CN105861657A - Cow mastitis resistance related CNV fragment and applications thereof - Google Patents
Cow mastitis resistance related CNV fragment and applications thereof Download PDFInfo
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Abstract
The invention relates to molecular biology, and concretely discloses a cow mastitis resistance related CNV (copy number variation) fragment and applications thereof. The CNV fragment is in Chinese Holstein cow TRAPPC9 gene, and the nucleotide sequence of the CNV fragment is represented by SEQ ID NO.1. The fragment is characterized in that the mastitis resistance of Chinese Holstein cows with a low copy number is substantially higher than that of Chinese Holstein cows with a high copy number. The invention also provides a primer used for detecting the fragment, and a kit containing the primer. The invention further provides an application of the CNV fragment in identification of mastitis resistance dominant varieties, and an application of the CNV fragment in Chinese Holstein cow molecular marker assisted breeding.
Description
Technical field
The present invention relates to molecular biology, specifically, relate to a kind of anti-with mammitis of cow
Property relevant CNV fragment.
Background technology
Mammitis of cow is one of disease that milch cow is most common, harm is maximum, is also to affect the world
One of principal disease of milk cattle cultivating, the Milk Production loss height that the whole world causes because of this disease every year
Reach 3,800,000 tons.Mammitis of cow not only can affect the milk yield of milch cow, reduces the product of raw material milk
Matter, also can affect milch cow normal physiological function, extends the post-partum estrus time, and serious meeting causes
Milch cow is eliminated too early.Mammitis of cow mainly uses antibiotic etc to treat, so at present
And effect is unsatisfactory and other can be caused to negatively affect, the most numerous experts and scholars seek
Ask and solve this problem by other approach, and genetic approach is considered as a kind of reduction milk Lac Bovis seu Bubali
The long term policy of room inflammation sickness rate.
But the heritability of mammitis of cow is relatively low, only 0.02-0.04, cause traditional breeding
Method genetic progress is slow, and Selection effect is inconspicuous.Along with molecular genetics, quantitative genetics and
Developing rapidly of Protocols in Molecular Biology, the molecular breeding skill with genome selection technique as representative
Art is progressively applied in Animal Breeding, and produces tremendous influence.Copy number variation (Copy
Number Variation, CNV) refer to compared with genome reference sequences, big in genome
DNA fragmentation in 1kb inserts, lacks or expand and be combined with each other the complicated variation derived.
CNVs and complex disease, the susceptibility etc. of disease is shown at model animal and physianthropy research
There is important relationship.Therefore, as a molecular genetic marker, CNVs can be applied to molecule educate
Kind.
Molecular breeding, i.e. molecular marker assisted selection breeding, refer to utilize DNA molecular heredity mark
Breeding material is selected by note, comprehensive improvement poultry important economical trait.Molecular breeding is comprehensive
Make use of genetic marker information, pedigree information and individual phenotypic information, be traditional genetic breeding with
The breeding method that modern molecular biology organically combines.Genetic marker refer to heredity, can
That identify and that genetic polymorphism can be accurately reflected biological characteristic.Molecular breeding is that Animal Breeding is opened
Ward off a new approach, along with the development of Protocols in Molecular Biology, the reduction of testing cost,
Molecular genetic marker has been applied to Animal Breeding at present, and achieves tremendous influence.At milk
In cattle breeding, by select associate significantly with milch cow important economical trait, and with quantitative trait base
Because of the closely linked DNA molecular marker of seat, realize Seedling selection, accelerate genetic progress and carry
The targets such as high selection accuracy, thus accelerate breeding process.
Summary of the invention
In order to solve problems of the prior art, it is an object of the invention to provide one and milk
The CNV fragment that Mammitis of cattle resistance is relevant.
In order to realize the object of the invention, technical scheme is as follows:
First aspect, present invention firstly provides a kind of CNV relevant to mammitis of cow resistance
Fragment, the nucleotide sequence of described CNV fragment is as shown in SEQ ID NO.1.This fragment is positioned at
, there is copy number variation in Chinese holstein cattle TRAPPC9 gene.
The present invention finds through test, and the Chinese Holstein Mammitis of cattle that described fragment copy number is low resists
Property is significantly higher than the Chinese holstein cattle that this fragment copy number is high.
Further, the present invention is provided to expand the primer of described CNV fragment, described primer
Nucleotide sequence as shown in SEQ ID NO.2-3, this primer can expand mesh with high specificity
Tap section.
Further, the invention provides the test kit containing aforementioned primer.
Described test kit may also include other conventional constituents, such as SYBR Green Master,
ddH2O etc..
In actual application, in order to realize the qualification to mammitis of cow resistance or detection, described examination
Agent box also includes the primer expanding reference gene BTF3 gene, in order to when detection by with interior
The comparison of ginseng gene, preferably judges result.Described reference gene BTF3 gene
Copy number is stable, the nucleotide sequence of its primer as shown in SEQ ID NO.4-5, this primer energy
Enough amplification BTF3 genes with high specificity.
Described reference gene can also use the gene conduct having been demonstrated have stable copy number
Reference gene.
Second aspect, the invention provides aforementioned CNV fragment and is identifying Mastitis resistance milch cow product
Application in kind.
The diagnosis of described application non-diseases and therapeutic purposes, be only used for identifying that the mastitis of milch cow resists
Property character.
Further, described application is the copy number variation (copy number utilizing aforementioned CNV fragment
Just) identify mammitis of cow resistance.
Concrete operations are: with the genomic DNA of testing sample as template, utilize aforementioned primer with
The primer of aforementioned reference gene BTF3 gene carries out quantitative fluorescent PCR reaction, utilizes 2-ΔΔctSide
Method calculates the copy number of testing sample CNV fragment, the milch cow that described CNV fragment copy number is low
Mastitis resistance is significantly higher than the milch cow that this fragment copy number is high, and the individuality of low copy number is breast
Scorching resistance advantage is individual.
Further, quantitative fluorescent PCR reaction offer is provided and preferably expands body
System and reaction condition.
Wherein, described amplification system is 20 μ L:50ng/ μ L genomic DNA template 1 μ L,
10pmol/ μ L forward primer F 1 μ L and reverse primer R 1 μ L, SYB Green Master
10 μ L, ddH2O 7μL。
Specifically, during amplification CNV, use the primer shown in SEQ ID NO.2-3, amplification
During reference gene, use the primer shown in SEQ ID NO.4-5.
Wherein, described reaction condition is: 1. denaturation 95 DEG C, 10min；2. amplified reaction:
95 DEG C of degeneration 10s, 60 DEG C of annealing 10s, 72 DEG C extend 10s, 45 circulations；3. melt curve analysis is raw
Become: 95 DEG C of 5s, 65 DEG C of 1min, 97 DEG C of 10s；4. 40 DEG C of coolings.
The third aspect, the invention provides aforementioned CNV fragment answering in milch cow assistant breeding
With.Fluorescence quantifying PCR method is specially utilized to detect the copy number of aforementioned CNV fragment, according to
The height screening Mastitis resistance Dominant variety of copy number carries out selection-breeding.
As preferably, described milch cow is Chinese holstein cattle.
The beneficial effects of the present invention is:
The copy number of the TRAPPC9 gene of china holstein cows is detected by the present invention, and
It is associated analyzing to the copy number of this gene and the Mastitis resistance of milch cow, passes through SAS9.0
The X 2 test of software and the T check analysis of Excel find, mastitis ill cattle TRAPPC9
The copy number of gene is significantly higher than healthy cattle (P < 0.05), utilizes SAS9.0 software to carry out variance
Analyze it has also been found that the copy number appreciable impact Contents in Cows cell score of TRAPPC9 gene.This copy
The molecular marker assisted selection that detection is china holstein cows Mastitis resistance of number variation carries
Supply scientific basis.
The CNV fragment that the present invention provides is not limited by milch cow Individual Age, sex etc., can be used for
The early stage selection-breeding of Chinese holstein cattle, thus the breeding being greatly accelerated china holstein cows is entered
Journey.
Accompanying drawing explanation
Fig. 1 is distributed in being detected Chinese holstein cattle colony by TRAPPC9 gene copy number.
Fig. 2 is different udder health state Chinese holstein cattle TRAPPC9 gene copy numbers.
Detailed description of the invention
Below in conjunction with embodiment, the preferred embodiment of the present invention is described in detail.Need
Being understood by providing merely to play descriptive purpose of following example, it is right to be not used to
The scope of the present invention limits.Those skilled in the art without departing substantially from spirit of the invention and
In the case of spirit, the present invention can be carried out various amendment and replacement.
Experimental technique used in following embodiment if no special instructions, is routine side
Method.
Material used in following embodiment, reagent etc., if no special instructions, all can be from business
Industry approach obtains.
The detection of the CNV labelling that embodiment 1 is relevant to china holstein cows Mastitis resistance
1.1 china holstein cows blood DNAs to be measured extract
Tail venous collection china holstein cows to be measured blood, room temperature is placed and is treated blood in 3-4 hour
Solidification, now blood is divided into serum and sludged blood two parts, and serum is clear yellow, sludged blood
For kermesinus, in bovine blood, only leukocyte is contained within DNA, is present in sludged blood.
Sky root blood/cell/tissue genome DNA sample is utilized to extract test kit from sludged blood
Middle extraction genomic DNA, specifically comprises the following steps that
Sterilized 2mL round bottom centrifuge tube is put into the sludged blood of eye scissors clip 0.2-0.3mL
In, add the cell pyrolysis liquid CL of 500 μ L；
Utilizing hand-held Syrup-homogenizing instrument to be fully homogenized by clot, vibration 15s, 12000rpm are centrifuged 1min,
Discard upper strata kermesinus supernatant；
Again adding 700 μ L cell pyrolysis liquid CL, fully vibration makes lower sediment scatter suspension,
12000rpm is centrifuged 1min, abandoning supernatant；
Adding 200 μ L buffer GS, abundant vortex fully suspends to precipitation；
Add 20 μ L E.C. 3.4.21.64s and 250 μ L buffer GB, fully mix；
Seal centrifuge tube sealed membrane to put in 56 DEG C of hybrid heaters and digest 3-4 hour, for really
Protecting fully digestion, need reverse mixing for several times in digestion process, final solution becomes limpid transparent,
Brief centrifugation；
Add 200 μ L and ice dehydrated alcohol, turn upside down mixing 15s, now it is possible that
Flocculent deposit；
Proceeding in adsorption column CB3 by previous step gained solution, adsorption column is put in collecting pipe,
12000rpm is centrifuged 30s, discards the waste liquid in collecting pipe, is put in collecting pipe by adsorption column；
In adsorption column CB3, add 500 μ L Deproteinization buffer GD, stand 2min,
12000rpm is centrifuged 30s, discards the waste liquid in collecting pipe, is put in collecting pipe by adsorption column；
Adding 700 μ L rinsing liquid PW in adsorption column CB3,12000rpm is centrifuged 30s, abandons
Remove the waste liquid in collecting pipe, adsorption column is put in collecting pipe；
Adding 500 μ L rinsing liquid PW in adsorption column CB3,12000rpm is centrifuged 30s, abandons
Remove the waste liquid in collecting pipe；
Being put into by adsorption column in collecting pipe, 12000rpm is centrifuged 2min, discards waste liquid, will inhale
Attached column is placed in room temperature and places several minutes, thoroughly remaining on volatilization adsorption column ethanol；
Adsorption column is proceeded to a new centrifuge tube, add the TE of 100 μ L 56 DEG C preheating
Buffer, room temperature place 5min, 12000rpm be centrifuged 2min, genomic DNA then exists
In centrifuge tube.
Utilize the matter of NanoDrop2000 UV spectrophotometer measuring extracting genome DNA
Amount and concentration, and take out a part of DNA, it is diluted to unified concentration.
1.2 design of primers
Use Primer 3.0 and Oligo 6.0 software separately design genes of interest TRAPPC9 and
Internal reference, with regard to the fluorescence quantification PCR primer of BTF3, is divided into for CNV section (CNVR)
Two sections, every section of sequence taking 10Kb is to screen suitable primer；For gene expression,
Take the method crossing over different exon to be designed, follow following former during design of primers
Then:
(1) between pcr amplified fragment length 100-200；
(2) primer G/C content is between 40-60%；
(3) primer length is between 18-24bp, and annealing temperature (Tm value) is at 58-62 DEG C；
(4) 3 ' ends of primer the most do not end up with A or T, and 3 ' ends are avoided sending out simultaneously
The situation that raw continuous 3 bases are connected, it is to avoid hairpin structure and primer dimer；
(5) avoid upstream and downstream primer or primer self that hairpin structure or primer dimerization occur as far as possible
Body.
For the primer of copy number, need to utilize UCSC PCR to carry out primer specificity detection.
1.3 primer detections
Grads PCR is utilized to carry out primer amplification specificity and annealing temperature detection, high specificity
Primer purpose band become clear, there is no dimer and non-specific miscellaneous band；Unwise to annealing temperature
The amplification change in the biggest temperature range of the primer of sense is little.
The standard substance of the doubling dilutions such as utilization carry out fluorescent quantitative PCR to draw mark
The amplification efficiency of directrix curve detection primer, in combination with amplification curve, melt curve analysis with melt peak
Carry out Comprehensive Evaluation primer the most suitable.Specifically comprise the following steps that
1. by DNA sample with Nuclease free water according to 4 gradients of 5 doubling dilution,
I.e. 1,1/5,1/25,1/125；
2. with this template as standard substance, every pair of primer is carried out quantitative fluorescent PCR reaction；
3. carry out the calculating of amplification efficiency, standard curve according to the operating instruction of instrument and melt song
The drafting of line.
Quantitative fluorescent PCR reaction system is: standard substance genomic DNA template 1 μ L,
10pmol/ μ L forward primer F 1 μ L and reverse primer R 1 μ L, SYB Green Master
10 μ L, ddH2O 7μL.Reaction condition is 1. denaturation 95 DEG C, 10min；2. amplified reaction:
95 DEG C of degeneration 10sec, 60 DEG C of annealing 10sec, 72 DEG C extend 10sec, 45 circulations；3. melt
Solution curve generates: 95 DEG C of 5sec, 65 DEG C of 1min, 97 DEG C of 10sec；4. 40 DEG C of coolings.
Specifically, during amplification CNV, use the primer shown in SEQ ID NO.2-3, amplification
During reference gene, use the primer shown in SEQ ID NO.4-5.
1.4 sample to be tested copy number variation detections
The DNA sample to be detected being diluted to uniform concentration is carried out genes of interest respectively
The quantitative fluorescent PCR reaction of TRAPPC9 and reference gene BTF3,3 weights of each sample
Multiple, reaction system is identical with previous step with condition.Operating instruction according to instrument obtains CT value,
Then 2 are utilized-ΔΔctMethod calculates the copy number of Chinese holstein cattle TRAPPC9 gene to be measured.
1.5 above-mentioned CNV are marked in Chinese holstein cattle Mastitis resistance Dominant variety selection-breeding
Application
This CNV labelling can as molecular genetic marker milch cow directly carried out gene Selection or
Marker assisted selection, thus accelerate the selection-breeding of Chinese holstein cattle Mastitis resistance Dominant variety.
Embodiment 2 china holstein cows TRAPPC9 gene copy number and the pass of Mastitis resistance
Connection is analyzed and detection application
According to the method for embodiment 1, to picking up from 133 of different breeding field, Northern Part of China
Chinese holstein cattle carries out the analysis of TRAPPC9 gene copy number, analysis result such as table 1,
Shown in Fig. 1, TRAPPC9 gene copy number is that the individuality of 4 is at Holstein cow colony intermediate frequency
Rate is the highest, reaches 36.8%, next to that the individuality of 5 copy numbers, accounts for Holstein cow colony
27.8%, minimum is the individuality of 8 copy numbers, frequency only 0.8%.
Utilize SAS9.0 software ANOVA program analyze TRAPPC9 gene copy number with
The incidence relation of somatic cell grade, during analysis using individual somatic number as the criteria for classifying be:
SCC≤200,000, grade is 1；200000 < SCC < 500,000, grade is 2；SCC >=500,000,
Grade is 3.Using grade as phenotypic number, use such as drag:
Y=μ+hys+p+m+g+e
Wherein, y: observed value, μ: colony's average, hys: season in field year effect, p: parity imitate
Should, m: lactation stage is imitated, g: copy number effect, e: random residual effect.Analysis result
As shown in table 2.
The copy number of table 1TRAPPC9 gene
Table 2 china holstein cows TRAPPC9 gene copy number is analyzed with Mastitis resistance trait associations
Numeric representation in table 2 is least square mean value ± standard error, and in table, same letter represents
Difference is not notable, and different letter representation significant differences (P < 0.05), * represents significant correlation
(P<0.05)。
Found by the T check analysis of Excel, mastitis ill cattle TRAPPC9 gene
Copy number is significantly higher than healthy cattle (P < 0.05), and result is as shown in Figure 2.
From table 2, Fig. 2, the copy number of TRAPPC9 gene is to Chinese holstein cattle breast
The impact of scorching resistance reaches significant level (P < 0.05).Low copy number Mammitis of cattle resistance is notable
Cattle higher than high copy number.
Embodiment 3 is for expanding the primer of CNV fragment of the present invention
Described primer includes:
Forward primer F1:5 '-CAAGACTCTGCTGAGATGACG-3 '；
Reverse primer R1:5 '-CGACCATCACCCAGACTATGC-3 '.
Embodiment 4 test kit
Described test kit includes:
1, the primer described in embodiment 3；
2, the primer of reference gene BTF3 gene:
BTF3 forward primer F2:5 '-TCAGGGAGATTACTAAGGGT-3 '；
BTF3 reverse primer R2:5 '-GAAGCAGAAGTCTAAGCAAC-3 '.
3, SYB Green Master: purchased from ROCHE company.
4、ddH2O。
Although, the most with a general description of the specific embodiments the present invention has been made in detail
Most description, but on the basis of the present invention, it can be made some modifications or improvements, this is to this
It is apparent from for skilled person.Therefore, on the basis without departing from spirit of the present invention
Upper these modifications or improvements, belong to the scope of protection of present invention.
Claims (10)
1. a CNV fragment relevant to mammitis of cow resistance, it is characterised in that described
The nucleotide sequence of CNV fragment is as shown in SEQ ID NO.1.
2. for expanding the primer of CNV fragment described in claim 1, it is characterised in that institute
State the nucleotide sequence of primer as shown in SEQ ID NO.2-3.
3. contain the test kit of primer described in claim 2.
4. the CNV fragment described in claim 1 is in identifying Mastitis resistance dairy bread
Application.
Application the most according to claim 4, it is characterised in that utilize claim 1 institute
The copy number height stating CNV fragment identifies mammitis of cow resistance.
Application the most according to claim 5, it is characterised in that with the gene of testing sample
Group DNA is template, utilizes primer described in claim 2 and reference gene primer to carry out fluorescence fixed
Amount PCR reaction, utilizes 2-△△ctMethod calculates the copy number of testing sample CNV fragment, described
The mammitis of cow resistance that CNV fragment copy number is low is significantly higher than the milk that this fragment copy number is high
Cattle；Described reference gene is BTF3 gene, the nucleotide sequence of reference gene primer such as SEQ ID
Shown in NO.4-5.
Application the most according to claim 6, it is characterised in that quantitative fluorescent PCR is anti-
The amplification system answered is 20 μ L:50ng/ μ L genomic DNA template 1 μ L, and 10pmol/ μ L is just
To primers F 1 μ L and reverse primer R 1 μ L, SYB Green Master 10 μ L, ddH2O
7μL；
The reaction condition of quantitative fluorescent PCR reaction is: 1. denaturation 95 DEG C, 10min；2. expand
Increase reaction: 95 DEG C of degeneration 10s, 60 DEG C of annealing 10s, 72 DEG C extend 10s, 45 circulations；3. melt
Solution curve generates: 95 DEG C of 5s, 65 DEG C of 1min, 97 DEG C of 10s；4. 40 DEG C of coolings.
8. the application in milch cow assistant breeding of the CNV fragment described in claim 1.
Application the most according to claim 8, it is characterised in that utilize quantitative fluorescent PCR
Method test right requires the copy number of CNV fragment described in 1, screens according to the height of copy number
Mastitis resistance Dominant variety carries out selection-breeding.
Application the most according to claim 8 or claim 9, it is characterised in that during described milch cow is
State's Holstein cow.
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| Application Number | Priority Date | Filing Date | Title |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110564829A (en) * | 2019-09-30 | 2019-12-13 | 西北农林科技大学 | Method for auxiliary detection of lactation traits of dairy cow NCAM2 gene CNV marker and special kit thereof |
| CN110592235A (en) * | 2019-09-30 | 2019-12-20 | 西北农林科技大学 | Method for detecting USP16 gene CNV marker of dairy cow and application thereof |
| CN111506881A (en) * | 2020-06-11 | 2020-08-07 | 中国农业大学 | System for predicting Chinese Holstein cow mastitis onset risk |
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| CN101942522A (en) * | 2010-10-14 | 2011-01-12 | 扬州大学 | Assay kit for identifying dairy cow mastitis resistance-related molecular marker |
| CN104611425A (en) * | 2015-01-15 | 2015-05-13 | 中国农业大学 | SNP (single nucleotide polymorphism) molecular marker related with staphylococcus aureus mastitis resistance of Chinese Holstein cows and application of SNP molecular marker |
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| Title |
|---|
| WANG X等: "Genome-wide association study in Chinese Holstein cows reveal two candidate genes for somatic cell score as an indicator for mastitis susceptibility.", 《BMC GENETICS》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564829A (en) * | 2019-09-30 | 2019-12-13 | 西北农林科技大学 | Method for auxiliary detection of lactation traits of dairy cow NCAM2 gene CNV marker and special kit thereof |
| CN110592235A (en) * | 2019-09-30 | 2019-12-20 | 西北农林科技大学 | Method for detecting USP16 gene CNV marker of dairy cow and application thereof |
| CN110564829B (en) * | 2019-09-30 | 2022-11-04 | 西北农林科技大学 | Method for auxiliary detection of lactation traits of dairy cow NCAM2 gene CNV marker and special kit thereof |
| CN111506881A (en) * | 2020-06-11 | 2020-08-07 | 中国农业大学 | System for predicting Chinese Holstein cow mastitis onset risk |
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