CN104928396B - A kind of method using EST SSR molecular marker Rapid identification capsicum variety gold green pepper purity - Google Patents

A kind of method using EST SSR molecular marker Rapid identification capsicum variety gold green pepper purity Download PDF

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CN104928396B
CN104928396B CN201510398026.0A CN201510398026A CN104928396B CN 104928396 B CN104928396 B CN 104928396B CN 201510398026 A CN201510398026 A CN 201510398026A CN 104928396 B CN104928396 B CN 104928396B
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cenospecies
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袁晓伟
李兴盛
韩永升
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Huasheng Agricultural Group Co.,Ltd.
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Abstract

The present invention relates to field of molecular marker, provide a kind of method using EST SSR molecular marker Rapid identification capsicum variety gold green pepper purity, this method is using the EST SSR primers specially designed, expanded using the leaves genomic DNA of golden green pepper as template, the variety of golden green pepper to be detected is detected by the specific spectruming belt to amplification, any period that can be grown in this way in golden pepper plant is identified, can distinguish cenospecies and female parent self-cross, male parent selfed seed；It is not high to DNA quality requirements and requirement is few；Cost is relatively low, easy to operate；Repeatability and stability are preferable；Rapidly and efficiently；Accuracy is high, and application has a extensive future, and scientific basis is provided to carry out the timely sale of the capsicum production of hybrid seeds and breeding.

Description

It is a kind of using EST-SSR molecular labeling Rapid identification capsicum variety gold green pepper purity Method
Technical field
The present invention relates to field of molecular marker, specifically provides one kind and utilizes EST-SSR molecular labeling Rapid identification capsicums The method of kind gold green pepper purity.
Background technology
Capsicum, belongs to Solanaceae, is 1 year or herbaceos perennial.Capsicum is with the distinctive pungent of its fruit, color and luster and battalion Form point becomes a kind of important worldwide vegetable.Numerous studies show that the anti-oxidant class material that contains in pepper fruit can be with Biologic artifact is protected from oxidative damage and improves immunity of organisms.In addition, Capsaicinoids can speed up energy in capsicum The metabolism of amount and lipid and the incidence for reducing cancer cell.As masses fill capsicum nutritional ingredient and medicinal ingredient Divide understanding, cultivated area increases year by year.
Seed quality directly affects the yield potential of agricultural product quality and improved seeds, carries out crop seed purity detecting pair Ensure that seed quality is of great significance.The common method of Identifying Crop Cultivars mainly includes seed morphology identification, field planting Morphological Identification and isozyme electrophoretic techique identification etc..Seed morphology is easily poor by environmental influence, qualification result accuracy；Field Between plant Morphological Identification it is longer there are the cycle, cost is higher, and workload is larger, and phenotype is easily by cultivation step and environmental condition Influence, seriously affect the efficiency and accuracy of cultivar identification, cannot increasingly meet breeding work and the needs of production and operation； Although accurate, reliable using isoenzyme technique identification crop varieties purity, isoenzyme mark has tissue and organ specificity, Information content is very limited, and polymorphism is not abundant enough.With being continuously increased for capsicum new varieties quantity, traditional identification method is difficult to Effectively distinguish the nearer cenospecies of genetic affinity.
Molecular marking technique can disclose the hereditary difference between filial generation and parent from DNA level, from environmental condition and The influence of cultivation step, inorganization, organ and development-specific, can detect small variation, from plant strain growth season Limitation, rich polymorphism, stability are high, can greatly shorten qualification time.DNA molecular marker technology develops rapidly so that The capsicum cenospecies true and false is detected from genomic level and purity is possibly realized.EST-SSR(expressed sequence Tag-simple sequence repeat) mark is that a kind of of simple tandem repetitive sequence exploitation in est sequence marks Remember type, be it is a kind of by several nucleotide for repeat unit group into tandem repetitive sequence, according to tandem repeat unit number Difference test polymorphism.EST-SSR marks with rich polymorphism, codominant inheritance, stability and repeatability preferably, to DNA Quality requirement is not high, operation is simple, from such environmental effects the advantages that, compared with genome SSR marker, EST-SSR Mark not only reduces development cost, improves development efficiency, and saves the development time, significantly improves its exploitation value Value.In recent years, with the completion of capsicum genome sequencing, the est sequence resource that capsicum is enriched is the exploitation of EST-SSR marks Provide convenience condition.The technology how is utilized to realize that the identification to capsicum variety purity becomes one of urgent problem to be solved.
The content of the invention
The present inventor is directed to the situation of the above-mentioned prior art, there is provided one kind is fast using EST-SSR molecular labelings The method of speed identification capsicum variety gold green pepper purity, this method is using the EST-SSR primers specially designed, with the blade gene of golden green pepper Group DNA is expanded for template, and the variety of golden green pepper to be detected is detected by the specific spectruming belt to amplification, is adopted It can be identified in this way in any period of golden pepper plant growth, can be by cenospecies and female parent self-cross, male parent Selfed seed distinguishes；It is not high to DNA quality requirements and requirement is few；Cost is relatively low, easy to operate；Repeatability and stability compared with It is good；Rapidly and efficiently；Accuracy is high, and application has a extensive future, and science is provided to carry out the timely sale of the capsicum production of hybrid seeds and breeding Foundation.
The targeted capsicum variety of the present invention is golden green pepper, and golden green pepper is that agriculture limited company is contained through grinding for many years by China of Shandong Province Study carefully peppery dry, fresh dual-purpose green pepper of getting bumper crops in selecting.The 35-50 days cross combination seedling stage, is colonized to dry green pepper and harvests 90-120 days.Plant 90-100 centimetres of plant height, 70-80 centimetres or so of strain width；Door green pepper tight knot position 10-13 sections；There is obvious suede in tender stem and blade Hair.Fruit erietiform, fruit is 12-15 centimetres long, 2.1-2.3 centimetres or so of fruit shoulder footpath, fresh green pepper single fruit weight 15-21g, dry green pepper single fruit weight 2.5-3.1g.Okra fruit green, mellow fruit peony, naturally dry speed are fast, commodity fruit rate is high.It is red equal inside and outside dry green pepper pericarp Even, dry green pepper color value value is high.Suitably the main load producing region of capsicum in all parts of the country, optimum are the thermophilic dry green pepper area such as the Inner Mongol to this kind.
In order to carry out the identification of variety to golden green pepper, maternal and male parent superior hybrid crosses, hair of the invention are obtained A person of good sense provide firstly a pair of of EST-SSR primers, be named as ClHS1, its positive sequence for 5 '- AGTTTTAAGAGCAAGGAGGCTC-3 ', its nucleotide sequence is as shown in SEQ ID No.1；Reverse sequence is 5 '- ATCCAACCCAATCCCCAGTC-3 ', its nucleotide sequence as shown in SEQ ID No.2,
Inventor utilizes the blade base of its cenospecies and its parent to kind gold green pepper to be measured after above-mentioned primer is obtained Because a group DNA is expanded, detailed process is as follows：
(1) golden green pepper cenospecies and its parent's leaves genomic DNA are extracted；
(2) using the genomic DNA of (1) step extraction as template, PCR amplification is carried out with EST-SSR primers ClHS1：
PCR reaction systems are 20 μ L, including 10 × PCR Buffer, 2 μ L, 0.4mM dNTPs, each 10 μM of primer, 1U Taq archaeal dna polymerases, template 100ng, sterile ultra-pure water polishing to 20 μ L；
Amplification program is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 are followed Ring；72 DEG C extend 10min eventually；PCR product is stored in 10 DEG C；
Inventor has found after test of many times, draws for the goal of the invention of the present invention, and the specificity specially designed Thing, when annealing temperature being arranged to 57 DEG C in amplification program, expanding effect is best, the result finally detected more close to reality Situation, so the annealing temperature of the present invention is set as 57 DEG C.
(3) amplified production detects：5 μ L amplified productions are mixed with 2 μ L sample-loading buffers, after through acrylamide quality volume Than the native polyacrylamide gel electrophoresis for 10%, 160v constant voltage electrophoresis 1.5h, silver staining colour developing carries out banding pattern statistics；
Acrylamide in wherein used non-denaturing polyacrylamide gel: the mass ratio of methylene diacrylamide is 29 ∶1；
Used sample-loading buffer is selected from 10 × glycerogel sample solution；
(4) purity of " golden green pepper " cenospecies is identified according to the amplification of specific spectruming belt, criterion is：
Female parent has 120bp, 200bp, 250bp, 270bp specific spectruming belt；Male parent have 120bp, 200bp, 250bp, 260bp specific spectruming belts；Cenospecies has 120bp, 200bp, 250bp, 270bp, 400bp, 450bp specific spectruming belt；
According to above-mentioned qualification result, can convert golden green pepper variety (%)=(1-n/N) × 100%, and wherein N is to be measured Pepper seed number, n are individually with male parent bands of a spectrum feature or individually with maternal bands of a spectrum feature or both different from " golden green pepper " spectrum Plant number with feature also different from Parent bands of a spectrum feature.
Utilize the above method, you can realize the quick detection to golden green pepper variety, can be from cultivation step and environment The influence of condition；Without organ, tissue and development-specific：This method can be identified in any period of plant strain growth；It is more State property enriches, codominant inheritance：Cenospecies and female parent self-cross, male parent selfed seed can be distinguished；To DNA quality requirements Not high and requirement is few；Cost is relatively low, easy to operate：This method can be identified in seedling stage, save a large amount of human and material resources and Land resource；Repeatability and stability are preferable；Rapidly and efficiently：Purity Identification work can be completed within 7d；Accuracy is high： This method discloses the hereditary difference between filial generation and parent from genomic level, overcomes the error that field phenotypic evaluation is brought； Application has a extensive future：" golden green pepper " purity of hybrid can quickly, be accurately detected, to carry out the timely of the capsicum production of hybrid seeds and breeding Sale provides scientific basis.
Brief description of the drawings
Fig. 1 is the PCR electrophoresis patterns of cenospecies of the present invention " golden green pepper " Seed inspection characteristic primer,
Wherein, P1 is maternal for " golden green pepper "；F1 is multigroup " golden green pepper " F1 cenospecies；P2 is " golden green pepper " male parent；M is molecular weight mark It is accurate；
The results show：" golden green pepper " F1 cenospecies possesses from father, wherein 6 maternal clear bands, F1 cenospecies and father Maternal band for apparent, can do purity differentiation compared to two at 400bp as characteristic strip；
Fig. 2 is influence electrophoresis pattern of the different PCR annealing temperatures to the purity detecting result of " golden green pepper " commodity seed sample；
Wherein, M is molecular weight standard, and P1 is maternal for " golden green pepper "；F1 is " golden green pepper " F1 cenospecies；P2 is " golden green pepper " male parent,
The results show that as Tm values increase, PCR bands gradually decrease, and characteristic strip is brighter at F1 generation cenospecies 400bp It is aobvious；Optimum annealing temperature may finally be determined at 57 DEG C；
Fig. 3 is influence electrophoresis pattern of the different DNA profiling amounts to the purity detecting result of " golden green pepper " commodity seed sample,
Wherein, M is molecular weight standard, and P1 is maternal for " golden green pepper "；F1 is " golden green pepper " F1 cenospecies；P2 is " golden green pepper " male parent,
The results show that DNA profiling amount is used equally for the purity detecting of " golden green pepper " commodity seed sample from 50ng to 500ng.
Embodiment
Further definition is of the invention in following embodiments, description and these embodiments more than, people in the art Member can determine the essential characteristic of the present invention, and without departing from the spirit and scope of the invention, can be to the present invention Make various changes and modifications, so that it is applicable in various uses and condition.In addition to special indicate, of the present invention is this The field prior art；Percentage in lower several embodiments is weight percentage in addition to special indicate.
The acquisition of 1 gold medal green pepper kind leaves genomic DNA of embodiment
Concrete operation method is as follows：
(1) " golden green pepper " parent and F1 seeds are put into the culture dish for being covered with damp filter paper, intensity of illumination 2000lx, illumination Cycle 16h light/8h is dark, and blade is taken after 28 DEG C of illumination cultivation 6d；
The acquisition process of the wherein described F1 seeds is as follows：After polliniferous maternal plant is pulled out when blooming, female parent is planted Strain and paternal plant 4：1, by artificial pollination, paternal pollen is pollinated onto female parent, finally the F1 generation seed on harvest female parent；
(2) a capsicum spire is put into each centrifuge tube, adds 500 μ L Extraction buffers of 60-65 DEG C of preheating, be put into Steel ball 4, in material is smashed on crusher, 60-65 DEG C of water-bath 20-30min；
The wherein described Extraction buffer is 2 × CTAB extracting solutions, its proportioning is as follows：
High-temperature sterilization, is cooled to room temperature, and adds 2mL mercaptoethanols, room temperature preservation,
(3) liquid that acquisition is walked on centrifuges 10min through 10000rpm/min, takes supernatant in the body such as another centrifuge tube, addition Product (500 μ L) phenol：Chloroform：Isoamyl alcohol (volume ratio 25:24:1) mixed liquor；
(4) mixed liquor is walked on and centrifuges 10min through 10000rpm/min, takes supernatant in another centrifuge tube, the body such as addition Long-pending chloroform：Isoamyl alcohol (volume ratio 24:1) mixed solution；
(5) mixed liquor is walked on and centrifuges 10min through 10000rpm/min, supernatant solution is taken in another centrifuge tube, adds 400 μ L isopropanols mix；
(6) mixed liquor is walked on and centrifuges 15min through 12000rpm/min, abandons supernatant, solid matter is washed with 70% ethanol, dried in the air It is dry；
(7) 200 μ L ddH are added into above-mentioned solid2O back dissolvings, add 1 μ LRNA enzymes, 37 DEG C of water-bath 30min, -20 DEG C standby With.
2 PCR amplification of embodiment
Using the RNase solution mixture of above-mentioned acquisition as template, PCR amplification is carried out using ClSH1 primers：
PCR reaction systems are 20 μ L, including 10 × PCR Buffer, 2 μ L, 0.4mM dNTPs, each 10 μM of primer, 1U Taq archaeal dna polymerases, template 100ng, sterile ultra-pure water polishing to 20 μ L；
Amplification program is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 are followed Ring；72 DEG C extend 10min eventually；PCR product is stored in 10 DEG C.
3 amplified production of embodiment detects
5 μ L amplified productions are mixed with 2 μ L sample-loading buffers, after through acrylamide mass volume ratio be 10% it is non denatured Polyacrylamide gel electrophoresis, 160v constant voltage electrophoresis 1.5h, silver staining colour developing carry out banding pattern statistics；
Used sample-loading buffer is selected from 10 × glycerogel sample solution；
The non-denaturing polyacrylamide gel formula (100mL) of wherein described 10% is as follows：
Wherein used 30% acrylamide/methylene diacrylamide configuration process is as follows：
Acrylamide 29g methylene diacrylamide 1g distilled water is settled to 4 DEG C of 100mL and is kept in dark place, the shelf-life one Month；
Used 10 ╳ TBE configuration process is as follows：
Tris Base 27g, boric acid 13.75g, 0.5M/L EDTA 10mL, distilled water are settled to 250mL, adjust pH extremely 8.3；
Used 10% ammonium persulfate (APS) is with the ratio matching while using of ammonium persulfate 0.1g and distilled water 1mL.
Electrophoretic buffer is 1 × TBE, and after electrophoresis, gel colour developing uses silver staining method, and detailed process is as follows：
1. washing：ddH2O is washed 2 times, each 1min；
2. dyeing：With 1 ‰ AgNO3Immersion, parallel shake 10-15min；
3. washing：Outwell nitrite ion, ddH2O is washed 2 times, each 1min；
4. development：Nitrite ion is added in disk and (contains 16g sodium hydroxides and 8mL formaldehyde in every liter of nitrite ion, surplus is pure Water purification), it is then parallel to shake until developing clear；
5. terminate：2min is soaked with terminate liquid (containing 7.5g carbonating sodium in every liter of terminate liquid, surplus is pure water), after Add ddH2O water cleans 1-2 times；Finally taken a picture and preserved with camera.
Embodiment 4 identifies the purity of " golden green pepper " cenospecies
The purity of " golden green pepper " cenospecies is identified according to the electrophoresis result of specific spectruming belt, criterion is：
Female parent has 120bp, 200bp, 250bp, 270bp specific spectruming belt；Male parent have 120bp, 200bp, 250bp, 260bp specific spectruming belts；Cenospecies has 120bp, 200bp, 250bp, 270bp, 400bp, 450bp specific spectruming belt；
According to above-mentioned qualification result, can convert golden green pepper variety (%)=(1-n/N) × 100%, and wherein N is to be measured Pepper seed number, n are individually with male parent bands of a spectrum feature or individually with maternal bands of a spectrum feature or both different from " golden green pepper " spectrum Plant number with feature also different from Parent bands of a spectrum feature.
Experimental example
By taking the golden green pepper seed of certain farm production in 2014 as an example, F1 generation seed is sprouted at once after September part harvest F1 generation seed Hair, takes tender tissue to extract capsicum DNA after about four days, Purity is carried out using the above-mentioned ClHS1 molecular labelings of the present invention, The seed purity result obtained after a week is 99%；
Utilize general survey method, that is, field planting Morphological Identification：After whole capsicum variety harvests, one, which plays Hainan, adds Generation identification purity, growth cycle is 100 days or so, and is subject to a variety of adverse weather conditions, needs repeat sowing to identify, final identification Purity result is also 99%.Specifically it is compared as follows table：
It can be seen that compared with general survey method, identify that golden green pepper hybrid seed purity is quickly high using the molecular labeling method Effect, easy to operate, repeated and stability is preferable, cost is relatively low.

Claims (2)

  1. A kind of 1. method using EST-SSR molecular labeling Rapid identification capsicum variety gold green pepper purity, it is characterised in that：Utilize EST-SSR primers are identified that the EST-SSR primers are named as ClHS1, its positive sequence for 5 '- AGTTTTAAGAGCAAGGAGGCTC-3 ', its nucleotide sequence is as shown in SEQ ID No.1；Reverse sequence is 5 '- ATCCAACCCAATCCCCAGTC-3 ', its nucleotide sequence is as shown in SEQ ID No.2；
    Comprise the following steps that：
    (1) golden green pepper cenospecies and its parent's leaves genomic DNA are extracted；
    (2) using the genomic DNA of (1) step extraction as template, PCR amplification is carried out with EST-SSR primers ClHS1：
    PCR reaction systems are 20 μ L, including 10 × PCR Buffer, 2 μ L, 0.4mM dNTPs, each 10 μM of primer, 1U Taq archaeal dna polymerases, template 100ng, sterile ultra-pure water polishing to 20 μ L；
    Amplification program is：94 DEG C of pre-degeneration 3min；94 DEG C of denaturation 30s, 57 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulate； 72 DEG C extend 10min eventually；PCR product is stored in 10 DEG C；
    (3) amplified production detects：5 μ L amplified productions are mixed with 2 μ L sample-loading buffers, after through acrylamide mass volume ratio be 10% native polyacrylamide gel electrophoresis, 160v constant voltage electrophoresis 1.5h, silver staining colour developing carry out banding pattern statistics；
    Acrylamide in wherein used non-denaturing polyacrylamide gel: the mass ratio of methylene diacrylamide is 29: 1；
    (4) purity of " golden green pepper " cenospecies is identified according to the amplification of specific spectruming belt, criterion is：
    Female parent has 120bp, 200bp, 250bp, 270bp specific spectruming belt；Male parent has 120bp, 200bp, 250bp, 260bp spy Different bands of a spectrum；Cenospecies has 120bp, 200bp, 250bp, 270bp, 400bp, 450bp specific spectruming belt；
    According to above-mentioned qualification result, can convert golden green pepper variety=(1-n/N) × 100%, and wherein N is pepper seed to be measured Number, n for individually there is male parent bands of a spectrum feature individually there is female parent bands of a spectrum feature or both different from " golden green pepper " bands of a spectrum feature or Different from the plant number of Parent bands of a spectrum feature.
  2. 2. the method according to claim 1 using EST-SSR molecular labeling Rapid identification capsicum variety gold green pepper purity, its It is characterized in that：
    Sample-loading buffer employed in step (3) is selected from 10 × glycerogel sample solution.
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CN108546774A (en) * 2018-06-01 2018-09-18 安徽省农业科学院园艺研究所 A kind of SSR primers and identification method for purple chili Hybrid Purity
CN109486991B (en) * 2018-12-06 2021-11-30 南京农业大学 Molecular marker primer composition for identifying intergeneric hybrid of pear and apple and application thereof
CN110643603A (en) * 2019-11-18 2020-01-03 安阳市农业科学院 Method for simply and rapidly extracting trace DNA of millet and application
CN112877453B (en) * 2021-01-26 2022-11-22 北京市农林科学院 Method for identifying authenticity of pepper variety
CN113801954A (en) * 2021-09-07 2021-12-17 云南省农业科学院质量标准与检测技术研究所 SSR core primer group for purity identification of pepper hybrid and screening method thereof

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CN103013986B (en) * 2011-09-20 2014-07-02 湖南省蔬菜研究所 Hot pepper SSR markers developed by using EST sequence redundancy, and method thereof
CN103642906B (en) * 2013-11-22 2015-07-22 中国热带农业科学院热带作物品种资源研究所 Pepper hybrid seed purity testing EST-SSR (expressed sequence tag-simple sequence repeat) molecular marker and application thereof

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Address after: No.6699, South Ring Road, Qingzhou City, Weifang City, Shandong Province

Patentee after: Huasheng Agricultural Group Co.,Ltd.

Address before: No.6699, South Ring Road, Qingzhou City, Weifang City, Shandong Province

Patentee before: SHANDONG HUASHENG AGRICULTURAL Co.,Ltd.

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Denomination of invention: A method for rapid identification of purity of Pepper Variety golden pepper by EST-SSR molecular marker

Effective date of registration: 20211224

Granted publication date: 20180413

Pledgee: Weifang Bank Co.,Ltd. Qingzhou Qicheng sub branch

Pledgor: Huasheng Agricultural Group Co.,Ltd.

Registration number: Y2021980016501