CN101423867B - Method for identifying celery cabbage-brassica oleracea alien addition lines - Google Patents
Method for identifying celery cabbage-brassica oleracea alien addition lines Download PDFInfo
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- CN101423867B CN101423867B CN200810055317XA CN200810055317A CN101423867B CN 101423867 B CN101423867 B CN 101423867B CN 200810055317X A CN200810055317X A CN 200810055317XA CN 200810055317 A CN200810055317 A CN 200810055317A CN 101423867 B CN101423867 B CN 101423867B
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Abstract
The invention discloses a method for identifying Chinese cabbage-cabbage addition lines. The method comprises the following steps: (1) selecting 91 pairs of primers in different linkage groups of cabbage according to a published cabbage genetic map; (2) extracting genetic groups DNA of different Chinese cabbages and cabbages to carry out PCR amplification; (3) electrophoretically separating an amplified product in modified polyacrylamide gel of 6 percent to carry out silver staining; (4) screening SSR primers in the linkage groups of the cabbage different from the Chinese cabbage; and (5) identifying the Chinese cabbage-cabbage addition lines by the SSR primers in the linkage groups of the cabbage different from the Chinese cabbage. The method has the advantages that the method can quickly and accurately identify important genetic analysis materials for the Chinese cabbage and the cabbage, such as coenospecies, addition lines, alien substitution lines, translocation lines and the like on molecular level, and lays foundation for modifying Chinese cabbage genetic background by cabbage gene.
Description
Technical field
The present invention relates to a kind of method of identifying Chinese cabbage-wild cabbage alien addition line, belong to molecular genetics theory application and breeding technical field.
Background technology
At present; About Chinese cabbage (Brassica campestris syn rapa ssp.Pekinensis; AA) with wild cabbage (B.oleracea var.L.; CC) chromosomal evaluation is limited to cell levels, because karyomit(e) is little and form is close, the method for karyotyping is difficult in and accurately identifies wild cabbage karyomit(e) under the Chinese cabbage karyomit(e) background.People such as Chen utilize Brassica campestris-B.oleracea var.alboglabra monomer alien addition line tip of a root somatocyte prometaphase chromosome karyotype analysis, have only identified the longest chromosomal 1 alien addition line of additional wild cabbage; Because Chinese cabbage and wild cabbage homology are stronger, genomic in situ hybridization can not be distinguished the two karyomit(e); People such as Robert are the fluorescence in situ hybridization of probe with 5SrDNA and 25SrDNA; Identified B.campestris-B.oleracea var.alboglabra monomer alien addition line No. 5, No. 8, No. 9, but can not accurately identify for the B.alboglabra karyomit(e) that does not have hybridization site.This has just restricted the evaluation and the application of Chinese cabbage-important genetic stockss such as wild cabbage alien addition line.Along with the fast development of Protocols in Molecular Biology, it is saturated that the wild cabbage linkage group has been tending towards, lays a good foundation for utilizing molecular marking technique evaluation wild cabbage karyomit(e).Because Chinese cabbage and wild cabbage homology are stronger, must select for use a kind of polymorphum by force, the molecule marker of good stability, and require easy and simple to handlely, the DNA consumption is little.Through retrieval, do not find the method for ideal technical evaluation Chinese cabbage-wild cabbage alien addition line in the existing document.
Summary of the invention
The object of the invention provides a kind of method of identifying Chinese cabbage-wild cabbage alien addition line, lays the foundation for utilizing wild cabbage improvement of genes Chinese cabbage genetic background.
Ultimate principle of the present invention is: the special SSR primer that utilizes the relative Chinese cabbage of wild cabbage linkage group; With cross-fertilize seed and parent Chinese cabbage, wild 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 all consistent with Chinese cabbage; Then show this linkage group of not adding wild cabbage in the alien addition line,, then show this linkage group of having added wild 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.Therefore, characteristics of the present invention are: utilize the SSR primer of the different linkage groups of wild cabbage with respect to Chinese cabbage differential, identify the external source wild cabbage linkage group of Chinese cabbage-wild cabbage alien addition line at molecular level.Particularly, method of the present invention may further comprise the steps:
(1), selects 91 pairs of SSR primers that are positioned at the different linkage groups of wild cabbage for use according to the wild cabbage genetic map of having delivered;
(2) the CTAB method is extracted the Chinese cabbage of different varieties and the genomic dna of wild cabbage, pcr amplification;
(3) amplified production electrophoretic separation in 6% denaturing polyacrylamide gel, silver dyes detection;
(4) screening wild cabbage linkage group is with respect to the SSR primer of Chinese cabbage differential;
Screening does not have amplified production in Chinese cabbage, and the primer of amplified production is arranged in the wild cabbage, or amplified production is all arranged in the two, but the amplified production of wild cabbage is different from the primer of Chinese cabbage amplified production, as the special SSR primer of wild cabbage linkage group with respect to Chinese cabbage;
(5) utilization screens the special SSR primer of wild cabbage linkage group with respect to Chinese cabbage, is that template is carried out pcr amplification with cross-fertilize seed and parent Chinese cabbage, wild cabbage and alien addition line genomic dna, identifies Chinese cabbage-wild cabbage alien addition line.
Particularly; Method of the present invention is specified as: according to the wild cabbage genetic map of having delivered; Selected 91 pairs of SSR primers that are positioned at the different linkage groups of wild cabbage for use, the linkage group distribution and the sequence of primer are as shown in table 1, and primer sequence is given birth to worker Bioisystech Co., Ltd by Shanghai and synthesized.With 8 head cabbage varieties (spring early maturing cabbage kind: in sweet 11,8312, the utmost point is wild cabbage early; Ripe head cabbage varieties in spring: rich No. one of capital; Spring and autumn dual-purpose early maturing cabbage kind: in sweet 17; Ripe head cabbage varieties in autumn: in sweet 19; Autumn late-maturing head cabbage varieties: late rich wild cabbage, in sweet 18) and 7 Chinese cabbage cultivars (anti-bolting bullet cut Chinese cabbage cultivars: strong Feng Chun Chinese cabbage, high anti-king; In the late-maturing straight tube Chinese cabbage cultivar of filling the span of a man's arms: how anti-75; The folded Chinese cabbage cultivar of embracing of extremely early mature: super Xiayang, precocious No. five; The straight tube of precocious high stake Chinese cabbage cultivar: autumn green 55; In the folded Chinese cabbage cultivar of embracing of ripe stump: Xinxiang parcel 23) planting seed coils in the cave, Routine Management, long during to 4～6 true leaves when seedling, the tender young leaves of clip children adopts 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 50ng, 2mmol/L Mg
2+, 1 * buffer, 200 μ mol/L dNTP, 1U Tap enzyme, each 2.5ng/ μ L of forward and reverse primer.The pcr amplification program is 94 ℃ of preparatory sex change 2min; 94 ℃ of sex change 1min, 60 ℃ of renaturation 30s, 72 ℃ are extended 45s, totally 10 circulations, each cycle annealing temperature reduces by 0.5 ℃; 94 ℃ of sex change 1min, 55 ℃ of renaturation 30s, 72 ℃ are extended 45s, totally 30 circulations; 72 ℃ are extended 5min., 4 ℃ of preservations.Amplified production is electrophoretic separation in 6% denaturing polyacrylamide gel, and silver dyes detection.Screening wild cabbage linkage group is with respect to the SSR primer of Chinese cabbage differential; Screening does not have amplified production in Chinese cabbage; And the primer of amplified production is arranged in the wild cabbage; Or amplified production is all arranged in the two, but the amplified production of wild cabbage is different from the primer of Chinese cabbage amplified production, as the SSR primer with respect to Chinese cabbage differential of the different linkage groups of wild cabbage.The result shows; 25 pairs of primers can amplify discrepant fragment in wild cabbage, Chinese cabbage; Fragment length is at 40-430bp, and wherein 7 pairs of primers have amplified production and in Chinese cabbage, do not have amplified production in cabbage, and 18 pairs of primers are different with the amplified production in the cabbage in Chinese cabbage; These 25 pairs of SSR primers have related to 9 linkage groups (seeing table 2) of wild cabbage, and the amplification in Chinese cabbage and wild cabbage is as shown in Figure 1.Utilize the special SSR primer of these 25 pairs of relative Chinese cabbages of wild cabbage linkage group; With cross-fertilize seed and parent Chinese cabbage, wild 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 Chinese cabbage; Then do not add this linkage group of wild cabbage in the alien addition line, if the amplified production of special primer in alien addition line in certain linkage group is all consistent with cross-fertilize seed, this linkage group of then having added wild cabbage in the alien addition line.
Table 1 SSR primer linkage group distributes and sequence
Table 2 is with respect to the wild cabbage linkage group SSR primer of Chinese cabbage differential
| SSR | Linkage group | Product fragment magnitude range (bp) | Repeat type |
| Na10H03 | 01 | 90-128 | (GGC) 6 |
| Ni4B10 | 01 | 165-185 | (CT) 20 |
| Na12C08 | 01 | 140-404 | (CT) 50 |
| Ol13G05 | 02 | 100-130 | (CCG) 5 |
| sORA43 | 02 | 100-198 | (GGC) 7 |
| Na12C03 | 02 | 123-250 | (GA) 30 |
| Na12H09 | 02 | 108-162 | (GA) 24 |
| Ol11G11 | 03 | 100-163 | (CCG) 5 |
| BN12A | 03 | 147-350 | (GA) 11(AAG) 4 |
| Na12E05 | 04 | 110-165 | (CA) 10 |
| Ol10F12 | 04 | 95-130 | (CT) 64 |
| Ol11H08 | 04 | 76-160 | (AG) 24 |
| Na10D11 | 05 | 123-220 | (GA) 27 |
| Ra2A04 | 05 | 40-67 | (GA) 58 |
| Ap1a5pr | 06 | 187-217 | —— |
| Na14F11 | 06 | 137-430 | (GT) 7 |
| Ol10B01 | 07 | 116-201 | (GA) 20 |
| BN72a | 07 | 140-309 | (TAA) 5(GA) 9 |
| Na12F03 | 07 | 147-309 | (GA) 35 |
| Ol10H04 | 07 | 120-201 | (CT) 29 |
| Ol12D05 | 08 | 103-140 | (CT) 32 |
| Ol12G04 | 08 | 90-135 | (TC) 24 |
| Ol12D09 | 09 | 95-135 | (CGG) 4 |
| Ra1F03 | 09 | 97-145 | (TCC\/GCC) 18 |
| Ol12A04 | 09 | 95-160 | (CT) 17 |
In the table 2: "---" expression it be unclear that.
The beneficial effect that the present invention obtains is: the present invention identifies Chinese cabbage-wild cabbage alien addition line from molecular level, has both brought up to the accuracy of identifying, makes the alien addition line of identifying by this method between different laboratories, have good interchange property again.
Description of drawings
The amplification that Fig. 1 is 25 pairs of special SSR primers in 8 head cabbage varieties and 7 Chinese cabbage cultivars.
The implication of label among Fig. 1 representative is: 1, the utmost point early wild cabbage 2, late rich wild cabbage 3, in sweet 17 4, rich No. one 5 of capital, in sweet 18 6, in sweet 11 7, in green 55 10, super Xiayang 11 of sweet 19 8,8,312 9, autumn, Xinxiang parcel 23 12, how anti-75 13, precocious No. five 14, high anti-king 15, strong Feng Chun Chinese cabbage.
Among Fig. 1: A, primer Na10H03 amplification; B, primer Ni4B10 amplification; C, primer Na12C08 amplification; D, primer Ol13G05 amplification; E, primer sORA43 amplification; F, primer Na12C03 amplification; G, primer Na12H09 amplification; H, primer Ol11G11 amplification; I, primer BN12A amplification; J, primer Na12E05 amplification; K, primer Ol10F12 amplification; L, primer Ol11H08 amplification; M, primer Na10D11 amplification; N, primer Ra2A04 amplification; O, primer Ap1a5pr amplification; P, primer Na14F11 amplification; Q, primer Ol10B01 amplification; R, primer BN72a amplification; S, primer Na12F03 amplification; T, primer Ol10H04 amplification; U, primer Ol12D05 amplification; V, primer Ol12G04 amplification; W, primer Ol12D09 amplification; X, primer Ra1F03 amplification; Y, primer Ol12A04 amplification.
Fig. 2 is the amplification of the special SSR primer of wild cabbage linkage group in cross-fertilize seed and parent and the two monomer alien addition lines of Chinese cabbage-wild cabbage.
Label implication among Fig. 2 is following: 1, Chinese cabbage 2, wild cabbage 3, cross-fertilize seed 4, alien addition line.
Among Fig. 2: A, primer Na10H03 amplification; B, primer Ni4B10 amplification; C, primer Na12C08 amplification; D, primer Ol13G05 amplification; E, primer sORA43 amplification; F, primer Na12C03 amplification; G primer Na12H09 amplification; H, primer Ol11G11 amplification; I, primer BN12A amplification; J, primer Na12E05 amplification; K, primer Ol10F12 amplification; L, primer Ol11H08 amplification; M, primer Na10D11 amplification; N, primer Ra2A04 amplification; O, primer Ap1a5pr amplification; P, primer Na14F11 amplification; Q, primer Ol10B01 amplification; R, primer BN72a amplification; S, primer Na12F03 amplification; T, primer Ol10H04 amplification; U, primer Ol12D05 amplification; V, primer Ol12G04 amplification; W, primer Ol12D09 amplification; X, primer Ra1F03 amplification; Y, primer Ol12A04 amplification.
Embodiment
Following examples are in order to explanation the present invention.
(tetraploid Chinese cabbage and the hybridization of diploid wild cabbage obtain the allotriploid cross-fertilize seed to embodiment with the two monomer alien addition lines of Chinese cabbage-wild cabbage; Cross-fertilize seed and diploid Chinese cabbage are backcrossed; The 2n=20A+2C=22 that in backcross population, screens) be accredited as example, the present invention will be described.
Principle explanation: the special SSR primer that utilizes the relative Chinese cabbage of wild cabbage linkage group; With cross-fertilize seed and parent Chinese cabbage, wild 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 all consistent with Chinese cabbage; Then show this linkage group of not adding wild cabbage in the alien addition line,, then show this linkage group of having added wild cabbage in the alien addition line if the amplified production of special primer in alien addition line in certain linkage group is all consistent with cross-fertilize seed.
Concrete grammar: according to the wild cabbage genetic map of having delivered, selected 91 pairs of SSR primers that are positioned at the different linkage groups of wild cabbage for use, primer sequence is given birth to worker Bioisystech Co., Ltd by Shanghai and is synthesized.With 8 head cabbage varieties (spring early maturing cabbage kind: in sweet 11,8312, the utmost point is wild cabbage early; Ripe head cabbage varieties in spring: rich No. one of capital; Spring and autumn dual-purpose early maturing cabbage kind: in sweet 17; Ripe head cabbage varieties in autumn: in sweet 19; Autumn late-maturing head cabbage varieties: late rich wild cabbage, in sweet 18) and 7 Chinese cabbage cultivars (anti-bolting bullet cut Chinese cabbage cultivars: strong Feng Chun Chinese cabbage, high anti-king; In the late-maturing straight tube Chinese cabbage cultivar of filling the span of a man's arms: how anti-75; The folded Chinese cabbage cultivar of embracing of extremely early mature: super Xiayang, precocious No. five; The straight tube of precocious high stake Chinese cabbage cultivar: autumn green 55; In the folded Chinese cabbage cultivar of embracing of ripe stump: Xinxiang parcel 23) planting seed coils in the cave, Routine Management, long during to 4～6 true leaves when seedling, the tender young leaves of clip children adopts 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 50ng, 2mmol/L Mg
2+, 1 * buffer, 200 μ mol/L dNTP, 1U Tap enzyme, each 2.5ng/ μ L of forward and reverse primer.The pcr amplification program is 94 ℃ of preparatory sex change 2min; 94 ℃ of sex change 1min, 60 ℃ of renaturation 30s, 72 ℃ are extended 45s, totally 10 circulations, each cycle annealing temperature reduces by 0.5 ℃; 94 ℃ of sex change 1min, 55 ℃ of renaturation 30s, 72 ℃ are extended 45s, totally 30 circulations; 72 ℃ are extended 5min., 4 ℃ of preservations.Amplified production is electrophoretic separation in 6% denaturing polyacrylamide gel, and silver dyes detection.Screening wild cabbage linkage group is with respect to the SSR primer of Chinese cabbage differential; Screening does not have amplified production in Chinese cabbage; And the primer of amplified production is arranged in the wild cabbage; Or amplified production is all arranged in the two, but the amplified production of wild cabbage is different from the primer of Chinese cabbage amplified production, as the SSR primer with respect to Chinese cabbage differential of the different linkage groups of wild cabbage.The result shows; 25 pairs of primers can amplify discrepant fragment in wild cabbage, Chinese cabbage; Fragment length is at 40-430bp, and wherein 7 pairs of primers have amplified production and in Chinese cabbage, do not have amplified production in cabbage, and 18 pairs of primers are different with the amplified production in the cabbage in Chinese cabbage; These 25 pairs of SSR primers have related to 9 linkage groups (table 2) of wild cabbage, the amplification in Chinese cabbage and wild cabbage such as Fig. 1.Utilize the special SSR primer of these 25 pairs of relative Chinese cabbages of wild cabbage linkage group; With cross-fertilize seed and parent Chinese cabbage, wild 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 Chinese cabbage; Then do not add this linkage group of wild cabbage in the alien addition line, if the amplified production of special primer in alien addition line in certain linkage group is all consistent with cross-fertilize seed, this linkage group of then having added wild cabbage in the alien addition line.The result shows; This alien addition line (is seen Fig. 2 to 3 couples of SSR primer Na10H03, Ni4B10, Na12C08 that are positioned at wild cabbage 01 linkage group; A-C) and 2 couples of SSR primer Ol12D05, Ol12G04 of 08 linkage group (see Fig. 2; U, V) amplify and the cross-fertilize seed identical segments, and to all consistent with the Chinese cabbage (see figure 2) of the amplification that is positioned at other linkage group primers, this just shows; This alien addition line is additional chromosomal pair of monomer alien addition line of 2 wild cabbages, and these 2 exogenous chromosomes are corresponding with wild cabbage 01,08 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), selects 91 pairs of SSR primers that are positioned at the different linkage groups of wild cabbage for use according to the wild cabbage genetic map;
(2) the CTAB method is extracted the Chinese cabbage of different varieties and the genomic dna of wild cabbage, pcr amplification;
(3) amplified production electrophoretic separation in 6% denaturing polyacrylamide gel, silver dyes detection;
(4) screening wild cabbage linkage group is with respect to the SSR primer of Chinese cabbage differential;
(5) utilization screens the special SSR primer of wild cabbage linkage group with respect to Chinese cabbage, identifies Chinese cabbage-wild cabbage alien addition line;
Wherein to screen the wild cabbage linkage group be Na10H03, Ni4B10, the Na12C08 of 01 linkage group with respect to the SSR primer of Chinese cabbage differential to step (4); The Ol13G05 of 02 linkage group, sORA43, Na12C03, Na12H09; The Ol11G11 of 03 linkage group, BN12A, the Na12E05 of 04 linkage group, Ol10F12, Ol11H08, the Na10D11 of 05 linkage group, Ra2A04; The Apla 5pr of 06 linkage group, Na14F11; The Ol10B01 of 07 linkage group, BN72a, Na12F03, Ol10H04, the Ol12D05 of 08 linkage group, Ol12G04, the Ol12D09 of 09 linkage group, Ra1F03, Ol12A04.
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| CN102876671B (en) * | 2010-07-26 | 2014-03-12 | 山东省农业科学院蔬菜研究所 | Chinese cabbage EST-SSR marking primer and application thereof in variety identification |
| CN102876670B (en) * | 2010-07-26 | 2013-12-11 | 山东省农业科学院蔬菜研究所 | Chinese cabbage EST-SSR (expressed sequence tag-simple sequence repeat) labeling primer and application thereof in variety identification |
| CN101956012B (en) * | 2010-09-30 | 2012-07-04 | 河北农业大学 | Method for identifying common head cabbage-Chinese cabbage alien addition line |
| CN102242219B (en) * | 2011-07-18 | 2013-01-23 | 北京市农林科学院 | Method and pair of special primers for identifying purple properties of Chinese cabbages |
| 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 |
| CN105506113B (en) * | 2016-01-06 | 2019-02-05 | 重庆市渝东南农业科学院 | The method of Rapid identification hybrid stem lump mustard purity |
| CN106701952B (en) * | 2017-01-04 | 2020-11-03 | 河北农业大学 | Method for identifying Chinese cabbage-common head cabbage translocation line based on collinear gene development marker |
| CN108739359A (en) * | 2018-06-06 | 2018-11-06 | 西南大学 | A method of carrying out plant back cross breeding using polyploid material |
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| CN1385059A (en) * | 2001-05-14 | 2002-12-18 | 东北师范大学 | Method for breeding isoaddition series of small ice-age wheat and translocation series |
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| CN1385059A (en) * | 2001-05-14 | 2002-12-18 | 东北师范大学 | Method for breeding isoaddition series of small ice-age wheat and translocation series |
Non-Patent Citations (2)
| Title |
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| 申书兴等.大白菜-结球甘蓝异附加系的获得及其部分单体异附加系连锁群归属研究.《中国园艺学会十届二次理事会暨学术研讨会论文摘要集》.2007,第104页. * |
| 顾爱侠等.二倍体大白菜与四倍体结球甘蓝杂种的获得及其SSR鉴定与GISH分析.《植物遗传资源学报》.2008,第9卷(第2期),第144-150页,特别是第145页左栏第二段、右栏第二段,第146页右栏最后一行到第147页左栏第一段. * |
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