CN1167328C - Method for utilizing cole intergenomic hybrid vigor - Google Patents
Method for utilizing cole intergenomic hybrid vigor Download PDFInfo
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- CN1167328C CN1167328C CNB991201272A CN99120127A CN1167328C CN 1167328 C CN1167328 C CN 1167328C CN B991201272 A CNB991201272 A CN B991201272A CN 99120127 A CN99120127 A CN 99120127A CN 1167328 C CN1167328 C CN 1167328C
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- type rape
- rape
- hybrid
- cabbage type
- plant
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Abstract
The present invention relates to a method for utilizing the superiority of rape inter-genomic hybrid, which belongs to the technical field of selection and breeding of a new rape strain. The present invention is characterized in that through interspecific crossing and molecular marker-assisted selection, genome A' of Chinese-cabbage type rape is combined together with genome C of cabbage type rape or genome C' of Ethiopian mustard to culture a new good cabbage type rape strain whose karyotype is A'A'CC or A'A'C'C', and thereby, cabbage type rape hybrid combinations with super-strength advantages are coordinated.
Description
Technical field
The invention belongs to rape heterosis and utilize technical field, be specifically related to a kind of heterotic method of rape genome of utilizing.Its international Patent classificating number is A01H 1/02.
Background technology
Genetics research is verified, the cabbage type rape of amphidiploid (AACC), mustard type rape (AABB) and brassicacarinata (BBCC) are three elementary species by Brassicas, double naturally to produce after promptly Chinese cabbage or turnip type rape (AA), black mustard (BB) and wild cabbage (CC) are hybridized in twos.It is of the remote past that this course takes place, and long-term isolation, selection and evolution make identical genome produce deep differentiation between different kinds.If the chromosome set of cabbage type rape is regarded as AACC,, the chromosome set of turnip type rape can be designated as A in order to reflect this genome differentiation
rA
r
Rape species hybrid exists extremely strong hybrid vigour.Cabbage type rape (AACC) and turnip type rape (A
rA
r) the F1 hybrid (be called for short sweet white hybrid, A
rAC) the plant nutrition advantage can surpass 152% (Qian etc., 10 of high parent's value
ThInternationalWorkshop on Gene, Genome and Isoenzyme, 1999, Beijing).Obviously, the hybrid vigour between sweet, white comes from turnip type rape A
rA genome in genome and the cabbage type rape or the mutual work between the C genome.Because this species hybrid advantage of rape derives from the mutual work between whole genome, is referred to as intergenomic hybrid vigor.
Because sweet white hybrid is aneuploid, chromosome pairing is undesired, and hybrid dysgenesis or fertility are extremely low, so this intergenomic hybrid vigor of rape can only show the aspect of nourishing and growing, but ripening rate is extremely low, can't be used for hybrid seed production.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, a kind of method that can obtain the normal rape gene of ripening rate species hybrid advantage is provided, particularly utilize the method for turnip type rape and cabbage type rape intergenomic hybrid vigor, improve the intensity of rape heterosis, and then the cabbage type rape hybrid combination of the superpower advantage of assembly.
The present invention is achieved through the following technical solutions:
A kind of method of utilizing rape heterosis, comprise conventional hybridization breeding and molecular biology breeding method, utilize rape species hybrid use of advantage methodological innovation for utilizing the heterotic method of rape genome traditional, by interspecific cross and molecular marker assisted selection, with the A of turnip type rape
rThe C genome of genome and cabbage type rape combines, and cultivating caryotype is A
rA
rThe cabbage type rape excellent strain of CC, and then the cabbage type rape hybrid combination of the superpower advantage of assembly, can produce caryotype is A
rThe F1 hybrid of ACC.
It comprises the following steps:
1) make female parent with low erucic acid, low-sulfur glycosides (two low) cabbage type rape variety, make male parent with turnip type rape, the acquisition caryotype is A
rThe F1 species hybrid seed of AC;
2) with the hybrid bagging and the supplementary pollination of cabbage type rape and turnip type rape, produce selfed seed, produce F2 for plant through plantation, eliminate growth deformity and the not high plant of fertility, obtaining the F3 strain is after plant does agronomy and quality selection, remake chromosome examination, select the fine individual plant that chromosome number is 2n=38;
3) plant that elected filial generation processing back is obtained carries out the dna fingerprint analysis, described method comprises utilization amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), restrictive fragment length polymerphism (RELP) method, according to the dna fingerprint of elected plant, in the filial generation of cabbage type rape and turnip type rape, select caryotype in conjunction with its parent's fingerprint and be mainly A
rA
rThe F4 strain system of CC;
4) with A
rA
rCC and Brassica napus male sterile line test cross are selected the hybrid combination of superpower advantage according to the performance of F1, when not containing in the selected strain system when recovering gene, adopt transgenic method to import TA29 or other male sterile gene.
Advantage of the present invention is:
1. solved the sterile problem of rape genome species hybrid, made its hybrid can be used for the production of rape F1 seed.
2. obtain the rape combination of superpower advantage.
Embodiment
Embodiment 1
1, interspecific cross:
Sweet white race intermolecular hybrid:
Make female parent with low erucic acid, low-sulfur glycosides (two low) cabbage type rape variety, make male parent with turnip type rape, the acquisition caryotype is A
rThe F1 species hybrid seed of AC.For example: with cabbage type rape strain 7S159 is female parent, and turnip type rape kind Zhejiang is for No. 1 male parent, castrates by female parent stripping flower bud, and sexual hybridization obtains the first generation of hybrid.It is tall and big that the first generation of hybrid shows as plant, and growth potential is strong, and full-bloom stage dry weight heterobeltiosis reaches 152.7%, and middle close advantage reaches 175%.
2, to the processing of filial generation:
Processing to sweet white hybrid generation
At sweet white hybrid (A
rAC) in the reduction division process, except that producing a large amount of non-multiple gametes, also will produce the A or the A of multiple
rGamete, and the AC or the A of multiple
rThe C gamete, therefore sweet white hybrid has certain fertility.Hybrid bagging and supplementary pollination with cabbage type rape and turnip type rape, produce selfed seed, produce F2 for plant through plantation, eliminate growth deformity and the not high plant of fertility, obtaining the F2 strain is seed, after the plant of each F3 strain system made agronomy and quality trait and select, remake chromosome examination, select the fine individual plant that chromosome number is 2n=38.
3, to genome analysis test when roguing system:
Carry out genomic fingerprinting to handling the plant that is obtained behind the filial generation.Described method comprises amplified fragment length polymorphism (AFLP), and simple sequence repeats (SSR), restriction fragment length polymorphism reliability height such as (RFLP), the dna fingerprint analytical method that stability is strong.And then according to the dna fingerprint of being elected to plant, selecting caryotype in conjunction with its parent's fingerprint in the filial generation of cabbage type rape and turnip type rape is A
rA
rThe F4 strain system of CC.
Described dna fingerprint analytical method concrete steps are:
The routine analyzer of AFLP: the method for Vos etc. (1995) is mainly adopted in the AFLP fingerprint analysis, and it has been carried out following correction: (1) adopts 250ng genomic DNA, 2.5U EcoRI, 2.5U MseI, 2.0 μ l double digestion buffer solution Y
+/ Tango
TM, add ultra-pure water and cut 3 hours to 37 ℃ of enzymes of 20 μ l; (2) after enzyme cuts, each reaction adds 5 μ l again and connects mixed liquor in 37 ℃ of connections 3 hours, this mixed liquor is made of following composition: EcoRI joint (5 μ M) 0.5 μ l, MseI joint (50 μ M) 0.5 μ l, T4DNA ligase 0.5U, 10x T4 dna ligase buffer solution 0.5 μ l transfers to 5 μ l to mixeding liquid volume with ultra-pure water; (3) pre-amplification adopts following system to carry out 10 * Taq dna polymerase buffer liquid, 2.0 μ l, MgCl
2(25mM) 1.5 μ l, dNTP (containing four kinds of each 2.0mM of deoxynucleotide of dATP dTTP dCTP dGTP) 2 μ l, band 1-base 3 '-the EcoRI primer 30ng of extending end, band 1-base 3 '-the MseI primer 30ng of extending end, Taq archaeal dna polymerase 0.5U, dilute 10 times enzyme and cut/connected nucleotide product 1 μ l, added ultra-pure water to 20 μ l.Pre-amplification PCR thermal cycle is: 1. 94 ℃ 2 minutes, 2. 94 ℃ 30 seconds, 3. 50 ℃ 30 seconds, 4. 72 ℃ 1 minute, 5. repeat 2. 6. the PCR thermal cycle to be stopped in 4 ℃ to 4. going on foot 34 times.(4) get 1 μ l after pre-expansion volume increase thing dilutes 10 times and carry out selective amplification again, and employing there is the primer of 3 '-extending end of 3 bases to control the dense degree of amplified band line.Its PCR thermal cycle is: 1. 94 ℃ 2 minutes, 2. 94 ℃ 30 seconds, 3. 65 ℃ 30 seconds, 4. 72 ℃ 1 minute, 5. repeat 2. to 4. going on foot 10 times, and the renaturation temperature of each circulation was all once being reduced on the basis of last circulation, make the renaturation temperature drop to 56 ℃ so always, keep 56 ℃ renaturation temperature to proceed 25 circulations then.6. at last the PCR thermal cycle is stopped in 4 ℃.(6) carry out polyacrylamide gel electrophoresis by the method in " molecular cloning " (second edition); (7) electrophoresis result adopts kit silver to dye demonstration, and method is seen silver-colored transfection reagent box specification.
The routine analyzer of SSR: SSRs (Simple Sequence Repeats, simple sequence repeats) is meant that the conserved sequence according to the simple repeated sequence two ends that have abundant variation in the eukaryotic gene group designs the polymorphism band line that the PCR primer amplification comes out.Our used primer is mainly synthetic according to report in 1996 such as Szewc-McFadden.The PCR mixed liquor is: dna profiling 10ng, each 15pmol of left and right sides primer, each 3.75 μ mol of four kinds of deoxynucleotides of dATP dTTP dCTP dGTP, MgCl
2(25mM) 1.875 μ l, 1.0U Taq enzyme, 10 * Taq dna polymerase buffer liquid, 1.5 μ l, 15 μ l reaction systems.The PCR thermal cycle adopts following system to carry out: 94 ℃ of sex change 2 minutes, then be the continuous alternating temperature pcr amplification that reduces of stringency of 20 circulations: 94 ℃ of sex change in 60 seconds, 30 seconds renaturation of alternating temperature, 72 ℃ were extended 45 seconds, the renaturation temperature is from 68 ℃ to 58 ℃, per 2 circulations reduce by 1 ℃, keep 58 ℃ renaturation temperature to increase 25 then again and circulate last 4 ℃ of storages.Pcr amplification product carries out polyacrylamide gel electrophoresis by the method in " molecular cloning " (second edition) and kit silver dyes the demonstration polymorphism.
The routine analyzer of RFLP: the RFLP fingerprint analysis is mainly undertaken by the method for " molecular cloning " (second edition), and it has been carried out a small amount of correction: (1) is got the first-class genomic DNA of 15 μ g quality and is carried out enzyme and cut, and 0.4N NaOH changes film; (2) 1-4 opens 10 * 20cm
2Film was got the 30ml prehybridization solution in 65 ℃ of prehybridization 8-10 hours; (the 30ml prehybridization solution comprises: dextran sulfate (Dextran sulfate) 0.3g is in 65 ℃ of dissolvings of 17.7ml ultra-pure water, 20 * SETS 6ml, 50 * Denharts 6ml, 10% SDS0.3ml, the salmon sperm dna 3mg of sex change fracture.) (3) 5-10ml hybridization solution in 65 ℃ hybridization 12 hours; (the 5ml hybridization solution comprises dextran sulfate 0.5g in 65 ℃ of dissolvings of 2.92ml ultra-pure water, 20 * SETS 1ml, and 50 * Denharts 1ml, 10%SDS0.05ml, the salmon sperm dna 0.1mg of sex change fracture, [α-
32P] the probe 40 μ l of dCTP mark; (4) probe mark adopted following system normal temperature mark 5 hours: dna probe template 4 μ l (about 400ng), 6 base random primers, 3 μ l (150ng), dna molecular amount mark 2 μ l (20ng), ultra-pure water 19 μ l, more than mixed the back boiling water bath 3 minutes, dithiothreitol (DTT) (DTT) 2ul (40mmol) is continued to add in the cooling back in frozen water, dATP, dGTP and dTTP mixed liquor 2 μ l (each 10mmol), 10 * Klenow polymerase buffer 4ul, the big fragment of dna polymerase i (Klenow fragment) 2ul, import [α-
32P] dCTP2ul (20 μ Ci).(5) washing film and compressing tablet is undertaken by the method for telling about in " molecular cloning " (second edition).
Adopt above various molecular labelings, the present invention is in turnip type rape or cabbage type rape inside and all detected abundant polymorphism between them, wherein more be no lack of the peculiar mark of turnip type rape or cabbage type rape,, now illustrate for assisted Selection provides bulk information.
Turnip type rape is peculiar to be marked with: PN138H15, PN67H9, PA14H3.4, PN54B6.5......
Cabbage type rape is peculiar to be marked with: PN25H2.2, PN25H3.3, PN31H3.3, PN159H10.....
4, coordinate force test
With A
rA
rCC and cabbage type rape male sterile line test cross are selected the hybrid combination of superpower advantage according to the performance of F1.When not containing the recovery gene in the roguing system of institute, adopt transgenic method to import TA29 or other male sterile gene.
Claims (2)
1, a kind of method of utilizing rape heterosis is characterized in that, utilizes rape genome hybrid vigour, by interspecific cross and molecular marker assisted selection, with the A of turnip type rape
rThe C genome of genome and cabbage type rape combines, and cultivating caryotype is A
rA
rThe cabbage type rape strain of CC, and then the hybrid combination of assembly cabbage type rape.
2, the method for utilizing rape heterosis according to claim 1 is characterized in that,
1) make female parent with low erucic acid, low-sulfur glycosides cabbage type rape variety, make male parent with turnip type rape, the acquisition caryotype is A
rThe F1 species hybrid seed of AC;
2) with the hybrid bagging and the supplementary pollination of cabbage type rape and turnip type rape, produce selfed seed, produce F2 for plant through plantation, eliminate growth deformity and the not high plant of fertility, obtaining the F3 strain is after plant does agronomy and quality selection, remake chromosome examination, select the fine individual plant that chromosome number is 2n=38;
3) plant that elected filial generation processing back is obtained carries out the dna fingerprint analysis, the utilization amplified fragment length polymorphism, simple sequence repeats, the restrictive fragment length polymerphism method, according to the dna fingerprint of elected plant, in the filial generation of cabbage type rape and turnip type rape, select caryotype in conjunction with its parent's fingerprint and be mainly A
rA
rThe F4 strain system of CC;
4) with A
rA
rCC and Brassica napus male sterile line test cross are selected hybrid combination according to the performance of F1, do not contain in selected strain is when recovering gene, adopt transgenic method to import TA29 or other male sterile gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991201272A CN1167328C (en) | 1999-12-15 | 1999-12-15 | Method for utilizing cole intergenomic hybrid vigor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991201272A CN1167328C (en) | 1999-12-15 | 1999-12-15 | Method for utilizing cole intergenomic hybrid vigor |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2003101001412A Division CN1533692A (en) | 1999-12-15 | 1999-12-15 | Method of utilizing hybridization superiority between rape A and C subgene group |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1303588A CN1303588A (en) | 2001-07-18 |
| CN1167328C true CN1167328C (en) | 2004-09-22 |
Family
ID=5281386
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB991201272A Expired - Fee Related CN1167328C (en) | 1999-12-15 | 1999-12-15 | Method for utilizing cole intergenomic hybrid vigor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1167328C (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101191132B (en) * | 2006-11-20 | 2010-04-07 | 上海市农业科学院 | Molecule mark linked to cabbage green bolting gene and establishing method thereof |
| CN101250524B (en) * | 2008-04-07 | 2011-05-11 | 华中农业大学 | Molecule marker of brassica napus self-incompatible maintenance line as well as preparation and uses thereof |
| CN102499056B (en) * | 2011-10-25 | 2014-08-13 | 西北农林科技大学 | Method for breeding new line of Brassica napus by utilizing biotechnology |
| AU2013224677B2 (en) | 2012-09-13 | 2017-05-11 | Plant Bioscience Limited | Brassica oleracea plants with improved nutritional value |
| CN110499383B (en) * | 2019-09-02 | 2022-05-31 | 中国农业科学院蔬菜花卉研究所 | Molecular marker for identifying segregation condition of interspecific hybrids and progeny materials A02 and C02 chromosomes of Chinese cabbages and brassica carinata |
-
1999
- 1999-12-15 CN CNB991201272A patent/CN1167328C/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| CN1303588A (en) | 2001-07-18 |
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