CN102505044B - Method for quickly identifying genetic purity of glutinous corn hybrid - Google Patents

Method for quickly identifying genetic purity of glutinous corn hybrid Download PDF

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CN102505044B
CN102505044B CN 201110351885 CN201110351885A CN102505044B CN 102505044 B CN102505044 B CN 102505044B CN 201110351885 CN201110351885 CN 201110351885 CN 201110351885 A CN201110351885 A CN 201110351885A CN 102505044 B CN102505044 B CN 102505044B
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glutinous
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srap
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柳李旺
刘君
龚义勤
徐良
陈莹
陆虎华
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Nanjing Agricultural University
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Abstract

The invention belongs to the technical field of biology, and relates to a method for quickly identifying the genetic purity of a glutinous corn hybrid. The method comprises the following steps of: extracting genomic deoxyribonucleic acid (DNA) from glutinous corn, performing polymerase chain reaction (PCR) amplification by using the screened sequence-related amplified polymorphism (SRAP) effective primer combination NAUSRem7/NAUSRpm1 and random amplified polymorphic DNA (RAPD) effective primers NAUSR709 and NAUSR712, respectively performing non-denaturing polyacrylamide gel electrophoresis and agarose gel electrophoresis on a PCR product obtained through amplification, and shooting a DNA electrophoresis pattern; and comparing and analyzing the size and position difference of polymorphism amplified fragments formed due to the difference of DNA fragment sequences in the electrophoresis pattern, and identifying the genetic purity of the glutinous corn F1 hybrid, namely a Suyunuo 2 seed. The detection method has the advantages of marking stability, high accuracy, no influence of the growth stage and environment of a sample to be detected, low cost, capability of being performed in the whole growth season, and the like.

Description

A kind of rapid identification method of crossbreeded glutiuous maize genetic purity
Technical field
The invention belongs to biological technical field, relate to a kind of rapid identification method of crossbreeded glutiuous maize genetic purity, utilize SRAP and RAPD molecular marking technique to carry out the detection of glutinous corn variety ' glutinous No. 2 of Su Yu ' seed genetic purity.
Background technology
Seed is the agriculture extremely important production means, and seed quality is directly connected to seed producers, manager and user's economic interests.The important evidence that the seed quality quality is passed judgment on is 4 indexs such as genetic purity, percentage of germination, cleanliness and moisture.3 index tests in back can carry out indoor, and genetic purity is identified relatively difficulty.Accurately, identify that rapidly, easily the seed genetic purity is the important content of modern agriculture production and seed work.Zea Gramineae Zea (Zea) corn kind (mays) (Gai Junyi. each opinion [M] of breeding of plants. Beijing: Chinese agriculture press, 2006,128-129).Can be divided into seed corn, silage corn, fresh edible maize, pop corn etc. according to results period and purposes corn.Wherein fresh edible maize can be as the edible fresh and tender fruit ear of vegetables and fruits and converted products thereof.Fresh edible maize is referred to as the vegetables corn abroad.Fresh edible maize comprises sweet corn, Glutinous Semen Maydis, bamboo shoot corn etc., wherein Glutinous Semen Maydis is because of the glutinous soft delicate fragrance of its seed, unique flavor, the thin no slag of skin, contain more rich nutrient substances and better palatability than sweet corn, and easily digest and assimilate, become all-ages snackfoods (Yang Hua etc. eat raw and pop corn breeding and cultivation [M]. Beijing: Scientia Agricultura Sinica technology press, 2008,3-5).
Cross-breeding occupies an important position in crop breeding work, because the cross-pollination of corn monoecism, be typical cross pollinated plant, than be easier to carry out hybrid seeding (Zhang Zemin. hybrid maize seed production general introduction [M]. Beijing: Chinese agriculture press, 2008,87-88).In production of hybrid seeds process, usually be mixed with in the cross-fertilize seed by maternal selfing and produce pseudostationary, also have mechanical admixture, cause great production risk, will bring heavy losses to cross-fertilize seed seed producers, retailer and grower.Therefore, before the cross-fertilize seed seed is sold, to the genetic purity of first generation cross-fertilize seed seed carry out fast, accurately evaluation seems particularly important.
Genetic purity detection to corn improved seeds seed mainly contains the field planting evaluation at present, isozyme is identified, but identifying, the field needs seeding and seedling raising, the field planting management, need observe morphological specificity, length consuming time, workload is big, and easily be subject to seasonal restrictions and such environmental effects (hole is extensively superfine. corn seed purity method of inspection research [J]. seed, 2000, (3): 26-28).Because morphologic difference performance is not clearly between the false cross-fertilize seed of corn and the true cross-fertilize seed before results, be difficult for differentiating with becoming the strain phase in seedling stage, especially when hereditary basis between the parent is very near, be difficult for more differentiating.Isozyme Analysis is subjected to the influence at environment and the position of drawing materials easily, and for the less Hybrid of some parent's hereditary differences, isozyme is difficult to carry out purity detecting and evaluation sometimes, accuracy not high (Liu Liwang etc. the application [J] of molecular marking technique in vegetable crop cultivar identification and purity detecting. Molecular Plant Breeding, 2004,2 (4): 563-568).Along with development of molecular biology, the dna molecular marker technology of PCR-based technology, because advantages such as it is fast and convenient, reliable and stable, cost is relatively low, become one of important technology of modern farm crop improved seeds seed genetic purity evaluation, begin to be applied in the various crop such as cotton, paddy rice, tomato, wild cabbage.
' glutinous No. 2 of Su Yu ' be the precocity of utilizing self-mating system T361 and T366 hybridization to breed, high yield Glutinous Semen Maydis Hybrid (state examines numbering: state examines beautiful 2003066) (Chen Guoqing etc. seed selection and the application [J] of precocious Good Quality Glutinous Corn cross-fertilize seed ' glutinous No. 2 of Su Yu '. the Jinling School of Science and Technology journal, 2006, (1): 71-74).Not only prematureness is good for this kind, output is high, and has good, the ripe advantage such as neat, nutritious of taste flavor, at present in Shandong, Henan, Hebei, Shaanxi, the north, Jiangsu, the northern Summer Maize Production district's large scale application in Anhui.
Summary of the invention
The objective of the invention is the above-mentioned deficiency at prior art, the rapid identification method of glutinous corn variety ' glutinous No. 2 of Su Yu ' seed genetic purity is provided.
Purpose of the present invention can be achieved through the following technical solutions:
The authentication method of glutinous corn variety ' glutinous No. 2 of Su Yu ' seed genetic purity may further comprise the steps:
1) extracts ' glutinous No. 2 of Su Yu ' seedling genomic dna.
2) utilize SRAP combination of primers NAUSRem7/NAUSRpm1, or RAPD primer NAURP709 and NAURP712, be that template is carried out pcr amplification with Glutinous Semen Maydis ' glutinous No. 2 of Su Yu ' genomic dna, also or simultaneously utilize SRAP combination of primers NAUSRem7/NAUSRpm1, with RAPD primer NAURP709 and NAURP712, be that template is carried out pcr amplification with corn ' glutinous No. 2 of Su Yu ' genomic dna;
Described SRAP combination of primers sequence is:
Forward primer NAUSRem7:GACTGCGTACGAATTATG (SEQ ID No.1);
Reverse primer NAUSRpm1:TGACGGCGAACAATCTCC (SEQ ID No.2);
The RAPD primer sequence:
NAURP709：AGTCCCCCTC(SEQ?ID?No.3)；
NAURP712：CAGCTCCTGT(SEQ?ID?No.4)。
3) dna fragmentation after will increasing utilizes native polyacrylamide gel electrophoresis to detect the SRAP amplified production respectively, utilizes agarose gel electrophoresis to detect the RAPD amplified production.
4) electrophoresis result is analyzed, the individual plant that has female parent, male parent specific marker band simultaneously is judged to be real cross-fertilize seed, calculate the seed genetic purity, wherein:
It is the maternal specific marker band of 170bp and 150bp male parent specific marker band that SRAP combination of primers NAUSRem7/NAUSRpm1 produces size;
It is the maternal specific marker band of 790bp that RAPD primer NAURP709 produces size;
It is 1250bp male parent specific marker band that NAURP712 produces size.
Wherein, the SRAP-PCR reaction system contains 10ng genomic dna, 2.5mmolL -1Mg 2+, 0.15mmolL -1DNTPs, 0.5 μ molL -1SRAP forward primer NAUSRem7 and reverse primer NAUSRpm1,0.75U Taq archaeal dna polymerase; Pcr amplification program: 90 ℃ of 3min; 94 ℃ of 1min, 35 ℃ of 1min, 72 ℃ of 1.5min, 5 circulations; 94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 1.5min, 33 circulations; 72 ℃ of 7min extend 10 ℃ of preservations.
The RAPD-PCR reaction system contains 10ng genomic dna, 2.0mmolL -1Mg 2+, 0.15mmolL -1DNTPs, 0.4 μ molL -1NAURP709 or NAURP712,0.75U Taq archaeal dna polymerase; 94 ℃ of 2min of pcr amplification program; 94 ℃ of 30s, 37 ℃ of 45s, 72 ℃ of 1.5min, 40 circulations; 72 ℃ of 10min extend 10 ℃ of preservations.
Beneficial effect
The present invention filters out to produce in half-blood has this specific mark of father and mother band and molecule marker not affected by environment, genetic purity for the identification of the Glutinous Semen Maydis seed, can improve the efficient that the cross-fertilize seed genetic purity is identified greatly, thereby for the Rapid identification of its seed genetic purity is set up efficiently, quality controlling means accurately.This detection method has that mark is stable, and the accuracy height is not subjected to sample growth phase and environmental influence, can carry out season in whole growth, and cost is low, and can identify advantage such as Glutinous Semen Maydis seed genetic purity at short notice exactly.
Description of drawings
Fig. 1 is the SRAP-PCR electrophoretogram of glutinous corn variety ' glutinous No. 2 of Su Yu ' seed genetic purity detection specificity primer NAUSRem7/NAUSRpm1.
Swimming lane F: ' glutinous No. 2 of Su Yu ' female parent; Swimming lane M: ' glutinous No. 2 of Su Yu ' male parent; Swimming lane 1-11: ' glutinous No. 2 of Su Yu ' F 1Cross-fertilize seed; Swimming lane L:100bp molecular criteria amount; Arrow shows the specific marker band.
Fig. 2 is the RAPD-PCR electrophoretogram of glutinous corn variety ' glutinous No. 2 of Su Yu ' seed genetic purity detection specificity primer NAURP709.
Swimming lane F: ' glutinous No. 2 of Su Yu ' female parent; Swimming lane M: ' glutinous No. 2 of Su Yu ' male parent; Swimming lane 1-11: ' glutinous No. 2 of Su Yu ' F 1Cross-fertilize seed; Swimming lane L:DL2000 plus molecular criteria amount; Arrow shows the specific marker band.
Fig. 3 is the RAPD-PCR electrophoretogram of glutinous corn variety ' glutinous No. 2 of Su Yu ' seed genetic purity detection specificity primer NAURP712.
Swimming lane F: ' glutinous No. 2 of Su Yu ' female parent; Swimming lane M: ' glutinous No. 2 of Su Yu ' male parent; Swimming lane 1-11: ' glutinous No. 2 of Su Yu ' F 1Cross-fertilize seed; Swimming lane L:DL2000 plus molecular criteria amount; Arrow shows the specific marker band.
Embodiment
Embodiment 1
The Rapid identification of crossbreeded glutiuous maize seed genetic purity.
It is as follows to utilize SRAP and RAPD molecular marking technique to carry out the main schedule of operation of Rapid identification of glutinous corn variety ' glutinous No. 2 of Su Yu ' seed genetic purity:
1 DNA extraction
(1) gets glutinous corn variety ' glutinous No. 2 of Su Yu ' seedling young tender leaf agreement that contracts a film or TV play to an actor or actress 0.1g, through liquid nitrogen grinding, add 500 μ l CTAB lysate (2%CTAB, 2molL -1NaCl, 20mmolL -1EDTA, 100mmolL -1Tris-HCl (pH 8.0) and 0.2% beta-mercaptoethanol V/V) change in the 1.5ml centrifuge tube 65 ℃ of water-bath 30-50min over to;
(2) take out at 16 ℃, centrifugal 10min under 12, the 000rpm condition, supernatant liquor change in the 1.5ml centrifuge tube of Amoxcillin, add the equal-volume chloroform: primary isoamyl alcohol (24: 1, volume ratio) mixed solution, jiggle room temperature 12, centrifugal 10min under the 000rmp condition then;
(3) supernatant liquor is sucked in the new centrifuge tube, repeating step (2) 1 times adds the sodium-acetate of 0.1 volume and Virahol and slow the upset 5-10 time of equal-volume precooling, is positioned over-20 ℃ more than the refrigerator 30min;
(4) 4 ℃, centrifugal 10min outwells supernatant liquor under 12, the 000rmp condition, with 70% pre-cooled ethanol washing DNA settling;
(5) 4 ℃, centrifugal 2-4min outwells supernatant liquor under 12, the 000rmp condition, and dry back adds 80 μ L sterilization TE damping fluid dissolving DNA, and agarose gel electrophoresis detects that to be stored in 4 ℃ or-20 ℃ behind the DNA that carries of institute standby.
2 molecular marker analysis
Utilize RAPD and SRAP primer that glutinous corn variety ' glutinous No. 2 of Su Yu ' and parent thereof are carried out pcr amplification, filter out the characteristic primer that can produce the target label band: SRAP combination of primers NAUSRem7 (SEQ ID No.1)/NAUSRpm1 (SEQ ID No.2), RAPD primer NAURP709 (SEQ ID No.3) and NAURP712 (SEQ ID No.4).Utilize the validity feature primer that obtains that glutinous corn variety ' glutinous No. 2 of Su Yu ' seedling genomic dna is carried out pcr amplification.SRAP and RAPD mark amplification reaction system and amplification program are:
The SRAP-PCR reaction system contains 10ng genomic dna, 2.5mmolL -1Mg 2+, 0.15mmolL -1DNTPs, 0.5 μ molL -1SRAP forward primer NAUSRem7 and reverse primer NAUSRpm1,0.75U Taq archaeal dna polymerase; Pcr amplification program: 90 ℃ of 3min; 94 ℃ of 1min, 35 ℃ of 1min, 72 ℃ of 1.5min, 5 circulations; 94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 1.5min, 33 circulations; 72 ℃ of 7min extend 10 ℃ of preservations.
The RAPD-PCR reaction system contains 10ng genomic dna, 2.0mmolL -1Mg 2+, 0.15mmolL -1DNTPs, 0.4 μ molL -1NAURP709 (SEQ ID No.3) or NAURP712 (SEQ ID No.4,0.75U Taq archaeal dna polymerase; 94 ℃ of 2min of pcr amplification program; 94 ℃ of 30s, 37 ℃ of 45s, 72 ℃ of 1.5min, 40 circulations; 72 ℃ of 10min extend 10 ℃ of preservations.
3 amplified productions detect
The SRAP-PCR amplified production adopts native polyacrylamide gel electrophoresis (PAGE).Contain in the 25ml gel: Arc: Bis (29: 1, mass ratio) 6.65ml, 5 * TBE 5ml, TEMED 20 μ l, 10%AP 0.3ml; The PCR product separates at 6.0% polyacrylamide gel.Go up sample, 120V electrophoresis 2.5-3.0h behind the prerunning 50-60V 30min; Gel adopts fixedly 12-20min of 0.5% Glacial acetic acid, 10% ethanol stationary liquid; 0.2%AgNO 3Solution-dyed 15min; Twice back of distilled water wash is in developing solution (1.5%NaOH, 0.4% formaldehyde, 0.002%Na 2S 2O 3) the middle development; Take pictures.
The RAPD-PCR amplified production adopts 1.2% agarose gel electrophoresis that contains ethidium bromide (EB).Adopt 1 times of TAE electrophoretic buffer; DNA sample after the amplification adds 3 μ l sample loading buffers (containing 0.25% tetrabromophenol sulfonphthalein, 30% sucrose), uses liquid-transfering gun application of sample 8 μ l behind the mixing, electrophoresis 40-50min under the 120V constant-pressure conditions; Utilize gel imaging system to take pictures, preserve.
4 hybrid seed genetic purities are identified
To Glutinous Semen Maydis ' glutinous No. 2 of Su Yu ' F 1Hybrid seedling genomic dna amplified production carries out electrophoretic analysis.The target label bar that produces according to 3 Auele Specific Primers brings differentiates true, pseudostationary.
Have only and have the father simultaneously, the seedling individual plant of maternal specific marker band is judged to be real cross-fertilize seed, and the seedling individual plant that only has maternal specific marker band or male parent specific marker band is judged to be pseudostationary.
Wherein utilize SRAP combination of primers NAUSRem7/NAUSRpm1 to carry out pcr amplification, producing size in cross-fertilize seed is the maternal specific marker band NAUSRem7/NAUSRpm1 of 170bp 170Male parent specific marker band NAUSRem7/NAUSRpm1 with 150bp 150The seedling individual plant be judged to be real cross-fertilize seed (Fig. 1).
Utilize RAPD primer NAURP709 to carry out pcr amplification, producing size in cross-fertilize seed is the maternal specific marker band NAURP709 of 790bp 790(Fig. 2), and utilize primer NAURP712 to carry out pcr amplification, producing size in cross-fertilize seed is the male parent specific marker band NAURP712 of 1250bp 1250Seedling individual plant (Fig. 3) is judged to be real cross-fertilize seed.
According to SRAP or RAPD molecule marker Auele Specific Primer analytical results, calculate for inspection seed genetic purity (%): [for inspection seed grain number (seedling number)-pseudostationary grain number (seedling number)]/for inspection seed grain number (seedling number) * 100.
SRAP primer code name NAUSRem7/NAUSRpm1 wherein, its implication is ' NAU ' representative " Agricultural University Of Nanjing "; " SR " represents the SRAP primer, and " NAUSR em7/NAUSR pm1 " represents laboratory SRAP primer numbering;
RAPD primer NAURP709, its implication is ' NAU ' representative " Agricultural University Of Nanjing "; " RP " represents the RAPD primer, and " 709 " represent laboratory RAPD random primer numbering;
RAPD primer NAURP712, its implication is ' NAU ' representative " Agricultural University Of Nanjing "; " RP " represents the RAPD primer, and " 712 " represent laboratory RAPD random primer numbering.
Figure IDA0000107153240000011
Figure IDA0000107153240000021

Claims (2)

1. the authentication method of glutinous corn variety ' glutinous No. 2 of Su Yu ' seed genetic purity is characterized in that may further comprise the steps:
1) extracts ' glutinous No. 2 of Su Yu ' seedling genomic dna;
2) utilize SRAP combination of primers NAUSRem7/NAUSRpm1, or RAPD primer NAURP709 and NAURP712, be that template is carried out pcr amplification with Glutinous Semen Maydis ' glutinous No. 2 of Su Yu ' genomic dna, also or simultaneously utilize SRAP combination of primers NAUSRem7/NAUSRpm1, with RAPD primer NAURP709 and NAURP712, be that template is carried out pcr amplification with Glutinous Semen Maydis ' glutinous No. 2 of Su Yu ' genomic dna;
Described SRAP combination of primers sequence is:
Forward primer NAUSRem7:SEQ ID No.1;
Reverse primer NAUSRpm1:SEQ ID No.2;
The RAPD primer sequence:
NAURP709：SEQ?ID?No.3；
NAURP712：SEQ?ID?No.4；
3) dna fragmentation after will increasing utilizes native polyacrylamide gel electrophoresis to detect the SRAP amplified production respectively, utilizes agarose gel electrophoresis to detect the RAPD amplified production;
4) electrophoresis result is analyzed, the individual plant that has female parent, male parent specific marker band simultaneously is judged to be real cross-fertilize seed, calculate the seed genetic purity, wherein:
It is the maternal specific marker band of 170bp and 150bp male parent specific marker band that SRAP combination of primers NAUSRem7/NAUSRpm1 produces size;
It is the maternal specific marker band of 790bp that RAPD primer NAURP709 produces size; It is 1250bp male parent specific marker band that NAURP712 produces size.
2. the authentication method of glutinous corn variety according to claim 1 ' glutinous No. 2 of Su Yu ' seed purity is characterized in that described SRAP-PCR reaction system contains 10ng genomic dna, 2.5mmolL -1Mg 2+, 0.15mmolL -1DNTPs, 0.5 μ molL -1SRAP forward primer NAUSRem7 and reverse primer NAUSRpm1,0.75U Taq archaeal dna polymerase; Pcr amplification program: 90 ℃ of 3min; 94 ℃ of 1min, 35 ℃ of 1min, 72 ℃ of 1.5min, 5 circulations; 94 ℃ of 1min, 50 ℃ of 1min, 72 ℃ of 1.5min, 33 circulations; 72 ℃ of 7min extend 10 ℃ of preservations;
Described RAPD-PCR reaction system contains 10ng genomic dna, 2.0mmolL -1Mg 2+, 0.15mmolL -1DNTPs, 0.4 μ molL -1NAURP709 or NAURP712,0.75U Taq archaeal dna polymerase; 94 ℃ of 2min of pcr amplification program; 94 ℃ of 30s, 37 ℃ of 45s, 72 ℃ of 1.5min, 40 circulations; 72 ℃ of 10min extend 10 ℃ of preservations.
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