CN104255433B - The initiative of a kind of allohexaploid Semen arachidis hypogaeae and authentication method - Google Patents
The initiative of a kind of allohexaploid Semen arachidis hypogaeae and authentication method Download PDFInfo
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Abstract
The present invention disclose and one cultivate peanut (Arachis hypogaeaL.) distant hybrid breeding method, the initiative of a kind of allohexaploid Semen arachidis hypogaeae and authentication method.First with cultigen (allotetraploid) for maternal, with the species hybrid F that wild species (diploid) are male parent1(triploid) tissue culture plant is material, utilizes the method for tissue culture to carry out chromosome doubling, cultivates allohexaploid peanut plant S0, and obtain its seed S1.Next utilizes codominant marker and genomic in situ hybridization technology from S1Middle initiative and identify the allohexaploid Semen arachidis hypogaeae that chromosome is 60.By the enforcement of above method, the most only Semen arachidis hypogaeae distant hybridization technology utilizes and provides effective chromosome doubling method, and obtains two allohexaploid Semen arachidis hypogaeae material E 4 and L 21, for breeding utilizationA. macedoiWithA. oteroiSpecific gene established material foundation.
Description
Technical field
The present invention relates to one cultivate peanut distant hybrid breeding method, the initiative of a kind of allohexaploid Semen arachidis hypogaeae and mirror
Determine method.
Background technology
Arachis includes that about 80 kinds, majority are annual or perennial diploid species.Based on morphology, hybridization parent
With property etc., Arachis is divided into 9 district's groups, 12 chromosome sets.These wildlife species be subject to various environment coerce and
Select, there is many favorable genes, and peanut cultivating variety (Arachis hypogaea L.) is allotetraploid, and most
Kind the most gradually loses under long-term domestication with cultivation or does not possess these favorable genes.Although China introduces and saves
Substantial amounts of wild-type peanut resource, but owing to wild-type peanut and cultigen Semen arachidis hypogaeae exist the most affine obstacle of hybridization in various degree, limit
Make the utilization of wild-type peanut resource.
Along with going deep into of Semen arachidis hypogaeae distant hybridization technical research, part Semen arachidis hypogaeae wild species the most successfully obtain with cultigen peanut hybridization
Obtain species hybrid F1, but Semen arachidis hypogaeae wild species and cultigen Semen arachidis hypogaeae species hybrid F1It mostly is triploid, the most sterile, it is impossible to sharp
With.Additionally, the crop distant hybridization breeding utilization practice such as Semen Tritici aestivi show, the intervarietal hybridization of incubation is wide in variety is containing progenitor species
Or the exogenous chromosome Small piece transposition of exogenic heredity resource or exogenous alleles gradually ooze.How Semen arachidis hypogaeae wild species there is niche
Because being transferred to cultivar, increase the genetic diversity of cultigen resource, have with cultivating new varieties formulating excellent new germ plasm
Significance.
To Semen arachidis hypogaeae wild species and cultigen species hybrid F1Triploid utilizes generally two kinds of approach: (1) triploid approach,
Triploid F1 under suitable natural conditions can solid without artificial treatment or directly with cultivation parents after
Again selfing obtain the available alien chromosome lines of breeding, this approach because of majority combination natural conditions under can not solid or obtain
Useful materials probability is low, applies less.(2) hexaploid approach, obtains six by triploid (3x) Hybrids F1 through colchicine treatment
Times body plant, then selfing or with cultivation parents after again selfing obtain the available alien chromosome lines of breeding, this approach
Both can the useful alien chromosome lines of autotelic acquisition, complete useful research material can be obtained again, such as a whole set of external source dyeing
Body adds system, substitution line etc..For development and utilization Semen arachidis hypogaeae Wild ornamental resources, applicant be engaged in for many years Semen arachidis hypogaeae wild resource and
The research of distant hybridization technology, establishes a set of effective chromosome doubling method, it is thus achieved that allohexaploid Semen arachidis hypogaeae.
Additionally, genomic in situ hybridization technology and molecular marking technique are species hybrid qualification and allohexaploid Semen arachidis hypogaeae
Acquisition provides technical foundation.Genomic in situ hybridization is the in situ hybridization making probe with complete genome DNA, educates in distant hybridization
In kind, with external source species full-length genome as probe, it can be used to detect exogenous chromosome or whether chromosome segment is included in
In detected species chromatin.Molecular marker is the heredity mark by between individuality based on hereditary material inner nucleotide sequence variations
Note, is the direct reflection of DNA level genetic polymorphism, can effectively identify that external source dyeing is added or penetrates into.
Summary of the invention
The technical problem to be solved in the present invention: overcome the defect in background technology, it is provided that a kind of allohexaploid Semen arachidis hypogaeae
Method for creating, utilizes method for tissue culture to formulate allohexaploid Semen arachidis hypogaeae；And utilize effective identification technology, identify allos
Hexaploid Semen arachidis hypogaeae, for shifting and utilizing the excellent genes of Semen arachidis hypogaeae wild species to provide technical guarantee.
Technical scheme:
The method for creating of a kind of allohexaploid Semen arachidis hypogaeae, spends 9331 for maternal, with diploid with tetraploid cultivar Henan
Wild species A.oteroi is that male parent hybridizes, and obtains triploid F through ovule and Hybrid embryo1, train with this triploid group
Seedling is material, carries out chromosome doubling, it is thus achieved that allohexaploid Semen arachidis hypogaeae under conditions of tissue culture, and concrete grammar is as follows:
(1) at F1Triploid tissue cultured seedling cuts from the 3rd leaf of top number, takes terminal bud part, is placed on solid training
Supporting in base, between 25 DEG C of constant temperature illumination cultivation, induced bud breaks up 14-16 days, obtains cultivating Seedling；
(2) transfer to by MS, 0.8 mg/L NAA and solid root media that 3% sucrose is component by cultivating Seedling, training
Support 15-30 days, until forming whole plant S0；
(3) by whole plant S0In being transplanted to experiment Tanaka；Continue to cultivate to observe and find that part side shoot Post flowering creates
Really pin, statistics has the pollen fertility of fruit pin side shoot and pin side shoot of having no result, and the pollen fertility that result shows fruit pin side shoot is the highest
In pin side shoot of having no result；Take the part branch having fruit pin side shoot that pollen fertility is high, with MS minimal medium and 0.8 mg/L NAA
The fluid medium formed carries out root culture, and then the tip of a root carries out cytological observation and chromosome counting, result display branch
Bar somatic chromosome is 60；Gather in the crops pod then；
(4) by the pod of gained with the sodium hypochlorite disinfection of 5%, take its seed, seed is inoculated into by 1/2
In the solid medium of MS and 5% sucrose composition, it is placed between 25 DEG C of constant temperature illumination cultivation cultivation, until developing into seedling S1；Described
The half that constituent content is MS in 1/2 MS culture medium.
Wherein the component of the solid medium in step (1) be MS, the NAA of 0.1 mg/L, the 6-BA of 0.4 mg/L, 3%
Sucrose and the Colchicine of 0.05%.
Described female parent is replaced with white sand 1016, and male parent replaces with A. macedoi, and other steps are constant.
A kind of authentication method of allohexaploid Semen arachidis hypogaeae, the seedling S that will obtain1Carry out genome breeding and divide
Sub-labeled analysis, specifically comprises the following steps that
A, respectively extract maternal Henan spend 9331, male parent wild species A. oteroi and S1The genomic DNA of plant；
B, take seedling S1The tip of a root, carry out film-making mitosis metaphase, then with the DNA of A. oteroi full-length genome be
Probe, to S1Carry out genome breeding, after having hybridized, drop to dyeing 3-4 min on slide with DAPI dye liquor, then
Take a picture under fluorescence microscope, it is thus achieved that hybridization picture, add up Chromosome number, it is thus achieved that chromosome is the allohexaploid Semen arachidis hypogaeae of 60
E-4；
C, further with Henan spend 9331, the allohexaploid of the codominant marker E420 of A. oteroi checking gained
The accuracy of Semen arachidis hypogaeae, with E420 Henan spent 9331, the DNA cloning slice result of A. oteroi and E-4 shows, E-4 wraps simultaneously
Contain Henan and spend the hereditary material of 9331 and A. oteroi.
Described molecular marker E420 includes SSR forward primer sequence and downstream primer sequence,
SSR primer upstream sequence is: TCCATCGTTAGTGGCACTGT；
SSR primer downstream sequence is: GTCGACTCCTGCCCAATCTA.
Described genome breeding is carried out by the following method: first preparing hybrid liquid, hybridization solution bag in centrifuge tube
Include deionized formamide analytical pure 7.5ml, 20 × SSC buffer 1.5 microlitre, dextran sulfate 2 microlitre of 50%, 10mg/ml
Salmon sperm dna 0.5 microlitre and biotin labeled A. oteroi DNA probe 2.5 microlitre, the white sand 1016 of 100 mg/ml
Blockade DNA 1 microlitre, at 103 DEG C of degeneration 13 min after hybridization solution mixing, then centrifuge tube is immediately placed on the wine of-20 DEG C of refrigerators
10-15min in essence；At film-making mitosis metaphase ,-70 DEG C of frost peels, after slide is dehydrated 6h in ethanol, it is placed on 70%
78 DEG C of degeneration 1 min 10 s of Methanamide, serial dehydration each 5 in the ethanol of 70%, 95%, 100% respectively at-20 DEG C afterwards
Min, dries up microscope slide；
Described hybridization solution is dripped on microscope slide, covered, hybridize 6-8 h in 37 DEG C；Respectively with 2 under the conditions of 42 DEG C
× SSC soaks 10 min, soaks 10 min with the Methanamide of 50%, soaks 10 min with 2 × SSC, the most at room temperature with 1 ×
TNT soaks 5 min；Film-making is drained, adds antibiotin FITC, put into room temperature in 37 DEG C of magazines and cultivate 30 min, with 1 ×
TNT solution room temperature washing film-making 3 times, each 5 min, finally film-making is dried up.
Described SSC buffer is by the trisodium citrate C of 0.3M6H5Na3O7.2H2The NaCl composition of O and 3M.
Described TNT solution is made up of the Tris-HCl of 0.1M, the NaCl of 0.15 M, the Tween-20 of 0.05%.
The positive beneficial effect of the present invention:
(1) the hexaploid Semen arachidis hypogaeae method for creating of the present invention, utilizes tissue culture technique, is being suitable to the culture medium of peanut growth
Middle addition Colchicine, successfully doubles into hexaploid Semen arachidis hypogaeae by species hybrid triploid, provides for Semen arachidis hypogaeae distant hybrid utilization
A kind of effective chromosome doubling method.
(2) present invention comprehensively utilize molecular marker and genome breeding technology successfully formulated out two different
Source hexaploid Semen arachidis hypogaeae, the desirable genes for breeding utilization wild species A. macedoi and A. oteroi has established material foundation.
(3) the allohexaploid Semen arachidis hypogaeae that the present invention obtains can be as the material of genetic breeding research, by returning with cultigen
Hand over, create chromosome addition system, substitution line or matter of evolving new breeds.
Accompanying drawing explanation
Fig. 1 aceto-camine detects plant S0There are fruit pin side shoot and the Pollen Activity figure of flower on pin side shoot of having no result.
Fig. 1 a: have the Pollen Activity of flower on fruit pin side shoot；Fig. 1 b: the Pollen Activity of flower on pin side shoot of having no result.Contrast
The Pollen Activity being found to have fruit pin branch flower is significantly higher；Fig. 1 c: have fruit pin branch to have silk to divide through cultivating produced root-tip cells
Split metaphase chromosome picture, result display 2n=60.
The fluorescence in situ hybridization qualification result of 9331, male parent A. oteroi and allohexaploid Semen arachidis hypogaeae is spent in Fig. 2 female parent Henan.
Fig. 2 a-2b: spend DAPI coloration result and 45S rDNA and the 5S rDNA probe in detecting result of 9331 for Henan, can see
Go out Henan spend 9331 be 42 chromosomes (actually 40, because of a pair macrosatellite chromosome of cultigen Semen arachidis hypogaeae, in cytology's film-making
Generally be drawn as thread due to secondary constriction, dyad shows as 4 on image, therefore is shown as 42)；
The DAPI coloration result of Fig. 2 c-2d:A. oteroi and 45S rDNA and 5S rDNA probe in detecting result, it can be seen that
A. oteroi has chromosome centromere band, has 22 chromosomes (to have 1 pair of macrosatellite chromosome, due to secondary in cytology's film-making
Contriction is generally drawn as thread, and item chromosome shows as 2 on image, actually 20)；
Fig. 2 e-2f:2e is DAPI coloration result and the A. oteroi whole genomic probe hybridization check result of E-4, can see
Go out E-4 be 64 chromosomes (comprise two pairs of macrosatellite chromosomes, actually 60) be hexaploid Semen arachidis hypogaeae, 2f green hybridization signal
For A. oteroi chromosome；
Fig. 3 specific mark E420 spends 9331, male parent A. oteroi and the amplification of allohexaploid E-4 in maternal Henan respectively
Result.
Can be seen that, maternal (Henan spends 9331) has specific band at about 450bp and 330bp, and male parent (A. oteroi) is about
There is specific band at 160bp, and E-4 all has specific band at 450bp, 330bp and 160bp, illustrate that E-4 is existing from female parent
Chromatin, have again the chromatin from male parent.
The tissue cultured seedling photo of allohexaploid Semen arachidis hypogaeae E-4 and L-21 of Fig. 4 present invention.
Detailed description of the invention
Following example are to further illustrate the present invention, are not offered as any limitation of the invention.If in literary composition
Being not particularly illustrated, percentage composition therein is weight percentage.
The method for creating of 1 one kinds of allohexaploid Semen arachidis hypogaeaes of example.
Material cultivar Henan flower 9331(allotetraploid 2n=4x=40) and wild species A. oteroi(diploid 2n=2x
=20) species hybrid F1, Economic Crops Research Inst., He'nan Prov. Agricultural Science Academy preserve.
Spend with cultigen Henan 9331 for maternal, hybridize for male parent with wild species A. oteroi, obtain F1Triploid
(2n=3x=30), with this triploid as material, the method for tissue culture is utilized to carry out chromosome doubling, it is thus achieved that allos six times
Body Semen arachidis hypogaeae, concrete grammar is as follows:
(1) at F1Triploid tissue cultured seedling cuts from the 3rd leaf of top number, takes terminal bud part, be placed on MS, 0.1
In mg/L NAA, 0.4 mg/L 6-BA, 3% sucrose and solid medium that 0.05% Colchicine is component, at 25 DEG C of constant temperature light
Break up 14-16 days according to induced bud between cultivating, obtain cultivating Seedling；
(2) transfer to by MS, 0.8 mg/L NAA and solid root media that 3% sucrose is component by cultivating Seedling, training
Support 15-30 days, until forming whole plant S0；
(3) the whole plant S that will be formed0It was transplanted to experiment field, academy of agricultural sciences of Henan Province to May 15 May 1 then,
Transplant 6 strains, survive 5 strains；August 1 then to August 20 it has been observed that the part side shoot Post flowering of wherein 1 strain creates
Really pin, statistics has fruit pin side shoot and pin side shoot pollen fertility of having no result；
Method is to choose S0The health having fruit pin branch and pin branch of having no result works as Radix Trichosanthis, is placed on microscope slide by pollen,
Add the aceto-camine dyeing of one 1%, covered in a moment, be placed in basis of microscopic observation, add up 5 area of visual field, every kind
Two flowers of Material Takeoff.It is generally believed that regular shape is circular, it is good to dye, it is pollen vibrant, that can educate.Result shows
The pollen fertility having fruit pin branch is 62.67%, and the pollen fertility of pin branch of having no result is 1.48%, has fruit pin branch pollen fertility
Being greatly improved, have no result pin branch pollen fertility and F1Fertility is close to (table 1, Fig. 1 a, 1b).
Table 1 Henan spends 9331 and A. oteroi hybrid F1、S0Pollen Activity situation
The branch fluid medium MS+0.8 mg/L NAA taking pollen fertility high carries out root culture, then to the tip of a root
Carrying out cytological observation and chromosome counting, result display branch somatic chromosome is 60 (Fig. 1 c).November then
Within about 15 days, press individual plant results, and record solid individual plant number, find 5 strains have 1 strain solid, account for 20%(table 2).
(4) pod of gained in step (3), with the sodium hypochlorite disinfection of 5%, is taken its seed, is connect by seed
Plant in 1/2 MS (minimal medium that a great number of elements halves) and 5% sucrose solids culture medium, be placed in 25 DEG C of constant temperature illumination cultivation
Between cultivate, until developing into seedling S1。
Example 2 method is essentially identical with example 1, and difference is:
Among Cultivated Peanuts white sand 1016 and wild species A. macedoi is used to carry out intervarietal hybridization, the triploid F that will obtain1
Formulating out allohexaploid Semen arachidis hypogaeae L-21 through the method, the tissue cultured seedling photo of L-21 sees Fig. 4.
With the method for example 1, gather in the crops by individual plant, and record solid individual plant number, find 6 strains have 1 strain solid, account for 16.7%(table
2).
Table 2 interspecific F_1 fertility chromosome doubling success rate1
| Numbering | Transplant survival strain number (strain) after process | Solid strain number (strain) | Double success rate (%) |
| F1(9331 × A. oteroi is spent in Henan) | 5 | 1 | 20% |
| F1(white sand 1016 × A. macedoi) | 6 | 1 | 16.7% |
Example 3, the authentication method of a kind of allohexaploid Semen arachidis hypogaeae
(1) extract respectively maternal Henan spend 9331, male parent wild species A. oteroi and S1The genomic DNA of plant, A.
Oteroi complete genome DNA biotinylated probe；
(2) seedling S in above-mentioned steps 4 is taken1The tip of a root, carry out film-making mitosis metaphase, then complete with A. oteroi
Genomic DNA is that probe is to S1Carry out genome breeding.
Genome breeding method: first preparing hybrid liquid in centrifuge tube, its component includes deionized formamide
7.5 microlitres, 20 × SSC buffer 1.5 microlitre, dextran sulfate 2 microlitre of 50%, 10mg/ml salmon sperm dna 0.5 micro-
Liter, biotin labeled A. oteroi DNA probe 2.5 microlitre, the white sand 1016 of 100 mg/ml are blockaded DNA 1 microlitre, will
At 103 DEG C of degeneration 13 min after hybridization solution mixing, then centrifuge tube is immediately placed on 10-15min in the ethanol of-20 DEG C of refrigerators；
In film-making mitosis metaphase ,-70 DEG C of frost peels, after dehydration of alcohol 70% 78 DEG C of degeneration 1 min 10 s of Methanamide, it
After be distributed in-20 DEG C 70%, 95%, 100% ethanol in each 5 min of serial dehydration, dry up microscope slide；By described hybridization drop
On microscope slide, covered, hybridize 6-8 h in 37 DEG C；10 min, use is soaked with 2 × SSC respectively under the conditions of 42 DEG C
The Methanamide of 50% soaks 10 min, soaks 10 min with 2 × SSC, soaks 5 min with 1 × TNT under room temperature；Film-making is drained,
Plus antibiotin FITC, put into room temperature in 37 DEG C of magazines and cultivate 30 min, with 1 × TNT room temperature washing film-making 3 times, each 5
Min, finally dries up film-making.
Described SSC buffer is by the trisodium citrate C of 0.3M6H5Na3O7.2H2The NaCl composition of O and 3M；TNT solution by
The Tris-HCl of 0.1M, the NaCl of 0.15 M, the Tween-20 composition of 0.05%.
With DAPI dyeing 3-4 min after having hybridized, take a picture under fluorescence microscope, it is thus achieved that hybridization picture, statistics dyeing
Body number, it is thus achieved that chromosome is the allohexaploid Semen arachidis hypogaeae E-4 of 60, is hybridized the dyeing that signal covers entirely in E-4 chromosome set
Body is A. oteroi chromosome (Fig. 2, Fig. 4).
(3) further with Henan spend 9331, the codominant marker E420 of A.oteroi checking allohexaploid Semen arachidis hypogaeae
Accuracy, Henan is spent 9331 by E420, the DNA cloning slice result of A. oteroi and E-4 shows, maternal (Henan spends 9331) is about
Have specific band, male parent (A. oteroi) to have specific band at about 160bp at 450bp and 330bp, and E-4 450bp,
All there is specific band (Fig. 3) at 330bp and 160bp, illustrate that allohexaploid Semen arachidis hypogaeae E-4 contains Henan simultaneously and spends 9331 and A.
The hereditary material of oteroi.
Wherein, codominant marker E420 includes that SSR forward primer and downstream primer, SSR forward primer sequence are:
TCCATCGTTAGTGGCACTGT；
SSR downstream primer sequence is: GTCGACTCCTGCCCAATCTA.
Claims (7)
1. the method for creating of an allohexaploid Semen arachidis hypogaeae, it is characterised in that spend 9331 for maternal with tetraploid cultivar Henan,
Hybridize for male parent with wild diploid species A.oteroi, obtain triploid F through ovule and Hybrid embryo1, with this three
Times body tissue cultured seedling is material, carries out chromosome doubling, it is thus achieved that allohexaploid Semen arachidis hypogaeae, concrete grammar under conditions of tissue culture
As follows:
(1) at F1Triploid tissue cultured seedling cuts from the 3rd leaf of top number, takes terminal bud part, is placed on solid medium
In, between 25 DEG C of constant temperature illumination cultivation, induced bud breaks up 14-16 days, obtains cultivating Seedling；Wherein the component of solid medium be MS,
The 6-BA of NAA, 0.4mg/L of 0.1mg/L, the sucrose of 3% and the Colchicine of 0.05%；
(2) transfer to, by MS, 0.8mg/L NAA and solid root media that 3% sucrose is component, cultivate by cultivating Seedling
15-30 days, until forming whole plant S0；
(3) by whole plant S0In being transplanted to experiment Tanaka；Continue to cultivate to observe and find that part side shoot Post flowering creates fruit pin,
Statistics has the pollen fertility of fruit pin side shoot and pin side shoot of having no result, and method is to choose S0There is the health of fruit pin branch and pin branch of having no result
Work as Radix Trichosanthis, pollen is placed on microscope slide, add the aceto-camine dyeing of one 1%, covered in a moment, be placed in micro-
Microscopic observation, adds up 5 area of visual field, two flowers of every kind of Material Takeoff；The pollen fertility that result shows fruit pin side shoot is obvious
Higher than having no result pin side shoot；Take the part branch having fruit pin side shoot that pollen fertility is high, with MS minimal medium and 0.8mg/L NAA
The fluid medium formed carries out root culture, and then the tip of a root carries out cytological observation and chromosome counting, result display branch
Bar somatic chromosome is 60；Gather in the crops pod then；
(4) by the pod of gained with the sodium hypochlorite disinfection of 5%, take its seed, seed is inoculated into by 1/2MS and
In the solid medium of 5% sucrose composition, it is placed between 25 DEG C of constant temperature illumination cultivation cultivation, until developing into seedling S1；Described 1/
Constituent content in 2MS culture medium is the half of MS.
2. method for creating as claimed in claim 1, it is characterised in that described female parent is white sand 1016, and male parent is
A.macedoi。
3. the authentication method of the allohexaploid Semen arachidis hypogaeae of one kind such as claim 1 method initiative, it is characterised in that: by claim
The seedling S obtained in 11Carry out genome breeding and molecular marker analysis, specifically comprise the following steps that
A, respectively extract maternal Henan spend 9331, male parent wild species A.oteroi and S1The genomic DNA of plant；
B, take seedling S1The tip of a root, carry out film-making mitosis metaphase, then with the DNA of A.oteroi full-length genome as probe, right
S1Carry out genome breeding, drop to dyeing 3-4min on slide with DAPI dye liquor after having hybridized, then show at fluorescence
Take a picture under micro mirror, it is thus achieved that hybridization picture, add up Chromosome number, it is thus achieved that chromosome is the allohexaploid Semen arachidis hypogaeae E-4 of 60；
C, further with Henan spend 9331, the allohexaploid Semen arachidis hypogaeae of the codominant marker E420 of A.oteroi checking gained
Accuracy, with E420 Henan spent 9331, the DNA cloning slice result of A.oteroi and E-4 shows, E-4 contains Henan flower simultaneously
The hereditary material of 9331 and A.oteroi.
4. authentication method as claimed in claim 3, it is characterised in that: described molecular marker E420 includes SSR forward primer sequence
Row and downstream primer sequence, SSR primer upstream sequence is: TCCATCGTTAGTGGCACTGT；
SSR primer downstream sequence is: GTCGACTCCTGCCCAATCTA.
5. authentication method as claimed in claim 3, it is characterised in that: described genome breeding enters by the following method
OK: first preparing hybrid liquid in centrifuge tube, hybridization solution includes deionized formamide analytical pure 7.5 microlitre, 20 × SSC buffer
1.5 microlitres, dextran sulfate 2 microlitre of 50%, salmon sperm dna 0.5 microlitre of 10mg/ml and biotin labeled
A.oteroi DNA probe 2.5 microlitre, the white sand 1016 of 100mg/ml are blockaded DNA 1 microlitre, 103 DEG C of changes after hybridization solution mixing
Property 13min, is then immediately placed on 10-15min in the ethanol of-20 DEG C of refrigerators by centrifuge tube；In film-making mitosis metaphase ,-70
DEG C frost peel, after slide is dehydrated 6h in ethanol, be placed on 78 DEG C of degeneration 1min 10s of Methanamide of 70%, afterwards-20
The each 5min of serial dehydration in the ethanol of 70%, 95%, 100% respectively at DEG C, dries up microscope slide；
Described hybridization solution is dripped on microscope slide, covered, hybridize 6-8h in 37 DEG C；Respectively with 2 × SSC under the conditions of 42 DEG C
Soak 10min, with 50% Methanamide soak 10min, with 2 × SSC immersion 10min, the most at room temperature with 1 × TNT immersion
5min；Film-making is drained, adds antibiotin FITC, put into room temperature in 37 DEG C of magazines and cultivate 30min, by 1 × TNT solution room temperature
Washing film-making 3 times, each 5min, finally film-making is dried up.
6. authentication method as claimed in claim 3, it is characterised in that: described SSC buffer is by the trisodium citrate of 0.3M
C6H5Na3O7.2H2The NaCl composition of O and 3M.
7. the authentication method as described in claim 5 or 6, it is characterised in that: described TNT solution by the Tris-HCl of 0.1M,
The NaCl of 0.15M, the Tween-20 composition of 0.05%.
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| CN102960237A (en) * | 2012-11-22 | 2013-03-13 | 河南省农业科学院 | Method for obtaining, breeding and storing peanut interspecies hybridization variety, and identifying molecular cytology |
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| AR067114A1 (en) * | 2007-06-22 | 2009-09-30 | Monsanto Technology Llc | METHODS AND COMPOSITIONS TO SELECT LOCI BASED ON PERFORMANCE AND EXCESSION OF CHARACTERISTICS |
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| CN102960237A (en) * | 2012-11-22 | 2013-03-13 | 河南省农业科学院 | Method for obtaining, breeding and storing peanut interspecies hybridization variety, and identifying molecular cytology |
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