CN116583177A

CN116583177A - Novel melon plant with long shelf life

Info

Publication number: CN116583177A
Application number: CN202180074092.6A
Authority: CN
Inventors: R·库马尔; M·奥利弗; J·I·阿尔瓦雷兹卡萨努瓦
Original assignee: Syngenta Crop Protection AG Switzerland
Current assignee: Syngenta Crop Protection AG Switzerland
Priority date: 2020-10-29
Filing date: 2021-10-26
Publication date: 2023-08-11

Abstract

The present invention relates to novel melon plants that produce fruits that exhibit a combination of long shelf life traits and maturity index traits. The invention also relates to seeds and parts of said plants, such as fruits. The invention further relates to methods of making and using such seeds and plants. The invention also relates to novel gene sequences associated with the transition to the crust phenotype at maturity which, when combined with long shelf life alleles, significantly alter the characteristics of mature melon fruits as a reliable maturation index while retaining suitable marketable characteristics and yielding novel melon plant types.

Description

Novel melon plant with long shelf life

Technical Field

Background

There are two types of fruit ripening for Cantaloupe planted in america (Cantaloupe melon): a jump type and a non-jump type. Traditionally, western Shipper cantaloupe planted in america is a mutant. The ripening of the fruit of the jump-type is characterized by respiratory burst and autocatalytic synthesis of ethylene at the beginning of ripening, which triggers a series of ethylene-dependent pathways or processes and expression of some ripening index phenotypes, if the skin changes from green to yellowish/yellow (chlorophyll degradation) and other characteristics such as the stem sliding off the vine (abscission layer formation, sliding melon) (for reviews see Pech et al, 2008). Thus, a change in peel color from green to yellow/yellowish can be an indicator of this melon type ripening, and when the peel color changes and the fruit slips off the vine, the harvester can pick the fruit off the stem.

In one aspect, these Western Shipper cantaloupes have a strong aroma and musk aroma. They are therefore highly appreciated by the end consumer. However, one disadvantage of this melon type is that they require frequent harvesting (10-12 times), almost two weeks of harvesting per day, and are therefore labor intensive. Indeed, if they are not picked in the full-slip phase, the subsequent delay shortens the shelf life of the fruit. In addition, as ethylene production increases, they also exhibit poor post-harvest shelf life and waste due to shrinkage during transportation or storage.

Thus, the presence of traditional Western Shipper cantaloupe (transition type), which is ubiquitous in most important plantation areas and most shops, begins to decline about 10-12 years ago. They are increasingly replaced by Long Shelf Life (LSL) cantaloupes (non-jump-type) which are now the most common local melons in the american market. LSL cantaloupe (also known as harpin) offers benefits to growers and retailers: growers can reduce harvest times and gain flexibility while retailers extend shelf life and reduce losses due to shrinkage. The single dominant gene (hereinafter LSL 10) contributes to the long shelf life of most of these melons. These types of melons comprising the LSL10 allele show non-jump-type fruit ripening, reduced levels of ethylene and/or reduced sensitivity to ethylene, and thus the fruits have excellent field and post-harvest shelf life. The fruits only need to be picked twice to three times, not 10-12 times.

However, LSL cantaloupes do not slip off the vine and their skin color does not change from green to yellow upon maturation as in the traditional Western Shipper variety. Thus, growers have difficulty determining ideal maturation harvest points because they lack reliable "maturation indicators," such as transition crust phenotypes, and thus some melons harvest before full maturation and lack aroma and flavor.

Thus, while the introduction of LSL-type cantaloupe has resulted in the conversion of most commercial growers to LSL melon over ten years ago due to several agronomic advantages described above, end consumers have simultaneously begun to move away from such melons exhibiting reduced organoleptic properties. In other words, the need for new melon types that can meet growers and retailers on the one hand and consumers on the other hand has not yet been met.

Disclosure of Invention

The present invention addresses the need for new and improved melon types that combine features that are beneficial to both growers and retailers on the one hand and consumers on the other hand. By identifying a QTL associated with a non-ethylene dependent transition coat phenotype and incorporating its corresponding sequence into the LSL cantaloupe plant background, the inventors obtained novel melon plant types exhibiting long shelf life and maturity index traits that enabled growers to harvest melon fruits at the proper maturity with minimal labor costs, retailers would benefit from preserved long shelf life features, and consumers could enjoy fully mature melon fruits. The transition-sheath QTL located on chromosome 6 and its potential introgression sequence (also referred to as transition-sheath QTL or QTL 6) have semi-dominant properties, so that one copy of this sequence already provides a transition-sheath, maturation index phenotype in the melon background (carrying at least one copy of the long shelf-life LSL10 allele).

In summary, the improved melon plant features disclosed herein provide new solutions to melon growers to improve economic and commercial efficiency when deploying cantaloupe varieties on the market.

In a first embodiment, the invention provides a method of growing melon plants, preferably cantaloupe plants, more preferably coarse-peel melon (cumis melo var. Reticulata) plants or melon (cumis melo. Reiciculatus) plants, comprising in their genome:

a) At least one copy of the LSL10 allele, and;

b) At least one copy of an introgression sequence from melon (c.melo var. Dudaim) associated with a transition sheath phenotype, located on chromosome 6 and comprising at least one of the following SNP markers:

i) Genotype A of heterozygous or homozygous state of SNP marker 1 at a position corresponding to position 61 in SEQ ID NO. 1;

ii) the G genotype of the heterozygous or homozygous state of SNP marker 2 at a position corresponding to position 55 in SEQ ID NO. 6;

iii) A G genotype of the heterozygous or homozygous state of SNP marker 3 at a position corresponding to position 65 in SEQ ID NO. 11;

iv) the G genotype of the heterozygous or homozygous state of SNP marker 4 at a position corresponding to position 49 in SEQ ID NO. 16;

v) genotype C of heterozygous or homozygous state of SNP marker 5 at a position corresponding to position 53 in SEQ ID NO. 21;

vi) the G genotype of the heterozygous or homozygous state of SNP marker 6 at a position corresponding to position 83 in SEQ ID NO. 26;

vii) a G genotype of the heterozygous or homozygous state of SNP marker 7 at a position corresponding to position 103 in SEQ ID NO. 31;

viii) genotype A of the heterozygous or homozygous state of SNP marker 8 at a position corresponding to position 115 in SEQ ID NO. 36;

ix) genotype A of the heterozygous or homozygous state of SNP marker 9 at a position corresponding to position 87 in SEQ ID NO. 41; and/or

x) the genotype C of the heterozygous or homozygous state of SNP marker 10 at a position corresponding to position 196 in SEQ ID NO. 46;

wherein the plant produces melon fruits that exhibit a long shelf life phenotype, and wherein the melon fruits further exhibit a transition crust phenotype upon reaching full maturity.

In further embodiments, the infiltration sequence comprises at least one of SEQ ID NO. 1, SEQ ID NO. 6, SEQ ID NO. 11, SEQ ID NO. 16, SEQ ID NO. 21, SEQ ID NO. 26, SEQ ID NO. 31, SEQ ID NO. 36, SEQ ID NO. 41 and/or SEQ ID NO. 46, or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identity to one or more of the sequences.

In a further embodiment, the present disclosure provides a plant according to any one of the preceding embodiments, wherein the transition sheath phenotype is characterized by a sheath color that changes from green when immature to yellow when full maturity is reached.

In further embodiments, the melon peel has a yellow color when fully ripe ranging from 15 to 20 (a-D) when measured using the color scale (color patch) of the uk royal gardening institute color chart (Royal Horticultural Society Colour Chart).

In further embodiments, the melon peel has a yellow color of 19A or 19B when fully ripe, as measured using the color scale of the uk royalty gardening institute color chart.

In further embodiments, the melon peel, when immature, has a green color range of 135 to 143 (a-D) when measured using the color scale of the uk royalty gardening institute color chart.

In further embodiments, the melon peel is green at green time of 138C, 138D or 139A when measured using the color scale of the uk royalty gardening institute color chart.

In further embodiments, the transition sheath phenotype is assessed as disclosed in example 2A.

In a further embodiment, the present invention provides a plant according to any one of the preceding embodiments, wherein the LSL10 allele comprises the following SNP markers:

a) G genotype of heterozygous or homozygous state of SNP marker 11 at a position corresponding to position 107 in SEQ ID NO: 51.

In further embodiments, the LSL10 allele comprises SEQ ID NO. 51, or a sequence having at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identity to the sequence.

In further embodiments of the invention, the at least one SNP marker 1-10 of the plant is homozygous. In a further embodiment, the present disclosure provides a plant according to any one of the preceding embodiments, wherein the LSL10 allele of the plant is homozygous.

In further embodiments of the invention, the LSL10 allele and the introgression sequence are contained in melon line 19MNA106815 (representative seed deposited under ATCC accession No. PTA-126875) or progeny or ancestors thereof.

In a further embodiment, the present invention provides a plant according to any one of the preceding embodiments, wherein said plant is obtained by crossing melon line 19MNA106815 (representative seed of which is deposited under ATCC accession No. PTA-126875) or progeny or ancestor thereof with a melon plant that does not contain the introgression sequence.

In a further embodiment, the present invention provides a plant according to any one of the preceding embodiments, wherein the plant is an inbred, a doubled haploid or a hybrid plant.

Further embodiments provide plant parts, organs or tissues obtainable from melon plants according to any of the preceding embodiments, including but not limited to leaves, stems, roots, flowers or flower parts, fruits, buds, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, fertilized eggs, embryos, meristematic regions, callus tissue, seeds, cuttings, cells or tissue culture, or any other part or product of a plant still exhibiting a transformed outer skin phenotype according to the invention (in particular when grown as a fruit producing plant).

In a further embodiment, the present invention provides a seed for producing a plant according to any one of the preceding embodiments.

In a further embodiment, the present invention provides a method for producing a cultivated melon plant, preferably a cantaloupe plant, more preferably a coarse-skin melon plant or a textured melon plant, wherein the plant produces melon fruits exhibiting a long shelf-life phenotype and a transition to a crust phenotype upon reaching full maturity, wherein the method comprises the steps of:

a) Crossing the plant according to any one of the preceding embodiments with a cultivated melon plant lacking the LSL10 allele and the introgression sequence;

b) Selecting a progeny plant comprising at least one copy of the LSL10 allele and at least one copy of the introgression sequence from cucumis sativus located on chromosome 6, the selecting step comprising detecting at least one of the following SNP markers:

thereby producing a plant that produces fruits having a long shelf life phenotype and that transition to the rind phenotype upon reaching full maturity.

In a further embodiment, the present disclosure is directed to a method according to any one of the preceding embodiments, wherein the method further comprises:

c) Selfing the selected progeny or crossing the selected progeny with another melon plant to produce additional progeny.

In a further embodiment, the present invention relates to a method according to the preceding embodiment, wherein further progeny are selected and selfed/crossed for 2 to 10 generations.

In a further embodiment, the present invention relates to a method according to any one of the preceding embodiments, wherein the plant of step a) is melon line 19MNA106815 (representative seed deposited under ATCC accession No. PTA-126875) or progeny or ancestor thereof.

In a further embodiment, the invention relates to a method for producing an F1 melon plant exhibiting a transformed outer skin phenotype, the method comprising crossing an inbred melon plant (which is a plant according to any one of the preceding embodiments) with a different inbred melon plant to produce an F1 hybrid progeny.

In a further embodiment, the present invention provides a method for identifying a melon plant, preferably a cantaloupe plant, more preferably a ground-network melon plant or a rough melon plant, wherein the plant produces melon fruits exhibiting a long shelf-life phenotype and a transition to a crust phenotype upon reaching full maturity, wherein the plant comprises at least one copy of the LSL10 allele and at least one copy of the introgression sequence from a melon located on chromosome 6, wherein the method comprises the steps of: detecting at least one of the following SNP markers:

a) Genotype A of heterozygous or homozygous state of SNP marker 1 at a position corresponding to position 61 in SEQ ID NO. 1;

b) A G genotype of the heterozygous or homozygous state of SNP marker 2 at a position corresponding to position 55 in SEQ ID NO. 6;

c) A G genotype of the heterozygous or homozygous state of SNP marker 3 at a position corresponding to position 65 in SEQ ID NO. 11;

d) A G genotype of the heterozygous or homozygous state of SNP marker 4 at a position corresponding to position 49 in SEQ ID NO. 16;

e) A genotype C of the heterozygous or homozygous state of SNP marker 5 at a position corresponding to position 53 in SEQ ID NO. 21;

f) A G genotype of the heterozygous or homozygous state of SNP marker 6 at a position corresponding to position 83 in SEQ ID NO. 26;

g) A G genotype of the heterozygous or homozygous state of SNP marker 7 at a position corresponding to position 103 in SEQ ID NO. 31;

h) A genotype A of the heterozygous or homozygous state of SNP marker 8 at a position corresponding to position 115 in SEQ ID NO. 36;

i) Genotype A of heterozygous or homozygous state of SNP marker 9 at a position corresponding to position 87 in SEQ ID NO. 41; and/or

j) A genotype C of the heterozygous or homozygous state of SNP marker 10 at a position corresponding to position 196 in SEQ ID NO. 46;

thereby identifying melon plants that produce fruits having a long shelf life phenotype and that transition to the crust phenotype upon reaching full maturity.

In a further embodiment, the present disclosure relates to a method according to the preceding embodiment, wherein the method further comprises the step of detecting the following SNP markers:

In a further embodiment, the present application relates to a method according to any one of the preceding embodiments, wherein the method further comprises selecting a melon plant comprising the one or more SNP markers, and crossing the selected melon plant with a second melon plant to produce a progeny melon plant comprising at least one of the SNP markers and exhibiting the transformed skin phenotype of the application.

Detailed Description

Definition of the definition

Technical terms and expressions used within the scope of the present application are generally given the meaning commonly applied thereto in the relevant field of plant breeding and cultivation, if not otherwise stated 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, etc.

"cultivated melon" or "elite melon" plants are understood within the scope of the application to mean plants which are no longer in a natural state but have been developed and domesticated by human care and are intended for agricultural use and/or human consumption. In the context of the present application, a "cultivated melon" or "elite melon" plant does not include wild melon germplasm. As an example, in an embodiment, a cultivated or elite melon plant according to the application is capable of growing fruits having a brix level exceeding 8, preferably exceeding 10, even more preferably exceeding 12. Alternatively, or in addition, the cultivated melon plant is a hybrid plant. Alternatively, or in addition, the cultivated melon plant is a cantaloupe plant. Alternatively, or in addition, the cultivated cantaloupe plant is a coarse-peel melon plant or a textured melon plant (Pitrat et al, 2000). In the context of an interspecific crossing between a rough-skinned melon plant or a textured melon plant and a wild melon germplasm, or a different melon variety (c.melo var.) (e.g. the melon germplasm), a cultivated melon plant is defined as a progeny plant of said interspecific crossing, wherein said progeny plant has been backcrossed at least three times with the rough-skinned melon plant or textured melon plant.

"allele" within the scope of the present invention is understood to mean a replaceable or variant form of different genetic units identical to or associated with a different form of a gene or of any type of identifiable genetic determinant, such as a QTL, which are replaceable in genetics in that they are at the same locus of a homologous chromosome. Such alternative or variant forms may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the result of genetic manipulation (caused by, for example, chemical or structural modification), transcriptional manipulation or post-translational modification/manipulation. In a diploid cell or organism, both alleles of a given gene or genetic element typically occupy corresponding loci on a homologous chromosome pair.

The term "shift of the skin" or "shift of the skin color" is understood herein to mean that a plant comprising the introgression sequence and comprising at least one of the SNP markers 1 to 10 and at least one copy of the LSL10 allele exhibits a shift of the skin phenotype compared to a plant lacking the introgression sequence from a melon plant located on chromosome 6. In particular, melon plants according to the invention produce fruits that exhibit a transition in the color of the outer skin from green when immature to yellowish or yellow when full maturity is reached. In contrast, plants lacking the introgression sequence of the invention produce fruits that do not change skin color (i.e., remain green or grayish green) between the immature stage to the fully mature stage.

"immature fruit" is understood within the scope of the present invention to mean melon fruit having a total soluble solids of less than 10 ° brix.

"fully ripe" or "fully ripe fruit" is understood within the scope of the present invention to mean melon fruits having a total soluble solids equal to or greater than 10 ° brix, preferably greater than 12 ° brix.

The term "long shelf life" phenotype is understood within the scope of the present invention to mean fruits that do not slip off the vine at maturity (i.e. do not form an upper delamination layer) and/or retain good marketability characteristics within 14 to 20 days after harvest at harvest.

"phenotype" is understood within the scope of the present invention to mean one or more distinguishable characteristics of a genetically controlled trait.

"control melon plant" means within the scope of the invention a melon plant having the same genetic background as the cultivated melon plant of the invention, wherein the control plant does not have an introgression sequence linked to the transition to the crust phenotype. In particular, the control melon plant is a melon plant belonging to the same plant variety and does not comprise the introgression sequence of the invention. In particular, the control melon plant may be a melon plant comprising the LSL10 allele but not comprising the introgression sequence located on chromosome 6. The control melon plants were grown under the same conditions and for the same length of time as the cultivated melon plants of the invention. Plant varieties are understood herein according to the definition of UPOV. Thus, the control melon plants may be near isogenic, inbred or hybrid, provided that they have the same genetic background as the melon plants of the invention, except that the control plants do not have the introgression sequences of the invention linked to the transition coat phenotype.

The term "trait" refers to a characteristic or phenotype. In the context of the present invention, a trait is a transition skin trait. The trait may be inherited in a dominant or recessive manner or in a partially or incompletely dominant manner. In the context of the present invention, the transition crust penetration sequence located on chromosome 6 is semi-dominant. Thus, the melon plants of the invention may be homozygous or heterozygous for the trait. Furthermore, a trait may be monogenic or polygenic, or may result from the interaction of one or more genes with the environment. In the context of the present invention, the transition sheath introgression sequence located on chromosome 6 confers a transition sheath trait when associated with the long shelf life LSL10 allele.

The terms "hybrid", "hybrid plant" and "hybrid progeny" refer to individuals produced from genetically diverse parents (e.g., individuals that are genetically heterozygous or predominantly heterozygous).

The term "inbred line" refers to a population that is homozygous or nearly homozygous in genes. For example, inbred lines may be obtained by several cycles of sibling/sister breeding or selfing or doubled haploid production.

The term "doubled haploid line" refers to a stable inbred line resulting from anther culture. Some pollen grains (haploids) cultivated in a specific medium and environment can develop into plantlets containing n chromosomes. These plantlets were then "doubled" and contained 2n chromosomes. The progeny of these plantlets are termed "doubled haploids" and are essentially no longer segregating (stable).

The term "genetically fixed" refers to a genetic sequence that has been stably incorporated into a plant genome that is typically free of the genetic sequence. When genetically fixed, genetic sequences can be transmitted to other plants in an easy and predictable manner by sexual crosses.

The term "stock" refers to a plant that serves as a scion plant support. Typically, the stock plant and the scion plant have different genotypes. In an embodiment, the plant according to the invention is used as a rootstock plant.

The term "plant" or "plant part" hereinafter refers to a plant part, organ or tissue obtainable from a melon plant according to the invention, including but not limited to leaves, stems, roots, flowers or flower parts, fruits, shoots, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, fertilized eggs, embryos, meristematic regions, callus tissue, seeds, cuttings, cells or tissue culture, or any other part or product of a plant still exhibiting the transformed skin trait according to the invention (in particular when grown as a fruit producing plant).

A "plant" is any plant at any stage of development.

Melon plant seeds are seeds grown into melon plants according to any of the embodiments.

"plant cells" are the structural and physiological units of plants (including protoplasts and cell walls). The plant cells may be in the form of isolated single cells or cultured cells, or as part of a higher organized unit (such as, for example, plant tissue, plant organs, or whole plants).

"plant cell culture" means a culture of plant units (such as, for example, protoplasts, cells of a cell culture, cells in a plant tissue, pollen tubes, ovules, embryo sacs, fertilized eggs, and embryos at different stages of development).

"plant organs" are unique and distinct structured and differentiated parts of plants, such as roots, stems, leaves, flower buds or embryos.

As used herein, "plant tissue" means a group of plant cells organized into structural and functional units. Including any plant tissue in a plant or in culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and/or functional units. The use of this term in combination or alone with any particular type of plant tissue as listed above or otherwise encompassed by this definition is not intended to exclude any other type of plant tissue.

As used herein, the term "breeding" and grammatical variations thereof refers to any process by which progeny individuals are produced. Breeding may be sexual or asexual, or any combination thereof. Exemplary non-limiting breeding types include crosses, selfing, doubled haploid derivative generation, and combinations thereof.

As used herein, the phrase "established breeding population" refers to a collection of potential breeding partners produced by and/or used as parents in a breeding program (e.g., a commercial breeding program). Members of an established breeding population are typically well characterized in terms of genes and/or phenotypes. For example, several phenotypic traits of interest may have been assessed, e.g., under different environmental conditions, at multiple locations, and/or at different times. Alternatively, or in addition, one or more genetic loci associated with expression of a phenotypic trait may have been identified, and one or more members of the breeding population may have been genotyped with respect to the one or more genetic loci and with respect to one or more genetic markers associated with the one or more genetic loci.

As used herein, the phrase "diploid individual" refers to an individual having two sets of chromosomes, typically one set from each of its two parents. However, it should be understood that in some embodiments, a diploid individual may receive chromosomes of its "female" and "male" sets from the same single organism, such as when the plant is self-pollinated to produce a plant's offspring.

"homozygous" is understood within the scope of the present invention to mean the same allele at one or more corresponding loci on a homologous chromosome. In the context of the present invention, two identical copies of a melon plant comprising a specific introgression sequence (e.g., an introgression sequence located on chromosome 6) at a specific locus are homozygous at the respective locus.

"heterozygous" is understood within the scope of the present invention to mean different alleles at one or more corresponding loci on homologous chromosomes. In the context of the present invention, melon plants comprising one copy of a specific introgression sequence (e.g., introgression sequence located on chromosome 6) at a specific locus are heterozygous at the corresponding locus.

"dominant" alleles are understood within the scope of the present invention to mean alleles which, when present in heterozygous or homozygous state, determine the phenotype.

"semi-dominant" alleles are understood within the scope of the invention as meaning alleles which, when present in heterozygous or homozygous state, determine the phenotype. However, the intensity of the phenotype is generally higher when the allele is present in a homozygous state.

"recessive" alleles refer to alleles that determine phenotype when present in homozygous state only.

"backcrossing" is understood within the scope of the present invention to mean a method of repeatedly crossing back hybrid progeny with one of the parents. Different recurrent parents may be used in subsequent backcrosses.

"locus" is understood within the scope of the present invention to mean a region on the chromosome which comprises genes, QTLs or their corresponding gene sequences contributing to a trait.

As used herein, a "marker locus" refers to a region on a chromosome that comprises a nucleotide or polynucleotide sequence that is present in the genome of an individual and that is associated with one or more loci of interest, which region may comprise a gene or any other genetic determinant or factor that contributes to a trait. "marker locus" also refers to a region on a chromosome that comprises a polynucleotide sequence complementary to a genomic sequence (e.g., a sequence of a nucleic acid used as a probe).

"genetic linkage" is understood within the scope of the present invention to mean the association of genetic features due to the proximity of genes at positions on the same chromosome, measured by the percentage of recombination between loci (centimorgan, cM).

For the purposes of the present invention, the term "co-separation" refers to the fact that: alleles for a trait and one or more alleles for one or more markers tend to pass together because they are physically close together on the same chromosome (recombination between them is reduced due to their physical proximity), resulting in their alleles not being randomly associated due to their proximity on the same chromosome. The term "associated with … …" may be used in the same sense.

As used herein, the phrases "sexual crossing" and "sexual reproduction" refer in the context of the presently disclosed subject matter to seed fusion to produce progeny (e.g., seed produced by fertilization, such as by pollination in a plant). In some embodiments, "sexual crosses" or "allofertilizations" are the fertilization of one individual by another (e.g., cross pollination in a plant). In some embodiments, the term "self-fertilization" refers to the production of seeds by self-fertilization or self-pollination; that is, the pollen and ovule are from the same plant.

As used herein, the phrase "genetic marker" or "DNA marker" refers to a feature in the genome of an individual that is associated with one or more loci of interest (e.g., a nucleotide or polynucleotide sequence present in the genome of the individual). In some embodiments, the genetic markers are polymorphic in the population of interest, or the locus is occupied by a polymorphism, depending on the context. Genetic markers include, for example, single Nucleotide Polymorphisms (SNPs), indels (i.e., insertions/deletions), simple Sequence Repeats (SSRs), restriction Fragment Length Polymorphisms (RFLP), random Amplified Polymorphic DNA (RAPD), cut Amplified Polymorphic Sequence (CAPS) markers, diversity array technology (DArT) markers, and Amplified Fragment Length Polymorphisms (AFLP), among many other examples. Genetic markers may be used, for example, to map genetic loci on chromosomes that contain alleles that contribute to variability in a phenotypic trait. The phrase "genetic marker" may also refer to a polynucleotide sequence complementary to a genomic sequence, such as the sequence of a nucleic acid used as a probe.

As used herein, the term "genotype" refers to the genetic makeup of a cell or organism. An individual's "genotype of a set of genetic markers" includes a particular allele of one or more genetic marker loci present in a haplotype of the individual.

As used herein, the term "progeny" refers to one or more offspring of a particular cross. Typically, progeny results from breeding of two individuals, but some species (particularly some plants and hermaphrodite animals) can selfe (i.e., the same plant serves as a donor for both male and female gametes). The one or more offspring may be, for example, F ₁ 、F ₂ Or any subsequent generation.

As used herein, the term "quantitative trait locus" (QTL) refers to the association between a genetic marker and a chromosomal region and/or gene and/or introgression sequence that affects the trait phenotype of interest. Typically, this is determined statistically, e.g., based on one or more methods disclosed in the literature. QTL may be a chromosomal region and/or genetic locus having at least two alleles that differentially affect a phenotypic trait.

The term "recipient melon plant" is used herein to indicate a melon plant that will receive DNA obtained from a donor melon plant that comprises an introgression sequence for transforming the crust trait. In the context of the present invention, the recipient plant may already comprise at least one copy of the LSL10 allele, in which case at least one copy of the introgression QTL6 is necessary for the expression of the transition crust phenotype. In the context of the present invention, the recipient plant may also be a plant lacking both QTL6 and LSL10 alleles, so that introgression of at least one copy of each allele is necessary for the transition of expression of the crust phenotype.

The term "natural genetic background" is used herein to indicate the original genetic background of a gene sequence. For example, such a background may be the genome of a wild melon germplasm. For example, the gene sequence of the invention is found at a specific position on chromosome 6 of the melon plant. In contrast, a method involving transferring DNA comprising the gene sequence from chromosome 6 of a melon plant to the same location on chromosome 6 of another melon species (preferably a melon plant or a cantaloupe plant, even more preferably a rough melon plant or a melon plant) via, for example, breeding, would result in the gene sequence not being in its natural genetic background. When the gene sequences of the invention are transferred from the melon background to another melon species, preferably a cultivated melon plant, even more preferably a rough-skinned melon or a textured melon plant, they are referred to as "introgression sequences" or "introgression gene sequences".

"donor melon plant" is understood within the scope of the present invention to mean a melon plant that provides at least an infiltration sequence for transforming the outer skin. In the context of the present invention, a donor plant may also comprise at least one copy of an LSL10 allele if such a LSL10 allele is absent from the recipient plant.

"marker-based selection" is understood within the scope of the present invention to mean that one or more nucleic acids are detected from plants, for example using genetic markers, wherein the nucleic acids are associated with a desired trait, to identify plants carrying alleles of the desired (or undesired) trait such that those plants can be used (or avoided) in a selective breeding program.

Single Nucleotide Polymorphisms (SNPs) are the single site variation in DNA and the most common type of variation in the genome. A Single Nucleotide Polymorphism (SNP) is a DNA sequence variation that occurs when a single nucleotide in the genome (or other consensus sequence) -A, T, C or G-differs between members of a biological species or pairs of chromosomes of an individual. For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a single nucleotide difference. In this case, there are two alleles: c and T. The basic principle of SNP array is the same as that of DNA microarray. These are fusions of DNA hybridization, fluorescence microscopy, and DNA capture. Three components of a SNP array are an array containing a nucleic acid sequence (i.e., amplified sequence or target), one or more labeled allele-specific oligonucleotide probes, and a detection system that records and interprets hybridization signals. The presence or absence of the desired SNP marker allele can be determined by real-time PCR or fluorescent reporter probe methods using double-stranded DNA dyes.

"PCR (polymerase chain reaction)" is understood within the scope of the present invention to mean a method of generating a relatively large amount of DNA of a genome or a specific region of one or more subsets, in order to carry out a possible different analysis based on those regions. "PCR primer" is understood within the scope of the present invention to mean a relatively short single-stranded DNA fragment used in the PCR amplification of a specific region of DNA.

As used herein, a "probe" refers to a set of atoms or molecules that are capable of recognizing and binding to a particular target molecule or cellular structure and thus allowing detection of the target molecule or structure. In particular, "probe" refers to a labeled DNA or RNA sequence that can be used to detect the presence of and quantify the complementary sequence by molecular hybridization.

"sequence identity". In the context of two or more nucleic acid or protein sequences, the term "identical" or "identity" refers 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 the two sequences to be compared to each other are different in length, sequence identity preferably relates to the percentage of nucleotide residues of the shorter sequence that have identity to the nucleotide residues of the longer sequence. As used herein, the percent identity/homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e.,% identity = number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap (they need to be introduced in an optimal alignment of the two sequences). Comparison of sequences and determination of percent identity between two sequences may be accomplished using mathematical algorithms, as described below. For example, sequence identity may be conventionally determined by using a computer program such as the Bestfit program (wisconsin sequence analysis package (Wisconsin Sequence Analysis Package), for version 8 of Unix, genetics computer group company (Genetics Computer Group), university research park (University Research Park), mason scientific precursor No. 575 (575Science Drive Madison,WI) 53711, wisconsin. Bestfit uses the local homology algorithm of Smith and Waterman, advances in Applied Mathematics [ applied math Advance ]2 (1981), 482-489 to find the segment with the greatest sequence identity between the two sequences. When using Bestfit or another sequence alignment program to determine whether a particular sequence has, for example, 95% identity with a reference sequence of the invention, the parameters are preferably adjusted to calculate the percentage of identity over the entire length of the reference sequence and to allow homology gaps in the reference sequence to account for up to 5% of the total number of nucleotides. When using Bestfit, so-called optional parameters are preferably kept at their preset ("default") values. Deviations that occur in the comparison between a given sequence and the above-described sequences of the invention may be caused by, for example, additions, deletions, substitutions, insertions or recombinations. Such sequence comparisons can also preferably be made using the program "fasta20u66" (version 2.0u66, 9 th month 1998, written by William R.Pearson and Virginia university; see also W.R.Pearson (1990), methods in Enzymology [ methods of enzymology ]183,63-98 appended examples and http:// workbench. Sps. Edu /). For this purpose, a "default" parameter setting may be used.

Examples

Plants, seeds, fruits.

In a first embodiment, the present invention provides a method of growing a melon plant, preferably a cantaloupe plant, more preferably a rough-skin melon plant or a reticulate melon plant, comprising in its genome:

a) At least one copy of the LSL10 allele, and;

b) At least one copy of an introgression sequence from a melon, the introgression sequence being associated with a transition to the crust phenotype, located on chromosome 6 and comprising at least one of the following SNP markers:

i) Genotype A of heterozygous or homozygous state of SNP marker 1 in SEQ ID NO. 1;

ii) the G genotype of the heterozygous or homozygous state of SNP marker 2 in SEQ ID NO. 6;

iii) G genotype of the heterozygous or homozygous state of SNP marker 3 in SEQ ID NO. 11;

iv) the G genotype of the heterozygous or homozygous state of SNP marker 4 in SEQ ID NO. 16;

v) genotype C of heterozygous or homozygous state of SNP marker 5 in SEQ ID NO. 21;

vi) the G genotype of the heterozygous or homozygous state of SNP marker 6 in SEQ ID NO. 26;

vii) the G genotype of the heterozygous or homozygous state of SNP marker 7 in SEQ ID NO. 31;

viii) genotype A of the heterozygous or homozygous state of SNP marker 8 in SEQ ID NO. 36;

ix) genotype A of the heterozygous or homozygous state of SNP marker 9 in SEQ ID NO. 41; and/or

x) genotype C of the heterozygous or homozygous state of SNP marker 10 in SEQ ID NO. 46;

In further embodiments, the invention provides for growing melon plants, preferably Hami melon plants, more preferably rough skin melon plants or reticulate melon plants, comprising in their genome:

a) At least one copy of the LSL10 allele, and;

Additionally, the plant of any of the preceding embodiments, wherein:

a) The A genotype of SNP marker 1 can be obtained by using oligonucleotide primer pairs in PCR: forward primer SEQ ID NO. 2 and reverse primer SEQ ID NO. 5, and probe SEQ ID NO. 3.

b) The G genotype of SNP marker 2 can be obtained by using oligonucleotide primer pairs in PCR: amplifying the nucleic acid fragments with the forward primer SEQ ID NO. 7 and the reverse primer SEQ ID NO. 10 and the probe SEQ ID NO. 8 for identification;

c) The G genotype of SNP marker 3 can be obtained by using oligonucleotide primer pairs in PCR: the forward primer SEQ ID NO. 12 and the reverse primer SEQ ID NO. 15 and the probe SEQ ID NO. 13 are used for amplifying the nucleic acid fragments for identification;

d) The G genotype of SNP marker 4 can be obtained by using oligonucleotide primer pairs in PCR: forward primer SEQ ID NO. 17 and reverse primer SEQ ID NO. 20, and probe SEQ ID NO. 18.

e) The C genotype of SNP marker 5 can be obtained by using oligonucleotide primer pairs in PCR: forward primer SEQ ID NO. 22 and reverse primer SEQ ID NO. 25, and probe SEQ ID NO. 23.

f) The G genotype of SNP marker 6 can be obtained by using oligonucleotide primer pairs in PCR: forward primer SEQ ID NO. 27 and reverse primer SEQ ID NO. 30, and probe SEQ ID NO. 28.

g) The G genotype of SNP marker 7 can be obtained by using oligonucleotide primer pairs in PCR: forward primer SEQ ID NO. 32 and reverse primer SEQ ID NO. 35, and probe SEQ ID NO. 33.

h) The A genotype of SNP marker 8 can be obtained by using oligonucleotide primer pairs in PCR: forward primer SEQ ID NO. 37 and reverse primer SEQ ID NO. 40, and probe SEQ ID NO. 38.

i) The A genotype of SNP marker 9 can be obtained by using oligonucleotide primer pairs in PCR: forward primer SEQ ID NO. 42 and reverse primer SEQ ID NO. 45, and probe SEQ ID NO. 43. And/or

j) The genotype C of SNP marker 10 can be detected in PCR by using oligonucleotide primer pairs: the forward primer SEQ ID NO. 47 and the reverse primer SEQ ID NO. 50 and the probe SEQ ID NO. 48 amplified nucleic acid fragments.

In further embodiments of the invention, the transition coat penetration sequence comprises at least one of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:41 and/or SEQ ID NO:46, or has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identity to the sequence while preserving the sequence of the corresponding SNP markers 1 to 10.

In further embodiments, the melon peel has a yellow color when fully ripe ranging from 15 to 20 (a-D) when measured using the color scale of the uk royalty gardening institute color chart.

a) G genotype of SNP marker 11 in heterozygous or homozygous state in SEQ ID NO. 51.

In addition, the plant according to the previous embodiment, wherein:

a) The G genotype of SNP marker 11 can be obtained by using oligonucleotide primer pairs in PCR: the forward primer SEQ ID NO. 52 and the reverse primer SEQ ID NO. 55, and probe SEQ ID NO. 53 amplified nucleic acid fragments.

In further embodiments, the LSL10 allele comprises SEQ ID NO. 51 or has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% identity with the allele while preserving the sequence of the corresponding SNP marker 11.

In another embodiment, the plant according to the invention is male sterile. In another embodiment, the plant according to the invention is cytoplasmic male sterile.

In another embodiment, a plant according to the invention grows a mature melon fruit, wherein the inner pulp of the mature fruit is orange.

In a further embodiment, the melon plant of the invention is a melon plant according to any of the preceding embodiments, wherein the transition coat introgression sequence located on chromosome 6 can be identified using any of the SNP markers 1-10 disclosed in table 4 below.

In a further embodiment, the melon plant of the invention is a melon plant according to any one of the preceding embodiments, wherein melon line 19MNA106815 or a progeny or ancestor thereof is the source of the transition coat introgression sequence and LSL10 allele, and wherein a representative seed of line 19MNA106815 has been deposited under ATCC accession No. PTA-126875.

In a further embodiment, the present invention relates to the use of a melon plant according to any one of the preceding embodiments as melon rootstock. In further embodiments, the invention relates to the use of melon line 19MNA106815 (representative seed deposited under ATCC accession No. PTA-126875) or progeny or ancestors thereof as melon rootstock.

In another embodiment, the use of a melon plant, plant part or seed according to any of the preceding embodiments for producing and harvesting melon fruits is contemplated.

In another embodiment, the invention relates to the use of a melon plant, plant part or seed according to any embodiment, wherein the melon plant, plant part or seed is melon line 19MNA106815 (representative seed of which is deposited under ATCC accession No. PTA-126875) or a progeny or ancestor thereof.

In a further embodiment, the present invention relates to the use of melon plants, plant parts or seeds according to any of the preceding embodiments for sowing seed fields, greenhouses or canopies.

In one embodiment, the present invention provides melon fruits produced by melon plants according to any one of the preceding embodiments.

The invention further relates to the use of a melon plant according to any of the preceding embodiments for introgressing a transformed skin trait into a melon plant lacking said transformed skin trait.

Gene sequence, and marker.

The invention further relates to introgression gene sequences linked to the transition coat trait in recipient melon plants comprising the LSL10 allele. In further embodiments, the introgression sequences of the invention are located on chromosome 6. In further embodiments of the invention, the gene sequence is comprised in, obtained from or obtainable from: melon line 19MNA106815 (representative seed deposited under ATCC accession No. PTA-126875) or a progeny or ancestor thereof, and comprises the introgression gene sequence.

In another embodiment, the introgression sequence of the invention is located on chromosome 6 and is characterized by at least one of the following SNP markers:

j) The genotype C of the heterozygous or homozygous state of SNP marker 10 at a position corresponding to position 196 in SEQ ID NO. 46.

The present invention discloses a kit for detecting a transitional crust trait in a melon plant (in particular a cultivated melon plant), wherein the kit comprises at least one PCR oligonucleotide primer pair and a probe selected from the group consisting of:

a) For SNP marker 1, forward primer SEQ ID NO. 2 and reverse primer SEQ ID NO. 5, and probe SEQ ID NO. 3;

b) For SNP marker 2, forward primer SEQ ID NO. 7 and reverse primer SEQ ID NO. 10, and probe SEQ ID NO. 8;

c) For SNP marker 3, the forward primer SEQ ID NO. 12 and the reverse primer SEQ ID NO. 15, and the probe SEQ ID NO. 13;

d) For SNP marker 4, forward primer SEQ ID NO. 17 and reverse primer SEQ ID NO. 20, and probe SEQ ID NO. 18;

e) For SNP marker 5, forward primer SEQ ID NO. 22 and reverse primer SEQ ID NO. 25, and probe SEQ ID NO. 23;

f) For SNP marker 6, forward primer SEQ ID NO. 27 and reverse primer SEQ ID NO. 30, and probe SEQ ID NO. 28;

g) For SNP marker 7, forward primer SEQ ID NO. 32 and reverse primer SEQ ID NO. 35, and probe SEQ ID NO. 33;

h) For SNP marker 8, forward primer SEQ ID NO. 37 and reverse primer SEQ ID NO. 40, and probe SEQ ID NO. 38;

i) For SNP marker 9, forward primer SEQ ID NO. 42 and reverse primer SEQ ID NO. 45, and probe SEQ ID NO. 43;

j) For SNP marker 10, forward primer SEQ ID NO. 47 and reverse primer SEQ ID NO. 50, and probe SEQ ID NO. 48.

The invention also discloses the use of at least one, at least two or at least three of the SNP markers according to the invention for the diagnostic selection and/or genotyping of the transformed crust trait locus in melon plants, in particular cultivated melon plants.

The invention also discloses the use of at least one, at least two or at least three of the SNP markers according to the invention for identifying the presence of genotypes associated with a transformed skin trait in a melon plant (in particular a cultivated melon plant, more particularly a melon plant according to the invention) and/or for monitoring the introgression of a transformed skin trait in a melon plant (in particular a cultivated melon plant, particularly a melon plant comprising an LSL10 allele).

The invention further discloses polynucleotides (amplification products) obtainable in a PCR reaction involving at least one oligonucleotide primer or PCR oligonucleotide primer pair (selected from table 4), wherein the amplification products correspond to amplification products comprising the converted skin penetration sequences of the invention obtainable from melon line 19MNA106815 (representative seed deposited under ATCC accession No. PTA-126875) or progeny or ancestors thereof.

Polynucleotides having at least 95%, in particular at least 96%, in particular at least 97%, in particular at least 98%, in particular at least 99% sequence identity to the sequence of the amplification product and/or polynucleotides exhibiting a nucleotide sequence which hybridizes to the nucleotide sequence of the amplification product obtainable in the above-described PCR reaction are also contemplated herein.

The amplification products according to the invention and described above can then be used to generate or develop new primers and/or probes that can be used to identify the locus of the altered crust trait.

Thus, in one embodiment, the invention further relates to derived markers, in particular derived primers or probes, which are developed from the amplification products according to the invention and as described above by methods known in the art, which are genetically linked to the transforming crust trait locus.

A breeding method.

a) Crossing the plant according to any one of the preceding embodiments with a cultivated melon plant comprising at least one copy of the LSL10 allele but lacking the introgression sequence associated with the transition to the crust phenotype;

b) Selecting a progeny plant comprising at least one copy of the introgression sequence from cucumis sativus located on chromosome 6, the selecting step comprising detecting a favorable genotype of at least one of the following SNP markers:

In another embodiment, the present invention relates to a method of providing a transformed muskmelon plant, plant part or seed, wherein the method comprises the steps of:

a) Crossing a first plant lacking the transformed husk penetrating sequence of the invention with a second melon plant according to any of the preceding embodiments,

b) Obtaining progeny melon plants, and

c) Optionally selecting said progeny plant, characterized in that said plant exhibits a transitional crust phenotype.

In a further embodiment, the present invention relates to a method according to the preceding embodiment, wherein the first melon plant is a plant that already comprises at least one copy of the LSL10 allele. In a further embodiment, the present invention relates to a method according to the preceding embodiment, wherein the second melon plant is melon line 19MNA106815 (representative seed deposited under ATCC accession No. PTA-126875) or progeny or ancestor thereof.

In another embodiment, the present invention relates to a method for producing a transformed skin melon plant, the method comprising the steps of:

a) Providing seeds of the melon plant according to any one of the preceding embodiments,

b) Germinating said seed and growing a mature fertile plant therefrom,

c) Inducing self-pollination of the plants under a), growing fruits and harvesting fertile seeds therefrom, and

d) Plants were grown from the seeds harvested under c) and transformed melon plants were selected.

In another embodiment, the invention relates to a method for providing a transformed outer skin phenotype to a melon plant, the method comprising the steps of:

a) Selecting melon comprising a transformed skin trait associated with one introgression sequence located on chromosome 6, wherein the trait can be identified by the presence of at least one of the SNP markers listed in table 4;

b) Crossing said plant comprising the transformed skin trait of step a) with a melon plant (in particular a cultivated melon plant which does not comprise the transformed skin trait and which does not exhibit the transformed skin phenotype compared to the plant of step a)), and

c) Selecting progeny from said crosses that exhibit a transition to the crust phenotype compared to the plants of step b).

In a further embodiment, the present invention relates to a method according to the preceding embodiment, wherein the recipient plant of step b) comprises at least one copy of the LSL10 allele.

A selection method.

In a further embodiment, the present invention relates to a method according to any one of the preceding embodiments, wherein the method further comprises selecting melon plants comprising an advantageous genotype at the one or more SNP markers, and crossing the selected melon plants with a second melon plant to produce progeny melon plants comprising at least one advantageous genotype at the SNP markers and exhibiting the transformed crust phenotype of the invention.

In another embodiment, the invention relates to a method of identifying melon plants comprising the transformed skin penetration sequence of the invention, wherein the method comprises the steps of:

a) Providing a population that is segregating for the transition crust trait,

b) Screening for members exhibiting a transition coat phenotype from an isolated population, wherein the trait can be identified by the presence of a transition coat introgression sequence of the invention,

c) Selecting a member of the isolated population, wherein the member comprises a transition skin trait.

In further embodiments, the present invention provides a method for identifying a cultivated melon plant comprising an introgression sequence on chromosome 6, wherein the introgression sequence confers a transition coat phenotype, the method comprising:

a) Providing a population that is segregating to transition the crust phenotype,

b) Screening the population using a kit for detecting at least one of the SNP markers set forth in Table 4, and

c) Identifying plants comprising the at least one SNP marker selected in the table 4 list.

In further embodiments, the present invention provides a method for identifying additional melon sources for transforming a crust trait on chromosome 6, the method comprising:

a) Providing a melon plant or melon germplasm or melon plants or germplasm,

b) Screening the one melon plant or melon germplasm or a plurality of melon plants or germplasm using a kit for detecting at least one of the SNP markers listed in table 4, and

c) Identifying melon plants or germplasm comprising the at least one SNP marker selected in the table 4 list.

In yet another embodiment, the present invention relates to the use of a SNP marker amplified by the genome of a melon plant according to any of the preceding embodiments, preferably by the genome of melon line 19MNA106815 (representative seed deposited under ATCC accession No. PTA-126875) or progeny or ancestors thereof, wherein the SNP marker is identified using one of the following kits:

a) Forward primer SEQ ID NO. 2 and reverse primer SEQ ID NO. 5, and probe SEQ ID NO. 3;

b) Forward primer SEQ ID NO. 7 and reverse primer SEQ ID NO. 10, and probe SEQ ID NO. 8;

c) Forward primer SEQ ID NO. 12 and reverse primer SEQ ID NO. 15, and probe SEQ ID NO. 13;

d) Forward primer SEQ ID NO. 17 and reverse primer SEQ ID NO. 20, and probe SEQ ID NO. 18;

e) Forward primer SEQ ID NO. 22 and reverse primer SEQ ID NO. 25, and probe SEQ ID NO. 23;

f) Forward primer SEQ ID NO. 27 and reverse primer SEQ ID NO. 30, and probe SEQ ID NO. 28;

g) Forward primer SEQ ID NO. 32 and reverse primer SEQ ID NO. 35, and probe SEQ ID NO. 33;

h) Forward primer SEQ ID NO. 37 and reverse primer SEQ ID NO. 40, and probe SEQ ID NO. 38;

i) Forward primer SEQ ID NO. 42 and reverse primer SEQ ID NO. 45, and probe SEQ ID NO. 43; and/or

j) Forward primer SEQ ID NO. 47 and reverse primer SEQ ID NO. 50, and probe SEQ ID NO. 48;

and wherein the SNP markers indicate the presence of a transformed skin trait in melon plants to identify melon plants that contain and exhibit the transformed skin trait.

In a further embodiment, the present invention relates to a method for assessing the genotype of a cultivated melon (melon) plant, preferably a cultivated melon (cucure melo) plant, more preferably a ground-network melon plant or a rough-skin melon plant, exhibiting a transformed skin phenotype, the method comprising the steps of:

a) Providing a sample from said plant, and

b) Detecting in the sample a QTL locus located on chromosome 6 and associated with the transition sheath phenotype, said QTL locus being flanked by SNP markers 1 and 10 and at least one of the following SNP markers:

x) the genotype C of the heterozygous or homozygous state of SNP marker 10 at a position corresponding to position 196 in SEQ ID NO. 46; and/or

xi) any other DNA markers associated with the QTL loci flanked by SNP markers 1 and 10.

In a further embodiment, the present invention relates to a method of identifying an introgression sequence associated with a transition to the crust phenotype in a cultivated melon (melon) plant, preferably a cultivated melon (cucure melo) plant, more preferably a ground-network melon plant or a coarse-skin melon plant, the method comprising the steps of: detecting in the plant an allele of at least one DNA marker genetically linked to a QTL locus associated with the transition sheath phenotype, wherein the allele is located within 10cM, preferably within 5cM, of the QTL locus located on chromosome 6 in the genomic region flanked by SNP markers 1 and 10.

In an alternative embodiment, the present invention relates to a method of identifying introgression sequences associated with a transition to the crust phenotype in growing melon (melon) plants, preferably melon (cucure melo) plants, more preferably in a mesh melon plant or in a coarse melon plant, the method comprising the steps of: detecting in the plant an allele of at least one DNA marker genetically linked to a QTL locus associated with the transition sheath phenotype, wherein the allele is located between position 25115792bp in public reference genome v CM3.5.1 and position 30769753bp in public reference genome v CM3.5.1 on the QTL locus on chromosome 6 in the genomic region flanked by SNP markers 1 and 10.

In a further embodiment, the present invention relates to a method according to the preceding embodiment, wherein the QTL locus is identifiable by at least one of the following SNP markers:

In a further embodiment, the invention relates to a method according to the preceding embodiment,

wherein the method further comprises the step of selecting a cultivated melon (melon) plant, preferably a cultivated melon (cusumis melo) plant, more preferably a ground network melon plant or a rough skin melon plant, comprising the introgression sequence.

In a further embodiment, the present disclosure relates to a method according to the preceding embodiment, wherein the QTL locus is identifiable by at least one of the following SNP markers

a) A genotype a in heterozygous or homozygous state of SNP marker 1 at a position corresponding to position 25115792bp in public reference genome v CM3.5.1;

b) A G genotype of heterozygous or homozygous state of SNP marker 2 at a position corresponding to position 25507734bp in public reference genome v CM3.5.1;

c) A G genotype of heterozygous or homozygous state of SNP marker 3 at a position corresponding to position 25660237bp in the public reference genome v CM3.5.1;

d) A G genotype of heterozygous or homozygous state of SNP marker 4 at a position corresponding to position 25714720bp in the public reference genome v CM3.5.1;

e) A genotype C in heterozygous or homozygous state of SNP marker 5 at a position corresponding to position 26327861bp in the public reference genome v CM3.5.1;

f) A G genotype of heterozygous or homozygous state of SNP marker 6 at a position corresponding to position 27206596bp in the public reference genome v CM3.5.1;

g) A G genotype of heterozygous or homozygous state of SNP marker 7 at a position corresponding to position 28161883bp in the public reference genome v CM3.5.1;

h) A genotype a in heterozygous or homozygous state of SNP marker 8 at a position corresponding to position 29331322bp in public reference genome v CM3.5.1;

i) A genotype a in heterozygous or homozygous state of SNP marker 9 at a position corresponding to position 30261023bp in public reference genome v CM3.5.1; and/or

j) The genotype C of the heterozygous or homozygous state of the SNP marker 10 at a position corresponding to position 30769753bp in the public reference genome v CM3.5.1.

In a further embodiment, the present invention relates to a method according to the preceding embodiment, wherein the method further comprises the step of selecting a cultivated melon (melon) plant, preferably a cultivated melon (cusumis melo) plant, more preferably a ground network melon plant or a coarse melon plant, comprising the introgression sequence.

In a further embodiment, the present invention relates to a method of identifying cultivated melon (melon) plants, preferably cultivated melon (Cucumis melo) plants, more preferably ground-pattern melon plants or coarse-peel melon plants, exhibiting a transition phenotype by identifying QTL associated with said transition phenotype, the method comprising the steps of:

a) Detecting at least one DNA marker from a melon plant, the DNA marker being linked to a chromosomal interval associated with a transition to the crust phenotype, wherein the chromosomal interval is flanked on both sides by SNP markers having at least 80% sequence identity to SEQ ID NOs 1 and 46; and

b) Identifying the melon plant comprising the at least one DNA marker.

Cultivation and use method.

The invention also relates to a method for reducing waste of fruit consumption during melon plant cultivation and melon fruit harvesting, the method comprising the steps of:

a) Seeding a melon field with seeds grown as melon plants according to any one of the preceding embodiments;

b) The determination as described in example 2 below is preferably used to monitor the change in skin color from green when immature to yellow when full maturity is reached;

c) Fruits were harvested at full maturity and the peel colour of these fruits ranged from 15 to 20 (a-D) when measured using the colour scale of the imperial gardening institute of united kingdom colour scale.

When used in such a way, the plants of the invention allow for an optimal harvest time point when the melon fruit has reached full maturity. Thus, melon fruits enter the market in their best consumption state, limiting the fruit waste observed for immature or over-ripe melon fruits.

The invention further relates to the use of a transformed skin propagation material obtainable from a melon plant according to any one of the preceding embodiments for growing a melon plant to produce a transformed skin melon plant, wherein the transformed skin phenotype can be assessed in a standard assay, in particular as described in example 2 below.

The invention also relates to the use of a transformed skin propagation material obtainable from a melon plant according to any one of the preceding embodiments for producing melon fruits.

In another embodiment, the present invention relates to the use of a cultivated melon (melon) plant, plant part or seed according to any of the preceding embodiments, more preferably a cultivated melon (cucure melo) plant, more preferably a ground network melon plant or a rough skin melon plant, plant part or seed for growing plants and producing and harvesting crops and/or fruits.

In another embodiment, the present invention relates to the use of a cultivated melon (melon) plant, more preferably a cultivated melon (cucure melo) plant, more preferably a ground net melon plant or a coarse skin melon plant according to any of the preceding embodiments for producing fruits for fresh market or for food processing.

In another embodiment, the present invention relates to the use of a cultivated melon (melon) plant, plant part or seed according to any of the preceding embodiments, preferably a cultivated melon (cusumis melo) plant, more preferably a ground net melon plant or a coarse melon plant, plant part or seed, wherein the cultivated melon (melon) plant, plant part or seed, preferably the cultivated melon (cusumis melo) plant, plant part or seed is melon line 19MNA106815 (representative seed of which is deposited under ATCC accession No. PTA-126875) or progeny or ancestor thereof.

In a further embodiment, the present invention relates to the use of a cultivated melon (melon) plant, plant part or seed according to any of the preceding embodiments, more preferably a cultivated melon (Cucumis melo) plant, more preferably a ground-line melon plant or a rough-skin melon plant, plant part or seed for sowing a seed field, a greenhouse or a greenhouse.

In another embodiment, the present invention relates to the use of a cultivated melon (melon) plant, more preferably a cultivated melon (cucure melo) plant, more preferably a ground network melon plant or a coarse skin melon plant according to any of the preceding embodiments for optimizing the harvest time of melon fruits by visually monitoring the peel color indicating complete ripeness of the fruits.

In a further embodiment, the present invention relates to the use of a melon plant according to any one of the preceding embodiments to confer a transformed skin trait on a melon plant lacking said trait. The invention further relates to the use of a melon plant according to any of the preceding embodiments to introgress a transformed skin trait into a melon plant lacking said trait.

In a further embodiment, the invention relates to the use of any one of SEQ ID NOs 1-50 for screening a population of melon plants for the presence of QTL loci located on chromosome 6 and associated with a transition to the crust phenotype.

In further embodiments, the invention relates to the use of any one of SEQ ID NOs 1, 6, 11, 16, 21, 26, 31, 36, 41 or 46 for screening a population of melon plants for the presence of a QTL locus located on chromosome 6 and associated with a transition to the crust phenotype.

Based on the description of the present invention, a skilled person possessing melon line 19MNA106815 (representative seed deposited under ATCC accession No. PTA-126875) or progeny or ancestors thereof (comprising the introgression gene sequence and at least one copy of the LSL10 allele) as described herein, can transfer the introgression gene sequence of the present invention into other various types of melon plants without difficulty using breeding techniques well known in the art with the support of the SNP markers disclosed herein.

Seed preservation details

Applicant has deposited at least 625 melon hybrid plant 19MNA106815 seeds with ATCC accession No. PTA-126875 at 10/19 in 2020 (american type culture collection (American Type Culture Collection, ATCC), university of 10801, ma, usa).

The applicant selects an expert solution and requires that only deposited material be released to the expert according to the corresponding laws and regulations of EPC clause 32 (1) or other countries or treaties (expert witness clauses) until the mention publication of patent authority or 20 years from the filing date if the application is refused, withdrawn or deemed withdrawn.

The LSL10 allele of the melon hybrid plant 19MNA106815 and the transition coat QTL on chromosome 6 are heterozygous, i.e. the line 19MNA106815 comprises one copy of the LSL10 allele and one copy of the introgression sequence from melon located on chromosome 6 and associated with the transition coat phenotype.

Examples

Example 1: germplasm and population development

Example 1A. Donor development.

Crosses were made between melon plants (which were transformed at maturity) and melon plants of the reticulate pattern ("09 MSP 006888") which were not transformed at maturity, to obtain F1 plants identified as "10MSP 002234". F1 plants were selfed to produce a population of F2 referred to herein as "12MSP 005549". Using the single pass method, 200F 2 plants were sown and advanced to the F3 family. In the field of california, the F3 family is assessed by phenotypic assessment of transition sheath traits. Self-pollination is used to advance the best F3 family, referred to herein as "13MSP005602", to the F4 generation. Optimal F4 progeny are selected based on optimal and stable trait expression. The transition skin trait was measured with a Royal gardening society of England color card and the phenotypic expression of the transition skin trait corresponds to a 15-20 (A-D) color range on the color card. An F4 strain was found to be stable for this trait and was designated "14MNA106599". This melon line of reticulate pattern was used as a donor to transform an elite parent line with the transformed crust trait.

Example 1B. Genetic mapping transformed population development of the crust trait.

The F2-localized population was developed using the reticulate melon F3 line designated "13MSP005602" (see example 1A) and the coarse-skin melon long shelf-life LSL10 inbred line. The F2 population was designated as "14CME-BWS3". 800F 2 individuals were sown in the field of California and phenotypically assessed for expression of the transition coat trait in the long shelf life LSL10 background.

Example 2: scheme (1).

Example 2A. Evaluation and scoring of the transition coat trait phenotype.

The outer skin of the fully ripe fruit remains grayish green to green in the normal long shelf life LSL10 melon background. This corresponds to the color range of 135-143 (A-D) when using the Royal gardening society color chart (Cuevas et al, 2010). Ripening of the fruit is monitored by a change in color from green to yellow of a long shelf life LSL10 melon plant comprising at least one copy of QTL 6. In the latter plants, the fruit color turns yellow upon ripening, the yellow range corresponding to 15-20 (A-D) using the Royal gardening institute color chart. The number of days to reach full maturity (and observe the associated skin color changes) typically varies from 82 days to 85 days, depending on the normal daily environmental conditions in the summer state of california.

Example 2B. Evaluation of long shelf life phenotype.

Assessing the ability of cantaloupe long shelf life LSL10 cantaloupe comprising at least one copy of QTL6 to produce fruits with preserved long shelf life characteristics at maturity. At maturity, no delamination forms at the pedicel attachment, so the fruit does not slip off the vine.

Example 2℃ Methods of identifying QTL's for transforming the skin trait and corresponding introgression sequences.

For QTL discovery, 800F 2 individuals of the "14CME-BWS3" population were pre-screened using the LSL10 allele markers (see example 6), and 372F 2 individuals containing at least one copy of the LSL10 locus were selected and periodically genotyped using 150 gene markers across the genome. Genetic maps were constructed and transformed skin phenotype data from 372 selected individuals were used for QTL detection.

QTL detection was performed using the R/QTL package in the R statistics framework. First, the genotype probability was calculated using the function `calc. Genolab` (step 1 cM). Haley-Knott regression was performed to provide an approximation of standard interval mapping results. Then, a function 'stepwiseqtl' is called, which provides a fully automated model selection forward/backward algorithm. The LOD threshold for the main effect is determined by 10,000 permutations. The algorithm takes into account the different possible interactions (e.g., superordinate). The function 'refiniqtl' is used to refine the QTL position in the context of a multiple QTL model (maximum likelihood estimation). The function 'fitqtl' is used to fit a defined QTL model and to obtain an estimate of QTL effects.

Example 3: identification of QTL associated with transition crust phenotype

Example 3A effect of QTL located on chromosome 6 on the transition to the crust at maturity.

One QTL was identified based on the transformed crust phenotype of 372 selected F2 individuals from the "14CME-BWS3" population. Table 1 shows the chromosomal location, the effect of the QTL measured as LOD score, and the percent variation of the transition sheath phenotype explained by the QTL on chromosome 6.

Table 1:important QTLs associated with a transition to the crust phenotype.

Chromosome of the human body	LOD	％var	P value (Chi 2)
				6	25.85	23.9	<0.001

"LOD" =log-likelihood score, "% var" =percent phenotypic variation explained by QTL, "P value (Chi 2)" =probability of QTL detection by Chi-square analysis random chance.

QTL showed additive or semi-dominant effects in the "14CME-BWS3" discovery population. The presence of only one copy of the donor allele at the QTL location shows a moderate transition crust phenotype compared to when two homozygous parent alleles are present.

Example 4: penetration of sequences conferring transformed outer skin into the LSL10 cantaloupe background

The reticulate melon LSL10 cantaloupe background does not change at maturity, whereas the melon peel changes color at maturity. The genetic sequences associated with the transition to the crust phenotype present in melon plants were introgressed into LSL10 cantaloupe breeding material by selecting plants as described in example 2 and backcrossing them into the LSL10 cantaloupe breeding line.

Advanced breeding lines highlight phenotypes similar to recurrent parents in terms of long shelf life and non-slip characteristics, while containing advantageous introgression sequences that shift the crust phenotype. The phenotypic results and the test results for the presence or absence of a representative marker in QTL6 and LSL10 alleles are summarized in table 2 below.

Table 2: whether or not the characteristic SNP markers of the QTL6 and the LSL10 are present and the corresponding phenotypes.

Existing commercial hybrids (Sweet Spring) and recurrent LSL10 cantaloupe recurrent parents (materials 3 and 4) are non-jumping, non-sliding and non-crust-changing. They carry only at least one copy of the LSL10 allele and their melon peel color remains green at maturity (RHS range 135 to 143 (a-D)). In contrast, all transformed cantaloupe lines (materials 5 to 10) comprising QTL6 and LSL10 alleles show a transformed crust phenotype (RHS range 15 to 20 (a-D)), while retaining the long shelf life and non-slip characteristics of the mature melon fruits. Furthermore, even though the experimental hybrids (including the deposited materials, materials 2, 11 and 12) contained only one copy of QTL6, they exhibited a transitional crust phenotype (RHS range 15 to 20 (a-D)). That is, all lines and hybrids comprising at least one copy of QTL6 (characterized by SNP markers spanning the gene interval 64.8-73.7 cM) and at least one copy of LSL10 SNP markers exhibit a transition-coat phenotype while retaining the long shelf-life and non-slip characteristics of melon fruits.

Within this region, ten SNPs within the QTL interval (SE 3980, SE3992, SE4033, SE4031, SE3981, SE4009, SE3978, SE3987, SE3729 and SE 3982) showed specificity for the selection of donor transition coat alleles and wherein the SNP markers SE4033, SE4031, SE3981, SE4009, SE3978, SE3987, SE3729 were most closely linked to the QTL. Table 3 below shows the positions of ten SNP markers closely linked to the transition-coat (RT) QTL allele, both the gene and the physical position of QTL 6.

TABLE 3 genetic map of QTL on chromosome 6

Example 5: sequence of QTL6 and SNP marker information

Sequence information of the SNP markers SE3980, SE3992, SE4033, SE4031, SE3981, SE4009, SE3978, SE3987, SE3729 and SE3982, and their respective PCR primers/probes (for detection) are summarized in table 4 below.

Table 4.

As an example, SNP marker 1 (SE 3980) at position 24564648bp/25225792bp on chromosome 6 (based on the reference PIT92 v7 sequence or the public genome version CM3.5.1, respectively) is characterized by a specific SNP marker (melon smelling donor relative to acceptor allele) at position 61 of the target sequence of SEQ ID NO: 1. Corresponding forward and reverse primers of SEQ ID NOs 2 and 5 are also disclosed, as are probes specific for the donor or acceptor alleles of SEQ ID NOs 3 and 4.

Example 6:LSL10allele sequence and SNP marker information

Genetic map information and sequence information of the SNP marker SE1787, as well as their respective PCR primers/probes (for detection) are summarized in tables 5 and 6 below. This marker can be used to detect the LSL10 allele when infiltrated into the background of a coarse-skinned melon or a textured melon. It may be from the deposited material 19MNA106815, or from other existing publicly available sources, such as PI 420176 (Perpina et al 2017).

TABLE 5.10 chromosomeLSL10Genetic map of alleles.

Table 6.

Bibliography of documents

·Cuevas et al.(2010),Euphytica 173:129-140.

·Pech et al.(2008),Plant Science 175:114-120.

·Perpina et al.(2017),HortScience 52(11):1633-1638.

·Pitrat et al.(2000),Proc.Cucurbitaceae 2000,Eds N.Katzir&H.S.Pqris,Acta Hort.510。

Sequence listing

<110> Xianzhengda crop protection Co., ltd (Syngenta Crop Protection AG)

Kumar, Rakesh

Oliver, Marc

Alvarez, Ignacio

<120> novel melon plants with long shelf life

<130> 82194-US-L

<160> 55

<170> patent In version 3.5

<210> 1

<211> 179

<212> DNA

<213> melon (Cucumis melo)

<220>

<221> feature not yet classified

<222> (167)..(167)

<223> n is a, c, g or t

<400> 1

taggagcaag ttcacacgat cctctcgtcc aacctatgtc actattaaat gcacctacat 60

agaaagacca aacaatttct aaagttagaa ggccaacccc aaatcccgta cgtcaagatg 120

aaaggaatca tcaacattcg caagaaggta acgattcata tccaganaaa cctcaaata 179

<210> 2

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 2

cctctcgtcc aacctatgtc act 23

<210> 3

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 3

atgcacctac atagaaagac c 21

<210> 4

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 4

atgcacctac atggaaagac 20

<210> 5

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 5

atgaatcgtt accttcttgc gaa 23

<210> 6

<211> 135

<212> DNA

<213> melon (Cucumis melo)

<400> 6

caagcccagt gcatgaaaga gaattgcggc tttggctagt gacattgatt ctacgactgt 60

gtagggtgac gtgtttgtaa tgggatgaag atcaacaaac atttccagct cctcctcgtt 120

gaattccaaa tcttc 135

<210> 7

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 7

gaattgcggc tttggctagt g 21

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 8

cattgattct acgactgtgt 20

<210> 9

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 9

cattgattct acaactgtgt a 21

<210> 10

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 10

ggaggagctg gaaatgtttg ttg 23

<210> 11

<211> 168

<212> DNA

<213> melon (Cucumis melo)

<400> 11

cctaaagcac tgagatattc ttggtacaca aggaaaacgt tagcctccca agtcccatct 60

atgtgataac aaacacaaga gatatggctg cttaattata actatgggat tgataatggt 120

gttttgttcg ttaaagagat gatccaccac cataagttcc aaagagaa 168

<210> 12

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 12

ttggtacaca aggaaaacgt tagc 24

<210> 13

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 13

tcccatctat gtgataaca 19

<210> 14

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 14

aagtcccatc tatgtaataa caa 23

<210> 15

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 15

ggtggatcat ctctttaacg aaca 24

<210> 16

<211> 122

<212> DNA

<213> melon (Cucumis melo)

<400> 16

gcctgagctc gaaatcccag cattgcctgg cgcacaaaac acagcatcgc aggaaggaga 60

gcgctgcaat gcatagcaaa gagcatgttc tcgccctcct ccaccaatca ccaatacaat 120

aa 122

<210> 17

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 17

cattgcctgg cgcacaaa 18

<210> 18

<211> 16

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 18

cacagcatcg caggaa 16

<210> 19

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 19

acacagcatc acaggaagg 19

<210> 20

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 20

ggaggagggc gagaacat 18

<210> 21

<211> 146

<212> DNA

<213> melon (Cucumis melo)

<220>

<221> feature not yet classified

<222> (19)..(19)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (43)..(43)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (130)..(130)

<223> n is a, c, g or t

<400> 21

tctgatagtc atttgatant tatgtgattg ctatttgatt gantttttaa accttaaggg 60

caagggtaat tgcaaatttg tcaaaaacat ttgaaaaaat taggcccata gcccaaagtt 120

tgcttttttn cgacaaaatt agaaat 146

<210> 22

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 22

tatgtgattg ctatttgatt ga 22

<210> 23

<211> 14

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 23

cccttgccct taag 14

<210> 24

<211> 14

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 24

cccttgccct taag 14

<210> 25

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 25

aaagcaaact ttgggctatg 20

<210> 26

<211> 164

<212> DNA

<213> melon (Cucumis melo)

<220>

<221> feature not yet classified

<222> (20)..(20)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (102)..(102)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (110)..(110)

<223> n is a, c, g or t

<400> 26

cccttgctgc atatccagcn gccactccac ctccaagaat caaatatttg aaacagttgg 60

gaaaaaagta gtttccttct tcgagtagct cttttgcctc ancaatcttn tctactacag 120

tccttccata aagataaaca gccactgtga aaagcttcca tttt 164

<210> 27

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 27

gccactccac ctccaagaat c 21

<210> 28

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 28

caaaagagct actcgaagaa gg 22

<210> 29

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 29

aggcaaaaga gctacttgaa ga 22

<210> 30

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 30

tggctgttta tctttatgga aggac 25

<210> 31

<211> 214

<212> DNA

<213> melon (Cucumis melo)

<220>

<221> feature not yet classified

<222> (13)..(14)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (45)..(45)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (73)..(73)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (212)..(212)

<223> n is a, c, g or t

<400> 31

cattattatc ccnngaggtt gtggatgatt gaaattgatt gtaanaacta ttttgaatat 60

atagcttatt ttnttagcat ttgttatgta gttataagat atgaactttg atatgatttt 120

tatctttgtt ttctttattt tttagttgta ccctcttaag gaagagatag ttctttcatt 180

ctcacagagc tcagataaat ttccttttta tntt 214

<210> 32

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 32

gtggatgatt gaaattgatt g 21

<210> 33

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 33

tatgtagtta taagatatga ac 22

<210> 34

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 34

agttataaga tatcaacttt g 21

<210> 35

<211> 18

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 35

ctgagctctg tgagaatg 18

<210> 36

<211> 180

<212> DNA

<213> melon (Cucumis melo)

<220>

<221> feature not yet classified

<222> (135)..(135)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (174)..(178)

<223> n is a, c, g or t

<400> 36

aagtcgctat ttttctaaag tggatgaaca tctgaagtca aggatacaac gatctcaata 60

cagaagagaa aactatagct ttcagatcca tgaacaaaac agaaaacaaa agcaaacggt 120

tatcaattgg catcnagtcc ctgtctttcc attgccctgt agcaaaaaaa aaannnnnga 180

<210> 37

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 37

tggatgaaca tctgaagtca agga 24

<210> 38

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 38

tgataaccgt ttgcttttg 19

<210> 39

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 39

ttgataaccg tgtgctttt 19

<210> 40

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 40

acagggcaat ggaaagacag g 21

<210> 41

<211> 150

<212> DNA

<213> melon (Cucumis melo)

<220>

<221> feature not yet classified

<222> (52)..(52)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (61)..(61)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (109)..(109)

<223> n is a, c, g or t

<400> 41

agatgaatct gtaagagaat tacaaagata gaaaacgagt ttagaaaaat gntagtccta 60

natatgttta agtaactagc atggtcattt taaaaatagt tcacgtacng tgagaggttg 120

gcagtggatg actgtctggg aagcaaaaga 150

<210> 42

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 42

tacaaagata gaaaacgagt ttag 24

<210> 43

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 43

agtaactagc atggtcatt 19

<210> 44

<211> 16

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 44

taactagcat ggtcgt 16

<210> 45

<211> 16

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 45

catccactgc caacct 16

<210> 46

<211> 254

<212> DNA

<213> melon (Cucumis melo)

<220>

<221> feature not yet classified

<222> (4)..(6)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (87)..(87)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (98)..(98)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (126)..(126)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (128)..(128)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (138)..(138)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (173)..(173)

<223> n is a, c, g or t

<220>

<221> feature not yet classified

<222> (175)..(175)

<223> n is a, c, g or t

<400> 46

aagnnntttt ttttgtctta gacagttaga gatagattgc tatctttgtc tatccaatag 60

ataaagatat caatttattt gttcctntct cagatagnca ttgatagatt gatatctata 120

tatatntnat gtatacanag attgaaatct atttcttctt attttattaa ttntncttat 180

ttgtgttatg tattacgttg atccaataag tacaactgaa taccaagtaa cggatggaaa 240

ttaacagttt attg 254

<210> 47

<211> 22

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 47

gacagttaga gatagattgc ta 22

<210> 48

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 48

acttattgga tcaacgtaa 19

<210> 49

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 49

acttattgga tcaacctaa 19

<210> 50

<211> 19

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 50

tccgttactt ggtattcag 19

<210> 51

<211> 214

<212> DNA

<213> melon (Cucumis melo)

<220>

<221> feature not yet classified

<222> (46)..(46)

<223> n is a, c, g or t

<400> 51

atttataggc catgttaggt caactcagcc ccttacttct acaagnagaa ggaaccatct 60

ctcttttact agtttttaat tcagattgat caccatcatc aagccagaga tgaatatcat 120

ctggcaaatt cgttgacaga aatgagaaaa atatatcatg tttatataga agaacgaatg 180

tccttacata atggcccctt aggttatgcc tcaa 214

<210> 52

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 52

caactcagcc ccttacttct acaag 25

<210> 53

<211> 16

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 53

tcatcaagcc agagat 16

<210> 54

<211> 17

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 54

catcaagcca tagatga 17

<210> 55

<211> 26

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis

<400> 55

ccattatgta aggacattcg ttcttc 26

PCT/RO/134 table

Claims

1. A melon plant, preferably a cantaloupe plant, more preferably a ground-network melon plant or a rough-skin melon plant, comprising in its genome:

a) At least one copy of the LSL10 allele, and;

b) At least one copy of an introgression sequence from a melon, said introgression sequence being associated with a transition to the crust phenotype, located on chromosome 6 and comprising at least one of the following SNP markers:

2. The plant of claim 1, wherein the transition sheath phenotype is characterized by a sheath color changing from green when immature to yellow when full maturity is reached.

3. The plant of claim 2, wherein the melon peel has a yellow color at full maturity ranging from 15 to 20 (a-D) when measured using the color scale of the imperial gardening institute color chart of uk.

4. A plant according to claim 3 wherein the melon peel has a yellow colour of 19A or 19B at full maturity when measured using the colour scale of the imperial gardening institute colour chart of uk.

5. The plant of any one of claims 1 to 4, wherein the green color of the immature melon peel ranges from 135 to 143 (a-D) when measured using the color scale of the uk's royalty gardening institute color chart.

6. The plant of any one of claims 1 to 5, wherein the green color of the immature melon peel is 138C, 138D or 139A when measured using the color scale of the uk royal gardening institute color chart.

7. The plant of any one of claims 1 to 6, wherein the transformed skin phenotype is assessed as disclosed in example 2A.

8. The plant of any one of claims 1 to 7, wherein

i) The A genotype of SNP marker 1 can be determined in PCR by using oligonucleotide primer pairs: forward primer SEQ ID NO. 2 and reverse primer SEQ ID NO. 5, and probe SEQ ID NO. 3.

ii) the G genotype of SNP marker 2 can be determined in PCR by using oligonucleotide primer pairs: amplifying the nucleic acid fragments with the forward primer SEQ ID NO. 7 and the reverse primer SEQ ID NO. 10 and the probe SEQ ID NO. 8 for identification;

iii) The G genotype of SNP marker 3 can be determined in PCR by using oligonucleotide primer pairs: the forward primer SEQ ID NO. 12 and the reverse primer SEQ ID NO. 15 and the probe SEQ ID NO. 13 are used for amplifying the nucleic acid fragments for identification;

iv) the G genotype of SNP marker 4 can be determined in PCR by using oligonucleotide primer pairs: forward primer SEQ ID NO. 17 and reverse primer SEQ ID NO. 20, and probe SEQ ID NO. 18.

v) the genotype C of SNP marker 5 can be detected in PCR by using oligonucleotide primer pairs: forward primer SEQ ID NO. 22 and reverse primer SEQ ID NO. 25, and probe SEQ ID NO. 23.

vi) the G genotype of SNP marker 6 can be determined in PCR by using oligonucleotide primer pairs: forward primer SEQ ID NO. 27 and reverse primer SEQ ID NO. 30, and probe SEQ ID NO. 28.

vii) the G genotype of SNP marker 7 can be determined in PCR by using oligonucleotide primer pairs: forward primer SEQ ID NO. 32 and reverse primer SEQ ID NO. 35, and probe SEQ ID NO. 33.

viii) genotype a of the SNP marker 8 can be determined in PCR by using oligonucleotide primer pairs: forward primer SEQ ID NO. 37 and reverse primer SEQ ID NO. 40, and probe SEQ ID NO. 38.

ix) the A genotype of SNP marker 9 can be determined in PCR by using oligonucleotide primer pairs: forward primer SEQ ID NO. 42 and reverse primer SEQ ID NO. 45, and probe SEQ ID NO. 43. And/or

x) the genotype C of the SNP marker 10 can be detected in PCR by using oligonucleotide primer pairs: the forward primer SEQ ID NO. 47 and the reverse primer SEQ ID NO. 50 and the probe SEQ ID NO. 48 amplified nucleic acid fragments.

9. The plant of any one of claims 1 to 8, wherein the introgression sequence comprises at least one of SEQ ID No. 1, SEQ ID No. 6, SEQ ID No. 11, SEQ ID No. 16, SEQ ID No. 21, SEQ ID No. 26, SEQ ID No. 31, SEQ ID No. 36, SEQ ID No. 41 and/or SEQ ID No. 46, or a sequence having at least 80% identity to one or more of said sequences.

10. The plant according to any one of claims 1 to 9, wherein the LSL10 allele comprises the following SNP markers:

11. The plant of claim 10, wherein

a) The G genotype of SNP marker 11 can be determined in PCR by using oligonucleotide primer pairs: the forward primer SEQ ID NO. 52 and the reverse primer SEQ ID NO. 55, and probe SEQ ID NO. 53 amplified nucleic acid fragments.

12. The plant of any one of claims 10 to 11, wherein the LSL10 allele comprises SEQ ID No. 51, or a sequence having at least 80% identity to said sequence.

13. The plant of any one of claims 1 to 12, wherein the LSL10 allele of the plant and/or the introgression sequence located on chromosome 6 is homozygous.

14. The plant according to any one of claims 1 to 13, wherein said LSL10 allele and said introgression sequence are comprised in melon line 19MNA106815, or progeny or ancestor thereof, a representative seed of said melon line 19MNA106815 deposited under ATCC accession No. PTA-126875.

15. The plant according to any one of claims 1 to 14, wherein said plant is obtained by crossing melon line 19MNA106815, or a progeny or ancestor thereof, with a melon plant that does not contain said introgression sequence, a representative seed of said melon line 19MNA106815 being deposited under ATCC accession No. PTA-126875.

16. The plant of any one of claims 1 to 15, wherein the plant is an inbred, doubled haploid or hybrid plant.

17. A plant of melon line 19MNA106815, representative seed of which is deposited under ATCC accession No. PTA-126875.

18. A plant part of a plant according to any one of claims 1 to 17.

19. A seed producing the plant or plant part of any one of claims 1 to 18.

20. A method for producing a cultivated melon plant, preferably a hami melon plant, more preferably a ground-network melon plant or a coarse-skin melon plant, wherein the plant produces melon fruits exhibiting a long shelf-life phenotype and a transition to a rind phenotype upon reaching full maturity, wherein the method comprises the steps of:

a) Crossing the plant of any one of claims 1 to 17 with a cultivated melon plant lacking the LSL10 allele and the introgression sequence;

thereby producing a plant that produces fruits having a long shelf life phenotype that transition to the rind phenotype upon reaching full maturity.

21. The method of claim 20, wherein the method further comprises:

22. The method of claim 21, wherein additional progeny are selected and selfed/crossed for 2 to 10 generations.

23. The method according to any one of claims 20 to 22, wherein the plant of step a) is melon line 19MNA106815 or a progeny or ancestor thereof, the representative seed of melon line 19MNA106815 being deposited under ATCC accession No. PTA-126875.

24. A method for producing an F1 melon plant exhibiting a transitional crust phenotype, the method comprising crossing an inbred melon plant that is a plant according to any one of claims 1 to 17 with a different inbred melon plant to produce an F1 hybrid progeny.

25. A method for identifying melon fruits that exhibit a long shelf life phenotype and that change the crust phenotype upon reaching full maturity, wherein the plant comprises at least one copy of the LSL10 allele and at least one copy of the introgression sequence from a melon located on chromosome 6, wherein the method comprises the steps of: detecting at least one of the following SNP markers:

26. The method of claim 25, wherein the method further comprises the step of detecting SNP markers as follows:

27. The method of any one of claims 25 or 26, wherein the method further comprises selecting a melon plant comprising the one or more SNP markers, and crossing the selected melon plant with a second melon plant to produce a progeny melon plant comprising at least one of the SNP markers and producing fruits having a long shelf-life phenotype and a transition to a crust phenotype upon reaching full maturity.

28. A method of producing melon seeds, the method comprising growing a melon plant from the seed of claim 19, and allowing the plant to produce additional melon seeds.

29. A method for assessing the genotype of a cultivated melon plant, preferably a cantaloupe plant, more preferably a ground-network melon plant or a coarse-skin melon plant, wherein the plant produces melon fruits exhibiting a long shelf-life phenotype and a transition to a crust phenotype upon reaching full maturity, the method comprising the steps of:

a) Providing a sample from said plant, and

b) Detecting in the sample QTL loci located on chromosome 6 that are associated with the long shelf life phenotype and the transition crust phenotype, flanked by SNP markers 1 and 10, and at least one of the following SNP markers:

30. A method of identifying an introgression sequence associated with a transition to a crust phenotype upon reaching full maturity in a cultivated melon plant, preferably a cantaloupe plant, more preferably a ground-network melon plant or a coarse-skin melon plant, the method comprising the steps of: detecting in the plant an allele of at least one DNA marker genetically linked to a QTL locus associated with the transition sheath phenotype, wherein the allele is located within 10cM, preferably within 5cM, of the QTL locus located on chromosome 6 in the genomic region flanked by SNP markers 1 and 10.

31. The method of claim 30, wherein the QTL locus is identifiable by at least one of the following SNP markers

32. The method according to claim 31, further comprising the step of selecting a cultivated melon plant, preferably a cantaloupe plant, more preferably a ground network melon plant or a coarse-peel melon plant, comprising the introgression sequence.

33. A method of identifying cultivated melon plants, preferably cultivated cantaloupe plants, more preferably ground-network melon plants or rough-skinned melon plants, exhibiting a long shelf-life phenotype and said transformed skin phenotype by identifying QTL associated with the transformed skin phenotype, comprising the steps of:

a) Detecting at least one DNA marker from a melon plant, said DNA marker being linked to a chromosomal interval associated with a transition to the crust phenotype, wherein said chromosomal interval is flanked on both sides by SNP markers having at least 80% sequence identity to SEQ ID nos. 1 and 46; and

b) Identifying the melon plant comprising the at least one DNA marker.

34. A method of identifying a melon source on chromosome 6 that converts a crust trait, the method comprising:

a) There is provided one or more melon materials,

b) Screening the one or more melon materials using a kit for detecting at least one of the SNP markers set forth in Table 4, and

c) Identifying the wild melon germplasm comprising the at least one SNP marker selected in the table 4 list.

Use of any one of seq ID NOs 1-50 for screening a population of melon plants for the presence of a QTL locus located on chromosome 6 and associated with a transition to the crust phenotype.