After determining candidate molecular DNA markers using the RAPD-analysis on the DNA-pools, these DNA-markers were checked on individuals of the segregating population, whereafter it was possible to determine which of the DNA-markers were best physically linked to the examined qualitative Dm-resistance gene with a broad-spectrum resistance to B. lactucae. 
For each of the 4 examined genes with a broad-spectrum Dm-resistance, the best linked molecular DNA-markers are shown in FIGS. 1-6. Marker A was identified with primer OPAF06 (451 bp); marker B was identified using primer OPAM10 (555 bp), marker C1 using primer OPW16 (750 bp). marker C2 using primer OPL03 (276 bp), marker C3 using primer OPAE19 (675 bp) and marker C4 using primer UBC711 (1083 bp); DNA-marker D1 was identified with primer OPW04 (520 bp), and marker D2 with primer OPW19 (963 bp).
Example 2
Crossing Schemes
In this example the crossing schemes for four different populations are shown. The following symbols/characters are used herein:
*=resistant plant, which means: resistance to all tested B. lactucae physios.
BC=“Back Crossing”
Z=Self−pollinating, the number of figures after Z indicates how many times self-pollination took place.
Population A, L. virosa CGN9365, (IVT1398) (marker A)
The BC3Z population was then tested and marker A identified. Individual BC3Z plants were self-pollinated and from the BC3Z2 populations the individual BC3Z2 plants homozygous for gene A were selected. The selected plant was used for linking analysis of the diverse identified DNA-markers (Example 3).
Population B, L. virosa CGN4683, (IVT280) (Marker B)
The BC3Z population was tested and marker B identified. Individual BC3Z plants were self-pollinated and from the obtained BC3Z2 populations the individual BC3Z2 plants homozygous for gene B were selected and used for linking analysis of the diverse identified DNA-markers (Example 3).
Population C, L. virosa CGN5148 (IVT1538) (Marker C1, C2, C3 and C4)
The BC3Z population was tested and markers C1, C2, C3 and C4 identified. The individual BC3Z plants were self-pollinated and from the BC3Z2 populations the individual BC3Z2 plants homozygous for gene C were selected and used for linking analysis of the diverse identified DNA-markers (Example 3).
Population D, L. serriola CGN5913 (IVT1308) (Marker D1 and D2)
The BC3Z population was tested and markers D1 and D2 identified. The individual BC3Z plants were self-pollinated and from the BC3Z2 populations the individual BC3Z2 plants homozygous for gene D were selected and used for linking analysis of the diverse identified DNA-markers (Example 3).
Example 3
Linking Analysis of the Identified DNA-Markers
There are different methods of demonstrating whether diverse qualitative resistance genes can be positioned in the same or in different linking groups (chromosomes).
A. Genetic Map
Determining of the position of DNA-markers can be carried out by generating a genetic map of the 9 chromosomes of lettuce. In order to generate a genetic map on which the position of the diverse molecular DNA-markers is indicated, crossings are made between lettuce plants which are highly polymorphic relative each other from a genetic viewpoint. For this type of crossing with a high degree of polymorphism a distinction can be made between:
intraspecific crossing:
This is a crossing between for instance butterhead lettuce and iceberg lettuce, a crossing is made within a species (L. sativa),
Interspecific crossing:
This is a crossing between two Lactuca species, for instance butterhead lettuce (L. sativa) with L. virosa. 
An F2 or BC1 population is generated of both types of crossing. By analysing this F2 or BC1 population with for instance RAPD-markers all plants can be individually analysed for the presence or absence of the polymorphic molecular DNA-markers. By analysing the obtained data using a computer program such as for instance JoinMap (Stam, Plant Journal 3, 739-744, 1993), linking groups can be constructed which place the diverse tested DNA-markers linearly relative to each other, separated by specific recombination distances denoted in centiMorgans. If a broad-spectrum Dm-resistance gene segregates in the used F2 or BC1 population, the broad-spectrum Dm-resistance gene can, after testing with B. lactucae, be placed within one of the linking groups shown on a detailed genetic map of lettuce. A genetic lettuce map with 9 linking groups has been described by Michelmore (Genetics 116, 331-337, 1987).
When the identified molecular DNA markers according to the present invention are polymorphic in the parents used to make an F2 or BC1 population, these DNA-markers can be placed on the genetic map, whereby it is possible to establish whether the DNA-markers originate from the same or from different linking groups,
B. Test Crossings
Another method for determining the position of the DNA-markers as applied in the present invention linked to the resistance genes consists of studying the dependent or independent segregation of the different DNA-markers. Selected for this purpose from the four populations were individual plants which are homozygous for the specific broad spectrum Dm-resistance genes from respectively population A, B, C or D. Specific crossings were then made for the generation of a segregating F2 population in which all Dm-resistance genes and their corresponding DNA-markers were present.
Selection of Plant with Gene A and B
A plant homozygous for Dm-resistance gene A (as demonstrated with marker A) was crossed with a plant homozygous for Dm-resistance gene B (marker B). The individual F1 plant with both Dm-resistance gene A and B (after analysis with the DNA-markers A and B), as well as the individual plants of the F2 population were subsequently self-pollinated. A selection was made from the F3 populations of plants which were homozygous for both Dm-resistance gene A and for Dm-resistance gene B, using the DNA-markers specific for Dm-resistance gene A and B.
Being able to select a plant with the qualitative Dm-resistance genes A and B each having a broad-spectrum Dm-resistance means that both resistance genes are localized in different linking groups.
Selection of a Plant with Both Genes C and D
A plant homozygous for Dm-resistance gene C (as demonstrated with markers C1, C2, C3 or C4) was crossed with a plant homozygous for Dm-resistance gene D (markers D1 or D2). The individual F1 plant with both Dm-resistance gene C and D (after analysis with the DNA-markers C1, C2, C3 or C4 and D1 or D2), as well as the individual plants of the F2 population were subsequently self-pollinated. A selection was made from the F3 populations of plants which were homozygous for Dm-resistance gene C and for Dm-resistance gene D, using the DNA-markers specific for the Dm-resistance genes C and D.
Being able to select a plant with the qualitative Dm-resistance genes C and D each having a broad-spectrum Dm-resistance means that both resistance genes are localized in different linking groups.
Example 4
Linking Analysis for the 4 Genes from the 4 Different Populations
The selected plant homozygous for Dm-resistance genes A and B was then crossed with the selected plant homozygoqs for Dm-resistance genes C and D. The F1 plants heterozygous for the Dm-resistance genes A, B, C and D (as determined with the DNA-markers specific to these genes) were self-pollinated.
The F2 population was tested in the B. lactucae disease test and analysed with the DNA-markers for the 4 broad-spectrum Dm-resistance genes.
For the disease test three to six leaf punches with a cross-section of 18 to 20 mm were taken from lettuce plants for testing with a cork drill, or 50 seeds were laid out on a filter paper. The punches or filter papers with lettuce seed were laid in a tray on wet thick filter paper and covered with a glass plate until the moment of inoculation. The punches were inoculated on the same day or a few days after the punching. The seeds were germinated and further cultivated in a climate cell of 12-16.degree. C. with 16 hours of light and 8 hours of darkness until the seed leaves were extended, whereafter inoculation took place.
The B. lactucae inoculum was prepared by arranging a determined physio of B. lactucae, (fresh or frozen) which sporulates on leaf material, in a small measured quantity of water, mixing it and sieving this solution. The concentration of living spores was then determined by means of fluorescence microscopy and adjusted if necessary. The optimal spore concentration is 10,000-50,000 virulent spores/ml water.
The inoculum was applied to the punches or seedlings with a plant spray until the punches were slightly moist. The tray was then covered again with a glass plate and set aside at 12-16.degree. C. and 16 hours light and 8 hours of darkness. After 10 to 14 days it was possible to assess the punches for the degree of development and sporulation and it was possible to state whether a tested plant or lettuce number is resistant or susceptible to the tested B. lactucae physio.
The DNA-marker analysis was performed as described in Example 1.
Of the made F2 population, 24 plants are shown in table 1 and FIGS. 7-14 which were tested in the B. lactucae disease test and analysed with the RAPD-markers. From this test it was found that the eight RAPD-markers can be split independently of each other and can therefore be positioned in four different linking groups.
CONCLUSION
FIGS. 7-14 show that the DNA-markers linked to the 4 broad-spectrum Dm-resistance genes segregate independently of each other and can thus be positioned in the four separate linking groups. Plants can hereby be selected which comprise at least 2, preferably 3, and most preferably 4 qualitative resistance genes (indicated with: * in table 1 below), have a broad-spectrum Dm-resistance and are therefore valuable for processing to a commercial lettuce variety.
Only application of the DNA-markers according to the invention makes such a selection possible because in the B. lactucae disease test no distinction can be made between the presence of one or more qualitative broad-spectrum Dm-resistance genes.
Corresponding results were obtained with the other wild lettuce species.

## Claims

1. An isolated DNA-marker for identifying a broad-spectrum Dm-resistance gene in a plant, wherein said DNA marker comprises (SEQ ID NO: 2).

2. A method for obtaining a plant with a lasting resistance to a pathogen, comprising of providing the DNA-marker of claim 1, determining the presence of one or more broad-spectrum resistance genes in a plant using said DNA-marker, subsequently crossing a first plant comprising one or more broad-spectrum resistance genes with a second plant comprising one or more broad-spectrum resistance genes, and selecting from the progeny a plant in which two or more broad-spectrum resistance genes are present using the DNA-markers.

3. The method as claimed in claim 1, wherein the broad-spectrum resistance genes are qualitative genes.

4. The method as claimed in claim 1, wherein the broad-spectrum resistance genes are located in different linking groups.

5. A method for obtaining a cultivated lettuce plant (L. sativa) with a lasting resistance to Bremia lactucae, comprising of providing the DNA-marker of claim 1, determining the presence of one or more broad-spectrum Dm-resistance genes in a cultivated lettuce plant and/or wild lettuce plant using said DNA-marker, subsequently crossing a cultivated lettuce plant comprising at least one or more broad-spectrum Dm-resistance genes with another cultivated lettuce plant, or a wild lettuce plant, comprising at least one or more broad-spectrum Dm-resistance genes, and selecting from the progeny thereof a cultivated lettuce plant with two or more broad-spectrum Dm-resistance genes using the DNA-markers.

6. The method as claimed in claim 5, wherein the broad-spectrum Dm-resistance genes are located in different linking groups.

7. The method as claimed in claim 5, wherein the wild lettuce plant is chosen from the group consisting of L. serriola, L. virosa, L. saligna, L. altaica, L. aculeata, L. homblei, L. indica, L. tenerrima, L. squarrosa, L. viminea, L. augustana, L. quercina, and L. cacadensis.

8. The method as claimed in claim 5, wherein the wild lettuce plant is L. serriola.

9. The method as claimed in claim 5, wherein the wild lettuce plant is L. virosa.

10. The method as claimed in claim 5, wherein the cultivated lettuce plant is chosen from the group consisting of head lettuce (L. sativa Lineaus capitata); leaf lettuce for picking (L. sativa Lineaus acephala); cos lettuce (L. sativa Lineaus romana); leaf lettuce for cutting (L. sativa Lineaus secalina) and asparagus lettuce (L. sativa Lineaus angustana).