CN104611414B - Utilize method and the application of ssr primer qualification pomegranate kind - Google Patents
Utilize method and the application of ssr primer qualification pomegranate kind Download PDFInfo
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Abstract
The present invention relates to field of molecular biotechnology, is a kind of SSR primer sets and the method for utilizing this primer qualification pomegranate kind thereof specifically; This SSR primer sets is made up of 20 primers; Authentication step comprises the extraction of (1) pomegranate leaf DNA; (2) pcr amplification; (3) electrophoresis detection; (4) data record; (5) result is judged. Primer specificity of the present invention is high, and amplification efficiency is good, and the different cultivars of energy Rapid identification pomegranate, has the cycle short, and cost is low, and reliable results is saved human and material resources and land resource characteristics. The present invention can be for to carrying out cultivar identification for examination pomegranate kind and known pomegranate kind, or structure pomegranate species data storehouse. The method is that precise Identification pomegranate germ plasm resource, especially synonym and homonym phenomenon are laid a good foundation, also for new varieties protection is laid a good foundation.
Description
Technical field: the present invention relates to field of molecular biotechnology, is to utilize ssr primer qualification pomegranate kind specificallyMethod and application.
Background technology:
Cultivar identification is the important method in agricultural production, crop breeding and Seed Inspection, is also cover crop kind, anti-Stop the important means that fake and forged kind comes into the market. Traditional fruit variety method of inspection has Morphological Identification and isodynamic enzyme mirrorFixed etc., still, the required cycle of Morphological Identification is long, expends a large amount of human and material resources and financial resources. The pure of pomegranate kind is that pomegranate is rawThe important step of producing. In pomegranate produces, various variet complexity phenomenons are serious, and homonym, synonym phenomenon are serious, routineIdentify consuming time longer, field shape learn qualification difficulty larger, along with the develop rapidly of Protocols in Molecular Biology, molecular marking techniqueConstantly ripe, open up approach to the qualification of fruit tree kind genomic level.
Summary of the invention:
For realizing the problem occurring in above-mentioned pomegranate cultivar identification, the object of the invention is to, propose to utilize ssr primer mirrorDetermine method and the application of pomegranate kind, the method is utilized molecular marking technique, realizes the qualification of pomegranate product in vegetative growth phase, hasCycle is short, and cost is low, and reliable results is saved human and material resources and land resource, has greatly improved the accuracy of cultivar identification.
To achieve these goals, the technical scheme that the present invention takes is: a kind of SSR primer sets, the order of this SSR primer setsBe listed as follows:
Pom04F:TCCTTTTTACCCAATTTTCA
R:TGCACATCTTTTGCTGTAAG
Pom06F:TACTAGGTGGAACCGAACTT
R:CCTTGACAACCTCATCTCAT
Pom10F:CCTCATTGCTGATGAATCTT
R:ACTCGAGAAGCTCTGTGAAG
Pom13F:CACACCCTTCATCAAAAGAT
R:GGACTAACAACCAGCCATAG
Pom14F:CGCATTTGGTTGTAGAAGAC
R:AGGAGCGTCTGTTTTAATCTT
Pom21F:GACTGGAAGAAGCAGAGACT
R:GAAAAGGAAGTAGCAGAGCA
Pom24F:GGAGATTTGAATTGGGAAGT
R:GTGGACTAACTCAAGCAAGG
Pom39F:TAGTTGAATAGGCCACATCC
R:CTATACAGTCCGAGGACCAC
Pom46F:CTTCCTCCTACCGAACTATG
R:CCCACTTTGACACTTCTACC
Pom55F:GAGACAATTGGGATCAGAAA
R:AGTCGACGAACTGTGAAATC
Pom56F:CTCGCCATACTACTGAAAGG
R:ATTGGGTGAGATATGTTTGG
Another object of the present invention is to provide a kind of method of the SSR of utilization primer sets qualification pomegranate kind, comprises as followsStep:
(1) extract each sample pomegranate germplasm DNA;
(2) utilize 11 pairs of pomegranate SR primers that design, the genomic DNA of amplification pomegranate;
(3) amplified band of the each sample of electrophoresis detection;
(4) record the DNA cloning banding pattern of every pair of primer of each sample;
(5) distinguish qualification pomegranate kind 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 renaturation45s, 72 DEG C are extended lmin, 35 circulations; Last 72 DEG C are extended lOmin, 4 DEG C of preservations.
Further, in described step (2), pcr amplification system comprises 20ng template DNA, 2.5mmol/LMg2+，0.2mmol/LdNTPs, lUTaqDNA polymerase, every primer 2 50nmol/L, 10XBuffero, in system, surplus is usedddH2O polishing.
Further, when described step (5) result is judged, if two kinds are at two or more SSR marker sitesThere are different equipotentials, be judged to be different cultivars.
Useful technique effect of the present invention is: primer specificity of the present invention is high, and amplification efficiency is good, can Rapid identification pomegranateDifferent cultivars, there is the cycle short, cost is low, reliable results is saved human and material resources and land resource characteristics.
Brief description of the drawings:
Fig. 1 the inventive method SSR primer detects figure through pcr amplification afterproduct. Primer Pom21 and Pom46 are at pomegranate germplasmIn amplification figure.
The different cultivars cluster analysis arborescence that Fig. 2 utilizes the inventive method to produce.
Fig. 3 utilizes primer Pom14 to detect different cultivars polyacrylamide gel electrophoresis result figure.
Fig. 4 utilizes primer Pom55 to detect different cultivars polyacrylamide gel electrophoresis result figure.
Detailed description of the invention:
Below in conjunction with concrete embodiment, the present invention is described further:
Embodiment
Materials and methods
Material
All pick up from pomegranate kind garden, fruit compbined test station, fruit industrial technology system Bengbu, Anhui Province for examination material. ReceiveThe pomegranate principal item of collection Bengbu Huaiyuan County cultivation. Comprising large stupid son and bud mutation new varieties (' 73-06 ') thereof. To each productPlant 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 ultraviolet light because of the phenyl ring that contains conjugation, and the maximal ultraviolet light absorption wavelength of DNA and RNA is 260nm,The maximum absorption wavelength of protein is 280nm. The roughly purity of dsDNA can be come by the ratio of the light absorption at 260nm and 280nm placeDetermine. The OD of pure dsDNA260/OD280Be 1.8, the OD of pure rna260/OD280Be 2.0, if the OD of DNA sample260/OD280Be greater than1.8 explanations have RNA to pollute. Be less than 1.8 explanations and have protein contamination. By every part, the DNA sample having extracted draw 15 μ L spend fromSub-water is diluted to 3mL, then on ultraviolet specrophotometer, measures respectively its OD260、OD280Value, conventionally OD260=1, DNA concentrationBe equivalent to 50 μ g/mL, therefore DNA concentration can be calculated with following formula: DNA concentration (μ g/mL)=OD260× 50 × 200(dilution doublyNumber)
Detected through gel electrophoresis
Get 5 μ LDNA extracts, in agarose gel electrophoresis 30 ~ 40min of 1.0%, voltage stabilizing 80V, takes a picture, and detection is carriedThe molecular size range of the genomic DNA of getting and the readability of band.
Analyze
Pomegranate SSR reaction system comprises 20ng template DNA, 2.5mmol/LMg2+，0.2mmol/LdNTPs，lUTaqDNA polymerase, every primer 2 50nmol/L, 10XBuffero, surplus ddH in system2O polishing.
On ultraviolet transilluminator, observe electrophoresis result, and take pictures (regulate time for exposure) with black and white camera, processing, andPicture is scanned.
According to the coding that has or not of band on collection of illustrative plates, there is band for " 1 ", be " 0 " without band. For weak band, the while in repeating for three timesOccur or twice appearance, think and have band, just once occur, think without band. For the very poor blanking bar of resolution ratio, weak band is not joinedAdd coding.
Electrophoretic
After coding, application SPSS11.0forWindows software, carries out Hierarchical Clustering (HierarchicalCluster), cluster coefficients adopts connection method between Euclidean distance square (SquaredEuclideandistance) and group(between-groupslinkage) analyze, obtain the Dendrogram of 10 analyzing samples of pomegranate.
Results and analysis
2.3.1SSR product detects
After pcr amplification reaction, product detects with 1.2% agarose gel electrophoresis. As seen from Figure 1, primer Pom21(left side) and the Pom46(right side) after pcr amplification, through agarose gel electrophoresis, near 100-200bp, there is band, order has been describedBand produce, reaction produced object product, can be through polyacrylamide gel electrophoresis test strip size and site.
Core primers screening
Different primers directly affects the result of test, in order result not to be exerted an influence, should choose band clear, repeatsProperty good, the result that goes out of primer amplification of being convenient to statistics is analyzed. This test, in 11 pairs of primers used, filters out applicable altogether, the good SSR primer 8 of polymorphism is to selected primer (be shown in table 2), for last polyacrylamide gel electrophoresis.
Table 2SSR primer numbering and sequence
Note: in table, black matrix person is 8 primers that filter out.
Cluster analysis
Band after electrophoresis is added up, utilized SPSS statistical software to carry out cluster analysis (table 3), the system that draws is tree-shapedFigure (Fig. 2).
Table 3 cluster analysis table
The pomegranate germ plasm resource system dendrogram (see figure 2) generating based on SSR, in an Euclidean distance square coefficient threshold value isWhen 14.00-15.00, it is 6 large classes that all kinds are gathered, and the 1st large class totally 3 kinds, by ' 73-06 ', large stupid son and rascal mixing groupBecome; The 2nd large class totally 2 kinds, are made up of carnelian seed and rouge and powder skin; The 3rd large class totally 2 kinds, by Yunnan pomegranate fruit, white jadeCarpolite composition; The 4th large class totally a kind, for thin skin rough; The 5th large class totally a kind is jade seed; The 6th large class totally 1 productKind, be the green seed of acid.
Clone kind ' 73-06 ' variation strip analysis
By ssr analysis, " large stupid son ' with its clone mutational variety ' 73-06 ' screening 8 primers in, have 5Primer amplification goes out 8 specific bands. Compared with ' large stupid son ', ' 73-06 ' be many 1 treaties 490 on primer Pom13 amplification figureThe band of bp; The band (see figure 3) of many 1 treaty 35bp on primer Pom14; Few 3 treaty 62bp, 110 on primer Pom21The band of bp, 180bp; The band of many 1 treaty 140bp on primer Pom39; Few 2 treaty 60bp, 87 on primer Pom55The band (see figure 4) of bp.
Conclusion
The Genetic relationship of pomegranate germ plasm resource
Pomegranate germ plasm resource cluster analysis based on SSR mark shows, is 14.00-in an Euclidean distance square coefficient threshold value15.00 o'clock, it was 6 large classes that all kinds are gathered, and has according to the trend of pedigree cluster between kind. And large stupid son and the variation of its clone' 73-06 ' can be classified as a class in an Euclidean distance square coefficient threshold value 10.00, and gathering is the earliest a class.
SSR cluster analysis thinks, large stupid son and its clone variation ' 73-06 ' are classified as a class, the heredity distance between themFrom short, there is very near affiliation.
" large stupid son " clone kind ' 73-06 ' variation strip analysis
Use SSR molecular marking technique, " large stupid son ' with the 8 pair SSR spies of its clone mutational variety ' 73-06 ' in screeningIn different primer, have 2 pairs of primer amplifications to go out 3 specific bands, variant sites is more. ' 73-06 ' " large stupid son " at DNA levelOn there is variation in a big way.
[0001]
<110>Academy of Agri-Science and Technology Anhui Province Horticultural Research Institute
<120>utilize ssr primer to identify method and the application of pomegranate bud mutation kind
<130>claims, description
<160>22
<170>PatentInversion3.3
<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. the method for utilizing ssr primer qualification pomegranate kind, is characterized in that, comprises the following steps:
(1) extract each sample pomegranate germplasm DNA;
(2) utilize 11 pairs of pomegranate SSR primers that design, the genomic DNA of amplification pomegranate;
(3) amplified band of the each sample of electrophoresis detection;
(4) record the DNA cloning banding pattern of every pair of primer of each sample;
(5) distinguish qualification pomegranate kind according to DNA cloning banding pattern;
11 pairs of described pomegranate SSR primer sequences are as follows:
Pom04F:TCCTTTTTACCCAATTTTCA
R:TGCACATCTTTTGCTGTAAG
Pom06F:TACTAGGTGGAACCGAACTT
R:CCTTGACAACCTCATCTCAT
Pom10F:CCTCATTGCTGATGAATCTT
R:ACTCGAGAAGCTCTGTGAAG
Pom13F:CACACCCTTCATCAAAAGAT
R:GGACTAACAACCAGCCATAG
Pom14F:CGCATTTGGTTGTAGAAGAC
R:AGGAGCGTCTGTTTTAATCTT
Pom21F:GACTGGAAGAAGCAGAGACT
R:GAAAAGGAAGTAGCAGAGCA
Pom24F:GGAGATTTGAATTGGGAAGT
R:GTGGACTAACTCAAGCAAGG
Pom39F:TAGTTGAATAGGCCACATCC
R:CTATACAGTCCGAGGACCAC
Pom46F:CTTCCTCCTACCGAACTATG
R:CCCACTTTGACACTTCTACC
Pom55F:GAGACAATTGGGATCAGAAA
R:AGTCGACGAACTGTGAAATC
Pom56F:CTCGCCATACTACTGAAAGG
R:ATTGGGTGAGATATGTTTGG。
2. the method that requires to utilize described in 1 ssr primer qualification pomegranate kind according to right, is characterized in that, in described step (2)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 are extended lmin, 35 circulations; Finally72 DEG C are extended l0min, 4 DEG C of preservations.
3. the method for utilizing according to claim 2 ssr primer qualification pomegranate kind, is characterized in that described step (5) resultWhen judgement, if two kinds have different equipotentials at two or more SSR marker sites, be judged to be different cultivars.
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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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
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| CN103667480A (en) * | 2013-12-06 | 2014-03-26 | 中国农业科学院油料作物研究所 | SSR core primer group developed based on sesame complete genomic sequence and application |
| CN103764842A (en) * | 2011-07-15 | 2014-04-30 | Acgt知识有限公司 | SSR markers for plants and uses thereof |
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| CN103764842A (en) * | 2011-07-15 | 2014-04-30 | Acgt知识有限公司 | SSR markers for plants and uses thereof |
| CN103667480A (en) * | 2013-12-06 | 2014-03-26 | 中国农业科学院油料作物研究所 | SSR core primer group developed based on sesame complete genomic sequence and application |
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| Title |
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| 石榴种质资源亲缘关系及其营养系芽变的RAPD、SSR分析;潘海发 等;《园艺学报》;20131231(第40期);全文 * |
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