CN107410011B - A kind of method of efficient quick separating cabbage type rape A and C subgenome - Google Patents
A kind of method of efficient quick separating cabbage type rape A and C subgenome Download PDFInfo
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- CN107410011B CN107410011B CN201710427217.4A CN201710427217A CN107410011B CN 107410011 B CN107410011 B CN 107410011B CN 201710427217 A CN201710427217 A CN 201710427217A CN 107410011 B CN107410011 B CN 107410011B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention belongs to field of plant genetic project technology, and in particular to a method of efficiently quickly separate cabbage type rape A and C subgenome.The method of the invention utilizes the double low kind (low erucic acids of self-compatibility, low-sulfur glycosides) cabbage type rape excellent character, the subgenome A and C that are loaded with its excellent character are separated, to create new turnip type rape and wild cabbage, isolated A or C genome new resources, on the one hand it can be hybridized with turnip type rape (or Chinese cabbage), for basic research (such as assignment of genes gene mapping and clone, chromosome evolution etc.) or the application study (beneficial traits in importing rape, it is such as disease-resistant, it is high-quality etc.), more excellent hybrid generations can be further obtained with this simultaneously.
Description
Technical field
The invention belongs to field of plant genetic project technology, and in particular to a kind of efficiently quickly to separate cabbage type rape A
With the method for C subgenome.
Background technique
Cabbage type rape (Brassica napus, AACC, 2n=38) is by turnip type rape (B.rapa, AA, 2n=
20) the allotetraploid product evolved with wild cabbage (B.oleracea, CC, 2n=18) by natural hybridization and chromosome doubling
Kind.Therefore, cabbage type rape contains turnip type rape and the ancestors of wild cabbage plant gene.In production, cabbage type rape is three kinds
The highest type of grain yield in rape (turnip type rape, mustard type rape, cabbage type rape) is the important oil in China
Expect crop.China realized the breeding of Brassica napus with double lowization (low erucic acid, low-sulfur glycosides) at 2000 or so, closed in production at present
The kind of method is double low kinds.The nutritive value and palatability for improving cabbage type rape of quality, make its function exist
Originally only expanded as on the basis of oil crops as seedling stage or elongation as vegetables or feed.
In practical breeding work or research, what researcher usually thought deeply is how to utilize turnip type rape (or Chinese cabbage)
With wild cabbage artificial synthesis Brassica napus, so as to by the beneficial traits of relationship kind (the anti-clubroot character of such as Chinese cabbage) import wild cabbage
In type rape.The present invention uses reverse thinking, by splitting cabbage type rape A, C genome, obtains double low-quality AA genes
Group (Chinese cabbage type) and CC genome (wild cabbage) original species.This original species, can be used as genome evolution research, the assignment of genes gene mapping with
The basic material of map based cloning can also be used as the donor of improvement turnip type rape (or Chinese cabbage) and wild cabbage.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in provide a kind of efficiently quick separation cabbage type rape A and C
The method of subgenome is the not affine double high kinds of selfing to solve parent species turnip type rape in the prior art and wild cabbage still
The problem of.
In order to solve the above technical problems, of the present invention efficiently quickly separate cabbage type rape A and C subgenome
Method includes the following steps:
(1) it takes Microspore of Brassica napus to be cultivated, after embryoid seedling, analyzes its ploidy feature by strain, screen
Monoploid and diploid out；
(2) by above-mentioned microspore transplantation of seedlings to crop field, male parent is made with gained diploid, gained monoploid is planted as female parent
It plants；
(3) in aobvious flower bud phase, gametocide processing is carried out to female parent seedling, to prevent maternal monoploid from generating trace-pollen；
(4) it after entering flowering stage, covers isolation curtain and carries out supple-mentary pollination, until the whole florescence removes bed-curtain, and female parent is done
Good label；
(5) it to harvest time, harvests seed in female parent strain and is denoted as F1, and plant in field, start to cover account and pollination in flowering stage,
Mixing harvest F2 seed；
(6) by F2 seed kind in field, it is in seedling stage coring leaf analysis single plant ploidy, while with Chinese cabbage and wild cabbage genome
Control, filters out the single plant as Chinese cabbage and wild cabbage ploidy respectively, and individually by its seedling replanting, Post flowering selfing is harvested
Novel Chinese cabbage or wild cabbage.
In the step (2), the planting patterns of the male parent row and maternal row is according to 1 row male parent: 4 rows are maternal: 1 row male parent
It is planted.
In the step (2), the male parent row kind is in maternal row two sides.
In the step (3), the gametocide processing step is to carry out when maternal bud about 2mm.
In the step (3), the gametocide processing step is that gametocide is primary.
In the step (4) and (5), the pollination step is the pollination of wall bee.
In the step (5), the genome of the F1 seed is A10+iC9+j, wherein i is A genome addition chromosome number,
J is C genome addition chromosome number, and i=0-10 item, j=0-9 item.
The step of analysis single plant ploidy is to utilize Flow Cytometry.
In the step (6), if there is single plant or the ploidy 2n=as wild cabbage for being 2n=20 as Chinese cabbage ploidy
18 single plant then individually chooses its seedling replanting；If the single plant of no 2n=20 or 2n=18 ploidy occurs, continue to cover account
And pollination processing, until selecting suitable euploid.
The invention also discloses the cabbage type rape A and C subgenomes isolated by the method.
The method of the invention utilizes the superiority of double low kind (the low erucic acid, low-sulfur glycosides) cabbage type rapes of self-compatibility
Shape separates the subgenome A and C that are loaded with its excellent character, isolated to create new turnip type rape and wild cabbage
On the one hand A or C genome new resources can be used for hybridizing it with turnip type rape (or Chinese cabbage), be used for basic research (such as base
Because of positioning and clone, chromosome evolution etc.) or application study (importing the beneficial traits in rape, such as disease-resistant, high-quality), simultaneously
More excellent hybridization products can be further obtained with this.
Separation method of the present invention is integrated with microspore culture, wall bee pollination technique, Chemical Castration Technology and stream
Formula cell technology, has the advantages that fast and efficiently.
Specific embodiment
Embodiment 1
The method that cabbage type rape A and C subgenome is efficiently quickly separated described in the present embodiment, includes the following steps:
(1) it takes double low selfing affinity type Microspore of Brassica napus cultures, after embryoid seedling, utilizes Flow Cytometry
Ploidy (monoploid or diploid) is analyzed by strain；
(2) by above-mentioned microspore transplantation of seedlings to crop field, diploid makees male parent, and monoploid makees maternal 1 row male parent of pressing: 4 rows are female
This: the mode of 1 row male parent is planted, and wherein male parent row kind is in maternal row two sides；
(3) flower bud phase is shown, it is primary to the spraying gametocide of female parent with male sterilant when maternal bud about 2mm, to prevent maternal single times
Body generates trace-pollen；
(4) enter flowering stage, cover isolation curtain, wall bee supple-mentary pollination is put into account, the whole florescence removes bed-curtain, and female parent carries out mark
Note；
(5) harvest time harvests seed in female parent strain, marks, be denoted as F1, genome A10+iC9+j.I is A gene
Group addition chromosome number (0-10 item), j are C genome addition chromosome number (0-9 item)；By F1 seed kind in field, flowering stage
Start to cover account, puts the pollination of wall bee, mixing harvest F2 seed；
(6) F2 seed kind is in field, and seedling stage coring leaf analyzes single plant ploidy with Flow Cytometry, while with Chinese cabbage and sweet
Blue Gene group is control, if there is the list for single plant or the ploidy 2n=18 as wild cabbage for being 2n=20 as Chinese cabbage ploidy
Strain, then individually choose its seedling, is transplanted to another field, and Post flowering selfing harvests novel Chinese cabbage or wild cabbage；If without 2n
The single plant of=20 or 2n=18 ploidy occurs, then continues to cover account, is put into the pollination of wall bee in the florescence, and harvest seed is denoted as F3, under F3
1 year same such as step 6 processing, the euploid until selecting 2n=20 and 2n=18.
It is identical as control Chinese cabbage and wild cabbage through flow cytometer detection blade ploidy, it is seen then that through the present embodiment the method
The single plant selected is to have the single plant of cabbage type rape A and C subgenome, it was demonstrated that the method for the invention can quick separating go out
Cabbage type rape A and C subgenome.
Embodiment 2
The method that cabbage type rape A and C subgenome is efficiently quickly separated described in the present embodiment, includes the following steps:
(1) it takes double low selfing affinity type Microspore of Brassica napus cultures, after embryoid seedling, utilizes Flow Cytometry
Ploidy (monoploid or diploid) is analyzed by strain；
(2) by above-mentioned microspore transplantation of seedlings to crop field, diploid makees male parent, and wherein male parent row kind is in maternal row two sides；
(3) flower bud phase is shown, it is primary to the spraying gametocide of female parent with male sterilant when maternal bud about 2mm, to prevent maternal single times
Body generates trace-pollen；
(4) enter flowering stage, cover isolation curtain, wall bee supple-mentary pollination is put into account, the whole florescence removes bed-curtain, and female parent carries out mark
Note；
(5) harvest time harvests seed in female parent strain, marks, be denoted as F1, genome A10+iC9+j.I is A gene
Group addition chromosome number (0-10 item), j are C genome addition chromosome number (0-9 item)；By F1 seed kind in field, flowering stage
Start to cover account, puts the pollination of wall bee, mixing harvest F2 seed；
(6) F2 seed kind is in field, and seedling stage coring leaf analyzes single plant ploidy with Flow Cytometry, while with Chinese cabbage and sweet
Blue Gene group is control, if there is the list for single plant or the ploidy 2n=18 as wild cabbage for being 2n=20 as Chinese cabbage ploidy
Strain, then individually choose its seedling, is transplanted to another field, and Post flowering selfing harvests novel Chinese cabbage or wild cabbage；If without 2n
The single plant of=20 or 2n=18 ploidy occurs, then continues to cover account, and the florescence is put into the pollination of wall bee, and harvest seed is denoted as F3, one under F3
Year is equally handled such as step 6, the euploid until selecting 2n=20 and 2n=18.
It is identical as control Chinese cabbage and wild cabbage through flow cytometer detection blade ploidy, it is seen then that through the present embodiment the method
The single plant selected is to have the single plant of cabbage type rape A and C subgenome, it was demonstrated that the method for the invention can quick separating go out
Cabbage type rape A and C subgenome.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (8)
1. a kind of method for separating cabbage type rape A and C subgenome, which comprises the steps of:
(1) it takes Microspore of Brassica napus to be cultivated, after embryoid seedling, analyzes its ploidy feature by strain, filter out list
Times body and diploid；
(2) by above-mentioned microspore transplantation of seedlings to crop field, male parent is made with gained diploid, gained monoploid is planted as female parent；
(3) in aobvious flower bud phase, gametocide processing is carried out to female parent seedling, to prevent maternal monoploid from generating trace-pollen；
(4) it after entering flowering stage, covers isolation curtain and carries out supple-mentary pollination, until the whole florescence removes bed-curtain, and mark is carried out to female parent
Note；
(5) it to harvest time, harvests seed in female parent strain and is denoted as F1, and plant in field, start to cover account and pollination, mixing in flowering stage
Harvest F2 seed；
It (6) is pair in seedling stage coring leaf analysis single plant ploidy, while with Chinese cabbage and wild cabbage genome by F2 seed kind in field
According to filtering out the single plant as Chinese cabbage and wild cabbage ploidy respectively, and individually by its seedling replanting, Post flowering selfing harvests new
Type Chinese cabbage or wild cabbage.
2. the method for separation cabbage type rape A and C subgenome according to claim 1, which is characterized in that the step
(2) in, the male parent row kind is in maternal row two sides.
3. the method for separation cabbage type rape A and C subgenome according to claim 2, which is characterized in that the step
(2) in, the planting patterns of the male parent row and maternal row is according to 1 row male parent: 4 rows are maternal: 1 row male parent is planted.
4. the method for separation cabbage type rape A and C subgenome according to claim 1-3, which is characterized in that
In the step (3), the gametocide processing step is to carry out when maternal bud about 2mm.
5. the method for separation cabbage type rape A and C subgenome according to claim 1-3, which is characterized in that
In the step (3), the gametocide processing step is that gametocide is primary.
6. the method for separation cabbage type rape A and C subgenome according to claim 1-3, which is characterized in that
In the step (4) and (5), the pollination step is the pollination of wall bee.
7. the method for separation cabbage type rape A and C subgenome according to claim 1-3, which is characterized in that
The step of analysis single plant ploidy is to utilize Flow Cytometry.
8. the method for separation cabbage type rape A and C subgenome according to claim 1-3, which is characterized in that
In the step (6), if there is the list for single plant or the ploidy 2n=18 as wild cabbage for being 2n=20 as Chinese cabbage ploidy
Strain, then individually choose its seedling replanting；If the single plant of no 2n=20 or 2n=18 ploidy occurs, continue to cover account and pollination
Processing, until selecting suitable euploid.
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| CN109197576A (en) * | 2018-11-16 | 2019-01-15 | 贵州省油料研究所 | A kind of method of Brassica napus recessive gms line and hybrid lily selectively breeding hybrid rape |
| CN110512022B (en) * | 2019-09-02 | 2022-05-31 | 中国农业科学院蔬菜花卉研究所 | Molecular marker and method for identifying segregation condition of intercede hybrids of brassica vegetables and progeny materials A06 and C05 chromosomes |
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| CN106134982A (en) * | 2016-07-13 | 2016-11-23 | 河南省农业科学院经济作物研究所 | The breeding method of cabbage type rape new lines |
| CN106171972A (en) * | 2016-06-30 | 2016-12-07 | 邢台市蔬菜种子公司 | A kind of cultural method of cabbage type rape Isolated microspore plant strain |
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| CN106171972A (en) * | 2016-06-30 | 2016-12-07 | 邢台市蔬菜种子公司 | A kind of cultural method of cabbage type rape Isolated microspore plant strain |
| CN106134982A (en) * | 2016-07-13 | 2016-11-23 | 河南省农业科学院经济作物研究所 | The breeding method of cabbage type rape new lines |
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| 甘蓝型油菜亚基因组间杂种(ArAnCcCn)的细胞学和育性研究;栗茂腾等;《作物学报》;20060331;第32卷(第3期);第351-357页 * |
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