CN1533692A - Method of utilizing hybridization superiority between rape A and C subgene group - Google Patents
Method of utilizing hybridization superiority between rape A and C subgene group Download PDFInfo
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- CN1533692A CN1533692A CNA2003101001412A CN200310100141A CN1533692A CN 1533692 A CN1533692 A CN 1533692A CN A2003101001412 A CNA2003101001412 A CN A2003101001412A CN 200310100141 A CN200310100141 A CN 200310100141A CN 1533692 A CN1533692 A CN 1533692A
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- rape
- type rape
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- brassicacarinata
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Abstract
A method for selectively culturing the new line of rape by use of the hybrid vigor between the sub-genomes of rape features that the genome Ar of Chinese cabbage-type rape and the genome Cc of Ethiopian mustard are combined together by interspecific hybridization and molecule label aided selection to obtain the new cabbage type rape whose kary-type is ArArCcCc.
Description
Technical field
The invention belongs to rape heterosis and utilize technical field, be specifically related to a kind of method of utilizing rape sub-gene group species hybrid advantage.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)) be 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.This course takes place of the remote past, 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, the brassicacarinata chromosome set is designated as B
cB
cC
cC
c
Rape species hybrid exists extremely strong hybrid vigour.Cabbage type rape and brassicacarinata (B
cB
cC
cC
c), also all show powerful hybrid vigour (Liu Houli, " heredity of rape and breeding ", Science and Technology of Shanghai publishing house, 1985 with the F1 hybrid of mustard type rape (AABB); Meng etc., Euphytica, 1998).Obviously, this sweet dust species hybrid advantage come from cabbage type rape A, C genome respectively with the B of dust mustard
cOr C
cMutual work between the sub-gene group.Because this species hybrid advantage of rape derives from the mutual work between whole genome or sub-gene group, is referred to as sub-gene group species hybrid advantage.
Because sweet dust hybrid is aneuploid, chromosome pairing is undesired, and hybrid dysgenesis or fertility are extremely low, so this sub-gene group species hybrid advantage 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 sub-gene of ripening rate group species hybrid advantage is provided, improve rape heterosis intensity, 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, this method comprises conventional hybridization breeding method and molecular biology breeding method, with the breeding method innovation of traditional species hybrid is the method for utilizing rape sub-gene group species hybrid advantage, by interspecific cross and molecular marker assisted selection, with the A of turnip type rape
rGenome and brassicacarinata C
cGenome combines, and cultivating caryotype is A
rA
rC
cC
cThe cabbage type rape excellent strain, and then the cabbage type rape hybrid combination of the superpower advantage of assembly.The value of these strains does not also lie in itself and may have certain hybrid vigour, and is that with they cabbage type rape combos with nature, can produce caryotype is A
rAC
cThe F1 hybrid of C.Owing to exist A in these hybrids
r/ A, A
r/ C and C
cDo mutually between mutual work of the genome between/C and sub-gene group, will produce superpower hybrid vigour.And on the other hand, these hybrids are again amphidiploids, and fertility is normal, thereby can be used in the production of rape seed.
The body embodiment
Embodiment 1
1, interspecific cross:
Make female parent with brassicacarinata, make male parent with turnip type rape, obtaining caryotype is A
rB
cC
cF1 species hybrid seed.But the cross compatibility of this combination is low, and ripening rate is different widely different because of making up.Being bordering on zero combination for ripening rate must adopt the embryo rescue method obtain the hybrid seedling.With the F1 hybrid seedling that 0.1% colchicine is handled brassicacarinata and turnip type rape, the acquisition caryotype is A
rA
rB
cB
cC
cC
cAllohexaploid.
2, to the processing of filial generation
With the allohexaploid hybridization of two low cabbage type rape varieties and brassicacarinata and turnip type rape hybridization, the generation caryotype is A
rA
rB
cC
cThe sesquialter pentaploid of C.In reduction division, the B in the sesquialter pentaploid
cGenome will be eliminated because of not matching, only A
r, A, C
C ', the genomic chromosome of C is delivered to F2 generation.F2 is for plant in plantation, checks the pollen mother cells behavior, selects reduction division and loose powder normal, and ripening rate height, agronomy and quality trait meet the breeding requirement and chromosome number is the individual plant of 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 brassicacarinata/turnip type rape and cabbage type rape filial generation is A
rA
rC
cC
cF3 strain system.
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, T4 dna 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 dTTPdCTP 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 (Dextransulfate) 0.3g is in 65 ℃ of dissolvings of 17.7ml ultra-pure water, 20 * SETS 6ml, 50 * Denharts 6ml, 10% SDS 0.3ml, 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% SDS 0.05ml, 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] dCTP 2ul (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 brassicacarinata inside and all detected abundant polymorphism between them, wherein more be no lack of the peculiar mark of turnip type rape or brassicacarinata,, now illustrate for assisted Selection provides bulk information.
Turnip type rape is peculiar to be marked with: PN138H15, PN67H9, PA14H3.4, PN54B6.5......
4, coordinate force test
Be mainly A with the genome composition
rA
rC
cC
cNew cabbage type rape and cabbage type rape male sterile line test cross, select 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.
Advantage of the present invention is:
1. solved the sterile problem of rape Intersubgenomic hybrid, made its hybrid can be used for the production of rape F1 seed.
2. obtain the rape combination of Heterobeltiosis.
Claims (6)
1, a kind of method of utilizing rape heterosis, comprise conventional hybridization breeding and molecular biology breeding method, it is characterized in that utilizing rape species hybrid use of advantage methodological innovation for utilizing the method for rape sub-gene group species hybrid advantage traditional, by interspecific cross and molecular marker assisted selection, with turnip type rape A
rGenome and brassicacarinata C
cGenome combines, and cultivating caryotype is A
rA
rC
cC
cThe cabbage type rape excellent strain, and then the cabbage type rape hybrid combination of the superpower advantage of assembly.
2, the method for utilizing rape heterosis according to claim 1 is characterized in that, makes female parent with brassicacarinata, makes male parent with turnip type rape, and obtaining caryotype is A
rB
cC
cF1 species hybrid seed.
3, the method for utilizing rape heterosis according to claim 2 is characterized in that, handles the seedling of the F1 hybrid of brassicacarinata and turnip type rape with the colchicine of 0.1% concentration, and the acquisition caryotype is A
rA
rB
cB
cC
cC
cAllohexaploid.
4, the method for utilizing rape heterosis according to claim 1 and 2 is characterized in that, with the allohexaploid hybridization of two low cabbage type rape varieties and brassicacarinata and turnip type rape hybridization, the production caryotype is A
rAB
cB
cCC
cThe sesquialter pentaploid, F2 is for plant in plantation, checks the pollen mother cells behavior, selects loose powder normal, the ripening rate height, agronomy and quality meet the individual plant of the chromosome number 2n=38 of breeding requirement.
5, the method for utilizing rape heterosis according to claim 1, it is characterized in that the plant that elected filial generation processing back is obtained is carried out the dna fingerprint analysis, described method comprises 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 brassicacarinata and turnip type rape, select caryotype in conjunction with its parent's fingerprint and be mainly A
rA
rC
cC
cF3 strain system.
6, utilize the method for rape heterosis according to claim 1 or 5, it is characterized in that, with A
rA
rC
cC
cWith cabbage type rape male sterile line test cross, select 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101001412A CN1533692A (en) | 1999-12-15 | 1999-12-15 | Method of utilizing hybridization superiority between rape A and C subgene group |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101001412A CN1533692A (en) | 1999-12-15 | 1999-12-15 | Method of utilizing hybridization superiority between rape A and C subgene group |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB991201272A Division CN1167328C (en) | 1999-12-15 | 1999-12-15 | Method for utilizing cole intergenomic hybrid vigor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1533692A true CN1533692A (en) | 2004-10-06 |
Family
ID=34303983
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2003101001412A Pending CN1533692A (en) | 1999-12-15 | 1999-12-15 | Method of utilizing hybridization superiority between rape A and C subgene group |
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Cited By (6)
| 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 |
| CN101822156A (en) * | 2010-03-16 | 2010-09-08 | 北京市农林科学院 | Method for quickly culturing homozygous transgenic ornamental collard with insect resistance and herbicide resistance |
| CN103477973A (en) * | 2013-09-22 | 2014-01-01 | 青海省农林科学院 | Method for creating anti-flea beetle brassia rapa type rape resource |
| CN110438254A (en) * | 2019-09-02 | 2019-11-12 | 中国农业科学院蔬菜花卉研究所 | Identify the molecular labeling and method of Vegetables in Brassica interspecific hybrid and progeny material A06 and C07 chromosome separation situation |
| CN110468228A (en) * | 2019-09-02 | 2019-11-19 | 中国农业科学院蔬菜花卉研究所 | Identify the molecular labeling of Chinese cabbage and brassicacarinata interspecific hybrid and progeny material A10 and C07 chromosome separation situation |
| CN110499383A (en) * | 2019-09-02 | 2019-11-26 | 中国农业科学院蔬菜花卉研究所 | Identify the molecular labeling of Chinese cabbage and brassicacarinata interspecific hybrid and progeny material A02 and C02 chromosome separation situation |
-
1999
- 1999-12-15 CN CNA2003101001412A patent/CN1533692A/en active Pending
Cited By (8)
| 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 |
| CN101822156A (en) * | 2010-03-16 | 2010-09-08 | 北京市农林科学院 | Method for quickly culturing homozygous transgenic ornamental collard with insect resistance and herbicide resistance |
| CN101822156B (en) * | 2010-03-16 | 2011-12-21 | 北京市农林科学院 | Method for quickly culturing homozygous transgenic ornamental collard with insect resistance and herbicide resistance |
| CN103477973A (en) * | 2013-09-22 | 2014-01-01 | 青海省农林科学院 | Method for creating anti-flea beetle brassia rapa type rape resource |
| CN103477973B (en) * | 2013-09-22 | 2015-04-22 | 青海省农林科学院 | Method for creating anti-flea beetle brassia rapa type rape resource |
| CN110438254A (en) * | 2019-09-02 | 2019-11-12 | 中国农业科学院蔬菜花卉研究所 | Identify the molecular labeling and method of Vegetables in Brassica interspecific hybrid and progeny material A06 and C07 chromosome separation situation |
| CN110468228A (en) * | 2019-09-02 | 2019-11-19 | 中国农业科学院蔬菜花卉研究所 | Identify the molecular labeling of Chinese cabbage and brassicacarinata interspecific hybrid and progeny material A10 and C07 chromosome separation situation |
| CN110499383A (en) * | 2019-09-02 | 2019-11-26 | 中国农业科学院蔬菜花卉研究所 | Identify the molecular labeling of Chinese cabbage and brassicacarinata interspecific hybrid and progeny material A02 and C02 chromosome separation situation |
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