Cross-breeding method using novel mutant gene imparting perpetual flowering property to rose

Abstract

The purpose of the present invention is to provide a novel mutant gene that imparts perpetual flowering properties to roses. Specifically, the present invention pertains to a KSN mutant gene comprising a base sequence (a) or (b): (a) a base sequence represented by SEQ ID NO: 1; and (b) a base sequence that is at least 90% identical in sequence to the base sequence represented by SEQ ID NO: 1, and maintains a transposon comprising the base sequence from position 729 to position 5812 in the base sequence represented by SEQ ID NO: 1.

Description

A breeding method using a novel mutant gene that gives roses repeat-blooming ability.

[0001] This invention relates to a novel mutant gene that gives roses (plants belonging to the genus Rosa of the family Rosaceae) the ability to bloom throughout the seasons, and to the use of the same.

[0002] Wild roses, like cherry blossoms, are "single-blooming" plants that form flower buds and bloom only once a year. However, most cultivated roses are "repeatable" plants that repeatedly form flower buds and bloom continuously throughout the year. This repeat-blooming characteristic has dramatically increased the value of roses as ornamental horticultural crops. Therefore, it is essential to create repeat-blooming roses when developing new varieties.

[0003] The ability of roses to bloom repeatedly throughout the seasons can sometimes be caused by a loss-of-function mutation in the KSN gene, a gene that suppresses flower bud formation (Patent Document 1). Since there is no established technology to artificially introduce mutations into specific genes in roses (for example, genome editing technology), a method is used to find roses that naturally possess a loss-of-function mutation in the KSN gene, and then crossbreed them to create new varieties that bloom repeatedly throughout the seasons.

[0004] There are two main types of KSN gene loss-of-function mutations known to date. One is a mutation in which a Copia-type retrotransposon (transposable element) is inserted into the KSN gene (ksn copia This is the result of an analysis of the KSN gene in Old Blush, a cultivar that blooms throughout the seasons and originates from China (Non-Patent Literature 1). The insertion of a retrotransposon inhibits the transcription of the normal KSN gene, preventing the production of the KSN protein that suppresses flower bud formation, thus resulting in continuous blooming. Another mutation is a KSN gene deletion mutation (ksn) caused by a partial inversion of the chromosome, which was discovered through the sequencing of the Old Blush genome (Non-Patent Literature 2). null ) In this mutation, the entire KSN gene is missing and therefore non-functional. Old Blush, which is diploid, has ksn copia / ksn null It has the genotype of ksn. copia / ksn copia and ksn null / ksn null It has been found that roses with the homozygous genotype of also bloom throughout the year (Non-Patent Document 3).

[0005] On the other hand, when having even one wild-type normal KSN gene (KSN W ), usually it does not bloom throughout the year but shows single-flowering habit. That is, roses with genotypes such as KSN W / ksn copia and KSN W / ksn null are single-flowering (Non-Patent Document 3). Also, the retrotransposon inserted into the ksn copia gene is as long as 8925 bases in full length, but may change to a state where only 856 bases of the terminal repetitive sequence (LTR, Long Terminal Repeat) remain. The KSN gene (KSN LTR ) with a shortened insertion sequence allows normal transcription of the gene and restores its function (Non-Patent Document 1). This structural change of the retrotransposon often occurs during the process of somatic cell division, causing the phenomenon that single-flowering branch mutants are generated from varieties that bloom throughout the year. When a branch mutation from blooming throughout the year to single-flowering occurs, the tip of the shoot continues to elongate without forming flower buds, thus becoming like a vine plant. Therefore, such branch mutant varieties are often named as the climbing type (Climbing) (for example, Peace Cl. (registered trademark), Iceberg Cl. (registered trademark), Old Blush Cl. (registered trademark)). Thus, in addition to the wild-type normal gene KSN W , even when having KSN LTR , roses will show single-flowering habit instead of blooming throughout the year.

[0006] In summary, for the KSN gene that controls the flowering habit (blooming throughout the year, single-flowering habit) of roses, there are two types of genes with loss-of-function (ksn copia , ksn null ), and two types of genes with normal functions (KSN W , KSN LTRIt is known that repeat-blooming roses only occur when they possess only the functionally deficient gene. Therefore, by examining the genotype of the KSN gene, it is possible to systematically breed repeat-blooming roses.

[0007] Thus, in conventional technology, a functional loss mutation of the KSN gene is called ksn copia yaksn null By using roses that possess the gene for crossbreeding, it is possible to create roses that bloom repeatedly throughout the seasons. However, roses that bloom repeatedly throughout the seasons produced by this method tend to have low genetic diversity and are similar to each other in other horticulturally important traits (e.g., pathogen resistance, flowering period, number of petals, tree shape, inflorescence morphology, etc.). This is because the chromosomal region where the KSN gene resides also contains genes that strongly influence pathogen resistance (Non-Patent Literature 4), flowering period (Non-Patent Literature 5), number of petals (Non-Patent Literature 6), tree shape (Non-Patent Literature 7), presence or absence of thorns (Non-Patent Literature 8), and self-incompatibility (Non-Patent Literature 3), and these genes are inherited in link with the KSN gene. As a result, specific KSN genotypes (ksn copia、 ksn null In breeding of repeat-blooming roses that rely solely on genetic factors, there is a strong tendency to create varieties with similar traits, which is thought to limit the diversification of traits. Furthermore, in populations with low genetic diversity, diseases and pests are more likely to spread, resulting in significant labor and costs for cultivation management.

[0008] WO 2004 / 070036

[0009] Iwata H. et al., The Plant Journal (2012) 69, 116-125Saint-Oyant LH et al., Nature Plants, VOL 4, JULY 2018, 473-484Kawamura K. et al., Horticulture Research, 2022, 9: uhac155Lopez Arias DC et al., Theoretical and Applied Genetics, Volume 133, pages 3299-3321, (2020)Kawamura K. et al., Theoretical and Applied Genetics, Volume 122, pages 661-675, (2011)Roman H. et al., Tree Genetics & Genomes, Volume 11, article number 85, (2015)Kawamura K. et al., Tree Genetics & Genomes, Volume 11, article number 22, (2015) Zhou NN et al., Theoretical and Applied Genetics, Volume 133, pages 3017-3035, (2020)

[0010] In view of the above circumstances, the present invention aims to provide a technology for identifying novel functional loss mutations in the KSN gene that differ from conventional ones, and for introducing them through crossbreeding.

[0011] In order to solve the above problem, we conducted intensive research and as a result, we discovered a novel KSN mutation gene (ksn) that confers repeat-blooming ability to roses. DT ) led to the discovery of this and the completion of the present invention.

[0012] In other words, the present invention encompasses the following: [1] A KSN mutant gene comprising the following nucleotide sequence: (a) the nucleotide sequence described in Sequence ID No. 1; (b) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence described in Sequence ID No. 1, and maintaining a transposon consisting of nucleotide sequences 729 to 5812 in the nucleotide sequence described in Sequence ID No. 1. [2] A primer set for amplifying part or all of a KSN mutant gene comprising the nucleotide sequence described in Sequence ID No. 1, the primer set for detecting the presence of a transposon consisting of nucleotide sequences 729 to 5812 in the nucleotide sequence described in Sequence ID No. 1 in the KSN mutant gene. [3] The primer set according to [2], comprising the following primers (1) and (2): (1) a primer comprising the nucleotide sequence described in Sequence ID No. 23; (2) a primer comprising the nucleotide sequence described in Sequence ID No. 24. [4] A kit for detecting ever-blooming roses comprising the primer set according to [2] or [3]. [5] Use of the KSN mutant gene according to [1] as a biomarker for detecting ever-blooming roses. [6] A biomarker containing the KSN mutant gene described in [1] for detecting repeat-blooming roses. [7] A method for selecting repeat-blooming roses, comprising the step of selecting repeat-blooming roses using the KSN mutant gene described in [1] as a biomarker. [8] A method for producing repeat-blooming roses having the KSN mutant gene described in [1] in a homozygous state, comprising the steps of: crossing the rose cultivar Summer Snow (registered trademark) with a diploid repeat-blooming rose cultivar as the pollen parent to produce an interspecific hybrid that inherits the KSN mutant gene described in [1] from the rose cultivar Summer Snow (registered trademark); and backcrossing the interspecific hybrid with pollen from the rose cultivar Summer Snow (registered trademark). [9] A diploid repeat-blooming rose cultivar having an S genotype S that controls self-incompatibility. C1 Possessing the S genotype, C2The method described in [8], which is a diploid repeat-blooming rose variety that does not possess the KSN mutant gene.

[10] A method for producing a repeat-blooming rose having the KSN mutant gene described in [1] in a homozygous state, comprising the steps of: crossing the rose cultivar Summer Snow (registered trademark) with Rosa multiflora as the pollen parent to produce a first-generation hybrid variety that inherits the KSN mutant gene described in [1] from the rose cultivar Summer Snow (registered trademark); backcrossing the pollen of the first-generation hybrid variety with Rosa multiflora; backcrossing the pollen of the first-generation backcross variety with Rosa multiflora; backcrossing the pollen of the second-generation backcross variety with Rosa multiflora; and crossing the third-generation backcross varieties with each other.

[11] A repeat-blooming rose plant or a part thereof having the KSN mutant gene described in [1] in a homozygous state.

[0013] This specification includes the disclosures of Japanese Patent Application No. 2024-189324, which forms the basis of the priority claim of this application.

[0014] According to the present invention, a novel KSN mutant gene (ksn) confers the ability to bloom repeatedly throughout the seasons to roses. DT By utilizing this technology, it is possible to provide repeat-blooming roses with high genetic diversity.

[0015] In particular, ksn DT By creating repeat-blooming rose varieties that possess the gene in a homozygous state, it becomes possible to cultivate a group of rose varieties that are genetically different from conventional repeat-blooming rose varieties and possess new characteristics in terms of pathogen resistance and flowering period. This will bring a new trend to conventional crossbreeding of repeat-blooming roses, which are becoming stagnant, and will be the catalyst for the birth of a new group of repeat-blooming rose varieties that are resistant to diseases and pests, with the wild rose (Rosa multiflora) adapted to the Japanese climate as their genetic background.

[0016] According to the present invention, ksn DT By creating a repeat-blooming wild rose that possesses the gene in a homozygous state, it will be possible to create a new line for breeding unprecedented repeat-blooming roses. In the history of rose breeding, spanning over 200 years, the breeding of repeat-blooming varieties originated from the R. chinensis line of China. copiagenes and KSN null This has been limited to methods utilizing genes. With this invention, ksn DT By creating wild roses that possess the gene, it becomes possible to create new strains of ever-blooming rose varieties that are genetically distinct from existing varieties, lacking the gene derived from R. chinensis. Furthermore, there are thornless strains of wild roses, which are vigorous growers, disease resistant, fragrant, and used as garden trees. This invention will further enhance the value of wild roses as ornamental horticultural crops by creating ever-blooming wild roses.

[0017] A schematic diagram of the KSN gene is shown. Electrophoretic maps of PCR products in the examples are shown. A phylogenetic tree of Summer Snow is shown. Specifically, the breeding history of the Summer Snow line is shown. The numbers in parentheses indicate the year of publication. This was created based on information from Roberts et al (2003) Encyclopedia of Rose Science and Helpmefind.com (https: / / www.helpmefind.com / rose / plants.php). Continuous-flowering includes Occasionally repeat-flowering. A molecular phylogenetic tree of the KSN gene sequence is shown. KSN of Summer Snow DT The base sequence is highly similar to that of wild rose. Old Blush's ksn copia The gene originates from the Chinese wild species Rosa chinensis var. spontanea, and ksn DT They belong to genetically distant groups. The results of the KSN gene expression levels in the examples are shown. A comparison of flowering cycles in the examples is shown. The distribution of functional loss mutations of the KSN gene related to repeat flowering in the examples is shown. The segregation ratio of S genotype and KSN genotype in the first generation (F1) hybrids in the examples is shown. By crossing repeat-flowering roses with Summer Snow, KSN DTThis shows a method for creating homozygous individuals and an estimate of its success rate (Case 1: Using a repeat-blooming rose that does not possess either SC1 or SC2 as a mating partner). (a) The frequency of occurrence of ovule genotypes (S genotype and the linked KSN genotype) and pollen genotypes is calculated based on the recombination frequency p between S and KSN (recombination is not considered on the ovule side because the KSN genotype is the same). Of the F1 generation roses produced by this cross, the following backcross will result in ksn DT (b) Next, the predictions for when F1 roses are backcrossed with Summer Snow pollen, among them, ksn DT The probability of obtaining a homozygous rose (BC1) was calculated. In this case, if the F1 rose possesses SC2, the SC2 pollen from Summer Snow will not fertilize due to self-incompatibility. The calculation for F1-(2) is almost the same as F1-(1), so it is omitted. The calculation for F1-(4) is almost the same as F1-(3), so it is omitted. Table 6 was created based on these calculation results. Crossing a repeat-blooming rose with Summer Snow results in ksn DT This section shows a method for creating homozygous roses and an estimate of their success rate (Case 2: Using a repeat-blooming rose that possesses SC1 as the mating partner). (a) The SC1 pollen of Summer Snow does not fertilize due to self-incompatibility. Of the F1 generation roses produced by this cross, the following backcross will produce ksn DT (b) Next, the predictions for when F1 roses are backcrossed with Summer Snow pollen, among them, ksn DT The probability of obtaining a homozygous rose (BC1) was calculated. Table 6 was created based on these calculation results. The cross between repeat-blooming roses and Summer Snow resulted in ksn DTThis section shows a method for creating homozygous roses and an estimate of their success rate (Case 3: Using a repeat-blooming rose with SC2 as the mating partner). (a) The SC2 pollen of Summer Snow does not fertilize due to self-incompatibility. Of the F1 generation roses produced by this cross, the following backcross will produce ksn DT (b) Next, the predictions for when F1 roses are backcrossed with Summer Snow pollen, among them, ksn DT The probability of obtaining Homo variegata (BC1) was calculated. Table 6 was created based on these calculation results. ksn DT The effect of F1 selection in homozygous breeding and ksn DT This shows the probability of obtaining homosexuality.

[0018] The present invention will be described in detail below.

[0019] The KSN mutant gene according to the present invention (hereinafter referred to as "ksn") DT A gene (sometimes referred to as a "gene") consists of or includes the following base sequences: (a) the base sequence described in Sequence ID No. 1; (b) a base sequence having at least 90% sequence identity with the base sequence described in Sequence ID No. 1, which maintains a transposon consisting of base sequences 729 to 5812 in the base sequence described in Sequence ID No. 1.

[0020] KSN according to the present invention DT The gene is a functionally deficient KSN mutant gene discovered in the ever-blooming rose cultivar Summer Snow®. It is caused by the insertion of a hAT-type DNA transposon (sometimes referred to as "RoDT1"), which inhibits the transcription of the normal KSN gene and prevents the expression (or coding) of the KSN protein that suppresses flower bud formation.

[0021] KSN according to the present invention DT The gene is a gene that has a genome sequence consisting of the base sequence described in Sequence ID No. 1. The base sequence from positions 729 to 5812 in Sequence ID No. 1 corresponds to the DNA transposon "RoDT1".

[0022] A ksn consisting of or containing the nucleotide sequence described in (b) above. DT The gene may be a nucleotide sequence having sequence identity with the nucleotide sequence described in Sequence ID No. 1 by, for example, at least 70%, at least 75%, at least 80%, at least 85%, preferably at least 90%, particularly preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, and consisting of a nucleotide sequence that maintains the DNA transposon "RoDT1", and may be a functionally deficient KSN mutant gene that does not express the KSN protein.

[0023] KSN according to the present invention DT The gene can be obtained, for example, by PCR using the genomic DNA of Summer Snow (registered trademark) as a template and a primer set designed based on the base sequence described in Sequence ID No. 1.

[0024] Furthermore, the present invention of ksn DT Genes can be used as biomarkers to detect ever-blooming roses.

[0025] In the present invention, the ever-blooming rose is the ksn according to the present invention. DT When the gene is present in a homozygous state, the ksn according to the present invention DT A loss-of-function mutant gene (ksn) is formed when a gene and a Copia-type retrotransposon (transposable element) are inserted into the KSN gene. copia Gene: A gene consisting of or containing the nucleotide sequence described in Sequence ID No. 35, or a KSN gene deletion mutant gene due to partial chromosomal inversion (ksn null The gene may be heterozygous for a gene consisting of or containing the nucleotide sequence described in Sequence ID No. 36.

[0026] Therefore, as a biomarker for detecting ever-blooming roses, the present invention provides ksn DT Genes and KSN copia gene or ksn null Combinations with genes may also be used.

[0027] Furthermore, the present invention relates to the ksn according to the present invention.DT This invention relates to a method for selecting repeat-blooming roses, which includes a step of selecting repeat-blooming roses using genes as biomarkers. In the selection step, for example, PCR is performed using genomic DNA derived from a repeat-blooming rose candidate as a template, using a primer set or kit according to the present invention as described below, and based on the presence or absence of amplification products, the repeat-blooming rose candidate is identified as having the ksn according to the present invention. DT The gene is present in a homozygous state, or the ksn according to the present invention. DT Genes and KSN copia gene or ksn null If a candidate possesses the gene in a heterozygous state, it can be selected as a repeat-blooming rose.

[0028] The primer set according to the present invention is used by PCR to obtain KSN DT A primer set for amplifying part or all of a gene, ksn DT This is a primer set for detecting the presence of the DNA transposon "RoDT1" in genes.

[0029] Using the primer set according to the present invention, the amplification product obtained from PCR using the target rose-derived genomic DNA as a template is used as an indicator for ksn DT By detecting the presence of genes, it is possible to confirm whether or not the target rose is a repeat-blooming rose.

[0030] As shown in Table 5, an example of a primer set according to the present invention is a primer set consisting of (1) primer KSN_DT_F1 (a primer consisting of or containing the nucleotide sequence described in SEQ ID NO: 23) and (2) primer KSN_Sn_R1 (a primer consisting of or containing the nucleotide sequence described in SEQ ID NO: 24), which are designed to specifically detect insertional mutations on the DNA transposon RoDT1 and the KSN gene, respectively.

[0031] Furthermore, the primer set according to the present invention is ksn copia gene, ksn null Genes and wild-type KSN genes (KSN WThe product may also contain a combination of primer sets for amplifying one or more (one, two, or three) of the genes (consisting of the base sequences described in Sequence ID No. 7 or 8) by PCR.

[0032] As shown in Table 5, ksn copia Examples of primer sets for amplifying part or all of a gene include a primer set consisting of primer ksncopia_F (a primer consisting of or containing the nucleotide sequence described in SEQ ID NO: 25) and primer ksncopia_R (a primer consisting of or containing the nucleotide sequence described in SEQ ID NO: 26).

[0033] Also, as shown in Table 5, ksn null Examples of primer sets for amplifying part or all of a gene include a primer set consisting of primer ksnnull_F (a primer consisting of or containing the nucleotide sequence described in SEQ ID NO: 27) and primer ksnnull_R (a primer consisting of or containing the nucleotide sequence described in SEQ ID NO: 28).

[0034] Furthermore, as shown in Table 5, KSN W Examples of primer sets for amplifying part or all of a gene include a primer set consisting of primer KSN_F4 (a primer consisting of or containing the nucleotide sequence described in SEQ ID NO: 29) and primer KSN_R16 (a primer consisting of or containing the nucleotide sequence described in SEQ ID NO: 30).

[0035] Examples of PCR reaction solutions using the primer set according to the present invention include a reaction solution prepared such that each primer included in the primer set according to the present invention is present at a final concentration of 0.1 to 0.4 μM, template DNA at 10 to 200 ng, DNA polymerase at 50 to 500 U, and dNTPs at a final concentration of 5 to 20 mM per 50 μL of reaction solution. Furthermore, examples of PCR thermal cycling conditions include denaturation at 95 to 98°C for 30 to 120 seconds, annealing at 54 to 64°C for 10 to 30 seconds, and extension at 68 to 72°C for 10 to 60 seconds, for a total of 30 to 40 cycles.

[0036] Next, after the PCR reaction, the reaction mixture is subjected to agarose gel electrophoresis, and the presence or absence of amplification products of the size expected from the primer set according to the present invention is confirmed by ethidium bromide staining.

[0037] Furthermore, the primer set according to the present invention can be provided as a kit for detecting ever-blooming roses. In addition to the primer set according to the present invention, the kit may further include, for example, DNA polymerase, nucleic acid synthesis substrate (dNTP), buffer, salts, container, etc. used for PCR; reagents necessary for detecting PCR amplification products (e.g., agarose gel, ethidium bromide, etc.); and instructions for use, etc.

[0038] Furthermore, the present invention relates to the ksn according to the present invention. DT This invention relates to a method for producing repeat-blooming roses that possess a homozygous gene (hereinafter referred to as "the production method according to the present invention").

[0039] In the first embodiment of the present invention, the rose cultivar Summer Snow (registered trademark) is used as the pollen parent and crossed with a diploid ever-blooming rose cultivar (seed parent), and ksn is produced from the rose cultivar Summer Snow (registered trademark). DT This process includes the steps of creating an interspecific hybrid (first-generation hybrid (F1) variety) that inherits genes, and backcrossing the said interspecific hybrid (seed parent) with pollen from the rose cultivar Summer Snow® (pollen parent).

[0040] The KSN genotype of Summer Snow (registered trademark) is heterozygous ksn. DT / ksn copia The inventors have found that in Summer Snow, the S genotype S controls self-incompatibility. C1 (The gene consisting of the base sequence described in Sequence ID No. 37) is ksn DT It is linked to the gene, while the S genotype S C2 (The gene consisting of the base sequence described in Sequence ID No. 38) is ksn copia They discovered that it is linked to genes.

[0041] Also, the S genotype of Summer Snow (registered trademark) is heterozygous S C1 / S C2 Therefore, even when crossed with roses having the same S genotype, seeds cannot be produced.

[0042] ksn DT To produce a homozygous form of the ksn gene, it is necessary to select individuals in which recombination has occurred between the ksn DT gene and the S C1 gene. In order to perform this efficiently, as the seed parent, a diploid remontant rose cultivar that is crossed with Summer Snow (registered trademark) has the S genotype S C1 but preferably does not have the S genotype S C2 Such rose cultivars include, for example, the old Chinese cultivar Rosa chinensis "Mutabilis", Slater's Crimson China, the polyantha Marie Pavie, or the bracteata Mermaid.

[0043] The production method according to the second embodiment of the present invention uses the rose cultivar Summer Snow (registered trademark) as the pollen parent and crosses it with Rosa multiflora (seed parent) to produce a first-generation hybrid (F1) cultivar that has inherited the ksn DT gene from the rose cultivar Summer Snow (registered trademark); a step of backcrossing Rosa multiflora (seed parent) to the pollen (pollen parent) of the first-generation hybrid cultivar; a step of backcrossing Rosa multiflora (seed parent) to the pollen (pollen parent) of the first-generation backcross (BC1) cultivar; a step of backcrossing Rosa multiflora (seed parent) to the pollen (pollen parent) of the second-generation backcross (BC2) cultivar; and a step of crossing the third-generation backcross (BC3) cultivars with each other.

[0044] The KSN genotype of Rosa multiflora is homozygous KSN W / KSN W is.

[0045] According to the production method of the second embodiment of the present invention, ksn DTBy possessing the gene in a homozygous state, it is possible to create rose varieties that are ever-blooming and whose other regions of genomic DNA are derived from wild roses.

[0046] In the breeding method according to the present invention, after each crossbreeding step, the following is performed in accordance with the selection method for repeat-blooming roses described above: ksn DT The process may include a selection step to identify varieties that possess the gene.

[0047] Furthermore, the present invention relates to ksn obtained by the production method according to the present invention. DT This refers to a repeat-blooming rose plant or a part thereof that possesses the gene in a homozygous state. Examples of plant parts include organs or tissues such as seeds, flowers, leaves, stems, and roots, or cells or cytoplasm from these organs or tissues.

[0048] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0049] To overcome the shortcomings of conventional techniques, a technique that involves identifying novel KSN gene loss-of-function mutations of different genetic origins and introducing them through crossbreeding is effective.

[0050] As a rose that may carry a loss-of-function mutation in the new KSN gene, Summer Snow (registered trademark) was focused on. Summer Snow (registered trademark) is a cultivar that blooms throughout the year and is said to be a variety created from a shoot mutation (somatic mutation) of Summer Snow CL (registered trademark), which is a once-blooming variety (Encyclopedia of Rose Science, 1st Edition, October 27, 2003). Usually, shoot mutations from year-round blooming to once-blooming occur frequently, but shoot mutations from once-blooming to year-round blooming, such as Summer Snow (registered trademark), are very rare examples. There is a possibility that some new loss-of-function mutation occurred in the KSN gene that is considered to function normally in the once-blooming Summer Snow CL (registered trademark) and mutated into the year-round blooming Summer Snow (registered trademark). Therefore, KSN gene analysis was performed on four varieties: Summer Snow and Summer Snow CL, and also Spring Pal and Harugasumi, which are shoot mutation varieties of their flower colors.

[0051] 〔Sequence analysis of the KSN gene in the Summer Snow (SS) line〕 Figure 1 shows a schematic diagram of the KSN gene. Primer KSN_F3 is on the second intron, and KSN_R6 is on the fourth exon, and the length of the PCR product is 476 bp. In the KSN gene of Old Blush, a 8925 bp copia-type retrotransposon is inserted 5' upstream of the KSN_F3 primer in the second intron (ksn copia ). In the KSN gene of Summer Snow, a 5084 bp hAT-type DNA transposon (RoDT1) is inserted into the third intron (ksn DT ). The length of the inserted sequence is shown after being reduced to one-tenth.

[0052] When PCR was performed using primers (KSN_F3 and KSN_R6) designed for the second intron and fourth exon of the KSN gene, polymorphism in PCR product length corresponding to ever-blooming and single-blooming varieties was observed (Figure 2). Specifically, Figure 2 shows the electrophoresis of the PCR products. The PCR products were electrophoresed on a 1% agarose gel. M: 1 kbp molecular marker, the number indicates the strain identification number. 111: Summer Snow, 259: Summer Snow, 124: Summer Snow Cl., 262: Summer Snow Cl., 121: Spring Pal, 265: Spring Pal, 266: Harugasumi, 440: Harugasumi. Polymorphism in PCR product length was observed between the ever-blooming Summer Snow and Spring Pal (bold identification numbers) and the single-blooming Summer Snow Cl. and Harugasumi.

[0053] The base sequence information of the primers used and the PCR conditions are shown below: KSN_F3: 5'-ACCAATTCTAAAAGCAAGTAGGAA-3' (SEQ ID NO: 2) KSN_R6: 5'-TCTGCGCATTGAAGTAAACG-3' (SEQ ID NO: 3) PCR conditions: 25 μL of TaKaRa(registered trademark) EmeraldAmp PCR Master Mix (2X) was mixed with 2 ng of template DNA and 0.2 μM of each primer, and the PCR reaction mixture was made up to 50 μL with MilliQ water. The reaction was carried out in the following thermal cycle: (1) 95°C for 2 minutes, (2) 95°C for 30 seconds, (3) 55°C for 30 seconds, (4) 72°C for 40 seconds, (5) return to step (2) 34 times, (6) 72°C for 5 minutes.

[0054] In the single-blooming variety, two bands of different PCR product lengths were visually observed, whereas in the ever-blooming variety, only one short band was visible. To confirm this, the PCR product was cloned and its base sequence was determined, and the short band was identified as the ksn of Old Blush. copia The long band matches the gene sequence and is the KSN of wild rose. WThe gene sequence matched (Table 1). Tracing the breeding history of Summer Snow Cl., it was found that wild rose was used three generations ago (Figure 3), and it was thought that this gene remained. In contrast, the ever-blooming Summer Snow and Spring Pal have KSN derived from this wild rose. W Since the gene was not amplified by PCR, it was thought that some kind of mutation had occurred.

[0055]

[0056] [Identification of DNA transposon insertion mutations] Therefore, KSN of wild rose W We designed primers to specifically amplify the gene, and used Summer Snow DNA as a template to obtain and analyze the surrounding base sequence information using the Genome Walking Kit (registered trademark).

[0057] The Genome Walking Kit is a method in which template DNA is restricted enzyme-treated, then adapter primers are added, and PCR is performed using gene-specific primers and adapter primers. KSN_gw_F1 was designed as the gene-specific primer on exon 2 (Figure 1). copia Because it contains a long insertion sequence, we hypothesized that it would be difficult to amplify using PCR with KSN_gw_F1 and an adapter primer. Furthermore, following the kit manual, we designed a gene-specific primer (KSN_gw_F2) for the 3' downstream region, performed nest PCR, and then determined the base sequence of the PCR product (Figure 1). Analysis of the results revealed that an insertion sequence, which had not been previously reported, was located in the third intron of the KSN gene. After determining the full length of the insertion sequence and analyzing it, it was identified as a 5084-base DNA transposon. It is an hAT-type DNA transposon with 20-base inverted repeat sequences (TIR, Terminal Inverted Repeat) at both ends, and was named RoDT1. A 3-base (TAA) target site duplication (TSD) was observed at the insertion site of the KSN gene.

[0058] Summer Snow's KSN gene (Summer Snow_ksnDT; ksn DTThe sample had the nucleotide sequence shown in Sequence ID No. 1. Furthermore, the positions of each exon, RoDT1, TIR, and TSD in the nucleotide sequence shown in Sequence ID No. 1 are shown in Table 2 below.

[0059]

[0060] rkksn DT [Origin of genes] ksn DT This is a gene in which the KSN gene of Rosa multiflora (wild rose) has mutated, and the KSN gene of R. chinensis var. spontanea (spontanea) has mutated. copia This is a different genetic resource from (Non-Patent Document 1 (Iwata H. et al., The Plant Journal (2012) 69, 116-125)). ksn DT and ksn copia The base sequence of the wild-type KSN gene, created by deleting the inserted sequence from the base sequence of the former, showed high homology to that of Rosa multiflora, and to that of Rosa spontanea, in the latter case (Figure 4). Examining the lineage of Summer Snow's crosses revealed that Rosa multiflora was used as a parent plant (Figure 3). Therefore, ksn DT This gene represents a new genetic resource with a different genetic background from R. chinensis, which has been frequently used in the breeding of ever-blooming varieties.

[0061] Furthermore, the KSN of Summer Snow determined in the above experiment DT We created a hypothetical wild-type KSN gene sequence by deleting the RoDT1 sequence from the gene's base sequence, and analyzed its similarity to the wild-type KSN gene sequence. The KSN base sequence data used in the analysis is shown in Table 3 below.

[0062]

[0063] [KSN Gene Expression Analysis] Figure 5 shows the KSN gene expression analysis. Specifically, it shows the results of comparing the KSN gene expression levels at shoot apex using the qPCR method. The RoTCTP gene was used as an endogenous control gene, and the KSN gene expression level was quantified and shown as a relative value with the SP expression level set to 1. The KSN gene expression levels were examined using different primers for exons 1 and 2 (Exon 1-2), 2 and 3 (Exon 2-3), and 3 and 4 (Exon 3-4). SSC: Summer Snow Cl., SS: Summer Snow, H: Harugasumi; SP: Spring Pal, F1: A F1-hybrid of SS. The number n in parentheses indicates the number of individuals. Total RNA was extracted from the shoot apex of 3-10 shoots per individual and used as a template for RT-qPCR. For SSC and SS, which have repeats from 4 individuals, the mean value and standard error (SE) of the expression level are shown. For the others, the data is from a single individual, so there is no estimation of the error. The expression level of the KSN gene in the single-blooming SSC and H varieties was several hundred times higher than that of the ever-blooming SS, SP, and F1 varieties, while KSN expression was undetectable or very low in the ever-blooming varieties.

[0064] Thus, when KSN gene expression analysis was performed, KSN gene expression could not be detected in the repeat-blooming varieties Summer Snow and Spring Pal (Figure 5). In contrast, normal KSN gene expression was detected in the once-blooming varieties Summer Snow CL and Harugasumi. The F1 roses obtained by crossing Summer Snow with Spring Pal and Summer Snow with R. chinennsis showed ksn copia / ksn DT While Summer Snow Cl. and Harugasumi have the genotype ksn copia / KSN W It has the genotype ksn. copia This is a loss-of-function mutation in which the transcription of the KSN gene does not proceed normally (Non-patent document 1 (Iwata H. et al., The Plant Journal (2012) 69, 116-125)). The results of this example show that ksnDT ksn copia This similarly suggests that gene transcription is being inhibited.

[0065] In this KSN gene expression analysis, rose seedlings grown in pots were pruned to 3-4 shoots, each pruned down to about 10 nodes from the base. These shoots were then left outdoors (Osaka) for three months from December to February, and then moved indoors in a constant temperature environment of 25°C under a plant growth fluorescent lamp (14 hours of illumination per day) from March onwards.

[0066] Within about a month, the first current year's branches formed flower buds at their tips, and secondary shoots developed from the axillary buds. When these secondary shoots had formed 3-5 leaves, the tips of the shoots were sampled. After removing the leaves, a 5mm portion of the stem containing the apical meristem was frozen in liquid nitrogen. 3-10 samples were collected from each plant, and after freezing, they were collected in a single tube, crushed, and total RNA was extracted using the NucleoSpinRNA Plant kit according to the manual.

[0067] Four Summer Snow specimens obtained from different garden centers were used as research material, as were four Summer Snow Cl. specimens obtained from different garden centers. One specimen each of Harugasumi, Spring Pal, and F1 (a hybrid of Summer Snow and Rh) were also investigated.

[0068] Total RNA was obtained by removing genomic DNA using the PrimeScript® RT reagent Kit with gDNA Eraser (TaKaRa), and then performing a reverse transcription reaction according to the manual using the Oligo dT Primer and Random Primer included with the kit to obtain cDNA.

[0069] Next, gene expression levels were quantified using the reagent PowerUp SYBR Green Master Mix and a StepOne real-time PCR instrument (Life Technologies). 10-μL PCR reaction mixtures were prepared (3 μL distilled water, 0.5 μM primer, 1 μL cDNA solution, and 5 μL Fast SYBR Green Master mix), and the reaction was performed using Fast cycling mode: (1) 50°C, 120s; (2) 95°C, 120s; (3) 95°C, 3s; (4) 64°C, 30s; (5) Back to (3) 39 times. Information on the primers used is shown in Table 4.

[0070] Remay et al. 2009: Remay, A., Lalanne, D., Thouroude, T. et al. A survey of flowering genes reveals the role of gibberellins in floral control in rose. Theor Appl Genet 119, 767-781 (2009). https: / / doi.org / 10.1007 / s00122-009-1087-1

[0071] [Investigation of Flowering Habits] Figure 6 shows a comparison of flowering cycles. The periodicity of flower bud formation in rose seedlings was investigated and compared. Rose seedlings cultivated indoors in a constant temperature environment of 25°C from April were investigated for shoot elongation, flower bud formation, and flowering. The seedlings used in the experiment were pruned in December of the previous year and kept outdoors in a low-temperature environment until March. In the first week of July, a large number of spider mites appeared, so the seedlings were severely pruned to a state of 2-3 shoots and about 10 nodes from the base to eradicate the spider mites.

[0072] Thus, when we examined the flowering periodicity, we found that Summer Snow and Spring Pal formed flower buds after vegetative growth of about 7-8 nodes, similar to conventional repeat-blooming varieties (Figure 6). Furthermore, in the first generation F1 hybrid roses produced using Summer Snow as a parent (details will be described later), ksn copia / ksn DTRoses with this genotype bloomed within a few weeks of germination. This indicates that the loss of the juvenile stage, characteristic of repeat-blooming varieties, occurred. Therefore, ksn DT The gene has been used until now (KSN) copia yaksn null It was found that it can be used as a loss-of-function mutation in the KSN gene equivalent to the gene itself.

[0073] rkksn DT [Rose varieties that possess the gene] ksn DT This gene is unique to the Summer Snow lineage, and there was no evidence of its introduction through breeding in other repeat-blooming varieties of the same lineage. For a total of 155 rose varieties, ksn DT ksn copia ksn null The results of PCR tests to determine whether the organism possessed the virus were summarized (Figure 7).

[0074] Specifically, Figure 7 shows the distribution of functional loss mutations in the KSN gene, which is related to ever-blooming characteristics. Three types of functional loss mutations (ksn copia ksn null ksn DT PCR tests were performed to specifically detect each of the mutated genes, and the probability of possessing the mutated gene was investigated. The roses investigated consisted of a total of 152 varieties, differing in class and breeding age (I, before 1850; II, 1850-1900; III, 1900-1940; IV, 1940-1980; V, 1980-2020). The numbers show the percentage of rose varieties that possessed the mutated gene and the number of varieties possessing the gene divided by the number of varieties investigated. The difference in color temperature represents the difference in the probability of possession.

[0075] As a result, out of 155 rose varieties from various lineages and breeding eras, ksn DT The only two rose varieties in which ksn was detected were Summer Snow (Breeding Year III: 1990-1940) and Spring Pal (Breeding Year V: 1980-2020). Other rose varieties, regardless of breeding year or lineage, were all free of ksn. DT No roses possessing this trait were found. In contrast, ksn copiaMost rose varieties possess this trait since its introduction from China. null ksn copia Although less frequent than other factors, it has been found that half to more than a quarter of roses possess it. From these analysis results, the ksn identified in this example DT This is the KSN that has been used conventionally. copia yaksn null It can be described as a "hidden genetic resource," distinct from the others.

[0076] Furthermore, an examination of the genotype of the KSN gene in Tausendschon (Figure 3), which is considered to be a parent of Summer Snow Cl., revealed that ksn DT It was not detected. This result indicates that the DNA transposon was transferred to the KSN gene, and ksn DT This supports the hypothesis that the phenomenon arose when Summer Snow emerged as a branch mutation from Summer Snow Cl.

[0077] This study investigated 7 additional rose varieties to the 148 varieties investigated in Non-Patent Document 3 (Kawamura K. et al., Horticulture Research, 2022, 9: uhac155; Kawamura et al. 2022), using ksn DT A PCR test was performed to determine the presence or absence of ksn. These roses were selected from varieties with different breeding eras and lineages. Table 5 shows the sequence information of the primers used in the PCR test. DT Primers for detecting insertion mutations were designed for the RoDT1 gene and the KSN gene, respectively, to specifically detect insertion mutations. PCR was performed by adding 5 μL of TaKaRa's EmeraldAmp PCR Master Mix, 0.2 μL each of primer solution (10 μM), 1 μL of template DNA solution (1 ng / μL), and 3.6 μL of distillate to make a PCR reaction mixture of 10 μL. The reaction was carried out in 34 cycles: (1) 95°C for 2 minutes; (2) 95°C for 30 seconds; (3) 60°C for 30 seconds; (4) 72°C for 30 seconds; (5) returning to (2). The presence or absence of bands was confirmed on a 1% agarose gel. Furthermore, using the same DNA solution and the primers shown in Table 5, KSN DTUsing a similar PCR method, ksn copia and ksn null Gene, wild-type KSN W The presence or absence of the gene was also confirmed.

[0078]

[0079] rkksn DT [Linkage between genes and the S gene] ksn DT This demonstrated that it is a hidden genetic resource that has not received any attention as breeding material until now. DT Summer Snow, which possesses ksn, is used as a parent in the cross. DT This demonstrates that it is inherited by subsequent generations. Furthermore, focusing on the fact that the KSN gene is located on the same chromosome as the S gene that controls self-incompatibility and is inherited in a linked manner (Non-patent Literature 3 (Kawamura et al. 2022)), we will demonstrate that ksn DT This will identify the genotype of the linked S gene and clarify its recombination frequency. Based on this information, ksn DT This paper predicts the crossbreeding of homozygous repeat-blooming roses and proposes methods to streamline the process.

[0080] According to the study in Non-Patent Document 3 (Kawamura et al. 2022), the S genotype of Summer Snow is S C1 / S C2 It has been found that... On the other hand, the KSN genotype of Summer Snow is ksn DT / ksn copia That is. ksn DT S C1 and S C2 It is unknown which of the two is linked. To quantify the degree of linkage (i.e., recombination frequency), Summer Snow was crossed with other diploid varieties, and the F1 generation was bred. Based on the segregation ratio of the S genotype and KSN genotype of the F1 generation, the KSN genotype of Summer Snow was determined. DT We estimated which S gene was linked to and the recombination frequency (Figure 8).

[0081] As a result, Figure 8 shows the segregation ratio of S genotype and KSN genotype in the first generation (F1) of hybrids. It shows the number of individuals with S genotype (a) and KSN genotype (b) in the F1 generation grown from seeds obtained by pollinating Rc46 (Rosa chinensis "white single eye") with Summer Snow pollen, and the number of individuals with S genotype (c) and KSN genotype (d) in the F1 generation grown from seeds obtained by pollinating The Fairy with Summer Snow pollen. When the S genotype was examined for 69 F1 generation individuals obtained by crossing Rc46 with Summer Snow (SS), all individuals were S C2 It possessed the gene (Figure 8a). This is because Rc46 is S C1 In order to possess SS's S C1 This is because fertilization is not possible due to self-incompatibility. Therefore, Summer Snow's S C1 The KSN gene (ksn DT Or ksn copia ) is predicted to be less likely to be inherited by the F1 generation (fertilization occurs only when recombination occurs between the S gene and the KSN gene). When the frequency of occurrence of the KSN gene genotype is examined, it is found that from summer snow to ksn DT Only 21 roses (30%) inherited the trait (Figure 8b). Therefore, the S of Summer Snow C1 The one linked to it is ksn DT It was estimated that recombination occurred with a probability of 30% (recombination frequency p=0.30). On the other hand, The Fairy (TF) was S C2 In order to possess S in SS pollen C2 The genotype of pollen cannot fertilize due to self-incompatibility. As predicted, all F1 generations grown by pollinating TF with SS pollen were S C1 It was thought that the individual was fertilized by pollen (Figure 8c). From the analysis of Rc46 described above, the S of summer snow C1 ksn DT and S C2 ksn copia It is highly likely that this is linked. In that case, in the hybrid population of TF and SS, S C1 KSN DTIt should be the dominant gene. In fact, ksn DT A large number of F1 individuals (40 individuals, 85%) possessed the gene (Figure 8d), and in this population, the recombination frequency between the S gene and the KSN gene was estimated to be p=0.15.

[0082] Regarding the method of this experiment, the S genotype of Rosa chinensis "white single eye" (Rc46) is S C1 / S 12 The Fairy (TF)'s S genotype is S C2 / S 21 This is the case (Non-Patent Literature 3 (Kawamura et al. 2022)). Therefore, each shares only one of the S genotypes with Summer Snow (semi-compatibility). These roses were used as seed parents and pollinated with pollen from Summer Snow (SS) to cultivate the F1 generation. 69 F1 individuals were produced from the cross between Rc46 and SS, and 47 F1 individuals were produced from the cross between TF and SS.

[0083] The gDNA of F1 roses was extracted, and the S genotype and KSN genotype were determined by PCR. The KSN genotype of Rc46 is KSN. W / ksn copia Therefore, the KSN genotype of TF is ksn copia / ksn copia That is the case.

[0084] rkksn DT [Efficient breeding methods for homozygous individuals] Summer Snow's KSN DT is S C1 It was shown that the gene is linked to the KSN gene. The recombination frequency between the S gene and the KSN gene differed depending on the population, and was estimated to be 0.15 or 0.3. Based on this fact, it was shown that the KSN gene is efficiently linked to the KSN gene. DT This paper proposes an efficient method for crossbreeding homozygous roses. The basic strategy involves using Summer Snow as the pollen parent and crossing it with a diploid repeat-blooming rose to produce ksn from Summer Snow. DT Select F1 individuals that inherited the trait (in the first or second year). Then, backcross these F1 individuals with pollen from Summer Snow, and ksn DTHomozygous BC1 individuals are created (in their second or third year). Since repeat-blooming roses have lost their juvenile stage, they form flower buds and bloom within a few weeks of germination. Therefore, crossbreeding can be carried out in a short period of time.

[0085] Furthermore, if you use the S genotype information in the method described below when selecting mating partners or F1 individuals, ksn DT This makes it possible to efficiently produce homozygous BC1 individuals.

[0086] First, choose a rose to be used as a crossbreeding partner for Summer Snow that meets the following requirements: (1) Diploid, (2) Repeat-blooming, (3) S genotype is S C1 / S C2 Not. Summer Snow's S genotype is S C1 / S C2 Therefore, even if you crossbreed roses that have the same S genotype, you will not be able to produce seeds. For the following three cases, see ksn DT The probability of obtaining homosexuality was calculated (Table 6): Case 1: S C1 S C2 When using repeat-blooming roses that you do not own; Case 2: S C1 They own S C2 When using repeat-blooming roses that you do not own; Case 3: S C2 They own S C1 This applies when using repeat-blooming roses that you do not own.

[0087]

[0088] Table 6 shows the results for each of the three cases, ksn DT The genotypes of F1 generation roses that can be used to create homozygous ksn, their occurrence frequency (F1-Genotype, Frequency of x), and the results when these F1 roses are backcrossed with Summer Snow pollen. DT This table summarizes the frequency of homozygous encephalopathy (Frequency of y).

[0089] The recombination frequency between the S gene and the KSN gene was denoted as p, and calculations were performed (details of the calculation process are shown in Figures 9-1, 9-2, and 9-3). The product z of x and y is the BC1 generation obtained by raising all the F1 generations that were generated without selection based on genotype information and backcrossing them with Summer Snow pollen. DT This is a formula for predicting the proportion of individuals that are homozygous. The probability z in case 1 is 0.5*p*(1-p), which is twice as large as z=0.25*p*(1-p) in cases 2 and 3, regardless of the value of p. Therefore, if selection of the F1 generation based on genetic information is not performed, S C1 S C2 The most efficient method is to use a diploid, ever-blooming rose that does not possess the necessary traits as a crossbreeding partner.

[0090] However, by selecting the F1 generation based on genetic information, the efficiency of crossbreeding can be significantly increased. In other words, at the breeding stage of the F1 generation, the S genotype and KSN genotype are examined, and the KSN genotype is selected. DT Only genotypes suitable for producing homozygous individuals (Table 6: F1-Genotype) are selected, and backcrossing is performed. At this time, among the BC1 individuals produced, ksn DT The probability w of homozygous individuals occurring is p*(1-p) in case 1, w=0.5*(1-p) in case 2, and w=0.5*p in case 3, indicating that the effect of F1 selection depends on p. Therefore, a graph was created showing the change in the value of w and the change in the selection effect of the F1 generation (1 / x) as p is varied from 0 to 0.5 (Figure 10). The effect of selecting F1 individuals is expressed as the reciprocal of the proportion of F1 individuals that can be used for backcrossing (x). This is because the smaller the value of x, the greater the waste when backcrossing without selection. As a result, regardless of the value of p, case 2 showed the highest effect of F1 selection, and ksn was found in the BC1 population. DT The high probability of obtaining homozygous individuals demonstrates that this is an efficient breeding method. Regardless of the p-value, the effect of F1 selection is highest in Case 2, approximately 13 times at p=0.15 and approximately 7 times at p=0.3 (Figure 10a). The rose used in Case 2 is S C1 In order to possess Summer Snow's S C1 Pollen cannot be fertilized. ksnDT is S C1 Because they are linked, the smaller the recombination frequency p, the smaller the ksn in the F1 generation. DT The probability of an individual possessing ksn being born decreases. Therefore, if backcrossing is performed without examining the genetic information, ksn DT The probability of obtaining homozygous BC1 individuals is low. However, ksn in the F1 generation DT If you select a small number of individuals that possess the trait, then most of the BC1 individuals produced by backcrossing will be ksn DT It can be expected to become homozygous. This is because, in the F1 generation, individuals with the S gene and KSN gene combination have been selected, so in backcrossing, the self-incompatibility of Summer Snow's KSN gene will be activated. copia Because it can effectively block S-genotype pollen linked to it, many BC1 individuals have ksn DT They become homosexual (Figure 9-2). When p=0.15, ksn in the BC1 group after F1 selection. DT The probability of homozygous development is as high as 42.5% in Case 2, significantly higher than the 12.75% in Case 1 and the 7.5% in Case 3. As the recombination frequency increases to p=0.3, this difference narrows, but Case 2 still has the highest probability at 35%, which is higher than the 21% in Case 1 and the 15% in Case 3 (Figure 10b).

[0091] rkksn DT Efficient breeding method for ever-blooming varieties that are homozygous for [the aforementioned ksn] DT Select a suitable variety for mating according to the efficient homozygous breeding method: When selecting the F1 generation based on genetic information, S C1 Possesses the gene but S C2 Diploid, ever-blooming varieties that do not possess the gene are the most efficient at producing KSN. DT It was predicted that homozygous strains could be produced. Therefore, referring to Non-Patent Document 3 (Kawamura et al. 2022), we searched for such varieties and found old Chinese cultivated varieties such as Rosa chinensis "Mutabilis" and Slater's Crimson China, as well as the polyantha variety Marie Pavie and the bracteata variety Mermaid. Note that S C1 and S C2Since the presence or absence of this trait can be easily confirmed by PCR, it should be relatively easy to find other varieties besides those mentioned as examples here.

[0092] Once the rose to be used for crossbreeding is selected, artificial pollination is performed with Summer Snow pollen, mature seeds are harvested (year 1), sown the following spring, and the F1 generation is cultivated (year 2). When the plant has grown to about 20 true leaves, a small amount of young leaves is collected and DNA is extracted, and ksn DT Individuals possessing the characteristic are selected, allowed to grow significantly until around summer, artificially pollinated with Summer Snow pollen in the fall, and then cultivated for about four months until the seeds are fully mature before harvesting. The following spring, the seeds are sown to cultivate the BC1 generation (3rd year), and ksn DT Homozygous individuals are selected. The frequency x of individuals to be selected in the F1 generation is predicted to be 0.5*p (Table 6). Therefore, if approximately 100 individuals are raised, when p=0.15, approximately 7 individuals will be ksn, and when p=0.3, approximately 15 individuals will be ksn. DT These are predicted to be selected individuals possessing the characteristic. Then, if these F1 roses are backcrossed with Summer Snow pollen and approximately 100 BC1 individuals are grown, approximately 42 individuals (p=0.15) or approximately 35 individuals (p=0.3) will be ksn. DT It is predicted that he will become homosexual. In this way, KSN is relatively short-term and small-scale. DT Homosexual repeat-blooming roses can be crossbred. The more individuals you cultivate, the more KSN you can obtain. DT As the number of homozygous individuals increases, the opportunities to select individuals with more desirable traits also expand.

[0093] rkksn DT [Breeding method for ever-blooming wild roses that are homozygous for [specific gene]] ksn DT Since it was thought to originate from the wild rose, it is also possible to breed wild roses that bloom throughout the year (Table 7).

[0094]

[0095] As shown in Table 7, wild roses were crossed with Summer Snow (year 1), and the F1 population was cultivated to produce ksn DTSelect F1 individuals that possess the trait (Year 2). Backcross these F1 roses with wild roses (Year 3) to cultivate a BC1 population, ksn DT Select a BC1 rose that possesses (4th year). Repeat this backcrossing two more times, ksn DT Select BC3 roses that possess the following traits (8th year). Backcross with wild rose and ksn DT Through repeated selection, it is predicted that over 93.75% of the BC3 roses have the genome of wild roses. Therefore, the BC3 roses are ksn DT It possesses [specific trait], but the rest of the area is mostly wild rose. These BC3 roses were crossbred (in the 9th year) and repeat-blooming roses were selected (in the 10th year). Individuals that exhibit repeat-blooming characteristics (flowering a few weeks after germination due to loss of juvenile stage) were selected as ksn DT It is homozygous. In this way, I have shown a method for crossbreeding and breeding ever-blooming wild roses, which takes about 10 years. Since wild roses are single-blooming, they can only flower and pollinate once a year, so it takes nearly 10 years.

[0096] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A KSN mutant gene comprising the following nucleotide sequence: (a) the nucleotide sequence described in Sequence ID No. 1; (b) a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence described in Sequence ID No. 1, and maintaining a transposon consisting of nucleotide sequences 729 to 5812 in the nucleotide sequence described in Sequence ID No.

1.

2. A primer set for amplifying part or all of a KSN mutant gene consisting of the nucleotide sequence described in Sequence ID No. 1, wherein the primer set detects the presence of a transposon consisting of nucleotide sequences 729 to 5812 in the nucleotide sequence described in Sequence ID No. 1 of the KSN mutant gene.

3. The primer set according to claim 2, comprising the following primers (1) and (2): (1) a primer comprising the nucleotide sequence described in SEQ ID NO: 23; (2) a primer comprising the nucleotide sequence described in SEQ ID NO:

24.

4. A kit for detecting ever-blooming roses, comprising the primer set according to claim 2 or 3.

5. Use of the KSN mutant gene according to claim 1 as a biomarker for detecting ever-blooming roses.

6. A biomarker comprising the KSN mutant gene according to claim 1 for detecting ever-blooming roses.

7. A method for selecting ever-blooming roses, comprising the step of selecting ever-blooming roses using the KSN mutant gene described in claim 1 as a biomarker.

8. A method for producing a repeat-blooming rose having the KSN mutant gene according to claim 1 in a homozygous state, comprising the steps of: crossing the rose cultivar Summer Snow (registered trademark) with a diploid repeat-blooming rose cultivar to produce an interspecific hybrid that inherits the KSN mutant gene described in claim 1 from the rose cultivar Summer Snow (registered trademark); and backcrossing the interspecific hybrid with the pollen of the rose cultivar Summer Snow (registered trademark).

9. Diploid repeat-blooming rose varieties have an S genotype that controls self-incompatibility. C1 Possessing the S genotype, C2 The method according to claim 8, wherein the rose variety is a diploid, ever-blooming rose variety that does not possess [specific characteristic].

10. A method for producing a repeat-blooming rose having the KSN mutant gene described in claim 1 in a homozygous state, comprising the steps of: crossing the rose cultivar Summer Snow (registered trademark) with Rosa multiflora as the pollen parent to produce a first-generation hybrid cultivar that inherits the KSN mutant gene described in claim 1 from the rose cultivar Summer Snow (registered trademark); backcrossing the pollen of the first-generation hybrid cultivar with Rosa multiflora; backcrossing the pollen of the first-generation backcross cultivar with Rosa multiflora; backcrossing the pollen of the second-generation backcross cultivar with Rosa multiflora; and crossing the third-generation backcross cultivars with each other.

11. A repeat-blooming rose plant or a part thereof having the KSN mutant gene described in claim 1 in a homozygous state.

Images