CN104611414A - Method for identification of pomegranate variety by SSR primers and application - Google Patents
Method for identification of pomegranate variety by SSR primers and application Download PDFInfo
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- CN104611414A CN104611414A CN201410612733.0A CN201410612733A CN104611414A CN 104611414 A CN104611414 A CN 104611414A CN 201410612733 A CN201410612733 A CN 201410612733A CN 104611414 A CN104611414 A CN 104611414A
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- pomegranate
- dna
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- identification
- ssr
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- 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
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- 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
- C12Q2600/13—Plant traits
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- 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
- C12Q2600/16—Primer sets for multiplex assays
Abstract
Relating to the technological field of molecular biology, the invention specifically relates to an SSR primer set and a method for identification of pomegranate variety by the primers. The SSR primer set is composed of 20 primers. The identification steps include: (1) pomegranate leaf DNA extraction; (2) PCR amplification; (3) electrophoresis detection; (4) data recording; and (5) result determination. The primers involved in the invention have high specificity and good amplification efficiency, can quickly identify different varieties of pomegranate, and have the characteristics of short period, low cost, reliable result, manpower saving, material resource and land resource saving, and the like. The method provided by the invention can be used for variety identification on test pomegranate varieties and known pomegranate varieties, or construction of a pomegranate variety database. The method lays the foundation for accurate identification of pomegranate germplasm resources, especially synonym and homonym phenomena, and also lays the foundation for new variety protection.
Description
technical field:
The present invention relates to field of molecular biotechnology, utilize ssr primer to identify method and the application of pomegranate kind specifically.
background technology:
Cultivar identification is the important method in agriculture production, crop breeding and Seed Inspection, is also cover crop kind, the important means that prevents fake and forged kind from coming into the market.Traditional fruit variety method of inspection has Morphological Identification and Identification of Isozyme etc., but needed for Morphological Identification, the cycle is long, at substantial human and material resources and financial resources.The pure important step being pomegranate and producing of pomegranate kind.In pomegranate produces, various variet complexity phenomenon is serious, homonym, synonym phenomenon are serious, general survey is consuming time longer, field shape qualification difficulty is larger, along with the develop rapidly of Protocols in Molecular Biology, molecular marking technique is constantly ripe, opens approach to fruit tree from the qualification kind genomic level.
summary of the invention:
For realizing produced problem in above-mentioned pomegranate cultivar identification, the object of the invention is to, propose to utilize ssr primer to identify method and the application of pomegranate kind, the method utilizes molecular marking technique, realizes the qualification of pomegranate product, have the cycle short in vegetative growth phase, cost is low, reliable results, saves human and material resources and land resources, substantially increases the accuracy of cultivar identification.
To achieve these goals, the technical scheme that the present invention takes is: a kind of SSR primer sets, and the sequence of this SSR primer sets is as follows:
Pom04 F:TCCTTTTTACCCAATTTTCA
R:TGCACATCTTTTGCTGTAAG
Pom06 F:TACTAGGTGGAACCGAACTT
R:CCTTGACAACCTCATCTCAT
Pom10 F:CCTCATTGCTGATGAATCTT
R:ACTCGAGAAGCTCTGTGAAG
Pom13 F:CACACCCTTCATCAAAAGAT
R:GGACTAACAACCAGCCATAG
Pom14 F:CGCATTTGGTTGTAGAAGAC
R:AGGAGCGTCTGTTTTAATCTT
Pom21 F:GACTGGAAGAAGCAGAGACT
R:GAAAAGGAAGTAGCAGAGCA
Pom24 F:GGAGATTTGAATTGGGAAGT
R:GTGGACTAACTCAAGCAAGG
Pom39 F:TAGTTGAATAGGCCACATCC
R:CTATACAGTCCGAGGACCAC
Pom46 F:CTTCCTCCTACCGAACTATG
R:CCCACTTTGACACTTCTACC
Pom55 F:GAGACAATTGGGATCAGAAA
R:AGTCGACGAACTGTGAAATC
Pom56 F:CTCGCCATACTACTGAAAGG
R:ATTGGGTGAGATATGTTTGG
Another object of the present invention is to provide a kind of method utilizing SSR primer sets to identify pomegranate kind, comprises the steps:
(1) each sample pomegranate kind matter DNA is extracted;
(2) 11 pairs of pomegranate SR primers of design are utilized, the genomic dna of amplification pomegranate;
(3) amplified band of each sample of electrophoresis detection;
(4) the DNA cloning banding pattern of each often pair, sample primer is recorded;
(5) qualification pomegranate kind is distinguished according to DNA cloning banding pattern;
Further, in described step (2), pcr amplification program is 94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 60 DEG C of renaturation 45s, 72 DEG C extend lmin, 35 circulations; Last 72 DEG C extend lOmin, 4 DEG C of preservations.
Further, in described step (2), PCR amplification system comprises 20ng template DNA, 2.5 mmol/L Mg
2+, 0.2mmol/L dNTPs, lU Taq DNA polymerase, every bar primer 2 50 nmol/L, 10 X Buffero, surplus ddH in system
2o polishing.
Further, when described step (5) result judges, if two kinds have different equipotential in two or more SSR marker sites, then different varieties is judged to be.
Advantageous Effects of the present invention is: primer specificity of the present invention is high, and amplification efficiency is good, and the different varieties of energy Rapid identification pomegranate, have the cycle short, cost is low, reliable results, saves human and material resources and land resource characteristics.
accompanying drawing illustrates:
Fig. 1 the inventive method SSR primer detects figure through pcr amplification after product.The amplification figure of primer Pom21 and Pom46 in pomegranate kind matter.
The different varieties cluster analysis tree derivation that Fig. 2 utilizes the inventive method to produce.
Fig. 3 utilizes primer Pom14 to detect different varieties polyacrylamide gel electrophoresis result figure.
Fig. 4 utilizes primer Pom55 to detect different varieties polyacrylamide gel electrophoresis result figure.
embodiment:
below in conjunction with concrete embodiment, the present invention is described further:
embodiment
materials and methods
material
Pomegranate kind garden, fruit comprehensive test station, fruit industry technical system Bengbu, Anhui Province is all picked up from for examination material.Collect the pomegranate principal item of Bengbu Huaiyuan County cultivation.Comprising large stupid son and bud mutation new variety (' 73-06 ') thereof.To each kind numbering mark in table 1.
Table 1 pomegranate variety name, numbering
method
the extraction of genomic dna
The extraction of pomegranate genomic dna adopts modified CTAB method.
pomegranate Genome DNA content and purity testing
spectrophotometer method
Nucleic acid absorbs UV-light because of the phenyl ring containing conjugation, and the maximal ultraviolet light absorption wavelength of DNA and RNA is 260 nm, and the maximum absorption wavelength of protein is 280 nm.The roughly purity of dsDNA can be determined by the ratio of the photoabsorption of 260 nm and 280 nm.The OD of pure dsDNA
260/ OD
280be 1.8, the OD of pure rna
260/ OD
280be 2.0, if the OD of DNA sample
260/ OD
280being greater than 1.8 explanations has RNA to pollute.Be less than 1.8 explanations and have protein contamination.The DNA sample every part extracted is drawn 15 μ L deionized waters and is diluted to 3 mL, then on ultraviolet spectrophotometer, measure its OD respectively
260, OD
280value, usual OD
260=1, DNA concentration is equivalent to 50 μ g/mL, and therefore DNA concentration can use following formulae discovery: DNA concentration (μ g/mL)=OD
260× 50 × 200(extension rate)
detected through gel electrophoresis
Get 5 μ L DNA extraction liquid, agarose gel electrophoresis 30 ~ 40 min in 1.0%, voltage stabilizing 80 V, take a picture, the molecular size range of the genomic dna of Detection and Extraction and the readability of band.
analyze
Pomegranate SSR reaction system comprises 20ng template DNA, 2.5 mmol/L Mg
2+, 0.2mmol/L dNTPs, lU Taq DNA polymerase, every bar primer 2 50 nmol/L, 10 X Buffero, surplus ddH in system
2o polishing.
Ultraviolet transilluminator observes electrophoresis result, and takes pictures (regulating the time shutter) with black and white camera, processing, and picture is scanned.
According to the presence or absence coding of band on collection of illustrative plates, having band for " 1 ", is " 0 " without band.For weak band, occur or twice appearance in repeating for three times simultaneously, think there is band, just once occur, think without band.For the blanking bar that resolving power is very poor, weak band does not participate in coding.
electrophoretic
[application SPSS 11.0 for Windows software after coding, carry out Hierarchical Clustering (Hierarchical cluster), cluster coefficients adopt Euclidean distance square between (Squared Euclidean distance) and group connection method (between-groups linkage) analyze, obtain the Dendrogram of pomegranate 10 analyzing samples.
Results and analysis
product detects
After pcr amplification reaction, product 1.2% agarose gel electrophoresis detects.As seen from Figure 1, primer Pom21(is left) and the Pom46(right side) after pcr amplification, through agarose gel electrophoresis, band is had near 100-200 bp, explanation has object band to produce, and reaction creates object product, can through polyacrylamide gel electrophoresis test strip size and site.
core primers screens
Different primers directly affects the result of test, in order to not have an impact to result, should choose band clear, reproducible, and the result that the primer amplification being convenient to add up goes out is analyzed.This test, in 11 pairs of primers used, filters out applicable altogether, the good SSR primer 8 of polymorphism to selected primer (see table 2), for last polyacrylamide gel electrophoresis.
Table 2 SSR primer numbers and sequence
Note: in table, black matrix person is 8 primers filtered out.
cluster analysis
Band after electrophoresis is added up, utilizes SPSS statistical software to carry out cluster analysis (table 3), draw system tree figure (Fig. 2).
Table 3 cluster analysis table
Based on the pomegranate germ plasm resource system tree figure (see figure 2) that SSR generates, when Euclidean distance squared modulus threshold value is 14.00-15.00, it is 6 large classes that all kinds are gathered, the 1st large class totally 3 kinds, is mixed form by ' 73-06 ', large stupid son and rascal; 2nd large class totally 2 kinds, are made up of carnelian seed and rouge and powder skin; 3rd large class totally 2 kinds, are made up of Yunnan Sweet fruit of Pomegranate, white jade carpolite; 4th large class totally a kind, for thin skin is rough; 5th large class totally a kind is jade seed; 6th large class totally a kind is the green seed of acid.
clone kind ' 73-06 ' makes a variation strip analysis
By ssr analysis, " large stupid son ' with its clone mutational variety ' 73-06 ' in 8 primers screened, have 5 primer amplifications to go out 8 specific bands.Compared with ' large stupid son ', the band of ' 73-06 ' 1 treaty 490 bp more than on primer Pom13 increases figure; The band (see figure 3) of many 1 treaty 35 bp on primer Pom14; The band of few 3 treaty 62 bp, 110 bp, 180 bp on primer Pom21; The band of many 1 treaty 140 bp on primer Pom39; The band (see figure 4) of few 2 treaty 60 bp, 87 bp on primer Pom55.
conclusion
the Genetic relationship of 3.1 pomegranate germ plasm resources
[0047] the pomegranate germ plasm resource cluster analysis based on SSR marker shows, when Euclidean distance squared modulus threshold value is 14.00-15.00, it is 6 large classes that all kinds are gathered, and has the trend according to pedigree cluster between kind.And large stupid son and its clone make a variation, ' 73-06 ' can be classified as a class in Euclidean distance squared modulus threshold value 10.00, and gathering the earliest is a class.
SSR cluster analysis is thought, large stupid son and its clone make a variation ' 73-06 ' be classified as a class, the genetic distance between them is short, has very near sibship.
" large stupid son " clone kind ' 73-06 ' makes a variation strip analysis
Use SSR molecular marker technology, " large stupid son ' with its clone mutational variety ' 73-06 ' in the 8 pairs of SSR special primers screened, have 2 pairs of primer amplifications to go out 3 specific bands, variant sites is more.Variation has in a big way been there is in ' 73-06 ' comparatively " large stupid son " on DNA level.
sequence table
[0001]
<110> Institute of Gardening, Anhui Academy of Agricultural Sciences
<120> utilizes ssr primer to identify method and the application of pomegranate bud mutation kind
<130> claims, specification sheets
<160>22
<170>PatentIn version 3.3
<210>1
<211>20
<212>DNA
<213>Pom04-F
<400>1
TCCTTTTTACCCAATTTTCA
<210>2
<211>20
<212>DNA
<213>Pom04-R
<400>2
TGCACATCTTTTGCTGTAAG
<210>3
<211>20
<212>DNA
<213>Pom06-F
<400>3
TACTAGGTGGAACCGAACTT
<210>4
<211>20
<212>DNA
<213>Pom06-R
<400>4
CCTTGACAACCTCATCTCAT
<210>5
<211>20
<212>DNA
<213>Pom10-F
<400>5
CCTCATTGCTGATGAATCTT
<210>6
<211>20
<212>DNA
<213>Pom10-R
<400>6
ACTCGAGAAGCTCTGTGAAG
<210>7
<211>20
<212>DNA
<213>Pom13-F
<400>7
CACACCCTTCATCAAAAGAT
<210>8
<211>20
<212>DNA
<213>Pom13-R
<400>8
GGACTAACAACCAGCCATAG
<210>9
<211>20
<212>DNA
<213>Pom14-F
<400>9
CGCATTTGGTTGTAGAAGAC
<210>1O
<211>20
<212>DNA
<213>Pom14-R
<400>10
AGGAGCGTCTGTTTTAATCTT
<210>11
<211>20
<212>DNA
<213>Pom21-F
<400>10
GACTGGAAGAAGCAGAGACT
<210>12
<211>20
<212>DNA
<213>Pom21-R
<400>10
GAAAAGGAAGTAGCAGAGCA
<210>13
<211>20
<212>DNA
<213>Pom24-F
<400>10
GGAGATTTGAATTGGGAAGT
<210>14
<211>20
<212>DNA
<213>Pom24-R
<400>10
GTGGACTAACTCAAGCAAGG
<210>15
<211>20
<212>DNA
<213>Pom39-F
<400>10
TAGTTGAATAGGCCACATCC
<210>16
<211>20
<212>DNA
<213>Pom39-R
<400>10
CTATACAGTCCGAGGACCAC
<210>17
<211>20
<212>DNA
<213>Pom46-F
<400>10
CTTCCTCCTACCGAACTATG
<210>18
<211>20
<212>DNA
<213>Pom46-R
<400>10
CCCACTTTGACACTTCTACC
<210>19
<211>20
<212>DNA
<213>Pom55-F
<400>10
GAGACAATTGGGATCAGAAA
<210>20
<211>20
<212>DNA
<213>Pom55-R
<400>10
AGTCGACGAACTGTGAAATC
<210>21
<211>20
<212>DNA
<213>Pom56-F
<400>10
CTCGCCATACTACTGAAAGG
<210>22
<211>20
<212>DNA
<213>Pom56-R
<400>10
ATTGGGTGAGATATGTTTGG
Claims (3)
1. utilize ssr primer to identify method and the application of pomegranate kind, it is characterized in that, comprise the following steps:
(1) each sample pomegranate kind matter DNA is extracted;
(2) 11 pairs of pomegranate SSR primers of design are utilized, the genomic dna of amplification pomegranate;
(3) amplified band of each sample of electrophoresis detection;
(4) the DNA cloning banding pattern of each often pair, sample primer is recorded;
(5) qualification pomegranate kind is distinguished according to DNA cloning banding pattern;
11 pairs of described pomegranate SSR primer sequences are as follows:
Pom04 F:TCCTTTTTACCCAATTTTCA
R:TGCACATCTTTTGCTGTAAG
Pom06 F:TACTAGGTGGAACCGAACTT
R:CCTTGACAACCTCATCTCAT
Pom10 F:CCTCATTGCTGATGAATCTT
R:ACTCGAGAAGCTCTGTGAAG
Pom13 F:CACACCCTTCATCAAAAGAT
R:GGACTAACAACCAGCCATAG
Pom14 F:CGCATTTGGTTGTAGAAGAC
R:AGGAGCGTCTGTTTTAATCTT
Pom21 F:GACTGGAAGAAGCAGAGACT
R:GAAAAGGAAGTAGCAGAGCA
Pom24 F:GGAGATTTGAATTGGGAAGT
R:GTGGACTAACTCAAGCAAGG
Pom39 F:TAGTTGAATAGGCCACATCC
R:CTATACAGTCCGAGGACCAC
Pom46 F:CTTCCTCCTACCGAACTATG
R:CCCACTTTGACACTTCTACC
Pom55 F:GAGACAATTGGGATCAGAAA
R:AGTCGACGAACTGTGAAATC
Pom56 F:CTCGCCATACTACTGAAAGG
R:ATTGGGTGAGATATGTTTGG
Required by right, utilize ssr primer to identify method and the application of pomegranate kind described in 1, it is characterized in that, in described step (2), pcr amplification program is 94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 60 DEG C of renaturation 45s, 72 DEG C extend lmin, 35 circulations; Last 72 DEG C extend lOmin, 4 DEG C of preservations.
2. utilize ssr primer to identify method and the application of pomegranate kind according to claim 3, it is characterized in that, in described step (2), PCR amplification system comprises 20ng template DNA, 2.5 mmol/L Mg
2+, 0.2mmol/L dNTPs, lU Taq DNA polymerase, every bar primer 2 50 nmol/L, 10 X Buffero, surplus ddH in system
2o polishing.
3. utilize ssr primer to identify method and the application of pomegranate kind according to claim 2, when it is characterized in that described step (5) result judges, if two kinds have different equipotential in two or more SSR marker sites, be then judged to be different varieties.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106834505A (en) * | 2017-03-15 | 2017-06-13 | 中国农业科学院郑州果树研究所 | A kind of method for building pomegranate kind identity card and its application |
| CN110172528A (en) * | 2019-07-12 | 2019-08-27 | 安徽省农业科学院园艺研究所 | Dedicated SSR primer and its application of a pair for pomegranate cultivar identification |
| CN110205403A (en) * | 2019-07-12 | 2019-09-06 | 安徽省农业科学院园艺研究所 | A pair of high polymorphism pomegranate SSR primer and its application |
| CN110205404A (en) * | 2019-07-12 | 2019-09-06 | 安徽省农业科学院园艺研究所 | SSR core primers group and its application based on the exploitation of pomegranate whole genome sequence |
| CN110283931A (en) * | 2019-07-12 | 2019-09-27 | 安徽省农业科学院园艺研究所 | The SSR finger-print and its construction method of 6 Northern Huaihe River Anhui pomegranate excellent variety and application |
| CN110283930A (en) * | 2019-07-12 | 2019-09-27 | 安徽省农业科学院园艺研究所 | The SSR finger-print and its construction method of 6 Huaiyuan pomegranate excellent variety and application |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106834505A (en) * | 2017-03-15 | 2017-06-13 | 中国农业科学院郑州果树研究所 | A kind of method for building pomegranate kind identity card and its application |
| CN110172528A (en) * | 2019-07-12 | 2019-08-27 | 安徽省农业科学院园艺研究所 | Dedicated SSR primer and its application of a pair for pomegranate cultivar identification |
| CN110205403A (en) * | 2019-07-12 | 2019-09-06 | 安徽省农业科学院园艺研究所 | A pair of high polymorphism pomegranate SSR primer and its application |
| CN110205404A (en) * | 2019-07-12 | 2019-09-06 | 安徽省农业科学院园艺研究所 | SSR core primers group and its application based on the exploitation of pomegranate whole genome sequence |
| CN110283931A (en) * | 2019-07-12 | 2019-09-27 | 安徽省农业科学院园艺研究所 | The SSR finger-print and its construction method of 6 Northern Huaihe River Anhui pomegranate excellent variety and application |
| CN110283930A (en) * | 2019-07-12 | 2019-09-27 | 安徽省农业科学院园艺研究所 | The SSR finger-print and its construction method of 6 Huaiyuan pomegranate excellent variety and application |
| CN110205404B (en) * | 2019-07-12 | 2021-07-30 | 安徽省农业科学院园艺研究所 | SSR core primer group developed based on pomegranate whole genome sequence and application thereof |
| CN110172528B (en) * | 2019-07-12 | 2022-03-22 | 安徽省农业科学院园艺研究所 | Special SSR primers for pomegranate variety identification and application thereof |
| CN110283931B (en) * | 2019-07-12 | 2022-04-26 | 安徽省农业科学院园艺研究所 | SSR fingerprint of 6 good varieties of pomegranate in Anhui, Huaihei and China, and construction method and application thereof |
| CN110283930B (en) * | 2019-07-12 | 2022-06-07 | 安徽省农业科学院园艺研究所 | SSR fingerprint of 6 Huaiyuan pomegranate excellent varieties and construction method and application thereof |
| CN110205403B (en) * | 2019-07-12 | 2022-08-09 | 安徽省农业科学院园艺研究所 | SSR primers for high-polymorphism pomegranate and application thereof |
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|---|---|
| CN104611414B (en) | 2016-05-11 |
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