CN102138515B - Method for introducing radish chromosomes into cabbage - Google Patents
Method for introducing radish chromosomes into cabbage Download PDFInfo
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- CN102138515B CN102138515B CN 201010104342 CN201010104342A CN102138515B CN 102138515 B CN102138515 B CN 102138515B CN 201010104342 CN201010104342 CN 201010104342 CN 201010104342 A CN201010104342 A CN 201010104342A CN 102138515 B CN102138515 B CN 102138515B
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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 wild cabbage distant hybrid utilization field.
Background technology
Distant hybridization refers to the hybridization between the type that affiliation becomes estranged, between comprising kind, belong between and the hybridization between section.It can create the 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 various crop such as cucumber (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 to the useful proterties of rape genus (Brassica) raise crop in Rhaphanus (Raphanus) plant, such as cytoplasmic male sterile gene and recover gene, to the resistant gene of the multiple diseases such as soft rot, black rot and club root, the Rhaphanus plant also has excellent specific property (Delourme etc., 1998 such as Nematode resistance, 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.In the world the distant hybridization research of rape genus and Rhaphanus started from the '20s in last century, 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 it is very difficult that majority studies show that radish and rape hybridization obtain hybrid, although the acquisition that has the 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 Vegetables in Brassica by Protoplast Fusion Technique, obtains the cytoplasmic male sterility material of wild cabbage and Chinese cabbage.Compare with sexual hybridization, although Protoplast Fusion Technique can overcome cross-incompatibility to a certain extent, obtain hybrid, but the randomness of hypochromatosis causes shifting targeted specifically the aspect such as specific proterties and all exists the problem (Song Shangwei etc., 2007) studying and solve demanded urgently in technology maturity that protoplast is cultivated, genetic stability, fertility (particularly in the edge somatic hybridization 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 effectively overcome 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, carry out sexual hybridization and utilize from the chromosomal cabbage type rape of additional radish d and the wild cabbage 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 (such as Nematode resistance etc.), for the wild cabbage genetic breeding is created more genetic variation.
Summary of the invention
The object 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 simultaneously cytology and molecular marking technique, selection contains the chromosomal cabbage plant of radish d, creates to carry 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 cell chromosome number and be 29 individual plant, carry out backcross transformation with wild cabbage, cultivate in conjunction with ovary, obtain two generations of transformation;
(3) utilize the RAPD Markers for Detection, selection contains the chromosomal plant of radish d, recycling cell microscopy, selection contains radish d chromosome and root tip cell chromosome number near 20, in recommendations for selection 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, and recycling cell microscopy selects to contain radish d chromosome and the root tip cell 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, are not repeated herein, 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 And Nomenclature: 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 of any one is arranged in the above-mentioned mark 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 cell chromosome to be checked 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: cultivated voluntarily by German federal raise crop breeding research center.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 the 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 only have 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 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 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 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: be maternal with cabbage type rape-radish d chromosome addition, wild cabbage (commercially available can the purchase obtains, and kind or the inbred line of isozygotying can be applied to the present invention, without specifically limiting) carries out hybridization pollination for male parent.Bloomed the respectively bagging isolation of Parent spray front 2～3 days.The maternal flower bud phase castrates, and the pollen of getting male parent is applied on the maternal column cap.Get rear 7～15 days ovary of pollination and organize cultivation.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) utilizes a detection transformation generation among radish d chromosomal RAPD mark OPF07-363bp and the OPG19-488bp whether to contain radish d chromosome, the plant of above-mentioned mark is arranged for containing radish d chromosome.As shown in Figures 2 and 3.
Fig. 2 is 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 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 cell 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 process 4h, after change in the Kano fixer (absolute ethyl alcohol: glacial acetic acid mixed with 3: 1) of now joining and fix 3～24h.The tip of a root of getting after fixing dissociates, and uses first 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 is used the sediments microscope inspection root tip cell chromosome, therefrom selective staining body number near 20, plant forms is near the plant of wild cabbage.
5, two generations of transformation backcross and cultivate with ovary: 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: same 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 is used the sediments microscope inspection root tip cell chromosome, and 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 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 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 (2)
1. the method with radish d chromosome importing wild cabbage 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 cell chromosome number and be 29 individual plant, carry out backcross transformation with wild cabbage, cultivate in conjunction with ovary, obtain two generations of transformation;
(3) utilize two generations of RAPD Markers for Detection transformation, selection contains the chromosomal plant of radish d, recycling cell microscopy, selection contains radish d chromosome and the root tip cell chromosome number is 19～29, Morphological Identification is the individual plant of wild cabbage, continue to carry out backcross transformation with wild cabbage, cultivate in conjunction with ovary, obtain the transformation three generations;
(4) utilize RAPD Markers for Detection transformation three generations, selection contains the chromosomal plant of radish d, and recycling cell microscopy selects to contain radish d chromosome and the root tip cell chromosome number is 19 or 20, Morphological Identification is the individual plant of wild cabbage, is to carry the chromosomal wild cabbage of radish d;
Described cabbage type rape-radish d chromosome addition is that deposit number is: CGMCC No.3569;
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 VB1,1.5mg/L IAA, 50g/L sucrose, pH=5.8;
Described RAPD Markers for Detection is: utilize a detection among the chromosomal RAPD mark of radish d OPF07-363bp, the OPG19-488bp, the plant of any one is arranged in the above-mentioned mark for containing radish d chromosome plant;
The primer sequence of described RAPD molecular labeling OPF07, OPG19 is as follows:
OPF07：5′-CCGATATCCC-3′；OPG19：5′-GTCAGGGCAA-3′。
2. the method with radish d chromosome importing wild cabbage according to claim 1 is characterized in that described cell microscopy is to utilize sediments microscope inspection plant root tip cell chromosome to be checked number.
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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 |
| 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 |
| 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 |
| CN112913681B (en) * | 2021-02-07 | 2022-09-06 | 北京市农林科学院 | Method for creating clubroot-resistant Chinese cabbage germplasm |
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| Title |
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| 李倩.甘蓝型油菜—萝卜d染色体附加系中d染色体导入甘蓝的初步研究.《中国优秀硕士学文论文全文数据库 农业科技辑》.2008, * |
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