CN116004880A - Cymbidium SSR primer group and method for constructing cymbidium variety fingerprint by using primer group - Google Patents
Cymbidium SSR primer group and method for constructing cymbidium variety fingerprint by using primer group 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 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 90 kinds of cymbidium SSR finger print, and can effectively identify cymbidium varieties. The method for constructing the cymbidium variety fingerprint has the advantages of high flux, high precision and low cost, and has 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
Cymbidium (Cymbidium goeringii) is a species of the genus Cymbidium (Cymbidium) of the family Orchidaceae. The plant type is small and exquisite, the leaf gesture is graceful, the inflorescence has single flower, 2 flowers are fewer, the flower type is larger, the flower type is fragrant, and the flower type is a traditional rare flower in China and is popular in domestic and foreign markets. Although cymbidium has long cultivation history and cultural inheritance, variety identification mostly depends on traditional morphological indexes, and it is difficult to accurately identify varieties through leaves and plant types when the cymbidium 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 good products and variety confusion 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.
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. However, the identification and evaluation of the characteristic resources in China are limited by regional limitation and variety collection difficulty, and no related report for high-throughput identification of cymbidium varieties based on capillary electrophoresis technology and SSR molecular markers is currently found in China. Therefore, the invention adopts SSR molecular markers to identify the cymbidium varieties, and has important significance in the aspects of germplasm resource evaluation, germplasm innovation utilization, commodity flower quality supervision, market standardization and the like.
Disclosure of Invention
Aiming at the technical defects of the prior art, the invention provides a 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 a cymbidium SSR primer group, which comprises the following 14 pairs of SSR primer sequences:
SSR_39969_c0_seq1 F:5’-TCGAACAAGGTCATTTGCCT-3’
R:5’-ACAAGGAGTGAAGGACCTTTT-3’；
SSR_71399_c0_seq1 F:5’-GCATGACTGCCGTTGTGTTT-3’
R:5’-GCAAGTGCATCAAAGCAGATG-3’；
SSR_256535_c0_seq1 F:5’-GGCGGCAGAAATGAACAGAA-3’
R:5’-CCACCCAATATTTTCTCCACCC-3’；
SSR_95792_c0_seq1 F:5’-TGAGGGTGACGCTTTGTTCA-3’
R:5’-TGCAACCAGAGCCAACTGAA-3’；
SSR_26057_c0_seq1 F:5’-AGAAGGTTGTGCGTCGACAT-3’
R:5’-GTTGCTGCAGTTGCGAAGAA-3’；
SSR_56999_c0_seq1 F:5’-TCTCACGCTGCATCCCATTT-3’
R:5’-GTGTTGCTTCCCTTGTTGCA-3’；
SSR_67726_c0_seq1 F:5’-GTGCAGTCGACGGGATCC-3’
R:5’-GCCGGAACAAGAAAACCCAG-3’；
SSR_281917_c0_seq1 F:5’-TGGCTTTGAGAGAGCATCGG-3’
R:5’-CGAAGCCATCATTGACCTGG-3’；
SSR_288485_c0_seq1 F:5’-GCGCAGATCATTTTTGCACG-3’
R:5’-TGACACACCAGTCTCCTTCC-3’；
SSR_81676_c0_seq1 F:5’-ATAGCCGCTGCAGCTTAACT-3’
R:5’-TGCTGCTTTTATTACACAAATATGCA-3’；
SSR_51709_c0_seq1 F:5’-ACCAATCAACCAAGCCCCAA-3’
R:5’-TGGTGGTTTTGCCTATGGGA-3’；
SSR_83667_c0_seq2 F:5’-GCACAAGCCCTCTCTACTCA-3’
R:5’-ACGAACGGACGAAATGGACA-3’；
SSR_937370_c0_seq1 F:5’-CGACACTGTCTCATTCCGAA-3’
R:5’-ACGTTCTGCTGTTCATCCTT-3’；
SSR_838285_c0_seq1 F:5’-CTTCAGCTTTGAACTCTGCAGA-3’
R:5’-TCCTCGACGATCAGGGCTAT-3’。
the second purpose of the invention is to provide the application of the cymbidium SSR primer group in constructing 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 cymbidium variety fingerprint, 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) Performing capillary electrophoresis on the amplified products, forming an amplified fragment length datamation code for the amplified results of the sample DNA of each variety of the cymbidium according to the capillary electrophoresis results, and constructing and obtaining the cymbidium 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 goeringii is subjected to data coding of the amplified fragment length according to the capillary electrophoresis result, specifically: according to the molecular markers of SSR_39969_c0_seq1, SSR_71399_c0_seq1, SSR_256535_c0_seq1, SSR_95792_c0_seq1, SSR_26057_c0_seq1, SSR_56999_c0_seq1, SSR_67726_c0_seq1, SSR_281917_c0_seq1, SSR_288485_c0_seq1, SSR_81676_c0_seq1, SSR_51709_c0_seq1, SSR_83667_c0_seq2, SSR_937370_c0_seq1 and SSR_838285_c0_seq1, the segment length of each spring orchid variety amplified by each pair of primers is recorded in sequence through capillary electrophoresis results, and the data coding is carried out according to the amplified segment length, namely the DNA fingerprint of the spring orchid variety.
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 variety fingerprint constructed by the method for constructing the cymbidium variety fingerprint.
The sixth object of the invention is to provide the application of the cymbidium varieties fingerprint in identifying cymbidium varieties.
The seventh object of the invention is to provide a kit for identifying cymbidium varieties, which contains the cymbidium 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 identification of cymbidium varieties.
(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, with sample sequences of Jiang Naxue, xiu lotus, queen lion, luyun, chinese yi plum from left to right.
Fig. 2 is a peak diagram of amplification results of different spring orchid varieties by using a primer pair SSR_81676_c0_seq1, wherein the peak diagram is formed by sequentially amplifying four different spring orchid varieties of Jiangnan snow, nelumbo nucifera, lvyun and Zhonghua Yipin plum from top to bottom.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1
1. Spring orchid variety harvesting
The samples were derived from Guangdong province, guangxi province and Taiwan province in China in the main production area of cymbidium, and included 90 varieties of different flower types, colors and leaf colors, as shown in Table 1 below.
Table 1 90 parts of spring orchid variety
2. Cymbidium genome DNA extraction
The 90 parts of the collected cymbidium germplasm resources were extracted with a plant genomic DNA kit (Tiangen). 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 (Gold 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
3.1SSR primer design
Based on the cymbidium genome-wide reference sequence, 50 SSR molecular markers were developed using software such as SSR Hunter, and 50 pairs of primers were designed to amplify each SSR using 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
3.2PCR reaction System comprising 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 designing fluorescent primers 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
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 90 qualified cymbidium samples by using 14 pairs of polymorphism SSR primers. The result shows that the primer pair SSR_81676_c0_seq1 can detect the band in 97.69% of samples, and the detection rate is highest; the average detection rate was 87.32% (Table 4), indicating 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_81676_c0_seq1 amplification result peak diagram as an example (figure 2).
Table 4 detection of 14 pairs of polymorphic SSR primers in 90 samples of cymbidium
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
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_39969_c0_seq1, ssr_71399_c0_seq1, ssr_256535_c0_seq1, ssr_95792_c0_seq1, ssr_26057_c0_seq1, ssr_56999_c0_seq1, ssr_67726_c0_seq1, ssr_281917_c0_seq1, ssr_288485_c0_seq1, ssr_81676_c0_seq1, ssr_51709_c0_seq1, ssr_83667_c0_seq2, ssr_937370_c0_seq1, ssr_838285_c0_seq1.
The fingerprint of 90 parts of cymbidium varieties is shown in Table 6.
TABLE 6 fingerprint of 90 parts of cymbidium varieties
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 (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 of claim 1 in constructing a cymbidium variety fingerprint.
3. The use of the cymbidium SSR primer set of claim 1 for identifying cymbidium varieties.
4. The method for constructing the spring orchid 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) Performing capillary electrophoresis on the amplified products, forming an amplified fragment length datamation code for the amplified results of the sample DNA of each variety of the cymbidium according to the capillary electrophoresis results, and constructing and obtaining the cymbidium variety fingerprint.
5. The cymbidium species fingerprint of claim 4The construction method is characterized in that the PCR reaction system in the step (2) comprises the following steps: 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 ℃.
6. The method for constructing a fingerprint of a variety of cymbidium of claim 4, wherein in step (3), the amplified products are subjected to capillary electrophoresis, and the amplified results of the sample DNA of each variety of cymbidium are subjected to capillary electrophoresis to form an amplified fragment length data encoding, specifically: according to the molecular markers of SSR_39969_c0_seq1, SSR_71399_c0_seq1, SSR_256535_c0_seq1, SSR_95792_c0_seq1, SSR_26057_c0_seq1, SSR_56999_c0_seq1, SSR_67726_c0_seq1, SSR_281917_c0_seq1, SSR_288485_c0_seq1, SSR_81676_c0_seq1, SSR_51709_c0_seq1, SSR_83667_c0_seq2, SSR_937370_c0_seq1 and SSR_838285_c0_seq1, the segment length of each spring orchid variety amplified by each pair of primers is recorded in sequence through capillary electrophoresis results, and the data coding is carried out according to the amplified segment length, namely the DNA fingerprint of the spring orchid variety.
7. The method for constructing a cymbidium variety fingerprint according to claim 6, wherein the step of performing data coding according to the length of each amplified fragment comprises the following steps: 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. The cymbidium variety fingerprint is characterized by being constructed by the cymbidium variety fingerprint construction method according to any one of claims 4-7.
9. The use of the cymbidium varieties fingerprint of claim 8 in identifying cymbidium varieties.
10. A kit for identifying a cymbidium variety, comprising the cymbidium SSR primer set of claim 1.
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