Reduced foliage celery

Abstract

The present invention relates to a cultivated Apium graveolens L. dulce plant comprising a QTL on chromosome 8 which is located between SEQ ID No. 1 and SEQ ID No. 2, which QTL when homozygously present is responsible for the presence of two internodes on average per leaf at the harvesting stage. The invention further relates to a cell, a seed and propagation material of the plant of the invention. The invention further relates to a marker for the identification in the plant of the invention comprising the SNPs presented in SEQ ID Nos. 1 or SEQ ID No. 2. In addition, the invention relates to methods for producing and selecting the plant of the invention.

Description

[0001] REDUCED FOLIAGE CELERY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a new type of celery (Apium graveolens L. dulce). The invention further relates to a method for producing such Apium graveolens L. dulce plant and to methods for identification and selection of such a celery plant. The invention also relates to progeny and seeds of such a celery plant, to propagation material suitable for producing such a celery plant, and to a food product comprising such a celery plant or part thereof. The invention further relates to a cell or a tissue culture that is produced from, or can be regenerated into such a celery plant. The invention also relates to markers for identification in such celery plant and to use of said markers.

[0004] BACKGROUND OF THE INVENTION

[0005] Apium graveolens is a plant species belonging to the Apiaceae family, which comprises two important vegetable crops, namely celery and celeriac. In Apium graveolens L. dulce (celery, also known as stalk celery), the plant part that is typically harvested for consumption are the first internodes of the leaves.

[0006] A single celery plant typically represents a large amount of biomass, and in particular market segments an entire celery plant is often too large for the consumer to store conveniently. Moreover, celery is a vegetable that has a relatively large amount of unusable parts. On average 50% of the leaves is not sold, but cut off as waste. Typically, celery plants comprise multiple leaves. The leaves of celery are compound leaves that are composed of a petiole or leaf stem (which is the first internode of the celery leaf, extending from the leaf base up to the first node) and a fully subdivided leaf blade. The leaf blade typically consists of several internodes, each with a pair of leaflets, and it ends in a terminal leaflet that sprouts from the uppermost node, along with two regular leaflets. Figure 1 illustrates that for industrial processing a celery plant is usually cut off just below the first pair of leaflets growing from the first node. The first internode is the marketable section of the celery plant that is processed and consumed, while everything above the first internode is usually discarded as waste. The first internode or petiole is also known as the stalk, and after removal of the leaflets it is usually called “stick”. The petiole carries a leaf blade, and a petiole and its leaf blade together form a leaf of the celery plant.

[0007] It is an object of the present invention to provide a new type of celery with a leaf architecture that allows for an optimized use of its biomass and a minimal amount of waste, and that is also better suited for machine-harvesting and for machine-processing than wildtype celery. In the research leading to the present invention, it was found that the homozygous presence of a specific Quantitative Trait Locus (QTL) on chromosome 8 of the celery genome causes a leaf architecture that is very different from the leaf architecture of a wildtype celery plant (Example 1). This research further revealed that said QTL is located between marker RF1 (SEQ ID No. 1) and marker RF2 (SEQ ID No. 2). The QTL is inherited in a recessive fashion.

[0008] The invention thus provides a celery plant of the species Apium graveolens L. dulce with a “reduced foliage” (Rf) architecture. Wildtype celery plants typically have three, four or even five internodes plus a terminal leaflet, whereby the largest part of the second internode and all internodes above that point (and the terminal leaflet) are usually cut off and discarded as waste after harvest. The Reduced foliage (Rf) celery type has only two internodes on average per leaf at the harvesting stage (Figure 3). Typically, its second internode is much shorter than that in wildtype celery plants. The first internode (the petiole, stalk or stick) is equally long or longer than in wildtype celery plants (Figure 4). As a result of this, Rf celery plants are generally shorter than wildtype celery plants when grown in identical conditions, but they have a much higher proportion of commercially usable biomass (Example 2).

[0009] The final number of internodes and nodes in a celery leaf is already established at an early developmental stage. Therefore, the Rf-type can already be recognized long before the harvesting stage, in young, immature leaves. It is therefore not necessary to wait until the plant is ready for harvest to assess the presence or absence of the Rf-type in any given celery plant.

[0010] The term “two internodes on average per leaf’ is intended to mean that all leaves of an Rf-type celery plant of the invention typically comprise two internodes. However, it may happen that an Rf-type plant has one or more leaves with only one and / or three internodes. When the total number of internodes on all leaves of a single celery plant is counted, and this number is divided by the number of leaves on that celery plant, this will result in an average number of internodes per leaf that is in any case significantly lower than three, and typically about two. The average number of internodes per leaf within a plant of the invention is thus about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5, but it is preferably about 2.0. In wildtype celery plants this average number of internodes per leaf within a plant is always about three or more (Figure 3).

[0011] In particular, the invention relates to a cultivated celery plant Apium graveolens L. dulce) comprising a QTL on chromosome 8 which is located between marker RF1 (SEQ ID No. 1) and marker RF2 (SEQ ID No. 2), which QTL when homozygously present is responsible for the presence of two internodes on average per leaf at the harvesting stage.

[0012] “Harvesting stage” is intended to mean the stage in the celery plant’s life cycle wherein its stalks are physiologically mature and ready for consumption. Typically, this is about four to six months after sowing the seeds, but this may depend on the environmental conditions, the plant’s genotype and the consumer’s preference. The person skilled in the art of celery breeding and / or celery growing knows best when his particular celery crop is at the optimal stage for harvesting, processing and consumption.

[0013] In one embodiment, the cultivated celery plant comprises a QTL on chromosome 8 that is genetically linked to at least one of the markers comprising a SNP as presented in the markers RF1 (SEQ ID No. 1) and marker RF2 (SEQ ID No. 2).

[0014] As used herein, a marker is genetically linked to, and can therefore be used for the identification of a QTL of the invention, when the marker and the Rf-type phenotype co-segregate in a segregating population resulting from a cross between a plant comprising a QTL of the invention and a plant lacking the QTL. A marker genetically linked to a QTL can be used for identification of that QTL because a linked marker is present in said QTL.

[0015] Table 1 provides the sequences of the SEQ ID Nos. that can be used as markers, or that can be used to develop markers, to identify the presence of the QTL of the invention in a celery plant. As used herein, the “SNP present in” or the “SNP presented in” a certain SEQ ID No. is the nucleotide of the SNP within the sequence that is indicative of the Rf-type. Markers RF1 (SEQ ID No. 1) and marker RF2 (SEQ ID No. 2) delimit the QTL region. Table 1 also lists the position of the SNP in each sequence, the derived (mutant) allele that is linked to the Rf trait, and the wildtype allele for each SNP, as well as the physical position of each SNP on the public Api- gra_Ventura_vl genome assembly (PMID 33095976; Song et al., 2020, Plant Biotechnol. 19: 731- 744). With reference to this genome assembly, the QTL region of the invention is located on chromosome 8 between positions 229,801,360 and 237,176,427. The genetic distance between the two flanking markers (RF1 and RF2) is about 0.1 cM (Example 1).

[0016] Table 1: Molecular markers delimiting the QTL region (RF1 and RF2), and markers genetically linked to the Rf trait in celery. In a further embodiment, the QTL of the invention is as comprised in the genome of a celery plant representative seed of which was deposited with the NCIMB under deposit number NCIMB 44381. Suitably, said QTL has been introgressed into the cultivated celery plant directly from NCIMB 44381, or from a progeny plant thereof that has retained the QTL.

[0017] “Introgression” as used herein is intended to mean introduction of a trait into a plant not carrying the trait by means of crossing and selection in the first generation in which the trait becomes phenotypically visible, or in any later generation, or in the Fl generation, or any further generation, if molecular markers are used for detecting the trait. For a dominant trait the trait becomes visible in the Fl generation of the cross between a plant with the trait and a plant without the trait. For a recessive trait this is suitably the F2 generation.

[0018] The invention also relates to progeny of a plant, a cell, a tissue, or a seed of a cultivated celery plant of the invention, which progeny comprises on chromosome 8 the QTL region of the invention as defined herein. Such progeny can in itself be a plant, a cutting, a seed, a cell, or a tissue.

[0019] As used herein, “progeny” is intended to mean the first and all further descendants, such as an Fl, F2, or further generation, 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 QTL of the invention as defined herein, that when present in a homozygous state is responsible for the presence of two internodes on average per leaf at the harvesting stage. The plant of the invention that is used in this cross is optionally a plant grown from seed of deposit NCIMB 44381, or from progeny seed thereof which is a direct or further descendant through crossing a plant grown from the deposited seed with itself or with another plant for one or more subsequent generations, wherein the progeny seed has retained the QTL of the invention.

[0020] Progeny also encompasses a cultivated celery plant that carries the QTL of the invention and has leaves with two internodes on average per leaf at the harvesting stage, and that is obtained from the plant, or progeny of a plant, of the invention by vegetative propagation or another form of multiplication.

[0021] The invention also relates to a celery seed comprising the QTL of the invention in its genome, wherein a plant grown from the seed is a celery plant with leaves with two internodes on average per leaf at the harvesting stage.

[0022] The current invention also relates to propagation material capable of developing into and / or being derived from a celery plant comprising the QTL of the invention in its genome. Preferably, the propagation material is suitable for sexual reproduction, and it is in particular selected from the group comprising a microspore, pollen, an ovary, an ovule, an embryo sac, and an egg cell; or it 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 propagation material and the plant produced from the propagation material comprises in its genome the QTL of the invention that, when homozygously present, is responsible for the presence of two internodes on average per leaf at the harvesting stage.

[0023] In one embodiment, said propagation material is derived from a celery plant, representative seed of which was deposited with the NCIMB under deposit number NCIMB 44381, or from a progeny plant thereof.

[0024] The invention further relates to a cell comprising the QTL of the invention as defined herein. A cell of the invention can be obtained from, or be present in, a plant of the invention. Such a cell may either be in isolated form, or a part of the complete plant, or from a part thereof, and still constitutes a cell of the invention because such a cell comprises the genetic information that determines the Rf trait as described herein. Each cell of a plant of the invention carries the QTL of the invention, and thereby the genetic information that leads to the presence of two internodes on average per leaf at the harvesting stage. A cell of the invention may also be a regenerable cell that can regenerate into a new plant of the invention. The presence of the genetic information in this context is the presence of the QTL of the invention, wherein the QTL is as defined herein.

[0025] The invention also relates to a food product comprising a celery plant of the invention, or part thereof. In particular, the invention relates to a food product comprising the stalks or sticks of such a celery plant, and to a packaging comprising such a food product.

[0026] The present invention further relates to a method for identifying a celery plant carrying the genetic trait of developing leaves comprising two internodes on average per leaf at the harvesting stage, wherein the method comprises screening a celery plant population for the presence of the QTL of the invention, and identifying a celery plant that comprises at least one of the SNP as presented in any one of the markers RE1 (SEQ ID No. 1) and RE2 (SEQ ID No. 2) as a celery plant of the invention. Optionally, said method may also comprise the step of phenotypically screening for the presence of the Rf trait of the invention by counting the average number of internodes per leaf at the harvesting stage, or at an earlier developmental stage in which the difference between wildtype and Rf-type leaf architecture is already visible.

[0027] The invention also relates to a marker for the identification of the QTL on chromosome 8 in a celery plant, which marker is any one of a group comprising the SNPs presented in markers RL1 (SEQ ID No. 1) and RE2 (SEQ ID No. 2). The SNPs presented in markers RL1 (SEQ ID No. 1) and RF2 (SEQ ID No. 2) are genetically linked to said QTL on chromosome 8 and to the Rf-trait in celery. The markers can be used to screen for the presence of the QTL in cultivated plants, such as celery varieties, but also in wild celery plants and accessions. The SNPs presented in markers RF1 (SEQ ID No. 1) and RF2 (SEQ ID No. 2) are genetically linked to the Rf-type phenotype in deposit NCIMB 44381.

[0028] This invention further relates to the use of a marker for the identification of a celery plant with two internodes on average per leaf at the harvesting stage. The markers are in particular useful for screening plants in an early developmental stage in which the phenotype is not yet visible.

[0029] This invention also relates to a method for selecting a celery plant carrying the trait of developing two internodes on average per leaf at the harvesting stage, comprising identifying the presence of the QTL on chromosome 8 of the invention, and selecting a plant that comprises said QTL in a homozygous state. Suitably, identifying the presence of the QTL on chromosome 8 is done by using a marker comprising a SNP presented in any one of SEQ ID Nos. 1 and 2 for the identification of said QTL.

[0030] 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. In some embodiments, 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.

[0031] In the context of the present invention, a marker allows the unambiguous detection of the QTL region that is genetically linked to the Rf-trait in celery, and the selection of celery plants that harbor in their genome the Rf-trait 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. 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, 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.

[0032] In particular examples, PCR detection and quantification is carried out using two labeled Anorogenic oligonucleotide forward primers and an unlabeled common reverse primer, for example, KASP™ (KBiosciences). Table 2 provides a list of KASP primers that are suitable for the identification of Ranking markers delimiting the QTL region, and for the identification of markers genetically linked to the Rf trait in celery. The person skilled in the art of molecular biology may of course also use other types of markers, and the primers listed here are merely illustrative and not limiting in any way.

[0033] The presence or absence of marker RF1 in the genome of a celery plant can be investigated in a KASP™ (KBiosciences) assay by using the primers listed in SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5. For investigating the presence or absence of marker RF2 the primers listed in SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8 can be used.

[0034] Table 2: KASP primers suitable for the identification of Ranking markers delimiting the QTL region, and for the identification of markers genetically linked to the Rf trait in celery.

[0035] This invention further relates to a method for producing a cultivated celery plant with two internodes on average per leaf at the harvesting stage, said method comprising: a) crossing a plant according to 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 homozygously comprises the QTL on chromosome 8 of the invention, which plant has the trait of developing two internodes on average per leaf at the harvesting stage (Example 3).

[0036] In one embodiment, selecting a plant comprising the QTL on chromosome 8 is done by using a molecular marker genetically linked to the QTL, preferably a marker comprising a SNP presented in any one of SEQ ID Nos. 1 and 2 for the identification of said QTL. In another embodiment, a plant with two internodes on average per leaf at the harvesting stage is phenotypically selected, in particular by determining the average number of internodes per leaf at the harvesting stage, or at an earlier developmental stage in which the difference between wildtype and Rf-type leaf architecture is already visible.

[0037] This invention also relates to a method for producing a cultivated celery plant with two internodes on average per leaf at the harvesting stage, said method comprising: a) crossing a plant grown from seed deposited under NCIMB accession number NCIMB 44381, or a progeny plant thereof that has retained the QTL on chromosome 8, 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 homozygously comprises the QTL on chromosome 8 of the invention, which plant has two internodes on average per leaf at the harvesting stage.

[0038] This invention also relates to a method for the production of cultivated hybrid celery seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and the second parent plant are plants comprising the QTL on chromosome 8 according to the invention in a homozygous state, wherein said QTL is responsible for the presence of two internodes on average per leaf at the harvesting stage in the hybrid plant that is grown from the seed. This invention also relates to the cultivated hybrid celery seed produced by this method.

[0039] The invention further relates to a method for the production of a celery plant comprising the QTL of the invention, by using tissue culture or by using vegetative propagation.

[0040] The invention further provides a method for the production of a celery plant comprising the QTL of the invention by using a doubled haploid generation technique to generate a doubled haploid line that is completely homozygous, and therefore homozygously comprises the QTL of the invention, and that has leaves with two internodes on average per leaf.

[0041] The invention further relates to a method for the production of a celery plant comprising the QTL of the invention, wherein the presence of said QTL leads to leaves with two internodes on average per leaf at the harvesting stage, which method comprises growing a seed comprising said QTL into the said plant.

[0042] The invention also relates to a method of growing a celery plant of the Rf-type and / or a celery plant comprising a QTL on chromosome 8 which is located between marker RF1 (SEQ ID No. 1) and marker RF2 (SEQ ID No. 2), comprising the step of germinating a celery seed of the invention under suitable conditions into a celery plant of the invention. Suitably, the QTL in said celery seed and celery plant is as present in the genome of a celery plant, representative seed of which was deposited with the NCIMB under deposit number NCIMB 44381. In one embodiment, said QTL is introgressed from NCIMB 44381 or from a progeny plant thereof.

[0043] This invention also relates to a QTL on chromosome 8 of the celery genome, which QTL when homozygously present is responsible for the presence of two internodes on average per leaf at the harvesting stage, and which QTL comprises a nucleotide sequence flanked by SEQ ID Nos. 1 and 2.

[0044] DEPOSIT

[0045] Seeds of Apium graveolens L. dulce homozygously comprising the quantitative trait locus (QTL) on chromosome 8 conferring the “reduced foliage” trait of the invention, were deposited with NCIMB Ltd, Wellheads Place, Dyce, Aberdeen, AB21 7GB Scotland on 12 April 2024 under deposit accession number NCIMB 44381.

[0046] SEQUENCE INFORMATION

[0047] SEQ ID No: 1 Genetic SNP marker RF1. A genomic fragment of Apium graveolens L. dulce chromosome 8 is presented (positive strand), wherein the position of the SNP comprising a change from T to A is indicated as [T / A]. In this sequence, as well as in all other sequences listed herein, Y refers to any pyrimidine (C or T), M refers to A or C, R refers to any purine (A or G), W refers to A or T, K refers to G or T, and S refers to C or G.

[0048] ATATAGTGAAAAGAATAAGAGGGTGAGGATGGGGCTGTAGCCTGTAGCACAAACGCA CACAGGAGGTAGCTTAGCTCTGCTGGACCGCTTCATGCCTCTC[T / A]TTTTTATGCCAA AAGTACAAACCAAAAAATAGTAAACAAAGCAATTGTATTTTCGAAAACATAAAACAT TAGCCGAAAGGAACTAAAACAATTGAGCATG SEQ ID No: 2 Genetic SNP marker RF2.

[0049] ATGCATATCACGTAACACATAATCATGTTATTCATATATCACGTAATCACATATTCCAT GTAACAYATAAGTCAGGTTGTCAAAAYATAGGTTTTAGGAC[G / A]TTCAGAATTGAAA TCGGGTCAATAACCGGGTTTATCGATCAGCTAYCGACTCAGTATAACTCACAAATCAA ATGACATTGCTATACAAAAGGAATTAGGT

[0050] SEQ ID No: 3 Forward primer that specifically recognizes the wildtype allele of the SNP in SEQ ID No: 1. Suitably it is labelled with FAM dye and used in a KASP™ marker assay in combination with SEQ ID No: 4 and SEQ ID No: 5.

[0051] GAAGGTGACCAAGTTCATGCTATTTTTTGGTTTGTACTTTTGGCATAAAAAA

[0052] SEQ ID No: 4 Forward primer that specifically recognizes the mutant allele of the SNP in

[0053] SEQ ID No: 1. Suitably it is labelled with VIC dye and used in a KASP™ marker assay in combination with SEQ ID No: 3 and SEQ ID No: 5.

[0054] GAAGGTCGGAGTCAACGGATTCTATTTTTTGGTTTGTACTTTTGGCATAAAAAT

[0055] SEQ ID No: 5 Common reverse primer (without a fluorophore label) that can be used in a

[0056] KASP™ marker assay in combination with SEQ ID No: 3 and SEQ ID No: 4, to distinguish between the wildtype and mutant alleles of the SNP in SEQ ID No: 1.

[0057] TGCTGGACCGCTTCATGCCTCT

[0058] SEQ ID No: 6 Forward primer that specifically recognizes the wildtype allele of the SNP in SEQ ID No: 2. Suitably it is labelled with FAM dye and used in a KASP™ marker assay in combination with SEQ ID No: 7 and SEQ ID No: 8.

[0059] GAAGGTGACCAAGTTCATGCTGTTATTGACCCGATTTCAATTCTGAAC

[0060] SEQ ID No: 7 Forward primer that specifically recognizes the mutant allele of the

[0061] SNP in SEQ ID No: 2. Suitably it is labelled with VIC dye and used in a KASP™ marker assay in combination with SEQ ID No: 6 and SEQ ID No: 8.

[0062] GAAGGTCGGAGTCAACGGATTGGTTATTGACCCGATTTCAATTCTGAAT

[0063] SEQ ID No: 8 Common reverse primer (without a fluorophore label) that can be used in a KASP™ marker assay in combination with SEQ ID No: 6 and SEQ ID No: 7, to distinguish between the wildtype and mutant alleles of the SNP in SEQ ID No: 2.

[0064] AGTCAGGTTGTCAAAAYATAGGTTTTAGGA FIGURES

[0065] The invention will be further illustrated in the Examples that follow. In the Examples reference is made to the following figures:

[0066] Figure 1 shows on the left side a typical wildtype celery leaf, with indication of the first internode (also known as the “stalk”), a second and third internode and a terminal leaflet. The dashed line indicates the usual position at which a celery leaf is cut during industrial processing, namely immediately below the first node. The part below the cutting level is commercially valuable, while the part above the cutting level is usually discarded as waste. On the right side, a typical leaf of a Rf-type celery plant of the invention is shown. It comprises two internodes on average, wherein the first internode is on average longer than in wildtype plants, and the second internode is always shorter than in wildtype plants. The overall result is that the proportion of leaf material that is discarded as waste is much lower than in wildtype celery plants.

[0067] Figure 2 shows a comparison between representative wildtype and Rf-type celery plants that had been grown alongside each other in identical conditions and for the same amount of time. In panel A, intact celery plants from both types are compared. In panel B, individual leaves are shown from representative wildtype and Rf-type celery plants. In two cases the first internode is indicated with a bar (“1st”), and arrows indicate the positions of nodes. The wildtype celery leaves in this figure have three internodes, whereas the Rf-type celery leaves have two internodes. It appears that the original first node is no longer present in the Rf-type and that the first internode thus comprises the original first and second internode.

[0068] Figure 3 shows the average number of internodes per leaf in Fl progeny plants derived from three different crosses (Wt x Wt, Rf x Wt, and Rf x Rf).

[0069] Figure 4 shows the average length (in millimeter) of the first and second leaf internodes, and the length of the total leaf, as measured in 15 wildtype (Wt) and in 15 Rf-type celery plants. One representative leaf was measured per plant.

[0070] EXAMPLES

[0071] EXAMPLE 1

[0072] Mapping of the Reduced Foliage trait on chromosome 8

[0073] In the research leading to the invention, plants with leaves of the Rf-type were observed.

[0074] A specific Rf-type celery plant (line 12.31110) was crossed (as a father) to a wildtype celery plant (Apium graveolens dulce) as a mother, and from this cross a segregating F2 progeny population of 282 plants was derived. In this population the Rf-type was observed to segregate in a monogenic recessive fashion, as determined by counting the total number of internodes per leaf petiole in the F2 progeny: of the 282 F2 plants, 77 had petioles with two internodes (= 27.3%), 193 plants had petioles with three internodes, and 12 plants had petioles with four internodes. This corresponds to a three-to-one ratio of wildtype plants (with three or more internodes) versus Rf-type plants (with two internodes).

[0075] Based on this segregating population, a QTL mapping study was performed in which the genetic linkage of the Rf-phenotype was investigated to a set of molecular markers that were distributed evenly across all eleven chromosomes of the celery genome. In this study, a QTL region was identified on chromosome 8 that was closely linked to the presence of the Rf-type in celery plants. The QTL region was flanked by marker RF1 (SEQ ID No. 1) and marker RF2 (SEQ ID No. 2). With reference to the public Api-gra_Ventura_vl genome assembly (PMID 33095976; Song et al., 2020, Plant Biotechnol. 19: 731-744), the QTL region of the invention was found to be located on chromosome 8 between positions 229,801,360 and 237,176,427. The genetic distance between the two flanking markers (RF1 and RF2) was observed to be about 0.1 cM.

[0076] Table 1 provides the sequences of the SEQ ID Nos. that can be used as markers, or that can be used to develop markers, to identify the presence of the QTL of the invention in a celery plant. Table 1 also lists the position of the SNP in each sequence, the derived (mutant) allele that is linked to the Rf trait, and the wildtype allele for each SNP, as well as the physical position of each SNP on the public Api-gra_Ventura_vl genome assembly.

[0077] To enable the unambiguous detection of the QTL region linked to the Rf-trait in celery breeding, and the selection of plants comprising said QTL region in their genome at any stage of their life cycle, primers were designed for the two flanking markers that were found to delimit said QTL region, for use in a KASP™ assay (KBiosciences). Table 2 provides a list of KASP primers that are suitable for the identification of the two flanking markers delimiting the QTL region.

[0078] EXAMPLE 2

[0079] Morphological characterization of the Reduced Foliage trait in celery

[0080] Fl progeny plants derived from three different crosses were grown in a field: Wt x Wt (five plots with five plants per plot), Rf x Rf (seven plots with five plants per plot), and Rf x Wt (five plots with five plants per plot). For each plant, the total number of internodes was counted in five individual leaves, resulting in a total of 125 measurements for both the Wt x Wt and Rf x Wt crosses, and 175 measurements for the Rf x Rf cross.

[0081] As shown in Figure 3, we observed that celery plants derived from the Wt x Wt cross had on average 3.4 + 0.5 internodes per leaf, while the offspring of the Rf x Rf cross had on average 2.0 + 0.0 internodes per leaf. For the progeny derived from the Rf x Wt cross the average was 3.0 ± 0.1 internodes per leaf. This experiment thus revealed that Rf-type celery plants that are homozygous for the Rf trait always have two internodes, whereas wildtype (Wt) celery plants usually have three or four internodes. Important to note is that the terminal leaflet has not been counted as an internode in this experiment.

[0082] In order to further describe and quantify the phenotype of Rf-type celery plants, 15 celery plants of the Rf-type were grown in a field alongside 15 wildtype celery plants. All leaves of the Rf-type plants had two internodes, and all leaves of the Wt plants had three internodes. The length of the first and second internodes of one representative leaf of each plant was measured, and the results are shown in Figure 4. The first internode, which is the commercial product known as the celery stalk or stick, was observed to be on average slightly longer in Rf-type celery plants than in Wt celery plants: 328 + 30 mm in Rf-type plants compared to 285 + 23 mm in Wt plants. However, the second internode was significantly shorter in Rf-type celery plants than in Wt celery plants: 43 + 6 mm in Rf-type plants compared to 137 + 10 mm in Wt plants. The average total leaf length of the Wt celery plants was measured to be 586 + 32 mm, as compared to 461 + 36 mm for Rf-type plants. In Wt celery plants the first internode thus constituted about 50 percent of the total leaf length, whereas this ratio was about 71 percent in Rf-type celery plants.

[0083] Taken together, the data from this experiment thus demonstrated that Rf-type celery plants have shorter leaves than Wt celery plants, but that they have a much higher commercially useful proportion than Wt celery plants, and that much less plant material needs to be discarded post-harvest. This is also illustrated in Figure 1 and Figure 2.

[0084] EXAMPLE 3

[0085] Introduction of the Reduced Foliage trait into other celery plants

[0086] Plants of the invention that were deposited under NCIMB accession number 44381 were crossed with wildtype celery plants that did not display the Rf phenotype. The F2 progeny segregated for plants that showed the same characteristics as the parent plant of NCIMB accession number 44381, more specifically having the Rf trait of the invention. These plants could be identified and selected from among the F2 progeny population by using the KASP primers developed in Example 1 and presented in Table 2. Further development of these plants resulted in lines and hybrid varieties with the Rf trait of the invention, as found in NCIMB accession number 44381.

Claims

CLAIMS1. A cultivated Apium graveolens L. dulce plant comprising a QTL on chromosome 8 which is located between SEQ ID No. 1 and SEQ ID No. 2, which QTL when homozygously present is responsible for the presence of two internodes on average per leaf at the harvesting stage.

2. A cultivated Apium graveolens L. dulce plant as claimed in claim 1 , wherein the average number of internodes per leaf at the harvesting stage is two.

3. A cultivated Apium graveolens L. dulce plant as claimed in claims 1 or 2, wherein the QTL on chromosome 8 is genetically linked to at least one of the SNPs as presented in SEQ ID No. 1 and SEQ ID No. 2.

4. A cultivated Apium graveolens L. dulce plant as claimed in any one of the claims 1 to 3, wherein the QTL is as comprised in the genome of an Apium graveolens L. dulce plant representative seed of which was deposited with the NCIMB under deposit number NCIMB 44381.

5. A cultivated Apium graveolens L. dulce plant as claimed in any one of the claims 1 to 4, wherein the QTL is introgressed from NCIMB 44381, or from a progeny plant thereof that has retained the QTL as defined in any one of claims 1 to 4.

6. A cell of a cultivated Apium graveolens L. dulce plant according to claims 1 to 5, which cell comprises a QTL on chromosome 8 as defined in any of the claims 1 to 5 in its genome.

7. An Apium graveolens L. dulce seed comprising a QTL on chromosome 8 as defined in any of the claims 1 to 5 in its genome, wherein a 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 an Apium graveolens L. dulce 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 produced from the propagation material comprises a QTL on chromosome 8 as defined in any of the claims 1 to 5 that is responsible for the presence of two internodes on average per leaf at the harvesting stage.

9. Marker for the identification in an Apium graveolens L. dulce plant of a QTL on chromosome 8 as defined in any of the claims 1 to 5, which marker is a marker comprising the SNPs presented in SEQ ID Nos. 1 or SEQ ID No. 2.

10. Use of a marker as claimed in claim 9 for identification of an Apium graveolens L. dulce plant with two internodes on average per leaf at the harvesting stage.

11. Method for selecting an Apium graveolens L. dulce plant with two internodes on average per leaf at the harvesting stage, comprising detecting the presence of a QTL on chromosome 8 as defined in any one of the claims 1 to 5, and selecting a plant that comprises said QTL in a homozygous state.

12. Method as claimed in claim 11, wherein the presence of the QTL on chromosome 8 is detected by using a marker comprising a SNP presented in SEQ ID No. 1 or SEQ ID No. 2..

13. Method for producing a cultivated Apium graveolens L. dulce plant with two internodes on average per leaf at the harvesting stage, 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 homozygously comprises a QTL on chromosome 8 as defined in any one of the claims 1 to 5, which plant has two internodes on average per leaf at the harvesting stage.

14. Method as claimed in claim 13, wherein the plant comprising the QTL on chromosome 8 is selected by using a molecular marker genetically linked to the QTL, preferably a marker comprising a SNP presented in SEQ ID No. 1 or SEQ ID No. 2.

15. Method for producing a cultivated Apium graveolens L. dulce plant with two internodes on average per leaf at the harvesting stage, 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 homozygously comprises a QTL on chromosome 8 as defined in any one of the claims 1 to 5, which plant has two internodes on average per leaf at the harvesting stage, by using a molecular marker genetically linked to the QTL, preferably a marker comprising a SNP presented in SEQ ID No. 1 or SEQ ID No. 2.

16. Method as claimed in any one of the claims 13-15, wherein the plant with two internodes on average per leaf at the harvesting stage is phenotypically selected, in particular by determining the average number of internodes per leaf.

17. Method as claimed in any one of the claims 13 to 16, wherein the plant according to any one of the claims 1 to 5 is a plant grown from seed deposited under NCIMB accession number NCIMB 44381, or a progeny plant thereof that has retained the QTL on chromosome 8.

18. Method for the production of cultivated hybrid Apium graveolens L. dulce seed comprising crossing a first parent plant with a second parent plant and harvesting the resultant hybrid seed, wherein the first parent plant and the second parent plant are plants comprising the QTL on chromosome 8 as defined in any one of the claims 1 to 5 in a homozygous state.

19. The cultivated hybrid Apium graveolens L. dulce seed produced by the method of claim 18.

20. A QTL on chromosome 8 of the Apium graveolens L. dulce genome, which when present in a homozygous state is responsible for the presence of two internodes on average per leaf at the harvesting stage, which QTL comprises a nucleotide sequence flanked by SEQ ID Nos. 1 and 2.

21. Method for growing a cultivated Apium graveolens L. dulce plant with two internodes on average per leaf at the harvesting stage, which method comprises the step of germinating a seed comprising the QTL according to claim 20 in its genome and allowing the germinated seed to grow into a plant.

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