CN106244713A - A kind of method detecting Beijing Fatty Chicken five toe character and application - Google Patents
A kind of method detecting Beijing Fatty Chicken five toe character and application Download PDFInfo
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Abstract
The present invention discloses a kind of method detecting Beijing Fatty Chicken five toe character.The method includes the step of the genotype by the rs80659072 mutational site in ZRS region in Real-Time Fluorescent Quantitative PCR Technique detection LMBR1 gene the 5th intron.The real time fluorescent quantitative TaqMan SNP typing method detection speed that the present invention uses is fast, and highly sensitive, automaticity is high, and can sentence type through a PCR reaction, easy and simple to handle, and reaction overall process is carried out in the pipe closed, and decreases cross-contamination.Breeding is carried out by molecular marker auxiliary, it is possible to rapid build Beijing Fatty Chicken five toe strain is sheerly on the basis of the present invention.
Description
Technical field
The present invention relates to field of biological detection, specifically, relate to a kind of method detecting Beijing Fatty Chicken five toe character and
Application.
Background technology
Beijing Fatty Chicken is one famous local varieties of China, originates in Beijing area, and this chicken kind meat Egg Quality is excellent, deeply
By raiser and consumers welcomed, listed in country's livestock and poultry species resource by the Ministry of Agriculture and lay special stress on protecting register.In recent years in north
There are popularization in Jing Shi and whole nation major part provinces and cities, and cultivation amount and cultivation scale rise year by year.This chicken appearance is unique, has five
Toe and the feature of three maos (phoenix head, beard, hair lower limbs).
Wherein, five toes are the most significantly to recognize character after Beijing Fatty Chicken butchers listing, can important as Beijing Fatty Chicken
Kind identifies, and helps consumer to take this to make consumption choice.Therefore the selection-breeding of Beijing Fatty Chicken five toe character is strengthened, no matter from promotion
For kind exploitation itself, or for culturist and consumer, the most significant.
But, five toes of chicken are relative complex qualitative traits, in incomplete dominant lnheritance, by the phenotype of toe number
Carry out selection development slowly, be difficult to obtain significant effect.
2010, Dorshorst etc. detected LMBR1 gene the 5th intron in Siklie and WhiteSultan chicken kind
Suddenly change in interior ZRS region rs80659072 and five toes relevant, wherein GG type shows as four toes, GT and TT type shows as five toes or more
Many toes.2016, Zhang etc. also illustrated that this site and the many toes of Beijing Fatty Chicken are perfectly correlated, and establishes based on enzyme action
This site is detected (number of patent application 201510044061.2) by PCR-RFLP method.
But, this polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method detecting step is complicated, bag
Include: 1. according to restriction enzyme site upstream and downstream primers, utilize polymerase chain reaction (PCR) amplification purpose fragment；2. amplified production
Agarose gel detection；3. the digestion with restriction enzyme of amplified production；4. the agarose gel electrophoresis detection of digestion products.
Not only complex steps, the longest, and will tube repeatedly and sample-adding, and DNA concentration, DNA mass, amplification condition, restriction endonuclease
Digestion condition, agarose gel quality etc. all can have influence on effectiveness and the accuracy sentencing type.
In the actual Breeding Process of Beijing Fatty Chicken five toe character, it is desirable to can quickly detect genotype, and result must
Accuracy must be ensured, because once decision error, heavy losses can be brought to breeding.Therefore, developing one can be quickly accurate
The method really detecting Beijing Fatty Chicken five toe character, the technical support strong as the selection-breeding offer of Beijing Fatty Chicken is particularly necessary.
Summary of the invention
It is an object of the invention to the step existed for existing employing PCR-RFLP method detection Beijing Fatty Chicken five toe character
The most loaded down with trivial details, the longest, sensitivity is low, the defect problem that testing result is easily affected by experiment condition, it is provided that one can be accurately high
The fluorescence quantifying PCR method of effect ground detection Beijing Fatty Chicken five toe character.
For achieving the above object, the present invention adopts the following technical scheme that
First the invention provides a kind of method detecting Beijing Fatty Chicken five toe character, the method includes passing through real-time fluorescence
The step of the genotype in the rs80659072 mutational site in ZRS region in quantitative PCR technique detection LMBR1 gene the 5th intron
Suddenly.
Preferably, said method includes, according to the sequence in ZRS region in LMBR1 gene the 5th intron, designing a pair fluorescence
Quantification PCR primer and with primer with the use of TaqMan probe, utilize these primers and probe, use real time fluorescent quantitative
The step of the genotype in round pcr detection rs80659072 mutational site；
Wherein, the nucleotide sequence such as SEQ ID NO:5 institute of ZRS region in described LMBR1 gene the 5th intron
Show.
A preferred aspect of the invention, the primer of described quantitative fluorescent PCR is: fixed for expanding the fluorescence in ZRS region
The nucleotide sequence of amount PCR primer pair is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2；With
With above-mentioned PCR primer with the use of TaqMan fluorescent probe nucleotides sequence be classified as: wild-type allele probe
Nucleotide sequence as shown in SEQ ID NO:3, the nucleotide sequence of mutant allele probe such as SEQ ID NO:4 institute
Show.
Preferably, the reaction system of above-mentioned quantitative fluorescent PCR is: genomic DNA 1 μ L, 40 times of probes concentrated and primer
Mixed liquor 0.25 μ L, universal PC R mixed liquor 5 μ L, with water polishing to 10 μ L.
Preferably, the reaction condition of above-mentioned quantitative fluorescent PCR is: 95 DEG C, denaturation 10min；95 DEG C of degeneration 15s, 60 DEG C
Annealing 1min, totally 40 circulations.
The invention provides a kind of primer for identifying Beijing Fatty Chicken five toe character, it is for expanding ZRS region
Primer pair, its nucleotide sequence is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2.
Further, present invention also offers a kind of Yu above-mentioned PCR primer with the use of TaqMan fluorescent probe, its
Including the nucleotide sequence of wild-type allele probe as shown in SEQ ID NO:3；The nucleoside of mutant allele probe
Acid sequence is as shown in SEQ ID NO:4.
Further, present invention also offers a kind of test kit, this test kit comprises above-mentioned primer and above-mentioned fluorescence is visited
The combination of pin.
Preferably, the test kit of the present invention application in detection Beijing Fatty Chicken five toe character.
Further, present invention also offers above-mentioned Beijing Fatty Chicken five toe character detection method is applied to selection-breeding Beijing
In a fine breed of chicken with thick brownish feathers five toe character.
The present invention by design specific primer to and with primer to the use of probe, use real-time fluorescence fixed
The genotype of amount TaqMan SNP typing method testing goal sequence.Use Real-Time Fluorescent Quantitative PCR Technique, it is possible to rapid sensitive
The genotype in detection mutational site, chicken target area to be measured, ground, and carry out breeding by molecular marker auxiliary on this basis,
Can rapid build Beijing Fatty Chicken five toe strain pure lines.Further, the real time fluorescent quantitative TaqMan SNP typing that the present invention uses
Technology for detection speed is fast, highly sensitive, and automaticity is high, can sentence type through a PCR reaction, easy and simple to handle, and reaction
Overall process is carried out in the pipe closed, and decreases cross-contamination.To meet breeding work, accuracy and promptness can be wanted
Ask.
Additionally, the present invention use with specific primer to the use of probe be TaqMan MGB probe, this spy
The quenching group of 3 ' ends of pin is non-luminous quenching group (Non-Fluorescent Quencher, NFQ), therefore works as cancellation
After the energy of group absorptions reporter group, luminescence, greatly reduces the interference of background signal；TaqMan MGB probe also has
Having higher sequence-specific, experimental result is more accurately reliable.
Accompanying drawing explanation
Fig. 1 is some individuals pcr amplification product agarose gel electrophoresis figure, and in figure, swimming lane 1-10 is Different Individual, swimming lane M
For 100bp Marker；
Fig. 2 is the genotype that fluorescence quantitative PCR method detection is individual；
Fig. 3 is different genotype individuality sequencing result.
Detailed description of the invention
The method of the detection many toes of Beijing Fatty Chicken is PCR-RFLP method pair based on enzyme action and Beijing Fatty Chicken five toe phase at present
The genotype closing site detects, the method not only complex steps, the longest, and accuracy can not meet the later stage and build
The requirement of Beijing Fatty Chicken five toe strain pure lines, the present invention is in order to overcome drawbacks described above, it is provided that one utilizes real-time fluorescence PCR technology
The method detecting Beijing Fatty Chicken five toe character efficiently.
Below in conjunction with specific embodiment, the present invention will be described, but should not be construed as limitation of the present invention.Do not carrying on the back
On the premise of present invention spirit and essence, modification made for the present invention or replacement, belong to scope of the invention:
The detection of embodiment 1 mutational site rs80659072
Reference mutational site rs80659072 (NCBI accession number) and about sequence (nucleotide sequence such as SEQ ID NO:
Shown in 5), design primer, forward primer: 5 '-ACATACCAAGAATGTGCATGTGC-3 ', downstream primer: 5 '-
TTTGAGGTAACTTCCTTGCTTAA-3 ', amplified production length 448bp.
The individuality of various toe type is randomly choosed from Beijing Fatty Chicken jumpbogroup, accumulative 155.Gather anticoagulation, use DNA to carry
Take test kit (sky root biochemical, Beijing) and extract genomic DNA, use this site of above-mentioned primer and about sequence carry out PCR expansion
Increase.Amplified production, after 2% agarose gel detection, is held up Ke Jiamei Bioisystech Co., Ltd by Beijing and is completed order-checking, reversely
Order-checking.
Fig. 1 gives the agarose gel electrophoresis testing result after the PCR amplification of above-mentioned some individuals, wherein swimming lane 8
The most weak with 10 corresponding individual products, it is impossible to for follow-up order-checking test.Use NanoDrop2000 ultraviolet spectrometry light light
Degree meter, measures genome concentration, finds that these 4 individual DNA concentration are relatively low, only 10-15ng/ μ L.DNA profiling addition increases
To 5 μ L, PCR amplification again, but still reach to expand concentration less than preferable PCR primer.Therefore, again these 4 individualities are carried
After taking genomic DNA, then PCR amplification just obtains satisfied result again.
The sequencing result of all 155 chickens is shown in Table 1: as can be seen from Table 1 in addition to a fine breed of chicken with thick brownish feathers for double four toes (4/4), remaining
No matter which kind of phenotype, left five-right four (5/4), left four-right five (4/5), double five toes (5/5), six toes (include double six toes and single six
Toe, 6/-), two kinds of genotype of the most only TT and GT.Result shows, T site be one of Beijing Fatty Chicken five toe/six toe phenotype must
Want condition, but be not necessary and sufficient condition.
The genotype distribution of table 1 different phenotype fine breed of chicken with thick brownish feathers rs80659072 mutational sites
The foundation of embodiment 2 fluorescent quantitative PCR detection method
The present invention through repeatedly comparison screen and checking, according to ZRS region in LMBR1 gene the 5th intron and near
Sequence (nucleotide sequence is as shown in SEQ ID NO:5) obtain a pair for the fluorescence detecting chicken rs80659072 mutational site
Quantification PCR primer and with primer with the use of TaqMan probe.
Forward primer F:5 '-TCAGTGGCAAAAAACGAGCAAAAAT-3 ' (SEQ ID NO.1)
Downstream primer R:5 '-CACACAGAAATGAGTAGGAAGTCCAA-3 ' (SEQ ID NO.2).
Forward primer is by 25 base compositions, correspondence chicken No. 2 chromosomes 8467207～8467231 base positions
(www.ensembl.org, galgal4), downstream primer by 26 base compositions, corresponding No. 2 chromosomes 8467272 of chicken～
8467299 base positions (www.ensembl.org, galgal4), the expanding fragment length of this primer pair is 93bp.
With above-mentioned specific primer with the use of TaqMan fluorescent probe nucleotides sequence be classified as:
Wild-type allele probe: 5 '-ATGCAATGAAAGCTC-3 ' (SEQ IN NO.3)；
Mutant allele probe: 5 '-CATGCAATTAAAGCTC-3 ' (SEQ IN NO.4).
Wild-type probe by 15 base compositions, saltant type probe by 16 base compositions, corresponding No. 2 chromosomes the
8467241～8467255 and 8467240～8467255 bit base sequences.Wherein, the probe of wild-type allele is for open country
Raw type bases G, probe 5 ' end is connected to fluorophor VIC.The probe of mutant allele is for mutating alkali yl T, probe 5 '
End is connected to fluorophor FAM；3 ' end marks of two probes are all marked with quenching group, and this group is non-luminous quenching group
(Non-Fluorescent Quencher, NFQ).
Bole (Bio-Rad) iQ5 type real-time fluorescence quantitative PCR instrument is used to carry out TaqMan probe real-time fluorescence quantitative PCR
Test.
In above-described embodiment 1, the genomic DNA of 155 individualities of known type is as template, sets up TaqMan fluorescence fixed
Amount PCR detection method.Reaction system is as follows:
Wherein, genomic DNA is the genomic DNA obtained in embodiment 1,40 × SNP Genotyping AssayMix
(upstream and downstream primer concentration each 36 μMs and each 8 μMs of concentration and probe concentration) is synthesized by ABI (U.S.), purchased from English Weihe River victory base (Shanghai) trade
Company limited),Genotyping Master Mix is synthesized by ABI (U.S.), purchased from English Weihe River victory base (Shanghai)
Trade Co., Ltd, ddH2O is sterilizing deionized water.
PCR reaction condition: 95 DEG C, denaturation 10min；95 DEG C of degeneration 15s, 60 DEG C of annealing 1min, totally 40 circulations, in real time
Detect the signal intensity of two kinds of fluorescent dyes in whole amplification.
During detection, every kind of reaction system all arranges 2 blanks being not added with template DNA, to facilitate software to the back of the body
Scenery is corrected.
After reaction terminates, it is also possible to use instrument to carry software, the Allelic Disc merit in data analysis module
Can directly carry out sentencing type in interface, sentence type result and see Fig. 2:
Pass through Fig. 2, it can be seen that together, wherein, what square represented is that TT isozygotys to the individual cluster of homologous genes type
Son；What triangle represented is GT heterozygote；Circular representative is GG homozygote.
The backward sequencing result of different genotype individuality is shown in Fig. 3, and a) only one of which peak at arrow indication mutational site, right
Answer A base, because being backward sequencing, so corresponding individual genotype is TT saltant type, b) at arrow indication mutational site
Having 2 peaks, corresponding A, two kinds of bases of C, because being backward sequencing, so corresponding idiotype is GT type, for heterozygote；C) exist
Having a peak at arrow indication mutational site, corresponding base is C, because being backward sequencing, so corresponding individual genotype is
GG, wild type.
To sum up result shows, to 155 individuality Taqman sonde method testing results and known PCR primer sequencing result pair
More completely the same than both displays, TaqMan probe real time fluorescence quantifying PCR method is described accurately and reliably.
When 155 individualities are detected by method provided by the present invention, use 96 orifice plates, only use two pieces of PCR plate
I.e. complete the judgement to result.Time-consuming only 4-5 hour.And comprise 4 occurred in embodiment 1 and cannot complete amplification
, it is relatively low to the requirement ratio of genome that this is primarily due to TaqMan SNP classifying method, can meet need from 10-100ng
Want, even if genome concentration is the lowest or it is the poorest to extract quality, all without again extracting genome, and do not interfere with detection
The judgement of genotyping result.
And if using method or the method for PCR-RFLP of the PCR primer direct Sequencing of embodiment 1, all to genome
There are the highest requirement, otherwise PCR amplification instability, or product amount is very few, affects follow-up enzyme action and examining order.Additionally,
In addition to PCR amplification step, the method for PCR primer direct Sequencing or the method for PCR-RFLP, in addition it is also necessary to increase order-checking or
The step of enzyme action, the most at least increases by more than 10 hours.So TaqMan SNP classifying method of the present invention has obviously
Advantage.
The different toe number phenotype of embodiment 3 and the genetic development research of genotype chicken
Select different toe number phenotype and the cock hen copulation of different genotype combination, the something lost of research different genotype respectively
Pass rule.Specific experiment operation is as follows:
(1) double four toe phenotypes: 7 GG type cocks, with 20 GG type hen copulation；3 TT cocks, and 7 TT hens, 7
GT hen；(2) double five toe phenotypes: 8 TT cocks, with 20TT hen and 6 GT hens；Two GT cocks and 2 GT hens.
Offspring to above-mentioned copulation type carries out the observation registration of toe type respectively.The results are shown in Table 2:
The different toe number phenotype of table 2 and genotype chicken post-coitum are for toe type result
By the offspring of CC type seen from table 2 Yu CC type, 100% is all double four toes, and this result of the test absolutely proves, if GG
The cock of genotype and hen copulation, then offspring is entirely double four toes individualities.
And offspring's great majority of AA Yu AA or AC type show as double five toes, make a concrete analysis of as follows:
Toe number phenotype is TT type and TT type or the GT type chicken copulation of double four toes, and its offspring still there will be the five of higher proportion
Toe chicken, double five toe ratios about 63%, single five toe ratios about 19%, certainly, colony yet suffers from about 18% ratio
Double four toes are individual.
But, even the TT type of double five toes and TT type or GT type chicken copulation, offspring still there will be double the four of low ratio
Toe phenotype, about 4%.
That is, it is understood that there may be some inhibitive factor can suppress it to express, but can't affect the heredity of five toes.Prominent
Displacement point rs80659072 can apply in breeding as a Beijing Fatty Chicken five toe character candidates, although offspring
Might not all show as five toes, but the ratio of colony offspring five toe phenotype can be greatly improved.
Claims (10)
1. the method detecting Beijing Fatty Chicken five toe character, it is characterised in that include being examined by Real-Time Fluorescent Quantitative PCR Technique
The step of the genotype in the rs80659072 mutational site in ZRS region in survey LMBR1 gene the 5th intron.
Method the most according to claim 1, it is characterised in that said method includes according in LMBR1 gene the 5th intron
The sequence in ZRS region, design a pair fluorescence quantification PCR primer and with primer with the use of TaqMan probe, utilize these
Primer and probe, use the step of the genotype in Real-Time Fluorescent Quantitative PCR Technique detection rs80659072 mutational site；
Wherein, in described LMBR1 gene the 5th intron, the nucleotide sequence of ZRS region is as shown in SEQ ID NO:5.
Method the most according to claim 2, it is characterised in that the primer of described quantitative fluorescent PCR is: be used for expanding ZRS
The nucleotide sequence of the fluorescence quantification PCR primer pair of the sequence in region is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2；
With
With above-mentioned PCR primer with the use of TaqMan fluorescent probe nucleotides sequence be classified as: the core of wild-type allele probe
Nucleotide sequence is as shown in SEQ ID NO:3, and the nucleotide sequence of mutant allele probe is as shown in SEQ ID NO:4.
Method the most according to claim 3, it is characterised in that the reaction system of described quantitative fluorescent PCR is: genome
DNA 1 μ L, 40 times of probes concentrated and primer mixed liquor 0.25 μ L, universal PC R mixed liquor 5 μ L, with water polishing to 10 μ L.
Method the most according to claim 3, it is characterised in that the reaction condition of described quantitative fluorescent PCR is: 95 DEG C, in advance
Degeneration 10min；95 DEG C of degeneration 15s, 60 DEG C of annealing 1min, totally 40 circulations.
6., for identifying the primer of Beijing Fatty Chicken five toe character, it is the primer pair for expanding ZRS region, its nucleotide sequence
Respectively as shown in SEQ ID NO:1 and SEQ ID NO:2.
7. with described in claim 6 PCR primer with the use of TaqMan fluorescent probe, it includes that wild-type allele is visited
The nucleotide sequence of pin is as shown in SEQ ID NO:3；The nucleotide sequence of mutant allele probe such as SEQ ID NO:4 institute
Show.
8. a test kit, it is characterised in that comprise the primer described in claim 6 and the fluorescent probe described in claim 7
Combination.
9. the method described in any one of Claims 1 to 5, it is characterised in that the application in selection-breeding Beijing Fatty Chicken five toe character.
10. the application in detection Beijing Fatty Chicken five toe character of the test kit described in claim 8.
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