Downy Mildew Resistant Spinach Plant

Abstract

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 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.

Claims

1. A spinach plant being resistant to downy mildew, wherein the spinach plant comprises a downy mildew resistance providing genomic fragment from Spinacia tetrandra.

2. The spinach plant according to claim 1, wherein the genomic fragment from Spinacia tetrandra is located on chromosome 4 of the spinach plant.

3. The spinach plant according to claim 1, wherein the genomic fragment from Spinacia tetrandra is obtainable, or obtained, from a plant, representative seeds thereof have been deposited under deposit number NCIMB 43379.

4. The spinach plant according to claim 1, wherein the spinach plant comprises in its genome a genomic fragment from Spinacia 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.

5. The spinach plant according to claim 1, wherein the spinach plant not comprises in its genome 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.

6. The spinach plant according to claim 1, wherein the spinach plant is a hybrid plant.

7. The spinach plant according to claim 1, wherein the spinach plant is an inbred plant.

8. The spinach plant according to claim 1, wherein the spinach plant is Spinacia oleracea.

9. A seed, edible parts, pollen, egg cells, callus, suspension culture, somatic embryos, embryos or plant parts of a spinach plant according to claim 1, comprising a downy mildew resistance providing genomic fragment from Spinacia tetrandra

10. A method 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.

11. (canceled)

12. A method for providing spinach plant being resistant to downy mildew comprising introgressing a downy mildew resistance providing genomic fragment from Spinacia tetrandra into a spinach plant.

13. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The present invention is detailed in the following examples and figures wherein:

[0038] FIG. 1: shows a representative microscopy image of P. farinosa f. sp. spinaciae;

[0039] FIG. 2: shows a representative photograph of leaves of spinach plants infected with the downy mildew causative pathogen Peronospora farinose;

[0040] FIG. 3: shows a representative photograph of a spinach plant resistant to downy mildew.

DESCRIPTION OF THE INVENTION

Examples

Example 1. Breeding Scheme: Introducing S. tetrandra Resistance Against Downy Mildew into S. oleracea

[0041] Initially, individual Spinacia tetrandra plants 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 the Spinacia tetrandra source. Blight A is an agriculturally elite line with round and dark leaves as its characteristics.

Result F1: Blight A×CGN120251.

[0042] Subsequently, downy mildew tests were performed with different races on the S. tetrandra-S. oleracea hybrids. 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.

Result BC1: Blight A(2)×CGN120251

[0043] 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.

Result BC2: (Blight A(2)×CGN120251)×Blight C

[0044] 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 to Spinacia 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.

Result BC3: (Blight A(2)×CGN120251)×Blight C(2)

[0045] 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 with S. oleracea. Selected plants were selfed.

Result BC3S1: ((Blight A(2)×CGN120251)×Blight C(2)) S1

Example 2. Description of Spinach Downy Mildew—Peronospora farinosa—Disease Trial

[0046] Resistance to Peronospora farinosa f. sp. spinaciae (synonym P. 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.

[0047] 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 on Peronospora 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-US-00001 TABLE 1 IWGP Spinach differential set for Pfs. Where “−” is resistant, “+” is susceptible and “(−)” indicates intermediate resistance. Race Pfs Variety/NIL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Viroflay + + + + + + + + + + + + + + + + + NIL5 − − + + + + + + + + + + + + + + + NIL3 − + − + − + + − − + − − + − + − + NIL4 − − − − + + + + + + + + + + − + + NIL6 − + − − − + − + + + − + (−) + − − + NIL1 − − − − − − − + − + − + − + − − + NIL2 − − − − − − − − − − + + + + − + + Whale − − − (−) − (−) (−) − − (−) − − + − (−) − + Pigeon − − − − − − − − − − − − − + − + + Caladonia − − − − − − − − − − − − − − + − + Meerkat − − − − − − − − − − − − − − − + (−) Hydrus − − − − − − − − − − − − − − − − −

TABLE-US-00002 TABLE 2 Resistance pattern of deposit NCIMB 43379. Where “−” is resistant and “+” is susceptible and “n.t.” is not tested. Race Pfs Variety 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Deposit 43379 n.t. n.t. n.t. − n.t. n.t. − n.t. − − − − − − − − −

Example 3. Novel Resistance Against Downy Mildew from Spinacia tetrandra-Marker Development

[0048] In the BC2 population derived from S. oleracea and S. tetrandra crosses 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-US-00003 TABLE 3 Number of plants showing correlation between disease scores and the genotype of a SNP in a BC2 population derived from S. oleracea and S. tetrandra. SNP* SNP* heterozygous homozygous (S. oleracea and Disease score Pfs11 (S. oleracea) S. tetrandra) Resistant 6 16 Susceptible 20 2 *chromosome 4 position 11,627,232 bp.

[0049] 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-US-00004 TABLE 4 Number of plants showing correlation between disease scores and the genotype of a genomic region in a BC3 population derived from S. oleracea and S. tetrandra. Downy Resistant Susceptible mildew homozygous homozygous Resistant Susceptible isolate S. oleracea* S. oleracea* heterozygous* Heterozygous* Pf4 1 7 9 0 Pf7 0 7 12 0 Pf9 0 5 11 0 Pf10 0 8 7 0 Pf12 0 7 9 0 Pf13 0 10 9 0 Pf14 0 11 7 0 Pf15 0 12 7 0 Pf16 0 10 6 0 Pf17 0 11 4 0 Total 1 88 81 0 *at identified genomic region.

[0050] The region co-segregating with the novel resistance from S. tetrandra is 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-US-00005 TABLE 5 SNPsfor the detection of the resistance against Peronospora farinosa Position Position Allele linked Alternative SNP Chromosome Chromosome* (bp) Scaffold Scaffold (bp) to resistance allele 1 chr4 8,104,487 000037 700,416 T A 2 chr4 8,204,126 000037 800,055 G A 3 chr4 8,305,539 000037 901,468 A T 4 chr4 8,500,200 000037 1,096,129 C T 5 chr4 8,502,319 000037 1,098,248 A C 6 chr4 8,502,334 000037 1,098,263 A G 7 chr4 8,502,394 000037 1,098,323 A G 8 chr4 8,508,716 000037 1,104,645 G C 9 chr4 8,508,834 000037 1,104,763 A G 10 chr4 8,508,984 000037 1,104,913 T C 11 chr4 8,508,996 000037 1,104,925 C T 12 chr4 8,509,556 000037 1,105,485 T A 13 chr4 8,509,737 000037 1,105,666 T C 14 chr4 8,510,680 000037 1,106,609 T G 15 chr4 8,510,715 000037 1,106,644 G C 16 chr4 8,510,919 000037 1,106,848 C G 17 chr4 8,510,962 000037 1,106,891 C T 18 chr4 8,804,303 000037 1,400,232 C G *The reference genome is : Xu, C., et al., Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions, Nature Communications 2017.

TABLE-US-00006 Genetic position* SEQ ID on Chr 4 Sequence No. (bp) -SNP nucleotide is highlighted bold and in brackets SEQ ID 8,104,487 AGGGAATACTGGATTGTTGGAAGCGTATAAATTGTCAAGGMCCACAACAA[T] No. 1 TATATGCCCACGAAAATAAACGCAAAAGTGATAACTGAATACTTTTCTAC SEQ ID 8,104,487 AGGGAATACTGGATTGTTGGAAGCGTATAAATTGTCAAGGMCCACAACAA[A] No. 2 TATATGCCCACGAAAATAAACGCAAAAGTGATAACTGAATACTTTTCTAC SEQ ID 8,204,126 ATTCAAATCCAAATCAAATCCAGAAAAATTCAAAAAAAAAATACAAAATC[G] No. 3 ACGCAGAAAACTTACAGAGAATGTCGCCGACGATTGACGCTGAAAATGCG SEQ ID 8,204,126 ATTCAAATCCAAATCAAATCCAGAAAAATTCAAAAAAAAAATACAAAATC[A] No. 4 ACGCAGAAAACTTACAGAGAATGTCGCCGACGATTGACGCTGAAAATGCG SEQ ID 8,305,539 ATTTCTACCTTTTTTACGTACCACAATTCCACAAACTTGTGTATTTATAT[A] No. 5 AACACTTTGGTCACACATTTCAACTACATTATTGCATTTCATTTCAATAY SEQ ID 8,305,539 ATTTCTACCTTTTTTACGTACCACAATTCCACAAACTTGTGTATTTATAT[T] No. 6 AACACTTTGGTCACACATTTCAACTACATTATTGCATTTCATTTCAATAY SEQ ID 8,500,200 AGTTTTCTCACCTGAGCTCTGATGATAGCACTTTTCTTGTCTTGCTCAGC[C] No. 7 TTTTCCACAATAAACTTGGCTCTTTCAGCATCTTGTGCAGCTACCTGTTT SEQ ID 8,500,200 AGTTTTCTCACCTGAGCTCTGATGATAGCACTTTTCTTGTCTTGCTCAGC[T] No. 8 TTTTCCACAATAAACTTGGCTCTTTCAGCATCTTGTGCAGCTACCTGTTT SEQ ID 8,502,319 TCCTTGATACCAGAGATGCGGTTGAAGACGATAGCTCGATGACCACCTTC[A] No. 9 ACATTGTACATACTRTGACTGACTCCATAAGCGGCAAGGCCAAGAGCACC SEQ ID 8,502,319 TCCTTGATACCAGAGATGCGGTTGAAGACGATAGCTCGATGACCACCTTC[C] No. 10 ACATTGTACATACTRTGACTGACTCCATAAGCGGCAAGGCCAAGAGCACC SEQ ID 8,502,334 ATGCGGTTGAAGACGATAGCTCGATGACCACCTTCMACATTGTACATACT[A] No. 11 TGACTGACTCCATAAGCGGCAAGGCCAAGAGCACCAAGAATTCCAACCTT SEQ ID 8,502,334 ATGCGGTTGAAGACGATAGCTCGATGACCACCTTCMACATTGTACATACT[G] No. 12 TGACTGACTCCATAAGCGGCAAGGCCAAGAGCACCAAGAATTCCAACCTT SEQ ID 8,502,394 CCATAAGCGGCAAGGCCAAGAGCACCAAGAATTCCAACCTTAACCARATT[A] No. 13 GCAGCAGCGCCACCGCCGCCACCAGGCATCTTGGGAACCTTAACGTTTTG SEQ ID 8,502,394 CCATAAGCGGCAAGGCCAAGAGCACCAAGAATTCCAACCTTAACCARATT[G] No. 14 GCAGCAGCGCCACCGCCGCCACCAGGCATCTTGGGAACCTTAACGTTTTG SEQ ID 8,508,716 AGATTTCTGATTTCCTGTAGTTGGGAGTTTGATTCTTCCAGTTTCGGACA[G] No. 15 AGAGGAAAATTTCTCAAATTATCACAAAATGTAATTAACAGATGAGAAAG SEQ ID 8,508,716 AGATTTCTGATTTCCTGTAGTTGGGAGTTTGATTCTTCCAGTTTCGGACA[C] No. 16 AGAGGAAAATTTCTCAAATTATCACAAAATGTAATTAACAGATGAGAAAG SEQ ID 8,508,834 AATGTGWTTGAAACCCTGCTACTTCTCGTGTCTCACCCATATCTGATTCT[A] No. 17 ATTTCCACCATCCTTCCAACTTTGGCATACTCCAAAGCTCAAGCTTTTCA SEQ ID 8,508,834 AATGTGWTTGAAACCCTGCTACTTCTCGTGTCTCACCCATATCTGATTCT[G] No. 18 ATTTCCACCATCCTTCCAACTTTGGCATACTCCAAAGCTCAAGCTTTTCA SEQ ID 8,508,984 CTCCTGATGTTGATGCAACCCCTTCTGCGCTGATAGTACTATTCTCCATA[T] No. 19 ACACCACTTCAYTCAAATATCGTAATGTAAGGAATTTCAAATGACGCAGT SEQ ID 8,508,984 CTCCTGATGTTGATGCAACCCCTTCTGCGCTGATAGTACTATTCTCCATA[C] No. 20 ACACCACTTCAYTCAAATATCGTAATGTAAGGAATTTCAAATGACGCAGT SEQ ID 8,508,996 ATGCAACCCCTTCTGCGCTGATAGTACTATTCTCCATAYACACCACTTCA[C] No. 21 TCAAATATCGTAATGTAAGGAATTTCAAATGACGCAGTTGACTCATCAAT SEQ ID 8,508,996 ATGCAACCCCTTCTGCGCTGATAGTACTATTCTCCATAYACACCACTTCA[T] No. 22 TCAAATATCGTAATGTAAGGAATTTCAAATGACGCAGTTGACTCATCAAT SEQ ID 8,509,556 GGCATATGYGACAAACTCTTACAGCCTTGTATATCTAGGTGCCTTAGATT[T] No. 23 ACTAATTTCCTCAAATCGCTTGGCAAAGTTTTTAGACCACGACACTCACG SEQ ID 8,509,556 GGCATATGYGACAAACTCTTACAGCCTTGTATATCTAGGTGCCTTAGATT[A] No. 24 ACTAATTTCCTCAAATCGCTTGGCAAAGTTTTTAGACCACGACACTCACG SEQ ID 8,509,737 GATATCTTAGATGCAACAAGTTACCTATCTCACTCGGCAACATTTCCAAA[T] No. 25 CAGAGTTATTCAAGCTTAAGACCCTTAAATATTCCACTTTTGATAACAAA SEQ ID 8,509,737 GATATCTTAGATGCAACAAGTTACCTATCTCACTCGGCAACATTTCCAAA[C] No. 26 CAGAGTTATTCAAGCTTAAGACCCTTAAATATTCCACTTTTGATAACAAA SEQ ID 8,510,680 TACATCATCCAAAACAAGGAGGTATATCTTCCCATCCAGTTCGTCCCGAA[T] No. 27 TTTCATCTGAAGCTGGTCCATACTCATACTCAGGSCATCGTGCTTTCTTC SEQ ID 8,510,680 TACATCATCCAAAACAAGGAGGTATATCTTCCCATCCAGTTCGTCCCGAA[G] No. 28 TTTCATCTGAAGCTGGTCCATACTCATACTCAGGSCATCGTGCTTTCTTC SEQ ID 8,510,715 CCAGTTCGTCCCGAAKTTTCATCTGAAGCTGGTCCATACTCATACTCAGG[G] No. 29 CATCGTGCTTTCTTCTAGGGTTTACGGCTGCAAGGATTTCAGCAACAAGA SEQ ID 8,510,715 CCAGTTCGTCCCGAAKTTTCATCTGAAGCTGGTCCATACTCATACTCAGG[C] No. 30 CATCGTGCTTTCTTCTAGGGTTTACGGCTGCAAGGATTTCAGCAACAAGA SEQ ID 8,510,919 CCAGCCCCCCAATCCCCACTATGCTGACAACAGAAATATCCTTCTCCTCA[C] No. 31 AAGAAGTGTTTAGTACCATATCTATCACTTTCCGCTTGTCATYATCCCTC SEQ ID 8,510,919 CCAGCCCCCCAATCCCCACTATGCTGACAACAGAAATATCCTTCTCCTCA[G] No. 32 AAGAAGTGTTTAGTACCATATCTATCACTTTCCGCTTGTCATYATCCCTC SEQ ID 8,510,962 CTCCTCASAAGAAGTGTTTAGTACCATATCTATCACTTTCCGCTTGTCAT[C] No. 33 ATCCCTCCCAATGATCATATCTTCCTCGCAAACAAAGGAATGAGTTTCCC SEQ ID 8,510,962 CTCCTCASAAGAAGTGTTTAGTACCATATCTATCACTTTCCGCTTGTCAT[T] No. 34 ATCCCTCCCAATGATCATATCTTCCTCGCAAACAAAGGAATGAGTTTCCC SEQ ID 8,804,303 TAATTGATGATTTGAAGATTTATTACTACAACRCAATGGATTTTGTTTTG[C] No. 35 AATTATGCATTAAGAAGAGACTCRCATTTTTCATATTGGTGTATGGTAGT SEQ ID 8,804,303 TAATTGATGATTTGAAGATTTATTACTACAACRCAATGGATTTTGTTTTG[G] No. 36 AATTATGCATTAAGAAGAGACTCRCATTTTTCATATTGGTGTATGGTAGT *The reference genome is Xu, C., et al., Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions, Nature Communications 2017. Odd sequences = linked to resistance, Even sequences = alternative allele.