CN114736977A - 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 PDFInfo
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Abstract
The invention discloses a cymbidium SSR primer group and a method for constructing a cymbidium variety fingerprint by using the primer group. The cymbidium SSR primer group comprises 14 pairs of SSR primer sequences. The 14 pairs of SSR primer sequences are utilized to carry out multiple PCR amplification on cymbidium genome DNA, the PCR products are detected by capillary fluorescence electrophoresis, SSR marker data are analyzed, 70 cymbidium SSR finger prints are constructed, and cymbidium varieties can be effectively identified. The method for constructing the cymbidium varieties fingerprint spectrum has the advantages of high flux, high precision, low cost and good application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a cymbidium SSR primer group and a method for constructing a cymbidium variety fingerprint by using the primer group.
Background
The distribution of orchids is wide in the whole world, and 801 plants and 27500 plants are available. In recent years, molecular markers, mainly including RAPD, ISSR, SSR, SRAP, ScoT, and the like, are widely used for the classification and identification of orchid species, species and varieties, the analysis of genetic diversity and genetic relationship, and the research of germplasm resources and populations. For example, the assay of 34 orchidaceae plants by Syrian researchers using CAPS molecular markers showed that the test material was extremely polymorphic, and that 5 species other than the 5 species such as spider orchid (Ophrys fucifora) could be identified. Brazilian researchers have studied two populations of dendrobium americanum (epiendenrum furgens) distributed in the atlantic rainforest on the brazilian coast using 9 new microsatellite markers. Aiming at native orchid, predecessors identify three Paphiopedilum (Paphiopedilum) species and hybrid species thereof by using SCAR molecular markers, and develop effective specific markers; utilizing SRAP molecular markers to perform molecular identification on 31 Dendrobium (Dendrobium) plants of 5 provinces in China, and obtaining 18 species specific markers; 129 Cymbidium (Cymbidium goeringii) varieties are researched by using EST-SSR molecular markers, and the varieties with the same horticultural type are found to have a common allele; the ISSR molecular marker technology is utilized to respectively carry out the related researches on genetic diversity, genetic relationship and population structure on 16 Cymbidium plants such as wild rhizoma gastrodiae (Gastrodia) habitata and cultivated rhizoma gastrodiae (Gastrodia), Cymbidium and spring sword, and 5 populations of wild rhizoma bletillae (Bletilla), 6 populations of Calanthe tsoongiana and Cymbidium japonicam. However, the current molecular marker technology is mainly applied to the genetic relationship and genetic relationship differentiation of the species or varieties of orchids, a relatively comprehensive molecular classification system is not formed, and the operability of direct application to breeding selection is low.
Cymbidium sinense (Cymbidium sinense) also known as Zealand Cymbidium, is a terrestrial species of Cymbidium of the orchidaceae family. The plant type is tall and big, the leaf posture is beautiful, the general inflorescence has 10-20 flowers, and the flower has orchid fragrance, is a traditional famous flower in the evening in China, and is popular in the markets at home and abroad. Although the cymbidium has a long cultivation history and cultural inheritance, the variety identification mostly depends on the traditional morphological indexes, and the variety is difficult to accurately identify through leaves and plant types when the cymbidium does not bloom. In recent years, with the popularization and market development of the cymbidium and the rapid development of the internet technology, various large e-commerce platforms, social media and forums provide more diversified transaction channels, the number of the cymbidium varieties appearing in the orchid market exceeds 500, but no unified and standard variety identification standard exists, the products are secondary and good, and the phenomenon of variety confusion generally exists. In order to promote the breeding, identification and popularization of varieties meeting the market demands, establishing an orchid variety identification standard which is suitable for the varieties is absolutely required. Therefore, the development of the cymbidium varieties identification work has important significance on the aspects of germplasm resource evaluation, germplasm innovation utilization, commodity flower quality supervision, market regulation and the like. At present, no report related to high-throughput identification of the cymbidium varieties based on the capillary electrophoresis technology and SSR molecular markers is seen in China.
Disclosure of Invention
The invention aims to provide a cymbidium SSR primer group and a method for constructing a cymbidium varietal fingerprint by combining a capillary electrophoresis technology and an SSR-PCR technology by using the primer group aiming at the technical defects of the prior art so as to quickly and efficiently identify the cymbidium varietal.
The first purpose of the invention is to provide a cymbidium SSR primer group, which comprises the following 14 pairs of SSR primer sequences:
the second purpose of the invention is to provide the application of the cymbidium SSR primer group in construction of the cymbidium varietal fingerprint.
The third purpose of the invention is to provide the application of the cymbidium SSR primer group in identifying cymbidium varieties.
The fourth purpose of the invention is to provide a construction method of the cymbidium sinense variety fingerprint spectrum, which comprises the following steps:
(1) extracting DNA of a cymbidium sample;
(2) establishing a PCR reaction system and carrying out PCR amplification by taking the extracted DNA as a template and the SSR primer group of claim 1 as an amplification primer;
(3) and performing capillary electrophoresis on the amplification product, forming an amplification fragment length datamation code according to the amplification result of the capillary electrophoresis on the sample DNA of each species of the cymbidium sinense, and constructing to obtain the cymbidium sinense species fingerprint.
Further, the PCR reaction system in the step (2) comprises: 10 XPCR Buffer 3. mu.L, 2.5mM dNTP 2. mu. L, MgCl2 2μL、Primer A 2μL、Primer B 2μL、Template 1μL、ddH2O18. mu. L, Taq enzyme 0.2. mu.L.
The reaction procedure of the PCR amplification is as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 30 s; 30s at 95 ℃, 30s at 55 ℃, 30s at 72 ℃ and 10 cycles; 30min at 60 ℃; storing at 4 ℃.
Further, in the step (3), capillary electrophoresis is performed on the amplification product, and an amplification fragment length datamation code is formed according to the amplification result of the capillary electrophoresis on the sample DNA of each variety of cymbidium sinense, which specifically comprises the following steps: according to the molecular marker sequence arrangement 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, the amplified segment length of each pair of cotton varieties is recorded in turn through capillary electrophoresis results, and the amplified segment of each pair of cotton varieties is subjected to datamation coding according to the amplified segment length, namely the cotton fingerprint DNA map.
Further, the performing of the data encoding according to the length of each amplified fragment specifically includes: the alleles of the amplified fragments at each locus are arranged according to the molecular weight, the alleles are marked by Arabic numerals 1-9 from small to large, more than 9 alleles are marked by 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 bits.
The fifth purpose of the invention is to provide a cymbidium sinense variety fingerprint spectrum which is constructed by the cymbidium sinense variety fingerprint spectrum construction method.
The sixth purpose of the invention is to provide the application of the cymbidium varieties fingerprint spectrum in identification of the cymbidium varieties.
The seventh purpose of the invention is to provide a kit for identifying the cymbidium varieties, which contains the cymbidium SSR primer group.
The invention has the beneficial effects that:
(1) the invention utilizes the capillary electrophoresis technology to realize the beneficial combination of the SSR molecular marker and the efficient and automatic capillary electrophoresis technology, the detection result is automatically stored by analysis software, the detection speed is high, the separation efficiency is high, the sensitivity is higher, the accuracy reaches within 1bp, the detection and the analysis of mass samples are facilitated, and a solid foundation is laid for the popularization and the application of the SSR molecular marker technology in the identification of the cymbidium varieties.
(2) The identification method has low cost, the market of reagent consumables is mature, the loss of manpower and instruments is not counted, 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 amplification results of different cymbidium varieties by using a primer pair SSR _445_400, wherein the top, middle and bottom are amplification peak diagrams of autumn leaderboard, green cloud and Japanese cymbidium respectively.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
1. Collection of cymbidium varieties
The samples are from Guangdong, Guangxi and Taiwan in the major production area of Maolan, and comprise 70 varieties with different flower types, flower colors and leaf colors, as shown in the following table 1.
Table 170 parts of Mozuku variety
2. Extraction of cymbidium genome DNA
70 collected cymbidium germplasm resources are extracted by using a plant genome DNA kit (Tiangen kit). Firstly, collecting young leaves in batches, storing 2-3 parts (about 2g each part) of each sample in a 2mL centrifuge tube, quickly freezing by liquid nitrogen, and then freezing and storing in a refrigerator at-80 ℃ for later use. The frozen plant tissue was ground thoroughly using a pre-cooled mortar and pestle to extract its genomic DNA. The DNA was electrophoresed on a 1% agarose gel, stained with GV (good view), and the bands were detected by a gel imaging system to judge the quality of DNA extraction and the DNA concentration thereof was measured by an ultraviolet spectrophotometer.
3. Whole genome SSR marker development and screening
3.1 SSR primer design
A total of 50 SSR molecular markers were developed based on the reference sequence of the whole genome of the cymbidium, using SSR Hunter et al software, and 50 pairs of upstream and downstream primers for amplifying each SSR were designed using Primer 5 software (Table 2). And carrying out PCR amplification on the developed 50 SSR molecular markers by taking various orchid genome DNAs as templates. Mix for PCR amplification (from Es Taq DNA Polymerase, Mg2+dNTPs and a premix system of PCR stabilizers and enhancers with blue dye added) from Tiangen corporation; the 50 pairs of SSR primers were purchased from Shanghai division, Biotechnology, Canada.
TABLE 250 primer pairs List
3.2 the PCR reaction system included 3. mu.L of 10 XPCR Buffer, 2. mu.L of 2.5mM dNTP, MgCl2 2μL，Primer A 2μL，Primer B 2μL，Template 1μL，H2O18. mu.L, Taq enzyme 0.2. mu.L.
The PCR reaction program is: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 30 s; 30s at 95 ℃, 30s at 55 ℃, 30s at 72 ℃ and 10 cycles; 30min at 60 ℃; storing at 4 ℃.
Detection by 3.32.0% agarose gel electrophoresis
Each well was loaded with 10. mu.L of PCR product, indicated by 100bp ladder, and polymorphic primers were preliminarily screened by 120v running electrophoresis for 60min (using the sizes of fragments amplified from different samples using the same primer as an index) (FIG. 1). And (3) screening 14 pairs of SSR molecular markers which are strong in band specificity and obvious in polymorphism in each sample according to the electrophoresis detection result, and designing a fluorescent primer for molecular identification of the cymbidium germplasm resources. The sequences of 14 pairs of SSR primers are shown in Table 3.
TABLE 314 pairs of SSR primer sequences
4. SSR-PCR and capillary electrophoresis detection
4.1 SSR-PCR reaction
The total volume of the PCR reaction system is 10. mu.L, and the PCR reaction system comprises 1.2. mu.L of DNA template (50 ng/. mu.L), 1.0. mu.L of 10 XBuffer I Buffer solution, 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 make up to 10. mu.L.
PCR reaction procedure: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 30 s; 30s at 95 ℃, 30s at 53 ℃, 30s at 72 ℃ and 10 cycles; 30min at 60 ℃; storing at 4 ℃.
4.2 capillary electrophoresis detection
Adding 1.0 mu L of amplification product, 9 mu L of ROX-500 molecular weight internal standard and formamide mixed liquor (volume ratio is 0.5:8.5) into each hole of a 96-hole plate, performing denaturation at 95 ℃ for 3min, detecting by using an ABI 3730XL detector, injecting sample at 1kV voltage for 10s, and performing electrophoresis at 15kV for 30 min.
5. Analysis of primer amplification results
And carrying out STR typing based on capillary electrophoresis fluorescence detection on 70 detected qualified cymbidium samples by using 14 screened pairs of 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), indicating that the 14 pairs of polymorphic SSR primers can be effectively used for molecular identification of cymbidium varieties. The peak image of the result of the primer pair SSR _445_400 amplification is used as an example for displaying (figure 2), the SSR molecular marker primer has good amplification effect and high detectable rate, and a stable DNA band can be amplified.
Table 4 shows the detection of 14 pairs of polymorphic SSR primers in 70 cymbidium samples
6. Data analysis
The original Data file collected by the Data cloning software is imported into GeneMapper 6.0 software for analysis, the position of each peak is compared with the molecular weight internal standard in the lane, and the accurate size of the target DNA fragment is calculated. Independently, 3 replicates of the capillary electrophoresis test were performed on each fluorescently labeled locus, and the 3 replicates were averaged and rounded up as the data for the test material at that locus.
POPGENE1.31 and PowerMarker software are adopted to analyze the following genetic diversity parameters, and software such as PAST3 and the like are adopted to calculate and analyze genetic diversity indexes, clusters and Polymorphic Information Content (PIC):
observation of allele factors (Na), gene observation heterozygosity (Ho), gene expectation heterozygosity (He), Polymorphism Information Content (PIC), effective allele factors (Ne), genetic deviation index (D), Shannon-Weaver diversity index (I), clustering spectrum, DNA fingerprint band information, molecular identity card information and the like. The STR typing data was subjected to genetic diversity and cluster analysis using bioinformatics software (POPGENE, MEGA, Joinmap, Structure, R, etc.). And (4) carrying out genetic diversity index, clustering and Polymorphic Information Content (PIC) calculation analysis by using software such as NTSYS and the like. The results are shown in Table 5.
TABLE 5 results of genetic diversity analysis
7. Molecular identity card construction
The method is constructed according to the diploid standard, and data encoding is carried out on fingerprint data according to SSR detection results (amplification fragments of each locus are arranged according to the molecular weight, the amplification fragments (alleles) are marked by Arabic numerals 1-9 from small to large, more than 9 alleles are marked by 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 bits. Wherein, the SSR molecular marker sequence of the molecular identity card 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_ g 3.
The finger prints of 70 cymbidium varieties are shown in Table 6.
Finger print of cymbidium varieties in 670 parts of table
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the 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 these modifications and adaptations should be considered within the scope of the invention.
Claims (10)
1. The cymbidium SSR primer group is characterized by comprising the following 14 pairs of SSR primer sequences:
2. the use of the cymbidium SSR primer group according to claim 1 for constructing a cymbidium variety fingerprint.
3. Use of the cymbidium SSR primer set of claim 1 for identifying cymbidium varieties.
4. A construction method of a cymbidium varieties fingerprint spectrum is characterized by comprising the following steps:
(1) extracting DNA of a cymbidium sample;
(2) establishing a PCR reaction system and carrying out PCR amplification by taking the extracted DNA as a template and the SSR primer group of claim 1 as an amplification primer;
(3) and performing capillary electrophoresis on the amplification product, forming an amplification fragment length datamation code according to the amplification result of the capillary electrophoresis on the sample DNA of each species of the cymbidium sinense, and constructing to obtain the cymbidium sinense species fingerprint.
5. The method for constructing the cymbidium varieties fingerprint as claimed in claim 4, wherein the PCR reaction system in the step (2) comprises: 10 XPCR Buffer 3. mu.L, 2.5mM dNTP 2. mu. L, MgCl2 2μL、Primer A 2μL、Primer B 2μL、Template 1μL、ddH2O18. mu. L, Taq enzyme 0.2. mu.L;
the reaction procedure of the PCR amplification is as follows: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 60 ℃ for 30s, and 72 ℃ for 30 s; 30s at 95 ℃, 30s at 55 ℃, 30s at 72 ℃ and 10 cycles; 30min at 60 ℃; storing at 4 ℃.
6. The method for constructing the fingerprint of the cymbidium varieties according to claim 4, wherein the amplification products are subjected to capillary electrophoresis in the step (3), and the amplification results of the sample DNA of each cymbidium variety form amplification fragment length datamation codes according to the capillary electrophoresis results, specifically: according to the molecular marker sequence arrangement 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, the amplified segment length of each pair of cotton varieties is recorded in turn through capillary electrophoresis results, and the amplified segment of each pair of cotton varieties is subjected to datamation coding according to the amplified segment length, namely the cotton fingerprint DNA map.
7. The method for constructing the fingerprint of the cymbidium varieties according to claim 6, wherein the data coding is performed according to the length of each amplified fragment, and specifically comprises the following steps: the alleles of the amplified fragments at each locus are arranged according to the molecular weight, the alleles are marked by Arabic numerals 1-9 from small to large, more than 9 alleles are marked by 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 bits.
8. A cymbidium varieties fingerprint, which is characterized in that the cymbidium varieties fingerprint is constructed by the cymbidium varieties fingerprint construction method of any one of claims 4 to 7.
9. Use of the cymbidium cultivar fingerprint of claim 8 for identifying cymbidium cultivar.
10. A kit for identifying a cymbidium strain, comprising the cymbidium SSR primer group according to claim 1.
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| CN116004880A (en) * | 2022-07-25 | 2023-04-25 | 广东省农业科学院环境园艺研究所 | Cymbidium SSR primer group and method for constructing cymbidium variety fingerprint by using primer group |
| CN117904353A (en) * | 2024-01-25 | 2024-04-19 | 广东省农业科学院环境园艺研究所 | KASP marker for identifying variety of cymbidium sinense piece odd flowers primer, kit and application |
| CN118547105A (en) * | 2024-07-25 | 2024-08-27 | 中国科学院昆明植物研究所 | Cypripedium macranthum DNA bar code and application thereof in species identification |
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