Powdery Mildew Resistance Genes in Carrot

Abstract

Provided herein are carrot plants resistant to powdery mildew, and especially powdery mildew caused by the plant pathogen Erysiphe heraclei, wherein the powdery mildew resistance is provided by one or two dominant powdery mildew resistance genes. Also provided herein are molecular markers genetically linked to the present powdery mildew, and especially powdery mildew caused by the plant pathogen Erysiphe heraclei, resistance providing genes and the use thereof for identifying carrots plants, or Daucus carota plants, being resistant to powdery mildew, and especially powdery mildew caused by the plant pathogen Erysiphe heraclei. Also provided herein are seeds, plant parts, pollen, egg cells, callus, suspension culture, somatic embryos and edible plant parts of the present plants.

Claims

1. A Daucus carota plant that is resistant to powdery mildew caused by the plant pathogen Erysiphe heraclei, said resistance provided by a first resistance gene located on chromosome 3 of said plant between SEQ ID No. 4 and SEQ ID No. 5.

2. The Daucus carota plant according to claim 1, further comprising a second resistance gene located on chromosome 3 of said plant between SEQ ID No. 11 and SEQ ID No. 12.

3. The Daucus carota plant according to claim 1, wherein said first resistance gene is located on chromosome 3 at 2.68 cM.

4. The Daucus carota plant according to claim 2, wherein said second resistance gene is located on chromosome 3 at 76.7 cM.

5. The Daucus carota plant according to claim 1, wherein said first resistance gene is obtainable from seeds of a Daucus carota plant deposited under deposit number NCIMB 42389.

6. The Daucus carota plant according to claim 2, wherein said second resistance gene is obtainable from seeds of a Daucus carota plant deposited under deposit number NCIMB 42397.

7. The Daucus carota plant according to claim 1, wherein said first resistance gene is identifiable by at least one molecular marker selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7.

8. The Daucus carota plant according to claim 2, wherein said second resistance gene is identifiable by at least one molecular marker selected from the group consisting of SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15.

9. The Daucus carota plant according to claim 1, comprising in its genome at least one genomic sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and SEQ ID No. 7.

10. The Daucus carota plant according to claim 2, comprising in its genome at least one genomic sequence selected from the group consisting of SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14 and SEQ ID No. 15.

11. The Daucus carota plant according to claim 1, wherein said plant is a hybrid plant.

12. The Daucus carota plant according to claim 11, wherein said hybrid plant is a sterile hybrid plant.

13. The Daucus carota plant according to claim 12, wherein said sterile hybrid plant is a sterile male.

14. The Daucus carota plant according to claim 1, wherein said plant is Daucus carota ssp. sativus.

15. Seeds, edible parts, pollen, egg cells, callus, suspension culture, somatic embryos, embryos or plant parts of a Daucus carota plant according to claim 1.

16.-19. (canceled)

20. The Daucus carota plant according to claim 13, wherein said sterile male plant is a cytoplasmic sterile male hybrid plant.

Description

[0038] The present invention will be further detailed in the following examples and figures wherein:

[0039] FIG. 1: shows a schematic physical map of chromosome 3 of Daucus carota showing both the present molecular markers and the present first resistance gene Eh 1 and the present second resistance gene Eh 2 providing resistance to the plant pathogen Erysiphe heraclei.

[0040] FIG. 2: shows sequences of the molecular markers SNP markers as shown in FIG. 1 and their position on chromosome 2 of the sequence of PRJNA268187.sup.(ref. 9).

[0041] SEQ ID No. 1-7 correspond with the SNP representing the resistant gene at locus Eh 1

[0042] SEQ ID No. 8-15 correspond with the SNP representing the resistant gene at locus Eh 2

[0043] SEQ ID No. 16-22 correspond with the SNP representing the susceptible gene at locus Eh 1

[0044] SEQ ID No. 23-30 correspond with the SNP representing the susceptible gene at locus Eh 2

[0045] Code usage according to the IUPAC nucleotide code.sup.(ref 10):

TABLE-US-00001 A = adenine T = thymine C = cytosine G = guanine K = G or T W = A or T Y = C or T

EXAMPLES

Example 1: Testing for Resistance Against Erysiphe heraclei in the Glasshouse

[0046] The fungus, as obligate parasite, was maintained on suitable susceptible carrot plants by placing infected leaves between them. Infection was spread among these plants by using a fan, by the air currents the spores were distributed among the plants.

[0047] Plants to be tested for resistance were sown in soil on tables, around 30 plants per row. Every 20 rows of plants to be assessed a row of resistant material for race 0 and race 1 and susceptible material each were inserted. When the plants were about 3 cm tall, inoculation took place by adding infected leaves, clearly showing fungal spores. Plants to be assayed for resistance were stroked first with these leaves, and then the inoculating leaves were placed between the young plants. Spores were spread further using a fan. Temperature was 162 C. at night; 222 C. during daytime; min. 16 hours light (or more if day length was longer) and max. 8 hours dark. Humidity was kept at a high level by spraying water between the tables a few times a week. After 6 weeks the plants were evaluated; infected leaves were covered with a white powdery mycelium and spores, and often turn chlorotic.

[0048] The severity of infection was reflected by scoring the symptoms between 0 (completely susceptible) and 9 (completely resistant). It was carefully checked that the susceptible control plants are indeed showing the symptoms of E. heraclei infection.

Example 2: Field Tests for Resistance Against E. heraclei

[0049] Field tests were performed under Dutch climatological conditions. The fungal inoculum was prepared as described above in Example 1. Material to be tested in the field is directly sown during the first half of May.

[0050] When plants were about 3 cm tall, the inoculum was spread by placing pots with sporulating plants in the field between the young materials to be tested. The wind will spread spores from the inoculating plants.

[0051] Plants were assessed for their resistance or susceptibility when symptoms were clearly visible during dry weather conditions. The severity of infection was reflected by scoring the symptoms between 0 (completely susceptible) and 9 (completely resistant).

Example 3: Molecular Characterization of Genomic DNA and Mapping of the Resistance Genes

[0052] Applying the two available genetic resources for resistance described above, two F1S1 populations were made by crossing the different sources of resistance to a susceptible carrot line, after which the resulting F1 plant was self-pollinated. The observed segregation of three resistant plants to one susceptible plant learned that indeed in both cases the resistance is based on a dominant trait.

Basic research lead to a partial genetic map of D. carota and also a near-complete sequence of its genome, submitted to NCBI as project PRJNA268187.sup.(ref. 9).

[0053] At least 2000 seeds were harvested from the F1S1 generation of a cross between the distinctive sources of resistance and a susceptible carrot line. To perform a QTL mapping, 1200 plants of each cross were grown in the glasshouse. From each individual plant, leaf material was used for DNA isolation and successive marker analysis.

[0054] Inbreds of selected individuals with crossovers nearby the resistance locus were tested in the greenhouse as described in example 1 and resistance was confirmed.

[0055] To develop more single nucleotide polymorphic (SNP) markers in the region of the resistance gene, a sequence project was started with the available sources of resistance against E. heraclei.

[0056] Using SNP markers covering the entire genome, both resistance genes were determined to be located on chromosome 3. Using sequences of the two resistance lines and one susceptible line, in combination with the genome sequence available, for both resistance loci many SNPs were discovered. Based on crossovers present in the mapping populations, each resistance locus could be located on the genome sequence, submitted to NCBI as PRJNA268187.sup.(ref. 9).

[0057] For D. carota accession NCIMB42389 the resistance locus (Eh 1) was located on chromosome 3 at 2.68 cM, corresponding to a fragment between position 1,648,619 and 1,739,519 bp.

[0058] By using the sequences described above, more SNP markers have been developed in the region of the resistance locus and used to genotype the resistance sources and the individuals with a crossover near the resistance locus, see Table 1 below:

TABLE-US-00002 physical Individual F1S1 plants marker position NCIMB42389 Susceptible S9342-13 S9342-19 S9342-05 S9342-12 S9342-21 9618 CHR3: 1,648,619 a b b h h h h 9620 CHR3: 1,654,801 a b h h h h h 9624 CHR3: 1,661,351 a b h h h h h 9703 CHR3: 1,661,662 a b h h h h h 9708 CHR3: 1,663,368 a b h h h h h disease r s r r r r r test 9625 CHR3: 1,672,079 a b h h h h h 9629 CHR3: 1,705,739 a b h h h h h 9635 CHR3: 1,734,335 a b h h h h h 9631* CHR3: 1,722,613 a b h h h h b 9636 CHR3: 1,739,519 a b h h h h b Individual F1S1 plants marker S9342-22 S9342-09 S9342-25 S9342-01 S9342-04 S9342-18 S9342-02 S9342-03 9618 h h h h h b b b 9620 h h h h h b b b 9624 h h h h h b b b 9703 h h h h h b b b 9708 h h h h h b b b disease r r r r r s s s test 9625 h h h h h b b b 9629 h h h h h b b b 9635 h h h h h b b b 9631* h h h h h b b b 9636 h h h h h b b b *regarding to marker 9631: based on crossover data, the physical map in this region was corrected for the order of markers

[0059] The resistance locus is located between markers 9618 and 9631.

[0060] Further, for D. carota accession NCIMB42397 the location of the second resistance locus (Eh 2) was determined on chromosome 3 around 76.7 cM, corresponding to a fragment between positions 45,210,264 bp and 45,845,221 bp.sup.(ref 9).

[0061] Also for genotype accession NCIMB42397 more markers could be developed with the information from the sequence project and used to genotype accession NCIMB42397 and individuals with a crossover, see Table 2 below:

TABLE-US-00003 physical Individual F1S1 plants marker position NCIMB42397 susceptible T9067-4 T9067-5 T9067-6 T9067-17 9659 CHR3: 45,210,264 a b h h h h 9666 CHR3: 45,264,585 a b h h h h 9669 CHR3: 45,290,166 a b h h h h 9670 CHR3: 45,295,089 a b h h h h 9671 CHR3: 45,302,019 a b h h h h disease r s r r r r test 9672 CHR3: 45,311,025 a b h h h h 6709 CHR3: 45,313,919 a b h h h h 9674 CHR3: 45,325,457 a b h h h h 9677 CHR3: 45,350,385 a b h h h h 9528 CHR3: 45,397,477 a b h h h h 6909 CHR3: 45,399,809 a b h h h h 4201 CHR3: 45,418,720 a b h h h h 6069 CHR3: 45,845,221 a b h h h h Individual F1S1 plants marker T9067-18 T9067-19 T9067-20 T9067-7 T9067-8 T9067-11 T9067-13 T9067-16 9659 h h h h h b b b 9666 h h h b b b b b 9669 h h h b b b b b 9670 h h h b b b b b 9671 h h h b b b b b disease r r r s s s s s test 9672 h h h b b b b b 6709 h h h b b b b b 9674 h h h b b b b b 9677 h h h b b b b b 9528 h h h b b b b b 6909 h h h b b b b b 4201 h h h b b b b b 6069 h h b b b h h b

[0062] As graph this situation can alternatively be illustrated as in FIG. 1.

As is clear both from the position in cM and base pair position and illustrated by FIG. 1, the present dominant resistance genes involved are located far apart on chromosome 3. This discovery of two separate resistance genes means that these resistance genes preferably can be stacked e.g. in a hybrid to have a more solid genetic base for a durable resistance.

Deposit Information

[0063] Seed samples of the sources of resistance mentioned above were deposited at the NCIMB, Ferguson Building; Craibstone Estate, Bucksburn, Aberdeen, Scotland, AB21 9YA, as: [0064] NCIMB 42389 (D. carota #954561), Mar. 19, 2015 [0065] NCIMB 42397 (D. carota #1360572), Apr. 16, 2015

REFERENCES

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