Patent Description:
Carrot, or Daucus carota, is a cultivated plant from the Umbelliferae (or Apiaceae) which is common in many parts of the world. The Umbelliferae family consists of many species which are in general aromatic plants with hollow stems; it is between the <NUM> largest families of flowering plants. Next to the genus Daucus other cultivated plants are known; e.g. caraway, celery, coriander, dill, fennel, parsley and parsnip. In total the Umbelliferae encompass more than <NUM>,<NUM> species.

Wild carrot, Daucus carota L. , is endemic in large parts of the world and has a white taproot which is edible when in a young stage, but becomes woody after prolonged growth. The cultivated carrot, Daucus carota and especially Daucus carota ssp. sativus, is a root vegetable, usually orange but also purple, red, yellow and white varieties are known.

Generally in more moderate climate zones, Daucus carota is a biennial plant which has a period of vegetative growth in the first year after sowing; after overwintering the plant will flower in the second year of cultivation. In tropical and subtropical areas carrot has an annual life cycle; the transfer from vegetative to generative stage occurs without vernalization. Further, a few wild species also have an annual life cycle.

Leaves are placed in a spiral composition. When the flower stalk elongates, the tip of the stem gets pointed and becomes a highly branched inflorescence. The stem can reach a length of <NUM> - <NUM>.

Flowers are placed in umbels with white, sometimes light green or yellow pedicels; individual flowers are borne on pedicels. The first umbel is present at the end of the main stem, additional umbels will grow from this main branch. Each flower has five petals, five stamens and one central stigma. The flowers are protrandrous, meaning that the anthers release their pollen first, before the stigma of the flower can be pollinated. This mechanism prevents self-pollination to a certain extent and promotes cross pollination. A nectar-containing disc is present at the upper surface of the carpels. The flowers attract pollinating insects, after fertilization the outer part of the umbel bends inward, changing the umbel in a convex, and later a cup shape. Seeds develop in about <NUM> days and consist of two mericarps, each containing a true seed.

The advent of male sterility in Daucus is a character which is very useful for producing hybrid seeds. Two types of male sterility are known in the genus Daucus (refs. <NUM>,<NUM>,<NUM>): the so-called brown anther type (anthers degenerate and shrivel before they can spread pollen) and the petaloid type where stamens are replaced by petal-like structures (ref.

Male sterility observed in carrot is generally cytoplasmic male sterility, meaning that the genetic determinant causing this trait is not located on the nuclear chromosomes but rather is encoded by the mitochondrial DNA. Since mitochondria are transferred to the offspring by egg cells only, this trait is maternally inherited. Since the occurrence of male sterility enables <NUM> % cross pollination, hybrids of Daucus are readily produced. Carrot is a crop which suffers from inbreeding depression but heterosis, or hybrid vigour, can be very strong.

Carrot is cultivated for its nutritious taproot. The major part of this root consists of an outer phloem cortex and an inner xylem core. A large proportion of cortex relative to the core is considered to be of high horticultural quality. Many shapes of the taproot are known, depending on usage a round, conical or more cylindrical shape is preferred. Root length varies from <NUM> to even <NUM>; the diameter can vary from <NUM> to <NUM>. The colour of the taproot is white in the wild type but cultivated forms are mostly orange, sometimes red, purple, black or yellow. The taproot is rich in carotene, especially β-carotene, an important anti-oxidant, which can be metabolized to vitamin A. Further, carrots are a source of dietary fibre, vitamins C, B6 and K, and the antioxidant falcarinol. Antioxidants (including carotenoids) have been studied for their ability to prevent chronic disease. Free sugars are mainly sucrose, glucose and fructose.

Cultivation of carrot is performed worldwide. In <NUM> more than <NUM> million tons of carrots were produced (ref. As is the case with any crop mankind is cultivating, there are also many threats to a good harvest of this crop. Many bacterial, fungal, viral and viroid diseases are known next to many insects and nematodes pests. Major bacterial and fungal diseases are caused by, among others, Xanthomonas campestris, Erwinia carotavora, Alternaria dauci, Alternaria radicina, Pythium spp. , Rhizoctonia spp. , Sclerotinia spp. , Fusarium spp, Botrytis cinerea and Phytophthora spp. Nematodes as Heterodera carotae, Meloidogyne spp. and Pratylenchus spp. cause severe damage to the taproot, resulting in yield loss and a product which is unsuitable for marketing. Further a vast array of viruses and viroids is known to have an adverse effect on plant health and yield of carrot.

The reduction in yield of carrot, caused by these pathogens, has led to dedicated breeding programs executed by companies and governmental institutes to introduce resistances against these pathogens. One of the diseases having a major effect on carrot cultivation is Erysiphe heraclei causing powdery mildew. Erysiphe heraclei belongs to the Ascomyceta, order Erysiphales and causes the foliar disease powdery mildew on several members of the Umbelliferae (ref. <NUM>, <NUM>). heraclei, several formae speciales are known which, in general, are specific to the various genera within this family.

Once infested with Erysiphe heraclei, carrot plants become covered with fungus and spores. Haustoria are applied by the fungus to penetrate the plant cells but do not cross the cell membrane; therefore the fungus is present intracellularly. Through these haustoria nutrients and water are absorbed from the extracellular space of the infected plant. Patches of the fungus appear on the lower leaves first, later spreading to the higher plant parts. These blotches spread out to a general colonization of the plant including, if present, the flower stalk. The disease is most severe at temperatures as found in summer and autumn. Severe infection results in loss of foliage, a reduced yield and, in seed crops, a poor seed quality. A yield loss of <NUM> % is not rare. Under moist conditions infected tissue could easily be invaded by other (secondary) pathogens causing a rapid collapse of the foliage. Due to loss of foliage, the affected crop cannot be harvested properly since modern top lifters pull the carrots from the ground by their foliage.

One approach to avoid the effects of an infection by Erysiphe heraclei can be the application of fungicides. However, the use of pesticides in general is more and more restrained and also public awareness is in favour of avoiding the application of these compounds. Moreover, organic growers do not apply fungicides in their cultivation. Accordingly, there is a clear need in the art to provide carrots which are resistant against Erysiphe heraclei.

In research, conducted by a Japanese institute, an attempt to introduce resistance against Erysiphe heraclei was made by a transgenic approach, utilizing a human lysozyme gene under control of the constitutive CaMV <NUM> promoter. Some transformants showed an improved resistance to Erysiphe heraclei. This resistance was confirmed in the offspring (ref.

It is an object of the present invention to partially, if not completely, provide a solution for the above problems of the art with genetic material from Daucus germplasm. Specifically, it is an object of the present invention amongst other objects, to provide carrot, or Daucus carota, plants being resistant to Erysiphe heraclei or to powdery mildew.

The above objects, amongst other objects, are met by providing plants as outlined in the appended claims.

Specifically, the above objects, amongst other objects are, according to a first aspect of the present invention, met by providing Daucus carota plants being resistant against powdery mildew caused by the plant pathogen Erysiphe heraclei, wherein the resistance is provided by a first resistance gene, or Eh <NUM>, located on chromosome <NUM> of the plant between SEQ ID No. <NUM>, also designated herein as <NUM>, and SEQ ID No. <NUM> also designated herein as <NUM>.

In the alternative, the above objects, amongst other objects are, according to a first aspect of the present invention, met by providing Daucus carota plants being resistant against powdery mildew caused by the plant pathogen Erysiphe heraclei, wherein the resistance is provided by a first resistance gene, or Eh <NUM>, located on chromosome <NUM> of the plant between any one of the molecular markers identified herein as <NUM>, <NUM>, <NUM>, <NUM> or <NUM> on one side and any one of the molecular markers identified herein as <NUM>, <NUM>, <NUM>, <NUM> or <NUM> on the other side.

In the alternative, the above objects, amongst other objects are, according to a first aspect of the present invention, met by providing Daucus carota plants being resistant against powdery mildew caused by the plant pathogen Erysiphe heraclei, wherein the resistance is provided by a first resistance gene, or Eh <NUM>, located on chromosome <NUM> of the plant between any one of SEQ ID Nos. <NUM>, <NUM>, <NUM>, or <NUM> on one side and any one of SEQ ID Nos. <NUM>, <NUM>, or <NUM> on the other side.

The genome of Daucus carota has been (partially) sequenced (ref. <NUM>) and this sequence is publically available at NCBI with sequence identification number PRJNA268187 (ref. In the sequence of PRJNA268187, the present first resistance gene, or Eh <NUM>, can be found between positions <NUM>,<NUM>,<NUM> and <NUM>,<NUM>,<NUM> of chromosome <NUM>. Using the sequences presented herein, a skilled person can readily identify the present first resistance gene, or Eh <NUM>, in other publically available sequence information of the genome of Daucus carota, and especially chromosome <NUM> thereof, such as between the position of SEQ ID No. <NUM> and SEQ ID No. <NUM> thereon. In the alternative the present first resistance gene, or Eh <NUM>, can be found between any one of SEQ ID Nos. <NUM>, <NUM>, <NUM>, or <NUM> on one side and any one of SEQ ID Nos. <NUM>, <NUM>, or <NUM> on the other side.

According to a preferred embodiment of this first aspect of the invention, in the present Daucus carota plants, the resistance is further provided by a second resistance gene, or Eh <NUM>, located on chromosome <NUM> of the plant between SEQ ID No. <NUM>, also designated herein as <NUM>, and SEQ ID No. <NUM> also designated herein as <NUM>.

In the alternative of this preferred embodiment of the present invention, the further resistance is provided by a second resistance gene, or Eh <NUM>, located on chromosome <NUM> of the plant between any one of the molecular markers identified herein as <NUM>, <NUM>, <NUM>, <NUM> or <NUM> on one side and any one of the molecular markers identified herein as <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or <NUM> on the other side.

In the alternative of this preferred embodiment of the present invention, the further resistance is provided by a second resistance gene, or Eh <NUM>, located on chromosome <NUM> of the plant between any one of SEQ ID Nos. <NUM>, <NUM>, <NUM>, or <NUM> on one side and any one of SEQ ID Nos. <NUM>, <NUM>, <NUM>, or <NUM> on the other side.

The genome of Daucus carota has been (partially) sequenced and this sequence is publically available at NCBI with sequence identification number PRJNA268187 (ref. In the sequence of PRJNA268187, the present second resistance gene, or Eh <NUM>, can be found between positions <NUM>,<NUM>,<NUM> and <NUM>,<NUM>,<NUM> of chromosome <NUM>. Using the sequences presented herein, a skilled person can readily identify the present second resistance gene, or Eh <NUM>, in other publically available sequence information of the genome of Daucus carota, and especially chromosome <NUM> thereof, such as between the position of SEQ ID No. <NUM> and SEQ ID No. <NUM> thereon. In the alternative the present second resistance gene, or Eh <NUM>, can be found between any one of SEQ ID Nos. <NUM>, <NUM>, <NUM>, or <NUM> on one side and any one of SEQ ID Nos. <NUM>, <NUM>, <NUM>, or <NUM> on the other side.

According to another preferred embodiment, the present first resistance gene, or Eh <NUM>, is located on chromosome <NUM> at <NUM> and the present second resistance gene, or Eh <NUM>, is located on chromosome <NUM> at <NUM>. As noted above, these chromosome positions in centimorgans correspond to, in the sequence of PRJNA268187, between positions <NUM>,<NUM>,<NUM> and <NUM>,<NUM>,<NUM> and between <NUM>,<NUM>,<NUM> and <NUM>,<NUM>,<NUM> of chromosome <NUM>, respectively.

The present first resistance gene, or Eh <NUM>, is obtainable, obtained or derived from seeds of a Daucus carota plant deposited on March <NUM>, <NUM> under deposit number NCIMB <NUM> (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom). Formulated differently, the present first resistance gene, or Eh <NUM>, is, preferably, the resistance gene to be found in seed deposit NCIMB <NUM>.

The present second resistance gene, or Eh <NUM>, is obtainable, obtained or derived from seeds of a Daucus carota plant deposited on April <NUM>, <NUM> under deposit number NCIMB <NUM> (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom). Formulated differently, the present second resistance gene, or Eh <NUM>, is, preferably, the resistance gene to be found in seed deposit NCIMB <NUM>.

The present first resistance gene, or Eh <NUM>, is identifiable by at least one molecular marker selected from the group consisting of SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, and SEQ ID No. <NUM>. Formulated differently, the present first resistance gene, or Eh <NUM>, is genetically linked to at least one molecular marker selected from the group consisting of SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, and SEQ ID No. <NUM>.

The present second resistance gene, or Eh <NUM>, is identifiable by at least one molecular marker selected from the group consisting of SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, and SEQ ID No. <NUM>. Formulated differently, the present second resistance gene, or Eh <NUM>, is genetically linked to at least one molecular marker selected from the group consisting of SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, and SEQ ID No. <NUM>.

The plants as defined above are preferably hybrid plants, more preferably sterile hybrid plants and most preferably male sterile hybrid plants such as a cytoplasmic male sterile.

The present Daucus carota plants are preferably Daucus carota ssp. sativus plants.

According to another aspect, the present invention relates to seeds, edible parts pollen, egg cells, callus, suspension culture, (somatic) embryos or plant parts of a Daucus carota plant as defined above.

The present molecular markers selected from the group consisting of SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, SEQ ID No. <NUM>, and SEQ ID No. <NUM> can be used for identifying a Daucus carota plant being resistant against powdery mildew caused by the plant pathogen Erysiphe heraclei.

The present invention will be further detailed in the following examples and figures wherein:.

Figure <NUM>:  shows a schematic physical map of chromosome <NUM> of Daucus carota showing both the present molecular markers and the present first resistance gene Eh <NUM> and the present second resistance gene Eh <NUM> providing resistance to the plant pathogen Erysiphe heraclei. Figure <NUM>:  shows sequences of the molecular markers SNP markers as shown in <FIG> and their position on chromosome <NUM> of the sequence of PRJNA268187 (ref.

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.

Plants to be tested for resistance were sown in soil on tables, around <NUM> plants per row. Every <NUM> rows of plants to be assessed a row of resistant material for race <NUM> and race <NUM> and susceptible material each were inserted. When the plants were about <NUM> 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 <NUM> ± <NUM>° C at night; <NUM> ± <NUM>° C during daytime; min. <NUM> hours light (or more if day length was longer) and max. <NUM> hours dark. Humidity was kept at a high level by spraying water between the tables a few times a week. After <NUM> weeks the plants were evaluated; infected leaves were covered with a white powdery mycelium and spores, and often turn chlorotic.

The severity of infection was reflected by scoring the symptoms between <NUM> (completely susceptible) and <NUM> (completely resistant). It was carefully checked that the susceptible control plants are indeed showing the symptoms of E. heraclei infection.

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

When plants were about <NUM> 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.

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 <NUM> (completely susceptible) and <NUM> (completely resistant).

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 (ref.

At least <NUM> 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, <NUM> plants of each cross were grown in the glasshouse. From each individual plant, leaf material was used for DNA isolation and successive marker analysis.

Inbreds of selected individuals with crossovers nearby the resistance locus were tested in the greenhouse as described in example <NUM> and resistance was confirmed.

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.

Using SNP markers covering the entire genome, both resistance genes were determined to be located on chromosome <NUM>. 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 (ref.

carota accession NCIMB42389 the resistance locus (Eh <NUM>) was located on chromosome <NUM> at <NUM>, corresponding to a fragment between position <NUM>,<NUM>,<NUM> and <NUM>,<NUM>,<NUM> bp.

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 <NUM> below:
<IMG>.

The resistance locus is located between markers <NUM> and <NUM>.

Further, for D. carota accession NCIMB42397 the location of the second resistance locus (Eh <NUM>) was determined on chromosome <NUM> around <NUM>, corresponding to a fragment between positions <NUM>,<NUM>,<NUM> bp and <NUM>,<NUM>,<NUM> bp (ref.

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 <NUM> below:
<IMG>.

As graph this situation can alternatively be illustrated as in <FIG>. As is clear both from the position in cM and base pair position and illustrated by <FIG>, the present dominant resistance genes involved are located far apart on chromosome <NUM>. 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.

Claim 1:
Daucus carota plant being resistant against powdery mildew caused by the plant pathogen Erysiphe heraclei, said resistance is provided by a first resistance gene located on chromosome <NUM> of said plant between SEQ ID No. <NUM> and SEQ ID No. <NUM>;
wherein said first resistance gene is located on chromosome <NUM> at <NUM>; and
wherein said first resistance gene is obtainable from seeds of a Daucus carota plant deposited under deposit number NCIMB42389.