RESISTANCE TO XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS (XCC) IN CAULIFLOWER

Abstract

The present invention relates to resistance to Xanthomonascampestris pv. campestris (Xcc) in cauliflower. According to the invention, the resistance is provided by DNA sequences, introgressed from a green cauliflower at specific loci in the genome of a white cauliflower. The introgressed sequences can be present homozygously or heterozygously in the genome of the white cauliflower, and they confer resistance to Xcc. The invention further relates to part of these cauliflowers, to seeds, to the progeny of these cauliflowers, and to method for producing cauliflowers resistant to Xcc.

Claims

1. A cauliflower plant that is resistant to Xanthomonas campestris pv. campestris (Xcc) and that does not have a green curd, said cauliflower plant: (i) comprising in its genome introgressed sequences from a green cauliflower plant conferring said resistance to Xcc; and (ii) not comprising in its genome a major Quantitative Trait Loci (QTL) on chromosome 5 conferring the green color of the curd.

2. The cauliflower plant according to claim 1, which has a white curd.

3. The cauliflower plant according to claim 1, wherein said introgressed sequences conferring said resistance to Xcc comprise one QTL that is present on chromosome 5 and one QTL that is present on chromosome 7.

4. The cauliflower plant according to claim 3, wherein: said QTL conferring resistance to Xcc that is present on chromosome 5 is located within a chromosomal region that is delimited by marker BN-0061002 and marker BO-0101641, and said QTL conferring resistance to Xcc that is present on chromosome 7 is located within a chromosomal region that is delimited by marker BO-0002582 and marker BN-0010593.

5. The cauliflower plant according to claim 1, wherein said major QTL on chromosome 5 conferring the green color of the curd is located within a chromosomal region that is delimited by marker BO-0103554 and marker BO-0101638.

6. The cauliflower plant according to claim 1, wherein said introgressed sequences from a green cauliflower conferring resistance to Xcc are selected from the introgressed sequences present in the genome of a plant of the line FLA1-116-02S (NCIMB accession number 42693), or RSF1-BC3-F3 (NCIMB accession number 43442).

7. The cauliflower plant according to claim 1, wherein said cauliflower plant is a progeny of a plant line FLA1-116-02S (NCIMB accession number 42693) or RSF1-BC3-F3 (NCIMB accession number 43442).

8. An isolated cell of the cauliflower plant according to claim 1.

9. A plant part obtained from a cauliflower plant as defined in claim 1.

10. The plant part according to claim 9, wherein said plant part is selected from the group consisting of a seed, a curd, a floret, a reproductive material, a root, and a flower.

11. A seed of a cauliflower plant, wherein the seed is cultivated to produce a cauliflower plant as defined in claim 1.

12. A hybrid plant of a cauliflower plant that is resistant to Xanthomonas campestris pv. campestris (Xcc) and that does not have a green curd, obtainable by crossing a cauliflower plant with a resistant cauliflower plant according to claim 1.

13. A method for detecting and/or selecting a cauliflower plant that is resistant to Xanthomonas campestris pv. campestris (Xcc) and that does not have a green curd, wherein said method comprises the step of detecting the presence or absence of: a QTL conferring resistance to Xcc on chromosome 5 located within a chromosomal region that is delimited by marker BN-0061002 and marker BO-0101641, a QTL conferring resistance to Xcc on chromosome 7 located within a chromosomal region that is delimited by marker BO-0002582 and marker BN-0010593, and a QTL conferring the green color of the curd on chromosome 5 located within a chromosomal region that is delimited by marker BO-0103554 and marker BO-0101638, and wherein (i) the presence of said QTLs conferring resistance to Xcc on chromosome 5 and on chromosome 7, and (ii) the absence of said QTL conferring the green color of the curd on chromosome 5, indicates that said cauliflower plant is resistant to Xanthomonas campestris pv. campestris (Xcc) and does not have a green curd.

14. A method for obtaining cauliflower plant resistant to Xanthomonas campestris pv. campestris (Xcc) that does not have a green curd comprising breeding a resistant cauliflower plant according to claim 1 with a second cauliflower plant.

15. A molecular marker that is linked to the QTL on chromosome 5 and/or on chromosome 7 conferring resistance to Xcc, wherein said marker is: one or more of the markers BN-0061002, BN-0060999, BN-0060988, BO-0101676, BN-0064638, BO-0101706 and BO-0101641, and/or one or more of the markers BO-0002582, BN-0010479, BO-0101656, BO-0101655, BO-0103553, BO-0101639, BO-0101640 and BN-0010593.

16. A method for improving the yield of cauliflower plants in an environment infested by Xanthomonas campestris pv. campestris (Xcc), comprising growing cauliflower plants resistant to Xcc and that does not have a green curd as defined in claim 1.

17. A method for improving the yield of cauliflower plants in an environment infested by Xanthomonas campestris pv. campestris (Xcc) comprising: a) identifying cauliflower plants resistant to Xcc and that does not have a green curd as defined in claim 1, and b) growing said resistant cauliflower plants in said infested environment.

18. A method for protecting a field from infestation and/or spread of Xanthomonas campestris pv. campestris (Xcc), comprising growing cauliflower plants resistant to Xcc and that does not have a green curd as defined in claim 1.

19. A method for increasing the number of harvestable or viable cauliflower plants in an environment infested by Xanthomonas campestris pv. campestris (Xcc), comprising growing cauliflower plants resistant to Xcc and that does not have a green curd as defined in claim 1.

20. A method for controlling infestation by Xanthomonas campestris pv. campestris (Xcc) comprising growing a resistant cauliflower plant that does not have a green curd as defined in claim 1.

21. A container comprising a cauliflower plant as defined in claim 1.

22. A method for the production of cauliflower plantlets or plants resistant to Xanthomonas campestris pv. campestris (Xcc), which method comprises: (i) culturing in vitro an isolated cell or tissue of the cauliflower plant as defined in claim 1 to produce cauliflower micro-plantlets resistant to Xanthomonas campestris pv. campestris (Xcc), and (ii) optionally further subjecting the cauliflower micro-plantlets to an in vivo culture phase to develop into cauliflower plant resistant to Xcc.

Description

FIGURE

[0465] FIG. 1 shows the LOD score determined for nine markers on chromosome 5, calculated as LOD=−log(p-value), plotted against the physical position of the markers on chromosome 5. The p-value was determined by performing a Kruskal-Wallis statistical test to assay the association between marker genotype and phenotype.

EXAMPLES

1. Material and Methods

1.1. Cauliflower Lines

[0466] The green cauliflower line FLA belongs to the Brassica oleracea L. var. botrytis species. This line has been identified by the inventors as being resistant to Xcc races 1 and/or 4 and has a curd with a green color at harvest maturity similar to the green color of the example varieties Alverda and Minaret cited in the table of characteristics of the in TG/45/7 document edited by the International Union for the Protection of new Variety of plants (UPOV) and dated 2009 Apr. 1 (characteristic 21 at page 13 of the document).

[0467] The white cauliflower line SOL5 and RST belong to the Brassica oleracea L. var. botrytis species. These lines have been identified by the inventors as being susceptible to Xcc races 1 and/or 4 and have a color at harvest maturity similar to the white color of the example varieties Aerospace, Aviron or Freebell.

1.2. Xcc Test (Field Test Conditions)

Xcc Inoculum Preparation

[0468] The Xcc strains of races 1 and 4 were stored at −80° C. Each strain is first grown on Petri dishes containing an LPGA medium during 48 h at 26° C. Then each strain is transferred into new LPGA agar plates and grown during 48 h at 26° C. to obtain a bacterial mat. For inoculum preparation, bacterial mat of each strain is pooled and adjusted to a final concentration of 10.sup.8 bacteria/ml. Then 3 drops/L of Tween 20 is added in the inoculum.

Plant Material Production

[0469] Plantlets are transplanted in the field when they bear 8-12 leafs. For each experiment, one susceptible check and one resistant check are also transplanted in the field and inoculated.

Experimental Procedure and Evaluation

[0470] One month after transplantation of the plants in the field, they are inoculated with each inoculum by spraying the inoculum on all leaves of each plant. Plant were the grown under natural field conditions, and the plants were evaluated for Xcc infection 1 months to 1.5/2 months after inoculation according to the following scale: 9=No symptoms, 8=1-12% of symptoms on leaves, 7=13-25% of symptoms on leaves, 5=26-50% of symptoms on leaves, 3=51-75% of symptoms on leaves and 1=>76% of symptoms on leaves. According to this scoring, a plant is considered as being highly resistant (HR) when the score is 9 or 8, a plant is considered as being intermediate resistant (IR) when the score is 7, and a plant is considered as being susceptible when the score is 5, 3 or 1.

1.3. DNA Extraction and Genotyping Protocol

[0471] Plants were sampled and DNA was isolated, using magnetic beads (NucleoMag® 96 Plant), according to the protocol of the manufacturer of the beads, Macherey-Nagel. DNA was eluted in 60 μL of PCR grade water.

[0472] Genotyping was done using KASP™ technology. KASP™ genotyping requires a KASP™ Assay mix which is specific to each marker and KASP™ Master mix was purchased at LGC (http://www.lgcgroup.com). Since genotyping was carried out in 1536-well plates, KASP V4.0 1×Mastermix 1536 Master mix was used.

[0473] The KASP™ Assay mix is specific to each marker and consists of the two competitive, allele-specific primers and one common primer (see tables 2 and 5). Each allele-specific primer incorporates an additional tail sequence that corresponds to one of two universal FRET (fluorescent resonance energy transfer) cassettes present in the KASP™ 1536 Master Mix. DNA strand and allele designation and orientation is done according to the TOP/BOT method developed by Illumina (https://www.illumina.com/documents/products/tech notes/technote_topbot.pdf).

[0474] For each marker and sample, 1.5 μl of 1:10 diluted DNA was aliquoted in 1536-well plates using a LGC repliKator™ robot. DNA was then dried overnight at room temperature. Genotyping reaction was prepared by dispensing, per DNA sample, 0.986 μl of Assay Mix and 0.014 μl of KASP™ 1536 Master Mix. Dispensing was performed using a LGC Meridian robot. Reaction plates were further sealed using LGC Fusion3™ laser welding system.

[0475] Thermal cycling was performed in LGC Hydrocycler™ water bath-based thermal cycler using the following thermal cycling touchdown program: Stage 1 (Hot start Taq activation): 94° C. for 15 minutes, Stage 2 (Touchdown): 10 cycles at 94° C. for 20 seconds and 65-57° C. for 60 seconds (65° C. decreasing 0.8° C. per cycle to achieve a final annealing/extension temperature of 57° C.), and Stage 3 (Amplification): 35 cycles at 94° C. for 20 seconds and 57° C. for 60 seconds.

[0476] Plate reading for fluorescence measurement was achieved by a BMG PHERAstar plate reader. Fluorescence data were further analyzed by LGC KlusterCaller™ software.

2. Introgression of the Resistance to Xcc from Green Cauliflower into White Cauliflower

[0477] A) Genetic Determinism of the Green Cauliflower Resistance to Xcc

[0478] The green cauliflower FLA was crossed with the white susceptible cauliflower

[0479] SOL5. The resultant F1 seeds were germinated, plants grown from the germinated seeds, and the resultant plants were selfed to produce F2 seeds/plants for further selection and breeding. F2 plants have been submitted in field to a pathological test for resistance to Xcc. Each plants of the F2 population have been scored individually, and the segregation ratio of the trait in the F2 population corresponded to one monogenic dominant gene.

[0480] Then a bulk segregant analysis was run on a resistant bulk versus susceptible one, using 384 SNPs spread over the whole genome. 24 SNPs discriminating the resistance and susceptibility were kept for further analysis. Among these 24 SNPs, one was located on chromosome 5 and eight were located on chromosome 7. A test for association to the resistance to Xcc was performed against these SNPs and allowed to confirm that one major QTL was located on chromosome 7 but that a second one was located on chromosome 5.

[0481] F3 families were produced from 200 new F2 plants randomly chosen.

[0482] Out of 200, 153 F3 families were evaluated for Xcc resistance races 1 and 4 in field trial. Parental lines FLA and SOL5 were included in this test. FLA had an intermediate level of resistance with a score of 7 and SOL5 was susceptible with a score of 5. F3 families had score of resistance comprised between 3 and 9.

[0483] The segregation ratio was still in accordance with the hypothesis of one major dominant gene.

[0484] Genotyping analysis of this population was further carried out to validate this hypothesis.

[0485] Genotyping of the 142 F2 plants out of 200 F2 was thus performed with 4 markers located on chromosome 7 (out of the 8 previously identified) and 3 located on chromosome 5. It enabled us to confirm that these two regions were each harboring a QTL of resistance to Xcc. As the markers physical position is known in the genome, we were able to localize the resistance region to a 33,521,178 bp wide region on chromosome 5 comprised between positions 9,354,311 and 42,875,489, and to a 2,773,439 bp wide region on chromosome 7 comprised between positions 34,714,403 and 38,690,572.

[0486] Eight additional markers, located in the defined resistance region on chromosome 5 were further identified and used to genotyped the 142 F2 plants. An association test between marker genotype and phenotype was performed using a Kruskal-Wallis statistical test. For each marker, a LOD score was calculated from the p-value of the test as LOD=−log(p-value) and plotted against the physical position of the markers on chromosome 5 (FIG. 1). The resistance region was thus further refined to a 1,044,654 bp wide region between positions 38,928,177 bp and 39,972,831 bp.

[0487] Seven additional markers (i.e. BO-0002582, BN-0010479, BO-0101656, BO-0101655, BO-0103553, BO-0101639, BO-0101640), located in the defined resistance region on chromosome 7 were further identified and used to genotyped the 142 F2 plants. An association test between marker genotype and phenotype was performed using a Kruskal-Wallis statistical test. For each marker, a LOD score was calculated from the p-value of the test as LOD=−log(p-value). The resistance region was thus further refined to a 1,550,367 bp wide region between positions 36,520,957 bp and 38,690,572 bp.

[0488] B) Genetic Determinism of the Green Curd Color

[0489] 229 cauliflower lines have been genotyped with 384 SNP markers well spread over the genome. Curd color of those 229 inbred lines was coded as a binary trait: white or green. An association study was performed on those dataset to identify markers linked to the green curd color trait.

[0490] 12 interesting markers (i.e. BN-0003844, BN-0002453, BN-0004278, BN-0010638, BN-0010246, BN-0009825, BN-0001304, BN-0001306, BN-0002268, BN-0003875, BN-0004457, BN-0003896, see Table 1 for the sequences of these markers) were kept for further analysis. Those 12 markers were the most tightly linked ones to the green curd color phenotype. Thanks to the mapping position information, it has been found that the 12 markers corresponded to 8 different QTLs, two QTLs being located on chromosome 1 (one QTL, named MiC1-2 for the purpose of the invention, encompassing the markers BN-0003844 and BN-0002453, and a second one, named MiC1-3 for the purpose of the invention, encompassing the marker BN-0004278), two QTLs being located on chromosome 2 (one QTL, named MiC2-1 for the purpose of the invention, encompassing the markers BN-0010638 and BN-0010246, and a second one, named MiC2-2 for the purpose of the invention, encompassing the marker BN-0009825), one QTL, named MiC4 for the purpose of the invention, being located on chromosome 4 and encompassing the markers BN-0001304 and BN-0001306, two QTLs being located on chromosome 5 (one major QTL, named MAC5 for the purpose of the invention, encompassing the marker BN-0004457 and one minor QTL, named MiC5 for the purpose of the invention, encompassing the markers BN-0002268 and BN-0003875), and one QTL, named MiC6 for the purpose of the invention, being located on chromosome 6 and encompassing the marker BN-0003896.

[0491] To validate the predictably of these 5 mostly associated markers among the 12 markers, two green cauliflower hybrids and 5 green cauliflower breeding lines not previously used for association study were used. The 5 alleles corresponding to the 5 markers define an haplotype allowing to predict the green color of the curd.

[0492] To further validate the identified haplotype to predict the green curd color in cauliflower, a Bulk Segregant Analysis on few FLA×SOL5 DH plants has been performed. Each DH plant had previously been phenotyped (visual scoring) for curd color on a 1 to 9 scale (1=white color similar to SOL5, 9=green color similar to FLA). One bulk of white curd color DH plants and 1 bulk of green curd color DH plants were tested with 384 SNPs. Five SNPs discriminating the green bulk from the white bulk were identified (i.e. markers BN-0004384, BN-0004457, BN-0000623, BN-0002182 and BO-0003450) and kept for further analysis. These five SNPs were located: [0493] in the previously highlighted regions on chromosome 1 (i.e. the MiC1-2 QTL with marker BN-0004384 that is located in the same region as markers BN-0003844 and BN-0002453) and chromosome 5 (i.e. the MAC5 QTL with marker BN-0004457 that is located in the same region as marker BO-0101638), [0494] on a new region on chromosome 1, named MiC1-1 for the purpose of the invention, encompassing the marker BN-0000623, and [0495] on a new region on chromosome 8, named MiC8 for the purpose of the invention, delimited by markers BN-0002182 and BO-0003450.

[0496] It is hypothesized that the QTL identified on chromosome 8 is specific to FLA because it was not found in the first association study. It has also been confirmed that chromosome 1 and 5 are involved in green curd trait in cauliflower. On chromosome 5, additional SNP markers polymorphic between FLA and SOL5 were used to genotype the DH population. Further analyses allowed to identify marker BO-0103554 located in the MAC5 QTL as tightly linked to the curd color.

[0497] Regarding these results and the ones obtained for the resistance to Xcc it has finally been found that there is a linkage on chromosome 5 between the “resistance to Xcc” and “green color of the curd” with a genetic distance of 6.1 cM>x>4.3 cM.

[0498] Once major «green color» QTL MAC5 and Xcc resistance» QTL position on chromosome 5 had been defined, occurrence of both QTLs in the original F2 (example 1, section A) population was analyzed. Due to the tight linkage, no plant carrying the right allele at both QTLs on chromosome 5 (respectively white color and resistance) at homozygous state could be found.

[0499] In the F2 population, one plant (FLA-116) heterozygous for the Xcc resistance QTL on chromosome 5 but homozygous for the white allele (coming from SOL5) at the «green color» QTL MAC5 on chromosome 5 has been identified. This plant represented a good starting point to break the linkage between the “green” color QTL MAC5 on chromosome 5 and the “Xcc resistance” QTL on chromosome 5. This plant was selfed to produce F3 seeds.

[0500] 95 F3 seeds were tested with markers to select the recombinants. 5 plants being homozygous resistant for Xcc QTLs on chromosome 5 and chromosome 7 and homozygous white for the green color QTL MAC5 on chromosome 5 were identified (FLA1-116-02, FLA1-116-38, FLA1-116-51, FLA1-116-62, FLA1-116-81) and selfed. Linkage drag was thus broken in F3 plants. These selected F3 plants were selfed to increase seeds set. F4 seeds were obtained and the F4 seeds (i.e. FLA1-116-02S seeds) obtained from the selfing of the FLA1-116-02 plants were deposited under the NCIMB number 42693. The inventors thus managed to obtain Xcc resistant plants not having a green curd due to the presence of (i) the resistant alleles at homozygous state for the Xcc resistance QTLs on chromosome 5 and chromosome 7 and (ii) the white alleles at homozygous state for the MAC5 QTL.

[0501] C) Introgression of Xcc Resistance into Elite White Genotype

[0502] In parallel the back-cross method has been used to introgress the two QTLs of resistance of the donor FLA plant into an elite recurrent white line named RST by breaking the linkage with the green color.

[0503] The green cauliflower FLA was crossed with the white susceptible cauliflower RST. The resultant F1 seeds (coded RSF1) were germinated, plants grown from the germinated seeds, and the resultant plants were back crossed with RST to produce the first backcross seeds (RSF1 Bc1). The 37 BC1 plants have been submitted to Marker Assisted Back-Cross (MABC). Two plants were selected (RSF1 Bc1 A and RSF Bc1 C), heterozygous resistant/susceptible for Xcc QTLs on chromosome 5 and chromosome 7; homozygous white for the green color QTL MAC5 on chromosome 5 were identified; with respectively 79.41% and 73.53% of the recurrent background. These two selected BC1 plants were backcrossed with the recurrent RST to produce the second backcross seeds (RSF1 Bc2 A and RSF1 Bc2 C). 88 plants from RSF1 Bc2 A seeds and 93 plants from RSF1 Bc2 C were tested with MABC. No plants from RSF1 Bc2 C were selected. But two plants from RSF1 Bc2 A seeds were selected (RSF1 Bc2 A1, RSF1 Bc2 A2). These two plants were heterozygous for Xcc QTLs on chromosomes 5 and 7, with the white cauliflower haplotype and with 95.42% and 94.51% of isogeny. These two selected BC2 plants were backcrossed with the recurrent RST to produce the third backcross seeds (RSF1 Bc3 A1 and RSF1 Bc3 A2) and selfed to produce the Bc2F2 seeds. The two Bc3 and the Bc2F2 populations were evaluated for Xcc resistance races 1 and 4 in field trial. Parental lines FLA and RST were included in this test. FLA had an high level of resistance with a score of 8 and RST was susceptible with a score of 5. Bc3 populations had score of resistance comprised between 5 and 7. Bc2F2 population had score of resistance comprised between 7 and 8. In parallel, 101 plants from RSF1 Bc3 A1 and 81 plants from RSF1 Bc3 A2 seeds were tested with Marker Assisted Selection. 2 plants were selected with the white cauliflower haplotype and the two FLA Xcc QTLs heterozygous (RSF1 Bc3 AlA and RSF1 Bc3 A2A). These two selected Bc3 plants were selfed to produce the Bc3F2 seeds. The two Bc3F2 populations were evaluated for Xcc resistance races 1 and 4 in field trial plant by plant. Parental lines FLA and RST were included in this test. FLA had a high level of resistance with a score of 8 and RST was susceptible with a score of 5. Bc3F2 plants had score of resistance comprised between 5 and 8. In parallel, 91 plants from the Bc3 selfed RSF1 Bc3 A1A and 90 plants from the BC3 selfed RSF1 Bc3 A2A were tested with MAS. One plant of each population being homozygous resistant for Xcc QTLs on chromosome 5 and chromosome 7 and with the white cauliflower haplotype were identified (RSF1 Bc3 A1A1 and RSF1 Bc3 A2A1). These two selected Bc3F2 plants were selfed to produce the Bc3F3 seeds, totally homozygous resistant for Xcc QTLs on chromosome 5 and chromosome 7 and with the white cauliflower haplotype (RSF1 Bc3 A1A1A and RSF1 Bc3 A2A1A). The RSF1 Bc3 A1A1A (re-named RSF1-BC3-F3) seeds were deposited under the NCIMB number 43442.

3. Genetic Modification of Cauliflower Seeds by Ethyl Methane Sulfonate (EMS)

[0504] Seeds of cauliflower plants are to be treated with EMS by submergence of approximately 2000 seeds into an aerated solution of either 0.5% (w/v) or 0.7% EMS for 24 hours at room temperature.

[0505] Approximately 1500 treated seeds per EMS dose are germinated and the resulting plants are grown, preferably in a greenhouse, for example, from March to September, to produce seeds.

[0506] Following maturation, M2 seeds are harvested and bulked in one pool per variety per treatment. The resulting pools of M2 seeds are used as starting material to identify the individual M2 seeds and the plants resistant to Xcc.