CN101956012A - Method for identifying common head cabbage-Chinese cabbage alien addition line - Google Patents
Method for identifying common head cabbage-Chinese cabbage alien addition line Download PDFInfo
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- CN101956012A CN101956012A CN 201010297749 CN201010297749A CN101956012A CN 101956012 A CN101956012 A CN 101956012A CN 201010297749 CN201010297749 CN 201010297749 CN 201010297749 A CN201010297749 A CN 201010297749A CN 101956012 A CN101956012 A CN 101956012A
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- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 title claims abstract description 53
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- 238000000034 method Methods 0.000 title claims abstract description 14
- 240000007124 Brassica oleracea Species 0.000 title claims abstract 11
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 claims abstract description 52
- 235000000536 Brassica rapa subsp pekinensis Nutrition 0.000 claims abstract description 52
- 241000499436 Brassica rapa subsp. pekinensis Species 0.000 claims abstract description 52
- 238000012216 screening Methods 0.000 claims abstract description 7
- 230000002068 genetic effect Effects 0.000 claims abstract description 6
- 238000000926 separation method Methods 0.000 claims abstract description 4
- 238000004519 manufacturing process Methods 0.000 claims description 23
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- 238000012408 PCR amplification Methods 0.000 claims description 7
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- 244000178937 Brassica oleracea var. capitata Species 0.000 abstract description 43
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- 108090000623 proteins and genes Proteins 0.000 abstract description 3
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- 238000012252 genetic analysis Methods 0.000 abstract 1
- 239000003550 marker Substances 0.000 abstract 1
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- 239000012634 fragment Substances 0.000 description 5
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
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- 238000009399 inbreeding Methods 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
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- 238000004153 renaturation Methods 0.000 description 2
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- 235000013311 vegetables Nutrition 0.000 description 2
- 235000005637 Brassica campestris Nutrition 0.000 description 1
- 244000060924 Brassica campestris Species 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a method for identifying a common head cabbage-Chinese cabbage alien addition line. The method comprises (1) selecting 207 SSR markers in 10 linkage groups of Chinese cabbage according to published cabbage genetic map, and synthesizing primer sequence by Shanghai Biotechnology Limited. (2) Extracting genome DNA of different common head cabbage and Chinese cabbage varieties and PCR amplifying. (3) And carrying out electrophoretic separation on the amplification product and detecting silver staining. (4) Screening the SSR markers of the Chinese cabbage linkage groups relative to the common head cabbage. (5) Identifying the common head cabbage-Chinese cabbage alien addition line by utilizing the SSR marker of the Chinese cabbage linkage group relative to the common head cabbage. Because the Chinese cabbage and the cabbage have small chromosomes and similar forms, and are difficult to accurately identify at a cell level, the invention can realize the rapid and accurate identification of important genetic analysis materials such as a Chinese cabbage-common head cabbage alien addition line, an alien substitution line, a translocation line and the like at a molecular level, and lays a foundation for utilizing the Chinese cabbage gene to improve the genetic background of the common head cabbage.
Description
Technical field
The present invention relates to a kind of method of identifying cabbage-Chinese cabbage alien addition line, belong to theoretical application of molecular genetics and breeding technical field.
Background technology
Cabbage (Brassica oleracea var.capitata L., CC) and Chinese cabbage (Brassica campestris L.ssp.pekinensis (Lour) Olsson, AA) all be important vegetables during rape belongs to, China vegetables produce and people's lives in occupy an important position.Creating cabbage-Chinese cabbage alien addition line is to carry out theory of heredity research and utilize the Chinese cabbage desirable genes cabbage to be carried out the important channel of breed improvement.Because the karyomit(e) of cabbage and Chinese cabbage is all less, adjacent chromosome morphology is closely similar again, only is difficult to accurately each bar karyomit(e) of identification by the conventional cell method.Development along with molecular cytogenetics, rDNA-fluorescence in situ hybridization and genomic in situ hybridization become the effective tool that exogenous chromosome is accurately identified in accurate identification of karyomit(e) and the alien addition line, but because the number of sites of rDNA in Chinese cabbage and cabbage genome is limited, only can identification division karyomit(e).As seen accurately the coloured differently body of identification Chinese cabbage is very difficult under the cabbage background, and this has also just restricted the application of cabbage-Chinese cabbage alien addition line in breeding practice and theory of heredity research.
Summary of the invention
The object of the invention provides a kind of method of identifying cabbage-Chinese cabbage alien addition line, this method had both improved efficient and the accuracy identified, make the alien addition line of acquisition between the laboratory, have good AC again, lay the foundation for utilizing Chinese cabbage improvement of genes cabbage genetic background.
Ultimate principle of the present invention is: utilize the special SSR mark of Chinese cabbage linkage group with respect to cabbage, with cross-fertilize seed and parent cabbage thereof, Chinese cabbage and alien addition line genomic dna are that template is carried out pcr amplification, the amplified production that compares them, if the amplified production of special primer in alien addition line in certain linkage group is consistent with cabbage, then show this linkage group of not adding Chinese cabbage in the alien addition line, if the amplified production of special primer in alien addition line in certain linkage group is consistent with cross-fertilize seed, then show this linkage group of having added Chinese cabbage in the alien addition line.
Characteristics of the present invention are: utilize the different linkage groups of the Chinese cabbage SSR mark special with respect to cabbage, identify additional Chinese cabbage linkage group in cabbage-Chinese cabbage alien addition line at molecular level.Particularly, the present invention finishes by following steps:
(1) belong to A genome genetic map according to the rape of having delivered, selected for use to be positioned at 207 SSR marks that rape belongs to 10 linkage groups of A genome, the SSR primer sequence is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized;
(2) the CTAB method is extracted the cabbage of different varieties and the genomic dna of Chinese cabbage, is the pcr amplification that template is carried out above-mentioned SSR mark with it.Each PCR reaction system is 20 μ L, contains template DNA 30ng, positive each 0.2 μ molL of anti-primer
-1, 10x PCR Buffer (contains Mg
2+), 2.5mmolL
-1DNTPs, 1U Taqase, surplus volume ddH
2The O polishing.The pcr amplification program is, is 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 1min, 55～60 ℃ of renaturation 30s, 72 ℃ are extended 45s, 35 circulations; 72 ℃ are extended 5min, 4 ℃ of preservations;
(3) amplified production electrophoretic separation in 10% non-denaturing polyacrylamide gel, silver dyes detection;
(4) the screening Chinese cabbage linkage group SSR mark special with respect to cabbage.Screening does not have amplified production in cabbage, and the mark of amplified production is arranged in the Chinese cabbage, or amplified production is all arranged in the two, but the amplified production of Chinese cabbage is different from the mark of cabbage amplified production, as the special SSR mark of Chinese cabbage linkage group with respect to cabbage;
(5) utilization screens the special SSR mark of Chinese cabbage linkage group with respect to cabbage, with cross-fertilize seed and parent's wild cabbage, Chinese cabbage and alien addition line genomic dna is that template is carried out pcr amplification, identify cabbage-Chinese cabbage alien addition line: if the amplified production of special primer in alien addition line in certain linkage group is consistent with cabbage, then show this linkage group of not adding Chinese cabbage in the alien addition line, if the amplified production of special primer in alien addition line in certain linkage group is consistent with cross-fertilize seed, then show this linkage group of having added Chinese cabbage in the alien addition line.
Technique effect of the present invention is: identify cabbage-Chinese cabbage alien addition line from molecular level, both brought up to the accuracy of identifying, make the alien addition line of identifying by this method have good AC again between different laboratories.
Description of drawings
Fig. 1 is that 33 special SSR are marked at the amplification in 10 head cabbage varieties and 9 Chinese cabbage cultivars.
The implication of label among Fig. 1 representative: 1, in sweet self-mating system 2, Japanese iron first number 3, the utmost point is wild cabbage 4 early, 83125, in sweet 11 6, rich No. one 7 of capital, in sweet 17 8, in sweet 18 9, in sweet 19 10, late rich wild cabbage 11,78-13 12, and capital spring doll's dish 13 is little assorted No. 56 14, capital summer king 15, Beijing improvement No. 67 16, the big OX-heart 17 in Beijing, Beijing No. 80 18, new No. one 19 of Beijing, new No. three of Beijing.
Fig. 2 is that the special SSR of Chinese cabbage linkage group is marked at the amplification in cross-fertilize seed and parent and the cabbage-Chinese cabbage alien addition line.
Label implication among Fig. 2 is as follows: 1, Chinese cabbage 2, cabbage 3, cross-fertilize seed 4, alien addition line.
The specific embodiment that provides below in conjunction with accompanying drawing and contriver further is elaborated to the present invention.
Embodiment
According to technical scheme of the present invention, the authentication method of cabbage-Chinese cabbage alien addition line, concrete implementation step is:
(1) belong to A genome genetic map according to the rape of having delivered, selected for use to be positioned at 207 SSR marks that rape belongs to A genome linkage group, the SSR primer sequence is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.
(2) with 10 head cabbage varieties (inbreeding of more generation parent: ' in sweet '; Spring early maturing variety: ' first number of Japanese iron ', ' utmost point early wild cabbage ', ' 8312 ', ' in sweet 11 '; Spring medium variety: ' rich No. one of capital '; Spring and autumn the dual-purpose early maturing variety: ' in sweet 17 '; ' in sweet 18 ' autumn medium variety: ' in sweet 19 '; Autumn late variety: ' late rich wild cabbage ') and 9 Chinese cabbage cultivar (inbreeding of more generation parents: ' 78-13 '; Extremely early variety: ' capital spring doll's dish '; Early maturing variety: ' little assorted No. 56 ', ' Jing Xiawang '; Mid-early maturity kind: ' No. 67, Beijing improvement '; Medium variety: ' the big OX-heart in Beijing '; Middle-late ripening variety: ' No. 80, Beijing '; Late variety: ' new No. one of Beijing ', ' new No. three of Beijing ') planting seed coils in the cave, Routine Management, and when seedling grew to 2～3 true leaves, the tender young leaves of clip children adopted the CTAB method to extract genomic dna.With these material genomic dnas is template, carries out pcr amplification with the wild cabbage linkage group SSR primer that obtains.Each PCR reaction system is 20 μ L, contains template DNA 30ng, positive each 0.2 μ molL of anti-primer
-1, 10x PCR Buffer (contains Mg
2+), 2.5mmolL
-1DNTPs, 1U Taqase, surplus volume ddH
2The O polishing.The pcr amplification program is, is 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 1min, 55～60 ℃ of renaturation 30s, 72 ℃ are extended 45s, 35 circulations; 72 ℃ are extended 5min, 4 ℃ of preservations.
(3) amplified production electrophoretic separation in 10% non-denaturing polyacrylamide gel adopts argentation to detect, and takes a picture.
(4) the screening Chinese cabbage linkage group SSR mark special with respect to cabbage, the standard of its screening is as follows: amplified fragments is arranged on the Chinese cabbage, and do not have amplified fragments on the cabbage; Or the two all has amplified fragments, but the amplified fragments size there are differences.The result shows that 33 SSR marks can amplify discrepant fragment between cabbage, Chinese cabbage.These 33 SSR marks have related to 10 linkage groups (table 2) of Chinese cabbage, amplification in cabbage and Chinese cabbage such as plate 1.
(5) utilize the special SSR marks of this 33 relative cabbage of Chinese cabbage linkage group, with cross-fertilize seed and parent cabbage, Chinese cabbage and alien addition line genomic dna is that template is carried out pcr amplification, the amplified production that compares them, if the amplified production of special primer in alien addition line in certain linkage group is consistent with balling, then do not add this linkage group of Chinese cabbage in the alien addition line, if the amplified production of special primer in alien addition line in certain linkage group is consistent with cross-fertilize seed, this linkage group of then having added Chinese cabbage in the alien addition line.The result shows, this alien addition line is to being positioned at 2 SSR mark Na12A02b, Ra2A05b (Fig. 2 of Chinese cabbage A7 linkage group, A7) amplify and the cross-fertilize seed identical segments, and to all consistent with the cabbage (Fig. 2 of the amplification that is positioned at other linkage group primers, A1-A6, A8-A10), this just shows that this alien addition line is additional 1 chromosomal monomer alien addition line of Chinese cabbage, and this 1 exogenous chromosome is corresponding with Chinese cabbage A7 linkage group.
Claims (1)
1. method of identifying Chinese cabbage-wild cabbage alien addition line is characterized in that may further comprise the steps:
(1) according to the Chinese cabbage genetic map of having delivered, 207 SSR marks that are positioned at 10 linkage groups of Chinese cabbage have been selected for use, synthetic SSR primer sequence;
(2) extracting the Chinese cabbage of different varieties and the genomic dna of wild cabbage, is the pcr amplification that template is carried out above-mentioned SSR mark with it;
(3) amplified production electrophoretic separation in 10% non-denaturing polyacrylamide gel, argentation detects;
(4) the screening Chinese cabbage linkage group SSR mark special with respect to cabbage, the standard of its screening is as follows: amplified production is arranged on the Chinese cabbage, and do not have amplified production on the cabbage, or the two all has amplified production, but mark that the amplified production clip size there are differences;
(5) utilize the Chinese cabbage linkage group screen special SSR mark with respect to cabbage, with cross-fertilize seed and parent Chinese cabbage, cabbage and alien addition line genomic dna is that template is carried out pcr amplification, the amplified production that compares them, if the amplified production of special primer in alien addition line in certain linkage group is consistent with cabbage, this linkage group of not adding Chinese cabbage in the alien addition line then is described, if the amplified production of special primer in alien addition line in certain linkage group is consistent with cross-fertilize seed, then show this linkage group of having added Chinese cabbage in the alien addition line.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102977499A CN101956012B (en) | 2010-09-30 | 2010-09-30 | Method for identifying common head cabbage-Chinese cabbage alien addition line |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102977499A CN101956012B (en) | 2010-09-30 | 2010-09-30 | Method for identifying common head cabbage-Chinese cabbage alien addition line |
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| CN101956012A true CN101956012A (en) | 2011-01-26 |
| CN101956012B CN101956012B (en) | 2012-07-04 |
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| CN2010102977499A Expired - Fee Related CN101956012B (en) | 2010-09-30 | 2010-09-30 | Method for identifying common head cabbage-Chinese cabbage alien addition line |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233077A (en) * | 2013-05-13 | 2013-08-07 | 浙江省农业科学院 | Molecular marking method as well as kit and primer for identifying purity of broccoli hybrid scarlet pimpernel variety |
| CN106191231A (en) * | 2016-01-15 | 2016-12-07 | 中国农业科学院蔬菜花卉研究所 | With common head cabbage wax powder-free viride nitens gene cgl 4 closely linked molecular marker and application thereof |
| CN115843681A (en) * | 2022-11-22 | 2023-03-28 | 河北农业大学 | Chinese cabbage heat-resistant variety identification and evaluation method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423867A (en) * | 2008-07-03 | 2009-05-06 | 河北农业大学 | Method for identifying Chinese cabbage-cabbage alien addition line |
| CN101755673A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Breeding method of alien addition line |
| CN101755683A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Applications of Elytrigia elongata to genetic improvement of wheat |
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2010
- 2010-09-30 CN CN2010102977499A patent/CN101956012B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101423867A (en) * | 2008-07-03 | 2009-05-06 | 河北农业大学 | Method for identifying Chinese cabbage-cabbage alien addition line |
| CN101755673A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Breeding method of alien addition line |
| CN101755683A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Applications of Elytrigia elongata to genetic improvement of wheat |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103233077A (en) * | 2013-05-13 | 2013-08-07 | 浙江省农业科学院 | Molecular marking method as well as kit and primer for identifying purity of broccoli hybrid scarlet pimpernel variety |
| CN106191231A (en) * | 2016-01-15 | 2016-12-07 | 中国农业科学院蔬菜花卉研究所 | With common head cabbage wax powder-free viride nitens gene cgl 4 closely linked molecular marker and application thereof |
| CN115843681A (en) * | 2022-11-22 | 2023-03-28 | 河北农业大学 | Chinese cabbage heat-resistant variety identification and evaluation method |
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| CN101956012B (en) | 2012-07-04 |
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