Lettuce plant with resistance to new isolates of fusarium oxysporum f. SP. lactucae

Abstract

The present invention relates to lettuce plants comprising a genomic fragment that provides resistance to new isolates of Fusarium oxysporum f. sp. lactucae. The present invention further relates to methods for identifying the present Fusarium -resistant lettuce plants and to methods for providing Fusarium-resistant lettuce plants. Specifically, the present invention relates to Lettuce plants resistant to Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471, wherein said resistance is encoded by a genomic fragment from Lactuca serriola located on chromosome 7, wherein the location of said resistance providing genomic fragment corresponds with positions 64.798.314 to 66.859.860 of chromosome 7 of the V8 lettuce reference genome, and wherein said resistance providing genomic fragment is obtainable from a lettuce plant of which representative seed is deposited under deposit number NCIMB 43937.

Description

[0001] LETTUCE PLANT WITH RESISTANCE TO NEW ISOLATES OF FUSARIUM OXYSPORUMF. SP. LACTUCAE

[0002] Description

[0003] The present invention relates to lettuce plants comprising a genomic fragment that provides resistance to new isolates of Fusarium oxysporum f. sp. lactucae. The present invention further relates to methods for identifying the present Fusarium -resistant lettuce plants and to methods for providing Fusarium-resistant lettuce plants.

[0004] Lettuce, also referred to as Lactuca saliva, is a cultivated plant that belongs to the daisy family (Asteraceae) . This family comprises over 23.000 species with a broad geographical distribution from the tropics to the polar region. The Asteraceae family includes, besides lettuce, several other food crops such as chicory, endive, sunflower, and artichoke.

[0005] Apart from lettuce, the genus Lactuca contains roughly 75 wild species. Although these species have undesirable agronomic traits, such as a bitter flavour, a high latex content, or leaf spines, they can be used to improve, for example, the taste, texture, and disease resistance of cultivated lettuce. The three wild species Lactuca saligna L. (least lettuce), Lactuca serriola L. (prickly lettuce), and Lactuca virosa L. (great lettuce) are, therefore, frequently used in cultivated lettuce breeding programs.

[0006] Lettuce is an annual crop mostly grown as a leaf vegetable. Lettuce produces crispy leaves and has an average size of 15-30 cm. The leaves are usually green, but red and other colours are also known. Leaves can have various shapes and textures. Breeding efforts have resulted in many different varieties of lettuce subdivided into seven different morphological types. The crisphead group contains iceberg and batavia lettuce types.

[0007] Iceberg lettuce has a large, firm head with a crisp texture and a white or creamy yellow interior. Batavia lettuce resembles the iceberg type but has a smaller and less firm head. The butterhead group has a small, soft head with an almost oily texture. The romaine, also known as cos lettuce, has elongated upright leaves forming a loose, loaf-shaped head and the outer leaves are usually dark green. Leaf lettuce, also known as cutting lettuce, comes in many varieties that do not form a head. Latin lettuce has a loose head with thick leathery leaves, dark green colour, and looks like a cross between romaine and butterhead. Stem lettuce has long, narrow leaves and thick, edible stems. Oilseed lettuce is a type grown for its large seeds that contain a high percentage of oil. Because of the bitter taste of its leaves, this type is not eaten as a vegetable.

[0008] Lettuce is commercially grown wherever environmental conditions permit the production of an economically viable yield. China is currently the largest producer of lettuce. Otherimportant markets are the USA and the EU. Lettuce is an increasingly popular crop as worldwide lettuce consumption continues to increase.

[0009] There are many plant pathogens that can affect lettuce. These pathogens can lead to significant financial losses for producers as they can reduce yield and make plants visually less appealing to the consumer. Examples of pathogens are bacteria (e.g., Sphingobium suberifaciens, Xanthomonas campestris pv. vitians), viruses (e.g., Lettuce mosaic virus, Mirafiori lettuce big-vein virus, Tomato spotted wilt virus), eukaryotic microorganisms (e.g., Bremia lactucae, Pythium, Fusarium oxysporum f. sp. lactucae, Sclerotinia sclerotiorum), nematodes (e.g., Meloidogyne incognita) and insects (e.g., aphids, thrips).

[0010] The fungus Fusarium oxysporum f. sp. lactucae (Fol) is the causal agent of Fusarium wilt and root rot in lettuce. The fungus is commonly found in soil and strains can be either pathogenic or non-pathogenic. Although F. oxysporum has a broad host range, strains display pathogenicity on a limited range of hosts. This observation has led to the concept of forma specials, which distinguish particular forms of the species (formae speciales. f. sp.) based on their adaptation to different hosts. For example, strains responsible for Fusarium wilt in lettuce belong to the forma specialis lactucae. This forma specialis contains four races that are distinguished from each other by their ability to cause disease on a differential set of lettuce cultivars, as well as by means of molecular tools developed to characterize different races of this pathogen. Race 1 (Fol: 1) is the most important race world-wide. Until recently, race 2 (Fol:2) and race 3 (Fol:3) were only found in Japan. Race 4 (Fol:4) was identified forthe first time in 2015 in The Netherlands and has spread to other countries including, Belgium, the U.K. and Italy.

[0011] The fungus Fusarium oxysporum f. sp. lactucae infects the plants through the root system. Accumulation of fungal biomass in the vascular system of the plant results in reduced water uptake and discolouration of the vascular tissue of the stem and taproot, which can be observed when plants are cut longitudinally. Symptoms of the infection include stunting and yellowing, progressive wilting, which eventually can lead to a complete loss of the plant. The severity of Fusarium wilt is linked to disease pressure and worsened by warm weather and heavy, wet soils.

[0012] The fungus F. oxysporum can spread to disease-free fields by the movement of contaminated soil, e.g., by farm equipment or by wind or water. The fungus can also be present on seeds and seedling transplants.

[0013] Soil-applied fungicides can prevent or reduce the effects of infection by F. oxysporum. However, these fungicides usually have limited efficacy. Moreover, an increasing number of countries have a policy aimed at reducing the use of crop protection agents, and there is a growing demand for organically grown crops due to public concerns about the effect of pesticides on human health and their impact on the environment.Alternative approaches, such as short-term fallowing of infested fields and crop rotation schemes using are not effective control measures. The fungus F. oxysporum can survive for long periods in the soil as dormant spores or even multiply in the roots of non- susceptible plants, including many weeds.

[0014] Improvements in the yield and quality of the crop, as well as a reduction in the application of fungicides, can be achieved by developing lettuce plants with genetically encoded tolerance, or resistance, to F. oxysporum.

[0015] The prior art discloses a number of lines and cultivars resistant to Fusarium. These include the varieties Banchu Red Fire and Romabella, which are used as differential hosts in the differential set used to validate F. oxysporum f. sp. lactucae races.

[0016] The resistance identified in Banchu Red Fire is located on chromosome 1. Aruga et al. 2012 developed RAPD and AFLP markers to detect Fol:2 resistance in lettuce line VP1013 and showed that the segregation patterns of resistant phenotypes indicated a single major locus. This resistance locus was also present in Banchu Red Fire, ChouyaNo. 37, and KikugawaNo. 102. Seki et al. 2021 developed aPCR-based marker to detect the presence of Fol:2 resistance in VI185. The targeted locus was broadly useful for identification of Fol:2-resistance, including the resistance in Banchu Red Fire confirming the results of Aruga et al. 2012 that Fol:2 resistance in these varieties is encoded by a single semi-dominant locus on chromosome 1.

[0017] Although Fusarium resistant lettuce varieties are available, breeding efforts continue because pathogen populations are under selection pressure for increased virulence. To counter this adaptation, breeders combine or stack resistances, i.e., introduce two or more different resistance genes into one plant to reduce the probability that the pathogen overcomes the resistance. There is thus a need in the field to provide new and alternative genetically encoded resistances against Fusarium. In this regard, dominant resistances are particularly preferred as a single copy of such a resistance is sufficient to obtain a strong resistance against disease, making it easier to introduce such resistances in combination with other resistances.

[0018] It is an object of the present invention, amongst other objects, to meet the above need in the art.

[0019] This object, amongst other objects, is met by the present invention as outlined in the appended claims.

[0020] Specifically, this object, amongst other objects, is met by providing a lettuce plant resistant to new Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471, wherein said resistance is encoded by a genomic fragment from Lactuca serriola located on chromosome 7, wherein the location of said resistance providing genomic fragment corresponds with positions 64.798.314 to 66.859.860 of chromosome 7 of the V8 lettuce reference genome, andwherein said resistance providing genomic fragment is obtainable from a lettuce plant of which representative seed is deposited under deposit number NCIMB 43937.

[0021] Representative seed is deposited on February 3rd2022 under deposit number NCIMB 43937 (NCIMB Ltd., Craibstone Estate, Ferguson Building, Bucksbum, Aberdeen AB21 9YA, United Kingdom). The Fusarium oxysporum isolates were deposited with NCIMB at NCIMB Ltd. Wellheads Place, Dyce, Aberdeen, AB21 7GB, Scotland on December 5th2024. The isolates have received the following deposit numbers: 1551, white and smooth,- NCIMB 44470 and 1552, purple and woolly - NCIMB 44471.

[0022] The present inventors have surprisingly identified a novel resistance on chromosome 7. Considering that the prior art resistances are located on chromosome 1, the present resistance provides not only an additional resistance to Fusarium but can also be readily combined, or stacked, with prior art resistances especially considering the present resistance inherits semidominant.

[0023] Specifically, the object of the invention is met by providing lettuce plant, wherein said lettuce plant comprises in its genome at least one nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31, preferably at least one nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13.

[0024] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 1.

[0025] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 3.

[0026] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 5.

[0027] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 7.

[0028] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 9.

[0029] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 11.

[0030] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 13.Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 1 and the nucleic acid sequence SEQ ID No. 3.

[0031] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 3 and the nucleic acid sequence SEQ ID No. 5.

[0032] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 5 and the nucleic acid sequence SEQ ID No. 7.

[0033] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 7 and the nucleic acid sequence SEQ ID No. 9.

[0034] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 9 and the nucleic acid sequence SEQ ID No. 11.

[0035] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequence SEQ ID No. 11 and the nucleic acid sequence SEQ ID No. 13.

[0036] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 1, SEQ ID No. 3 and SEQ ID No. 5.

[0037] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7.

[0038] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 9.

[0039] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11.

[0040] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13.Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7.

[0041] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 9.

[0042] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11.

[0043] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13.

[0044] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 9.

[0045] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11.

[0046] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13.

[0047] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 and SEQ ID No. 11.

[0048] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13.

[0049] Specifically, the present application relates to a lettuce plant, wherein said lettuce plant comprises in its genome the nucleic acid sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13.

[0050] In one of the embodiments the object of the invention, amongst other objects, is met by providing a lettuce plant, wherein said genomic fragment is obtained, or derived or is from a lettuce plant of which representative seed is deposited under deposit number NCIMB 43937.

[0051] Specifically, the embodiment relates to a lettuce plant, wherein said lettuce plant is heterozygous or homozygous for said genomic fragment.In another embodiment the application relates to a method for identifying a genomically encoded resistance against Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471 found in a lettuce plant of which representative seed is deposited under deposit number NCIMB 43937, wherein said method comprises the step of identifying a genomic fragment by detecting at least one genomic sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31.

[0052] In more detail, the above mentioned method comprises the step of introgressing a genomic fragment as defined above into a lettuce plant.

[0053] Furthermore, the method comprises the step of introducing a genomic fragment as defined above into a lettuce plant, wherein the resulting lettuce plant is not exclusively obtained by means of an essentially biological process.

[0054] The application also relates to seed, or progeny, of a lettuce plant as described above comprising a genomic fragment as defined above.

[0055] In a preferred embodiment said seed is polished, coated, encrusted, pelleted or primed.

[0056] Further the application also relates to edible parts, pollen, egg cell, callus, suspension culture, somatic embryo, clone, or plant part of a lettuce plant as described above.

[0057] Additionally, the object of this inventions is met by providing a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31.

[0058] According to another preferred embodiment the application relates to the use of at least one resistance providing genomic fragment, wherein the resistance is resistance against Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471, comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID No.

[0059] 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31 foridentifying, or providing, a lettuce plant resistant to Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471.

[0060] In yet another preferred embodiment the application relates to the use at least one nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31 for identifying, or providing, a lettuce plant resistant to Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471.

[0061] According to another embodiment, the application relates to Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471, specifically, wherein the isolates are capable of causing Fusarium wilt in lettuce plants.

[0062] More specifically, wherein the lettuce plant is selected from Lactuca sativa, Lactuca virosa, Lactuca saligna, Lactuca serriola, Lactuca aculeate, Lactuca georgica, Lactuca perennis, Lactuca tatarica, Lactuca viminea, preferably Lactuca sativa.

[0063] In yet another embodiment, the application relates to a container or a sterilized solution or a severed plant part comprising the Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471.

[0064] Additionally, in another embodiment, the application also relates to a method of using the Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471 for identifying plants of the genus Lactucae comprising resistance against the Fusarium isolate(s), wherein the method comprises the steps of:

[0065] a) providing one or more plants;

[0066] b) providing inoculum comprising Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471;

[0067] c) inoculating plants obtained in step (a) with the inoculum obtained in step(b);

[0068] d) incubating the inoculated plants.

[0069] Additionally, the method further comprises the step:

[0070] e ) assessing symptoms on plants or plant parts.

[0071] In yet another embodiment, the method further comprises the step of identifying plants which have no or reduced symptoms.

[0072] Additionally, the application relates to a method for assessing the presence of Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471,wherein the method comprises the step of visually inspecting lettuce plants for the presence or absence of Fusarium wilt symptoms

[0073] The present invention will be further detailed in the examples below.

[0074] EXAMPLES

[0075] Example 1. Identification of new Fusarium isolates and disease trial

[0076] The new Fusarium isolate has been collected in the vicinity of Murcia, Spain. The isolate was transported to the laboratory in Warmenhuizen, the Netherlands. The isolate was grown on potato dextrose agar (PDA) plates for 5 days at 20 °C. When growing the isolate, two different morphologies were observed leading to the detection of two different strains. One displayed a white color and was smooth, and the other displayed a purple color and was woolly. The isolates were named 1551 and 1552, respectively.

[0077] To perform a disease trial, the Fusarium isolates were transferred from PDA to liquid CDBY medium that is incubated on a shaker at 25 °C for 5 days. The spores were harvested, washed and the concentration was adjusted to obtain a. Fusarium spore suspension with lxlOA6 spores / mL. The Fusarium disease trial was performed in the greenhouse at 25 °C / 23 °C during 16 / 8 hours day / night periods, respectively. In short, lettuce seeds were sown in potting soil and are left to germinate at 16 °C. After 10 days, the seedlings were uprooted and inoculated by dipping the roots in the Fusarium spore suspension described above. Fusarium-inoculated and mock-inoculated seedlings were then transplanted in tables containing potting soil in the greenhouse at 25 °C / 23 °C during 16 / 8 hours day / night periods, respectively. Assessment of the trial was performed between 2 and 3 weeks after inoculation depending on the severity of the disease symptoms. Four official races (Fol:l, Fol:2, Fol:3, Fol:4) have been described for Fusarium oxysporum f. sp. lactucae (Fol). The official differential set is included in the disease trial to confirm the race of the used isolate. The results of the disease trial are displayed in Table 1.Table 1. Results of the Fusarium oxysporum f. sp. lactucae disease trial. Fol: 1 to Fol: 4 are the four official denominated races. 1551 and 1552 are the new isolates. The plants were scored on a scale of 1 (susceptible) to 9 (fully resistant). For the official races, the disease scores were obtained from the ISF (International Seed Federation) website. A generalized approach is used for the determination set: S (susceptible) 1,0 - 3,9; IR (intermediate resistant) 4,0 - 7,9; HR (high resistant) 8,0 - 9, 0. ND = no data.

[0078]

[0079]

[0080] numbers: 1551, white and smooth,- NCIMB 44470 and 1552, purple and woolly - NCIMB 44471

[0081] Example 2. Development of markers to detect resistance to the new Fusarium isolates on chromosome 7

[0082] Genotypes were screened for resistance against the Fusarium isolates 1551 and 1552 and a lettuce breeding line, containing L. serriola introgressions, was found to be resistant. A segregating Fl SI population was developed to map the resistance. A population derived from the Fusarium resistant breeding line and a susceptible L. sativa was analyzed to identify the genomic location(s) of the resistance.

[0083] The Fl SI population consisted of 1017 plants. Of the 1017, 63 plants had no disease symptoms at all and 393 showed severe symptoms. All other F1S1 plants (n=561) had an intermediate disease phenotype. All plants of the susceptible parent showed severe symptoms while the resistant parent showed intermediate or no disease symptoms. The genotype wasdetermined for all Fl SI plants with informative markers within the L. serriola introgressions. The used reference genome was V8 (Reyes-Chin-Wo et al., 2017 https: / / lgr.genomecenter.ucdavis.edu).

[0084] All Fl SI plants without disease symptoms contained a / .. serriola introgression on chromosome 7. Of the 561 F 1 S 1 plants with an intermediate disease phenotype only six lacked the introgression on chromosome 7. These six plants most likely escaped the disease pressure. The resistance was mapped to a / .. serriola intogression on chromosome 7 between 60.0 and 69.2 Mbp. With the identification of crossing overs within the introgression, the resistance could be mapped to chromosome 7 between 64.7 and 66.9 Mbp.

[0085] The locus was validated in another Fl SI population that was derived from the Fusarium resistant breeding line and another susceptible L. sativa. In this population of 190 plants the identified locus co-segregated with the disease phenotype.

[0086] It is concluded that the resistance is a monogenic resistance which localizes on chromosome 7 between 60.0 and 69.2 Mbp and more specifically between 64.7 and 66.9 Mbp in lettuce. Plants, containing the allele that confers resistance on chromosome 7 between 64.7 and 66.9 Mbp, show resistance against the new Fusarium isolates 1551 and 1552.

[0087] A lettuce line containing the resistance was sequenced and additional SNP markers were developed within the introgression. The newly developed markers within the locus cosegregated with the resistance in all breeding material tested.

[0088] Table 2. SNPsfor the detection of the resistance against the new Fusarium isolates.

[0089]

[0090] * The reference genome is: V8 (Reyes-Chin-Wo et al., 2017; https: / / lgr.genomecenter.ucdavis.edu)Table 3. Sequences of the SNPs for the detection of the resistance against the new Fusarium isolates. Odd sequences = linked to resistance (from L. serriola), Even sequences = alternative allele (from L. sativa cv. Salinas / The SNP for detection of resistance is located in the middle of the sequence.

[0091]

[0092]

[0093] * The reference genome is: V8 (Reyes-Chin-Wo et al., 2017; https: / / lgr.genomecenter.ucdavis.edu)Table 4. Sequences linked to the resistance against the new Fusarium race isolates.

[0094]

[0095]

[0096] Table 5. Nucleotide base codes according to the International Union of Pure and Applied Chemistry (IUPAC) code.

[0097]

[0098] Example 3. Fusarium resistant varieties from the prior art comprise a different Fusarium resistance.

[0099] Banchu Red Fire, the deposit NCIMB 43937, Costa RicaNo.4, Patriot and Romabella were genotyped with KASP markers targeting the SNPs described in Table 2 and Table 4. Only the deposit NCIMB 43937 contains the novel resistance on chromosome 7. Banchu Red Fire contains the publicly known Fusarium resistance on chromosome 1 but lacks the novel resistance on chromosome 7. Romabella, Costa RicaNo.4 and Patriot also do not contain the novel resistance on chromosome 7.

[0100] Table 6. Genotype results of two resistant and two susceptible lettuce lines of the SNPs for the detection of the resistance against the Fusarium isolates.

[0101]

[0102] R = resistant; IR = intermediately resistant; S = susceptible

[0103] Example 4. Introduction of the genetic fragments providing the new Fusarium resistance into a lettuce plant with Agrobacterium.

[0104] Transformation of plants with resistance genes using the Agrobacterium tumefaciens system can be a useful way of creating plants resistant to pathogens. To this end constructs harbouring the genetically encoded resistance according to the present invention can be designed and synthesized or otherwise obtained with molecular biology techniques.

[0105] Susceptible lettuce plants can be transformed with a construct containing the new Fusarium resistance on chromosome 7 using co-cultivation with Agrobacterium. Upon completed transformation, stable transformants can be subjected to a disease test using the new Fusarium isolates. It is expected that the stable transformants will be resistant to infection with said new Fusarium isolates, while the non-transformed plants are susceptible.

Claims

CLAIMS1. Letuce plant resistant to Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471, wherein said resistance is encoded by a genomic fragment from Lactuca serriola located on chromosome 7, wherein the location of said resistance providing genomic fragment corresponds with positions 64.798.314 to 66.859.860 of chromosome 7 of the V8 letuce reference genome, and wherein said resistance providing genomic fragment is obtainable from a lettuce plant of which representative seed is deposited under deposit number NCIMB 43937.

2. Letuce plant according to claim 1, wherein said letuce plant comprises in its genome at least one nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31, preferably at least one nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 and SEQ ID No. 13.

3. Letuce plant according to claim 1 or claim 2, wherein said genomic fragment is obtained, or derived or is from a letuce plant of which representative seed is deposited under deposit number NCIMB 43937.

4. Letuce plant according to any one of the claims 1 to 3, wherein said lettuce plant is heterozygous or homozygous for said genomic fragment.

5. Method for identifying a genomically encoded resistance against Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471 found in a letuce plant of which representative seed is deposited under deposit number NCIMB 43937, wherein said method comprises the step of identifying a genomic fragment by detecting at least one genomic sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31.

6. Method for providing a lettuce plant according to any one of claims 1 to 4, wherein said method comprises the step of introgressing a genomic fragment as defined in any one of the claims 1 to 3 into a lettuce plant.

7. Method for providing a lettuce plant resistant to Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471, wherein said method comprises the step of introducing a genomic fragment as defined in any one of the claims 1 to 3 into a lettuce plant, wherein the resulting lettuce plant is not exclusively obtained by means of an essentially biological process.

8. Seed, or progeny, of a lettuce plant according to any one of claims 1 to 4 comprising a genomic fragment as defined in any one of the claims 1 to 3.

9. Seed according to claim 8 wherein said seed is polished, coated, encrusted, pelleted or primed.

10. Edible part, pollen, egg cell, callus, suspension culture, somatic embryo, clone, or plant part of a lettuce plant according to any one of claims 1 to 4.

11. Nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31.

12. Use of at least on resistance providing genomic fragment, wherein the resistance is resistance against Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471, comprising one or more nucleic acid sequences selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31 for identifying, or providing, a lettuce plant resistant to Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471.

13. Use of at least one nucleic acid sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, and SEQ ID No. 31 for identifying, or providing, a lettuce plant resistant to Fusarium oxysporum f. sp. lactucae isolate NCIMB 44470 and / or isolate NCIMB 44471.

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