A Cymbidium SSR primer set and a method for constructing a Cymbidium variety fingerprint using the primer set

Abstract

The present invention discloses a Cymbidium SSR primer set and a method for constructing a Cymbidium variety fingerprint map using the primer set. The Cymbidium SSR primer set includes 14 pairs of SSR primer sequences. The present invention uses the 14 pairs of SSR primer sequences to perform multiple PCR amplification on Cymbidium genomic DNA, uses capillary fluorescence electrophoresis to detect PCR products, analyzes SSR marker data, constructs 79 Cymbidium SSR fingerprint maps, and can effectively identify Cymbidium varieties. The method for constructing a Cymbidium variety fingerprint map of the present invention has the advantages of high throughput, high precision, and low cost, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a cymbidium SSR primer set and a method for constructing a cymbidium variety fingerprint map by using the primer set. Background Art

[0002] Cymbidium faberi is a terrestrial species of the Orchidaceae family. It has a large plant shape, beautiful leaves, and a raceme with 5-10 flowers. The flowers are large, broad, and gorgeous. It is a traditional precious flower in my country and is very popular in domestic and foreign markets. Although Cymbidium has a long history of cultivation and cultural heritage, variety identification mostly depends on traditional morphological indicators. It is difficult to accurately identify varieties by leaves and plant shape before they bloom. In recent years, with the promotion and market development of Cymbidium, coupled with the rapid development of Internet technology, major e-commerce platforms, social media, and forums have provided more diversified trading channels. There are more than 500 varieties of Cymbidium on the orchid market. However, there is no unified and standardized variety identification standard, and products are inferior and confused with varieties. In order to promote the selection, identification, and promotion of varieties that meet market demand, it is imperative to establish orchid variety identification standards that are compatible with them.

[0003] Orchidaceae plants are widely distributed around the world, with 801 genera and more than 27,500 species. In recent years, molecular markers, mainly including RAPD, ISSR, SSR, SRAP and ScoT, have been widely used in the classification and identification of orchid species, interspecies and varieties, analysis of genetic diversity, kinship, and research on germplasm resources and populations. However, the identification and evaluation of my country's characteristic resources are limited by geographical limitations and the difficulty of variety collection. At present, there are no reports on high-throughput identification of Cymbidium orchid varieties based on capillary electrophoresis technology and SSR molecular markers in China. Therefore, the use of SSR molecular markers in the present invention to identify Cymbidium orchid varieties will be of great significance to the evaluation of germplasm resources, innovative utilization of germplasm, and supervision of commercial flower quality and market regulations. Summary of the invention

[0004] The present invention aims to address the technical defects of the prior art and provide a Cymbidium SSR primer set and a method for constructing a Cymbidium variety fingerprint using the primer set by combining capillary electrophoresis technology with SSR-PCR technology, so as to quickly and efficiently identify Cymbidium varieties.

[0005] The first object of the present invention is to provide a Cymbidium orchid SSR primer set, which comprises the following 14 pairs of SSR primer sequences:

[0006] SSR_21319_c0_g2i1 F:5'-GGCACCTCTGGAATTTCTGGT-3'

[0007] R:5'-GTGCGTGAGAGAGAGAAAGACA-3';

[0008] SSR_71852_c0_g2i1 F:5'-GGCACCTCTGGAATTTCTGGT-3'

[0009] R:5'-GTGCGTGAGAGAGAGAAAGACA-3';

[0010] SSR_92040_c0_g1i7 F:5'-AGGTTGCCAAGCTAGCTACT-3'

[0011] R:5'-TGAGGCTTGACTAACCCGTC-3';

[0012] SSR_12201_c0_g1i10 F:5'-CACGCGAACGCAATACAGAG-3'

[0013] R:5'-ACAGAGATGCGTGCATGTGA-3';

[0014] SSR_42275_c0_g1i1 F:5'-TTCTTGTCTGCTCGCTCCAG-3'

[0015] R:5'-CCCATTGCCTTCACCTCCTT-3';

[0016] SSR_52366_c0_g1i1 F:5'-GGGTGAGAGGAAAAGTGGACT-3'

[0017] R:5'-TCACATCACCAGTGCTGCAT-3';

[0018] SSR_32581_c0_g1i1 F:5′-CACGGCTGCTCATCAGAATG-3′

[0019] R:5'-GTCCACAGAGGCAGAGACAT-3';

[0020] SSR_22677_c0_g2i1 F:5'-TTGTCATCGGCATTGACCAA-3'

[0021] R:5'-TGTGGGAAAACATGGGAATTGG-3';

[0022] SSR_12742_c0_g7i1 F:5'-AACAGGAGCTAATCACGGGC-3'

[0023] R:5'-CAGCCCAGCGTTCAAAAGAC-3';

[0024] SSR_62892_c1_g1i6 F:5'-GCCTCATGTAGGGGAGCTTC-3'

[0025] R:5'-ATCGTCCACCTGTTATCCGC-3';

[0026] SSR_33144_c2_g1i2 F:5'-AGCATCTCGCAGAACTTTACTG-3'

[0027] R:5'-CTTGCTCTCTCCCTCTCCCT-3';

[0028] SSR_33179_c0_g1i2 F:5'-CATCCACGGACCCAAATCCA-3'

[0029] R:5'-TCGACGATGAGTAATGGTGGT-3';

[0030] SSR_73252_c3_g1i1 F:5'-AGCCATCAATCACTGTGCGA-3'

[0031] R:5'-TGGATATGCCTACTGCCCTG-3';

[0032] SSR_53283_c0_g5i3 F:5'-TCTGTTGCTAGCCTTCTCCAC-3'

[0033] R:5'-TCTCGTCCTTTCCCTCCCTT-3'.

[0034] The second object of the present invention is to provide the application of the above-mentioned Cymbidium SSR primer set in constructing a fingerprint map of Cymbidium varieties.

[0035] The third object of the present invention is to provide the application of the above-mentioned Cymbidium SSR primer set in identifying Cymbidium varieties.

[0036] The fourth object of the present invention is to provide a method for constructing a fingerprint of Cymbidium orchid varieties, which comprises the following steps:

[0037] (1) Extract DNA from Cymbidium orchid samples;

[0038] (2) using the extracted DNA as a template and the SSR primer set described in claim 1 as an amplification primer, establishing a PCR reaction system and performing PCR amplification;

[0039] (3) The amplified products are subjected to capillary electrophoresis, and the amplified fragment length data encoding is formed for the sample DNA of each variety of Cymbidium orchid according to the capillary electrophoresis results, so as to construct a fingerprint map of Cymbidium orchid varieties.

[0040] Furthermore, the PCR reaction system in step (2) includes: 3 μL of 10×PCR Buffer, 2 μL of 2.5 mM dNTP, MgCl 2 2μL, Primer A 2μL, Primer B 2μL, Template 1μL, ddH 2 O 18μL, Taq enzyme 0.2μL.

[0041] The reaction procedure of the PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 30 cycles; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 10 cycles; 60°C for 30 min; and storage at 4°C.

[0042] Furthermore, in step (3), the amplified product is subjected to capillary electrophoresis, and the amplification result of the sample DNA of each variety of Cymbidium is digitized into the length of the amplified fragment according to the capillary electrophoresis result, specifically: according to SSR_21319_c0_g2i1, SSR_71852_c0_g2i1, SSR_92040_c0_g1i7, SSR_12201_c0_g1i10, SSR_42275_c0_g1i1, SSR_52366_c0_g1i1, SSR_32581_c0_g1i1, SSR_ The molecular markers of SSR_22677_c0_g2i1, SSR_12742_c0_g7i1, SSR_62892_c1_g1i6, SSR_33144_c2_g1i2, SSR_33179_c0_g1i2, SSR_73252_c3_g1i1, and SSR_53283_c0_g5i3 were arranged in sequence, and the length of the fragments amplified by each pair of SSR primers for each Cymbidium variety was recorded in turn through capillary electrophoresis results. The lengths of the amplified fragments were digitized and encoded, which was the DNA fingerprint of the Cymbidium variety.

[0043] Furthermore, the data encoding is performed based on the length of each amplified fragment, specifically: the alleles of the amplified fragments of each site are arranged according to the molecular weight, the alleles are labeled with Arabic numerals 1-9 from small to large, and more than 9 alleles are labeled with capital letters AZ. If the site is not amplified in a certain variety, it is recorded as 0, and each site occupies two bits.

[0044] The fifth object of the present invention is to provide a fingerprint map of Cymbidium orchid varieties, which is constructed by the above-mentioned Cymbidium orchid variety fingerprint map construction method.

[0045] The sixth object of the present invention is to provide the application of the above-mentioned Cymbidium variety fingerprint map in identifying Cymbidium variety.

[0046] The seventh object of the present invention is to provide a kit for identifying Cymbidium orchid varieties, which contains the above-mentioned Cymbidium orchid SSR primer set.

[0047] The beneficial effects of the present invention are:

[0048] (1) The present invention utilizes capillary electrophoresis technology to achieve a beneficial combination of SSR molecular markers and efficient, automated capillary electrophoresis technology. The detection results are automatically saved by the analysis software, with fast detection speed, high separation efficiency, high sensitivity, and an accuracy of less than 1 bp, which is conducive to the detection and analysis of large quantities of samples and lays a solid foundation for the promotion and application of SSR molecular marker technology in the identification of Cymbidium orchid varieties.

[0049] (2) The identification method of the present invention is low-cost, the market for reagents and consumables is mature, and the detection cost of one sample is less than 10 yuan, excluding labor and instrument losses. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Some representative images of SSR marker screening. The sample order from left to right is Lao Ranzi, Dayipin, Nanyangmei, Jin Aosu and Zheng Xiaohe.

[0051] Figure 2 This is the peak graph of the amplification results of different Cymbidium orchid varieties using primer pair SSR_62892_c1_g1i6. From top to bottom, they are the amplification peak graphs of four different Cymbidium orchid varieties: Lao Ranzi, Dayipin, Nanyangmei and Zheng Xiaohe. DETAILED DESCRIPTION

[0052] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0053] Example 1

[0054] 1. Collection of Cymbidium orchid varieties

[0055] The samples came from the main producing areas of Cymbidium orchids, namely Guangdong Province, China, Guangxi Zhuang Autonomous Region, China and Taiwan Province, China, and included 79 varieties of different flower shapes, flower colors and leaf colors, as shown in Table 1 below.

[0056] Table 1 79 Cymbidium varieties

[0057]

[0058]

[0059]

[0060]

[0061] 2. Extraction of Cymbidium genomic DNA

[0062] The 79 collected Cymbidium germplasm resources were extracted using a plant genomic DNA kit (Tiangen Company). First, young leaves were collected in batches, and each sample was divided into 2-3 portions (about 2g each) and stored in a 2mL centrifuge tube. After quick freezing in liquid nitrogen, they were frozen in a -80℃ refrigerator for later use. The frozen plant tissues were fully ground with a pre-cooled mortar and pestle to extract their genomic DNA. The quality of DNA extraction was determined by electrophoresis on a 1% agarose gel, GV (Gold view) staining, and bands were detected using a gel imaging system. The DNA concentration was determined using a UV spectrophotometer.

[0063] 3. Development and screening of whole-genome SSR markers

[0064] 3.1 SSR primer design

[0065] Based on the reference sequence of the whole genome of Cymbidium orchid, 50 SSR molecular markers were developed using software such as SSR Hunter, and 50 pairs of upstream and downstream primers for amplifying each SSR were designed using Primer 5 software (Table 2). The 50 SSR molecular markers developed were amplified by PCR using various orchid genomic DNA as templates. The Mix used for PCR amplification (composed of Es Taq DNA Polymerase, Mg 2+ The premix system consisting of PCR stabilizer and enhancer, dNTPs, and blue dye was purchased from Tiangen Company; 50 pairs of SSR primers were purchased from the Shanghai Branch of Canada Biotechnology Company.

[0066] Table 2 List of 50 pairs of primers

[0067]

[0068]

[0069]

[0070]

[0071] 3.2 PCR reaction system includes 10×PCR Buffer 3μL, 2.5mM dNTP 2μL, MgCl 2 2μL, PrimerA2μL, Primer B 2μL, Template 1μL, H 2O 18 μL, Taq enzyme 0.2 μL.

[0072] The PCR reaction program was: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 30 cycles; 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, 10 cycles; 60°C for 30 min; and stored at 4°C.

[0073] 3.3 2.0% agarose gel electrophoresis detection

[0074] 10 μL of PCR product was loaded into each well, with 100 bp ladder as indicator, and electrophoresis was performed at 120 V for 60 min to preliminarily screen polymorphic primers (the size of the fragment amplified by the same primer in different samples was used as an indicator) Figure 1 ). According to the electrophoresis test results, 14 pairs of SSR molecular markers with strong band specificity and obvious polymorphism in each sample were screened out, and fluorescent primers were designed for molecular identification of Cymbidium germplasm resources. The sequences of the 14 pairs of SSR primers are shown in Table 3.

[0075] Table 3 14 pairs of SSR primer sequences

[0076]

[0077]

[0078] 4. SSR-PCR and capillary electrophoresis detection

[0079] 4.1 SSR-PCR reaction

[0080] The total volume of the PCR reaction system was 10 μL, including 1.2 μL DNA template (50 ng / μL), 1.0 μL 10×Buffer Ι buffer, 0.1 μL TAKARA HS Taq enzyme (5 U / μL), 0.6 μL forward primer (5 μM), 0.6 μL reverse primer (5 μM), 0.8 μL 2.5 mM dNTP, 0.5 μL TP-M13 (5 μM), and deionized water was added to 10 μL.

[0081] PCR reaction program: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 30 cycles; 95°C for 30 s, 53°C for 30 s, 72°C for 30 s, 10 cycles; 60°C for 30 min; store at 4°C.

[0082] 4.2 Capillary electrophoresis detection

[0083] 1.0 μL of amplified product, 9 μL of ROX-500 molecular weight internal standard and formamide mixture (volume ratio 0.5:8.5) were added to each well of a 96-well plate. After denaturation at 95°C for 3 min, the sample was detected on an ABI 3730XL detector, with injection at 1 kV for 10 s and electrophoresis at 15 kV for 30 min.

[0084] 5. Analysis of primer amplification results

[0085] The 14 pairs of polymorphic SSR primers screened were used to perform STR typing based on capillary electrophoresis fluorescence detection on 79 qualified Cymbidium orchid samples. The results showed that the primer pair SSR_62892_c1_g1i6 could detect bands in 96.87% of the samples, with the highest detection rate; the average detection rate was 88.60% (Table 4), indicating that these 14 pairs of polymorphic SSR primers can be effectively used for molecular identification of Cymbidium orchid varieties. The peak graph of the amplification result of the primer pair SSR_62892_c1_g1i6 is used as an example for display ( Figure 2 ), the SSR molecular marker primers described in the present invention have good amplification effect, high detection rate, and can amplify stable DNA bands.

[0086] Table 4 Detection of 14 pairs of polymorphic SSR primers in 79 Cymbidium orchid samples

[0087]

[0088] 6. Data Analysis

[0089] The raw data files collected by Data Colletion software were imported into GeneMapper 6.0 software for analysis, and the positions of each peak were compared with the molecular weight internal standard in its lane to calculate the exact size of the target DNA fragment. Capillary electrophoresis was performed three times independently on each fluorescent marker locus, and the average of the three repetitions was taken and rounded off as the data of the experimental material at that locus.

[0090] POPGENE1.31 and PowerMarker software were used to analyze the following genetic diversity parameters, and PAST3 and other software were used to calculate and analyze genetic diversity indicators, clustering, and polymorphism information content (PIC):

[0091] Observed allele number (Na), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), effective allele number (Ne), genetic deviation index (D), Shannon–Weaver diversity index (I), cluster map, DNA fingerprint band information and molecular ID information, etc. Bioinformatics software (POPGENE, MEGA, Joinmap, Structure, R, etc.) was used to perform genetic diversity and cluster analysis on STR typing data. NTSYS and other software were used to calculate and analyze genetic diversity indicators, clustering and polymorphism information content (PIC). The results are shown in Table 5.

[0092] Table 5 Genetic diversity analysis results

[0093]

[0094] 7. Molecular ID card construction

[0095] According to the diploid standard, the fingerprint data is digitally encoded according to the SSR test results (the amplified fragments of each site are arranged according to the molecular weight, and the amplified fragments (alleles) are marked with Arabic numerals 1-9 from small to large, and alleles with more than 9 are marked with capital letters AZ). If the site is not amplified in a certain variety, it is recorded as 0, and each site occupies two bits. Among them, the order of SSR molecular markers for the molecular ID card is: SSR_21319_c0_g2i1, SSR_71852_c0_g2i1, SSR_92040_c0_g1i7, SSR_12201_c0_g1i10, SSR_42275_c0_g1i1, SSR_52366_c0_g1i1, SSR_3258 1_c0_g1i1, SSR_22677_c0_g2i1, SSR_12742_c0_g7i1, SSR_62892_c1_g1i6, SSR_ 33144_c2_g1i2, SSR_33179_c0_g1i2, SSR_73252_c3_g1i1, SSR_53283_c0_g5i3.

[0096] The fingerprints of 79 Cymbidium orchid varieties are shown in Table 6.

[0097] Table 6 Fingerprints of 79 Cymbidium varieties

[0098]

[0099]

[0100]

[0101] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Cymbidium orchid SSR primer set, characterized in that: Including the following 13 pairs of SSR primer sequences: SSR_21319_c0_g2i1 F:5’-GGCACCTCTGGAATTTCTGGT-3’ R:5’-GTGCGTGAGAGAGAGAAAGACA-3’; SSR_92040_c0_g1i7 F:5’-AGGTTGCCAAGCTAGCTACT-3’ R:5’-TGAGGCTTGACTAACCCGTC-3’; SSR_12201_c0_g1i10 F:5’-CACGCGAACGCAATACAGAG-3’ R:5’-ACAGAGATGCGTGCATGTGA-3’; SSR_42275_c0_g1i1 F:5’-TTCTTGTCTGCTCGCTCCAG-3’ R:5’-CCCATTGCCTTCACCTCCTT-3’; SSR_52366_c0_g1i1 F:5’-GGGTGAGAGGAAAAGTGGACT-3’ R:5’-TCACATCACCAGTGCTGCAT-3’; SSR_32581_c0_g1i1 F:5’-CACGGCTGCTCATCAGAATG-3’ R:5’-GTCCACAGAGGCAGAGACAT-3’; SSR_22677_c0_g2i1 F:5’-TTGTCATCGGCATTGACCAA-3’ R:5’-TGTGGGAAAAACATGGGAATTGG-3’; SSR_12742_c0_g7i1 F:5’-AACAGGAGCTAATCACGGGC-3’ R:5’-CAGCCCAGCGTTCAAAAGAC-3’; SSR_62892_c1_g1i6 F:5’-GCCTCATGTAGGGGAGCTTC-3’ R:5’-ATCGTCCACCTGTTATCCGC-3’; SSR_33144_c2_g1i2 F:5’-AGCATCTCGCAGAACTTTACTG-3’ R:5’-CTTGCTCTCTCCCTCTCCCT-3’; SSR_33179_c0_g1i2 F:5’-CATCCACGGACCCAAATCCA-3’ R:5’-TCGACGATGAGTAATGGTGGT-3’; SSR_73252_c3_g1i1 F:5’-AGCCATCAATCACTGTGCGA-3’ R:5’-TGGATATGCCTACTGCCCTG-3’; SSR_53283_c0_g5i3 F:5’-TCTGTTGCTAGCCTTCTCCAC-3’ R:5'-TCTCGTCCTTTCCCTCCCTT-3'.

2. Application of the Cymbidium SSR primer set described in claim 1 in constructing a fingerprint map of Cymbidium varieties.

3. Use of the Cymbidium SSR primer set of claim 1 in identifying Cymbidium varieties; the Cymbidium varieties include Laoranzi, Dangzi, Chengmei, Nanyangmei, Zhengxiaohe, Changshoumei, Cui'e, Jiepeimei, Laojipin, Jinaosu, Jiangnanxinjipin, Duanhuimei, Shiyuan, Lushixiongshi, Zishaxing, Dinghongruidie, Xiuxiangjin, Hybrid Cymbidium tiger stripes, Guanding, Jinbian Cymbidium, Lu Mudan, Yinbian Cymbidium, Dachenzi, Dadiecai, Yuandong Qilin, Yuandong Qidie, Xianlu, Yuanzi, Lufengqiao, Wenzhousu, Cuidinghesu, Qinghua plum, Liu plum, Pan Lu, crystal dragon, Shanghai plum, dyed words, building plum, top quality, Duan plum, Rongmei, Tianjiao peony, Sixi peony, Green cloud peony, Lu Yiqi butterfly, tower butterfly, Ying butterfly, step by step, gathering of heroes, top quality fairy lotus, beauty of Jianhu, Quanzhou plum, green peach offering longevity, lion butterfly, Lingnan white crane, golden bell, cymbidium crystal flower, purple azalea, Yuewang plum, polyflower element, Duoyun, Zhejiang top, Duanxiu plum, Diecui, Chunhui Thousand-Handed Guanyin, imperial concubine, pink Monroe, Nanxi, dream, jasper, phantom, waltz, jade Zen, dawn, sunset.

4. A method for constructing a fingerprint of Cymbidium orchid varieties, characterized in that: The following steps are involved: (1) Extract DNA from Cymbidium orchid samples; (2) using the extracted DNA as a template and the SSR primer set described in claim 1 as an amplification primer, establishing a PCR reaction system and performing PCR amplification; (3) The amplified products are subjected to capillary electrophoresis, and the amplified fragment length data encoding is formed for the sample DNA of each variety of Cymbidium orchid according to the capillary electrophoresis results, so as to construct a fingerprint map of Cymbidium orchid varieties.

5. The method for constructing the fingerprint of Cymbidium orchid varieties according to claim 4, characterized in that: The PCR reaction system in step (2) includes: 10×PCR Buffer 3μL, 2.5mM dNTP 2μL, MgCl2 2μL, Primer A 2μL, PrimerB 2μL, Template 1μL, ddH2O 18μL, Taq enzyme 0.2μL; The reaction program of the PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, 30 cycles; 95℃30s, 55℃30s, 72℃30s, 10 cycles; 60℃30min; store at 4℃.

6. The method for constructing the fingerprint of Cymbidium orchid varieties according to claim 4, characterized in that: In step (3), the amplified product is subjected to capillary electrophoresis, and the amplification result of the sample DNA of each variety of Cymbidium is digitized into the length of the amplified fragment according to the capillary electrophoresis result, specifically: according to SSR_21319_c0_g2i1, SSR_92040_c0_g1i7, SSR_12201_c0_g1i10, SSR_42275_c0_g1i1, SSR_52366_c0_g1i1, SSR_32581_c0_g1i1, SSR_22677_c0_g2i 1. The molecular markers SSR_12742_c0_g7i1, SSR_62892_c1_g1i6, SSR_33144_c2_g1i2, SSR_33179_c0_g1i2, SSR_73252_c3_g1i1, and SSR_53283_c0_g5i3 were arranged in sequence. The length of the fragments amplified by each pair of SSR primers for each Cymbidium variety was recorded in turn through the capillary electrophoresis results. The length of each amplified fragment was digitized and encoded, which was the DNA fingerprint of the Cymbidium variety.

7. The method for constructing the fingerprint of Cymbidium orchid varieties according to claim 6, characterized in that: The digital encoding is performed based on the length of each amplified fragment, specifically: the alleles of the amplified fragments of each site are arranged according to the molecular weight, the alleles are labeled with Arabic numerals 1-9 from small to large, and alleles above 9 are labeled with capital letters AZ. If the site is not amplified in a certain variety, it is recorded as 0, and each site occupies two bits.

8. Application of the Cymbidium variety fingerprint obtained by the construction method according to any one of claims 4 to 7 in identifying Cymbidium variety; the Cymbidium variety comprises Lao Ranzi, Dangzi, Cheng Mei, Nanyang Mei, Zheng Xiaohe, Changshou Mei, Cui E, Jie Pei Mei, Lao Jipin, Jin Aosu, Jiangnan Xinjipin, Duan Huimei, Shiyuan, Lushi Xiongshi, Zishaxing, Dinghong Ruidie, Xiuxiangjin, Hybrid Cymbidium Tiger Stripe, Guanding, Gold Edge Cymbidium, Green Peony, Silver Edge Cymbidium, Da Chenzi, Da Diecai, Far East Qilin, Far East Qidie, Xianlu, Yuanzi, Lufengqiao, Wen The best flowers in the world are as follows: Zhousu, Cuidinghesu, Qinghuamei, Liumei, Panlu, Shuijinglong, Shanghaimei, dyed characters, Loumei, top-quality, Duanmei, Rongmei, Tianjiao peony, Sixi peony, Green cloud peony, Luyiqidie, Loutaidie, Yingdie, BBK, a gathering of heroes, the best fairy lotus, the beauty of Jianhu, Quanzhoumei, Cuitaoxianshou, Shidie, Lingnan white crane, Jinling, Huilan crystal flower, Zijuan, Yuewangmei, Juhuasu, Duoyun, Zhejiangding, Duanxiumei, Diecui, Chunhui Thousand-Handed Guanyin, Guifei, Pink Monroe, Nanxi, Dream, Jasper, Phantom, Waltz, Yuchan, Dawn, and Sunset.

9. A kit for identifying Cymbidium orchid varieties, characterized in that: Contains the Cymbidium SSR primer set as described in claim 1.

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