CN106834505A - A kind of method for building pomegranate kind identity card and its application - Google Patents
A kind of method for building pomegranate kind identity card and its application Download PDFInfo
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Abstract
The invention discloses a kind of method for building pomegranate kind identity card and its application, the method is comprised the following steps：The blade of different Punica granatum L. Germplasm Resources Baseds is won in seedling stage, for the extraction of DNA；Using the SSR molecular marker primer pair, the DNA to extracting enters performing PCR reaction；Electrophoresis detection；Data analysis.The present invention constructs the SSR fingerprint network databases of tool query function, realization is accurately distinguished to different regions pomegranate resource, successfully molecular marker assisted selection breeding technique is applied in the pomegranate breeding work of reality, is that detection unit and breeder provide easily network data exchange and a shared platform.It is that hybridization pomegranate variety authentication and Purity are provided fundamental basis in the seed selection work of various trait pomegranate, the workload of pomegranate breeding can be significantly reduced, improve the accuracy and purpose of Juvenile stage, shorten breeding time, is greatly enhanced breeding efficiency.
Description
Technical field
The invention belongs to gene engineering technology field, it is related to a kind of method for building pomegranate kind identity card and its application,
Specifically, it is related to one kind to be combined using the information such as SSR molecular fingerprints and kind morphological feature characteristic, building has inquiry
The pomegranate SSR fingerprint network databases of function.
Background technology
Pomegranate (Punica granatum L.) is if if alias pomegranate, pomegranate, pellet, day slurry, painting woods etc., are Punicaceae
(Punicaceae) Punica, machaka or dungarunga, originate in the Central Asia such as Iran, Afghanistan and Caucasia, at me
Existing more than the 2000 years cultivation history of state, is one of excellent small miscellaneous fruit fruit tree that China quickly grows in recent years, is currently formed
Main Cultivation centered on Xinjiang Yecheng, Lintong, Kaifeng, Anhui Huaiyuan, Shandong Yi Cheng and Mengzi Yunnan etc.
Region, it is increasingly favored with its economy higher, nutrition, pharmaceutical value and healthcare function by consumption market.But due to
Introducing a fine variety between cross-pollination and different geographical exchanges with kind, causes variet complexity, pedigree unclear, synonym or of the same name different
Thing phenomenon is serious.In addition the preference of long-term vegetative propagation and the mankind, makes pomegranate genotype tend to single, causes gene diversity
Reduce, the phenomenon such as variety deterioration.At present, the distant hybridization of pomegranate germ plasm resource has turned into an important way of Germplasm enhancement
Footpath.Therefore, the affiliation for specifying various trait kind and the genetic diversity for evaluating pomegranate germplasm, to pomegranate germ plasm resource
Protection, high-efficient breeding and effective utilization have important scientific meaning.But there is no a kind of accurate and effective in pomegranate breeding work
Molecular labeling be used for various trait kind affiliation and evaluate pomegranate germplasm genetic diversity.Domestic and foreign scholars are main
Pomegranate is studied with morphological method and molecular marking technique.But morphological method is easily influenceed by season and environment,
Qualification result is inaccurate.Pomegranate cultivar identification mainly is carried out using technologies such as RAPD, AFLP, SRAP on a molecular scale, and
Majority is concentrated on to be studied the pomegranate kind in a certain area, comes that source range is narrower, and the kind quantity being related to is few, not enough system
System.Therefore, using it is more stable, more accurately molecular marking technique identification pomegranate kind affiliation and evaluate genetic diversity gesture
Must go.
And as pomegranate cultivated area expands rapidly, nursery stock variet complexity phenomenon especially severe causes pole to production
Big confusion.By carrying out cultivar identification, can be likelihood and body between kind essential information, kind molecular identity card, kind
Part card verification etc. is inquired about, it is ensured that the authenticity and purity of kind, and property right protection tool is implemented to excellent pomegranate kind or material
There is very great meaning.Can also instruct to hybridize heredity between pomegranate variety authentication and Purity, analysis parent simultaneously similar
Property, the reference of parental apolegamy is provided for the seed selection for hybridizing pomegranate Combination nova.This promotes industry hair to accelerating pomegranate improved variety process
Exhibition, safeguards that the interests of breeder and the producer are significant.
The content of the invention
It is an object of the invention to provide a kind of method for building pomegranate kind identity card and its application.
An object of the present invention is to provide a kind of a kind of and pomegranate cultivar identification and use based on SSR sites exploitation
In the primer pair for expanding the mark.
The second object of the present invention is the fingerprint that pomegranate kind is obtained using Capillary Electrophoresis SSR Fluorometric assay of fluorescence-labeled
Information, finger print information is combined with the information such as kind morphological feature characteristic etc. of pomegranate, build pomegranate kind identity card and
Its application in the work of pomegranate breeding of new variety.
Its concrete technical scheme is：
A kind of method for building pomegranate kind identity card, comprises the following steps：
Step 1, the blade that different Punica granatum L. Germplasm Resources Baseds are won in seedling stage, for the extraction of DNA；
The DNA of the fresh blade of pomegranate is extracted using modified CTAB method
(1) 2 × CTAB Extraction buffers (0.1MTris-HCl, the 0.02M for configuring in advance are preheated in 65 DEG C of water-baths
EDTA, 1.4M NaCl, 2%PVP, 2%CTAB)；
(2) pomegranate blade is ground in liquid nitrogen to powdered, in going to 2.0mL centrifuge tubes；
(3) 2 × CTAB Extraction buffers 1.0mL is rapidly added, 10 μ l beta -mercaptoethanols are mixed, 65 DEG C of water-bath 60min,
Overturned every 10-15min and shaken up once；
(4) 12000rpm centrifugations 5min under normal temperature, sucts and is transferred to clearly in new 2.0mL centrifuge tubes；
(5) isometric chloroform/isoamyl alcohol is added, 5min is fully mixed, 5min, the centrifugation of 12000rpm normal temperature is placed
10min；
(6) suct clear in new 2.0mL centrifuge tubes, extracting is repeated once with chloroform/isoamyl alcohol；
(7) it is centrifuged, sucts clear in new centrifuge tube, the isometric isopropanol mixing of addition, -20 DEG C of placement 2h, precipitation
DNA；
(8) 10000rpm normal temperature centrifugation 10min；
(9) isopropanol is outwelled, precipitation is washed twice with 500 μ l ethanol, with 100 μ lTE dissolving DNAs after drying；
(10) 10mg/mlRNaseA2 μ l final concentrations (200ng/ul) is added to mix, 37 DEG C of insulation more than 2h；
(11) 500 μ lTE are added, isometric chloroform/isoamyl alcohol (24 is added:1), gently overturn and shake up 2min；
(12) 12000rpm normal temperature centrifugation 10min；
(13) clear in new centrifuge tube, the isometric isopropanol mixing of addition, -20 DEG C of placement 2h, precipitation DNA are sucted
(14) 12000rpm normal temperature centrifugation 10min；
(15) isopropanol is outwelled, precipitation is cleaned with 500 μ l ethanol twice, with 50 μ lTE dissolving DNAs after drying；4 DEG C of refrigerators
Middle preservation；
(16) quality of DNA is detected using 1.5% agarose gel electrophoresis.
Step 2, using the SSR molecular marker primer pair, the DNA to extracting enters performing PCR；
For 13 pairs of SSR core labeled primers of pomegranate varieties systematics analysis, Microbial Technics Ltd is read by Beijing
Synthesis, and respectively with FAM (blueness), HEX (green), TAMRA (yellow), 4 kinds of fluorophors modification sense primers of ROX (red)
5 ' ends.
Step 3, PCR reaction systems (15 μ l) are as follows：10 × Buffer I, 1.5 μ l, 2.5mM dNTP 1.2 μ l, TP-
The μ l of M13 (5 μM) 1,1 μ l, TAKARA HS Taq of special primer (5 μM) 0.1 μ l, DNA1.2 μ l, plus ddH2O polishings are to 15 μ l.
Step 4, PCR response procedures：(1) primer 2 363,14870,68159,16262,16942,5347 PCR reaction intervals
Sequence is 95 DEG C of 5min, 94 DEG C of 30s, 56 DEG C of 30s, and 35 circulate, then 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, 15 circulations, most
60 DEG C of 30min afterwards, totally 50 circulations.(2) primer 34768,18616,27944,37300,41575,47743,61814 PCR
Response procedures are 95 DEG C of 5min, 94 DEG C of 30s, and 58 DEG C drop to 48 DEG C of 30s, 72 DEG C of 30s, and 10 circulate, 94 DEG C of 30s, 48 DEG C of 30s,
72 DEG C of 30s, 20 circulations, 95 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, 10 circulations, last 60 DEG C of 30min, totally 40 circulations.
Step 5, electrophoresis detection
Molecular weight internal standard and the μ l of formamide mixed liquor 9, wherein the μ l of PCR primer 1.0, the molecular weight are added in 96 orifice plates per hole
The volume ratio of both internal standard and formamide mixed liquor is 0.5：8.5.PCR primer is diluted 40 times, in each Kong Zhongfen of 96 orifice plates
The PCR primer after 8.5 μ l deionized formamides, 0.5 μ l LIZ-500 molecular weight internal standards and 1 μ l dilution, 95 DEG C of denaturation are not added
3min, 10min, 4000 turns of min are placed in 4 DEG C-1Centrifugation 1min, in carrying out Capillary Electrophoresis on ABI3730DNA analyzers.In advance
Electrophoresis 15kV, 3min；2kV electricity sample introductions 10s；Electrophoresis 15kV；20min.
Step 6, data analysis
Initial data is collected with Data Collection softwares, the initial data collected with Genemapper software analysis,
Compare the LIZ-500 molecular weight internal standards in the position and its swimming lane of each peak value, obtain the accurate molecular weight bp of SSR amplified fragments；
The DNA fingerprint information of pomegranate kind is encoded, the information such as morphological feature characteristic in conjunction with kind and may have it is special
Gene information, builds 136 parts of identity cards of different regions pomegranate varieties of resources, and utilize online barcode generator fhttp://
barcode.tecit.combarcodege.nerator.aspxLANG=zhcn) and Quick Response Code generation software Encoder2 with
The pomegranate kind identity card that bar code statement builds.
Application of the method for building pomegranate kind identity card of the present invention in pomegranate breeding process.
Compared with prior art, beneficial effects of the present invention：
The method of the invention can be simple and quick the different pomegranate kinds of acquisition clip size, by above-mentioned pomegranate kind body
Part card, on seedling packing, the number of each kind is unique to labeling, is so both reached in the form of bar code or Quick Response Code
With the purpose of minimum mark zone graded kind, while reducing the workload of fingerprint analysis, it is ensured that the authenticity and purity of kind,
Implementing property right protection to excellent pomegranate kind or material has very great meaning.The section to kind seedling quality can also be realized
Review, Rapid identification and standardized administration.Simultaneously in the seed selection work of various trait pomegranate, the work of breeding is significantly reduced
Amount, shortens breeding time, is greatly enhanced breeding efficiency.Can also be the authenticity and Purity of hybridization pomegranate kind, analysis
Genetic similarity between parent, the reference of parental apolegamy is provided for the seed selection for hybridizing pomegranate Combination nova.Enter to accelerating pomegranate improved variety
Journey, promotes industry development, safeguards that the interests of breeder and the producer are significant.
Specific embodiment
Technical scheme is described in more detail with reference to specific embodiment.
The present invention is realized by following technology path：
SSR primers mentioned by the present invention are the Stepwise Screenings from 7810 SSR sequences that pomegranate gene order-checking is obtained
Go out.60 SSR fluorescence labelings with polymorphism are filtered out first to be expanded in for examination pomegranate kind, are produced according to amplification
Readability, the polymorphism of thing in Capillary Electrophoresis etc., finally filters out 13 SSR markers that polymorphism is higher, amplification is stable.
It is that, using 13 pairs of SSR primers, the 136 parts of pomegranate resources that will be collected from national different regions are all distinguished in the present invention
Open, expand the primer pair such as table 1 of the SSR marker：
Table 1
Using the SSR molecular marker, by simple and effective PCR, Capillary Electrophoresis operation etc. is realized to different regions stone
, successfully be applied to molecular marker assisted selection breeding technique in the pomegranate breeding work of reality by accurately distinguishing for pomegranate resource.
The method of the present invention can significantly reduce the workload of pomegranate breeding in the seed selection work of various trait pomegranate,
The accuracy and purpose of Juvenile stage are improved, shortens breeding time, be greatly enhanced breeding efficiency.
1st, first, the blade of different Punica granatum L. Germplasm Resources Baseds is won in seedling stage, for the extraction of DNA.
2nd, using the SSR molecular marker primer pair, the DNA to extracting enters performing PCR analysis.
3rd, PCR reaction systems are as shown in table 2:
System (15 μ l)：
Table 2
4th, PCR response procedures：
Primer：2363,14870,68159,16262,16942,5347.
Table 3
Primer：34768,18616,27944,37300,41575,47743,61814.
Table 4
5th, electrophoresis detection
Molecular weight internal standard and formamide mixed liquor (0.5 are added in (1) 96 orifice plate per hole：8.5) 9 μ l, the μ l of PCR primer 1.0；
(2) 95 DEG C of denaturation 3min, upper machine testing；
6th, data analysis
Initial data is collected with Data Collection softwares, the initial data collected with Genemapper software analysis,
Compare the LIZ-500 molecular weight internal standards in the position and its swimming lane of each peak value, obtain the accurate molecular weight of SSR amplified fragments
(bp).The DNA fingerprint information of pomegranate kind is encoded, morphological feature characteristic in conjunction with kind and the special base that may have
Because of information, 136 parts of identity cards of different regions pomegranate varieties of resources are built, and utilize online barcode generator fhttp://
barcode.tecit.combarcodege.nerator.aspxLANG=zhcn) and Quick Response Code generation software Encoder2 with
The pomegranate kind identity card that bar code statement builds.
The present invention constructs the SSR fingerprint network databases of tool query function, is that detection unit and breeder provide
One easily network data exchange and shared platform.In the seed selection work of various trait pomegranate, stone can be significantly reduced
The workload of pomegranate breeding, improves the accuracy and purpose of Juvenile stage, shortens breeding time, is greatly enhanced breeding efficiency.
The above, preferably specific embodiment only of the invention, protection scope of the present invention not limited to this are any ripe
Those skilled in the art are known in the technical scope of present disclosure, the letter of the technical scheme that can be become apparent to
Altered or equivalence replacement are each fallen within protection scope of the present invention.
Sequence table
SEQUENCE LISTING（Sequence table）
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Claims (3)
1. it is a kind of build pomegranate kind identity card method, it is characterised in that comprise the following steps：
Step 1, the blade that different Punica granatum L. Germplasm Resources Baseds are won in seedling stage, for the extraction of DNA；
Step 2, using the SSR molecular marker primer pair, the DNA to extracting enters performing PCR analysis；
For 13 pairs of SSR label primers of pomegranate varieties systematics analysis, Microbial Technics Ltd is read by Beijing and is synthesized, and point
Yong not FAM bluenesss, HEX greens, the red 4 kinds of fluorophors modification sense primer 5 ' end of TAMRA yellow, ROX；
Step 3, PCR reaction systems are as follows：10 × Buffer I, 1.5 μ l, 2.5mM dNTP 1.2,1 μ l of μ l, TP-M13, specifically
1 μ l, TAKARA HS Taq of primer 0.1,1.2 μ l of μ l, DNA, plus ddH2O polishings are to 15 μ l；
Step 4, PCR response procedures：(1) primer 2 363,14870,68159,16262,16942,5347 PCR response procedures are
95 DEG C of 5min, 94 DEG C of 30s, 56 DEG C of 30s, 35 circulate, then 94 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, 15 circulations, last 60
DEG C 30min, totally 50 circulations；(2) primer 34768,18616,27944,37300,41575,47743,61814 PCR reactions
Program is 95 DEG C of 5min, 94 DEG C of 30s, and 58 DEG C drop to 48 DEG C of 30s, 72 DEG C of 30s, 10 circulations, 94 DEG C of 30s, 48 DEG C of 30s, 72 DEG C
30s, 20 circulations, 95 DEG C of 30s, 53 DEG C of 30s, 72 DEG C of 30s, 10 circulations, last 60 DEG C of 30min, totally 40 circulations；
Step 5, electrophoresis detection
Molecular weight internal standard and the μ l of formamide mixed liquor 9, wherein the μ l of PCR primer 1.0, the molecular weight internal standard are added in 96 orifice plates per hole
It is 0.5 with the volume ratio of both formamide mixed liquors：8.5；PCR primer is diluted 40 times, is added respectively in each hole of 96 orifice plates
Enter the PCR primer after 8.5 μ l deionized formamides, 0.5 μ l LIZ-500 molecular weight internal standards and 1 μ l dilution, 95 DEG C are denatured 3min,
10min, 4000 turns of min are placed in 4 DEG C-1Centrifugation 1min, in carrying out Capillary Electrophoresis on ABI3730 DNA analysis instrument；Prerunning
15kV, 3min；2kV electricity sample introductions 10s；Electrophoresis 15kV, 20min；
Step 6, data analysis
Initial data is collected with Data Collection softwares, the initial data collected with Genemapper software analysis compares
LIZ-500 molecular weight internal standards in the position of each peak value and its swimming lane, obtain the accurate molecular weight bp of SSR amplified fragments；By stone
The DNA fingerprint information coding of pomegranate kind, morphological feature characteristic in conjunction with kind and the specific gene information that may have, structure
136 parts of molecular identity cards of different regions pomegranate varieties of resources are built, and utilizes online barcode generator http://
barcode.tecit.combarcodege.nerator.aspxLANG=zhcn and Quick Response Code generation software Encoder2 with
The pomegranate kind identity card that bar code statement builds.
2. it is according to claim 1 build pomegranate kind identity card method, it is characterised in that in step 1 using improvement
CTAB methods extract the fresh blade of pomegranate DNA, specifically include following steps：
(1) 2 × CTAB Extraction buffers for configuring in advance are preheated in 65 DEG C of water-baths, 0.1MTris-HCl, 0.02M EDTA,
1.4M NaCl, 2%PVP, 2%CTAB；
(2) pomegranate blade is ground in liquid nitrogen to powdered, in going to 2.0mL centrifuge tubes；
(3) 2 × CTAB Extraction buffers 1.0mL is rapidly added, 10 μ l beta -mercaptoethanols are mixed, 65 DEG C of water-bath 60min, every 10-
15min is reverse to be shaken up once；
(4) 12000rpm centrifugations 5min under normal temperature, sucts and is transferred to clearly in new 2.0mL centrifuge tubes；
(5) isometric chloroform/isoamyl alcohol is added, 5min is fully mixed, 5min, 12000rpm normal temperature centrifugation 10min is placed；
(6) suct clear in new 2.0mL centrifuge tubes, extracting is repeated once with chloroform/isoamyl alcohol；
(7) it is centrifuged, sucts clear in new centrifuge tube, the isometric isopropanol mixing of addition, -20 DEG C is placed 2h, precipitate DNA；
(8) 10000rpm normal temperature centrifugation 10min；
(9) isopropanol is outwelled, precipitation is washed twice with 500 μ l ethanol, with 100 μ lTE dissolving DNAs after drying；
(10) the μ l final concentrations 200ng/ul of 10mg/mlRNaseA 2 are added to mix, 37 DEG C of insulation more than 2h；
(11) 500 μ lTE are added, isometric chloroform/isoamyl alcohol is added, is gently overturned and is shaken up 2min；
(12) 12000rpm normal temperature centrifugation 10min；
(13) clear in new centrifuge tube, the isometric isopropanol mixing of addition, -20 DEG C of placement 2h, precipitation DNA are sucted；
(14) 12000rpm normal temperature centrifugation 10min；
(15) isopropanol is outwelled, precipitation is cleaned with 500 μ l ethanol twice, with 50 μ lTE dissolving DNAs after drying；Protected in 4 DEG C of refrigerators
Deposit.
3. application of the method for the structure pomegranate kind identity card described in claim 1 in pomegranate breeding process.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728572A (en) * | 2018-06-08 | 2018-11-02 | 棕榈生态城镇发展股份有限公司 | A kind of labeling method of calibration four seasons camellia hybrid new breed molecular identity card |
| 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 |
| 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 |
| 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 |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728572A (en) * | 2018-06-08 | 2018-11-02 | 棕榈生态城镇发展股份有限公司 | A kind of labeling method of calibration four seasons camellia hybrid new breed molecular identity card |
| CN108728572B (en) * | 2018-06-08 | 2021-10-22 | 棕榈生态城镇发展股份有限公司 | Marking method for marking molecular identity card of new hybrid variety of camellia at four seasons |
| 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 |
| 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 |
| 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 |
| 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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