CN102181540B - Method for verifying seed purity of cotton - Google Patents
Method for verifying seed purity of cotton Download PDFInfo
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- CN102181540B CN102181540B CN 201110079445 CN201110079445A CN102181540B CN 102181540 B CN102181540 B CN 102181540B CN 201110079445 CN201110079445 CN 201110079445 CN 201110079445 A CN201110079445 A CN 201110079445A CN 102181540 B CN102181540 B CN 102181540B
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Abstract
The invention discloses a method for verifying the seed purity of cotton, and in particular relates to a method for verifying the seed purity of insect-resistant hybrid cotton, namely Guofeng cotton 198 by using simple sequence repeat (SSR) molecular markers. The method comprises the following steps of: amplifying genome deoxyribose nucleic acid (DNA) of male and female parents and first-filial generation of a cotton hybrid variety, namely the Guofeng cotton 198 by using a pair of SSR primers P1 and P2; performing polyacrylamide gel electrophoresis separation to find the characteristic spectral band of the difference between the first-filial generation and the male and female parents; verifying first-filial generation of seed samples of the Guofeng cotton 198 by calculating the purity of verified seeds; and comparing the verified purity with the purity verified by field implantation, wherein a comparison result is that the verified purity is consistent with the purity verified by field implantation. The method is suitable for verifying the authenticity and the seed purity of the Guofeng cotton 198 and the male and female parents of the Guofeng cotton 198.
Description
Technical field
The invention belongs to the Seed Testing Technology field, relate to the method for hybrid cotton Seed purity assessment,
Rich cotton 198 cotton seeds of a kind of SSR of the utilization molecule marker Insect-resistant Hybrid Cotton state method of carrying out purity test particularly.
Background technology
State rich cotton 198 is high yield and high quality hybrid cotton new variety that Fengle Seed Breeding Co., Ltd., Hefei cultivates, and has the anti insect gene patent of China's independent intellectual property right.This kind needs to award with paternal pollen and finish crossover process by maternal artificial emasculation in the middle of the hybrid seeding process, and maternal castration is thorough or perforated is male, will greatly affect seed production purity.Traditional purity of hybrid detection method is field plot test, depends on the kind phenotypic difference, and by strain face shaping Identification, qualification time is long, expense is high, difficulty is large and poor accuracy.
SSR (Simple Sequence Repeat) is a kind of new molecular marking technique that grows up on PCR (Polymerase Chain Reaction) basis, have height polymorphism, codominance, conservative property and only need a small amount of DNA, easy and simple to handle, good reproducibility, the advantage such as reliable and stable, in the research fields such as animal, plant, microorganism, be widely applied, and begun to be applied to the analysis of genetic diversity of cotton and the identification research of purity of hybrid.Zhu Meixia etc. utilize No. 8621,15 pairs of SSR primer pairs, 5 cotton varieties such as middle cotton 19 to carry out presentation of results SSR Primer combination that individual plant and Primer combination detect to have high efficiency and reliability detecting variety.The fingerprint map construction that the 2 pairs of current Xinjiang of SSR primer pair of the usefulness such as Qin Li main breed carries out and the result of Hybrid seed purity test show that the SSR mark can be distinguished 3 cross-fertilize seed and their parent.Rich cotton 198 cross-fertilize seed of this research and utilization state and the difference of parents on some SSR sites are sought the stable band that suitable primer amplification goes out cross-fertilize seed and parents' obvious difference, are used for the indoor purity detecting of state rich cotton 198.For rich cotton 198 Purities of state provide accurate, stable, quick, a practical detection method.
Summary of the invention
The object of the present invention is to provide a kind ofly can carry out accurately rich cotton 198 cotton seeds of state, stablize, quick, the practical method that detects.
Its technical scheme is: the method for rich cotton 198 verifying seed purity of cottons of a kind of state is characterized in that its concrete steps are:
1) DNA extraction:
When placing nutrition pot germination and emergence to seedling that 2-3 sheet true leaf is arranged in seed, get blade about 4cm in mortar, add 1.5 * CTAB Extraction buffer it is ground, change 65 ℃ of water-baths of centrifuge tube half an hour over to; Add and the isopyknic chloroform of Extraction buffer/primary isoamyl alcohol mixed solution, hand 10 minutes, centrifugal 10 minutes of 10000rpm; Get its supernatant, add freezing 95% ethanol, place 30 minutes precipitation DNA in-20 ℃ of refrigerator-freezers; Centrifugal 10 minutes of 10000rpm abandons supernatant, adds 75% ethanol, leaves standstill and desalts in 4 minutes; Abandon 75% ethanol, centrifuge tube is inverted on the thieving paper air-dry; Add 200ul sterilization distilled water dissolving DNA stand-by;
2) PCR reaction:
In the PCR pipe, add respectively dna profiling, 10 * PCR damping fluid (contains MgCl
2Magnesium chloride), dNTP triphosphoric acid dezyribonucleoside (2.0mM), primer P1 and P2, Taq enzyme and ddH
2The O distilled water; 94 ℃ of denaturations 4 of elder generation '; Again 94 ℃ of sex change 30 ", 55 ℃ annealing 30 ", 72 ℃ extend 1 ', 32 circulations; 72 ℃ extend 7 ', 95 ℃ of water-bath 4 ' sex change postposition are on ice;
3) injecting glue electrophoresis detection:
Separation gel is slowly injected between two sheet glass, leave standstill and solidify more than half an hour; Sheet glass is loaded onto electrophoresis chamber, 2000V, 90W preheating half an hour; Insert comb, point sample; 2000V, 85W electrophoresis approximately 3 hours; Behind the lower glue, offset plate is placed in the box of 10% Glacial acetic acid, jog 20 minutes to glue all decolours; With clear water rinsing twice, each 5 minutes; Put into dyeing basin staining fluid, shake gently dyeing 15-20 minute; Take out offset plate, with clear water rinsing 10 seconds; Put into developing solution develop be with to DNA clear; Washed 1 minute.
4) being examined seed purity calculates:
Obtain respectively rich cotton 198 first-filial generations of state and parental seed electrophoretic band with aforesaid method, counting is subjected to that the clearly seed number of two strip-types is arranged in the sample product, and calculates the clearly seed number of two strip-types/examined seed sum * 100 of the seed purity (%) be subjected to the sample product=be subjected to have in the sample product with following formula.
Its technique effect is: the present invention utilizes a pair of SSR primer P1 and P2 that the genomic dna of rich cotton 198 the Parent of cotton hybrid kind state and first-filial generation is increased to separate with polyacrylamide gel electrophoresis, find out the key band of first-filial generation and Parent difference, calculated by examining seed purity, state's rich cotton 198 assorted generation seed samples are tested, the purity of its check through and the purity identified of field planting compare the as a result height of the two consistent (for details see attached table 1).
Subordinate list 1: use the indoor detection purity of SSR molecule marker and field planting and identify the purity synopsis
| Sample number into spectrum | Field test result (%) | Indoor qualification result (%) |
| 1 | 97.6 | 98.0 |
| 2 | 98.5 | 98.0 |
| 3 | 97.1 | 98.3 |
| 4 | 97.8 | 97.0 |
| 5 | 96.9 | 96.5 |
| 6 | 98.3 | 98.0 |
| 7 | 98.3 | 97.5 |
| 8 | 98.7 | 98.0 |
| 9 | 98.1 | 97.0 |
| 10 | 99.8 | 99.0 |
| 11 | 98.7 | 97.5 |
| 12 | 97.4 | 97.5 |
| 13 | 98.7 | 97.5 |
| 14 | 98.8 | 97.0 |
| 15 | 97.9 | 97.5 |
| 16 | 98.4 | 97.0 |
| 17 | 99.1 | 97.0 |
| 18 | 98.3 | 97.5 |
| 19 | 98.9 | 96.5 |
| 20 | 99.3 | 98.5 |
| 21 | 98.5 | 98.0 |
| 22 | 97.2 | 98.4 |
| 23 | 99.5 | 97.5 |
| 24 | 99.1 | 96.5 |
| 25 | 99.4 | 96.5 |
| 26 | 99.4 | 97.0 |
| 27 | 99.5 | 98.5 |
| 28 | 98.7 | 97.0 |
| 29 | 98.1 | 98.0 |
| 30 | 98.6 | 98.5 |
| On average | 98.5 | 97.6 |
By upper table analysis, the mean value that two kinds of methods detect purity be respectively 98.5 and 97.6, t to test the two difference not remarkable, illustrated that different methods detects purity result's consistence.Thereby the SSR molecular marking technique is applicable to the check of rich cotton 198 kinds of state and parent's verity and seed purity.
Description of drawings
Fig. 1 is with rich cotton 198 parents of state and F thereof
1Key band.1,27 is the rich cotton 198 male parent banding patterns (400bp) of state among the figure; 2,28 is the rich cotton 198 maternal banding patterns (620bp) of state; 3-26 is rich cotton 198 first generation of hybrid seed banding patterns (banding pattern that comprises male parent 400bp and maternal 620bp) of state.
Embodiment
The method of rich cotton 198 verifying seed purity of cottons of a kind of state, its concrete steps are as follows:
1) DNA extraction:
Seed is placed the nutrition pot germination and emergence, when by the time seedling has 2-3 sheet true leaf, get blade about 4cm in little mortar, the 1.5 * CTAB Extraction buffer that adds 700ul grinds it, changes the 1.5ml centrifuge tube over to.1.5 * CTAB Extraction buffer is by the hexadecyl front three brometo de amonio CTAB of 1.5% (w/v), 1.4M sodium chloride nacl, the Tris-Cl damping fluid (pH8.0) of 100mM, the edta edta of 20mM and 0.2%(v/v) alpha-mercapto ethanol consist of.
A) 65 ℃ of water-bath half an hour; Add the chloroform isopyknic with Extraction buffer/primary isoamyl alcohol mixed solution.Chloroform/primary isoamyl alcohol mixed solution is mixed by chloroform and primary isoamyl alcohol, and its mixed volume ratio is 24 ︰ 1;
B) 10000rpm is centrifugal 10 minutes;
C) suct clearly 300ul in another centrifuge tube, add freezing 95% ethanol of 600ul, place 30 minutes precipitation DNA in-20 ℃ of refrigerator-freezers;
D) 10000rpm is centrifugal 10 minutes, abandons supernatant;
E) add 400ul 75% ethanol, leave standstill and desalted in 4 minutes;
F) abandon 75% ethanol, centrifuge tube is inverted on the thieving paper air-dry;
G) add 200ul sterilization distilled water dissolving DNA stand-by;
1% sepharose detects the DNA product.
2) PCR reaction:
⑴ reaction system (20ul): in the PCR pipe, add respectively 2ul dna profiling, 10 * PCR damping fluid (magnesium chloride containing MgCl
2), primer P1 and the Taq enzyme of P2,0.2ul and the distilled water ddH of 13ul of triphosphoric acid dezyribonucleoside dNTP (2.0mM), the 1.0ul of 1.8ul
2O.
Primer P1 sequence is 5 '-CGGTCAGATTCCAACAGA-3 ',
Primer P2 sequence is 5 '-GCCATCTCAGAGAGGACA-3 ';
⑵ response procedures: 94 ℃ of denaturations 4 of elder generation ';
Again 94 ℃ of sex change 30 ", 55 ℃ annealing 30 ", 72 ℃ extend 1 ', 32 circulations;
72 ℃ extend 7 ', 95 ℃ of water-bath 4 ' sex change postposition are on ice.
3) injecting glue electrophoresis detection
1. clean sheet glass: firmly clean with 95% alcohol and be coated with that two panels of peeling off silane and affine silane.
2. be coated with affine silane: the affine liquid of 1ml (being mixed by 995ul 95% ethanol and 5ul glacial acetic acid) and the affine silane of 15ul are applied to equably do not have on the reeded sheet glass.
3. be coated with and peel off silane: smear equably 1ml on the reeded sheet glass and peel off silane.
4. add edge strip, dress plate: a sheet glass is scribbled one of affine silane face up and lie against on the experiment table, edge strip is placed in the both sides alignment, another piece sheet glass is scribbled peel off one of silane and face down and be placed on it upper clip of zygomorphy folder.
5. fill with separation gel: separation gel is slowly injected between two sheet glass, leave standstill and solidify more than half an hour.Separation gel is by the polyacrylamide of 65ml 4%, the ammonium persulphate APS of 400ul10% and the N of 40ul, N, and N', N'-Tetramethyl Ethylene Diamine TEMED mixes.
6. electrophoresis: sheet glass is loaded onto electrophoresis chamber, 2000V, 90W preheating half an hour; Insert comb, point sample; 2000V, 85W electrophoresis approximately 3 hours.
7. silver dyes:
Under a behind the glue, offset plate is placed in the box of 10% Glacial acetic acid of 1 liter of new preparation, jog 20 minutes to glue all decolours.B clear water rinsing twice, each 5 minutes.C puts into dyeing basin staining fluid, shakes gently dyeing 15-20 minute.Staining fluid is by 37% formaldehyde of 1L deionized water, 1.5ml and the Silver Nitrate AgNO of 1g
3Mix.D took out offset plate, with clear water rinsing 10 seconds.E puts into to develop in the developing solution (adding 30g sodium acetate, anhydrous, 37% formaldehyde of 1.5ml and 200ul Sulfothiorine (10mg/ml) at the 1L deionized water) to DNA and is with clear (approximately 5 minutes); F washing 1 minute.
8. observe: offset plate dries afterwards observed and recorded result under the lamp box of reading X-ray.
4) being examined seed purity calculates: obtain respectively rich cotton 198 first-filial generations of state and the parental seed electrophoretic band (is seen Fig. 2 with aforesaid method.Cross-fertilize seed has clearly two strip-types, is 620bp and 400bp; Female parent has the banding pattern of a 620bp; Male parent has the banding pattern of a 400bp), with being subjected to clearly counting of two strip-types arranged in the sample product, can calculate with following formula by the seed purity of sample product:
Be subjected to the clearly seed number of two strip-types/examined seed sum * 100 of the seed purity (%) of sample product=be subjected to have in the sample product
Claims (3)
1. the method for rich cotton 198 verifying seed purity of cottons of state is characterized in that its concrete steps are:
1) DNA extraction:
When placing nutrition pot germination and emergence to seedling that 2-3 sheet true leaf is arranged in seed, get blade about 4cm in mortar, add 1.5 * CTAB Extraction buffer it is ground, change 65 ℃ of water-baths of centrifuge tube half an hour over to; Add and the isopyknic chloroform of Extraction buffer/primary isoamyl alcohol mixed solution, hand 10 minutes, centrifugal 10 minutes of 10000rpm; Get its supernatant, add freezing 95% ethanol, place 30 minutes precipitation DNA in-20 ℃ of refrigerator-freezers; Centrifugal 10 minutes of 10000rpm abandons supernatant, adds 75% ethanol, leaves standstill and desalts in 4 minutes; Abandon ethanol, centrifuge tube is inverted on the thieving paper air-dry; Add 200ul sterilization distilled water dissolving DNA stand-by;
Described 1.5 * CTAB Extraction buffer is by the hexadecyl front three brometo de amonio of 1.5% (w/v), the sodium-chlor of 1.4M, and the Tris-Cl damping fluid of 100mM, the ethylenediamine tetraacetic acid (EDTA) of 20mM, alpha-mercapto ethanol 0.2%(v/v) consists of;
2) PCR reaction:
In the PCR pipe, add respectively dna profiling, 10 * PCR magnesium chloride containing damping fluid, triphosphoric acid dezyribonucleoside, primer P1 and P2, Taq enzyme and distilled water; 94 ℃ of denaturations of elder generation 4 minutes; 94 ℃ of sex change are 30 seconds again, 55 ℃ of annealing 30 seconds, and 72 ℃ were extended 32 circulations 1 minute; 72 ℃ were extended 7 minutes, and 4 minutes sex change postposition of 95 ℃ of water-baths on ice;
Described primer P1 sequence is 5 '-CGGTCAGATTCCAACAGA-3 ', and primer P2 sequence is 5 '-GCCATCTCAGAGAGGACA-3 '
3) injecting glue electrophoresis detection:
Separation gel is slowly injected between two sheet glass, leave standstill and solidify more than half an hour; Sheet glass is loaded onto electrophoresis chamber, 2000V, 90W preheating half an hour; Insert comb, point sample; 2000V, 85W electrophoresis approximately 3 hours; Behind the lower glue, offset plate is placed in the box of 10% Glacial acetic acid, jog 20 minutes to glue all decolours; With clear water rinsing twice, each 5 minutes; Put into dyeing basin staining fluid, shake gently dyeing 15-20 minute; Take out offset plate, with clear water rinsing 10 seconds; Put into developing solution develop be with to DNA clear; Washed 1 minute;
4) being examined seed purity calculates:
Obtain respectively rich cotton 198 first-filial generations of state and parental seed electrophoretic band with aforesaid method, counting is subjected to that the clearly seed number of two strip-types is arranged in the sample product, and calculates the clearly seed number of two strip-types/examined seed sum * 100 of the seed purity (%) be subjected to the sample product=be subjected to have in the sample product with following formula.
2. the method for rich cotton 198 verifying seed purity of cottons of a kind of state according to claim 1, it is characterized in that: described chloroform/primary isoamyl alcohol mixed solution is mixed by chloroform and primary isoamyl alcohol, and its mixed volume ratio is 24 ︰ 1.
3. the method for rich cotton 198 verifying seed purity of cottons of a kind of state according to claim 1, it is characterized in that: described separation gel is by 4% polyacrylamide, 10% ammonium persulphate and N, N, N', the N'-Tetramethyl Ethylene Diamine mixes, and its mixed volume is than being 65:0.4:0.04.
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| CN102640592A (en) * | 2012-02-29 | 2012-08-22 | 河南省农业科学院 | Method for identifying purity of pigment-gland-marked hybrid cotton seeds |
| CN102640591A (en) * | 2012-02-29 | 2012-08-22 | 河南省农业科学院 | Seedling identification method of purity of palmatum leaf pigment gland double labeled hybrid cotton seeds |
| CN103627786A (en) * | 2013-07-19 | 2014-03-12 | 合肥丰乐种业股份有限公司 | Inspection method for purity of melon seeds |
| CN103884848B (en) * | 2014-04-02 | 2015-08-05 | 江苏省农业科学院 | A kind of method predicting transgenic Bt cotton insect resistace intensity |
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| CN100587078C (en) * | 2007-07-03 | 2010-02-03 | 江苏省农业科学院 | Molecule marking method for identifying variety purity of insect resistance hybrid cotton Suza No.3 |
| CN101768639A (en) * | 2009-12-15 | 2010-07-07 | 华中农业大学 | Rapid detection method of cabbage type rape variety SSR fingerprint |
| CN101824484B (en) * | 2010-06-09 | 2012-02-29 | 中国农业科学院棉花研究所 | DNA fingerprint detection method for authenticity identification of cotton variety |
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Granted publication date: 20130123 Termination date: 20140331 |