Melon plants resistant to geminivirus

Abstract

The present invention relates to a melon plant that is resistant to a Geminivirus, wherein the plant comprises a TDG1 resistance gene encoding a TDG1 resistance protein. The present invention further relates to the TDG1 resistance gene for providing Geminivirus resistance in Cucurbita or Solanum plants, preferably a melon plant, and a resistance locus providing Geminivirus resistance in Cucurbita or Solanum plants, wherein said resistance locus comprises a TDG1 resistance gene. Additionally the present invention relates to methods for identifying and / or selecting a Geminivirus resistant melon plant and a method for providing said resistant melon plant.

Description

[0001] MELON PLANTS RESISTANT TO GEMINIVIRUS

[0002] Description

[0003] The present invention relates to a melon plant that is resistant to a Geminivirus, wherein the plant comprises a TDG1 resistance gene encoding a TDG1 resistance protein. The present invention further relates to the TDG1 resistance gene for providing Geminivirus resistance in Cucurbita or Solanum plants, preferably a melon plant, and a resistance locus providing Geminivirus resistance in Cucurbita or Solanum plants, wherein said resistance locus comprises a TDG1 resistance gene. Additionally, the present invention relates to methods for identifying and / or selecting a Geminivirus resistant melon plant and a method for providing said resistant melon plant.

[0004] Geminiviruses are a family of plant viruses characterized by their twinned (geminate) particles and a single-stranded DNA genome. They belong to the family Gemini viridae. Geminiviruses are among the most economically important plant viruses worldwide, causing significant losses in crop yields. Some of the common plants that geminiviruses infect include: tomato, pepper, cucurbits including squash, melon, and cucumber, potato and tobacco. Since 2013, Tomato leaf curl New Delhi virus (ToLCNDV) is one of the most dominant geminivirus present causing high economical losses in crop production, especially in Asia and Europe.

[0005] ToLCNDV is a plant virus that infects tomatoes and other crops, including melon, cucumber and pumpkin. ToLCNDV belongs to the Begomo virus genus in the family Geminiviridae. It was first reported in New Delhi, India, hence its name, but has since spread to other regions, including the Middle East, North Africa, and parts of Europe. ToLCNDV is transmitted by whiteflies, such as Bemisia tabaci, which are difficult to control. Whiteflies can rapidly spread the virus within and between fields, exacerbating the problem of ToLCNDV infections.

[0006] Symptoms of ToLCNDV infection in plants typically include the curling or rolling upwards of the leaves, especially the younger ones. Leaves may also show yellowing (chlorosis) and clearing of the veins. Infected plants may display stunted growth resulting in smaller overall size compared to healthy plants. The overall yield of the infected plants is often reduced due to the impact on growth and development. Fruits from infected plants may be smaller, deformed, or exhibit uneven ripening. Some varieties of tomato and melon plants may show necrotic spots on leaves and stems.

[0007] ToLCNDV is a serious disease that can cause significant losses in melon. At present, there are no commercially available ToLCNDV resistant melon plants. As the use of pesticides is becoming scrutinized ever more strictly, Geminiviruses including ToLCNDV are impacting crop yield, such as melon, and fruit quality more severely every year. Management strategies for ToLCNDV typically involve controlling whitefly populations, continual development of resistant cultivars, and practicing good agricultural practices to minimize the spread of the virus. Control or prevention of ToLCNDV infection is at present focused on limiting the spread of virus including limiting virus sources and vector populations or interfering with vector activities to regulate the population of white fly vectors and / or their virus load. Breeding for resistance is the most attractive and durable method of controlling ToLCNDV.

[0008] Considering the above, there is a need in the art for a more diverse and durable resistance in Cucurbitaceae crops against Geminivirus, preferably against ToLCNDV infection in melon. Furthermore, it is an object of present invention to provide a method to obtain such Geminivirus resistant melon plants. There is a need for new or more diversity of resistance genes such that genetic variation can be achieved in commercial crops, making it harder for Gemini viruses to adapt and break resistance.

[0009] It is an object of the present invention, amongst other objects, to address the above need in the art. The object of present invention, amongst other objects, is met by the present invention as outlined in the appended claims.

[0010] Specifically, the above object, amongst other objects, is met, according to a first aspect, by the present invention by a Melon plant (Cucumis Melo) that is resistant to a Geminivirus, wherein the plant comprises a TDG1 resistance gene encoding a protein having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 3. The Geminivirus resistant melon plant of present invention comprising the TDG1 resistance gene is an agronomical elite plant and provides melon fruit that are edible and have a sweet taste due to its elevated Brix levels of the melon fruit. Although resistant to the Geminivirus, the plant of present invention may not show resistance to whitefly, which is known as the primary viral vector. Therefore, the TDG1 resistance gene does not seem to affect virus transmission due to whitefly but provides Geminivirus resistance to the plant trough a different, yet to be elucidated mechanism. A single dominant ToLCNDV resistance has not been described before in melon. The TDG1 resistance gene is a single dominant ToLCNDV resistance gene, providing a high level of disease resistance in melon plants. It is the first R gene identified in Cucurbits for ToLCNDV resistance.

[0011] According to a preferred embodiment, the present invention relates to the melon plant wherein the TDG1 resistance gene encodes for a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.2.

[0012] According to yet another preferred embodiment, the present invention relates to the Melon plant wherein the resistance locus comprises a genomic sequence having at least 98%, preferably at least 99%, more preferably 100% sequence identity with SEQ ID No.l. The resistance locus comprises the TDG1 resistance gene providing Geminivirus resistance to the melon plant.

[0013] According to another preferred embodiment, the present invention relates to the plant wherein the plant comprises a resistance locus on chromosome 5 comprising said TDG1 resistance gene, wherein said resistance locus is linked to marker of SEQ ID No.10 having a C to G on position 26142771, as determined in Table 1 below in view of the public reference genome (DHL92 V4.0, http: / / cucurbitgenomics.org / ) which is susceptible to ToLCNDV. The marker is unique for selecting a melon plant that comprises the TDG1 resistance gene and is resistant to Geminivirus, more specifically ToLCNDV.

[0014] According to a preferred embodiment, the present invention relates to the Melon plant wherein the plant is one or more selected from the group consisting of C. melo subsp. melo, C. melo subsp. agrestis, preferably C. melo subsp. melo. The term "melon" as used herein refers to the species Cucumis melo (C. melo) including subspecies melo (vars. cantalupensis, reticulatus, adana, chandalok, ameri, inodorus, flexuosus, chate, tibish, dudaim and chito), and subspecies agrestis (vars. conomon, makuwa, momordica and acidulous). Preferably the plant of the present in invention is a plant of any Cucumis melo subsp. melo var, including cultivated varieties of the Cucumis melo var. cantalupensis (i.e. Charantais or Cantaloupe fruit types), Cucumis melo var. reticulatis (i.e. Galia or Ananas fruit types), and Cucumis melo var. inodorus (i.e. Piel de Sapo, Amarillo, and Branco fruit types).

[0015] According to a preferred embodiment, the present invention relates to the Melon plant wherein the plant is a melon of the type selected from the group consisting of Galia, Piel de Sapo, Charentais, Cantaloupe, Amarillo, and Brancho, preferably Cantaloupe, more preferably Piel de Sapo, even more preferably Amarillo, most preferably Cantaloupe and Piel de Sapo.

[0016] According to a preferred embodiment, the present invention relates to the Melon plant, wherein the plant provides melon fruit having a Brix value of at least 5 degrees, preferably at least 10 degrees, more preferably at least 15 degrees, most preferably at least 20 degrees. The plant of present invention is preferably an agronomical elite plant providing edible, sweat tasting fruits that can be commercially sold on the consumer market.

[0017] According to another preferred embodiment, the present invention relates to the melon plant wherein the Geminivirus is one of more selected from the group consisting of Tomato leaf curl New Delhi virus (ToLCNDV), Squash leaf curl virus (SLCV), Cucumber leaf curl virus (CuLCV), preferably ToLCNDV.

[0018] According to yet another preferred embodiment, the present invention relates to the melon plant wherein the TDG1 resistance gene is heterozygously or homozygously present in the genome of said plant. It is sufficient that the TDG1 resistance gene is heterozygously present as the resistance trait is largely dominant, but in some case, it may be preferred to have the TDG1 resistance gene homozygously in the plant. In the case of heterozygous, the TDG1 resistance gene is also more easily combined with other known resistances on Chr 5, such as powdery mildew resistance or aphid resistance present on Chr 5. Homozygous presence of the TDG1 resistance gene may provide improved Geminivirus resistance and / or increased resistance to disease pressure in the Melon plant, however you may lose other resistances on Chr 5, as indicated above.

[0019] According to a preferred embodiment, the present invention relates to the melon plant wherein the TDG1 resistance gene and / or resistance locus is obtainable from deposit NCIMB 44205 seeds of a Geminivirus resistant melon plant according to present invention comprising the TDG1 resistance gene on Chr5 have been deposited at NCIMB Ltd, Aberdeen, Scotland on 9 August 2023 under reference NCIMB 44205.

[0020] According to a preferred embodiment, the present invention relates to the melon plant wherein the plant further comprises a TDG2 resistance gene encoding a TDG2 resistance protein having at least 95% sequence identity with SEQ ID No. 6, and / or a TDG3 resistance gene encoding a TDG3 resistance protein having at least 95% preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 9. Disease tests show that melon plants of present invention comprising the TDG1 resistance gene are resistant to ToLCNDV.

[0021] According to a preferred embodiment, the present invention relates to the melon plant wherein the TDG2 resistance gene comprises a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with with SEQ ID No.5, and / or wherein the TDG3 resistance gene comprises a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.8. The TDG2 and TDG3 resistance genes are located on chromosome 5 and are located very closely together on the resistance locus between Chr5: 26159606-26176476. Marker 2 (SEQ ID No.l l) and Marker 3 (SEQ ID No.12) are used to determine the presence of a locus comprising both the TDG2 and TDG3 gene of present invention on said resistance locus, see Table 1.

[0022] According to a preferred embodiment, the present invention relates to the Melon plant wherein the plant further comprises a Vat (Virus Aphid Transmission) gene encoding a Vat protein that provides resistance to aphid. The Vat gene is known to limit virus transmission by aphids in melon. Vat genes are part of an elaborate Vat gene cluster and characterized as R-genes, including CC-NBS-LRR, and many Vat genes have been identified in cucurbits and linked to limiting virus transmission by aphid. Copy-number polymorphisms and single-nucleotide polymorphisms have also been identified in the Vat cluster (for example V. Chovelon et al., Horticulture Research Vol.8, 72 (2021)). The role of these polymorphisms in plant / aphid interactions remains unclear. Disease tests show that melon plants of present invention comprising the TDG1 resistance gene are resistant to ToLCNDV. Furthermore, on a separate note, preliminary experiments show that melon plants comprising TDG2 and / or TDG3 resistance gene may also provide ToLCNDV resistance. The TDG2 and TGD3 resistance genes are found in the deposit NCIMB 44205, as disclosed herein.

[0023] According to a further aspect, the present invention relates to a Melon plant (Cucumis Melo) that is resistant to a Geminivirus, wherein the plant comprises a TDG2 resistance gene encoding a protein having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 6.

[0024] According to a preferred embodiment, the present invention relates to the melon plant wherein the TDG2 resistance gene comprises a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.5.

[0025] According to yet another preferred embodiment, the present invention relates to the Melon plant wherein the resistance locus comprises a genomic sequence having at least 98%, preferably at least 99%, more preferably 100% sequence identity with SEQ ID No.4. The resistance locus comprises the TDG2 resistance gene providing Geminivirus resistance to the melon plant.

[0026] According to a further aspect, the present invention relates to a Melon plant (Cucumis Melo) that is resistant to a Geminivirus, wherein the plant comprises a TDG3 resistance gene encoding a protein having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 9.

[0027] According to a preferred embodiment, the present invention relates to the melon plant wherein the TDG3 resistance gene encodes for a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.8.

[0028] According to yet another preferred embodiment, the present invention relates to the Melon plant wherein the resistance locus comprises a genomic sequence having at least 98%, preferably at least 99%, more preferably 100% sequence identity with SEQ ID No.7. The resistance locus comprises the TDG3 resistance gene providing Geminivirus resistance to the melon plant.

[0029] The present invention, according to a further aspect, relates to a TDG1 resistance gene for providing Geminivirus resistance in a Cucurbita or Solanum, preferably a Cucumis Melo plant, wherein the TDG1 resistance gene encodes for a TDG1 resistance protein having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.3.

[0030] According to a preferred embodiment, the present invention relates to the TDG1 resistance gene, wherein the TDG1 resistance gene encodes for a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.2.

[0031] The present invention, according to a further aspect, relates to a protein for Geminivirus resistance in a Cucurbita or Solanum plant, preferably a Cucumis Melo plant, wherein the protein has at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.3.

[0032] The present invention, according to a further aspect, relates to a combination of two or more TDG resistance genes for providing Geminivirus resistance in a Cucurbita or Solanum plant, preferably a Cucumis Melo plant, wherein the two or more TDG resistance genes encodes for a TDG resistance protein selected from the group consisting of TDG1 having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%sequence identity with SEQ ID No. 3, TDG2 having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 6, and TDG3 having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 9, preferably all three of said TDG genes are combined for providing Geminivirus resistance in a Cucurbita or Solanum plant.

[0033] The present invention, according to a further aspect, relates to a resistance locus providing Geminivirus resistance in a Cucurbita or Solanum plant, preferably a Cucumis Melo plant, wherein said resistance locus comprises a TDG1 resistance gene, wherein said resistance locus is linked to marker of SEQ ID No.10 having a C to G on position 26142771, as determined in Table 1 (in view of reference genome that is susceptible to ToLCNDV), and / or said resistance locus comprises a genomic sequence having at least 98%, preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.1.

[0034] According to a preferred embodiment, the present invention relates to the resistance locus, further comprising a TDG2 resistance gene encoding a TDG2 resistance protein having at least 95% sequence identity with SEQ ID No. 6 and / or a TDG3 resistance gene encoding a TDG3 resistance protein having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 9, and / or said resistance locus further comprises a genomic sequence having at least 98%, preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.4 and / or SEQ ID No.7. The present invention, according to a further aspect, relates to a seed, plant cell, plant tissue, fruit, or plants parts of a melon plant of present invention comprising the TDG1 resistance gene providing resistance to a Gemini virus.

[0035] The present invention, according to a further aspect, relates to a method for identifying and / or selecting a Melon plant, preferably a Cucumis Melo plant, that is resistant to a Geminivirus, preferably resistant to ToLCNDV, wherein said method comprises the step of establishing the presence of a TDG1 resistance gene, and optionally further the presence of a TDG2 and / or TDG3 resistance gene, providing said resistance to Geminivirus in said plant, wherein said method comprises the step of establishing in said plant the presence of; a resistance locus that comprises the TDG1 resistance gene wherein the TDG1 resistance gene encodes for a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.2, preferably the resistance locus further comprises a TDG2 resistance gene encoding a TDG2 resistance protein having at least 95% preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 6 and / or a TDG3 resistance gene encoding a TDG3 resistance protein having at least 95% sequence identity with SEQ ID No. 9, and / or said resistance locus further comprises a genomic sequence having at least 98% preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.4 and / or SEQ ID No.7 ; and / or a genomic sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.1 ; and / or a TDG1 resistance gene comprised of a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.2; and / or, a TDG1 resistance protein having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.3; and / or a TDG2 resistance gene comprised of a coding sequence having at least 95% preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.5, and / or a TDG3 resistance gene comprised of a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.8.

[0036] According to a preferred embodiment, the present invention relates to the method for identifying and / or selecting a Melon plant that is resistant to a Geminivirus, wherein the step of establishing in said plant the presence of said locus that comprises the TDG1 resistance gene and / or said TDG1 resistance gene comprises the TDG1 resistance gene and / or said TDG1 resistance gene comprises the determination of the presence of a SNPs on Chr5 of a C to G on position 26142771, as determined in Table 1 below in view of a susceptible melon plant as reference genome (DHL92 V4.0.).

[0037] The present invention, according to a further aspect, relates to a method for providing a Melon plant that is resistant to a Geminivirus, wherein the method comprises the steps of, a) crossing a Melon plant comprising a TDG1 resistance gene, and optionally further comprising a TDG2 and / or TDG3 resistance gene, as defined herein with a Melon plant that is susceptible to said Geminivirus and does not comprise said TDG1 resistance gene, and optionally not further comprising said TDG2 and / or TDG3 resistance gene, b) optionally, selfing the plant obtained in step a) for at least one time, c) selecting the plants that are resistant to said Geminivirus, wherein said plant comprises said TDG1 resistance gene.

[0038] According to a preferred embodiment, the present invention relates to the method for providing a Melon plant that is resistant to a Geminivirus, wherein the selecting of step c is done by establishing in said plant the presence of a locus that comprises the TDG1 resistance gene, preferably the determination of the presence of the SNPs on Chr5 as described in Table 1 using the marker of SEQ ID No.10, and / or, a genomic sequences having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.1 ; and / or a TDG1 resistance gene comprised of a coding sequence having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.2; and / or a resistance protein having at least 95%, preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.3; and / or a TDG2 resistance gene encoding a TDG2 resistance protein having at least 95% preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% sequence identity with SEQ ID No. 6; and / or a TDG3 resistance gene encoding a TDG3 resistance protein having at least 95% sequence identity with SEQ ID No. 9, and / or said resistance locus further comprises a genomic sequence having at least 98% preferably at least 99%, most preferably 100% sequence identity with SEQ ID No.4 and / or SEQ ID No.7. The present invention, according to a further aspect, relates to the use of a TDG1 resistance gene or resistance locus as defined herein for providing Geminivirus resistance in a Cucurbita or Solanum plant, preferably a Cucumis Melo plant.

[0039] The present invention will be further detailed in the following examples and figures wherein:

[0040] Figure 1: shows a Geminivirus resistant melon plant according to the present invention comprising the TDG1 resistance gene, approximately about three weeks (23 days post inoculation) after inoculation with ToLCNDV. The left panel shows the plant that was not inoculated with virus, the right panel shows the plant that is inoculated with virus. The inoculated melon plant remains green and free of disease symptoms, resulting in a disease score of 9, comparable to the control.

[0041] Figure 2: shows a Geminivirus susceptible melon plant not comprising the TDG1 resistance gene of present invention, approximately three weeks after inoculation with TolCNDV. The susceptible plant inoculated with virus (right panel) shows severe spotting of the leaves, and the plant is very affected and cannot develop, resulting in a disease score of 3. The control plant (left panel) that was not inoculated remained green and free of disease symptoms, resulting in a disease score of 9.

[0042] Examples

[0043] Example 1- Fine mappins of the Geminivirus TDG1 resistance sene in Melon

[0044] Geminivirus resistance, more specifically ToLCNDV resistance, was identified in nonedible melon accession. This wild melon is not edible due to poor taste and low to no Brix value of the melon fruit. The Geminivirus resistance locus was identified in a wild melon species after resistance screening in ToLCNDV inoculated melon plants (See example 2). The resistance locus was subsequently crossed into F2 melons of C. melo, type Galia. The Geminivirus resistance locus was subsequently screened for the identification of candidate resistance gene(s) involved. About 400 F2 melon plants were initially used to rough map the region to chromosome 5, and validation of this QTL narrowed to a region of 3,3Mb. Following the identification of this single QTL, 2500 additional F2 plants we were screened to generate 140 F3 recombinant families. Using these F3 families it was possible to reduce the region to 100Kb on the De Novo genome. On the public reference genome (DHL92 V4.0, http: / / cucurbitgenomics.org / ) this region is equivalent to a 34Kb region between positions Chr5: 26142771-26176476. In the public reference there are two full genes in this region. Expression data based on RNA expression levels from the ToLCNDV resistant melon plant of present invention indicated three full length genes in the De Novo genome in the identified region. In the previously identified region only three regions, corresponding to the three identified TDG genes, showed increased RNA expression levels. The full genes annotated herein as the herein identified TDG1 resistance gene encoding for a TDG1 resistance protein, TDG2 resistance gene encoding for a TDG2 resistance protein, TDG3 resistance gene encoding for a TDG3 resistance protein. The genomic-, coding- and protein sequences of the identified TDG1 resistance gene are as included in the sequence listing herein, SEQ ID No’s. 1 to 3, respectively, and for TDG2 (SEQ ID No. 4 to 6, gDNA, cDNA and protein respectively), for TDG3 (SEQ ID No. 7 to 9, respectively). A unique marker, Marker 1 (SEQ ID No.10) is used to determine the presence of the locus comprising the TDG1 resistance gene of present invention, on the basis of SNP (underlined and bold) linked to the Geminivirus resistance, see Table 1. TDG2 and TDG3 are located very closely together on the resistance locus between Chr5: 26159606-26176476. Marker 2 (SEQ ID No.11) and Marker 3 (SEQ ID No.12) are used to determine the presence of a locus comprising both the TDG2 and TDG3 gene of present invention on said locus, see Table 1.

[0045] Table 1. Marker for identification of TDG1 resistance gene in resistant melon plants

[0046] Disease tests show that melon plants of present invention comprising the TDG1 resistance gene are resistant to ToLCNDV. Furthermore, on a separate note, preliminary experiments show that melon plants comprising TDG2 and / or TDG3 resistance gene may also provide ToLCNDV resistance.

[0047] Seeds of a Geminivirus resistant melon plant according to present invention comprising the TDG1 resistance gene on Chr5 have been deposited at NCIMB Ltd, Aberdeen, Scotland on 9 August 2023 under reference NCIMB 44205. Example 2 - Disease tests of melon plants comprising the TDG1 resistance gene

[0048] Melon plants were screened to identify for Geminivirus resistance, more specifically ToLCNDV resistance, in a test performed in controlled inoculation (controlled white fly test) with the virus and scored for disease symptoms. This test was confirmed by a controlled infection using the white fly vector, wherein the disease symptoms were scored by rating on a scale of 1 being susceptible to 9 being resistant. Plants having a score of 9 were identified as fully resistant and selected for further analysis.

[0049] Briefly, young melon plants at the 2-4 true leaf stage, were infected with ToLCNDV using Bemisia tabaci in a Greenhouse (~24 °C) inside a net cage for inoculation. Disease observations was initiated at about 21 days post inoculation and continued for additional 3 to 4 weeks or until no new disease progression was observed, detecting spotting and curling of the leaves. Disease symptoms are scored according to a scale 1 to 9, wherein;

[0050] 9. Fully resistant. No symptoms (0% symptoms on leaf surface).

[0051] 8. Very resistant. There may be signs of symptoms appearing or one or two spots are visible (<2%).

[0052] 7. Resistant. It can present some sporadic spotting (<5%) but it does not affect the development of the plant.

[0053] 6. High intermediate resistance. Symptoms are present (35%) and the plant is somewhat affected.

[0054] 5. Low intermediate resistance. It presents enough symptoms, on the border of being a susceptible plant (60%). The development of the plant is affected.

[0055] 4. Susceptible. Many symptoms (75%). The plant is quite affected in its development.

[0056] 3. Susceptible (>75%). The plant is very affected and cannot develop.

[0057] 2. Very susceptible (>95%), plant almost dead.

[0058] 1. Plant dead

[0059] Disease resistance was tested on a melon plant according to the present invention (plant 1), comprising the TDG1 resistance gene. Plants were tested and scored in Spain and Thailand. A Vedrantais melon plant was included as positive control which is known to be susceptible to ToLCNDV, see Table 2.

[0060] Table 2. Disease score of melon plants inoculated with ToLCNDV In a further experiment, Plant 1 was again tested in a disease experiment, including the susceptible Vedrantais melon plant, and furthermore including the Fl of a crossing of plant 1 x Vedrantais. The Fl plants were shown to be fully resistant to ToLCNDV, having a disease score of 9, comparable to their resistant patent Plant 1, see Table 3.

[0061] Table 3. Disease score monitored over time of melon plants inoculated with ToLCNDV

[0062] PCT

[0063] (Original in Electronic Form)

[0064] (This sheet is not part of and does not count as a sheet of the international application)

[0065] FOR RECEIVING OFFICE USE ONLY

[0066] FOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS1. Melon (Cucumis Melo) plant that is resistant to a Gemini virus, wherein the plant comprises a TDG1 resistance gene encoding a TDG1 resistance protein having at least 95% sequence identity with SEQ ID No. 3.

2. Melon plant according to claim 1, wherein the TDG1 resistance gene encodes for a coding sequence having at least 95% sequence identity with SEQ ID No.2.

3. Melon plant according to claim 1 or 2, wherein the plant comprises a resistance locus on chromosome 5 comprising a genomic sequence having at least 98% sequence identity with SEQ ID No.1.

4. Melon plant according to any one of the claims 1 to 3, wherein TDG1 resistance gene and / or said resistance locus is linked to marker of SEQ ID No.10 having a C to G on position 26142771, as determined in Table 1.

5. Melon plant according to any one of the claims 1 to 4, wherein the plant is one or more selected from the group consisting of C. melo subsp. melo, C. melo subsp. agrestis, preferably C. melo subsp. melo.

6. Melon plant according to any one of the claims 1 to 5, wherein the plant is a melon of the type selected from the group consisting of Galia, Piel de Sapo, Charentais, Cantaloupe, Amarillo, and Brancho, preferably Cantaloupe, more preferably Piel de Sapo, even more preferably Amarillo, most preferably Cantaloupe and Piel de Sapo.

7. Melon plant according to any one of the claims 1 to 6, wherein the plant provides melon fruit having a Brix value of at least 5 degrees, preferably at least 10 degrees, more preferably at least 15 degrees, most preferably at least 20 degrees.

8. Melon plant according to any one of the claims 1 to 7, wherein the Gemini virus is one of more selected from the group consisting of Tomato leaf curl New Delhi virus (ToLCNDV), Squash leaf curl virus (SLCV), Cucumber leaf curl virus (CuLCV), preferably ToLCNDV9. Melon plant according to any one of the claims 1 to 8, wherein the TDG1 resistance gene is heterozygous or homozygous present in the genome of said plant, preferably heterozygous.

10. Melon plant according to any one of the claims 1 to 9, wherein the TDG1 resistance gene and / or resistance locus is obtainable from deposit NCIMB 44205.

11. Melon plant according to any one of the claims 1 to 10, wherein the plant further comprises a TDG2 resistance gene encoding a TDG2 resistance protein having at least 95% sequence identity with SEQ ID No. 6, and / or a TDG3 resistance gene encoding a TDG3 resistance protein having at least 95% sequence identity with SEQ ID No. 9.

12. Melon plant according to claim 11, wherein the TDG2 resistance gene encodes for a coding sequence having at least 95% sequence identity with SEQ ID No.5, and / or wherein the TDG2 resistance gene encodes for a coding sequence having at least 95% sequence identity with SEQ ID No.8.

13. Melon plant according to any one of the claims 1 to 12, wherein the plant further comprises a Vat gene encoding a Vat protein that provides resistance to aphid.

14. A TDG1 resistance gene for providing Geminivirus resistance in a Cucurbita or Solanum plant, preferably a Cucumis Melo plant, wherein the TDG1 resistance gene encodes for a TDG1 resistance protein having at least 95% sequence identity with SEQ ID No.3.

15. The TDG1 resistance gene of claim 14, wherein the TDG1 resistance gene encodes for a coding sequence having at least 95% sequence identity with SEQ ID No.2.

16. A TDG1 resistance protein for providing Geminivirus resistance in a Cucurbita or Solanum, preferably a Cucumis Melo plant, wherein the protein has at least 95% sequence identity with SEQ ID No.3.

17. A resistance locus providing Geminivirus resistance in a Cucurbita or Solanum plant, preferably a Cucumis Melo plant, wherein said resistance locus comprises a TDG1 resistance gene of claim 14 or 15, wherein said resistance locus comprises a genomic sequence having at least 98% sequence identity with SEQ ID No.1 , and / or said resistance locus is linked to marker of SEQ ID No.10 having a C to G on position 26142771, as determined in Table 1.

18. A Seed, plant cell, plant tissue, fruit, or plants parts of a melon plant according to any of claim 1 to 13 comprising a TDG1 resistance gene providing resistance to a Geminivirus according to claims 14 or 15.

19. Method for identifying and / or selecting a Melon plant, preferably a Cucumis Melo plant, that is resistant to a Geminivirus, preferably resistant to ToLCNDV, wherein said method comprises the step of establishing the presence of a TDG1 resistance gene, and optionally further the presence of a TDG2 and / or TDG3 resistance gene, providing said resistance to Geminivirus in said plant, wherein said method comprises the step of establishing in said plant the presence of; a resistance locus that comprises the TDG1 resistance gene according to claim 17; and / or, a genomic sequences having at least 95% sequence identity with SEQ ID No.1; and / or a TDG1 resistance gene comprised of a coding sequence having at least 95% sequence identity with SEQ ID No.2; and / or a TDG1 resistance protein having at least 95% sequence identity with SEQ ID No.3; and / or a TDG2 resistance gene comprised of a coding sequence having at least 95% sequence identity with SEQ ID No.5, and / or a TDG3 resistance gene comprised of a coding sequence having at least 95% sequence identity with SEQ ID No.8.

20. Method according to claim 19, wherein the step of establishing in said plant the presence of said locus that comprises the TDG1 resistance gene and / or said TDG1 resistance gene comprises the determination of the presence of a SNPs on Chr5 of a C to G on position 26142771, as determined in Table 1.

21. Method for providing a melon plant that is resistant to a Geminivirus, wherein the method comprises the steps of, a) crossing a melon plant comprising a TDG1 resistance gene, and optionally further comprising a TDG2 and / or TDG3 resistance gene, as defined in any one of the claims 1 to 13 with a melon plant that is susceptible to said Geminivirus and does not comprise said TDG1 resistance gene, and optionally further not comprising said TDG2 and / or TDG3 resistance gene, b) optionally, selfing the plant obtained in step a) for at least one time, c) selecting the plants that are resistant to said Geminivirus, wherein said plant comprises said TDG1 resistance gene.

22. Method according to claim 21, wherein the selecting of step c is done by the method of claim 19 or 20.

23. Use of a TDG1 resistance gene or a resistance locus as defined in any one of the claims 14, 15, and 17 for providing Geminivirus resistance in a Cucurbita or Solanum plant, preferably a Cucumis Melo plant.

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