CN100587078C - Molecule marking method for identifying variety purity of insect resistance hybrid cotton Suza No.3 - Google Patents
Molecule marking method for identifying variety purity of insect resistance hybrid cotton Suza No.3 Download PDFInfo
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- CN100587078C CN100587078C CN200710024785A CN200710024785A CN100587078C CN 100587078 C CN100587078 C CN 100587078C CN 200710024785 A CN200710024785 A CN 200710024785A CN 200710024785 A CN200710024785 A CN 200710024785A CN 100587078 C CN100587078 C CN 100587078C
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Abstract
The invention relates to a SSR labeling method to appraisal the authenticity and /or variety purity insect-resistant hybrid cotton suza3, which belongs to biological technology field. The invention takes a commodity of insect-resistant hybrid cotton <suza3> and parental DNA as template to screen with SSR molecular marker, which can differentiate two SSR markers of male parent, female parent and hybrid at the same time. A band size of female parent specific marker JZSSSR259205 produced by SSR probe JZSSSR259 is 205bp; female parent specific marker JZSSSR259245 band size is 245bp; a band size offemale parent specific marker JZSSSR413125 produced by SSR probe JZSSSR413 is 125bp; male parent specific marker JZSSSR413135 band size is 135bp. The two SSR marker can not only identify the hybrid cotton variety purity and authenticity, but also can do mutual identification which is good for controlling quality accurately and efficiently of the hybrid cotton breed and quicken the quality inspection process to identify quickly to mass sample in short time with a reliable result.
Description
Technical field
The present invention identifies the SSR marking method of bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " verity and/or variety, to cotton F
1The seed of Hybrid " assorted No. 3 of Soviet Union " carries out real and fake discrimination and/or variety is identified, belongs to biological technical field.
Background technology
Improved seeds are bases of high crop yield, and variet complexity and purity drop then can obviously reduce output.Rapidly and accurately identification of species and carry out purity check for corresponding seed quality criteriaization, variety certification, false plant distinguish, property right dispute all plays an important role.At present, China seed is produced and operation control standard not too also, underproof seed is sneaked into market repeatedly and is used for producing, cause the underproduction and financial loss, therefore variety identify seem particularly important (Wu Minsheng, Wang Shoucai wear the application [J] of the auspicious .DNA fingerprint pattern technology of scape on cultivar identification and purity check. Journal of Agricultural Biotechnology, 1998,6 (1): 51-56; Wang Zhong China's .DNA fingerprint pattern technology and the application in the crop varieties resource [J] thereof. Molecular Plant Breeding, 2006,4 (3): 425-430.).Producing at present and going up most of high advantage hybrid cotton kinds all is by the artificial emasculation production of hybrid seeds.In the production of hybrid seeds process, maternal castration is thorough or perforated is male, and the selfing bell that forms can have a strong impact on purity of hybrid.As the untimely purity of hybrid of identifying effectively, then can bring heavy losses to Cotton Production.Therefore, to cotton crossbreed carry out fast, accurately and efficiently purity is identified, applying of hybrid cotton kind had important practical significance.The detection method of traditional cotton variety purity has multiple: (Gao Xiangs such as seed morphology method, seedling evaluation, protein electrophorese method and the plantation evaluation of sub-district, field, Cha Chunhong, the Ai Ke Baeyer, etc. cotton seed purity authenticate technology [J]. seed science and technology, 2006 (3): 52-53.).Comparatively speaking, based on the molecular marking technique of DNA such as simple repeated sequence (SSR) etc., have that detection site quantity is many, polymorphism is high, inheritance stability, be not subjected to advantages such as environmental influence, be applied at cotton variety evaluation, seed purity detection and aspects such as kind sibship and classification, and bringing into play more and more important effect.
(original name: be the homemade high-intensity fiber divalent insect-resistant hybrid cotton by Commercial Crop Inst., Jiangsu Prov. Academy of Agricultural Sciences and Biological Technology institute, Chinese Academy of Agricultural Sciences's cooperation seed selection rather assorted 602, SGKz9), the breed parent source is TB005/SGK321 to assorted No. 3 of Soviet Union.Obtain Ministry of Agriculture's genetically modified crops safety certificate in December, 2004, in April, 2005 is by national crop varietal approval committee.The assorted No. 3 outstanding performances of Soviet Union are that fiber strength is good, product are of fine quality, output is high, comprehensive disease and insect resistance shape is good.For guaranteeing that this improved seeds produce maximum economic benefits, need a kind of authority, accurately, the stable detection method carries out Rapid identification to the true and false and the purity thereof of its commodity seed.
Summary of the invention
Technical problem
The objective of the invention is to find the method for the SSR mark of identifying bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " verity and/or variety, this method has polymorphism height, inheritance stability, is not subjected to advantages such as environmental influence, can remedy the deficiency of existing purity detecting work, thereby set up the quality controlling means of efficiently and accurately for the Rapid identification of its seed real and fake discrimination and purity.
Technical scheme
1, identify the SSR marking method of bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " variety, may further comprise the steps:
(1) extracts bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " cotyledon genomic dna;
With the genomic dna is template, selects for use 2 pairs of SSR primers to carry out pcr amplification simultaneously, and 2 pairs of SSR primer sequences are
Primer JZSSSR259
F：TGAAGATTTGGAGGCAATTG
R：GAAATCAAGCCTCAATTCGG
With primer JZSSSR413
F：CCTCCCTCACTTAAGGTGCA
R：ATGTTGTAAGGGTGCAAGGC；
(2) amplified production is carried out gel electrophoresis;
(3) electrophoresis result is analyzed, wherein:
The maternal specific mark JZSSSR259 that SSR primer JZSSSR259 produces
205Stripe size is 205bp, the male parent specific mark JZSSSR259 of generation
245Stripe size is 245bp;
The maternal specific mark JZSSSR413 that SSR primer JZSSSR413 produces
125Stripe size is 125bp, the male parent specific mark JZSSSR413 of generation
135Stripe size is 135bp.
By using 2 characteristic primers the marker gene type of hybrid individual plant seedling is analyzed, have only the individual plant that has parent's specific band simultaneously just to be real " assorted No. 3 of Soviet Union " cross-fertilize seed, any one band that lacks wherein all is regarded as pseudostationary, and the mean value that detects the purity result who obtains is the purity of " assorted No. 3 of Soviet Union " variety seeds.
The extracting method of cotton genomic dna wherein: the fresh cotton cotyledon of 1 thumb lid size is put into the centrifuge tube of 1.5ml, add 0.6ml and extract damping fluid, electricity is gone round and round a millstone broken.10, the centrifugal 10min of 000rpm abandons supernatant; In precipitation, add the lysis buffer of 65 ℃ of preheatings of 0.6ml, and use the toothpick stirring and evenly mixing, 65 ℃ of water-bath 30min; The chloroform that adds 0.6ml: primary isoamyl alcohol, its volume ratio are 24: 1, overturns more than 50 times, and 12, the centrifugal 15min of 000rpm; Supernatant is changed in the centrifuge tube of another new 1.5ml, add the chloroform of 0.6ml: primary isoamyl alcohol, its volume ratio are 24: 1, overturn more than 50 times, and 12, the centrifugal 15min of 000rpm; Add behind the Virahol of 0.6 times of volume precooling upset 30 times, place 30min for-20 ℃, 10, the centrifugal 5min of 000rpm abandons supernatant; Add 0.5ml 70% ethanol and wash DNA, 10, centrifugal 5 minutes of 000rpm abandons supernatant, natural draft drying DNA; The TE damping fluid dissolving DNA that adds 100 μ l sterilization, pH 8.0, after sepharose detected quality, it was standby to be stored in 4 ℃ or-20 ℃.
Pcr amplification reaction and gel electrophoresis step comprise: the SSR reaction system is 10ul, wherein contains genomic dna 10～30ng, Mg
2+2.5mmolL
-1, dNTPs 0.2mmoL
-1, primer 0.6 μ molL
-1, 0.3U Taq archaeal dna polymerase.Pcr amplification reaction carries out on the pcr gene amplification instrument, and amplification program is: 95 ℃ of 2min; 94 ℃ of 40s, 57 ℃ of 45s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 7min; Amplified production is to carry out electrophoresis on two vertical panel non-denaturing polyacrylamide gels of 8% in mass volume ratio concentration, after 120V voltage stabilizing electrophoresis 1.2～1.5h, electrophoresis finish the back gel and wash slightly with distilled water, fixes 12～15min; Silver dyes 12min; Colour developing; Use 0.75% aqueous sodium carbonate color development stopping at last, under visible lamp box, observe and take pictures.
Beneficial effect
The present invention filters out can be in half-blood to produce simultaneously from used a large amount of primers have father, maternal specific mark band, and banding pattern is clear, the primer of good reproducibility, good reliability: the characteristic primer that JZSSSR259 and JZSSSR413 identify as bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " the seed true and false and purity.
The application of primer of the present invention, it is easy to have accuracy height, result influence, statistics stable, that be not subjected to envrionment conditions and developmental stage, can detect the true and false of cotton variety seed quickly and accurately.
Primer of the present invention all can identify has this special complementary band of father and mother.2 codominant markers' SSR primer (accompanying drawing), performance is stable in repeatedly repeating, can be effective to differentiate the true and false and the purity of cotton crossbreed seed, and can verify mutually, help the quality control of cotton commodity seed efficiently and accurately, accelerated the quality examination process of cotton commodity seed.
Description of drawings
Accompanying drawing is the SSR-PCR electrophoretogram of bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " seed detected characteristics primer.
P
1: " assorted No. 3 of Soviet Union " female parent; P
2: " assorted No. 3 of Soviet Union " male parent; F
1: " assorted No. 3 of Soviet Union " F
1Cross-fertilize seed; The L:50bp molecular weight standard.Arrow is represented the specific mark between the parent respectively.
Embodiment
SSR labeled analysis embodiment below by the PCR-based technology further specifies the present invention.
The experiment material of present embodiment is bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " commodity seed and a parent material seed thereof.Under 25-30 ℃ of condition of the husky bed of indoor employing, cultivate seedling 5-7 days, get its fresh cotyledon.
Method: extract the DNA of cotton cotyledon, adopt the SSR molecule marker to carry out pcr amplification, amplified production is carried out gel electrophoresis, statistics is also screened characteristic primer.
1. cotton genomic dna extracts
(1) 1 thumb is covered the centrifuge tube that big or small fresh cotton cotyledon is put into 1.5ml, add 0.6ml and extract damping fluid [0.35M Glucose, 0.1M Tris-HCl (pH8.0), 5mM Na-EDTA (pH8.0), 2%PVP, 1% (V/V) β-Me], electricity is gone round and round a millstone broken.10, the centrifugal 10min of 000rpm abandons supernatant;
(2) lysis buffer [1.4MNaCl, 0.1M Tris-HCl (pH8.0), the 20mM Na-EDTA (pH8.0) of 65 ℃ of preheatings of adding 0.6ml in precipitation, 2% (V/V) CTAB, 2%PV P, 1% (V/V) β-Me], and use toothpick stirring and evenly mixing, 65 ℃ of water-bath 30min;
(3) chloroform of adding 0.6ml: primary isoamyl alcohol (24: 1), overturn more than 50 times, 12, the centrifugal 15min of 000rpm;
(4) supernatant is changed in the centrifuge tube of another new 1.5ml, behind the repeating step (3) 1 times, add behind the Virahol of 0.6 times of volume precooling upset 30 times, place 30min for-20 ℃, 10, the centrifugal 5min of 000rpm abandons supernatant;
(5) add 0.5ml 70% ethanol and wash DNA, 10, centrifugal 5 minutes of 000rpm abandons supernatant, natural draft drying DNA;
(6) add 100 μ l sterilization TE damping fluid (pH 8.0) dissolving DNA, after sepharose detected quality, it was standby to be stored in 4 ℃ or-20 ℃.
2. molecular marker analysis
The SSR reaction system is 10ul, wherein contains genomic dna 10～30ng, Mg
2+2.5mmolL
-1, dNTPs0.2mmoL
-1, primer 0.6 μ molL
-1, 0.3U Taq archaeal dna polymerase.Pcr amplification reaction carries out on the pcr gene amplification instrument.Amplification program is: 95 ℃ of 2min; 94 ℃ of 40s, 57 ℃ of 45s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 7min.Amplified production carries out electrophoresis at two vertical panel non-denaturing polyacrylamide gels (concentration 8%), 120V voltage stabilizing electrophoresis 1.2～1.5h.After gel washed slightly with distilled water after electrophoresis finished, fix (10% ethanol, 0.5% glacial acetic acid) 12～15min; Silver dyes (0.2%AgNO
3The aqueous solution) 12min; Colour developing (1.5%NaOH, 0.4% formaldehyde); Use 0.75% aqueous sodium carbonate color development stopping at last.Under visible lamp box, observe and take pictures.
3, the characteristic primer of screening purity evaluation
From used primer, filter out and in half-blood, to produce father, maternal specific mark band simultaneously, and banding pattern is clear, totally 2 of the primers of good reproducibility, good reliability, be respectively JZSSSR259 and JZSSSR413, as the characteristic primer (accompanying drawing) of bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " seed authenticity and/or variety evaluation.2 pairs of SSR primer sequences are:
Primer JZSSSR259
F：TGAAGATTTGGAGGCAATTG
R：GAAATCAAGCCTCAATTCGG
With primer JZSSSR413
F：CCTCCCTCACTTAAGGTGCA
R：ATGTTGTAAGGGTGCAAGGC；
The maternal specific mark JZSSSR259 that SSR primer JZSSSR259 produces
205Stripe size is 205bp, the male parent specific mark JZSSSR259 of generation
245Stripe size is 245bp; The maternal specific mark JZSSSR413 that SSR primer JZSSSR413 produces
125Stripe size is 125bp, the male parent specific mark JZSSSR413 of generation
135Stripe size is 135bp.
4, identify carrying out the seed genetic purity with characteristic primer
2 characteristic primers that application screens carry out purity to bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " and identify the same 1-3 of step.Have only the individual plant that has parent's specific mark band simultaneously just to be real cross-fertilize seed, any one band that lacks wherein all is regarded as pseudostationary.By using 2 characteristic primers the marker gene type of hybrid individual plant seedling is analyzed, the mean value that detects the purity result who obtains can be designated as the genetic purity of cross-fertilize seed.Performance was stablized during these 2 SSR were marked at and repeatedly repeat, and analytical results can be verified mutually, can identify cotton crossbreed purity exactly.
Wherein its implication of SSR primer code name JZSSSR259 is: " JZS " represents cash crop institute; " SSR " represents the simple repeated sequence mark; " 259 " represent this laboratory cotton SSR primer numbering.
Claims (2)
1, identify the SSR marking method of bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " variety, may further comprise the steps:
(1) extracts bollworm-resistance hybrid cotton " assorted No. 3 of Soviet Union " cotyledon genomic dna;
With the genomic dna is template, selects for use 2 pairs of SSR primers to carry out pcr amplification simultaneously, and 2 pairs of SSR primer sequences are
Primer JZSSSR259
F：TGAAGATTTGGAGGCAATTG
R：GAAATCAAGCCTCAATTCGG
With primer JZSSSR413
F：CCTCCCTCACTTAAGGTGCA
R：ATGTTGTAAGGGTGCAAGGC；
(2) amplified production is carried out gel electrophoresis;
(3) electrophoresis result is analyzed, wherein:
The maternal specific mark JZSSSR259 that SSR primer JZSSSR259 produces
205Stripe size is 205bp, the male parent specific mark JZSSSR259 of generation
245Stripe size is 245bp;
The maternal specific mark JZSSSR413 that SSR primer JZSSSR413 produces
125Stripe size is 125bp, the male parent specific mark JZSSSR413 of generation
135Stripe size is 135bp;
By using described 2 SSR primers the marker gene type of hybrid individual plant seedling is analyzed, have only the individual plant that has parent's specific band simultaneously just to be real " assorted No. 3 of Soviet Union " cross-fertilize seed, any one band that lacks wherein all is regarded as pseudostationary, and the mean value that detects the purity result who obtains is the purity of " assorted No. 3 of Soviet Union " variety seeds.
2, according to the SSR marking method of the described evaluation bollworm-resistance hybrid cotton of claim 1 " assorted No. 3 of Soviet Union " variety, the wherein extraction of cotton genomic dna, pcr amplification reaction and gel electrophoresis step comprise:
The extraction of cotton genomic dna: 1 thumb is covered the centrifuge tube that big or small fresh cotton cotyledon is put into 1.5ml, add 0.6ml and extract damping fluid, electricity is gone round and round a millstone broken; 10, the centrifugal 10min of 000rpm abandons supernatant; In precipitation, add the lysis buffer of 65 ℃ of preheatings of 0.6ml, and use the toothpick stirring and evenly mixing, 65 ℃ of water-bath 30min; The chloroform that adds 0.6ml: primary isoamyl alcohol, its volume ratio are 24: 1, overturns more than 50 times, and 12, the centrifugal 15min of 000rpm; Supernatant is changed in the centrifuge tube of another new 1.5ml, add the chloroform of 0.6ml: primary isoamyl alcohol, its volume ratio are 24: 1, overturn more than 50 times, and 12, the centrifugal 15min of 000rpm; Add behind the Virahol of 0.6 times of volume precooling upset 30 times, place 30min for-20 ℃, 10, the centrifugal 5min of 000rpm abandons supernatant; Add 0.5ml 70% ethanol and wash DNA, 10, centrifugal 5 minutes of 000rpm abandons supernatant, natural draft drying DNA; The TE damping fluid dissolving DNA that adds 100 μ l sterilization, pH 8.0, after sepharose detected quality, it was standby to be stored in 4 ℃ or-20 ℃;
Pcr amplification reaction and gel electrophoresis: the SSR reaction system is 10ul, wherein contains cotton genomic dna 10～30ng, Mg
2+2.5mmolL
-1, dNTPs 0.2mmoL
-1, primer 0.6 μ molL
-1, 0.3U Taq archaeal dna polymerase; Pcr amplification reaction carries out on pcr gene amplification instrument, and amplification program is: 95 ℃ of 2min; 94 ℃ of 40s, 57 ℃ of 45s, 72 ℃ of 1min, 30 circulations; 72 ℃ of 7min; Amplified production is to carry out electrophoresis on two vertical panel non-denaturing polyacrylamide gels of 8% in mass volume ratio concentration, after 120V voltage stabilizing electrophoresis 1.2～1.5h, electrophoresis finish the back gel and wash slightly with distilled water, fixes 12～15min; Silver dyes 12min; Colour developing; Use 0.75% aqueous sodium carbonate color development stopping at last, under visible lamp box, observe and take pictures.
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| CN102181540B (en) * | 2011-03-31 | 2013-01-23 | 合肥丰乐种业股份有限公司 | Method for verifying seed purity of cotton |
| CN105039525A (en) * | 2015-06-30 | 2015-11-11 | 山东棉花研究中心 | Rapid screening method of SSR (Simple Sequence Repeat) molecular markers for genetic diversity analysis of cotton |
| CN107475395B (en) * | 2017-09-05 | 2020-10-30 | 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） | Identification method for assisting in identifying hybrid cotton wing 1518 and special kit thereof |
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| CN1286902A (en) * | 2000-11-03 | 2001-03-14 | 南京农业大学 | Breeding process for gene transfering cotton hybrid resisting bollworm |
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| CN1286902A (en) * | 2000-11-03 | 2001-03-14 | 南京农业大学 | Breeding process for gene transfering cotton hybrid resisting bollworm |
Non-Patent Citations (2)
| Title |
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| 陆地棉品种SSR标记的多态性及用于杂交种纯度检测的研究. 武耀廷等.棉花学报，第13卷第3期. 2001 * |
| 陆地棉数量性状遗传分析和产量性状轮回选择的研究. 金骏培.中国博士学位论文全文数据库，第2004卷第4期. 2004 * |
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