Identification of hydrangea macrophylla plants producing phyllodulcin and phyllodulcin precursors

Abstract

The present invention relates to methods for the marker-assisted identification of plants producing phyllodulcin and / or its precursors hydrangenol and thunberginol G. Furthermore, the present invention provides oligonucleotides for the identification of such QTLs as well as transgenic plants comprising the QTLs associated with phyllodulcin, hydrangenol and / or thunberginol G production.

Description

[0001] Eisenfuhr Speiser

[0002] Munich, 24 January 2025

[0003] Our Ref.: SM 7035-01 WO SOE / HGR

[0004] Applicant: Symrise AG

[0005] Serial Number: New Application

[0006] Symrise AG

[0007] MiihlenfeldstraBe 1, 37603 Holzminden, Germany

[0008] Identification of Hydrangea macrophylla plants producing phyllodulcin and phyllodulcin precursors

[0009] The present invention relates to methods for the marker-assisted identification of plants producing phyllodulcin and / or its precursors hydrangenol and thunberginol G. Furthermore, the present invention provides oligonucleotides for the identification of such QTLs as well as transgenic plants comprising the QTLs associated with phyllodulcin, hydrangenol and / or 5 thunberginol G production.

[0010] Consumers generally have a strong preference for foodstuffs or indulgence foods, which have a large amount of high caloric sugar, in particular sucrose (saccharose), glucose, fructose or mixtures thereof, due to the pleasant sweetness and sweetness profile associated therewith. On the other hand, it is generally known that a large content of readily 0 metabolizable carbohydrates causes a steep rise in blood sugar levels, leads to the formation of fat deposits and ultimately can result in health problems such as overweight, obesity, insulin resistance, age-onset diabetes and complications thereof. Another particular aggravating factor is that many of the above-mentioned carbohydrates can also have an adverse effect on dental health, as they are decomposed by specific types of bacteria in the oral cavity into lactic acid, for example, and can attack the enamel of milk teeth or adult teeth (caries).

[0011] Therefore, it has long been an objective to reduce the high caloric sugar content of food or beverage products and replace it partly or entirely by other substances that impart a sweet taste or which can positively affect the sweet taste in a low concentration without exhibiting 0 sweet taste itself at these low concentration (taste modulators).

[0012] The use of phyllodulcin as taste modulator for sweetener-reduced products, flavoring mixtures for same, and method of producing such products was described in EP 2,298,084-B1.

[0013] Phyllodulcin is a natural compound occurring exclusively in Hydrangea macrophylla. Phyllodulcin cannot be economically produced by chemical synthesis or biotechnological approaches.

[0014] The leaves of Hydrangea macrophylla are mainly used for preparing tea, particularly Amacha, a sweet tasting Japanese tea, which contains tannins and dihydroisocoumarins including phyllodulcin. The leaves of Hydrangea macrophylla are typically used in Japan and Korea for ceremonial purposes (Buddhas birthday). The phyllodulcin content could reach 0.03% of leaf fresh weight and ranges between 0.9 and 4.4% in dried, fermented leaves (Ujihara, M., et al. (1995). "Accumulation of Phyllodulcin in Sweet-Leaf Plants of Hydrangea serrata and Its Neutrality in the Defence Against a Specialist Leafmining Herbivore." Res. Popul. Ecol. 37(2): 249-257).

[0015] The climate conditions between the origin of Hydrangea macrophylla in Southeast Asia and Europe differ significantly. Nevertheless, some Hydrangea macrophylla plants have been bred to be cultivated as horticultural plants in Europe. These plants are used as decorative plants only and most of them do not exhibit any phyllodulcin presence. Commercial cultivation of Amacha plants of Hydrangea macrophylla outside Japan and Korea is not known to date. A method for the cultivation of Hydrangea macrophylla in the greenhouse is described in WO2023 / 078553.

[0016] It is known that not all plants of Hydrangea macrophylla produce phyllodulcin (Wellmann et al. (2022), Comprehensive Metabolite Profiling of Hydrangea macrophylla ssp. serrata Extracts Using Liquid Chromatography Coupled with Electrospray Ionization Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry, J. Agric. Food Chem. 2002, 70, 11823-11831) and that in general the production of phyllodulcin as well as its precursors is highly dependent on the plant genotype and less pronounced by environmental conditions. It was found, that wild plants reproduced by free pollination have a phyllodulcin content, which varies from plant to plant (Ujihara, M., et al. (1995). "Accumulation of Phyllodulcin in Sweet-Leaf Plants of Hydrangea serrata and Its Neutrality in the Defence Against a Specialist Leafmining Herbivore." Res. Popul. Ecol. 37(2): 249-257). It is thus required to identify varieties for breeding, which have appropriated alleles at the QTL controlling phyllodulcin biosynthesis, and for selecting such favorable plants for a breeding program to yield plantsthat have a robust high production of phyllodulcin for the industrial extraction of phyllodulcin.

[0017] A first attempt for developing trait-associated markers for Hydrangea macrophylla was done by Waki et al. (2018) “Development of DNA markers linked to double-flower and hortensia traits in Hydrangea macrophylla (Thunb.) Ser", The Horticulture Journal, 87, 164-273. This scientific paper focuses on the development of markers for the traits doubleflowering and the inflorescence type, but does not provide markers associated with phyllodulcin, hydrangenol and / or thunberginol G.

[0018] It was thus the primary object of the present invention to provide methods for the identification of a plant of the species Hydrangea macrophylla. Further objects of the present invention are visible to the person skilled in the art by the following detailed description as well as the claims.

[0019] The primary object of the present invention was solved by providing a method for identifying a plant of the species Hydrangea macrophylla comprising a chromosomal section associated with production of the metabolites phyllodulcin, hydrangenol and thunberginol G, which comprises the following steps:

[0020] 1) Providing a plant or plant part of the species Hydrangea macrophylla;

[0021] 2) Using one or more markers to identify a genomic region between and including

[0022] i) markers HS127 and AF195, especially preferable between and including markers HS127 and AF006 in linkage group 15, and / or

[0023] ii) markers BZ063 and BZ095, preferably between and including markers AF102 and HS272 and especially preferable between and including markers AF086 and HS272 in linkage group 16, and / or

[0024] iii) markers AF209 and AF181 , preferably between and including markers AF184 and HS602 and especially preferable between and including markers BZ019b and BZ052 in linkage group 6, and / or

[0025] iv) markers BZ040 and AF188, preferably between and including markers BZ094 and BZ003, in linkage group 12, and / orv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205 and especially preferably between and including markers AF343 and AF004, in linkage group 16.

[0026] The method according to the present invention provides marker for identifying QTLs, which are associated with the overall production of hydrangenol, thunberginol G and phyllodulcin. All of these three metabolites are members of the same pathway, wherein hydrangenol can be reacted to thunberginol G and is then reacted to phyllodulcin. The metabolites are present as glycosides in the plant. Whenever the present application refers to hydrangenol, thunberginol G and phyllodulcin, also the glycosides are meant. Preferably, the markers linked to these QTL are associated with an increased production of at least one of the metabolites hydrangenol, thunberginol G and phyllodulcin.

[0027] It was discovered in terms of the present invention that certain “linkage groups” exist, which comprise the QTLs for the production of the sum of hydrangenol, thunberginol G and phyllodulcin. These “linkage groups” are chromosomal or genomic intervals in which the QTLs associated with the metabolite expression of the sum of hydrangenol, thunberginol G and phyllodulcin are present. The markers according to the present invention can be used for identifying those QTLs with the method according to the invention.

[0028] A “QTL” or “quantitative trait locus” describes a specific region of the genome associated with a quantitative trait — one that varies continuously, like height, weight or metabolite expression. These traits are typically influenced by multiple genes, making them complex and often polygenic. In terms of the present invention, it was possible to identify the QTLs associated with the sum of the dihydroisocoumarins hydrangenol, thunberginol G and phyllodulcin as well as for the production of phyllodulcin alone. Furthermore, the inventors have developed suitable markers which are linked with QTLs associated with the sum of the dihydroisocoumarins hydrangenol, thunberginol G and phyllodulcin as well as for the production of phyllodulcin alone and making them available for commercial breeding or genetic engineering.

[0029] The terms “chromosomal fragment”, “chromosomal interval” and “chromosomal segment” are used interchangeably unless otherwise specified and refer to a specific chromosomal DNA segment of a particular chromosome comprising at least one gene. An integrated chromosomal fragment originates from a donor source. For the purposes of the invention, the sequential order of the genes within an integrated chromosome fragment may correspond to the order present in the original chromosome fragment of the donor source.A chromosome fragment or a part thereof may represent a specific “haplotype”, the chromosome fragment then having certain sequence polymorphisms such as SNPs (single nucleotide polymorphisms) or InDeis (insertion / deletions) by which the haplotype is also uniquely specified and can be identified.

[0030] In terms of the present invention, it is needed that at least one marker in the area between the specified markers is identified. However, also the end points of the range between the specified markers, are suitable for identifying the QTL of interest associated with the production of hydrangenol, thunberginol G and phyllodulcin.

[0031] The term “marker” refers to a nucleotide sequence that is used as a reference or landmark. A marker for detecting a recombination event should be capable of monitoring differences or polymorphisms within a plant population. For markers, these differences are found at the DNA level and are, for example, polynucleotide sequence differences such as SSRs (simple sequence repeats), RFLPs (restriction fragment length polymorphisms), FLPs (fragment length polymorphisms) or SNPs. Markers may be derived from genomic or expressed nucleic acids such as spliced RNA, cDNA or ESTs and may also refer to nucleic acids that are used as probes or primer pairs and as such are capable of amplifying a sequence fragment using PCR-based methods.

[0032] Preferably, at least one, two, three, four or more markers from the following group can be used in terms of the present invention for identifying a plant of the species Hydrangea macrophylla comprising a chromosomal section associated with production of the metabolites phyllodulcin, hydrangenol and thunberginol G:

[0033] HS127, AF006, AF247, BZ024, AF262, BZ053, BZ032, BZ004, AF240, AF195 in linkage group 15,

[0034] BZ063, AF102, BZ087, AF162, BZ059, BZ041 , AF086, HS272, HS412, AF224, BZ062, AF131 , BZ043, BZ095 in linkage group 16,

[0035] AF209, AF184, HS468, BZ019b, BZ052, HS602, HS129, AF200, AF181 , AF104, AF255, AF019, AF320, BZ022, AF014, BZ036 in linkage group 6a and 6b,

[0036] BZ040, BZ094, BZ003, BZ074, AF188 in linkage group 12,AF062, AF065, BZ063, AF128, AF343, AF004, AF055, AF301 , BZ041 , BZ059, AF205, AF028, AF285, AF148, AF217, HS272, BZ062, BZ043, BZ095 in linkage group 16.

[0037] The primer sequences for detecting these as well as all disclosed marker loci in this application are listed in the sequence protocol and the corresponding primer sequences for the marker loci are listed under “Short description of sequences”.

[0038] It is preferred in terms of the method for identifying a plant of the species Hydrangea macrophylla comprising a chromosomal section associated with production of the metabolites phyllod ulcin , hydrangenol and thunberginol G, that the method comprises the following steps:

[0039] 1) Providing a plant or plant part of the species Hydrangea macrophylla;

[0040] 2) Using the following two markers

[0041] i) markers HS127 and AF006, or

[0042] ii) markers AF086 and HS727, or

[0043] iii) markers BZ019b and BZ052, or

[0044] iv) markers BZ094 and BZ003, or

[0045] v) markers AF343 and AF004.

[0046] Another aspect relates to a method for identifying a plant of the species Hydrangea macrophylla comprising a chromosomal section associated with production of phyllodulcin, which comprises the following steps:

[0047] 1) Providing a plant or plant part of the species Hydrangea macrophylla;

[0048] 2) Using one or more markers to identify a genomic region between and includingi) markers HS468 and BZ036, preferably between and including markers HS602 and AF302 and especially preferable between and including markers AF326 and AF218 in linkage group 6, and / or

[0049] ii) markers AF209 and HS602, preferably between and including markers BZ052 and HS602 in linkage group 6, and / or

[0050] iii) markers BZ063 and BZ095, preferably between and including markers AF102 and BZ062, especially preferable between and including AF224 and BZ062 in linkage group 16, and / or

[0051] iv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205, especially preferably between and including HS272 and BZ062 in linkage group 16, and / or

[0052] v) markers BZ049 and BZ006, preferably between and including markers AF275 and AF347, especially preferably between and including markers AF258 and AF024 in linkage group 18.

[0053] It was also discovered in terms of the present invention that certain “linkage groups” exist, which comprise the QTLs for the production of phyllodulcin. These “linkage groups” are chromosomal or genomic sections in which the QTLs associated with the metabolite expression of phyllodulcin are present. Such “linkage groups” are particularly of interest for identifying such plants producing phyllodulcin or preferably a higher amount of phyllodulcin compared to other Hydrangea macrophylla plants.

[0054] The markers according to the present invention can be used for identifying those QTLs with the method according to the invention.

[0055] In terms of the present invention, it is needed that at least one marker in the area between the specified markers in the phyllodulcin linkage group is identified. However, also the end points of the range between the specified markers, are suitable for identifying the QTL of interest associated with the production of phyllodulcin.

[0056] It is preferred in terms of the method for identifying a plant of the species Hydrangea macrophylla comprising a chromosomal section associated with production of phyllodulcin,, that the method comprises the following steps:1) Providing a plant or plant part of the species Hydrangea macrophylla

[0057] 2) Using the following two markers

[0058] i) markers AF326 and AF128, or

[0059] ii) markers AF224 and BZ062, or

[0060] iii) markers BZ052 and HS602, or

[0061] iv) markers HS272 and BZ062, or

[0062] v) markers AF258 and AF024.

[0063] Preferably, at least one, two, three, four or more markers from the following group can be used in terms of the present invention for identifying a plant of the species Hydrangea macrophylla comprising a chromosomal section associated with production of phyllodulcin:

[0064] HS468, BZ019b, BZ052, HS602, AF278, AF283, AF326, AF218, AF302, HS129, AF022, AF079, AF040, AF229, BZ022, BZ036 in linkage group 6,

[0065] AF209, AF184, HS468, BZ019b, BZ052, HS602, HS129, AF200, AF181, AF104, AF255, AF019, AF320, BZ022, AF014, BZ036 in linkage group 6a and 6b,

[0066] BZ063, AF185, AF102, BZ087, AF330, AF162, BZ059, BZ041, AF086, HS272, HS412, AF224, BZ062, AF131, BZ043, BZ095 in linkage group 16,

[0067] AF062, AF065, BZ063, AF128, AF343, AF004, AF055, AF301, BZ041, BZ059, AF205, AF028, AF285, AF148, AF217, HS272, BZ062, BZ043, BZ095 in linkage group 16.

[0068] BZ049, BZ066, AF275, BZ013, AF258, HS071, AF024, AF042, AF347, BZ006 in linkage group 18.It is preferred that the plant of the species Hydrangea macrophylla is a plant of Hydrangea macrophylla ssp. serrata, preferably of the varieties Yae Amacha, Odoriko Amacha, Hortensia serrata var. thunbergii, Hydrangea macrophylla subsp. Serrata, Hydrangea thunbergii and Platycrater serrata.

[0069] It is especially preferred that the plant is Hydrangea macrophylla ssp. serrata variety Yae Amacha or Odoriko Amacha.

[0070] The marker development underlying this invention is based on the varieties Yae Amacha and Odoriko Amacha as paternal plants, but could be applied to all varieties of Hydrangea macrophylla.

[0071] It is preferred in terms of the present invention that one or more oligonucleotides for identifying the markers selected from the group consisting of

[0072] SEQ ID NO.: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 119, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143, 145 or 147

[0073] and / or

[0074] SEQ ID NO.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146 or 148,

[0075] or an oligonucleotide having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereto,

[0076] are used.

[0077] Preferably, the following combinations of oligonucleotides are used for identifying the corresponding marker positions:

[0078] SEQ ID NO.: 3 and 4 for identifying marker AF195,

[0079] SEQ ID NO.: 5 and 6 for identifying marker AF006,SEQ ID NO.: 7 and 8 for identifying marker BZ032, SEQ ID NO.: 9 and 10 for identifying marker BZ063, SEQ ID NO.: 11 and 12 for identifying marker AF102, SEQ ID NO.: 15 and 16 for identifying marker AF086, SEQ ID NO.: 17 and 18 for identifying marker AF209, SEQ ID NO.: 19 and 20 for identifying marker AF181, SEQ ID NO.: 21 and 22 for identifying marker AF184, SEQ ID NO.: 23 and 24 for identifying marker BZ019b, SEQ ID NO.: 27 and 28 for identifying marker BZ040, SEQ ID NO.: 29 and 30 for identifying marker AF188, SEQ ID NO.: 31 and 32 for identifying marker BZ094, SEQ ID NO.: 33 and 34 for identifying marker BZ003, SEQ ID NO.: 35 and 36 for identifying marker AF062, SEQ ID NO.: 37 and 38 for identifying marker AF205, SEQ ID NO.: 39 and 40 for identifying marker AF343, SEQ ID NO.: 41 and 42 for identifying marker BZ052, SEQ ID NO.: 43 and 44 for identifying marker BZ095, SEQ ID NO.: 45 and 46 for identifying marker AF347, SEQ ID NO.: 47 and 48 for identifying marker AF065, SEQ ID NO.: 49 and 50 for identifying marker AF004, SEQ ID NO.: 53 and 54 for identifying marker BZ036, SEQ ID NO.: 55 and 56 for identifying marker AF302, SEQ ID NO.: 57 and 58 for identifying marker AF326, SEQ ID NO.: 59 and 60 for identifying marker AF218, SEQ ID NO.: 61 and 62 for identifying marker BZ062, SEQ ID NO.: 63 and 64 for identifying marker AF224, SEQ ID NO.: 65 and 66 for identifying marker BZ049, SEQ ID NO.: 67 and 68 for identifying marker BZ006, SEQ ID NO.: 69 and 70 for identifying marker AF275, SEQ ID NO.: 71 and 72 for identifying marker AF258, SEQ ID NO.: 75 and 76 for identifying marker AF278, SEQ ID NO.: 77 and 78 for identifying marker AF283, SEQ ID NO.: 81 and 82 for identifying marker AF022, SEQ ID NO.: 83 and 84 for identifying marker AF079, SEQ ID NO.: 85 and 86 for identifying marker AF040, SEQ ID NO.: 87 and 88 for identifying marker AF229, SEQ ID NO.: 89 and 90 for identifying marker BZ022,SEQ ID NO.: 91 and 92 for identifying marker AF247,

[0080] SEQ ID NO.: 93 and 94 for identifying marker AF271,

[0081] SEQ ID NO.: 95 and 96 for identifying marker BZ024,

[0082] SEQ ID NO.: 97 and 98 for identifying marker AF262,

[0083] SEQ ID NO.: 99 and 100 for identifying marker BZ053,

[0084] SEQ ID NO.: 101 and 102 for identifying marker AF023,

[0085] SEQ ID NO.: 103 and 104 for identifying marker BZ004,

[0086] SEQ ID NO.: 105 and 106 for identifying marker AF240,

[0087] SEQ ID NO.: 107 and 108 for identifying marker AF185,

[0088] SEQ ID NO.: 109 and 110 for identifying marker BZ087,

[0089] SEQ ID NO.: 111 and 112 for identifying marker AF330,

[0090] SEQ ID NO.: 113 and 114 for identifying marker AF162,

[0091] SEQ ID NO.: 115 and 116 for identifying marker BZ059,

[0092] SEQ ID NO.: 117 and 118 for identifying marker BZ041 ,

[0093] SEQ ID NO.: 121 and 122 for identifying marker AF131,

[0094] SEQ ID NO.: 123 and 124 for identifying marker BZ043,

[0095] SEQ ID NO.: 125 and 126 for identifying marker BZ074,

[0096] SEQ ID NO.: 127 and 128 for identifying marker AF128,

[0097] SEQ ID NO.: 129 and 130 for identifying marker AF055,

[0098] SEQ ID NO.: 131 and 132 for identifying marker AF301,

[0099] SEQ ID NO.: 133 and 134 for identifying marker AF028,

[0100] SEQ ID NO.: 135 and 136 for identifying marker AF285,

[0101] SEQ ID NO.: 137 and 138 for identifying marker AF148,

[0102] SEQ ID NO.: 139 and 140 for identifying marker AF217,

[0103] SEQ ID NO.: 141 and 142 for identifying marker BZ066,

[0104] SEQ ID NO.: 143 and 144 for identifying marker BZ013,

[0105] SEQ ID NO.: 147 and 148 for identifying marker AF024.

[0106] Another aspect of the present invention relates to a method for producing a plant of the species Hydrangea macrophylla having a production of the metabolites phyllodulcin, hydrangenol and thunberginol G, wherein the method comprises

[0107] (1) Removing a chromosomal interval between and including

[0108] i) markers HS127 and AF195 and especially preferable between and including markers HS127 and AF006 in linkage group 15, and / orii) markers BZ063 and BZ096, preferably between and including markers AF102 and HS272 and especially preferable between and including markers AF086 and HS272 in linkage group 16, and / or

[0109] iii) markers AF209 and HS602, preferably between and including markers AF184 and HS602 and especially preferable between and including markers BZ019b and BZ052 in linkage group 6, and / or

[0110] iv) markers BZ040 and AF188, preferably between and including markers BZ094 and BZ003, in linkage group 12, and / or

[0111] v) markers AF062 and BZ095, preferably between and including markers AF065 and AF205 and especially preferably between and including markers AF343 and AF004, in linkage group 16,

[0112] from a donor plant of the species Hydrangea macrophylla

[0113] (2) introducing said chromosomal interval of (1) into a plant,

[0114] wherein the method is not an essentially biological method.

[0115] The trait of interest is connected with the production of the sum of the metabolites hydrangenol, thunberginol G and phyllodulcin. Thus, the obtained plant contains the genomic section comprising the QTL associated with the sum of the metabolites. The obtained plant is a transgenic plant.

[0116] A “transgenic plant” refers to a plant in whose genome at least one polynucleotide, preferably a heterologous polynucleotide, is integrated. Preferably, the polynucleotide is stably integrated, which means that the integrated polynucleotide is stably maintained in the plant, is expressed and can also be stably inherited by the progeny. The stable introduction of a polynucleotide into the genome of a plant also includes integration into the genome of a plant of the previous parent generation, whereby the polynucleotide can be stably inherited. The term “heterologous” means that the polynucleotide introduced originates, for example, from a cell or an organism with a different genetic background of the same species or a different species, or is homologous to the prokaryotic or eukaryotic host cell, but is then localized in a different genetic environment and thus differs from anynaturally present corresponding polynucleotide. A heterologous polynucleotide can be present in addition to a corresponding endogenous gene.

[0117] The step of introduction the genomic element into a target plant can be performed with methods known to the person skilled in the art. Such methods could be the use of TALE nucleases (TALENs), Zinc finger nucleases (ZFNs) or CRISPR / Cas systems. The present invention also includes the use of conventional breeding and modern biotechnology.

[0118] Preferably, the target plant is also a plant of the species Hydrangea macrophylla.

[0119] Another aspect of the present invention relates to a method for producing a plant of the species Hydrangea macrophylla having a production of phyllodulcin, wherein the method comprises

[0120] (1) Removing a chromosomal interval between and including

[0121] i) markers HS468 and BZ036, preferably between and including markers HS602 and AF302 and especially preferable between and including markers AF326 and AF218 in linkage group 6, and / or

[0122] ii) markers AF209 and HS602, preferably between and including markers BZ052 and HS602 in linkage group 6, and / or

[0123] iii) markers BZ063 and BZ095, preferably between and including markers AF102 and BZ062, especially preferable between and including markers AF224 and BZ062 in linkage group 16, and / or

[0124] iv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205, especially preferably between and including markers HS272 and BZ062 in linkage group 16, and / or

[0125] v) markers BZ049 and BZ006, preferably between and including markers AF275 and AF347, especially preferably between and including markers AF258 and AF024 in linkage group 18

[0126] from a donor plant of the species Hydrangea macrophylla(2) introducing said chromosomal interval of (1) into a plant,

[0127] wherein the method is not an essentially biological method.

[0128] Another trait of interest is connected with the production of phyllodulcin. Thus, the obtained plant contains the genomic section comprising the QTL associated with the production of phyllodulcin. The obtained plant is a transgenic plant.

[0129] It is preferred that the method for producing a plant comprise the following steps:

[0130] (I) providing a part of a plant as a target structure containing a nucleic acid target region;

[0131] (II) providing one or more recombinant constructs which together comprise or encode the components of a genome editing tool;

[0132] (III) providing at least one vector for introducing the recombinant construct(s) comprising a chromosomal interval as defined in in terms of the present invention;

[0133] (IV) transforming the recombinant constructs from (II) and (III) into the plant target structure; and

[0134] (V) culturing the plant target structure under conditions that cause activation of the components of the genome editing tool and thereby permit targeted modification of the nucleic acid target region in the plant target structure to obtain a plant target structure comprising at least one cell comprising the targeted modification of the nucleic acid target region; and

[0135] (VI) regenerating a plant from the at least one cell.

[0136] A “part of a plant” in terms of the present invention includes, but is not limited to, the shoot axis or stem, leaves, flowers, inflorescences, roots, fruits and seeds as well as pollen. Plant “parts” also refer to a combination of several organs, e.g. a flower or a seed, or a part of an organ, e.g. a cross-section of the shoot axis. Thus also includes plant “tissues”, for example, callus tissue, storage tissue, meristematic tissue, leaf tissue, shoot tissue, root tissue, plant tumor tissue or reproductive tissue as well as the forming tissue, ground tissue (the so-called parenchyma), leading tissue, firming tissue and the covering tissue (the so-called epidermis). However, the tissue is not limited by this list. The term “part of a plant”also includes plant cells. Plant “cells” are, for example, isolated cells with a cell wall or aggregates thereof or protoplasts.

[0137] In terms of the present invention, the use of a genome editing tool is preferred. Such a genome editing tool could be the use of a CRISPR / Cas system, which comprises specific elements for targeted modification of the target plant. Suitable genome editing tools as well as the required genetic elements are known to the person skilled in the art.

[0138] The term “vector” or “vector system”, as used here in connection with genome editing, refers to a means of transport for introducing a recombinant construct, comprising nucleic acids or polypeptides and, where appropriate, other sequences such as regulatory sequences or localization sequences, directly or indirectly into a desired target cell or plant target structure in the desired cellular compartment.

[0139] These vectors can be transformed into the plant part of interest, wherein transforming means direct introduction or indirect introduction.

[0140] Direct introduction takes place directly into a plant target cell or plant target structure which contains nucleic acids that are to be specifically modified according to the present disclosure.

[0141] Indirect introduction comprises introduction into a structure, e.g. cells of leaves or further plant organs and tissues, which do not directly comprise the target plant cell of interest, but which ensures systemic spread and onward delivery of the vector comprising a recombinant construct according to the present disclosure into the target plant structure, e.g. meristematic tissues or cells or stem cells. In the context of transfection of amino acid sequences, the term vector includes suitable agents for peptide or protein transfection, such as ionic lipid mixtures or agents suitable for transfection of a nucleic acid, such as carrier materials through which nucleic acid and amino acid sequences can be introduced into a cell by particle bombardment, such as gold and tungsten particles.

[0142] It is preferred that the donor plant of the species Hydrangea macrophylla is a plant of Hydrangea macrophylla ssp. serrata, preferably of the varieties Yae Amacha, Odoriko Amacha, Hortensia serrata var. thunbergii, Hydrangea macrophylla subsp. serrata, Hydrangea thunbergii and Platycrater serrata. Preferably, the donor and receiver plant, in which the chromosomal interval is introduced in, are both of the species Hydrangea macrophylla.Another aspect of the present invention relates to an oligonucleotide comprising one of the following nucleic acids

[0143] (i) SEQ ID NO.: 3, 5, 7, 9, 11 , 13, 15, 17, 21 , 23, 25, 27, 29, 33, 35, 37, 39, 41 , 43, 45, 47, 49, 53, 55, 57, 59, 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143, 147, or a complement thereof,

[0144] or

[0145] (ii) SEQ ID NO.: 4, 6, 8, 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, 60, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 148 or a complement thereof,

[0146] or an oligonucleotide having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereto.

[0147] The oligonucleotides according to the present invention have a maximum length of preferably 50 nucleotides and can be used for identifying the markers for the QTLs of interest.

[0148] Another aspect of the present invention relates to an expression cassette, recombinant DNA or vector, comprising a chromosomal segment of a plant of the species Hydrangea macrophylla between and including

[0149] i) markers HS127 and AF195 and especially preferable between and including markers AF006 and HS127 in linkage group 15, and / or

[0150] ii) markers BZ063 and BZ096, preferably between and including markers AF102 and HS272 and especially preferable between and including markers AF086 and HS272 in linkage group 16, and / or

[0151] iii) markers AF209 and AF181 , preferably between and including markers AF184 and HS602 and especially preferable between and including markers BZ019b and BZ062 in linkage group 6, and / oriv) markers BZ040 and AF188, preferably between and including markers BZ094 and BZ003, in linkage group 12, and / or

[0152] v) markers AF062 and BZ095, preferably between and including markers AF065 and AF205 and especially preferably between and including markers AF343 and AF004, in linkage group 16.

[0153] Such an expression cassette comprises a chromosomal segment associated with the sum of the metabolites hydrangenol, thunberginol G and phyllodulcin and can be used for manufacturing a transgenic plant comprising the desired QTLforthe sum of the metabolites hydrangenol, thunberginol G and phyllodulcin.

[0154] Yet another aspect of the present invention relates to an expression cassette, recombinant DNA or vector, comprising a chromosomal segment of a plant of the species Hydrangea macrophylla between and including

[0155] i) markers HS468 and BZ036, preferably between and including markers HS602 and AF302 and especially preferable between and including markers AF326 and AF218 in linkage group 6, and / or

[0156] ii) markers AF209 and HS602, preferably between and including markers BZ052 and HS602 in linkage group 6a, and / or

[0157] iii) markers BZ063 and BZ095, preferably between and including markers AF102 and BZ062, especially preferable between and including markers AF224 and BZ062 in linkage group 16, and / or

[0158] iv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205, especially preferably between and including markers HS272 and BZ062 in linkage group 16, and / or

[0159] iv) markers BZ049 and BZ006, preferably between and including markers AF275 and AF347, especially preferably between and including markers AF258 and AF024 in linkage group 18.Such an expression cassette comprises a chromosomal segment associated with the production of phyllodulcin and can be used for manufacturing a transgenic plant comprising the desired QTL for the production of phyllodulcin.

[0160] Thus, another aspect of the present invention is a host cell or plant cell comprising the expression cassette, the recombinant DNA as transgene or the vector according to the invention as well as a transgenic plant or seed thereof comprising a host or plant cell according to the invention.

[0161] Yet another aspect of the present invention relates to a method for the production of phyllodulcin, comprising the following steps:

[0162] i. Providing plant material of a plant of the species Hydrangea macrophylla comprising a genomic section as defined in claim 1 or 2;

[0163] ii. Extracting the metabolites hydrangenol and / or thunberginol G and / or phyllodulcin from the plant material;

[0164] Hi. Obtaining a mixture comprising hydrangenol and / or thunberginol G and / or phyllodulcin;

[0165] iv. Optionally further purifying the obtained mixture and / or enzymatically reacting the metabolites hydrangenol and / or thunberginol G to phyllodulcin.

[0166] The method for producing phyllodulcin according to the invention relates to the extraction or recovery of the phyllodulcin bound as glycosides in the leaves as well as the enzymatic conversion of the metabolites hydrangenol as well as thunberginol G to phyllodulcin.

[0167] Furthermore, preferably also an enzymatic conversion can be used for obtaining phyllodulcin from the metabolites hydrangenol and thunberginol G. The term “enzymatic conversion” or “enzymatic reaction” in terms of the present invention includes all methods either with an isolated enzyme or a whole cell biocatalyst. The latter may be used in a fermentation process.

[0168] Hydrangenol can be used for example as a precursor for phyllodulcin. It has the following general structure:

[0169]

[0170] Hydrangenol is preferably hydroxylated by the enzyme 4-coumarate 3-hydroxylase and 4- hydroxyphenylacetate 3-monooxygenase, which are known in the art. Suitable enzymes are for example described in the patent application PCT / EP2023 / 069755.

[0171] The resulting product is thunberginol G, which has the following general structure:

[0172]

[0173] Thunberginol G, either synthesized from the educt hydrangenol or obtained from the leaves of the Hydrangea macrophylla plants of the present invention, can then be reacted with an O-methyltransferase for obtaining phyllodulcin. The thus obtained phyllodulcin can then be used as a non-caloric sweetener for various applications in the food and pharma industry.

[0174] The term “extracting” means that the metabolites are isolated from the plant material by methods known in the art. It can be interchangeably used with “recovery”.

[0175] It is preferred that the plant of the species Hydrangea macrophylla is a plant of Hydrangea macrophylla ssp. serrata, preferably of the varieties Yae Amacha or Odoriko Amacha.

[0176] The invention is furthermore characterized with illustrative, non-limiting examples.

[0177]

[0178] SEQ ID NO.: 1 and 2 are forward and reverse primer nucleic acid sequences for identifying marker HS127.

[0179] SEQ ID NO.: 3 and 4 are forward and reverse primer nucleic acid sequences for identifying marker AF195.

[0180] SEQ ID NO.: 5 and 6 are forward and reverse primer nucleic acid sequences for identifying marker AF006.

[0181] SEQ ID NO.: 7 and 8 are forward and reverse primer nucleic acid sequences for identifying marker BZ032.

[0182] SEQ ID NO.: 9 and 10 are forward and reverse primer nucleic acid sequences

[0183]

[0184] identifying marker BZ063.

[0185] SEQ ID NO.: 11 and 12 are forward and reverse primer nucleic acid sequences

[0186]

[0187] identifying marker AF102.

[0188] SEQ ID NO.: 13 and 14 are forward and reverse primer nucleic acid sequences for identifying marker HS272.

[0189] SEQ ID NO.: 15 and 16 are forward and reverse primer nucleic acid sequences

[0190]

[0191] identifying marker AF086.

[0192] SEQ ID NO.: 17 and 18 are forward and reverse primer nucleic acid sequences for identifying marker AF209.

[0193] SEQ ID NO.: 19 and 20 are forward and reverse primer nucleic acid sequences

[0194]

[0195] identifying marker AF181.

[0196] SEQ ID NO.: 21 and 22 are forward and reverse primer nucleic acid sequences for identifying marker AF184.

[0197] SEQ ID NO.: 23 and 24 are forward and reverse primer nucleic acid sequences

[0198]

[0199] identifying marker BZ019b.

[0200] SEQ ID NO.: 25 and 26 are forward and reverse primer nucleic acid sequences for identifying marker HS602.

[0201] SEQ ID NO.: 27 and 28 are forward and reverse primer nucleic acid sequences

[0202]

[0203] identifying marker BZ040.

[0204] SEQ ID NO.: 29 and 30 are forward and reverse primer nucleic acid sequences for identifying marker AF188.

[0205] SEQ ID NO.: 31 and 32 are forward and reverse primer nucleic acid sequences

[0206]

[0207] identifying marker BZ094.

[0208] SEQ ID NO.: 33 and 34 are forward and reverse primer nucleic acid sequences for identifying marker BZ003.SEQ ID NO.: 35 and 36 are forward and reverse primer nucleic acid sequences for identifying marker AF062.

[0209] SEQ ID NO.: 37 and 38 are forward and reverse primer nucleic acid sequences for identifying marker AF205.

[0210] SEQ ID NO.: 39 and 40 are forward and reverse primer nucleic acid sequences for identifying marker AF343.

[0211] SEQ ID NO.: 41 and 42 are forward and reverse primer nucleic acid sequences for identifying marker BZ052.

[0212] SEQ ID NO.: 43 and 44 are forward and reverse primer nucleic acid sequences for identifying marker BZ095.

[0213] SEQ ID NO.: 45 and 46 are forward and reverse primer nucleic acid sequences for identifying marker AF347.

[0214] SEQ ID NO.: 47 and 48 are forward and reverse primer nucleic acid sequences for identifying marker AF065.

[0215] SEQ ID NO.: 49 and 50 are forward and reverse primer nucleic acid sequences for identifying marker AF004.

[0216] SEQ ID NO.: 51 and 52 are forward and reverse primer nucleic acid sequences for identifying marker HS468.

[0217] SEQ ID NO.: 53 and 54 are forward and reverse primer nucleic acid sequences for identifying marker BZ036.

[0218] SEQ ID NO.: 55 and 56 are forward and reverse primer nucleic acid sequences for identifying marker AF302.

[0219] SEQ ID NO.: 57 and 58 are forward and reverse primer nucleic acid sequences for identifying marker AF326.

[0220] SEQ ID NO.: 59 and 60 are forward and reverse primer nucleic acid sequences for identifying marker AF218.

[0221] SEQ ID NO.: 61 and 62 are forward and reverse primer nucleic acid sequences for identifying marker BZ062.

[0222] SEQ ID NO.: 63 and 64 are forward and reverse primer nucleic acid sequences for identifying marker AF224.

[0223] SEQ ID NO.: 65 and 66 are forward and reverse primer nucleic acid sequences for identifying marker BZ049.

[0224] SEQ ID NO.: 67 and 68 are forward and reverse primer nucleic acid sequences for identifying marker BZ006.

[0225] SEQ ID NO.: 69 and 70 are forward and reverse primer nucleic acid sequences for identifying marker AF275.SEQ ID NO.: 71 and 72 are forward and reverse primer nucleic acid sequences for identifying marker AF258.

[0226] SEQ ID NO.: 75 and 76 are forward and reverse primer nucleic acid sequences for identifying marker AF278.

[0227] SEQ ID NO.: 77 and 78 are forward and reverse primer nucleic acid sequences for identifying marker AF283.

[0228] SEQ ID NO.: 79 and 80 are forward and reverse primer nucleic acid sequences for identifying marker HS129.

[0229] SEQ ID NO.: 81 and 82 are forward and reverse primer nucleic acid sequences for identifying marker AF022.

[0230] SEQ ID NO.: 83 and 84 are forward and reverse primer nucleic acid sequences for identifying marker AF079.

[0231] SEQ ID NO.: 85 and 86 are forward and reverse primer nucleic acid sequences for identifying marker AF040.

[0232] SEQ ID NO.: 87 and 88 are forward and reverse primer nucleic acid sequences for identifying marker AF229.

[0233] SEQ ID NO.: 89 and 90 are forward and reverse primer nucleic acid sequences for identifying marker BZ022.

[0234] SEQ ID NO.: 91 and 92 are forward and reverse primer nucleic acid sequences for identifying marker AF247.

[0235] SEQ ID NO.: 93 and 94 are forward and reverse primer nucleic acid sequences for identifying marker AF271.

[0236] SEQ ID NO.: 95 and 96 are forward and reverse primer nucleic acid sequences for identifying marker BZ024.

[0237] SEQ ID NO.: 97 and 98 are forward and reverse primer nucleic acid sequences for identifying marker AF262.

[0238] SEQ ID NO.: 99 and 100 are forward and reverse primer nucleic acid sequences for identifying marker BZ053.

[0239] SEQ ID NO.: 101 and 102 are forward and reverse primer nucleic acid sequences for identifying marker AF023.

[0240] SEQ ID NO.: 103 and 104 are forward and reverse primer nucleic acid sequences for identifying marker BZ004.

[0241] SEQ ID NO.: 105 and 106 are forward and reverse primer nucleic acid sequences for identifying marker AF240.

[0242] SEQ ID NO.: 107 and 108 are forward and reverse primer nucleic acid sequences for identifying marker AF185.SEQ ID NO.: 109 and 110 are forward and reverse primer nucleic acid sequences for identifying marker BZ087.

[0243] SEQ ID NO.: 111 and 112 are forward and reverse primer nucleic acid sequences for identifying marker AF330.

[0244] SEQ ID NO.: 113 and 114 are forward and reverse primer nucleic acid sequences for identifying marker AF162.

[0245] SEQ ID NO.: 115 and 116 are forward and reverse primer nucleic acid sequences for identifying marker BZ059.

[0246] SEQ ID NO.: 117 and 118 are forward and reverse primer nucleic acid sequences for identifying marker BZ041.

[0247] SEQ ID NO.: 119 and 120 are forward and reverse primer nucleic acid sequences for identifying marker HS412.

[0248] SEQ ID NO.: 121 and 122 are forward and reverse primer nucleic acid sequences for identifying marker AF131.

[0249] SEQ ID NO.: 123 and 124 are forward and reverse primer nucleic acid sequences for identifying marker BZ043.

[0250] SEQ ID NO.: 125 and 126 are forward and reverse primer nucleic acid sequences for identifying marker BZ074.

[0251] SEQ ID NO.: 127 and 128 are forward and reverse primer nucleic acid sequences for identifying marker AF128.

[0252] SEQ ID NO.: 129 and 130 are forward and reverse primer nucleic acid sequences for identifying marker AF055.

[0253] SEQ ID NO.: 131 and 132 are forward and reverse primer nucleic acid sequences for identifying marker AF301.

[0254] SEQ ID NO.: 133 and 134 are forward and reverse primer nucleic acid sequences for identifying marker AF028.

[0255] SEQ ID NO.: 135 and 136 are forward and reverse primer nucleic acid sequences for identifying marker AF285.

[0256] SEQ ID NO.: 137 and 138 are forward and reverse primer nucleic acid sequences for identifying marker AF148.

[0257] SEQ ID NO.: 139 and 140 are forward and reverse primer nucleic acid sequences for identifying marker AF217.

[0258] SEQ ID NO.: 141 and 142 are forward and reverse primer nucleic acid sequences for identifying marker BZ066.

[0259] SEQ ID NO.: 143 and 144 are forward and reverse primer nucleic acid sequences for identifying marker BZ013.SEQ ID NO.: 145 and 146 are forward and reverse primer nucleic acid sequences for identifying marker HS071.

[0260] SEQ ID NO.: 147 and 148 are forward and reverse primer nucleic acid sequences for identifying marker AF024.

[0261] Short description of figures

[0262] Figure 1 shows a schematic overview of the linkage groups identified in relation to the phyllodulcin metabolism. In this context, the abbreviation PD shows the suspected QTL responsible for phyllodulcin production and “Sum” means the QTL, which is responsible for the sum of the metabolites phyllodulcin, hydrangenol and thunberginol G.Examples

[0263] 1. Cultivation of Hydrangea plants

[0264] A Fi population was produced from a hand-cross between the tea-hortensia varieties H. macrophylla (Thunb.) Ser. ssp. serrata (Thunb.) Makino ‘Odoriko Amacha’ and ‘Yae Amacha’. Both of these cultivars were obtained from the Botanical Collection Pirna-Zuschendorf, Technical University Dresden, Germany via the sub-network Hydrangea of the German Genebank Ornamentals (www.bundessortenamt.de, accession numbers DEU156HYDRN0261 and DEU156HYDRN0263). Young leaves of ‘Odoriko Amacha’ contained on average 1.2% phyllodulcin and 2.3% hydrangenol per leaf dry weight, whereas leaves of the ‘Yae Amacha’ contained on average 2.2% phyllodulcin and 1.6% hydrangenol. The hand-cross was performed using ‘Odoriko’ as seed bearer and ‘Yae Amacha’ as pollinizer.

[0265] Seeds were sown onto standard propagation soil. Germinated plants were transferred in Quick Pot 35 Trays (HerkuPlast Kubern) filled with substrate “Einheitserde ED73” (Einheitserdewerke Werkverband) and cultivated at a research station in Erfurt, Germany. After growth was observable, Fi plants were potted in 3 I pots filled with substrate “Einheitserde ED73” and cultivated as described by Moll et al. (2022) “Dihydroisocoumarin Content and Phenotyping of Hydrangea macrophylla subsp. serrata Cultivars under Different Shading Regimes" Agronomy 11 , 1743. Irrigation, fertilization and plant protection treatments were done as necessary.

[0266] 2. Determination of phyllodulcin, hydrangenol and thunberqinol G content

[0267] The content of phyllodulcin, hydrangenol and thunberginol G is the sum of the metabolite contents and besides the content of phyllodulcin one target of the present invention.

[0268] Youngest fully developed leaves were harvested, dried for at least 48 hours at 40 °C and subsequently homogenized to a fine powder using a mortar and pistil. Per sample, 10 mg leaf powder was transferred in a 2 mL Eppendorf tube and resuspended in 200 pL distilled water for endogenous enzymatic cleavage of glycosides. Enzymatic cleavage was stopped by adding 1.8 mL methanol. Extraction of aglycones was done by ultrasonication for 30 min at room temperature, followed by filtration using the membrane filter Chromafile QtraPTFE-20 / 25. UPLC analyses were performed on a Acquity UPLC® l-Class System (Waters, Milford, MA, United States) equipped with a Acquity UPLC sA PDA detector and a polarreverse phase C18 column (Luna Omega Polar RP-18 50 x 2.1 mm, 1.6 pm, 100 A, Phenomenex, Torrance, CA, United States). Samples were analysed under following conditions: column temperature 50 °C; injection volume 2 pL; mobile phase A - water + 0.1% formic acid, B - acetonitrile; gradient mode 0.00 min - 30% B, 2.00 min - 34% B, 2.01 min - 95% B, 3.01 min - 95% B; flow rate 0.8 mL / min; pre-injection time 1 min. Phyllodulcin, hydrangenol and thunberginol G were detected at wavelength 254 nm and quantified using external calibration. The software Empower™ 3 Pro 2010 was used for instrument control, data acquisition and data evaluation.

[0269] 3. Development of molecular markers

[0270] Genomic DNA was extracted from leaf samples using the DNeasy® Plant Mini-Kit (Qiagen, Germany) or the NucleoMag® Plant kit (Macherey-Nagel, Germany), following the manufacturer’s protocols.

[0271] Population-specific markers were developed based on whole genome sequence data of ‘Odoriko Amacha’ and ‘Yae Amacha’. Genomic DNA of each parental plant was sequenced on an Illumina NovaSeq 600 system (LGC Genomics GmbH, Berlin, Germany). In total, 542,294,822 and 469,499,284250 bp paired-end reads were generated. These reads were mapped against the draft reference genome L10642 of H. macrophylla ssp. macrophylla cultivar ‘Sir Joseph Banks’ using the software CLC Genomics Workbench 10.1.1 (QIAGEN). Parameters were: no masking, mismatch cost 2, insertion and deletion cost 1 , length fraction 0.4, and similarity fraction 0.9. Only uniquely mapped reads were considered to detect polymorphic positions in each parent. To detect 3 to 4 alleles within the Fi population, loci with length polymorphisms within and between ‘Odoriko Amacha’ and ‘Yae Amacha’ were selected to develop SSR and InDei markers. Primers were designed using the OligoCalc software tool (www.basic.northwestern.edu / biotools / oligocalc.html). Forward primers were 5'-tailed with the M13R sequence 5’-caggaaacagctatgacc-3’ (SEQ ID NO.: 149) and reverse primers with the pigtail sequence 5’-gtttctt-3’ (SEQ ID NO.: 150) to permit PCR product labelling. In addition, previously published SSR and InDei markers were used for marker enrichment.

[0272] For detection of SSR and InDei markers with tailed primers, multiplex single-tube, tailed PCRs were performed in combination with fluorescence dye labelled M13R primers using the QIAGEN Multiplex PCR Plus Kit (QIAGEN). Per multiplex tailed PCR assay, 2 to 6 primer pairs that produced marker fragments with distinguishable lengths were combined. Multiplex tailed PCR assays were done in a total volume of 10 pL containing 2x MultiplexPCR Master Mix, 2 to 6 forward primers (0.025 pM) and corresponding reverse primers (0.25 pM), 0.1 pM M13R primer either labelled with the fluorescence dye 6FAM (Sigma-Aldrich), VIC, PET or NED (Thermo Fisher Scientific) and 10 ng DNA. The PCR reaction included initial denaturation at 95°C 5 min, 15 cycles of denaturation at 95°C 30 sec, annealing at 56°C 90 sec and elongation at 72°C 40 sec, followed by 23 cycles of denaturation at 95°C 30 sec, annealing at 53°C 90 sec and elongation at 72°C 40 sec and final elongation at 68°C 30 min. For detection of SSR and InDei markers with labelled primers, PCR assays were done in a total volume of 10 pL containing 1x Multiplex PCR Master Mix, 3 or 5 forward primers (0.2 pM) labelled with the fluorescence dye 6FAM, JOE or TAMRA (Sigma-Aldrich) and corresponding reverse primers (0.2 pM) and 10 ng DNA. The PCR reaction included initial denaturation at 95°C 5 min, 35 cycles of denaturation at 95°C 30 sec, annealing at 60°C 90 sec and elongation at 72°C 40 sec and final elongation at 68°C 30 min. Subsequently, differently labelled PCR fragments of up to 21 markers were pooled. Capillary gel electrophoresis and visualisation were done on a SeqStudio™ Genetic Analyzer (Applied Biosystems by Thermo Fisher Scientific) using the GeneScan™ 600 LIZ™ Size Standard v2.0 (Applied Biosystems by Thermo Fisher Scientific) as internal standard. Peaks were recognized and sized using the software GeneMapper® version 6 (Applied Biosystems by Thermo Fisher Scientific).

[0273] 4. Statistical analysis

[0274] All phenotypic data are based on the analysis of single Fi individuals. Paired t-test and correlation analyses were done in Excel. Normal distribution was tested with Lilliefors-corrected Kolmogorov-Smirnov test using the software program IBM SPSS Statistics v29.0. One-way analysis of variance (ANOVA) followed by Bonferroni post-hoc tests were performed using the software IBM SPSS Statistics v29.0. The marker genotype was considered as fixed factor.

[0275] 5. Results

[0276] QTL mapping was performed based on the phyllodulcin, hydrangenol and thunberginol G contents of 282 Fi individuals. Using the mean phyllodulcin content as phenotypic trait, significant QTL were detected on chromosomes 6 and 16 based on the genetic map of ‘Odoriko Amacha’, while significant QTL on chromosomes 6, 16 and 18 were mapped for ‘Yae Amacha’. These QTL were named according to the studied trait, parental linkage group and mapped position.In addition, a QTL detection based on the sum of phyllodulcin, hydrangenol and thunberginol G contents per individual as phenotypic trait was performed. As summarized in Table 1 , significant QTL were detected.

[0277] The developed molecular markers and their position in relation to the identified QTL can be seen in the genetic map of figure 1.

[0278] Table 1 shows the identified QTLs as well as the flanking markers.

[0279] Table 1 : Overview of identified QTLs

[0280]

Claims

Claims1. Method for identifying a plant of the species Hydrangea macrophylla comprising a chromosomal section associated with production of the metabolites phyllodulcin, hydrangenol and thunberginol G, which comprises the following steps:1) Providing a plant or plant part of the species Hydrangea macrophylla.2) Using one or more markers to identify a genomic region between and includingi) markers HS127 and AF195, preferably between and including markers and especially preferable between and including markers HS127 and AF006 in linkage group 15, and / orii) markers BZ063 and BZ095, preferably between and including markers AF102 and HS272 and especially preferable between markers AF086 and HS272 in linkage group 16, and / oriii) markers AF209 and AF181 , preferably between and including markers AF184 and HS602 and especially preferable between and including markers BZ019b and BZ052 in linkage group 6a and 6b, and / oriv) markers BZ040 and AF188, preferably between and including markers BZ094 and BZ003, in linkage group 12, and / orv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205 and especially preferably between and including markers AF343 and AF004, in linkage group 16.

2. Method for identifying a plant of the species Hydrangea macrophylla comprising a chromosomal section associated with production of phyllodulcin, which comprises the following steps:1) Providing a plant or plant part of the species Hydrangea macrophylla’,2) Using one or more markers to identify a genomic region between and includingi) markers HS468 and BZ036, preferably between and including markers HS602 and AF302 and especially preferable between and including markers AF326 and AF218 in linkage group 6, and / orii) markers AF209 and HS602, preferably between and including markers BZ052 and HS602 in linkage group 6a and 6b, and / oriii) markers BZ063 and BZ095, preferably between and including markers AF102 and BZ062, especially preferable between and including AF224 and BZ062 in linkage group 16, and / oriv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205, especially preferably between and including HS272 and BZ062 in linkage group 16, and / orv) markers BZ049 and BZ006, preferably between and including markers AF275 and AF347, especially preferably between and including markers AF258 and AF024 in linkage group 18.

3. Method according to claim 1 or 2, wherein the plant of the species Hydrangea macrophylla is a plant of Hydrangea macrophylla ssp. serrata, preferably of the varieties Yae Amacha, Odoriko Amacha, Hortensia serrata var. thunbergii, Hydrangea macrophylla subsp. Serrata, Hydrangea thunbergii and Platycrater serrata.

4. Method of any one of the previous claims, wherein one or more oligonucleotides for identifying the markers selected from the group consisting ofSEQ ID NO.: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 119, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143, 145 or 147and / orSEQ ID NO.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146 or 148,or an oligonucleotide having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereto,are used.

5. Method for producing a plant of the species Hydrangea macrophylla having a production ofthe metabolites phyllodulcin, hydrangenol and thunberginol G, wherein the method comprises(1) Removing a chromosomal interval between and includingi) markers HS127 and AF195 and especially preferable between and including markers HS127 and AF006 in linkage group 15, and / orii) markers BZ063 and BZ096, preferably between and including markers AF102 and HS272 and especially preferable between and including markers AF086 and HS272 in linkage group 16, and / oriii) markers AF209 and HS602, preferably between and including markers AF184 and HS602 and especially preferable between and including markers BZ019b and BZ052 in linkage group 6a and 6b, and / oriv) markers BZ040 and AF188, preferably between and including markers BZ094 and BZ003, in linkage group 12, and / orv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205 and especially preferably between and including markers AF343 and AF004, in linkage group 16from a donor plant of the species Hydrangea macrophylla(2) introducing said chromosomal interval of (1) into a plant,wherein the method is not an essentially biological method.

6. Method for producing a plant of the species Hydrangea macrophylla having a production of phyllodulcin, wherein the method comprises(1) Removing a chromosomal interval between and includingi) markers HS468 and BZ036, preferably between and including markers HS602 and AF302 and especially preferable between and including markers AF326 and AF218 in linkage group 6, and / orii) markers AF209 and HS602, preferably between and including markers BZ052 and HS602 in linkage group 6a and 6b, and / oriii) markers BZ063 and BZ095, preferably between and including markers AF102 and BZ062, especially preferable between and including markers AF224 and BZ062 in linkage group 16, and / oriv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205, especially preferably between and including markers HS272 and BZ062 in linkage group 16, and / orv) markers BZ049 and BZ006, preferably between and including markers AF275 and AF347, especially preferably between and including markers AF258 and AF024 in linkage group 18from a donor plant of the species Hydrangea macrophylla(2) introducing said chromosomal interval of (1) into a plant,wherein the method is not an essentially biological method.

7. Method according to claim 5 or 6, wherein the method comprises the following steps:(I) providing a part of a plant as a target structure containing a nucleic acid target region;(II) providing one or more recombinant constructs which together comprise or encode the components of the genome editing tool;(III) providing at least one vector for introducing the recombinant constructs) comprising a chromosomal interval as defined in claim 1 ;(IV) transforming the recombinant constructs from (II) and (III) into the plant target structure; and(V) culturing the plant target structure under conditions that cause activation of the components of the genome editing tool and thereby permit targeted modification of the nucleic acid target region in the plant target structure to obtain a plant target structure comprising at least one cell comprising the targeted modification of the nucleic acid target region; and(VI) regenerating a plant from the at least one cell.

8. Method according to any one of claims 5 to 7, wherein the plant of the species Hydrangea macrophylla is a plant of Hydrangea macrophylla ssp. serrata, preferably of the varieties Yae Amacha, Odoriko Amacha, Hortensia serrata var. thunbergii, Hydrangea macrophylla subsp. serrata, Hydrangea thunbergii and Platycrater serrata.

9. Oligonucleotide comprising one of the following nucleic acids(i) SEQ ID NO.: 3, 5, 7, 9, 11 , 13, 15, 17, 21 , 23, 25, 27, 29, 33, 35, 37, 39, 41 , 43, 45, 47, 49, 53, 55, 57, 59, 63, 65, 67, 69, 71 , 73, 75, 77, 79, 81 , 83, 85, 87, 89, 91 , 93, 95, 97, 99, 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 121 , 123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143, 147, or a complement thereof,or(ii) SEQ ID NO.: 4, 6, 8, 10, 12, 14, 16, 18, 22, 24, 26, 28, 30, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 58, 60, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 148 or a complement thereof,or an oligonucleotide having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% thereto.

10. Expression cassette, recombinant DNA or vector, comprising a chromosomal segment of a plant of the species Hydrangea macrophylla between and includingi) markers HS127 and AF195 and especially preferable between and including markers AF006 and HS127 in linkage group 15, and / orii) markers BZ063 and BZ096, preferably between and including markers AF102 and HS272 and especially preferable between and including markers AF086 and HS272 in linkage group 16, and / oriii) markers AF209 and AF181 , preferably between and including markers AF184 and HS602 and especially preferable between and including markers BZ019b and BZ062 in linkage group 6, and / oriv) markers BZ040 and AF188, preferably between and including markers BZ094 and BZ003, in linkage group 12, and / orv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205 and especially preferably between and including markers AF343 and AF004, in linkage group 16.

11. Expression cassette, recombinant DNA or vector, comprising a chromosomal segment of a plant of the species Hydrangea macrophylla between and includingi) markers HS468 and BZ036, preferably between and including markers HS602 and AF302 and especially preferable between and including markers AF326 and AF218 in linkage group 6, and / orii) markers AF209 and HS602, preferably between and including markers BZ052 and HS602 in linkage group 6a, and / oriii) markers BZ063 and BZ095, preferably between and including markers AF102 and BZ062, especially preferable between and including markers AF224 and BZ062 in linkage group 16, and / oriv) markers AF062 and BZ095, preferably between and including markers AF065 and AF205, especially preferably between and including markers HS272 and BZ062 in linkage group 16, and / oriv) markers BZ049 and BZ006, preferably between and including markers AF275 and AF347, especially preferably between and including markers AF258 and AF024 in linkage group 18.

12. Host cell or plant cell comprising the expression cassette, the recombinant DNA as transgene or the vector according to claim 10 or 11.

13. Transgenic plant or seed thereof comprising a host or plant cell according to claim 12.

14. Method for the production of phyllodulcin, comprising the following steps:i. Providing plant material of a plant of the species Hydrangea macrophylla comprising a genomic section as defined in claim 1 or 2;ii. Extracting the metabolites hydrangenol and / or thunberginol G and / or phyllodulcin from the plant material;Hi. Obtaining a mixture comprising hydrangenol and / or thunberginol G and / or phyllodulcin;iv. Optionally further purifying the obtained mixture and / or enzymatically reacting the metabolites hydrangenol and / or thunberginol G to phyllodulcin.

15. Method according to claim 14, wherein the plant of the species Hydrangea macrophylla is a plant of Hydrangea macrophylla ssp. serrata, preferably of the varieties Yae Amacha or Odoriko Amacha.

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