A cymbidium ssr primer set and a method for constructing a cymbidium variety fingerprint by using the primer set

Abstract

The application discloses a Cymbidium SSRR primer group and a method for constructing a Cymbidium variety fingerprint by using the primer group. The Cymbidium SSRR primer group comprises 14 pairs of SSR primer sequences. The 14 pairs of SSR primer sequences are used for carrying out multiplex PCR amplification on Cymbidium genomic DNA, the PCR products are detected by using capillary electrophoresis fluorescence, SSR marker data are analyzed, 70 kinds of Cymbidium SSR fingerprint maps are constructed, and Cymbidium varieties can be effectively identified. The method for constructing the Cymbidium variety fingerprint has the advantages of high throughput, high precision and low cost, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a Cymbidium ensifolium SSR primer set and a method for constructing a Cymbidium ensifolium variety fingerprint using the primer set. BACKGROUND

[0002] Cymbidium ensifolium is a terrestrial species of the Orchidaceae family. It has a moderate plant type, beautiful leaf posture, and a total inflorescence with 5-10 flowers, and has a delicate fragrance. It is a rare multi-season flowering species among traditional national orchids in China, and is therefore also known as the four-season orchid. It is deeply welcomed on the domestic and international markets. Although Cymbidium ensifolium has a long history of cultivation and cultural heritage, the identification of varieties is mostly dependent on traditional morphological indicators, and it is difficult to accurately identify the variety through leaf and plant type before flowering. In recent years, with the popularization and market development of Cymbidium ensifolium, and the rapid development of Internet technology, major e-commerce platforms, social media and forums provide more diversified trading channels. There are more than 500 varieties of Cymbidium ensifolium on the orchid market, but there is no uniform and standardized variety identification standard. The phenomenon of product substitution and variety confusion is widespread. In order to promote the breeding, identification and promotion of varieties that meet market demand, it is necessary to establish a corresponding orchid variety identification standard.

[0003] Orchid 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, genetic diversity, genetic relationship analysis, and germplasm resources and population research. However, the identification and evaluation of China's characteristic resources are limited by geographical limitations and the difficulty of collecting varieties. Currently, there is no related report on high-throughput identification of Cymbidium ensifolium varieties based on capillary electrophoresis technology and SSR molecular markers in China. Therefore, the present application uses SSR molecular markers to identify Cymbidium ensifolium varieties, which has important significance for germplasm identification, innovative utilization of germplasm, quality supervision of commodity flowers and market standardization. SUMMARY

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

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

[0006]

[0007]

[0008] A second object of the present application is to provide the use of the Cymbidium SSR primer set as described above in the construction of a Cymbidium variety fingerprint.

[0009] A third object of the present application is to provide the use of the Cymbidium SSR primer set as described above in the identification of a Cymbidium variety.

[0010] A fourth object of the present application is to provide a method for constructing a Cymbidium variety fingerprint, comprising the following steps:

[0011] (1) extracting DNA from a Cymbidium sample;

[0012] (2) using the extracted DNA as a template and the Cymbidium SSR primer set as described in claim 1 as amplification primers, establishing a PCR reaction system and performing PCR amplification;

[0013] (3) performing capillary electrophoresis on the amplification product, forming an amplification fragment length data code according to the capillary electrophoresis results of the amplification results of the sample DNA of each Cymbidium variety, and constructing a Cymbidium variety fingerprint.

[0014] Further, the PCR reaction system in step (2) comprises: 10x PCR Buffer 3 μL, 2.5 mM dNTP 2 μL, MgCl2 2 μL, Primer A 2 μL, Primer B 2 μL, Template 1 μL, ddH2O 18 μL, Taq enzyme 0.2 μL.

[0015] The reaction procedure for the PCR amplification is: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 30 cycles; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 10 cycles; 60℃ for 30 min; 4℃ storage.

[0016] Further, the amplification product in step (3) is subjected to capillary electrophoresis, and the amplification result of the sample DNA of each variety of Cymbidium is formed into an amplification fragment length data code according to the capillary electrophoresis result, specifically: according to the order of the molecular markers of SSR_41756_c0_g2, SSR_44148_c1_g1, SSR_44775_c0_g1, SSR_44824_c2_g1, SSR_45085_c1_g2, SSR_47270_c0_g2, SSR_49506_c0_g1, SSR_50081_c2_g1, SSR_51250_c1_g6, SSR_52068_c3_g3, SSR_54963_c0_g1, SSR_55127_c0_g1, SSR_56903_c1_g2, and SSR_43897_c1_g1, the fragment length of each Cymbidium variety amplified by each pair of SSR primers is recorded in sequence through the capillary electrophoresis result, and the data code is formed according to the length of each amplification fragment, that is, the DNA fingerprint of the Cymbidium variety.

[0017] Further, the data code formed according to the length of each amplification fragment is specifically: the alleles of each locus amplification fragment are arranged according to the molecular weight, and the alleles are marked with Arabic numerals 1-9 from small to large, and more than 9 alleles are marked with capital English letters A-Z, and if there is no amplification in a certain variety, it is recorded as 0, and each locus occupies two bits.

[0018] The fifth object of the present application is to provide a Cymbidium variety fingerprint constructed by the Cymbidium variety fingerprint construction method.

[0019] The sixth object of the present application is to provide the application of the above-mentioned Cymbidium variety fingerprint in identifying Cymbidium varieties.

[0020] The seventh object of the present application is to provide a kit for identifying Cymbidium varieties, which contains the above-mentioned Cymbidium SSR primer group.

[0021] The beneficial effects of the present application are:

[0022] (1) The present application realizes the beneficial combination of SSR molecular markers and high-efficiency and automatic capillary electrophoresis technology by using capillary electrophoresis technology, the detection result is automatically saved by the analysis software, the detection speed is fast, the separation efficiency is high, the sensitivity is relatively high, the accuracy is within 1 bp, which is beneficial to the detection and analysis of a large number of samples, and lays a solid foundation for the popularization and application of SSR molecular marker technology in Cymbidium variety identification.

[0023] (2) The identification method of the present application has low cost, mature reagent consumables market, and does not count labor and instrument loss, and the detection cost of one sample is less than 10 yuan. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Part of the representative figure for SSR marker screening, sample order from left to right in turn is the island fairy, color green, jade peony, Emei mountain strange butterfly, king of lotus.

[0025] Figure 2 For the peak figure of amplification results of different Cymbidium varieties with primer pair SSR_54963_c0_g1, from top to bottom in turn is the amplification peak figure of four different Cymbidium varieties of island fairy, jade peony, Emei mountain strange butterfly, king of lotus. DETAILED DESCRIPTION

[0026] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0027] Example 1

[0028] 1. Cymbidium variety collection

[0029] The samples are derived from Cymbidium main producing areas of Guangdong Province, Guangxi Zhuang Autonomous Region and Taiwan Province of China, and include 70 varieties of different flower types, colors and leaf colors, as shown in Table 1 below.

[0030] Table 1 70 Cymbidium varieties

[0031]

[0032]

[0033]

[0034]

[0035] 2. Cymbidium genomic DNA extraction

[0036] The collected 70 Cymbidium germplasm resources are extracted with plant genomic DNA kit (Tiangen Company). First, collect young leaves in batches, and divide each sample into 2-3 parts (about 2g each) in a 2mL centrifuge tube, freeze in liquid nitrogen, and store in a-80℃ refrigerator for standby. Use pre-cooled mortar and pestle to grind the frozen plant tissue thoroughly, and extract its genomic DNA. Electrophorese on 1% agarose gel, stain with GV (Gold view), detect the band with a gel imaging system, judge the quality of DNA extraction, and determine the DNA concentration with a UV spectrophotometer.

[0037] 3. Whole genome SSR marker development and screening

[0038] 3.1 SSR primer design

[0039] According to the Cymbidium sinense genome reference sequence, 40 SSR molecular markers were developed using SSR Hunter and other software, and 40 pairs of upstream and downstream primers for amplifying each SSR were designed using Primer 5 software (Table 2). The 40 SSR molecular markers developed were used as templates for PCR amplification of various orchid genomic DNA. The PCR amplification mix (a premix system composed of Es Taq DNA Polymerase, Mg 2+ , dNTPs, and PCR stabilizers and enhancers, with the addition of blue dye) was purchased from Tiangen; 40 pairs of SSR primers were purchased from Sangon Shanghai Branch in Canada.

[0040] Table 2: List of 40 pairs of primers

[0041]

[0042]

[0043]

[0044]

[0045] 3.2 PCR reaction system includes 10x PCR Buffer 3μL, 2.5mM dNTP 2μL, MgCl22μL, Primer A 2μL, Primer B 2μL, Template 1μL, H2O 18μL, Taq enzyme 0.2μL.

[0046] The PCR reaction program is as follows: 95℃ for 5min; 95℃ for 30s, 60℃ for 30s, 72℃ for 30s, 30 cycles; 95℃ for 30s, 55℃ for 30s, 72℃ for 30s, 10 cycles; 60℃ for 30min; 4℃ storage.

[0047] 3.3 2.0% agarose gel electrophoresis detection

[0048] Load 10μL PCR product per well, 100bp ladder as an indicator, run electrophoresis at 120v for 60min to preliminarily screen polymorphic primers (use the size of fragments amplified by the same primer in different samples as an indicator) Figure 1 ). According to the electrophoresis detection results, 14 pairs of SSR molecular markers with strong band specificity and obvious polymorphism in each sample were selected for fluorescence primer design for molecular identification of Cymbidium germplasm resources. The sequences of the 14 pairs of SSR primers are shown in Table 3.

[0049] Table 3: Sequences of 14 pairs of SSR primers

[0050]

[0051] 4. SSR-PCR and capillary electrophoresis detection

[0052] 4.1 SSR-PCR reaction

[0053] The total volume of the PCR reaction system is 10 μL, including 1.2 μL of DNA template (50 ng / μL), 1.0 μL of 10x Buffer I buffer, 0.1 μL of TAKARA HS Taq enzyme (5 U / μL), 0.6 μL of forward primer (5 μM), 0.6 μL of reverse primer (5 μM), 0.8 μL of 2.5 mM dNTP, 0.5 μL of TP-M13 (5 μM), and deionized water to make up to 10 μL.

[0054] The PCR reaction program is as follows: 95℃ for 5 min; 95℃ for 30 s, 60℃ for 30 s, 72℃ for 30 s, 30 cycles; 95℃ for 30 s, 53℃ for 30 s, 72℃ for 30 s, 10 cycles; 60℃ for 30 min; and 4℃ storage.

[0055] 4.2 Capillary electrophoresis detection

[0056] 1.0 μL of the amplification product, 9 μL of a ROX-500 molecular weight marker and formamide mixture (volume ratio 0.5:8.5) are added to each well of a 96-well plate, and after denaturation at 95℃ for 3 min, detection is performed on an ABI 3730XL detector, 1 kV voltage for 10 s, electrophoresis at 15 kV for 30 min.

[0057] 5. Analysis of primer amplification results

[0058] The 70 qualified Cymbidium samples are subjected to STR typing based on capillary electrophoresis fluorescence detection by using the screened 14 pairs of polymorphic SSR primers. The results show that the primer pair SSR_54963_c0_g1 can detect bands in 95.29% of the samples, and the detection rate is the highest; the average detection rate is 85.58% (Table 4), indicating that the 14 pairs of polymorphic SSR primers can be effectively used for molecular identification of Cymbidium varieties. The peak chart of the amplification results of the primer pair SSR_54963_c0_g1 is shown as an example (Fig. 8), and the SSR molecular marker primer amplification effect is good, the detection rate is high, and stable DNA bands can be amplified. Figure 2

[0059] Table 4 Detection of the 14 pairs of polymorphic SSR primers in 70 Cymbidium samples

[0060]

[0061] 6. Data analysis

[0062] ​The original data file collected by the Data Collection software was imported into the GeneMapper 6.0 software for analysis. The position of each peak was compared with the molecular weight internal standard in its lane, and the accurate size of the target DNA fragment was calculated. The capillary electrophoresis detection of each fluorescently labeled locus was independently repeated three times, and the average value of the three repetitions was taken and rounded off to the nearest whole number as the data of the experimental material on the locus.

[0063] The following genetic diversity parameters were analyzed using POPGENE 1.31 and PowerMarker software, and the genetic diversity index, clustering, and polymorphic information content (PIC) were calculated and analyzed using PAST3 and other software:

[0064] The observed allele number (Na), observed gene heterozygosity (Ho), expected gene heterozygosity (He), polymorphic information content (PIC), effective allele number (Ne), genetic deviation index (D), Shannon-Weaver diversity index (I), clustering map, DNA fingerprint band information, and molecular ID information were observed. The STR typing data were analyzed for genetic diversity and clustering using bioinformatics software (POPGENE, MEGA, Joinmap, Structure, R, etc.). The genetic diversity index, clustering, and polymorphic information content (PIC) were calculated and analyzed using NTSYS and other software. The results are shown in Table 5.

[0065] Table 5. Results of genetic diversity analysis

[0066]

[0067] 7. Construction of molecular ID

[0068] According to the standard construction of diploid, the fingerprint data is coded according to the results of SSR detection (the amplified fragments of each locus are arranged according to the molecular weight, and the amplified fragments (alleles) are marked with Arabic numerals 1-9 from small to large, and more than 9 alleles are marked with capital English letters A-Z), if the locus has no amplification in a certain variety, it is recorded as 0, and each locus occupies two bits. The order of the molecular identity card SSR molecular markers is: SSR_41756_c0_g2, SSR_44148_c1_g1, SSR_44775_c0_g1, SSR_44824_c2_g1, SSR_45085_c1_g2, SSR_47270_c0_g2, SSR_49506_c0_g1, SSR_50081_c2_g1, SSR_51250_c1_g6, SSR_52068_c3_g3, SSR_54963_c0_g1, SSR_55127_c0_g1, SSR_56903_c1_g2, SSR_43897_c1_g1.

[0069] The fingerprint of 70 Cymbidium varieties is shown in Table 6.

[0070] Table 6 Fingerprint of 70 Cymbidium varieties

[0071]

[0072]

[0073]

[0074] The above is only the preferred embodiment of the present application, it should be noted that the above preferred embodiment should not be considered as a limitation of the present application, the protection scope of the present application should be limited by the scope defined by the claims. For ordinary skilled in the art, without departing from the spirit and scope of the present application, a number of improvements and refinements can also be made, which should be considered as the protection scope of the present application.

Claims

1. A Cymbidium ensifolium SSR primer set, characterized in that, 14-year-old SSR: SSR_41756_c0_g2 F:5'-TTGCAGTCGTGGAAGAGAGA-3'; R:5'-TGCAAATGTCAGCGAGAAA-3'; SSR_44148_c1_g1 F:5'-AACAATTTTCAAACGAGCCG-3'; R:5'-ATTCGCACTTCCATCTCCAT-3': SSR_44775_c0_g1 F:5'-GCCCTTTGTGCAATTTAGGA-3'; R:5'-ACATCCAGATCCAAACCTCG-3'; SSR_44824_c2_g1 F:5'-TCCAATTCGATCGTCACGTA-3'; R:5'-TGGACGAAGGAGACCTTGAC-3'; SSR_45085_c1_g2 F:5'-ACCCAGTCCCCTCTCCTTAG-3'; R:5'-TGGCAATTGAGAGGTAGGGA-3'; SSR_47270_c0_g2 F:5'-TTGTTTCTTGTGTGATTTTGAGG-3'; R:5'-CCGGAATGTTATGATGGGAG-3'; SSR_49506_c0_g1 F:5'-AAAAGCATTCAAATCGGGTG-3'; R:5'-TGATGGGGGTCTCGTATCTC-3'; SSR_50081_c2_g1 F:5'-TTCCAACAACCCCTTCTGTC-3'; R:5'-AGCGAAGTGAACTGGGAGAA-3'; SSR_51250_c1_g6 F:5'-TGCTAATTGCCTGTGACCAA-3'; R:5'-CTTCTATCAGAGCACACTTGACA-3';SSR_52068_c3_g3 F:5'-TCTTAAAGCACCTCACTGGAGA-3'; R:5'-AGAAGCAGTTGGGCTCAGAA-3'; SSR_54963_c0_g1 F:5'-CAATCAGCCTAGGGTTCCAA-3'; R:5'-AAGGAGAAACTTCCTGCGGT-3'; SSR_55127_c0_g1 F:5'-CGATGGAGGACAGCACTTTT-3'; R:5'-GCACCTTCTTCTCCCTCACA-3': SSR_56903_c1_g2 F:5'-GCGTTGAAGAAAGGCAAAAG-3'; R:5'-TCGTGTATTTGGCACGAAAG-3'; SSR_43897_c1_g1 F:5'-TTGTACGGGGAGGAGTTTTG-3'; R:5'-ACCAGGTTGTAGCCCTCCTT-3'.

2. The application of the Cymbidium ensifolium SSR primer set as described in claim 1 in constructing fingerprint profiles of Cymbidium ensifolium varieties.

3. The application of the Cymbidium SSR primer set described in claim 1 in the identification of Cymbidium varieties; wherein the Cymbidium varieties include Babao Qizhen, Baodao Jinlong, Baodao Xiannv, Baodao Yanzhi, Caifeng, Caifengmei, Caihongzhao, Cailv, Caiyunfei, Chaotianhe, Cuiyu Mudan, Diehuang, Duoshehua, Duoyun, Emeishan Qidie, Emei Xue, Fuse Dahe, Fuxing Qidie, Fushan Jin, Fushan Qidie, Gaishimei, Guanshanhe, Guifuren, Hewang, Hongfen Jiaren, Hongniang, Hongshuixian, Hongxingmei, Hongyipin, Huamulan, Huang Flag, Yellow First Rank, Jiaozhou White Plum, Jianlan Spring, Delicate Crane, Golden Lotus, Golden Lotus Cloud Well, Golden Silk Horse Tail Claw, Golden Wrinkled Rainbow Lotus, Brocade Flag, Crystal Dragon Wonderful Butterfly, National Celebration, Jun Lotus, Exquisite Lotus, Lingnan Wonderful Butterfly, Longyan Plain, Arhat Lotus, Green Bird Beak, Green Narcissus, Hemp Shell Plain, Horse Ear Claw, Horse Ear Claw Art, Penglai Lion, Penglai Flower, Seven Fairies, Thousand Buddha Golden Top, Millennium Ruyi, Green Mountain Jade Spring, Sun and Moon Treasure, Samo Brocade, Shaoguan Plain Wonderful, Holy Plum, Mayor Red, Crystal Lotus, Crystal Royal Plum, Four Seasons Three Brotherhoods, Four Seasons Crystal, Four Seasons Jade Concubine.

4. A method for constructing a fingerprint spectrum of Cymbidium ensifolium varieties, characterized in that, Includes the following steps: (1) Extracting DNA from Cymbidium ensifolium samples; (2) Using the extracted DNA as a template and the SSR primer set described in claim 1 as amplification primers, a PCR reaction system was established and PCR amplification was performed; (3) The amplification products were subjected to capillary electrophoresis. Based on the capillary electrophoresis results, the amplification fragment length data of each variety of Cymbidium ensifolium was digitized and encoded to construct the fingerprint map of Cymbidium ensifolium varieties.

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

6. The method for constructing fingerprint profiles of Cymbidium ensifolium varieties according to claim 4, characterized in that, In step (3), the amplification products are subjected to capillary electrophoresis. Based on the capillary electrophoresis results, the amplification fragment length data of the sample DNA for each variety of Cymbidium ensifolium is digitized and encoded, specifically as follows: according to SSR_41756_c0_g2, SSR_44148_c1_g1, SSR_44775_c0_g1, SSR_44824_c2_g1, SSR_45085_c1_g2, SSR_47270_c0_g2, SSR_49506_c0_g1, SSR_500 The molecular markers 81_c2_g1, SSR_51250_c1_g6, SSR_52068_c3_g3, SSR_54963_c0_g1, SSR_55127_c0_g1, SSR_56903_c1_g2, and SSR_43897_c1_g1 were sequentially arranged. The length of the fragments amplified by each pair of SSR primers for each Cymbidium ensifolium variety was recorded sequentially by capillary electrophoresis. The DNA fingerprint of the Cymbidium ensifolium variety was obtained by digitizing and encoding each amplified fragment length.

7. The method for constructing fingerprint profiles of Cymbidium ensifolium varieties according to claim 6, characterized in that, The data encoding based on the length of each amplified fragment is as follows: the alleles of each amplified fragment are arranged according to molecular weight, and the alleles are labeled with Arabic numerals 1-9 from smallest to largest. Alleles with more than 9 alleles are labeled with uppercase English letters AZ. If the locus is not amplified in a certain variety, it is recorded as 0. Each locus occupies two bits.

8. The application of the fingerprint spectrum of Cymbidium ensifolium varieties constructed by the construction method according to any one of claims 4-7 in the identification of Cymbidium ensifolium varieties; wherein the Cymbidium ensifolium varieties include Babao Qizhen, Baodao Jinlong, Baodao Xiannv, Baodao Yanzhi, Caifeng, Caifengmei, Caihongzhao, Cailv, Caiyunfei, Chaotianhe, Cuiyu Mudan, Diehuang, Duoshehua, Duoyun, Emeishan Qidie, Emei Xue, Fuse Dahe, Fuxing Qidie, Fushan Jin, Fushan Qidie, Gaishimei, Guanshanhe, Guifuren, Hewang, Hongfen Jiaren, Hongniang, Hongshuixian, Hongxingmei, Hong First-class, Mulan, Imperial Flag, Yellow First-class, Jiaozhou White Plum, Jianlan Spring, Delicate Crane, Golden Lotus, Golden Lotus Cloud Well, Golden Silk Horse Tail Claw, Golden Wrinkled Rainbow Lotus, Brocade Flag, Crystal Dragon Wonderful Butterfly, National Celebration, Junhe, Exquisite Lotus, Lingnan Wonderful Butterfly, Longyan Plain, Luohan Lotus, Green Bird Beak, Green Narcissus, Hemp Shell Plain, Horse Ear Claw, Horse Ear Claw Art, Penglai Lion, Penglai Flower, Seven Fairies, Thousand Buddha Golden Top, Millennium Ruyi, Green Mountain Jade Spring, Sun and Moon Treasure, Samo Brocade, Shaoguan Plain Wonder, Holy Plum, Mayor Red, Crystal Lotus, Crystal Imperial Plum, Four Seasons Three Brotherhoods, Four Seasons Crystal, Four Seasons Jade Concubine.

9. A kit for identifying Cymbidium ensifolium varieties, characterized in that, It contains the SSR primer set of *Cymbidium goeringii* as described in claim 1.

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