CN105543352A - Method of detecting copy number variation of Qinchuan cattle FGF13 genes and application thereof - Google Patents
Method of detecting copy number variation of Qinchuan cattle FGF13 genes and application thereof Download PDFInfo
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Abstract
The invention discloses a method of detecting copy number variation of Qinchuan cattle FGF13 genes and application thereof. According to the method, based on real-time quantitative PCR, Qinchuan cattle genome DNA is used as a template, a pair of specific PCR primers is utilized for amplifying a copy number variation region of the Qinchuan cattle FGF13 genes, another pair of specific PCR primers is utilized for amplifying cattle general transcription factor 3 genes as reference at the same time, and lastly a 2-delta delta Ct method is utilized for calculating and determining the individual copy number variation type. The method lays a foundation for establishment of correlation between copy number variation of the Qinchuan cattle FGF13 genes and growth traits, and is beneficial to accelerating Qinchuan cattle molecular marker assisted selective breeding work, simple, quick and convenient to apply and popularize.
Description
Technical field
The invention belongs to molecule genetics research field, be specifically related to a kind of method detecting Qinchuan Cattle FGF13 gene copy number variation, the method utilizes genomic dna real-time quantitative PCR, with BTF3 gene for reference, according to 2
-Δ Δ Ctvalue thus determine individual copy number type.
Background technology
In the middle of the beef raising development of China, the growth rate of beef cattle and meat are two main restraining factors.And the molecular mechanism of research regulation and control Beef Cattle Growth and muscle development, theoretical foundation can be provided for the development of Chinese beef raising.Numerous research shows, in the middle of the process that somatomedin is grown at conditioner bulk-growth, play vital effect, and fibroblast growth factor (FibroblastGrowthFactor, FGF) just plays such effect.This family protein participates in various physiological processes, as the growth of the growth of embryo, cell, form occur and tissue repair.As one of the important member of FGF family, FGF13 is positioned on X chromosome, as the growth of a microtubule bindin nervous system regulation, and participate in mitogen activated protein kinase (MitogenActivatedProteinKinase, MAPK) signal path.But the FGF family protein particularly detailed mechanism of FGF13 albumen in the growth and development process of ox is not yet studied clear.
Nearly ten years, along with the develop rapidly of biotechnology, the research of copy number variation entered flourish period.So-called copy number makes a variation the insertion being greater than 50bp genome sequence or deletion mutation that refer between species two individualities, is a kind of genome structure variation type.He can affect the function of gene and the phenotype of individuality by dosage effect, position effect, block function gene, fusion gene, exposure Recessive alleles and potential transition effect.Along with completing of ox genome sequencing work, cow genome group CNVs research also becomes focus.Research shows, some CNV site is positioned at functional gene inside, and some is relevant to the various economic characters of ox, or the QTL site relevant to healthy proterties overlaps.These researchs illustrate that the normal growth of CNV and ox is grown closely bound up.In the various methods detecting known CNV, qPCR uses more a kind of technology.The method is simple to operate, and susceptibility is high, and speed is fast.Choose the gene of single copy in PCR, as the ox single copy gene BTF3 gene found with reference to checkings such as Liu, as reference gene, then utilize 2
-Δ Δ Ctmethod judge individual copy number variation type and Relative copy number.
But so far, there is not yet about detection Qinchuan Cattle desmocyte growth factor-21 3 (FibroblastGrowthFactor13, FGF13) gene copy number variation (CopyNumberVariations, the method report of genomic dna real-time quantitative PCR (quantitiveReal-TimePCR, qPCR) CNVs).
Summary of the invention
The object of the present invention is to provide a kind of method and the application thereof that detect Qinchuan Cattle FGF13 gene copy number variation.
For achieving the above object, present invention employs following technical scheme:
A kind of method detecting Qinchuan Cattle FGF13 gene copy number variation; comprise the following steps: with Qinchuan Cattle genomic dna for template; with primer pair P1 and primer pair P2 for primer; respectively by the copy number variable region of real-time quantitative PCR amplification FGF13 gene and the Partial Fragment of BTF3 gene in contrast, then according to the copy number variation type of quantitative result qualification Qinchuan Cattle FGF13 gene.
The copy number variable region of described FGF13 gene is positioned at 621003 to 622778 of FGF13 gene reference genome sequence AC_000187.1.
Described copy number variation type is three classes that quantitative result is divided into by basis-Δ Δ Ct: insert type ,-Δ Δ Ct>0.5; Absence type ,-Δ Δ Ct<-0.5; Normal type ,-0.5≤-Δ Δ Ct≤0.5.
Described primer pair P1 is:
Upstream primer F1:5 '-AAGAGGGTGTTTGCTAT-3 '
Downstream primer R1:5 '-CAGTAACGCTGAAGAAG-3 ';
Described primer pair P2 is:
Upstream primer F2:5 '-AACCAGGAGAAACTCGCCAA-3 '
Downstream primer R2:5 '-TTCGGTGAAATGCCCTCTCG-3 '.
Described real-time quantitative PCR amplification system used comprises in 25 μ L: the primer pair P1 of 50ng/ μ L template DNA 2 μ L, 10 μm of ol/L or each 1 μ L of upstream and downstream primer corresponding to primer pair P2 and
premixExTaq
tMiI12.5 μ L.
Described real-time quantitative PCR response procedures used is: (1) 95 DEG C of denaturation 1min; (2) 95 DEG C of sex change 10s, 60 DEG C of annealing 30s, totally 40 circulations.
The PCR primer clip size increased based on primer pair P1 is 236bp, and the PCR primer clip size increased based on primer pair P2 is 166bp.
The application of method in the breeding of Qinchuan Cattle molecular marker assisted selection of above-mentioned detection Qinchuan Cattle FGF13 gene copy number variation.
In described copy number variation type, the individuality with insert type copy number variation type is significantly better than the individuality with normal type copy number variation type in growth traits.
Described growth traits is that body is long or point of the buttocks is wide.
The method of detection Qinchuan Cattle FGF13 gene copy number variation disclosed by the invention, compared with the method such as high-flux sequence method, gene chip, fast simple, cost is low, can identify individual copy number type accurately.
The CNV type of the present invention to Qinchuan Cattle FGF13 gene (the copy number variable region of FGF13 gene) has carried out detection and type frequencies statistics, and the growth traits of this site and Qinchuan Cattle is carried out association analysis.Result shows that, in this site, the frequency of Loss type is the highest, and the body of Gain type to Qinchuan Cattle in this site is long and point of the buttocks is wide significant positive-effect.
Accompanying drawing explanation
Fig. 1 is that FGF13 gene C NV detects primer PCR amplified production electrophorogram; In Fig. 1: swimming lane 1 is DNAmarkerI, swimming lane 2 and 3 is FGF13 gene-specific primer (primer pair P1) PCR primer, and clip size is 236bp.
Embodiment
Elaborate to invention below in conjunction with drawings and Examples, the explanation of the invention is not limited.
In the middle of the ox genome in early stage resurveys sequence research; find that 621003 to 622778 of ox FGF13 genome sequence there occurs copy number variation; therefore; based on the physiological action of FGF13 gene and the regulation mechanism of CNV; the copy number variation of research FGF13 gene is very necessary with the cognation of growth traits, can be the theoretical foundation that ox molecular breeding provides important.
The region that copy number variation occurs in the Qinchuan Cattle FGF13 genome sequence that the present invention obtains according to sequence of resurveying is stencil design Auele Specific Primer, then with Qinchuan Cattle genomic dna for template, carry out qPCR amplification, and with BTF3 gene for reference gene, utilize 2
-Δ Δ Ctmethod, judges individual copy number type.
The present invention is in utilizing qPCR technology, detect the copy number variation situation of Qinchuan Cattle FGF13 gene, and different copy number variation types and growth traits are carried out association analysis, find the copy number type with dominant growth proterties, thus provide basic data for the molecular breeding work of Qinchuan Cattle, accelerate the germ plasm resource improvement work of Chinese Qinchuan Cattle.
1. the collection of sample and extracting genome DNA
(1) collection of blood sample
The Qinchuan Cattle gathered in the present invention comes from Qinchuan Cattle original seed ox conservation field, Shaanxi, is for 24 monthly ages, and amount to 132 individualities, the acquisition method of blood is jugular vein blood collection.And record their growth traits data, as height, body length, chest measurement, buttocks is long, point of the buttocks is wide, hip cross is high, for follow-up association analysis.
(2) extraction of blood sample DNA
1. freezing blood sample (being mainly hemocyte) thaw at RT, draw 500 μ L blood in 1.5mL centrifuge tube, add isopyknic phosphoric acid buffer (PBS) mixing, gentle shake, 4 DEG C, the centrifugal 5min of 12000r/min, abandoning supernatant, repeat above-mentioned steps transparent to supernatant liquor, precipitate transparent look.
2. in centrifuge tube, add DNA extraction buffer 500 μ L, blow and beat gently, hemocyte is precipitated and departs from centrifugal tube wall, 37 DEG C of water-bath 1h.
3. add Proteinase K to 5 μ L (20mg/mL), and mix, digest spend the night (about 16h) and lose to flocks in 55 DEG C of water-baths, solution is clarified, still unclarified, can add 10 μ L Proteinase K mixings and continue digestion until clarification.
4. reaction solution is cooled to room temperature, adds the saturated phenol of 500 μ LTris, gentle shake 15min, make it fully mix, 4 DEG C, the centrifugal 10min of 12000r/min, proceeds to another sterile centrifugation tube by upper strata aqueous phase, repeats above-mentioned steps 1 time.
5. add chloroform 500mL, gentle shake 20min, make it fully mix, 4 DEG C, the centrifugal 15min of 12000r/min, proceeds to the 1.5mL centrifuge tube of another sterilizing by upper strata aqueous phase.
6. add chloroform, primary isoamyl alcohol mixed solution (24:1) 500mL, fully mix 20min, 4 DEG C, the centrifugal 10min of 12000r/min, supernatant liquor is proceeded in another 1.5mL centrifuge tube.
7. add the NaAc damping fluid of 0.1 times of volume and the ice-cold dehydrated alcohol of 2 times of volumes, centrifuge tube is rotated in mixing until the flocks of white is separated out.
8. 4 DEG C, the centrifugal 10min of 12000r/min, abandoning supernatant, precipitates 2 times with the ice cold ethanol rinsing DNA of 70%.
9. 4 DEG C, the centrifugal 10min of 12000r/min, abandons supernatant liquor, makes ethanol volatilize clean under room temperature.
10. the TE adding 80 ~ 100 μ L in dried DNA solution dissolves, and preserves until DNA dissolves completely for 4 DEG C, utilizes UV detector to swim and detects its quality ,-80 DEG C of preservations.
2. the design of the amplification Auele Specific Primer of goal gene and reference gene
The ox FGF13 gene (AC_000187.1) announced with NCBI is reference sequences, find the sequence of the copy number variable region filtered out in sequence of resurveying, namely FGF13 gene (goal gene) organizes 621003 of sequence to 622778, utilize Prime5.0 software design to be comprised in the primer in this region, and compare in NCBI_BLAST.Its primer sequence following (primer pair P1):
Upstream primer F1:5 '-AAGAGGGTGTTTGCTAT-3 ' (SEQ.ID.NO.1)
Downstream primer R1:5 '-CAGTAACGCTGAAGAAG-3 ' (SEQ.ID.NO.2)
Simultaneously, the ox BTF3 gene order (AC_000177.1) announced with NCBI is reference sequences, identical method design is adopted to increase the primer of specific fragment (166bp) in this reference gene (BTF3 gene order), its primer sequence following (primer pair P2):
Upstream primer F2:5 '-AACCAGGAGAAACTCGCCAA-3 ' (SEQ.ID.NO.3)
Downstream primer R2:5 '-TTCGGTGAAATGCCCTCTCG-3 ' (SEQ.ID.NO.4)
Regular-PCR is utilized to increase and the agarose electrophoresis of 1% demonstrates the specificity of primer pair P1 and P2 amplified production, such as, see Fig. 1.
3. real-time quantitative PCR
QPCR reaction system is as shown in table 1 below.
The reaction system of table 1qPCR
PCR response procedures is:
(1) 95 DEG C of denaturation 1min, then carries out amplified reaction according to (2);
(2) 95 DEG C of sex change 10s, 60 DEG C of annealing 30s, totally 40 circulations.
4. individual CNV type decision
Experimental result adopts 2
-△ △ Ctmethod calculates, and concrete method of calculation are: Δ Δ Ct=Δ Ct
(experiment group)-Δ Ct
(reference group), Δ Ct
(experimental group)=Ct
(experimental group goal gene)-Ct
(experimental group reference gene), Δ Ct
(reference group)=Ct
(reference group goal gene)-Ct
(ginseng according to group reference gene)
In formula, experimental group is the individual specimen with or without copy number variation to be detected.Reference group is the known individual specimen without copy number variation, and the reference group Qinchuan Cattle resurveyed selected in sequence test can be adopted individual.
-△ △ the Ct of each test individual is drawn according to formulae discovery, and according to the criterion of CNV type :-△ △ Ct>0.5 is Gain type (insert type);-0.5≤-△ △ Ct≤0.5 is Median type (normal type), and-△ △ Ct<-0.5 is Loss type (absence type), judges the copy number type of the Qinchuan Cattle individuality detected.
5. data processing
The number of individuals of all kinds (Gain, Median and Loss) in statistic mixed-state colony, and add up various types of frequency.Calculation formula is as follows:
P
C＝N
C/N
Wherein, P
crepresent the frequency of certain copy number type; N
crepresent in colony the number of individuals with this CNV type of C; N representative detects the total quantity of colony.
SPSS (18.0) is utilized to carry out correlation analysis.In data handling, according to the difference of factor affecting body measurement trait index, consider environmental effect, age, sex, hereditary effect and reciprocal effects thereof, adopt fixed model to analyze, simplify according to practical situation simultaneously.Complete model is as follows:
Y
ijk＝μ+G
j+E
ijk
Wherein, Y
ijkfor individual phenotype record; μ is colony's average; G
jfor the copy number type in each site; E
ijkfor random error.
The result of data processing is as shown in table 2.
The correlation analysis of table 2 Qinchuan Cattle FGF13 gene copy number variation and growth traits
Note: mean value shoulder is put on to be had same letter and represent difference significantly (P>0.05), mean value shoulder puts on that letter is different represents significant difference (P<0.05).
*P<0.05。The frequency of the numeric representation copy number type inside bracket.
Result shows, the copy number variant sites of Qinchuan Cattle FGF13 gene and body grow and point of the buttocks these two growth traitss wide have significant cognation.Wherein, the growth traits of Gain type is significantly better than Median type individuality.Therefore, the Gain copy number type of FGF13 gene can the molecule marker of and point of the buttocks wide proterties Seedling selection long as Qinchuan Cattle body, for the quick breeding of Qinchuan Cattle.
Claims (10)
1. one kind is detected the method for Qinchuan Cattle FGF13 gene copy number variation; it is characterized in that: comprise the following steps: with Qinchuan Cattle genomic dna for template; with primer pair P1 and primer pair P2 for primer; respectively by the copy number variable region of real-time quantitative PCR amplification FGF13 gene and the Partial Fragment of BTF3 gene in contrast, then according to the copy number variation type of quantitative result qualification Qinchuan Cattle FGF13 gene.
2. a kind of method detecting Qinchuan Cattle FGF13 gene copy number variation as claimed in claim 1, is characterized in that: the copy number variable region of described FGF13 gene is positioned at 621003 to 622778 of FGF13 gene reference genome sequence AC_000187.1.
3. a kind of method detecting Qinchuan Cattle FGF13 gene copy number variation as claimed in claim 1, is characterized in that: described copy number variation type is three classes that quantitative result is divided into by basis-Δ Δ Ct: insert type ,-Δ Δ Ct>0.5; Absence type ,-Δ Δ Ct<-0.5; Normal type ,-0.5≤-Δ Δ Ct≤0.5.
4. a kind of method detecting Qinchuan Cattle FGF13 gene copy number variation as claimed in claim 1, is characterized in that: described primer pair P1 is:
Upstream primer F1:5 '-AAGAGGGTGTTTGCTAT-3 '
Downstream primer R1:5 '-CAGTAACGCTGAAGAAG-3 ';
Described primer pair P2 is:
Upstream primer F2:5 '-AACCAGGAGAAACTCGCCAA-3 '
Downstream primer R2:5 '-TTCGGTGAAATGCCCTCTCG-3 '.
5. a kind of method detecting Qinchuan Cattle FGF13 gene copy number variation as claimed in claim 1, is characterized in that: the amplification system that described real-time quantitative PCR is used comprises: the primer pair P1 of 50ng/ μ L template DNA 2 μ L, 10 μm of ol/L or each 1 μ L of upstream and downstream primer corresponding to primer pair P2 and
premixExTaq
tMiI12.5 μ L.
6. a kind of method detecting Qinchuan Cattle FGF13 gene copy number variation as claimed in claim 1, is characterized in that: described real-time quantitative PCR response procedures used is: (1) 95 DEG C of denaturation 1min; (2) 95 DEG C of sex change 10s, 60 DEG C of annealing 30s, totally 40 circulations.
7. a kind of method detecting Qinchuan Cattle FGF13 gene copy number variation as claimed in claim 1, is characterized in that: the PCR primer clip size increased based on primer pair P1 is 236bp, and the PCR primer clip size increased based on primer pair P2 is 166bp.
8. as the application of the method in claim 1-7 as described in any one claim in the breeding of Qinchuan Cattle molecular marker assisted selection.
9. apply as claimed in claim 8, it is characterized in that: in described copy number variation type, the individuality with insert type copy number variation type is significantly better than the individuality with normal type copy number variation type in growth traits.
10. apply as claimed in claim 9, it is characterized in that: described growth traits is that body is long or point of the buttocks is wide.
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Cited By (5)
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| CN107119117A (en) * | 2017-04-25 | 2017-09-01 | 西北农林科技大学 | A kind of method and its application for detecting Qinchuan Cattle GBP2 gene Cs NV marks |
| CN107400720A (en) * | 2017-09-08 | 2017-11-28 | 西北农林科技大学 | A kind of method and its dedicated kit of KLF3 gene Cs NV marks auxiliary detection ox growth traits |
| CN110157810A (en) * | 2019-05-14 | 2019-08-23 | 西北农林科技大学 | A kind of detection method and its application of CNV label relevant to Xia Nanniu growth traits |
| 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 |
| CN111394474A (en) * | 2020-03-24 | 2020-07-10 | 西北农林科技大学 | Method for detecting copy number variation of cattle GA L3 ST1 gene and application thereof |
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| CN107119117A (en) * | 2017-04-25 | 2017-09-01 | 西北农林科技大学 | A kind of method and its application for detecting Qinchuan Cattle GBP2 gene Cs NV marks |
| CN107119117B (en) * | 2017-04-25 | 2019-10-29 | 西北农林科技大学 | A kind of method and its application of detection Qinchuan Cattle GBP2 gene C NV label |
| CN107400720A (en) * | 2017-09-08 | 2017-11-28 | 西北农林科技大学 | A kind of method and its dedicated kit of KLF3 gene Cs NV marks auxiliary detection ox growth traits |
| CN107400720B (en) * | 2017-09-08 | 2020-04-21 | 西北农林科技大学 | Method for detecting growth traits of cattle under assistance of KLF3 gene CNV marker and special kit thereof |
| CN110157810A (en) * | 2019-05-14 | 2019-08-23 | 西北农林科技大学 | A kind of detection method and its application of CNV label relevant to Xia Nanniu growth traits |
| 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 |
| 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 |
| CN111394474A (en) * | 2020-03-24 | 2020-07-10 | 西北农林科技大学 | Method for detecting copy number variation of cattle GA L3 ST1 gene and application thereof |
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