CN113528693B - Primer and method for identifying torreya grandis variety torreya grandis, syzygium aromaticum and torreya grandis - Google Patents
Primer and method for identifying torreya grandis variety torreya grandis, syzygium aromaticum and torreya grandis Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention relates to a molecular characteristic fluorescent SSR (simple sequence repeat) marker primer and a method for rapidly identifying new species of torreya grandis, syzygium aromaticum and torreya grandis. The molecular characteristic fluorescent SSR marker primer has the nucleotide sequence as follows: the upstream primer tg_u15f:5'-FAM-GCACAAACATCCATGCAAAC-3'; downstream primer tg_u15r:5'-AACAAGGGTCCAGGGAGAGT-3'. The beneficial effects of the invention are mainly as follows: the molecular characteristic fluorescent SSR marker primer can be used for rapidly and early identifying the Chinese torreya of new species, namely the Chinese torreya, the Daxiang and the Pan Chinese torreya simultaneously by utilizing needle DNA, and the method is simple, rapid and accurate, and is a molecular means which cannot be replaced by the traditional method for identifying the Chinese torreya variety according to the morphological characteristics of fruits.
Description
Field of the art
The invention relates to a molecular characteristic fluorescent SSR (simple sequence repeat) marker primer and a method for rapidly identifying torreya grandis, grand clove and rock torreya.
(II) background art
The Torreya grandis belonging to Torreya (Torreya) of Taxaceae, which is an excellent variety cultivated by grafting and propagating after artificial breeding of excellent variation in Torreya grandis species (Torreya grandis), and is the only variety cultivated in the Torreya grandis at present on an artificial scale. Chinese torreya is a special precious economic tree species in China, and has multiple purposes of eating, medicinal use, material use, greening and the like.
The Chinese torreya has a cultivation history of 1300 years so far and is mainly distributed in 10 provinces of Zhejiang, jiangsu, anhui, fujian, jiangxi, hunan, hubei, sichuan, yunnan, guizhou and the like in the Yangtze river basin. The real-time grafting propagation of the torreya grandis produces rich variation types, and a plurality of torreya grandis varieties are bred by a forest breeder in Zhejiang province through excellent strain breeding and mass propagation tests for many years. In 2011, the improved variety 'cephalotaxus' (t. Grandis 'Xifei') was approved by the national forestry office forest variety approval committee. In recent years, zhejiang province examined (believed) new varieties of 'pearl torreya', 'grand torreya', 'ivory torreya', 'eastern white pearl', 'crisp kernel torreya', 'Zhu Yanfei', 'Ding Shanfei', 'torreya No. 1', 'torreya No. 2', 'torreya No. 3', 'grand lobal species torreya', 'grand ampere torreya', 'yushan torreya', 'strong torreya', and the like are defined. With the continuous breeding of new varieties of torreya trees and the development of the torreya industry, the identification and intellectual property protection of the new varieties are also in urgent need of corresponding molecular technical means to follow.
The variety identification of Chinese torreya is based on traditional morphological classification, namely, the Chinese torreya is classified into round seed type (big, middle and small Chinese torreya and the like) and long seed type (fine torreya, sesame torreya, rice torreya, eggplant torreya, raised torreya, cephalotaxus and the like) according to the characters of fruits or seeds of Chinese torreya in full bearing period. The identification method needs to be able to identify and select seeds after the fruits are ripe in one year, and cannot be used for early identification. Since 2000, several PCR-based molecular marker technologies such as RAPD (Random Amplified Polymorphic DNA ), ISSR (Inter-Simple Sequence Repea, simple Sequence repeat interval amplified polymorphism) and SRAP (Sequence-Related Amplified Polymorphism, related Sequence amplified polymorphism) were used successively for Chinese torreya variety classification, genetic variation and resident genetic diversity analysis.
However, all the molecular markers are universal primers, and the PCR amplification patterns are complex, have poor repeatability and low specificity, and need a large amount of screening work, so that the PCR amplification patterns are not suitable for variety identification. SSR (Simple Sequence Repeat, simple repeated sequence; or Microsatellite) is a co-dominant marker, can distinguish homozygote and heterozygote, can detect multiple alleles, and has the advantages of abundant polymorphism, simple operation, reliable result, good repeatability and the like. In recent years, there are reports of developing SSR markers based on torreya transcriptome sequencing, but the availability of the SSR markers is based on traditional polyacrylamide gel electrophoresis, so that the manual interpretation error of the stripes is large, and the difference of alleles on SSR sites on a plurality of bases is difficult to accurately distinguish; moreover, the SSR markers have not been subjected to polymorphism screening work, so that whether the SSR markers can be used for accurately and rapidly identifying Chinese torreya varieties is unknown.
The development of fluorescent SSR markers is a capillary electrophoresis detection method based on a DNA sequencing platform, wherein fluorescent groups (such as FAM, HEX, TAMRA and the like) with different colors are used for marking the tail end of one primer in a pair of SSR primers, and a fluorescent detector is used for detecting products, so that the size of amplified products can be automatically identified, the defect of polyacrylamide gel electrophoresis detection by the traditional silver staining method is overcome, and the method has the technical advantages of rapidness, high efficiency, accuracy, sensitivity and the like. Therefore, the fluorescent SSR marking technology is utilized to develop the characteristic SSR fingerprint (genotype) of the new or excellent and special Chinese torreya varieties, so that the genotype difference among the varieties is revealed, and the fluorescent SSR marking technology has important significance for variety identification and intellectual property protection. However, no report for identifying torreya varieties by utilizing a fluorescent SSR marking technology to develop characteristic SSR fingerprints exists at home and abroad.
(III) summary of the invention
The invention aims to provide a molecular characteristic fluorescent SSR (simple sequence repeat) marker primer and a method for rapidly identifying torreya grandis, syzygium aromaticum and torreya grandis of torreya grandis varieties.
The technical scheme adopted by the invention is as follows:
a pair of molecular characteristic fluorescent SSR primers for identifying torreya yushanensis, dasyringa grandis and torreya grandis varieties, wherein the nucleotide sequence of the primers is as follows:
the upstream primer tg_u15f:5'-FAM-GCACAAACATCCATGCAAAC-3'; (upstream primer plus fluorescent labeling group FAM)
Downstream primer tg_u15r:5'-AACAAGGGTCCAGGGAGAGT-3'.
The primer pair is obtained based on a fluorescence SSR marking technology through carrying out polymorphism screening test on a large number of SSR primers which are independently developed among torreya varieties to be detected. By using the primer pair to carry out fluorescent SSR detection on 9 Chinese torreya varieties, 3 Chinese torreya new varieties (Yushan fish Chinese torreya, daxiang and Pan Chinese torreya) can stably obtain 3 different SSR characteristic fingerprints (genotypes), and other Chinese torreya varieties do not have the 3 characteristics, so that the primer can be used for rapidly identifying Chinese torreya varieties Yushan fish Chinese torreya, daxiang and Pan Chinese torreya. It should be noted that the molecular characteristic fluorescent SSR primer is only limited to the identification of the torreya varieties (whether the torreya is Yushanyu torreya, daxiang and Dayan torreya is identified), namely, the sample to be detected is only limited to the torreya varieties.
The invention also relates to a method for rapidly identifying the torreya grandis, the lilac and the pan torreya, which comprises the following steps: extracting genomic DNA of a torreya variety to be detected needle leaf as a template, carrying out PCR amplification by taking a molecular characteristic fluorescent SSR primer pair Tg_U15F and Tg_U15R as amplification primers, and carrying out capillary electrophoresis detection on the amplification product, wherein if the genotype appearing on a capillary electrophoresis peak diagram is 231/231, 229/231/233 or 233/233, the torreya variety to be detected is the torreya yushanensis, the large clove or the large-leaf torreya grandis, otherwise, the torreya grandis is not detected;
the molecular characteristic fluorescent SSR marker primer has the nucleotide sequence as follows:
the upstream primer tg_u15f:5'-FAM-GCACAAACATCCATGCAAAC-3';
downstream primer tg_u15r:5'-AACAAGGGTCCAGGGAGAGT-3'.
The key point of the method is that the selection of the amplified SSR primer, DNA extraction, PCR reaction system and reaction condition determination, fluorescent capillary electrophoresis detection and data statistical analysis can be carried out according to the conventional method in the field.
Preferably, the PCR amplification reaction conditions are as follows: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 59℃for 10s, extension at 72℃for 10s for 30 cycles; and finally, filling in at 72 ℃ for 2min, wherein the termination temperature is 4 ℃.
Preferably, the fluorescence capillary electrophoresis detection method comprises the following steps: diluting PCR amplified product, denaturing at 96 deg.C for 5min, rapidly freezing the denatured diluted product at-20deg.C for 2min, and adding ABI 3730 XL Genetic Analyzer (ABI, CA, USA) and internal standard GeneScan TM -500LIZ Size Standard (ABI) capillary electrophoresis detection was performed simultaneously and Data was collected using Data Collection 3.0 software (ABI).
Specifically, the method comprises the following steps:
(1) Taking tender needles of a torreya variety to be detected, adding nitrogen, grinding, and extracting genomic DNA of the torreya;
(2) Taking the genomic DNA extracted in the step (1) as a template, and taking the molecular characteristic SSR primer as an amplification primer for PCR amplification:
the PCR reaction system consisted of, per 20. Mu.L:
the PCR conditions were as follows:
pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 59℃for 10s, extension at 72℃for 10s for 30 cycles; finally, filling the mixture into a flat state for 2min at 72 ℃ and keeping the termination temperature at 4 ℃;
(3) And (3) preparing an internal standard: 10ml Hi-Di and 80 mu LGene scan were taken TM Mixing-500 and LIZ Size Standard, centrifuging, and sub-packaging 10 mu L of the mixture in 96-well internal standard plates, and centrifuging; diluting and centrifuging PCR amplification products; adding the diluted product into the allocated 96-well internal standard plate according to 0.5 mu L/well, uniformly mixing, centrifuging, placing into a PCR instrument, denaturing at 96 ℃ for 5min, rapidly freezing at-20 ℃ for 2min, and centrifuging to obtain a denatured PCR product; the denatured PCR product was then subjected to internal reactionPlacing the label into ABI 3730 XL Genetic Analyzer synchronously for capillary electrophoresis detection, and collecting Data by using Data Collection 3.0 software;
(4) Data analysis: raw Data collected by Data Collection 3.0 software was analyzed by GeneMapper 4.1 software (ABI), the software system will be based on the position of the target peak and the internal standard GeneScan in the same lane TM -500LIZ Size Standard comparing, directly reading out the exact peak (bp number) of the SSR fragment of interest, the allelic variation data for the homozygous site being recorded as X/X, the allelic variation data for the heterozygous site being recorded as X/Y or X/Y/Z/(polyploid);
(5) And (3) judging results: if the genotype displayed on the fluorescence capillary electrophoresis peak diagram is 231/231, the torreya variety to be detected is the torreya yushanensis torreya; if the genotype displayed on the fluorescence capillary electrophoresis peak diagram is 229/231/233, the torreya variety to be detected is Daxiang; if the genotype displayed on the fluorescence capillary electrophoresis peak diagram is 233/233, the torreya variety to be detected is the large torreya, otherwise, whether the torreya variety to be detected is the large torreya is judged.
The beneficial effects of the invention are mainly as follows: the molecular characteristic fluorescent SSR primer can be used for rapidly and early identifying the Chinese torreya of new species of Chinese torreya simultaneously by utilizing needle DNA, is simple, rapid and accurate, and is a molecular means which cannot be replaced by the traditional method for identifying Chinese torreya varieties according to morphological characteristics of fruits.
(IV) description of the drawings
FIG. 1 shows the result of fluorescence capillary electrophoresis detection after PCR amplification of 9 Torreya grandis variety DNAs; a is a multi-eye Chinese torreya (Yushan fish torreya) (from dry village of mountain Shang Huzhen), B is a Chinese torreya (from dry village of mountain Shang Hu), C is a Pan-An Chinese torreya (from dry village of mountain large plate of mountain), D is a Dashan Chinese torreya (from village of mountain Shang Hu), E is a Daeugenoma (from village of mountain large plate of mountain), F is a Fang ya (from front village of yellow rock of mountain Shang Hu), G is a Pan Chinese torreya (from dry village of mountain lake plate of mountain), H is a Yuan Chinese torreya (from dry village of mountain lake plate of mountain), and I is a Zhong Yuan Chinese torreya (from village of mountain Shang Hu).
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1:
(1) Extraction of genomic DNA of torreya grandis varieties:
taking 0.03g of young leaves stored in 9 Chinese torreya varieties to be detected in silica gel, adding liquid nitrogen, thoroughly grinding, and extracting genomic DNA by using a novel rapid plant genomic DNA extraction box (DP 3111, beijing baitaike) to extract and obtain a Chinese torreya variety genomic DNA crude extract.
The crude DNA extract was checked for integrity, purity and concentration by 1.5% agarose gel electrophoresis and DNA/RNA uv spectrophotometry (Nanodrop Technologies, USA). OD (optical density) 260 /OD 280 >1.8 for subsequent PCR amplification. The DNA extract is stored in a refrigerator at-20 ℃ for standby.
(2) Molecular characteristic SSR marking primer, the sequence of the primer pair is:
the upstream primer tg_u15f:5'-FAM-GCACAAACATCCATGCAAAC-3';
downstream primer tg_u15r:5'-AACAAGGGTCCAGGGAGAGT-3'.
Synthesized by Hangzhou Kangshen Biotechnology Co.
(3) The PCR amplification, 20. Mu.L PCR reaction system consisted of: :
the PCR reaction solution comprises the following components: 2×T5 Super PCR Mix (PAGE) (TSE 006, beijing Optraceae) 10. Mu.L, tg_U15F and Tg_U15R primers (10. Mu.M) 1.5. Mu.L each, template DNA (20 ng/. Mu.L) 3. Mu.L, ddH 2 O 4μL。
The amplification reaction was performed on a Life ECO type amplification apparatus (Bioer, hangzhou Bosch technology). Amplification conditions: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 59℃for 10s, extension at 72℃for 10s for 30 cycles; and finally, filling in at 72 ℃ for 2min. The termination temperature was 4 ℃.
(4) And (3) preparing an internal standard: 10ml Hi-Di and 80. Mu.L GeneScan were taken TM Mixing-500 and LIZ Size Standard, centrifuging, and packaging into 96-well internal standard plate with 10 μl per well, and centrifuging.
(5) Fluorescent capillary electrophoresis detection: taking 5 mu L of PCR amplified product in the step (3), diluting 100 times, and taking 0.5 mu L/holeAdding into the distributed internal standard plate, mixing, centrifuging, placing into a PCR instrument, denaturing at 96 deg.C for 5min, rapidly freezing at-20 deg.C for 2min, and centrifuging to obtain denatured PCR product. The denatured PCR products and internal standard GeneScan were used TM -500LIZ Size Standard was simultaneously placed into ABI 3730 XL Genetic Analyzer for capillary electrophoresis detection and Data were collected using Data Collection 3.0 software.
(6) Genotype analysis and variety identification: raw Data collected by Data Collection 3.0 software was analyzed using GeneMapper 4.1 software. The software system will be based on the location of the target peak and the internal standard GeneScan in the same lane TM -500LIZ Size Standard, directly giving the exact peak (bp) of the SSR fragment of interest. Allelic variation data for homozygous sites are recorded as X/X, allelic variation data for heterozygous sites are recorded as X/Y or X/Y/Z/(polyploid). Different torreya varieties can be identified according to the difference comparison of the allelic variation data. Genotype data of the 9 torreya varieties are shown in table 1.
Table 1: genotype of nine Chinese torreya varieties
| Chinese torreya variety Cultivar | Genotype Genotype |
| A multiple eye torreya (Yushan fish torreya) | 231/231 |
| B cephalotaxus sinensis | 231/233 |
| C pan Chinese torreya | 231/233 |
| D Chinese torreya | 231/233 |
| E big clove | 229/231/233 |
| F-bucktooth Chinese torreya | 231/233 |
| G pan Chinese torreya | 233/233 |
| H Chinese torreya | 231/233 |
| Chinese torreya | 231/233 |
Of the 9 Chinese torreya varieties shown in Table 1, the genotypes of Yushan fish torreya, dasyringa grandis and Datorreya are 231/231, 229/231/233 and 233/233, respectively, which are clearly different from the genotypes of the other 6 varieties 231/233, and the genotypes of these 3 new varieties are also different from each other. The method shows that U15 is an SSR marker with high polymorphism among Chinese torreya varieties, tg_U15 can be used as a molecular characteristic SSR primer to identify 3 Chinese torreya new varieties at one time, and is a powerful tool for Chinese torreya variety identification and intellectual property protection.
Sequence listing
<110> Zhejiang province forestry science institute
Strong natural resource and planning bureau
Strong and safe county traditional Chinese medicine industry development promotion center
<120> primers and method for identifying torreya yushanensis, syzygium aromaticum and torreya in large pan
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 20
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<213> Unknown (Unknown)
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gcacaaacat ccatgcaaac 20
<210> 2
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 2
aacaagggtc cagggagagt 20
Claims (4)
1. A method for rapidly identifying new species of torreya grandis, syzygium aromaticum and torreya in a pan, the method comprising: extracting genomic DNA of a torreya grandis variety needle leaf to be detected as a template, carrying out PCR (polymerase chain reaction) amplification by taking a molecular characteristic fluorescent SSR primer as an amplification primer, and carrying out capillary electrophoresis detection on an amplification product, wherein if the genotype displayed on a capillary electrophoresis peak diagram is 231/231, the torreya grandis variety to be detected is torreya yushanensis; if the genotype displayed on the capillary electrophoresis peak diagram is 229/231/233, the torreya varieties to be detected are respectively Daxiang; if the genotype displayed on the capillary electrophoresis peak diagram is 233/233, the torreya variety to be detected is the large torreya, otherwise, the torreya variety to be detected is not;
the nucleotide sequence of the molecular characteristic fluorescent SSR primer is as follows:
the upstream primer tg_u15f:5'-FAM-GCACAAACATCCATGCAAAC-3';
downstream primer tg_u15r:5'-AACAAGGGTCCAGGGAGAGT-3'.
2. The method of claim 1, wherein the PCR amplification reaction conditions are as follows: pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 59℃for 10s, elongation at 72℃for 10s for 30 cycles; and finally, filling in at 72 ℃ for 2min, wherein the termination temperature is 4 ℃.
3. The method of claim 1 wherein said bristles are of the typeThe tubule electrophoresis detection method comprises the following steps: diluting PCR amplified product, denaturing at 96 deg.C for 5min, rapidly freezing the denatured diluted product at-20deg.C for 2min, and placing into ABI 3730 XL Genetic Analyzer analyzer together with internal standard GeneScan TM -500LIZ Size Standard simultaneous capillary electrophoresis detection and Data Collection with Data Collection 3.0 software.
4. The method according to claim 1, characterized in that the method is as follows:
(1) Taking tender needles of a torreya variety to be detected, adding nitrogen, grinding, and extracting genomic DNA of the torreya;
(2) Taking the genome DNA extracted in the step (1) as a template, and taking the molecular characteristic fluorescent SSR primer as an amplification primer for PCR amplification:
the PCR reaction system consisted of, per 20. Mu.L:
2×T5 Super PCR Mix 10 μL
Tg_U15F and Tg_U15R primers (10. Mu.M) 1.5. Mu.L each
Template DNA (20 ng/. Mu.L) 3. Mu.L
ddH 2 O 4 μL
The PCR conditions were as follows:
pre-denaturation at 98℃for 2min; denaturation at 98℃for 10s, annealing at 59℃for 10s, elongation at 72℃for 10s for 30 cycles; finally, filling the mixture into a flat state for 2min at 72 ℃ and keeping the termination temperature at 4 ℃;
(3) And (3) preparing an internal standard: 10ml Hi-Di and 80. Mu.L GeneScan were taken TM Mixing-500 and LIZ Size Standard, centrifuging, and sub-packaging 10 mu L of the mixture in 96-well internal standard plates, and centrifuging; diluting and centrifuging PCR amplification products; adding 0.5 mu L/hole of diluted product into the allocated 96-hole internal standard plate, uniformly mixing, centrifuging, placing into a PCR instrument, denaturing at 96 ℃ for 5min, rapidly freezing at-20 ℃ for 2min, centrifuging to obtain a denatured PCR product; synchronously placing the denatured PCR product and an internal standard into a DNA analyzer for capillary electrophoresis detection, and collecting Data by using Data Collection 3.0 software;
(4) Data analysis: data Colle with GeneMapper 4.1 softwareAnalysis of raw data collected by the action 3.0 software, the software system was based on the position of the target peak and the internal standard GeneScan in the same lane TM -500LIZ Size Standard, directly reading out the accurate peak value of the target SSR fragment, wherein the allelic variation data of the homozygous site is recorded as X/X, and the allelic variation data of the heterozygous site is recorded as X/Y or X/Y/Z;
(5) And (3) judging results: if the genotype displayed on the capillary electrophoresis peak diagram is 231/231, the torreya species to be detected is the torreya yushanensis torreya; if the genotype displayed on the capillary electrophoresis peak diagram is 229/231/233, the torreya variety to be detected is Daxiang; if the genotype displayed on the capillary electrophoresis peak diagram is 233/233, the torreya variety to be detected is the large torreya, otherwise, the torreya variety to be detected is not.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108165652A (en) * | 2018-02-12 | 2018-06-15 | 杭州师范大学 | For the specific molecular marker TGMI001 of Chinese torreya seedling stage sex identification |
| CN108359738A (en) * | 2018-02-06 | 2018-08-03 | 浙江农林大学 | A kind of Chinese torreya EST-SSR primers and Variety fingerprinting construction method |
| CN111304356A (en) * | 2020-04-17 | 2020-06-19 | 宁波市农业科学研究院 | Molecular marker primer combination for rapidly identifying sex traits of Chinese torreya in high throughput manner and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108359738A (en) * | 2018-02-06 | 2018-08-03 | 浙江农林大学 | A kind of Chinese torreya EST-SSR primers and Variety fingerprinting construction method |
| CN108165652A (en) * | 2018-02-12 | 2018-06-15 | 杭州师范大学 | For the specific molecular marker TGMI001 of Chinese torreya seedling stage sex identification |
| CN111304356A (en) * | 2020-04-17 | 2020-06-19 | 宁波市农业科学研究院 | Molecular marker primer combination for rapidly identifying sex traits of Chinese torreya in high throughput manner and application thereof |
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