CN108546775B - InDel mark of Chinese cabbage burrs as well as detection primer and application thereof - Google Patents
InDel mark of Chinese cabbage burrs as well as detection primer and application thereof Download PDFInfo
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Abstract
The invention relates to an InDel marked detection primer and application thereof in burrless Chinese cabbage breeding. The detection primers of the Chinese cabbage InDel marked TR-1 are a pair, the nucleotide sequence of an upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2. The invention discloses an InDel mark TR-1 related to Chinese cabbage burrs and a detection primer thereof for the first time, through carrying out PCR amplification on DNA of F2 generation separation population materials generated after hybridization of a hairy self-bred line and a hairless Chinese cabbage self-bred line and the two self-bred lines and detecting by using 8% denatured polyacrylamide gel electrophoresis, the InDel mark TR-1 related to Chinese cabbage burrs can obviously distinguish hairy and hairless Chinese cabbage varieties and can be used for breeding new hairless Chinese cabbage varieties.
Description
Technical Field
The invention relates to an InDel marker of Chinese cabbage burrs as well as a detection primer and application thereof, belonging to the technical field of molecular biology.
Background
Molecular marker-assisted selection (MAS) is not limited by environmental conditions and can be performed at the DNA molecular level at any growth stage of plant development (Tanksley, 1989). The method overcomes the defects of long breeding time, low speed, uncertainty, limitation by environmental conditions and the like of the traditional plants. At present, a plurality of plant molecular marker technical systems are established internationally and applied to genetic improvement of crops, which opens up a new way for crop breeding. The rapid screening by utilizing the molecular marker technology, particularly the appearance of the molecular marker, enables the selection in the breeding work to be changed from the traditional phenotype selection into the genotype selection, and greatly improves the accuracy and the efficiency of the selection. The development and utilization of molecular markers based on PCR, such as RAPD (random amplified polymorphic DNA), SSR (simple sequence repeat), AFLP (amplified fragment length polymorphism), InDel (infection-deletion) and the like, are spread over various crops due to the characteristics of simplicity, rapidness, high efficiency and the like. The InDel mark is characterized in that the insertion/deletion of nucleotide segments occurs among different individuals due to DNA sequences at the same site, specific primers are designed according to sequences at two sides of a target site for PCR amplification, and the length polymorphism of amplified segments is the InDel mark. Because the InDel marker is a co-dominant marker, the method has the advantages of good stability, rich polymorphism and the like, and is widely applied to polymorphism analysis, fine positioning of genes, map-based cloning and the like.
Chinese cabbage (Brassica rapa ssp. pekinensis, AA genome, 2n is 20) originates from China, is one of the vegetable crops with the largest cultivation area and yield in China, has important economic value, and is called as Chinese cabbage. The Chinese cabbage leaf and hair is a protective tissue on the surface of a plant body and is the outermost layer of the plant in contact with the environment. The Chinese cabbage seedling growing liquid has the effects of preventing and treating diseases and insect damage and ultraviolet ray damage, reducing plant transpiration, increasing freezing tolerance and the like, but with the improvement of living standard of people, most consumers prefer a hairless Chinese cabbage variety due to the palatability and the touch, and therefore, the existence of leafy furs is an important target character and commodity character of disease-resistant breeding and quality breeding of the Chinese cabbage at present. However, so far, the lack of markers closely linked with the burr character seriously hinders the breeding process of the Chinese cabbage, which means that the development of molecular markers closely linked with the burr character of the Chinese cabbage has important guiding and practical significance for quickly breeding new varieties of the disease-resistant Chinese cabbage without hairs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an InDel mark of Chinese cabbage burrs, and a detection primer and application thereof.
The technical scheme of the invention is as follows:
an InDel marker TR-1 of Chinese cabbage burr has a nucleotide sequence shown in SEQ ID NO.3 or SEQ ID NO. 4.
The detection primers of the InDel mark TR-1 of the burs of the Chinese cabbages are a pair, the nucleotide sequence of an upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2.
The detection primer of the InDel mark TR-1 of the Chinese cabbage burrs is applied to the selection of hairless Chinese cabbage varieties.
According to a preferred embodiment of the present invention, the steps of the application are as follows:
(1) extracting the genome DNA of the Chinese cabbage variety to be detected to obtain a genome DNA solution;
(2) taking the genome DNA prepared in the step (1) as a template, and carrying out PCR amplification on the genome DNA by using the detection primer to prepare a PCR amplification product;
(3) after the PCR amplification product prepared in the step (2) is subjected to gel electrophoresis, when the electrophoresis pattern of the PCR product shows that the detected sample only has a strip of 156bp, the detected sample is a hairy material; when the electrophoresis pattern of the PCR product shows that only one band of 144bp exists, the detected sample is a hairless material; when the PCR product electrophoretogram shows that the PCR product contains 156bp and 144bp bands, the detected sample is a hairy and hairless hybrid material and is phenotypically represented as a hairy material.
According to a further preferred embodiment of the present invention, in the step (1), the genomic DNA of the to-be-detected Chinese cabbage variety is extracted by using an improved CTAB method.
According to a further preferred embodiment of the present invention, in the step (2), the PCR reaction system is as follows, and the total system is 20 μ L:
20 ng/. mu.L template DNA 2. mu.L, containing 20mM MgCl 210 XPCR buffer2 uL, 2.5mM dNTPs0.5 uL, 10 uM upstream primer 0.4 uL, 10 uM downstream primer 0.4 uL, 1U Taq DNA polymerase 0.2 uL, plus ddH2O to 20. mu.L.
According to a further preferred embodiment of the present invention, in the step (2), the PCR amplification procedure is:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, for a total of 36 cycles; extension at 72 ℃ for 10 min.
According to a further preferred embodiment of the present invention, in the step (3), the gel electrophoresis is a denaturing polyacrylamide gel electrophoresis with a mass percentage of 8%.
Advantageous effects
The invention discloses an InDel mark TR-1 related to Chinese cabbage burrs and a detection primer thereof for the first time, through carrying out PCR amplification on DNA of F2 generation separation population materials generated after hybridization of a hairy self-bred line and a hairless Chinese cabbage self-bred line and the two self-bred lines and detecting by using 8% denatured polyacrylamide gel electrophoresis, the InDel mark TR-1 related to Chinese cabbage burrs can obviously distinguish hairy and hairless Chinese cabbage varieties and can be used for breeding new hairless Chinese cabbage varieties.
Drawings
FIG. 1 is a nucleotide sequence comparison diagram of an InDel marker TR-1 related to Chinese cabbage burs;
the marked area marked by a chain line in the figure is an Indel mark TR-1; compared with the hairless Chinese cabbage (ZHB), the hairless Chinese cabbage (G291) has 12bp DNA insertion;
FIG. 2 is a photograph showing the PCR amplification electrophoresis result of detection primers of InDel marker TR-1 related to Chinese cabbage burrs in F2 generation separated population materials generated after the hybridization of hairless (ZHB) and hairy (G291) Chinese cabbages and the two;
in the figure: lane 1: ZHB (hairless), lane 2: g291 (haired), lanes 3-14: ZHB and G291 crosses the F2 hairless population, lanes 15-26: ZHB and G291 hybrid F2 generation haired population;
FIG. 3 is a photograph showing the result of PCR amplification electrophoresis of InDel marker TR-1 detection primers related to Chinese cabbage burrs in the material of Chinese cabbage with or without hair;
in the figure: lane 1: ZHB (hairless), lane 2: g291 (hairy), lanes 3-17 (hairless): respectively AM4-1, AM3-1, AM5-2, AM9-1, AM94, AM30, AM54, AM72-1, AM10-1, AM56-1, Qing Jiangchun No.2, Degao lily, Xibai 45, Y2, L41, lanes 18-32 (hairy): respectively, West Bai No. 8, China No.1, Qing Jiang CR20, Qing Jiang Xibai No.2, Guangdong Zao, L46, 2013-33, AM2-1, Gao kang No.2, Xibai No. 5, Xiyou No. two, Jintongyuyu, attacking guan No. two, Fengcang No. 60 and De Gao xi kang 65.
FIG. 4 is a photograph showing the result of PCR amplification electrophoresis of the detection primers of the InDel marker TR-1 related to the Chinese cabbage burr and other 7 pairs of InDel marker primer pairs designed by the same method, wherein the detection primers are in the material of hairy and hairless Chinese cabbages;
in the figure: lanes 1-4 (hairless): respectively ZHB, AM3-1, Degao lily, AM10-1, lanes 5-8 (haired): g291, China No.1, West Bai No. 8 and attacking Guo No. two respectively; in the figure, A1-A7 are InDel markers with poor screening effect respectively.
Detailed Description
The technical solutions of the present invention are further described below with reference to the following embodiments and the drawings of the specification, but the scope of the present invention is not limited thereto.
Source of biological material
AM4-1, AM3-1, AM5-2, AM9-1, AM94, AM30, AM54, AM72-1, AM10-1, AM56-1, Y2, L41, AM2-1, Guangdong morning, L46, Gao kang No.2, 2013-33 and attacking second variety are purchased from Shandong province agricultural science institute;
the De-Gao lily and the De-Gaoxiang 65 variety are purchased from the De-Gao vegetable seedling research institute of Dezhou city, Shandong province;
xibai No. 45 and Xibai No. 8 were purchased from Shandong Shanghai breed Ltd;
zhonghua No.1 was purchased from Demin breed Co., Ltd, Shandong province, Lin \, 26384;
qing Jiang CR20, Qing Jiang Xiabai No.2 and Qing Jiang Chunbai No.2 were purchased from the institute of agricultural science in Qingdao city, Shandong province;
xibai No. 5 and Fengcang No. 60 varieties were purchased from vegetable seedling institute of agricultural academy of sciences, Laizhou, Shandong province;
the Xiuyou No. two variety is purchased from the research institute of Chinese cabbage in spring and summer in the Hill of the Hill city;
the golden boy and jade girl varieties are purchased from agriculture science and technology limited, Aoshuofeng, Beijing.
Example 1 development of InDel-labeled primers
And (3) taking the Chinese cabbage burr formation related gene as a candidate gene, and performing whole genome re-sequencing on the Chinese cabbage with hairs (G291) and without hairs (ZHB) by using a re-sequencing technology. And (3) carrying out sequence comparison on candidate genes in the hairy Chinese cabbage and the hairless Chinese cabbage according to a re-sequencing result, designing primers for 1 InDel marker TR-1, wherein a sequence comparison chart of the InDel marker TR-1 of the Chinese cabbage is shown in figure 1. The marked area marked by the dashed line in the figure is an InDel mark TR-1.
The haired cabbage (G291) has a 12bp DNA insertion compared with the hairless cabbage (ZHB). Designing a detection primer of the Chinese cabbage InDel mark TR-1, wherein the primer sequence is shown as SEQ ID NO.1 and SEQ ID NO. 2.
Example 2 application of InDel labeled primers
2.1 extraction of plant Material and template DNA
Selecting commercial varieties of first filial generation of Chinese cabbage, namely Qing-Jiang-Chun-Bai No.2, De-Gao-Bai, Xibai No. 45, Xibai No. 8, Chinese No.1, Qing-Jiang-CR 20, Qing-Jiang-Xibai No.2, Chinese No.1, Gao-kang No.2, Xibai No. 5, Xiyou No. two, golden boy jade girl, Gongguan No. two, Feng-kang 60, Huayang white, De-Gao-Xikang 65, and leaves of hybrid lines of Chinese cabbage Guangdong-Zao, Y2, L41, L46, AM4-1, AM3-1, AM5-2, AM9-1, AM94, AM30, AM54, AM72-1, AM10-1, AM56-1, and hybrid F2 generation leaves of ZHB and G291 as materials.
The method comprises the following steps of extracting total DNA by an improved CTAB method:
1. taking 1-10g of fresh Chinese cabbage leaves, and quickly grinding into powder in liquid nitrogen;
2. transferring the frozen powder into a precooled centrifugal tube, adding an isovolumetric (w/v, g/ml)2 xCTAB extraction buffer solution, and preserving the temperature for 20 minutes at 65 ℃;
3. adding chloroform/isoamylol (volume ratio is 24:1) with the same volume, inverting the centrifuge tube, mixing uniformly, and centrifuging at room temperature of 12000r/min for 10 minutes;
4. transferring the supernatant into another centrifuge tube, adding chloroform/isoamylol (volume ratio is 24:1) with the same volume, reversing the centrifuge tube, uniformly mixing, and centrifuging at room temperature of 12000r/min for 10 minutes;
5. transferring the upper-layer water phase into a new centrifugal tube, adding isopropanol with the same volume, slightly reversing and uniformly mixing, and standing at room temperature for 30 minutes;
6.12000r/min, centrifuging at room temperature for 5-10 min, and removing supernatant;
rinsing with 7.70% ethanol, centrifuging at 12000r/min at room temperature for 5-10 min, and removing supernatant;
8. repeating the step 7 once;
9. air drying the precipitate, adding 100 μ l TE buffer solution or ultrapure water to dissolve DNA, and storing at-20 deg.C;
10. mu.l of the solution was detected by electrophoresis on a 1% agarose gel.
2.2PCR amplification
According to the detection primers (nucleotide sequences of SEQ ID NO.1 and SEQ ID NO. 2) of the Chinese cabbage InDel marker TR-1 designed above, the primers of the Chinese cabbage InDel marker TR-1 are synthesized by the company Biotechnology engineering (Shanghai) GmbH, and are diluted to 10 mu M for standby after dissolution.
And (3) performing PCR amplification by using the extracted DNA as a template and Chinese cabbage InDel marked primers respectively to prepare PCR products.
The PCR reaction was as follows, 20. mu.L total:
20 ng/. mu.L template DNA 2. mu.L, containing 20mM MgCl 210 XPCR buffer2 uL, dNTPs0.5 uL at concentration of 2.5mM, upstream primer 0.4 uL at concentration of 10 uM, downstream primer 0.4 uL at concentration of 10 uM, 1U of Taq DNA polymerase 0.2 uL, plus ddH2O to 20. mu.L.
The amplification procedure was:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57 ℃ for 30s, and extension at 72 ℃ for 30s, for a total of 36 cycles; extension at 72 ℃ for 10 min.
2.3 Polyacrylamide gel electrophoresis separation and polymorphism detection
And (3) carrying out electrophoresis on the PCR product by using a modified polyacrylamide gel with the mass concentration of 8%, and carrying out development detection by using a silver staining method.
Mixing the PCR product with the sample buffer solution, performing pre-denaturation at 95 ℃ for 10min, sampling 4 μ l of the mixture, and performing amplification reaction in Sequi-
The GT nucleic acid electrophoresis system (Bio-Rad, USA) was used for electrophoretic separation with an electrode buffer of 1 XTBE at 25 ℃ under constant power of 55W for 1.5h, developed by silver staining, photographed and recorded.
FIG. 2 is a diagram showing the results of 8% denaturing polyacrylamide gel electrophoresis of PCR amplification products of the F2 population material hybridized with ZHB, G291, ZHB and G291 by using a primer pair of Chinese cabbage InDel marker TR-1.
FIG. 3 is a diagram showing the results of denaturing polyacrylamide gel electrophoresis at a mass concentration of 8% using primers of Chinese cabbage InDel labeled TR-1 for PCR amplification products of hairless materials (AM4-1, AM3-1, AM5-2, AM9-1, AM94, AM30, AM54, AM72-1, AM10-1, AM56-1, Qing Jiang Chun Bai No.2, Degao lily, Xibai 45, Y2, L41) and hairy materials (Xibai No. 8, Zhonghua No.1, Qing Jiang CR20, Qing Jiang Xibai No.2, Guangdong Zao, L46, Zhonghua No.1, Gao No.2, Xibai No. 5, Xiyou No. two, Jintongyuyu, Gouyi, Fenggang No. 60, Huayang Bai, Degao Xiyan 65).
FIG. 4 is a graph showing the results of electrophoresis of PCR amplification products of hairless materials (Zaohuangbai, AM3-1, Degao lily, AM10-1) and hairless materials (crown 291, Zhonghua No.1, Xibai No. 8, and Chaoguan No. two) on denatured polyacrylamide gel with a mass concentration of 8% using Chinese cabbage InDel labeled TR-1 primers and other 7 pairs of InDel labeled primers designed by the same method.
As can be seen from the graphs in FIGS. 2, 3 and 4, the primer of the Chinese cabbage InDel marked TR-1 can effectively amplify DNA of the hairy and hairless Chinese cabbages, has clear band types, and has good polymorphism in the hairy and hairless Chinese cabbage materials, wherein the amplification band of the hairy material is 156bp, and the amplification band of the hairless material is 144bp, so that the hairy and hairless Chinese cabbages can be well distinguished, and the primer can be used for breeding new varieties of the hairless Chinese cabbages.
SEQUENCE LISTING
<110> institute of vegetables and flowers of academy of agricultural sciences of Shandong province
<120> InDel mark of Chinese cabbage burr as well as detection primer and application thereof
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 1
ttccatacct ccctcactgg ta 22
<210> 2
<211> 22
<212> DNA
<213> Artificial Synthesis
<400> 2
ttcaactgtc cacaaccctt tc 22
<210> 3
<211> 144
<212> DNA
<213> ZHB
<400> 3
ttccatacct ccctcactgg tagcaatctt tataagtaga gtctctatca cagataataa 60
tctgagaatc aaaggatgag aacgaggaga agaacagagg aagagaatca tcaagaatac 120
aagaaagggt tgtggacagt tgaa 144
<210> 4
<211> 156
<212> DNA
<213> G291
<400> 4
ttccatacct ccctcactgg tagcaatctc tttctctctc tttataagta gagtctctat 60
cacagataat aatctgagaa tcaaaggatg agaacgagga gaagaacaga ggaagagaat 120
catcaagaat acaagaaagg gttgtggaca gttgaa 156
Claims (7)
1. An application of an InDel marker of Chinese cabbage burrs in selection of hairless Chinese cabbage varieties is disclosed, wherein a nucleotide sequence of a detection primer of the InDel marker is as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2; the characteristic band of the detection primer amplified hairless Chinese cabbage genome DNA is 144bp, and the nucleotide sequence is shown as SEQ ID NO. 3.
2. The application of the InDel marked detection primer for Chinese cabbage burrs in the selection of hairless Chinese cabbage varieties is characterized in that the nucleotide sequence of the InDel marked detection primer is as follows: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2.
3. Use according to claim 2, characterized by the steps of:
(1) extracting the genome DNA of the Chinese cabbage variety to be detected to obtain a genome DNA solution;
(2) taking the genome DNA prepared in the step (1) as a template, and carrying out PCR amplification on the genome DNA by using the detection primer to prepare a PCR amplification product;
(3) after the PCR amplification product prepared in the step (2) is subjected to gel electrophoresis, when the electrophoresis pattern of the PCR product shows that the detected sample only has a strip of 156bp, the detected sample is a hairy material; when the electrophoresis pattern of the PCR product shows that only one band of 144bp exists, the detected sample is a hairless material; when the PCR product electrophoretogram shows that the PCR product contains 156bp and 144bp bands, the detected sample is a hairy and hairless hybrid material and is phenotypically represented as a hairy material.
4. The use according to claim 3, wherein in the step (1), the genomic DNA of the Chinese cabbage variety to be detected is extracted by a modified CTAB method.
5. The use of claim 3, wherein in step (2), the PCR reaction system is as follows, and the total volume is 20 μ L:
20 ng/. mu.L template DNA 2. mu.L, containing 20mM MgCl210 XPCR buffer2 uL, 2.5mM dNTPs0.5 uL, 10 uM upstream primer 0.4 uL, 10 uM downstream primer 0.4 uL, 1U Taq DNA polymerase 0.2 uL, plus ddH2O to 20. mu.L.
6. The use of claim 3, wherein in step (2), the PCR amplification procedure is:
pre-denaturation at 95 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, for a total of 36 cycles; extension at 72 ℃ for 10 min.
7. The use according to claim 3, wherein in the step (3), the gel electrophoresis is 8% by mass of denaturing polyacrylamide gel electrophoresis.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103184A (en) * | 2013-01-28 | 2013-05-15 | 西北农林科技大学 | SSR (Simples sequence repeats) and InDel (insertion/deletion) molecular marker primer linked with brassica campestris orange head gene Br-or, and application thereof |
| CN103172715A (en) * | 2011-12-21 | 2013-06-26 | 中国科学院上海生命科学研究院 | Plant epidermal hair controlling gene and application thereof |
| CN103757018A (en) * | 2014-02-20 | 2014-04-30 | 山东省农业科学院蔬菜花卉研究所 | InDel mark relating to mutation of untranslated region of Chinese cabbage PHYB gene mRNA5'and application thereof |
| CN104774951A (en) * | 2015-04-02 | 2015-07-15 | 山东省农业科学院蔬菜花卉研究所 | Chinese cabbage EST-SSR (expressed sequence tag-simple sequence repeat) markers as well as primers and applications thereof |
| CN107164502A (en) * | 2017-06-15 | 2017-09-15 | 西南大学 | The molecular labeling being closely related and its application are whether there is with cabbage leaves epidermal hair |
-
2018
- 2018-07-04 CN CN201810725802.7A patent/CN108546775B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172715A (en) * | 2011-12-21 | 2013-06-26 | 中国科学院上海生命科学研究院 | Plant epidermal hair controlling gene and application thereof |
| CN103103184A (en) * | 2013-01-28 | 2013-05-15 | 西北农林科技大学 | SSR (Simples sequence repeats) and InDel (insertion/deletion) molecular marker primer linked with brassica campestris orange head gene Br-or, and application thereof |
| CN103757018A (en) * | 2014-02-20 | 2014-04-30 | 山东省农业科学院蔬菜花卉研究所 | InDel mark relating to mutation of untranslated region of Chinese cabbage PHYB gene mRNA5'and application thereof |
| CN104774951A (en) * | 2015-04-02 | 2015-07-15 | 山东省农业科学院蔬菜花卉研究所 | Chinese cabbage EST-SSR (expressed sequence tag-simple sequence repeat) markers as well as primers and applications thereof |
| CN107164502A (en) * | 2017-06-15 | 2017-09-15 | 西南大学 | The molecular labeling being closely related and its application are whether there is with cabbage leaves epidermal hair |
Non-Patent Citations (1)
| Title |
|---|
| Fine mapping and candidate gene analysis of Brtri1, a gene controlling trichome development in Chinese cabbage (Brassica rapa L. ssp pekinensis);X.L. Ye等;《Genetics and Molecular Research》;20161202;第15卷(第4期);摘要、第10页第2段、第11页第1-2段 * |
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| CN108546775A (en) | 2018-09-18 |
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