KASP-SNP molecular marker primer for identifying modern Chinese rose variety, composition and application of KASP-SNP molecular marker primer

Abstract

The invention relates to a KASP-SNP molecular marker primer for identifying modern Chinese rose varieties, a composition and application of the KASP-SNP molecular marker primer and the composition. The molecular marker primer comprises KASP-SNP1-KASP-SNP35, and nucleotide sequences of the KASP-SNP1-KASP-SNP35 are shown as SEQ ID NO.1-105. On the basis of whole genome re-sequencing data, 35 high-polymorphism SNP loci are screened out, specific primers are designed, fluorescence signals of a sample to be detected are detected through a KASP technical platform, genotypes can be automatically interpreted, and a DNA fingerprint spectrum unique to varieties can be constructed. The marking system has the advantages of high flux, strong specificity, objective and stable result, no environmental influence and the like, and can be used for precise identification of modern Chinese rose varieties, genetic relationship and genetic diversity analysis, DUS test assistance and molecular marker-assisted selective breeding.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to a KASP-SNP molecular marker primer, composition, and application for identifying modern rose varieties. Background Technology

[0002] Modern garden roses (primarily *Rosa hybrida*) are a large horticultural group within the genus *Rosa* of the family Rosaceae, developed through long-term and complex hybridization and selection. Their ancestors mainly originated from various wild species of the *Rosa* genus in China, Europe, and the Middle East. Modern garden roses are renowned worldwide for their rich variety of flower shapes (such as high-centered, cup-shaped blooms), vibrant colors (covering all hues except pure blue), continuous flowering (multi-season blooming), and diverse plant forms (shrubs, vines, miniatures, etc.). They are among the most important ornamental plants globally, widely used in landscaping, cut flower production, potted plants, and garden design, earning them the title of "Queen of Flowers" and holding an irreplaceable core position in the economic, cultural, and horticultural industries.

[0003] As one of the most important commercial ornamental flowers globally and in China, the protection of variety rights for modern roses has long been a focus of attention and has been included in the "List of Protected Plant Varieties of the People's Republic of China (Forestry Section)". With the continuous expansion and upgrading of the domestic ornamental horticulture market, the market demand for new rose varieties with independent intellectual property rights and excellent traits is increasingly urgent, and breeders' awareness of new variety protection and rights enforcement has also significantly increased. However, the phenotypic traits among modern rose varieties are easily affected by the environment, and relying solely on traditional DUS testing for variety specificity identification sometimes has limitations such as long cycles and strong subjectivity. Therefore, developing a high-resolution, stable KASP-SNP molecular marker system suitable for the identification of modern rose varieties, and constructing accurate DNA fingerprint profiles based on it, has significant technical support and practical application value for efficiently and objectively assisting DUS testing, clarifying variety identity, protecting the legitimate rights and interests of breeders, standardizing the seedling market, and promoting the healthy and orderly development of the rose industry. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of this invention is to provide a KASP-SNP molecular marker primer, composition and application for identifying modern rose varieties.

[0005] The technical solution adopted by this invention to solve its technical problem is: a KASP-SNP molecular marker primer for identifying modern rose varieties, wherein the modern roses and their closely related species or important wild parents of the genus Rosa used for hybridization breeding include, but are not limited to: wild rose (Rosa multiflora), French rose (Rosa gallica), Damask rose (Rosa damascena), and glossy-leaved rose (Rosa wichurana).

[0006] The KASP-SNP molecular markers used to identify modern rose varieties include KASP-SNP1, KASP-SNP2, KASP-SNP3, KASP-SNP4, KASP-SNP5, KASP-SNP6, KASP-SNP7, KASP-SNP8, KASP-SNP9, KASP-SNP10, KASP-SNP11, KASP-SNP12, KASP-SNP13, KASP-SNP14, KASP-SNP15, KASP-SNP16, and KASP-SNP17. 17. KASP-SNP18, KASP-SNP19, KASP-SNP20, KASP-SNP21, KASP-SNP22, KASP-SNP23, KASP-SNP24, KASP-SNP25, KASP-SNP2 6. KASP-SNP27, KASP-SNP28, KASP-SNP29, KASP-SNP30, KASP-SNP31, KASP-SNP32, KASP-SNP33, KASP-SNP34, KASP-SNP35;

[0007] The KASP-SNP molecular marker primer sequences for identifying modern rose varieties are as follows:

[0008] Primers for the molecular marker KASP-SNP1:

[0009] SEQ ID NO.1: KASP-SNP1-F1:5 '-aaattcttcatatttgtttacttttgtacA-3';

[0010] SEQ ID NO.2: KASP-SNP1-F2:5 '-aaattcttcatatttgtttacttttgtacG-3';

[0011] SEQ ID NO.3: KASP-SNP1-R:5'-agtctatgaccgtagaagagtTG-3';

[0012] Primers for the molecular marker KASP-SNP2:

[0013] SEQ ID NO.4: KASP-SNP2-F1:5 '-gggataaacgattgaggtgtcctA-3';

[0014] SEQ ID NO.5: KASP-SNP2-F2:5 '-gggataaacgattgaggtgtcctG-3';

[0015] SEQ ID NO.6: KASP-SNP2-R:5'-agtccttacctaatacaataccgacT-3';

[0016] Primers for the molecular marker KASP-SNP3:

[0017] SEQ ID NO.7: KASP-SNP3-F1:5'-aCAatttgggacAtataatttacgtaaagT-3';

[0018] SEQ ID NO.8: KASP-SNP3-F2:5'-aCAatttgggacAtataatttacgtaaagC-3';

[0019] SEQ ID NO.9: KASP-SNP3-R:5'-tctgaaggaacatgttcattaagttT-3';

[0020] Primers for the molecular marker KASP-SNP4:

[0021] SEQ ID NO.10: KASP-SNP4-F1:5'-cccttttcactccaaaagattgagT-3';

[0022] SEQ ID NO.11: KASP-SNP4-F2:5'-cccttttcactccaaaagattgagC-3';

[0023] SEQ ID NO.12: KASP-SNP4-R:5'-tgagtctggatttatcttttggcaT-3';

[0024] Primers for the molecular marker KASP-SNP5:

[0025] SEQ ID NO.13: KASP-SNP5-F1:5'-tttccagactgctgatcctA-3';

[0026] SEQ ID NO.14: KASP-SNP5-F2:5'-tttccagactgctgatcctG-3';

[0027] SEQ ID NO.15: KASP-SNP5-R:5'-tgctttgtggagtttctaATtTTCtA-3';

[0028] Primers for the molecular marker KASP-SNP6:

[0029] SEQ ID NO.16: KASP-SNP6-F1:5'-gtccggactatctgttgaagtctA-3';

[0030] SEQ ID NO.17: KASP-SNP6-F2:5'-gtccggactatctgttgaagtctT-3';

[0031] SEQ ID NO.18: KASP-SNP6-R:5'-tggagcttgtgaaagagacG-3';

[0032] Primers for the molecular marker KASP-SNP7:

[0033] SEQ ID NO.19: KASP-SNP7-F1:5'-tgcacatggaAgggttcataA-3';

[0034] SEQ ID NO.20: KASP-SNP7-F2:5'-tgcacatggaAgggttcataG-3';

[0035] SEQ ID NO.21: KASP-SNP7-R:5'-actttggagggctAGCTTCT-3';

[0036] Primers for the molecular marker KASP-SNP8:

[0037] SEQ ID NO.22: KASP-SNP8-F1:5'-aGttgccatgaaatcattgatccT-3';

[0038] SEQ ID NO.23: KASP-SNP8-F2:5'-aGttgccatgaaatcattgatccC-3';

[0039] SEQ ID NO.24: KASP-SNP8-R:5'-ggcaTtcTacttaagtgatcttcttctC-3';

[0040] Primers for the molecular marker KASP-SNP9:

[0041] SEQ ID NO.25: KASP-SNP9-F1:5'-ccGctcttctcttcaTcttacaaaA-3';

[0042] SEQ ID NO.26: KASP-SNP9-F2:5'-ccGctcttctcttcaTcttacaaaT-3';

[0043] SEQ ID NO.27: KASP-SNP9-R:5'-AgtgaattggtttcaaaacttgTTGG-3';

[0044] Primers for the molecular marker KASP-SNP10:

[0045] SEQ ID NO.28: KASP-SNP10-F1:5'-cagtGtCctaaactactagaCtgtctA-3';

[0046] SEQ ID NO.29: KASP-SNP10-F2:5'-cagtGtCctaaactactagaCtgtctC-3';

[0047] SEQ ID NO.30: KASP-SNP10-R:5'-GTGACTCtattggtttTacatttcacTaC-3'.

[0048] Primers for the molecular marker KASP-SNP11:

[0049] SEQ ID NO.31: KASP-SNP11-F1:5'-tcaatatgaggtgaatattaatggtaacaA-3';

[0050] SEQ ID NO.32: KASP-SNP11-F2:5'-tcaatatgaggtgaatattaatggtaacaG-3';

[0051] SEQ ID NO.33: KASP-SNP11-R:5'-gcAaatataattagtccacatgcatacaG-3';

[0052] Primers for the molecular marker KASP-SNP12:

[0053] SEQ ID NO.34: KASP-SNP12-F1:5 '-caatttccctAtttctaattaggttgtttT-3';

[0054] SEQ ID NO.35: KASP-SNP12-F2:5'-caatttccctAtttctaattaggttgtttC-3';

[0055] SEQ ID NO.36: KASP-SNP12-R:5'-gcaacctagctaTAtccaaaactagC-3';

[0056] Primers for the molecular marker KASP-SNP13:

[0057] SEQ ID NO.37: KASP-SNP13-F1:5'-ggtgcgaggcaagagacT-3';

[0058] SEQ ID NO.38: KASP-SNP13-F2:5'-ggtgcgaggcaagagacC-3';

[0059] SEQ ID NO.39: KASP-SNP13-R:5'-ttgtcaatatcgttGgTTAGAAcAttA-3';

[0060] Primers for the molecular marker KASP-SNP14:

[0061] SEQ ID NO.40: KASP-SNP14-F1:5'-aacttGaacacAAAaCCCAAAaT-3';

[0062] SEQ ID NO.41: KASP-SNP14-F2:5'-aacttGaacacAAAaCCCAAAaC-3';

[0063] SEQ ID NO.42: KASP-SNP14-R:5'-attgaaggaaacctttatTTTActtttgtaT-3';

[0064] Primers for the molecular marker KASP-SNP15:

[0065] SEQ ID NO.43: KASP-SNP15-F1:5'-acagtcaaacattctcaccttcT-3';

[0066] SEQ ID NO.44: KASP-SNP15-F2:5'-acagtcaaacattctcaccttcC-3';

[0067] SEQ ID NO.45: KASP-SNP15-R:5'-tgcatttatgtgagtaaaaactgttgatT-3';

[0068] Primers for the molecular marker KASP-SNP16:

[0069] SEQ ID NO.46: KASP-SNP16-F1:5'-aattgatctacttaatgtatcgatcgaA-3';

[0070] SEQ ID NO.47: KASP-SNP16-F2:5'-aattgatctacttaatgtatcgatcgaG-3';

[0071] SEQ ID NO.48: KASP-SNP16-R:5'-tctgtaaaataaggggtGTActccaT-3';

[0072] Primers for the molecular marker KASP-SNP17:

[0073] SEQ ID NO.49: KASP-SNP17-F1:5'-agctagatccaccaaatcccA-3';

[0074] SEQ ID NO.50: KASP-SNP17-F2:5'-agctagatccaccaaatcccG-3';

[0075] SEQ ID NO.51: KASP-SNP17-R:5'-CggttggtggttcgattggA-3';

[0076] Primers for the molecular marker KASP-SNP18:

[0077] SEQ ID NO.52: KASP-SNP18-F1:5'-cagggcgttttgtgagaagT-3';

[0078] SEQ ID NO.53: KASP-SNP18-F2:5'-cagggcgttttgtgagaagC-3';

[0079] SEQ ID NO.54: KASP-SNP18-R:5'-agcaccaccaCAGatTGaaAA-3';

[0080] Primers for the molecular marker KASP-SNP19:

[0081] SEQ ID NO.55: KASP-SNP19-F1:5'-gaacaccgagtcttgccgT-3';

[0082] SEQ ID NO.56: KASP-SNP19-F2:5'-gaacaccgagtcttgccgC-3';

[0083] SEQ ID NO.57: KASP-SNP19-R:5'-ggcttttagcaccgaagcG-3';

[0084] Primers for the molecular marker KASP-SNP20:

[0085] SEQ ID NO.58: KASP-SNP20-F1:5'-gggagactttacctaagaaatgggtA-3';

[0086] SEQ ID NO.59: KASP-SNP20-F2:5'-gggagactttacctaagaaatgggtC-3';

[0087] SEQ ID NO.60: KASP-SNP20-R:5'-tcagatctattcccatgttccG-3';

[0088] Primers for the molecular marker KASP-SNP21:

[0089] SEQ ID NO .61:KASP-SNP21-F1:5 '-ggttttttattagctaaaacggtaaagtgA-3';

[0090] SEQ ID NO .62:KASP-SNP21-F2:5 '-ggttttttattagctaaaacggtaaagtgG-3';

[0091] SEQ ID NO .63:KASP-SNP21-R:5′-tcacaagttctaccaGTGTGaaA-3′;

[0092] Description of the active ingredient of KASP-SNP22:

[0093] SEQ ID NO .64:KASP-SNP22-F1:5′-CCcTGcaatccatttggcaaaA-3′;

[0094] SEQ ID NO .65:KASP-SNP22-F2:5′-CCcTGcaatccatttggcaaaC-3′;

[0095] SEQ ID NO .66:KASP-SNP22-R:5′-tgctGtttataatggaAagcagTGTT-3′;

[0096] Description of the active ingredient of KASP-SNP23:

[0097] SEQ ID NO .67:KASP-SNP23-F1:5′-tgcataaacgtttctttgaagggtT-3′;

[0098] SEQ ID NO .68:KASP-SNP23-F2:5′-tgcataaacgtttctttgaagggtA-3′;

[0099] SEQ ID NO .69:KASP-SNP23-R:5′-ccatttctgcattctcaagAGTCAA-3′;

[0100] Recommendations for the KASP-SNP24 catalyst:

[0101] SEQ ID NO .70:KASP-SNP24-F1:5′-ccaggtatGttggaagtgggA-3′;

[0102] SEQ ID NO.71: KASP-SNP24-F2:5'-ccaggtatGttggaagtgggG-3';

[0103] SEQ ID NO.72: KASP-SNP24-R:5'-gtgaggTagttattcCaaacgtcA-3';

[0104] Primers for the molecular marker KASP-SNP25:

[0105] SEQ ID NO.73: KASP-SNP25-F1:5'-AgacttaattgttgaaacttgaaaggT-3';

[0106] SEQ ID NO.74: KASP-SNP25-F2:5'-AgacttaattgttgaaacttgaaaggC-3';

[0107] SEQ ID NO.75: KASP-SNP25-R:5'-gggaattataatccagccaaaggtC-3';

[0108] Primers for the molecular marker KASP-SNP26:

[0109] SEQ ID NO.76: KASP-SNP26-F1:5'-TCtTTCTTtcattactagttgcaaatgT-3';

[0110] SEQ ID NO.77: KASP-SNP26-F2:5'-TCtTTCTTtcattactagttgcaaatgC-3';

[0111] SEQ ID NO.78: KASP-SNP26-R:5'-aGacatcatcatcaAaaCGACCaT-3';

[0112] Primers for the molecular marker KASP-SNP27:

[0113] SEQ ID NO.79: KASP-SNP27-F1:5'-ACACTagtcttactttctgaatccaaG-3';

[0114] SEQ ID NO.80: KASP-SNP27-F2:5'-ACACTagtcttactttctgaatccaaC-3';

[0115] SEQ ID NO.81: KASP-SNP27-R:5'-acttgctgtcttctGAGTtACAG-3';

[0116] Primers for the molecular marker KASP-SNP28:

[0117] SEQ ID NO.82: KASP-SNP28-F1:5'-aGTcGAtctattttcCattaacatcaA-3';

[0118] SEQ ID NO.83: KASP-SNP28-F2:5'-aGTcGAtctattttcCattaacatcaC-3';

[0119] SEQ ID NO.84: KASP-SNP28-R:5'-GTCCAGTGTaCAcctcaACaTATataC-3';

[0120] Primers for the molecular marker KASP-SNP29:

[0121] SEQ ID NO.85: KASP-SNP29-F1:5'-caaccgccgttgttaaatgtT-3';

[0122] SEQ ID NO.86: KASP-SNP29-F2:5'-caaccgccgttgttaaatgtC-3';

[0123] SEQ ID NO.87: KASP-SNP29-R:5'-aggtactgaacatgatgtctctatatcA-3';

[0124] Primers for the molecular marker KASP-SNP30:

[0125] SEQ ID NO.88: KASP-SNP30-F1:5 '-tgttactagggttaggtgttatggT-3';

[0126] SEQ ID NO.89: KASP-SNP30-F2:5 '-tgttactagggttaggtgttatggA-3';

[0127] SEQ ID NO.90: KASP-SNP30-R:5'-AgagaatttttgatgcgtcaagT-3';

[0128] Primers for the molecular marker KASP-SNP31:

[0129] SEQ ID NO.91: KASP-SNP31-F1:5'-tcaggaaacaagcccgaaaaA-3';

[0130] SEQ ID NO.92: KASP-SNP31-F2:5'-tcaggaaacaagcccgaaaaG-3';

[0131] SEQ ID NO.93: KASP-SNP31-R:5'-tgcatttgaagatgattctttatgtgtT-3';

[0132] Primers for the molecular marker KASP-SNP32:

[0133] SEQ ID NO.94: KASP-SNP32-F1:5'-accaacaatactctgcgagaA-3';

[0134] SEQ ID NO.95: KASP-SNP32-F2:5'-accaacaatactctgcgagaG-3';

[0135] SEQ ID NO.96: KASP-SNP32-R:5'-gcaggccacatatTcctaaatcA-3';

[0136] Primers for the molecular marker KASP-SNP33:

[0137] SEQ ID NO.97: KASP-SNP33-F1:5'-ctcagttgaGgtgttgacactA-3';

[0138] SEQ ID NO.98: KASP-SNP33-F2:5'-ctcagttgaGgtgttgacactG-3';

[0139] SEQ ID NO.99: KASP-SNP33-R:5'-accAgcgttagggttttctG-3';

[0140] Primers for the molecular marker KASP-SNP34:

[0141] SEQ ID NO.100: KASP-SNP34-F1:5'-TGaGgtcataattttgacaaGtgcA-3';

[0142] SEQ ID NO.101: KASP-SNP34-F2:5'-TGaGgtcataattttgacaaGtgcT-3';

[0143] SEQ ID NO.102: KASP-SNP34-R:5'-tcacActCAtttcctctcaacT-3';

[0144] Primers for the molecular marker KASP-SNP35:

[0145] SEQ ID NO.103: KASP-SNP35-F1:5'-tctcttcttttgatttggccgT-3';

[0146] SEQ ID NO.104: KASP-SNP35-F2:5'-tctcttcttttgatttggccgA-3';

[0147] SEQ ID NO.105: KASP-SNP35-R:5'-cgcccaattactttttGTAtAtTtATTTTG-3'.

[0148] As a further improvement of the present invention, the method for screening KASP-SNP molecular marker primers includes the following steps: extracting high-quality genomic DNA from the tested modern rose varieties, screening SNP sites with high polymorphism information content based on publicly available high-quality SNP datasets from whole-genome retesting, and designing KASP-SNP primers using bioinformatics tools, then verifying and screening candidate markers for polymorphism using a standardized KASP-SNP genotyping experimental procedure, and finally determining a set of core KASP-SNP markers for subsequent analysis based on the genotyping success rate, allele frequency distribution, and their ability to distinguish between varieties.

[0149] As a further improvement of the present invention: the total DNA of the modern rose variety is extracted by using a modified CTAB method to extract the DNA from fresh leaves.

[0150] As a further improvement of the present invention: the selection of KASP-SNP sites and the design and screening of primers based on the SNP database obtained from the resequencing of 233 Rosa species are carried out. Specifically, from the 114 sequences containing KASP-SNP sites obtained, 70 pairs of primers were designed according to the following principles, wherein the selection principles for KASP-SNP primers are as follows:

[0151] 1) Allele-specific forward primers are designed to be 30–45 nucleotides in length, and their 3' terminal bases must be precisely complementary to the allele of the target SNP;

[0152] 2) The common reverse primer is designed to be 18-25 nucleotides in length, and the GC content of all primers is controlled at 40%-60%, while avoiding continuous single base repetitions;

[0153] 3) The calculated melting temperature (Tm value) of the primers should be between 55℃ and 65℃, and the difference in Tm value between the same set of primers should not exceed 2℃;

[0154] 4) Primer design must be rigorously evaluated using bioinformatics tools to ensure the absence of primer dimers, hairpin structures, and non-specific amplification sites.

[0155] As a further improvement of the present invention: the polymorphism verification and screening of candidate KASP-SNP markers is specifically performed as follows: using a standardized DNA sample containing 193 modern rose varieties on a real-time quantitative PCR instrument, amplification and endpoint fluorescence signal acquisition are carried out according to the standard KASP-SNP reaction system and procedure. By analyzing the clustering and separation of each data point in the allele discrimination scatter plot, the markers are initially screened based on the genotyping clarity (Call Rate ≥ 95%), intra-cluster aggregation degree, and consistency between the cluster and the expected genotype. For markers that pass the initial screening, their polymorphism information content is further calculated, their allele frequency distribution in the population is evaluated, and the sample size is expanded to verify their genotyping stability and repeatability, thereby screening out core KASP-SNP markers with high resolution and high discriminative power.

[0156] The present invention also includes a KASP-SNP molecular marker primer composition for identifying modern rose varieties, the composition comprising the KASP-SNP molecular markers KASP-SNP1 to KASP-SNP35 described above, the sequences of the molecular marker primers KASP-SNP1 to KASP-SNP35 being shown in the sequence listing SEQ ID NO. 1 to SEQ ID NO. 105, respectively.

[0157] This invention also includes the application of KASP-SNP molecular marker primers, as described above, in the analysis of phylogenetic relationships in rose germplasm resources, DUS testing, and marker-assisted breeding. For example, due to their long breeding history and complex hybrid lineages, modern roses exhibit numerous synonyms or homonyms. KASP-SNP markers can accurately resolve the true genetic relationships between famous varieties such as "Peace," "Princess Monaco," and "Iceberg," and their sister lines or bud mutations, providing molecular evidence for clarifying varietal lineages and classification. Secondly, given the large number of new rose varieties and their phenotypes' susceptibility to environmental influences, a high-throughput DNA fingerprint database constructed using KASP-SNP markers can rapidly and objectively screen for similar varieties in DUS testing, significantly improving testing efficiency and accuracy. Finally, by association analysis with important ornamental or resistance traits, the selected KASP-SNP markers can be used for early tracking and assisted selection of target genes, thereby directional aggregation of superior alleles, accelerating the breeding of new varieties with specific flower colors, plant shapes, fragrances or disease resistance, and providing key technical support for the efficient and precise breeding of modern roses.

[0158] Compared with the prior art, the beneficial effects of the present invention are:

[0159] The KASP-SNP molecular marker primers and their composition for identifying modern rose varieties of this invention have the advantages of high throughput, strong specificity, objective and automated result interpretation, compatibility with high-throughput platforms, and relatively low cost per detection compared with conventional molecular marker technology. The identification of the selected rose varieties is not limited by the plant's growth stage, cultivation environment, or sampling site. DNA extraction and typing can be performed on young leaves, mature leaves, petals, and even dried or silica gel-dried samples without affecting the accuracy and stability of the identification results.

[0160] In the screening and verification of KASP-SNP molecular marker primers for identifying modern rose varieties in this invention, a high-throughput genotyping platform based on real-time fluorescence PCR was adopted. This technology system has the characteristics of closed-tube detection, no need for subsequent electrophoresis, automatic allele interpretation, and flexible and scalable throughput. It plays a key role in establishing a rapid, accurate, and standardized DNA fingerprinting technology for rose varieties, and significantly shortens the variety identification cycle and improves testing efficiency. Attached Figure Description

[0161] To more clearly illustrate the technical solution, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0162] Figure 1This is a typing diagram of some modern rose varieties using some KASP-SNP primer pairs of this invention.

[0163] Figure 2 This is a cluster diagram of the DUS phenotypic data of 193 modern rose varieties in this invention.

[0164] Figure 3 This is a scatter plot showing the allele discrimination of genotyping of 193 modern rose varieties based on 35 KASP-SNP markers, according to the present invention.

[0165] Figure 4 This is a scatter principal component analysis plot of allele discrimination for genotyping of 193 modern rose varieties based on 35 KASP-SNP markers, according to the present invention. Detailed Implementation

[0166] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0167] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0168] Modern roses (Rosa hybrida) are core ornamental flowers bred through long-term, complex hybridization of multiple wild species in the Rosa genus. Revered as the "Queen of Flowers," they hold significant economic, cultural, and varietal conservation value in the global horticulture industry. With the rapid development of my country's ornamental horticulture market, new varieties with independent intellectual property rights are constantly emerging, making the need for varietal rights protection and precise identification increasingly urgent.

[0169] However, modern rose varieties are numerous and have complex genetic backgrounds. Traditional identification methods, mainly relying on morphological observation and DNA fingerprinting (DUS) testing, have limitations such as long cycles, high subjectivity, and susceptibility to environmental factors, making it difficult to meet the needs for rapid, accurate, and standardized variety identification. The ornamental traits of modern roses (such as flower color, plant shape, and flowering habits) vary under different cultivation conditions or growth stages, resulting in poor repeatability and stability of phenotype-based DUS testing. Because modern roses are hybrids of multiple wild species, their genomes are highly heterozygous, and the phylogenetic relationships between varieties are close. Conventional molecular markers (such as RAPD and AFLP) have limitations in terms of resolution, repeatability, and high throughput. Currently, molecular marker identification methods for rose varieties have not yet formed a standardized, large-scale applicable technical system, making it difficult to achieve rapid and accurate identification of a large number of varieties. With the continuous emergence of new rose varieties and the internationalization of the market, there is an urgent need to establish a high-resolution, repeatable, and automated variety DNA fingerprinting system to support variety rights confirmation, seedling market supervision, and breeder rights protection.

[0170] To address the aforementioned issues, this invention provides a KASP-SNP molecular marker primer, composition, and its application for identifying modern rose varieties. It features high throughput, strong specificity, objective and automated result interpretation, compatibility with high-throughput platforms, and relatively low cost per test. The identification of selected rose varieties is not limited by plant growth stage, cultivation environment, or sampling location. DNA extraction and typing can be performed on young leaves, mature leaves, petals, and even dried or silica gel-preserved samples without affecting the accuracy and stability of the identification results.

[0171] Example 1:

[0172] Extraction of total DNA from modern rose varieties:

[0173] 1) The 193 modern rose varieties selected were provided by the Yunnan Flower Technology Extension Center;

[0174] 2) Using the selected modern rose varieties as materials, total DNA was extracted using the CTAB method. The specific operation process is as follows:

[0175] ① Take a 2mL centrifuge tube, add 0.1g of ground modern rose powder, and add 700mL of CTAB extract (preheated to 65℃ beforehand);

[0176] ② Place the ground sample in a 65℃ water bath for 20 min to 1 h, shaking and inverting the centrifuge tube every 10 min during the process. After the water bath, remove the centrifuge tube and centrifuge at 12000×g for 10 min.

[0177] ③ Carefully aspirate the supernatant (about 600 μL), transfer it to a new 2.0 mL centrifuge tube, add an equal volume (600 μL) of chloroform / isoamyl alcohol (24:1) solution, vortex to mix, and centrifuge at 12000×g for 10 min.

[0178] ④ Transfer the supernatant to a new 2.0 mL centrifuge tube, add an equal volume of isopropanol, slowly invert to mix, and store at -20°C for at least half an hour to precipitate genomic DNA, or store at -20°C overnight.

[0179] ⑤ Remove the sample from the -20℃ refrigerator, centrifuge at 12000×g for 10min, and discard the supernatant;

[0180] ⑥ Add 1 mL of pre-cooled 70% ethanol to a 2.0 mL centrifuge tube, wash the precipitate, centrifuge at 12000×g for 10 min, and discard the supernatant;

[0181] ⑦ Repeat step ⑥ once;

[0182] ⑧ Open the cap of the centrifuge tube, carefully absorb the ethanol from the tube wall with a sterile filter paper strip, and place the centrifuge tube in a fume hood to dry for 5-10 minutes;

[0183] ⑨ Dissolve in 50-100 μL of 1×TE solution or ddH2O and store at -20℃ for later use.

[0184] Example 2:

[0185] KASP-SNP molecular marker primers for screening and identifying modern rose varieties:

[0186] 1) Based on 233 publicly available whole genome reference sequences and retesting data of the Rosa genus, bioinformatics methods were used to scan and screen for SNP sites with high polymorphic potential across the whole genome;

[0187] Primers were designed for candidate SNP loci using professional KASP-SNP primer design software (such as Kraken or PrimerPicker), and initial evaluation was performed based on multiple screening criteria. From a large number of candidate loci, KASP-SNP primer combinations suitable for variety identification were successfully designed and screened according to the following core principles:

[0188] ① Allele-specific primers are typically 30-45 nucleotides in length, and their 3' end bases must precisely correspond to the allele genotype of the target SNP to ensure specific recognition;

[0189] ② The length of a common reverse primer is generally 18-25 nucleotides. The overall GC content of all primers should be controlled between 40% and 60%, and consecutive single-base repeats or sequences that may form stable secondary structures should be avoided.

[0190] ③ The calculated melting temperature (Tm value) of the primers should be between 55℃ and 65℃, and the Tm values ​​of the two allele-specific primers used in the same reaction should be as close as possible, with a difference of no more than 2℃, in order to ensure consistent amplification efficiency.

[0191] ④ Primer sequences must be rigorously evaluated using bioinformatics tools to ensure there is no significant risk of primer dimer or hairpin formation, and no nonspecific binding sites, in order to guarantee the specificity and reliability of amplification;

[0192] Sample amplification: The total reaction volume is 5 μL, consisting of 2.5 μL DNA template, 2.5 μL 2×PAGE Master Mix, 0.008 μL F1 and F2 solutions, and 0.02 μL of [unspecified solution]. Each experiment should include 4-6 negative controls, where the 2.5 μL DNA template is replaced with 2.5 μL DEPC water. (Reagents used are from Shanghai Sangon Biotech Co., Ltd.)

[0193] The PCR reaction program is as follows: 94℃ pre-denaturation for 15 min, 1 cycle; 94℃ denaturation for 20 s, 10 cycles; 65-57℃ annealing / extension for 60 s, 10 cycles; 94℃ denaturation for 20 s, 30 cycles; 65-57℃ annealing / extension for 60 s, 30 cycles; store at 4℃. The annealing temperature is determined according to each primer.

[0194] KASP-SNP genotyping reaction and initial screening of fluorescence signals. KASP-SNP amplification was performed on a real-time quantitative PCR instrument. After the reaction, the fluorescence signal values ​​of the FAM and HEX channels were collected by the instrument's built-in endpoint fluorescence detection module to generate a preliminary allele discrimination scatter plot for initial screening of genotyping quality.

[0195] Markers with clear genotyping, obvious cluster separation of data points in scatter plots, and high genotyping success rate (call rate) were selected for a large-sample-based secondary screening and validation stage. By calculating their polymorphism information content, observing allele frequency distribution, and evaluating their discriminative ability in the population, core KASP-SNP markers with high polymorphism and high stability were screened out. For example... Figure 1 As shown, the KASP-SNP molecular marker primers and their composition for identifying modern rose varieties of the present invention have the characteristics of high throughput, automated interpretation, strong specificity, and flexible and scalable throughput compared with other molecular markers.

[0196] The specific steps for automating data interpretation and statistical analysis of KASP-SNP genotyping results are as follows:

[0197] 1. Data file preparation:

[0198] ① Export the raw data file containing the endpoint fluorescence signals of all samples in the FAM and HEX channels from the real-time quantitative PCR instrument;

[0199] ② Prepare a sample information mapping table, which includes metadata such as sample number, variety name, and category;

[0200] ③Prepare a detection information file containing the KASP-SNP marker name and its corresponding allele fluorescent marker combination;

[0201] 2. Automated allele interpretation:

[0202] ① Import the above files using the corresponding genotyping analysis software (such as Kluster Caller, SNPviewer, etc.);

[0203] ② The software automatically performs normalization based on a preset algorithm and interprets alleles according to the distribution of fluorescent signal clusters, providing genotyping results (AA, AB, BB, or No Call) for each sample at each marker site.

[0204] 3. Quality control and statistical analysis of typing results:

[0205] ① Establish and apply unified quality control standards, such as removing samples or markers with a typing success rate (Call Rate) of less than 95%;

[0206] ② Calculate the allele frequency, observed heterozygosity, expected heterozygosity, and polymorphism information content for each marker;

[0207] ③ Export the high-quality final typing data matrix for subsequent phylogenetic analysis, population structure analysis and fingerprint map construction.

[0208]

[0209] Table 1. Genetic diversity information of 35 KASP-SNP molecular markers

[0210]

[0211]

[0212] Table 2 Fingerprint profiles of some modern rose varieties marked with KASP-SNP1-6

[0213]

[0214]

[0215] Table 3 Fingerprint profiles of some modern rose varieties marked with KASP-SNP7-12

[0216]

[0217]

[0218] Table 4. Fingerprint patterns of some modern rose varieties marked with KASP-SNP13-18

[0219]

[0220]

[0221] Table 5. Fingerprint patterns of some modern rose varieties marked with KASP-SNP19-24.

[0222]

[0223]

[0224] Table 5. Fingerprint patterns of some modern rose varieties marked with KASP-SNP25-30.

[0225]

[0226]

[0227] Table 6. Fingerprint patterns of some modern rose varieties marked with KASP-SNP31-35.

[0228] As shown in Tables 2-6, the 35 KASP-SNP molecular marker primer combinations and germplasm identification method described in this invention can accurately identify 193 modern rose varieties with an accuracy rate of 100%.

[0229] Due to the large number of KASP-SNP molecular marker primers and modern rose varieties, only the genotyping results of some markers for some varieties are shown, such as... Figure 1 As shown in the figure, the KASP-SNP molecular marker primers described in this invention have good typing effect and strong usability.

[0230] like Figure 3As shown in Table 2, the genetic relationship analysis and cluster diagram of modern roses based on KASP-SNP molecular markers are presented. The analysis, combined with Table 2, shows that the average polymorphism information content of the selected KASP-SNP primers is 0.46, indicating that the selected markers have high polymorphism and good genetic identification ability for modern rose varieties, suitable for variety identification and kinship analysis. From the clustering results, the 193 tested modern rose varieties can be clearly divided into five main groups: Group I is characterized by strong double petals and rich fragrance; Group II is characterized by upright plant form and standard flower shape; Group III is characterized by clustered flowering and strong disease resistance; and Group IV is characterized by compact plants or creeping branches. This molecular clustering result is consistent with... Figure 2 The cluster analysis based on DUS phenotypic data shown is largely consistent, further validating the effectiveness and reliability of KASP-SNP markers in modern rose genetic analysis.

[0231] In summary, the KASP-SNP molecular marker primers of this invention can be used for assisted selection breeding to achieve early selection at the seedling stage, thereby accelerating the breeding process of modern roses.

[0232] Working principle of the invention:

[0233] Based on the principle of competitive allele-specific PCR (KASP), specific primer combinations were designed for 35 highly polymorphic SNP loci screened across the entire genome of modern roses. Each locus contains two allele-specific forward primers (their 3' ends are precisely complementary to the two SNP alleles, and their 5' ends are linked to unique tag sequences) and one universal reverse primer. During PCR amplification, the sample genomic DNA first binds to and extends with the perfectly matched allele-specific primers, integrating the tag sequence into the amplification product and releasing a specific fluorescent signal. After the reaction, the homozygous or heterozygous genotype of each SNP locus can be automatically determined by the combination of FAM and HEX fluorescence signals through endpoint fluorescence detection. This transforms the genotype data of the 35 labeled genotypes into a high-resolution DNA fingerprint map unique to each variety. This not only enables accurate and rapid identification of modern rose varieties and detection of seedling purity, but also reveals the kinship between varieties and the genetic structure of the population through genetic distance analysis, fundamentally overcoming the technical limitations of traditional morphological identification methods, such as being greatly affected by environmental factors, being highly subjective, and having a long cycle.

[0234] The main functions of this invention are:

[0235] Using 35 KASP-SNP molecular marker primer combinations, a high-resolution DNA fingerprint map unique to each variety is constructed by detecting the genotype of specific SNP loci in the modern rose genome. This enables rapid and accurate identification of modern rose varieties, effectively solving the problem of synonyms or homonyms. Based on the genotyping data of KASP-SNP markers, genetic distance between varieties can be calculated to conduct phylogenetic analysis, population genetic structure research, and cluster analysis of modern rose germplasm resources, providing molecular evidence for clarifying the hybrid lineage and genetic background of varieties. In the DUS test of new plant varieties, this marker system can be used to quickly screen similar varieties and construct a DNA fingerprint database of reference varieties, improving the efficiency and objectivity of specificity, uniformity, and stability testing, overcoming the limitations of traditional morphological testing, such as long testing cycles and susceptibility to environmental influences. Through association analysis with important horticultural traits, the screened KASP-SNP markers can be used for early tracking and assisted selection of target traits, enabling early screening at the seedling stage and accelerating the breeding of new rose varieties with excellent traits such as specific flower color, plant type, and disease resistance.

[0236] In summary, after reading this invention document, those skilled in the art can make various other corresponding modifications to the technical solutions and concepts based on this invention without creative mental effort, and all of these modifications fall within the scope of protection of this invention.

Claims

1. A KASP-SNP molecular marker primer for identifying modern rose varieties, characterized in that, The modern rose varieties mentioned include wild rose, French rose, Damask rose, and glossy rose; The KASP-SNP molecular markers used to identify modern rose varieties include KASP-SNP1 to KASP-SNP35. The KASP-SNP molecular marker primer sequences for identifying modern rose varieties are as follows: Primers for the molecular marker KASP-SNP1: SEQ ID NO.1: KASP-SNP1-F1:5 '-aaattcttcatatttgtttacttttgtacA-3'; SEQ ID NO.2: KASP-SNP1-F2:5 '-aaattcttcatatttgtttacttttgtacG-3'; SEQ ID NO.3: KASP-SNP1-R:5'-agtctatgaccgtagaagagtTG-3'; Primers for the molecular marker KASP-SNP2: SEQ ID NO.4: KASP-SNP2-F1:5 '-gggataaacgattgaggtgtcctA-3'; SEQ ID NO.5: KASP-SNP2-F2:5 '-gggataaacgattgaggtgtcctG-3'; SEQ ID NO.6: KASP-SNP2-R:5'-agtccttacctaatacaataccgacT-3'; Primers for the molecular marker KASP-SNP3: SEQ ID NO.7: KASP-SNP3-F1:5'-aCAatttgggacAtataatttacgtaaagT-3'; SEQ ID NO.8: KASP-SNP3-F2:5'-aCAatttgggacAtataatttacgtaaagC-3'; SEQ ID NO.9: KASP-SNP3-R:5'-tctgaaggaacatgttcattaagttT-3'; Primers for the molecular marker KASP-SNP4: SEQ ID NO.10: KASP-SNP4-F1:5'-cccttttcactccaaaagattgagT-3'; SEQ ID NO.11: KASP-SNP4-F2:5'-cccttttcactccaaaagattgagC-3'; SEQ ID NO.12: KASP-SNP4-R:5'-tgagtctggatttatcttttggcaT-3'; Primers for the molecular marker KASP-SNP5: SEQ ID NO.13: KASP-SNP5-F1:5'-tttccagactgctgatcctA-3'; SEQ ID NO.14: KASP-SNP5-F2:5'-tttccagactgctgatcctG-3'; SEQ ID NO.15: KASP-SNP5-R:5'-tgctttgtggagtttctaATtTTCtA-3'; Primers for the molecular marker KASP-SNP6: SEQ ID NO.16: KASP-SNP6-F1:5'-gtccggactatctgttgaagtctA-3'; SEQ ID NO.17: KASP-SNP6-F2:5'-gtccggactatctgttgaagtctT-3'; SEQ ID NO.18: KASP-SNP6-R:5'-tggagcttgtgaaagagacG-3'; Primers for the molecular marker KASP-SNP7: SEQ ID NO.19: KASP-SNP7-F1:5'-tgcacatggaAgggttcataA-3'; SEQ ID NO.20: KASP-SNP7-F2:5'-tgcacatggaAgggttcataG-3'; SEQ ID NO.21: KASP-SNP7-R:5'-actttggagggctAGCTTCT-3'; Primers for the molecular marker KASP-SNP8: SEQ ID NO.22: KASP-SNP8-F1:5'-aGttgccatgaaatcattgatccT-3'; SEQ ID NO.23: KASP-SNP8-F2:5'-aGttgccatgaaatcattgatccC-3'; SEQ ID NO.24: KASP-SNP8-R:5'-ggcaTtcTacttaagtgatcttcttctC-3'; Primers for the molecular marker KASP-SNP9: SEQ ID NO.25: KASP-SNP9-F1:5'-ccGctcttctcttcaTcttacaaaA-3'; SEQ ID NO.26: KASP-SNP9-F2:5'-ccGctcttctcttcaTcttacaaaT-3'; SEQ ID NO.27: KASP-SNP9-R:5'-AgtgaattggtttcaaaacttgTTGG-3'; Primers for the molecular marker KASP-SNP10: SEQ ID NO.28: KASP-SNP10-F1:5'-cagtGtCctaaactactagaCtgtctA-3'; SEQ ID NO.29: KASP-SNP10-F2:5'-cagtGtCctaaactactagaCtgtctC-3'; SEQ ID NO.30: KASP-SNP10-R:5'-GTGACTCtattggtttTacatttcacTaC-3'.

2. The KASP-SNP molecular marker primer for identifying modern rose varieties according to claim 1, characterized in that, Primers for the molecular marker KASP-SNP11: SEQ ID NO.31: KASP-SNP11-F1:5'-tcaatatgaggtgaatattaatggtaacaA-3'; SEQ ID NO.32: KASP-SNP11-F2:5'-tcaatatgaggtgaatattaatggtaacaG-3'; SEQ ID NO.33: KASP-SNP11-R:5'-gcAaatataattagtccacatgcatacaG-3'; Primers for the molecular marker KASP-SNP12: SEQ ID NO.34: KASP-SNP12-F1:5 '-caatttccctAtttctaattaggttgtttT-3'; SEQ ID NO.35: KASP-SNP12-F2:5'-caatttccctAtttctaattaggttgtttC-3'; SEQ ID NO.36: KASP-SNP12-R:5'-gcaacctagctaTAtccaaaactagC-3'; Primers for the molecular marker KASP-SNP13: SEQ ID NO.37: KASP-SNP13-F1:5'-ggtgcgaggcaagagacT-3'; SEQ ID NO.38: KASP-SNP13-F2:5'-ggtgcgaggcaagagacC-3'; SEQ ID NO.39: KASP-SNP13-R:5'-ttgtcaatatcgttGgTTAGAAcAttA-3'; Primers for the molecular marker KASP-SNP14: SEQ ID NO.40: KASP-SNP14-F1:5'-aacttGaacacAAAaCCCAAAaT-3'; SEQ ID NO.41: KASP-SNP14-F2:5'-aacttGaacacAAAaCCCAAAaC-3'; SEQ ID NO.42: KASP-SNP14-R:5'-attgaaggaaacctttatTTTActtttgtaT-3'; Primers for the molecular marker KASP-SNP15: SEQ ID NO.43: KASP-SNP15-F1:5'-acagtcaaacattctcaccttcT-3'; SEQ ID NO.44: KASP-SNP15-F2:5'-acagtcaaacattctcaccttcC-3'; SEQ ID NO.45: KASP-SNP15-R:5'-tgcatttatgtgagtaaaaactgttgatT-3'; Primers for the molecular marker KASP-SNP16: SEQ ID NO.46: KASP-SNP16-F1:5'-aattgatctacttaatgtatcgatcgaA-3'; SEQ ID NO.47: KASP-SNP16-F2:5'-aattgatctacttaatgtatcgatcgaG-3'; SEQ ID NO.48: KASP-SNP16-R:5'-tctgtaaaataaggggtGTActccaT-3'; Primers for the molecular marker KASP-SNP17: SEQ ID NO.49: KASP-SNP17-F1:5'-agctagatccaccaaatcccA-3'; SEQ ID NO.50: KASP-SNP17-F2:5'-agctagatccaccaaatcccG-3'; SEQ ID NO.51: KASP-SNP17-R:5'-CggttggtggttcgattggA-3'; Primers for the molecular marker KASP-SNP18: SEQ ID NO.52: KASP-SNP18-F1:5'-cagggcgttttgtgagaagT-3'; SEQ ID NO.53: KASP-SNP18-F2:5'-cagggcgttttgtgagaagC-3'; SEQ ID NO.54: KASP-SNP18-R:5'-agcaccaccaCAGatTGaaAA-3'; Primers for the molecular marker KASP-SNP19: SEQ ID NO.55: KASP-SNP19-F1:5'-gaacaccgagtcttgccgT-3'; SEQ ID NO.56: KASP-SNP19-F2:5'-gaacaccgagtcttgccgC-3'; SEQ ID NO.57: KASP-SNP19-R:5'-ggcttttagcaccgaagcG-3'; Primers for the molecular marker KASP-SNP20: SEQ ID NO .58:KASP-SNP20-F1:5'-gggagactttacctaagaaatgggtA-3'; SEQ ID NO .59:KASP-SNP20-F2:5′-gggagactttacctaagaaatgggtC-3′; SEQ ID NO .60:KASP-SNP20-R:5′-tcagatctattcccatgttccG-3′.

3. The KASP-SNP molecular marker primer for identifying modern rose varieties according to claim 1, characterized in that, Recommendations for the KASP-SNP21 catalyst: SEQ ID NO .61:KASP-SNP21-F1:5 '-ggttttttattagctaaaacggtaaagtgA-3'; SEQ ID NO .62:KASP-SNP21-F2:5 '-ggttttttattagctaaaacggtaaagtgG-3'; SEQ ID NO .63:KASP-SNP21-R:5′-tcacaagttctaccaGTGTGaaA-3′; Description of the active ingredient of KASP-SNP22: SEQ ID NO .64:KASP-SNP22-F1:5′-CCcTGcaatccatttggcaaaA-3′; SEQ ID NO .65:KASP-SNP22-F2:5′-CCcTGcaatccatttggcaaaC-3′; SEQ ID NO .66:KASP-SNP22-R:5′-tgctGtttataatggaAagcagTGTT-3′; Description of the active ingredient of KASP-SNP23: SEQ ID NO .67:KASP-SNP23-F1:5′-tgcataaacgtttctttgaagggtT-3′; SEQ ID NO .68:KASP-SNP23-F2:5′-tgcataaacgtttctttgaagggtA-3′; SEQ ID NO .69:KASP-SNP23-R:5′-ccatttctgcattctcaagAGTCAA-3′; Recommendations for the KASP-SNP24 catalyst: SEQ ID NO .70:KASP-SNP24-F1:5′-ccaggtatGttggaagtgggA-3′; SEQ ID NO .71:KASP-SNP24-F2:5′-ccaggtatGttggaagtgggG-3′; SEQ ID NO .72:KASP-SNP24-R:5′-gtgaggTagttattTcCaaacgtcA-3′; Primers for the molecular marker KASP-SNP25: SEQ ID NO.73: KASP-SNP25-F1:5'-AgacttaattgttgaaacttgaaaggT-3'; SEQ ID NO.74: KASP-SNP25-F2:5'-AgacttaattgttgaaacttgaaaggC-3'; SEQ ID NO.75: KASP-SNP25-R:5'-gggaattataatccagccaaaggtC-3'; Primers for the molecular marker KASP-SNP26: SEQ ID NO.76: KASP-SNP26-F1:5'-TCtTTCTTtcattactagttgcaaatgT-3'; SEQ ID NO.77: KASP-SNP26-F2:5'-TCtTTCTTtcattactagttgcaaatgC-3'; SEQ ID NO.78: KASP-SNP26-R:5'-aGacatcatcatcaAaaCGACCaT-3'; Primers for the molecular marker KASP-SNP27: SEQ ID NO.79: KASP-SNP27-F1:5'-ACACTagtcttactttctgaatccaaG-3'; SEQ ID NO.80: KASP-SNP27-F2:5'-ACACTagtcttactttctgaatccaaC-3'; SEQ ID NO.81: KASP-SNP27-R:5'-acttgctgtcttctGAGTtACAG-3'; Primers for the molecular marker KASP-SNP28: SEQ ID NO.82: KASP-SNP28-F1:5'-aGTcGAtctattttcCattaacatcaA-3'; SEQ ID NO.83: KASP-SNP28-F2:5'-aGTcGAtctattttcCattaacatcaC-3'; SEQ ID NO.84: KASP-SNP28-R:5'-GTCCAGTGTaCAcctcaACaTATataC-3'; Primers for the molecular marker KASP-SNP29: SEQ ID NO.85: KASP-SNP29-F1:5'-caaccgccgttgttaaatgtT-3'; SEQ ID NO.86: KASP-SNP29-F2:5'-caaccgccgttgttaaatgtC-3'; SEQ ID NO.87: KASP-SNP29-R:5'-aggtactgaacatgatgtctctatatcA-3'; Primers for the molecular marker KASP-SNP30: SEQ ID NO.88: KASP-SNP30-F1:5 '-tgttactagggttaggtgttatggT-3'; SEQ ID NO.89: KASP-SNP30-F2:5 '-tgttactagggttaggtgttatggA-3'; SEQ ID NO.90: KASP-SNP30-R:5'-AgagaatttttgatgcgtcaagT-3'.

4. The KASP-SNP molecular marker primer for identifying modern rose varieties according to claim 1, characterized in that, Primers for the molecular marker KASP-SNP31: SEQ ID NO.91: KASP-SNP31-F1:5'-tcaggaaacaagcccgaaaaA-3'; SEQ ID NO.92: KASP-SNP31-F2:5'-tcaggaaacaagcccgaaaaG-3'; SEQ ID NO.93: KASP-SNP31-R:5'-tgcatttgaagatgattctttatgtgtT-3'; Primers for the molecular marker KASP-SNP32: SEQ ID NO.94: KASP-SNP32-F1:5'-accaacaatactctgcgagaA-3'; SEQ ID NO.95: KASP-SNP32-F2:5'-accaacaatactctgcgagaG-3'; SEQ ID NO.96: KASP-SNP32-R:5'-gcaggccacatatTcctaaatcA-3'; Primers for the molecular marker KASP-SNP33: SEQ ID NO.97: KASP-SNP33-F1:5'-ctcagttgaGgtgttgacactA-3'; SEQ ID NO.98: KASP-SNP33-F2:5'-ctcagttgaGgtgttgacactG-3'; SEQ ID NO.99: KASP-SNP33-R:5'-accAgcgttagggttttctG-3'; Primers for the molecular marker KASP-SNP34: SEQ ID NO.100: KASP-SNP34-F1:5'-TGaGgtcataattttgacaaGtgcA-3'; SEQ ID NO.101: KASP-SNP34-F2:5'-TGaGgtcataattttgacaaGtgcT-3'; SEQ ID NO.102: KASP-SNP34-R:5'-tcacActCAtttcctctcaacT-3'; Primers for the molecular marker KASP-SNP35: SEQ ID NO.103: KASP-SNP35-F1:5'-tctcttcttttgatttggccgT-3'; SEQ ID NO.104: KASP-SNP35-F2:5'-tctcttcttttgatttggccgA-3'; SEQ ID NO.105: KASP-SNP35-R:5'-cgcccaattactttttGTAtAtTtATTTTG-3'.

5. The KASP-SNP molecular marker primer for identifying modern rose varieties according to claim 1, characterized in that, The screening method for KASP-SNP molecular marker primers includes the following steps: 1) Extract high-quality genomic DNA from leaf samples of the modern Lunar Species to be tested; 2) Based on the published whole-genome resequencing data of roses, SNP sites were screened on chromosomes using bioinformatics methods, and specific KASP-SNP primers were designed; 3) Use a high-throughput genotyping platform to verify and screen primers for polymorphism, and screen out core KASP-SNP markers.

6. The KASP-SNP molecular marker primer for identifying modern rose varieties according to claim 1, characterized in that, Step 2) describes identifying 17,567,247 SNP variant sites in the SNP database obtained from the resequencing of 233 Rosa species. The sites are then screened for quality control. Specifically, KASP-SNP primers are designed from the selected candidate SNP sites based on the following principles: 1) The allele-specific forward primers are designed to be 30–45 nucleotides in length; 2) The universal reverse primer is designed to be 18-25 nucleotides in length, and the GC content of all primers is controlled at 40%-60%, while avoiding continuous single base repetitions; 3) The calculated melting temperature of the primers is between 55℃ and 65℃, and the difference in Tm value between the same set of primers does not exceed 2℃.

7. The KASP-SNP molecular marker primer for identifying modern rose varieties according to claim 1, characterized in that, Step 3) The verification and screening of the high-throughput genotyping platform includes: preliminary verification of KASP-SNP primers using standardized DNA samples containing different varieties; preparation of the reaction system according to the standard KASP-SNP assay protocol, and amplification and endpoint fluorescence reading on a real-time quantitative PCR instrument; preliminary screening based on the degree of aggregation of data points in the allele discrimination scatter plot, the clarity of genotyping, and the concordance rate with the expected genotype; and selection of high-resolution core KASP-SNP markers suitable for large-scale variety identification from the primers that pass the preliminary screening.

8. A KASP-SNP molecular marker primer composition for identifying modern rose varieties, characterized in that, The composition comprises the KASP-SNP molecular markers KASP-SNP1 to KASP-SNP35 as described in claim 1, wherein the sequences of the molecular marker primers for KASP-SNP1 to KASP-SNP35 are shown in the sequence listing SEQ ID NO. 1 to SEQ ID NO. 105, respectively.

9. The application of the KASP-SNP molecular marker primers for identifying modern rose varieties as described in any one of claims 1-7 in the analysis of phylogenetic relationships and genetic diversity of modern rose varieties, the identification of varieties and the analysis of trait associations in DUS testing, and the application of molecular marker-assisted selection breeding for target horticultural traits.

Images