# Patent

## Title
QTL responsible for tomato fruit firmness

## Abstract

This invention relates to QTL responsible for significantly increased firmness in tomato fruit in the cultivated plant producing said tomato fruit, compared to fruit from a control tomato plant which does not have said genetic elements. A cultivated tomato plant producing tomato fruit with significantly increased fruit firmness and a method for detecting QTLs linked to significantly increased fruit firmness are also provided.

## Background
RELATED APPLICATION INFORMATION
This application claims priority under 35 U.S.C. §371 from InternationalApplication No. PCT/EP2013/062071, filed 11 Jun. 2013, which claims priority to European Patent Application No. EP12163971.0, filed 12 Apr. 2012, the contents of which are incorporated herein by reference herein in their entirety.
STATEMENT REGARDING ELECTRONIC SUBMISSION OF A SEQUENCE LISTING
A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled “SEQLTXT.txt”, 748 bytes in size, generated on Apr. 19, 2013 and filed via EFS-Web is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated by reference into the specification for its disclosures.
Tomato is a well known source of vitamins, minerals and antioxidants, which make up the essential components of a balanced healthy diet. It is also widely accepted that quality attributes such as colour, flavour and firm texture strongly influence consumer choice in the purchase of this expensive and readily perishable crop.
Harvesting tomato fruit when ripening has set in would make maturity determination easier as it would be based on visible peel color and would assure full quality development. After harvest, ripening continues and softening advances, increasing the susceptibility of the fruit to handling damage and limiting the marketing period. Slowing down the ripening and softening stages would allow harvesting, transport and storage of partially ripe but firm fruit (T. Chanthasombath et al, 2008).
Ripening mutants in tomato such as Colourless non-ripening and ripening inhibitor have yielded important insights into an emerging genetic framework which regulates ripening and modulates fruit firmness (Thompson et al, 1999; Vrebalov et al, 2002; is Eriksson et al, 2004; Manning et al, 2006). Delaying ripening and softening may be achieved by employing modified atmosphere packaging (MAP) which has been extensively studied as a simple and cheap method of prolonging shelf life of many fruits and vegetables including tomato (Batu & Thompson, 1998, Exama et al, 1993, Geeson et al, 1985), however it increases the cost of packaging and handling of fruits. Existing methods to enhance fruit firmness in conventional plant breeding programs rely on screening fruit firmness differences in fruit harvested from mature plants. Any identification of enhanced fruit firmness in this scenario will largely be down to chance. Currently it is not financially viable or efficient to breed for enhanced fruit firmness due to the cost and complexity of growing and phenotyping large numbers of plants.
To bridge the gap between the emerging model for the regulation of fruit ripening and a full knowledge of the components involved in controlling fruit firmness will require additional strategies to those based on either targeting genes for known cell wall-related proteins or investigating pleiotropic ripening mutants. Fruit firmness is a quantitative trait involving many genes and yet the identity of the majority of these genes remains elusive.
Wild tomato species offer a rich and largely unexplored source of new genetic variation for breeders. Tanksley and Zamir (Frary et al, 2000; Fridman et al, 2004) have demonstrated that this source of genetic diversity can be used to understand the molecular basis of important fruit quality traits and provide new material for breeding.
The need clearly exists for the efficient and early selection of genotypes that are likely to display firmer fruit when mature. However, previous work in the texture area has been hampered by problems of reproducibility and precision.
SUMMARY OF THE INVENTION
The inventors have defined a genetic element (or QTL) which is linked to significantly increased fruit firmness in tomato.
The genetic element is on chromosome 3 of tomato and narrowed to a 2.1 MB region (between DNA markers TG599 and TG42). Further analysis has revealed that the genetic element contains the tomato pectate lyase gene (PL). Results from the tomato EST programme (available on the World Wide Web at tigr.org/tdb/lgi/index.html) has previously suggested a high level of PL-like gene expression in ripe tomato fruits. However, the present inventors have surprisingly shown that expression levels of the PL gene, when introgressed into cultivated tomato from a wild tomato source, is around 20 fold lower than that found in control plants. It has also been shown that, when the region comprising the PL gene is introgressed, there is a corresponding increase in fruit firmness. This increase is only observed after the breaker stage. The current application thus demonstrates for the first time the importance of developmental stage specific fruit firmness associated with PL from a wild source in a cultivated background.
The present invention therefore relates in a first aspect to a tomato fruit with significantly increased fruit firmness at the harvesting stage, preferably at the breaker plus 7 days stage, wherein said increased fruit firmness is linked to said genetic elements in the cultivated tomato plant producing said tomato fruit, wherein said firmness is up to 4 times greater or, more preferably, up to 2 times greater than that of fruit from a control tomato plant which does not have this genetic element. Introgression lines IL3-4 (deposited under accession number LA3489), and which bears this genetic element, showed a significant increase in fruit firmness compared to an M82 parental control. There is a dominant effect of this genetic element in the F1 generation and the trait is most apparent in ripe tomato fruit.
There is also provided a cultivated tomato plant which produces tomato fruit with increased firmness as described above, wherein said plant can be characterised by a) the at least one genetic element is linked to at least one DNA marker selected from the group consisting of TG599, NT3374FM, TG246, NT5880VC and TG42 and/or b) the at least one genetic element is complementary to the corresponding genetic element in S. pennellii lines IL3-4, wherein the said genetic element in IL3-4 is linked to at least one DNA marker selected from the said group in (a).
There is also provided a cultivated tomato plant which produces tomato fruit with increased firmness as described above, wherein said plant can be characterised by a) the at least one genetic element is linked to at least one DNA marker selected from the group consisting of NT3374FM, TG246 and NT5880VC and/or b) the at least one genetic element is complementary to the corresponding genetic element in S. pennellii lines IL3-4, wherein the said genetic element in IL3-4 is linked to at least one DNA marker selected from the said group in (a).
There is also provided a cultivated tomato plant wherein the at least one genetic element is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
There is also provided a cultivated tomato plant as described above wherein the QTL is QTL1 linked to at least one of the DNA markers NT3374FM, TG246 and NT5880VC; which is present only in the outer pericarp.
There is also provided a cultivated tomato plant as described above wherein said plant is an inbred, a diploid or a hybrid. In a specific embodiment, the plant is male sterile.
There is also provided a tomato seed which produces a cultivated tomato plant of the present invention.
There is also provided a plant part of a cultivated tomato plant as described herein. In a specific embodiment there is provided plant material obtainable from a plant part of a cultivated tomato plant as described herein.
The above identified DNA markers represent a rapid assay and research tool to select for cultivated tomato plants containing a defined genetic region. These DNA markers yield the potential to manipulate a very small and well defined genetic region which is linked to increased fruit firmness. This has the advantage of allowing the screening of large numbers (1000's) of plants at a very early stage of development for desirable combinations of DNA markers and will allow the enhancement of fruit is firmness in commercial varieties of tomato.
There is also provided a method for detecting a QTL linked to significantly increased fruit firmness in fruit from a cultivated tomato plant as described above compared to a control tomato plant comprising the steps of a) crossing a donor tomato plant with a recipient tomato plant to provide offspring plants, b) quantitatively determining the firmness in the fruit of said offspring plants c) establishing a genetic linkage map that links the observed increased fruit firmness to the presence of at least one DNA marker from said donor plant in said offspring plants and d) assigning to a QTL the DNA markers on said map that are linked to significantly increased fruit firmness.
Preferably said donor plant has fruit with a significantly increased fruit firmness compared to said recipient plant.
Preferably, the donor plant is S. pennellii and the recipient plant is S. lycopersicum. The fruit firmness range in the offspring plants is up to 4 times greater, or alternatively up to 2 times greater, than that of fruit produced from a control tomato plant at the harvesting stage. Preferably, the harvesting stage is the breaker stage plus 7 days. Preferably the at least one DNA marker is selected from TG599, NT3374FM, TG246, NT5880VC and TG42. Preferably said QTL is QTL1 linked to at least one of the DNA markers NT3374FM, TG246 and NT5880VC.
In one embodiment, said QTL is QTL1 linked to at least one of the DNA markers NT3374FM, TG246 and NT5880VC; which is present only in the outer pericarp.
There is also provided a QTL responsible for increased fruit firmness in fruit of a cultivated tomato plant detected by a method as herein described.
In one embodiment, said QTL is located on chromosome 3.
In one embodiment there is provided a QTL as herein described linked to at least one to DNA marker selected from the group consisting of TG599, NT3374FM, TG246, NT5880VC and TG42.
In one embodiment there is provided a QTL as herein described wherein said QTL is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
In one embodiment there is provided a QTL as herein described wherein said QTL is QTL1 linked to at least one of the DNA markers NT3374FM, TG246 and NT5880VC; which is present only in the outer pericarp.
There is also provided an isolated DNA sample obtained from a tomato plant comprising a QTL as herein described.
There is also provided a method of producing a tomato plant which provides fruit with increased fruit firmness as herein described.
There is also provided a method of producing an offspring cultivated tomato plant which provides fruit with increased fruit firmness comprising the steps of performing a method for detecting a QTL linked to increased fruit firmness, and transferring a nucleic acid comprising at least one QTL thus detected, from a donor tomato plant to a recipient tomato plant, wherein said increased fruit firmness is measured in fruit from an offspring cultivated tomato plant compared to fruit from a control tomato plant. The transfer of nucleic acid can be performed by any of several methods known in the art e.g. transformation, by protoplast fusion, by a doubled haploid technique or by embryo rescue.
Preferably, the fruit firmness range in the offspring tomato plant is greater than fruit from a control tomato plant at the breaker plus 7 days stage. The fruit firmness range could be up to 4 times greater or, alternatively, up to 3 times greater or, alternatively, up to 2 times greater. Preferably, the donor plant is S. pennellii and the recipient plant is S. lycopersicum. Preferably, said QTL is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
In one embodiment said QTL is QTL1 linked to at least one of the DNA markers to NT3374FM, TG246 and NT5880VC; which is present only in the outer pericarp.
There is also provided a tomato plant, or part thereof, obtainable by a method as described herein.
There is also provided a cultivated tomato plant comprising a QTL responsible for increased fruit firmness as described herein.
There is also provided a hybrid tomato plant, or part thereof, obtainable by crossing a tomato plant as described herein with a tomato plant that exhibits commercially desirable characteristics.
There is also provided tomato seed produced by growing a tomato plant as described herein.
There is also provided tomato seed produced by crossing a tomato plant as described herein with a plant having desirable phenotypic traits to obtain a plant that has significantly increased fruit firmness compared to a control plant.
There is also provided the use of a QTL as described herein for the production of a tomato plant having increased fruit firmness compared to a control plant.
There is also provided the use of a tomato plant for expanding the harvesting slot of tomato fruit and/or for use in the fresh cut market or for food processing.
There is also provided processed food made from a tomato plant comprising the at least one QTL as described herein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 Mechanical measurements on the outer pericarp of IL3-4 lines reveals significantly enhanced fruit firmness at the breaker plus 7 day stage and the breaker plus 14 day stage in comparison with the M82 controls. The graph shows the maximum load (N) on the Y-axis and fruit developmental stage on the X-axis.
FIG. 2 Mechanical measurements reveal that the texture effect from S. pennelii appears to be semi-dominant in heterozygous individuals. The graph shows the maximum load (N) on the Y-axis and parental and F1 populations used on the X-axis.
FIG. 3 Location of QTL for increased fruit firmness on tomato chromosome 3F as determined by measuring fruit firmness in the outer pericarp. QTL1 is shown as a black solid bar. Marker positions and the distance between them are shown on the X-axis. LOD score is shown on the Y-axis.
FIG. 4 Mean fruit firmness determinations on the outer pericarp of selected QTL-NILs and parent lines. The Y axis shows maximum load (N) and the X-axis represents the line number.
FIG. 5 Genotyping of randomly selected lines of IL3-4 to show distribution of recombination events with respect to Syngenta SSR and published RFLP and PCR-based markers. Key: A=M82; B=S. pennellii; *=missing data. The SSR markers are shown in the left hand column. The selected lines are shown on the top row.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
The technical terms and expressions used within the scope of this application are generally to be given the meaning commonly applied to them in the pertinent art of plant breeding and cultivation if not otherwise indicated herein below.
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a plant” includes one or more plants, and reference to “a cell” includes mixtures of cells, tissues, and the like.
As used herein, the term “about” when referring to a value or to an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.
An “allele” is understood within the scope of the invention to refer to alternative or is variant forms of various genetic units identical or linked to different forms of a gene or of any kind of identifiable genetic element, which are alternative in inheritance because they are situated at the same locus in homologous chromosomes. Such alternative or variant forms may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, chemical or structural modification, transcription regulation or post-translational modification/regulation. In a diploid cell or organism, the two alleles of a given gene or genetic element typically occupy corresponding loci on a pair of homologous chromosomes.
An allele linked to a quantitative trait may comprise alternative or variant forms of various genetic units including those that are identical or linked to a single gene or multiple genes or their products or even a gene disrupting or controlled by a genetic factor contributing to the phenotype represented by said QTL.
As used herein, the term “backcross”, and grammatical variants thereof, refers to a process in which a breeder crosses a hybrid progeny back to one of the parents, for example, a first generation hybrid F1 with one of the parental genotypes of the F1 hybrid. In some embodiments, a backcross is performed repeatedly, with a progeny individual of one backcross being itself backcrossed to the same parental genotype.
As used herein, the term “breeding”, and grammatical variants thereof, refer to any process that generates a progeny individual. Breedings can be sexual or asexual, or any combination thereof. Exemplary non-limiting types of breedings include crossings, selfings, doubled haploid derivative generation, and combinations thereof.
For the purpose of the present invention, the term “co-segregation” refers to the fact that the allele for the trait and the allele(s) for the markers) tend to be transmitted together because they are physically close together on the same chromosome (reduced recombination between them because of their physical proximity) resulting in a non-random association of their alleles. “Co-segregation” also refers to the presence of two or more traits within a single plant of which at least one is known to be genetic and which cannot be readily explained by chance.
A “cultivated tomato plant” is understood within the scope of the invention to refer to is a plant that is no longer in the natural state but has been developed by human care and for human use and/or consumption. “Cultivated tomato plants” are further understood to exclude those wild-type species which comprise the trait being subject of this invention as a natural trait and/or part of their natural genetics. Cultivated tomato plants also typically display resistance to Tobacco mosaic virus, whereas wild-type species do not. For the purposes of the present invention, the S. pennellii introgression line IL3-4 is not regarded as a cultivated line, whereas parental line M82 and recombinant lines e.g. Q7774 are regarded as cultivated lines.
As used herein, the term “dihaploid line”, refers to stable inbred lines issued from anther culture. Some pollen grains (haploid) cultivated on specific medium and circumstances can develop plantlets containing n chromosomes. These plantlets are then “doubled” and contain 2n chromosomes. The progeny of these plantlets are named “dihaploid” and are essentially not segregating anymore (stable).
As used herein, the term “gene” refers to a hereditary unit including a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristic or trait in an organism.
As used herein, the term “genetic architecture at the QTL” refers to a genomic region which is statistically correlated to the phenotypic trait of interest and represents the underlying genetic basis of the phenotypic trait of interest.
“Genetic engineering”, “transformation” and “genetic modification” are all used herein as synonyms for the transfer of isolated and cloned genes into the DNA, usually the chromosomal DNA or genome, of another organism.
As used herein, the phrases “genetic marker”, “DNA marker” or “molecular marker” are interchangeable and refer to a feature of an individual's genome (e.g. a nucleotide or a polynucleotide sequence that is present in an individual's genome) that is linked to one or more loci of interest. In some embodiments, a genetic marker is polymorphic in a population of interest, or the locus occupied by the polymorphism, depending on context. 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 is 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 phrase “genetic marker” can also refer to a polynucleotide sequence complementary to a genomic sequence, such as a sequence of a nucleic acid used as probes. A genetic or molecular marker can be physically located in a position on a chromosome that is distal or proximal to the genetic loci with which it is linked (i.e. is intragenic or extragenic, respectively). Stated another way, whereas genetic markers are typically employed when the location on a chromosome of the gene or of a functional mutation, e.g. within a control element outside of a gene, that corresponds to the locus of interest has not been identified and there is a very low rate of recombination between the genetic marker and the locus of interest, the presently disclosed subject matter can also employ genetic markers that are physically within the boundaries of a genetic locus (e.g. inside a genomic sequence that corresponds to a gene such as, but not limited to a polymorphism within an intron or an exon of a gene). In some embodiments of the presently disclosed subject matter, the one or more genetic markers comprise between one and ten markers, and in some embodiments the one or more genetic markers comprise more than ten genetic markers.
As used herein, the term “genotype” refers to the genetic constitution of a cell or organism. An individual's “genotype for a set of genetic markers” includes the specific alleles, for one or more genetic marker loci, present in the individual's haplotype. As is known in the art, a genotype can relate to a single locus or to multiple loci, whether the loci are related or unrelated and/or are linked or unlinked. In some embodiments, an individual's genotype relates to one or more genes that are related in that the one or more of the genes are involved in the expression of a phenotype of interest (e.g. a quantitative trait as defined herein). Thus, in some embodiments a genotype comprises a summary of one or more alleles present within an individual at one or more genetic loci of a quantitative trait. In some embodiments, a genotype is expressed in terms of a haplotype (defined herein below).
“Heterozygous” is understood within the scope of the invention to refer to unlike alleles at one or more corresponding loci on homologous chromosomes.
“Homozygous” is understood within the scope of the invention to refer to like alleles at one or more corresponding loci on homologous chromosomes.
As used herein, the term “hybrid” in the context of plant breeding refers to a plant that is the offspring of genetically dissimilar parents produced by crossing plants of different lines or breeds or species, including but not limited to the cross between two inbred lines.
The term “hybridize” as used herein refers to conventional hybridization conditions, preferably to hybridization conditions at which 5×SSPE, 1% SDS, 1×Denhardts solution is used as a solution and/or hybridization temperatures are between 35° C. and 70° C., preferably 65° C. After hybridization, washing is preferably carried out first with 2×SSC, 1% SDS and subsequently with 0.2×SSC at temperatures between 35° C. and 75° C. particularly between 45° C. and 65° C., but especially at 59° C. (regarding the definition of SSPE, SSC and Denhardts solution see Sambrook et al. (2001)). High stringency hybridization conditions as for instance described in Sambrook et al. (2001), are particularly preferred. Particularly preferred stringent hybridization conditions are for instance present if hybridization and washing occur at 65° C. as indicated above. Non-stringent hybridization conditions for instance with hybridization and washing carried out at 45° C. are less preferred and at 35° C. even less.
As used herein, the phrase “inbred line” refers to a genetically homozygous or nearly homozygous population. An inbred line, for example, can be derived through several cycles of brother/sister breedings or of selfing or in dihaploid production. In some embodiments, inbred lines breed true for one or more phenotypic traits of interest. An “inbred”, “inbred individual”, or “inbred progeny” is an individual sampled from an inbred line.
As used herein, the terms “introgression”, “introgressed” and “introgressing” refer to the process whereby genes, a QTL or haplotype of one species, variety or cultivar are moved into the genome of another species, variety or cultivar, by crossing those species. The crossing may be natural or artificial. The process may optionally be completed by backcrossing to the recurrent parent, in which case introgression refers to infiltration of the genes of one species into the gene pool of another through repeated backcrossing of an interspecific hybrid with one of its parents. An introgression may also be described as a heterologous genetic material stably integrated in the genome of a recipient plant.
As used herein, the term “linkage”, and grammatical variants thereof, refers to the tendency of alleles at different loci on the same chromosome to segregate together more often than would be expected by chance if their transmission were independent, in some embodiments as a consequence of their physical proximity. Linkage is measured by percent recombination between loci (centimorgan, cM).
As used herein, the phrase “linkage group” refers to all of the genes or genetic traits that are located on the same chromosome. Within the linkage group, those loci that are close enough together can exhibit linkage in genetic crosses. Since the probability of crossover increases with the physical distance between loci on a chromosome, loci for which the locations are far removed from each other within a linkage group might not exhibit any detectable linkage in direct genetic tests. The term “linkage group” is mostly used to refer to genetic loci that exhibit linked behavior in genetic systems where chromosomal assignments have not yet been made. Thus, in the present context, the term “linkage group” is synonymous with the physical entity of a chromosome, although one of ordinary skill in the art will understand that a linkage group can also be defined as corresponding to a region of (i.e. less than the entirety) of a given chromosome. Preferably, markers of the present invention are no further than 1 cM from the genetic element conferring fruit firmness.
“Locus” is understood within the scope of the invention to refer to a region on a chromosome, which comprises a gene or any other genetic element or factor contributing to a trait.
As used herein, the term “marker allele” refers to an alternative or variant form of a genetic unit as defined herein above, when used as a marker to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits.
“Marker-based selection” is understood within the scope of the invention to refer to e.g. the use of genetic markers to detect one or more nucleic acids from the plant, where the nucleic acid is linked to a desired trait to identify plants that carry genes, QTLs or haplotypes for desirable (or undesirable) traits, so that those plants can be used (or avoided) in a selective breeding program.
“Marker assisted selection” refers to the process of selecting a desired trait or desired traits in a cultivated plant or cultivated plants by detecting one or more nucleic acids from the cultivated plant, where the nucleic acid is linked to the desired trait.
As used herein, “marker locus” refers to a region on a chromosome, which comprises a nucleotide or a polynucleotide sequence that is present in an individual's genome and that is linked to one or more loci of interest, which may which comprise a gene or any other genetic element or factor contributing to a trait. “Marker locus” also refers to a region on a chromosome, which comprises a polynucleotide sequence complementary to a genomic sequence, such as a sequence of a nucleic acid used as a probe.
“Microsatellite or SSRs (Simple sequence repeats) marker” is understood within the scope of the invention to refer to a type of genetic marker that consists of numerous repeats of short sequences of DNA bases, which are found at loci throughout the plant's genome and have a likelihood of being highly polymorphic.
“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammatical equivalents thereof used herein means at least two nucleotides covalently linked together. Oligonucleotides are typically from about 7, 8, 9, 10, 12, 15, 1820 25, 30, 40, 50 or up to about 100 nucleotides in length. Nucleic acids and polynucleotides are polymers of any length, including longer lengths, e.g. 200, 300, 500, 1000, 2000, to 3000, 5000, 7000, 10000, etc. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have alternate backbones comprising, e.g. phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages (see Eckstein, 1991), and peptide nucleic acid backbones and linkages. Mixtures of is naturally occurring nucleic acids and analogs can be used. Particularly preferred analogs for oligonucleotides are peptide nucleic acids (PNA).
As used herein, the term “offspring” plant refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof. For instance an offspring plant can be obtained by cloning or selfing of a parent plant or by crossing two parent plants and include selfings as well as the F1 or F2 or still further generations. An F1 is a first-generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offspring of second generation (F2) or subsequent generations (F3, F4, and the like) are specimens produced from selfings of F1s, F2s and the like. An F1 can thus be (and in some embodiments is) a hybrid resulting from a cross between two true breeding parents (true-breeding is homozygous for a trait), while an F2 can be (and in some embodiments is) an offspring resulting from self-pollination of the F1 hybrids.
“PCR (Polymerase chain reaction)” is understood within the scope of the invention to refer to a method of producing relatively large amounts of specific regions of DNA or subset(s) of the genome, thereby making possible various analyses that are based on those regions.
“PCR primer” is understood within the scope of the invention to refer to relatively short fragments of single-stranded DNA used in the PCR amplification of specific regions of DNA.
As used herein, the expression “phenotype” or “phenotypic trait” refers to the appearance or other detectable characteristic of an individual, resulting from the interaction of its genome, proteome and/or metabolome with the environment.
A “plant” is any plant at any stage of development, particularly a seed plant.
A “plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.
“Plant cell culture” means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
“Plant material” refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
As used herein, the phrase “plant part” refers to a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps, and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as scions, rootstocks, protoplasts, calii, and the like.
“Plant tissue” as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
“Polymorphism” is understood within the scope of the invention to refer to the presence in a population of two or more different forms of a gene, genetic marker, or inherited trait or a gene product obtainable, for example, through alternative splicing, DNA methylation, etc.
As used herein, the term “population” means a genetically heterogeneous collection of plants sharing a common genetic derivation.
“Probe” as used herein refers to a group of atoms or molecules which is capable of recognizing and binding to a specific target molecule or cellular structure and thus allowing detection of the target molecule or structure. Particularly, “probe” refers to a labeled DNA or RNA sequence which can be used to detect the presence of and to is quantitate a complementary sequence by molecular hybridization.
As used herein, the term “progeny” refers to the descendant(s) of a particular cross. Typically, progeny result from breeding of two individuals, although some species (particularly some plants and hermaphroditic animals) can be selfed (i.e. the same plant acts as the donor of both male and female gametes). The descendant(s) can be, for example, of the F1, the F2, or any subsequent generation.
The term “QTL” is used herein in its art-recognised meaning. The term “QTL linked to increased fruit firmness in tomato” as well as the shorter term “QTL for increased fruit firmness” refer to a region located on a particular chromosome of tomato that is linked to at least one gene that is responsible for increased fruit firmness or at least a regulatory region, i.e. a region of a chromosome that controls the expression of one or more genes involved in increased fruit firmness. A QTL may for instance comprise one or more genes, the products of which confer increased fruit firmness. Alternatively, a QTL may for instance comprise regulatory genes or sequences, the products of which influence the expression of genes on other loci in the genome of the plant thereby conferring the increased fruit firmness. The QTLs of the present invention may be defined by indicating their genetic location in the genome of the respective wild tomato accession using one or more molecular genomic markers. One or more markers, in turn, indicate a specific locus. Distances between loci are usually measured by frequency of crossing-over between loci on the same chromosome. The farther apart two loci are, the more likely that a crossover will occur between them. Conversely, if two loci are close together, a crossover is less likely to occur between them. As a rule, one centimorgan (cM) is equal to 1% recombination between loci (markers). When a QTL can be indicated by multiple markers the genetic distance between the end-point markers is indicative of the size to of the QTL.
As used herein, the term “QTL1” refers to the genomic region linked to increased tomato firmness as defined by the markers TG599, NT3374FM, TG246, NT5880VC and TG42. For the purposes of the instant disclosure, these markers are said to be present on S. pennellii chromosome 3.
The term “quantitatively determining” is defined herein as establishing or assessing in a manner involving measurement, in particular the measurement of aspects measurable in terms of amounts and number. Determinations in degrees of severity and indications of greater, more, less, or equal or of increasing or decreasing magnitude, are not comprised in the present term “quantitatively determining”, which term ultimately implies the presence of objective counting mechanism for determining absolute values.
The term “recipient tomato plant” is used herein to indicate a tomato plant that is to receive DNA obtained from a donor tomato plant that comprises a QTL for increased fruit firmness. Said “recipient tomato plant” may or may not already comprise one or more QTLs for fruit firmness, in which case the term indicates a plant that is to receive an additional QTL.
The term “natural genetic background” is used herein to indicate the original genetic background of a QTL. Such a background may for instance be the genome of a wild accession of tomato. For instance, the QTLs of the present invention were found at specific locations on chromosome 3 of S. pennellii. As an example, S. pennellii represents the natural genetic background of QTL1 on chromosome 3 of S. pennellii. Conversely, a method that involves the transfer of DNA comprising this QTL, or a fruit firmness-conferring part thereof, from Chromosome 3 of S. pennellii to the same position on chromosome 3 of another tomato species, preferably cultivated S. lycopersicum, will result in this QTL, or said fruit firmness-conferring part thereof, not being in its natural genetic background.
In this application, a “recombination event” is understood to mean a meiotic crossing-over.
“Sequence Homology” or “sequence Identity” is used herein interchangeably. The terms “identical” or “percent identity” in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection, if two sequences which are to be compared with each other differ in length, sequence identity preferably relates to the percentage of the nucleotide residues of the shorter sequence which are identical with the nucleotide residues of the longer sequence. Sequence identity can be determined conventionally with the use of computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive Madison, WI 53711). Bestfit utilizes the local homology algorithm of Smith and Waterman (1981) in order to find the segment having the highest sequence identity between two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has for instance 95% identity with a reference sequence of the present invention, the parameters are preferably so adjusted that the percentage of identity is calculated over the entire length of the reference sequence and that homology gaps of up to 5% of the total number of the nucleotides in the reference sequence are permitted. When using Bestfit, the so called optional parameters are preferably left at their preset (“default”) values. The deviations appearing in the comparison between a given sequence and the above described sequences of the invention may be caused for instance by addition, deletion, substitution, insertion or recombination. Such a sequence comparison can preferably also be carried out with the program fasta20u66′′ (version 2.0u65, September 1998 by William R. Pearson and the University of Virginia; see also W. R. Pearson (1990), appended examples and at workbench.sdsc.edu/). For this purpose, the “default” parameter settings may be used.
Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g. total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
As used herein, the phrases “sexually crossed” and “sexual reproduction” in the context of the presently disclosed subject matter refers to the fusion of gametes to produce progeny (e.g. by fertilization, such as to produce seed by pollination in plants). A “sexual cross” or “cross-fertilization” is in some embodiments fertilization of one individual by another (e.g. cross-pollination in plants). The term “selfing” refers in some embodiments to the production of seed by self-fertilization or self-pollination i.e. pollen and ovule are from the same plant.
A “single nucleotide polymorphism” (SNP) is a DNA sequence variation occurring when a single nucleotide A, C, G, T in the genome (or other shared sequences as mitochondrial DNA) differs between a set (paired) chromosomes of an individual or differs between members of a species.
The term “standard greenhouse conditions” and “standard conditions” refer to the conditions of light, humidity, temperature, etc whereupon plants are grown or incubated, for instance for the purpose of phenotypic characterization of disease fruit firmness, as being standard. Growth conditions can for example be a photoperiod of 16 h (photosynthetic photon flux (PPF) 50 to 1000 μmol nv2 s1), preferably a regime of 8 hours dark, an air temperature of about 20° C. during the day and 18° C. at night, a water vapour pressure deficit of about 4.4 g m3 corresponding to a relative humidity (RH) of about 60%-85%, at atmospheric oxygen concentration and at atmospheric air pressure (generally 1008 hPa). Water and nutrients may be given drop wise near the stem, or in the form of spray or mist.
“Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part 1 chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York. Generally, highly stringent hybridization and wash conditions are selected to be is about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Typically, under “stringent conditions” a probe will hybridize to its target subsequence, but to no other sequences.
The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42° C., with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2 times SSC wash at 65° C. for 15 minutes (see Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of for example more than 100 nucleotides, is 1 times SSC at 45° C. for 15 minutes. An example low stringency wash for a duplex of, e.g. more than 100 nucleotides, is 4-6 times SSC at 40° C. for 15 minutes. For short probes (e.g. about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0M Na ion concentration, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2 times (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs for example when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
As used herein, the term “tomato” means any plant, line or population within the species Solanum lycopersicum (synonyms are Lycopersicon lycopersicum or Lycopersicon esculentum) or formerly known under the genus name of Lycopersicon including but not limited to L. cerasiforme, L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum (now S. pennellii), L. hirsutum, L. parviborum, L. pennellii, L. peruvianum, L. pimpinellifolium, or S. lycopersicoides. The newly proposed scientific name for L. esculentum is S. pennellii. Similarly, the names of the wild species may be altered. L. pennellii has become S. pennellii, L. hirsutum may become S. habrochaites, L. peruvianum may be split into S. ‘N peruvianum’ and S. ‘Callejon de Huayles’, S. peruvianum, and S. corneliomuelleri, L. parviflorum may become S. neorickii, L. chmielewskii may become S. chmielewskii, L. chilense may become S. chilense, L. cheesmaniae may become S. cheesmaniae or S. galapagense, and L. pimpinellifolium may become S. pimpinellifolium (Knapp (2005)).
“Trait” is understood within the scope of the invention to refer to a characteristic or phenotype, for example increased fruit firmness. A trait may be inherited in a dominant or recessive manner, or may be monogenic or polygenic.
“Monogenic” is understood within the scope of the invention to refer to being determined by a single locus.
“Polygenic” is understood within the scope of the invention to refer to being determined by more than one locus.
“Dominant” is understood within the scope of the invention to refer to an allele which determines the phenotype when present in the heterozygous or homozygous state.
A “recessive” allele is only displayed when present in the homozygous state.
“Harvesting stage” is understood within the scope of the invention to mean the date of harvesting ie the date the tomato fruit is removed from the plant.
“Immature Green stage” is defined as when the fruits are unripe and still growing in size. This stage is understood to be the first stage in the ripening process.
“Mature green stage” is defined as when the fruit is fully expanded mature, but unripe and follows the “immature green stage” in the ripening process. Mature green tomatoes have a white to yellow “star” on the blossom end. Traditional tomatoes harvested at the mature green stage are best suited for the commercial fresh market because they tolerate rough handling better than the riper stages and hold their shape the longest in storage, shipping, and on the supermarket shelf; however they somehow lack full aroma and taste.
“Breaker stage” is defined as first sign of red colour in the fruit, typically it occurs within 24 hours of the mature green stage. Tomatoes that are harvested at the “Breaker stage” usually have better flavor and taste but they have reduced firmess and are slighty less suitable for handling, packaging and transportation than tomatoes at the mature green stage.
“Red ripe stage” is defined as when the fruits are fully red, with no sign of green colour. These fruits have reached their optimum in taste and flavor but they cannot be transported because of their lack of firmness and they do not tolerate much handling.
“Genetic element” and “genetic element, or part thereof” are understood within the scope of the invention to mean a QTL or part thereof (in particular, a gene residing on the chromosome under the QTL) that is capable of contributing to the firmness of the fruits of the plant by influencing expression of the firmness trait at the level of the DNA itself, at the level of translation, transcription and/or activation of a final polypeptide product, i.e., to regulate metabolism in tomato fruit flesh leading to the phenotypic expression of the genotype.
“Inner pericarp” and “outer pericarp” are understood within the scope of the invention to mean fruit tissue where the outer pericarp is the layer (approximately 2 mm) immediately below the outer epidermis and above the vascular tissue layer. The inner pericarp is from 3 mm up to 10 mm below the vascular layer and before the inner epidermis.
“Commercially desirable characteristics” are understood within the scope of the invention to include but not be limited to superior fruit quality, disease resistance, insect resistance, uniform shape and size
“Harvesting slot” is understood within the scope of the invention to mean the period of time from the harvesting stage until when the fruit is too ripe to be harvested for is the purposes of commercial sale. Typically, the harvesting slot starts at mature green stage and continues until the breaker stage plus two to five days, depending on the cultivar and environmental conditions.
“donor tomato plant” is understood within the scope of the invention to mean the tomato plant which provides at least one genetic element linked to significantly increased fruit firmness.
“linked to” and “characterized by” or “associated with” at least one of the DNA markers of the present invention is understood within the scope of the invention to mean a DNA sequence that is genetically linked, to the genetic element responsible for the increased fruit firmness trait and wherein a specific marker sequence is linked to a particular allele of that gene. When two markers/sequences are said to be genetically linked, the recombination frequency between the two markers/sequences are low and it can be expected that both these markers/sequences are inherited jointly. For the population of plants described herein, markers named as linked to the QTLs are a distance of 1 cM or less away. Markers that are 1 cM distance apart from each other have a 1% chance of being separated from each other due to a recombination event in a single generation.
“fruit firmness conferring parts of QTLs” is understood within the scope of the invention to mean the genetic element(s) or part(s) thereof responsible for increased tomato fruit firmness as determined by mechanical measurement as described in the examples.
“Increase in fruit firmness” and “increased fruit firmness” are understood within the scope of the invention to mean tomato fruit which has an increased maximum load value (for example as described in Example 1), statistically significant at P<0.05 or P<0.01 compared to fruit from a control plant.
Maximum load is defined as the value that represents the greatest load (in Newtons (N)) required to cause failure of tissue integrity.
“control tomato plant” is understood within the scope of the invention to mean a tomato plant that has the same genetic background as the cultivated tomato plant of is the present invention wherein the control plant does not have any of the at least one genetic elements—or part thereof—of the present invention linked to increased fruit firmness. In particular a control tomato plant is a tomato plant belonging to the same plant variety and does not comprise any of the at least one genetic element, or part thereof. The control tomato plant is grown for the same length of time and under the same conditions as the cultivated tomato plant of the present invention. Plant variety is herein understood according to definition of UPOV. Thus a control tomato plant may be an inbred line or a hybrid provided that they have the same genetic background as the tomato plant of the present invention except the control plant does not have any of the at least one genetic element—or part thereof—of the present invention linked to increased fruit firmness.
“anthesis” is understood within the scope of the invention to mean the period during which the flower is fully open and pollen is released.
“Processed food” is understood within the scope of the invention to mean food which has been altered from its natural state. Methods used for processing food include but are not limited to canning, freezing, refrigeration, dehydration and asceptic processing.
“Fresh cut market” is understood within the scope of the invention to mean vegetables on the market which have been minimally processed.
“first true leaf” is understood within the scope of the invention to mean when the first leaf emerges after emergence of the seed leaves or cotyledons.
Plant Breeding
The purpose of breeding programs in agriculture and horticulture is to enhance the performance of plants by improving their genetic composition. In essence, this improvement accrues by increasing the frequency of the most favorable alleles for the genes influencing the performance characteristics of interest.
Wild plant lines provide a rich resource of genetic and phenotypic variation. Traditionally, agricultural or horticultural practice makes use of this variation by is selecting a wild plant line or its offspring for having desired genotypic or potential phenotypic properties, crossing it with a line having additional desired genotypic or potential phenotypic properties and selecting from among the offspring plants those that exhibit the desired genotypic or potential phenotypic properties (or an increased frequency thereof).
A growing understanding and utilization of the laws of Mendelian inheritance in combination with molecular genetic tools have in the past century facilitated this selection process. For example, methods for selecting plants for having desired genotypic or potential phenotypic properties have become available based on testing the plant for the presence of a quantitative trait locus (QTL); i.e. for the presence of a genetic element containing alleles linked to the expression of a continuously distributed (quantitative) phenotypic trait. Usually a QTL is characterized by one or more markers that statistically associate to the quantitative variation in the phenotypic trait and is essentially synonymous to a gene. QTL mapping allows for the identification of genetic element(s) affecting the expression of a trait of interest. In plant breeding, it allows for marker-assisted selection (MAS); i.e. the selection of plants having favorable alleles by detecting in those plants the QTL-associated markers.
Knowledge of the inheritance of various traits would allow for the selection of lines homozygous for a QTL linked to increased fruit firmness. Use of the knowledge of the genetic origin and location of a desired trait in a breeding program can increase the accuracy of the predicted breeding outcome and can enhance the rate of selection compared to conventional breeding programs. For instance, the fact that the genetic basis of a desired trait is heritably linked to another trait can help to increase uniformity for those two traits among the offspring since a parent homozygous for the desired alleles will pass them to most if not all offspring, resulting in a reduced segregation in the offspring.
The presently disclosed subject matter provides for better models for marker-assisted selection (MAS). The presently disclosed subject matter relates to methods is of plant breeding and to methods to select tomato plants, particularly cultivated tomato plants as breeder plants for use in breeding programs or cultivated tomato plants having desired genotypic or potential phenotypic properties, in particular those which produce tomato fruit with increased fruit firmness at the harvesting stage.
Accordingly, there is provided a tomato fruit with significantly increased fruit firmness at the harvesting stage linked to a genetic element in the cultivated tomato plant producing said tomato fruit, wherein said firmness is up to 4 times greater than that of fruit from a control tomato plant which does not have the said genetic element.
The harvesting stage is preferably the breaker stage plus 7 days. Alternatively, the harvesting stage can be any chosen point in the development of the tomato fruit, which includes but is not limited to the immature green stage, rapid expansion stage, mature green stage, breaker stage or red ripe stage or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after any of these stages. In one embodiment, the harvesting stage is the breaker stage plus 14 days.
In a specific embodiment there is provided a tomato fruit providing increased fruit firmness at the mature green stage linked to a genetic element in the cultivated tomato plant producing said tomato fruit, wherein said firmness is up to 4 times greater than that of fruit from a control tomato plant which does not have the said genetic element. In another embodiment, said firmness is up to 2 times greater than that of fruit from a control tomato plant which does not have the said genetic element.
In a specific embodiment, there is provided a tomato fruit providing increased fruit firmness at the breaker stage plus 7 days caused by a genetic element in the cultivated tomato plant producing said tomato, wherein said firmness is up to 4 times greater than that of fruit from a control tomato plant which does not have the said at least one genetic element. In another embodiment, said firmness is up to 2 times greater than that of fruit from a control tomato plant which does not have the said at least one genetic element.
Fruit firmness is preferably measured in the outer pericarp and inner pericarp of the cultivated tomato plant. Alternatively, fruit firmness can be measured in the outer pericarp only. Preferably, fruit firmness is measured by mechanical means. Typically, is a Lloyd LRX machine is used as described in the Example section. Such measurements represent a sensitive mechanical measure of fruit firmness that correlates very well with conventional penetrometer assays, but gives substantially more sensitive and reproducible results. In addition these measurements also correlate well with organoleptic/sensory descriptions of fruit firmness (King et al, 2001). However, alternative methods for measuring fruit firmness which are known to the skilled person may also be employed such as those described in Causse et al (2002).
Preferably the control tomato plant is M82 with deposit number NCIMB 41661.
Alternatively, the control plant can be any tomato plant which differs from its offspring essentially due to the absence of at least one genetic element, or part thereof, responsible for increased fruit firmness in the cultivated tomato plant. The control tomato plant may be selected from any plant, line or population within the species Solanum lycopersicum (synonyms are Lycopersicon lycopersicum or Lycopersicon esculentum) or formerly known under the genus name of Lycopersicon including but not limited to L. cerasiforme, L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum (now S. pennellii), L. hirsutum, L. parviborum, L. pennellii, L. peruvianum, L. pimpinellifolium, or S. lycopersicoides. The newly proposed scientific name for L. esculentum is S. pennellii. Similarly, the names of the wild species may be altered. L. pennellii has become S. pennellii, L. hirsutum may become S. habrochaites, L. peruvianum may be split into S. ‘N peruvianum’ and S. ‘Callejon de Huayles’, S. peruvianum, and S. corneliomuelleri, L. parviflorum may become S. neorickii, L. chmielewskii may become S. chmielewskii, L. chilense may become S. chilense, L. cheesmaniae may become S. cheesmaniae or S. galapagense, and L. pimpinellifolium may become S. pimpinellifolium (Knapp (2005))
Preferably the genetic element(s) responsible for significantly increased fruit firmness of the present invention is present on chromosome 3 of tomato. Preferably, the genetic element is present on chromosome 3 of S. pennellii. 
In a further aspect, there is provided a cultivated tomato plant which produces tomato fruit as described herein wherein said plant comprises a genetic element which is characterized by at least one DNA marker selected from the group consisting of TG599, NT3374FM, TG246, NT5880VC and TG42.
In a further aspect, there is provided a cultivated tomato plant which produces tomato fruit wherein said plant comprises a genetic element which is complementary to the corresponding genetic element in S. pennellii lines IL3-4 deposited under accession number LA3489, wherein said genetic element in S. pennellii IL3-4 can be characterized by at least one DNA marker selected from the group consisting of TG599, NT3374FM, TG246, NT5880VC and TG42.
There is also provided a cultivated tomato plant which produces tomato fruit with increased fruit firmness compared to fruit from a control tomato plant wherein said cultivated tomato plant has a genetic element which is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
There is also provided a cultivated tomato plant which produces tomato fruit with increased fruit firmness compared to fruit from a control tomato plant wherein said cultivated tomato plant comprises a genetic element, wherein the genetic element is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
There is also provided a cultivated tomato plant produces tomato fruit with increased fruit firmness compared to a control tomato plant wherein said cultivated tomato plant comprises a genetic element QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
There is also provided a cultivated tomato plant produces tomato fruit with increased fruit firmness compared to a control tomato plant wherein said cultivated tomato plant comprises a genetic element, wherein the genetic element is QTL1 linked to at least to one of the DNA markers NT3374FM, TG246 and NT5880VC; which is present only in the outer pericarp.
In a further aspect of the invention, there is provided a cultivated tomato plant as herein described wherein said plant is an inbred, a dihaploid or a hybrid. In a specific embodiment, the cultivated tomato plant is male sterile.
In a further aspect of the invention, there is provided tomato seed which produces a cultivated tomato plant as herein described.
In a further aspect of the invention, there is provided a plant part of a cultivated tomato plant as herein described.
In a further aspect, there is provided plant material obtainable from a plant part of a cultivated tomato plant as herein described.
Identification of QTLs Linked to Increased Fruit Firmness in Tomato
The presently disclosed subject matter also provides methods for selecting a cultivated tomato plant with fruit having increased firmness compared to fruit from a control tomato plant. Such methods comprise detecting in the cultivated tomato plant the presence of QTL1 as defined herein. In general, the methods comprise providing a sample of genomic DNA from a tomato plant; and (b) detecting in the sample of genomic DNA at least one molecular marker linked to QTL1. In some embodiments, the detection step may comprise detecting at least one molecular marker from the group, the at least one molecular markers detecting QTL1. The providing of a sample of genomic DNA from a tomato plant can be performed by standard DNA isolation methods well known in the art.
In some embodiments, the detecting of a molecular marker (step (b)) may comprise the use of a nucleic acid probe having a base sequence that is substantially complementary to the nucleic acid sequence defining the molecular marker and which nucleic acid probe specifically hybridizes under stringent conditions with a nucleic acid sequence defining the molecular marker. A suitable nucleic acid probe can for instance be a single strand of the amplification product corresponding to the marker. The detecting of a molecular marker can also comprise performing a nucleic acid amplification reaction on the genomic DNA to detect one or more QTLs. This can be done by performing a PCR reaction using a set of marker-specific primers. In is some embodiments, the detecting can comprise the use of at least one set of primers defining one or more markers linked to QTL1, or a set of primers which specifically hybridize under stringent conditions with nucleic acid sequences of one or more markers linked to QTL1.
The presently disclosed methods can also include detecting an amplified DNA fragment linked to the presence of a QTL. In some embodiments, the amplified fragment linked to presence of a QTL has a predicted length or nucleic acid sequence, and detecting an amplified DNA fragment having the predicted length or the predicted nucleic acid sequence is performed such that the amplified DNA fragment has a length that corresponds (plus or minus a few bases; e.g. a length of one, two or three bases more or less) to the expected length as based on a similar reaction with the same primers with the DNA from the plant in which the marker was first detected or the nucleic acid sequence that corresponds (has a homology of in some embodiments more than 80%, in some embodiments more than 90%, in some embodiments more than 95%, in some embodiments more than 97%, and in some embodiments more than 99%) to the expected sequence as based on the sequence of the marker linked to that QTL in the plant in which the marker was first detected. One of ordinary skill in the art would appreciate that markers that are absent in plants providing fruit with increased fruit firmness, while they were present in the control parent(s) (so-called trans-markers), can also be useful in assays for detecting increased fruit firmness among offspring plants, although testing the absence of a marker to detect the presence of a specific trait is not optimal. The detecting of an amplified DNA fragment having the predicted length or the predicted nucleic acid sequence can be performed by any of a number of techniques, including but not limited to standard gel-electrophoresis techniques or by using automated DNA sequencers. These methods are well known to the skilled person.
Molecular Markers and QTLs
Molecular markers are used for the visualization of differences in nucleic acid sequences. This visualization can be due to DNA-DNA hybridization techniques after digestion with a restriction enzyme (RFLP) and/or due to techniques using the is polymerase chain reaction (e.g. STS, SSR/microsatellites, AFLPs and the like). In some embodiments, all differences between two parental genotypes segregate in a mapping population based on the cross of these parental genotypes. The segregation of the different markers can be compared and recombination frequencies can be calculated. Methods for mapping markers in plants are disclosed in, for example, Glick & Thompson, 1993; Zietkiewicz et al., 1994.
The recombination frequencies of molecular markers on different chromosomes are generally 50%. Between molecular markers located on the same chromosome, the recombination frequency generally depends on the distance between the markers. A low recombination frequency corresponds to a low genetic distance between markers on a chromosome. Comparing all recombination frequencies results in the most logical order of the molecular markers on the chromosomes. This most logical order can be depicted in a linkage map (Paterson, 1996). A group of adjacent or contiguous markers on the linkage map that is linked to an increased level of fruit firmness can provide the position of a QTL linked to increased fruit firmness.
The markers identified herein can be used in various aspects of the presently disclosed subject matter. Aspects of the presently disclosed subject matter are not to be limited to the use of the markers identified herein, however. It is stressed that the aspects can also make use of markers not explicitly disclosed herein or even yet to be identified. Other than the genetic unit “gene”, on which the phenotypic expression depends on a large number of factors that cannot be predicted, the genetic unit “QTL” denotes a region on the genome that is directly related to a phenotypic quantifiable trait.
QTL1 identified herein is located on chromosome 3 of tomato and its location can be characterized by a number of otherwise arbitrary markers. In the present investigations, microsatellite markers (e.g. SSRs) and single nucleotide polymorphisms (SNPs) were used, although restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, insertion mutation markers, sequence-characterized amplified region is (SCAR) markers, cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of these markers might also have been used.
In general, a QTL can span a region of several million bases. Therefore, providing the complete sequence information for the QTL is practically unfeasible but also unnecessary, as the way in which the QTL is first detected—through the observed correlation between the presence of a string of contiguous genomic markers and the presence of a particular phenotypic trait—allows one to trace among a population of offspring plants those plants that have the genetic potential for exhibiting a particular phenotypic trait. By providing a non-limiting list of markers, the presently disclosed subject matter thus provides for the effective use of the presently disclosed QTL in a breeding program.
In general, a marker is specific for a particular line of descent. Thus, a specific trait can be linked to a particular marker. The markers as disclosed herein not only indicate the location of the QTL, they also correlate with the presence of the specific phenotypic trait in a plant. It is noted that the contiguous genomic markers that indicate the location of the QTL on the genome are in principal arbitrary or non-limiting. In general, the location of a QTL is indicated by a contiguous string of markers that exhibit statistical correlation to the phenotypic trait. Once a marker is found outside that string (i.e. one that has a LOD-score below a certain threshold, indicating that the marker is so remote that recombination in the region between that marker and the QTL occurs so frequently that the presence of the marker does not correlate in a statistically significant manner to the presence of the phenotype) the boundaries of the QTL can be considered set. Thus, it is also possible to indicate the location of the QTL by other markers located within that specified region. It is further noted that the contiguous genomic markers can also be used to indicate the to presence of the QTL (and thus of the phenotype) in an individual plant, which sometimes means that they can be used in marker-assisted selection (MAS) procedures. In principle, the number of potentially useful markers is limited but can be very large, and one of ordinary skill in the art can easily identify markers in addition to those specifically disclosed in the present application. Any marker that is is linked to the QTL (e.g. falling within the physical boundaries of the genomic region spanned by the markers having established LOD scores above a certain threshold thereby indicating that no or very little recombination between the marker and the QTL occurs in crosses, as well as any marker in linkage disequilibrium to the QTL, as well as markers that represent the actual causal mutations within the QTL) can be used in MAS procedures. This means that the markers identified in the application as being linked with QTL1 (markers TG599, NT3374FM, TG246, NT5880VC and TG42), are mere examples of markers suitable for use in MAS procedures. Moreover, when the QTL, or the specific trait-conferring part thereof, is introgressed into another genetic background (i.e. into the genome of another tomato or another plant species), then some markers might no longer be found in the offspring although the trait is present therein, indicating that such markers are outside the genomic region that represents the specific trait-conferring part of the QTL in the original parent line only and that the new genetic background has a different genomic organization. Such markers of which the absence indicates the successful introduction of the genetic element in the offspring are called “trans markers” (see above).
Upon the identification of a QTL, the QTL effect (i.e. for increased fruit firmness) can for instance be confirmed by assessing fruit firmness in progeny segregating for the QTL under investigation. The assessment of the fruit firmness can suitably be performed by measuring fruit firmness as known in the art.
The markers provided by the presently disclosed subject matter can be used for detecting the presence of one or more increased fruit firmness alleles at QTLs of the presently disclosed subject matter in a tomato plant with increased fruit firmness, and can therefore be used in methods involving marker-assisted breeding and selection of tomato plants with increased fruit firmness. In some embodiments, detecting the presence of a QTL of the presently disclosed subject matter is performed with at least one of the markers for a QTL as defined herein. The presently disclosed subject matter therefore relates in another aspect to a method for detecting the presence of a QTL for increased fruit firmness, comprising detecting the presence of a nucleic acid sequence of the QTL in a tomato plant, which presence can be detected by the use is of the herein disclosed markers.
The nucleotide sequence of a QTL of the presently disclosed subject matter can for instance be resolved by determining the nucleotide sequence of one or more markers linked to the QTL and designing internal primers for the marker sequences that can then be used to further determine the sequence of the QTL outside of the marker sequences. For instance, the nucleotide sequence of the SSR markers disclosed herein can be obtained by isolating the markers from the electrophoresis gel used in the determination of the presence of the markers in the genome of a subject plant, and determining the nucleotide sequence of the markers by, for example, dideoxy chain termination sequencing methods, which are well known in the art. In embodiments of such methods for detecting the presence of a QTL in a tomato plant, the method can also comprise providing a oligonucleotide or polynucleotide capable of hybridizing under stringent hybridization conditions to a nucleic acid sequence of a marker linked to the QTL, in some embodiments selected from the markers disclosed herein, contacting the oligonucleotide or polynucleotide with digested genomic nucleic acid of a tomato plant, and determining the presence of specific hybridization of the oligonucleotide or polynucleotide to the digested genomic nucleic acid.
In some embodiments, the method is performed on a nucleic acid sample obtained from the tomato plant, although in situ hybridization methods can also be employed. Alternatively, one of ordinary skill in the art can, once the nucleotide sequence of the QTL has been determined, design specific hybridization probes or oligonucleotides capable of hybridizing under stringent hybridization conditions to the nucleic acid sequence of the QTL and can use such hybridization probes in methods for detecting the presence of a QTL disclosed herein in a tomato plant.
In a further aspect of the invention, there is provided a method for detecting a QTL linked to significantly increased fruit firmness in fruit from a cultivated tomato plant compared to a control tomato plant comprising the steps of a) crossing a donor tomato plant with a recipient tomato plant to provide offspring plants, b) quantitatively determining the fruit firmness in the fruit of said offspring plants c) establishing a genetic linkage map that links the observed increased fruit firmness to the presence of at least one DNA marker from said donor plant in said offspring plants and d) assigning to a QTL the DNA markers on said map that are linked to significantly increased fruit firmness.
In a specific embodiment, the donor tomato plant has fruit with a significantly increased fruit firmness compared to said recipient tomato plant.
The donor plant is preferably S. pennellii and the recipient plant is preferably S. lycopersicum.
The donor plant or the recipient plant could be any one of the following: any plant, line or population within the species Solanum lycopersicum (synonyms are Lycopersicon lycopersicum or Lycopersicon esculentum) or formerly known under the genus name of Lycopersicon including but not limited to L. cerasiforme, L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum (now S. pennellii), L. hirsutum, L. parviborum, L. pennellii, L. peruvianum, L. pimpinellifolium, or S. lycopersicoides. The newly proposed scientific name for L. esculentum is S. pennellii. Similarly, the names of the wild species may be altered. L. pennellii has become S. pennellii, L. hirsutum may become S. habrochaites, L. peruvianum may be split into S. ‘N peruvianum’ and S. ‘Callejon de Huayles’, S. peruvianum, and S. corneliomuelleri, L. parviflorum may become S. neorickii, L. chmielewskii may become S. chmielewskii, L. chilense may become S. chilense, L. cheesmaniae may become S. cheesmaniae or S. galapagense, and L. pimpinellifolium may become S. pimpinellifolium (Knapp (2005))
In a specific embodiment, the fruit firmness range in offspring plants is up to 4 times greater than that of fruit produced from a control tomato plant at the harvesting stage.
In a specific embodiment, the fruit firmness range in offspring plants is up to 2 times greater than that of fruit produced from a control tomato plant at the harvesting stage.
The harvesting stage is preferably the breaker stage plus 7 days. Alternatively, the harvesting stage can be any chosen point in the development of the tomato fruit, which includes but is not limited to the immature green stage, rapid expansion stage, mature green stage, breaker stage, red ripe stage or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after any of these stages, preferably 10 days after the breaker stage.
In a specific embodiment, the at least one DNA marker is found in S. pennellii. Alternatively, the at least one DNA marker could be found in any tomato plant, line or population.
There is also provided at least one DNA marker selected from TG599, NT3374FM, TG246, NT5880VC and TG42.
A QTL of the present invention is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
In a specific embodiment, the QTL is QTL1 linked to at least one of the DNA markers NT3374FM, TG246 and NT5880VC; which is present only in the outer pericarp.
In a further aspect there is provided a QTL linked to increased fruit firmness in fruit provided by a cultivated tomato plant. Preferably the QTL is detected by a method as herein described. Alternatively, the QTL may be detected by any method known to a person skilled in the art.
Preferably, the QTL of the present invention is present on chromosome 3 of S. pennellii. More preferably, the QTL is present on Chromosome 3F of S. pennellii. 
In a specific embodiment, the QTL of the present invention is linked to at least one DNA marker selected from the group consisting of NT3374FM, TG246 and NT5880VC.
The QTL of the present invention is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
In a specific embodiment, the QTL is QTL1 linked to at least one of the DNA markers NT3374FM, TG246 and NT5880VC; which is present only in the outer pericarp.
There is further provided an isolated DNA sample obtained from a tomato plant comprising QTL1. The DNA sample may be isolated as described in the examples or by any other means familiar to the skilled person.
Production of Tomato Fruit with Increased Firmness by Transgenic Methods
According to another aspect of the presently disclosed subject matter, a nucleic acid (in some embodiments a DNA) sequence comprising QTL1 or fruit firmness conferring parts thereof, can be used for the production of a tomato plant providing fruit with increased firmness compared to a control tomato plant. In this aspect, the presently disclosed subject matter provides for the use of a QTL as defined herein or increased fruit firmness conferring parts thereof, for producing a tomato plant providing fruit with increased firmness compared to a control tomato plant, which use involves the introduction of a nucleic acid sequence comprising the QTL into a suitable recipient plant. As stated, the nucleic acid sequence can be derived from a suitable donor plant with increased fruit firmness compared to a control tomato plant. A suitable source for the increased fruit firmness locus identified herein as QTL1 is S. pennellii. A number of tomato cultivars that have varying degrees of increased fruit firmness are commercially available.
The source of the increased fruit firmness loci described herein are introgression lines IL-3, which were originally generated by Dani Zamir and colleagues (Eshed & Zamir, 1994).This line was obtained from the Tomato Genetics Resource Centre at Davis, California (available at tgrc.ucdavis.edu/) or from Dani Zamir at the Hebrew University of Jerusalem, Israel. Once identified in a suitable donor plant, the nucleic acid sequence that comprises a QTL for increased fruit firmness, or increased fruit firmness -conferring part thereof, can be transferred to a suitable recipient plant by any method available. For instance, the nucleic acid sequence can be transferred by crossing a donor tomato plant with a recipient plant i.e. by introgression, by transformation, by protoplast fusion, by a doubled haploid technique, by embryo rescue, or by any other nucleic acid transfer system, followed by selection of offspring plants comprising the presently disclosed QTL and exhibiting increased fruit firmness. For transgenic methods of transfer, a nucleic acid sequence comprising a QTL for increased fruit firmness, or increased fruit firmness -conferring part thereof, can be isolated from the donor plant using methods known in the art, and the thus isolated nucleic acid sequence can be transferred to the recipient plant by transgenic methods, for instance by means of a vector, in a gamete, or in any other suitable transfer element, such as a ballistic particle coated with the nucleic acid sequence.
Plant transformation generally involves the construction of an expression vector that will function in plant cells. In the presently disclosed subject matter, such a vector comprises a nucleic acid sequence that comprises a QTL for increased fruit firmness, or increased fruit firmness-conferring part thereof, which vector can comprise an increased fruit firmness conferring gene that is under control of, or operatively linked to, a regulatory element such as a promoter. The expression vector can contain one or more such operably linked gene/regulatory element combinations, provided that at least one of the genes contained in the combinations encodes increased fruit firmness. The vector(s) can be in the form of a plasmid, and can be used, alone or in combination with other plasmids, to provide transgenic plants that have increased fruit firmness using transformation methods known in the art, such as the Agrobacterium transformation system.
The inventors have characterized QTL1 at the molecular level and identified several putative candidate genes for use in an expression vector. The list of candidate genes is as follows: cathepsin B-like cysteine proteinase, betaine aldehyde dehydrogenase, pectate lyase, helix-loop-helix DNA-binding. The genomic co-ordinates of these candidate genes are 56434422 . . . 56432592, 57902443 . . . 57894787, 56396337 . . . 56398488, and 57713477 . . . 57715822 respectively.
Expression vectors can include at least one marker gene, operably linked to a regulatory element (such as a promoter) that allows transformed cells containing the marker to be either recovered by negative selection (by inhibiting the growth of cells that do not contain the selectable marker gene), or by positive selection (by screening for the product encoded by the marker gene). Many commonly used selectable marker genes for plant transformation are known in the art, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent that can be an antibiotic or a herbicide, or genes that encode an altered target which is insensitive to the inhibitor. Several positive selection methods are known in the art, such as mannose selection. Alternatively, marker-less transformation can be used to obtain plants without the aforementioned marker genes, the techniques for which are also known in the art.
One method for introducing an expression vector into a plant is based on the natural transformation system of Agrobacterium (see e.g., Horsch et al., 1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria that genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant (see e.g., Kado, 1991). Methods of introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant cells with Agrobacterium tumefaciens (Horsch et al., 1985). Descriptions of Agrobacterium vectors systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber & Crosby, 1993, Moloney et al., 1989, and U.S. Pat. No. 5,591,616. General descriptions of plant expression vectors and reporter genes and transformation protocols and descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer can be found in Gruber & Crosby, 1993. General methods of culturing plant tissues are provided for example by Miki et al., 1993 and by Phillips et al., 1988. A reference handbook for molecular cloning techniques and suitable expression vectors is Sambrook & Russell, (2001).
Another method for introducing an expression vector into a plant is based on microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles. The expression vector is introduced into plant tissues with a ballistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes (see e.g., Sanford et al., 1987; Klein et al., 1988; Sanford, 1988; Sanford, 1990; Klein et al., 1992; Sanford et al., to 1993). Another method for introducing DNA into plants is via the sonication of target cells (see Zhang et al., 1991). Alternatively, liposome or spheroplast fusion can be used to introduce expression vectors into plants (see e.g., Deshayes et al., 1985 and Christou et al., 1987). Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol, or poly-L-ornithine has also been reported (see e.g., is Hain et al. 1985 and Draper et al., 1982). Electroporation of protoplasts and whole cells and tissues has also been described (D'Halluin et al., 1992 and Laursen et al., 1994).
Other well known techniques such as the use of BACs, wherein parts of the tomato genome are introduced into bacterial artificial chromosomes (BACs), ie., vectors used to clone DNA fragments (100- to 300-kb insert size; average 150 kb) in Escherichia coli cells, based on naturally occurring F-factor plasmid found in the bacterium E. coli. (Zhao & Stodoisky, 2004) can be employed for example in combination with the Bi BAC system (Hamilton, 1997) to produce transgenic plants. One example of an over-expression vector is pGWB405 (Nakagawa T, Suzuki T, Murata S at el. Improved Gateway Binary Vectors: High-performance Vectors for CREATION of Fusion Constructs in Transgenic Analysis of Plants. Bioscience biotechnology Biochemistry, 71(8)2095-2010, 2007). For overexpression construct production, sequence corresponding to the complete open reading frame of the candidate gene involved in modulating tomato fruit firmness can be cloned in front of the CaMV 35S promoter using the Gateway clone system which avoids the need for restriction sites. Such a construct also contains the CaMV terminator at the opposite end. For RNAi construct production, a fragment of the coding sequence unique to the candidate gene involved in modulating tomato fruit firmness can be cloned, for example, in the Gateway system RNAi vector pK7GWIWG2(I).
Following transformation of tomato target tissues, expression of the above described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using standard regeneration and selection methods.
There is also provided 4 genes the expression levels of which are altered in recombinants with greater fruit firmness. The 4 genes code for cathepsin B-like cysteine proteinase, betaine aldehyde dehydrogenase, pectate lyase, helix-loop-helix DNA-binding. The genomic co-ordinates of these candidate genes are 56434422 . . . 56432592, 57902443 . . . 57894787, 56396337 . . . 56398488, and 57713477 . . . 57715822 respectively.
The present invention therefore provides an isolated nucleotide sequence selected is from the group consisting of: a) a nucleotide sequence corresponding to the promoter region and/or open reading frame or part thereof corresponding to the following preferably derived from S. pennellii: cathepsin B-like cysteine proteinase, betaine aldehyde dehydrogenase, pectate lyase, helix-loop-helix DNA-binding; b) a nucleotide sequence that is at least 80% identical to the nucleotide sequence of a); c) a nucleotide sequence comprising at least 21 consecutive nucleotides of the nucleotide sequence of a); d) a nucleotide sequence that hybridises under stringent conditions to the complement of any of nucleotide sequences a) to c); and e) a nucleotide sequence that is the complement to the nucleotide sequences of any one of a) to d). In one embodiment, the nucleotide sequence of step b) is at least 90% identical to the nucleotide sequence of a).
In one embodiment, the isolated nucleotide sequence of the invention is cathepsin B-like cysteine proteinase. In another embodiment, the isolated nucleotide sequence of the invention is betaine aldehyde dehydrogenase. In another embodiment, the isolated nucleotide sequence of the invention is pectate lyase. In another embodiment, the isolated nucleotide sequence of the invention is helix-loop-helix DNA-binding. The isolated nucleotide sequence of the invention is preferably derived from S. pennellii. 
There is also provided a vector comprising the isolated nucleotide sequence of the invention. In one embodiment, the isolated nucleotide sequence is in the sense orientation. In another embodiment, the isolated nucleotide sequence is in the antisense orientation.
There is also provided a host cell which expresses a vector of the invention.
There is also provided a transgenic plant or part thereof comprising a host cell of the invention. In one embodiment, the transgenic plant or part thereof is a monocot. In another embodiment, the plant or part thereof is a dicot, for example a tomato.
There is also provided a method for producing a transgenic plant comprising regenerating a plant from a host cell according to the invention.
There is also provided a cultivated plant or part thereof produced by a method according to the invention.
There is also provided a method of manipulating the texture of fruit of a transgenic plant, for example a tomato plant comprising transforming said plant with a vector of the invention. In one embodiment, the speed of fruit ripening is increased when compared with fruit from an untransformed plant. In another embodiment, the speed of fruit ripening is decreased when compared with fruit from an untransformed plant. In one embodiment, the speed of tomato ripening is measured at breaker stage.
There is also provided a plant, for example a tomato plant or part thereof obtained by a method of the invention.
There is also provided a method of detecting genetic markers indicative of tomato texture of a plant of the Solanaceae family, comprising isolating DNA from said plant and from one or both parents of said plant; screening for genetic markers in a region of said DNA at or near sequence corresponding to an isolated sequence of the invention; and determining co-inheritance of said markers from one or both parents to said individual.
There is also provided a genetic marker detectable by a method of detecting genetic markers of the invention.
There is also provided use of a genetic marker of the invention for the production of a cultivated tomato plant capable of bearing tomato fruit.
There is also provided a cultivated tomato plant or part thereof produced by a method of the invention.
There is also provided use of a cultivated tomato plant or part thereof according to the invention in the fresh cut market or for food processing.
There is also provided use of an isolated nucleotide sequence of the invention in the manipulation of speed of ripening of fruit of a plant, preferably a tomato plant, wherein said manipulation is effected by genetic modification of said plant.
There is also provided use of a method according to the invention, wherein said genetic modification is introduced into a plant of the invention by a method selected from the list consisting of transposon insertion mutagenesis, T-DNA insertion mutagenesis, TILLING, site-directed mutagenesis, directed evolution, and homologous recombination. In one embodiment, genetic modification is introduced by TILLING.
Production of Tomato Plants with Increased Fruit Firmness by Non Transgenic Methods
In some embodiments for producing a tomato plant with increased fruit firmness, protoplast fusion can be used for the transfer of nucleic acids from a donor plant to a recipient plant. Protoplast fusion is an induced or spontaneous union, such as a somatic hybridization, between two or more protoplasts (the cell walls of which are removed by enzymatic treatment) to produce a single bi- or multi-nucleate cell. The fused cell, which can even be obtained with plant species that cannot be interbred in nature, is tissue cultured into a hybrid plant exhibiting the desirable combination of traits. More specifically, a first protoplast can be obtained from a tomato plant or other plant line that exhibits increased fruit firmness. A second protoplast can be obtained from a second tomato plant or other plant variety, preferably a tomato plant line that comprises commercially valuable characteristics. The protoplasts are then fused using traditional protoplast fusion procedures, which are known in the art.
Alternatively, embryo rescue can be employed in the transfer of a nucleic acid comprising one or more QTLs as described herein from a donor tomato plant to a recipient tomato plant. Embryo rescue can be used as a procedure to isolate embryos from crosses wherein plants fail to produce viable seed. In this process, the fertilized ovary or immature seed of a plant is tissue cultured to create new plants (Pierik, 1999). The presently disclosed subject matter also relates to methods for producing tomato plant with increased fruit firmness comprising performing a method for detecting the presence of a QTL linked to increased fruit firmness in a donor tomato plant as described herein, and transferring a nucleic acid sequence comprising at least one QTL thus detected, or an increased fruit firmness-conferring part thereof, from the donor plant to a recipient tomato plant. The transfer of the nucleic acid sequence can be performed by any of the methods previously described herein.
An exemplary embodiment of such a method comprises the transfer by introgression of the nucleic acid sequence from a donor tomato plant into a recipient tomato plant by crossing the plants. This transfer can thus suitably be accomplished by using is traditional breeding techniques. QTLs are introgressed in some embodiments into commercial tomato varieties using marker-assisted selection (MAS) or marker-assisted breeding (MAB). MAS and MAB involves the use of one or more of the molecular markers for the identification and selection of those offspring plants that contain one or more of the genes that encode for the desired trait. In the context of the presently disclosed subject matter, such identification and selection is based on the QTL of the presently disclosed subject matter or markers associated therewith. MAS can also be used to develop near-isogenic lines (NIL) harboring the QTL of interest, allowing a more detailed study of each QTL effect and is also an effective method for development of backcross inbred line (BIL) populations (see e.g., Nesbitt & Tanksley, 2001; van Berloo et al., 2001). Tomato plants developed according to these embodiments can advantageously derive a majority of their traits from the recipient plant, and derive increased fruit firmness from the donor plant. As discussed herein, traditional breeding techniques can be used to introgress a nucleic acid sequence encoding for increased fruit firmness into a recipient tomato plant. In some embodiments, a donor tomato plant that exhibits increased fruit firmness and comprising a nucleic acid sequence encoding for increased fruit firmness is crossed with a recipient tomato plant that in some embodiments exhibits commercially desirable characteristics.
The resulting plant population (representing the F1 hybrids) is then self-pollinated and set seeds (F2 seeds). The F2 plants grown from the F2 seeds are then screened for increased fruit firmness by methods known to the skilled person.
Tomato plant lines with increased fruit firmness can be developed using the techniques of recurrent selection and backcrossing, selfing, and/or dihaploids, or any other technique used to make parental lines. In a method of recurrent selection and backcrossing, increased fruit firmness can be introgressed into a target recipient plant (the recurrent parent) by crossing the recurrent parent with a first donor plant, which differs from the recurrent parent and is referred to herein as the “non-recurrent parent”. The recurrent parent is a plant that does not have increased fruit firmness but does possess commercially desirable characteristics.
In some embodiments, the non-recurrent parent exhibits increased fruit firmness and is comprises a nucleic acid sequence that encodes for increased fruit firmness. The non-recurrent parent can be any plant variety or inbred line that is cross-fertile with the recurrent parent.
The progeny resulting from a cross between the recurrent parent and non-recurrent parent are backcrossed to the recurrent parent. The resulting plant population is then screened for increased fruit firmness. Marker-assisted selection (MAS) can be performed using one or more of the herein described molecular markers, or by using hybridization probes, or polynucleotides to identify those progeny that comprise a nucleic acid sequence encoding for increased fruit firmness. Also, MAS can be used to confirm the results obtained from the quantitative fruit firmness measurements.
Following screening, the F1 hybrid plants that exhibit an increased fruit firmness phenotype or, in some embodiments, genotype and thus comprise the requisite nucleic acid sequence encoding for increased fruit firmness, are then selected and backcrossed to the recurrent parent for a number of generations in order to allow for the tomato plant to become increasingly inbred. This process can be performed for two, three, four, five, six, seven, eight, or more generations. In principle, the progeny resulting from the process of crossing the recurrent parent with the increased fruit firmness non-recurrent parent are heterozygous for one or more genes that encode for increased fruit firmness.
In general, a method of introducing a desired trait into a hybrid tomato variety may comprise:
(a) crossing an inbred tomato parent, preferably S. lycopersicum, with another tomato plant, preferably S. pennellii, that comprises one or more desired traits, to produce F1 progeny plants, wherein the desired trait is increased fruit firmness;
(b) selecting the F1 progeny plants that have the desired trait to produce selected F1 progeny plants, in some embodiments using molecular markers as described herein;
(c) backcrossing the selected progeny plants with the inbred tomato parent plant to produce backcross progeny plants;
(d) selecting for backcross progeny plants that have the desired trait and morphological and physiological characteristics of the inbred tomato parent plant, wherein the selection comprises the isolation of genomic DNA and testing the DNA for the presence of at least one molecular marker for QTL1, in some embodiments as described herein;
(e) repeating steps (c) and (d) two or more times in succession to produce selected third or higher backcross progeny plants;
(f) optionally selfing selected backcross progeny in order to identify homozygous plants; and
(g) crossing at least one of the backcross progeny or selfed plants with another inbred tomato parent plant to generate a hybrid tomato variety with the desired trait and all of the morphological and physiological characteristics of hybrid tomato variety when grown in the same environmental conditions.
As indicated, the last backcross generation can be selfed in order to provide for homozygous pure breeding (inbred) progeny having increased fruit firmness. Thus, the result of recurrent selection, backcrossing, and selfing is the production of lines that are genetically homogenous for the genes linked to increased fruit firmness, and in some embodiments as well as for other genes linked to traits of commercial interest.
Accordingly, there is provided a method of producing a tomato plant which provides fruit with increased fruit firmness as herein described.
In a specific embodiment there is provided a method of producing a tomato plant which provides fruit with increased fruit firmness as herein described comprising the steps of performing a method for detecting a QTL linked to increased fruit firmness as herein described, and transferring a nucleic acid comprising at least one QTL thus detected, from a donor tomato plant to a recipient tomato plant, wherein said increased fruit firmness is measured in fruit from an offspring cultivated tomato plant compared to fruit from a control tomato plant.
In a specific embodiment there is provided a method of producing a cultivated tomato plant which provides fruit with increased fruit firmness as herein described wherein said transfer of nucleic acid is performed by transformation, by protoplast fusion, by a doubled haploid technique or by embryo rescue.
In a specific embodiment there is provided a method of producing a cultivated tomato plant which provides fruit with increased fruit firmness as herein described, wherein the fruit firmness range in the offspring tomato plant is up to 4 times greater than fruit of a control tomato plant at the harvesting stage. Alternatively, the fruit firmness range in the offspring tomato plant is up to 2 times greater than fruit from a control tomato plant at the harvesting stage. Preferably the harvesting stage is the breaker stage plus 7 days. Alternatively, the harvesting stage can be the immature green stage, rapid expansion stage, breaker stage or red ripe stage or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after any of these stages.
In a specific embodiment there is provided a method of producing a tomato plant which provides fruit with increased fruit firmness as herein described, wherein the fruit firmness range remains up until breaker plus 7 days.
In a further aspect there is provided a method of producing a tomato plant which provides fruit with increased fruit firmness as herein described, wherein the donor plant is S. pennellii and the recipient plant is S. lycopersicum. 
There is also provided a method of producing a tomato plant which provides fruit with increased fruit firmness as herein described, wherein said QTL is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
There is also provided a method of producing a tomato plant which provides fruit with increased fruit firmness as herein described, wherein said QTL is QTL1 linked to at least one of the DNA markers NT3374FM, TG246 and NT5880VC; which is present only in the outer pericarp.
There is also provided a method of producing a tomato plant which provides fruit with increased fruit firmness as herein described, wherein said QTL is linked to at least one of the DNA markers NT3374FM and TG246; which is present only in the outer pericarp
In a further aspect there is provided a tomato plant, or part thereof, obtainable by a method as herein described.
In a further aspect there is provided a cultivated tomato plant comprising a QTL responsible for increased fruit firmness as herein described.
In a further aspect there is provided a hybrid tomato plant, or part thereof, obtainable by crossing a tomato plant as herein described with a tomato plant that exhibits commercially desirable characteristics.
In a further aspect there is provided a tomato seed produced by growing the tomato plant as herein described.
In a further aspect there is provided a tomato seed produced by crossing the cultivated tomato plant as herein described with a plant having desirable phenotypic traits to obtain a plant that has significantly increased fruit firmness compared to a control plant.
In a further aspect there is provided use of a QTL as herein described for the production of tomato plants having increased fruit firmness compared to control plants.
In a further aspect there is provided use of a tomato plant having increased fruit firmness as herein described for expanding the harvesting slot of tomato fruit. The harvesting slot can be expanded by any one of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
In a further aspect there is provided use of a tomato plant having increased fruit firmness as herein described in the fresh cut market or for food processing.
In a further aspect there is provided processed food made from a tomato plant comprising the at least one QTL as herein described.
Embodiments of the Invention
Embodiment 1: A tomato fruit with significantly increased fruit firmness at the harvesting stage linked to a genetic element in the cultivated tomato plant producing is said tomato fruit, wherein said firmness is up to 4 times greater than that of fruit from a control tomato plant which does not have the said genetic element.
Embodiment 2: A tomato fruit according to embodiment 1 wherein the harvesting stage is the breaker stage plus 7 days
Embodiment 3: A tomato fruit according to embodiment 1 or 2 wherein the fruit firmness is up to 3 times greater than that produced from a control tomato plant which does not have the said genetic element and preferably wherein the fruit firmness is less at the breaker stage than that produced from a control tomato plant which does not have the said genetic element.
Embodiment 4: A tomato fruit according to embodiment 1 to 3 wherein the fruit firmness is up to 2 times greater than that produced from a control tomato plant which does not have the said at least one genetic element.
Embodiment 5: A tomato fruit according to any one of embodiments 1 to 4, wherein said firmness range is measured at breaker plus 14 days.
Embodiment 6: A tomato fruit according to any preceding embodiment wherein the at least one genetic element is located on chromosome 3.
Embodiment 7: A cultivated tomato plant which produces tomato fruit according to any of embodiments 1 to 6 wherein said plant can be characterised by a) the genetic element is linked to at least one DNA marker selected from the group consisting of TG599, NT3374FM, TG246, NT5880VC and TG42 and/or b) the genetic element is complementary to the corresponding genetic element in Solanum pennellii line IL3-4 deposited under accession number LA3489, wherein the said genetic element in LA3489 is linked to at least one DNA marker selected from the group consisting of TG599, NT3374FM, TG246, NT5880VC and TG42.
Embodiment 8: A cultivated tomato plant according to embodiment 7 wherein the at least one genetic element is QTL1 linked to at least one of the DNA markers is NT3374FM, TG246 and NT5880VC.
Embodiment 9: A cultivated tomato plant according to embodiment 7 or 8 wherein the QTL is QTL1 which is present only in the outer pericarp.
Embodiment 10: A cultivated tomato plant according to any previous embodiment wherein the genetic element, or part thereof, corresponds to the S. pennellii pectate lyase promoter region or open reading frame sequence.
Embodiment 11: A cultivated tomato plant according to any one of embodiments 7 to 10 wherein said plant is an inbred, a dihaploid or a hybrid.
Embodiment 12: A cultivated tomato plant according to embodiment 11 wherein said plant is male sterile.
Embodiment 13: A cultivated tomato plant according to any previous embodiment, wherein the tomato fruit with significantly increased fruit firmness and fruit from the control tomato plant have no significant difference in fruit colour at the breaker plus 7 day stage.
Embodiment 14: A tomato seed which produces a cultivated tomato plant according to any one of embodiments 7 to 13
Embodiment 15: Plant part of a cultivated tomato plant according to embodiment 7 to 13.
Embodiment 16
Plant material obtainable from a plant part of a cultivated tomato plant according to embodiment 15.
Embodiment 17: A method for detecting a QTL linked to significantly increased fruit firmness in fruit from a cultivated tomato plant compared to a control tomato plant comprising the steps of a) crossing a donor tomato plant with a recipient tomato plant to provide offspring cultivated tomato plants, b) quantitatively determining the firmness in the fruit of said offspring plants c) establishing a genetic linkage map that links the observed increased fruit firmness to the presence of at least one DNA marker from said donor plant in said offspring plants and d) assigning to a QTL the DNA markers on said map that are linked to significantly increased fruit firmness.
Embodiment 18: The method according to embodiment 17 wherein said donor plant has a significantly increased fruit firmness compared to said recipient plant.
Embodiment 19: The method of embodiment 17 or 18 wherein the donor plant is Solanum pennellii and the recipient plant is Solanum lycopersicum. 
Embodiment 20: The method of any one of embodiments 17 to 19 wherein the fruit firmness range in offspring plants is up to 4 times greater than that of fruit produced from a control tomato plant at the harvesting stage.
Embodiment 21: The method of embodiment 20 wherein the fruit firmness range in offspring plants is up to 2 times greater than that of fruit produced from a control tomato plant at the harvesting stage.
Embodiment 22: The method of any one of embodiments 17 to 21 wherein the harvesting stage is the breaker stage plus 7 days.
Embodiment 23: The method of any one of embodiments 17 to 22 wherein the at least one DNA marker is found in Solanum pennellii. 
Embodiment 24: The method of any one of embodiments 17 to 23 wherein the at least one DNA marker is selected from TG599, NT3374FM, TG246, NT5880VC and TG42.
Embodiment 25: The method of any one of embodiments 17 to 24 wherein said QTL QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
Embodiment 26: The method of embodiment 25 wherein said QTL is QTL1 which is present only in the outer pericarp.
Embodiment 27: A QTL responsible for increased fruit firmness in fruit provided by a cultivated tomato plant detected by a method according to any one of embodiments 17 to 26.
Embodiment 28: A QTL according to embodiment 27 located on chromosome 3.
Embodiment 29: A QTL according to any one of embodiments 27 to 28 associated with at least one DNA marker selected from the group consisting of TG599, is NT3374FM, TG246, NT5880VC and TG42.
Embodiment 30: A QTL according to any one of embodiments 27 to 29 wherein said QTL is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
Embodiment 31: A QTL according to embodiment 30 wherein said QTL is QTL1 which is present only in the outer pericarp.
Embodiment 32: An isolated DNA sample obtained from a tomato plant comprising a QTL according to any one of embodiments 27 to 31.
Embodiment 33: A method of producing a cultivated tomato plant which provides fruit with significantly increased fruit firmness according to any one of embodiments 1 to 6
Embodiment 34: A method of producing a cultivated tomato plant which provides fruit with increased fruit firmness according to embodiment 33 comprising the steps of performing a method for detecting a QTL associated with significantly increased fruit firmness according to any one of embodiments 17 to 26, and transferring a nucleic acid comprising at least one QTL thus detected, from a donor tomato plant to a recipient tomato plant, wherein said increased fruit firmness is measured in fruit from an offspring cultivated tomato plant compared to fruit from a control tomato plant.
Embodiment 35: A method of producing a cultivated tomato plant which provides fruit with increased fruit firmness according to any one of embodiments 33 to 34 wherein said transfer of nucleic acid is performed by transformation, by protoplast fusion, by a doubled haploid technique or by embryo rescue.
Embodiment 36: A method of producing a cultivated tomato plant which provides fruit with increased fruit firmness according to any one of embodiments 33 to 35, wherein the fruit firmness range in the donor tomato plant is up to 4 times greater than fruit of a control tomato plant at the breaker stage plus 7 days, and preferably wherein the fruit firmness range in the donor tomato plant is less than that of fruit of a control tomato plant at the breaker stage.
Embodiment 37A method of producing a cultivated tomato plant which provides fruit with increased fruit firmness according to any one of embodiments 33 to 36, wherein the fruit firmness range is measured at breaker stage plus 14 days.
Embodiment 38A method of producing a cultivated tomato plant which provides fruit with increased fruit firmness according to any one of embodiments 33 to 37, wherein the donor plant is Solanum pennellii and the recipient plant is Solanum lycopersicum. 
Embodiment 39: A method of producing a cultivated tomato plant which provides fruit with increased fruit firmness according to any one of embodiments 33 to 38 wherein said QTL is QTL1 linked to at least one of the DNA markers TG599, NT3374FM, TG246, NT5880VC and TG42.
Embodiment 40: A method of producing a cultivated tomato plant which provides fruit with increased fruit firmness according to embodiment 39 wherein said QTL is QTL1 which is present only in the outer pericarp.
Embodiment 41: A cultivated tomato plant, or part thereof, obtainable by a method according to any one of embodiments 33 to 40.
Embodiment 42: A cultivated tomato plant comprising a QTL responsible for increased fruit firmness according to any one of embodiments 27 to 31.
Embodiment 43: A hybrid tomato plant, or part thereof, obtainable by crossing a cultivated tomato plant according to embodiments 7 to 13 with a tomato plant that exhibits commercially desirable characteristics.
Embodiment 44: Tomato seed produced by growing the tomato plant of embodiment 43.
Embodiment 45: Tomato seed produced by crossing the tomato plant of any one of embodiments 7 to 13 and 43 to 44 with a plant having desirable phenotypic traits to obtain a plant that has significantly increased fruit firmness compared to a control plant.
Embodiment 46: Use of a QTL according to any one of embodiments 27 to 31 for the production of tomato plants having significantly increased fruit firmness compared to control tomato plants.
Embodiment 47: Use of a tomato plant according to any one of embodiments 7 to 13 and 43 to 44 for expanding the harvesting slot of tomato fruit.
Embodiment 48: Use of a tomato plant according to any one of embodiments 7 to 13 and 43 to 44 in the fresh cut market or for food processing.
Embodiment 49: Use of a tomato fruit according to any one of embodiments 1 to 6 in the fresh cut market or for food processing
Embodiment 50: Processed food made from a tomato plant comprising the at least one genetic element according to embodiments 7 to 13.
Deposited Lines
The following seed samples were deposited at NCIMB, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, Scotland, UK under the provisions of the Budapest Treaty in the name of Syngenta Participations AG: Solanum lycopersicum CV M82, deposit number NCIMB 41661 on Oct. 22, 2009. Solanum lycopersicum Q7774, deposit number NCIMB 41949 on Mar. 21, 2012.
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EXAMPLES
Example 1
Identification of Fruit Firmness QTL in the S. Pennellii ILs
In order to identify new texture QTLs, S. pennellii introgression lines were screened using the pericarp puncture test (PPT) for fruit firmness, with the parental line S. lycopersicum ‘M82’ as a reference. Increases (P<0.001) in firmness were recorded for the S. pennellii ILs including Chr 3 (IL3-4). This indicated a robust QTL, which is independent of environmental influence. Fruit firmness in IL3-4 plants (Chr 3) was consistent across two independent glasshouse trials in the UK demonstrating reproducibility with a statistical significance of P<0.001. Interestingly we found no significant differences in fruit colour between IL3-4 and M82 (P<0.05). The texture effects in IL3-4 are confined to the outer pericarp and the magnitude of the effects are shown in FIG. 1.
The texture effect from S. pennelii appears to be semi-dominant in heterozygous individuals (FIG. 2).
Example 2
Fine Mapping of Texture QTL in the S. Pennellii ILs
The Chr 3 QTL was mapped to bin 3F (Eshed and Zamir, 1995) and fine mapped using 41 IL 3-4 QTL-Nils (FIG. 3). PCR based markers designed to RFLP sequences were used to construct a genetic map of Chr 3, bin 3F using JoinMap® 3.0 and this was linked to the physical map using the tomato genome sequence, v2.31 (available at solgenomics.net/). Interval mapping was also used to fine map the Chr 3 outer and inner pericarp texture QTLs. A significant (P<0.001) texture QTL was resolved and was apparent only in the outer pericarp. This texture QTL was mapped to a 2.1 Mb interval between markers TG599 and TG42. To our knowledge this is the first fruit firmness Chr 3 QTL to be described in S. pennellii. Further, QTL-NIL lines were texture tested and mechanical measurements based on the maximum load were obtained for the outer pericarp of red ripe (breaker +7 days) fruits (FIG. 4). Substantial variation in fruit firmness was apparent between QTL-NILs and parental lines M82 and IL 3-4 (FIG. 4). QTL-Nils were genotyped using, CAPS, SSR and PCR based markers and for each QTL-NIL, the genomic location and size of the introgressed S. pennellii segment was visualized in Excel (FIG. 5).
A QTL-Map was generated for the outer pericarp using marker TG246 and using SSR markers NT1819, NT1434, NT1743, NT3374, NT5880, NT4324, NT0247, NT1234. The forward and reverse primer sequences for each of these markers are shown below in table 1, together with their physical position on chromosome 3.











TABLE 1






primer
primer




SSR
forward
reverse
START
END







NT1819
CATCTTGGGA
AGCTTTCCAG
60959804
60960031



ATCTGACCC
TTCTGACCG





(SEQ ID 
(SEQ ID 





NO: 1)
NO: 2)




 


NT1434
TGGTAGAAGC
AAAGCATCTA
61572820
61573046



TGAATTGGG
TGGCTGTCG





(SEQ ID 
(SEQ ID 





NO: 3)
NO: 4)




 


NT1743
ATGGGCGCA
GGGTTCAAC

57086487



CTATTATGA
CGATCTCA





(SEQ ID 
(SEQ ID 





NO: 5)
NO: 6)




 


NT3374
AGCATACTGT
TTTTACCGGT
57086348
57086500



GATGGGTTC
TTGACCTTG





(SEQ ID 
(SEQ ID 





NO: 7)
NO: 8)




 


NT5880
ATCGGTGCTT 
TAGCTGCAAT
57216738
57216840



GATAAACGGT
TGCCAAGAAA





(SEQ ID 
(SEQ ID 





NO: 9)
NO: 10)




 


NT4324
TGCGTACGTA
AGGCGTATGTA
57454274
57454207



CTCTCTTTC
ATAAGCTAAG





(SEQ ID 
(SEQ ID 





NO: 11)
NO: 12)




 


NT0247
TGGTTCTGGAA
TTTGAGTAAA
57510882
57511291



CTTGGCTATC
AACGCCATCG





(SEQ ID 
(SEQ ID 





NO: 13)
NO: 14)




 


NT1234
AACGATGCAC
ATGTAGCCAG
57689859
57690052



CAGTTTCAC
GTCCATTTG





(SEQ ID 
(SEQ ID 





NO: 15)
NO: 16)









Primer sequences for marker TG246 are as follows:











(SEQ ID NO: 17)



TG246F
TTCCTCATCCGAAAAGCAAC



 




(SEQ ID NO: 18)



TG246R
TCTCATTGCAATTAACGATTCC






Primer sequences for TG599 are as follows:











(SEQ ID NO: 19)



TG599F
TCATGAACAAAATTGCGACA



 




(SEQ ID NO: 20)



TG599R
TCCTTCTCAATTGACCAAACC






Primers were also used to amplify the pectate lyase gene for the generation of transgenic plants. These are PL2F (ACGCGTTTAATTGGACGTATG)[SEQ ID NO: 21] and PL2R (AACAGAGGAAGTGCCCATTG) [SEQ ID NO: 22].
More markers were generated and used to genotype the QTL-NILs but not used to generate the outer pericarp QTL map.
Example 3
Candidate Genes Under the Mapping Interval
An array experiment was performed using the public 10,000 element tomato GeneChip. This experiment indicated genes that are differentially expressed in the IL3-4 texture QTL mapping region (see Table 2 below). The most likely candidate gene for increased fruit firmness found under the mapping interval is pectate lyase.











TABLE 2







Replicate
Replicate





mean (M82) ±
mean (IL3-4) ±




standard
standard
Fold


Annotation
Locus ID
error
error
change



















Solyc03g111730
Cathepsin B-like
 2742.6 ± 58.66
32.65 ± 5.57 
−84.01


56434422 . . . 56432592
cysteine



proteinase


Solyc03g113800
Betaine aldehyde
5890.77 ± 66.36
99.55 ± 24.87
−59.17


57902443 . . . 57894787
dehydrogenase


No clear fit to

   9245 ± 352.59
303.65 ± 117.65
−30.45


sequence


assembly


Solyc03g111690
Pectate lyase
13182.67 ± 197.72
690.17 ± 133.15
−19.1


56396337 . . . 56398488


Solyc03g113560
Helix-loop-helix
120.13 ± 6.94
11.59 ± 3.35 
−10.37


57713477 . . . 57715822
DNA-binding









Example 4
Plant Material
The S. pennellii introgression lines were generated by Eshed and Zamir (1995) by repeated backcrossing of the S. pennellii wild species (LA716) with the S. lycopersicum ‘M82’ recurrent parent. Seeds for these ILs were obtained from D. Zamir (Hebrew University of Jerusalem). The population (IL-pen) consisted of 75 lines each containing a single introgression fragment from S. pennellii LA716 in the genetic background of M82, a processing tomato variety with determinate growth to (Eshed and Zamir, 1995). The lines were grown in the UK under standard glasshouse conditions (16 h day length, day temp 20° C. and night temp 18° C.). Plants were grown in 7.5 litre pots of Levington M2 pot/bedding compost. Irrigation supplemented with Vitax 214. The population was planted in four blocks each containing one plant. Four fruits from each line were tagged at breaker and is harvested seven days later at the red ripe stage. The environmental conditions within the glasshouse were recorded throughout the experiment and included in the statistical analysis.
Example 5
IL 3-4 Mapping Population
A total of 41 QTL-NILs derived from an IL 3-4×M82 F2 mapping population were used for the fine mapping of the texture QTL. Plants were grown in Autumn 2009/Summer 2010 under standard glasshouse conditions. At least four fruits per line were tagged at breaker and harvested seven days later.
Example 6
Pericarp Penetration Test (PPT)
Phenotypic analysis was carried out on the S. pennellii ILs grown under glasshouse conditions in the UK. A 6 mm transverse section was cut from each fruit and the maximum load (force required to penetrate the pericarp tissue at 10 mm/minute) was measured using a Lloyd Instrument LRF+ machine (Lloyd, UK) equipped with a 10 N load cell and 1.6 mm probe. Measurements were taken separately from the outer and inner pericarp in duplicate. Fruit weight, truss position and colour were also recorded. Colour was measured using a Chroma Meter (Minolta).
Example 7
Genomic DNA Extraction
Genomic DNA was extracted using the protocol described by Fulton et al. 1995. In brief, fresh and young leaf material was collected in 1.5 ml eppendorf tubes and grinded together with a miniprep solution containing DNA extraction buffer, nuclei lyse buffer, sarkosyl (5%) and sodium bisulfite. Crushed samples were incubated at 65° C. for 1 to 2 hours, and chloroform isoamyl (24:1) was added immediately. After centrifugation, only the supernatant aqueous phase was maintained. Subsequently, isopropanol was used to precipitate the DNA. After a second centrifugation step, ethanol (70%) was used to clean the DNA pellets before a final dilution in TE (10:1). DNA concentration (10 ng/μl) was estimated by enzymatic digestion, using two DNA samples of known concentrations as controls. Alternatively, small unexpanded leaves were collected into Qiagen 96 well collection plants with a 3 mm stainless steel ball and 600 μl of extraction buffer (1M Tris-HCl pH 7.5, 4M NaCl, 0.5M EDTA, 10% SDS) before milling in a ball mill for 3 minutes. The supernatant was cleared by centrifugation at 5600 g for 20 minutes before 300 μl was precipitated with an equal volume of isopropanol at room temperature for 3 mins. The nucleic acids were collected by centrifugation at 5600 g for 20 minutes, washed in 70% and 100% EtOH before drying and resuspension in TE (10:1) buffer containing 12 mg/ml of RNase.
Example 8
Molecular Characterization of Plant Material
Molecular analysis was performed mostly using PCR-based markers. Primers were designed using sequences of RFLP, CAPS and COS markers available at the SOL genomics network (available at solgenomics.net/), using the tomato genetic map EXP-2000 as a reference for the location of new markers. This genetic map was constructed based on an interspecific cross between two Solanum species, lycopersicum and pennellii (Fulton et al. 2002; Tanksley et al. 1992). PCR products were either sequenced or examined by single strand conformational polymorphism (SSCP) assay using Sequa Gel® MD (National Diagnostics, U.K Ltd) and visualised by silver staining (Bassam et al. 1991). dCAPS markers were designed as described by Neff et al. (2002). For other markers, a SNP detection protocol was used to detect specific nucleotide substitutions in the recombinant QTL-NILs. Analysis of these SNPs was performed using Genalys 2.0 (Takahashi and Matsuda, 2001). The general PCR reaction mixture included 1.5 microliters of genomic DNA (10 ng/μl), 5 units of 5× Reaction Buffer, 2.5 units of MgCl2 25mM, 1.25 units of dNTPs 5 mM, 0.5 units of each forward and reverse primers 10 μM, 0.25 units of goTaq (5 u/μl) enzyme and miliQ water making a total volume of 25 microliters. A general amplification protocol for all PCR-based markers was as follows: hot start at 95° for 4 minutes; 35 cycles of 30 seconds at 95°, 30 seconds at annealing temperature and 30 seconds of extension at 75° C.; with a final extension at 75° C. for 4 minutes. Digested fragments of dCAPS markers were visualized using UV light after migration in a 2.5% agarose gel, stained in an ethidium bromide solution. Microsatellite markers (SSRs) were also used in this experiment. Before fragment amplification, reverse primers were labeled with 33P as described by Hodgson and Fisk (1987). Subsequently, a similar amplification protocol was used before fragment separation in a polyacrylamide gel; and visualization using a Kodak film developer. One RFLP marker was used following the labeling procedure described by Hodgson and Fisk (1987). Labelling, hybridization and film development were similar as described by Cheng et al. (2003).
Example 9
Statistical and QTL Analysis
S. pennellii based populations were analysed using Residual Maximum Likelihood (REML) (Patterson and Thompson, 1971) variance components analysis taking environmental factors such as light intensity, temperature and humidity into account along with plot position in a randomised plot, plant truss number, date of collection, is weight and colour for texture traits using GenStat Release 9.1 (Lawes Agricultural Trust, Rothamsted Experimental Station). The Maximum load was analysed separately for the inner and outer pericarp following log transformation of the data. Significance was determined from the t-stat based on the population means with respect to the recurrent parent. Chi-squared test for dominance traits recovered a two tailed P value with one degree of freedom.
IL 3-4 mapping population were analysed as above, predicted means generated from the REML for each QTL-NIL line were used in the analysis. Linkage maps were calculated from recombination frequencies (0.4) and a LOD of 3.0 in JoinMap® 3.0 (van Ooijen and Voorips 2001). QTL analysis was carried out using MapQTL® version 5.0. (van Ooijen, 2004). Interval mapping was carried out on one linkage group created following the map construction of Chr 3 using JoinMap® 3.0 (van Ooijen and Voorips 2001). LOD scores were generated by a permutation test (1,000 cycles) to determine the genomic location of QTLs with a confidence interval of 95%.

## Claims

1. A solanum lycopersicum tomato fruit with significantly increased fruit firmness at the harvesting stage linked to a genetic element in a cultivated solanum lycopersicum tomato plant producing said tomato fruit, wherein said firmness is up to 4 times greater than that of fruit from a control Solanum lycopersicum tomato plant which does not have the said genetic element, wherein said genetic element is the genetic element of solanum lycopersicum Q7774 deposited with NCIMB under accession number 41949, and wherein the said genetic element of Q7774 is linked to at least one DNA marker in the cultivated plant, said at least one DNA marker is selected from the group consisting of:
a) TG599, which can be detected by a forward primer of SEQ ID NO:19 and a reverse primer of SEQ ID NQ:20;
b) NT3374, which can be detected by a forward primer of SEQ ID NO:7 and a reverse primer of SEQ ID NO:8;
c) TG246, which can be detected by a forward primer of SEQ ID NO:17 and a reverse primer of SEQ ID NO:18: and
d) NT5880, which can be detected by a forward primer of SEQ ID NO:9 and a reverse primer of SEQ ID NO:10.

2. The tomato fruit according to claim 1 wherein the harvesting stage is the breaker stage plus 7 days.

3. A cultivated solanum lycopersicum tomato plant which produces the tomato fruit according to claim 1.

4. A tomato seed which produces the cultivated tomato plant according to claim 3.

5. Plant part of the cultivated tomato plant according to claim 3, wherein the plant part comprises the genetic element.

6. A method of producing a cultivated tomato plant which provides fruit with significantly increased fruit firmness by cultivating the tomato seed according to claim 4.

7. A cultivated tomato plant, or part thereof, obtained by the method according to claim 6, wherein the plant or part thereof comprises the genetic element.

8. A cultivated solanum lycopersicum tomato plant comprising a QTL responsible for increased fruit firmness at the harvesting stage compared to that of fruit from a control solanum lycopersicum tomato plant, wherein said QTL can be detected by at least one DNA marker, said tomato plant is produced by the method comprising:
a) crossing a donor tomato plant with a recipient tomato plant to provide offspring cultivated tomato plants, wherein said donor plant has the genetic element of solanum lycopersicum Q7774 deposited with NCIMB under accession number 41949,
b) quantitatively determining the firmness in the fruit of said offspring plants,
c) establishing a genetic linkage map that links the observed increased fruit firmness to the presence of at least one DNA marker from said donor plant in said offspring plants and
d) assigning to a QTL the DNA markers on said map that are linked to the increased fruit firmness;
and wherein said genetic element of Q7774 is linked to at least one DNA marker in the donor plant, said at least one DNA marker is selected from the group consisting of:
TG599, which can be detected by a forward primer of SEQ ID NO:19 and a reverse primer of SEQ ID NQ:20;
NT3374, which can be detected by a forward primer of SEQ ID NO:7 and a reverse primer of SEQ ID NO:8:
TG246, which can be detected by a forward primer of SEQ ID NO:17 and a reverse primer of SEQ ID NO:18; and
NT5880, which can be detected by a forward primer of SEQ ID NO:9 and a reverse primer of SEQ ID NQ:10.

9. A hybrid tomato plant, or part thereof, obtained by crossing the cultivated tomato plant according to claim 3 with a second tomato plant, wherein the hybrid tomato plant or part thereof comprises the genetic element.

10. Tomato seed produced by growing the tomato plant of claim 3, wherein the tomato seed comprises the genetic element.

11. Tomato seed produced by crossing the tomato plant of claim 3 with a second tomato plant to obtain a seed that produces a plant that comprises the genetic element and has significantly increased fruit firmness compared to a control tomato plant.