A SSR molecular marker primer for identifying Cattleya species and its application

Abstract

The present invention belongs to the technical field of molecular biology, and specifically relates to an SSR molecular marker primer for identifying Cattleya varieties and its application. The Cattleya includes closely related genera such as Phalaenopsis, Dendrobium, and Cymbidium; the SSR molecular marker for identifying Cattleya varieties includes SSR1 to SSR50, and their nucleic acid sequences are shown in the sequence table SEQ ID NO.1 to SEQID NO.100, respectively. Compared with other molecular markers, the SSR molecular marker primer for identifying Cattleya varieties of the present invention and its composition have the characteristics of high polymorphism, large quantity and low cost. The selected Cattleya materials are not limited by season, environment and test time, and DNA can be extracted from any tissue in any growth period of the variety or samples after refrigerated storage for a period of time without affecting the identification results.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to an SSR molecular marker primer for identifying Cattleya varieties and an application thereof. Background Art

[0002] Cattleya belongs to the genus Cattleya of the family Orchidaceae. It is a tropical compound stem epiphytic orchid plant that is distributed from Mexico in Central America to Brazil in South America, about 5,000 kilometers from north to south. They are famous for their large and colorful flowers and are widely used for hybridization and potted cultivation in the cut flower trade. They are also an ideal component of floral decorations such as bouquets and flower arrangements. Among orchids, Cattleya is the first choice for most people's home garden orchids. It has a long flowering period and exudes a beautiful fragrance, and is known as the "King of Orchids".

[0003] As a characteristic tropical flower in South China, Cattleya was included in the 7th batch of the List of New Plant Varieties of the People's Republic of China for Protection (forestry part) in December 2020. Therefore, DUS testing is needed to protect the legitimate rights and interests of breeders. In recent years, with the continuous expansion of the ornamental market, the increasing demand for excellent flower varieties in my country and the continuous successful cultivation of new varieties of Cattleya, more and more breeders have become more aware of the protection of new varieties and actively apply for new variety protection. Therefore, it is particularly necessary to provide technical support for the protection of new varieties of Cattleya plants by screening a set of SSR molecular core markers and primer combinations of Cattleya and constructing DNA fingerprints of varieties. Summary of the invention

[0004] In view of the above problems, the object of the present invention is to provide an SSR molecular marker primer for identifying Cattleya varieties and its application.

[0005] The technical contents of the present invention are as follows:

[0006] The present invention provides an SSR molecular marker primer for identifying Cattleya species, wherein the Cattleya species include closely related genera such as Phalaenopsis, Dendrobium and Cymbidium;

[0007] The SSR molecular markers for identifying Cattleya varieties include SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR22, SSR23, SSR24, SSR25, SSR26, SSR27, SSR28, SSR29, SSR30, SSR31, SSR32, SSR33, SSR34, SSR35, SSR36, SSR37, SSR38, SSR39, SSR40, SSR41, SSR42, SSR43, SSR44, SSR45, SSR46, SSR47, SSR48, SSR49, SSR50;

[0008] The sequences of the SSR molecular marker primers for identifying Cattleya species are as follows:

[0009] Primers for the molecular marker SSR1:

[0010] SEQ ID NO.1: SSR1-F:5'-GGGAGGGAGGAATAGGAAGA-3';

[0011] SEQ ID NO.2: SSR1-R:5'-TGAGCTGCGATATCAAAGGA-3';

[0012] Primers for the molecular marker SSR2:

[0013] SEQ ID NO.3: SSR2-F:5'-TCAAGGCCTGCACATAGAGA-3';

[0014] SEQ ID NO.4: SSR2-R:5'-AAGAGGA AGGCTTCGTTGC-3';

[0015] Primers for the molecular marker SSR3:

[0016] SEQ ID NO.5: SSR3-F:5'-AACAGGCATTTGGAGCTTTT-3';

[0017] SEQ ID NO.6: SSR3-R:5'-CCTCATTTCTCTCACCCTCTTT-3';

[0018] Primers for the molecular marker SSR4:

[0019] SEQ ID NO.7: SSR4-F:5'-TGGATCTTACCTTGGGCTTCTA-3';

[0020] SEQ ID NO.8: SSR4-R:5'-CCCTGCTTCTCTTTCTCACTCAT-3';

[0021] Primers for the molecular marker SSR5:

[0022] SEQ ID NO.9: SSR5-F:5'-GGGAGGACTATCAACCTCAA-3';

[0023] SEQ ID NO.10: SSR5-R:5'-CAGCCTCTCCAGGTAGGTTT-3';

[0024] Primers for the molecular marker SSR6:

[0025] SEQ ID NO.11: SSR6-F:5'-AATGCGACGAAGAAAGCCTA-3';

[0026] SEQ ID NO.12: SS6-R:5'-AGCACTTGCCCTTGTCAGTT-3';

[0027] Primers for the molecular marker SSR7:

[0028] SEQ ID NO. 13: SSR7-F: 5'-TTTGGTAAGGGTGGACCAAG- 3';

[0029] SEQ ID NO.14: SSR7-R:5'-TTACCGAGTCCTTCCAAACA-3';

[0030] Primers for the molecular marker SSR8:

[0031] SEQ ID NO.15: SSR8-F:5'-ATCAAGCCATGGCAGTCTTT-3';

[0032] SEQ ID NO.16: SSR8-R:5'-TCGGCTTGACTGCAAAGTTA-3';

[0033] Primers for the molecular marker SSR9:

[0034] SEQ ID NO.17: SSR9-F:5'-CATGAGACACGTTGGGCTTA-3';

[0035] SEQ ID NO.18: SSR9-R:5'-ATGCGACGAAGAAAGCCTAC-3';

[0036] Primers for the molecular marker SSR10:

[0037] SEQ ID NO.19: SSR10-F:5'-CTCCGATGGGTCTATATCAAG-3';

[0038] SEQ ID NO.20: SSR10-R:5'-TGGGAGCTTGTACCCAAGA-3';

[0039] Primers for the molecular marker SSR11:

[0040] SEQ ID NO.21: SSR11-F:5'-CTCAGTCCTGCGATCCAAT-3';

[0041] SEQ ID NO.22: SSR11-R:5'-GGTAACCCGTGCAACCAT-3';

[0042] Primers for the molecular marker SSR12:

[0043] SEQ ID NO.23: SSR12-F:5'-AGAATCAATGCCCCAAGC-3';

[0044] SEQ ID NO.24: SSR12-R:5'-GGTAACCCGTGCAACCAT-3';

[0045] Primers for the molecular marker SSR13:

[0046] SEQ ID NO.25: SSR13-F:5'-TGTGGTCTGTCTAACGCAGAG-3';

[0047] SEQ ID NO.26: SSR13-R:5'-TCTTCAGCTCAGTCCTGCAA-3';

[0048] Primers for the molecular marker SSR14:

[0049] SEQ ID NO.27: SSR14-F:5'-TCCCTTGACCCTAGGAAGGT-3';

[0050] SEQ ID NO.28: SSR14-R:5'-TGAGTCCCTCCCCTTTGACTT-3';

[0051] Primers for the molecular marker SSR15:

[0052] SEQ ID NO.29: SSR15-F:5'-GGCATCAAACCACTTACATCC-3';

[0053] SEQ ID NO.30: SSR15-R:5'-CGCAAGCCTAAGTGAGATCC-3';

[0054] Primers for the molecular marker SSR16:

[0055] SEQ ID NO.31: SSR16-F:5'-GTGGCTTAGTCTTACATTGGATA GTT-3';

[0056] SEQ ID NO.32: SSR16-R:5'-TATTAGAGAGTAAATCAAAAAG GGCT-3';

[0057] Primers for the molecular marker SSR17:

[0058] SEQ ID NO.33: SSR17-F:5'-GGGGATCATTAGGGCATATAACT AGAA-3';

[0059] SEQ ID NO.34: SSR17-R:5'-AGAAGGGTCTTAAAATAGGGAT TAGGG-3';

[0060] Primers for the molecular marker SSR18:

[0061] SEQ ID NO.35: SSR18-F:5'-ACGTGGCTTCTCCTTCTTGAAT -3';

[0062] SEQ ID NO.36: SSR18-R:5'-CAGGGAGAAAAAGGGATAGAA CC-3';

[0063] Primers for the molecular marker SSR19:

[0064] SEQ ID NO.37: SSR19-F:5'-TAACAGGCTGACCATGCTTTCTA T-3';

[0065] SEQ ID NO.38: SSR19-R:5'-TGAAGTCGAGTATTCCAAACAC AA-3';

[0066] Primers for the molecular marker SSR20:

[0067] SEQ ID NO.39: SSR20-F:5'-CCCCTCTTGTCTGTCACACATAA -3';

[0068] SEQ ID NO.40: SSR20-R:5'-ATGAACTGAATACTCATGGATAC TGA-3';

[0069] Primers for the molecular marker SSR21:

[0070] SEQ ID NO.41: SSR21-F:5'-CAAGCTTTTATTAGCTTATTTTAT TTAC-3';

[0071] SEQ ID NO.42: SSR21-R:5'-TCTTTGACCGCCACAGTAGG-3';

[0072] Primers for the molecular marker SSR22:

[0073] SEQ ID NO.43: SSR22-F: 5'-GTTGTTTGTAGATTGATTCTCT TTCATCC-3';

[0074] SEQ ID NO.44: SSR22-R:5'-TTCATTAGCGATTTCTTCTCAAATGC-3';

[0075] Primers for the molecular marker SSR23:

[0076] SEQ ID NO.45: SSR23-F:5'-AAAGTGACTAGGAAAAAAATAA CTGAG-3';

[0077] SEQ ID NO.46: SSR23-R:5'-TTGTGTGGGTTATGTAGGAAAGT -3';

[0078] Primers for the molecular marker SSR24:

[0079] SEQ ID NO.47: SSR24-F:5'-TAAAATGGTCTATTCAAATAAAA ATCGT-3';

[0080] SEQ ID NO.48: SSR24-R:5'ACATTGTCTATGACCCCCTAATAA AA-3';

[0081] Primers for the molecular marker SSR25:

[0082] SEQ ID NO.49: SSR25-F:5'-TAAATAAGAGGCCTTGGAATGT C-3';

[0083] SEQ ID NO.50: SSR25-R:5'-GGGTGTAGAAAACTTAAGGAAT TGA-3';

[0084] Primers for the molecular marker SSR26:

[0085] SEQ ID NO.51: SSR26-F:5'-TTCTTGCTTCTTTTTGTGGA-3';

[0086] SEQ ID NO.52: SSR26-R:5'-:ATTTGTTTGATACGCCATTG-3';

[0087] Primers for the molecular marker SSR27:

[0088] SEQ ID NO.53: SSR27-F:5'-GTGCTAATTCAACGCAAA-3';

[0089] SEQ ID NO.54: SSR27-R:5'-TTAAAAGCCGAGTACTCTACC-3';

[0090] Primers for the molecular marker SSR28:

[0091] SEQ ID NO.55: SSR28-F:5'-TGAGATTGGATTGGAAGAAGA-3';

[0092] SEQ ID NO.56: SSR28-R:5'-TGAGGGTTCAAGTCCCTCTA-3';

[0093] Primers for the molecular marker SSR29:

[0094] SEQ ID NO.57: SSR29-F:5'-TAGGATGATGCACGGGAAA-3';

[0095] SEQ ID NO.58: SSR29-R:5'-GGGGGTTTTTATCATTGAGGA-3';

[0096] Primers for the molecular marker SSR30:

[0097] SEQ ID NO.59: SSR30-F:5'-AGAGAAAGCCCTTGTGTTGG-3';

[0098] SEQ ID NO.60: SSR30-R:5'-TCAGCTCTTCCGATTCTGGT-3';

[0099] Primers for the molecular marker SSR31:

[0100] SEQ ID NO.61: SSR31-F:5'-GCTTCAAGCAAGTGCAGAAA-3';

[0101] SEQ ID NO.62: SSR31-R:5'-AAGACAGGCCAACCAGAGAA-3';

[0102] Primers for the molecular marker SSR32:

[0103] SEQ ID NO.63: SSR32-F:5'-TGGTTTGCTAAGTATTTCTTCAA GTT-3';

[0104] SEQ ID NO.64: SSR32-R:5'-TATGCAAAGCTTCCCCAAGT-3';

[0105] Primers for the molecular marker SSR33:

[0106] SEQ ID NO.65: SSR33-F:5'-TGAATGCAAAATCCAATTGCT-3';

[0107] SEQ ID NO.66: SSR33-R:5'-GAAATATTCAAGCACGATGATCC-3';

[0108] Primers for the molecular marker SSR34:

[0109] SEQ ID NO.67: SSR34-F:5'-TGCATACGTAACAACTGGAGGT-3';

[0110] SEQ ID NO.68: SSR34-R:5'-GGAAGGTCATTCTAACCAGGAA -3';

[0111] Primers for the molecular marker SSR35:

[0112] SEQ ID NO.69: SSR35-F:5'-ACGCTCTTTGGCTGGAACT-3';

[0113] SEQ ID NO.70: SSR35-R:5'-CTCACATGCCTTTAGCCTCAC-3';

[0114] Primers for the molecular marker SSR36:

[0115] SEQ ID NO.71: SSR36-F:5'-TGCCCCAGACAATTAAGC-3';

[0116] SEQ ID NO.72: SSR36-R:5'-CCTCGATGGAACCCCATAAT-3';

[0117] Primers for the molecular marker SSR37:

[0118] SEQ ID NO.73: SSR37-F:5'-TTGGGTTTCGTCTCACATCA-3';

[0119] SEQ ID NO.74: SSR37-R:5'-CCCTCAGTATCCGCCACTT-3';

[0120] Primers for the molecular marker SSR38:

[0121] SEQ ID NO.75: SSR38-F:5'-CTTGTCTACGTGAGGGCACTG-3';

[0122] SEQ ID NO.76: SSR38-R:5'-TCAACAACGTGAAAAGCCATC-3';

[0123] Primers for the molecular marker SSR39:

[0124] SEQ ID NO.77: SSR39-F:5'-CCCTCCACCCACCAAAATC-3';

[0125] SEQ ID NO.78: SSR39-R:5'-CAACACCTTGAGGAACGCC-3';

[0126] Primers for the molecular marker SSR40:

[0127] SEQ ID NO.79: SSR40-F:5'-TGGCATTTGGGAGCAACAG-3';

[0128] SEQ ID NO.80: SSR40-R:5'-TGACGCACACCAGAGATCC-3';

[0129] Primers for the molecular marker SSR41:

[0130] SEQ ID NO.81: SSR41-F:5'-CATGTCAATACCGACAAC-3';

[0131] SEQ ID NO.82: SSR41-R:5'-CATATATGTTCATATCGT-3';

[0132] Primers for the molecular marker SSR42:

[0133] SEQ ID NO.83: SSR42-F:5'-GTGCTCAACCCACAGGAACT-3';

[0134] SEQ ID NO.84: SSR42-R:5'-AATAAGTATGAATAGAAC-3';

[0135] Primers for the molecular marker SSR43:

[0136] SEQ ID NO.85: SSR43-F:5'-TCATTCATAGTCCATAT-3';

[0137] SEQ ID NO.86: SSR43-R:5'-GCATCATCCTAGCAGAGT-3';

[0138] Primers for the molecular marker SSR44:

[0139] SEQ ID NO.87: SSR44-F:5'-TGGTATAGGTTCAAATCC-3';

[0140] SEQ ID NO.88: SSR44-R:5'-AGCTATTCAGGAACAGAT-3';

[0141] Primers for the molecular marker SSR45:

[0142] SEQ ID NO.89: SSR45-F:5'-GAAAGCAATCCAACAAC-3';

[0143] SEQ ID NO.90: SSR45-R:5'-GTAAAGGAGAATAATGACC-3';

[0144] Primers for the molecular marker SSR46:

[0145] SEQ ID NO.91: SSR46-F:5'-ATGGGCGGTTTCTCATAACG-3';

[0146] SEQ ID NO.92: SSR46-R:5'-TTTCTTTACGCGTCATTCTTTGG-3';

[0147] Primers for the molecular marker SSR47:

[0148] SEQ ID NO.93: SSR47-F:5'-ATTGGCATTCTTCTGT-3';

[0149] SEQ ID NO.94: SSR47-R:5'-TAAGCCCTTATGTTTG-3';

[0150] Primers for the molecular marker SSR48:

[0151] SEQ ID NO.95: SSR48-F:5'-TCAGACACTTTGGGTCAAAGA-3';

[0152] SEQ ID NO.96: SSR48-R:5'-GAGGTGCTCCAAAGATCAAACT-3';

[0153] Primers for the molecular marker SSR48:

[0154] SEQ ID NO.97: SSR49-F:5'-AAAAATAAATGCATGAATATGAGTATG-3';

[0155] SEQ ID NO.98: SSR49-R:5'-GGATTATAAGAAATCGATGATGT GA-3';

[0156] Primers for the molecular marker SSR50:

[0157] SEQ ID NO.99: SSR50-F:5'-TTTTGAACATAGAAAGCAATCC-3';

[0158] SEQ ID NO. 100: SSR50-R:5'-GATTCGGATAGAGAAGCAAAA-3'.

[0159] The screening steps of the above SSR molecular markers are as follows: extraction of total DNA of Cattleya, selection of SSR loci and design and screening of primers based on 33 microsatellite sequences published by Cattleya on NCBI, PCR amplification and FragementAnalyzer TM The fully automatic capillary electrophoresis system was used for initial screening, and core primers were selected based on the electrophoresis results;

[0160] The extraction of the total DNA of Cattleya is carried out by using a modified CTAB method to extract the fresh leaves taken as materials;

[0161] The 33 microsatellite sequences published by Cattleya on NCBI were used to select SSR sites and design and screen primers. The specific operation was to design 120 pairs of primers according to the following principles from the 20 sequences containing SSR sites, where the screening principles of SSR primers are as follows:

[0162] 1) The length of the primer is 18-27 bp, and the target fragment is about 250 bp to 380 bp;

[0163] 2) The GC content is 40% to 60%, and three or four consecutive bases should be avoided in the primer sequence;

[0164] 3) Annealing temperature is 55-65℃, preferably around 50℃, upstream and downstream primers T m The value difference does not exceed 4 to 6°C;

[0165] 4) Avoid more than three consecutive bases at the 3' end of the primer, and try to avoid primer dimers and hairpin structures.

[0166] The PCR Amplification and Fragment Analyzer TM The fully automatic capillary electrophoresis system was used for initial screening. The specific operation was to draw 2 μL of PCR product for initial screening by agarose gel electrophoresis, and to perform initial screening of primers by comparing with the target fragment. PCR products with high amplification rate, clear bands, and good peaks were selected by Fragment Analyzer. TM The fully automatic capillary electrophoresis system for multiple screening can accurately reflect the differences between allelic variation sites and screen primers with high polymorphism.

[0167] The present invention also provides an SSR molecular marker primer composition for identifying Cattleya varieties, wherein the sequences of the individual SSR molecular marker primers in the composition are respectively shown in sequence listings SEQ ID NO.1 to SEQ ID NO.100.

[0168] The present invention also provides an application of SSR molecular marker primers for identifying Cattleya varieties in Cattleya kinship analysis, DUS specificity test and molecular marker assisted breeding. For example, the naming of Cattleya varieties has undergone three major changes, resulting in the mixing of varieties. SSR molecular markers are used to identify varieties such as C.guttata, C.tigrina and C.leopoldii. The results show that C.guttata and C.leopoldii have very close homology and are far away from C.tigrina, exploring the genetic relationship and cluster analysis of Cattleya germplasm resources. Secondly, Cattleya belongs to asexually propagated plants, and there are a large number of new and old varieties, which is easy to cause confusion of homonyms or homonyms. Therefore, SSR markers are used to construct DNA fingerprints for screening similar varieties in DUS tests, which provides the possibility of improving work efficiency and accelerating variety identification. Finally, by detecting molecular markers, the presence of target genes can be detected and target traits can be selected. The level of phenotypic genetic diversity can be studied through correlation analysis, and breeders can be helped to carry out targeted breeding, which is the basis for breeding of excellent plant varieties.

[0169] The beneficial effects of the present invention are as follows:

[0170] Compared with other molecular markers, the SSR molecular marker primers and the composition thereof for identifying Cattleya species of the present invention have the characteristics of high polymorphism, large quantity, low cost, easy reading, etc.; the selected Cattleya species are not restricted by season, environment and test time, and DNA can be extracted from any tissue in any growth period of the species or samples after being refrigerated and stored at 22° C. for a period of time without affecting the identification result;

[0171] In the screening of SSR molecular marker primers for identifying Cattleya species of the present invention, FragmentAnalyzer TM The fully automatic capillary electrophoresis system has the characteristics of high throughput, safety, convenience and high sensitivity. It plays a great role in building a set of fast and accurate SSR fingerprint technology and shortens the identification cycle of Cattleya varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0172] Figure 1 This is a comparison of SSR and ISSR primers in amplification of Cattleya varieties;

[0173] Figure 2 Agarose gel electrophoresis of DNA extracted from 12 Cattleya species with large phenotype differences;

[0174] Figure 3 The peak diagram of SSR molecular marker primer amplification results of 35 varieties of Cattleya species;

[0175] Figure 4 Genetic analysis and cluster diagram. DETAILED DESCRIPTION

[0176] The present invention is further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art are all within the scope of the claims attached to this application.

[0177] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents from the conventional market.

[0178] Example 1

[0179] Extraction of Total DNA from Cattleya Species

[0180] 1) The 35 Cattleya species selected were provided by Times Viagra (Guangzhou) Co., Ltd.;

[0181] 2) Using the selected Cattleya species as materials, the total DNA was extracted using the CTAB method. The specific operation process is as follows:

[0182] ① Take a 2mL centrifuge tube, add 0.1g of ground Cattleya powder, and add 700mL CTAB extract (preheated to 65℃ in advance);

[0183] ②Put the ground sample in a 65℃ water bath for 20min to 1h, shaking the centrifuge tube upside down every 10min. After the water bath, take it out and centrifuge it at 12000×g for 10min.

[0184] 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 and mix well, and centrifuge at 12,000 × g for 10 min;

[0185] ④ Pipette the supernatant into a new 2.0mL centrifuge tube, add an equal volume of isopropanol, slowly invert to mix, and store in a -20℃ refrigerator for more than half an hour to precipitate genomic DNA, or store in a -20℃ refrigerator overnight;

[0186] ⑤Take out the sample from the -20℃ refrigerator, centrifuge at 12000×g for 10 min, and discard the supernatant;

[0187] 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;

[0188] ⑦Repeat step ⑥ once;

[0189] ⑧ Open the cap of the centrifuge tube, carefully absorb the ethanol on the tube wall with a sterilized filter paper strip, and place the centrifuge tube in a fume hood to dry for 5 to 10 minutes;

[0190] ⑨ Add 50-100 μL of 1×TE solution or ddH2O to dissolve and store in a -20℃ refrigerator for later use.

[0191] Example 2

[0192] Screening of SSR Molecular Marker Primers for Identification of Cattleya Species

[0193] 1) NCBI was used to search for 33 template sequences of Cattleya walkeriana for amplified genes. The 33 gene sequences were opened with SSRHunter software and the SSR loci of each gene sequence were analyzed;

[0194] Primers were designed using Primer 5.0. A total of 50 primers with a rating ratio of more than 60 points were screened according to the primer design principles. SSR loci were selected and primers were designed and screened based on the 33 microsatellite sequences published by Cattleya on NCBI. Specifically, 120 pairs of primers were successfully designed based on the following principles from the 20 sequences containing SSR loci. The principles for SSR primer screening are as follows:

[0195] ① The length of the primer is 18-27 bp, and the target fragment is about 250 bp to 380 bp;

[0196] ②GC content is 40% to 60%, and three or four consecutive bases should be avoided in the primer sequence;

[0197] ③ Annealing temperature is 55-65℃, preferably around 50℃, upstream and downstream primers T m The value difference does not exceed 4 to 6°C;

[0198] ④ Avoid more than 3 consecutive bases at the 3' end of the primer, and try to avoid primer dimers and hairpin structures;

[0199] Sample amplification: The total reaction system is 10 μL, including 1.0 μL of 2 ng / μL DNA solution, 1.0 μL of 4 pmol / μL upstream and downstream primers, 5.0 μL of 2×Taq PCR Master Mix, and 2.0 μL of ddH2O; (The reagents used are from Shanghai Shenggong Biological Company)

[0200] The PCR reaction program was as follows: 94°C pre-denaturation for 1 min, 35 cycles, 94°C denaturation for 1 min, 50°C annealing for 45 s, 72°C extension for 1 min, and 72°C extension for 10 min; 4°C storage, where the annealing temperature was determined according to each primer;

[0201] PCR amplification and agarose gel electrophoresis initial screening, 1.2% agarose gel electrophoresis to detect the amplified product, specifically, 3.0 μL of PCR product was aspirated and added to 2.0 μL of 6× Loading buffer for initial screening;

[0202] Select PCR products with high amplification rate, bright bands, and good peak shape for rescreening through the Fragment AnalyzerTM fully automatic capillary electrophoresis system, which can accurately reflect the differences between alleles and screen out primers with high polymorphism. Figure 1 As shown, the SSR molecular marker primers and the composition thereof for identifying Cattleya varieties of the present invention have the characteristics of high polymorphism, large quantity, low cost, easy reading, etc. compared with other molecular markers.

[0203] The specific operation of the Fragment AnalyzerTM fully automatic capillary electrophoresis system for rescreening is as follows:

[0204] 1. Preparation of reagents:

[0205] ① Preparation of gel: add 2μL of Intercalating Dye to 40ml dsDNA 800Separation Gel and mix thoroughly;

[0206] ②5×930dsDNA Inter Buffer: dilute 5×Inter Buffer 5 times;

[0207] ③5×Capillary conditioning solution: dilute the 5×Capillary conditioning solution 5 times and add 1 mL of Capillary conditioning solution to each well of the 96-well plate to avoid bubbles;

[0208] ④Marker: Add 30 μL of 1bp-500bp markers to each well of a 96-well plate, seal each well with 20 μL of mineral oil, and centrifuge;

[0209] ⑤Sample preparation: Add 20% Dilution buffer and 2% PCR product to each of the 96 wells, add 24% 100-500bp Rang DNA Ladder to the last well, and centrifuge to avoid bubbles;

[0210] 2. Operation steps: Place the prepared reagent into the designated position of the instrument and click the running program of the instrument;

[0211] Data recording and result analysis: Select the band with the highest peak value in each band and record the specific value;

[0212] The requirements for primer rescreening are as follows: 1) the main band is clear, without extra bands, 2) the polymorphism value is high, and there are many alleles, 3) the band difference between two adjacent alleles is greater than 5bp, 4) the primers that meet the first three points are amplified three times, and the primers with good repeatability and stability are selected. Finally, 50 pairs of primers are selected as the core primers for identifying Cattleya varieties. The sorted data are used to calculate and analyze genetic diversity indicators, clustering and polymorphic information content PIC using NTSYS software.

[0213] The molecular marker primers screened and their sequences and annealing temperatures are shown in Table 1.

[0214] Table 1 SSR molecular marker primers and annealing temperature

[0215]

[0216]

[0217]

[0218] The SSR molecular marker primer amplification results are shown in Table 2:

[0219] Table 2 SR molecular marker primer amplification results

[0220]

[0221]

[0222]

[0223] As shown in Table 2, by using the SSR molecular marker primer composition and germplasm identification method of the present invention, 35 Cattleya species can be accurately identified with an accuracy rate of 100%.

[0224] Since there are many SSR molecular marker primers and Cattleya species, the peak graph of SSR12 amplification results is shown as follows: Figure 2The figure shows the agarose gel electrophoresis of DNA extracted from 12 Cattleya varieties with large phenotypic differences. It can be seen that primer SSR12 can distinguish the 12 Cattleya varieties into 5 categories. The molecular clustering results are consistent with the morphological classification, which further reflects the accuracy of SSR molecular markers. Figure 3 The peak diagram of the primer amplification results of 35 varieties of SSR molecular markers. Figure 3 It can be seen that the SSR molecular marker primers described in the present invention have good amplification effect, high detection rate, and can amplify stable DNA bands.

[0225] like Figure 4 The results are shown as genetic analysis and cluster diagram. Combined with the analysis in Table 2, the polymorphic information content (PIC) analysis shows that the SSR primers selected in the experiment are all highly polymorphic primers, which have high polymorphism detection capabilities for Cattleya varieties and are suitable for identification and kinship analysis of Cattleya varieties. From the clustering results, it can be seen that the 25 Cattleya varieties are clustered into 5 categories: Category I is composed of C.lueddemanniana and C.maxima, which are small and medium-sized plants with large flowers; Category II is mostly natural hybrids and artificial hybrids, with rich flower types; Category III is C.eldorado, a single-leaf variety with small flowers and a distinct yellow throat; Category IV is mainly C.trianae, with many flower color varieties and rich lip types; Category V is C.guttata, a two-leaf species with medium-sized flowers and spots, which is basically consistent with the morphological clustering results.

[0226] In summary, it can be seen that the SSR molecular marker primers of the present invention can be used to assist in selective breeding and achieve early selection at the seedling stage, thereby accelerating the breeding process of Cattleya.

Claims

1. An application of SSR molecular marker primers in Cattleya phylogenetic analysis, DUS specificity test and molecular marker-assisted breeding, characterized in that: The genus Cattleya includes the closely related genera Phalaenopsis, Dendrobium, and Cymbidium; The SSR molecular markers for identifying Cattleya species include SSR1~SSR50; The sequences of the SSR molecular marker primers for identifying Cattleya species are as follows: Primers for the molecular marker SSR1: SEQ ID NO.1: SSR1-F:5'-GGGAGGGAGGAATAGGAAGA-3'; SEQ ID NO.2: SSR1-R:5'-TGAGCTGCGATATCAAAGGA-3'; Primers for the molecular marker SSR2: SEQ ID NO.3: SSR2-F:5'-TCAAGGCCTGCACATAGAGA-3'; SEQ ID NO.4: SSR2-R:5'-AAGAGGA AGGCTTCGTTGC-3'; Primers for the molecular marker SSR3: SEQ ID NO.5: SSR3-F:5'-AACAGGCATTTGGAGCTTTT-3'; SEQ ID NO.6: SSR3-R:5'-CCTCATTTCTCTCACCCTCTTT-3'; Primers for the molecular marker SSR4: SEQ ID NO.7: SSR4-F:5'-TGGATCTTACCTTGGGCTTCTA-3'; SEQ ID NO.8: SSR4-R:5'-CCCTGCTTCTCTTTCTCACTCAT-3'; Primers for the molecular marker SSR5: SEQ ID NO.9: SSR5-F:5'-GGGAGGACTATCAACCTCAA-3'; SEQ ID NO.10: SSR5-R:5'-CAGCCTCTCCAGGTAGGTTT-3'; Primers for the molecular marker SSR6: SEQ ID NO.11: SSR6-F:5'-AATGCGACGAAGAAAGCCTA-3'; SEQ ID NO.12: SS6-R:5'-AGCACTTGCCCTTGTCAGTT-3'; Primers for the molecular marker SSR7: SEQ ID NO.13: SSR7-F: 5'-TTTGGTAAGGGTGGACCAAG-3'; SEQ ID NO.14: SSR7-R:5'-TTACCGAGTCCTTCCAAACA-3'; Primers for the molecular marker SSR8: SEQ ID NO.15: SSR8-F:5'-ATCAAGCCATGGCAGTCTTT-3'; SEQ ID NO.16: SSR8-R:5'-TCGGCTTGACTGCAAAGTTA-3'; Primers for the molecular marker SSR9: SEQ ID NO.17: SSR9-F:5'-CATGAGACACGTTGGGCTTA-3'; SEQ ID NO.18: SSR9-R:5'-ATGCGACGAAGAAAGCCTAC-3'; Primers for the molecular marker SSR10: SEQ ID NO.19: SSR10-F:5'-CTCCGATGGGTCTATATCAAG-3'; SEQ ID NO.20: SSR10-R:5'-TGGGAGCTTGTACCCAAGA-3'; SEQ ID NO.21: SSR11-F:5'-CTCAGTCCTGCGATCCAAT-3'; SEQ ID NO.22: SSR11-R:5'-GGTAACCCGTGCAACCAT-3'; Primers for the molecular marker SSR12: SEQ ID NO.23: SSR12-F:5'-AGAATCAATGCCCCAAGC-3'; SEQ ID NO.24: SSR12-R:5'-GGTAACCCGTGCAACCAT-3'; Primers for the molecular marker SSR13: SEQ ID NO.25: SSR13-F:5'-TGTGGTCTGTCTAACGCAGAG-3'; SEQ ID NO.26: SSR13-R:5'-TCTTCAGCTCAGTCCTGCAA-3'; Primers for the molecular marker SSR14: SEQ ID NO.27: SSR14-F:5'-TCCCTTGACCCTAGGAAGGT-3'; SEQ ID NO.28: SSR14-R:5'-TGAGTCCCTCCCCTTTGACTT-3'; Primers for the molecular marker SSR15: SEQ ID NO.29: SSR15-F:5'-GGCATCAAACCACTTACATCC-3'; SEQ ID NO.30: SSR15-R:5'-CGCAAGCCTAAGTGAGATCC-3'; Primers for the molecular marker SSR16: SEQ ID NO.31: SSR16-F:5'-GTGGCTTAGTCTTACATTGGATAGTT-3'; SEQ ID NO.32: SSR16-R:5'-TATTAGAGAGTAAATCAAAAAGGGCT-3'; Primers for the molecular marker SSR17: SEQ ID NO.33: SSR17-F:5'-GGGGATCATTAGGGCATATAACTAGAA-3'; SEQ ID NO.34: SSR17-R:5'-AGAAGGGTCTTAAAATAGGGATTAGGG-3'; Primers for the molecular marker SSR18: SEQ ID NO.35: SSR18-F:5'-ACGTGGCTCTTCCTTCTTGAAT-3'; SEQ ID NO.36: SSR18-R:5'-CAGGGAGAAAAAGGGATAGAACC-3'; Primers for the molecular marker SSR19: SEQ ID NO.37: SSR19-F:5'-TAACAGGCTGACCATGCTTTCTAT-3'; SEQ ID NO.38: SSR19-R:5'-TGAAGTCGAGTATTCCAAACACAA-3'; Primers for the molecular marker SSR20: SEQ ID NO.39: SSR20-F:5'-CCCCTCTTGTCTGTCACACATAA-3'; SEQ ID NO.40: SSR20-R:5'-ATGAACTGAATACTCATGGATACTGA-3'; SEQ ID NO.41: SSR21-F:5'-CAAGCTTTTATTAGCTTATTTTATTTAC-3'; SEQ ID NO.42: SSR21-R:5'-TCTTTGACCGCCACAGTAGG-3'; Primers for the molecular marker SSR22: SEQ ID NO.43: SSR22-F: 5'-GTTGTTTGTAGATTGATTCTCTTTCATCC-3'; SEQ ID NO.44: SSR22-R:5'-TTCATTAGCGATTTCTTCTCAAATGC-3'; Primers for the molecular marker SSR23: SEQ ID NO.45: SSR23-F:5'-AAAGTGACTAGGAAAAAAATAACTGAG-3'; SEQ ID NO.46: SSR23-R:5'-TTGTGTGGGTTATGTAGGAAAGT-3'; Primers for the molecular marker SSR24: SEQ ID NO.47: SSR24-F:5'-TAAAATGGTCTATTCAAATAAAAATCGT-3'; SEQ ID NO.48: SSR24-R:5'-ACATTGTCTATGACCCCCTAATAAAA-3'; Primers for the molecular marker SSR25: SEQ ID NO.49: SSR25-F:5'-TAAATAAGAGGCCTTGGAATGTC-3'; SEQ ID NO.50: SSR25-R:5'-GGGTGTAGAAAACTTAAGGAATTGA-3'; Primers for the molecular marker SSR26: SEQ ID NO.51: SSR26-F:5'-TTCTTGCTTCTTTTTGTGGA-3'; SEQ ID NO.52: SSR26-R:5'-ATTTGTTTGATACGCCATG-3'; Primers for the molecular marker SSR27: SEQ ID NO.53: SSR27-F:5'-GTGCTAATTCAACGCAAA-3'; SEQ ID NO.54: SSR27-R:5'-TTAAAAGCCGAGTACTCTACC-3'; Primers for the molecular marker SSR28: SEQ ID NO.55: SSR28-F:5'-TGAGATTGGATTGGAAGAAGA-3'; SEQ ID NO.56: SSR28-R:5'-TGAGGGTTCAAGTCCCTCTA-3'; Primers for the molecular marker SSR29: SEQ ID NO.57: SSR29-F:5'-TAGGATGATGCACGGGAAA-3'; SEQ ID NO.58: SSR29-R:5'-GGGGGTTTTTATCATTGAGGA-3'; Primers for the molecular marker SSR30: SEQ ID NO.59: SSR30-F:5'-AGAGAAAGCCCTTGTGTTGG-3'; SEQ ID NO.60: SSR30-R:5'-TCAGCTCTTCCGATTCTGGT-3'; SEQ ID NO.61: SSR31-F:5'-GCTTCAAGCAAGTGCAGAAA-3'; SEQ ID NO.62: SSR31-R:5'-AAGACAGGCCAACCAGAGAA-3'; Primers for the molecular marker SSR32: SEQ ID NO.63: SSR32-F:5'-TGGTTTGCTAAGTATTTCTTCAAGTT-3'; SEQ ID NO.64: SSR32-R:5'-TATGCAAAGCTTCCCCAAGT-3'; Primers for the molecular marker SSR33: SEQ ID NO.65: SSR33-F:5'-TGAATGCAAAATCCAATTGCT-3'; SEQ ID NO.66: SSR33-R:5'-GAAATATTCAAGCACGATGATCC-3'; Primers for the molecular marker SSR34: SEQ ID NO.67: SSR34-F:5'-TGCATACGTAACAACTGGAGGT-3'; SEQ ID NO.68: SSR34-R:5'-GGAAGGTCATTCTAACCAGGAA-3'; Primers for the molecular marker SSR35: SEQ ID NO.69: SSR35-F:5'-ACGCTCTTTGGCTGGAACT-3'; SEQ ID NO.70: SSR35-R:5'-CTCACATGCCTTTAGCCTCAC-3'; Primers for the molecular marker SSR36: SEQ ID NO.71: SSR36-F:5'-TGCCCCAGACAATTAAGC-3'; SEQ ID NO.72: SSR36-R:5'-CCTCGATGGAACCCCATAAT-3'; Primers for the molecular marker SSR37: SEQ ID NO.73: SSR37-F:5'-TTGGGTTTCGTCTCACATCA-3'; SEQ ID NO.74: SSR37-R:5'-CCCTCAGTATCCGCCACTT-3'; Primers for the molecular marker SSR38: SEQ ID NO.75: SSR38-F:5'-CTTGTCTACGTGAGGGCACTG-3'; SEQ ID NO.76: SSR38-R:5'-TCAACAACGTGAAAAGCCATC-3'; Primers for the molecular marker SSR39: SEQ ID NO.77: SSR39-F:5'-CCCTCCACCCACCAAAATC-3'; SEQ ID NO.78: SSR39-R:5'-CAACACCTTGAGGAACGCC-3'; Primers for the molecular marker SSR40: SEQ ID NO.79: SSR40-F:5'-TGGCATTTGGGAGCAACAG-3'; SEQ ID NO.80: SSR40-R:5'-TGACGCACACCAGAGATCC-3'; SEQ ID NO.81: SSR41-F:5'-CATGTCAATACCGACAAC-3'; SEQ ID NO.82: SSR41-R:5'-CATATATGTTCATATCGT-3'; Primers for the molecular marker SSR42: SEQ ID NO.83: SSR42-F:5'-GTGCTCAACCCACAGGAACT-3'; SEQ ID NO.84: SSR42-R:5'-AATAAGTATGAATAGAAC-3'; Primers for the molecular marker SSR43: SEQ ID NO.85: SSR43-F:5'-TCATTCATAGTCCATAT-3'; SEQ ID NO.86: SSR43-R:5'-GCATCATCCTAGCAGAGT-3'; Primers for the molecular marker SSR44: SEQ ID NO.87: SSR44-F:5'-TGGTATAGGTTCAAATCC-3'; SEQ ID NO.88: SSR44-R:5'-AGCTATTCAGGAACAGAT-3'; Primers for the molecular marker SSR45: SEQ ID NO.89: SSR45-F:5'-GAAAAGCAATCCAACAAC-3'; SEQ ID NO.90: SSR45-R:5'-GTAAAGGAGAATAATGACC-3'; Primers for the molecular marker SSR46: SEQ ID NO.91: SSR46-F:5'-ATGGGCGGTTTCTCATAACG-3'; SEQ ID NO.92: SSR46-R:5'-TTTCTTTACGCGTCATTCTTTGG-3'; Primers for the molecular marker SSR47: SEQ ID NO.93: SSR47-F:5'-ATTGGCATTCTTCTGT-3'; SEQ ID NO.94: SSR47-R:5'-TAAGCCCTTATGTTTG-3'; Primers for the molecular marker SSR48: SEQ ID NO.95: SSR48-F:5'-TCAGACACTTTGGGTCAAAGA-3'; SEQ ID NO.96: SSR48-R:5'-GAGGTGCTCCAAAGATCAAACT-3'; Primers for the molecular marker SSR48: SEQ ID NO.97: SSR49-F:5'-AAAAATAAATGCATGAATATGAGTATG-3'; SEQ ID NO.98: SSR49-R:5'-GGATTATAAGAAATCGATGATGTGA-3'; Primers for the molecular marker SSR50: SEQ ID NO.99: SSR50-F:5'-TTTTGAACATAGAAAGCAATCC-3'; SEQ ID NO. 100: SSR50-R:5'-GATTCGGATAGAGAAGCAAAA-3'.

2. An SSR molecular marker primer for identifying Cattleya species, characterized in that: The screening method of the SSR molecular marker primers comprises the following steps: 1) Extraction of total DNA from Cattleya; 2) Select SSR loci and design and screen primers based on the 33 microsatellite sequences published by Cattleya on NCBI; 3) PCR amplification and initial screening using the Fragement AnalyzerTM fully automated capillary electrophoresis system, and selecting core primers based on the electrophoresis results; The sequences of the molecular marker primers obtained by screening are shown in SEQ ID NO.1 to SEQ ID NO.100 respectively.

3. The SSR molecular marker primer for identifying Cattleya species according to claim 2, characterized in that: Step 2) The 33 microsatellite sequences published by Cattleya on NCBI were used to select SSR sites and design and screen primers. Specifically, 120 pairs of primers were designed according to the following principles from the 20 sequences containing SSR sites obtained, wherein the screening principles of SSR primers are as follows: 1) The length of the primer is 18~27bp, and the target fragment is about 250bp~380bp; 2) The GC content is 40% to 60%, and three or four consecutive bases should be avoided in the primer sequence; 3) The annealing temperature is between 55 and 65°C, preferably around 50°C, and the Tm value difference between the upstream and downstream primers should not exceed 4 to 6°C; 4) Avoid more than three consecutive bases at the 3' end of the primer, and try to avoid primer dimers and hairpin structures.

4. The SSR molecular marker primer for identifying Cattleya species according to claim 2, characterized in that: Step 3) The PCR amplification and Fragment AnalyzerTM fully automatic capillary electrophoresis system initial screening specifically involves aspirating 2 μL of the PCR product for initial screening by agarose gel electrophoresis, initial screening of primers by comparison with the target fragment, and selecting PCR products with high amplification rate, clear bands, and good peak shape for rescreening by the Fragment AnalyzerTM fully automatic capillary electrophoresis system, which can accurately reflect the differences between allelic variation sites and screen primers with high polymorphism.

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