CN107475395A - Auxiliary differentiates the authentication method and its dedicated kit of hybrid cotton Ji 1518 - Google Patents
Auxiliary differentiates the authentication method and its dedicated kit of hybrid cotton Ji 1518 Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a kind of authentication method and its dedicated kit for aiding in differentiating hybrid cotton Ji 1518, the method that auxiliary differentiates hybrid cotton, it is made up of following steps：1）Extract the male parent Ji 567 of Ji 1518, maternal Ji 228, Ji 1518（F1）With the genomic DNA of hybrid cotton to be identified；2）Using the genomic DNA of said extracted as template, enter performing PCR with every pair of primers in the kit respectively and expand, obtain the pcr amplification product per pair of primers；3）Detect the collection of illustrative plates of the pcr amplification product per pair of primers.Experiment proves：Kit, SSR collection of illustrative plates and the authentication method of the present invention can effectively, accurately and rapidly distinguish whether hybrid cotton to be measured is Ji 1518（F1）The Parent without hybridization whether is mixed, and other most of mixed hybrid cotton varieties can be detected.
Description
Technical field
The present invention relates to the authentication method and its dedicated kit that auxiliary differentiates hybrid cotton Ji 1518.
Background technology
Cotton is Chang Yihua crops, and the upper most of high advantage hybrid cotton varieties of production at present are by artificial emasculation system
Kind.During the production of hybrid seeds, because the intersection certainly that maternal emasculation is not thorough or perforated is male and formed has a strong impact on purity of hybrid, cause
Yield, quality and the disease resistance of hybrid cotton reduce.With cotton hybrid kind be on the increase and the hereditary basis day of parent
It is gradually narrow, the purity of cenospecies and authenticity identification certain difficulty is occurred.Traditional cotton crossbreed authenticity and pure
Degree is identified by the method for field planting, and time-consuming, cost height, poor in timeliness, is easily influenceed by environmental factor.Therefore,
Establish that quick, accurately and efficiently authentication method is to protecting the power of the intellectual property and breeding man of improved variety to target hybrid cotton
Benefit has great importance.Ji 1518 is that the transform insect-resistant gene that Cotton Inst., Academy of Agriculture and Forestry Sciences of Hebei Prov. cultivates hybridizes spring cotton product
Kind, therefore, can be quick and precisely the invention provides the authentication method and its dedicated kit that auxiliary differentiates hybrid cotton Ji 1518
Discriminating hybrid cotton Ji 1518 and other hybrid cottons.
The content of the invention
It is an object of the invention to provide a kind of authentication method and its dedicated kit for aiding in differentiating hybrid cotton Ji 1518, with
Solve the problems, such as to propose in above-mentioned background technology.
The present invention uses following technical scheme：
A kind of dedicated kit for aiding in differentiating hybrid cotton Ji 1518, is drawn to 7 Dui shown in 1a to primer pair 7a as following primer
Thing forms：
Primer pair 1a：One primer sequence is GCCTTCAATCAATTCATACG, and another primer sequence is
GAAGGAGAAAGCAACGAATTAG；
Primer pair 2a：One primer sequence is GTAGTCTTCTCAACTCCACTGTT, and another primer sequence is
GGTGACATCAGTGTTGTTC；
Primer pair 3a：One primer sequence is TTCCTACTGCTCCTCCTCAG, and another primer sequence is
ATATTTGTGAGGGGCAAATG；
Primer pair 4a：One primer sequence is TTTGAACGAACACATTACGG, and another primer sequence is
ATGGGTTTTTACCAGAGCAG；
Primer pair 5a：One primer sequence is GGGTTTTTGCATTTTGTTCT, and another primer sequence is
ATGACAGATTCCACCGTTCT；
Primer pair 6a：One primer sequence is AATGGTGTCCGGTATGTAGG, and another primer sequence is
CATTCTTTCGATCATCACCA；
Primer pair 7a：One primer sequence is TCAGCTCCGATTCTTCAAACCCT, and another primer sequence is
TCGTTGAGGACAGCTTCGCC。
A kind of authentication method for aiding in differentiating hybrid cotton Ji 1518, comprises the following steps：
S1, DNA are extracted
Male parent Ji 567, maternal Ji 228, hybrid cotton Ji 1518 and other hybrid cottons to be identified of hybrid cotton Ji 1518 are extracted respectively
Genomic DNA, genomic DNA integrality and impurity are detected using 1% agarose gel electrophoresis, using spectrophotometer
Nanodrop2000 detects the concentration of genomic DNA, and most genomic DNA is diluted to 25 ~ 40ng/ul of final concentration at last；
S2, SSR amplification, electrophoresis and silver staining
Respectively using the genomic DNA after above-mentioned dilution as template, enter performing PCR respectively with above-mentioned primer and expand, obtain every a pair
The pcr amplification product of primer；Specifically PCR amplification method is：PCR reaction systems 10uL, PCR reaction system include template DNA
1uL, Tiangeng 2 × Taq RCR Master Mix 5ul, sense primer 0.5ul, anti-sense primer 0.5ul, ddH2O 3ul, then
By 95 DEG C of pre-degeneration 5min of PCR reaction systems, then 94 DEG C of denaturation 45s, then 55 DEG C of annealing 45s, then 72 DEG C of extension 1min, enter repeatedly
28 circulations of row, rear 72 DEG C of standings 10min, are then separated, most using 8% vertical panel native polyacrylamide gel electrophoresis
After carry out silver staining；
S3, the detection pcr amplification product per pair of primers collection of illustrative plates, differentiate the kind of hybrid cotton Ji 1518 and it is mixed other
Hybrid cotton varieties.
A kind of combination S SR collection of illustrative plates for aiding in differentiating hybrid cotton Ji 1518, aid in differentiating hybrid cotton Ji 1518 by a kind of
Authentication method obtains；Combination S SR collection of illustrative plates is made up of following 7 groups of SSR collection of illustrative plates：
1）The following SSR collection of illustrative plates that primer pair 1a PCR amplification cotton varieties obtain：
By adjacent 3 DNA bands that size from top to bottom is 90bp, 92bp, 95bp and from top to bottom size be 130bp,
The SSR collection of illustrative plates of 160bp, 190bp adjacent 3 DNA bands composition, size 90bp, 92bp, 95bp adjacent 3 DNA bands
It is non-conterminous for 130bp, 160bp, 190bp adjacent 3 DNA bands with size；
2）The following SSR collection of illustrative plates that primer pair 2a PCR amplification cotton varieties obtain：
The SSR being made up of adjacent 5 DNA bands that size from top to bottom is 170bp, 185bp, 195bp, 210bp, 220bp schemes
Spectrum；
3）The following SSR collection of illustrative plates that 3a PCR amplification cotton varieties obtain：
The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 200bp, 205bp；
4）The following SSR collection of illustrative plates that primer pair 4a PCR amplification cotton varieties obtain：
The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 145bp, 180bp；
5）The following SSR collection of illustrative plates that primer pair 5a PCR amplification cotton varieties obtain：
Be 400bp, 420bp by adjacent 2 DNA bands and size that size from top to bottom is 210bp, 230bp, 440bp,
The SSR collection of illustrative plates of 480bp adjacent 4 DNA bands composition, the adjacent 2 DNA bands and size of size 210bp, 230bp are
400bp, 420bp, 440bp, 480bp adjacent 4 DNA bands are non-conterminous；
6）The following SSR collection of illustrative plates that primer pair 6a PCR amplification cotton varieties obtain：
The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 180bp, 185bp；
7）The following SSR collection of illustrative plates that primer pair 7a PCR amplification cotton varieties obtain：
The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is respectively 390bp and 420bp.
Further, in step S3, if the collection of illustrative plates of the pcr amplification product of each detection seed corresponding to each primer pair
It is consistent with the collection of illustrative plates corresponding to above-mentioned same primers, then hybrid cotton seed to be identified is judged for the seed of hybrid cotton Ji 1518, if
The collection of illustrative plates corresponding to each detection seed and above-mentioned same primers corresponding at least one pair of primer pair is inconsistent, then judges to wait to reflect
Other hybrid cotton seed is other hybrid cotton seeds.
Further, the quantity of hybrid cotton Ji 1518 is 8, and other hybrid cottons to be identified include common miscellaneous of 14 kinds of in the markets
Hand over cotton seed.
The beneficial effects of the present invention are：
1st, hybrid cotton Ji 1518 can fast and accurately be differentiated（F1）With other hybrid cotton varieties.
2nd, the intellectual property of breeder's kind and the rights and interests of breeding man are protected.
Brief description of the drawings
The application is described in further detail with reference to the accompanying drawings and detailed description.
Fig. 1 is respectively the primer pair 1a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）And Ji
1518（F1）Codominance/dominant marker's testing result.
Fig. 2 is respectively the primer pair 2a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）And Ji
1518（F1）Codominance/dominant marker's testing result.
Fig. 3 is respectively the primer pair 3a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）And Ji
1518（F1）Codominance/dominant marker's testing result.
Fig. 4 is respectively the primer pair 4a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）And Ji
1518（F1）Codominance/dominant marker's testing result.
Fig. 5 is respectively the primer pair 5a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）And Ji
1518（F1）Codominance/dominant marker's testing result.
Fig. 6 is respectively the primer pair 6a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）And Ji
1518（F1）Codominance/dominant marker's testing result.
Fig. 7 is respectively the primer pair 7a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）And Ji
1518（F1）Codominance/dominant marker's testing result.
Fig. 8 is respectively the primer pair 1a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）, 8 Jis
1518 F1Individual plant and the testing result of 14 other hybrid cottons.
Fig. 9 is respectively the primer pair 2a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）, 8 Jis
1518 F1 Individual plant and the testing result of 14 other hybrid cottons.
Figure 10 is respectively the primer pair 3a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）, 8 Jis
1518 F1Individual plant and the testing result of 14 other hybrid cottons.
Figure 11 is respectively the primer pair 4a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）, 8 Jis
1518 F1Individual plant and the testing result of 14 other hybrid cottons.
Figure 12 is respectively the primer pair 5a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）, 8 Jis
1518 F1Individual plant and the testing result of 14 other hybrid cottons.
Figure 13 is respectively the primer pair 6a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）, 8 Jis
1518 F1Individual plant and the testing result of 14 other hybrid cottons.
Figure 14 is respectively the primer pair 7a male parent of Ji 1518 from left to right（Ji 567）, Ji 1518 it is maternal（Ji 228）, 8 Jis
1518 F1Individual plant and the testing result of 14 other hybrid cottons.
In Fig. 1-14 marked as：1st, the male parent Ji 567 of Ji 1518；2nd, the maternal Ji 228 in Ji 1518；3rd, the F of Ji 15181Individual plant
1；4th, the F of Ji 15181Individual plant 2；5th, the F of Ji 15181Individual plant 3；6th, the F of Ji 15181Individual plant 4；7th, the F of Ji 15181Individual plant 5；8th, Ji 1518
F1Individual plant 6；9th, the F of Ji 15181Individual plant 7；10th, the F of Ji 15181Individual plant 8；11st, miscellaneous No. 2 of Ji；12nd, auspicious miscellaneous 816；13rd, Ji H239；14、
Ji H156；15th, Ji miscellaneous 999；16th, rich 202 are closed；17th, Ji You 01；18th, Ji H170；19th, Handan miscellaneous 306；20th, Handan miscellaneous 301；21st, Ji
Excellent miscellaneous 69；22nd, miscellaneous cotton 20 is created；23rd, miscellaneous No. 1 of Ji；24th, general 3 are wished.
Embodiment
In order to facilitate the understanding of those skilled in the art, below in conjunction with embodiment, the present invention will be further described.
Embodiment is only to the invention for example, not being limitation of the invention, and the step of not illustrated in embodiment is equal
It is prior art, is not described in detail herein.
Embodiment one
The present invention completes the structure of SSR electrophoresis fingerprints, that is, completes the polymorphism to hybrid cotton Ji 1518 and its parent
Detection.The male parent Ji 567 of Ji 1518, maternal Ji 228, Ji 1518（F1）, in -2015 years end of the year 2014 kind in agricultural section of Hebei province
Institute's Cotton Research Institute greenhouse.
The polymorphic detection of hybrid cotton Ji 1518 and its parent is comprised the following steps herein：
S1, DNA are extracted
The male parent Ji 567, maternal Ji 228, the genomic DNA of hybrid cotton Ji 1518 of hybrid cotton Ji 1518 are extracted respectively, using 1%
Agarose gel electrophoresis detects genomic DNA integrality and impurity, and genome is detected using spectrophotometer Nanodrop2000
DNA concentration, and most genomic DNA is diluted to 25 ~ 40ng/ul of final concentration at last；
S2, SSR amplification, electrophoresis and silver staining
Respectively using the genomic DNA after above-mentioned dilution as template, enter performing PCR respectively with 308 pairs of primers and expand, obtain every a pair and draw
The pcr amplification product of thing；Specifically PCR amplification method is：PCR reaction systems 10uL, PCR reaction system include template DNA 1uL,
Tiangeng 2 × Taq RCR Master Mix 5ul, sense primer 0.5ul, anti-sense primer 0.5ul, ddH2O 3ul, then by PCR
95 DEG C of pre-degeneration 5min of reaction system, then 94 DEG C of denaturation 45s, then 55 DEG C of annealing 45s, then 72 DEG C of extension 1min, are repeated 28
Individual circulation, rear 72 DEG C of standings 10min, is then separated using 8% vertical panel native polyacrylamide gel electrophoresis, most laggard
Row silver staining；
S3, the detection pcr amplification product per pair of primers collection of illustrative plates, 7 primers are obtained altogether two parents with more
State property.
A kind of above-mentioned dedicated kit for aiding in differentiating hybrid cotton Ji 1518 of 7 pairs of primers composition, the dedicated kit is under
State 7 pairs of primers composition shown in primer pair 1a to primer pair 7a：
Primer pair 1a：One primer sequence is GCCTTCAATCAATTCATACG, and another primer sequence is
GAAGGAGAAAGCAACGAATTAG；
Primer pair 2a：One primer sequence is GTAGTCTTCTCAACTCCACTGTT, and another primer sequence is
GGTGACATCAGTGTTGTTC；
Primer pair 3a：One primer sequence is TTCCTACTGCTCCTCCTCAG, and another primer sequence is
ATATTTGTGAGGGGCAAATG；
Primer pair 4a：One primer sequence is TTTGAACGAACACATTACGG, and another primer sequence is
ATGGGTTTTTACCAGAGCAG；
Primer pair 5a：One primer sequence is GGGTTTTTGCATTTTGTTCT, and another primer sequence is
ATGACAGATTCCACCGTTCT；
Primer pair 6a：One primer sequence is AATGGTGTCCGGTATGTAGG, and another primer sequence is
CATTCTTTCGATCATCACCA；
Primer pair 7a：One primer sequence is TCAGCTCCGATTCTTCAAACCCT, and another primer sequence is
TCGTTGAGGACAGCTTCGCC。
Polymorphic detection is carried out using 308 pairs of primer pair hybrid cotton Jis 1518 and its parent, obtains 7 pairs of primers altogether at two
There is polymorphism, as shown in figs. 1-7 between parent.In Fig. 1-7, from left to right, three row SSR electrophoresis fingerprints, three row are schemed altogether
Spectrum is respectively from left to right the male parent Ji 567 of Ji 1518, maternal Ji 228, Ji 1518（F1）Finger-print.Wherein 5 pairs of primers
（1a, 2a, 3a, 5a, 6a）Band of different sizes is amplified between two parents, and these marker sites are in F1In show as heterozygosis
Band, it is codominant marker.These codominant markers can make a distinction hybrid cotton Ji 1518 and male parent, female parent respectively, from theory
On say, these marker sites can be used to carry out cultivar identification and purity detecting to hybrid cotton Ji 1518；Other 2 pairs of primers（4a,
7a）The banding pattern of Ji 1518 it is consistent with maternal or male parent, be dominant marker.Wherein primer pair 4a F1Banding pattern is consistent with male parent, draws
F of the thing to 7a1Banding pattern is consistent with female parent.Though this 2 dominant markers directly can not make a distinction cenospecies and parent, but if
Inclined male parent is marked and marks collocation cooperation detection with partially maternal, still can be used for the identification to the cenospecies.
Above-mentioned codominant marker can separate hybrid cotton Ji 1518 and its parent, but due to cotton hereditary basis more
It is narrow, only by primer as a pair be difficult to distinguish Ji 1518 and other cenospecies, this just need using primer combination come
The differentiation of the Hybrid and other Hybrids is realized, this research makes the finger-print of Ji 1518 using 7 pairs of primer combinations,
The breeding true identity of the kind can more truly be reflected, avoid the error of identification.
Embodiment two
The present invention this is mixed into Ji 1518 from 14 parts of other hybrid cottons, using above-mentioned 7 pairs of primer pair Jis 1518 and 14 other
Hybrid cotton varieties are tested.Can be by hybrid cotton Ji 1518 and other mixed 14 hybrid cottons by the electrophoresis pattern of combination
Make a distinction.The electrophoresis pattern of above-mentioned 7 pairs of primers is as seen in figures 8-14.
The auxiliary used herein differentiates that the authentication method of hybrid cotton Ji 1518 comprises the following steps：
S1, DNA are extracted
Male parent Ji 567, maternal Ji 228, hybrid cotton Ji 1518 and other hybrid cottons to be identified of hybrid cotton Ji 1518 are extracted respectively
Genomic DNA, genomic DNA integrality and impurity are detected using 1% agarose gel electrophoresis, using spectrophotometer
Nanodrop2000 detects the concentration of genomic DNA, and most genomic DNA is diluted to 25 ~ 40ng/ul of final concentration at last；
S2, SSR amplification, electrophoresis and silver staining
Respectively using the genomic DNA after above-mentioned dilution as template, enter performing PCR respectively with 7 pairs of above-mentioned primers and expand, obtain each
To the pcr amplification product of primer；Specifically PCR amplification method is：PCR reaction systems 10uL, PCR reaction system include template DNA
1uL, Tiangeng 2 × Taq RCR Master Mix 5ul, sense primer 0.5ul, anti-sense primer 0.5ul, ddH2O 3ul, then
By 95 DEG C of pre-degeneration 5min of PCR reaction systems, then 94 DEG C of denaturation 45s, then 55 DEG C of annealing 45s, then 72 DEG C of extension 1min, enter repeatedly
28 circulations of row, rear 72 DEG C of standings 10min, are then separated, most using 8% vertical panel native polyacrylamide gel electrophoresis
After carry out silver staining；
S3, the detection pcr amplification product per pair of primers collection of illustrative plates, differentiate hybrid cotton Ji 1518（F1）With it is mixed other
Hybrid cotton varieties.
In Fig. 8-14, from left to right, 24 row SSR electrophoresis fingerprints, 24 row collection of illustrative plates are respectively from left to right altogether
The male parent Ji 567 of Ji 1518, the Ji 1518 of maternal Ji 228,8（F1）The finger-print of individual plant and 14 other hybrid cottons etc. 24
Electrophoresis pattern of the cotton variety after primer expands, 14 other hybrid cottons be respectively from left to right Ji it is miscellaneous No. 2, it is auspicious miscellaneous 816,
Ji H239, Ji H156, Ji are miscellaneous 999, close rich 202, Ji You 01, Ji H170, Handan are miscellaneous 306, Handan is miscellaneous 301, Jiyouza 69, the miscellaneous cotton of wound
20th, Ji is miscellaneous No. 1, wishes general 3.
As shown in figure 8, this figure is the electrophoresis pattern made using pair of primers 1a, the 1st is classified as the electrophoresis of male parent Ji 567
Collection of illustrative plates, the 2nd is classified as the electrophoresis pattern of maternal Ji 228, and the 3rd row to the 10th are classified as 8 Jis 1518（F1）The electrophoresis pattern of individual plant, the
11-24 is classified as the electrophoresis pattern of other hybrid cottons.3rd arranges to 8 Jis 1518 of the 10th row（F1）Individual plant electrophoresis pattern it is identical,
The cotton of 11,13,14,16,18,20,21,22,23,24 kinds can be defined as other cotton product using pair of primers 1a
Kind.
As shown in figure 11, this figure is the electrophoresis pattern made using the 4th couple of primer 4a, and the 1st is classified as the electricity of male parent Ji 567
Swimming collection of illustrative plates, the 2nd is classified as the electrophoresis pattern of maternal Ji 228, and the 3rd row to the 10th are classified as 8 Jis 1518（F1）Individual plant electrophoretogram
Spectrum, 11-24 are classified as the electrophoresis pattern of other kinds, the kind of 11-20,22,24 substantially can be defined as into other kinds.
With reference to Fig. 8 and Figure 11, it may be determined that 11-24 is other kinds, and 3-10 is first generation hybrid cotton Ji 1518, such a
Method is efficient to identify target hybrid cotton.Simultaneously with such a method, effectively differentiated using the electrophoresis pattern of other primer pairs
Hybrid cotton Ji 1518, efficient, degree of accuracy height.
Add frame in Fig. 8-14 is the electrophoresis pattern of the individual plant of Ji 1518.The description of DNA bands is equal in this paper electrophoresis patterns
To describe from bottom to up.A kind of combination S SR collection of illustrative plates for aiding in differentiating hybrid cotton Ji 1518, combination S SR collection of illustrative plates is by following 7 groups of SSR
Collection of illustrative plates forms：
1）The following SSR collection of illustrative plates that primer pair 1a PCR amplification cotton varieties obtain, as shown in Fig. 1,8：
By adjacent 3 DNA bands that size from top to bottom is 90bp, 92bp, 95bp and from top to bottom size be 130bp,
The SSR collection of illustrative plates of 160bp, 190bp adjacent 3 DNA bands composition, size 90bp, 92bp, 95bp adjacent 3 DNA bands
It is non-conterminous for 130bp, 160bp, 190bp adjacent 3 DNA bands with size；
2）The following SSR collection of illustrative plates that primer pair 2a PCR amplification cotton varieties obtain, as shown in Fig. 2,9：
The SSR being made up of adjacent 5 DNA bands that size from top to bottom is 170bp, 185bp, 195bp, 210bp, 220bp schemes
Spectrum；
3）The following SSR collection of illustrative plates that primer pair 3a PCR amplification cotton varieties obtain, as shown in Fig. 3,10：
The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 200bp, 205bp；
4）The following SSR collection of illustrative plates that primer pair 4a PCR amplification cotton varieties obtain, as shown in Figure 4 and 11：
The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 145bp, 180bp；
5）The following SSR collection of illustrative plates that primer pair 5a PCR amplification cotton varieties obtain, as shown in Fig. 5,12：
By adjacent 2 DNA bands that size from top to bottom is 210bp, 230bp and from top to bottom size be 400bp, 420bp,
440bp, 480bp adjacent 4 DNA bands composition SSR collection of illustrative plates, adjacent 2 DNA bands of size 210bp, 230bp and
Size is that 400bp, 420bp, 440bp, 480bp adjacent 4 DNA bands are non-conterminous；
6）The following SSR collection of illustrative plates that primer pair 6a PCR amplification cotton varieties obtain, as shown in Fig. 6,13：
The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 180bp, 185bp；
7）The following SSR collection of illustrative plates that primer pair 7a PCR amplification cotton varieties obtain, as shown in Fig. 7,14：
The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is respectively 390bp and 420bp.
In the authentication method step S3 that auxiliary differentiates hybrid cotton Ji 1518, if each detection corresponding to each primer pair
The collection of illustrative plates of the pcr amplification product of seed is consistent with the collection of illustrative plates corresponding to same primers, then judges cotton seeds to be identified for hybridization
The seed of cotton Ji 1518, if the collection of illustrative plates corresponding to each detection seed and same primers corresponding at least one pair of primer pair is inconsistent,
It is other cotton seeds then to judge cotton seeds to be identified.
Test result indicates that：Each pair primer will can effectively differentiate target cenospecies and its Parent.Pass through the electricity of combination
Swimming collection of illustrative plates can make a distinction hybrid cotton Ji 1518 and other mixed 14 hybrid cottons.In 14 of selection other hybrid cottons
In, primer pair 4a differentiation efficiency highest, only kind 21 and kind 23 are shown with hybrid cotton Ji 1518 in 14 kinds
Identical banding pattern, primer pair 7a or primer pair 1a is coordinated to make a distinction every other kind from target hybrid cotton.This
14 of selected works other hybrid cottons, popularizing area is larger, there is stronger representativeness, therefore the polymorphism primer filtered out
With broad applicability.This research and utilization 7 is preferable to polymorphism, the SSR primers that band is easily read, constructs the fingerprint image of Ji 1518
Spectrum, can by Ji 1518, other cenospecies make a distinction with mixed major part, can be applied to the detection of the seed purity of Ji 1518
With the identification of unknown species identity.Reference is provided for the registration of the kind of Ji 1518 and intellectual property protection, and is other hybrid cotton varieties
Research work offer reference effect and theoretical foundation.
Applied to the detection of the Molecular Identification and seed purity of cenospecies, compared with growing identification, have detection time it is short,
The advantages of speed is soon and detection is accurate.For perforated is male, emasculation is not thorough or other reasons caused by carry parental autocopulation
Specific admixture, only a pair of codominant SSR primers are needed just to be enough accurately to detect.But if it is mixed with its of non-parent
Its seed, the probability for only being distinguished the parent of cenospecies from other kinds with a SSR marker are relatively small.Such as primer
From excessive, then cost is too high, efficiency reduces.This is just needed when establishing cenospecies finger-print, on the one hand with a large amount of primers
Screened, another aspect consideration combines or developed new mark with different primers to screen the polymorphic of hybrid parentses
Property.7 marks that screening herein obtains largely ensure that the accuracy of identification Ji 1518, can root in actually differentiating
Wherein several marks are chosen according to demand to be identified, improve accuracy rate.
Claims (5)
- A kind of 1. dedicated kit for aiding in differentiating hybrid cotton Ji 1518, it is characterised in that：By following primer to 1a to primer pair 7 pairs of primers composition shown in 7a：Primer pair 1a：One primer sequence is GCCTTCAATCAATTCATACG, and another primer sequence is GAAGGAGAAAGCAACGAATTAG；Primer pair 2a：One primer sequence is GTAGTCTTCTCAACTCCACTGTT, and another primer sequence is GGTGACATCAGTGTTGTTC；Primer pair 3a：One primer sequence is TTCCTACTGCTCCTCCTCAG, and another primer sequence is ATATTTGTGAGGGGCAAATG；Primer pair 4a：One primer sequence is TTTGAACGAACACATTACGG, and another primer sequence is ATGGGTTTTTACCAGAGCAG；Primer pair 5a：One primer sequence is GGGTTTTTGCATTTTGTTCT, and another primer sequence is ATGACAGATTCCACCGTTCT；Primer pair 6a：One primer sequence is AATGGTGTCCGGTATGTAGG, and another primer sequence is CATTCTTTCGATCATCACCA；Primer pair 7a：One primer sequence is TCAGCTCCGATTCTTCAAACCCT, and another primer sequence is TCGTTGAGGACAGCTTCGCC。
- A kind of 2. authentication method for aiding in differentiating hybrid cotton Ji 1518, it is characterised in that：Comprise the following steps：S1, DNA are extractedMale parent Ji 567, maternal Ji 228, hybrid cotton Ji 1518 and other hybrid cottons to be identified of hybrid cotton Ji 1518 are extracted respectively Genomic DNA, genomic DNA integrality and impurity are detected using 1% agarose gel electrophoresis, using spectrophotometer Nanodrop2000 detects the concentration of genomic DNA, and most genomic DNA is diluted to 25 ~ 40ng/ul of final concentration at last；S2, SSR amplification, electrophoresis and silver stainingRespectively using the genomic DNA after above-mentioned dilution as template, enter performing PCR respectively with the primer described in claim 1 and expand, Obtain the pcr amplification product per pair of primers；Specifically PCR amplification method is：PCR reaction systems 10uL, PCR reaction system includes Template DNA 1uL, Tiangeng 2 × Taq RCR Master Mix 5ul, sense primer 0.5ul, anti-sense primer 0.5ul, ddH2O 3ul, then by 95 DEG C of pre-degeneration 5min of PCR reaction systems, then 94 DEG C of denaturation 45s, then 55 DEG C of annealing 45s, then 72 DEG C of extensions 1min, is repeated 28 circulations, rear 72 DEG C of standings 10min, then using 8% vertical panel non-denaturing polyacrylamide gel Electrophoretic separation, finally carries out silver staining；S3, the detection pcr amplification product per pair of primers collection of illustrative plates, differentiate the kind of hybrid cotton Ji 1518 and it is mixed other Hybrid cotton varieties.
- A kind of 3. combination S SR collection of illustrative plates for aiding in differentiating hybrid cotton Ji 1518, it is characterised in that：Pass through one kind in claim 2 Auxiliary differentiates that the authentication method of hybrid cotton Ji 1518 obtains；Combination S SR collection of illustrative plates is made up of following 7 groups of SSR collection of illustrative plates：1）The following SSR collection of illustrative plates that primer pair 1a PCR amplification cotton varieties in the claim 1 obtain：By adjacent 3 DNA bands that size from top to bottom is 90bp, 92bp, 95bp and from top to bottom size be 130bp, The SSR collection of illustrative plates of 160bp, 190bp adjacent 3 DNA bands composition, size 90bp, 92bp, 95bp adjacent 3 DNA bands It is non-conterminous for 130bp, 160bp, 190bp adjacent 3 DNA bands with size；2）The following SSR collection of illustrative plates that primer pair 2a PCR amplification cotton varieties in the claim 1 obtain：The SSR being made up of adjacent 5 DNA bands that size from top to bottom is 170bp, 185bp, 195bp, 210bp, 220bp schemes Spectrum；3）The following SSR collection of illustrative plates that primer pair 3a PCR amplification cotton varieties in the claim 1 obtain：The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 200bp, 205bp；4）The following SSR collection of illustrative plates that primer pair 4a PCR amplification cotton varieties in the claim 1 obtain：The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 145bp, 180bp；5）The following SSR collection of illustrative plates that primer pair 5a PCR amplification cotton varieties in the claim 1 obtain：Be 400bp, 420bp by adjacent 2 DNA bands and size that size from top to bottom is 210bp, 230bp, 440bp, The SSR collection of illustrative plates of 480bp adjacent 4 DNA bands composition, the adjacent 2 DNA bands and size of size 210bp, 230bp are 400bp, 420bp, 440bp, 480bp adjacent 4 DNA bands are non-conterminous；6）The following SSR collection of illustrative plates that primer pair 6a PCR amplification cotton varieties in the claim 1 obtain：The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is 180bp, 185bp；7）The following SSR collection of illustrative plates that primer pair 7a PCR amplification cotton varieties in the claim 1 obtain：The SSR collection of illustrative plates being made up of adjacent 2 DNA bands that size from top to bottom is respectively 390bp and 420bp.
- A kind of 4. authentication method for aiding in differentiating hybrid cotton Ji 1518 according to claim 2, it is characterised in that：Step S3 In, if the collection of illustrative plates of the pcr amplification product of each detection seed corresponding to each primer pair and identical described in claim 3 are drawn Collection of illustrative plates corresponding to thing is consistent, then hybrid cotton seed to be identified is judged for the seed of hybrid cotton Ji 1518, if at least one pair of primer pair The collection of illustrative plates corresponding to same primers described in corresponding each detection seed and claim 3 is inconsistent, then judges to be identified miscellaneous It is other hybrid cotton seeds to hand over cotton seed.
- A kind of 5. authentication method for aiding in differentiating hybrid cotton Ji 1518 according to claim 2, it is characterised in that：Hybrid cotton 1518 individual plant quantity of Ji 8, other hybrid cottons to be identified include the common hybrid cotton seed in 14 kinds of markets.
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