CN114736977B - Cymbidium SSR primer group and method for constructing cymbidium variety fingerprint by using same - Google Patents

Cymbidium SSR primer group and method for constructing cymbidium variety fingerprint by using same Download PDF

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CN114736977B
CN114736977B CN202210170607.9A CN202210170607A CN114736977B CN 114736977 B CN114736977 B CN 114736977B CN 202210170607 A CN202210170607 A CN 202210170607A CN 114736977 B CN114736977 B CN 114736977B
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杨凤玺
任锐
陆楚桥
金建鹏
朱根发
高洁
魏永路
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Environmental Horticulture Institute of Guangdong Academy of Agricultural Sciences
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Abstract

The invention discloses an ink orchid SSR primer group and a method for constructing ink orchid variety fingerprint by using the primer group. The cymbidium SSR primer group comprises 14 pairs of SSR primer sequences. The invention uses the 14 pairs of SSR primer sequences to carry out multiplex PCR amplification on the cymbidium genome DNA, uses capillary fluorescent electrophoresis to detect PCR products, analyzes SSR marker data, constructs 70 cymbidium SSR finger print, and can effectively identify cymbidium varieties. The method for constructing the cymbidium sinense variety fingerprint has the advantages of high flux, high precision and low cost, and has good application prospect.

Description

Cymbidium SSR primer group and method for constructing cymbidium variety fingerprint by using same
Technical Field
The invention relates to the technical field of biology, in particular to an ink orchid SSR primer group and a method for constructing ink orchid variety fingerprint by using the primer group.
Background
Orchids are widely distributed worldwide, with 801 genera and more than 27500 species. In recent years, molecular markers, mainly comprising RAPD, ISSR, SSR, SRAP, scoT and the like, are widely used for classification and identification among orchid seeds, varieties and varieties, genetic diversity analysis, genetic relationship analysis and research on germplasm resources and populations. If the researcher in Syrian uses CAPS molecular markers to identify 34 orchid plants, the result shows that the polymorphism of the test materials is extremely high, and other species can be identified except 5 species such as late spider orchid (Ophrys fucifura). Two populations of dendrobium americanum (Epidendrum fulgens) distributed in the atlantic rainforest of the coast of brazil were studied by brazil researchers using 9 new microsatellite markers. Aiming at orchids which are originally produced by the former, three species of Paphiopedilum (Paphiopedilum) and hybrid seeds thereof are identified by utilizing SCAR molecular markers, and effective specific markers are developed; carrying out molecular identification on 31 Dendrobium (Dendrobium) plants of 5 provinces in China by using an SRAP molecular marker, and obtaining 18 species-specific markers; 129 cymbidium (Cymbidium goeringii) varieties are researched by using EST-SSR molecular markers, and the varieties of the same gardening type are found to have common alleles; the ISSR molecular marker technology is used to develop the relative researches of genetic diversity, genetic relationship and population structure of 16 kinds of orchid plants such as wild Gastrodia elata (Gastrodia), cymbidium plants such as Cymbidium and spring sword, etc., wild Bletilla (Bletilla) plants, 6 kinds of non-distance calanthe (Calanthe tsoongiana) plants, 5 kinds of Paenitaking cypripedium (Cypripedium japonicum) and the like. However, the molecular marker technology is mainly applied to the differentiation of the genetic relationship and genetic relationship of the species or varieties of orchid plants at present, a relatively comprehensive molecular classification system is not formed yet, and the molecular marker technology can be directly applied to breeding selection and has low operability.
The chinese ink (Cymbidium sinense) is also known as cymbidium, a cymbidium species. The plant type flower has high size, beautiful leaf appearance, 10-20 flowers in the total inflorescence, and the flower has the fragrance of orchid, is a flower in the traditional rare year of China, and is popular in the markets at home and abroad. Although cymbidium sinense has long cultivation history and cultural inheritance, variety identification mostly depends on traditional morphological indexes, and the variety is difficult to accurately identify through leaves and plant types when the cymbidium sinense is not yet flowering. In recent years, along with the popularization and market development of cymbidium, and the rapid development of internet technology, various large electronic commerce platforms, social media and forums provide more diversified transaction channels, and cymbidium varieties appearing in the cymbidium market are more than 500 varieties, however, no unified and standard variety identification standard exists, and the phenomena of secondary filling and variety confusion of products are common. In order to promote variety breeding, identification and popularization which are suitable for market demands, it is imperative to establish orchid variety identification standards which are suitable for the variety breeding, identification and popularization. Therefore, developing the cymbidium variety identification work has important significance in the aspects of germplasm resource evaluation, germplasm innovation utilization, commodity flower quality supervision, market standardization and the like. At present, no related report on high-throughput identification of cymbidium varieties based on capillary electrophoresis technology and SSR molecular markers is found in China.
Disclosure of Invention
Aiming at the technical defects of the prior art, the invention provides an cymbidium SSR primer group and a method for constructing a cymbidium variety fingerprint by combining a capillary electrophoresis technology and an SSR-PCR technology by using the primer group so as to rapidly and efficiently identify the cymbidium variety.
The first object of the invention is to provide an ink blue SSR primer group, which comprises the following 14 pairs of SSR primer sequences:
Figure BDA0003517972440000021
Figure BDA0003517972440000031
the second purpose of the invention is to provide the application of the cymbidium SSR primer group in the construction of the cymbidium variety fingerprint.
The third object of the invention is to provide the application of the cymbidium SSR primer group in identifying cymbidium varieties.
The fourth object of the invention is to provide a method for constructing a finger print of a cymbidium sinense variety, which comprises the following steps:
(1) Extracting DNA of a cymbidium sample;
(2) Using the extracted DNA as a template and the SSR primer set as an amplification primer as set forth in claim 1, establishing a PCR reaction system and performing PCR amplification;
(3) And (3) performing capillary electrophoresis on the amplified products, forming an amplified fragment length datamation code for the amplified result of the sample DNA of each variety of the cymbidium sinense according to the capillary electrophoresis result, and constructing and obtaining the cymbidium sinense variety fingerprint.
Further, the PCR reaction system in the step (2) comprises: 10 XPCR Buffer 3. Mu.L, 2.5mM dNTP 2. Mu. L, mgCl 2 2μL、Primer A 2μL、Primer B 2μL、Template 1μL、ddH 2 O18. Mu. L, taq enzyme 0.2. Mu.L.
The PCR amplification 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; 30min at 60 ℃; preserving at 4 ℃.
Further, in the step (3), the amplified product is subjected to capillary electrophoresis, and the amplified result of the sample DNA of each variety of cymbidium sinense is subjected to data coding of the amplified fragment length according to the capillary electrophoresis result, specifically: the method comprises the steps of sequentially recording the fragment lengths of each cotton variety amplified by each pair of SSR primers according to the molecular markers of SSR_23012_c0_g1, SSR_47035_c6_g1, SSR_45085_c2_g1, SSR_53453_c0_g1, SSR_55630_c0_g1, SSR_39257_c3_g1, SSR_51432_c6_g1, SSR_46859_c0_g2, SSR_50940_c5_g2, SSR_445_400, SSR_44145_c1_g1, SSR_44775_c0_g1, SSR_48838_c2_g1 and SSR_46319_c4_g3, and performing data coding according to the amplified fragment lengths of each cotton variety by capillary electrophoresis results.
Further, the data coding is performed according to the length of each amplified fragment, specifically: alleles of amplified fragments at each site are arranged according to the molecular weight, the alleles are marked with Arabic numerals 1-9 from small to large, more than 9 alleles are marked with capital English letters A-Z, if the site is not amplified in a certain variety, the site is marked as 0, and each site occupies two places.
The fifth object of the invention is to provide a cymbidium sinense variety fingerprint constructed by the method for constructing the cymbidium sinense variety fingerprint.
The sixth object of the invention is to provide the application of the cymbidium sinense variety fingerprint in identifying cymbidium sinense varieties.
The seventh object of the invention is to provide a kit for identifying cymbidium sinense varieties, which contains the cymbidium sinense SSR primer group.
The beneficial effects of the invention are as follows:
(1) The invention utilizes capillary electrophoresis technology to realize the beneficial combination of SSR molecular marker and high-efficiency and automatic capillary electrophoresis technology, and the detection result is automatically stored by analysis software, so that the detection speed is high, the separation efficiency is high, the sensitivity is higher, the accuracy is within 1bp, the detection and analysis of a large number of samples are facilitated, and a solid foundation is laid for the popularization and application of SSR molecular marker technology in the detection of cymbidium sinense variety.
(2) The identification method has low cost, mature reagent consumable market, no manual and instrument loss, and the detection cost of one sample is lower than 10 yuan.
Drawings
FIG. 1 is a partial representation of SSR marker screening.
FIG. 2 is a peak diagram of the result of amplifying different cymbidium varieties by using a primer pair SSR_445_400, wherein the top, the middle and the bottom are respectively the peak diagrams of amplifying the flower autumn placard, the green cloud and the day oriented cymbidium.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1
1. Cymbidium sinense variety acquisition
Samples were derived from the major regions of cymbidium, guangdong, guangxi and taiwan, encompassing 70 varieties of different flower types, colors, and leaf colors, as shown in table 1 below.
TABLE 1 70 parts of cymbidium sinense variety
Figure BDA0003517972440000061
Figure BDA0003517972440000071
2. Extraction of genomic DNA from cymbidium sinense
70 parts of the collected cymbidium germplasm resources were extracted with a plant genomic DNA kit (Tiangen kit). Firstly, young leaves are collected in batches, each sample is stored in 2-3 parts (about 2g each part) in a 2mL centrifuge tube, and after quick freezing by liquid nitrogen, the young leaves are frozen in a refrigerator at-80 ℃ for later use. The frozen plant tissue is sufficiently ground by using a precooled mortar and pestle to extract the genomic DNA. Electrophoresis on 1% agarose gel, GV (Good view) staining, detection of bands by gel imaging system, determination of DNA extraction quality, and determination of DNA concentration by UV spectrophotometer.
3. Whole genome SSR marker development and screening
3.1 SSR primer design
Based on the whole genome reference sequence of cuttlefish, 50 SSR molecular markers were developed in total with software such as SSR Hunter, and 50 pairs of upstream and downstream primers for amplifying each SSR were designed with Primer 5 software (Table 2). And carrying out PCR amplification on the developed 50 SSR molecular markers by taking various orchid genome DNA as templates. Mix for PCR amplification (Es Taq DNA Polymerase, mg 2+ Premix system composed of dNTPs, PCR stabilizer and enhancer, and blue dye added) was purchased from Tiangen corporation; 50 pairs of SSR primers were purchased from Shanghai, canada.
TABLE 2 list of 50 pair primers
Figure BDA0003517972440000081
Figure BDA0003517972440000091
Figure BDA0003517972440000101
Figure BDA0003517972440000111
3.2 The PCR reaction system comprises 10 XPCR Buffer 3. Mu.L, 2.5mM dNTP 2. Mu.L, mgCl 2 2μL，Primer A 2μL，Primer B 2μL，Template 1μL，H 2 O18. Mu.L, taq enzyme 0.2. Mu.L.
The PCR reaction procedure was: 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; 30min at 60 ℃; preserving at 4 ℃.
3.3 2.0% agarose gel electrophoresis detection
Each well was loaded with 10. Mu.L of PCR product, 100bp ladder indicated, and run for 60min at 120v to initially screen polymorphic primers (using the size of fragments amplified from different samples of the same primer as an indicator) (FIG. 1). And (3) screening out 14 pairs of SSR molecular markers with strong band specificity and obvious polymorphism in each sample according to an electrophoresis detection result, and carrying out fluorescent primer design for molecular identification of cymbidium germplasm resources. The 14 pairs of SSR primer sequences are shown in Table 3.
TABLE 3 SSR primer sequences for 14 pairs
Figure BDA0003517972440000121
Figure BDA0003517972440000131
4. SSR-PCR and capillary electrophoresis detection
4.1 SSR-PCR reaction
The total volume of the PCR reaction system was 10. Mu.L, including 1.2. Mu.L of DNA template (50 ng/. Mu.L), 1.0. Mu.L of 10 XBuffer I Buffer, 0.1. Mu. L TAKARA HS Taq enzyme (5U/. Mu.L), 0.6. Mu.L of forward primer (5. Mu.M), 0.6. Mu.L of reverse primer (5. Mu.M), 0.8. Mu.L of 2.5mM dNTP, 0.5. Mu.L of TP-M13 (5. Mu.M), and deionized water to 10. Mu.L.
PCR reaction procedure: 95 ℃ for 5min;95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 30s,30 cycles; 95 ℃ for 30s, 53 ℃ for 30s,72 ℃ for 30s,10 cycles; 30min at 60 ℃; preserving at 4 ℃.
4.2 capillary electrophoresis detection
1.0 mu L of amplification product, 9 mu L of mixture of ROX-500 molecular weight internal standard and formamide (volume ratio of 0.5:8.5) are added into each well of a 96-well plate, after denaturation at 95 ℃ for 3min, detection is carried out by an ABI 3730XL detector, sample injection is carried out for 10s at 1kV voltage, and electrophoresis is carried out for 15kV and 30min.
5. Primer amplification result analysis
And (3) performing STR typing based on capillary electrophoresis fluorescence detection on 70 parts of cymbidium samples which are qualified by detection by using 14 pairs of screened polymorphic SSR primers. The result shows that the primer pair SSR_445_400 can detect the strip in 99.47% of samples, and the detection rate is highest; the average detection rate was 80.94% (Table 4), demonstrating that these 14 pairs of polymorphic SSR primers can be effectively used for molecular identification of cymbidium varieties. The SSR molecular marker primer provided by the invention has good amplification effect and high detection rate, and can amplify a stable DNA band by taking a primer pair SSR_445_400 amplification result peak diagram as an example (figure 2).
Table 4 detection of 14 pairs of polymorphic SSR primers in 70 samples of cymbidium sinense
Figure BDA0003517972440000132
Figure BDA0003517972440000141
6. Data analysis
The original Data file collected by the Data collection software was imported into the GeneMapper 6.0 software for analysis, and the position of each peak was compared with the molecular weight internal standard in its lane to calculate the exact size of the target DNA fragment. Capillary electrophoresis measurements were performed independently for 3 replicates at each fluorescent-labeled seat, and the average of 3 replicates was taken and rounded off to serve as data for the experimental material at that seat.
The following genetic diversity parameters are analyzed by using POPGENE1.31 and PowerMarker software, and genetic diversity indexes, clustering and Polymorphic Information Content (PIC) calculation and analysis are performed by using PAST3 and other software:
observing allele factors (Na), gene observing heterozygosity (Ho), gene expected heterozygosity (He), polymorphism Information Content (PIC), effective allele factors (Ne), genetic deviation indexes (D), shannon-Weaver diversity index (I), clustering patterns, DNA fingerprint strip information, molecular identity card information and the like. Genetic diversity and cluster analysis was performed on STR typing data using bioinformatics software (POPGENE, MEGA, joinmap, structure, R, etc.). And (3) carrying out genetic diversity index, clustering and Polymorphic Information Content (PIC) calculation analysis by using NTSYS and other software. The results are shown in Table 5.
TABLE 5 genetic diversity analysis results
Figure BDA0003517972440000151
7. Construction of molecular identity card
According to diploid standard construction, fingerprint data are subjected to data coding (amplified fragments of all loci are arranged according to molecular weight, amplified fragments (alleles) are marked with Arabic numerals 1-9 from small to large, more than 9 alleles are marked with capital English letters A-Z), if the locus is not amplified in a certain variety, the locus is marked as 0, and each locus occupies two positions. Wherein, the molecular identification card SSR molecular marking sequence is as follows: ssr_23012_c0_g1, ssr_47035_c6_g1, ssr_45085_c2_g1, ssr_53453_c0_g1, ssr_55630_c0_g1, ssr_39257_c3_g1, ssr_51432_c6_g1, ssr_46859_c0_g2, ssr_50940_c5_g2, ssr_445_400, ssr_44145_c1_g1, ssr_44775_c0_g1, ssr_48838_c2_g1, ssr_46319_c4_g3.
The fingerprint of 70 parts of cymbidium sinense variety is shown in Table 6.
TABLE 6 fingerprint of 70 parts of cymbidium sinense variety
Figure BDA0003517972440000161
Figure BDA0003517972440000171
Figure BDA0003517972440000181
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (9)

1. The ink blue SSR primer group is characterized by comprising the following 14 pairs of SSR primer sequences:
SSR_23012_c0_g1 F：5'-TTTCCATGAACCATGATGTGA-3'
R：5'-GAATGGATAGCACAACTGCG-3'；
SSR_47035_c6_g1 F：5'-TTCTGTTGAACACGTCGAGC-3'
R：5'-ATAAGTGTAGGGGCAGCGTG-3'；
SSR_45085_c2_g1 F：5'-TTGATTTCTTTAACAAAATGATGC-3'
R：5'-TGTTGCAATCTAAGATCATTGAAGA-3'；
SSR_53453_c0_g1 F：5'-TTAAGCAGATAAGATCAACACGG-3'
R：5'-CACTCCAAGGGGTGGTAGAAG-3'；
SSR_55630_c0_g1 F：5'-ATCCTACCTGAAAGATGCCAAGA-3'
R：5'-ATCTCTTCTGAAGGTTTTCCAGC-3'；
SSR_39257_c3_g1 F：5'-TGCAGGGCACATCTTGTAAA-3'
R：5'-CCCAGGGCTGAAACTAATCA-3'；
SSR_51432_c6_g1 F：5'-TCTCAACTTGCGTACCCTGA-3'
R：5'-TTTGCCTTCATTTTCTCGCT-3'；
SSR_46859_c0_g2 F：5'-AATTGCGAAAGAGATGGGTG-3'
R：5'-GCCAAAGTCTTAAAAGGCCA-3'；
SSR_50940_c5_g2 F：5'-AGAGGAACAGCAGAACACGG-3'
R：5'-CAGCATCTTGCGGAGTTGTA-3'；
SSR_445_400F：5'-GCAAGAGCATCCCTAACGAG-3'
R：5'-CTCTGCTCGACTCCCCTATG-3'；
SSR_44145_c1_g1 F：5'-AACAATTTTCAAACGAGCCG-3'
R：5'-ATTCGCACTTCCATCTCCAT-3'；
SSR_44775_c0_g1 F：5'-GCCCTTTGTGCAATTTAGGA-3'
R：5'-ACATCCAGATCCAAACCTCG-3'；
SSR_48838_c2_g1 F：5'-CTTTCTTCCAATGGCGTGAT-3'
R：5'-CAACGCCAAAACACACAAAC-3'；
SSR_46319_c4_g3 F：5'-TCTCCTGAGCTGCTCTCCTC-3'
R：5'-AACGTAGAGGCGTAGACCGA-3'。
2. the use of the cymbidium SSR primer group of claim 1 in constructing a cymbidium variety fingerprint.
3. Use of the cymbidium SSR primer set of claim 1 for identifying cymbidium varieties; the cymbidium variety comprises the following 70 varieties: the Chinese medicinal composition comprises the following raw materials of largeseed, baoshan claw, verdigris, color butterfly, color dragon, damo claw, damo Zhongtong, daqian mountain, dasheng, dashimen onyx, dashimen Ruiyu, datun kylin, dagao Ji, dagao guan, dagao Ji, dazhu Ji, dayin side, dai fu, fenghuan Ji, fenghuang, fulong, fulu shou, fugui red plum, fugui long, fugui line, fucui, fuzui green, guogong, hejinjian Bao, red moon, hubi heart, hua Qiu Bang the Chinese herbal medicines comprise Cistanchis herba, royal jelly, golden dragon, ji Fulong plum, golden bird crystal, golden bird claw, golden Ruyi, jin Sique, jin Xiangrui, golden tripod, golden eagle imogolian, blue butterfly, ling nan Damei, six fairy, longquan butterfly, lv Yun, nanhai plain, ning Fu, ganlai mountain, qinglong sword, qing Ye Dajian, qong, qiu Bang, ning, ruibao, rui Hua Yi, three-sided tooth, sanxiong butterfly, eighteen jiao claw, double dragon crown, double American, crystal dragon, sihong Kong water, peach Ji claw, tianlong art and Tianshan mountain.
4. The method for constructing the cymbidium sinense variety fingerprint is characterized by comprising the following steps of:
(1) Extracting DNA of a cymbidium sample;
(2) Using the extracted DNA as a template and the SSR primer set as an amplification primer as set forth in claim 1, establishing a PCR reaction system and performing PCR amplification;
(3) And (3) performing capillary electrophoresis on the amplified products, forming an amplified fragment length datamation code for the amplified result of the sample DNA of each variety of the cymbidium sinense according to the capillary electrophoresis result, and constructing and obtaining the cymbidium sinense variety fingerprint.
5. The method for constructing a finger print of cymbidium sinense variety according to claim 4, wherein the PCR reaction system in step (2) comprises: 10 XPCR Buffer 3. Mu.L, 2.5mM dNTP 2. Mu. L, mgCl 2 2μL、Primer A2μL、Primer B 2μL、Template 1μL、ddH 2 O18 mu L, taq enzyme 0.2 mu L;
the PCR amplification 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; 30min at 60 ℃; preserving at 4 ℃.
6. The method for constructing a finger print of cymbidium sinense variety according to claim 4, wherein in step (3), capillary electrophoresis is performed on the amplified products, and the amplified results of the sample DNA of each cymbidium variety are subjected to capillary electrophoresis results to form amplified fragment length databased codes, specifically: the DNA fingerprint of the cymbidium variety is obtained by sequentially recording the fragment lengths of each cymbidium variety amplified by each pair of SSR primers according to the molecular marker sequences of ssr_23012_c0_g1, ssr_47035_c6_g1, ssr_45085_c2_g1, ssr_53453_c0_g1, ssr_55630_c0_g1, ssr_39257_c3_g1, ssr_51432_c6_g1, ssr_46859_c0_g2, ssr_50940_c5_g2, ssr_445_400, ssr_44145_c1_g1, ssr_44775_c0_g1, ssr_48838_c2_g1 and ssr_46319_c4_g3 and performing data coding according to the amplified fragment lengths of each cymbidium variety through capillary electrophoresis results.
7. The method for constructing a finger-print of cymbidium variety according to claim 6, wherein said encoding is performed according to the length of each amplified fragment, specifically: alleles of amplified fragments at each site are arranged according to the molecular weight, the alleles are marked with Arabic numerals 1-9 from small to large, more than 9 alleles are marked with capital English letters A-Z, if the site is not amplified in a certain variety, the site is marked as 0, and each site occupies two places.
8. Use of a cymbidium variety fingerprint constructed by the cymbidium variety fingerprint construction method of any one of claims 4-7 for identifying cymbidium varieties.
9. A kit for identifying cymbidium varieties, which is characterized by comprising the cymbidium SSR primer group of claim 1.
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