CN104928396A - Method for rapidly identifying hot pepper species and golden pepper purity degree by using EST-SSR molecular markers - Google Patents
Method for rapidly identifying hot pepper species and golden pepper purity degree by using EST-SSR molecular markers Download PDFInfo
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Abstract
The invention relates to the field of molecular markers and provides a method for rapidly identifying hot pepper species and golden pepper purity degrees by using the EST-SSR molecular markers. The method adopts the specially designed EST-SSR primers, augmentation is conducted with the leaf genome DNA of golden pepper, the species purity degree of the golden pepper to be detected is tested through the specific bands of the augmentation result, by the adoption of the method, the identification can be conducted at any period during the growth of golden pepper, and the coeno-species can be separated from female parent selfed seeds and male parent selfed seeds; the requirement on DNA quality is not high, and the needing amount is less; the cost is low, the operation is simple; the repeatability and the stability are good; the fastness and the high efficiency are achieved; the accuracy is high, the application and the popularization prospect is wide, and scientific basis is provided for the development of pepper seed production and good seeds and the in-time selling.
Description
Technical field
The present invention relates to field of molecular marker, specifically provide a kind of method utilizing EST-SSR molecule marker Rapid identification capsicum variety gold green pepper purity.
Background technology
Capsicum, belongs to Solanaceae, is 1 year or per nnial herb.Capsicum has become a kind of important worldwide vegetables with the distinctive pungent of its fruit, color and luster and nutritive ingredient.Large quantity research shows that the anti-oxidant class material contained in pepper fruit can be protected biological organism from oxidative damage and improve immunity of organisms.In addition, in capsicum Capsaicinoids can acceleration energy and lipid metabolism and reduce the incidence of cancer cell.Along with masses' fully realizing capsicum nutritive ingredient and medicinal ingredients, 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 to ensureing that seed quality is significant.The common method of Identifying Crop Cultivars mainly comprises seed morphology qualification, field planting identification of morphology and isozyme electrophoretic techique qualification etc.Seed morphology is subject to environmental influence, and qualification result accuracy is poor; It is longer to there is the cycle in field planting identification of morphology, and cost is higher, and workload is comparatively large, and phenotype is subject to the impact of cultivation step and envrionment conditions, has a strong impact on efficiency and the accuracy of cultivar identification, more and more can not meet the needs of breeding work and production and operation; Although crop varieties purity is accurate, reliable to utilize isoenzyme technique to identify, isoenzyme mark has tissue and organ specificity, and quantity of information is very limited, and polymorphism is abundant not.Along with the continuous increase of capsicum new variety quantity, traditional authentication method is difficult to effectively distinguish the nearer cross-fertilize seed of genetic affinity.
Molecular marking technique can disclose hereditary difference between filial generation and parent from DNA level, be not subject to the impact of envrionment conditions and cultivation step, inorganization, organ and development-specific, small variation can be detected, not by the restriction in plant strain growth season, rich polymorphism, stability are high, greatly can shorten qualification time.Developing rapidly of DNA molecular marker technology, makes to detect the capsicum cross-fertilize seed true and false from genomic level and purity becomes possibility.EST-SSR (expressed sequence tag-simplesequence repeat) marks, it is a kind of labeling pattern according to the simple tandem repetitive sequence exploitation in est sequence, be a class be the tandem repetitive sequence that repeating unit forms by several Nucleotide, according to the Difference test polymorphism of tandem repeat unit number.EST-SSR mark have rich polymorphism, codominant inheritance, stability and repeatability better, not high to DNA specification of quality, operation is simple, not by advantages such as such environmental effects, compared with genome SSR marker, EST-SSR mark not only reduces cost of development, improves development efficiency, and save the development time, significantly improve its utility value.In recent years, along with completing of capsicum genome sequencing, the est sequence resource that capsicum is enriched is that the exploitation of EST-SSR mark is provided convenience condition.This technology how is utilized to realize becoming one of problem demanding prompt solution to the qualification of capsicum variety purity.
Summary of the invention
The present inventor is for the situation of above-mentioned prior art, provide a kind of method utilizing EST-SSR molecule marker Rapid identification capsicum variety gold green pepper purity, the method adopts custom-designed EST-SSR primer, with the leaves genomic DNA of golden green pepper for template increases, by detecting the variety of specific spectruming belt to golden green pepper to be detected of amplification, adopt any period that can grow at golden pepper plant in this way to identify, cross-fertilize seed and female parent self-cross, male parent selfed seed can be made a distinction; To DNA specification of quality is not high and requirement is few; Cost is lower, simple to operate; Repeatability and stability better; Rapidly and efficiently; Accuracy is high, and application has a extensive future, for the timely sale carrying out the capsicum production of hybrid seeds and breeding provides scientific basis.
The present invention for capsicum variety be golden green pepper, China of Jin Jiaoshi Shandong Province contains that agriculture limited-liability company peppery high yield in research for many years selects is dry, fresh dual-purpose green pepper.This cross combination 35-50 days in seedling stage, field planting to dry green pepper is gathered 90-120 days.Plant height 90-100 centimetre, strain width 70-80 cm; Door green pepper tight knot position 10-13 and saves; Tender stem and blade there is obvious fine hair.Fruit erietiform, the long 12-15 centimetre of fruit, fruit shoulder footpath 2.1-2.3 cm, fresh green pepper single fruit weight 15-21g, dry green pepper single fruit weight 2.5-3.1g.Tender fruit green, mellow fruit scarlet, dries that speed is fast, commodity fruit rate is high naturally.Dry green pepper pericarp is inside and outside evenly red, and dry green pepper look is worth high.This kind is suitable for capsicum master in all parts of the country and carries producing region, and optimum is that Inner Mongol etc. is addicted to dry green pepper area.
In order to carry out the qualification of variety to golden green pepper, obtain superior hybrid crosses that is maternal and male parent, the present inventor provide firstly a pair EST-SSR primer, called after ClHS1, its forward sequence is 5 '-AGTTTTAAGAGCAAGGAGGCTC-3 ', and 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,
After the above-mentioned primer of acquisition, contriver utilizes it to increase to the kind gold cross-fertilize seed of green pepper to be measured and the leaves genomic DNA of parent thereof, and detailed process is as follows:
(1) golden green pepper cross-fertilize seed and parent's leaves genomic DNA thereof is extracted;
(2) with (1) step extract genomic dna for template, carry out pcr amplification with EST-SSR primer ClHS1:
PCR reaction system is 20 μ L, comprising 10 × PCR Buffer 2 μ L, 0.4mM dNTPs, and each 10 μMs of primer, 1U Taq archaeal dna polymerase, template 100ng, aseptic ultrapure water polishing to 20 μ L;
Amplification program is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 1min, 35 circulations; 72 DEG C of ends extend 10min; PCR primer is stored in 10 DEG C;
Contriver finds after test of many times, for goal of the invention of the present invention, and custom-designed Auele Specific Primer, when annealing temperature being set in amplification program 57 DEG C, expanding effect is best, the result of final detection is pressed close to and practical situation more, so annealing temperature of the present invention is set as 57 DEG C.
(3) amplified production detects: 5 μ L amplified productions mixs with 2 μ L sample-loading buffers, after be the native polyacrylamide gel electrophoresis of 10% through acrylamide mass volume ratio, 160v constant voltage electrophoresis 1.5h, silver-colored contaminate colour developing and carries out banding pattern statistics;
Acrylamide in wherein adopted non-denaturing polyacrylamide gel: the mass ratio of methylene diacrylamide is 29: 1;
The sample-loading buffer adopted is selected from 10 × glycerogel sample solution;
(4) identify the purity of " golden green pepper " cross-fertilize seed 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 specific spectruming belt; Cross-fertilize seed has 120bp, 200bp, 250bp, 270bp, 400bp, 450bp specific spectruming belt;
According to above-mentioned qualification result, can be converted golden green pepper variety (%)=(1-n/N) × 100%, wherein N is pepper seed number to be measured, and n has separately male parent bands of a spectrum feature or has separately maternal bands of a spectrum feature or be both different from the plant number that " golden green pepper " bands of a spectrum feature is also different from Parent bands of a spectrum feature.
Utilize aforesaid method, the rapid detection to golden green pepper variety can be realized, the impact of cultivation step and envrionment conditions can not be subject to; Without organ, tissue and development-specific: the method all can be identified in any period of plant strain growth; Rich polymorphism, codominant inheritance: cross-fertilize seed and female parent self-cross, male parent selfed seed can be made a distinction; To DNA specification of quality is not high and requirement is few; Cost is lower, simple to operate: the method can be identified in seedling stage, saves a large amount of human and material resources and land resources; Repeatability and stability better; Rapidly and efficiently: within 7d, Purity Identification work can be completed; Accuracy is high: the 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: can detect " golden green pepper " purity of hybrid fast, accurately, for the timely sale carrying out the capsicum production of hybrid seeds and breeding provides scientific basis.
Accompanying drawing explanation
Fig. 1 is the PCR electrophoretogram of cross-fertilize seed of the present invention " golden green pepper " Seed inspection characteristic primer,
Wherein, P1 is that " golden green pepper " is maternal; F1 is many groups of " golden green pepper " F1 cross-fertilize seed; P2 is " golden green pepper " male parent; M is molecular weight standard;
Result shows: " golden green pepper " F1 cross-fertilize seed has from father, 6 maternal clear bands, and wherein F1 cross-fertilize seed two generations of 400bp place compared with Parent band are more clear, can be used as feature and brings and do purity and distinguish;
Fig. 2 is that different PCR annealing temperature affects electrophoretogram to the purity detecting result of " golden green pepper " commodity seed sample;
Wherein, M is molecular weight standard, and P1 is that " golden green pepper " is maternal; F1 is " golden green pepper " F1 cross-fertilize seed; P2 is " golden green pepper " male parent,
Result shows, and along with Tm value increases, PCR band reduces gradually, and F1 generation cross-fertilize seed 400bp place characteristic strip is more obvious; Finally can determine that optimum annealing temperature is at 57 DEG C;
Fig. 3 is that different DNA profiling amount affects electrophoretogram to the purity detecting result of " golden green pepper " commodity seed sample,
Wherein, M is molecular weight standard, and P1 is that " golden green pepper " is maternal; F1 is " golden green pepper " F1 cross-fertilize seed; P2 is " golden green pepper " male parent,
Result shows, and DNA profiling amount all can be used for the purity detecting of " golden green pepper " commodity seed sample from 50ng to 500ng.
Embodiment
The present invention is defined further in following examples, according to above description and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and when not departing from spirit and scope of the invention, various change and amendment can be made, to make its applicable various uses and condition to the present invention.Except special indicating, be of the present inventionly state of the art; Per-cent in lower several embodiment, except special indicating, is all weight percentage.
The acquisition of embodiment 1 gold medal green pepper kind leaves genomic DNA
Concrete operation method is as follows:
(1) " golden green pepper " parent and F1 seed are put into the culture dish being covered with damp filter paper, intensity of illumination 2000lx, periodicity of illumination 16h light/8h is dark, gets blade after 28 DEG C of illumination cultivation 6d;
The procurement process of wherein said F1 seed is as follows: after being pulled out by polliniferous maternal plant when blooming, maternal plant and paternal plant 4:1, and by artificial pollination, paternal pollen is pollinated on female parent, finally gathers in the crops the F1 generation seed on female parent;
(2) put into a capsicum spire in each centrifuge tube, add 500 μ L Extraction buffers of 60-65 DEG C of preheating, put into steel ball 4, on crusher, material is smashed, 60-65 DEG C of water-bath 20-30min;
Wherein said Extraction buffer is 2 × CTAB extracting solution, and its proportioning is as follows:
High-temperature sterilization, is cooled to room temperature, adds 2mL mercaptoethanol, room temperature preservation,
(3) liquid of upper step acquisition is through the centrifugal 10min of 10000rpm/min, gets supernatant in another centrifuge tube, adds equal-volume (500 μ L) phenol: chloroform: primary isoamyl alcohol (volume ratio is 25:24:1) mixed solution;
(4) upper step mixed solution is through the centrifugal 10min of 10000rpm/min, gets supernatant liquor in another centrifuge tube, adds isopyknic chloroform: primary isoamyl alcohol (volume ratio is 24:1) mixing solutions;
(5) upper step mixed solution is through the centrifugal 10min of 10000rpm/min, gets supernatant solution in another centrifuge tube, adds 400 μ L Virahol mixings;
(6) upper step mixed solution is through the centrifugal 15min of 12000rpm/min, and abandon supernatant, solid matter 70% washing with alcohol, dries;
(7) in above-mentioned solid, 200 μ L ddH are added
2o back dissolving, add 1 μ LRNA enzyme, 37 DEG C of water-bath 30min ,-20 DEG C for subsequent use.
Embodiment 2 pcr amplification
With the RNA enzymolysis mixture of above-mentioned acquisition for template, ClSH1 primer is utilized to carry out pcr amplification:
PCR reaction system is 20 μ L, comprising 10 × PCR Buffer 2 μ L, 0.4mM dNTPs, and each 10 μMs of primer, 1U Taq archaeal dna polymerase, template 100ng, aseptic ultrapure water polishing to 20 μ L;
Amplification program is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 1min, 35 circulations; 72 DEG C of ends extend 10min; PCR primer is stored in 10 DEG C.
Embodiment 3 amplified production detects
5 μ L amplified productions mix with 2 μ L sample-loading buffers, after through acrylamide mass volume ratio be the native polyacrylamide gel electrophoresis of 10%, 160v constant voltage electrophoresis 1.5h, silver dye colour developing carry out banding pattern statistics;
The sample-loading buffer adopted is selected from 10 × glycerogel sample solution;
The non-denaturing polyacrylamide gel formula (100mL) of wherein said 10% is as follows:
30% wherein adopted acrylamide/methylene diacrylamide layoutprocedure is as follows:
Acrylamide 29g methylene diacrylamide 1g distilled water is settled to 100mL 4 DEG C and keeps in Dark Place, one month quality guaranteed period;
The 10 ╳ TBE layoutprocedures adopted are as follows:
Tris Base 27g, boric acid 13.75g, 0.5M/L EDTA 10mL, distilled water is settled to 250mL, adjusts pH to 8.3;
10% ammonium persulphate (APS) adopted is with the ratio matching while using of ammonium persulphate 0.1g and distilled water 1mL.
Electrophoretic buffer is 1 × TBE, and after electrophoresis terminates, gel colour developing adopts silver staining method, and detailed process is as follows:
1. wash: ddH
2o washes 2 times, each 1min;
2. dye: with 1 ‰ AgNO
3soak, parallel shake 10-15min;
3. wash: outwell nitrite ion, ddH
2o washes 2 times, each 1min;
4. develop: in dish, add nitrite ion (containing 16g sodium hydroxide and 8mL formaldehyde in often liter of nitrite ion, surplus is pure water), then parallel shake is until develop clear;
5. stop: soak 2min with stop buffer (in often liter of stop buffer containing 7.5g carbonating sodium, surplus is pure water), after add ddH
2o water cleaning 1-2 time; Finally take a picture with camera and preserve.
Embodiment 4 identifies the purity of " golden green pepper " cross-fertilize seed
Identify the purity of " golden green pepper " cross-fertilize seed according to the electrophoresis result of specific spectruming belt, criterion is:
Female parent has 120bp, 200bp, 250bp, 270bp specific spectruming belt; Male parent has 120bp, 200bp, 250bp, 260bp specific spectruming belt; Cross-fertilize seed has 120bp, 200bp, 250bp, 270bp, 400bp, 450bp specific spectruming belt;
According to above-mentioned qualification result, can be converted golden green pepper variety (%)=(1-n/N) × 100%, wherein N is pepper seed number to be measured, and n has separately male parent bands of a spectrum feature or has separately maternal bands of a spectrum feature or be both different from the plant number that " golden green pepper " bands of a spectrum feature is also different from Parent bands of a spectrum feature.
Experimental example
For the golden green pepper seed of certain farm production in 2014, after gathering in the crops F1 generation seed September, horse back is by F1 generation seed germination, get tender tissue after about four days and extract capsicum DNA, the ClHS1 molecule marker utilizing the present invention above-mentioned carries out Purity, and the seed purity result obtained after a week is 99%;
Utilize general survey method and field planting identification of morphology: after waiting whole capsicum variety to gather in the crops, one plays Hainan adds generation qualification purity, and growth cycle is about 100 days, and is subject to multiple adverse weather conditions, need repeat sowing to identify, finally identify that purity result is also 99%.Specifically be compared as follows table:
Visible compared with general survey method, utilize this molecular labeling method identify golden green pepper hybrid seed purity rapidly and efficiently, simple to operate, repeated and stability better, cost is lower.
Claims (3)
1. one kind utilizes the method for EST-SSR molecule marker Rapid identification capsicum variety gold green pepper purity, it is characterized in that: utilize EST-SSR primer to identify, described EST-SSR primer called after ClHS1, its forward sequence is 5 '-AGTTTTAAGAGCAAGGAGGCTC-3 ', and its nucleotide sequence is as shown in SEQ ID No.1; Reverse sequence is 5 '-ATCCAACCCAATCCCCAGTC-3 ', and its nucleotide sequence is as shown in SEQ IDNo.2.
2. the method utilizing EST-SSR molecule marker Rapid identification capsicum variety gold green pepper purity according to claim 1, is characterized in that: concrete steps are as follows:
(1) golden green pepper cross-fertilize seed and parent's leaves genomic DNA thereof is extracted;
(2) with (1) step extract genomic dna for template, carry out pcr amplification with EST-SSR primer ClHS1:
PCR reaction system is 20 μ L, comprising 10 × PCR Buffer 2 μ L, 0.4mM dNTPs, and each 10 μMs of primer, 1U Taq archaeal dna polymerase, template 100ng, aseptic ultrapure water polishing to 20 μ L;
Amplification program is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 1min, 35 circulations; 72 DEG C of ends extend 10min; PCR primer is stored in 10 DEG C;
(3) amplified production detects: 5 μ L amplified productions mixs with 2 μ L sample-loading buffers, after be the native polyacrylamide gel electrophoresis of 10% through acrylamide mass volume ratio, 160v constant voltage electrophoresis 1.5h, silver-colored contaminate colour developing and carries out banding pattern statistics;
Acrylamide in wherein adopted non-denaturing polyacrylamide gel: the mass ratio of methylene diacrylamide is 29: 1;
(4) identify the purity of " golden green pepper " cross-fertilize seed 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 specific spectruming belt; Cross-fertilize seed has 120bp, 200bp, 250bp, 270bp, 400bp, 450bp specific spectruming belt;
According to above-mentioned qualification result, can be converted golden green pepper variety=(1-n/N) × 100%, wherein N is pepper seed number to be measured, and n has separately male parent bands of a spectrum feature or has separately maternal bands of a spectrum feature or be both different from the plant number that " golden green pepper " bands of a spectrum feature is also different from Parent bands of a spectrum feature.
3. the method utilizing EST-SSR molecule marker Rapid identification capsicum variety gold green pepper purity according to claim 2, is characterized in that:
The sample-loading buffer adopted in step (3) is selected from 10 × glycerogel sample solution.
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Cited By (5)
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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 |
| CN109486991A (en) * | 2018-12-06 | 2019-03-19 | 南京农业大学 | Identify molecular labeling primer composition and its application of pears and apple intergeneric conjugal transfer |
| 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 |
| CN112877453A (en) * | 2021-01-26 | 2021-06-01 | 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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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN109486991A (en) * | 2018-12-06 | 2019-03-19 | 南京农业大学 | Identify molecular labeling primer composition and its application of pears and apple intergeneric conjugal transfer |
| 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 |
| CN112877453A (en) * | 2021-01-26 | 2021-06-01 | 北京市农林科学院 | Method for identifying authenticity of pepper variety |
| 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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| CN104928396B (en) | 2018-04-13 |
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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 |