CN113718050A - Molecular characteristic SSR primer and method for identifying Chinese torreya polyploid varieties - Google Patents
Molecular characteristic SSR primer and method for identifying Chinese torreya polyploid varieties Download PDFInfo
- Publication number
- CN113718050A CN113718050A CN202110846256.4A CN202110846256A CN113718050A CN 113718050 A CN113718050 A CN 113718050A CN 202110846256 A CN202110846256 A CN 202110846256A CN 113718050 A CN113718050 A CN 113718050A
- Authority
- CN
- China
- Prior art keywords
- polyploid
- primer
- chinese torreya
- torreya
- varieties
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a molecular characteristic SSR marker primer for rapidly identifying Chinese torreya polyploid varieties and a Chinese torreya polyploid identification method. The molecular characteristic fluorescent SSR primer has the following nucleotide sequence: primer Tg _ U33: upstream primer Tg _ U33F: 5 '-FAM-TAAGAGGATTGACATGGCCC-3' downstream primer Tg _ U33R: 5'-TATTCTGGAGTTTGGACCCG-3', respectively; the molecular characteristic fluorescent SSR primer can utilize coniferous DNA to quickly identify the Chinese torreya polyploid varieties, has simple, quick, accurate and efficient method and good repeatability of detection results, is not influenced by seasons, plant development periods and growth environments, not only provides reference and basis for quick identification of the Chinese torreya polyploid varieties, but also provides references and basis for researches on new variety protection, polyploid breeding and the like. Compared with the traditional morphological identification method, the karyotype analysis method and the flow cytometry method, the method has obvious advantages and is an advanced molecular means for identifying the Chinese torreya polyploid variety.
Description
(I) technical field
The invention relates to a molecular characteristic SSR marker primer for rapidly identifying Chinese torreya polyploid varieties, and also relates to application of the primer in Chinese torreya polyploid identification.
(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 Zhejiang, Jiangsu, Anhui, Fujian, Jiangxi, Hunan, Hubei, Sichuan, Yunnan and Guizhou provinces in the south of Yangtze river basin. Due to the influence of long and complex historical environment and natural hybridization, a plurality of variations are generated, and the diversity of natural torreya resource resources is evolved. In addition, the seedling grafting propagation of the Chinese torreya generates rich variation types. These variation types are the fundamental guarantee for developing new and improved Chinese torreya seed selection. Through years of excellent plant breeding and mass propagation tests, Chinese forestry breeding workers breed a plurality of Chinese torreya excellent varieties, including 'Chinese torreya', 'pearl torreya', 'long torreya', 'elephant torreya', 'east white pearl', and the like. Development of resource evaluation, germplasm protection and improved variety breeding of Chinese torreya has great significance for promoting sustainable development of the Chinese torreya frame.
Polyploid breeding is an important means for plant breeding. Polyploid plants contain three or more sets of chromosome groups, are very common in nature, and generally have the characteristics of high growth speed, high biological yield, strong stress resistance and the like. Triploid torreya is ubiquitous in large adult trees and seedlings. These triploid plants may be fused from a haploid genome of the male and a diploid genome of the female (Chenophoresis, 1990; Huangpau et al, 1990). So far, no tetraploid and other polyploid torreya are found. The identification method of plant polyploids mainly includes plant morphology identification, chromosome counting, i.e., karyotype analysis, and Flow Cytometry (FCM). The morphological identification is simple and extensive, but the accuracy is not high; the chromosome counting method is a classical and reliable identification method, but has high requirements on the sampling parts of plants and the period of cells, and has the disadvantages of complicated operation process, large workload and low speed; although the flow cytometry method can quickly identify the ploidy level of the chromosome of the plant, the method also has some defects that the preparation time of a sample is not longer than 20 minutes, otherwise, the position of a DNA peak value is shifted, so that the measurement result is influenced; secondly, different internal standards are used for detection, and the result has errors; the results of detection by using different nuclear fluorescent dyes are also obviously different; and high measuring cost. Therefore, a simple, quick and efficient method for identifying the polyploidy of the Chinese torreya is urgently needed to be developed, which is beneficial to quickly screening the polyploidy germplasm material of the natural Chinese torreya population and provides powerful technical support for the excellent polyploidy breeding of the Chinese torreya. In recent years, identification of polyploids by using SSR molecular markers has been successfully applied to plants such as poplar (gaja, etc. 2015), jujube (changeable, etc. 2018), acacia (maoxihong, etc. 2018), and populus deltoides (lugwei, etc. 2019), but there is no report on rapid identification of ploidy level of torreya grandis by using molecular-characteristic fluorescent SSR markers.
Disclosure of the invention
The invention aims to provide a molecular characteristic SSR marker primer for rapidly identifying Chinese torreya polyploid varieties and also relates to application of the primer in identification of the Chinese torreya polyploid varieties.
The technical scheme adopted by the invention is as follows:
the molecular characteristic fluorescent SSR marker primer for identifying Chinese torreya polyploid varieties has the following nucleotide sequence:
primer Tg _ U33:
upstream primer Tg _ U33F: FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer Tg _ U33R: 5'-TATTCTGGAGTTTGGACCCG-3', respectively;
the molecular characteristic primer Tg _ U33 is obtained by screening a large number of autonomously developed EST-SSR primers among polyploid varieties of the Chinese torreya to be detected based on a fluorescent SSR labeling technology, has high amplification efficiency, good stability and high heterozygosity, can accurately identify whether the Chinese torreya variety is polyploid, and can identify the Chinese torreya variety as the polyploid as long as more than 3 (including 3) alleles can be amplified by utilizing the primer. The primer modified by the fluorescent group FAM is used for detecting 9 Chinese torreya varieties (8 of which are triploid and 1 of which is diploid as proved by flow cytometry), and the polyploid detection rate is up to 100%, so that Tg _ U33 is a molecular characteristic primer for rapidly identifying the Chinese torreya polyploid varieties.
The invention also relates to a method for identifying the Chinese torreya polyploid variety, which comprises the following steps:
extracting genome DNA of coniferous leaves of a Chinese torreya variety to be detected as a template, performing PCR amplification by using molecular characteristic fluorescent SSR primer pairs Tg _ U33F and Tg _ U33R as amplification primers, performing capillary electrophoresis detection on an amplification product, and determining that the plant is polyploid if the number of allele factors detected on a capillary electrophoresis peak map is more than or equal to 3.
The molecular characteristic fluorescent SSR primer has the following nucleotide sequence:
primer Tg _ U33:
upstream primer Tg _ U33F: 5 '-FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer Tg _ U33R: 5'-TATTCTGGAGTTTGGACCCG-3', respectively;
preferably, the PCR amplification reaction conditions are as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 15s for 35 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 ABI3730XL 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 the Chinese torreya variety to be detected, adding liquid nitrogen and 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 PCR reaction was as follows:
the PCR reaction conditions were as follows:
pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 15s, and extension at 72 ℃ for 15s for 35 cycles; finally, filling for 2min at 72 ℃, wherein the termination temperature is 4 ℃;
(1) preparing an internal standard: 10ml of Hi-Di and 80 mu of LGeneScan are takenTM-500LIZ SizeStandard, centrifuged, aliquoted 10 μ L per well into 96 well standard plates, centrifuged; diluting and centrifuging the PCR amplification product; adding the diluted product into a well-distributed 96-pore standard plate according to 0.5 mu L/pore, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then rapidly freezing at-20 ℃ for 2min, centrifuging to obtain a denatured PCR product, synchronously placing the denatured PCR product and an internal standard into an ABI3730 XLANetic Analyzer for capillary electrophoresis detection, and collecting Data by using Data Collection 3.0 software;
(2) and (3) data analysis: data were aligned using GeneMapper 4.1 software (ABI)The raw data collected by the Collection 3.0 software was analyzed and the software system would compare the position of the target peak to the internal standard GeneScan in the same laneTMComparing the 500LIZ Size Standard, directly reading the accurate peak value (bp number) 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/;
(3) and (5) judging a result: if the detected allelic base factors on the capillary electrophoresis peak image are more than or equal to 3, the Chinese torreya variety to be detected is polyploid, otherwise, the Chinese torreya variety to be detected is not polyploid.
The invention has the following beneficial effects: the molecular characteristic fluorescent SSR primer can utilize coniferous DNA to quickly identify the Chinese torreya polyploid varieties, has simple, quick, accurate and efficient method and good repeatability of detection results, is not influenced by seasons, plant development periods and growth environments, not only provides reference and basis for quick identification of the Chinese torreya polyploid varieties, but also provides references and basis for researches on new variety protection, polyploid breeding and the like. Compared with the traditional morphological identification method, the karyotype analysis method and the flow cytometry method, the method has obvious advantages and is an advanced molecular means for identifying the Chinese torreya polyploid variety.
(IV) description of the drawings
FIG. 1 shows the flow cytometry detection results and fluorescence capillary electrophoresis detection results for identifying 9 varieties of torreya grandis, wherein A1-I1 are the flow cytometry detection results, and A2-I2 are the fluorescence capillary electrophoresis detection results; a is Yushan fruit torreya (from Huanglin Tu village in Shang lake town), B is big torreya (from deep Cang shan mountain in Yushan village, Sichuan village), C is big torreya (from deep Yushan mountain in Yushan village), D is big clove (from Wanwen street Wang hidden pit village), E is Pan big torreya (from Shang lake Zhen Tu Torreya village), F is big clove (from Dapan Zhen Chang Tu village), G is big torreya (from Anwen street Yunshan community in dock), H is big torreya (from Anwen street Yunshan community in dock), and I is small clove (from Shang lake town Huanglin Ji front 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 and tender leaves preserved by 9 Chinese torreya varieties to be detected (identified by a flow cytometry method, wherein 8 are triploid and 1 is diploid), adding liquid nitrogen for thoroughly grinding, and extracting the genome DNA by using a novel rapid plant genome DNA extraction box (DP3111, Beijing Baitaike) to extract and obtain a genome DNA crude extract 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 sequences of molecular characteristic SSR primers and primer pairs are as follows:
primer Tg _ U33:
upstream primer Tg _ U33F: 5 '-FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer Tg _ U33R: 5'-TATTCTGGAGTTTGGACCCG-3', respectively;
synthesized by Hangzhou Youkang Biotechnology Co.
(3) PCR amplification, 20. mu.L PCR reaction system consisted of:
| reaction components | Volume (μ L) |
| 2×T5 Super PCR Mix(PAGE) | 10.0 |
| Tg_U33F(10μM) | 1.5 |
| Tg_U33R(10μM) | 1.5 |
| 20 ng/. mu.L template DNA | 3.0 |
| ddH2O | 4.0 |
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 55 ℃ for 15s, and extension at 72 ℃ for 15s for 35 cycles; finally, the temperature is leveled off at 72 ℃ for 2min, and the termination temperature is 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 a well-distributed 96-hole standard plate, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then quickly freezing at-20 ℃ for 2min, and centrifuging. Mixing the denatured PCR product with an internal standard GeneScanTMThe-500 LIZ Size Standard was synchronously set in ABI3730XL 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 laneTMAnd (3) comparing the SSR fragments with-500 LIZ Size Standard, and directly reading the accurate peak value (bp) of the target SSR fragment. 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/. And judging whether the Chinese torreya variety is polyploid or not according to the quantity of amplified allelic variation (allele). Genotype number of the above 9 Torreya grandis varietiesSee table 1.
Table 1: genotypes of nine Chinese torreya varieties
Among the 9 torreya species shown in table 1, 8 torreya species numbered from a to H all had a genotype of 204/207/225, and the lilac genotype of the torreya species numbered I was 207/225. Shows that the variety numbered from A to H has 3 alleles and is triploid; the variety with the I number has 2 alleles and is diploid, which is completely consistent with the ploidy level identified by the flow cytometry method. This shows that the U33 locus of the Chinese torreya variety is highly heterozygous, and Tg _ U33 can be used as a molecular characteristic SSR primer to identify the ploidy level of the Chinese torreya variety, and is an advanced molecular means for identifying the polyploid variety of the Chinese torreya.
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> molecular characteristic SSR primers and method for identifying Chinese torreya polyploid varieties
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 2
Claims (5)
1. A molecular characteristic SSR primer for identifying Chinese torreya polyploid varieties has the following nucleotide sequence: primer Tg _ U33:
upstream primer Tg _ U33F: FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer Tg _ U33R: 5'-TATTCTGGAGTTTGGACCCG-3' are provided.
2. A method of identifying a torreya grandis polyploid variety, the method comprising: extracting genome DNA of needle leaves of a Chinese torreya variety to be detected as a template, performing PCR amplification by using molecular characteristic fluorescent SSR primer pairs Tg _ U33F and Tg _ U33R as amplification primers, performing capillary electrophoresis detection on an amplification product, and if the number of allele factors detected on a capillary electrophoresis peak map is more than or equal to 3, determining that the plant is polyploid, otherwise, determining that the plant is not polyploid;
the molecular characteristic fluorescent SSR primer has the following nucleotide sequence:
primer Tg _ U33:
upstream primer Tg _ U33F: 5 '-FAM-TAAGAGGATTGACATGGCCC-3'
Downstream primer Tg _ U33R: 5'-TATTCTGGAGTTTGGACCCG-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 55 ℃ for 15s, and extension at 72 ℃ for 15s for 35 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, adding ABI3730XL Genetic Analyzer, and mixing with internal standard GeneScanTM-500 LIZ SThe ize Standard was used to perform capillary electrophoresis in parallel and Data Collection 3.0 software was used to collect the Data.
5. The method of claim 2, characterized in that the method is as follows:
(1) taking young and tender coniferous leaves of the Chinese torreya variety to be detected, adding liquid nitrogen and 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 55 ℃ for 15s, and extension at 72 ℃ for 15s for 35 cycles; finally, filling for 2min at 72 ℃, wherein the termination temperature is 4 ℃;
(3) 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-pore standard plate according to 0.5 mu L/pore, uniformly mixing, centrifuging, placing into a PCR instrument, performing denaturation at 96 ℃ for 5min, then rapidly freezing at-20 ℃ for 2min, centrifuging to obtain a denatured PCR product, synchronously placing the denatured PCR product and an internal standard into an ABI3730XL 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 the GeneMapper 4.1 software, and the software system would compare the internal standard GeneScan in the same lane with the target peak positionTM-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 detected allelic base factors on the capillary electrophoresis peak image are more than or equal to 3, the Chinese torreya variety to be detected is polyploid, otherwise, the Chinese torreya variety to be detected is not polyploid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110846256.4A CN113718050B (en) | 2021-07-26 | 2021-07-26 | Molecular characteristic SSR primer and method for identifying torreya polyploid variety |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110846256.4A CN113718050B (en) | 2021-07-26 | 2021-07-26 | Molecular characteristic SSR primer and method for identifying torreya polyploid variety |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113718050A true CN113718050A (en) | 2021-11-30 |
| CN113718050B CN113718050B (en) | 2023-05-26 |
Family
ID=78674010
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110846256.4A Active CN113718050B (en) | 2021-07-26 | 2021-07-26 | Molecular characteristic SSR primer and method for identifying torreya polyploid variety |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113718050B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118581274A (en) * | 2024-08-05 | 2024-09-03 | 云南师范大学 | SSR (simple sequence repeat) marker detection primer for potato homologous chromosome typing identification and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2021
- 2021-07-26 CN CN202110846256.4A patent/CN113718050B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118581274A (en) * | 2024-08-05 | 2024-09-03 | 云南师范大学 | SSR (simple sequence repeat) marker detection primer for potato homologous chromosome typing identification and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113718050B (en) | 2023-05-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113584217A (en) | Rhododendron hybrid variety identification method based on EST-SSR molecular marker | |
| CN108588255A (en) | The Indel marker developments of one differentiation, five pepper cultivation kinds and its application | |
| CN109486991B (en) | Molecular marker primer composition for identifying intergeneric hybrid of pear and apple and application thereof | |
| CN113718050B (en) | Molecular characteristic SSR primer and method for identifying torreya polyploid variety | |
| CN107058494B (en) | Method for simplifying purity identification of common vetch variety by adopting SCoT molecular marker | |
| CN117512176A (en) | SNP molecular marker closely linked with brassica napus pod length and grain weight gene BnARF18 and application thereof | |
| CN104673790A (en) | Molecular-specificity labeling primer for oil-tea good-variety longlin 18 and identification method | |
| CN112029894A (en) | Fingerprint of Chinese arborvitae SSR marker as well as construction method and application thereof | |
| CN116240207A (en) | Primer group for identifying Sanhua plums and early plums and application thereof | |
| CN102618555B (en) | Nucleotide sequence of gamma-alcohol-soluble protein gene and application thereof | |
| CN115927731A (en) | SNP (Single nucleotide polymorphism) site combination for constructing litchi SNP fingerprint, application and identification method | |
| CN104946641A (en) | Specificity labeling primer for camellia oleifera improved varieties changlin number three and twenty one and detection method | |
| CN108517373A (en) | It one InDel labeled primer pair for distinguishing five pepper cultivation kinds and its applies | |
| CN113481313B (en) | Multiple fluorescence SSR (simple sequence repeat) labeled primers and method for identifying three Chinese torreya varieties | |
| CN113493852B (en) | Primer and method for identifying Yushan fish torreya, fine Chinese torreya and large Chinese torreya | |
| CN105631245A (en) | Dedicated primer used for identifying superior clones of Populus deltoides Marsh. and identification method for superior clone of Populus deltoides Marsh. | |
| CN113718049B (en) | Multiple-labeled primer for identifying torreya grandis, syzygium aromaticum and torreya grandis of torreya grandis varieties | |
| TWI447227B (en) | Method and kit for identifying phalaenopsis varieties | |
| CN113528693B (en) | Primer and method for identifying torreya grandis variety torreya grandis, syzygium aromaticum and torreya grandis | |
| CN113846176A (en) | Multi-labeled primer for identifying variety of torreya grandis, cephalospora grandis and cephalotaxus grandis | |
| CN104975010A (en) | High-specificity molecular specific labeled primers of oil-tea camellia fine breed Changlin No. 21 and detection method thereof | |
| CN114058724B (en) | Multiple SSR (simple sequence repeat) marker primer and method for identifying premature yuhuan shaddock' early yuwendan | |
| CN112831593B (en) | SNP molecular marker primer for variety identification of red peach trees in early summer and identification and application method thereof | |
| CN116411127B (en) | Molecular marker primer combination for rapidly identifying mature-period characters of peach fruits and application thereof | |
| CN110129469B (en) | Characteristic nucleotide sequence, specific primer and identification method of improved camellia oleifera variety GanYong 5 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |