CN113528693A - Primer and method for identifying variety of torreya grandis, namely Yushan torreya grandis, Chinese lilac and Pan torreya grandis - Google Patents
Primer and method for identifying variety of torreya grandis, namely Yushan torreya grandis, Chinese lilac and Pan torreya grandis Download PDFInfo
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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 varieties of torreya grandis, namely Yushan torreya grandis, Syringa oblata and Panti torreya grandis. The molecular characteristic fluorescent SSR marker primer has the following nucleotide sequence: upstream primer Tg _ U15F: 5 '-FAM-GCACAAACATCCATGCAAAC-3'; downstream primer Tg _ U15R: 5'-AACAAGGGTCCAGGGAGAGT-3' are provided. The invention has the following beneficial effects: the molecular characteristic fluorescent SSR marker primer can utilize coniferous DNA to rapidly and early identify the Chinese torreya new varieties Yushan Chinese torreya, big clove and Pan Chinese torreya at the same time, 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
(I) technical field
The invention relates to a molecular characteristic fluorescent SSR (simple sequence repeat) marker primer and a method for rapidly identifying variety Yushan fish torreya, cloves and Panti torreya grandis.
(II) background of the invention
Torreya grandis belongs to Taxaceae (Taxaceae) Torreya genus (Torreya) and is a good variety of Torreya grandis species (Torreya grandis) which is cultivated by grafting after artificial breeding of good variation and is also the only variety cultivated in the current Torreya grandis on a manual scale. Chinese torreya is a unique precious economic tree species in China and has multiple purposes of eating, medicinal use, material use, greening and the like.
Torreya grandis has been cultivated for over 1300 years to date, and is mainly distributed in 10 provinces of Zhejiang, Jiangsu, Anhui, Fujian, Jiangxi, Hunan, Hubei, Sichuan, Yunnan, Guizhou and the like in the south of Yangtze river basin. The seedling grafting propagation of Chinese torreya generates rich variation types, and through years of excellent plant breeding and mass propagation tests, forestry breeding workers in Zhejiang province breed a plurality of Chinese torreya varieties. In 2011, the improved variety of the cephalotaxus (t. grandis 'Xifei') is approved by the forest variety approval committee of the national forestry agency. In recent years, new varieties of 'pearl torreya', 'changchun torreya', 'elephant torreya', 'east white bead', 'crisp kernel torreya', 'Zhuyan torreya', 'butyl torreya', 'Torreya No. 1', 'Torreya No. 2', 'Torreya No. 3', 'big leaf seed torreya', 'Pan an long torreya', 'Yushan torreya' and 'Qian torreya' were examined (acknowledged) in Zhejiang province. Along with the continuous breeding of new varieties of torreya and the development of torreya industry, the identification and intellectual property protection of the new varieties also urgently need to follow up by corresponding molecular technical means.
The identification of Chinese torreya varieties is always based on the traditional morphological classification, namely, the Chinese torreya varieties are classified into round-seed types (large, medium and small round torreya, etc.) and long-seed types (cephalotaxus sinensis, sesamotorreya grandis, Chinese torreya, solanaceae torreya, cephalotaxus fortunei, cephalotaxus turczii, etc.) according to the characters of Chinese torreya fruits or seeds in full bearing period. The identification method needs to identify seeds after fruits mature in one year, and early identification cannot be carried out. Since 2000, some PCR-based molecular marker technologies such as RAPD (Random Amplified Polymorphic DNA), ISSR (Inter-Simple Sequence repeat Polymorphism) and SRAP (Sequence-Related Amplified Polymorphic) were successively used for variety classification, genetic variation and population genetic diversity analysis of Torreya grandis.
However, the molecular markers all adopt universal primers, and the PCR amplification map is not only complex and poor in repeatability, but also low in specificity, and needs a lot of screening work, so that the molecular markers are not suitable for variety identification. SSR (Simple Sequence Repeat, or Microrecelite, Microsatellite) is a codominant marker, can distinguish homozygote from heterozygote, can detect multiple alleles, and has the advantages of rich polymorphism, Simple operation, reliable result, good repeatability and the like. In recent years, SSR markers are reported to be developed based on torreya grandis transcriptome sequencing, but the usability of the SSR markers is based on traditional polyacrylamide gel electrophoresis, the artificial interpretation error of a band is large, and the difference of alleles on SSR loci on a plurality of bases is difficult to accurately distinguish; moreover, polymorphism screening work is not carried out on the SSR markers, so that whether the SSR markers can be used for accurately and quickly 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 fluorescence detector is used for detecting products, so that the size of an amplification product is automatically identified, the defects of polyacrylamide gel electrophoresis detection by the traditional silver staining method are 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 fingerprints (genotypes) of new varieties or excellent and special varieties of the Chinese torreya, reveal the genotype difference among the varieties and have important significance for the identification of the varieties and the protection of intellectual property rights. However, so far, there is no report of identifying Chinese torreya varieties by developing characteristic SSR fingerprints by using a fluorescent SSR marking technology at home and abroad.
Disclosure of the invention
The invention aims to provide a molecular characteristic fluorescent SSR (simple sequence repeat) marker primer and a method for rapidly identifying variety Yushan grand torreya, large clove and Pan grand torreya.
The technical scheme adopted by the invention is as follows:
a pair of molecular characteristic fluorescent SSR primers for identifying variety Yushan grand torreya grandis, big clove and Pan grand torreya grandis have the following nucleotide sequences:
upstream primer Tg _ U15F: 5 '-FAM-GCACAAACATCCATGCAAAC-3'; (upstream primer plus fluorescent labeling group FAM)
Downstream primer Tg _ U15R: 5'-AACAAGGGTCCAGGGAGAGT-3' are provided.
The primer pair is obtained by carrying out polymorphism screening test on a large number of self-developed SSR primers among Chinese torreya varieties to be tested based on a fluorescent SSR marking technology. By utilizing the primer to perform fluorescent SSR detection on 9 Chinese torreya varieties, 3 different SSR characteristic fingerprints (genotypes) can be stably obtained for 3 Chinese torreya new varieties (Yushan Chinese torreya, big clove and Pan Chinese torreya), and other Chinese torreya varieties do not have the 3 characteristics, so the primer can be used for rapidly identifying the Chinese torreya varieties Yushan Chinese torreya, big clove and Pan Chinese torreya. It should be noted that the molecular-characteristic fluorescent SSR primers are only limited to identification of Chinese torreya varieties (whether the Chinese torreya is Yushan Chinese torreya, big clove and Panti torreya grandis or not is identified), namely, samples to be detected are only limited to Chinese torreya varieties.
The invention also relates to a method for rapidly identifying the variety Yushan grand torreya, cloves and Panti grand torreya, which comprises the following steps: extracting genome DNA of coniferous needles of the Chinese torreya variety to be detected as a template, performing PCR amplification by using molecular characteristic fluorescent SSR primer pairs Tg _ U15F and Tg _ U15R as amplification primers, performing capillary electrophoresis detection on an amplification product, and if the genotypes appearing on a capillary electrophoresis peak map are 231/231, 229/231/233 or 233/233, respectively determining that the Chinese torreya variety to be detected is Yushanyu torreya, Dasyringa grandis or Pan torreya grandis, or else, determining that the Chinese torreya variety to be detected is not;
the molecular characteristic fluorescent SSR marker primer has the following nucleotide sequence:
upstream primer Tg _ U15F: 5 '-FAM-GCACAAACATCCATGCAAAC-3';
downstream primer Tg _ U15R: 5'-AACAAGGGTCCAGGGAGAGT-3' are provided.
The key point of the method is that the selection of the SSR primer, the DNA extraction, the determination of the PCR reaction system and reaction conditions, the fluorescence capillary electrophoresis detection and the 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 2 min; denaturation at 98 ℃ for 10s, annealing at 59 ℃ for 10s, and extension at 72 ℃ for 10s for 30 cycles; finally, the temperature is leveled off at 72 ℃ for 2min, and the termination temperature is 4 ℃.
Preferably, the fluorescence capillary electrophoresis detection method is as follows: diluting PCR amplification product, denaturing at 96 deg.C for 5min, quickly freezing at-20 deg.C for 2min, adding ABI 3730XL Genetic Analyzer (ABI, CA, USA), and internal standard GeneScanTMCapillary electrophoresis detection was performed simultaneously with 500LIZ Size Standard (ABI), and Data Collection 3.0 software (ABI) was used to collect Data.
Specifically, the method comprises the following steps:
(1) taking young and tender coniferous leaves of a Chinese torreya variety to be detected, adding liquid nitrogen, grinding, and extracting genome DNA of the Chinese torreya;
(2) performing PCR amplification by taking the genomic DNA extracted in the step (1) as a template and the molecular characteristic SSR primer as an amplification primer:
the composition of each 20. mu.L of the PCR reaction system was as follows:
the PCR reaction conditions were as follows:
pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 59 ℃ for 10s, and extension at 72 ℃ for 10s for 30 cycles; finally, filling for 2min at 72 ℃, wherein the termination temperature is 4 ℃;
(3) preparing an internal standard: 10ml of Hi-Di and 80 mu of LGeneScan are takenTM-500LIZ Size Standard, centrifuged, aliquoted 10 μ L per well into a 96 well Standard plate, centrifuged; diluting and centrifuging the PCR amplification product; adding the diluted product into a well-distributed 96-well standard plate according to 0.5 mu L/well, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then rapidly freezing at-20 ℃ for 2min, and centrifuging to obtain a denatured PCR product; synchronously placing the denatured PCR product and an internal standard into an ABI 3730XL Genetic Analyzer for capillary electrophoresis detection, and collecting Data by using Data Collection 3.0 software;
(4) and (3) data analysis: the raw Data collected by the Data Collection 3.0 software was analyzed by GeneMapper 4.1 software (ABI), and the software system would compare the internal standard GeneScan in the same lane with the position of the target peakTM-500LIZ Size Standard for comparison, directly reading the exact peak (bp number) of the target SSR fragment, and recording the allelic variation data of homozygous locus as X/X and the allelic variation data of heterozygous locus as X/Y or X/Y/Z/. once (polyploidy);
(5) and (5) judging a result: if the genotype displayed on the fluorescence capillary electrophoresis peak image is 231/231, the variety of the Chinese torreya to be detected is the Yushan Chinese torreya; if the genotype displayed on the fluorescence capillary electrophoresis peak image is 229/231/233, the Chinese torreya variety to be detected is the big clove; if the genotype displayed on the fluorescence capillary electrophoresis peak image is 233/233, the Chinese torreya variety to be detected is Pan torreya grandis, otherwise, the Chinese torreya variety to be detected is not Pan torreya grandis.
The invention has the following beneficial effects: the molecular characteristic fluorescent SSR primer can utilize coniferous DNA to rapidly and early identify the Chinese torreya new varieties such as Yushan Chinese torreya, big clove and Pan Chinese torreya at the same time, has simple, rapid and accurate method, and is a molecular means which can not be replaced by the traditional method for identifying the Chinese torreya variety according to the morphological characteristics of fruits.
(IV) description of the drawings
FIG. 1 shows the result of detection of DNA of 9 varieties of Torreya grandis by fluorescence capillary electrophoresis after PCR amplification; a is Torreya grandis (Yushan Torreya grandis) (from Torpan county lake town dry village), B is Torreya grandis (from Torpan county lake town lake mouth village), C is Torpan Torreya grandis (from Torpan county large disc Zhengchang village), D is Torreya grandis (from Torpan county Shang lake town lake mouth village), E is Ganggui (from Torpan county large disc Zhengchang village), F is Torreya grandis (from Pan county lake Zhenghuangyan village), G is Torreya grandis (from Pan county Shang lake dry village), H is Torreya grandis (from Pan county lake green dry village), and I is Torreya grandis (from Pan county lake town Shandong village).
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1:
(1) extracting the genome DNA of the Chinese torreya variety:
taking 0.03g of young leaves preserved by silica gel of 9 Chinese torreya varieties to be detected, adding liquid nitrogen to thoroughly grind, and extracting the genome DNA by using a novel rapid plant genome DNA extraction box (DP3111, Beijing Baitaike) to extract and obtain a crude extract of the genome DNA of the Chinese torreya varieties.
Crude DNA extracts were checked for integrity, purity and concentration by 1.5% agarose gel electrophoresis and DNA/RNA UV spectrophotometer (Nanodrop Technologies, USA). OD260/OD280>1.8 DNA samples were used for subsequent PCR amplification. The DNA extract was stored at-20 ℃ in a refrigerator for further use.
(2) The SSR marker primer with molecular characteristics has the following sequences:
upstream primer Tg _ U15F: 5 '-FAM-GCACAAACATCCATGCAAAC-3';
downstream primer Tg _ U15R: 5'-AACAAGGGTCCAGGGAGAGT-3' are provided.
Synthesized by Hangzhou Youkang Biotechnology Co.
(3) PCR amplification, 20. mu.L PCR reaction system consisted of: :
the PCR reaction solution comprises: 2 XT 5 Super PCR Mix (PAGE) (TSE006, New Prov. Peking Okagaku) 10. mu.L, Tg _ U15F and Tg _ U15R primers (10. mu.M) each 1.5. mu.L, template DNA (20 ng/. mu.L) 3. mu.L, ddH2O 4μL。
The amplification reaction was performed on a Life ECO type amplification apparatus (Bioer, Hangzhou Bori technology). Amplification conditions: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 59 ℃ for 10s, and extension at 72 ℃ for 10s for 30 cycles; finally, filling for 2min at 72 ℃. The termination temperature was 4 ℃.
(4) Preparing an internal standard: 10ml of Hi-Di and 80 mu L of GeneScan are takenTM-500LIZ Size Standard, centrifuged, aliquoted 10. mu.L per well into 96 well plates, and centrifuged.
(5) And (3) detecting by fluorescence capillary electrophoresis: and (3) taking 5 mu L of PCR amplification product in the step (3), diluting by 100 times, adding 0.5 mu L/hole into the well-distributed internal standard plate, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then rapidly freezing at-20 ℃ for 2min, and centrifuging to obtain a denatured PCR product. Mixing the denatured PCR product with an internal standard GeneScanTMThe-500 LIZ Size Standard was synchronously placed in ABI 3730XL Genetic Analyzer for capillary electrophoresis detection, and Data Collection 3.0 software was used to collect Data.
(6) Genotype analysis and variety identification: the raw Data collected by the Data Collection 3.0 software was analyzed by GeneMapper 4.1 software. The software system will compare the position of the target peak with the internal standard GeneScan in the same laneTMComparison was performed on-500 LIZ Size Standard, which directly gives the exact peak (bp) of the SSR fragment of interest. Allelic variation data for homozygous loci were recorded as X/X, and allelic variation data for heterozygous loci were recorded as X/Y or X/Y/Z/. Different Chinese torreya varieties can be identified according to the difference comparison of the equipotential variation data. The genotype data of the above 9 varieties of torreya grandis are shown in table 1.
Table 1: genotypes of nine Chinese torreya varieties
| Cultivar of Chinese torreya variety | Genotype |
| A polypore Chinese torreya (Yushan fish Chinese torreya) | 231/231 |
| B Chinese torreya | 231/233 |
| C Strong safety torreya grandis | 231/233 |
| D big Chinese torreya | 231/233 |
| E Dalianxiang (E) | 229/231/233 |
| F platya grandis | 231/233 |
| G Strong torreya grandis | 233/233 |
| H big Torreya grandis | 231/233 |
| Chinese torreya | 231/233 |
Among the 9 varieties of torreya grandis shown in table 1, genotypes of yushan torreya grandis, syzygium aromaticum and grandma torreya were 231/231, 229/231/233 and 233/233, respectively, apparently different from 231/233 of the other 6 varieties, and genotypes among the 3 new varieties were also different from each other. This indicates that U15 is an SSR marker with high polymorphism among torreya grandis varieties, Tg _ U15 can be used as a molecular characteristic SSR primer to identify 3 torreya grandis new varieties at one time, and is a powerful tool for torreya grandis variety identification and intellectual property protection.
Sequence listing
<110> scientific institute of forestry in Zhejiang province
Natural resources and planning bureau in Pan-an county
Strong county traditional Chinese medicine industry development promotion center
<120> primers and method for identifying variety of torreya grandis, Yushan torreya grandis, Syringa oblata and Panti torreya grandis
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<170> SIPOSequenceListing 1.0
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gcacaaacat ccatgcaaac 20
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<211> 20
<212> DNA
<213> Unknown (Unknown)
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aacaagggtc cagggagagt 20
Claims (5)
1. The molecular characteristic fluorescent SSR labeled primers for identifying new varieties of torreya grandis, Yushan torreya grandis, Syringa oblata and Pan torreya grandis have the following nucleotide sequences:
upstream primer Tg _ U15F: 5 '-FAM-GCACAAACATCCATGCAAAC-3';
downstream primer Tg _ U15R: 5'-AACAAGGGTCCAGGGAGAGT-3' are provided.
2. A method for rapidly identifying Yushan fish torreya, lilac and Panti torreya of new varieties of torreya grandis, comprising: extracting genome DNA of coniferous needles of the Chinese torreya variety to be detected as a template, taking a molecular characteristic fluorescent SSR primer as an amplification primer, carrying out PCR amplification, and carrying out capillary electrophoresis detection on an amplification product, wherein if the genotype displayed on a capillary electrophoresis peak diagram is 231/231, 229/231/233 or 233/233, the Chinese torreya variety to be detected is Yushanyu torreya, big clove or big torreya, and if not, the Chinese torreya variety to be detected is not;
the molecular characteristic fluorescent SSR primer has the following nucleotide sequence:
upstream primer Tg _ U15F: 5 '-FAM-GCACAAACATCCATGCAAAC-3';
downstream primer Tg _ U15R: 5'-AACAAGGGTCCAGGGAGAGT-3' are provided.
3. The method of claim 2, wherein the PCR amplification reaction conditions are as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 59 ℃ for 10s, and extension at 72 ℃ for 10s for 30 cycles; finally, the temperature is leveled off at 72 ℃ for 2min, and the termination temperature is 4 ℃.
4. The method of claim 2, wherein the fluorescence capillary electrophoresis detection method comprises: diluting PCR amplification product, denaturing at 96 deg.C for 5min, quickly freezing at-20 deg.C for 2min, placing into ABI 3730XL Genetic Analyzer, and mixing with internal standard GeneScanTMThe capillary electrophoresis detection was performed simultaneously with-500 LIZ Size Standard, and Data Collection 3.0 software was used to collect Data.
5. The method of claim 2, characterized in that the method is as follows:
(1) taking young and tender coniferous leaves of a Chinese torreya variety to be detected, adding liquid nitrogen, grinding, and extracting genome DNA of the Chinese torreya;
(2) performing PCR amplification by taking the genomic DNA extracted in the step (1) as a template and the molecular characteristic SSR primer as an amplification primer:
the composition of each 20. mu.L of the PCR reaction system was as follows:
the PCR reaction conditions were as follows:
pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 59 ℃ for 10s, and extension at 72 ℃ for 10s for 30 cycles; finally, filling for 2min at 72 ℃, wherein the termination temperature is 4 ℃;
(3) preparing an internal standard: 10ml of Hi-Di and 80 mu L of GeneScan are takenTM-500LIZ Size Standard, centrifuged, aliquoted 10 μ L per well into a 96 well Standard plate, centrifuged; diluting and centrifuging the PCR amplification product; adding the diluted product into a well-distributed 96-well standard plate according to 0.5 mu L/well, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then rapidly freezing at-20 ℃ for 2min, and centrifuging to obtain a denatured PCR product; synchronously placing the denatured PCR product and the internal standard into a DNA analyzer for capillary electrophoresis detection, and collecting Data by using Data Collection 3.0 software;
(4) and (3) data analysis: the raw Data collected by the Data Collection 3.0 software was analyzed by the GeneMapper 4.1 software, the software system was aligned with the internal standard GeneScan in the same lane according to the position of the target peakTM-500LIZ Size Standard for comparison, directly reading the accurate peak value of the target SSR fragment, recording the allelic variation data of homozygous locus as X/X, and recording the allelic variation data of heterozygous locus as X/Y or X/Y/Z;
(5) and (5) judging a result: if the genotype displayed on the capillary electrophoresis peak picture is 231/231, the variety of the Chinese torreya to be detected is the Yushan Chinese torreya; if the genotype displayed on the capillary electrophoresis peak image is 229/231/233, the Chinese torreya variety to be detected is big clove; if the genotype displayed on the capillary electrophoresis peak image is 233/233, the Chinese torreya variety to be detected is Pan grand torreya, otherwise, the Chinese torreya variety to be detected is not Pan grand torreya.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
|---|---|---|---|---|
| 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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