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CN-121992142-A - KASP-SNP molecular marker primer and composition for identifying modern China rose varieties and application thereof

CN121992142ACN 121992142 ACN121992142 ACN 121992142ACN-121992142-A

Abstract

A KASP-SNP molecular marker primer for identifying modern China rose varieties, a composition and application thereof, wherein the molecular marker primer comprises KASP-SNP 1-KASP-SNP 35, and the nucleotide sequence of the molecular marker primer is shown as SEQ ID NO. 1-105. According to the invention, based on whole genome resequencing data, 35 SNP loci with high polymorphism are screened out, specific primers are designed, fluorescent signals of a sample to be detected are detected through a KASP (KASP sequencing technology) platform, and genotypes can be automatically interpreted and DNA fingerprint patterns 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 the precise identification, genetic relationship and genetic diversity analysis, DUS test assistance and molecular marker assistance selection breeding of modern China rose varieties.

Inventors

  • TAN BIN
  • ZHOU YINLONG
  • TAN WANXIANG
  • WANG JUNYUN
  • XU ZHENJIANG
  • LIU HONG

Assignees

  • 华南农业大学
  • 云南省花卉技术培训推广中心

Dates

Publication Date
20260508
Application Date
20260319

Claims (9)

  1. 1. A KASP-SNP molecular marker primer for identifying modern Chinese rose varieties, which is characterized in that the modern Chinese rose varieties comprise wild rose, french rose, damascus rose and Rosa glabra; The KASP-SNP molecular marker for identifying the modern China rose variety comprises KASP-SNP 1-KASP-SNP 35; The sequence of the KASP-SNP molecular marker primer for identifying the modern China rose variety is as follows: the primer of the molecular marker KASP-SNP 1: 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 of the molecular marker KASP-SNP 2: 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'; the primer of the molecular marker KASP-SNP 3: 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'; The primer of the molecular marker KASP-SNP 4: 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'; the primer of the molecular marker KASP-SNP 5: 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-SNP 6: 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'; the primer of the molecular marker KASP-SNP 7: 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'; the primer of the molecular marker KASP-SNP 8: 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'; The primer of the molecular marker KASP-SNP 9: 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'; the primer of the molecular marker KASP-SNP 10: 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. 2. The molecular marker primer for identifying the KASP-SNP of modern rose varieties according to claim 1, wherein the primer for the molecular marker KASP-SNP 11: 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'; The primer of the molecular marker KASP-SNP 12: 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'; the primer of the molecular marker KASP-SNP 13: 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'; the primer of the molecular marker KASP-SNP 14: 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 '-attgaaggaaaccttatTTTActtttgtaT-3'; primers for the molecular marker KASP-SNP 15: 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'; the primer of the molecular marker KASP-SNP 16: 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'; the primer of the molecular marker KASP-SNP 17: 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-SNP 18: 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'; the primer of the molecular marker KASP-SNP 19: 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-SNP 20: 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. 3. The molecular marker primer for identifying the KASP-SNP of modern rose varieties according to claim 1, wherein the primer for the molecular marker KASP-SNP 21: SEQ ID NO .61:KASP-SNP21-F1:5 '-ggtttttattagctaaaacggtaaagtgA-3'; SEQ ID NO .62:KASP-SNP21-F2:5 '-ggtttttattagctaaaacggtaaagtgG-3'; SEQ ID NO .63:KASP-SNP21-R:5 '-tcacaagttctaccaGTGTGaaA-3'; primers for the molecular marker KASP-SNP 22: 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'; primers for the molecular marker KASP-SNP 23: 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'; primers for the molecular marker KASP-SNP 24: 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-SNP 25: 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-SNP 26: 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'; The primer of the molecular marker KASP-SNP 27: 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-SNP 28: 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'; the primer of the molecular marker KASP-SNP 29: 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 of the molecular marker KASP-SNP 30: 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. 4. the molecular marker primer for identifying the KASP-SNP of modern rose varieties according to claim 1, wherein the primer for the molecular marker KASP-SNP 31: 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 '-tgcatttgaagatgattcttatgtgtT-3'; primers for the molecular marker KASP-SNP 32: 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-SNP 33: 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-SNP 34: 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-SNP 35: 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. 5. the KASP-SNP molecular marker primer for identifying modern rose varieties according to claim 1, wherein the screening method of the KASP-SNP molecular marker primer comprises the following steps: 1) Extracting high-quality genome DNA from leaf samples of modern month varieties to be detected; 2) Screening SNP loci on chromosomes by using a bioinformatics method based on published China rose genome-wide re-sequencing data, and designing specific KASP-SNP primers; 3) And (3) carrying out polymorphism verification and screening on the primers by using a high-throughput genotyping platform, and screening out core KASP-SNP markers.
  6. 6. The KASP-SNP molecular marker primer for identifying modern China rose varieties according to claim 1, wherein 17,567,247 SNP variation sites are found in the published SNP database obtained based on the resequencing of 233 rose varieties in the step 2), and site quality control screening is carried out, and the specific operation is that the KASP-SNP primer is designed according to the following principle from screened candidate SNP sites, wherein the screening principle is as follows: 1) The length of the allele-specific forward primer is designed to be 30-45 nucleotides; 2) The length of the universal reverse primer is designed to be 18-25 nucleotides, the GC content of all the primers is controlled to be 40-60%, and continuous single base repetition is avoided; 3) The calculated melting temperature of the primers is 55-65 ℃, and the difference of Tm values among the same group of primers is not more than 2 ℃.
  7. 7. The KASP-SNP molecular marker primer for identifying modern China rose varieties according to claim 1, wherein the high-throughput genotyping platform is characterized by comprising the steps of carrying out preliminary verification on KASP-SNP primers by using standardized DNA samples containing different varieties, preparing a reaction system by referring to a standard KASP-SNP assay protocol, carrying out amplification and end point fluorescence reading on a real-time fluorescence quantitative PCR instrument, carrying out primary screening according to the aggregation degree of data points in an allele discrimination scatter diagram, the definition of typing and the coincidence rate with expected genotypes, and screening the primers passing through the primary screening to obtain the high-resolution core KASP-SNP marker suitable for large-scale variety identification.
  8. 8. A KASP-SNP molecular marker primer composition for identifying modern China rose varieties is characterized by comprising KASP-SNP molecular markers KASP-SNP 1-KASP-SNP 35 according to claim 1, wherein the sequences of the molecular marker primers of the KASP-SNP 1-KASP-SNP 35 are respectively shown in sequence tables SEQ ID NO. 1-SEQ ID NO. 105.
  9. 9. Use of a KASP-SNP molecular marker primer for identifying modern rose varieties according to any one of claims 1-7 in modern month variety genetic relationship and genetic diversity analysis, variety identity identification and trait association analysis in DUS testing, and molecular marker-assisted selection breeding of target horticultural traits.

Description

KASP-SNP molecular marker primer and composition for identifying modern China rose varieties and application thereof Technical Field The invention relates to the technical field of molecular biology, in particular to a KASP-SNP molecular marker primer for identifying modern China rose varieties, a composition and application thereof. Background Modern China rose (Modern Garden Roses, the subject of which is mainly Rosa hybrid) is a large gardening group formed by long-term and complex hybridization breeding in Rosa (Rosa) of Rosaceae. The ancestors are mainly derived from wild stock of various rose genus in China, europe, middle east and the like. Modern China rose is known to the world by rich flowers (such as high-core wane and cup-shaped), gorgeous colors (all color systems except pure blue are covered), continuous flowering (multi-season flowering) and various plant types (shrubs, vines, miniature and the like), is one of the most important ornamental plants worldwide, is widely applied to courtyard beautification, cut flower production, potted ornamental and garden landscaping, is known as "queen in flowers" and occupies an irreplaceable core position in the economic, cultural and horticultural industries. Modern China rose is one of the most important commercial ornamental flowers worldwide and China, and the protection of the species right thereof has been paid attention for a long time, and has been incorporated into the protection directory (forestry part) of new species of plants in the people's republic of China. Along with the continuous expansion and consumption upgrading of domestic ornamental gardening markets, the demands of the markets on new rose varieties with independent intellectual property rights and excellent properties are increasingly urgent, and the awareness of breeders on the protection and maintenance of the new varieties is also obviously enhanced. However, modern inter-rose cultivars are susceptible to environmental impact, and the limitation of long period, strong subjectivity and the like sometimes exists in the process of performing variety specificity identification by only relying on the traditional DUS test. Therefore, a set of high-resolution stable KASP-SNP molecular marker system suitable for modern China rose variety identification is developed, and an accurate DNA fingerprint is constructed according to the high-resolution stable KASP-SNP molecular marker system, so that the method has important technical support significance and practical application value for efficiently and objectively assisting DUS test, defining variety identity, protecting legal rights and interests of breeders, standardizing seedling markets and promoting healthy and orderly development of China rose industry. Disclosure of Invention In order to overcome the defects in the prior art, the invention aims to provide a KASP-SNP molecular marker primer for identifying modern China rose varieties, a composition and application thereof. The invention solves the technical problems by adopting a technical scheme that a KASP-SNP molecular marker primer for identifying modern China rose varieties is adopted, wherein the modern China rose and related species thereof or rose important wild parents used for hybridization breeding include but are not limited to wild rose (Rosa multiflora), french rose (Rosa gallica), damascus rose (Rosa damascena) and Rosa glabra (Rosa wichurana). The KASP-SNP molecular marker for identifying modern China rose varieties comprises 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、KASP-SNP17、KASP-SNP18、KASP-SNP19、KASP-SNP20、KASP-SNP21、KASP-SNP22、KASP-SNP23、KASP-SNP24、KASP-SNP25、KASP-SNP26、KASP-SNP27、KASP-SNP28、KASP-SNP29、KASP-SNP30、KASP-SNP31、KASP-SNP32、KASP-SNP33、KASP-SNP34、KASP-SNP35; The sequence of the KASP-SNP molecular marker primer for identifying the modern China rose variety is as follows: the primer of the molecular marker KASP-SNP 1: 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 of the molecular marker KASP-SNP 2: 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'; the primer of the molecular marker KASP-SNP 3: 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'; The primer of the molecular marker KASP-SNP 4: 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'; the primer of the molecular marker KASP-SNP 5: SEQ ID NO .