SSR molecular marker primer for identifying orchid varieties and application thereof

Abstract

The application discloses a kind of SSR molecular marker primer for identifying Cymbidium varieties, and its application, the Cymbidium includes Cymbidium kanran, Cymbidium ensifolium, Cymbidium goeringii, Cymbidium sinense, Cymbidium devonianum, Cymbidium lancilobatum, Cymbidium kanran, hybrid orchid and hybrid Cymbidium. The SSR molecular marker includes SSR1-SSR23, and the primer sequence is shown as SEQ ID NO.1-SEQ ID NO.46 in the sequence table. The screening method of the SSR molecular marker primer is also included, which comprises the following steps of total DNA extraction of Cymbidium, SSR site selection and primer design based on Cymbidium reference genome, PCR amplification, polyacrylamide gel electrophoresis primary screening and ABI3730XL gene analyzer secondary screening. The application also provides a composition comprising the primer and its application in Cymbidium genetic relationship analysis, DUS specificity test and molecular marker assisted breeding. The SSR molecular marker primer provided by the application has the characteristics of high polymorphism, large quantity, low cost, easy reading, and is not limited by environment, and can quickly and accurately construct the DNA fingerprint of Cymbidium varieties.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to an SSR molecular marker primer for identifying Orchid species and its application. Background Technology

[0002] The genus *Cymbidium* belongs to the subfamily Epidendroideae within the family Orchidaceae. It possesses significant scientific, economic, cultural, and social value, and is widely loved by scholars and the public throughout history and across the world. Ancient people frequently used orchids as a creative image, expressing their emotions and aspirations in poetry and painting, leaving behind many precious artistic treasures. Today, orchid culture is deeply integrated into national culture, folk traditions, economic development, and daily life, becoming a unique and important part of traditional Chinese culture. The cultivation history of orchids in my country can be traced back more than two thousand years. From ancient times to the present, they have been widely admired and praised, deeply ingrained in people's minds with beautiful names such as "the gentleman among flowers" and "the king of fragrance," holding a pivotal position in markets throughout China.

[0003] Orchids were included in the first batch of protected plant varieties of the People's Republic of China in 2001, thus requiring DUS testing to protect the legitimate rights and interests of breeders. In recent years, with the continuous expansion of the ornamental market, the increasing demand for superior flower varieties in my country, and the successful cultivation of new Orchid varieties, more and more breeders are becoming more aware of the importance of new variety protection and are actively applying for such protection. Therefore, it is particularly necessary to utilize a set of SSR molecular core markers for Orchids and their primer combinations, and to construct the DNA fingerprint profiles of the varieties, to provide technical support for the protection of new Orchid varieties. 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 an SSR molecular marker primer for identifying Orchid species and its application.

[0005] The technical solution adopted by this invention to solve its technical problem is: an SSR molecular marker primer for identifying orchid species, wherein the orchid species include Cymbidium sinense, Cymbidium ensifolium, Cymbidium goeringii, Cymbidium kanran, Cymbidium faberi, Cymbidium lancifolium, Cymbidium lancifolium, Cymbidium lancifolium, hybrid orchids, and hybrid Chinese orchids;

[0006] The SSR molecular markers for identifying Orchid species include SSR1, SSR2, SSR3, SSR4, SSR5, SSR6, SSR7, SSR8, SSR9, SSR10, SSR11, SSR12, SSR13, SSR14, SSR15, SSR16, SSR17, SSR18, SSR19, SSR20, SSR21, SSR22, and SSR23.

[0007] The SSR molecular marker primer sequences for identifying Orchid species are as follows:

[0008] Primers for the molecular marker SSR1:

[0009] Primers for the molecular marker SSR1:

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

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

[0012] Primers for the molecular marker SSR2:

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

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

[0015] Primers for the molecular marker SSR3:

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

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

[0018] Primers for the molecular marker SSR4:

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

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

[0021] Primers for the molecular marker SSR5:

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

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

[0024] Primers for the molecular marker SSR6:

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

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

[0027] Primers for the molecular marker SSR7:

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

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

[0030] Primers for the molecular marker SSR8:

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

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

[0033] As a further improvement of the present invention: the primers for the molecular marker SSR9:

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

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

[0036] Primers for the molecular marker SSR10:

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

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

[0039] Primers for the molecular marker SSR11:

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

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

[0042] Primers for the molecular marker SSR12:

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

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

[0045] Primers for the molecular marker SSR13:

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

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

[0048] Primers for the molecular marker SSR14:

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

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

[0051] Primers for the molecular marker SSR15:

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

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

[0054] Primers for the molecular marker SSR16:

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

[0056] SEQ ID NO. 32: SSR16-R:5'-GGCTTGTTGGATGGCAGAGA-3'.

[0057] As a further improvement of the present invention: the primers for the molecular marker SSR17:

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

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

[0060] Primers for the molecular marker SSR18:

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

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

[0063] Primers for the molecular marker SSR19:

[0064] SEQ ID NO.37: SSR19-F:5'-TTGGTTCACTTCTGCACAACTT-3';

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

[0066] Primers for the molecular marker SSR20:

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

[0068] SEQ ID NO. 40: SSR20-R:5'-CTCCCCTCTCCCATTGCATTTA-3'.

[0069] Primers for the molecular marker SSR21:

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

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

[0072] Primers for the molecular marker SSR22:

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

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

[0075] Primers for the molecular marker SSR23:

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

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

[0078] As a further improvement of the present invention, the screening steps for the above-mentioned SSR molecular markers are as follows: extraction of total DNA of Orchid, selection of SSR sites and design and screening of primers based on the Cymbidium reference genome published on NCBI, PCR amplification, initial screening by polyacrylamide gel electrophoresis and secondary screening by ABI3730XL gene analyzer, and selection of core primers based on electrophoresis results.

[0079] As a further improvement of the present invention: the total DNA of the Orchid genus is extracted by using a modified CTAB method to extract the DNA from fresh leaves.

[0080] As a further improvement of the present invention: SSR loci were selected and primers were designed and screened using the Cymbidium reference genome published on NCBI. 120 primer pairs were designed according to the following principles, wherein the selection principles for SSR primers are as follows:

[0081] 1) The primer length is 18-27 bp, and the target fragment is approximately 100-280 bp;

[0082] 2) The GC content should be 40%–60%, and the primer sequence should avoid three or four consecutive bases;

[0083] 3) The annealing temperature should be between 55 and 65℃, preferably around 55℃, and the difference in Tm values ​​between the upstream and downstream primers should not exceed 4 to 6℃;

[0084] 4) Avoid having more than 3 consecutive bases at the 3' end of the primer, and try to avoid primer dimers and hairpin structures.

[0085] As a further improvement of the present invention: the PCR amplification and polyacrylamide gel electrophoresis initial screening and ABI3730XL gene analyzer secondary screening are specifically performed as follows: 2 μL of PCR product is aspirated and subjected to polyacrylamide gel electrophoresis for initial screening. Primers are initially screened by comparison with the target fragment. PCR products with high amplification rate, clear bands, and good peak shape are selected for secondary screening by ABI3730XL gene analyzer, which can accurately reflect the differences between allelic variation sites and screen primers with high polymorphism.

[0086] The present invention also provides an SSR molecular marker primer composition for identifying Orchid species, wherein the sequences of each SSR molecular marker primer in the composition are shown in the sequence listing SEQ ID NO.1 to SEQ ID NO.46.

[0087] This invention also provides an application of SSR molecular marker primers for identifying Orchid varieties in Orchid phylogenetic analysis, DUS specificity testing, and marker-assisted breeding. Secondly, Orchids are asexually reproducing plants with numerous new and old varieties, easily leading to confusion regarding synonyms or different species with the same name. Therefore, using SSR markers to construct DNA fingerprints for screening similar varieties in DUS testing offers the possibility of improving efficiency and accelerating variety identification. Finally, by detecting molecular markers, the presence of target genes can be detected and target traits selected. Correlation analysis can be used to study the level of phenotypic genetic diversity, and this can help breeders conduct targeted selection, forming the basis for breeding superior plant varieties.

[0088] Compared with the prior art, the beneficial effects of the present invention are:

[0089] The SSR molecular marker primers and their composition for identifying Orchid species of the present invention have the characteristics of high polymorphism, large quantity, low cost and easy reading compared with other molecular markers. The selected Orchid species are not limited by season, environment and test time. DNA can be extracted from any tissue at any stage of the growth of the species or from samples that have been refrigerated at 22°C for a period of time without affecting the identification results.

[0090] In the screening of SSR molecular marker primers for identifying Orchid species in this invention, the ABI3730XL gene analyzer was used. This system has the characteristics of high throughput, safety and convenience, and high sensitivity. It plays a significant role in constructing a rapid and accurate SSR fingerprinting technique and shortens the identification cycle of Orchid species. Attached Figure Description

[0091] 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.

[0092] Figure 1 This is a polyacrylamide gel electrophoresis image of 12 orchid species with significant phenotypic differences extracted in this invention.

[0093] Figure 2 The above are capillary electrophoresis peak diagrams of the SSR4 primers of this invention in different Orchid species.

[0094] Figure 3 This is the genetic analysis and cluster diagram of the present invention. Detailed Implementation

[0095] 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.

[0096] 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.

[0097] Orchids possess scientific, economic, and cultural value, boasting a long cultivation history and profound cultural significance in my country, and are hailed as "gentlemen among flowers" and "kings of fragrance." In 2001, orchids were included in China's first batch of protected plant varieties, thus requiring DUS testing (specificity, uniformity, and stability testing) to protect breeders' legal rights. In recent years, with the expansion of the orchid market and advancements in breeding technology, new varieties are constantly emerging, breeders' awareness of new variety protection is increasing, and the number of applications for plant variety protection is also rising. Therefore, there is an urgent need to establish an efficient, accurate, and stable molecular identification technology system to support DUS testing, phylogenetic analysis, DNA fingerprinting construction, and assisted breeding work for orchid varieties.

[0098] Traditional DUS testing relies on morphological observation, which is time-consuming, susceptible to environmental factors, and struggles to distinguish closely related varieties, especially at the seedling stage. The vast number of Orchid species, many of which are propagated asexually, easily leads to confusion regarding synonyms or homonyms, posing challenges to variety management and rights protection. Current research on SSR molecular markers for Orchids is limited, lacking a core primer set with high polymorphism, good reproducibility, and broad coverage, failing to meet the needs of large-scale variety identification. Traditional electrophoresis methods have limited resolution, making it difficult to accurately distinguish allelic variations, and have low throughput, failing to meet the demands for rapid, high-throughput identification.

[0099] To address the aforementioned issues, this application provides an SSR molecular marker primer for identifying Orchid species and its application, which features high polymorphism, large quantity, low cost, and easy reading. The selected Orchid species are not limited by season, environment, or testing time, and DNA can be extracted from any tissue at any stage of the species' growth or from samples that have been refrigerated at 22°C for a period of time without affecting the identification results.

[0100] Example 1:

[0101] Extraction of total DNA from Orchid Species:

[0102] 1) The 30 selected Orchid varieties were provided by the Flower Innovation Team of the National Engineering Research Center for Space Breeding of Plants, the Flower Research Institute of the Guangdong Academy of Agricultural Sciences, and the Plant Variety Testing (Kunming) Branch of the Crop Research Institute of the Fujian Academy of Agricultural Sciences;

[0103] 2) Using the selected Orchid cultivars as materials, total DNA was extracted using the CTAB method. The specific procedure is as follows:

[0104] ① Take 1 g of fresh leaves, put them into a frozen mortar, add 0.2 g of insoluble PVP, and grind them into powder quickly in liquid nitrogen; then put the powder into a centrifuge tube containing 1.5 ml of 650 µL high-salt extraction buffer, add 80 µL of β-mercaptoethanol, mix well, and incubate in a 65 ℃ water bath for 75 min, mixing 2 to 3 times during the incubation period. Remove the sample and cool it to room temperature.

[0105] ② Add 650µL chloroform / isoamyl alcohol (24:1) and 50µL saturated phenol, shake to mix, and centrifuge at 12,000 r / min for 10 min.

[0106] ③ Take the supernatant, transfer it to another 1.5 ml centrifuge tube, add 700 µL of chloroform / isoamyl alcohol (24:1), shake to mix, and centrifuge at 12,000 r / min for 10 min;

[0107] ④ Take the supernatant and transfer it to another 1.5 ml centrifuge tube. Add 200 µL of 5 mol / L NaCl and 600 µL of frozen anhydrous ethanol. Mix thoroughly, let stand, and allow the precipitate to coagulate at -20 °C for 2 h. Centrifuge at 10,000 r / min for 10 min.

[0108] ⑤ Discard the supernatant, wash 2-3 times with 75% ethanol, and finally dry the precipitate in a 40 ℃ oven. After drying, dissolve in 200 µL of ultrapure water, and briefly centrifuge after complete dissolution. Measure the concentration of the DNA stock solution using a micro spectrophotometer, and add ultrapure water to prepare 500 µL of DNA working solution, which is stored at -20 ℃. The stock solution is stored at -80 ℃ for later use.

[0109] Example 2:

[0110] Screening primers for SSR molecular markers to identify Orchid species

[0111] 1) Use NCBI to search the reference genome of *Mexican*;

[0112] Primers were designed using Primer 5.0. Based on primer design principles, 50 primers with a rating ratio of 60 or higher were selected. SSR loci were selected and primers were designed and screened based on the Cymbidium reference genome published on NCBI. A total of 120 primer pairs were successfully designed according to the following principles, with the SSR primer selection principles being as follows:

[0113] ①The primer length is 18-27 bp, and the target fragment is about 250-380 bp;

[0114] ② The GC content should be 40%–60%, and the primer sequence should avoid three or four consecutive bases;

[0115] ③ The annealing temperature is 55-65℃, preferably around 50℃, and the difference between the Tm values ​​of the upstream and downstream primers should not exceed 4-6℃;

[0116] ④ Avoid having more than 3 consecutive bases at the 3' end of the primer, and try to avoid primer dimers and hairpin structures;

[0117] Sample amplification: The total reaction volume was 10 μL, including 1.0 μL of 2 ng / μL DNA solution, 1.0 μL each of 4 pmol / μL forward and reverse primers, 5.0 μL of 2×Taq PCR Master Mix, and 2.0 μL of ddH2O; (Reagents used were from Shanghai Sangon Biotech Co., Ltd.)

[0118] The PCR reaction program is as follows: 94℃ pre-denaturation for 1 min, 35 cycles, 94℃ denaturation for 1 min, 50℃ annealing for 45 s, 72℃ extension for 1 min, 72℃ extension for 10 min; store at 4℃, where the annealing temperature is determined according to each primer.

[0119] PCR amplification and polyacrylamide gel electrophoresis were used for initial screening, and 1.2% agarose gel electrophoresis was used to detect the amplification products. Specifically, 3.0 μL of PCR product was aspirated and added to 2.0 μL of 6× Loading buffer for initial screening.

[0120] PCR products with high amplification rates, bright bands, and good peak shapes were selected and re-screened using an ABI 3730XL gene analyzer. This accurately reflects the differences between alleles and allows for the selection of primers with high polymorphism. Figure 1 As shown, the SSR molecular marker primers and their compositions for identifying Orchid species of the present invention have the characteristics of high polymorphism, low cost, and easy reading compared with other molecular markers.

[0121] The specific procedures for secondary screening using the ABI3730XL gene analyzer are as follows:

[0122] ① Take 1.1 µL of the product from the primer combination and mix it, then dilute it 100-120 times with ultrapure water.

[0123] ② Electrophoresis preparation: Mix deionized formamide and molecular weight internal standard LIZ500 at a ratio of 100:1. Then, take 9 µL of the mixture and 1 µL of the fluorescent mixture, and add them to a capillary electrophoresis deep-well plate. After thorough mixing, centrifuge for 2 min, then denature the sample at 95 °C for 5 min, cool, centrifuge again, and finally place it in an ABI 3730XL DNA analyzer.

[0124] ③ Electrophoresis detection: Strictly follow the operation manual for standardized operation. First, initialize the system, establish the sample attribute database and electrophoresis plate parameter matrix, then start the detection program and store the electrophoresis pattern data file.

[0125] ④ Data Reading: Use SSR Analyser to read the exported data. "X / X" indicates that the locus is homozygous, "X / Y" indicates that the locus is heterozygous, and ". / ." indicates that the locus is missing.

[0126] The criteria for primer rescreening were as follows: 1) high polymorphism value and numerous alleles; 3) stable amplification; and 4) easily readable peak shape. Ultimately, 23 primer pairs were selected as the core primers for identifying Orchid species. The processed data were then analyzed using NTSYS software to calculate and analyze genetic diversity indicators, clustering, and polymorphic information content (PIC).

[0127] The molecular marker primers obtained through screening, their sequences, and annealing temperatures are shown in Table 1.

[0128]

[0129] Table 1. SSR molecular marker primers and annealing temperatures

[0130] The amplification results of the SSR molecular marker primers are shown in Table 2:

[0131]

[0132] Table 2. SSR molecular marker primer amplification results

[0133] As shown in Table 2, the SSR molecular marker primer composition and germplasm identification method described in this invention can accurately identify 30 orchid varieties with an accuracy rate of 100%.

[0134] Due to the large number of SSR molecular marker primers and Orchid species, the peak diagrams of SSR1, SSR2, and SSR3 amplification results are shown as examples. Figure 1 The image shown is a polyacrylamide gel electrophoresis diagram of 12 orchid species with significant phenotypic differences. Figure 2 The capillary electrophoresis peak diagrams for different Orchid cultivars using SSR4 primer pairs are shown in Table 2. Figure 2 It can be seen that the SSR molecular marker primers described in this invention have good amplification effect, high detection rate, and can amplify stable peak patterns.

[0135] like Figure 3The results show the genetic analysis and cluster diagram. Combined with the analysis in Table 2, the polymorphic information content (PIC) analysis indicates that the SSR primers used in the experiment are all highly polymorphic primers, which have a high polymorphic detection capability for Orchid varieties and are suitable for the identification and phylogenetic analysis of Orchid varieties. The clustering results show that the 30 Orchid varieties are clustered into 3 major categories, which is basically consistent with the morphological clustering results.

[0136] In summary, the SSR molecular marker primers of this invention can be used to assist in selective breeding, enabling early selection at the seedling stage, thereby accelerating the breeding process of Orchids.

[0137] Working principle of the invention:

[0138] Based on simple repeat sequence (SSR) molecular marker technology, and taking advantage of the high polymorphism of SSR sites widely present in the Orchid genome among different varieties, this study designed multiple candidate primer pairs covering the entire genome, referencing the whole genome sequence of Cymbidium goeringii published on NCBI and adhering to strict bioinformatics primer design principles (including primer length, GC content, annealing temperature, and avoidance of secondary structures). Subsequently, total DNA was extracted from Orchid varieties for PCR amplification, and polyacrylamide gel electrophoresis was used for initial screening to evaluate amplification efficiency and band clarity. Further high-precision secondary screening was performed using an ABI 3730XL gene analyzer, and capillary electrophoresis was used to accurately detect subtle differences between allelic variant sites. Finally, 23 core primer pairs (SSR1 to SSR23) with high polymorphism, stable amplification, and easily readable peak shapes were selected. In practical applications, these 23 primer pairs are used to perform PCR amplification on samples of unknown Orchid species. The differences in fragment length of the amplified products constitute the unique DNA fingerprint of the species. By comparing it with the standard fingerprint in the database, the species can be quickly identified. At the same time, these data can also be used for genetic similarity calculation and cluster analysis, thereby supporting applications such as phylogenetic analysis of Orchid species, DUS (specificity, uniformity, and stability) testing, and molecular marker-assisted breeding.

[0139] The main functions of this invention are:

[0140] PCR amplification of DNA from Orchid samples was performed using 23 pairs of core SSR primers (SSR1–SSR23). Fragment length polymorphism of the amplified products was used to construct cultivar-specific DNA fingerprints, enabling rapid and accurate identification of nine Orchid genera, including Cymbidium sinense, Cymbidium ensifolium, Cymbidium goeringii, and Cymbidium kanran, with an accuracy rate of up to 100%. This effectively resolves the confusion caused by synonyms or homonyms. Based on the codominant genetic characteristics of SSR markers, genetic similarity between varieties was calculated and cluster diagrams were generated by detecting allele differences. This can be used to analyze the phylogenetic relationships and genetic diversity among different Orchid varieties, providing a scientific basis for germplasm resource classification and conservation. In plant variety protection, it assists in DUS (specificity, uniformity, and stability) testing, allowing for rapid screening of similar varieties through molecular markers, improving the efficiency and accuracy of new variety review, and protecting the legitimate rights and interests of breeders. By detecting SSR markers associated with target traits, individuals carrying superior genes can be screened in advance at the seedling stage, accelerating the breeding process and enabling early prediction and targeted selection.

[0141] 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. An SSR molecular marker primer for identifying Orchid species, characterized in that, The orchid genus mentioned includes Cymbidium sinense, Cymbidium ensifolium, Cymbidium goeringii, Cymbidium kanran, Cymbidium faberi, Cymbidium lancifolium ... hybrid orchids, and hybrid Chinese orchids; The SSR molecular markers for identifying Orchid species include SSR1 to SSR23; The SSR molecular marker primer sequences for identifying Orchid species are as follows: Primers for the molecular marker SSR1: SEQ ID NO.1: SSR1-F:5'-TTTCTTGCTGAGCCTTTTATGTC-3'; SEQ ID NO.2: SSR1-R:5'-CCACTCCTTTCTTCATCATTTG-3'; Primers for the molecular marker SSR2: SEQ ID NO.3: SSR2-F:5'-TTAAATTCAAAGTTTCACTCGCC-3'; SEQ ID NO.4: SSR2-R:5'-AACTCCCCAGTAGCTTTCAGTTT-3'; Primers for the molecular marker SSR3: SEQ ID NO.5: SSR3-F:5'-CTTTCTTCGCGCATATGTACTTT-3'; SEQ ID NO.6: SSR3-R:5'-ATCAAAGCTCACCATTTGTTCAT-3'; Primers for the molecular marker SSR4: SEQ ID NO.7: SSR4-F:5'-TCTGTGCTTGTGTAGGGCTTAAAT-3'; SEQ ID NO.8: SSR4-R:5'-TTGAACCAAAAATAAATCATCGC-3'; Primers for the molecular marker SSR5: SEQ ID NO.9: SSR5-F:5'-TAATGGAATGATGCGTAG-3'; SEQ ID NO.10: SSR5-R:5'-CAGTAATTGGACCCTAAC-3'; Primers for the molecular marker SSR6: SEQ ID NO.11: SSR6-F:5'-TTAAAAGTTTTCAAGCAGAATGCC-3'; SEQ ID NO.12: SS6-R:5'-AGACAAAGAGAGACCTGGGAGTT-3'; Primers for the molecular marker SSR7: SEQ ID NO.13: SSR7-F:5'-CAAATGAATTCCGGTGATGTAT-3'; SEQ ID NO.14: SSR7-R:5'-ACCATTCAAAAGAAGTGTTGGAA-3'; Primers for the molecular marker SSR8: SEQ ID NO.15: SSR8-F:5'-CTCTTCTCCTCTCGATTTCCAAT-3'; SEQ ID NO. 16: SSR8-R:5'-GGCTTCCGTAATATTTCCTTTG-3'.

2. The SSR molecular marker primer for identifying Orchid species according to claim 1, characterized in that, Primers for the molecular marker SSR9: SEQ ID NO.17: SSR9-F:5'-GAAGAAGATAGGGAAGGTGGAAA-3'; SEQ ID NO.18: SSR9-R:5'-AACGATGCTTATTGGGTTGACTA-3'; Primers for the molecular marker SSR10: SEQ ID NO.19: SSR10-F:5'-GATGATGATGAAGACCCTGAAGA-3'; SEQ ID NO.20: SSR10-R:5'-ATTGTCCTGATCCTCCTCCTCTA-3'; Primers for the molecular marker SSR11: SEQ ID NO.21: SSR11-F:5'-AGGGTTCGGTCAATTTCTTAAAC-3'; SEQ ID NO.22: SSR11-R:5'-TCCCTTCTCACTTGCATTCTAAC-3'; Primers for the molecular marker SSR12: SEQ ID NO.23: SSR12-F:5'-AATGTCCTTCCGCTTAATCCCTAC-3'; SEQ ID NO.24: SSR12-R:5'-GCTTATTCTTCTCCGTTCCTCTC-3'; Primers for the molecular marker SSR13: SEQ ID NO.25: SSR13-F:5'-ACGCTGGTCCGTTTATG-3'; SEQ ID NO.26: SSR13-R:5'-CACCCATCCTTGCTCTC-3'; Primers for the molecular marker SSR14: SEQ ID NO.27: SSR14-F:5'-TATGCGTCTCTCCCAACCG-3'; SEQ ID NO.28: SSR14-R:5'-AAGCTAGTGGCCTTTGGTG-3'; Primers for the molecular marker SSR15: SEQ ID NO.29: SSR15-F:5'-GGAACCCTCGTCGATCTTGA-3'; SEQ ID NO.30: SSR15-R:5'-GAGGGAAGCACAGTAAAAGCG-3'; Primers for the molecular marker SSR16: SEQ ID NO.31: SSR16-F:5'-CCCAGCACAGATAGCTCCTT-3'; SEQ ID NO. 32: SSR16-R:5'-GGCTTGTTGGATGGCAGAGA-3'.

3. The SSR molecular marker primer for identifying Orchid species according to claim 1, characterized in that, Primers for the molecular marker SSR17: SEQ ID NO.33: SSR17-F:5'-AGGATACCGTGTGGAGGAGAA-3'; SEQ ID NO.34: SSR17-R:5'-AGATCAGCGAGCATCTGAGG-3'; Primers for the molecular marker SSR18: SEQ ID NO.35: SSR18-F:5'-ACTGGGGACATTGCGTTAGG-3'; SEQ ID NO.36: SSR18-R:5'-AAGATGACGTGCGCAATGAG-3'; Primers for the molecular marker SSR19: SEQ ID NO.37: SSR19-F:5'-TTGGTTCACTTCTGCACAACTT-3'; SEQ ID NO.38: SSR19-R:5'-CATATAGCCGACCCCACTTAGT-3'; Primers for the molecular marker SSR20: SEQ ID NO.39: SSR20-F:5'-TGAGTAACCCATCAATGTAGCT-3'; SEQ ID NO.40: SSR20-R:5'-CTCCCTCCTCCCATTGCATTTA-3'; Primers for the molecular marker SSR21: SEQ ID NO.41: SSR21-F:5'-ATCTTTATTGGCAGAAAAGGCA-3'; SEQ ID NO.42: SSR21-R:5'-ATTCTTGCTCCAAATTCTCATCA-3'; Primers for the molecular marker SSR22: SEQ ID NO.43: SSR22-F: 5'-CCTGCATCATAACACCGTCG-3'; SEQ ID NO.44: SSR22-R:5'-TGAGAGCTCATGGGGTAAGC-3'; Primers for the molecular marker SSR23: SEQ ID NO.45: SSR23-F:5'-GCTCTGTCATTCCCTCT-3'; SEQ ID NO. 46: SSR23-R:5'-GTAACCAAGAATTTCAACAT-3'.

4. The SSR molecular marker primer for identifying Orchid species according to claim 1, characterized in that, The method for screening SSR molecular marker primers includes the following steps: 1) Extraction of total DNA from Orchids; 2) Based on the Cymbidium reference genome published on NCBI, SSR loci were selected and primers were designed and screened; 3) PCR amplification, polyacrylamide gel electrophoresis for initial screening, and ABI3730XL gene analyzer for secondary screening, and core primers were selected based on the electrophoresis results; The sequences of the molecular marker primers obtained by screening are shown in SEQ ID NO.1-SEQ ID NO.46, respectively.

5. The SSR molecular marker primer for identifying Orchid species according to claim 1, characterized in that, Step 2) The SSR loci were selected and primers were designed and screened using the Cymbidium reference genome published on NCBI. 120 primer pairs were designed according to the following principles, with the SSR primers selected based on the following criteria: 1) The primer length is 18-27 bp, and the target fragment is about 100-280 bp; 2) The GC content should be 40%–60%, and the primer sequence should avoid three or four consecutive bases; 3) The annealing temperature is 50-65℃, preferably around 55℃, and the difference between the Tm values ​​of the upstream and downstream primers should not exceed 4-6℃; 4) Avoid having more than 3 consecutive bases at the 3' end of the primer, and try to avoid primer dimers and hairpin structures.

6. The SSR molecular marker primer for identifying Orchid species according to claim 1, characterized in that, Step 3) describes PCR amplification, polyacrylamide gel electrophoresis for initial screening, and ABI3730XL gene analyzer for secondary screening. Specifically, 2 μL of the PCR product is aspirated and subjected to polyacrylamide gel electrophoresis for initial screening. Primers are then screened by comparing them with the target fragment. PCR products with high amplification rates, clear bands, and good peak shapes are selected for secondary screening using the ABI3730XL gene analyzer. This process accurately reflects the differences between allelic variation sites and allows for the screening of primers with high polymorphism.

7. The SSR molecular marker primer for identifying Orchid species according to claim 1, characterized in that, The composition includes the SSR molecular markers SSR1 to SSR23 as described in claim 1, wherein the sequences of the molecular marker primers for SSR1 to SSR23 are shown in the sequence listing SEQ ID NO.1 to SEQ ID NO.46, respectively.

8. The application of the SSR molecular marker primer for identifying Orchid species as described in any one of claims 1-7 in Orchid phylogenetic analysis, DUS specificity testing, and molecular marker-assisted breeding.

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