Spinach Plants Resistant to Peronospora Farinosa and Stemphylium Vesicarium

Abstract

Provided herein are spinach plants resistant to Peronospora farinosa and Stemphylium vesicarium, and spinach plants additionally resistant to Cucumber Mosaic Virus (CMV). Also provided herein are genomic fragments providing the present resistances and use thereof for identifying spinach resistant to Peronospora farinosa and Stemphylium vesicarium, and spinach plants additionally resistant to Cucumber Mosaic Virus (CMV). Provided herein are a spinach plant resistant to Peronospora farinosa and Stemphylium vesicarium: including a genomic fragment of Spinacia tetrandra located on chromosome 4 between positions 8255074 and 8620598 of the spinach reference genome, the genomic fragment of Spinacia tetrandra lacking a lethal factor and providing Peronospora farinosa resistance and including a genomic fragment located on chromosome 3 and between positions 1177586 and 1271037 of the spinach reference genome providing Stemphylium vesicarium resistance.

Claims

1. A spinach plant resistant to Peronospora farinosa and Stemphylium vesicarium, comprising a genomic fragment of Spinacia tetrandra located on chromosome 4 of said spinach plant and between positions 8255074 and 8620598 of a spinach reference genome, said genomic fragment of Spinacia tetrandra lacking a lethal factor and providing Peronospora farinosa resistance; and comprising a genomic fragment located on chromosome 3 and between positions 1177586 and 1271037 of the spinach reference genome providing Stemphylium vesicarium resistance.

2. The spinach plant according to claim 1 comprising in its genome one or more nucleotide sequences selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21.

3. The spinach plant according to claim 1, comprising in its genome one or more nucleotide sequences selected from the group consisting of SEQ ID Nos. 31, 33, 35, 37, 39, 41, and 43.

4. The spinach plant according to claim 1, comprising in its genome SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 31, 33, 35, 37, 39, 41, and 43.

5. The spinach plant according to claim 1, further comprising a genomic fragment located on chromosome 3 and between positions 1201575 and 1220905 of the spinach reference genome providing Cucumber Mosaic Virus (CMV) resistance.

6. The spinach plant according claim 5, comprising in its genome one or more nucleotide sequences selected from the group consisting of SEQ ID Nos. 23, 25, 27, and 29.

7. The spinach plant according to claim 1, comprising in its genome SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43.

8. The spinach plant according to claim 5, wherein said genomic fragment of Spinacia tetrandra providing Peronospora farinosa resistance, said genomic fragment providing Stemphylium vesicarium resistance, and said genomic fragment providing Cucumber Mosaic Virus (CMV) resistance are obtained from a spinach plant deposited under deposit number NCIMB 43676.

9. The spinach plant according to claim 1, wherein said Peronospora farinosa resistance comprises resistance to at least Peronospora farinosa isolates 1 to 17, UA2020-01E, and SP1924.

10-13. (canceled)

14. A method for identifying a spinach plant resistant to Peronospora farinosa and Stemphylium vesicarium comprising: isolating genomic DNA from said spinach plant; subjecting said genomic DNA to nucleic acid amplification using one or more nucleotide sequences selected from the group consisting of SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 17, 19 and 21 for establishing resistance to Peronospora farinosa; subjecting said genomic DNA to nucleic acid amplification using one or more nucleotide sequences selected from the group consisting of SEQ ID Nos. 31, 33, 35, 37, 39, 41, and 43 for establishing resistance to Stemphylium vesicarium; thereby identifying a spinach plant as resistant to Peronospora farinosa and Stemphylium vesicarium.

15. A plant cell, tissue, plant part or seed of a spinach plant according to claim 1.

16. (canceled)

17. The method according to claim 14, further comprising subjecting said genomic DNA to nucleic acid amplification using one or more nucleotide sequences selected from the group consisting of SEQ ID Nos. 23, 25, 27, and 29 for establishing Cucumber Mosaic Virus (CMV) resistance, thereby further identifying said spinach plant resistant to Cucumber Mosaic Virus (CMV).

18. A method of providing a spinach plant resistant to Peronospora farinosa and/or Stemphylium vesicarium, comprising introducing into the spinach plant a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 31, 33, 35, 37, 39, 41, and 43.

19. The method according to claim 18, further comprising providing resistance to Cucumber Mosaic Virus (CMV) by introducing into the spinach plant a nucleic acid sequence selected from the group consisting of SEQ ID Nos. 23, 25, 27, and 29.

Description

BRIEF DESCRIPTION OF THE INVENTION

[0055] The present invention will be further detailed in the following examples. In the examples, reference is made to figures wherein:

[0056] FIG. 1: shows a representative microscopy image of P. farinosa f sp. Spinaciae.

[0057] FIG. 2: shows a representative photograph of leaves of spinach plants infected with the downy mildew causative pathogen Peronospora farinose.

[0058] FIG. 3: shows a representative photograph of a spinach plant resistant to downy mildew.

[0059] FIG. 4: shows a representative photograph of Stemphylium conidospores.

[0060] FIG. 5: shows a representative photograph of lesions on spinach leaves caused by Stemphylium.

DESCRIPTION OF THE INVENTION

Examples

Example 1. Breeding Scheme: Introducing S. tetrandra Genomic Fragment Comprising Pathogen Resistance into S. oleracea. Removal of the Lethal Factor

[0061] Initially, individual plants of S. tetrandra were crossed with SuscA100168 (S. Oleracea). SuscA100168 is a female line which does not contain any downy mildew resistance. SuscA100168 has commercially desired characteristics like round and dark green leaves.

Result F1: SuscA100168 x S. tetrandra

[0062] Subsequently, disease assays were performed with different races of downy mildew on the hybrids. Per individual hybrid plant, resistant and vital plants were put together in a pollen-free bag together with SuscA100168. The hybrid-plants were flowering very female and therefore we waited for the pollen production of SuscA100168. In this generation we used SuscA100168 as a father, instead of a mother. The seeds were harvested in bulk from the hybrid plants.

Result BC1: SuscA100168 (2) x S. tetrandra

[0063] The seed harvested from the hybrid had the unfavorable property that all seeds harvested were sharp and formed in clusters. At first the seeds did not geminate at all, even when the embryos were grown in vitro. This is why we mechanically cut the clusters open, carefully got the individual seeds out, sowed these in normal potting soil and incubated the planted individual seeds in 4-6 degrees Celsius. Hereafter, the seeds were put at 20 degrees Celsius and the embryos were germinating. Hence, we performed downy mildew disease assay on the plants with race 11. The resistant plants were selected and crossed individually with SuscA100168 (S. oleracea).

Result BC2: SuscA100168 (3) x S. tetrandra

[0064] In the BC2, populations were selected for round seeds and the amount of seed available per cross. On the selected BC2 lots a disease assay with downy mildew race 11 was performed. Resistant plants were selected and genotyped selected for highest genetic resemblance with SuscA100168. Selected plants are individually crossed with SuscA100168.

Result BC3: SuscA100168(4) x S. tetrandra

[0065] A downy mildew disease assay with race 11 was performed on the BC3 plants. A selection was made in the resistant plants for plant that genetically resembled SuscA100168. The selected plants were selfed. In addition a second batch of seedlings was tested for resistance against race 15. Again, resistant plants genetically resembling SuscA100168 were selected for selfing.

[0066] Surprisingly, in all but one population of the BC3S1, about 25% of the plants died just after germination indicative for a lethal factor. Genetic tests showed a strong linkage between the lethality and the region of the genome that gives rise to the downy mildew resistance. This is likely because of incompatibility between the S. oleracea and S. tetrandra genome. A possible solution to overcome this lethality is reducing the size of the S. tetrandra introgression. Therefore, a plant with a unique crossover event that removes the linkage between the resistance and the lethal factor had to be identified.

[0067] Remarkably, one plant with a specific crossover event left of the resistance locus was surprisingly identified in the BC3 populations that showed resistance in a race 15 downy mildew disease assay (plant A7-13). As expected in a BC3 population, the resistance locus was heterozygous.

Result BC3S1: SuscA100168(4) x S. tetrandra c.o. S1 (=A86)

[0068] Next, 21 BC3S1 plants originating from this unique BC3 plant were tested for Pf 16 resistance. No sign of a lethal factor was observed. Overall, it can be concluded that a lethal factor was present at the beginning of chromosome 4 that is not present anymore in this lineage. Out of the 21 plant, 14 plants showed resistance. All 14 plants were selfed.

Result BC3S2: SuscA100168(4) x S. tetrandra c.o. S2 (=A172)

[0069] Genotyping the BC3S2 population showed vital plants that contained the resistance locus in a homozygous state. Offspring of one of these 14 plants had an additional crossover event right of the resistance locus, further reducing linkage drag. This population was A172-8 named Bejo T1 C0-2-1. In conclusion, the line Bejo T1 C0-2-1 comprises a genomic fragment of S. tetrandra that comprises resistance to pathogen incorporated in the genome of S. oleracea. In this line the lethal factor is no longer present. Representative seeds of Bejo T1 C0-2-1 were deposited on 28 Oct. 2020 under deposit number NCIMB 43676 (Craibstone Estate, Ferguson Building, Bucksburn, Aberdeen AB21 9YA, United Kingdom).

Example 2. Description of Spinach Downy MildewPeronospora farinosaDisease Trial

[0070] 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.

[0071] 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, The Netherlands, naktuinbouw.com).

TABLE-US-00001 TABLE 1 IWGP Spinach differential set for Pfs. 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 Where is resistant, + is susceptible and () indicates intermediate resistance.

TABLE-US-00002 TABLE 2 Resistance pattern of deposit NCIMB 43676. Where is resistant and + is susceptible and n.t. is not tested. Deposit NCIMB 43676 Race Pfs 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Isolate Pfs UA2020-01E SP1924

Example 4. Description of SpinachStemphyliumDisease Trial

[0072] Resistance to Stemphylium vesicarium is tested in a qualitative disease assay. In short, 7 to 10 days after untreated seed is sown in soil, a minimum of 20 plants per line is transplanted in pots. Plants are inoculated between 14 and 20 days after sowing when the first true leaves are fully expanded. S. vesicarium is maintained in glycerol at 80 C. and multiplied on potato dextrose agar (PDA). Spores are harvested and counted to result in a spore suspension with a concentration of 1*10.sup.4 spores/mL Inoculated plants are incubated under plastic at high humidity (80-100%) and at a temperature ranging from 20 C.-22 C. After 24 hours plastic is removed, plants are assessed at 4 days after inoculation. When leaf spots are observed on the true leaves a plant is considered susceptible and when no leaf spots are observed a plant is considered resistant.

TABLE-US-00003 Variety Score Deposit NCIMB 43676 R Responder S Patton S
R: resistant; S: susceptible.

Example 5. Description of SpinachCMVDisease Trial

[0073] Resistance to CMV is tested in a qualitative disease assay. In short, 7 to 10 days after untreated seed is sown in soil, a minimum of 20 plants per line is transplanted in pots. Plants are inoculated between 14 and 20 days after sowing when the first true leaves are fully expanded. CMV is maintained as lyophilized spinach leafs at 4 C. CMV is first mechanically inoculated on Nicotiana benthamiana followed by a multiplication on a susceptible spinach variety. Spinach plants are assessed 10 days post inoculation. Plants with leaf yellowing are considered susceptible whereas plant without leaf yellowing are considered resistant.

TABLE-US-00004 Variety Score Deposit NCIMB 43676 R Responder S
R: resistant; S: susceptible.

Example 6. Novel Resistance Against Downy Mildew from Spinacia tetrandraMarker Development

[0074] An inbred population was used to validate the hypothesis that the introgression from S. tetrandra provides resistance against Peronospora. The introgression fully segregated with the resistance in a disease test for race 17 (Table 3).

TABLE-US-00005 TABLE 3 Number of plants showing correlation between disease scores and the genotype of a SNP in an inbred population derived from S. oleracea and S. tetrandra. SNP* SNP* heterozygous SNP* Disease homozygous (S. oleracea and homozygous score Pfs17 (S. tetrandra) S. tetrandra) (S. oleracea) Resistant 20 50 0 Susceptible 0 0 25 *chromosome 4 position 1,108,440 bp.
Selected plants with a crossover close to the resistance locus were used to further confirm the genetic location of the resistance. The region co-segregating with the resistance from S. tetrandra is located on chromosome 4 between 8.3 and 8.6 Mbp and can be identified with several nucleotide sequences (Table 4). Abbreviations are according to IUPAC Nucleotide code:

TABLE-US-00006 Symbol Nucleotide Base A Adenine C Cytosine G Guanine T Thymine N A or C or G or T M A or C R A or G W A or T S C or G Y C or T K G or T V Not T H Not G D Not C B Not A

TABLE-US-00007 TABLE 4 SNPs for the detection of the resistance against Peronospora farinosa. Position Allele Chromo- Position linked Chromo- some* Scaffold to Alternative SNP some (bp) Scaffold (bp) resistance allele 1 chr4 8255074 37 851003 G A 2 chr4 8293796 37 889725 C T 3 chr4 8456792 37 1052721 C T 4 chr4 8486432 37 1082361 T A 5 chr4 8508790 37 1104719 T A 6 chr4 8508969 37 1104898 A G 7 chr4 8509446 37 1105375 T G 8 chr4 8512290 37 1108219 T C 9 chr4 8512511 37 1108440 G A 10 chr4 8605622 37 1201551 T A 11 chr4 8620598 37 1216527 G T *The reference genome is : Xu, C., et al., Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions, Nature Communications 2017.

TABLE-US-00008 Genetic SEQID position* Sequence No. onChr4(bp) -SNPnucleotideishighlightedboldandinbrackets SEQID 8255074 TCTGGGGTTTTCTGTAAATTGAAAAATTGCAGGAGGTTCCTAAAGCATTGCTCCCAGTTG No.1 GGAATCGGCCATTAGTGTCGTATGTATTGGACCTTTTGGA[G]CAAAGCAATCTCAAGGA TATTATTGTGGTTCGTTTCTTtgcttttgattgttttttaWttgGGAAAATGGCGAAATT GKGTTGCAATATACTTGTATTTG SEQID 8255074 TCTGGGGTTTTCTGTAAATTGAAAAATTGCAGGAGGTTCCTAAAGCATTGCTCCCAGTTG No.2 GGAATCGGCCATTAGTGTCGTATGTATTGGACCTTTTGGA[A]CAAAGCAATCTCAAGGA TATTATTGTGGTTCGTTTCTTtgcttttgattgttttttaWttgGGAAAATGGCGAAATT GKGTTGCAATATACTTGTATTTG SEQID 8293796 GACCTTTTCAACTTTAAGAAGAGATTGGACCTTATCAGCTTTAAGAATCTGTTATTGTGA No.3 CTTGAATCTAGCGAAGAAGAGGAAAGACTGAAATTCARGC[C]AAGTGAAGTGCCCCAGA AGGACACGTAATAAGATCCTAGGTGGGATGACAAAGGTGCAAAGTCCCTCGTCTATCATA AGGCACCCTGGGCTGGCTATAAT SEQID 8293796 GACCTTTTCAACTTTAAGAAGAGATTGGACCTTATCAGCTTTAAGAATCTGTTATTGTGA No.4 CTTGAATCTAGCGAAGAAGAGGAAAGACTGAAATTCARGC[T]AAGTGAAGTGCCCCAGA AGGACACGTAATAAGATCCTAGGTGGGATGACAAAGGTGCAAAGTCCCTCGTCTATCATA AGGCACCCTGGGCTGGCTATAAT SEQID 8456792 TCATTCTGATCATAATCATCTGAGAATAAACATTTCACCAAATACATGagacaattcaat No.5 caaatactGCCATATTATGCTTATTAGTTATTAGCCCGCA[C]ATCCTTTAAAGGCAATA TATACTACACCTTTCACAAATGAATGAGCCACAAACAATAATTTCTCAATCAAGCAATAA GTTAATTTACACATGCWATAACT SEQID 8456792 TCATTCTGATCATAATCATCTGAGAATAAACATTTCACCAAATACATGagacaattcaat No.6 caaatactGCCATATTATGCTTATTAGTTATTAGCCCGCA[T]ATCCTTTAAAGGCAATA TATACTACACCTTTCACAAATGAATGAGCCACAAACAATAATTTCTCAATCAAGCAATAA GTTAATTTACACATGCWATAACT SEQID 8486432 ATMAATCCATCTGCTCATTTATCGATTTCAGATAAACATCTTTYACCAGGTTCGtgattc No.7 ttctctttctctctcttcgattTTTCTTCAGAATTAGTTa[T]Yttttctttaattattt ggtCGCGATTGGTATTTTATGCCCTAATTACGTGATTGAATTGCGTTTTGAGCKTCAATT TKGGATGTATTGTTTGTAGAAAG SEQID 8486432 ATMAATCCATCTGCTCATTTATCGATTTCAGATAAACATCTTTYACCAGGTTCGtgattc No.8 ttctctttctctctcttcgattTTTCTTCAGAATTAGTTa[A]Yttttctttaattattt ggtCGCGATTGGTATTTTATGCCCTAATTACGTGATTGAATTGCGTTTTGAGCKTCAATT TKGGATGTATTGTTTGTAGAAAG SEQID 8508790 GAGTTTGATTCTTCCAGTTTCGGACASAGAGGAAAATTTCTCAAATTATCACAAAATGTA No.9 ATTAACAGATGAGAAAGGTGATGGAACGAATAYGAATGTG[T]TTGAAACCCTGCTACTT CTCGTGTCTCACCCATATCTGATTCTRATTTCCACCATCCTTCCAACTTTGGCATACTCC AAAGCTCAAGCTTTTCAAGGGAT SEQID 8508790 GAGTTTGATTCTTCCAGTTTCGGACASAGAGGAAAATTTCTCAAATTATCACAAAATGTA No.10 ATTAACAGATGAGAAAGGTGATGGAACGAATAYGAATGTG[A]TTGAAACCCTGCTACTT CTCGTGTCTCACCCATATCTGATTCTRATTTCCACCATCCTTCCAACTTTGGCATACTCC AAAGCTCAAGCTTTTCAAGGGAT SEQID 8508969 AAGCTCAAGCTTTTCAAGGGATGGAAAGAAAACCAAGTCAACATCTGCTCCTCCATGATT No.11 TGACCCTCCTGATGTTGATGCAACCCCTTCTGCGCTGATA[A]TACTATTCTCCATAYAC ACCACTTCAYTCAAATATCGTAATGTAAGGAATTTCAAATGACGCAGTTGACTCATCAAT GGAAGATGCTCCAACCTTGTACA SEQID 8508969 AAGCTCAAGCTTTTCAAGGGATGGAAAGAAAACCAAGTCAACATCTGCTCCTCCATGATT No.12 TGACCCTCCTGATGTTGATGCAACCCCTTCTGCGCTGATA[G]TACTATTCTCCATAYAC ACCACTTCAYTCAAATATCGTAATGTAAGGAATTTCAAATGACGCAGTTGACTCATCAAT GGAAGATGCTCCAACCTTGTACA SEQID 8509446 TTTTTTKACATAAATTACCATATAGTGGCTATGATTGCTGAAATTTTTCAAATCTCCCAG No.13 CTCACCAACACCGTTTGACCCCCGATTCCAGCTATTTCTT[T]TTCTGCTCACTACAAAC CCTGTCAGTYTATGCAGAGATGTCATGCTATTCATCCCCCGRGGCATATGYGACAAACTC TTACAGCCTTGTATATCTAGGTG SEQID 8509446 TTTTTTKACATAAATTACCATATAGTGGCTATGATTGCTGAAATTTTTCAAATCTCCCAG No.14 CTCACCAACACCGTTTGACCCCCGATTCCAGCTATTTCTT[G]TTCTGCTCACTACAAAC CCTGTCAGTYTATGCAGAGATGTCATGCTATTCATCCCCCGRGGCATATGYGACAAACTC TTACAGCCTTGTATATCTAGGTG SEQID 8512290 CCTTTCAAGTAATACAGAGTTAGGTCCAAGTTTAGGATCAGGTCAAGCAAAACAACTGTA No.15 TAACCATACTCAACACAGCCCCACCGGAGTCAAGTCGAAA[T]AACCAAGTCTTTGAAAG AGAAGACATGACCTTATGTTCGGRCCCAACGCACAAAATCAATAGAGTAGAAATGTACAA TTCATTTCCATTGCTATGtaKtg SEQID 8512290 CCTTTCAAGTAATACAGAGTTAGGTCCAAGTTTAGGATCAGGTCAAGCAAAACAACTGTA No.16 TAACCATACTCAACACAGCCCCACCGGAGTCAAGTCGAAA[C]AACCAAGTCTTTGAAAG AGAAGACATGACCTTATGTTCGGRCCCAACGCACAAAATCAATAGAGTAGAAATGTACAA TTCATTTCCATTGCTATGtaKtg SEQID 8512511 ATAGGTTGTTTAGAACTTTATCAATTACTactaactactccgtactctTCACTGataagt No.17 tgtcaattactaacTAATTACTACATAGGTTTTGTTAAAA[G]TTGTCAATTATTAGCTA TTTTTCTAACAAWGGAGGTCAATTACTAATTCGTTGTCAATTAATACGTTTTCCGCCCAA TTAGTCGACTAATCACATCATAA SEQID 8512511 ATAGGTTGTTTAGAACTTTATCAATTACTactaactactccgtactctTCACTGataagt No.18 tgtcaattactaacTAATTACTACATAGGTTTTGTTAAAA[A]TTGTCAATTATTAGCTA TTTTTCTAACAAWGGAGGTCAATTACTAATTCGTTGTCAATTAATACGTTTTCCGCCCAA TTAGTCGACTAATCACATCATAA SEQID 8605622 AGATATTTTAACCGATAATTCGATATTATCCAGCTTgtactaataataaataataaatta No.19 tataatcaaatacaaaaaatatttacaattttaaaatcaa[T]tttcagTGTTATATTGT TATCCAAASCCAATAAGGATAAGTTAATTATCTTATAAACGTGCAATTAATACATACAAT CCTTGTAAATTCAGTTTTACTTC SEQID 8605622 AGATATTTTAACCGATAATTCGATATTATCCAGCTTgtactaataataaataataaatta No.20 tataatcaaatacaaaaaatatttacaattttaaaatcaa[A]tttcagTGTTATATTGT TATCCAAASCCAATAAGGATAAGTTAATTATCTTATAAACGTGCAATTAATACATACAAT CCTTGTAAATTCAGTTTTACTTC SEQID 8620598 tttctttacttatatatttctcttttttcacCCTTTTACTTCTCACTACCCACATAATAA No.21 CAMTCTCCCACAGTCCCACCCTTTCCCCCTCCATAWCCCA[G]CAAAATTACAAGTTCAT TTCTCGTCTTTCGGGTGAGATTCTCACTGCAAAGTGACCACATTACTCTTCCaaccacaa aattaattaatcaaattactaCA SEQID 8620598 tttctttacttatatatttctcttttttcacCCTTTTACTTCTCACTACCCACATAATAA No.22 CAMTCTCCCACAGTCCCACCCTTTCCCCCTCCATAWCCCA[T]CAAAATTACAAGTTCAT TTCTCGTCTTTCGGGTGAGATTCTCACTGCAAAGTGACCACATTACTCTTCCaaccacaa aattaattaatcaaattactaCA *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.

Example 7. Markers for Detection of CMV Resistance

[0075] We have used the following markers to identify plants that are resistant to CMV.

TABLE-US-00009 TABLE 5 SNPs for the detection of the resistance against CMV. Position Allele Chromo- Position linked Chromo- some* Scaffold to Alternative SNP some (bp) Scaffold (bp) resistance allele 12 Chr3 1201575 31 1201575 T A 13 Chr3 1216490 31 1216490 A T 14 Chr3 1219873 31 1219873 A C 15 Chr3 1220905 31 1220905 G A *The reference genome is : Xu, C., et al., Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions, Nature Communications 2017.

TABLE-US-00010 Genetic SEQID position* Sequence No. onChr3(bp) -SNPnucleotideishighlightedboldandinbrackets SEQID 1201575 ccctccgtattttttctTAATGTTATAATTGCACTATTTGAtgtttcacgtttgtcaatg No.23 cgaAACTTTAGCACAATTRGTTAGTAAGACATCACATGTC[T]GATRTCTTTATCCATTT GAATGGGCCATATGTTGGCCATATACATTTCTAAAAAGGTATGGGCTACAACGCCTACAA GTAACAACTATCATAATTATAAG SEQID 1201575 ccctccgtattttttctTAATGTTATAATTGCACTATTTGAtgtttcacgtttgtcaatg No.24 cgaAACTTTAGCACAATTRGTTAGTAAGACATCACATGTC[A]GATRTCTTTATCCATTT GAATGGGCCATATGTTGGCCATATACATTTCTAAAAAGGTATGGGCTACAACGCCTACAA GTAACAACTATCATAATTATAAG SEQID 1216490 ATGGRAAATGTAAGTTARTTGGGGATGCACataaggtgtttgMtgaaatgtctATKAgaa No.25 atgttgtttcttggacttAGAATGATATACACTGTCGTCC[A]TTGGTTTCCAATCTTAC AtttggtttKtgttttcttaGTTTGTTTCTTTAATCAACACCARcccattttttttaaac tacCTGCAACTAYTAAWTTTCAT SEQID 1216490 ATGGRAAATGTAAGTTARTTGGGGATGCACataaggtgtttgMtgaaatgtctATKAgaa No.26 atgttgtttcttggacttAGAATGATATACACTGTCGTCC[T]TTGGTTTCCAATCTTAC AtttggtttKtgttttcttaGTTTGTTTCITTAATCAACACCARcccattttttttaaac tacCTGCAACTAYTAAWTTTCAT SEQID 1219873 CAAACTTGGAAAATAATGACAGAAAAATGCTAAGTTAATATAGGAAACTACATCATTTTC No.27 ACTCGAAAAGATTAGAGGAATTCATWTTCGTCTTCCTCCC[A]TTGAYTTCATGTGCCGT AAATCTTYTGACTGTCTATATTGGTACAAGTRATTTCAGGTATATACATTGGTTAATCCA TTTTAATCTGTATCTGCCGGTGT SEQID 1219873 CAAACTTGGAAAATAATGACAGAAAAATGCTAAGTTAATATAGGAAACTACATCATTTTC No.28 ACTCGAAAAGATTAGAGGAATTCATWTTCGTCTTCCTCCC[C]TTGAYTTCATGTGCCGT AAATCTTYTGACTGTCTATATTGGTACAAGTRATTTCAGGTATATACATTGGTTAATCCA TTTTAATCTGTATCTGCCGGTGT SEQID 1220905 TATTATGAMGGGTCAACAATTATGTAATATATAGGGAAGTAGAAAGTCTGGGATGTTACC No.29 TGAAGAGTCTCCTTGCCTGCAAAGTTTTAAGCTGCAAGAA[G]GTATTTGRCAATAATTA ATRACATTKCACAACAAAGATTATGcacatttttctttaaaaatcaaGAGAAACTTGTAG ATGTACCGTAAATAGGAACATCT SEQID 1220905 TATTATGAMGGGTCAACAATTATGTAATATATAGGGAAGTAGAAAGTCTGGGATGTTACC No.30 TGAAGAGTCTCCTTGCCTGCAAAGTTTTAAGCTGCAAGAA[A]GTATTTGRCAATAATTA ATRACATTKCACAACAAAGATTATGcacatttttctttaaaaatcaaGAGAAACTTGTAG ATGTACCGTAAATAGGAACATCT *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.

Example 8. Markers Used to Evaluate Stemphylium Resistance

[0076] In an F1S1 population derived from a Stemphylium susceptible and resistant S. oleracea line, 15 Single Nucleotide Polymorphisms (SNPs) informative between both parents were used to genotype the population. The 15 SNPs were all located on chromosome 3. A strong correlation was found between the disease score and one of the SNPs on chromosome 3 (Table 6).

TABLE-US-00011 TABLE 6 Number of plants showing correlation between disease scores and the genotype of a SNP in a F1S1 population derived from a Stemphylium susceptible and resistant S. oleracea line. Diseas SNP* SNP* score homozygous SNP* homozygous Stemphylium (Resistant parent) heterozygous (Susceptible parent) Resistant 107 0 1 Susceptible 0 179 107 *chromosome 3 position 1,216,584 bp.
The region co-segregating with Stemphylium resistance is located on chromosome 3 between 1.1 and 1.3 Mbp and can be identified with several nucleotide sequences (Table 7). Abbreviations are according to IUPAC Nucleotide code.

TABLE-US-00012 TABLE 7 SNPs for the detection of the resistance against Stemphylium. Position Allele Chromo- Position linked Chromo- some* Scaffold to Alternative SNP some (bp) Scaffold (bp) resistance allele 16 Chr3 1177586 31 1177586 C A 17 Chr3 1206382 31 1206382 T A 18 Chr3 1216394 31 1216394 G A 19 Chr3 1227120 31 1227120 A C 20 Chr3 1231945 31 1231945 T C 21 Chr3 1253108 31 1253108 A G 22 Chr3 1271037 31 1271037 G T *The reference genome is : Xu, C., et al., Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions, Nature Communications 2017.

TABLE-US-00013 Genetic SEQID position* Sequence No. onChr4(bp) -SNPnucleotideishighlightedboldandinbrackets SEQID 1177586 gGCAAACGTGTMCAGTCAAAACAGTGYGAATATTACGGGACGAAGGGCAGTAAGtagttt No.31 ttaatatatttttatttatttatttatggtaRAAAATCaa[C]taatttWtatttttgag tGTGAACCGTTTCCTAATTAGAATAAAAAACTCTTGGGAAAATATTTCCATAAATACGAG Attactaaaaattaaattaacat SEQID 1177586 gGCAAACGTGTMCAGTCAAAACAGTGYGAATATTACGGGACGAAGGGCAGTAAGtagttt No.32 ttaatatatttttatttatttatttatggtaRAAAATCaa[A]taatttWtatttttgag tGTGAACCGTTTCCTAATTAGAATAAAAAACTCTTGGGAAAATATTTCCATAAATACGAG Attactaaaaattaaattaacat SEQID 1206382 TTAGTACAGTCGTCAATTAAAAATACTCCCGTTATACACGtgaataaaattttgttttca No.33 ctCRTTCGAGTGGTTAGtgagaatttattttWaaaaaaaa[T]tctagttTCTACCAAAA CTAAATTCTTTCCATtataactacggagtaatttgtatagcgattYccatttttttaagG AGATCTAATAGGAGACYTACTCA SEQID 1206382 TTAGTACAGTCGTCAATTAAAAATACTCCCGTTATACACGtgaataaaattttgttttca No.34 ctCRTTCGAGTGGTTAGtgagaatttattttWaaaaaaaa[A]tctagttTCTACCAAAA CTAAATTCTTTCCATtataactacggagtaatttgtatagcgattYccatttttttaagG AGATCTAATAGGAGACYTACTCA SEQID 1216394 TYTTTTYGTTGGGGTTGGWTTTCATGTATATGTTGCTGATTAAATACSagactgatgatg No.35 atKatgtgttTATGGGTTTTAAATCAGATTAAATATATGG[G]AAATGTAAGTTARTTGG GGATGCACataaggtgtttgMtgaaatgtctATKAgaaatgttgtttcttggacttAGAA TGATATACACTGTCGTCCWTTGG SEQID 1216394 TYTTTTYGTTGGGGTTGGWTTTCATGTATATGTTGCTGATTAAATACSagactgatgatg No.36 atKatgtgttTATGGGTTTTAAATCAGATTAAATATATGG[A]AAATGTAAGTTARTTGG GGATGCACataaggtgtttgMtgaaatgtctATKAgaaatgttgtttcttggacttAGAA TGATATACACTGTCGTCCWTTGG SEQID 1227120 gATGGGARCACTATACATTGAGGTCGACTCAARTAACATATGATGCACGCATAACACCAA No.37 GAGTCSAAGACTCTGTTTTCTTGTataaagagagaagaaa[A]gaacacaGGTAACTGCA ACAATCACTTCATATGCACAACACARAGTTTAAGATAAACTAAATTTTAGATTTAGGCAA TCTGGAAAATTTATTACTTCATT SEQID 1227120 gATGGGARCACTATACATTGAGGTCGACTCAARTAACATATGATGCACGCATAACACCAA No.38 GAGTCSAAGACTCTGTTTTCTTGTataaagagagaagaaa[C]gaacacaGGTAACTGCA ACAATCACTTCATATGCACAACACARAGTTTAAGATAAACTAAATTTTAGATTTAGGCAA TCTGGAAAATTTATTACTTCATT SEQID 1231945 tatgCTRACCACAATGCTGTAAAATATTGGGCCCCATGTGCTTAWATAGGGAAATTTYCA No.39 TTGAGaagattRaaaattataacGAAGCCAAATGTTGTGA[T]CCCWCAGTAGTGTGTAC CCTKGCTTTTGTATRCTAACAGTTACACATCAATAATACAGAATRCCTCTAGACTCCAAG CAATGACATTCTGCAAGAGTACC SEQID 1231945 tatgCTRACCACAATGCTGTAAAATATTGGGCCCCATGTGCTTAWATAGGGAAATTTYCA No.40 TTGAGaagattRaaaattataacGAAGCCAAATGTTGTGA[C]CCCWCAGTAGTGTGTAC CCTKGCTTTTGTATRCTAACAGTTACACATCAATAATACAGAATRCCTCTAGACTCCAAG CAATGACATTCTGCAAGAGTACC SEQID 1253108 TGAGGGAGATAYGGGATTAATTCATATTGTCTTGGTGGAGAAGACGGCCGGCATAGATGG No.41 GAActtgagagggagagagagaaagattgagttaaaaaaa[A]tcagtaacaACATTGAA YGTTTTTCTTAAGctttttgtgaaaataaaataaacaaMtaaaTATTGAAGGATCTCATT TTTTAGCTCCGGTGAGTTAAACC SEQID 1253108 TGAGGGAGATAYGGGATTAATTCATATTGTCTTGGTGGAGAAGACGGCCGGCATAGATGG No.42 GAActtgagagggagagagagaaagattgagttaaaaaaa[G]tcagtaacaACATTGAA YGTTTTTCTTAAGctttttgtgaaaataaaataaacaaMtaaaTATTGAAGGATCTCATT TTTTAGCTCCGGTGAGTTAAACC SEQID 1271037 CGGTTGACCACACATTTGAGAAGCACTAATTASTCATAATGACAAACATATACGRAACCG No.43 ACCATGCATGTTTCTTGTCTCWATATTTATGCTTAATTAA[G]TCTTAACACAAAGGTTA ATTAAGGGGATGTGAAACTAATCACGTTCGATATAAGGTAATAGGGAGAATCATWGGYTT AAAATTAGTCATATAGCTACGCA SEQID 1271037 CGGTTGACCACACATTTGAGAAGCACTAATTASTCATAATGACAAACATATACGRAACCG No.44 ACCATGCATGTTTCTTGTCTCWATATTTATGCTTAATTAA[T]TCTTAACACAAAGGTTA ATTAAGGGGATGTGAAACTAATCACGTTCGATATAAGGTAATAGGGAGAATCATWGGYTT AAAATTAGTCATATAGCTACGCA *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.