CN109852717A - It is a kind of for identifying the molecule labelling method of the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star - Google Patents
It is a kind of for identifying the molecule labelling method of the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star Download PDFInfo
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Abstract
The invention discloses a kind of for identifying the molecule labelling method of the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star, and the SSR molecular marker primer pair and its detection method of exploitation can distinguish three the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star kinds simultaneously.This method is not influenced by vine growth and development stage and environment, has many advantages, such as that detection is quick, accurate, effectively can be supervised and be arbitrated when there is kind personation or disputable situation, is worth a wide range of and is promoted.
Description
Technical field
The invention belongs to vegetable variety identifications and breeding technical field, and in particular to one kind is for identifying Chinese cabbage oil
Bright short-foot Huang, Autumn Gold, green star molecule labelling method.
Background technique
Chinese cabbage germ plasm resource is abundant, type is more, distribution is wide, and with a long history, and economic value is high.With not balling
The continuous development of Chinese cabbage breeding process, the excellent variety newly cultivated are more and more.But it is also brought to Seed Market huge
Challenge.One kind needs before determining through national (reflect, recognize) or provincial (reflect, recognize) through single plant screening, variety test of lines, kind region
Test, it is time-consuming very long.And differential variety authenticity is gone according to morphological feature and biochemical composition, also arranged vulnerable to stage of development, cultivation
Apply the influence with environmental condition.
Summary of the invention
The technical issues of solution: the present invention is for heavy workload, at high cost, time present in existing field plot test method
The technical problems such as long, result lag, provide it is a kind of using the glossy short-foot Huang of SSR molecular marker identification Chinese cabbage, Autumn Gold,
The method of green star kind, this method have many advantages, such as detection quickly, it is accurate, not by environmental influence.
Technical solution: a kind of glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star SSR molecular marker primer, sequence
Are as follows:
SSR03118-F:5 '-GACTCTCAGCCTCCTTTTGC -3 ' (SEQ ID NO.1),
SSR03118-R:5 '-TGACTGGGTTCATGCGTAAT -3 ' (SEQ ID NO.2).
Above-mentioned primer is used to identify the molecule labelling method of the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star, including with
Lower step:
Step 1, the DNA of Chinese cabbage to be identified is extracted；
Step 2, using the DNA of step 1 extraction as template, with above-mentioned primer pair, it carries out PCR amplification；
Step 3, the pcr amplification product of step 2 is separated by electrophoresis, obtains the separation banding pattern of each sample；
The banding pattern of each sample in step 3 is counted, comparison determines that as a result judgment criteria is as follows: if not balling to be measured is white
The PCR amplification sample electrophoresis band of vegetable kind is consistent with a band in glossy short-foot Huang, Autumn Gold, green star, and and its
There are polymorphisms for his two bands, then prove the detection sample and the consistent kind of band is same kind；If it is inconsistent,
Show kind to be measured not and be glossy short-foot Huang, Autumn Gold, any one in green star.
Further, the PCR amplification system in step 2 includes: 2 × Taq MasterMix, 10 μM of forward direction SSR primers, 10
μM reversed SSR primer, 50ng/ μ L DNA profiling, deionized water.
Further, the PCR amplification system in step 2 is 10 μ L amplification systems, is specifically included: 2 × Taq MasterMix
5 μ L, 10 μM of 1 μ L of forward direction SSR primer, 10 μM of reversed 1 μ L of SSR primer, 1 μ L of 50ng/ μ L DNA profiling, 2 μ L of deionized water.
Further, PCR amplification program is 94 DEG C of initial denaturation 5min in step 2, then 94 DEG C of denaturation 30s, 56 DEG C of annealing
30s, 72 DEG C of extension 1min20s, totally 28 recycle, last 72 DEG C of extensions 7min, 4 DEG C of preservations.
Further, it is detected in step 3 using 8% native polyacrylamide gel electrophoresis.
Application of the above-mentioned primer in the glossy short-foot Huang of Chinese cabbage, Autumn Gold, the identification of green star.
The utility model has the advantages that the SSR molecular marker primer pair and its detection method of exploitation of the present invention can will not balling it is white
Three the bright short-foot Huang of rape oil, Autumn Gold, green star kinds distinguish simultaneously.This method is not by vine growth and development stage and environment
Influence, have many advantages, such as that detection is quick, accurate, when there is kind personation or when disputable situation can effectively be supervised and
Arbitration is worth a wide range of and promotes.
Detailed description of the invention
Fig. 1 is the native polyacrylamide gel electrophoresis result figure after glossy short-foot Huang, Autumn Gold, green star PCR amplification,
Wherein: Marker is DL1000 Marker, 1 is the glossy short-foot Huang swimming lane of Chinese cabbage, 2 is Chinese cabbage Huang rose
Rare swimming lane, 3 are the green star swimming lane of Chinese cabbage.
A, b, c, 1,2,3 are respectively tetraploid SUZHOUQING(sic), diploid SUZHOUQING(sic), fragrant green vegetables, glossy short-foot Huang, Huang in Fig. 2
Rose, six Chinese cabbage kinds of green star PCR amplification after native polyacrylamide gel electrophoresis result figure.
Wherein: Marker is DL1000 Marker, a is Chinese cabbage tetraploid SUZHOUQING(sic) swimming lane, b is that balling is not white
Dish diploid SUZHOUQING(sic) swimming lane, c are Chinese cabbage perfume (or spice) green vegetables swimming lane, 1 are the glossy short-foot Huang swimming lane of Chinese cabbage, 2 for not
Chinese cabbage Autumn Gold swimming lane, 3 are the green star swimming lane of Chinese cabbage.
Specific embodiment
Below in conjunction with specific embodiment, technical scheme is described further.
The present invention utilizes to be reported not in Chinese cabbage database, SSR molecular marker primer database and bibliography
It is purposive in Chinese cabbage SSR primer to filter out 59 pairs of SSR primers, primer will be carried out after the synthesis of above-mentioned 59 pairs of SSR primers
Polymorphism screening.
The detailed process of screening are as follows: the DNA for extracting glossy short-foot Huang, Autumn Gold, green star first, then with glossy short-foot
Huang, Autumn Gold, green star genomic DNA be template, PCR amplifications are carried out to 59 pairs of SSR primers respectively, are gathered after amplification
Acrylamide gel electrophoresis, by the aobvious dyeing of gel after electrophoresis, the band finally come out to every a pair of of primer amplification is sentenced
It is disconnected, if the amplified band of certain pair of primers polymorphism occurs in these three different cultivars, determine that this pair of primers can be with
For subsequent accurate qualification test, otherwise, indicate that this pair of primers is not suitable for identifying glossy short-foot Huang, Autumn Gold, green
These three Chinese cabbage kinds of star.Finally, by this selection course, SSR points for subsequent qualification test have been selected
Sub- labeled primer SSR03118.
The present invention also provides a kind of for identifying the molecule labelling method of the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star,
It the described method comprises the following steps:
(1) DNA of Chinese cabbage to be identified is extracted；
(2) using Chinese cabbage genomic DNA to be detected as template, using SSR03118 as primer, PCR amplification is carried out；
(3) pcr amplification product is separated by electrophoresis, obtains the separation banding pattern of each sample, if Chinese cabbage kind to be measured
PCR amplification sample electrophoresis band it is consistent with a band in glossy short-foot Huang, Autumn Gold, green star, and with other two
There are polymorphisms for band, then prove that the detection sample and the consistent kind of band are same kind, if it is inconsistent, show to
Surveying kind is not glossy short-foot Huang, Autumn Gold, any one of in green star.
Embodiment 1
The molecule labelling method provided in this embodiment for identifying the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star, including it is following
Step:
(1) extraction of Chinese cabbage genomic DNA
It is extracted according to plant genome DNA rapidly extracting kit (being purchased from Hangzhou Bao Sai Biotechnology Co., Ltd) specification
DNA, briefly as follows:
1) it takes appropriate Chinese cabbage young leaflet tablet (100mg) in 2mL centrifuge tube, steel ball is added and is placed in cooling in liquid nitrogen,
It is fully ground using plant tissue beveller；
0.2%) and 5 μ L RNaseA 2) PL1(that 500 μ L have been preheated at 65 DEG C is added and has been added to beta -mercaptoethanol to, acutely
Be vortexed concussion, the warm bath 10min in 65 DEG C of water-bath；
3) the Buffer PL2 solution of 170 μ L is added, mixes well, is placed at room temperature for 5min, 12000rpm is centrifuged 5min, by supernatant
It is transferred in a new centrifuge tube；
4) the PL3 solution of 750 μ L is added in Xiang Shangshu solution, mixes at once, and acquired solution is transferred in adsorption column RB,
12000rpm is centrifuged 1min, outwells the waste liquid in collecting pipe；
5) 700 μ L rinsing liquid WB, 12000rpm centrifugation 1min are added into adsorption column, outwell waste liquid；
6) it is primary that 5 steps are repeated；
7) adsorption column RB is put back in collecting pipe, 12000rpm is centrifuged 2min, eliminates rinsing liquid；
8) adsorption column RB is taken out, is put into new 1.5mL centrifuge tube, 100 μ L distilled waters is added in adsorption column, are stored at room temperature
3min, 12000rpm are centrifuged 1min, elute genomic DNA；
9) DNA is diluted to 50ng/ μ L through purity and Concentration Testing, deposits in spare in -20 DEG C of refrigerators.
Glossy short-foot Huang sequence is as shown in SEQ ID NO.3.
Autumn Gold sequence is as shown in SEQ ID NO.4.
Green star sequence is as shown in SEQ ID NO.5.
(2) Chinese cabbage genome SSR is expanded
Use SSR-PCR total volume to specifically include for the amplification system of 10 μ L: 2 × Taq MasterMix is 5 μ L, concentration 10
μM forward and reverse SSR primer is respectively 1 μ L, and concentration is that 50ng/ μ L template DNA is 1 μ L, supplies 10 μ L systems with deionization sterile water.
Wherein forward and reverse primer is SSR03118, and forward primer SSR03118-F sequence is 5 '-GACTCTCAGCCTCCTTTTGC -3 '；
Reverse primer SSR03118-R sequence is 5 '-TGACTGGGTTCATGCGTAAT -3 '.
PCR amplification program are as follows: 94 DEG C of initial denaturation 5min, then 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extensions
1min20s, totally 28 recycle, last 72 DEG C of extensions 7min, 4 DEG C of preservations.
(3) polyacrylamide gel electrophoresis detects
After PCR amplification, using 8% native polyacrylamide gel electrophoresis, electrophoretic buffer is 1 × TBE, on 1.5 μ L
Sample, after 180v electrophoresis 1.5h, silver staining colour developing takes pictures and acquires electrophoretic image and count diversity.Specific gel solution includes: poly-
Acrylamide solution (prepare: weighing 142.5g acrylamide and 7.5g methene-bisacrylamide, is dissolved in water by specific solution
And be settled to 500mL) 7.5mL, 10 × TBE 3mL, deionization sterile water 20mL, 10% 200 μ L of ammonium persulfate solution and
TEMED is 40 μ L.
Specific dye glue process are as follows:
(1) 0.2%AgNO is put into after tearing glue open3Solution, which is placed on shaking table, slowly rocks 8 ~ 10min.
(2) AgNO is outwelled3Solution is simultaneously added deionized water and slowly rocks 1min on shaking table.
(3) it is added containing 1.5% sodium hydroxide and the developing solution of 1.0% formaldehyde, is slowly rocked on shaking table, until aobvious
Color success.
(4) developing solution is outwelled, deionized water is cleaned twice, wrapped up using preservative film, dry observation.
If the product band of detection is consistent with glossy short-foot Huang, Autumn Gold, the one of band of green star, and and its
There are polymorphisms for his two bands, then prove the detection sample and the consistent kind of band is same kind, if it is inconsistent,
Show kind to be measured not and be glossy short-foot Huang, Autumn Gold, any one in green star.
Fig. 1 is the electrophoresis result of glossy short-foot Huang, Autumn Gold, green star, and Marker is DL1000 Marker, 1 is not balling
The glossy short-foot Huang swimming lane of Chinese cabbage, 2 be Chinese cabbage Autumn Gold swimming lane, 3 be the green star swimming lane of Chinese cabbage.
Fig. 2 is the electrophoresis knot of tetraploid SUZHOUQING(sic), diploid SUZHOUQING(sic), fragrant green vegetables, glossy short-foot Huang, Autumn Gold, green star
Fruit.See that picture can obtain tetraploid SUZHOUQING(sic), diploid SUZHOUQING(sic), fragrant three groups of items of green vegetables with consistency, with glossy short-foot Huang, Huang
Rose, green star three groups of items have a notable difference, and glossy short-foot Huang, Autumn Gold, green star this three groups of bands also significant difference.
By Fig. 1 and Fig. 2 it is found that the glossy short-foot Huang of primer pair of the invention, Autumn Gold, these three Chinese cabbages of green star tool
There is specificity, can be used to identify three kinds.
Sequence table
<110>Agricultural University Of Nanjing
<120>a kind of for identifying the molecule labelling method of the glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gactctcagc ctccttttgc 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
tgactgggtt catgcgtaat 20
<210> 3
<211> 322
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gactctcagc ctccttttgc attaaacaaa tgacgtttta gtgtggcatt tctttgctac 60
ttgatgtacg atttcacgta gacttaactc tctcccacca gttcttctat atattgaagt 120
agtttaactc tctcattttc tcactctcat accaaaatac tttgctctct ctctctctct 180
ctctctctct ctgatcgggt gtgtaagaat gtccgattca catagccttc aagatatatt 240
tcacgagaca aatgctggta aactaatccc cttaacctca ttttgtaagc aattgccaat 300
agattacgca tgaacccagt ca 322
<210> 4
<211> 306
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gactctcagc ctccttttgc attaaacaaa tgacgtttta gtgtggcatt tctttgctac 60
ttgatgtacg atttcacgta gacttaactc tctcccacca gttcttctat atattgaagt 120
agtttaactc tctcattttc tcactctcat accaaaatac tttgctctct ctctctgatc 180
ggttgtgtaa gaatgtccga ctcacatagc cttcaagata tatttcacga gacaaatgct 240
ggtaaactaa tccccttaac ctcattttgt aagcaattgc caatagatta cgcatgaacc 300
cagtca 306
<210> 5
<211> 320
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gactctcagc ctccttttgc attaaacaaa tgacgtttta gtgtggcatt tctttgctac 60
ttgatgtacg atttcacgta gacttaactc tctcccacca gttcttctat atattgaagt 120
agtttaactc tctcattttc tcactctcat accaaaatac tttgctctct ctctctctct 180
ctctctctct gatcggttgt gtaagaatgt ccgattcaca tagccttcaa gatatatttc 240
acgagacaaa tgctggtaaa ctaatcccct taacctcatt ttgtaagcaa ttgccaatag 300
attacgcatg aacccagtca 320
Claims (7)
1. the SSR molecular marker primer of a kind of glossy short-foot Huang of Chinese cabbage, Autumn Gold, green star, it is characterised in that: sequence
Are as follows:
SSR03118-F:5 '-GACTCTCAGCCTCCTTTTGC -3 ',
SSR03118-R:5 '-TGACTGGGTTCATGCGTAAT -3 '.
2. utilizing the molecular labeling side of the glossy short-foot Huang of primer identification Chinese cabbage described in claim 1, Autumn Gold, green star
Method, it is characterised in that: the following steps are included:
Step 1, the DNA of Chinese cabbage to be identified is extracted；
Step 2, using the DNA of step 1 extraction as template, with above-mentioned primer pair, it carries out PCR amplification；
Step 3, the pcr amplification product of step 2 is separated by electrophoresis, obtains the separation banding pattern of each sample；
The banding pattern of each sample in step 3 is counted, comparison determines that as a result judgment criteria is as follows: if not balling to be measured is white
The PCR amplification sample electrophoresis band of vegetable kind is consistent with any one band in glossy short-foot Huang, Autumn Gold, green star, and
There are polymorphisms with other two bands, then prove the detection sample and the consistent kind of band is same kind；If different
It causes, then shows kind to be measured not and be glossy short-foot Huang, Autumn Gold, any one of in green star.
3. molecule labelling method according to claim 2, it is characterised in that: the PCR amplification system in step 2 includes: 2
× Taq MasterMix, 10 μM of forward direction SSR primers, 10 μM of reversed SSR primers, 50ng/ μ L DNA profilings, deionized water.
4. molecule labelling method according to claim 2, it is characterised in that: the PCR amplification system in step 2 is 10 μ L
Amplification system specifically includes: 2 × Taq MasterMix, 5 μ L, 10 μM of 1 μ L of forward direction SSR primer, 10 μM of reversed 1 μ L of SSR primer,
1 μ L of 50ng/ μ L DNA profiling, 2 μ L of deionized water.
5. molecule labelling method according to claim 2, it is characterised in that: PCR amplification program is 94 DEG C of pre- changes in step 2
Property 5min, then 94 DEG C of denaturation 30s, 56 DEG C of annealing 30s, 72 DEG C of extension 1min20s, totally 28 circulations, last 72 DEG C of extensions
7min, 4 DEG C of preservations.
6. molecule labelling method according to claim 2, it is characterised in that: use 8% non denatured polyacrylamide in step 3
Amine gel electrophoresis is detected.
7. application of the primer described in claim 1 in the glossy short-foot Huang of Chinese cabbage, Autumn Gold, the identification of green star.
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| CN201811566959.6A CN109852717B (en) | 2018-12-19 | 2018-12-19 | Molecular marking method for identifying non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star |
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| CN110804675A (en) * | 2019-11-21 | 2020-02-18 | 南京农业大学 | Microsatellite DNA marker fingerprint spectrum of non-heading Chinese cabbage and application thereof |
| CN112575117A (en) * | 2021-01-07 | 2021-03-30 | 南京农业大学 | SSR molecular marker primers of non-heading Chinese cabbage and red-flowering Chinese cabbage and application thereof |
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| CN110804675A (en) * | 2019-11-21 | 2020-02-18 | 南京农业大学 | Microsatellite DNA marker fingerprint spectrum of non-heading Chinese cabbage and application thereof |
| CN110804675B (en) * | 2019-11-21 | 2022-07-22 | 南京农业大学 | Microsatellite DNA marker fingerprint spectrum of non-heading Chinese cabbage and application thereof |
| CN112575117A (en) * | 2021-01-07 | 2021-03-30 | 南京农业大学 | SSR molecular marker primers of non-heading Chinese cabbage and red-flowering Chinese cabbage and application thereof |
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