CN109852717B - Molecular marking method for identifying non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star - Google Patents
Molecular marking method for identifying non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star Download PDFInfo
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Abstract
The invention discloses a molecular marker method for identifying non-heading Chinese cabbage glossy bantam yellow, yellow rose and green star. The method is not influenced by the growth and development stage of the plant and the environment, has the advantages of quick and accurate detection and the like, can effectively supervise and arbitrate when variety counterfeiting or disputed conditions occur, and is worthy of large-scale popularization.
Description
Technical Field
The invention belongs to the technical field of vegetable variety identification and improved variety, and particularly relates to a molecular marking method for identifying non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star.
Background
The non-heading Chinese cabbage has the advantages of abundant germplasm resources, various varieties, wide distribution, long history and high economic value. With the continuous development of the breeding process of the non-heading Chinese cabbage, more and more excellent varieties are newly cultivated. However, it also presents a significant challenge to the seed market. Before being identified by the state (identification and recognition) or provincial level (identification and recognition), one variety needs to be subjected to single plant screening, strain comparison tests and variety region tests, and the time consumption is long. The authenticity of the variety is identified according to morphological characteristics and biochemical components, and the variety is also easily influenced by the development stage, cultivation measures and environmental conditions.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the technical problems of large workload, high cost, long time, lag result and the like in the existing planting identification method, the invention provides a method for identifying varieties of non-heading Chinese cabbage with glossy and dwarf yellow foot yellow, yellow rose and greenstar by utilizing SSR molecular markers, and the method has the advantages of rapidness and accuracy in detection, no influence of environmental conditions and the like.
The technical scheme is as follows: an SSR molecular marker primer of non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star has the sequence as follows:
SSR03118-F：5’- GACTCTCAGCCTCCTTTTGC -3’（SEQ ID NO.1），
SSR03118-R：5’- TGACTGGGTTCATGCGTAAT -3’（SEQ ID NO.2）。
the molecular marking method of the primers for identifying the non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star comprises the following steps:
counting the band types of the samples in the step 3, comparing and judging, wherein the result judgment standard is as follows: if the electrophoresis band of the PCR amplification sample of the non-heading Chinese cabbage variety to be detected is consistent with one band of the glossy bantam yellow, the yellow rose and the green star and is polymorphic with the other two bands, the variety of the detection sample and the variety with the consistent bands are proved to be the same variety; if the two are not consistent, the variety to be detected is not any one of the glossy dwarf yellow, yellow rose and green star.
Further, the PCR amplification system in step 2 comprises: 2 XTaq MasterMix, 10 muM forward SSR primer, 10 muM reverse SSR primer, 50 ng/. mu.L DNA template and deionized water.
Further, the PCR amplification system in step 2 is a 10 μ L amplification system, which specifically includes: 2 XTaq MasterMix 5. mu.L, 10. mu.M forward SSR primer 1. mu.L, 10. mu.M reverse SSR primer 1. mu.L, 50 ng/. mu.L DNA template 1. mu.L, and deionized water 2. mu.L.
Further, the PCR amplification procedure in step 2 was pre-denaturation at 94 ℃ for 5min, followed by denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 1min for 20s, for 28 cycles, final extension at 72 ℃ for 7min, and storage at 4 ℃.
Further, 8% native polyacrylamide gel electrophoresis was used for detection in step 3.
The primer is applied to identification of non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star.
Has the advantages that: the invention can simultaneously distinguish three varieties of the non-heading Chinese cabbage, namely the glossy dwarf yellow foot, the yellow rose and the green star by utilizing the developed SSR molecular marker primer pair and the detection method thereof. The method is not influenced by the growth and development stage of the plant and the environment, has the advantages of quick and accurate detection and the like, can effectively supervise and arbitrate when variety counterfeiting or disputed conditions occur, and is worthy of large-scale popularization.
Drawings
FIG. 1 is a graph showing the results of non-denaturing polyacrylamide gel electrophoresis after PCR amplification of yellow-yellow dwarf, yellow rose and lunar star,
wherein: the Marker is DL1000 Marker, 1 is a lane of non-heading Chinese cabbage, glossy and short foot yellow, 2 is a lane of non-heading Chinese cabbage, yellow rose, and 3 is a lane of non-heading Chinese cabbage, green star.
In FIG. 2, a, b, c, 1, 2, 3 are graphs of PCR amplified results of six non-heading Chinese cabbage varieties, namely tetraploid Suzhou green, diploid Suzhou green, caraway, yellow dwarf yellow, yellow rose and Luxingxing, on native polyacrylamide gel electrophoresis.
Wherein: the Marker is DL1000 Marker, a is tetraploid Suzhou cyan lane of non-heading Chinese cabbage, b is diploid Suzhou cyan lane of non-heading Chinese cabbage, c is fragrant green lane of non-heading Chinese cabbage, 1 is bright short foot yellow lane of non-heading Chinese cabbage, 2 is yellow rose lane of non-heading Chinese cabbage, and 3 is green star lane of non-heading Chinese cabbage.
Detailed Description
The technical solution of the present invention is further described below with reference to the specific embodiments.
The invention utilizes non-heading Chinese cabbage database, SSR molecular marker primer database and SSR primers reported in reference documents to purposefully screen 59 pairs of SSR primers, and carries out primer polymorphism screening after synthesizing the 59 pairs of SSR primers.
The screening process comprises the following specific steps: firstly, DNA of the oleanolic dwarf yellow, the yellow rose and the aventurine is extracted, then genome DNA of the oleanolic dwarf yellow, the yellow rose and the aventurine is taken as a template, PCR amplification is respectively carried out on 59 pairs of SSR primers, polyacrylamide gel electrophoresis is carried out after the amplification is finished, the gel is dyed after the electrophoresis is finished, finally, the band amplified by each pair of primers is judged, if polymorphism occurs in the amplified bands of a certain pair of primers in three different varieties, the pair of primers can be determined to be used for subsequent accurate identification tests, otherwise, the pair of primers is indicated to be not suitable for identifying three varieties of the oleanolic dwarf yellow, the yellow rose and the aventurine. Finally, through the selection process, an SSR molecular marker primer SSR03118 for subsequent identification tests is selected.
The invention also provides a molecular marking method for identifying the non-heading Chinese cabbage glossy bantam yellow, yellow rose and green star, which comprises the following steps:
(1) extracting DNA of the non-heading Chinese cabbage to be identified;
(2) performing PCR amplification by using the genomic DNA of the non-heading Chinese cabbage to be detected as a template and the SSR03118 as a primer;
(3) and (3) carrying out electrophoretic separation on the PCR amplification product to obtain the separation band type of each sample, if the electrophoretic band of the PCR amplification sample of the non-heading Chinese cabbage variety to be detected is consistent with one band of the oil-bright bangboot yellow, the yellow rose and the aventurine, and polymorphism exists between the electrophoretic band and the other two bands, the variety with the consistent band of the detection sample is proved to be the same variety, and if the electrophoretic band is not consistent with the other two bands, the variety to be detected is proved not to be any one of the oil-bright bangboot yellow, the yellow rose and the aventurine.
Example 1
The molecular marking method for identifying the non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star provided by the embodiment comprises the following steps of:
(1) extraction of genome DNA of non-heading Chinese cabbage
DNA was extracted according to the instructions of the kit for rapid extraction of plant genomic DNA (purchased from Hangzhou BaoSai Biotechnology Co., Ltd.), as follows:
1) taking a proper amount of non-heading cabbage young leaf (100 mg) in a 2mL centrifuge tube, adding steel balls, placing in liquid nitrogen for cooling, and fully grinding by using a plant tissue grinder;
2) adding 500 μ L of PL1 (which had been preheated to 65 deg.C (beta-mercaptoethanol had been added to 0.2%) and 5 μ L of RNaseA, vortexing vigorously, and warm-bathing in a 65 deg.C water bath for 10 min;
3) adding 170 mu L of Buffer PL2 solution, mixing well, standing at room temperature for 5min, centrifuging at 12000rpm for 5min, and transferring the supernatant into a new centrifuge tube;
4) adding 750 μ L of PL3 solution into the above solution, mixing immediately, transferring the obtained solution into adsorption column RB, centrifuging at 12000rpm for 1min, and removing waste liquid in the collection tube;
5) adding 700 μ L of rinsing liquid WB into the adsorption column, centrifuging at 12000rpm for 1min, and pouring off the waste liquid;
6) repeating the step 5 once;
7) putting the adsorption column RB back into the collection pipe, centrifuging at 12000rpm for 2min, and removing rinsing liquid;
8) taking out the adsorption column RB, putting the adsorption column RB into a new 1.5mL centrifuge tube, adding 100 mu L double distilled water into the adsorption column, standing the adsorption column for 3min at room temperature, centrifuging the adsorption column for 1min at 12000rpm, and eluting the genomic DNA;
9) the DNA was diluted to 50 ng/. mu.L by purity and concentration detection, and stored in a freezer at-20 ℃ for further use.
The sequence of the glossy dwarf foot yellow is shown in SEQ ID NO. 3.
The sequence of the yellow rose is shown in SEQ ID NO. 4.
The sequence of the Lvxing is shown in SEQ ID NO. 5.
(2) SSR amplification of genome of non-heading Chinese cabbage
An amplification system with the total volume of 10 mu L of SSR-PCR is adopted, and the method specifically comprises the following steps: 2 xTaq MasterMix is 5 μ L, forward and reverse SSR primers are 1 μ L each at a concentration of 10 μ M, template DNA is 1 μ L at a concentration of 50 ng/. mu.L, and 10 μ L of the system is complemented with deionized sterile water. Wherein the forward primer is SSR03118, and the forward primer is SSR03118-F sequence 5'-GACTCTCAGCCTCCTTTTGC-3'; the reverse primer SSR03118-R has the sequence of 5'-TGACTGGGTTCATGCGTAAT-3'.
The PCR amplification procedure was: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, annealing at 56 deg.C for 30s, extension at 72 deg.C for 1min for 20s, 28 cycles, final extension at 72 deg.C for 7min, and storage at 4 deg.C.
(3) Polyacrylamide gel electrophoresis detection
After the PCR amplification is finished, 8% non-denaturing polyacrylamide gel electrophoresis is adopted, the electrophoresis buffer solution is 1 xTBE, 1.5 mu L of sample is loaded, after 180v electrophoresis is carried out for 1.5h, silver staining is carried out for color development, electrophoresis images are photographed and collected, and the diversity is counted. Specific gel solutions include: 7.5mL of polyacrylamide solution (specific solution preparation: 142.5g of acrylamide and 7.5g of methylene-bisacrylamide are weighed and dissolved by adding water to be 500 mL), 10 XTBE 3mL, 20mL of deionized sterile water, 200 muL of 10% ammonium persulfate solution and 40 muL of TEMED.
The specific glue dyeing process comprises the following steps:
(1) after the glue is removed, 0.2 percent AgNO is added3The solution is placed on a shaking table and slowly shaken for 8-10 min.
(2) Pouring AgNO3The solution was then added with deionized water and shaken slowly on a shaker for 1 min.
(3) A color developing solution containing 1.5% of sodium hydroxide and 1.0% of formaldehyde is added, and the mixture is slowly shaken on a shaking table until the color development is successful.
(4) Pouring off the color developing solution, washing twice with deionized water, wrapping with preservative film, air drying and observing.
If the detected product band is consistent with one band of the oil bright yellow dwarf, the yellow rose and the green star and has polymorphism with other two bands, the variety of the detected sample with the consistent band is proved to be the same variety, and if the detected product band is not consistent with the other two bands, the variety to be detected is proved to be not any one of the oil bright yellow dwarf, the yellow rose and the green star.
FIG. 1 shows the electrophoresis results of "Youliangponghuang", "Huang Rose", and "Luxing", wherein the Marker is DL1000 Marker, 1 is lane of "Youliangponghuang" in non-heading Chinese cabbage, 2 is lane of "Huang Rose in non-heading Chinese cabbage, and 3 is lane of" Luxing "in non-heading Chinese cabbage.
FIG. 2 shows the electrophoresis results of tetraploid Suzhou green, diploid Suzhou green, caraway, glossy bantam yellow, yellow rose and green star. Looking at the picture, the tetraploid Suzhou green, the diploid Suzhou green and the caraway have consistency, are obviously different from the three groups of the oily dwarf yellow, the yellow rose and the green star, and are also obviously different from the three groups of the oily dwarf yellow, the yellow rose and the green star.
As can be seen from FIGS. 1 and 2, the primers of the present invention are specific to three non-heading Chinese cabbages, such as yellow-rose yellow, green-star, and can be used to identify three varieties.
Sequence listing
<110> Nanjing university of agriculture
<120> a molecular marking method for identifying non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star
<160> 5
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gactctcagc ctccttttgc 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tgactgggtt catgcgtaat 20
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<211> 322
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<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 (5)
1. A molecular marking method for identifying the non-heading Chinese cabbage glossy dwarf yellow, yellow rose and green star is characterized in that: the method comprises the following steps:
step 1, extracting DNA of non-heading Chinese cabbage to be identified;
step 2, taking the DNA extracted in the step 1 as a template, and performing PCR amplification on the DNA by using SSR molecular marker primers SSR03118-F, SSR 03118-R:
SSR03118-F：5’- GACTCTCAGCCTCCTTTTGC -3’
SSR03118-R：5’- TGACTGGGTTCATGCGTAAT -3’；
step 3, carrying out electrophoretic separation on the PCR amplification product obtained in the step 2 to obtain a separation band pattern of each sample;
counting the band types of the samples in the step 3, comparing and judging, wherein the result judgment standard is as follows: if the electrophoresis band of the PCR amplification sample of the non-heading Chinese cabbage variety to be detected is consistent with any one band of the glossy bantam yellow, the yellow rose and the green star and is polymorphic with other two bands, the variety of the detection sample and the variety with the consistent bands are proved to be the same variety; if the two are not consistent, the variety to be detected is not any one of the glossy dwarf yellow, yellow rose and green star.
2. The molecular labeling method of claim 1, wherein: the PCR amplification system in the step 2 comprises: 2 XTaq MasterMix, 10 muM forward SSR primer, 10 muM reverse SSR primer, 50 ng/. mu.L DNA template and deionized water.
3. The molecular labeling method of claim 1, wherein: the PCR amplification system in the step 2 is a 10 mu L amplification system, and specifically comprises: 2 XTaq MasterMix 5. mu.L, 10. mu.M forward SSR primer 1. mu.L, 10. mu.M reverse SSR primer 1. mu.L, 50 ng/. mu.L DNA template 1. mu.L, and deionized water 2. mu.L.
4. The molecular labeling method of claim 1, wherein: the PCR amplification procedure in step 2 is pre-denaturation at 94 ℃ for 5min, then denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 20s, which are carried out for 28 cycles, and finally extension at 72 ℃ for 7min, and storage at 4 ℃.
5. The molecular labeling method of claim 1, wherein: and 3, detecting by using 8% non-denaturing polyacrylamide gel electrophoresis.
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| 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 |
| CN112575117B (en) * | 2021-01-07 | 2023-03-21 | 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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| NL1036531C2 (en) * | 2009-02-06 | 2010-08-09 | Bejo Zaden Bv | XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS RESISTANT BRASSICA PLANT AND METHODS FOR OBTAINING IT. |
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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 |
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| NL1036531C2 (en) * | 2009-02-06 | 2010-08-09 | Bejo Zaden Bv | XANTHOMONAS CAMPESTRIS PV. CAMPESTRIS RESISTANT BRASSICA PLANT AND METHODS FOR OBTAINING IT. |
| CN108165647A (en) * | 2018-01-02 | 2018-06-15 | 南京农业大学 | A kind of molecule labelling method for being used to identify Chinese cabbage SUZHOUQING(sic), short-foot Huang, Wuta-tsai |
| 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 |
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