CN102138515A - Method for introducing radish chromosomes into cabbage - Google Patents
Method for introducing radish chromosomes into cabbage Download PDFInfo
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- CN102138515A CN102138515A CN201010104342XA CN201010104342A CN102138515A CN 102138515 A CN102138515 A CN 102138515A CN 201010104342X A CN201010104342X A CN 201010104342XA CN 201010104342 A CN201010104342 A CN 201010104342A CN 102138515 A CN102138515 A CN 102138515A
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Abstract
The invention discloses a method for introducing radish chromosomes d into cabbage. The method comprises the following steps of: performing sexual hybridization on a cabbage type rape-radish chromosome d addition line and cabbage; introducing the radish chromosomes d into the cabbage; selecting a cabbage plant containing the radish chromosomes d by adopting a molecular marker detection and cell microscopy method; and creating a new cabbage germplasm carrying the radish chromosomes d. In the method, cabbage type rape is taken as a carrier of the radish chromosome d, and is hybridized with cabbage to transfer the radish chromosome d to the cabbage, so that direct hybridization between the cabbage and radish is avoided. Hybridization in brassica alboglabra succeeds easier than intergeneric hybridization between brassica alboglabra and radish. Moreover, only one pair of radish chromosomes is integrated with a cabbage genome, so that more genetic drag caused by the hybridization of an entire radish genome can be avoided, and the transfer and selection efficiencies of target traits can be improved. By adopting the method, a novel thought is provided for distant hybridization utilization.
Description
Technical field
The present invention relates to a kind of breeding method of wild cabbage, specifically radish d chromosome is imported the method for wild cabbage, belong to the wild cabbage distant hybridization and utilize the field.
Background technology
Source far away hybridization is meant the hybridization between the type that affiliation becomes estranged, between comprising kind, belong between and the hybridization between section.It can create existing unexistent special type of plant or new germ plasm, so distant hybridization become the important means that solves the plant gene scarcity of resources, is widely used in plant genetics and breeding, as in wheat (Bris B. etc., 1983; Kozub NA etc., 2004), cotton (Vmh B I etc., 1999), rape (Liu Zhongsong etc., 2001; Chen Shuzhong etc., 2000), on the cucumber various crop such as (Chen J F etc., 1997) by between planting or intergeneric cross carry out breed improvement, obtained abundant genetic variation type.
As everyone knows, exist abundant anti-source and many in Rhaphanus (Raphanus) plant to the useful proterties of rape genus (Brassica) raise crop, as cytoplasmic male sterile gene and recover gene, to the resistant gene of multiple diseases such as soft rot, black rot and club root, the Rhaphanus plant also has excellent specific property (Delourme etc., 1998 such as nematicide disease, drought resisting, cold-resistant, acid and alkali-resistance; Dolstra, 1982; Baukloh, 1976).Therefore, Rhaphanus is that rape belongs to and other Cruciferae relative genus plant carries out genetic improvement and the important donor resource of creating new variation.Distant hybridization research to rape genus and Rhaphanus starts from the '20s in last century in the world, so far carried out for eight more than ten years, wherein wild cabbage (Brassica oleracea) and Chinese cabbage (Brassica campestris) are many with the distant hybridization research of radish, but majority studies show that radish and rape hybridization and obtains very difficulty of hybrid, though the acquisition that has hybrids such as radish rape, raphanobrassica or radish black mustard, but hybrid is highly sterile, is difficult to continue to backcross.In order to overcome brassica plant and radish cross incompatibility, many scholars attempt with different technology and method, as: hybridization, pollen mixture pollination, chemicals processing etc. after the plant grafting, but these technology and method almost do not have effect (Li Xufeng etc., 1995) to the distant hybridization of brassica plant and radish.Tissue culture technique and Protoplast Fusion Technique had promoted the development in this field afterwards, the most successful is successfully to change the cytoplasmic male sterile gene of radish over to rape genus vegetables by Protoplast Fusion Technique, obtains the cytoplasmic male sterility material of wild cabbage and Chinese cabbage.Compare with sexual hybridization, though Protoplast Fusion Technique can overcome cross-incompatibility to a certain extent, obtain hybrid, but the randomness of hypochromatosis causes having and shifts specific aspects such as proterties at ground and all exist the problem (Song Shangwei etc., 2007) studying and solve demanded urgently in technology maturity that protoplast is cultivated, genetic stability, fertility (in the edge somatic hybridization particularly far away) and asymmetric hybridization burdo.
Based on above-mentioned situation, those skilled in the art are still actively seeking a kind of distant hybridization obstacle that can overcome effectively between wild cabbage and radish, radish chromosome or gene are imported to the approach of wild cabbage.The present invention intends adopting a kind of new thinking and method, avoid the direct cross between wild cabbage and the radish, and utilization is from the chromosomal cabbage type rape of additional radish d and the wild cabbage sexual hybridization of external introduction, thereby radish d chromosome is transferred to wild cabbage from cabbage type rape, make wild cabbage obtain the entrained merit of radish d chromosome (as the nematicide disease etc.), for the wild cabbage genetic breeding is created more genetic variation.
Summary of the invention
The objective of the invention is to, a kind of distant hybridization obstacle that can overcome between radish and the wild cabbage is provided, radish d chromosome is imported the method for wild cabbage by sexual hybridization.
Invention thinking of the present invention is: in order to overcome the distant hybridization obstacle between wild cabbage and the radish, the inventor utilizes cabbage type rape-radish d chromosome addition system (CGMCC No.3596) as the bridge between wild cabbage and the radish, sexual hybridization by this addition line and wild cabbage, radish d chromosome is imported to wild cabbage, utilize cytology and molecular marking technique simultaneously, selection contains the chromosomal cabbage plant of radish d, creates and carries the chromosomal wild cabbage new germ plasm of radish d.This method with cabbage type rape as the chromosomal carrier of radish d, hybridization by it and wild cabbage passes to wild cabbage with radish d chromosome, avoided the direct cross between wild cabbage and the radish, the hybridization in this rape belongs to is than the easier success of intergeneric cross between rape genus and the Rhaphanus.In the evaluation of progeny material, utilize cytology and molecular marking technique, fast and efficiently the select target material.
In order to reach above-mentioned purpose, the present invention adopts following concrete technical scheme:
The method that described acquisition carries the chromosomal wild cabbage of radish d comprises the steps:
(1) it is maternal utilizing cabbage type rape-radish d chromosome addition, and wild cabbage is that male parent is carried out artificial flower bud phase hybridization pollination, cultivates in conjunction with ovary, obtains a transformation generation;
(2) utilize RAPD Markers for Detection and cell microscopy to identify a transformation generation, select to contain radish d chromosome and root-tip cells chromosome number and be 29 individual plant, carry out backcross transformation, cultivate, obtain two generations of transformation in conjunction with ovary with wild cabbage;
(3) utilize the RAPD Markers for Detection, selection contains the chromosomal plant of radish d, utilize the cell microscopy again, selection contains radish d chromosome and root-tip cells chromosome number near 20, recommend to select in 19~29 scopes, plant forms continues to carry out backcross transformation with wild cabbage near the individual plant of wild cabbage, cultivate in conjunction with ovary, obtain the transformation three generations;
(4) utilize the RAPD Markers for Detection, selection contains the chromosomal plant of radish d, utilizes the cell microscopy again, and selection contains radish d chromosome and the root-tip cells chromosome number is 19 or 20, plant forms is and carries the chromosomal wild cabbage of radish d like the individual plant of wild cabbage.
Cabbage plant form and biological property are content as well known to those skilled in the art, do not give unnecessary details at this, and final products of the present invention contain radish d chromosome and possess wild cabbage form and biological property through Molecular Identification.
Described cabbage type rape-radish d chromosome addition lies on January 27th, 2010, be preserved in Chinese microbial preservation administration committee common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is: CGMCC No.3596, classification name: rape brassica napus.
Above-mentioned RAPD Markers for Detection is: utilize a detection among the chromosomal RAPD mark of radish d OPF07-363bp, the OPG19-488bp, the plant that any one is arranged in the above-mentioned mark is for containing radish d chromosome plant.The primer sequence of OPF07 and OPG19 is as follows.
OPF07-363bp:5’-CCGATATCCC-3’;OPG19-488bp:5’-GTCAGGGCAA-3’。
Described cell microscopy is to utilize sediments microscope inspection plant root-tip cells to be checked chromosome number.
Cabbage type rape involved in the present invention-radish d chromosome addition is that CGMCC No.3596 direct sources is: German federal raise crop breeding research center; Primary source: cultivate voluntarily by German federal raise crop breeding research center.The direct sources and the primary source of male parent wild cabbage are: cultivate voluntarily the Beijing City Agriculture and Forestry Institute.
Advantage of the present invention and beneficial effect:
Compare with the direct distant hybridization method of radish with traditional wild cabbage, adopt additional chromosomal cabbage type rape of radish d and wild cabbage to hybridize, with the middle bridge of cabbage type rape-radish d chromosome addition system (genome AACC+RR) as wild cabbage (genome C C) and radish (genome RR), and have only 1 pair of radish chromosome and wild cabbage genome conformity, can avoid the more heredity that causes with whole radish genomic hybridization burdensome, therefore this method not only can overcome the distant hybridization obstacle between wild cabbage and the radish, can also improve the transfer and the efficiency of selection of objective trait.This method provides a new thinking and method for the distant hybridization of wild cabbage and radish.
Description of drawings
Fig. 1 is the technology of the present invention flow chart;
Fig. 2 is a RAPD molecular labeling OPF07-363bp testing result, and swimming lane from left to right is respectively Marker, 2 addition line parents, and 2 wild cabbage parents, 3 transformation generation, Marker, 1 transformation generation, in 1 two generation of transformation, all the other are the transformation three generations;
Fig. 3 is a RAPD molecular labeling OPG19-488bp testing result, and swimming lane from left to right is respectively Marker, the addition line parent, and 3 wild cabbage parents, 3 transformation generation, 1 two generation of transformation, 1 transformation three generations, Marker, all the other are the transformation three generations.
Embodiment
As shown in Figure 1, concrete steps are as follows:
1, hybridization pollination and ovary are cultivated: with cabbage type rape-radish d chromosome addition is maternal, and wild cabbage (commercially available can the purchase obtains, and kind or the inbred line of isozygotying can be applied to the present invention, do not have specifically to limit) carries out hybridization pollination for male parent.Bloomed preceding 2~3 days, this spray of father and mother bagging respectively isolates.The maternal flower bud phase castrates, and the pollen of getting male parent is applied on the maternal column cap.Get 7~15 days the ovary in pollination back and carry out tissue culture.Medium is: 1/2MS+ inositol (10g/L)+nicotinic acid (100mg/L)+VB6 (100mg/L)+VB1 (1g/L)+IAA (1.5mg/L)+sucrose (50g/L), pH=5.8.Cultivated about one month, the seed in the ovary is mature on the whole, and obtains a transformation generation.
2, a transformation generation is identified:
(1) utilize a detection transformation generation among radish d chromosomal RAPD mark OPF07-363bp and the OPG19-488bp whether to contain radish d chromosome, the plant that above-mentioned mark is arranged is for containing radish d chromosome.As shown in Figures 2 and 3.
Fig. 2 is a RAPD molecular labeling OPF07-363bp testing result, and swimming lane from left to right is respectively Marker, 2 addition line parents, and 2 wild cabbage parents, 3 transformation generation, Marker, 1 transformation generation, in 1 two generation of transformation, all the other are the transformation three generations;
Fig. 3 is a RAPD molecular labeling OPG19-488bp testing result, and swimming lane from left to right is respectively Marker, the addition line parent, and 3 wild cabbage parents, 3 transformation generation, 1 two generation of transformation, 1 transformation three generations, Marker, all the other are the transformation three generations.
Wherein the base sequence of primer OPF07, OPG19 is as follows respectively, is specially 5 '-CCGATATCCC-3 ', 5 '-GTCAGGGCAA-3 '.A transformation generation contains radish d chromosome.
The RAPD molecular markers for identification adopts the SDS method to extract parent and hybrid seedling leaves DNA, carry out pcr amplification with the RAPD primer, reaction system is: 16ng DNA, 0.25mmol/L dNTP, 1 * Buffer, 0.1U Taq archaeal dna polymerase, 2.5mmol/L MgCL2,0.4mmol/L primer, final volume 8 μ L.Reaction cycle is: 94 ℃ (2min), 45 circulations in ℃ (30s)-72,94 ℃ of (30s)-36 ℃ (1min), 72 ℃ of 10min afterwards, 4 ℃ of preservations.The PCR product separates with 6% denaturing polyacrylamide gel electrophoresis, and silver dyes detection.
(2) with the root-tip cells chromosome number of sediments microscope inspection transformation generation plant, the chromosome number of a transformation generation is 29.
Chromosome microscopy method: the first fixing tip of a root, get 2~3mm left and right sides tip of a root lucifuge in saturated paracide solution and handle 4h, after change in the Kano fixer (absolute ethyl alcohol: glacial acetic acid was with mixing in 3: 1) of now joining and fix 3~24h.The tip of a root of getting after fixing dissociates, and earlier with the distilled water rinsing tip of a root 2~3 times, then the tip of a root is put into 60 ℃ of water-baths of 1mol/L hydrochloric acid 6~8 minutes of preheating, changes in the distilled water again and soaks more than 10 minutes.Cut the tip of a root with carbolfuchsin dye liquor dyeing 4~6 minutes, behind the conventional compressing tablet at OLYMPUS BH-2 microscopically microscopy chromosome.
3, back pollinating and ovary are cultivated: the transformation one that goes out with evaluation and screening is on behalf of female parent, and wild cabbage is that male parent is carried out back pollinating.Pollinating method and ovary cultural method are with step 1, and the seed that obtains is two generations of transformation.
4, in two generations of transformation, identified: with step 2 method (1), whether contain radish d chromosome with in two generations of Markers for Detection transformation.The chromosomal transformation of select tape radish d two generations plant, with sediments microscope inspection root-tip cells chromosome, therefrom selective staining body number near 20, plant forms is near the plant of wild cabbage.
5, two generations of transformation backcross and ovary is cultivated: the transformation two that obtains with evaluation and screening is on behalf of female parent, and wild cabbage is that male parent continues back pollinating.Pollination and ovary cultural method obtain the transformation three generations with step 1.
6, the transformation three generations identifies: with second step (1) method, detect among the transformation three generations whether contain radish d chromosome.The chromosomal transformation three generations of select tape radish d plant, with sediments microscope inspection root-tip cells chromosome, therefrom selective staining body number is 19 or 20, plant forms is like the plant of wild cabbage, is the chromosomal wild cabbage of radish d that carries of the present invention.
The wild cabbage that obtains through this method contains radish d chromosome through the RAPD molecular markers for identification, and has the morphology and the biological property of wild cabbage.The present invention has overcome distant hybridization obstacle between wild cabbage and the radish by simple method, improves the transfer and the efficiency of selection of objective trait.The present invention has good market prospects for the distant hybridization of wild cabbage and radish provides a new thinking and method.
Claims (8)
1. method that radish d chromosome is imported wild cabbage, it is characterized in that, it is to utilize cabbage type rape-radish d chromosome addition system and wild cabbage sexual hybridization, radish d chromosome is imported to wild cabbage, utilize Markers for Detection and cell microscopy method simultaneously, selection contains the chromosomal cabbage plant of radish d, creates and carries the chromosomal wild cabbage new germ plasm of radish d.
2. the method with radish d chromosome importing wild cabbage according to claim 1 is characterized in that described cabbage type rape-radish d chromosome addition is that deposit number is: CGMCC No.3596.
3. the method with radish d chromosome importing wild cabbage according to claim 1 and 2 is characterized in that described method comprises the steps:
(1) it is maternal utilizing cabbage type rape-radish d chromosome addition, and wild cabbage is that male parent is carried out artificial flower bud phase hybridization pollination, cultivates in conjunction with ovary, obtains a transformation generation;
(2) utilize RAPD Markers for Detection and cell microscopy to identify a transformation generation, select to contain radish d chromosome and root-tip cells chromosome number and be 29 individual plant, carry out backcross transformation, cultivate, obtain two generations of transformation in conjunction with ovary with wild cabbage;
(3) utilize two generations of RAPD Markers for Detection transformation, selection contains the chromosomal plant of radish d, utilize the cell microscopy again, selection contains radish d chromosome and the root-tip cells chromosome number is 19~29, morphology is accredited as the individual plant of wild cabbage, continue to carry out backcross transformation, cultivate, obtain the transformation three generations in conjunction with ovary with wild cabbage;
(4) utilize RAPD Markers for Detection transformation three generations, selection contains the chromosomal plant of radish d, utilizes the cell microscopy again, and selection contains radish d chromosome and the root-tip cells chromosome number is 19 or 20, morphology is accredited as the individual plant of wild cabbage, is to carry the chromosomal wild cabbage of radish d.
4. the method with radish d chromosome importing wild cabbage according to claim 3 is characterized in that described cabbage type rape-radish d chromosome addition is that deposit number is: CGMCC No.3596.
5. the method that radish d chromosome is imported wild cabbage according to claim 2, it is characterized in that, the culture medium prescription that described step (1) ovary is cultivated is: 1/2MS, 10g/L inositol, 100mg/L nicotinic acid, 100mg/L VB6,1g/L VB 1,1.5mg/L IAA, 50g/L sucrose, pH=5.8.
6. according to claim 4 or the 5 described methods that radish d chromosome imported wild cabbage, it is characterized in that, described RAPD Markers for Detection is: utilize a detection among the chromosomal RAPD mark of radish d OPF07-363bp, the OPG19-488bp, the plant that any one is arranged in the above-mentioned mark is for containing radish d chromosome plant.
7. the method with radish d chromosome importing wild cabbage according to claim 6 is characterized in that the primer sequence of described RAPD molecular labeling OPF07, OPG19 is as follows:
OPF07:5′-CCGATATCCC-3′;OPG19:5′-GTCAGGGCAA-3′。
8. the method with radish d chromosome importing wild cabbage according to claim 7 is characterized in that described cell microscopy is to utilize sediments microscope inspection plant root-tip cells to be checked chromosome number.
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Cited By (9)
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CN106472300A (en) * | 2016-10-26 | 2017-03-08 | 北京市农林科学院 | A kind of method that initiative can stablize the Chinese cabbage germplasm carrying Radix Raphani chromosome of heredity |
CN109349099A (en) * | 2018-11-20 | 2019-02-19 | 贵州省油料研究所 | Brassica napus recessive gms line is close, distant hybridization selectively breeding hybrid rape selection |
CN110499384A (en) * | 2019-09-03 | 2019-11-26 | 中国农业科学院蔬菜花卉研究所 | Identification cabbage mustard and brassica campestris var purpurea interspecific hybrid and the molecular labeling for tracking its offspring's materials A 08 and C08 chromosome separation situation |
CN110499384B (en) * | 2019-09-03 | 2022-05-31 | 中国农业科学院蔬菜花卉研究所 | Molecular markers for identifying interspecific hybrids of cabbage mustard and red cabbage moss and tracking the segregation of A08 and C08 chromosomes in progeny materials thereof |
CN111235300A (en) * | 2020-03-23 | 2020-06-05 | 北京市农林科学院 | Method for identifying authenticity of cabbage variety and special SSR primer combination thereof |
CN111235300B (en) * | 2020-03-23 | 2021-04-23 | 北京市农林科学院 | Method for identifying authenticity of cabbage variety and special SSR primer combination thereof |
CN112251460A (en) * | 2020-10-28 | 2021-01-22 | 中国农业科学院蔬菜花卉研究所 | Method for researching radish functional genome and verifying gene function |
CN112322650A (en) * | 2020-10-28 | 2021-02-05 | 中国农业科学院蔬菜花卉研究所 | Radish and cabbage homologous gene function research method capable of eliminating background difference |
CN112913681A (en) * | 2021-02-07 | 2021-06-08 | 北京市农林科学院 | Method for creating clubroot-resistant Chinese cabbage germplasm |
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