CN100460521C - SCAR marking method for identifying Chinese cabbage variety and detecting seed purity - Google Patents

SCAR marking method for identifying Chinese cabbage variety and detecting seed purity Download PDF

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CN100460521C
CN100460521C CNB2006101047424A CN200610104742A CN100460521C CN 100460521 C CN100460521 C CN 100460521C CN B2006101047424 A CNB2006101047424 A CN B2006101047424A CN 200610104742 A CN200610104742 A CN 200610104742A CN 100460521 C CN100460521 C CN 100460521C
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scar
primer
seed
mark
chinese cabbage
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CN1944677A (en
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张鲁刚
王绮
张明科
惠麦侠
张战风
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Northwest A&F University
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Northwest A&F University
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Abstract

The present invention discloses SCAR marking method for identifying Chinese cabbage species and detecting the seed purity. The method adopts the genome DNA of new spring Chinese cabbage variety, Guanchun, and its parents as the template in RAPD analysis to establish the specific RAPD fingerprint map of Guanchun hybrid species and its parents, and obtains one specific RAPD female parent marker and one specific RAPD hybrid marker, which may be used in the species identification and seed purity detection of Guanchun hybrid species. Random identification on 134 Guanchun colonies shows the uniform results with field identification.

Description

A kind of SCAR of utilization mark carries out Chinese cabbage cultivar to be identified and the seed purity method of inspection
Technical field
The vegetable variety that the invention belongs to agricultural is identified and the breeding field, relates to the cultivar identification and the seed purity method of inspection of Chinese cabbage, and the particularly a kind of SCAR of utilization mark carries out the cultivar identification and the seed purity method of inspection of " hat spring " Chinese cabbage.
Background technology
To the check of hybrid seed genetics purity, more and more cause the special attention of seed production unit.Though it is reliable that field shape is learned qualification result, enforcement is simple, and sense cycle is long, and consuming time taking a lot of work is difficult to satisfy results then, then with the requirement of planting.Isozyme and protein electrophorese technology have obtained application in some brassicaceous vegetable crop varieties and hybrid purity check, but because there is tissue specificity in it, in factors such as Different Organs, different growing stage banding pattern difference instabilities, and polymorphism is not abundant, bands of a spectrum are less, defectives such as difference is limited are difficult to satisfy increasing kind and detect requirement.
Molecule marker is the popular domain of Recent study, it is a research object with DNA, has following superiority: (1) directly with form performance of DNA, all can detect at each tissue, each developmental stage of plant materials, be not subjected to season, environmental restraint, do not have expression whether problem; (2) numbers of poles is many, spreads all over whole genome, can detected locus almost be unlimited; (3) polymorphism height, nature exists many allelic variations, does not need the special special genetic stocks of creating; (4) show as " neutrality ", do not influence the expression of objective trait, do not have the chain of certainty with bad proterties; (5) there are many molecule markers to show as codominance (codominance), can identify homozygous genotype and heterozygous genes type, complete genetic information is provided, thereby now formed many molecule markers system.
Round pcr is one of dna molecular marker of getting up of development in recent years, compares with RFLP, and the RAPD mark has following characteristics: the primer of (1) RAPD has versatility, and same set of primer is applicable to the analysis of different biological gene groups.Opposite RFLP mark has ethnic specificity, only can use mutually in certain kind scope; (2) the RAPD technology is simply ripe, and the programmable operation is particularly suitable for mass detection; (3) owing to the randomness of primer, the RAPD mark almost can cover whole genome; (4) the RAPD mark generally shows as dominant inheritance, can not distinguish heterozygous and homozygous; (5) the RAPD mark generally can detect a plurality of sites simultaneously, is suitable for making up fast very much the screening of genetic map and differing materials polymorphism; (6) RAPD analyzes and does not use isotropic substance, thereby compares safety, also is not subjected to special regional prescribed limits; (7) RAPD technology DNA consumption is few, is not subjected to the restriction in plant strain growth condition, period.So RAPD analyzes the numerous research fields such as plant breeding, population genetics and species diversity that are very suitable for extensive sample.
The SCAR mark, promptly the sequence specific amplified band is based on the class mark that RAPD grows up, and on the basis to the order-checking of polymorphism RAPD product, designs a pair of new primer, increases specifically one and be defined as the DNA section in a site in heredity.On basis to the order-checking of polymorphism RAPD product, design a pair of new primer, a dna fragmentation at specific site specifically increases, generally 3 ' and 5 ' end at former RAPD primer prolongs 14 bases, utilize the primer of each 24 base of two ends to carry out specific amplified, therefore, this method is compared with RAPD has following advantage: owing to use long primer and high annealing temperature, therefore have repeatability and good stability, advantages such as banding pattern is simple, therefore, the SCAR mark becomes the first-selected mark of molecular breeding, cultivar identification and seed purity check.
Summary of the invention
The purpose of this invention is to provide a kind of SCAR of utilization mark and carry out the Chinese cabbage cultivar evaluation and the seed purity method of inspection.
To achieve these goals, the technical solution used in the present invention is, a kind of SCAR of utilization mark carries out Chinese cabbage cultivar to be identified and the seed purity method of inspection, and this method is with " hat spring " father and mother and F 1For seed is research material, at first carries out the PCR reaction system optimization, the screening Auele Specific Primer, further, be converted into the SCAR mark, be applied to " hat spring " Chinese cabbage cultivar and identify and the seed purity check the specific fragment cloning and sequencing, it is characterized in that, specifically may further comprise the steps:
1) preparation of material:
To transplant to the land for growing field crops behind spring Chinese cabbage new variety " hat spring " and the parental seed Pregermination and seedling breeding thereof, in seedling phase individual plant numbering, picked at random 100-200 strains, it is standby to get young leaflet tablet extraction DNA seedling stage, becomes the strain phase according to breediness characterization of biological proterties;
2) DNA extraction:
Adopt the CTAB method, its process is to get the 0.2g young leaflet tablet, grind into powder under the liquid nitrogen; Change over to the 1.5mL centrifuge tube after adding 750 μ L CTAB extracting solutions; 65 ℃ of water-bath 30min then, mixing therebetween vibrates; Use the saturated phenol of isopyknic Tris-, chloroform, primary isoamyl alcohol mixing solutions and isopyknic chloroform, each extracting of primary isoamyl alcohol mixing solutions respectively once, under the normal temperature under the 12000r/min condition centrifugal 2 times, each 10min; Get the dehydrated alcohol deposit D NA that supernatant liquor adds 2 times of volume precoolings, centrifugal 15min under 4 ℃ of conditions; Centrifugal rotational speed is 12000r/min, and centrifugal back is with 70% ethanol washing and precipitating 2～3 times; Add the DNA stock solution, confirm purification degrees, be stored in-20 ℃ of refrigerators standby through the DNA purity detecting;
3) SCAR design of primers:
Maternal Auele Specific Primer SC134-1200
fS134M：TGCTGCAGGTGAAGGCTGGTTC
rS134M：TGCTGCAGGTTATCCTTTTTGGTTG
Hybrid Auele Specific Primer SC266-260
fS266F：AGGCCCGATGGAGTTTGTGTAAG
rS266F：AGGCCCGATGATGATAAATCATTG
4) SCAR-pcr amplification:
Adopt the PCR reaction system of 25 μ L, comprising 1 * damping fluid, 2.0mmol/L MgCl 2, 2.5mmol/L dNTPs, 0.3 μ mol/L primer, 1U TaqDNA polysaccharase, 30～50ng template DNA; Response procedures: the SCAR amplification program is, 94 ℃ of pre-sex change 4min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 90s, circulates 30 times, last 72 ℃ of extension 10min;
Amplified production is at 1.5% agarose gel that contains 5 μ g/mLEB, and electrophoresis 1.0h under the 120V observes under ultraviolet lamp and photograph behind the electrophoresis;
5) feature of SCAR mark:
A, SC134-1200 marks:
SCAR primer with SC134-1200 is identified " hat spring " colony, in maternal and cross-fertilize seed, can amplify the specific band of S134-1200, and can not expand the specific band that S134-1200 in male parent and the self-crossing seedling thereof, therefore utilize evaluation " hat spring " the reciprocal cross seed that this relatively can be correct; Identify that in the colony ratio of amplified band individual plant being arranged is exactly the hybrid rate of reciprocal cross seed;
B, SC266-260 mark:
SCAR primer with SC266-260 identifies that to " hat spring " colony maternal, male parent does not all have amplified band, has only true hybrid that amplified band is arranged, and what do not have amplified band may be pseudostationary; Utilize evaluation " hat spring " quadrature, the reciprocal cross list that this relatively can be correct to receive and mix the receipts seed; Identify that in the colony ratio of amplified band individual plant being arranged is exactly the hybrid rate of reciprocal cross seed.
Method of the present invention is by the evaluation of 134 hat spring random populations, and qualification result is identified consistent with the field.
Description of drawings
Fig. 1 is primer S 47To " hat spring " and parent's thereof amplification, wherein, M.DL2000 Marker; 1, female parent, 2, male parent, 3～15, cross-fertilize seed;
Fig. 2 is primer S 134To " hat spring " cross-fertilize seed and parent's thereof amplification, wherein, M.DL2000Marker; 1, female parent, 2, male parent, 3～18, cross-fertilize seed;
Fig. 3 is primer S 4To " hat spring " cross-fertilize seed and parent's thereof amplification, wherein, M.DL2000Marker; 1, female parent, 2, male parent, 3～18, cross-fertilize seed;
Fig. 4 is primer S 73To " hat spring " cross-fertilize seed and parent's thereof amplification collection of illustrative plates, wherein, M.DL2000Marker; 1. maternal; 2. male parent; 3～18. cross-fertilize seed;
Fig. 5 is primer S 494To " hat spring " cross-fertilize seed and parent's thereof amplification collection of illustrative plates, wherein, M.DL2000Marker; 1. maternal; 2. male parent; 3～19. cross-fertilize seed
Fig. 6 is primer S 266To " hat spring " cross-fertilize seed and parent's thereof amplification collection of illustrative plates, wherein, M.DL2000Marker; 1. maternal; 2. male parent; 3～19. cross-fertilize seed;
The result that Fig. 7 SC134-1200 primer increases to " hat spring " colony, wherein, M.DL2000Marker; 1. maternal; 2. male parent; 3～17. cross-fertilize seed;
The result that Fig. 8 SC266-260 primer increases to " hat spring " colony, wherein, M.DL2000Marker; 1～2. female parent; 3～4. male parents; 5～20. cross-fertilize seed;
The present invention is described in further detail to carry out the cultivar identification of " hat spring " Chinese cabbage and seed purity check below in conjunction with accompanying drawing and contriver.
Embodiment
Concrete enforcement of the present invention is carried out according to the following steps:
1, the preparation of material:
To transplant to the land for growing field crops behind spring Chinese cabbage new variety " hat spring " and the parental seed Pregermination and seedling breeding thereof, in seedling phase individual plant numbering, picked at random 100-200 strains, it is standby to get young leaflet tablet extraction DNA seedling stage.Become the strain phase according to breediness characterization of biological proterties.
2, DNA extraction
Adopt the CTAB method, process is to get the tender true leaf of 0.2g children, the liquid nitrogen grinding powdered; Change over to the 1.5mL centrifuge tube after adding 750 μ LCTAB extracting solutions; 65 ℃ of water-bath 30min vibrate mixing therebetween several times then; Use isopyknic (saturated phenol of Tris-: chloroform: primary isoamyl alcohol=25:24:1) and isopyknic (chloroform: each extracting one of primary isoamyl alcohol=24:1) is inferior to the centrifugal 10min of 12000r/min (2 times, normal temperature) respectively.Get the dehydrated alcohol deposit D NA that supernatant liquor adds 2 times of volume precoolings, in the centrifugal 15min of 12000r/min (4 ℃).With 70% ethanol washing and precipitating 2～3 times; After adding DNA stock solution (ethanol of 70% volume fraction wherein adds 0.3moL/L NaAc solution),, confirm that purifying is better through the DNA purity detecting.Be stored in-20 ℃ of refrigerators standby.
3, RAPD amplification
PCR adopts the reaction system of 25 μ L, comprising 1 * damping fluid, and 2.0mmol/L MgCl 2, 2.5mmol/L dNTPs, 0.3 μ mol/L primer, 1U TaqDNA polysaccharase, 30～50ng template DNA.Reaction conditions: pre-94 ℃/4min of sex change, 94 ℃/60s, 36 ℃/60s, 72 ℃/90s, 45 circulations, 72 ℃ are extended 10min.Last RAPD amplified production is at 1.5% agarose gel that contains 5 μ g/mLEB, and electrophoresis 1.0h under the 120V observes under ultraviolet lamp and photograph behind the electrophoresis.
4, the screening of Auele Specific Primer
Randomly draw each 10 strain of male parent, female parent and cross-fertilize seed, set up mixing pit respectively, carry out the RAPD primer screening.In 320 primers, there are 208 primers can amplify stable and band clearly, the RAPD band number of different primer amplifications is 0～11 not wait, the RAPD clip size is between 0.4kb～3.0kb, wherein the primer of performance amplification polymorphism has 96 in parent and cross-fertilize seed, accounts for 30% of total primer.Through repeating screening more than 4 times, there are 6 primers to stablize and amplify specific band, be respectively S4, S47, S73, S134, S266, S494.
5, Auele Specific Primer is to the evaluation of hat spring colony
1) S47 and S134 are to the evaluation of " hat spring " colony
With primer S47 134 " hat spring " cross-fertilize seed individual plant DNA are carried out the RAPD amplification, in maternal and 132 individual plants, all expand the characteristic strip that S47-700, wherein 2 individual plants " 13 " (accompanying drawing 1) and " 56 " (not shown) and male parent do not amplify S47-700 characteristic strip, and its amplification bands of a spectrum are similar to the male parent banding pattern, can think that individual plant " 13 " and " 56 " may be the male parent self-crossing seedlings, detecting hybrid rate is 98.5%.
When carrying out identical amplification with primer S134, the result that performance is consistent.Maternal and 132 individual plants all expand the characteristic strip that S134-1200,2 individual plants " 17 " (accompanying drawing 2 wherein, with " 13 " of accompanying drawing 1 is same individual plant) and " 56 " (not shown) do not expand the characteristic strip that S134-1200, and individual plant " 17 " is similar to the amplification banding pattern of male parent with " 56 ", verified that once more these 2 individual plants may be the male parent self-crossing seedlings, detecting hybrid rate also is 98.5%.
Find out that thus the maternal key band that utilizes primer S47 and S134 to amplify all can detect miscellaneous male parent seed, but can't distinguish female parent and cross-fertilize seed, therefore for identifying that the reciprocal cross seed is effective.
2) primer S4 is to the evaluation of " hat spring " colony
With primer S4 134 " hat spring " cross-fertilize seed individual plant DNA are carried out the RAPD amplification, the result shows, male parent and 130 individual plants all expand and S4-370 characteristic strip, wherein 4 individual plants " 17 " (accompanying drawing 3), " 20 ", " 66 " and " 78 " (not shown) all do not expand and this characteristic strip, the banding pattern that amplifies is similar to maternal banding pattern, illustrate that this 4 strain may be maternal self-crossing seedling, is not found with S47, S134 the time.And the male parent selfing strain of finding previously " 13 " (accompanying drawing 1) does not show specificity, shows that primer S4 is the male parent specificity amplification primer, can distinguish with cross-fertilize seed maternal, and it detects hybrid rate is 97%.But male parent and cross-fertilize seed can't be distinguished, therefore the evaluation for the quadrature seed is effectively, if unite use with maternal specificity amplification primer, then can identify male parent selfing strain and maternal selfing strain simultaneously.
3) primer S73 and S494 are to the evaluation of " hat spring " colony
134 hat spring cross-fertilize seed individual plant DNA are increased with primer S73, there are 128 individual plants to amplify simultaneously and male parent and maternal band, be the characteristic strip S73-730 and the maternal characteristic strip S73-660 (accompanying drawing 4 of male parent, the part picture), there is 4 strains (" 17 " of accompanying drawing 4 and " 20 ", " 66 " and " 78 " (not shown)) amplification banding pattern similar to maternal banding pattern; There is 2 strains (being " 13 " of accompanying drawing 1, " 56 ") amplification banding pattern similar, verified once more in selected 134 individual plants, have the maternal self-crossing seedling of 4 strains to the male parent banding pattern, 2 strain male parent self-crossing seedling (not shown)s, it detects hybrid rate is 95.5%.
In accompanying drawing 4, the amplification banding pattern of individual plant " 17 " (being " 13 " in the accompanying drawing 1, " 17 " in the accompanying drawing 2) is all similar to the male parent banding pattern, does not expand maternal characteristic strip S73-660, consistent with primer S47 and S134 assay, provide this individual plant may be the evidence of male parent self-crossing seedling once more; With primer S73 the individual plant in the accompanying drawing 3 " 17 " is increased, the maternal banding pattern of result is similar, verifies that once more this individual plant may be maternal self-crossing seedling (picture does not show).
4) primer S266 is to the evaluation of " hat spring " colony
134 hat spring cross-fertilize seed individual plant DNA are increased with primer S266, there are 128 individual plants to amplify and are different from male parent and maternal band, it is characteristic strip S266-260 (accompanying drawing 6, the part picture), there is 4 strains (" 17 " of accompanying drawing 4 and " 20 ", " 66 " and " 78 " (not shown)) amplification banding pattern similar to maternal banding pattern; It (is " 13 " of accompanying drawing 1 that 2 strains are arranged, " 56 ") amplification banding pattern similar to the male parent banding pattern, verified once more in selected 134 individual plants, has the maternal self-crossing seedling of 4 strains, 2 strain male parent self-crossing seedling (not shown)s, wherein the individual plant in the accompanying drawing 6 " 14,15 " is other Chinese cabbage seed of artificial miscellaneous, and it detects hybrid rate is 95.5%.
6, the order-checking of specific band and SCAR design of primers
Required band is dug out under ultraviolet lamp, put into the 1.5mL pipe, reclaim test kit with agarose and reclaim fragment, carry out the conversion and the screening of recombinant plasmid with reference to the method in " molecular cloning experiment guide ".Detect with PCR.The purpose segment is entrusted the order-checking of learned company and the result is provided.8 pairs of SCAR primers and synthetic have been designed according to sequence results.The result is as follows:
1) random primer S 4: GGACTGGAGT
Order-checking tense marker title: S4-414F, measured length: 383bp
GGACTGGAGTGCTAGTTTGAGAACAAAAACTTGGTTTAGTGCTATTTTGGTCTTTTTCTCTTATT
TAAACTCAAGCTTGTACTCTTCATTCTTCTATGGCAAAGATTATATTTTCAAACTGAATTTTTTA
TATACATAGCCAAAAAAAATATGTTTAATAGTCCGTCCGTAGAGATTCAGATTAATCCATATACC
TAATCTGCATTGTTCTAAGCTCATGAGATATCTCAAGCACCAACCCTTGCATAATATTTTCAACT
ACATCAGAGACTACACCTTCAGATTCTCCACCACAATCCAACCAAAGACATGGCTTCATCAGATC
AACCTCGACGACCTGATCCAATGTCCTTGGAGAGT CACAGCAGAGTAAACTCCAGTCC
The SCAR primer:
fS4F：GGACTGGAGTGCTAGTTTGAGAAC
rS4F：GGACTGGAGTTTACTCTGCTGTG
2) random primer S 47: TTGGCACGGG
Order-checking tense marker title: S47-700M, measured length: 666bp
TTGGCACGGGATGTTGAGGTTGGTAAGCATCTATACCTAGCTCAGCCATTAGAGTTAGAGTCCAT
TCACGGCTAACTTACCACCAACGAAGAATTCCTCGCATAAAATGATTCTTACATTCTATAGATAC
TGTAAAAGCCAACTCATGTTTCTGCGAAAATAAATAAGAAAGCTCATTTTTTTCTTTTATTATGC
TAGCCTTTCTAAATATCCTTCTCTTGTTGATGAAGCGCCCCCTTATGGAGTTGAGCCTGACTTGG
GCAGCCGCTTTCATTGTTGTGCCATTATATTCTTTTTTCATGTCTTTTATGGATGGCTGTTCTCA
TTCCGTCGGAGGCGATGGAAGCGGTGCCAGCTCTTCGAAGCGACCGCGTCTCGACCTGCCCAGAA
CAACAGGAATAGTTTAATCTAAAAAAAAAATATGAAGAAGTAAGAAAGATCAAAATGGAGTTAGC
CGAACTGATTGGCTCCCTAATTGACCAAGCGAAAGAGGGGCTGCAATTTTCCGCCCATAAGGGGA
GTGCCTTCGCCACCGGTAATGTGTGGAAGAGCTAATAAACCGAATTGGGAACAATGAGAAGGCCA
GACCAGAAGGGAAAACGACCCCAATGGGGAGATTACCGATTTTATAAATAAAAAGAA GTTCTTAA
AGCGGGCCCGTGCCAA
The SCAR primer:
fs47-700m：TTGGCACGGGATGTTGAGGTTG
rs47-700m：TTGGCACGGGCCCGCTTTAAGAAC
3) random primer S 134: TGCTGCAGGT
Order-checking tense marker title: S134-1200M, measured length: 1091bp
TGCTGCAGGTGAAGGCTGGTTCAATATTTGACAATATATTGATCAGTGATGACCCGG
AATATGCAAGGAGCATGGTGGATGACTATTTTGCTCAGCACAGAGAGGTAGGCTTC
TCCATGCCTTTGGGATACCTTCTTTTTGCTTCTTTTTAGACAAAGTTAAATGAGTTTC
TGCATGATTTTATTATAGAAAATGTCTTATTAGTAATCTGATGTGCTTATATATACTCTT
CTGACCAGTCTGAGAAGGAGTTATTTGCGGAAGCTGAGAAAGAAAAAAAAGCTAG
AGAAGATGAGGTTTGTAGTTTTCAAAACAAGTCCTTACTTGCTTTTAAAATCTTCTC
TGTTTATCTTAATCATGATCTCATACAATTTGACGATTGCAGGAAGCTAGGATAGCAA
GAGAAGAAGGGGAACGTAGACGAAAAGAGAGGGGTGACCGGTATGGACACAGGG
ACAGGAGGCATCGTTACAAACGGGTACATACTCAATGAGACCAACTCAGTTCTACA
GTTTTCTTTCTCCTTCTCAACACTGTTAGTTCTGGAATATAAGTGAGTGACTAAGTAT
AAACGTTTTGTTTTGCAGCACCACCGACGTGGTTACATGGACGATTACCATGTAAGT
AAACTAAGTGGTTTTCTATTCAACCACTTTTAAATGCTTTATGCGTTTTAGTACTGAA
TCATCTTAACCGTTTTCGTGTAGGATGAGCTATAAGGCGCAGTGGTGGCCTGGTGG
GGGATTTGGCAATGACAACCAATAAAAGCAAGTATTCATTAAAGTCATCATACCTAG
AGAGCTGGAAACTATATGGTTGGTTTTTGTAGTTTGTATGATTGTAATATGTATCATAT
CACTTCTTTTTTTTTTTTGTCATCTGAAGTTTCATATATCACAAAGGTTAACATTGCA
AACACCTCCTTTACACGGATCCAACACAAAAAGCAATTCTAAACCAAGGACAAAC
TAACACTCCAAAGTCTTTAGTGTACCAAAAAATTTTCGAAGGAGACAACTTCAAGT
CAAAGAGAGGCCAAACGTTTCTCATTTTCAAAGACGTCCTG CAACCAAAAAGGATA
ACCTGCAGCA
SCAR primer: TGCTGCAGGT
fS134M：TGCTGCAGGTGAAGGCTGGTTC
rS134M：TGCTGCAGGTTATCCTTTTTGGTTG
4) random primer S 73: AAGCCTCGTC
Order-checking tense marker title: S73-671M, measured length: 660bp
AAGCCTCGTCTGTGGTAAGAAATTAACCATCATGTTATCTTTGTCCTTGTGTTTTGGTCTCTGGG
TCCTAAGATTTACTCAATCATGTCTGACGTTCTCAGGATCTTAGCAAGAAGCAGTTTAAGAAGTC
TATGACCAAGAAATCACATTCATGGTGGTGGGACAGTCACAATTGTCCCAAGAACTCGAAGTGGC
TTGCTCAGAATCTGGAGAGTATGTTGCCTCACTTATCATTGAGATACCTATCTCCACTTAAATGT
AGATCTTGAACTTGTTCTTATTATGGCTTCTCAGAGATGGATGACCGTGTGAAACACATGTTAAA
GTTGATTGAAGAAGACGCAGACTCTTTTGCCAAGAAAGCTCAGATGTATTACCAGAAGCGTCCCG
AGTTAATCCACCTTGTGGAGGAGTTTTACCGCATGTATCGCGCTTTGGCTGAGCGTTATGATCAA
GCTAGTGGTGAACTTCAGAAAATTCATCTCTCTGGGATACAGTCACAGGGCTCTCTTGAGAAATC
ATCTCCTACTAATTCCCAAGAGAAATCGAGTCACCCTCATAAGGAAGAGGAAGACTCATCATCTC
TGTCTGATTCTGATTCTGACTCTAAATCGGTTCCTGATCATTCCTCTGTTAATGAT GAAGATGGT
GACGAGGCTT
The SCAR primer:
fs73F：AAGCCTCGTCTGTGGTAAG
rs73F：AAGCCTCGTCACCATCTTC
5) random primer S 73: AAGCCTCGTC
Order-checking tense marker title: S73-750F, measured length: 728bp
AAGCCTCGTCTGATGTAAAACACACCGCTTGTGCTGTTTTTTGAAAGAAGCAGTGTCCATTTTCT
TCCTTTCTCGTTGACTAGATCTATCTCACAGGGCTTGTTGTGCTCATTAAACGACACAACCGTAA
AGTCTTTAGAAAGATCCTGGAAAAAAAACAAAGAAATAATACATCACTCACAGTATCATACACTT
GACTACTTACAAAAGGTAAACGAGGAGTGTAGTAATTAGTTACCAAACGATCTTTGATGGGGCTA
TAAGGTGTGACACGTGCTCTGAGGATACAAGAGAAACCTGCTTTTGTGCTCTTGTTCACCAAAAT
GTTCCCTGAATCACAACAATCTTCATTTACATTTTACTACCAAACCAAAAACTCAAAACATATAC
CACTTATGTCGTTTCAAGAAACGAAAACACCTTACCTGCGTCATCCTCTCCTCCATCGTCATCAT
CATCATATTCGTCTTCTCCATCCTCAGATTCAGAATAGTCATCATCAGTCTCAACATCGTCAGTA
TCAACCAAGCTAGGAGACGCGTGGTGACTTATGTCACAAGAATCCGACCGAGGGGACACAGATAC
ATGAAAGATCTCGTCTCCATCGTGTCTGAAAACCAAGAGGTCTCCGTCTCTGAAACTATGAGCTT
TGGCGAACTCTTCCCAGCCGCCGCCGGCGAGCCTGCTTCCCTCTAGTTTCACTTTC CAAATCTTG
TCCGACGAGGCTT
The SCAR primer:
fS73-750F：AAGCCTCGTCTGATGTAAAACAC
rS73-750F：AAGCCTCGTCGGACAAGATTTG
6) random primer S 494: GGACGCTTCA
Order-checking tense marker title: S494-399M, measured length: 376bp
GGACGCTTCAATAATATTGAAATATATTTTTTAAAAAAATATAATAATTGGAATATTGCGTAATT
AAGTTTACGTTTTTGGGAATTTCACATATTAAATAATTAATTACATATTTTGAGGTAAATATTCA
AGAGACTGTGATTTTCATTAATCAATCATTAGAAGAGAATAAAAGCATTAAAAAAATCTTCAGTT
TATTTTAAAAGAAACAGTTGTGACAAATGTTTTTATCAGACAAGACAACAACAAATAATTAAAAA
GACAGTGTCCACCGCCAAAAGAAACTCAACTCTAAAGCTTAAATATGAAGAGAATCAAGTCAAAG
CCGGAGTCTCTGCAAACATATTGGAACACTTT GTCTGAACCTGAAGCGTCC
The SCAR primer:
fS494M：GGACGCTTCAATAATATTG
rS494M：GGACGCTTCAGGTTCAGAC
7) random primer S 494: GGACGCTTCA
Order-checking tense marker title: S494-1770F, measured length: 1771bp
S494-1775F：(-47)
GGACGCTTCAGAAATATTTAATAAAATAAAAAAAAGACCACTAAAATCTTAAAGACTAAATAGTA
AAAAGATAAAGATACAAACGTGTGAAGCAAAACCCAAGCCGACACGTTCAATTCCATCGTTTTGA
TCCGGTTCCGGCAGGCACGAGCTTTAGGCCGGTTGGTTAAAACACGCGCTTTGGACCATTTAGGA
AACACAGTCAGGTTCTCGTGATTTCTTGCTTATAATGGGCCCCTCCCACCCATATGGTCAGCTAA
ACGAGTGAAATTCTAGGGCACACCTTTGATTATTTTTTTTAAATAATCAAAAAATTATTATTAGA
AACCGAAAATTATTATTTATTCCAAAGACATGAATTCCTACAAGTCCACAACTGTTGTTTATGGT
CAAATCATAAAGCAAAACTAATGTTTTCCAAATAGTTAATGGAAAAAAAGAAAAACAAAAGCAAA
TGCATCTTGCGTTGTTGGAATGCAAAAAGAGAGAAAACGATACGTACCTAATGTTAATTCTAATT
AGTTAGTTATAGAATGAGGAGGTACATTAAAAAGGAAAATTAGTATTAGTACTCAGTTAAGTATG
CACGTGTGTGGGAAGTATGGTGGCGAAACTATACAACCAACCCCTAGTAGATGGCTAGTTGTACA
TGTCTCGTGACGTATACATCATATATATAATAAATACGAACATAGAAATTAATAGGACTTAGTTG
TTGCGAATATACGTAAAATCAAATAAAACCAACCATTCAATGTTCTTGGTAAGTAACGAAATAAA
ACGGAAGCAAGAAGGAGAACCACATAAAAGGGAGCTAACTCCTGTACACGTTGTGCTTCATCTGC
CCGTCCGTCAAACTACCACACTCCCTTCTCTTTTCCCCTTTGTCATCTTGTATAATAATATCTCA
CTCTCCCATGCTTTCTAACAT
S494-1775F：(-48)
GGACGCTTCACGGAAGGGCGAAGGCCAAGGAGCTTGGTGAGCTGGTAGTCAGCACCAGGCATGTC
AACTCCAGAGGTGGTGCAGAAGACAACGTGTGTGACCTTGGACTTAGGCTGACCCCACTCCTTGA
TGGCCTTCACTGCTGCATCTTTGCCTAGCTTAGGGACTTCAACCACCACGAGGTCTTGTCTAGCG
TCGAGAGAAGGAGCCATGTAGGCGCACATGTTAGGGTTCTCTTTCAAGAACTCCTCGGTCAAGTG
CATGTGGCGTTTTCTTATGGTCGACTTATCGCCTATTAGAGTCATAATCAAGTTAACTATGTGAA
CATACTAATAAGTACAGTTTAAGGTAGAAGACTATTAAATTTTCGGTTTTATCTATTTAATACAT
TTTTTTTTGTTAGTTAATGAACTAAAAGATACGGATAAAACTGGTGAATTATTTAAATGTATTTT
GAAAATGTACAACAAATTTTAAAAACTAAAAATGATAAACGGATGAATTATATGCCAAAATTCTA
AATGGAAAAATACAAAGTAGGGTTTGTTAAGACATACACATGCGCTTGAACTTCTCCTTAAGGTC
GGTCTTGTGTTCACTGTTGGTGATGCGGAAGTAGTAGTCTGGATACTCAGCTTGGAGCACATGGT
TCGCAGGGTTGGCCGTACCTATCGCTAAGATGCCTGCAGGACCGTCTGCTCTCTGTGCCTTTCTG
ATCTCATCCAACGAAGAACGCCCCATCACCATATTAAACCAACTACGTTTTAACTAAGTGTGATC
GATGTGTTACTTTGCGTAATAAGCAAGTTATGTATGTATGTTTAGAAAGCATGCAGAGTA
The SCAR primer:
fS494F：GGACGCTTCAGAAATATTTAATA
rS494F：GGACGCTTCACGGAAGGGCGAAG
8) random primer S 266: AGGCCCGATG
Order-checking tense marker title: S266-260F, measured length: 294bp
AGGCCCGATGGAGTTTGTGTAAGAAAATGGGTTTCGGCCGTTTTTAAAATAAAACATTGACGACA
AAAAAAAAAAAATAATTTGGCATCTCCCTCCATCTAAATCATGCATGACAAATGAATAAAAAGCT
TTTATTATCATATGACGTCTATACAATGCGTATAATATGTTTCCTTTACTTTATTTACTTGTCGT
CTTTATCTTATTATATTATATGCTACATTATAGTCATTGTTAAAGTCAGAGACTACAGGTATCAT
AAGGTTGCAA CAATGATTTATCATCATCGGGCCT
The SCAR primer:
fS266260F：AGGCCCGATGGAGTTTGTGTAAG
rS266260F：AGGCCCGATGATGATAAATCATTG
7, the checking of SCAR amplification and SCAR mark
SCAR-pcr amplification:
Adopt the PCR reaction system of 25 μ L, comprising 1 * damping fluid, 2.0mmol/L MgCl 2, 2.5mmol/L dNTPs, 0.3 μ mol/L primer, 1U TaqDNA polysaccharase, 30ng～50ng template DNA.Response procedures: the SCAR amplification program is 94 ℃ of pre-sex change 4min, 94 ℃ of sex change 1min, and 55 ℃ of annealing 1min, 72 ℃ are extended 90s, circulate 30 times, and last 72 ℃ are extended 10min.Amplified production is at 1.5% agarose gel that contains 5 μ g/mLEB, and electrophoresis 1.0h under the 120V observes under ultraviolet lamp and photograph behind the electrophoresis.
The SCAR primer is used for two parents and F 1Amplification, if identical with the purpose clip size position of RAPD amplification then illustrate that the SCAR mark transforms successfully.In 8 pairs of SCAR primers, SC 4-370, SC 47-700, SC 73-730 and SC 73-660, SC 494-400 and SC 494-1770 primer does not show polymorphism when " hat spring " cross-fertilize seed colony and parent thereof increase, have only SC134-1200 with 2 pairs of primers of SC266-260 mark the result that colony increases to be conformed to fully with the result of RAPD, illustrates that the SCAR mark transforms successfully.Its primer is as follows:
Maternal Auele Specific Primer SC134-1200
fS134M：TGCTGCAGGTGAAGGCTGGTTC
rS134M：TGCTGCAGGTTATCCTTTTTGGTTG
Hybrid Auele Specific Primer SC266-260
fS266F：AGGCCCGATGGAGTTTGTGTAAG
rS266F：AGGCCCGATGATGATAAATCATTG
The feature of SCAR mark:
A, SC134-1200 marks:
SCAR primer with SC134-1200 is identified " hat spring " colony, from accompanying drawing 7 as can be seen, in maternal and cross-fertilize seed, can amplify the specific band of S134-1200, and can not expand the specific band that S134-1200 in male parent and the self-crossing seedling thereof, therefore utilize evaluation " hat spring " the reciprocal cross seed that this relatively can be correct.Identify that in the colony ratio of amplified band individual plant being arranged is exactly the hybrid rate of reciprocal cross seed.
B, SC266-260 mark:
With the SCAR primer of SC266-260 " hat spring " colony is identified, in accompanying drawing 8, can find out that female parent, male parent all do not have amplified band, have only true hybrid that amplified band is arranged, not having " 10 " and " 15 " etc. of amplified band may be pseudostationary.134 " hat spring " colonies are identified, find to have 6 strain pseudostationaries, identify consistent with the field.Utilize evaluation " hat spring " quadrature, the reciprocal cross list that this relatively can be correct to receive and mix the receipts seed.Identify that in the colony ratio of amplified band individual plant being arranged is exactly the hybrid rate of reciprocal cross seed.
8, the SCAR mark is to the checking of " hat spring " colony
With SCAR primer SC134-1200 " hat spring " colony is identified, wherein male parent does not expand the specific band that S134-1200, the specific band that S134-1200 is not expanded in same " 11 " and " 14 " in cross-fertilize seed, and two male parent self-crossing seedlings that this 2 strain also identifies with the RAPD primer just coincide with the RAPD amplification.
With SCAR primer SC266-260 " hat spring " colony is identified, all do not expand the specific band that S266-260 female parent, male parent and hybrid " 10 " and " 15 ", this result and RAPD amplification are found relatively " 10 " are maternal self-crossing seedling, " 15 " are the male parent self-crossing seedling.
Identify with RAPD qualification result consistent with S134-1200 to whole " hat spring " colony with SCAR primer S266-260, the character observation result matches with the field, promptly, find to have 2 strain male parent self-crossing seedlings, the maternal self-crossing seedling of 4 strains in that 134 " hat spring " colonies are identified.
Special RAPD segment nucleotide sequence and SCAR primer sequence
1) random primer S 4: GGACTGGAGT
Order-checking tense marker title: S4-414F, measured length: 383bp;
GGACTGGAGTGCTAGTTTGAGAACAAAAACTTGGTTTAGTGCTATTTTGGTCTTTTTCTCTTATT
TAAACTCAAGCTTGTACTCTTCATTCTTCTATGGCAAAGATTATATTTTCAAACTGAATTTTTTA
TATACATAGCCAAAAAAAATATGTTTAATAGTCCGTCCGTAGAGATTCAGATTAATCCATATACC
TAATCTGCATTGTTCTAAGCTCATGAGATATCTCAAGCACCAACCCTTGCATAATATTTTCAACT
ACATCAGAGACTACACCTTCAGATTCTCCACCACAATCCAACCAAAGACATGGCTTCATCAGATC
AACCTCGACGACCTGATCCAATGTCCTTGGAGAGT CACAGCAGAGTAAACTCCAGTCC
The SCAR primer:
fS4F：GGACTGGAGTGCTAGTTTGAGAAC
rS4F：GGACTGGAGTTTACTCTGCTGTG
2) random primer S 47: TTGGCACGGG
Order-checking tense marker title: S47-700M, measured length: 666bp;
TTGGCACGGGATGTTGAGGTTGGTAAGCATCTATACCTAGCTCAGCCATTAGAGTTAGAGTCCAT
TCACGGCTAACTTACCACCAACGAAGAATTCCTCGCATAAAATGATTCTTACATTCTATAGATAC
TGTAAAAGCCAACTCATGTTTCTGCGAAAATAAATAAGAAAGCTCATTTTTTTCTTTTATTATGC
TAGCCTTTCTAAATATCCTTCTCTTGTTGATGAAGCGCCCCCTTATGGAGTTGAGCCTGACTTGG
GCAGCCGCTTTCATTGTTGTGCCATTATATTCTTTTTTCATGTCTTTTATGGATGGCTGTTCTCA
TTCCGTCGGAGGCGATGGAAGCGGTGCCAGCTCTTCGAAGCGACCGCGTCTCGACCTGCCCAGAA
CAACAGGAATAGTTTAATCTAAAAAAAAAATATGAAGAAGTAAGAAAGATCAAAATGGAGTTAGC
CGAACTGATTGGCTCCCTAATTGACCAAGCGAAAGAGGGGCTGCAATTTTCCGCCCATAAGGGGA
GTGCCTTCGCCACCGGTAATGTGTGGAAGAGCTAATAAACCGAATTGGGAACAATGAGAAGGCCA
GACCAGAAGGGAAAACGACCCCAATGGGGAGATTACCGATTTTATAAATAAAAAGAA GTTCTTAA
AGCGGGCCCGTGCCAA
The SCAR primer:
fs47-700m：TTGGCACGGGATGTTGAGGTTG
rs47-700m：TTGGCACGGGCCCGCTTTAAGAAC
3) random primer S 134: TGCTGCAGGT
Order-checking tense marker title: S 134-1200M, measured length: 1091bp;
TGCTGCAGGTGAAGGCTGGTTCAATATTTGACAATATATTGATCAGTGATGACCCGGAATATGCA
AGGAGCATGGTGGATGACTATTTTGCTCAGCACAGAGAGGTAGGCTTCTCCATGCCTTTGGGATA
CCTTCTTTTTGCTTCTTTTTAGACAAAGTTAAATGAGTTTCTGCATGATTTTATTATAGAAAATG
TCTTATTAGTAATCTGATGTGCTTATATATACTCTTCTGACCAGTCTGAGAAGGAGTTATTTGCG
GAAGCTGAGAAAGAAAAAAAAGCTAGAGAAGATGAGGTTTGTAGTTTTCAAAACAAGTCCTTACT
TGCTTTTAAAATCTTCTCTGTTTATCTTAATCATGATCTCATACAATTTGACGATTGCAGGAAGC
TAGGATAGCAAGAGAAGAAGGGGAACGTAGACGAAAAGAGAGGGGTGACCGGTATGGACACAGGG
ACAGGAGGCATCGTTACAAACGGGTACATACTCAATGAGACCAACTCAGTTCTACAGTTTTCTTT
CTCCTTCTCAACACTGTTAGTTCTGGAATATAAGTGAGTGACTAAGTATAAACGTTTTGTTTTGC
AGCACCACCGACGTGGTTACATGGACGATTACCATGTAAGTAAACTAAGTGGTTTTCTATTCAAC
CACTTTTAAATGCTTTATGCGTTTTAGTACTGAATCATCTTAACCGTTTTCGTGTAGGATGAGCT
ATAAGGCGCAGTGGTGGCCTGGTGGGGGATTTGGCAATGACAACCAATAAAAGCAAGTATTCATT
AAAGTCATCATACCTAGAGAGCTGGAAACTATATGGTTGGTTTTTGTAGTTTGTATGATTGTAAT
ATGTATCATATCACTTCTTTTTTTTTTTTGTCATCTGAAGTTTCATATATCACAAAGGTTAACAT
TGCAAACACCTCCTTTACACGGATCCAACACAAAAAGCAATTCTAAACCAAGGACAAACTAACAC
TCCAAAGTCTTTAGTGTACCAAAAAATTTTCGAAGGAGACAACTTCAAGTCAAAGAGAGGCCAAA
CGTTTCTCATTTTCAAAGACGTCCTGCAACCAAAAAGGATAACCTGCAGCA
SCAR primer: TGCTGCAGGT
fS134M：TGCTGCAGGTGAAGGCTGGTTC
rS134M：TGCTGCAGGTTATCCTTTTTGGTTG
4) random primer S 73: AAGCCTCGTC
Order-checking tense marker title: S 73-671M, measured length: 660bp;
AAGCCTCGTCTGTGGTAAGAAATTAACCATCATGTTATCTTTGTCCTTGTGTTTTGGTCTCTGGG
TCCTAAGATTTACTCAATCATGTCTGACGTTCTCAGGATCTTAGCAAGAAGCAGTTTAAGAAGTC
TATGACCAAGAAATCACATTCATGGTGGTGGGACAGTCACAATTGTCCCAAGAACTCGAAGTGGC
TTGCTCAGAATCTGGAGAGTATGTTGCCTCACTTATCATTGAGATACCTATCTCCACTTAAATGT
AGATCTTGAACTTGTTCTTATTATGGCTTCTCAGAGATGGATGACCGTGTGAAACACATGTTAAA
GTTGATTGAAGAAGACGCAGACTCTTTTGCCAAGAAAGCTCAGATGTATTACCAGAAGCGTCCCG
AGTTAATCCACCTTGTGGAGGAGTTTTACCGCATGTATCGCGCTTTGGCTGAGCGTTATGATCAA
GCTAGTGGTGAACTTCAGAAAATTCATCTCTCTGGGATACAGTCACAGGGCTCTCTTGAGAAATC
ATCTCCTACTAATTCCCAAGAGAAATCGAGTCACCCTCATAAGGAAGAGGAAGACTCATCATCTC
TGTCTGATTCTGATTCTGACTCTAAATCGGTTCCTGATCATTCCTCTGTTAATGAT GAAGATGGT
GACGAGGCTT
The SCAR primer:
fs73F：AAGCCTCGTCTGTGGTAAG
Rs73F:AAGCCTCGTCACCATCTTC5) random primer S 73: AAGCCTCGTC
Order-checking tense marker title: S 73-750F, measured length: 728bp;
AAGCCTCGTCTGATGTAAAACACACCGCTTGTGCTGTTTTTTGAAAGAAGCAGTGTCCATTTTCT
TCCTTTCTCGTTGACTAGATCTATCTCACAGGGCTTGTTGTGCTCATTAAACGACACAACCGTAA
AGTCTTTAGAAAGATCCTGGAAAAAAAACAAAGAAATAATACATCACTCACAGTATCATACACTT
GACTACTTACAAAAGGTAAACGAGGAGTGTAGTAATTAGTTACCAAACGATCTTTGATGGGGCTA
TAAGGTGTGACACGTGCTCTGAGGATACAAGAGAAACCTGCTTTTGTGCTCTTGTTCACCAAAAT
GTTCCCTGAATCACAACAATCTTCATTTACATTTTACTACCAAACCAAAAACTCAAAACATATAC
CACTTATGTCGTTTCAAGAAACGAAAACACCTTACCTGCGTCATCCTCTCCTCCATCGTCATCAT
CATCATATTCGTCTTCTCCATCCTCAGATTCAGAATAGTCATCATCAGTCTCAACATCGTCAGTA
TCAACCAAGCTAGGAGACGCGTGGTGACTTATGTCACAAGAATCCGACCGAGGGGACACAGATAC
ATGAAAGATCTCGTCTCCATCGTGTCTGAAAACCAAGAGGTCTCCGTCTCTGAAACTATGAGCTT
TGGCGAACTCTTCCCAGCCGCCGCCGGCGAGCCTGCTTCCCTCTAGTTTCACTTTC CAAATCTTG
TCCGACGAGGCTT
The SCAR primer:
fS73F：AAGCCTCGTCTGATGTAAAACAC
rS73F：AAGCCTCGTCGGACAAGATTTG
6) random primer S 494: GGACGCTTCA
Order-checking tense marker title: S494-399M, measured length: 376bp;
GGACGCTTCAATAATATTGAAATATATTTTTTAAAAAAATATAATAATTGGAATATTGCGTAATT
AAGTTTACGTTTTTGGGAATTTCACATATTAAATAATTAATTACATATTTTGAGGTAAATATTCA
AGAGACTGTGATTTTCATTAATCAATCATTAGAAGAGAATAAAAGCATTAAAAAAATCTTCAGTT
TATTTTAAAAGAAACAGTTGTGACAAATGTTTTTATCAGACAAGACAACAACAAATAATTAAAAA
GACAGTGTCCACCGCCAAAAGAAACTCAACTCTAAAGCTTAAATATGAAGAGAATCAAGTCAAAG
CCGGAGTCTCTGCAAACATATTGGAACACTTT GTCTGAACCTGAAGCGTCC
The SCAR primer:
fS494M：GGACGCTTCAATAATATTG
rS494M：GGACGCTTCAGGTTCAGAC
7) random primer S 494: GGACGCTTCA
Order-checking tense marker title: S494-1770F, measured length: 1771bp;
S494-1775F：(-47)
GGACGCTTCAGAAATATTTAATAAAATAAAAAAAAGACCACTAAAATCTTAAAGACTAAATAGTA
AAAAGATAAAGATACAAACGTGTGAAGCAAAACCCAAGCCGACACGTTCAATTCCATCGTTTTGA
TCCGGTTCCGGCAGGCACGAGCTTTAGGCCGGTTGGTTAAAACACGCGCTTTGGACCATTTAGGA
AACACAGTCAGGTTCTCGTGATTTCTTGCTTATAATGGGCCCCTCCCACCCATATGGTCAGCTAA
ACGAGTGAAATTCTAGGGCACACCTTTGATTATTTTTTTTAAATAATCAAAAAATTATTATTAGA
AACCGAAAATTATTATTTATTCCAAAGACATGAATTCCTACAAGTCCACAACTGTTGTTTATGGT
CAAATCATAAAGCAAAACTAATGTTTTCCAAATAGTTAATGGAAAAAAAGAAAAACAAAAGCAAA
TGCATCTTGCGTTGTTGGAATGCAAAAAGAGAGAAAACGATACGTACCTAATGTTAATTCTAATT
AGTTAGTTATAGAATGAGGAGGTACATTAAAAAGGAAAATTAGTATTAGTACTCAGTTAAGTATG
CACGTGTGTGGGAAGTATGGTGGCGAAACTATACAACCAACCCCTAGTAGATGGCTAGTTGTACA
TGTCTCGTGACGTATACATCATATATATAATAAATACGAACATAGAAATTAATAGGACTTAGTTG
TTGCGAATATACGTAAAATCAAATAAAACCAACCATTCAATGTTCTTGGTAAGTAACGAAATAAA
ACGGAAGCAAGAAGGAGAACCACATAAAAGGGAGCTAACTCCTGTACACGTTGTGCTTCATCTGC
CCGTCCGTCAAACTACCACACTCCCTTCTCTTTTCCCCTTTGTCATCTTGTATAATAATATCTCA
CTCTCCCATGCTTTCTAACAT
S494-1775F：(-48)
GGACGCTTCACGGAAGGGCGAAGGCCAAGGAGCTTGGTGAGCTGGTAGTCAGCACCAGGCATGTC
AACTCCAGAGGTGGTGCAGAAGACAACGTGTGTGACCTTGGACTTAGGCTGACCCCACTCCTTGA
TGGCCTTCACTGCTGCATCTTTGCCTAGCTTAGGGACTTCAACCACCACGAGGTCTTGTCTAGCG
TCGAGAGAAGGAGCCATGTAGGCGCACATGTTAGGGTTCTCTTTCAAGAACTCCTCGGTCAAGTG
CATGTGGCGTTTTCTTATGGTCGACTTATCGCCTATTAGAGTCATAATCAAGTTAACTATGTGAA
CATACTAATAAGTACAGTTTAAGGTAGAAGACTATTAAATTTTCGGTTTTATCTATTTAATACAT
TTTTTTTTGTTAGTTAATGAACTAAAAGATACGGATAAAACTGGTGAATTATTTAAATGTATTTT
GAAAATGTACAACAAATTTTAAAAACTAAAAATGATAAACGGATGAATTATATGCCAAAATTCTA
AATGGAAAAATACAAAGTAGGGTTTGTTAAGACATACACATGCGCTTGAACTTCTCCTTAAGGTC
GGTCTTGTGTTCACTGTTGGTGATGCGGAAGTAGTAGTCTGGATACTCAGCTTGGAGCACATGGT
TCGCAGGGTTGGCCGTACCTATCGCTAAGATGCCTGCAGGACCGTCTGCTCTCTGTGCCTTTCTG
ATCTCATCCAACGAAGAACGCCCCATCACCATATTAAACCAACTACGTTTTAACTAAGTGTGATC
GATGTGTTACTTTGCGTAATAAGCAAGTTATGTATGTATGTTTAGAAAGCATGCAGAGTA
The SCAR primer:
fS494F：GGACGCTTCAGAAATATTTAATA
rS494F：GGACGCTTCACGGAAGGGCGAAG
8) random primer S 266: AGGCCCGATG
Order-checking tense marker title: S266-260F, measured length: 294bp;
AGGCCCGATGGAGTTTGTGTAAGAAAATGGGTTTCGGCCGTTTTTAAAATAAAACATTGACGACA
AAAAAAAAAAAATAATTTGGCATCTCCCTCCATCTAAATCATGCATGACAAATGAATAAAAAGCT
TTTATTATCATATGACGTCTATACAATGCGTATAATATGTTTCCTTTACTTTATTTACTTGTCGT
CTTTATCTTATTATATTATATGCTACATTATAGTCATTGTTAAAGTCAGAGACTACAGGTATCAT
AAGGTTGCAA CAATGATTTATCATCATCGGGCCT
The SCAR primer:
fS266F：AGGCCCGATGGAGTTTGTGTAAG
rS266F：AGGCCCGATGATGATAAATCATTG。

Claims (2)

1. one kind is utilized the SCAR mark to carry out the Chinese cabbage cultivar evaluation and the seed purity method of inspection, it is characterized in that, this method is with " hat spring " father and mother's basis and F 1For seed is that research material carries out the PCR reaction system optimization, the screening Auele Specific Primer further to the specific fragment cloning and sequencing, is converted into the SCAR mark, and this SCAR mark is used for " hat spring " Chinese cabbage cultivar is identified and seed purity is checked, specifically may further comprise the steps:
1) preparation of material:
To transplant to the land for growing field crops behind spring Chinese cabbage new variety " hat spring " and the parental seed Pregermination and seedling breeding thereof, in seedling phase individual plant numbering, picked at random 100-200 strains, it is standby to get young leaflet tablet extraction DNA seedling stage, becomes the strain phase according to breediness characterization of biological proterties;
2) DNA extraction:
Adopt the CTAB method, its process is to get the 0.2g young leaflet tablet, grind into powder under the liquid nitrogen; Change over to the 1.5mL centrifuge tube after adding 750 μ L CTAB extracting solutions; 65 ℃ of water-bath 30min then, mixing therebetween vibrates; Use the saturated phenol of isopyknic Tris-, chloroform, primary isoamyl alcohol mixing solutions and isopyknic chloroform, each extracting of primary isoamyl alcohol mixing solutions respectively once, under the normal temperature under the 12000r/min condition centrifugal 2 times, each 10min; Get the dehydrated alcohol deposit D NA that supernatant liquor adds 2 times of volume precoolings, centrifugal 15min under 4 ℃ of conditions; Centrifugal rotational speed is 12000r/min, and centrifugal back is with 70% ethanol washing and precipitating 2～3 times; Add the DNA stock solution, confirm purification degrees, be stored in-20 ℃ of refrigerators standby through the DNA purity detecting;
3) SCAR design of primers:
Hybrid Auele Specific Primer SC266-260
fS266—260F：AGGCCCGATGGAGTTTGTGTAAG
rS266—260F：AGGCCCGATGATGATAAATCATTG
4) SCAR-pcr amplification:
Adopt the PCR reaction system of 25 μ L, comprising 1 * damping fluid, 2.0mmol/L MgCl 2, 2.5mmol/L dNTPs, 0.3 μ mol/L primer, 1U TaqDNA polysaccharase, 30～50ng template DNA; Response procedures: the SCAR amplification program is, 94 ℃ of pre-sex change 4min, and 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 90s, circulates 30 times, last 72 ℃ of extension 10min;
Amplified production is at 1.5% agarose gel that contains 5 μ g/mLEB, and electrophoresis 1.0h under the 120V observes under ultraviolet lamp and photograph behind the electrophoresis;
5) feature of SCAR mark:
The SC266-260 mark:
SCAR primer with SC266-260 identifies that to " hat spring " colony maternal, male parent does not all have amplified band, has only true hybrid that amplified band is arranged, and what do not have amplified band may be pseudostationary; Utilize evaluation " hat spring " quadrature, the reciprocal cross list that this relatively can be correct to receive and mix the receipts seed; Identify that in the colony ratio of amplified band individual plant being arranged is exactly the hybrid rate of reciprocal cross seed.
2. the SCAR of utilization mark as claimed in claim 1 carries out Chinese cabbage cultivar to be identified and the seed purity method of inspection, it is characterized in that, and the special segment total length 294bp of described SC266-260 mark, its nucleotide sequence is:
AGGCCCGATGGAGTTTGTGTAAGAAAATGGGTTTCGGCCGTTTTTAAAATAAAACATTG
ACGACAAAAAAAAAAAAATAATTTGGCATCTCCCTCCATCTAAATCATGCATGACAAAT
GAATAAAAAGCTTTTATTATCATATGACGTCTATACAATGCGTATAATATGTTTCCTTT
ACTTTATTTACTTGTCGTCTTTATCTTATTATATTATATGCTACATTATAGTCATTGTT
AAAGTCAGAGACTACAGGTATCATAAGGTTGCAA CAATGATTTATCATCATCGGGCCT。
CNB2006101047424A 2006-10-17 2006-10-17 SCAR marking method for identifying Chinese cabbage variety and detecting seed purity Expired - Fee Related CN100460521C (en)

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