WO1998042866A1

WO1998042866A1 - MARKER OF RESISTANCE TO $i(SCLEROTINIA SCLEROTIORUM)

Info

Publication number: WO1998042866A1
Application number: PCT/FR1998/000616
Authority: WO
Inventors: Saïd MOUZEYAR; Laurent Gentzbittel; Saloua Badaoui; Marc Cock; Christian Dumas; Denis Tourvieille De Labrouhe; Félicity VEAR; Paul Nicolas
Original assignee: Institut National De La Recherche Agronomique; Universite Blaise Pascal
Priority date: 1997-03-26
Filing date: 1998-03-26
Publication date: 1998-10-01
Also published as: AR012170A1; FR2761361B1; FR2761361A1; HUP0002542A2; EP0970248A1; AU7051898A

Abstract

The invention relates to the indentification of a resistance marker to Sclerotinia sclerotiorum and its application to determine the genotype in a plant resistant to Sclerotinia sclerotiorum.

Description

Sclerotinia sclerotiorum resistance marker

The present invention relates to the identification of a marker of resistance to Sclerotinia sclerotiorum.

Cultivated plants can be attacked by many pathogenic organisms including viruses, bacteria, fungi, nematodes and parasitic plants. The methods of infection and development of the disease depend on the plant and the parasite involved. However, plants have developed resistance systems that allow them to defend themselves against these parasites. Generally, there are two types of interaction between plants and their parasites: interactions obeying a so-called "gene-for-gene" model and interactions where the existence of a gene-for-gene system is not demonstrated.

The concept "gene-for-gene" was developed by Flor by focusing on flax rust and postulates that a resistance gene in the plant corresponds to an avirulence gene in the parasite (Flor, 1971). Resistance in plants is generally dominant over sensitivity. In parasites, avululence is dominant over virulence. The interaction between the resistance gene product and that of the avirulence gene leads to a rapid and localized reaction at the site of penetration of the parasite. This reaction is called hypersensitive reaction (HR). In this system, the triggering of the resistance process is under monogenic control and a plant gene gives confers resistance to a specific race of the country. It is also said that this resistance is race- SDeCifique

In addition, there are plantcs-paihogenes interactions where π does not have recognition depending on a specificity genotype of the parasite / genotype of the host All the strains of the parasite can attack all the genotypes of the plant. In this case, the resistance is generally partial (often we speak of tolerance) and above all under genetic control. According to Van der Planc, this resistance is called "horizontal" (1968) In this case, there would be no avirulence gene in the parasite

For several years, many resistance genes have been cloned in different plant species. Among these resistance genes, the Hm l gene in corn is particular because it codes for an εnz mc αui inac e the toxin produced by the parasitic fungus Ilelmiπ tosponum carbonum (Johal and Bπggs, 1992). On the other hand, the other genes of resistance do not cost for

detoxification measures and can be classified into different categories • the Pto gene in tomatoes, which is a protein with serine threonine kinase function (Martin et al., 1993);

• genes containing an LRR motif (Leucin-rich repeats) and an NBS motif (Nucleotide Binding Site), this is the case of the N gene in tobacco (VVhitham et al., 1994), L6 in flax (Lawrence and al., 1995), RPS2 and RPM1 in

Arabidopsis thaliana (Bent et al., 1994; Mindrinos et al., 1994; Grant et al., 1995);

• genes containing an LRR motif but no NBS motif, this is the case for the Cf9 and CΩ genes in tomatoes (Jones et al., 1994; Di on et al., 1996); • the Xa21 gene in rice which contains a serine threonine kinase motif (such as Pro) and an LRR motif (such as Cf9) (Song et al., 1995).

It should be noted that these genes (apart from Hm l) intervene in resistance mechanisms requiring specific recognition between the product of the resistance gene and the product of the avirulence gene in the parasite. So far, no direct relationship has been established between an identified gene sequence and a chromosomal region involved in triggering a process of resistance under polygenic control.

The genus Sclerotinia groups together parasite species capable of attacking many species of cultivated plants. Indeed, only the grasses seem to be spared by this parasite.

More particularly, Sclerotinia sclerotiorum is the agent of white rot in sunflowers. Resistance in the latter is partial and polygenic (Robert et al., 1987; Vear and Tourvieille, 1988). However, Pirvu et al. (1985) proposed the existence of a recessive gene controlling resistance to infection of the flower head. Each organ of the sunflower can have a different level of resistance (Castano et al., 1993).

The present invention therefore relates to the discovery and sequencing of a genomic fragment of a co-aggregating plant with a major QTL (Quantitative Trait Loci) of resistance to Sclerotinia sclerotiorum. First of all, the invention has for obj and two nucleotide sequences corresponding to SEQ. 1D N ° 1 and SEQ ID N "2 respectively, as well as their use for the demonstration of the aforesaid genomic fragment.

More particularly, in the context of the present invention, one and / or the other of these two sequences can be used as primer (s) for the implementation of a chain amplification process by polymerase (PCR) applied to plant DNA. Indeed, the sequences in question constitute degenerate primers of which the number of possible combinations of sequences is such that there always exists at least one in each "series" of primers having sufficient homology (of at least 80%) with the DNA of the plant to be studied likely to contain the marker for resistance to Scleroϋnia sclerotiorum.

The invention also relates to the DNA fragment (s) obtained by implementing the above polymerase chain reaction method. In the context of the present invention, it has been found that a DNA fragment thus obtained was of particular interest at a site corresponding to a QTL of resistance to sunflower, this fragment corresponding to SEQ ID No. 3.

Another object of the present invention is to use the above DNA fragment as a marker of resistance in a plant to Sclerotinia sclerotiorum. As such, the DNA fragment in question can be used for carrying out a Southern type molecular hybridization. This technique is entirely within the reach of those skilled in the art and consists mainly in labeling the DNA fragment in question, for example radioactively, in placing the labeled fragment in contact with the genomic DNA of the plant previously digested with using restriction enzymes under hybridization conditions and determining the presence of molecular hybridization by, for example, autoradiography.

The use of the above DNA fragment as a marker of resistance to Sclerotinia sclerotiorum therefore makes it possible to identify resistant genotypes with the aim of identifying plants containing the sensitive or resistant allele of the fragment, but also to implement programs selection assisted by said marker to introgress resistance to this parasite in susceptible varieties. This type of approach consists in fact in crossing a plant material which we know to be resistant to Sclerotinia sclerotiorum with a sensitive plant material but having sought-after agronomic qualities, then to select the plant material resulting from this crossing both having a resistance to Sclerotinia sclerotiorum and having the above agronomic qualities. These programs include, for example, building ideal genotypes and marker-assisted back cross programs. From the sequence corresponding to SEQ ID N "3, an open reading frame has been identified whose translation into amino acids corresponds at SEQ. ID No 4. Consequently, the present invention also relates to this peptide sequence as well as the peptide sequences having a homology of at least 50% with SE ID No 4. Indeed, such a limit in the degree of homology is entirely acceptable in the context of the present invention insofar as the parts of the peptide sequence at the origin of the essential functions of the peptide are preserved, such as for example the serine / threonine kinase domain. Finally, the invention relates to the nucleotide sequences coding for the above peptide sequences. The present invention will be better understood in the light of the example below which, however, is only given for purely illustrative purposes and does not in any way constitute a limitation to the invention. EXAMPLE

I / Cloning of the gene sequence (P) in sunflower a) Amplification conditions:

To clone said sequence, we used the PCR (Polvmerase Chain Reaction) technique with degenerate primers, RKl and RK2 (SEQ ID No. 1 and SEQ. ID No. 2 respectively).

For the amplification reaction, DNA extracted from the public sunflower line RHA266 was used under the following conditions:

DNA '50 ng diJTP 200 μM

RKL (10 _μ M) 2.5 .mu.l RK2 (10 microM) 2.5 .mu.l

Taq Pol (5U / μl; Appligène France) 0.5 U

H ₂ 0 QSP 25 μl

Initial denaturation 94 ° C for 5 minutes. 40 cycles: 9- ^ 'for 30 seconds,

55'C for 30 seconds, and 72 ° C for 1 minute 30 seconds. After the 40 cycles, a final elongation step at 72 ° C for 10 minutes. The amplification products were checked on agarose gel according to standard methods (Sambrook et al., 1989). b) Cloning and sequencing:

An approximately 300 bp fragment was cloned (Clone PK) using the PCRII cloning kit (InVitrogen, Netherland). It was then sequenced using the Dye Terminator method by the company Génome Express (Grenoble, France). The sequence obtained corresponds to SE ID No. 3. 1 1 / Characteristics of the PK sequence

From the sequence (SEQ ID No. 3), an open reading frame has been identified (ORF) whose translation into amino acids gives the sequence corresponding to SEQ. ID N ³ 4. Using the BLAST program at the NCBI (National Center of Biolnformatique, USA) we detected homologies with threonine kinase genes from other plants, notably the Atlec gene in Arabidopsis thaliana (Swarp et al, 1996) and AthLecRK also in Arabidopsis thaliana (Hervé et al., 1996). 1 1 1 / Localization of a PK locus The PK clone thus obtained was used as a probe in Southern hybridization experiments, under the following conditions: a) Crossing:

A cross between public lines was used: Cross Cl: between the line SD, resistant to the extension of ms cehum and the line PACl resistant to the penetration of the fungus. 139 F2 plants from this crossing were studied.

Crossover C3: between the sensitive GU line and the resistant P \ C2 line 161 F2 plants resulting from this crossover were studied b) Extraction, digestion and transfer of DNA: Four restriction enzymes were used to digest the DNA of F2 plants as well as that of the parents. EcoRl, EcoRV, Hmdlll and BglU. The DNA was digested, analyzed on agarose gel and transferred onto a Hybond N ^{, d} - nylon membrane (Amersham, France) according to standard methods (Sa brook et al., 1989). The Pk probe was radioactively labeled using the "megapπme DNA labellmg labeling kit

stems "(Amersham, France) c) Hybridization and rinsing:

The conditions for hybridization of the PK probe with genomic DNA and for rinsing are those already published (Gentzbittel et al., 1992) I V / l localization of PK on the genetic map of the sunflower

We used the MΛPMAKER / EXP program (Lander et al, 1987) to build a consensus genetic map in two crosses with the following parameters: minimum LOD score = 3.0; maximum recombinant fraction = 0.40. Probes already located have been used as anchors and the nomenclature followed is that of Gentzbittel et al., 1995.

A polymorphic locus was detected by this PK probe on linkage group 1 of the genetic map of the sunflower obtained in the laboratory (Gentzbittel et al., 1995), located between loci S003H3 and S 145H3-2. V / Resistance to S. sclerotiorum: QTL analysis

The resistance tests were carried out on plants of the F3 and F4 generations from two crosses. a) Crosses used

C l: between the line SD resistant to the extension of the parasite and the line PACl resistant to the penetration of the parasite.

C2: between the sensitive CP73 line and the PACl line described above. C3: between the sensitive GH line and the resistant PAC2 line. b) Stress tests

The plants F3 and F4 were tested in 1994 (for SD X PAC l) and 1995 (for CP73 X PAC l) and in 5 different places. Several resistance tests, corresponding to different forms of attack on S. sclerotiorum, were used. For the GH x PAC2 cross, the plants were tested in 1994

(generation F3) and 1995 (generation F4), in one place, in Clermont-Ferrand. c) Ascospore test on flower head (Tourvieille and Vear, 19S4)

Contamination occurs when the fertile side of the flower head has 2 or 3 rows of open florets. About 25,000 ascospores are then sprayed on the flower head. About 3 weeks after contamination, each flower head is observed individually. Two parameters are measured:

- "latency index" which expresses the latency period between the date of infection and the date of appearance of the first symptoms. This test makes it possible to estimate the resistance to the extension of the parasite,

- the "attack percentage" which expresses the percentage of plants attacked by family in comparison with control plants. This test makes it possible to estimate the resistance to penetration of the parasite. For this ascospore test on the flower head, two repetitions were carried out in Clermont-Ferrand and one repetition in each of the 4 other places of experimentation. d) Mycelium leaf test (Castano et al., 1992)

The infection is made in late June-early July by depositing a mycelium pellet (8 mm in diameter), at the end of the main vein on two leaves per plant. Symptom scoring is done a week after infection. The parameter measured is the length of necrosis on the underside of the limbus, which reflects the speed of extension of the parasite. The leaves are then removed to allow the plant to continue its normal development. This test was carried out on two replicates of five plants in Clermont-Ferrand. c) Mycelium test on flower head (Vear and Guillaumin, 1977)

The flower heads to be infected are harvested when the sterile side of the flower head is yellow and the bracts are brown (stage M2, 4 to 6 weeks after flowering). Three 10 mm diameter mycelial implants are placed on the sterile side of each flower head and held in place by sticky paper. The infected flower heads are then placed in survival conditions, in an air-conditioned room at 18 ± 2 ° C (the stems soak in tubs filled with water) and covered by black polyethylene tarpaulins. The ratings are made 3 days after infection by measuring the area of rot spots that appeared at the sites of infection. The measured parameter is a "mycelium index" which is calculated by relating the average of the areas of the 3 spots to the average of the spots of the sensitive control. This test was carried out on two replicates of five plants in Clermont-Ferrand. V I / Analysis of the results:

The normality and the equality of the variances of the experimental data were checked and when it was necessary, these data were transformed using logl O or arcsin of the square root.

The QJL analysis was performed either by a one-factor analysis of variance (ANOVA) with a significance level of 0.001, or by the "interval-mapping" method (MAPMAKER / QTL, Lincoln et al., 1992) with a LOD threshold score of 3.0. The part of the phenotypic variability of the character explained by the segregation of the significant marker is estimated by FJ = sum of the squares due to the factor / sum of the total squares. When a probe is not polymorphic in a crossover, its QTL effect is estimated using the polymorphic markers closest to the interval at which the probe was assigned. The QTLs were analyzed for the 4 resistance tests, for each test (place of experimentation) and on the means of all the tests. A significant correlation (r = 0.76) was found between the latency index and the percentage of attack. Vil / Detection of QTL, co-localization of QTL and PK

The methods of analysis presented above made it possible to detect a major QTL for resistance to S. sclerotiorum analyzed by the latency index and by the percentage of attack on the linkage group 1 of the genetic map of the sunflower. It is also possible to detect a major QTL for the leaf mycelium index, on linkage group 1 of the sunflower genetic map. Figure 1 :

Scan showing the colocalization between the PK probe and the QTL peak of resistance to S. sclerotiorum in the C l crossing. Figure 2

Scan showing the colocalization between the PK probe and the QTL peak of resistance to S. sclerotiorum in the C2 crossing. Figure 3 Scan showing the colocalization between the PK probe and the QTL peak of resistance to S. sclerotiorum in the C3 crossing.

The QTL in question covers the region of this group encompassing the PK locus. The latter corresponds to the QTL peak in the crossing C l (Figure 1); (LOD score = 5.1 for the attack percentage and LOD score = 5.28 for the latency index); in this crossover this QTL e. explains 16% of the phenotypic variance. In crossover C2, the QTL effect of the PK locus was estimated using the neighboring markers S003H3 and S 145H3-1 (Figure 2); this QTL explains more than 50% of the phenotypic variance. In crossover C3, the QTL also includes the PK locus, which is located approximately at the peak of the QTL. In this crossover, the QTL explains 50% of the phenotypic variance (LOD score = 8.5) for the F4 generation.

The colocation, on the link group 1, between the PK locus and the QTL corresponding to the resistance to 5. sclerotiorum analyzed by the latency index and by the percentage of attack was found not only by using the mean value of each character in each cross but also by doing the multi-place analysis, in three places of experimentation out of five in the Cl crossing (with LOD score between 3.03 and 7.12) and in the five places of the C2 crossing (with LOD score between 3.51 and 8.66). For the C3 crossover, this QTL appears in the F3 generations (LOD score = 4.78) and F4 (see above).

The tables collect all of these results.

TABLES Connection between the PK probe and QTLs of sunflower resistance to Sclerotinia sclerotiorum

Cl: SDx PAC 1

C2: CP73 xPΛCl

C3: Gli PΛC2 lintre hooks: in the case where the probe is not polymorphic in the crossover studied, the effect of the locus is estimated using adjacent markers.

VIII / Conclusions

All the results presented above demonstrate the existence of a statistically significant association between a PK locus and a QTL of resistance to Sclerotinia sclerotiorum on the linkage group 1 of sunflower. This locus explains up to 50% of the variance of this resistance, for the latency index and attack percentage tests.

For the resistance to progression of the fungus in the leaves, this locus explains up to 50% of the variance of this resistance.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

(i) DEPOSITOR:

(A) NAME: NATIONAL INSTITUTE OF AGRONOMIC RESEARCH

(INRA)

(B) STREET: 147 STREET OF THE UNIVERSITY

(C) CITY: PARIS CEDEX 07

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75338

(G) TELEPHONE: 01 42 75 90 00

(A) NAME: UNIVERSITY B AISE PASCAL

(B) STREET: 34 AVENUE CARNOT

(C) CITY: CLERMONT-FERRAND

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 63000

(II) TITLE OF THE INVENTION: MARKER FOR RESISTANCE TO SCLEROTINIA SCLEROTIORUM

(m) NUMBER OF SEQUENCES: 4

(îv) COMPUTER-DETACHABLE FORM:

(A) TYPE OF SUPPORT: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: not known

(n) TYPE OF MOLECULE: DNA

(x) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 3

(D) OTHER INFORMATION: / note = "N means Inosine"

(ix) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 6

(D) OTHER INFORMATION: / note = "N means Inosine"

(ix) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATIONS

(D) OTHER INFORMATION: / note = "N means Cytosme or Thy ine"

(îx) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 12

(D) OTHER INFORMATION: / note = "N means Inosine" (ιx) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 15

(D) OTHER INFORMATION: / note = "N means Adenme or Cytosine or

Thymine "

(îx) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 18

(D) OTHER INFORMATION: / note = "N means Inosine"

(îx) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 20

(D) OTHER INFORMATION: / note = "N means Adenme or Thymine"

(îx) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 21

(D) OTHER INFORMATION: / note = "N means Cytosine or Thymine"

(ix) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 24

(D) OTHER INFORMATION: / note = "N means Adenme or Guanine"

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1. GGNGGNTTNG GNATNGTNTN NAANGG 26

(2) INFORMATION FOR SEQ ID NO- 2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: not known

(n) TYPE OF MOLECULE: DNA

(ix) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATIONS

(D) OTHER INFORMATION: / note = "N means Inosine"

(î) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATIONS

(D) OTHER INFORMATION: / note = "N means Inosine"

(ix) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATIONS

(D) OTHER INFORMATION: / note = "N means Guanine or Thymine" (ix) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION

(D) OTHER INFORMATION: / note = "N means Inosine"

(îx) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 15

(D) OTHER INFORMATION: / note = "N means Inosine"

(îx) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 18

(D) OTHER INFORMATION: / note = "N means Adenine or Guanine"

(îx) CHARACTERISTIC:

(A) NAME / KEY: special characteristic

(B) LOCATION: 21

(D) OTHER INFORMATION: / note = "N means Adenme or Guanine"

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: AANATNCNNG CCATNCCNAA NTC 23

(2) INFORMATION FOR SEQ ID NO: 3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 277 base pairs

(B) TYPE: nucleotide

(C) NUMBER OF STRANDS: double

(D) CONFIGURATION: not known

(il) TYPE OF MOLECULE: DNA (genomics)

(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3:

GGGGGGTTCG GGATTGTGTT CAAGGGTGAG TTGTTGTTGG TTTATGAGTT TATGGCTAAT 60

GGAAGTTTGG ATAAGTGTAT ATATAGTGGT AATAAGCCTA AΛ.TTGGTTTT GAGTTGGGAA 120

CAAAGGTTTA AGGTTATAAA AGATGTGGCA AATGGGTTAC TTTATTTGCC TGAAGGGTGG 180

GGAAAAACTG TGGTGCATAG AGATATTAAA GCAGGAAATG TGTTGTTGGA TTCGGATTTA 240

AATGGTAAGT TAGGCGACTT CGGAATGGCC CGCATCT 277 (2) INFORMATION FOR SEQ ID NO: 4:

(l) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 92 amino acids

(B) TYPE: amino acid

(C) NUMBER OF STRANDS: single

(D) CONFIGURATION: not known

_(The MOLECULE TYPE: Deotide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:

Gly Gly Phe Gly Ile Val Phe Lys Gly Glu Leu Leu Leu Val Tyr Glu 1 5 10 15

Phe Met Ala Asn Gly Ser Leu Asp Lys Cys Ile Tyr Ser Gly Asn Lys - 20 25 30

Pro Lys Leu Val Leu Ser Trp Glu Gin Arg Phe Lys Val Ile Lys Asp 35 40 45

Val Ala Asn Gly Leu Leu Tyr Leu Pro Glu Gly Trp Gly Lys Thr Val 50 55 60 ^*

Val His Arg Asp Ile Lys Ala Gly Asn Val Leu Leu Asp Ser Asp Leu

65 70 75 ^'80

Asn Gly Lys Leu Gly Asp Phe Gly Met Ala Arg Ile 85 90

References

• Bent, AF, Kunkel, BN, Dahlbeck, D., Brown, KL, Schmidt, R., Giraudat, J., Leung, J. and Staskawicz, BJ (1994) RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes. Science 265,

1856-1860.

• Castano, F., Hemery-Tardin, M-C, Tourvieille de Labrouhe, D. and Vear, F. (1992). The inheritance and biochemistry of resistance to Sclerotinia sclerotiorum leaf infections in sunflower (Helian th us ann uus L.). Euphyϋca 58, 209-219.

• Dixon, M.S., Jones, D.A., Keddie, J.S., Thomas, CM., Harrison, K. and Jones, J.D.G. (1996). The tomato Cf-2 disease resistance locus included two functional genes encoding leucine-rich repeats proteins. CellS4, 451-459.

• Gentzbittel, L, Vear, F., Zhang, Y-X., Bervillé, A. and Nicolas, P. (1995) Development of a consensus linkage RFLP map of cultivated sunflower

(Helianthus annuus L) Theor. Appl. Broom. 90, 1079-1086.

• Hervé, C, Dabos, P., Galaud, J.P., Rouge, P. and Lescure, B. (1996). Characterization of an Arabidopsis thaliana gene that defines a new class of putative plant receptor kinases with an extracellular lectin-like domain. J. Mol. Biol. 258, 77S-78S.

• Johal, G. S., Briggs, S. P. 1992. Reductase activity encoded by th HM 1 disease resistance gene in maize. Science. 258: 985-987.

• Jones, D.A., Thomas, CM., Hammond-Kosack, K.E., Balint-Kurti, P.J. and Jones, J. D.G. (1994). Isolation of the tomato Cf-9 gene for resistance to Cladosporfum fuivu by transposon tagging. Science 266, 789-793.

• Lander, E.S., Green, P., Abrahamson, J., Barlow, A., Daly, M.J., Lincoln, S.E and Newburg, L (1987). Mapmaker: an interactive computer package for constructing primary genetic linkage maps of experimental and natural population. Genomics 1, 174-181. • Lawrence, G.J., Finnegan, E.J., Ayliffe, M. A. and Ellis, J.G. (1995). The L6 gene for flax rust resistance is related to the Arabidopsis bacterial resistance gene RPS2 and tobacco viral resistance gcne N. Plan t Cell 7, 1195-1206.

• Lincoln, E., Daly, MJ and Lander, ES (1992). Mapping genes controlling quantitative traits using MapMaker / QTL version 1. 1: a tutorial and reference manual. Whitchead Institute Technical Report, Cambridge, 2nd edition. • Martin, GB, Brommonschenkel, SH, Chunwongse, J., Frary, A., Ganal, MW, Spivey, R., VVu, T., Earle, ED. and Tanksley, SD (1993). Map-based cloning of a protein kinase gene conferring disease resistance in tomato. Science 262, 1432-1436. • Mindrinos, M., Katagiri, F., Yu, GL and Ausubel, FM (1994). The A. thaliana disease resistance gene SPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats. Cell 78, 1089-1099.

• Pirvu N., Vranceanu A., Stoenesc, U.F. 1985. Genetic mechanisms of sunflower resistance to white rot (Sclerotinia sclerotiorum Lib. De By). 2. Pflanzenzuchtg. 95: 157-163.

• Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: A laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

• Song, WY., Wang, GL, Chen, LL, Kim, HS., Holsten, T., Wang, B., Zhai, W- X., Zhu, LH., Fanquet, C. and Ronald, P. (1995). A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21. Science 270, 1804-1806.

• Swarup.R., Dumas, C. and Cock, J.M. (1996). A new class of receptor-like protein kinase gene from Arabidopsis thaliana possessing a domain with similarity to plant lectin genes (Accession N0. X95909) Plant Physiol. 1 1 1,

347-347.

• Vear F., Tourvieille D. 1988. Heredity of resistance to Sclero tin ia sclerotiorum in sunflowers. II. Study of capitulum resistance to natural and artificial ascospore infections. Agronomy. S: 503-50S. • Whitha, S., Dinesh-Khumar, S.P., Choi, D., Hehl, R., Corr, C. and Baker, B. (1994). The product of the tobacco mosaic virus resistance gene N: similarity to Toll and the interleukin- 1 receptor. Cell 78, 1 101-1 1 15.

Claims

1 / Nucleotide sequence corresponding to SEQ ID No. 1. 2 / Nucleotide sequence corresponding to SEQ. ID No "2. 3 / Use of the nucleotide sequence according to claim 1 and / or the nucleotide sequence according to claim 2, as primer (s) for the implementation of a chain amplification process by polymerase (PCR) applied to plant DNA.

4 / DNA fragment obtained by using the polymerase chain reaction (PCR) method according to claim 3. 5 / DNA fragment corresponding to SEQ ID N "3.

6 / Use, as a probe, of a DNA fragment according to claim 4 or 5 for the identification in a plant of the genotype resistant to Sclerotinia sclerotiorum, said identification consisting in: - labeling the DNA fragment,

- place the labeled DNA fragment in contact with the plant's DNA under hybridization conditions,

- determine the presence of molecular hybridization.

7 / Use according to claim 6, characterized in that the plant is sunflower.

8 / Peptide sequence corresponding to SEQ ID N "4 or having at least 50% homology with it.

9 / Nucleotide sequences coding for a peptide sequence according to claim S.