TolCNDV resistant melon plants

Abstract

The present invention relates to ToLCNDV resistant melon plants comprising one or two introgression fragments in their genome.

Claims

1. A cultivated melon plant or plant cell comprising an introgression fragment from chromosome 11 of a ToLCNDV resistant donor plant, wherein the introgression fragment on chromosome 11 comprises a sequence of the ToLCNDV resistant donor melon plant in-between SNP_01 at nucleotide 101 of SEQ ID NO: 1 and SNP_04 at nucleotide 101 of SEQ ID NO: 4, and wherein the introgression fragment comprises a QTL which confers ToLCNDV resistance in a recessive manner and wherein the introgression fragment comprises a Thymine for SNP_03 at nucleotide 101 of SEQ ID NO: 3, and wherein the introgression fragment on chromosome 11 is obtainable from seeds deposited under accession number NCIMB 42625.

2. The cultivated melon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 11 further comprises a Thymine at nucleotide 101 of SEQ ID NO: 4 and/or a Guanine at nucleotide 101 of SEQ ID NO: 2.

3. A cultivated melon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 11 comprises a Adenine at nucleotide 101 of SEQ ID NO: 1 or of a sequence comprising at least 90% sequence identity to SEQ ID NO: 1, and a Guanine at nucleotide 101 of SEQ ID NO: 2 or of a sequence comprising at least 90% sequence identity to SEQ ID NO: 2 and a Thymine at nucleotide 101 of SEQ ID NO: 3 or of a sequence comprising at least 90% sequence identity to SEQ ID NO: 3 and a Thymine at nucleotide 101 of SEQ ID NO: 4 or of a sequence comprising at least 90% sequence identity to SEQ ID NO: 4.

4. The cultivated melon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 11 is in homozygous form.

5. The cultivated melon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 11 is from a wild melon or wild relative of melon.

6. The cultivated melon plant or plant cell according to claim 1, wherein the introgression fragment on chromosome 11 comprises a recessive QTL which confers an average TolCNDV resistance score of at least 7 on a scale of 1 (dead plant) to 9 (no symptoms) when the QTL is in homozygous form.

7. The cultivated melon plant or plant cell according to claim 1, wherein the plant or plant cell is an inbred plant or plant cell or an F1 hybrid plant or plant cell.

8. A seed which grows into a plant according to claim 1.

9. A cultivated melon fruit or fruit part comprising plant cells according to claim 1.

10. A cultivated melon plant propagation material comprising a cultivated melon plant cell according to claim 1.

11. A method of crossing an inbred line according to claim 7 to another inbred line and collecting the F1 hybrid seed produced from said cross.

12. A method for producing hybrid cultivated melon seeds comprising the following steps a) providing a first ToLCNDV resistant inbred melon plant comprising two chromosomes 11 each having an introgression fragment from chromosome 11 of a ToLCNDV resistant donor plant, the introgression fragment comprising QTL11 and a Thymine at nucleotide 101 of SEQ ID NO: 3 for SNP_03; or the introgression fragment comprising QTL11 and the resistant donor genotype for one or more or all of SNP_01 at nucleotide 101 of SEQ ID NO: 1, SNP_02 at nucleotide 101 of SEQ ID NO: 2, SNP_03 at nucleotide 101 of SEQ ID NO: 3 and/or SNP_04 at nucleotide 101 of SEQ ID NO: 4; b) providing a second ToLCNDV resistant inbred melon plant comprising two chromosomes 11 each having an introgression fragment from chromosome 11 of a ToLCNDV resistant donor plant, the introgression fragment comprising QTL11 and a Thymine at nucleotide 101 of SEQ ID NO: 3 for SNP_03; or the introgression fragment comprising QTL11 and the resistant donor genotype for one or more or all of SNP_01 at nucleotide 101 of SEQ ID NO: 1, SNP_02 at nucleotide 101 of SEQ ID NO: 2, SNP_03 at nucleotide 101 of SEQ ID NO: 3 and/or SNP_04 at nucleotide 101 of SEQ ID NO: 4; c) crossing the inbred melon plant provided in step a) with the inbred melon plant provided in step b); and d) collecting seeds obtained from the cross of step c).

13. A method of screening plants or plant parts, or DNA derived therefrom, for the presence of a fragment on chromosome 11 conferring ToLCNDV resistance comprises the steps of: i) screening the genomic DNA for the SNP genotype of one or more or all of SNP_01 01 at nucleotide 101 of SEQ ID NO: 1, SNP_02 at nucleotide 101 of SEQ ID NO: 2, SNP_03 at nucleotide 101 of SEQ ID NO: 3 and/or SNP_04 at nucleotide 101 of SEQ ID NO: 4; ii) and optionally selecting a plant or plant part comprising an Adenine at nucleotide 101 of SEQ ID NO: 1 and/or a Guanine at nucleotide 101 of SEQ ID NO: 2 and/or a Thymine at nucleotide 101 of SEQ ID NO: 3 and/or a Thymine at nucleotide 101 of SEQ ID NO: 4.

14. The cultivated melon plant or plant cell according to claim 1, wherein the donor plant is not Ag-WM7Ind/WM7.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Shown are the average symptom levels (of three replicates) at 35 days post infection (dpi) with ToLCNDV by whitefly transmission of a donor plant line being ToLCNDV resistant (Wild Donor), a recurrent plant line (Recurrent) and of plant line NCIMB 42625. The average symptom levels were determined as described herein under “General Methods”.

GENERAL METHODS

(2) 1. Determination of Symptom Level on ToLCNDV Infected Plants

(3) 1.1 Plants and Pathogens (Virus)

(4) A melon plant (Cucumis melo) infecting strain of ToLCNDV was used for infection of melon plants. In the present invention a ToLCNDV strain isolated in Murcia, Spain was used as inoculum.

(5) 1.2 ToLCNDV Inoculum

(6) The ToLCNDV inoculum source was maintained on living infected melon plants. It must be ensured, that pure virus isolates are used and that neither the virus source, nor the whiteflies are contaminated with other diseases, in particular with other viruses (e.g. CGMMV, CYSDV, CYVY, SqMV). For pre-multiplication of the ToLCNDV inoculum whiteflies (Bemisia tabaci) were fed on ToLCNDV sensitive (susceptible), infected melon plants in an insect proof cage. Before infection of test plants, the ToLCNDV infected plants were placed into an insect proof cage, whiteflies were released into the same cage and allowed to feed for approximately 3 days on the ToLCNDV infected plants.

(7) 1.3 Inoculation of Plants to be Tested

(8) For each genotype of melon plants to be analysed 14 plants were grown until the first true leaf is expanded (normally 12-15 days after sowing), 12 of which were infected and 2 were mock infected. Also 12 plants of susceptible varieties were included, in this experiment variety Gandalf F1 (Hild Samen) and variety Vedantrais. The 12 plants per genotype to be tested for ToLCNDV resistance were placed into an insect proof cage, infected whiteflies (obtained as described under 1.2 above) were released into the cage to infect the plants. It has to be ensured that at least 5-10 whiteflies are available for each test plant in the cage. Whiteflies and test plants are kept in the cage for approximately 48 hours, then the whiteflies are eliminated with an appropriate insecticide. Also two plants per genotype were mock infected, i.e. they were treated in the same manner as the test plants apart from the fact that the whiteflies used for infection were free of ToLCNDV.

(9) 1.4 Growing Infected Test Plants

(10) Infected test plants obtained as described under 1.3 were transplanted to bigger pots, transferred into a greenhouse with cooling equipment. The plants were grown at approximately 18° C. night temperature and approximately 25° C. day temperature in a timeframe of 14 to 16 hours daylight. The infected plants for each infected genotype were grown in two replicates in two different plots, each of which comprised 6 ToLCNDV infected plants and 1 mock infected plant. The plots are randomized in respect to the growing area.

(11) 1.5. Scoring the Symptom Level of ToLCNDV Infection

(12) The scoring of the symptom level may already be done approximately 15 days post infection (dpi) with ToLCNDV but is preferably done approximately 30 days post infection (dpi) with ToLCNDV, or later, e.g. 35 dpi. In case plants are present which show recovery from the virus infection, a further scoring of the symptom can optionally be done approximately 45 days post infection (dpi) with ToLCNDV.

(13) The following symptom levels are to be used according to the phenotypes indicated in the following:

(14) TABLE-US-00005 Symptom level Observed phenotype 1 Dead plant 2 Severe mosaic and curling, chlorosis and growth reduction. No recovery 3 Strong mosaic and curling, chlorosis and growth reduction. No recovery 4 Curling and mosaic, chlorosis, no or mild growth reduction. No recovery 5 Curling and mosaic, chlorosis, no growth reduction. Slight recovery of the upper plant zone 6 Mild curling, mosaic and chlorosis, no growth reduction. Recovery of the upper middle plant 7 Mild curling, mosaic and chlorosis, no growth reduction. Symptoms appear only in the lower plant zone 8 Faint mosaic 9 No symptoms

(15) 1.6 Optional Additional Tests

(16) It is recommended to use at least one genotype highly resistant to ToLCNDV (e.g. average symptom level 8-9) and/or one genotype highly sensitive to ToLCNDV (e.g. average symptom level 1 or 2) in each experimental setup as controls. It is further recommended to also include a genotype having intermediate resistance to ToLCNDV infection in each test setup. When these control genotypes show the expected symptom level, this gives a clear indication that the experimental conditions are right to evaluate the test plants. It is especially important to include a susceptible control line or variety, which shows the expected symptoms at the time of scoring, e.g. 30 dpi or later (e.g. 35 dpi).

(17) Furthermore, it is advisable to check infection and spreading of ToLCNDV in infected plants and control plants. This can be done by checking for the presence and/or amount of virus DNA in upper parts of the plants. A suitable way to check for the presence and/or amount of ToLCNDV DNA in upper plant parts is hybridization of plant material with a probe hybridizing with the DNA of the ToLCNDV strain used. Various hybridization techniques are well known in the art. A simple so called Dot Blot analysis is sufficient for obtaining valuable results. Additionally or alternatively qPCR can be used.

(18) 1. Selection of ToLCNDV Resistant Donor Plants

(19) The symptom level of wild accessions of melon plants were tested for ToLCNDV resistance according to the test described under “General Methods”. A wild donor plant was identified which has a high resistance to ToLCNDV infection, having an average disease score of 9 (no symptoms) at 15 dpi, 25 dpi and 35 dpi, while the susceptible control line had an average disease score of 2.8 at 15 dpi, 2.0 at 25 dpi and 2.0 at 35 dpi.

(20) Also-blot hybridization and qPCR (quantitative PCR) were carried out on infected donor plants and a susceptible control line after 30 dpi. No ToLCNDV virus was detected in the donor tissue at 30 dpi, while ToLCNDV virus was detected in the susceptible control. (Note that a low Ct-value indicates a high virus titer, while a high Ct-value indicates a low amount of virus)

(21) TABLE-US-00006 qPCR- Dot-blot average Ct signal value ng/μl Donor line − 33.8 3.76E−07 Susceptible line + 13.1 0.19 qPCR negative control Ct = 34

(22) Another real time qPCR (quantitative PCR) experiment was carried out, using a protocol described in Simon et al. 2018 (supra). In short, the primers and probes ToLA-up (forward primer), ToLA-Low (reverse primer) and ToLA-Probe (TaqMan probe) of Table 1 of Simon et al 2018 (supra) were used in a qPCR analysis as described therein, with thermos cycling conditions being incubation at 95° C. for 2 minutes followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 1 minute.

(23) Plants of the donor and of the susceptible variety Vedantrais were sown and inoculated with ToLCNDV as described herein above. Three replicates of six plants per genotype were used and three mock inoculated Vedantrais plants were included per replicate. Phenotypic scoring of each plant was done at 15 dpi, 30 dpi and 45 dpi. Samples for qPCR were taken at 60 dpi from each plant.

(24) TABLE-US-00007 Average Average ToLCNDV Ct-value Genotype disease score STDV (60 dpi) STDV Donor line 9.0 0.0 30.59 3.12 Susceptible line  2.75  0.49 10.07 1.32 (Vedantrais) Mock inoculated 9.0 0.0 39.97 0.42 susceptible line (Vedantrais) STDV = standard deviation

(25) Results confirmed that the symptom free donor line had extremely low ToLCNDV virus levels in the leaves at 60 dpi.

(26) 2. Identification of Genomic Location of ToLCNDV Resistance

(27) Three mapping populations were developed using the donor plant obtained in Example 1 to map the position of the ToLCNDV resistance conferring loci (QTL). Phenotyping of the mapping populations was carried out as described above, using a TolCNDV scale of 1 to 9. Genotyping was initially carried out using 192 SNP markers, and later additional SNP markers were run to saturate the QTL regions.

(28) Analysis in these mapping populations revealed two major QTLs associated with resistance, located on chromosome 11 and 12.

(29) From resistant material the inventors developed backcross lines to fine map and further investigate resistance from the donor.

(30) The markers identified during fine mapping and their respective positions according to publicly known data from Diaz et al. (2015, Mol Breeding 35, 188) is shown in Table 2 (in the description).

(31) 3. Development of KASP-Assays

(32) A KASP-assay was developed for identifying the SNPs linked to QTL11 (SNP_01, SNP_02, SNP_03 and SNP_04) and QTL12 (SNP_05, SNP_06 and SNP_07). The SNPs associated with QTL11 and QTL12 can be determined by use of the following primers in a KASP-assay:

(33) TABLE-US-00008 SNP FAM allele VIC allele Common Primer SNP_01 SEQ ID NO 8 SEQ ID NO 9 SEQ ID NO 10 SNP_02 SEQ ID NO 11 SEQ ID NO 12 SEQ ID NO 13 SNP_03 SEQ ID NO 14 SEQ ID NO 15 SEQ ID NO 16 SNP_04 SEQ ID NO 17 SEQ ID NO 18 SEQ ID NO 19 SNP_05 SEQ ID NO 20 SEQ ID NO 21 SEQ ID NO 21 SNP_06 SEQ ID NO 23 SEQ ID NO 24 SEQ ID NO 25 SNP_07 SEQ ID NO 26 SEQ ID NO 27 SEQ ID NO 28

(34) 4. Introgression of ToLCNDV into a Cultivated Melon Plant

(35) Backcrossing was performed with a recurrent parent elite breeding line of a Piel de Sapo type to develop a line comprising both QTL11 and QTL12 in homozygous form.

(36) Seeds of an introgression line comprising both QTL11 and QTL12 in homozygous form were deposited under NCIMB 42625. The donor genotype is present for SNP_01 to SNP_04 and for SNP_05 to SNP_07, as verified using the KASP assay.

(37) FIG. 1 and the Table below shows that the donor and the introgression line deposited have an average disease score of 9.0 when scored 35 dpi, while the susceptible recurrent parent line has an average disease score of 2.4 at 35 dpi.

(38) TABLE-US-00009 AVG STDV Wild Donor 9.0 0.0 Recurrent (susceptible) parent 2.4  0.43 NCIMB 42625 (melon plant comprising 9.0 0.0 introgression fragment on chr. 11 and chr. 12) AVG: average value of tested genotype, STDV: standard deviation

(39) Additional backcrossing was done with the donor to introgress one or both QTLs into elite Galia, Cantaloup and Charentais backgrounds.