SSR molecular marker primer based on transcriptome sequence of huahua shai and application thereof
Patent Information
- Application Number
- CN202610682787.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-05-18
- Publication Date
- 2026-09-29
AI Technical Summary
尽管SSR分子标记技术已广泛应用于草类植物的种质遗传多样性分析及DNA指纹图谱构建，但在花花柴中尚未见报道
本发明基于花花柴转录组数据开发的SSR分子标记，该标记多态性水平高、稳定性好，能够高效应用于花花柴遗传多样性分析、种质资源鉴定、品种真实性检测，为花花柴种质资源保护、遗传育种及分子辅助选择提供可靠、高效的分子工具。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular marker technology, and more specifically, this invention relates to SSR molecular markers, primers and their applications in *Hylocereus undatus*. Background Technology
[0002] Flower Tree ( Karelinia caspia *Ilex chinensis* is a perennial herbaceous plant belonging to the genus *Ilex* in the family Asteraceae, mainly distributed in saline-alkali sandy lands and Gobi environments in the arid regions of Northwest China. *Ilex chinensis* possesses extremely strong tolerance to abiotic stresses, exhibiting excellent characteristics such as drought resistance, salt and alkali tolerance, high temperature resistance, wind erosion resistance, and sand burial resistance. It can effectively play an ecological role in soil and water conservation and improving saline-alkali land, occupying an irreplaceable position in the restoration of degraded grasslands and sand control in arid and semi-arid regions. Furthermore, the branches and leaves of *Ilex chinensis* are rich in crude protein, various amino acids, and mineral elements, making it suitable for use as forage. At the same time, the flavonoids, polysaccharides, chlorogenic acid, and other active ingredients in the leaves endow it with potential medicinal development value, making it a unique species with both ecological and economic value.
[0003] Molecular marker technology is a core tool for accelerating the breeding process. Among them, SSR (simple sequence repeat) markers have significant advantages such as high polymorphism, strong co-dominance, good specificity, wide distribution, low cost, and stable reproducibility, and have become a commonly used technique for genetic mapping, germplasm identification, gene localization, marker-assisted selection, and genetic diversity analysis. Although SSR molecular marker technology has been widely used in the analysis of genetic diversity and DNA fingerprinting of herbaceous plants, it has not yet been reported in *Pterocarya stenoptera*. Analyzing the genetic diversity of *Pterocarya stenoptera* germplasm and developing efficient molecular markers are of great significance for the screening and identification of *Pterocarya stenoptera* germplasm resources and for the breeding of *Pterocarya stenoptera* varieties. Summary of the Invention
[0004] To address the shortcomings and challenges of existing technologies, this invention aims to provide a combination of SSR molecular marker primers for *Cymbidium goeringii* and their applications.
[0005] The primer combination for *Cymbidium goeringii* SSR molecular markers provided by this invention is any combination of one or more of the following primer pairs 1-17: The sequence of the upstream primer of primer pair 1 is: 5'-TGCTTCATTTCCCTACCAAAA-3', and the sequence of the downstream primer is: 5'-AGGTCCTCAGCGAAGCAGT-3'; The sequence of the upstream primer of primer pair 2 is: 5'- GACAATCATTCCAACGGCTT -3', and the sequence of the downstream primer is: 5'- GGGCTTGTGACAAATGTTGA -3'; The sequence of the upstream primer of primer pair 3 is: 5'-AGAATGGGACAAGCAAAAGAG-3', and the sequence of the downstream primer is: 5'-TGGTGGTAAGTTGGGTTGAA-3'; The sequence of the upstream primer for primer pair 4 is: 5'-CAAGTACATCAAAACCGAACCA -3', and the sequence of the downstream primer is: 5'-CTTCCCTTTTTTTTTTGGGC -3'. The sequence of the upstream primer of primer pair 5 is: 5'- AGAGAGGAGTTGCGATGATGA -3', and the sequence of the downstream primer is: 5'- GCTTTTCCAGTGCATCCTGT -3'; The sequence of the upstream primer of primer pair 6 is: 5'- CCCAAGCACTGGAAATTGTT-3', and the sequence of the downstream primer is: 5'- CTCCTCGTGAACCCATTGTT-3'; The sequence of the upstream primer of primer pair 7 is: 5'- GGGCTTTAATGAAGGTGGTG -3', and the sequence of the downstream primer is: 5'- TGGTTTACAGCAATGACCTCC -3'; The sequence of the upstream primer of primer pair 8 is: 5'- TCCACACCGCCTAAATTTTC -3', and the sequence of the downstream primer is: 5'- AAACCTCCTGATGGCAAGTG -3'; The sequence of the upstream primer of primer pair 9 is: 5'-CTCGATTCGCTTTGCATTTT-3', and the sequence of the downstream primer is: 5'-CCTCATTGACCCAATCTCGT-3'; The sequence of the upstream primer of primer pair 10 is: 5'- TGGGCTTTGCTGTTATCTCTC-3', and the sequence of the downstream primer is: 5'- TGGAAGGGTGCAAGTAGGTT-3'; The sequence of the upstream primer of primer pair 11 is: 5'- TCTGGCCTTGATTTTGGAAC -3', and the sequence of the downstream primer is: 5'- CTCTTCCAGGCATGGTTTTC -3'; The sequence of the upstream primer of primer pair 12 is: 5'-CAAGAAGCTGTCTTTTCGCA-3', and the sequence of the downstream primer is: 5'-TCACCCATTCACCCCTATGT-3'; The sequence of the upstream primer of primer pair 13 is: 5'- AGGGCCAAGAGCCTACATCT -3', and the sequence of the downstream primer is: 5'- TGCATGAGCAATGGAATTGT -3'; The sequence of the upstream primer of primer pair 14 is: 5'- CAAATCTCTGCCTTGGCTTC -3', and the sequence of the downstream primer is: 5'- AATCCCATTTCGTCCTCTCC -3'; The sequence of the upstream primer of primer pair 15 is: 5'- TGAAAGGGAAGGGGTTTTCT -3', and the sequence of the downstream primer is: 5'- GCATTCTCGCATTTATGTGG -3'; The sequence of the upstream primer of primer pair 16 is: 5'- TTATTCCCATACCGTGCCAT -3', and the sequence of the downstream primer is: 5'- AACCAAACCCAAGAGACCAA -3'; The sequence of the upstream primer of primer pair 17 is: 5'- TTCACAGCTTTTGATGCAGG -3', and the sequence of the downstream primer is: 5'- AACCATGGTGGAGTTTTTGG -3'.
[0006] The sequence of the upstream primer of primer pair 18 is: 5'-CGTCAAATGGGAATTGCTTT-3', and the sequence of the downstream primer is: 5'-AGGTACGTCCATGAATTGGC-3'; The sequence of the upstream primer of primer pair 19 is: 5'- GCTTCGAGGTAGAGGACGTG -3', and the sequence of the downstream primer is: 5'- CGAACCTAAAATGACCGGAG -3'; The sequence of the upstream primer of primer pair 20 is: 5'- AAACGAACGGTGGAGATACG -3', and the sequence of the downstream primer is: 5'- GTCGATTCAGGCTTCTCGTC -3'; The sequence of the upstream primer of primer pair 21 is: 5'- GTCCGTGGTTTCACGTTTCT -3', and the sequence of the downstream primer is: 5'- AAATGAATTACAGGTCGCCG -3'; The sequence of the upstream primer of primer pair 22 is: 5'- CCAAATCGGAGGTACAGGAA -3', and the sequence of the downstream primer is: 5'- AGCTAGAACTGCCACTCCCA -3'; The sequence of the upstream primer of primer pair 23 is: 5'- AAGATGAGTCAGACTGGCCG -3', and the sequence of the downstream primer is: 5'- GGTGTGATGGTGGAGCTTTT -3'; The sequence of the upstream primer of primer pair 24 is: 5'- AATGCGTACCTCCACCTGAG -3', and the sequence of the downstream primer is: 5'- TTCATGCTCTTTTCCGGTTC 3'; The sequence of the upstream primer of primer pair 25 is: 5'-GTCTCCTTCCCACAACCTCA-3', and the sequence of the downstream primer is: 5'-AAAAAGAACCCATTACCCGC-3'; The sequence of the upstream primer of primer pair 26 is: 5'- AATGTTTCGTCCGATTGAGG -3', and the sequence of the downstream primer is: 5'- AGAACAGACGGACGGAGAGA -3'; The sequence of the upstream primer for primer pair 27 is: 5'- TTCGTTGTTGCATCATGGTT -3', and the sequence of the downstream primer is: 5'- GTTAGTTGTTGAAGGGGCCA -3'.
[0007] Preferably, the *Cymbidium goeringii* SSR molecular marker primer combination consists of primer pair 1 and primer pair 2; the sequence of the upstream primer of primer pair 1 is: 5'-TGCTTCATTTCCCTACCAAAA-3', and the sequence of the downstream primer is: 5'-AGGTCCTCAGCGAAGCAGT-3'; the sequence of the upstream primer of primer pair 2 is: 5'-GACAATCATTCCAACGGCTT-3', and the sequence of the downstream primer is: 5'-GGGCTTGTGACAAATGTTGA-3'.
[0008] The application of the primers described herein in distinguishing germplasm and identifying varieties of *Cypripedium pratum* is also within the scope of protection of this invention.
[0009] The application of the primers described herein in the identification of kinship among *Cercidiphyllum japonicum* is also within the scope of protection of this invention.
[0010] The application of the primers described herein in the analysis of genetic diversity of *Pterocarya stenoptera* germplasm is also within the scope of protection of this invention.
[0011] The application of the primers described herein in molecular marker-assisted breeding of *Cypripedium pratense* is also within the scope of protection of this invention.
[0012] Compared with the prior art, the beneficial effects of the present invention include: This invention relates to an SSR molecular marker developed based on *Pterocarya stenoptera* transcriptome data. This marker exhibits high polymorphism and good stability, and can be efficiently applied to *Pterocarya stenoptera* genetic diversity analysis, germplasm resource identification, and variety authenticity detection, providing a reliable and efficient molecular tool for *Pterocarya stenoptera* germplasm resource protection, genetic breeding, and molecular-assisted selection. Attached Figure Description
[0013] Figure 1 This is an agarose gel electrophoresis image of some primers.
[0014] Figure 2 This is a capillary electrophoresis image of some primers.
[0015] Figure 3 UPGMA cluster analysis of 25 *Pterocarya stenoptera* germplasm resources.
[0016] Figure 4 A line graph showing the ΔK value.
[0017] Figure 5 Population structure diagrams for 25 *Cinnamomum camphora* germplasms.
[0018] Figure 6 This is a fingerprint map of 25 *Pterocarya stenoptera* germplasm accessions constructed based on SSR markers. Detailed Implementation
[0019] The technical solution of the present invention will be further described in detail below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.
[0020] In this invention, the equipment and raw materials used are all commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.
[0021] Example 1: Screening of SSR molecular markers in *Cypripedium praecox* 1. Test materials The 25 *Gnaphalium affine* germplasm resources used for identification in this patent were obtained from the National Important Wild Plant Germplasm Resource Bank of Kunming Institute of Botany, Chinese Academy of Sciences, self-collected germplasm, and air-induced mutation germplasm. The germplasm distribution covers three provincial-level administrative regions in China: Xinjiang Uygur Autonomous Region, Gansu Province, and Inner Mongolia Autonomous Region (Table 1).
[0022] Table 1 Germplasm Resource Information of *Hylocereus undatus* ; Table 1 (continued)
[0023] 2. DNA extractand SSR Primer Screening Adopting an improved version CTAB DNA was extracted from 25 *Cinnamomum camphora* germplasms using the following method: 50 mg of fresh leaves were weighed into a 2 mL centrifuge tube, a steel bead was added, and the sample was rapidly frozen using liquid nitrogen. The sample was then ground into powder using a grinder. The powder was transferred to a 2 mL centrifuge tube containing 800 μL of 2×CTAB, and 60 μL of mercaptoethanol was added. The mixture was preheated at 60 °C for 30 min (inverting and mixing 2-3 times during this period). After cooling to room temperature, 800 μL of chloroform:isoamyl alcohol (24:1) was added, and the mixture was centrifuged at 12000 r·min⁻¹ for 15 min. The supernatant was transferred to a new tube, 1.5 times the volume of 1×CTAB precipitation buffer was added, and the mixture was incubated at room temperature for 20-30 min, centrifuged for 15 min, the supernatant was discarded, and the precipitate was dried and dissolved in 200 μL of LTE-buffer. Add 400 μL of 95% ethanol and 20 μL of 3M NaAC, precipitate at -20℃ for 1 h, centrifuge at 4℃ for 15 min, discard the supernatant, wash with 500 μL of 75% ethanol, and centrifuge for 10 min. Finally, air-dry the precipitate, dissolve it in 30-50 μL of TE buffer, and determine the DNA quality by 0.8% agarose gel electrophoresis. Store samples that meet the standards at -20℃ for later use.
[0024] From the previous transcriptome data (NCBI-SRA accession number: PRJNA744046), 178 pairs of SSR primers were screened according to tetranucleotide type 5-6 repeat sequences, pentanucleotide type 5 repeat sequences, and hexanucleotide type 5-8 repeat sequences for preliminary identification. Eleven *Cypripedium praecox* samples were randomly selected for PCR amplification. The reaction system was 25 μl: 10×PCR buffer 2.5 μl, template DNA 2 μl (20 ng·μL-1), forward and reverse primers 0.5 μl each (10 pmol·μL-1), dNTP 0.5 μl (5 μmol / L), Taq DNA polymerase 0.5 μl (2.5 U·μL-1), and ddH2O 18.5 μl. PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50-60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 10 min; PCR products were stored at 4℃. Agarose gel electrophoresis was performed on the above PCR products, and 27 pairs of primers with clear bands and good polymorphism were initially screened, including 18 pairs of tetranucleotide repeat unit primers and 9 pairs of hexanucleotide repeat unit primers. Agarose gel images of some primer bands are shown below. Figure 1 .
[0025] 3. PCR amplification and capillary electrophoresis of 27 *Cinnamomum camphora* germplasm The 27 pairs of primers with good polymorphism selected (shown in Table 2) were used to perform PCR amplification and capillary electrophoresis on 25 *Cinnamomum camphora* germplasm accessions. Figure 2 The reaction was performed in a 25 μl system: 2.5 μl of 10×PCR buffer and 2 μl of template DNA (20 ng / μL). -1 0.5 μL (10 pmol·μL) each of the forward and reverse primers -1 ), dNTP 0.5 μL (5 μmol / L), Taq DNA polymerase 0.5 μL (2.5 U·μL) -1 ), ddH2O 18.5 μl. PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 50~60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 35 cycles; 72℃ extension for 10 min, PCR products stored at 4℃. Before loading, the molecular weight internal standard and deionized formamide were prepared at a ratio of 0.5 µl: 9.5 µl to form a loading system. 10 µl of this system was added to the sample plate, followed by 1 µl of PCR product diluted 10-fold. The mixture was then analyzed by capillary electrophoresis using an ABI 3730XL sequencer.
[0026] 4. SSR molecular marker polymorphism analysis The peak plots were read and analyzed using Genescan 3.1 and Binthere software, and the results were exported and saved in XLS format. Non-zero values in the table were converted to 1 (zero values were not converted), thus generating the original matrix composed of "1"s and "0"s. Powermarker software was used to calculate the number of alleles (Na), effective number of alleles (Ne), Shannon information index (I), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information index (PIC), and genetic distances between various types of alleles for each tested sample. A total of 192 alleles were detected in 25 *Cinnamomum camphora* germplasm materials using 27 SSR primer pairs, with an average number of alleles (Na) of 7.111; the total number of effective alleles (Ne) was 90.585, with an average of 3.355. The Shannon Information Index (I) ranged from 0.606 to 2.950, with an average of 1.259. The observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.120 to 0.920 and 0.285 to 0.838, respectively, with averages of 0.487 and 0.578. Among the 27 primer pairs, 6 pairs had a mean observed heterozygosity higher than the mean expected heterozygosity, while the other 21 pairs showed the opposite. The polymorphism information content (PIC) ranged from 0.271 to 0.935, with an average of 0.541; 15 sites had PIC values greater than 0.5, and 12 primer pairs had PIC values between 0.25 and 0.50. This indicates that the selected primers possess high polymorphism and good discriminative ability.
[0027] 5. Cluster analysis Genetic similarity coefficients were calculated for 25 *Pterocarya stenoptera* germplasms using the unweighted group average method, and cluster analysis was performed based on genetic distance. When the genetic distance threshold was set to 0.77, all tested materials could be divided into 5 groups. Group I included 18 germplasms (Kc1, Kc2, Kc4, Kc6, Kc7, Kc8, Kc9, Kc10, Kc11, Kc12, Kc13, Kc15, Kc16, Kc18, Kc20, Kc21, Kc22, and Kc25); Group II included 3 germplasms (Kc14, Kc23, and Kc24); Group III consisted of 2 germplasms (Kc17 and Kc19); and Kc3 and Kc5 constituted Group IV and Group V, respectively. Figure 3 ).
[0028] 6. Population genetic structure analysis The genetic structure of the tested *Pterocarya stenoptera* germplasms was analyzed using Structure 2.3.4 software. The predicted population size K was set to 1–10. The initial MCMC (Markov chain monte carlo) iterations were set to 10,000 without counting, and then the MCMC iterations after the no-count iterations were set to 100,000. This was repeated 5 times. The results were imported into the Structureharvester online program to calculate ΔK, and then graphically output in Structure 2.3.4. The ΔK value showed a clear peak characteristic with the change of K value, reaching its highest peak when K=5. Based on this, the optimal number of genetic groups for the 25 *Pterocarya stenoptera* germplasms was determined to be 5. Figure 4 Based on this optimal K value, the population structure map of 25 *Pterocarya stenoptera* germplasms was drawn using Structure software. Using the maximum Q value in each germplasm as the basis for population division, the 25 *Pterocarya stenoptera* germplasms were divided into 5 groups (Pop I ~ Pop V). Figure 5 PopⅠ (Red Dominant): Includes Kc3, Kc5, Kc9, Kc8, Kc10, Kc13; PopⅡ (Green Dominant): Includes Kc18 and Kc16; PopⅢ (Blue Dominant): Includes Kc23, Kc14, Kc24, Kc21, Kc12, Kc2, Kc1; PopⅣ (Yellow Dominant): Includes Kc6, Kc20, Kc25; PopⅤ (Pink Dominant): Includes Kc19, Kc17, Kc22, Kc15, Kc7, Kc11, Kc4.
[0029] 7. Construction of fingerprint spectrum of *Cyprinus praecox* Fingerprint maps of *Pterocarya stenoptera* were constructed based on the genotyping results of SSR molecular markers. P156 could identify 20 *Pterocarya stenoptera* accessions at once, P58 could identify 15, P45 could identify 13, P124, P158, and P162 could identify 9, P43, P66, P100, and P151 could identify 5, while P76, P98, P108, P153, P175, and P176 could only identify one sample at a time. High-discrimination primers were preferred for fingerprint map construction; using primer pairs P156 and P58 was sufficient to identify 25 *Pterocarya stenoptera* accessions. Therefore, these two primer pairs were used to construct the *Pterocarya stenoptera* fingerprint map. The presence or absence of each allele in each germplasm is assigned two values, "1" and "0". The combination of primer site data for each germplasm is the DNA fingerprint of that sample. Figure 6 ).
[0030] Table 2 Information on 27 SSR primer pairs P1 (AAAT)5 AGAATGGGACAAGCAAAAGAG(SEQ ID No.5) TGGTGGTAAGTTGGGTTGAA(SEQ ID No.6) P4 (GAAA)6 CAAGTACATCAAAACCGAACCA(SEQ ID No.7) CTTCCCTTTTTCTTTTGGGC(SEQ ID No.8) P8 (TTTA)5 AGAGAGGAGTTGCGATGATGA(SEQ ID No.9) GCTTTTCCAGTGCATCCTGT(SEQ ID No.10) P43 (TTTA)5 CCCAAGCACTGGAAATTGTT(SEQ ID No.11) CTCCTCGTGAACCCATTGTT(SEQ ID No.12) P45 (TTCT)8 GGGCTTTAATGAAGGTGGTG(SEQ ID No.13) TGGTTTACAGCAATGACCTCC(SEQ ID No.14) P47 (AATT)5 TCCACACCGCCTAAATTTTC(SEQ ID No.15) AAACCTCCTGATGGCAAGTG(SEQ ID No.16) P58 (GATA)5 GACAATCATTCCAACGGCTT(SEQ ID No.3) GGGCTTGTGACAAATGTTGA(SEQ ID No.4) P66 (AATA)5 CTCGATTCGCTTTGCATTTT(SEQ ID No.17) CCTCATTGACCCAATCTCGT(SEQ ID No.18) P72 (ATTT)5 TGGGCTTTGCTGTTATCTCTC(SEQ ID No.19) TGGAAGGGTGCAAGTAGGTT(SEQ ID No.20) P76 (AAAT)5 TCTGGCCTTGATTTTGGAAC(SEQ ID No.21) CTCTTCCAGGCATGGTTTTC(SEQ ID No.22) P88 (TTAT)6 CAAGAAGCTGTCTTTTCGCA(SEQ ID No.23) TCACCCATTCACCCCTATGT(SEQ ID No.24) P98 (TTAT)5 AGGGCCAAGAGCCTACATCT(SEQ ID No.25) TGCATGAGCAATGGAATTGT(SEQ ID No.26) P100 (TATT)5 CAAATCTCTGCCTTGGCTTC(SEQ ID No.27) AATCCCATTTCGTCCTCTCC(SEQ ID NO.28) P108 (AAAT)5 TGAAAGGGAAGGGGTTTTCT(SEQ ID No.29) GCATTCTCGCATTTATGTGG(SEQ ID NO.30) P109 (AAAG)6 TTATTCCCATACCGTGCCAT(SEQ ID NO.31) AACCAAACCCAAGAGACCAA(SEQ ID No.32) P99 (AAAG)5 TTCACAGCTTTTGATGCAGG(SEQ ID NO.33) AACCATGGTGGAGTTTTTGG(SEQ ID No.34) P124 (CTTT)6 CGTCAAATGGGAATTGCTTT(SEQ ID No.35) AGGTACGTCCATGAATTGGC(SEQ ID NO.36) P128 (AAAT)5 GCTTCGAGGTAGAGGACGTG(SEQ ID NO.37) CGACCTAAAATGACCGGAG(SEQ ID No.38) P151 (GAAAAC)5 AAACGAACGGTGGAGATACG(SEQ ID NO.39) GTCGATTCAGGCTTCTCGTC(SEQ ID NO.40) . P153 (TGCTGT)5 GTCCGTGGTTTCACGTTTCT(SEQ ID NO.41) AAATGAATTACAGGTCGCCG(SEQ ID No.42) P156 (TTATTG)6 TGCTTCATTTCCCTACCAAAA(SEQ ID NO.1) AGGTCCTCAGCGAAGCAGT(SEQ ID NO.2) P158 (ACTGGT)8 CCAAATCGGAGGTACAGGAA(SEQ ID No.43) AGCTAGAACTGCCACTCCCA(SEQ ID NO.44) P162 (PRICE)5 AAGATGAGTCAGACTGGCCG(SEQ ID No.45) GGTGTGATGGTGGAGCTTTT(SEQ ID No.46) P164 (GATGAC)5 AATGCGTACCTCCACCTGAG(SEQ ID NO.47) TTCATGCTCTTTTCCGGTTC(SEQ ID NO.48) P175 (CCCAAA)5 GTCTCCTTCCCACAACCTCA(SEQ ID NO.49) AAAAAGAACCCATTACCGCC(SEQ ID NO.50) P176 (AGGGTT)5 AATGTTTCGTCCGATTGAGG(SEQ ID NO.51) AGAACAGACGGACGGAGAGA(SEQ ID No.52) P177 (GATGGT)5 TTCGTTGTTGCATCATGGTT(SEQ ID No.53) GTTAGTTGTTGAAGGGGCCA(SEQ ID No.54)
Claims
1. The primer combination for the *Cypripedium lancifolium* SSR molecular marker is any combination of one or more primer pairs from primer pairs 1 to primer pairs 17 below: The sequence of the upstream primer of primer pair 1 is: 5'-TGCTTCATTTCCCTACCAAAA-3', and the sequence of the downstream primer is: 5'-AGGTCCTCAGCGAAGCAGT-3'; The sequence of the upstream primer of primer pair 2 is: 5'- GACAATCATTCCAACGGCTT -3', and the sequence of the downstream primer is: 5'- GGGCTTGTGACAAATGTTGA -3'; The sequence of the upstream primer of primer pair 3 is: 5'-AGAATGGGACAAGCAAAAGAG-3', and the sequence of the downstream primer is: 5'-TGGTGGTAAGTTGGGTTGAA-3'; The sequence of the upstream primer for primer pair 4 is: 5'-CAAGTACATCAAAACCGAACCA -3', and the sequence of the downstream primer is: 5'-CTTCCCTTTTTTTTTTGGGC -3'. The sequence of the upstream primer of primer pair 5 is: 5'- AGAGAGGAGTTGCGATGATGA -3', and the sequence of the downstream primer is: 5'- GCTTTTCCAGTGCATCCTGT -3'; The sequence of the upstream primer of primer pair 6 is: 5'- CCCAAGCACTGGAAATTGTT-3', and the sequence of the downstream primer is: 5'- CTCCTCGTGAACCCATTGTT-3'; The sequence of the upstream primer of primer pair 7 is: 5'- GGGCTTTAATGAAGGTGGTG -3', and the sequence of the downstream primer is: 5'- TGGTTTACAGCAATGACCTCC -3'; The sequence of the upstream primer of primer pair 8 is: 5'- TCCACACCGCCTAAATTTTC -3', and the sequence of the downstream primer is: 5'- AAACCTCCTGATGGCAAGTG -3'; The sequence of the upstream primer of primer pair 9 is: 5'-CTCGATTCGCTTTGCATTTT-3', and the sequence of the downstream primer is: 5'-CCTCATTGACCCAATCTCGT-3'; The sequence of the upstream primer of primer pair 10 is: 5'- TGGGCTTTGCTGTTATCTCTC-3', and the sequence of the downstream primer is: 5'- TGGAAGGGTGCAAGTAGGTT-3'; The sequence of the upstream primer of primer pair 11 is: 5'- TCTGGCCTTGATTTTGGAAC -3', and the sequence of the downstream primer is: 5'- CTCTTCCAGGCATGGTTTTC -3'; The sequence of the upstream primer of primer pair 12 is: 5'-CAAGAAGCTGTCTTTTCGCA-3', and the sequence of the downstream primer is: 5'-TCACCCATTCACCCCTATGT-3'; The sequence of the upstream primer of primer pair 13 is: 5'- AGGGCCAAGAGCCTACATCT -3', and the sequence of the downstream primer is: 5'- TGCATGAGCAATGGAATTGT -3'; The sequence of the upstream primer of primer pair 14 is: 5'- CAAATCTCTGCCTTGGCTTC -3', and the sequence of the downstream primer is: 5'- AATCCCATTTCGTCCTCTCC -3'; The sequence of the upstream primer of primer pair 15 is: 5'- TGAAAGGGAAGGGGTTTTCT -3', and the sequence of the downstream primer is: 5'- GCATTCTCGCATTTATGTGG -3'; The sequence of the upstream primer of primer pair 16 is: 5'- TTATTCCCATACCGTGCCAT -3', and the sequence of the downstream primer is: 5'- AACCAAACCCAAGAGACCAA -3'; The sequence of the upstream primer for primer pair 17 is: 5'- TTCACAGCTTTTGATGCAGG -3', and the sequence of the downstream primer is: 5'- AACCATGGTGGAGTTTTTGG -3'; The sequence of the upstream primer of primer pair 18 is: 5'-CGTCAAATGGGAATTGCTTT-3', and the sequence of the downstream primer is: 5'-AGGTACGTCCATGAATTGGC-3'; The sequence of the upstream primer of primer pair 19 is: 5'- GCTTCGAGGTAGAGGACGTG -3', and the sequence of the downstream primer is: 5'- CGAACCTAAAATGACCGGAG -3'; The sequence of the upstream primer of primer pair 20 is: 5'- AAACGAACGGTGGAGATACG -3', and the sequence of the downstream primer is: 5'- GTCGATTCAGGCTTCTCGTC -3'; The sequence of the upstream primer of primer pair 21 is: 5'- GTCCGTGGTTTCACGTTTCT -3', and the sequence of the downstream primer is: 5'- AAATGAATTACAGGTCGCCG -3'; The sequence of the upstream primer of primer pair 22 is: 5'- CCAAATCGGAGGTACAGGAA -3', and the sequence of the downstream primer is: 5'- AGCTAGAACTGCCACTCCCA -3'; The sequence of the upstream primer of primer pair 23 is: 5'- AAGATGAGTCAGACTGGCCG -3', and the sequence of the downstream primer is: 5'- GGTGTGATGGTGGAGCTTTT -3'; The sequence of the upstream primer of primer pair 24 is: 5'- AATGCGTACCTCCACCTGAG -3', and the sequence of the downstream primer is: 5'- TTCATGCTCTTTTCCGGTTC 3'; The sequence of the upstream primer of primer pair 25 is: 5'-GTCTCCTTCCCACAACCTCA-3', and the sequence of the downstream primer is: 5'-AAAAAGAACCCATTACCCGC-3'; The sequence of the upstream primer of primer pair 26 is: 5'- AATGTTTCGTCCGATTGAGG -3', and the sequence of the downstream primer is: 5'- AGAACAGACGGACGGAGAGA -3'; The sequence of the upstream primer for primer pair 27 is: 5'- TTCGTTGTTGCATCATGGTT -3', and the sequence of the downstream primer is: 5'- GTTAGTTGTTGAAGGGGCCA -3'.
2. The primer pair for *Cypripedium lancifolium* SSR molecular marker consists of primer pair 1 and primer pair 2. The sequence of the upstream primer of primer pair 1 is 5'-TGCTTCATTTCCCTACCAAAA-3', and the sequence of the downstream primer is 5'-AGGTCCTCAGCGAAGCAGT-3'. The sequence of the upstream primer of primer pair 2 is 5'-GACAATCATTCCAACGGCTT-3', and the sequence of the downstream primer is 5'-GGGCTTGTGACAAATGTTGA-3'.
3. The application of the primer combination according to claim 1 or 2 in distinguishing germplasm of *Cypripedium pratum*.
4. The application of the primer combination described in claim 1 or 2 in the identification of *Cypripedium spp.* varieties or kinship.
5. The application of the primer combination described in claim 1 or 2 in the analysis of genetic diversity of *Cypripedium pratum* germplasm.
6. The application of the primer combination according to claim 1 or 2 in molecular marker-assisted breeding of *Cypripedium pratum*.