Downy Mildew Resistant Spinach Plant

The present invention relates to downy mildew, and especially downy mildew caused by the plant pathogen Peronospora farinosa, resistant spinach plants (Spinacia oleracea). The present spinach plants include a downy mildew resistance providing genomic fragment from Spinacia tetrandra. Specifically, the present invention relates to spinach plants being resistant to downy mildew, wherein the spinach plant includes a downy mildew resistance providing genomic fragment from Spinacia tetrandra such as spinach plants including in their genomes one or more nucleic acid sequences selected from the groups consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No. 35.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to downy mildew, and especially downy mildew caused by the plant pathogenPeronospora farinosa, resistant spinach plants (Spinacia oleracea). The present spinach plants comprise a downy mildew resistance providing genomic fragment fromSpinacia tetrandra. The present invention further relates to seeds and plant parts of the present plants, methods for providing, or identifying, the present plants and downy mildew resistance providing nucleic acid sequences.

Description of Related Art

Spinach is commercially grown worldwide for its attractive and nutritious leaves. In 2017, production of spinach was close to 28 million tons worldwide. Spinach (Spinacia oleracea) is a member of the Amaranthaceae family, subfamily Chenopodioideae. Other well-known family members include such plants as quinoa and beet. The latter is a cultivated plant of big importance for agriculture with sugar beet, red beet and Swiss chard as examples.

Regarding nutritional value, while providing only a small amount of calories (only 23 for 100 grams of cooked spinach), spinach is a rich source of vitamins A, B2 (or folate), B6, C, E and K; magnesium, manganese, calcium, potassium, iron and dietary fibre.

Spinach flowering is induced by (long) day length and under optimal conditions can reach even up to 4 generations in a year with a life cycle from seed to new harvest completed within 3 months. A bottleneck can be caused by seed dormancy.

Spinach is a wind pollinator and its pollen can reach far. A line is considered male if it converts from female or mixed flowering to (all) male flowering within a week. Female lines stay so for at least three weeks without producing any pollen. Hybrids of spinach can readily be produced making use of plants which have a female flowering phase and plants which have a male flowering phase as pollinator. Before the female plants develop male flowers, all female flowers are fertilized by the male plant. The setting of seeds occurs rapidly within 3 days and after that the ripening of the seed takes approximately a month.

Under optimal conditions commercial elite spinach lines are grown and harvested within 25 days to obtain baby leaf spinach.

Breeding resulted in spinach plants which are rapid growing without premature flowering. Older varieties tend to have narrower leaves and have a stronger, somewhat bitter taste; newer varieties have broader leaves and a milder taste. Also, recent types have little tendency for bolting in warm conditions and therefore will not prematurely flower and produce seeds.

Spinach is cultivated for the leaves. Commercial spinach can have round leaves of dark green color. The leaf morphology is of interest to spinach breeders. A significant share of the market of cultivated spinach is the early harvested baby leaf spinach. For spinach growers it is important that the leaves stand straight up which facilitates easy harvest, dark green colour is desired.

Spinach originates from middle Asia but it is now produced all over the world. Traditional areas where spinach was grown as a crop are Europe and Northern America, however contemporarily the biggest volume of spinach is produced in China. Spinach is produced for the food processing industry (canned or frozen spinach) as well as for the fresh market, where especially baby leaf spinach is in demand. Breeders develop lines with characteristics best suited for the location or the purpose.

An important development in the production and sales of fresh spinach was the introduction of bagged spinach. For this application the desired leaf morphology is such that the leaves are not too closely packed together that are found in the partly savoyed types.

Basic types of spinach are on the market:A savoy type with dark green, curly and crinkly leaves (for the fresh market);A flat, or smooth, leaf spinach with broad, smooth leaves that can be cleaned easily. This type is used for industry (canned or frozen spinach, as well as processed food and baby food;Semi savoy is an intermediate type of spinach with a comparable texture as the savoy type but easy to clean as the smooth type of spinach. It is cultivated both for fresh market and industry.An oriental type which is heat tolerant, has long petioles, pointed leaves with several side lobes and as plant has an upright growth.

Most spinach is produced at high plant density for fresh market production which creates the ideal environment for disease development. Additionally, there is an increasing demand to produce organic vegetables. Consumers are looking for vegetables that are obtained without the use of pesticides, fungicides, insecticides and without chemical treatment of the seed. The challenge here is that such production conditions often lead to the development of plant disease. This creates a need for spinach cultivars that encompass natural, genetically encoded resistance against pathogens.

The most common pathogens causing diseases of spinach arePeronospora, Fusarium, Stemphyllium, Colletotrichum, Cercosporaand Cucumber Mosaic Virus. The most important disease in spinach is downy mildew caused by the oomycete pathogenPeronospora effusa(=P. farinosa f.sp.spinaciae[hereafter Pfs]). The short lifecycle of Pfs results in rapid multiplication of the pathogen on susceptible cultivars. At first, small pale yellow irregular spots appear on the upper surface of the leafs and a purple downy growth on the lower surface of the spots. Spores develop on the leaves 9-12 days after first infection and are spread by wind and splashes of water. Infected leafs are no longer attractive for consumption and prone to other, secondary (microbial) infections.

One way to combat downy mildew is to spray the plants with fungicide. This approach is highly undesirable due to its heavy impact on the environment and because it is cost and labour intense. Moreover, half of the agriculturally produced spinach is meant for the organic market and this approach is not suitable for this application. There is thus a strong need in the field for spinach with resistance against the pathogen.

Peronospora farinosais a pathogen that rapidly overcomes resistance. Within 2 to 3 years newly introduced resistance genes can be broken by the pathogen and therefore there is a constant demand to identify new resistance sources. Seventeen official races have been described by the International Working group onPeronospora effusa/farinosa/Pfs (IWGP). Since only a limited set of Resistance toPeronospora(RPF) genes have been described that originate fromS. oleracea, wild relatives are a potential interesting source of novel and alternative RPF genes.

Spinacia tetrandraandSpinacia turkestanicaare wild relatives of the contemporary spinach. It is possible to crossSpinacia oleraceawith these wild relatives and as such they can be important sources of genetically encoded resistance to plant pathogens. Morphologically they resemble ancient spinachSpinacia oleracea. They are also either male or female with pointy leaves with sharp angles.

Considering the above, there is a need in the art for novel resistance sources providing resistance to downy mildew.

SUMMARY OF THE INVENTION

It is an object of present invention to provide a solution for the above problems and needs of the art.

This object of the present invention, amongst other objects, is met as outlined in the appended claims.

Specifically, this object of the present invention, amongst other objects is met, according to a first aspect of the present invention by spinach plants being resistant to downy mildew, wherein the spinach plants comprise a downy mildew resistance providing genomic fragment fromSpinacia tetrandra.

The present inventors have surprisingly discovered that a resistance in a wild relative of spinach could be successfully transferred to commercial spinach plants thereby providing downy mildew resistance to these plants.

According to a preferred embodiment, the present invention relates to spinach plants, wherein the genomic fragment fromSpinacia tetrandrais located on chromosome 4 of the spinach plant.

According to another preferred embodiment, the present invention relates to a spinach plant, wherein the genomic fragment fromSpinacia tetrandrais obtainable, or obtained, from a plant, representative seeds thereof have been deposited on 4 Apr. 2019 under deposit number NCIMB 43379 (National Collection of Industrial Food and Marine Bacteria, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK).

According to an especially preferred embodiment, the present spinach plants comprise in their genomes a genomic fragment fromSpinacia tetrandra, the genomic fragment comprises one or more nucleic acid sequences selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No. 35.

According to another especially preferred embodiment, the present spinach plants do not comprise in their genomes a genomic fragment comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No.28, SEQ ID No. 30, SEQ ID No. 32, SEQ ID No. 34 and SEQ ID No. 36.

The present spinach plants preferably are hybrids or inbred plants.

According to the present invention, the present spinach plants are preferablySpinacia oleraceaplants.

According to a second aspect, the present invention relates to seeds, edible parts, pollen, egg cells, callus, suspension culture, somatic embryos, embryos or plant parts of the spinach plants defined above comprising a downy mildew resistance providing genomic fragment fromSpinacia tetrandra.

According to a third aspect, the present invention relates to methods for identifying a spinach plant being resistant to downy mildew, the method comprises the step of establishing the presence of a genomic fragment comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No. 35 in the genome of the spinach plant.

According to a fourth aspect, the present invention relates to the use of one or more genomic DNA sequences selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No. 35 for identifying or providing a spinach plant being resistant to downy mildew.

According to a fifth aspect, the present invention relates to methods for providing the present spinach plant being resistant to downy mildew, wherein the methods comprise introgressing a downy mildew resistance providing genomic fragment fromSpinacia tetrandrainto a spinach plant.

According to a sixth aspect, the present invention relates to nucleic acid sequences selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33 and SEQ ID No. 35.

DESCRIPTION OF THE INVENTION

EXAMPLES

Initially, individualSpinacia tetrandraplants from CGN number CGN120251 were crossed with Blight A (S. oleracea). The latter is a female line which does not harbor any downy mildew resistance and therefore resistance in the offspring has to originate from theSpinacia tetrandrasource. Blight A is an agriculturally elite line with round and dark leaves as its characteristics.

Subsequently, downy mildew tests were performed with different races on theS. tetrandra,-S. oleraceahybrids. Per hybrid population, resistant and vital plants were put together in a pollen-tight bag with Blight A. The hybrid-plants were female and therefore it was necessary to wait for the pollen production of Blight A. In this generation Blight A was used as a father instead of being used as a mother. The seeds were harvested in bulk from the hybrid plants.

The seeds harvested from the hybrid were prickly. At first, the BC1 seeds did not germinate at all even when the embryos were grown in vitro. To overcome the dormancy the seeds were first incubated at 4° C. Hereafter, the pots were kept at 20° C. and the seeds were germinating. Subsequently, a Pfs race 11 downy mildew disease test was performed on the plants. The resistant plants were selected and individually crossed with Blight C (S. oleracea). Blight C is a male line that also lacks mildew resistance.

To continue backcrossing, BC2 seed lots were selected on seed smoothness and yield. On the selected BC2 lots, another Pfs race 11 downy mildew test was performed. Resistant plants were selected and after that genotyped. Plants with highest resemblance toSpinacia oleracea(originating from Blight A or C) were selected. These plants were individually crossed with Blight A or C depending on the flowering behaviour of the BC2. In this example Blight C was used in this cross. Seeds were harvested from the backcross plant.

A Pfs strain 11 downy mildew test was performed on the BC3 plants. Resistant plants were genotyped to be able to select plants with the highest resemblance withS. oleracea. Selected plants were selfed.

Description of Spinach Downy Mildew—Peronospora farinosa—Disease Trial

Resistance toPeronospora farinosa f. sp.spinaciae(synonymP. effusa[hereafter Pfs]) is tested in a qualitative disease assay. In short, 10 to 14 days after untreated seed is sown in soil, a minimum of 8 plants is inoculated with a spore suspension of a single Pfs race or isolate. Pfs is maintained on a living susceptible host plant e.g. Viroflay or Blight or plant material with spores is stored for a maximum of 1 year at −20° C. Inoculated plants are incubated under plastic at high humidity (80-100%) and at a temperature ranging from 16° C.-20° C. After 24 hours plastic is removed, plants are assessed at 9 to 12 days after inoculation. When sporulation is observed on the cotyledons or true leaves a plant is considered susceptible and when no sporulation is observed a plant is considered resistant.

A differential set as described in Table 1 is included in each disease trial under the same environmental conditions to confirm the race. This differential set for Pfs was developed by the International Working Group onPeronospora farinosa(IWGP) and can be found on the website of the International Seed Federation (ISF). This differential set that consists of spinach varieties and near-isogenic lines (NILs) is used to determine the Pfs race. In this table “−” indicates resistance (no sporulation), “+” indicates susceptibility (sporulation), “(−)” indicates intermediate resistance (sparse sporulation on the tips of cotyledons), “n.t.” indicates that the current strain was not tested. Seeds of this differential set and Pfs races can be obtained at Naktuinbouw (P.O. Box 40, NL-2370 AA, Roelofarendsveen, Netherlands, naktuinbouw.com).

TABLE 1IWGP Spinach differential set for Pfs. Where “−” is resistant,“+” is susceptible and “(−)” indicates intermediate resistance.Race PfsVariety/NIL1234567891011121314151617Viroflay+++++++++++++++++NIL5−−+++++++++++++++NIL3−+−+−++−−+−−+−+−+NIL4−−−−++++++++++−++NIL6−+−−−+−+++−+(−)+−−+NIL1−−−−−−−+−+−+−+−−+NIL2−−−−−−−−−−++++−++Whale−−−(−)−(−)(−)−−(−)−−+−(−)−+Pigeon−−−−−−−−−−−−−+−++Caladonia−−−−−−−−−−−−−−+−+Meerkat−−−−−−−−−−−−−−−+(−)Hydrus−−−−−−−−−−−−−−−−−

TABLE 2Resistance pattern of deposit NCIMB 43379. Where “−”is resistant and “+” is susceptible and “n.t.” is not tested.Race PfsVariety1234567891011121314151617Deposit 43379n.t.n.t.n.t.−n.t.n.t.−n.t.−−−−−−−−−

Novel Resistance Against Downy Mildew FromSpinacia tetrandra—Marker Development

In the BC2 population derived fromS. oleraceaandS. tetrandracrosses 56 Single Nucleotide Polymorphisms (SNPs) informative between both parents were used to genotype the population. The 56 SNPs were distributed across all six chromosomes. A correlation was found between the disease score and a SNP on chromosome 4 (Table 3). The correlation was not absolute, showing the identified SNP did not fully segregate with the resistance.

TABLE 3Number of plants showing correlation between disease scores and thegenotype of a SNP in a BC2 population derived fromS. oleraceaandS. tetrandra.SNP*SNP* heterozygoushomozygous(S. oleraceaandDisease score Pfs11(S. oleracea)S. tetrandra)Resistant616Susceptible202*chromosome 4 position 11,627,232 bp.

The genomic region identified in the BC2 was genotyped for additional SNPs informative between both original parents. Resistant plants, heterozygous for the identified SNP, were selected and used to create BC3 populations. One BC3 population was challenged with several downy mildew isolates. A genomic region close to the SNP identified in the BC2 population segregated well with the disease scores (Table 4).

TABLE 4Number of plants showing correlation between disease scores and thegenotype of a genomic region in a BC3 population derived fromS. oleraceaandS. tetrandra.DownyResistantSusceptiblemildewhomozygoushomozygousResistantSusceptibleisolateS. oleracea*S. oleracea*heterozygous*Heterozygous*Pf41790Pf707120Pf905110Pf100870Pf120790Pf1301090Pf1401170Pf1501270Pf1601060Pf1701140Total188810*at identified genomic region.

The region co-segregating with the novel resistance fromS. tetrandrais located on chromosome 4 between 8.0 and 8.9 Mbp and can be identified with several nucleotide sequences (Table 5). Abbreviations are according to IUPAC Nucleotide code.

TABLE 5SNPsfor the detection of the resistance against Peronospora farinosaPositionPositionAlleleChromosome*Scaffoldlinked toAlternativeSNPChromosome(bp)Scaffold(bp)resistanceallele1chr48,104,487000037700,416TA2chr48,204,126000037800,055GA3chr48,305,539000037901,468AT4chr48,500,2000000371,096,129CT5chr48,502,3190000371,098,248AC6chr48,502,3340000371,098,263AG7chr48,502,3940000371,098,323AG8chr48,508,7160000371,104,645GC9chr48,508,8340000371,104,763AG10chr48,508,9840000371,104,913TC11chr48,508,9960000371,104,925CT12chr48,509,5560000371,105,485TA13chr48,509,7370000371,105,666TC14chr48,510,6800000371,106,609TG15chr48,510,7150000371,106,644GC16chr48,510,9190000371,106,848CG17chr48,510,9620000371,106,891CT18chr48,804,3030000371,400,232CG*The reference genome is : Xu, C., et al., Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions, Nature Communications 2017.