TolCNDV resistant melon plants

Abstract

The application concerns melon plants (Cucumis melo) resistant to infection with tomato leaf curl New Dehli virus (ToLCNDV). The resistant melon plants have a genomic introgression fragment on chromosome 5 which confers tolerance to ToLCNDV in a dominant manner. Also disclosed are markers for identifying those fragments, methods for identifying or producing resistant melon plants.

Claims

1. A cultivated melon plant cell of the species Cucumis melo comprising an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment confers ToLCNDV resistance and comprises a Cytosine (C) for SNP_07 at nucleotide 101 of SEQ ID NO: 25, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 25, and/or a Guanine (G) for SNP_08 at nucleotide 101 of SEQ ID NO: 26, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 26, and wherein the introgression fragment is obtainable from seeds, a representative sample of which has been deposited under accession number NCIMB 42585.

2. The cultivated melon plant cell of claim 1, wherein the introgression fragment comprises the sequence of the ToLCNDV resistant donor melon plant in-between nucleotide 25832880 and nucleotide 25065080 of chromosome 5, and wherein the introgression fragment is obtainable from seeds, a representative sample of which has been deposited under accession number NCIMB 42585.

3. The cultivated melon plant cell according to claim 1, wherein the introgression fragment comprises a Cytosine (C) for SNP_07 at nucleotide 25361598 of chromosome 5 and/or a Guanine (G) for SNP_08 at nucleotide 25257353 of chromosome 5.

4. The cultivated melon plant cell according to claim 1, comprising the introgression fragment in homozygous form and comprising the genotype CC for SNP_07 and/or the genotype GG for SNP_08.

5. The cultivated melon plant cell according to claim 1, wherein the introgression fragment further comprises a Thymine (T) for SNP_03 at nucleotide 68 of SEQ ID NO: 3 or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 3 and/or a Adenine (A) for SNP_04 at nucleotide 227 of SEQ ID NO: 4 or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 4.

6. A cultivated melon plant comprising melon plant cells according to claim 1.

7. A cultivated melon seed comprising plant cells according to claim 1.

8. A cultivated melon fruit comprising plant cells according to claim 1.

9. Cultivated melon plant propagation material comprising plant cells according to claim 1.

10. A method for producing a ToLCNDV resistant melon plant comprising the following steps a) selecting a ToLCNDV resistant donor plant comprising a Cytosine (C) for SNP_07 at nucleotide 101 of SEQ ID NO: 25, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 25, and/or a Guanine (G) for SNP_08 at nucleotide 101 of SEQ ID NO: 26, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 26, b) crossing the donor plant selected in step a) with a plant sensitive to ToLCNDV, c) obtaining seeds from the plants crossed in step b), and optionally d) verifying if the plants grown from the seeds obtained in step c) are resistant to ToLCNDV and/or comprise a Cytosine (C) for SNP_07 at nucleotide 101 of SEQ ID NO: 25, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 25, and/or a Guanine (G) for SNP_08 at nucleotide 101 of SEQ ID NO: 26, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 26.

11. A method for producing melon seeds comprising the following steps: a) growing a melon plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment confers ToLCNDV resistance and comprises a Cytosine (C) for SNP_07 at nucleotide 101 of SEQ ID NO: 25, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 25, and/or a Guanine (G) for SNP_08 at nucleotide 101 of SEQ ID NO: 26, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 26, and wherein the introgression fragment is obtainable from seeds, a representative sample of which has been deposited under accession number NCIMB 42585, b) harvesting the fruits of the melon plants grown in step a), and optionally c) collecting the seeds from the fruits obtained in step b).

12. A method for producing hybrid melon seeds comprising the following steps a) crossing a first inbred melon plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment confers ToLCNDV resistance and comprises a Cytosine (C) for SNP_07 at nucleotide 101 of SEQ ID NO: 25, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 25, and/or a Guanine (G) for SNP_08 at nucleotide 101 of SEQ ID NO: 26, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 26, and wherein the introgression fragment is obtainable from seeds, a representative sample of which has been deposited under accession number NCIMB 42585, with a second inbred melon plant with or without a chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment comprises a Cytosine (C) for SNP_07 at nucleotide 101 of SEQ ID NO: 25, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 25, and/or a Guanine (G) for SNP_08 at nucleotide 101 of SEQ ID NO: 26, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 26, and wherein the introgression fragment is obtainable from seeds, a representative sample of which has been deposited under accession number NCIMB 42585, and b) selecting seeds obtained from the cross of step a).

13. A method for producing a melon fruit comprising the following steps: a) growing a plant comprising at least one chromosome 5 having an introgression fragment from chromosome 5 of a ToLCNDV resistant donor plant, wherein the introgression fragment confers ToLCNDV resistance and comprises a Cytosine (C) for SNP_07 at nucleotide 101 of SEQ ID NO: 25, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 25, and/or a Guanine (G) for SNP_08 at nucleotide 101 of SEQ ID NO: 26, or at the equivalent nucleotide of a sequence comprising at least 94% sequence identity to SEQ ID NO: 26, and wherein the introgression fragment is obtainable from seeds, a representative sample of which has been deposited under accession number NCIMB 42585, and b) harvesting the fruits produced by the plants grown in step a).

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Shown is a ToLCNDV sensitive recurrent plant (upper picture) and a recurrent plant into which ToLCNDV resistance was integrated (lower picture) by introgression of a fragment comprising the sequence of the donor in-between SNP_01 and SNP_06. The picture was taken 25 days post infection (dpi) with ToLCNDV by whitefly transmission.

(2) FIG. 2: Shown are the symptom levels 35 days post infection (dpi) with ToLCNDV by whitefly transmission of a donor plant being ToLCNDV resistant (Wild Donor), a recurrent plant (Recurrent) and a plant obtained after introgression of the ToLCNDV resistance from the donor plant into the recurrent plant (Introgression). The symptom levels were determined as described herein under “General Methods”.

GENERAL METHODS

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

(4) 1.1 Plants and Pathogens (Virus)

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

(6) 1.2 ToLCNDV Propagation

(7) The ToLCNDV inoculum source is 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) are fed on ToLCNDV sensitive (susceptible), infected melon plants in an insect proven cage. Before infection of test plants, put ToLCNDV infected plants into an insect proven cage, release whiteflies to the same cage and allow the whiteflies to feed for approximately 3 days on the ToLCNDV infected plants.

(8) 1.3 Inoculation of Plants to be Tested

(9) 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 on ToLCNDV resistance were put 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, before 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 that the whiteflies used for infection were free of ToLCNDV.

(10) 1.4 Growing Infected Test Plants

(11) 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 comprises 6 ToLCNDV infected plants and 1 mock infected plant. The plots are randomized in respect to the growing area.

(12) 1.5. Scoring the Symptom Level of ToLCNDV Infection

(13) 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. In case plants are present which show recovery from the virus infection, a further scoring of the symptom is done approximately 45 days post infection (dpi) with ToLCNDV.

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

(15) TABLE-US-00004 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

(16) 1.6 Optional Additional Tests

(17) It is recommended to use at least one genotype highly resistant to ToLCNDV (symptom level 8-9) and one genotype highly sensitive to ToLCNDV (symptom level 1) in each experimental setup. It is further recommended to also include a genotype being intermediate resistant to ToLCNDV infection in each test setup. Best results are obtained when the just mentioned genotypes are included and the symptom levels of each genotype is scored relative to the results obtained for the highly resistant, highly sensitive and intermediate resistant genotypes. These genotypes also give a clear indication on the amount of infection of melon plants with ToLCNDV by the whiteflies.

(18) 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 amount of virus DNA in upper parts of the plants. A suitable way to check for the presence and 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. Likewise PCR or quantitative PCR techniques can be used.

EXAMPLES

(19) 1. Selection of ToLCNDV Resistant Donor Plants

(20) 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 a resistance level of about 7 (as seen further below the average disease score was 7.4, while the susceptible plant had an average score of 2.0).

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

(22) Three mapping populations were developed including the use of the donor plant obtained in Example 1 to map the position of the ToLCNDV resistance conferring fragment (QTL) in the genome of donor melon plant.

(23) Analysis in these mapping populations revealed one major QTL associated with resistance, located on chromosome 5 and showing dominant inheritance patterns.

(24) The magnitude of the detected fragment QTL and observed inheritance patterns suggested a single locus dominant gene. From resistant material the inventors developed BC (back cross) lines to fine map and further investigate resistance from the donor. Genotypic results in 10 advanced BC families developed through phenotypic selection showed >93% agreement with individual phenotypes.

(25) 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 the following Table:

(26) TABLE-US-00005 Pseudo- molecule SNP coor- SNP Marker ID position dinates AI_13-H12 CM3.5_scaffold00003 7996720 27276249 SNP_01 mME11320_k CM3.5_scaffold00003 7824960 SNP_02 mME43070_k CM3.5_scaffold00003 6950286 SNP_03 mME10621_k CM3.5_scaffold00003 6553350 SNP_04 mME50729_k CM3.5_scaffold00003 5785550 SNP_05 mME32395_k CM3.5_scaffold00003 5202092 CMGAAN144 CM3.5_scaffold00003 5123876 24403405 CMPSNP682 CM3.5_scaffold00009 4703882 15854444 SNP_06 mME49184_k CM3.5_scaffold00009 2607437 CMPSNP460 CM3.5_scaffold00009 1816109 12966671

(27) 3. Development of KASP-Assay

(28) A KASP-assay was developed for identifying the SNPs flanking the QTL. The SNPs associated with the QTL can be determined by use of the following primers in a KASP-assay:

(29) TABLE-US-00006 SNP FAM allele VIC allele Common Primer SNP_01 SEQ ID NO 7 SEQ ID NO 8 SEQ ID NO 9 SNP_02 SEQ ID NO 10 SEQ ID NO 11 SEQ ID NO 12 SNP_03 SEQ ID NO 13 SEQ ID NO 14 SEQ ID NO 15 SNP_04 SEQ ID NO 16 SEQ ID NO 17 SEQ ID NO 18 SNP_05 SEQ ID NO 19 SEQ ID NO 20 SEQ ID NO 21 SNP_06 SEQ ID NO 22 SEQ ID NO 23 SEQ ID NO 24

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

(31) Backcrossing has been performed with cultivated melon plants and recurrent melon plants having an increased resistance to ToLCNDV have been obtained. The presence of the QTL in these lines was established by use of the KASP-assay sequences shown in Example 3. A representative result for the ToLCNDV symptom level achieved in those plants is given in the following Table and in FIG. 2. The symptom levels have been determined 35 days post infection (dpi) according to the method described herein under “General Methods”.

(32) TABLE-US-00007 AVG STDV Wild Donor 7.40 2.07 Recurrent 2.00 0.00 Introgression 5.11 1.45

(33) AVG: Average Value of Tested Genotype, STDV: Standard Deviation

(34) Additional backcrossing is done to introgress the resistance in elite Piel de Sapo, Galia, Cantaloup and Charentais backgrounds. Genotypic results in 10 advanced BC families developed through phenotypic selection show >93% agreement with individual phenotypes.

(35) 5. Additional SNP Markers Closely Linked to the Resistance

(36) Two additional markers (SNP_07 and SNP_08) were found closely linked to the resistance QTL, located in between the previous SNP_03 and SNP_04 on chromosome 5. Previously SNP_03 was found to be most closely linked to the QTL. In the further analysis SNP_08 was found most closely linked and SNP_07 was found to be closely linked and also to be unique in a large panel of melon germplasm.

(37) TABLE-US-00008 TABLE 3 Strand Position of orientation SNP haplotype SNP on of marker of introgression Melon sequence fragment, reference with comprising the genome respect of SNP donor (DHL92) reference genotype nucleotide for v3.5.1 genome of resistant one or more or (cucurbitgeno Marker SNP DHL92 donor all of the SNP mics.org name marker v3.5.1 SNP (homozygous) markers database) mME49184 SNP_06 (nt Forward (+ A/G AA A 13758001 445 of SEQ strand) ID NO 6) mME32395 SNP_05 (nt Reverse (− C/T CC (GG when C (G when 24481621 839 of SEQ strand) referring to referring to ID NO 5) forward strand) forward strand) mME50729 SNP_04 (nt Forward (+ A/G AA A 25065080 227 of SEQ strand) ID NO 4) mME72209 SNP_08 (nt Reverse (− A/G GG (CC when G (C when 25257353 101 of SEQ strand) referring to referring to ID NO: 26) forward strand) forward strand) mME72207 SNP_07 (nt Reverse (− C/T CC (GG when C (G when 25361598 101 of SEQ strand) referring to referring to ID NO: 25) forward strand) forward strand) mME10621 SNP_03 (nt Forward (+ C/T TT T 25832880 68 of SEQ strand) ID NO 3) mME43070 SNP_02 (nt Forward (+ G/T TT T 26229815 945 of SEQ strand) ID NO 2) mME11320 SNP_01 (nt Reverse (− C/T CC (GG when C (G when 27104489 101 of SEQ strand) referring to referring to ID NO 1 forward strand) forward strand)

(38) It is noted that the DNA sequences for SNP_01, SNP_05, SNP_07 and SNP_08 are the reverse strand (− strand) with respect of the reference genome sequence found on the world wide web at cucurbitgenomics.org for melon (DHL92) v3.5.1. This can be seen by Blast analysis of the sequence against the reference genome database, resulting in an alignment with either the forward strand (wherein nucleotide numbering is counting upwards) or with the reverse strand/complement strand whereby nucleotide numbering is counting downwards. It is understood that this is just a matter of naming. So a nucleotide C for SNP_01 at nucleotide 101 in SEQ ID NO: 1 corresponds to the nucleotide G for SNP_01 at nucleotide 101 of the complementary sequence of SEQ ID NO: 1. When referring to the donor SNP nucleotide in a reverse strand sequence, such reference thus also comprises a reference to the complement donor SNP nucleotide in the complement/forward strand of the sequence (and vice versa, reference to a donor nucleotide in a forward strand sequence can also be referred to by referring to the complement donor nucleotide in the reverse strand).

(39) It is also noted that for SEQ ID NO: 26, when BLAST analysis against the database of the reference genome Melon (DHL92) v3.5.1 (world wide web at cucurbitgenomics.org) is done, shows that in this reference genome there are 10 additional nucleotides inserted between nucleotide 27 and 28 of SEQ ID NO: 26. There is, therefore, only a sequence identity of 94.79% given between SEQ ID NO: 26 and the reference genome region. However, an ‘A’ is present in the reference genome at the SNP position corresponding to nucleotide 101 of SEQ ID NO: 26 (which is a ‘G’).

(40) The donor nucleotide of any of the SNP markers herein may thus be present in the SEQ ID NO: mentioned, or at the corresponding nucleotide of a sequence comprising at least 94% (or at least 95%, 96%, 97%, 98%, 99%) sequence identity to the given sequence. This applies to all SNP markers. The ‘corresponding nucleotide’ can be easily identified by pairwise alignment using e.g. Emboss-Needle pairwise alignment (using default parameters).

(41) The above SNP markers, including markers SNP_07 (mME72207) and SNP_08 (mME72209), were analyzed in a KASP assay (provided below) for 210 diverse melon material. The donor SNP nucleotide for SNP_07 (mME72207), i.e. a ‘C’ at nucleotide 101 of SEQ ID NO: 25 (corresponding to a G at nucleotide 101 of the strand complementary to SEQ ID NO: 25) was unique to the donor and not found in any of the other melon accessions analyzed.

(42) SNP_08 (mME72209) had the best correlation to the phenotype in the mapping population.

(43) A KASP-assay was developed for identifying the SNPs flanking the QTL. The SNPs associated with the QTL can be determined by use of the following primers in a KASP-assay:

(44) TABLE-US-00009 SNP FAM allele VIC allele Common Primer SNP_07 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SNP_08 SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32

(45) A total of 210 diverse melon lines, accessions and varieties were analyzed in a KASP assay for SNP_01 to SNP_06. Below the SNP haplotype for some of the material is given. It is noted that for SNP_01, SNP_05, SNP_07 and SNP_08, the assay was based on detection of the SNP nucleotide in the reverse strand, which was converted into the SNP nucleotide in the complementary (forward) strand in the data presented below.

(46) TABLE-US-00010 SNP Haplotype of ToLCNDV donor 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 SNP_06 A A A G G A A A A G A A G A A G SNP_05 G A A G A A G G A A G G A G G G SNP_04 A A A A A A A A A A A A A A G A SNP_08 C C C C T T T T T T T T T T T T SNP_07 G A A A A A A A A A A A A A A A SNP_03 T C C T C C C T T T T T T T C T SNP_02 T G T G T T T G T T G G G T G T SNP_01 G A A G G A A G A G A A G G A G

(47) SNP_07 and/or SNP_08 are markers closely linked to the QTL and can be used to detect the introgression fragment comprising the QTL and comprising the donor nucleotide for SNP_07 and/or SNP_08, or other applications described herein.