CN106319088A - Specific kit for detecting cow HH5 haplotype and detecting method thereof - Google Patents
Specific kit for detecting cow HH5 haplotype and detecting method thereof Download PDFInfo
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- C12Q1/6858—Allele-specific amplification
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Abstract
The invention discloses a specific kit for detecting cow HH5 haplotype and a detecting method thereof. The kit is composed of a primer 1, a primer 2 and a primer 3; the primer 1 is a single stranded DNA molecule presented in the sequence 1; the primer 2 is a stranded DNA molecule presented in the sequence 2; and the primer 3 is a stranded DNA molecule presented in the sequence 3. The experiment shows that: the built method for detecting whether the milk cow carries the HH5 haplotype is fast and accurate, and is a method and idea provided for the molecule screening of the milk cow HH5 haplotype. The technical support is provided for designedly reducing the HH5 haplotype frequency in the later breeding work, the evidence is provided for the scientific seed selection and apolegamy in a dairy farm.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of dedicated kit detecting cattle HH5 haplotype and inspection thereof
Survey method.
Background technology
In cattle breeding, due to the application of the GENERALIZATION OF MODERN BREEDING TECHNIQUE such as progeny testing, genome selection, it is being greatly enhanced
While selection accuracy, improve selection intensity the most potentially.Due to answering of the breeding biotechnology such as artificial insemination (AI)
With, make indivedual outstanding breeding oxen be used widely, add the coefficient of inbreeding of colony, reduce effective population size, recessive
The probability of gene pure strengthens, and makes bad gene constantly occur in milch cow colony, along with deepening continuously of genomics research
With the fast development of high throughput sequencing technologies, accelerate milk cow recessive genetic defect dig according to, identify and apply.
Haplotype, refers to carry out allele on multiple locus of coinheritance on same chromosome on hereditism
Combination.Harmful haplotype is the new model of the new recessive inheritance's defect occurred in cows in recent years, in Holstein cow group
Having been found that more than 20 kind of haplotype, the haplotype successfully carrying out deciding field has 15 kinds.When these haplotype recessiveness are isozygotied, just
Different types of clinical symptoms, embryo's Deaths etc. can be shown, be the Silent Killer affecting milk cows breeding health, seriously
The economic benefit of the person that affects milk cattle cultivating.
Haplotype 5 (HH5, olstein Haplotype 5) is newfound a kind of recessive harmful in Holstein cow group
Haplotype, when HH5 haplotype recessiveness is isozygotied, causes cow embryo early lethality, has had a strong impact on cow reproduction rate, to milch cow
Culturist brings huge economic loss.
The Molecular and genetic basis of HH5 is to have lacking of 138kb (BTA9:93,233kb-93,371kb) on No. 9 chromosomes of cattle
Losing, mitochondrial transcription factor B1 gene (transcription factor B1mitochondrial, TFB1M) is just at this
In deleted segment, the translation of TFB1M gene pairs mitochondrial protein starts most important, and its major function is to make mitochondrion 12s rRNA
Adenine residue in the hairpin loop of 3' end methylates, and the 12s rRNA little ribosomal subunit that is mitochondrion synthesis and play merit
Can be necessary, so TFB1M is the reason gene causing the lethal haplotype of HH5, it is verified by mouse model.Germany
In Holstein cow group, HH5 carrier ratio is 5.5%, has higher ratio in cows.
Cow frozen semen and the universal and extensively application of technology of artificial insemination, cause the impact of outstanding breeding oxen and use model
It is global for enclosing.One outstanding breeding oxen can produce the most up to a million doses of frozen semens of hundreds of thousands agent all one's life, its heredity shadow
Ring and well imagine.Due to the circulation in worldwide of the outstanding breeding oxen frozen semen be widely used, in Holstein cow group
What the harmful haplotype found brought is all global problem.This causes cattle breeding worker in recent years paying attention to outstanding kind
While the performance of bull production performance, more pay attention to whether outstanding breeding oxen carries the impact of some undesirable recessive genes.Therefore,
Set up the molecular biology method of a kind of effective detection HH5 haplotype, formulate reasonable seed selection and selective pairing scheme, be that reduction HH5 is pure
The best method of zygote.At present, China not yet carries out relevant research and detection work, and HH5 haplotype is at China's Holstein cow
Carriage in Qun is unclear.
Summary of the invention
First purpose of the present invention is a kind of to detect whether milch cow to be measured carries the primer set of HH5 haplotype.
The primer set that the present invention provides is made up of primer 1, primer 2 and primer 3；
Described primer 1 is the single strand dna shown in sequence 1；
Described primer 2 is the single strand dna shown in sequence 2；
Described primer 3 is the single strand dna shown in sequence 3.
In above-mentioned primer set, the mole of described primer 1, described primer 2 and described primer 3 is than for 1:1:1.
Second object of the present invention is to provide and a kind of detects whether milch cow to be measured carries the PCR reagent of HH5 haplotype.
The PCR reagent that the present invention provides includes above-mentioned primer set.
In above-mentioned PCR reagent, described primer 1, described primer 2, the described primer 3 final concentration in described PCR reagent are equal
For 20pmol/L.
Third object of the present invention is to provide and a kind of detects whether milch cow to be measured carries the test kit of HH5 haplotype.
What the present invention provided detect whether milch cow to be measured the carry test kit of HH5 haplotype includes above-mentioned primer set or on
State PCR reagent.
Fourth object of the present invention is to provide the new use of above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box
On the way.
The invention provides above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box following 1) or 2) in any one
In application:
1) detect whether milch cow to be measured carries HH5 haplotype；
2) preparation detects whether milch cow to be measured carries the product of HH5 haplotype.
5th purpose of the present invention is to provide and a kind of detects the method whether milch cow to be measured carries HH5 haplotype.
Whether the detection milch cow to be measured that the present invention provides carries the method for HH5 haplotype comprises the steps:
With the genomic DNA of milch cow to be measured as template, use above-mentioned primer set to carry out PCR amplification, obtain PCR amplification and produce
According to described pcr amplification product, thing, judges whether milch cow to be measured carries HH5 haplotype:
If described pcr amplification product contains only the band that size is 442bp, milch cow the most to be measured does not carry HH5 haplotype；
If described pcr amplification product contains band that size is 442bp and 256bp or contains only the bar that size is 256bp
Band, milch cow the most to be measured carries HH5 haplotype.
In said method, if the genotype of milch cow is HH5 isozygoty haplotype time, pcr amplification product contains only size and is
The band of 256bp, but genotype is HH5, and the isozygoty milch cow of haplotype will be miscarried, so examining in reality brephic time
In survey, pcr amplification product is generally the following two kinds situation: (1) pcr amplification product contains only the band that size is 442bp；(2)
Containing the band that size is 442bp and 256bp.
In said method, the annealing temperature of described PCR is 58 DEG C.
In above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box or above-mentioned application or said method,
Described HH5 haplotype is that HH5 isozygotys haplotype or HH5 heterozygosis haplotype；
The described HH5 haplotype that isozygotys refers to the 93,232,651-of two articles of homologous chromosomes on individual No. 9 chromosomes of cattle
All there is the genotype of disappearance in 93,370,998 nucleic acid molecules；
Described HH5 heterozygosis haplotype refers to the 93,232,651-of one article of homologous chromosome on individual No. 9 chromosomes of cattle
93,370,998 nucleic acid molecules lack, and the 93rd, 232,651-93 of another article of homologous chromosome the, 370,998
There is not the genotype of disappearance in nucleic acid molecule.
In above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box or above-mentioned application or said method,
Described milch cow is Holstein cow.
The present invention establishes a kind of method whether milch cow of detection fast and accurately carries HH5 haplotype, for milch cow HH5
The Molecular screening of haplotype provides a kind of method and thinking, in a planned way reducing HH5 haplotype in breeding work from now on
Frequency provides technical support, and the seed selection and selective pairing carrying out science for cattle farm provides foundation.
Accompanying drawing explanation
Fig. 1 is the gel electrophoresis figure of grads PCR annealing temperature.Wherein, swimming lane 3:53.1 DEG C；Swimming lane 4:54.6 DEG C；Swimming lane
5:56.4 DEG C；Swimming lane 6:58.5 DEG C；Swimming lane 7:60.7 DEG C；Swimming lane 8:62.9 DEG C；Swimming lane 9:64.9 DEG C；Swimming lane 10:66.6 DEG C；Swimming
11:67.8 DEG C of road；Swimming lane 12:68.5 DEG C；M:100bp DNA ladder Marker.
Fig. 2 is the PCR electrophoretogram of detection cattle HH5 haplotype carrier.Wherein, the first swimming lane and the second swimming lane: healthy
Cattle (does not carry HH5 haplotype)；3rd swimming lane: HH5 haplotype carrier；4th swimming lane: 100bp DNA ladder
Marker。
Fig. 3 is healthy cattle and the order-checking peak figure of HH5 haplotype carrier.Wherein, Fig. 3 A is the survey that healthy cattle is individual
Sequence peak figure；Fig. 3 B is the order-checking peak figure of HH5 haplotype carrier.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Quantitative test in following embodiment, is respectively provided with three times and repeats experiment, results averaged.
HH5 haplotype in following embodiment refers to 93,232,651-93,370,998 on individual No. 9 chromosomes of cattle
Position nucleic acid molecule occurs disappearance to cause allelic combination, two homologous chromosomes on individual No. 9 chromosomes of cattle
The 93rd, 232,651-93,370,998 nucleic acid molecules when all there is disappearance, the individual genotype referred to as HH5 of cattle isozygotys list
Times type；As the 93,232,651-only having one article of homologous chromosome in two articles of homologous chromosomes on individual No. 9 chromosomes of cattle
When 93,370,998 nucleotide molecule fragments occur disappearance, the genotype of cattle individuality is referred to as HH5 heterozygosis haplotype (following enforcement
HH5 haplotype in example is HH5 heterozygosis haplotype), this cattle is HH5 haplotype carrier.
Embodiment 1, a kind of dedicated kit detecting cattle HH5 haplotype and detection method thereof
One, the primer of cattle HH5 haplotype is detected
According to gene order on 93233kb-93371kb section on No. 9 chromosomes of cattle on GenBank, and with reference to Sch ü tz
The primer sequence designed Deng (2016), it is determined that the specific primer sequence of detection HH5 carrier:
Forward primer HH5-F:5 '-AGATATGCTAAAGTTTACCTAGAAGAA-3 ' (sequence 1)；
Downstream primer HH5-R1:5 '-CTGAAGCTCCATTCTGAGTCAT-3 ' (sequence 2)；
Downstream primer HH5-R2:5 '-TGCTCTATGAATTTTGTGAATGGT-3 ' (sequence 3).
Wherein, F/R1 primer pair amplifies fragment length is 442bp；F/R2 primer pair amplifies fragment length is 256bp.
Two, the method detecting cattle HH5 haplotype
1, the extraction of cattle STb gene
Select the He Sitan carrying HH5 haplotype (genotype identification is HH5 heterozygosis haplotype) of Beijing's Breeding bull station
Cattle and healthy cattle are experiment material, extract STb gene respectively from blood, and STb gene can also be (such as, fresh or cold from seminal fluid
Freeze), tissue sample (such as ear tissue etc.) or containing hair follicle ox hair sample in extracting and developing and purification.
2, PCR amplification
Cattle STb gene in step 1, as template, uses primer HH5-F, HH5-R1 and the HH5-R2 in step one, with not
Same annealing temperature gradient (10 gradients: 53.1 DEG C；54.6℃；56.4℃；58.5℃；60.7℃；62.9℃；64.9℃；
66.6℃；67.8℃；68.5 DEG C) carry out multiplexed PCR amplification, obtain pcr amplification product.
PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 60 ± 8 DEG C of annealing 30s, 72 DEG C extend 30s, enter
Row 35 circulation；72 DEG C extend 7min, then 4 DEG C of preservations.
PCR reaction system (cumulative volume 25 μ l): dNTP mixture (4 × 2.5mmol/L) 2.0 μ l, HH5-F (20pmol/L)
0.5 μ l, HH5-R1 (20pmol/L) 0.5 μ l, HH5-R2 (20pmol/L) 0.5 μ l, Taq archaeal dna polymerase (3U/ μ l) 0.5 μ l,
Template DNA (50ng/ μ l) 0.5 μ l, ddH2O 18.0μl。
Through screening and the optimization of PCR amplification condition, the preferable annealing region of the PCR reaction system of above-mentioned primer is
54.5-60.5 DEG C, optimum annealing temperature is defined as T=58 DEG C.Fig. 1 is the gel electrophoresis figure of grads PCR annealing temperature.
3, genotype judges
Take 4 μ L pcr amplification products with 2% agarose gel electrophoresis detection and it to be checked order.
The electrophoretogram of PCR amplification as in figure 2 it is shown, sequencing result as shown in Figure 3.In Fig. 2, swimming lane 1 and swimming lane 2 are strong
The electrophoretogram of the PCR primer of the cattle of health, swimming lane 3 is the electrophoretogram of the cattle carrying HH5 haplotype.As can be seen from the figure: all
The pcr amplification product of healthy cattle contains only the band that size is 442bp, and the PCR of all cattle carrying HH5 haplotype expands
Containing size in product is 442bp (nucleotides sequence is classified as sequence 4) and the band of 256bp (nucleotides sequence is classified as sequence 5).
Therefore can detect milch cow to be measured with the following method and whether carry HH5 haplotype:
With the genomic DNA of milch cow to be measured as template, primer HH5-F, HH5-R1 and HH5-R2 is used to carry out PCR amplification,
Obtain pcr amplification product, judge whether milch cow to be measured carries HH5 haplotype according to pcr amplification product:
If pcr amplification product contains only the band that size is 442bp, milch cow the most to be measured does not carry HH5 haplotype；
If pcr amplification product contains band that size is 442bp and 256bp or contains only the band that size is 256bp, then
Milch cow to be measured carries HH5 haplotype.
Embodiment 2, the checking of method of detection cattle HH5 haplotype
1, genotype detection
The method utilizing the detection milch cow to be measured of step 2 in embodiment 1 whether to carry HH5 haplotype, plants public affairs to Beijing
The genotype of 40 Holstein cow at cattle station detects.
Result shows: 40 Holstein cow have the pcr amplification product of 2 Holstein cow contain size be 442bp and
The band of 256bp, is HH5 carrier (genotype is HH5 haplotype), and carrying rate is 5%.
2, sequence verification
The cattle individuality of 2 HH5 carriers is carried out sequence verification, and sequencing result shows: No. 9 dyeing of 2 HH5 carriers
On body the 93rd, 232,651-93 of one article of homologous chromosome the, 370,998 nucleic acid molecules lack, and another homology
The 93,232,651-93,370,998 position nucleic acid molecule of chromosome lacks.Illustrate that the present invention detects milch cow to be measured
Whether carry the method for HH5 haplotype accurately, reliably, and rate of accuracy reached 100%, can be used for detecting whether milch cow to be measured carries
HH5 haplotype.
Sequence table
<110>Beijing Milk Cow Center
<120>a kind of dedicated kit detecting cattle HH5 haplotype and detection method thereof
<160>5
<210>1
<211>27bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>1
agatatgcta aagtttacct agaagaa 27
<210>2
<211>22bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>2
ctgaagctcc attctgagtc at 22
<210>3
<211>24bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>3
tgctctatga attttgtgaa tggt 24
<210>4
<211>442bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>4
agatatgcta aagtttacct agaagaatat ataagcaaga agaagcaaaa aaaaagtgaa 60
aaggaaaaga gaagaaagaa agaaaaaagg agaagaaaga aagaagaaca gaacagaaag 120
aaagagaaga taagaaagag aaaaggaaat tcagcatcca gaagcatcat tgtaaaacct 180
aagatcataa agtaaccaga aagataacac taaattactt acaaataaac aacaataaat 240
ttgacagctg ccttctcaga agcagaagta gagaccaaat gataatgaag taatatcttc 300
aatgtgctga aggaacataa ctataaattg agatttttgt gcttagtgaa gttattgggc 360
aaaagtggag gccaaaaaac aaaagtgaag gcaaaataac agatattttc aaacaaaaga 420
atgactcaga atggagcttc ag 442
<210>5
<211>256bp
<212>DNA
<213>artificial sequence
<220>
<223>
<400>5
agatatgcta aagtttacct agaagaatat ataagcaaga agaagcaaaa aaaaagtgaa 60
aaggaaaaga gaagaaagaa agaaaaaagg agaagaaaga aagaagaaca gaacagaaag 120
aaagagaaga taagaaagag aaaaggaaat tcagcatcca gaagcatcat tgtaattgta 180
atcattgaat ggttctatga agacctacaa gacattttag aacctacacc ataccattca 240
caaaattcat agagca 256
Claims (9)
1. detecting whether milch cow to be measured carries a primer set for HH5 haplotype, it is made up of primer 1, primer 2 and primer 3；
Described primer 1 is the single strand dna shown in sequence 1；
Described primer 2 is the single strand dna shown in sequence 2；
Described primer 3 is the single strand dna shown in sequence 3.
Primer set the most according to claim 1, it is characterised in that: described primer 1, described primer 2 and described primer 3
Mole is than for 1:1:1.
3. detect whether milch cow to be measured carries the PCR reagent of HH5 haplotype, draw including complete described in claim 1 or 2
Thing.
PCR reagent the most according to claim 3, it is characterised in that: described primer 1, described primer 2, described primer 3 are in institute
The final concentration stated in PCR reagent is 20pmol/L.
5. detect whether milch cow to be measured carries the test kit of HH5 haplotype, draw including complete described in claim 1 or 2
PCR reagent described in thing or claim 3 or 4.
6. described in the primer set described in claim 1 or 2 or the PCR reagent described in claim 3 or 4 or claim 5
Test kit is following 1) or 2) in application in any one:
1) detect whether milch cow to be measured carries HH5 haplotype；
2) preparation detects whether milch cow to be measured carries the product of HH5 haplotype.
7. detect the method whether milch cow to be measured carries HH5 haplotype, comprise the steps:
With the genomic DNA of milch cow to be measured as template, the primer set described in claim 1 or 2 is used to carry out PCR amplification,
To pcr amplification product, judge whether milch cow to be measured carries HH5 haplotype according to described pcr amplification product:
If described pcr amplification product contains only the band that size is 442bp, milch cow the most to be measured does not carry HH5 haplotype；
If described pcr amplification product contains band that size is 442bp and 256bp or contains only the band that size is 256bp, then
Milch cow to be measured carries HH5 haplotype.
Method the most according to claim 7, it is characterised in that: the annealing temperature of described PCR is 58 DEG C.
PCR reagent described in primer set the most according to claim 1 and 2 or claim 3 or 4 or claim 5 institute
Application described in the test kit stated or claim 6 or the method described in claim 7 or 8, it is characterised in that:
Described milch cow is Holstein cow.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611029923.5A CN106319088A (en) | 2016-11-15 | 2016-11-15 | Specific kit for detecting cow HH5 haplotype and detecting method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611029923.5A CN106319088A (en) | 2016-11-15 | 2016-11-15 | Specific kit for detecting cow HH5 haplotype and detecting method thereof |
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| Publication Number | Publication Date |
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| CN106319088A true CN106319088A (en) | 2017-01-11 |
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ID=57816594
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611029923.5A Pending CN106319088A (en) | 2016-11-15 | 2016-11-15 | Specific kit for detecting cow HH5 haplotype and detecting method thereof |
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| CN (1) | CN106319088A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564862A (en) * | 2019-08-20 | 2019-12-13 | 中国农业大学 | Method for rapidly screening Holstein cattle HH5 genetic defect gene carrier |
-
2016
- 2016-11-15 CN CN201611029923.5A patent/CN106319088A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| EKKEHARD SCHÜTZ等: "The Holstein Friesian Lethal Haplotype 5(HH5) Results from a Complete Deletion of TBF1M and Cholesterol Deficiency (CDH) from an ERV-(LTR) Insertion into the Coding Region of APOB", 《PLOS ONE》 * |
| SE´BASTIEN FRITZ等: "Detection of Haplotypes Associated with Prenatal Death in Dairy Cattle and Identification of Deleterious Mutations in GART, SHBG and SLC37A2", 《PLOS ONE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564862A (en) * | 2019-08-20 | 2019-12-13 | 中国农业大学 | Method for rapidly screening Holstein cattle HH5 genetic defect gene carrier |
| CN110564862B (en) * | 2019-08-20 | 2020-09-01 | 中国农业大学 | Method for rapidly screening Holstein cattle HH5 genetic defect gene carrier |
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Application publication date: 20170111 |