Iceberg lettuce with improved harvesting characteristics

Abstract

The present invention relates to an iceberg lettuce plant exhibiting an ease of harvest phenotype, wherein said plant comprises a QTL on chromosome 3, wherein the QTL comprises marker LS15315, marker LS15316, and marker LS15317 and wherein said lettuce plant further comprises a QTL on chromosome 2 and a QTL on chromosome 7, wherein the QTL on chromosome 2 comprises marker LS03054, marker LS15311, and marker LS15313 and wherein the QTL on chromosome 7 comprises marker LS14073. The invention further relates to cell, a seed, and propagation material of said iceberg lettuce plant and methods for producing, identifying and selecting said iceberg lettuce plant. In addition, the invention relates to markers for the identification of the presence of said QTLs.

Description

[0001] ICEBERG LETTUCE WITH IMPROVED HARVESTING CHARACTERISTICS

[0002] The present invention relates to an iceberg lettuce plant exhibiting an ease of harvest phenotype. The invention further relates to a cell of said plant and to a seed wherein the plant grown from the seed is an iceberg lettuce plant exhibiting an ease of harvest phenotype. The invention further relates to propagation material capable of developing into and / or being derived from an iceberg lettuce plant exhibiting an ease of harvest phenotype. The invention also relates to a method for growing an iceberg plant and harvesting an iceberg lettuce plant. The invention relates to markers for the identification of an iceberg lettuce plant exhibiting an ease of harvest phenotype, and to the use of said markers. The invention further relates to a method for selecting and a method for producing an iceberg lettuce plant exhibiting an ease of harvest phenotype.

[0003] Iceberg lettuce is part of Lactuca sativa L. (Capitata Group) and is also indicated as crisphead lettuce, although crisphead also involves the Batavia subtype. Iceberg lettuce became one of the most popular lettuce types and its leaves with a crunchy texture are used for instance in salads or sandwiches, or are cooked.

[0004] The production of iceberg lettuce is constantly increasing, in the Netherlands the production of iceberg lettuce increased from 55.0 million kg in 1998 to 90.4 million kg in 2023 (https: / / www.cbs.nl / en-gb / figures / detail / 37738ENG). However, harvesting an iceberg lettuce head is labor intensive and time consuming as it is mostly done manually. One of the challenges of harvesting the head is that an iceberg lettuce head is covered by many large outer leaves. When harvesting the head, said head is cut from the stem and the outer leaves are removed by using a knife. Ideally, the stem is cut between the lower part of the stem, where the outer leaves are attached, and the upper part of the stem, where the head leaves are attached, and which upper part of the stem is part of the head. This requires the use of a tool i.e. a knife and the person harvesting the head needs to know where to cut the stem in order not to damage the head and, at the same time, leave as much as possible of the stem with outer leaves attached behind in the field. In most cases the cutting of the stem takes place below the ideal cutting position, leaving part of the outer leaves attached to the head. These remaining outer leaves are removed by a second cut with the knife.

[0005] In order to improve the harvestability of iceberg lettuce a fast and easy harvesting is desired. For that, iceberg lettuce plants with an ease of harvest phenotype can be used. One type of iceberg lettuce plant that is easier to harvest is already known. This type of plant has a longer stem segment below the head. It is described that this phenotype makes it easier for the person harvesting the head to cut the longer stem segment between soil and head. However, the ideal cutting position remains equally small, and in most cases part of the outer leaves of this plant are still present on the harvested head and still need to be removed by using a knife. Another disadvantage of this type of iceberg is that the longer stem segment leads to less support of the outer leaves to the lettuce head, the head can tilt due to wind or due to one-sided pressure of the harvesting knife.

[0006] It is an object of the present invention to provide an iceberg lettuce plant that has an ease of harvest phenotype without the above stated drawbacks.

[0007] In the research that led to the present invention an iceberg lettuce plant exhibiting an ease of harvest phenotype was developed. The ease of harvest phenotype in this iceberg lettuce plant lies in that the leaves can just be bent until they detach as they break from the stem more easily than in a regular iceberg lettuce plant. It was found that this phenotype was caused by the presence of three QTLs, a QTL on chromosome 2, a QTL on chromosome 3 and a QTL on chromosome 7.

[0008] Thus, the invention relates to an iceberg lettuce plant exhibiting an ease of harvest phenotype, wherein said plant comprises a QTL on chromosome 3, wherein the QTL comprises marker LS15315 comprising a G at position 51 of SEQ ID NO: 8, or a G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 8, and marker LSI 5316 comprising a C at position 51 of SEQ ID NO: 10, or a C at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 10, and marker LS15317 comprising a G at position 51 of SEQ ID NO: 12, or a G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 12; and wherein said lettuce plant further comprises a QTL on chromosome 2 and a QTL on chromosome 7, wherein the QTL on chromosome 2 comprises marker LS03054 comprising a G at position 51 of SEQ ID NO: 2, or a G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 2, and marker LS15311 comprising a T at position 51 of SEQ ID NO: 4, or a T at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 4, and marker LS15313 comprising an A at position 51 of SEQ ID NO: 6, or an A at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 6; and wherein the QTL on chromosome 7 comprises marker LS14073 comprising an insertion having SEQ ID NO: 15 at position 51 of SEQ ID NO:13, or an insertion having SEQ ID NO: 15 at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 13.

[0009] This plant is herein interchangeably referred to as ‘plant of the invention’ and ‘iceberg lettuce plant of the invention’

[0010] The large outer leaves of an iceberg lettuce plant with an ease of harvest phenotype of the invention can be easily removed without using a knife, the leaves can just be bend until they detach as they break from the stem more easily than for a regular iceberg lettuce plant not having the ease of harvest phenotype. Thereby there is no need to use a knife for removing the leaves thus reducing the risk of damaging the head. The ease of harvest iceberg is also more suitable for mechanical harvesting of the iceberg lettuce head, because outer leaves of a regular iceberg plant usually remain on the head after being harvested mechanically and still have to be removed manually with a knife. With the invention, the redundant outer leaves can easily be removed by peeling away the outer layers of the head. The plant of the invention does not have an elongated stalk, in contrast to plants as described in WO2020 / 125925.

[0011] As used herein iceberg is defined as a subtype of cultivated lettuce with closed head, which is round-shaped, and which head has a strong degree of overlapping of the upper part of the leaves. The leaf of the iceberg lettuce plant is thick and has no divisions or very few divisions as well as an undulation of margin which is absent to medium. The venation of the leaf is flabellate (“CPVO, Protocol for tests on distinctness, uniformity and stability, Lactuca sativa L.” Entry into force on 01 / 01 / 2024 https: / / cpvo.europa.eu / sites / default / files / documents / 2024-04 / lactuca-6.4_0.pdf).

[0012] The iceberg lettuce plant having an ease of harvest phenotype according to the invention has a height / width ratio of the head of 0.8 to 1.5, leaves with flabellate to semi-flabellate venation, and outer leaves that can be easily detached from the stem. The ease of harvest phenotype comprises the detachment of the 12thleaf from the stem when peeled off as shown in figure 2.

[0013] As used herein, the height / width ratio of a lettuce head is measured when the non-bolting plant is at mature stage and when its head has a weight of at least 400 grams. The stem part within such a head should not exceed 8 cm. The width corresponds to the largest width of the head and the height corresponds to highest height of the head.

[0014] Measurements and observations on the plants as described herein are done on at least 20 plants for each genotype that grown under the same conditions.. Preferably, the plant is grown outdoor under long-day conditions: sowing 75-85 days before the longest day in a mild midlatitude climate with a warm summer (Koppen-classification: Csb or Cfb; McKnight & Hess, 2000. Physical Geography: A Landscape Appreciation. Upper Saddle River, NJ: Prentice Hall).

[0015] A head of the iceberg lettuce plant of the invention is shown in Fig. 1.

[0016] In one embodiment the invention relates to an iceberg lettuce plant exhibiting an ease of harvest phenotype, wherein said plant comprises a QTL on chromosome 3, wherein the QTL comprises marker LS15315 comprising a G at position 51 of SEQ ID NO: 8, and marker LS15316 comprising a C at position 51 of SEQ ID NO: 10, and marker LS15317 comprising a G at position 51 of SEQ ID NO: 12,; and wherein said lettuce plant further comprises a QTL on chromosome 2 and a QTL on chromosome 7, wherein the QTL on chromosome 2 comprises marker LS03054 comprising a G at position 51 of SEQ ID NO: 2, and marker LS15311 comprising a T at position 51 of SEQ ID NO: 4, and marker LS15313 comprising an A at position 51 of SEQ ID NO: 6; and wherein the QTL on chromosome 7 comprises marker LS 14073 comprising an insertion having SEQ ID NO: 15 at position 51 of SEQ ID NO:13.

[0017] In one embodiment the plant of the invention comprises the QTLs on chromosome 2, 3 and 7 as defined herein homozygously. As used herein, a QTL comprising a marker means that the marker is genetically linked to said QTL.

[0018] The invention further relates to an iceberg lettuce plant of the invention wherein the 12tholdest leaf detaches when the leaf is peeled off. The skilled person knows how to peel off a leaf from an iceberg lettuce head. However, more in particular, as used herein, peeling off is removing the 12tholdest leaf by holding it in between two fingers at 20 cm from the stem and bend the leaf until it detaches from the stem completely at the point where it is attached to the stem before reaching 180 degrees. The movement to bend the leaf is circular and downward i.e leaves are bend in the direction to the cut stem away from the head of the lettuce plant as represented in Fig. 2.

[0019] The iceberg lettuce plant of the invention has a height / width ratio between 0.8 and 1.5, preferably 0.9 and 1.45, more preferably between 0.92 and 1.4, most preferably between 0.95 and 1.35, wherein the head has a weight of at least 400 grams.

[0020] The iceberg lettuce plant of the invention has flabellate to semi-flabellate venation of the leaves. The term flabellate is defined in “CPVO, Protocol for tests on distinctness, uniformity and stability, Lactuca sativa L.” Entry into force on 01 / 01 / 2024 https: / / cpvo.europa.eu / sites / default / files / documents / 2024-04 / lactuca-6.4_0.pdf.

[0021] The invention also relates to an iceberg lettuce plant grown from seed deposited under NCIMB accession number NCIMB 44406, NCIMB 44407, or NCIMB 44408.

[0022] The invention further relates to an iceberg lettuce plant comprising the QTLs on chromosome 2, 3 and 7 as defined herein, wherein the QTLs are introgressed from a plant, from seed deposited with the NCIMB under deposit number NCIMB 44406, NCIMB 44407, or NCIMB 44408 or a progeny plant of seed of NCIMB accession NCIMB 44406, NCIMB 44407, or NCIMB 44408 that has retained said QTLs.

[0023] In a particular embodiment, the plant of the invention is an agronomically elite iceberg lettuce plant.

[0024] In the context of this invention, an agronomically elite plant is a plant having a genotype that, as a result of human intervention, comprises an accumulation of distinguishable and desirable agronomic traits which allow a producer to harvest a product of commercial significance, preferably the agronomically elite plant of the invention is a plant of an inbred line or a hybrid.

[0025] As used herein, a plant of an inbred line is a plant of a population of plants that is the result of three or more rounds of selfing, or backcrossing; or which plant is a double haploid. An inbred line may e.g. be a parent line used for the production of a commercial hybrid.

[0026] As used herein, a hybrid plant is a plant which is the result of a cross between two different plants having different genotypes. More in particular, a hybrid plant is the result of a cross between plants of two different inbred lines, such a hybrid plant may e.g. be a plant of an Li hybrid variety. Another aspect of the invention relates to a seed capable of growing into a plant of the invention wherein said plant comprises the QTLs on chromosome 2, 3 and 7 as defined herein. The invention also relates to use of said seed for the production of a plant of the invention, by growing said seed into a plant.

[0027] The invention also relates to propagation material suitable for producing a plant of the invention, wherein the propagation material is suitable for sexual reproduction, and is in particular selected from a microspore, a pollen, an ovary, an ovule, an embryo sac and an egg cell, or is suitable for vegetative reproduction, and is in particular selected from a cutting, a root, a stem a cell, and a protoplast, or is suitable for tissue culture of regenerable cells or protoplasts, and is in particular selected from a leaf, a pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root, a root tip, an anther, a flower, a seed and a stem, wherein the plant produced from the propagation material is a plant of the invention.

[0028] The invention further relates to a cell of a plant of the invention. Such a cell may either be in isolated form, or a part of the complete plant or parts thereof and still forms a cell of the invention because such a cell comprises the QTLs on chromosome 2, 3 and 7 as defined herein. Each cell of a plant of the invention carries the QTLs on chromosome 2, 3 and 7 as defined herein. A cell of the invention may also be a regenerable cell that can regenerate into a new plant of the invention.

[0029] The invention further relates to plant tissue of a plant of the invention. The tissue can be undifferentiated tissue or already differentiated tissue. Undifferentiated tissue is for example a stem tip, an anther, a petal, or pollen, and can be used in micro propagation to obtain new plantlets that are grown into new plants of the invention. The tissue can also be grown from a cell of the invention.

[0030] The invention further relates to a method for the production of a plant of the invention, by using tissue culture or by using vegetative propagation.

[0031] The invention moreover relates to progeny of a plant, a cell, a tissue, or a seed of the invention, which progeny comprises QTLs on chromosome 2, 3 and 7 as defined herein. Such progeny can in itself be a plant, a cell, a tissue, or a seed. The progeny can in particular be progeny of a plant of the invention, representative seeds of which were deposited under NCIMB 44406, NCIMB 44407, and NCIMB 44408. As used herein, progeny comprises the first and all further descendants from a cross with a plant of the invention, wherein a cross comprises a cross with itself or a cross with another plant, and wherein a descendant that is determined to be progeny comprises the QTLs on chromosome 2, 3 and 7 as defined herein. Descendants can be obtained through selfing and / or further crossing of the deposit. Progeny also encompasses material that is obtained by vegetative propagation or another form of multiplication. The invention further relates to germplasm of a plant of the invention. The germplasm is constituted by all inherited characteristics of an organism and according to the invention encompasses at least the resistance trait of the invention. The germplasm can be used in a breeding program for the development of plants that exhibit the ease of harvest phenotype. The use of germplasm that comprises the QTLs on chromosome 2, 3 and 7 as defined herein in breeding is also part of the present invention.

[0032] The invention also relates to a method for growing an iceberg lettuce plant of the invention, which method comprises the step of germinating a seed and cultivating the germinated seed to a plant of the invention.

[0033] In a particular embodiment the iceberg lettuce plant is grown from a seed deposited under NCIMB accession number NCIMB 44406, NCIMB 44407, or NCIMB 44408, or a progeny plant thereof that has retained the QTLs on chromosome 2, 3 and 7 as defined herein.

[0034] The invention also relates to a method for harvesting an iceberg lettuce plant of the invention, which method comprises the step of cultivating an iceberg lettuce plant of the invention and harvesting the head of said plant, preferably when the head has a weight of at least 400 grams. In a further embodiment said method comprises the step of peeling off the outer leaves that are still attached to the lettuce head. Preferably the peeling off of the outer leaves is done manually.

[0035] In yet another embodiment the iceberg head is harvested mechanically before the outer leaves are peeled off.

[0036] Another aspect of the invention relates to a harvest product of an iceberg lettuce plant of the invention wherein the harvest product is a leaf or head of lettuce and wherein the harvested product can be either in natural or processed form.

[0037] In a preferred embodiment the harvest product is a head of iceberg lettuce of at least 400 grams.

[0038] The invention also relates to the use of the QTLs on chromosome 2, 3 and 7 as defined herein for producing an iceberg lettuce plant having an ease of harvest phenotype. The QTLs can be used for producing an iceberg lettuce plant by means of transgenesis or by introgression from a plant comprising the QTLs to a plant without the QTLs.

[0039] The current invention also relates to the use of a plant of the invention as a crop, as a source of seed or as a source of propagation material.

[0040] The invention also relates to a marker and / or the use thereof for the identification of the presence of the QTLs on chromosome 2, 3 and / or 7 as defined herein in an iceberg lettuce plant, wherein the marker is selected from any of the markers presented in Table 1.

[0041] The invention also relates to the use of markers as described herein for the identification of an iceberg lettuce plant comprising the ease of harvest phenotype. This invention also relates to a method for selecting a plant of the invention, comprising identifying the presence of QTLs on chromosome 2, 3 and / or 7 as defined herein, and selecting a plant that comprises said QTL. Suitably, identifying the presence of any one of the QTLs on chromosome 2, 3 and 7 is done by using any one of the markers in Table 1 as described herein. The identification or selection of a plant of the invention can also be done phenotypically as described herein

[0042] A marker can be defined as a reference sequence that comprises the modification(s) that can be detected using any suitable method known. The term “marker”, “molecular marker”, "genetic marker" or “DNA marker” refers to a feature of an organism’s genome (e.g. a nucleotide or a polynucleotide sequence that is present in an organism’s genome) that is associated with one or more loci of interest. A genetic marker is polymorphic in a population of interest. Genetic markers include, for example, single nucleotide polymorphisms (SNPs), indels (i.e. insertions / deletions), simple sequence repeats (SSRs), restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), cleaved amplified polymorphic sequence (CAPS) markers, Diversity Arrays Technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs), among many other examples. Genetic markers can, for example, be used to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits. The term “marker” or “genetic marker” can also refer to a polynucleotide sequence complementary to a genomic sequence, such as a sequence of a nucleic acid used as a probe. The term “marker” then refers to a physical entity that can be used in molecular biological techniques for detecting the mutation.

[0043] In the context of the present invention, a marker of Table 1 as described herein allows for the detection of the QTLs on chromosome 2, 3 and 7 as defined herein responsible for the ease of harvest phenotype, and the selection of iceberg lettuce plants that harbor in their genome the QTLs on chromosome 2, 3 and 7 as defined herein at any stage of their life cycle, even when the plants are only in the seedling stage. Marker-assisted breeding and selection greatly increases the speed with which a trait can be introduced into different genetic backgrounds, and with which it can be commercialized.

[0044] The markers of Table 1 as described herein can also be used to develop other markers that are genetically linked to the QTLs on chromosome 2, 3 and 7 as defined herein.

[0045] Methods for detecting markers and specific alleles are abundantly known in the field. In general, these methods allow to distinguish between two different alleles of a marker, or the presence or absence of a marker on a specific chromosome. Detection of a polymorphism can be achieved by electrophoretic techniques, but the widespread availability of DNA sequencing often makes it easier to simply sequence amplified products directly. Once the polymorphic sequence difference is known, rapid assays for the detection of a polymorphism can be designed for progeny testing, generally involving some version of PCR amplification of specific alleles.

[0046] The invention further relates to a method for producing an iceberg lettuce plant comprising the ease of harvest phenotype, said method comprising: a) crossing a plant of the invention with another plant to obtain a first generation population; b) performing one or more rounds of selfing and / or crossing of the plant resulting from the cross to obtain a further generation population; c) selecting from among the plants resulting from the further generation population of step b) a plant that comprises the QTLs on chromosome 2, 3 and 7 as defined herein, which plant comprises the ease of harvest phenotype.

[0047] In one embodiment, selecting a plant the invention is done by using one or more molecular markers genetically linked to the QTLs on chromosome 2, 3 and 7 as defined herein, preferably a marker of Table 1 as described herein.

[0048] In another embodiment, a plant of the invention is phenotypically selected, in particular by determining the ease of harvest phenotype as defined herein.

[0049] In a specific embodiment the plant of the invention in step a) for producing an iceberg lettuce plant comprising the ease of harvest phenotype is a plant grown from seed deposited under NCIMB accession number NCIMB 44406, NCIMB 44407, or NCIMB 44408, or a progeny plant thereof that has retained the QTLs on chromosome 2, 3 and 7 as defined herein.

[0050] The invention further relates to a hybrid iceberg lettuce seed and to a method for the production of a hybrid iceberg lettuce seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and / or the second parent plant is a plant comprising the QTLs on chromosome 2, 3 and 7 as defined herein.

[0051] In a particular embodiment, the first and / or second parent plant is a plant grown from seed deposited under NCIMB accession number NCIMB 44406, NCIMB 44407, or NCIMB 44408, or a progeny plant thereof that has retained the QTLs on chromosome 2, 3 and 7 as defined herein.

[0052] FIGURES

[0053] Fig. 1 : Plant and harvested heads of an iceberg plant of the invention.

[0054] Fig. 2: Representation of the detachment of the outer leaf as described in Example 2.

[0055] DEPOSIT

[0056] Seeds of an iceberg lettuce plant of the invention were deposited with NCIMB Ltd, Wellheads Place, Dyce, Aberdeen AB21 7GB, UK, on 29 July, 2024, under deposit accession numbers NCIMB 44406, NCIMB 44407 and NCIMB 44408. The deposits were made pursuant to the terms of the Budapest Treaty. Upon issuance of a patent, all restrictions upon the deposits will be removed, and the deposits are intended to meet the requirements of 37 CFR § 1.801-1.809. The deposits will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposits will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced if necessary during that period.

[0057] SEQUENCE INFORMATION

[0058] Table 1: sequence information of the molecular markers linked to the three QTLs on chromosome

[0059] 2, 3 and 7.

[0060] EXAMPLES

[0061] EXAMPLE 1

[0062] Measurement of the head height / width ratio

[0063] The height (H) and the width (W) of harvested heads of Calesita RZ (crunchy), Crunchita RZ (crunchy), Evionas RZ (iceberg), Scandinas RZ (iceberg), Rictus (cos) and three different iceberg lines with the ease of harvest phenotype (Line 1 (NCIMB 44406), Line 2 (NCIMB 44407) and Line 3(NCIMB 44408)) grown under the same conditions were measured. The plants were grown outdoor under long-day conditions: sowing 75-85 days before the longest day in a mild mid-latitude climate with a warm summer (Koppen-classification: Csb or Cfb; McKnight & Hess, 2000. Physical Geography: A Landscape Appreciation. Upper Saddle River, NJ: Prentice Hall). The core within such a head should not exceed 8 cm. The measurements were taken when the plants were at mature stage and when the heads had a weight of at least 400 grams. The width corresponds to the largest width of the head and the height corresponds to highest height of the head. Based on the measurements the height / width ratio was measured. Table 2 shows the average of the measurements: iceberg plants have a height / width ratio of 1.0 and 0.9 (average 0.9), ease of harvest iceberg plants have a height / width ratio between 1.2 and 1.4 (average 1.30), crunchy lettuce plants have a height / width ratio between 2.1 and 2.4 (average 2.2) and cos lettuce plants have a height / width ratio of 2.6. The results show that iceberg lettuce plants and ease of harvest iceberg plants have a height / width ratio between 0.9 and 1.4 which is lower than the ratio of cos lettuce and crunchy lettuce plants when the plants are grown under the same conditions.

[0064] Table 2: Measurements of the height and width in centimeters and height / width (H / W) ratio

[0065] EXAMPLE 2

[0066] Detachment of the outer leaf

[0067] The lettuce head was cut from the stem at mature stage with a head weight of at least 400 grams of plants grown under the same conditions. The plants were grown outdoor under long-day conditions: sowing 75-85 days before the longest day in a mild mid-latitude climate with a warm summer (Koppen-classification: Csb or Cfb; McKnight & Hess, 2000. Physical Geography: A Landscape Appreciation. Upper Saddle River, NJ: Prentice Hall). The core within such a head should not exceed 8 cm. The whole head was held in upright position. The measurement was performed with the 12tholdest leaf which is the 12thleaf counting from the roots to the top of the plant. The 12thleaf was hold by two fingers at 20 cm from the stem and bend until it detached from the stem completely at the point where it is attached to the stem before reaching 180 degrees. The movement to bend the leaf is circular and downward i.e leaves are bent in the direction to the cut stem away from the head of the lettuce plant as represented in Fig. 2.

[0068] The 12thouter leaf of the 12 tested iceberg lettuce heads of the variety Scandinas RZ didn’t detach i.e. the probability of successful detachment was 0%. For iceberg lettuce plants with the QTLs on chromosome 2, 3 and 7 as defined herein, successful detachment of the 12thleaf was observed. The results show that iceberg plants with the QTLs on chromosome 2, 3 and 7 as defined herein had a 12ththat detached as it is required for the ease of harvest phenotype.

[0069] EXAMPLE 3

[0070] QTL mapping and marker development

[0071] The first mapping population was derived from a cross between an iceberg lettuce plant and a crunchy lettuce variety. 405 F2 plants were phenotyped for traits that are relevant for the ease of harvest phenotype of an iceberg lettuce plant.. DNA was extracted from young leaf tissue from the F2 plants. 78 polymorph KASP markers were run for mapping distributed with an average spacing of 14.8 cM. 12 markers were removed due to segregation distortion. QTL analysis was performed, and mapping of the data resulted in the identification of a QTL on chromosome 2 and a QTL on chromosome 7.

[0072] An additional population was developed from a cross between an iceberg lettuce with the ease of harvest phenotype and the iceberg lettuce Kyoto RZ. 250 F2 plants were phenotyped for traits that are relevant for the ease of harvest phenotype of an iceberg lettuce plant. DNA was extracted from young leaf tissue from the F2 plants. 391 polymorph KASP markers were run for mapping distributed with an average spacing of 2.8 cM. The mapping of the data resulted in the identification of a QTL on chromosome 3 and the confirmation of the QTL on chromosome 2.

[0073] A third population was developed from a cross between a crunchy lettuce variety and an iceberg lettuce plant. 260 F2 plants were phenotyped for traits that are relevant for the ease of harvest phenotype of an iceberg lettuce plant. DNA was extracted from young leaf tissue from the F2 plants. 312 polymorph KASP markers were run for mapping distributed with an average spacing of 4 cM. The QTLs on chromosome 2, 3 and 7 were confirmed.

[0074] The markers developed for the ‘ease of harvest’ phenotype can be found in Table 1.

[0075] EXAMPLE 4

[0076] Introducing the ease of harvest phenotype in a lettuce iceberg plant

[0077] A lettuce iceberg plant comprising the QTLs on chromosome 2, 3 and 7 as defined herein, of which a representative sample of seed was deposited with the NCIMB under accession numbers NCIMB NCIMB 44406, NCIMB 44407, and NCIMB 44408 was crossed with a plant of the iceberg variety Scandinas RZ to obtain an Fl generation. The resulting Fl from this cross had the same phenotype as Scandinas RZ i.e. the 12thleaf didn’t detach from the stem.

[0078] From the Fl population a plant was selected which was selfed to obtain a population of F2 plants. The F2 segregates in a manner that corresponds with a recessive inheritance of the trait. Genomic DNA of each plant of the F2 population was isolated and the markers as described in Example 3 were tested.

[0079] Plants comprising the QTLs on chromosome 2, 3 and 7 as defined herein homozygously were assayed as described in Example 2 for the presence of the ease of harvest phenotype. A plant having the QTLs on chromosome 2, 3 and 7 as defined herein and the phenotype of the invention was thereby selected.

[0080] Plants comprising the QTL on chromosome 3 or on chromosome 2 homozygously were selfed to obtain a F3 population. Genomic DNA of each plant of the F3 population was isolated and the markers as described in Example 3 were tested. Plants comprising the QTLs on chromosome 2, 3 and 7 homozygously were assayed as described in Example 2 for the presence of the ease of harvest phenotype. A plant having the QTLs on chromosome 2, 3 and 7 as defined herein and the phenotype of the invention was thereby selected.

[0081] Alternatively, a plant of the Fl population was selected and backcrossed with a plant of the deposits in order to obtain a F2 population. Genomic DNA of each plant of the F2 population was isolated and the markers as described in Example 3 were tested. Plants comprising the QTLs on chromosome 2, 3 and 7 homozygously were assayed as described in Example 2 for the presence of the ease of harvest phenotype. A plant having the QTLs on chromosome 2, 3 and 7 as defined herein and the phenotype of the invention was thereby selected.

Claims

CLAIMS1. An iceberg lettuce plant exhibiting an ease of harvest phenotype, wherein said plant comprises a QTL on chromosome 3, wherein the QTL comprises marker LS15315 comprising a G at position 51 of SEQ ID NO: 8, or a G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 8, and marker LSI 5316 comprising a C at position 51 of SEQ ID NO: 10, or a C at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 10, and marker LS15317 comprising a G at position 51 of SEQ ID NO: 12, or a G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 12; and wherein said lettuce plant further comprises a QTL on chromosome 2 and a QTL on chromosome 7, wherein the QTL on chromosome 2 comprises marker LS03054 comprising a G at position 51 of SEQ ID NO: 2, or a G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 2, and marker LS15311 comprising a T at position 51 of SEQ ID NO: 4, or a T at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 4, and marker LS15313 comprising an A at position 51 of SEQ ID NO: 6, or an A at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 6; and wherein the QTL on chromosome 7 comprises marker LSI 4073 comprising an insertion having SEQ ID NO: 15 at position 51 of SEQ ID NO: 13, or an insertion having SEQ ID NO: 15 at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 13.

2. The plant of claim 1 wherein the QTL on chromosome 3 comprises marker LS15315 having SEQ ID NO: 8, and marker LSI 5316 having SEQ ID NO: 10 and marker LSI 5317 having SEQ ID NO: 12, and wherein said lettuce plant further comprises a QTL on chromosome 2 and a QTL on chromosome 7, wherein the QTL on chromosome 2 comprises marker LS03054 having SEQ ID NO: 2, and marker LS15311 having SEQ ID NO: 4, and marker LS15313 having SEQ ID NO: 6; and wherein the QTL on chromosome 7 comprises marker LS 14073 having SEQ ID NO:13.

3. The plant of claim 1 and / or 2, wherein the ease of harvest phenotype is defined as the 12tholdest leaf detaches when the leaf is peeled off.

4. The plant as claimed in any one of the claim 1 to 3, wherein the head of the iceberg lettuce plant has a height / width ratio between 0.8 and 1.5.

5. The plant of any of the claim 1 to 4, wherein said QTLs are introgressed from a plant, which is grown from seed deposited with the NCIMB under deposit number NCIMB 44406,NCIMB 44407, or NCIMB 44408, or a progeny plant of seed of NCIMB accession NCIMB 44406, NCIMB 44407, or NCIMB 44408 that has retained said QTLs.

6. A cell of an iceberg lettuce plant according to any of the claims 1 to 5.

7. An iceberg lettuce seed wherein the plant grown from the seed is a plant as claimed in any one of the claims 1 to 5.

8. Propagation material capable of developing into and / or being derived from a Lactuca sativa plant according to claims 1 to 5, wherein the propagation material is suitable for sexual reproduction, and is in particular selected from the group comprising a microspore, pollen, an ovary, an ovule, an embryo sac, and an egg cell; or is suitable for vegetative reproduction, and is in particular selected from the group comprising a cutting, a root, a stem, a cell, and a protoplast; or is suitable for tissue culture of regenerable cells, and is in particular selected from the group comprising a leaf, pollen, an embryo, a cotyledon, a hypocotyl, a meristematic cell, a root, a root tip, an anther, a flower, a seed, and a stem; wherein the plant developed from the propagation material is a plant according to any of the claims 1 to 5.

9. A method for growing an iceberg lettuce plant as claimed in any of the claims 1 to 5, which method comprises the step of germinating a seed and cultivating the germinated seed to a plant as claimed in any of the claims 1 to 5.

10. A method for harvesting an iceberg lettuce plant as claimed in any of the claims 1 to 4, which method comprises the step of cultivating an iceberg lettuce plant as claimed in any one of the claims 1 to 5 and harvesting the lettuce and peeling off the outer leaves.

11. The method of claim 10, wherein the iceberg head is harvested mechanically before the outer leaves are peeled off.

12. Marker for the identification of the presence of the QTLs as defined in claim 1 in an iceberg lettuce plant, which marker is selected from the group comprising: marker LS03054 comprising a G at position 51 of SEQ ID NO: 2, or an G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 2; marker LS15311 comprising a T at position 51 of SEQ ID NO: 4, or a T at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 4;17 marker LS15313 comprising an A on position 51 of SEQ ID NO: 6, or an A at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 6 marker LSI 15315 comprising a G on position 51 of SEQ ID NO: 8, or a G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 8 marker LS15316 comprising a C on position 51 of SEQ ID NO: 10, or a C at position 511 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 10 marker LS15317 comprising a G at position 51 of SEQ ID NO: 12, or a G at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 12; marker LS14073 comprising an insertion having SEQ ID NO: 15 at position 51 of SEQ ID NO:13, or an insertion having SEQ ID NO: 15 at position 51 of a sequence comprising at least 95% sequence identity to SEQ ID NO: 13.

13. Use of a marker as claimed in claim 12 for identification of an iceberg lettuce plant comprising the QTLs as defined in claim 1.

14. Use of a marker as claimed in claim 12 for identification of an iceberg lettuce plant comprising the ease of harvest phenotype.

15. Method for selecting an iceberg lettuce plant comprising the ease of harvest phenotype, comprising detecting the presence of the QTLs a as defined in claims 1 and selecting a plant that comprises a combination of QTLs as defined in claim 1.

16. Method for producing an iceberg lettuce plant comprising the ease of harvest phenotype, said method comprising: a) crossing a plant according to any one of the claims 1 to 5 with another plant to obtain a first generation population; b) performing one or more rounds of selfing and / or crossing of the plant resulting from the cross to obtain a further generation population; c) selecting from among the plants resulting from the further generation population of step b) a plant that comprises a combination of QTLs as defined in claim 1 , which plant comprises the ease of harvest phenotype.

17. Method as claimed in claim 16, wherein the plant comprising the combination of QTLs as claimed in claim 1 is selected by using a molecular marker genetically linked to said QTLs, preferably a marker as claimed in claim 12.

18. Method as claimed in claim 17, wherein the plant as claimed in any of the claims 1 to 5 is phenotypically selected.

19. Method as claimed in any one of the claims 16 to 18, wherein the plant according to any one of the claims 1 to 4 is a plant grown from seed deposited under NCIMB accession number NCIMB 44406, NCIMB 44407, or NCIMB 44408, or a progeny plant thereof that has retained the QTLs as defined in claim 1.

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