CN104145807A - Method for obtaining genus hybrid of forsythia and abeliophyllum - Google Patents
Method for obtaining genus hybrid of forsythia and abeliophyllum Download PDFInfo
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- CN104145807A CN104145807A CN201410366170.1A CN201410366170A CN104145807A CN 104145807 A CN104145807 A CN 104145807A CN 201410366170 A CN201410366170 A CN 201410366170A CN 104145807 A CN104145807 A CN 104145807A
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Abstract
The invention discloses a method for obtaining genus hybrid of forsythia and abeliophyllum. The method comprises the following specific steps: (1) carrying out generic artificial hybridization by taking fructus forsythiae of the forsythia as a female parent and taking abeliophyllum distichum of the abeliophyllum as a male parent, and gathering the seeds obtained after the hybridization; and (2) planting the seeds to obtain filial generation materials, carrying out morphological observation statistics by referring to the male parent and the female parent, carrying out hybrid identification by using an AFLP (Amplified Fragment Length Polymorphism) molecular marker and selecting the genus hybrid of the forsythia and the abeliophyllum. According to the method, distant hybridization is carried out on fructus forsythiae of the forsythia and abeliophyllum distichum of the abeliophyllum, so that the generic gene exchange between the forsythia and the abeliophyllum is realized and a generic hybridization new germplasm is obtained. Based on the method, the genetic information of the forsythia plants can be enriched, the new variety cultivation of fructus forsythiae via generic hybridization is possible and the research of affinis genus genetic relationship can be also carried out.
Description
Technical field
The invention belongs to field of plant breeding, be specifically related to a kind of method that Forsythia Vahl and Abelia biflora leaf are bigenered that obtains.
Background technology
Forsythia Vahl (Forsythia) plant is and sees flower machaka early spring, first leaf or open with leaf, brightly yellowish look, mitriform, distyly, capsule (Chinese Plants will, 1992).
In 19th century, western countries introduce forsythia eastwardly, have carried out selecting widely breeding, cultivate a collection of kind, due to strong stress resistance and early spring flower dense, these kinds obtain extensively cultivation application (Rosati et al., 2007) in Temperate Region in China.Germany
the Natural hybrid gold clock capsule of weeping forsythia (Forsythia × intermedia) is found in botanical garden, becomes the main parent of modern capsule of weeping forsythia kind, and (Wyman, 1961) appear in new varieties in a large number.Capsule of weeping forsythia conventional hybridization and spontaneous mutation select breeding engineering mainly to concentrate on the U.S., Canada, Poland and the country such as German.Main target proterties concentrates on the aspects such as the improvement of cold resistance (using Forsythia ovata and the northeast capsule of weeping forsythia as breeding parent), plant type and flower portion proterties.
As ornamental plants, self also comes with some shortcomings forsythia, as: the florescence concentrates on April to May, and colony's florescence is only about 25 days; Pattern is single, and only having yellow is kind; Flower British plain spirits etc.In traditional Forsythia Vahl, crossbreeding cannot address these problems.Abelia biflora leaf belongs to the samara capsule of weeping forsythia (Abeliophyllum distichum) floral white or pink colour, light perfume (or spice) (Suk-Pyo Hong & Mae-Ja Han, 2002), different obviously with Flos Forsythiae aberration, relatively near with capsule of weeping forsythia affiliation.Adopt the samara capsule of weeping forsythia and the capsule of weeping forsythia to carry out intergeneric cross, be expected to enrich the proterties such as pattern, florescence of the capsule of weeping forsythia.Have great importance to inquiring into the capsule of weeping forsythia and relative genus distant hybridization compatibility, gene exchange mechanism simultaneously.
Summary of the invention
The present situation single for current forsythia pattern, the florescence is concentrated, the object of this invention is to provide a kind of method that Forsythia Vahl and Abelia biflora leaf are bigenered that obtains, and views and admires characteristic to improve forsythia.
A kind of method that Forsythia Vahl and Abelia biflora leaf are bigenered that obtains provided by the invention, concrete steps are as follows:
1) select the capsule of weeping forsythia of long pistillate Forsythia Vahl for maternal, the samara capsule of weeping forsythia that selects brachystylous Abelia biflora leaf to belong to is male parent, and artificial hybridization between belonging to, collects the seed after hybridization;
2) plantation seed obtains filial generation material, carries out morphological observation statistics, and adopt AFLP molecular labeling to carry out hybrid identification with reference to male parent and female parent, chooses Forsythia Vahl and Abelia biflora leaf is bigenered.
The samara capsule of weeping forsythia that Abelia biflora leaf belongs to is heterostyled, chooses brachystylous plant as female parent of the present invention.The capsule of weeping forsythia of same Forsythia Vahl is also heterostyled, chooses long pistillate plant as male parent of the present invention.
Wherein, step 1) artificial hybridization between described genus, comprise the steps:
A gathers the fresh flower powder that brachystylous Abelia biflora leaf belongs to the samara capsule of weeping forsythia;
B is by the emasculation of the long pistillate Forsythia Vahl capsule of weeping forsythia;
The pollen that c gathers step a is pollinated on the column cap of the step b Forsythia Vahl capsule of weeping forsythia and bagging;
After d seed development maturation, gather.
Wherein, the time of pollinating described in step c is morning when 11-12, every day 1 time, repeats 3 times.
Wherein, bagging described in step c, cover sulfuric acid paper bag after pollination, style changes mesh bag into after wilting.
Wherein, gather described in steps d, for plucking after seed development maturation, drying in the shade.The seed room temperature drying condition of gathering is preserved.
Wherein, step 2) described plantation seed, concrete steps are as follows:
Seed adopts after 100mg/L gibberellin seed soaking 6h, and point is sowed in the cave dish that peat is housed, and overlay film, is placed in greenhouse, after sprouting, obtains filial generation material.
Wherein, step 2) described in carry out morphological observation statistics, within continuous 3 years, carrying out Phenotypic Observation statistics.
Wherein, step 2) described in carry out morphological observation statistics, for the phenotype of stem, leaf, branch, flower.
Wherein, step 2) described employing AFLP molecular labeling carries out hybrid identification, and primer pair used is:
E38/M47
Upstream primer E38:5 '-gactgcgtaccaattcact-3 ' (as shown in SEQ ID No.1)
Downstream primer M47:5 '-gatgagtcctgagtaacaa-3 ' (as shown in SEQ ID No.4)
E38/M55
Upstream primer E38:5 '-gactgcgtaccaattcact-3 ' (as shown in SEQ ID No.1)
Downstream primer M55:5 '-gatgagtcctgagtaacga-3 ' (as shown in SEQ ID No.7)
E40/M52
Upstream primer E40:5 '-gactgcgtaccaattcagc-3 ' (as shown in SEQ ID No.2)
Downstream primer M52:5 '-gatgagtcctgagtaaccc-3 ' (as shown in SEQ ID No.6)
And/or E44/M49
Upstream primer E44:5 '-gactgcgtaccaattcatc-3 ' (as shown in SEQ ID No.3)
Downstream primer M49:5 '-gatgagtcctgagtaacag-3 ' (as shown in SEQ ID No.5).
Wherein, step 2) described in choose the standard that Forsythia Vahl and Abelia biflora leaf bigener and be: between Parent, and there is the new proterties that Parent does not have in morphologic phenotypic characteristic, has the peculiar AFLP band of Parent.
The present invention also provides the application in breeding of method that described acquisition Forsythia Vahl and Abelia biflora leaf bigener.
The present invention is also provided for the bigener primer pair of AFLP molecular markers for identification of Forsythia Vahl and Abelia biflora leaf, and it is:
E38/M47
Upstream primer E38:5 '-gactgcgtaccaattcact-3 ' (as shown in SEQ ID No.1)
Downstream primer M47:5 '-gatgagtcctgagtaacaa-3 ' (as shown in SEQ ID No.4)
E38/M55
Upstream primer E38:5 '-gactgcgtaccaattcact-3 ' (as shown in SEQ ID No.1)
Downstream primer M55:5 '-gatgagtcctgagtaacga-3 ' (as shown in SEQ ID No.7)
E40/M52
Upstream primer E40:5 '-gactgcgtaccaattcagc-3 ' (as shown in SEQ ID No.2)
Downstream primer M52:5 '-gatgagtcctgagtaaccc-3 ' (as shown in SEQ ID No.6)
And/or E44/M49
Upstream primer E44:5 '-gactgcgtaccaattcatc-3 ' (as shown in SEQ ID No.3)
Downstream primer M49:5 '-gatgagtcctgagtaacag-3 ' (as shown in SEQ ID No.5).
The method of utilizing hybridization pollination to obtain the capsule of weeping forsythia and samara Forsythia Vahl species hybrid provided by the invention, has the following advantages and beneficial effect:
(1) the present invention utilizes the Forsythia Vahl capsule of weeping forsythia and Abelia biflora leaf to belong to the samara capsule of weeping forsythia to carry out distant hybridization, realized Forsythia Vahl and Abelia biflora leaf belong between gene exchange, obtained intergeneric cross new germ plasm.
(2) on basis of the present invention, can enrich the hereditary information of forsythia, become possibility for utilizing intergeneric cross to carry out capsule of weeping forsythia rearing new variety, can carry out the research of relative genus affiliation simultaneously.
Brief description of the drawings
Fig. 1 be the embodiment of the present invention 1 capsule of weeping forsythia (left side), hybrid (in) with the contrast photo of the samara capsule of weeping forsythia (right side) current-year branch.
Fig. 2 be the embodiment of the present invention 1 capsule of weeping forsythia (left side), hybrid (in) with the contrast photo of the samara capsule of weeping forsythia (right side) blade.
Fig. 3 be the embodiment of the present invention 1 capsule of weeping forsythia (left side), hybrid (in) with the contrast photo of the biennial branch of the samara capsule of weeping forsythia (right side).
Fig. 4 is that the separation of primer E38/M47 in parent and filial generation detects (70-110bp).
Fig. 5 is that the separation of primer E38/M55 in parent and filial generation detects (80-130bp).
Fig. 6 is that the separation of primer E40/M52 in parent and filial generation detects (70-120bp).
Fig. 7 is that the separation of primer E44/M49 in parent and filial generation detects (45-80bp).
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Experimental technique in following embodiment, if no special instructions, is conventional method.
Embodiment 1
1, the capsule of weeping forsythia and Abelia biflora leaf belong to the acquisition of intergeneric cross progeny material
1.1 materials are selected
Select the capsule of weeping forsythia of Forsythia Vahl and the samara capsule of weeping forsythia that relative genus Abelia biflora leaf belongs to.
1.2 cross methods and hybrid obtain
Spring in 2011, in nursery, the national flowers little Tang of engineering center mountain, carry out the isolation of artificial emasculation bagging taking the long style capsule of weeping forsythia as female parent, gather the fresh pollen of the short style samara capsule of weeping forsythia, pollinate when 11-12 the morning, once a day, pollinate 3 times; After hybridization, after column cap is wilted, change mesh bag into.After seed physical maturity, gather, dry in the shade, under dry room temperature condition, preserve.
Seed is sowed at point after 100mg/L gibberellin aqueous solution seed soaking 6h in the cave dish that peat is housed, and overlay film, obtains intergeneric cross offspring plant after sprouting.
2, the qualification of the capsule of weeping forsythia and samara capsule of weeping forsythia hybrid
Phenotypic evaluation: will hybridize Parent and filial generation in open country field planting in July, and carry out conventional maintenance management.Spring in next year, Parent and offspring are carried out to morphology mensuration and statistics, carry out continuously 3 years.
The results are shown in Figure 1-3, show: the morphological features such as the plant type of filial generation, hat width, plant height, branch, leaf, between parents, have Parent Comprehensive Traits.The capsule of weeping forsythia (female parent): capsule of weeping forsythia growth potential is strong, annual shoot hollow, the long 5.4cm ± 0.01cm of internode, the long oval of blade, rough sawn tooth, many trilobated leaves, blade size 6.05cm × 4.04cm; The samara capsule of weeping forsythia (male parent): growth potential is weak, annual shoot marrow is solid, the long 3.42cm ± 0.02cm of internode, the wealthy oval of blade, Quan Yuan, without trilobated leaf, blade size 5.14cm × 3.56cm; Filial generation: growth potential is placed in the middle, annual shoot hollow, old branch tool sheet marrow, the long 3.46cm ± 0.01cm of annual shoot internode, blade oval, rough sawn tooth, many trilobated leaves, the big or small 5.36cm × 3.86cm of blade.
AFLP Molecular Identification: (primer sequence is referring to table 1 to have designed 4 couples of primer pair that polymorphism is good: E38/M47, E38/M55, E40/M52, E44/M49,2), parent and filial generation are carried out to selective amplification, and specific band is carried out to statistical analysis.
Table 1 aflp analysis joint used and primer sequence
| Joint and primer | Restriction endonuclease | Sequence (5 '-3 ') |
| Adaptor | EcoR?Ⅰ | 5’-GACTGCGTACC-3’ |
| ? | ? | 3’-CTGACGCATGGTTAA-5’ |
| ? | Mse?Ⅰ | 5’-GACGTAGAGTCCTGAG-3’ |
| ? | ? | 3’-TGCTACTCAGGACTCAT-5’ |
| Primer | EcoR?Ⅰ-00 | 5’-AGACTGCGTACCAATT-3’ |
| ? | Mse?Ⅰ-00 | 5’-GACGATGAGTCCTGAGTAA-3’ |
| ? | EcoR?Ⅰ+*** | 5’-AGACTGCGTACCAATT+***-3’ |
| ? | Mse?Ⅰ+*** | 5’-GACGATGAGTCCTGAGTAA+***-3’ |
Table 2 AFLP primer sequence used
| Primer numbering | Primer sequence (5 '-3 ') | Corresponding sequence table numbering |
| E38 | GAC?TGC?GTA?CCA?ATT?C?ACT | SEQ?ID?No.1 |
| E40 | GAC?TGC?GTA?CCA?ATT?C?AGC | SEQ?ID?No.2 |
| E44 | GAC?TGC?GTA?CCA?ATT?C?ATC | SEQ?ID?No.3 |
| M47 | GAT?GAG?TCC?TGA?GTA?A?CAA | SEQ?ID?No.4 |
| M49 | GAT?GAG?TCC?TGA?GTA?A?CAG | SEQ?ID?No.5 |
| M52 | GAT?GAG?TCC?TGA?GTA?A?CCC | SEQ?ID?No.6 |
| M55 | GAT?GAG?TCC?TGA?GTA?A?CGA | SEQ?ID?No.7 |
Found that, in the AFLP mark band of filial generation, occurred father, maternal peculiar mark, also occur the band (referring to table 3, Fig. 4-Fig. 7) that Parent does not have simultaneously.
The AFLP fingerprint strip analysis of table 3 capsule of weeping forsythia and samara capsule of weeping forsythia filial generation
Phenotypic character and AFLP molecular labeling result all judge that filial generation is as true hybrid, not only heredity parent's feature, also produced variation.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. obtain the method that Forsythia Vahl and Abelia biflora leaf are bigenered, it is characterized in that, concrete steps are as follows:
1) select the capsule of weeping forsythia of long pistillate Forsythia Vahl for maternal, the samara capsule of weeping forsythia that selects brachystylous Abelia biflora leaf to belong to is male parent, and artificial hybridization between belonging to, collects the seed after hybridization;
2) plantation seed obtains filial generation material, carries out morphological observation statistics, and adopt AFLP molecular labeling to carry out hybrid identification with reference to male parent and female parent, chooses Forsythia Vahl and Abelia biflora leaf is bigenered.
2. the method for claim 1, is characterized in that step 1) artificial hybridization between described genus, comprise the steps:
A gathers the fresh flower powder that brachystylous Abelia biflora leaf belongs to the samara capsule of weeping forsythia;
B is by the emasculation of the long pistillate Forsythia Vahl capsule of weeping forsythia;
The pollen that c gathers step a is pollinated on the column cap of the step b Forsythia Vahl capsule of weeping forsythia and bagging;
After d seed development maturation, gather.
3. the method for claim 1, is characterized in that, the time of pollinating described in step c is morning when 11-12, every day 1 time, repeats 3 times.
4. the method for claim 1, is characterized in that, bagging described in step c, and cover sulfuric acid paper bag after pollination, style changes mesh bag into after wilting.
5. the method for claim 1, is characterized in that, gathers described in steps d, for plucking after seed development maturation, drying in the shade.
6. the method for claim 1, is characterized in that step 2) described plantation seed, concrete steps are as follows:
Seed adopts after 100mg/L gibberellin seed soaking 6h, and point is sowed in the cave dish that peat is housed, and overlay film, is placed in greenhouse, after sprouting, obtains filial generation material.
7. the method for claim 1, is characterized in that, wherein, step 2) described in carry out morphological observation statistics, for the phenotype of stem, leaf, branch, flower.
8. the method for claim 1, is characterized in that step 2) described employing AFLP molecular labeling carries out hybrid identification, and primer pair used is:
E38/M47
Upstream primer E38:5 '-gactgcgtaccaattcact-3 ' (as shown in SEQ ID No.1)
Downstream primer M47:5 '-gatgagtcctgagtaacaa-3 ' (as shown in SEQ ID No.4)
E38/M55
Upstream primer E38:5 '-gactgcgtaccaattcact-3 ' (as shown in SEQ ID No.1)
Downstream primer M55:5 '-gatgagtcctgagtaacga-3 ' (as shown in SEQ ID No.7)
E40/M52
Upstream primer E40:5 '-gactgcgtaccaattcagc-3 ' (as shown in SEQ ID No.2)
Downstream primer M52:5 '-gatgagtcctgagtaaccc-3 ' (as shown in SEQ ID No.6)
And/or E44/M49
Upstream primer E44:5 '-gactgcgtaccaattcatc-3 ' (as shown in SEQ ID No.3)
Downstream primer M49:5 '-gatgagtcctgagtaacag-3 ' (as shown in SEQ ID No.5).
9. described in claim 1-8, obtain the application in breeding of method that Forsythia Vahl and Abelia biflora leaf bigener.
10. for the bigener primer pair of AFLP molecular markers for identification of Forsythia Vahl and Abelia biflora leaf, it is:
E38/M47
Upstream primer E38:5 '-gactgcgtaccaattcact-3 ' (as shown in SEQ ID No.1)
Downstream primer M47:5 '-gatgagtcctgagtaacaa-3 ' (as shown in SEQ ID No.4)
E38/M55
Upstream primer E38:5 '-gactgcgtaccaattcact-3 ' (as shown in SEQ ID No.1)
Downstream primer M55:5 '-gatgagtcctgagtaacga-3 ' (as shown in SEQ ID No.7)
E40/M52
Upstream primer E40:5 '-gactgcgtaccaattcagc-3 ' (as shown in SEQ ID No.2)
Downstream primer M52:5 '-gatgagtcctgagtaaccc-3 ' (as shown in SEQ ID No.6)
And/or E44/M49
Upstream primer E44:5 '-gactgcgtaccaattcatc-3 ' (as shown in SEQ ID No.3)
Downstream primer M49:5 '-gatgagtcctgagtaacag-3 ' (as shown in SEQ ID No.5).
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Cited By (1)
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| CN112946113A (en) * | 2021-02-01 | 2021-06-11 | 湖北医药学院 | Method for identifying genuine medicinal material Shiweir fructus forsythiae basal source |
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| CN101914525A (en) * | 2010-08-03 | 2010-12-15 | 河南中医学院 | Method for extracting DNA from weeping forsythia leaves |
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| EP2041289B1 (en) * | 2006-07-07 | 2010-11-17 | Enza Zaden Beheer B.V. | Resistance to powdery mildew and absence of necrosis in cucumis sativus |
| CN101914525A (en) * | 2010-08-03 | 2010-12-15 | 河南中医学院 | Method for extracting DNA from weeping forsythia leaves |
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| CN112946113A (en) * | 2021-02-01 | 2021-06-11 | 湖北医药学院 | Method for identifying genuine medicinal material Shiweir fructus forsythiae basal source |
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