CN110178720A - A method of being quickly obtained and identify Maloideae distant hybrids - Google Patents
A method of being quickly obtained and identify Maloideae distant hybrids Download PDFInfo
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- CN110178720A CN110178720A CN201910410587.6A CN201910410587A CN110178720A CN 110178720 A CN110178720 A CN 110178720A CN 201910410587 A CN201910410587 A CN 201910410587A CN 110178720 A CN110178720 A CN 110178720A
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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
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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Abstract
The present invention relates to a kind of methods for being quickly obtained and identifying Maloideae distant hybrids.To grow fine, the healthy and strong apple tree of development is maternal plant, it pollinates using the good pear tree of pollen activity as paternal plant, the distant hybridization positive seedling of apple and pears is obtained by paternal pollen acquisition, vigor identification, artificial hybridization pollination, hybrid seed lamination and hybrid seedling management, the flow high efficiency that leaf DNA is extracted and specific primer PCR is identified.Wherein, obtaining after cenospecies efficient nursery strategy and carrying out specific primer PCR screening according to S-RNase sequence difference on apple, pears S locus is the effective means for improving distant hybridization positive seedling and obtaining efficiency.The present invention can be relatively simple acquisition with apple be maternal distant hybridization positive seedling, provide effective means for initiative apple new species, transformation past heritage kind, and reform to obtain with new genetic stocks for Apple breeding mode and thinking and solution are provided.
Description
Technical field
The present invention relates to plant biotechnology fields, and specifically one kind is quickly obtained and identifies Maloideae distant hybridization
The method of kind.It is espespecially a kind of efficiently to obtain and identify Maloideae distant hybrids, and stalwartness is efficiently obtained by quickly breeding
The method of seedling.
Background technique
China is apple cultivation big country, and area and yield occupy first place in the world, but Main Cultivars are still with " Fuji " apple
Based on fruit, with the continuous improvement of China's Living consumption, wanted to meet people to tasting and consuming variety classes apple
It asks, also for the plantation efficiency for improving apple and reduces management cost, the work of breeding Variety of Apple is always without any confusion
Progress.However, tradition crossbreeding means carry out Variety of Apple breeding by way of over time gradually into
Enter bottleneck, it is especially urgent to develop new breed breeding means.
At present in addition to crossbreeding, Variety of Apple selection also includes genetic transformation breeding, ploidy breeding etc.,
In, the former is also difficult to be received by general public since Agrobacterium-mediated Genetic Transformation of Apple is more difficult, and at present, and the latter's breeding efficiency it is low and
It is unobvious to the change of Apple character, it is still being difficult at this stage as main breeding mode.At present studies have found that, parent
Mutually pollination is that have the ability for generating filial generation between the closer different plant species of edge relationship, the breeding plan based on the exploitation of this phenomenon
Slightly applied in Chinese cabbage etc. crops, but it is this improvement past heritage kind, create new species mode in fruit tree simultaneously
Do not carry out.The present invention will be mainly male parent to Apple Pollination with the pears of apple affiliation relative close, it is desirable to provide a kind of
Apple new species based on distant hybridization technology are quickly formulated and identification technology system, provide help for Apple breeding and research.
Summary of the invention
In view of the deficiencies in the prior art, apple Asia is quickly obtained and identified the purpose of the present invention is to provide one kind
The method of section's distant hybrids.Using the acquisition that the method for the invention can be relatively simple with apple is maternal distant hybridization
Positive seedling provides effective means for initiative apple new species, transformation past heritage kind, and reform for Apple breeding mode and
New genetic stocks, which obtains, provides thinking and solution.
To achieve the above objectives, the technical solution adopted by the present invention is that:
A method of being quickly obtained and identify Maloideae distant hybrids, specifically includes the following steps:
Step 1, paternal pollen acquires；
In the big flower bud phase of male parent flower, anther is gently pushed smooth paper with tweezers by the flower of acquisition hybridization ordinal number 2 times or more
On, choose sundries, is placed on shady and cool dry place；After anther dehiscence, the pollen in anther is packed into bottle, pollen on label
Title is placed in spare in drier；The male parent is the pears with apple affiliation relative close；
Step 2, pollen activity is identified；
The pollen acquired in step 1 is equably sowed at 10% liquid glucose media surface, greenhouse sealing marks；It puts
It is checked after hour for 24 hours in 15-25 DEG C of incubator, three visuals field of every observation, pollen grain number is no less than 100, calculates flower
Powder germination rate, pollen germination rate should keep 80% or more；
Step 3, artificial hybridization pollination and seedling fast breeding；
Selection develops normal, robust growth, no disease and pests harm and the plant with kind characteristic feature as maternal plant；Flower
Piece choosing growth conditions is the flower that petal will protrude sepal package, carefully pushes petal emasculation aside with tweezers, tweezers when emasculation
Filigree is gently clamped, anther is all removed, but do not injure style, pollen is dipped on column cap with rubber head immediately and applies repeatedly
It smears；Isolation paper bag is put on after pollination immediately, is marked, extracts paper bag after petal is completely fallen off after a week, investigates fruit-setting rate；
Reinforce allocarpy field management, prevent disease pest from endangering, avoid damaging, harvesting takes kind after fruit maturation；Pass through layer
Product completes the After-ripening of embryo and breaking dormancy promotes to sprout；It is packed into after cenospecies and wet sand are puddled uniformly by a certain percentage husky
Mesh bag, it is overwintering to be embedded in outdoor shady spot, keeps ventilation；The vernalization when Stratification time was up to 80 days or more, room temperature are controlled at 10 DEG C -15
DEG C, humidity 60%-80% avoids direct sunlight, when embryo, which shows money or valuables one carries unintentionally, reaches 85%, sowing in time；Plumule is sprouted and is showed money or valuables one carries unintentionally
Seed, plumule is placed in downward in nursery plug, and nursery plug is put into hole tray, covers the fine sand of 2cm thickness, sprinkles profoundly water, every two
It checks that once timely moisturizing, weeding are cultivated in cold canopy；When 5 true leaves of seed germination and growth, take out with nursery plug
Seed seedling is transplanted in the temporary planting garden of field and cultivates, prevents and treats pest and disease damage；
Step 4, the positive plant identification based on S genotype；
There is the S-RNase base singly copied on self-incompatible multiple equipotential S locus due to controlling in apple and pears
Cause, gene order have differences in apple and pears, therefore whether there is or not i.e. for the identification distinctive S-RNase sequence of pears in seed seedling
It can efficiently identify and obtain positive distant hybridization seedling.
Based on the above technical solution, in step 3, the every sequence of the flower of the selection only stays 2 flowers, and flower chooses life
Long status is that petal will protrude sepal package.
Based on the above technical solution, in step 3, the moisture condition of the wet sand are as follows: holding is agglomerating, and a touching is
It dissipates.
Based on the above technical solution, in step 3, the Mixed sample Proportion of the wet sand and cenospecies is 3~5:1.
Based on the above technical solution, in step 4, in seed seedling identify the distinctive S-RNase sequence of pears whether there is or not
It can efficiently identify that the method for obtaining positive distant hybridization seedling is as follows: seed seedling young leaflet tablet be won, using CTAB method
Leaf DNA is extracted, according to primer shown in the S-RNase sequence design sequence table 1,2,3,4 that the pears as male parent include, primer
Specific requirements avoid the homologous sequence with apple S-RNase, and PCR amplification is carried out in DNA, have had the filial generation of amplified band both
For the positive seedling of distant hybridization pollination.
The method for being quickly obtained and identifying Maloideae distant hybrids through the invention, can pass through Distant pollination
Mode overcome inter-species pollination compatibility difference phenomenon, efficiently obtain new rosaceae germ plasm resource, and according to S genotype identification
Method high frequency zone filial generation has great importance to research related with Apple breeding and practice.
Detailed description of the invention
The present invention has following attached drawing:
Apple seed photo after distant hybridization pollination in Fig. 1 (a) embodiment 1.
Apple seed photo after distant hybridization pollination in Fig. 1 (b) embodiment 2.
Apple seed photo after distant hybridization pollination in Fig. 1 (c) embodiment 3.
Apple seed photo after distant hybridization pollination in Fig. 1 (d) embodiment 4.
Agarose gel photograph one after specific primer PCR in Fig. 2 (a) embodiment 1.
Agarose gel photograph two after specific primer PCR in Fig. 2 (b) embodiment 1.
Agarose gel photograph one after specific primer PCR in Fig. 3 embodiment 3.
Agarose gel photograph two after specific primer PCR in Fig. 4 embodiment 3.
Agarose gel photograph one after specific primer PCR in Fig. 5 (a) embodiment 4.
Agarose gel photograph two after specific primer PCR in Fig. 5 (b) embodiment 4.
Fig. 6 (a) distant hybridization seedling photo one.
Fig. 6 (b) distant hybridization seedling photo two.
Fig. 6 (c) distant hybridization seedling photo three.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Following concentration of component are the final concentration of mentioned reagent unless otherwise specified.
Embodiment 1, with " pear " be male parent to " Jin Guan " apple petal pollinate when offspring acquisition and Molecular Identification
1, " pear " pollen collection
In April, 2017 spends big flower bud phase at " pear ", the flower of acquisition hybridization ordinal number 2 times or more, with tweezers by anther gently
It pushes on smooth paper, chooses sundries, be placed on shady and cool dry place.After anther dehiscence, pollen is packed into bottle, on label
Pollen title is placed in spare in drier.
2, pollen activity is identified
The pollen of acquisition is equably sowed at 10% liquid glucose media surface, greenhouse sealing marks.It is put in 15-25
It is checked after hour for 24 hours in DEG C incubator, three visuals field of every observation, pollen grain number is no less than 100, pollen germination rate, flower
Powder germination rate is 80.6%.
3, artificial hybridization pollination and seedling fast breeding
Selection develops normal, robust growth, no disease and pests harm and the plant " Jin Guan " with kind characteristic feature as female parent
Plant.The bud emasculation of large balloon phase is selected, for convenience of counting, every sequence only stays 2 flowers.Flower chooses growth conditions as petal
By the flower of prominent sepal package.Filigree is gently clamped with tweezers when emasculation, anther is all removed, but do not injure style, immediately
Pollen is dipped on column cap with rubber head to smear repeatedly.Isolation paper bag is put on after pollination immediately, is marked, works as petal after a week
Paper bag is extractd after completely falling off, investigates fruit-setting rate, and inflorescence fruit-setting rate is 85.56%, and flower fruit-setting rate is 62.22%.
Reinforce allocarpy field management, prevent disease pest from endangering, avoid damaging, harvesting takes kind after fruit maturation.Pass through layer
Product completes the After-ripening of embryo and breaking dormancy promotes to sprout.Cenospecies 120 are puddled uniformly rear loading sand net bag with wet sand, are buried
It is overwintering in outdoor shady spot, keep ventilation.Sand humidity is agglomerating to hold, and a touching, which dissipates, to be advisable, and is 3~5:1 with seed rate.
The vernalization when Stratification time was up to 80 days or more, room temperature are controlled at 10 DEG C -15 DEG C, and it is straight to avoid sunlight in 60%-80% for humid control
It penetrates, when embryo, which shows money or valuables one carries unintentionally, reaches 85%, sowing in time sows 113.Plumule is sprouted the seed to show money or valuables one carries unintentionally, plumule is placed downward
In nursery plug, nursery plug is put into hole tray, is covered the fine sand of 2cm thickness, is sprinkled profoundly water, and is checked within every two days once, timely moisturizing,
Weeding is cultivated in cold canopy.When 5 true leaves of seed germination and growth, the seed seedling with nursery plug is taken out, is transplanted to field temporary planting
It is cultivated in garden, prevents and treats pest and disease damage.
4,20 seed seedling young leaflet tablets are won and extract DNA, steps are as follows:
(1) 1g blade liquid nitrogen grinding is transferred in a 2.0mL centrifuge tube, 800 2 × CTAB of μ L of addition (2%CTAB,
10mM Tris-HCl, 1.4M NaCl, 1%PVP), slightly oscillation mixes, and is put in 20-40min in 65 DEG C of water-baths, during which jog is several
It is secondary.
(2) it is cooled to room temperature, isometric chloroform/isoamyl alcohol (volume ratio 24:1), room temperature, 12000rpm centrifugation is added
10min。
(3) supernatant is taken, isometric chloroform/isoamyl alcohol (volume ratio 24:1) is added, 12000rpm is centrifuged 10min.
(4) 2 times of volume dehydrated alcohols, -20 DEG C of precipitating 30min are added in supernatant.
(5) 12000rpm is centrifuged 10 minutes, abandons supernatant, and precipitating is washed twice with 75% ethyl alcohol, dries precipitating, is dissolved in appropriate double
It steams in water.
(6) if desired, add the ratio of 1 μ L RNA enzyme to carry out RNA in 100 μ L to handle, 37 DEG C 30 minutes.With CI extracting one
Time, then with ethanol precipitation, precipitating is dissolved in appropriate distilled water.
(7) 1% agarose gel electrophoresis detect integrality, and it is dense that ultraviolet specrophotometer surveys absorbance calculating DNA at 260nm
Degree.
5, " pear " S genotype is S21S34, and " Jin Guan " S genotype is S2S3, designs S21 nuclease and S34 core accordingly
The specific primer of sour enzyme is as follows:
| YL21 identifies F | GTGGCCTTCAAACAAAATAGGACGTG |
| YL21 identifies R | CTGTATGATT GGTTCGATCT AGGAC |
| YL34 identifies F | GGCCCTCAAACTGGAATGGATCCC |
| YL34 identifies R | ATCGTTTCGA TTGAGTACAT TCGGCC |
PCR reaction system: Prime STARTM HS archaeal dna polymerase (Takara company, DR010S), Takara LA
PCRTM in vitro Cloning Kit (Takara company, DRR015)
PCR response procedures are as follows:
94 DEG C of initial denaturation 3min；
94℃40s；
62℃30s；
72℃1min；
30 circulations.
72 DEG C of last extension 2min.
PCR product detection: according to big 1% Ago-Gel of little makings of target fragment, 0.1%TAE electrophoretic buffer, 70-
110v electrophoresis about 15min, Ethidum Eremide dye, and PCR product clip size is detected under ultraviolet lamp.Wherein, it is examined in offspring
It measures 3 seedlings and contains S21 genotype, 5 seedlings contain S34 genotype.
Embodiment 2 take " pear " as acquisition and Molecular Identification that male parent blooms offspring when flower is pollinated to " Jin Guan " apple
1, " pear " pollen collection
In April, 2017 spends big flower bud phase at " pear ", the flower of acquisition hybridization ordinal number 2 times or more, with tweezers by anther gently
It pushes on smooth paper, chooses sundries, be placed on shady and cool dry place.After anther dehiscence, pollen is packed into bottle, on label
Pollen title is placed in spare in drier.
2, pollen activity is identified
The pollen of acquisition is equably sowed at 10% liquid glucose media surface, greenhouse sealing marks.It is put in 15-25
It is checked after hour for 24 hours in DEG C incubator, three visuals field of every observation, pollen grain number is no less than 100 or more, calculates pollen and sprouts
Hair rate, pollen germination rate 80.6%.
3, artificial hybridization pollination and seedling fast breeding
Selection develops normal, robust growth, no disease and pests harm and the plant " Jin Guan " with kind characteristic feature as female parent
Plant.The bud emasculation of large balloon phase is selected, for convenience of counting, every sequence only stays 2 flowers.It pollinates when flowering.Emasculation
When with tweezers gently clamp filigree, anther is all removed, but do not injure style, dips pollen on column cap with rubber head immediately
It smears repeatedly.Isolation paper bag is put on after pollination immediately, is marked, extracts paper bag after petal is completely fallen off after a week, is investigated
Fruit-setting rate, inflorescence fruit-setting rate are 40.00%, and flower fruit-setting rate is 10.50%.
Reinforce allocarpy field management, prevent disease pest from endangering, avoid damaging, harvesting takes kind after fruit maturation.Pass through layer
Product completes the After-ripening of embryo and breaking dormancy promotes to sprout.Cenospecies 10 are puddled uniformly rear loading sand net bag with wet sand, are buried
It is overwintering in outdoor shady spot, keep ventilation.Sand humidity is agglomerating to hold, and a touching, which dissipates, to be advisable, and is 3~5:1 with seed rate.
The vernalization when Stratification time was up to 80 days or more, room temperature are controlled at 10 DEG C -15 DEG C, and it is straight to avoid sunlight in 60%-80% for humid control
It penetrates, when embryo, which shows money or valuables one carries unintentionally, reaches 85%, sowing in time sows 7.Plumule is sprouted the seed to show money or valuables one carries unintentionally, plumule is placed in downward
In nursery plug, nursery plug is put into hole tray, is covered the fine sand of 2cm thickness, is sprinkled profoundly water, and checks within every two days that once timely moisturizing removes
Grass is cultivated in cold canopy.When 5 true leaves of seed germination and growth, the seed seedling with nursery plug is taken out, is transplanted to field temporary planting garden
Interior cultivation prevents and treats pest and disease damage.
4,5 seed seedling young leaflet tablets are won and extract DNA, steps are as follows:
(1) 1g blade liquid nitrogen grinding is transferred in a 2.0mL centrifuge tube, 800 2 × CTAB of μ L of addition (2%CTAB,
10mM Tris-HCl, 1.4M NaCl, 1%PVP), slightly oscillation mixes, and is put in 20-40min in 65 DEG C of water-baths, during which jog is several
It is secondary.
(2) it is cooled to room temperature, isometric chloroform/isoamyl alcohol (volume ratio 24:1), room temperature 12000rpm centrifugation is added
10min。
(3) supernatant is taken, isometric chloroform/isoamyl alcohol (volume ratio 24:1) is added, 12000rpm is centrifuged 10min.
(4) 2 times of volume dehydrated alcohols, -20 DEG C of precipitating 30min are added in supernatant.
(5) 12000rpm is centrifuged 10 minutes, abandons supernatant, and precipitating is washed twice with 75% ethyl alcohol, dries precipitating, is dissolved in appropriate double
It steams in water.
(6) if desired, add the ratio of 1 μ L RNA enzyme to carry out RNA in 100 μ L to handle, 37 DEG C 30 minutes.With CI extracting one
Time, then with ethanol precipitation, precipitating is dissolved in appropriate distilled water.
(7) 1% agarose gel electrophoresis detect integrality, and it is dense that ultraviolet specrophotometer surveys absorbance calculating DNA at 260nm
Degree.
5, " pear " S genotype is S21S34, and " Jin Guan " S gene is S2S3, designs S21 nuclease and S34 nucleic acid accordingly
The specific primer of enzyme is as follows:
| YL21 identifies F | GTGGCCTTCAAACAAAATAGGACGTG |
| YL21 identifies R | CTGTATGATT GGTTCGATCT AGGAC |
| YL34 identifies F | GGCCCTCAAACTGGAATGGATCCC |
| YL34 identifies R | ATCGTTTCGA TTGAGTACAT TCGGCC |
PCR reaction system: Prime STARTM HS archaeal dna polymerase (Takara company, DR010S), Takara LA
PCRTM in vitro Cloning Kit (Takara company, DRR015)
PCR response procedures are as follows:
94 DEG C of initial denaturation 3min；
94℃40s；
62℃30s；
72℃1min；
30 circulations.
72 DEG C of last extension 2min.
PCR product detection: according to big 1% Ago-Gel of little makings of target fragment, 0.1%TAE electrophoretic buffer, 70-
110v electrophoresis about 15min, Ethidum Eremide dye, and PCR product clip size is detected under ultraviolet lamp.Wherein, it is examined in offspring
It measures 0 seedling and contains S21 genotype, 0 seedling contains S34 genotype.
Embodiment 3, be with " pear " offspring when male parent is pollinated to " cold richness " apple petal acquisition and Molecular Identification
1, " pear " pollen collection
In April, 2017 spends big flower bud phase at " pear ", the flower of acquisition hybridization ordinal number 2 times or more, with tweezers by anther gently
It pushes on smooth paper, chooses sundries, be placed on shady and cool dry place.After anther dehiscence, pollen is packed into bottle, on label
Pollen title is placed in spare in drier.
2, pollen activity is identified
The pollen of acquisition is equably sowed at 10% liquid glucose media surface, greenhouse sealing marks.It is put in 15-25
It is checked after hour for 24 hours in DEG C incubator, three visuals field of every observation, pollen grain number is no less than 100, calculates pollen germination
Rate, pollen germination rate 80.6%.
3, artificial hybridization pollination and seedling fast breeding
Selection develops normal, robust growth, no disease and pests harm and " cold richness " plant with kind characteristic feature as female parent
Plant.The bud emasculation of large balloon phase is selected, for convenience of counting, every sequence only stays 2 flowers.Flower chooses growth conditions as petal
By the flower of prominent sepal package.Filigree is gently clamped with tweezers when emasculation, anther is all removed, but do not injure style, immediately
Pollen is dipped on column cap with rubber head to smear repeatedly.Isolation paper bag is put on after pollination immediately, is marked, works as petal after a week
Paper bag is extractd after completely falling off, investigates fruit-setting rate, and inflorescence fruit-setting rate is 68.75%, and flower fruit-setting rate is 54.69%.
Reinforce allocarpy field management, prevent disease pest from endangering, avoid damaging, harvesting takes kind after fruit maturation.Pass through layer
Product completes the After-ripening of embryo and breaking dormancy promotes to sprout.Cenospecies 47 are puddled uniformly rear loading sand net bag with wet sand, are buried
It is overwintering in outdoor shady spot, keep ventilation.Sand humidity is agglomerating to hold, and a touching, which dissipates, to be advisable, and is 3~5:1 with seed rate.
The vernalization when Stratification time was up to 80 days or more, room temperature are controlled at 10 DEG C -15 DEG C, and it is straight to avoid sunlight in 60%-80% for humid control
It penetrates, when embryo, which shows money or valuables one carries unintentionally, reaches 85%, sowing in time sows 36.Plumule is sprouted the seed to show money or valuables one carries unintentionally, plumule is placed in downward
In nursery plug, nursery plug is put into hole tray, is covered the fine sand of 2cm thickness, is sprinkled profoundly water, and checks within every two days that once timely moisturizing removes
Grass is cultivated in cold canopy.When 5 true leaves of seed germination and growth, the seed seedling with nursery plug is taken out, is transplanted to field temporary planting garden
Interior cultivation prevents and treats pest and disease damage.
4,15 seed seedling young leaflet tablets are won and extract DNA, steps are as follows:
(1) 1g blade liquid nitrogen grinding is transferred in a 2.0mL centrifuge tube, 800 2 × CTAB of μ L of addition (2%CTAB,
10mM Tris-HCl, 1.4M NaCl, 1%PVP), slightly oscillation mixes, and is put in 20-40min in 65 DEG C of water-baths, during which jog is several
It is secondary.
(2) it is cooled to room temperature, isometric chloroform/isoamyl alcohol (volume ratio 24:1), room temperature 12000rpm centrifugation is added
10min。
(3) supernatant is taken, isometric chloroform/isoamyl alcohol (volume ratio 24:1) is added, 12000rpm is centrifuged 10min.
(4) 2 times of volume dehydrated alcohols, -20 DEG C of precipitating 30min are added in supernatant.
(5) 12000rpm is centrifuged 10 minutes, abandons supernatant, and precipitating is washed twice with 75% ethyl alcohol, dries precipitating, is dissolved in appropriate double
It steams in water.
(6) if desired, add the ratio of 1 μ L RNA enzyme to carry out RNA in 100 μ L to handle, 37 DEG C 30 minutes.With CI extracting one
Time, then with ethanol precipitation, precipitating is dissolved in appropriate distilled water.
(7) 1% agarose gel electrophoresis detect integrality, and it is dense that ultraviolet specrophotometer surveys absorbance calculating DNA at 260nm
Degree.
5, " pear " S genotype is S21S34, and " cold richness " S gene is S1S9, designs S21 nuclease and S34 nucleic acid accordingly
The specific primer of enzyme is as follows:
| YL21 identifies F | GTGGCCTTCAAACAAAATAGGACGTG |
| YL21 identifies R | CTGTATGATT GGTTCGATCT AGGAC |
| YL34 identifies F | GGCCCTCAAACTGGAATGGATCCC |
| YL34 identifies R | ATCGTTTCGA TTGAGTACAT TCGGCC |
PCR reaction system: Prime STARTM HS archaeal dna polymerase (Takara company, DR010S), Takara LA
PCRTM in vitro Cloning Kit (Takara company, DRR015)
PCR response procedures are as follows:
94 DEG C of initial denaturation 3min；
94℃40s；
62℃30s；
72℃1min；
30 circulations.
72 DEG C of last extension 2min.
PCR product detection: according to big 1% Ago-Gel of little makings of target fragment, 0.1%TAE electrophoretic buffer, 70-
110v electrophoresis about 15min, Ethidum Eremide dye, and PCR product clip size is detected under ultraviolet lamp.Wherein, it is examined in offspring
It measures 4 seedlings and contains S21 genotype, 5 seedlings contain S34 genotype.
Embodiment 4, with " pear " be male parent to " Wang Shanhong " apple petal pollinate when offspring acquisition and Molecular Identification
1, " pear " pollen collection
In April, 2017 spends big flower bud phase at " pear ", the flower of acquisition hybridization ordinal number 2 times or more, with tweezers by anther gently
It pushes on smooth paper, chooses sundries, be placed on shady and cool dry place.After anther dehiscence, pollen is packed into bottle, on label
Pollen title is placed in spare in drier.
2, pollen activity is identified
The pollen of acquisition is equably sowed at 10% liquid glucose media surface, greenhouse sealing marks.It is put in 15-25
It is checked after hour for 24 hours in DEG C incubator, three visuals field of every observation, pollen grain number is no less than 100, calculates pollen germination
Rate, pollen germination rate 80.6%.
3, artificial hybridization pollination and seedling fast breeding
Selection develops normal, robust growth, no disease and pests harm and " Wang Shanhong " plant with kind characteristic feature as mother
This plant.The bud emasculation of large balloon phase is selected, for convenience of counting, every sequence only stays 2 flowers.It is petal that flower, which chooses growth conditions,
The flower of sepal package will be protruded.Filigree is gently clamped with tweezers when emasculation, anther is all removed, but do not injure style, with
Pollen is dipped on column cap with rubber head to smear repeatedly.Isolation paper bag is put on after pollination immediately, is marked, works as flower after a week
Valve extracts paper bag after completely falling off, investigate fruit-setting rate, and inflorescence fruit-setting rate is 89.02%, and flower fruit-setting rate is 68.90%.
Reinforce allocarpy field management, prevent disease pest from endangering, avoid damaging, harvesting takes kind after fruit maturation.Pass through layer
Product completes the After-ripening of embryo and breaking dormancy promotes to sprout.Cenospecies 77 are puddled uniformly rear loading sand net bag with wet sand, are buried
It is overwintering in outdoor shady spot, keep ventilation.Sand humidity is agglomerating to hold, and a touching, which dissipates, to be advisable, and is 3~5:1 with seed rate.
The vernalization when Stratification time was up to 80 days or more, room temperature are controlled at 10 DEG C -15 DEG C, and it is straight to avoid sunlight in 60%-80% for humid control
It penetrates, when embryo, which shows money or valuables one carries unintentionally, reaches 85%, sowing in time sows 65.Plumule is sprouted the seed to show money or valuables one carries unintentionally, plumule is placed in downward
In nursery plug, nursery plug is put into hole tray, is covered the fine sand of 2cm thickness, is sprinkled profoundly water, and checks within every two days that once timely moisturizing removes
Grass is cultivated in cold canopy.When 5 true leaves of seed germination and growth, the seed seedling with nursery plug is taken out, is transplanted to field temporary planting garden
Interior cultivation prevents and treats pest and disease damage.
4,18 seed seedling young leaflet tablets are won and extract DNA, steps are as follows:
(1) 1g blade liquid nitrogen grinding is transferred in a 2.0mL centrifuge tube, 800 2 × CTAB of μ L of addition (2%CTAB,
10mM Tris-HCl, 1.4M NaCl, 1%PVP), slightly oscillation mixes, and is put in 20-40min in 65 DEG C of water-baths, during which jog is several
It is secondary.
(2) it is cooled to room temperature, isometric chloroform/isoamyl alcohol (volume ratio 24:1), room temperature 12000rpm centrifugation is added
10min。
(3) supernatant is taken, isometric chloroform/isoamyl alcohol (volume ratio 24:1) is added, 12000rpm is centrifuged 10min.
(4) 2 times of volume dehydrated alcohols, -20 DEG C of precipitating 30min are added in supernatant.
(5) 12000rpm is centrifuged 10 minutes, abandons supernatant, and precipitating is washed twice with 75% ethyl alcohol, dries precipitating, is dissolved in appropriate double
It steams in water.
(6) if desired, add the ratio of 1 μ L RNA enzyme to carry out RNA in 100 μ L to handle, 37 DEG C 30 minutes.With CI extracting one
Time, then with ethanol precipitation, precipitating is dissolved in appropriate distilled water.
(7) 1% agarose gel electrophoresis detect integrality, and it is dense that ultraviolet specrophotometer surveys absorbance calculating DNA at 260nm
Degree.
5, " pear " S genotype is S21S34, and " Wang Shanhong " S gene is S1S9, designs S21 nuclease and S34 core accordingly
The specific primer of sour enzyme is as follows:
| YL21 identifies F | GTGGCCTTCAAACAAAATAGGACGTG |
| YL21 identifies R | CTGTATGATT GGTTCGATCT AGGAC |
| YL34 identifies F | GGCCCTCAAACTGGAATGGATCCC |
| YL34 identifies R | ATCGTTTCGA TTGAGTACAT TCGGCC |
PCR reaction system: Prime STARTM HS archaeal dna polymerase (Takara company, DR010S), Takara LA
PCRTM in vitro Cloning Kit (Takara company, DRR015)
PCR response procedures are as follows:
94 DEG C of initial denaturation 3min；
94℃40s；
62℃30s；
72℃1min；
30 circulations.
72 DEG C of last extension 2min.
PCR product detection: according to big 1% Ago-Gel of little makings of target fragment, 0.1%TAE electrophoretic buffer, 70-
110v electrophoresis about 15min, Ethidum Eremide dye, and PCR product clip size is detected under ultraviolet lamp.Wherein, it is examined in offspring
It measures 3 seedlings and contains S21 genotype, 4 seedlings contain S34 genotype.
The content being not described in detail in this specification belongs to the prior art well known to professional and technical personnel in the field.
Sequence table
<110>China Agricultural University
<120>a kind of method for being quickly obtained and identifying Maloideae distant hybrids
<130> 1
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>artificial sequence ()
<400> 1
gtggccttca aacaaaatag gacgtg 26
<210> 2
<211> 25
<212> DNA
<213>artificial sequence ()
<400> 2
ctgtatgatt ggttcgatct aggac 25
<210> 3
<211> 24
<212> DNA
<213>artificial sequence ()
<400> 3
ggccctcaaa ctggaatgga tccc 24
<210> 4
<211> 26
<212> DNA
<213>artificial sequence ()
<400> 4
atcgtttcga ttgagtacat tcggcc 26
Claims (5)
1. a kind of method for being quickly obtained and identifying Maloideae distant hybrids, which is characterized in that specifically includes the following steps:
Step 1, paternal pollen acquires；
In the big flower bud phase of male parent flower, the flower of acquisition hybridization ordinal number 2 times or more is gently pushed anther on smooth paper with tweezers,
Choose sundries, is placed on shady and cool dry place；After anther dehiscence, the pollen in anther is packed into bottle, pollen name on label
Claim, is placed in spare in drier；The male parent is the pears with apple affiliation relative close；
Step 2, pollen activity is identified；
The pollen acquired in step 1 is equably sowed at 10% liquid glucose media surface, greenhouse sealing marks；It is put in 15-
It is checked after hour for 24 hours in 25 DEG C of incubators, three visuals field of every observation, pollen grain number is no less than 100, calculates pollen germination
Rate, pollen germination rate should keep 80% or more；
Step 3, artificial hybridization pollination and seedling fast breeding；
Selection develops normal, robust growth, no disease and pests harm and the plant with kind characteristic feature as maternal plant；Flower choosing
Taking growth conditions is the flower that petal will protrude sepal package, carefully pushes petal emasculation aside with tweezers, when emasculation gently with tweezers
Filigree is clamped, anther is all removed, but do not injure style, pollen is dipped on column cap with rubber head immediately and smears repeatedly；It awards
Isolation paper bag is put on after powder immediately, is marked, extracts paper bag after petal is completely fallen off after a week, investigates fruit-setting rate；
Reinforce allocarpy field management, prevent disease pest from endangering, avoid damaging, harvesting takes kind after fruit maturation；It is complete by lamination
Promote to sprout at the After-ripening and breaking dormancy of embryo；Husky net is packed into after cenospecies and wet sand are puddled uniformly by a certain percentage
Bag, it is overwintering to be embedded in outdoor shady spot, keeps ventilation；The vernalization when Stratification time was up to 80 days or more, room temperature are controlled at 10 DEG C -15
DEG C, humidity 60%-80% avoids direct sunlight, when embryo, which shows money or valuables one carries unintentionally, reaches 85%, sowing in time；Plumule is sprouted and is showed money or valuables one carries unintentionally
Seed, plumule is placed in downward in nursery plug, and nursery plug is put into hole tray, covers the fine sand of 2cm thickness, sprinkles profoundly water, every two
It checks that once timely moisturizing, weeding are cultivated in cold canopy；When 5 true leaves of seed germination and growth, take out with nursery plug
Seed seedling is transplanted in the temporary planting garden of field and cultivates, prevents and treats pest and disease damage；
Step 4, the positive plant identification based on S genotype；
There is the S-RNase gene singly copied on self-incompatible multiple equipotential S locus due to controlling in apple and pears,
Gene order has differences in apple and pears, therefore whether there is or not can be high for the identification distinctive S-RNase sequence of pears in seed seedling
Effect identification obtains positive distant hybridization seedling.
2. the method for being quickly obtained and identifying Maloideae distant hybrids as described in claim 1, which is characterized in that step
In 3, the every sequence of the flower of the selection only stays 2 flowers, and it is that petal will protrude sepal package that flower, which chooses growth conditions,.
3. the method for being quickly obtained and identifying Maloideae distant hybrids as described in claim 1, which is characterized in that step
In 3, the moisture condition of the wet sand are as follows: holding is agglomerating, and a touching dissipates.
4. the method for being quickly obtained and identifying Maloideae distant hybrids as claimed in claim 3, which is characterized in that step
In 3, the Mixed sample Proportion of the wet sand and cenospecies is 3~5:1.
5. the method for being quickly obtained and identifying Maloideae distant hybrids as described in claim 1, which is characterized in that step
In 4, identify that whether there is or not can efficiently identify to obtain positive distant hybridization seedling for the distinctive S-RNase sequence of pears in seed seedling
Method it is as follows: win seed seedling young leaflet tablet, using CTAB method extract leaf DNA, the S- for including according to the pears as male parent
Primer shown in RNase sequence design sequence table 1,2,3,4, primer specificity require to avoid the homologous sequence with apple S-RNase
Column, and PCR amplification is carried out in DNA, the filial generation for having amplified band had both been the positive seedling of distant hybridization pollination.
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| CN115369125A (en) * | 2022-09-28 | 2022-11-22 | 西藏自治区农牧科学院农业研究所 | Highland barley genetic transformation system and transgenic highland barley cultivation method |
| CN115369125B (en) * | 2022-09-28 | 2024-02-09 | 西藏自治区农牧科学院农业研究所 | Highland barley genetic transformation system and transgenic highland barley cultivation method |
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