CN110760597A - Method for detecting copy number variation of cattle NCSTN gene and application thereof - Google Patents
Method for detecting copy number variation of cattle NCSTN gene and application thereof Download PDFInfo
- Publication number
- CN110760597A CN110760597A CN201911159100.8A CN201911159100A CN110760597A CN 110760597 A CN110760597 A CN 110760597A CN 201911159100 A CN201911159100 A CN 201911159100A CN 110760597 A CN110760597 A CN 110760597A
- Authority
- CN
- China
- Prior art keywords
- gene
- copy number
- ncstn
- cattle
- number variation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000283690 Bos taurus Species 0.000 title claims abstract description 66
- 101150036168 ncstn gene Proteins 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 30
- 108020004414 DNA Proteins 0.000 claims abstract description 13
- 238000011529 RT qPCR Methods 0.000 claims abstract description 12
- 239000012634 fragment Substances 0.000 claims abstract description 11
- 101150095259 BTF3 gene Proteins 0.000 claims abstract description 9
- 239000003147 molecular marker Substances 0.000 claims abstract description 5
- 239000013643 reference control Substances 0.000 claims abstract 2
- 230000003321 amplification Effects 0.000 claims description 12
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 12
- 238000012217 deletion Methods 0.000 claims description 9
- 230000037430 deletion Effects 0.000 claims description 9
- 238000011144 upstream manufacturing Methods 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000009395 breeding Methods 0.000 claims description 5
- 230000001488 breeding effect Effects 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 238000000137 annealing Methods 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 238000003752 polymerase chain reaction Methods 0.000 claims 1
- 238000009394 selective breeding Methods 0.000 abstract description 2
- 230000002349 favourable effect Effects 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 19
- 238000003753 real-time PCR Methods 0.000 description 9
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000010219 correlation analysis Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000000038 chest Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 101000578062 Homo sapiens Nicastrin Proteins 0.000 description 3
- 102100028056 Nicastrin Human genes 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 101000984916 Homo sapiens Butyrophilin subfamily 3 member A3 Proteins 0.000 description 1
- 101000933542 Homo sapiens Transcription factor BTF3 Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- -1 Tris saturated phenol Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000004731 jugular vein Anatomy 0.000 description 1
- 102000046701 nicastrin Human genes 0.000 description 1
- 108700022821 nicastrin Proteins 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for detecting copy number variation of cattle NCSTN gene and application thereof: based on real-time quantitative PCR, using the genomic DNA of a Yunnan cattle as a template, using one pair of specific PCR primers to amplify partial fragments of the copy number variation region of the cattle NCSTN gene, simultaneously using the other pair of specific PCR primers to amplify partial fragments of the cattle BTF3 gene as an internal reference control, and finally calculating and judging the copy number variation type of an individual by using-delta Ct. The method provided by the invention lays a foundation for establishing the correlation between the copy number variation and the growth traits of the NCSTN gene of the Yunnan cattle, is favorable for accelerating the molecular marker-assisted selective breeding work of the cattle, and is simple, rapid and convenient to popularize and apply.
Description
Technical Field
The invention belongs to the field of molecular genetics, and particularly relates to a method for detecting copy number variation of NCSTN (negative resistance to high-temperature shock) genes of cattle (such as a Yunnan cattle).
Background
Copy Number Variations (CNVs) refer to insertion and deletion Variations of genomic sequences greater than 50bp between two individuals of a species, and belong to genomic structural Variations. CNVs can affect gene function as well as individual phenotype by dose effects, location effects, blocking functional genes, fusing genes, exposing recessive alleles and potential transition effects. With the completion of the sequencing work of the bovine whole genome, the research of the bovine genome CNVs also becomes a hotspot. Research shows that some CNV sites are located inside functional genes, and some CNV sites are related to various economic traits of cattle. These studies suggest that CNVs are closely related to normal growth and development in cattle. Among the various methods for detecting the known CNV, quantitative real-Time PCR (qPCR) is a widely used technique, which is simple in operation, high in sensitivity and high in speed, and single copy is selected from PCRThe shellfish gene is used as an internal reference gene (such as a single copy gene BTF3 found by Liu et al), and then 2 is utilized-ΔΔCtThe method of (3) determines the type of copy number variation and the relative copy number of the individual.
However, no Real-Time quantitative PCR (qPCR) method for detecting copy number variation of a cloudy cow stubborn protein (Nicastrin) gene has been reported so far.
Disclosure of Invention
The invention aims to provide a method for detecting copy number variation of cattle NCSTN gene and application thereof, so as to quickly establish cattle (such as Yunnan cattle) population with excellent genetic resources and accelerate the improved breeding speed of cattle.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting copy number variation of cattle NCSTN gene comprises the following steps:
the method comprises the steps of using the whole genome DNA of a to-be-detected Yunnan cattle individual as a template, using a primer pair P1 and P2 as primers, amplifying a copy number variation region of the NCSTN gene and a partial fragment of the BTF3 gene as an internal reference through real-time quantitative PCR, and identifying the copy number variation type of the NCSTN gene of the Yunnan cattle individual according to the quantitative result.
Preferably, the copy number variation region of the NCSTN gene is located from position 9345960 to position 9349559 of the reference genomic sequence NC — 037330.1 of the bovine NCSTN gene.
Preferably, the copy number variation types are three types of quantitative results according to- Δ Δ Ct: multicopy, - Δ Δ Ct > 0.5; deletion type, -delta Ct < -0.5; normal type, -0.5 ≤ and-delta-Ct ≤ 0.5.
Preferably, the primer pair P1 is:
the upstream primer F1: 5'-ACCTACTACCCTCATCGCCT-3'
The downstream primer R1: 5'-CTGTGTTCGTGGCTGTTGAC-3', respectively;
the primer pair P2 is as follows:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3' are provided.
Preferably, the amplification system for real-time quantitative PCR comprises 12.5 μ L: 50 ng/. mu.L of template DNA 1. mu.L, 10. mu. mol/L of upstream and downstream primers corresponding to the primer pair P1 or P2, each 0.5. mu.L of pure water 4.25. mu.L, andPremixEx TaqTMII 6.25μL。
preferably, the reaction procedure for real-time quantitative PCR is: (1) pre-denaturation at 95 ℃ for 1 min; (2) denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 30s for 40 cycles.
The method for detecting copy number variation of cattle NCSTN gene is applied to cattle molecular marker-assisted selective breeding.
Preferably, of the copy number variation types, individuals having a multi-copy type copy number variation type (e.g., a Yunnan bovine individual) are significantly superior in growth traits to individuals having a normal type, deletion type copy number variation type.
Preferably, the growth trait is selected from one or two of breast size and tube size.
A real-time quantitative PCR detection kit for NCSTN gene copy number variation related to cattle growth traits comprises the primer pair P1 and P2.
The invention has the beneficial effects that:
compared with methods such as a high-throughput sequencing method and a gene chip, the method for detecting the copy number variation of the NCSTN gene of the cattle, disclosed by the invention, is rapid, simple and low in cost, and can accurately identify the copy number variation type of an individual. According to the invention, the detection and type frequency statistics of the CNV variation type of the NCSTN gene of cattle (such as Yunnan cattle) and the correlation analysis of the CNV and the growth traits of cattle (such as the Yunnan cattle) show that the specific type in the CNV can be used as a molecular marker for early selection of the growth traits of cattle for the rapid breeding of cattle.
Drawings
FIG. 1 shows a melting curve obtained by qPCR (NCSTN gene) in the present invention.
Detailed Description
The invention is described in detail below with reference to the drawings and examples, which are illustrative of the invention and are not intended to limit the scope of the invention.
In the previous cattle genome re-sequencing research, Copy Number Variation (CNV) occurs from 9345960 bit to 9349559 bit of a cattle NCSTN genome sequence (reference genome sequence NC-037330.1), but based on the physiological action of the NCSTN gene, the regulation mechanism of the CNV in important economic traits of cattle cannot be clarified.
The invention designs specific primers according to the region with copy number variation in the bovine NCSTN genome sequence obtained by the resequencing, then uses the genomic DNA of the Yunnan cattle as a template to carry out qPCR amplification, uses BTF3 gene as an internal reference gene, and utilizes 2-ΔΔCtMethod, determining the type of copy number of an individual.
According to the invention, the copy number variation condition of the NCSTN gene of the Yunnan cattle is detected by utilizing a qPCR technology, different copy number variation types and individual growth traits are subjected to correlation analysis, and the copy number variation type with the dominant growth trait is found, so that the germplasm resource improvement work of the Yunnan cattle can be accelerated, and basic data is provided for the molecular breeding work of the cattle.
1. Sample collection and genomic DNA extraction
(1) Collection of blood samples
The collected Yunling cattle are from the small Yangxiang grassland animal science research institute in Kunming city, Yunnan province, are collected in 8 months in 2018, are all 24 months old, and count 119 individuals, and the blood collection method is jugular vein blood collection. And meanwhile, collecting growth character data records of the individuals, such as body height, body length, chest circumference, tube circumference, nojiri length, width of sitting bone end, high cross part and the like, so as to be used for subsequent correlation analysis.
(2) Extraction of DNA from blood samples
① frozen blood sample (mainly blood cells) is thawed at room temperature, 500 μ L of blood is absorbed into a 1.5mL centrifuge tube, Phosphate Buffer Solution (PBS) with the same volume is added for even mixing, the mixture is gently shaken, centrifuged at 12000r/min at 4 ℃ for 5min, supernatant is discarded, and the steps are repeated until the supernatant is transparent.
② adding DNA extraction buffer solution 500 μ L into the centrifuge tube, gently blowing to make the blood cell precipitate to separate from the wall of the centrifuge tube, and bathing in water at 37 deg.C for 1 h.
③ adding 5 μ L (20mg/mL) of proteinase K, mixing, digesting in 55 deg.C water bath overnight (about 16 h) until no flocculent precipitate is observed, clarifying the solution, and adding 10 μ L proteinase K, mixing, and digesting until it is clear.
④ cooling the reaction solution to room temperature, adding 500 μ L Tris saturated phenol, gently shaking for 15min to mix well, centrifuging at 4 deg.C and 12000r/min for 10min, transferring the upper aqueous phase to another sterilized centrifuge tube, and repeating the steps for 1 time.
⑤ adding chloroform 500mL, gently shaking for 20min to mix well, centrifuging at 12000r/min at 4 deg.C for 15min, and transferring the upper aqueous phase to another sterilized 1.5mL centrifuge tube.
⑥ adding chloroform and isoamyl alcohol mixture (24:1)500mL, mixing thoroughly for 20min, centrifuging at 4 deg.C and 12000r/min for 10min, transferring the supernatant to another 1.5mL centrifuge tube.
⑦ Add 0.1 volume NaAc buffer and 2 volumes of ice cold absolute ethanol and mix and rotate the tube until a white flocculent precipitate separates.
⑧ 4 deg.C, 12000r/min centrifugation for 10min, discarding the supernatant, using 70% ice cold ethanol rinse DNA precipitation 2 times.
⑨ 4 deg.C, centrifuging at 12000r/min for 10min, discarding supernatant, and volatilizing ethanol at room temperature.
⑩ adding 80-100 μ L TE to dissolve the dried DNA, storing at 4 deg.C until the DNA is completely dissolved, detecting its quality by ultraviolet spectrophotometer, and storing at-80 deg.C.
2. Design of specific primers for amplification of target gene and reference gene
Using the bovine NCSTN gene (NC-037330.1) published by NCBI as a reference sequence, the sequence of the copy number variation region screened out in the re-sequencing, namely 9345960 bits to 9349559 bits of the NCSTN gene (target gene) group sequence, was found, primers contained in this region were designed using Prime 5.0 software, and aligned in NCBI _ BLAST. The primer sequences are specifically as follows (primer pair P1):
the upstream primer F1: 5'-ACCTACTACCCTCATCGCCT-3'
The downstream primer R1: 5'-CTGTGTTCGTGGCTGTTGAC-3', respectively;
based on the BTF3 gene having neither CNV sequence nor segmented repeats, the BTF3 gene was used as an internal reference gene. The designed primer sequences were as follows (primer pair P2):
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3' are provided.
The amplification specificity of the primer pair is verified by common PCR amplification and 1% agarose gel electrophoresis. The size of the PCR product fragment amplified based on the primer pair P1 is 100bp, and the size of the PCR product fragment amplified based on the primer pair P2 is 166 bp.
3. Real-time quantitative PCR
The qPCR reaction system is shown in table 1.
TABLE 1 reaction System for qPCR
The qPCR reaction procedure was:
(1) pre-denaturation at 95 ℃ for 1min, and then carrying out amplification reaction according to (2);
(2) denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 30s for 40 cycles.
Primers were determined to be suitable for qPCR analysis by plotting amplification curves and melting peaks. According to the drawn melting curve, the curves of the samples are matched together, and the curves are smooth in trend, high and sharp in peak, and free from primer dimer or a hybrid peak caused by nonspecific amplification (see figure 1).
4. Individual CNV type determination
Experimental results 2-△△CtThe method carries out calculation, and the specific calculation method comprises the following steps: Δ Δ Ct ═ Δ Ct(Experimental group)-ΔCt(reference group)In the formula,. DELTA.Ct(Experimental group)＝Ct(Experimental group target Gene)-Ct(Experimental group internal reference gene)，ΔCt(reference group)＝Ct(reference group target Gene)-Ct(reference group internal reference gene)(ii) a The experimental group is an individual sample to be detected for the existence of copy number variation, the reference group is an individual sample known to have no copy number variation, and a reference group of the individuals of the Yunnan cattle selected in the resequencing can be adopted; ct, Cycle threshold, is the number of amplification cycles that pass when the fluorescence signal of the amplified product reaches a set threshold during PCR amplification.
Calculating to obtain-delta Ct of each individual to be detected according to a formula, and according to the CNV type judgment standard: - Δ Δ Ct >0.5 is multicopy (replication); normal type (Median) with-delta Ct less than or equal to 0.5 and less than or equal to 0.5; and-delta Ct < -0.5 is a Deletion type (Deletion), so that the copy number variation type of the detected individual Yunling cattle is judged.
5. Data processing
The number of individuals of each type (Deletion, media and Duplication) in the test population is counted, and the frequencies of each type are counted. The calculation formula is as follows:
PC＝NC/N
wherein, PCFrequency representing a certain type of copy number variation; n is a radical ofCRepresents the number of individuals in the population with C, the CNV type; n represents the total number of detection populations.
The correlation analysis was performed using SPSS (18.0). In the data processing, according to different factors influencing growth character indexes, considering environmental effect, age, sex, genetic effect and interaction effect thereof, a fixed model is adopted for analysis, and simplification is carried out according to actual conditions. The complete model is as follows:
Yijk＝μ+Gj+Eijk
wherein, Yijk(ii) recording the phenotype of the individual; μ is the population mean; gjThe copy number type of each site; eijkIs a random error.
The data processing results are shown in table 2, with the highest frequency of deletion individuals.
TABLE 2 correlation analysis of copy number variation and growth traits of the NcSTN gene of Yunnan cattle
Note: mean shoulder marks with the same letter indicate no significant difference (P)>0.05), the average value is marked with a difference in letters on the shoulder to indicate significant difference (P)<0.05)；*P<0.05; the numbers in parentheses indicate the frequency of the copy number variation type.
The results show (table 2) that the copy number variation site of the Yunnan cattle NCSTN gene (9345960-9349559 of the reference genome sequence NC-037330.1 of the cattle NCSTN gene) has obvious relevance with the growth traits of the tube circumference and the chest circumference (the multicopy type of the site has obvious positive effect on the tube circumference and the chest circumference of the Yunnan cattle). Wherein, the growth traits of the individuals with multiple copy types are obviously better than those of the individuals with normal types and deletion types. Therefore, the multi-copy type of the NCSTN gene copy number variation sites can be used as a molecular marker for early selection of growth traits (such as tube size and chest size traits) of the Yunnan cattle and is used for rapid breeding of the Yunnan cattle.
<110> northwest agriculture and forestry science and technology university
<120> method for detecting copy number variation of cattle NCSTN gene and application thereof
<160>4
<210>1
<211>20
<212>DNA
<213> Artificial Synthesis
<400>1
acctactacc ctcatcgcct 20
<210>2
<211>20
<212>DNA
<213> Artificial Synthesis
<400>2
ctgtgttcgt ggctgttgac 20
<210>3
<211>20
<212>DNA
<213> Artificial Synthesis
<400>3
aaccaggaga aactcgccaa 20
<210>4
<211>20
<212>DNA
<213> Artificial Synthesis
Claims (10)
1. A method for detecting copy number variation of cattle NCSTN gene is characterized in that: the method comprises the following steps:
and (2) taking the genomic DNA of the Yunnan cattle as a template, amplifying the copy number variation region of the NCSTN gene and a partial fragment of the BTF3 gene serving as an internal reference through real-time quantitative PCR (polymerase chain reaction), and identifying the copy number variation type of the NCSTN gene of the Yunnan cattle according to the quantitative result.
2. The method of claim 1, wherein the variation in copy number of the NCSTN gene of cattle is detected by: the copy number variation region of the NCSTN gene is positioned from 9345960 to 9349559 of a reference genome sequence NC-037330.1 of the bovine NCSTN gene.
3. The method of claim 1, wherein the variation in copy number of the NCSTN gene of cattle is detected by: the copy number variation types are divided into three types according to-delta-Ct: multicopy, - Δ Δ Ct > 0.5; deletion type, -delta Ct < -0.5; normal type, -0.5 ≤ and-delta-Ct ≤ 0.5.
4. The method of claim 1, wherein the variation in copy number of the NCSTN gene of cattle is detected by: the amplification primer pair of the partial fragment of the copy number variation region of the NCSTN gene comprises:
the upstream primer F1: 5'-ACCTACTACCCTCATCGCCT-3'
The downstream primer R1: 5'-CTGTGTTCGTGGCTGTTGAC-3', respectively;
the amplification primer pair of the partial fragment of the BTF3 gene comprises the following components:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3' are provided.
5. The method of claim 1, wherein the variation in copy number of the NCSTN gene of cattle is detected by: the reaction procedure of the real-time quantitative PCR is as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 30s for 40 cycles.
6. Use of the method according to any one of claims 1 to 5 in molecular marker assisted selection breeding of cattle.
7. The use of claim 6, wherein: among the copy number variation types, individuals having a multi-copy type copy number variation type are superior in growth traits.
8. The use of claim 7, wherein: the growth trait is selected from one or two of chest circumference and tube circumference.
9. A real-time quantitative PCR detection kit for NCSTN gene copy number variation related to cattle growth traits is characterized in that: the kit comprises a primer pair for amplifying a copy number variation region of a Yunnan cattle NCSTN gene and a partial fragment of a BTF3 gene serving as an internal reference control; the NCSTN gene copy number variation region is positioned at 9345960-9349559 of a bovine NCSTN gene reference genome sequence NC-037330.1.
10. The real-time quantitative PCR detection kit for the NCSTN gene copy number variation associated with cattle growth traits as claimed in claim 9, characterized in that: the amplification primer pair of the partial fragment of the NCSTN gene copy number variation region is as follows:
the upstream primer F1: 5'-ACCTACTACCCTCATCGCCT-3'
The downstream primer R1: 5'-CTGTGTTCGTGGCTGTTGAC-3', respectively;
the amplification primer pair of the partial fragment of the BTF3 gene comprises the following components:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3' are provided.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911159100.8A CN110760597B (en) | 2019-11-22 | 2019-11-22 | Method for detecting copy number variation of cattle NCSTN gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911159100.8A CN110760597B (en) | 2019-11-22 | 2019-11-22 | Method for detecting copy number variation of cattle NCSTN gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110760597A true CN110760597A (en) | 2020-02-07 |
| CN110760597B CN110760597B (en) | 2022-08-23 |
Family
ID=69338940
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911159100.8A Expired - Fee Related CN110760597B (en) | 2019-11-22 | 2019-11-22 | Method for detecting copy number variation of cattle NCSTN gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110760597B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321232A (en) * | 2020-03-18 | 2020-06-23 | 西北农林科技大学 | Method for rapidly detecting copy number variation of EIF4A2 gene of beef cattle and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2083826A2 (en) * | 2006-11-07 | 2009-08-05 | Protelogiecs, Ltd. | Pyrimidine derivatives as posh and posh-ap inhibitors |
| CN102618549A (en) * | 2011-01-30 | 2012-08-01 | 深圳华大基因科技有限公司 | NCSTN mutant gene, and its identification method and tool |
| CN103333920A (en) * | 2013-06-18 | 2013-10-02 | 西北农林科技大学 | Culture medium for establishing pig iPS cell line and culture method thereof |
| WO2017031353A1 (en) * | 2015-08-19 | 2017-02-23 | Rutgers, The State University Of New Jersey | Novel methods of generating antibodies |
| CN107523643A (en) * | 2017-10-20 | 2017-12-29 | 西北农林科技大学 | A kind of method and its dedicated kit of ox KCNJ12 gene Cs NV marks auxiliary detection growth traits |
| CN108513582A (en) * | 2015-06-18 | 2018-09-07 | 布罗德研究所有限公司 | Novel C RISPR enzymes and system |
| CN109943646A (en) * | 2019-04-29 | 2019-06-28 | 西北农林科技大学 | The method and its application of ox PLAG1 gene C NV molecular labeling auxiliary detection growth traits |
| CN109943647A (en) * | 2019-04-29 | 2019-06-28 | 西北农林科技大学 | A kind of method and its application of quick detection ox MLLT10 gene C NV label |
-
2019
- 2019-11-22 CN CN201911159100.8A patent/CN110760597B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2083826A2 (en) * | 2006-11-07 | 2009-08-05 | Protelogiecs, Ltd. | Pyrimidine derivatives as posh and posh-ap inhibitors |
| CN102618549A (en) * | 2011-01-30 | 2012-08-01 | 深圳华大基因科技有限公司 | NCSTN mutant gene, and its identification method and tool |
| CN103333920A (en) * | 2013-06-18 | 2013-10-02 | 西北农林科技大学 | Culture medium for establishing pig iPS cell line and culture method thereof |
| CN108513582A (en) * | 2015-06-18 | 2018-09-07 | 布罗德研究所有限公司 | Novel C RISPR enzymes and system |
| WO2017031353A1 (en) * | 2015-08-19 | 2017-02-23 | Rutgers, The State University Of New Jersey | Novel methods of generating antibodies |
| CN107523643A (en) * | 2017-10-20 | 2017-12-29 | 西北农林科技大学 | A kind of method and its dedicated kit of ox KCNJ12 gene Cs NV marks auxiliary detection growth traits |
| CN109943646A (en) * | 2019-04-29 | 2019-06-28 | 西北农林科技大学 | The method and its application of ox PLAG1 gene C NV molecular labeling auxiliary detection growth traits |
| CN109943647A (en) * | 2019-04-29 | 2019-06-28 | 西北农林科技大学 | A kind of method and its application of quick detection ox MLLT10 gene C NV label |
Non-Patent Citations (2)
| Title |
|---|
| MD MESBAH-UDDIN等: ""Genome-wide mapping of large deletions and their population-genetic properties in dairy cattle"", 《DNA RESEARCH》 * |
| MESBAH-UDDIN等: ""Gene: NCSTN ENSBTAG00000004334"", 《ENSEMBL数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111321232A (en) * | 2020-03-18 | 2020-06-23 | 西北农林科技大学 | Method for rapidly detecting copy number variation of EIF4A2 gene of beef cattle and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110760597B (en) | 2022-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109943647B (en) | Method for rapidly detecting MLLT10 gene CNV marker of cattle and application thereof | |
| CN107400720B (en) | Method for detecting growth traits of cattle under assistance of KLF3 gene CNV marker and special kit thereof | |
| CN109943646B (en) | Method for CNV molecular marker of cattle PLAG1 gene and application thereof | |
| CN107523643B (en) | Method for auxiliary detection of growth traits of cattle KCNJ12 gene CNV marker and special kit thereof | |
| CN112695106B (en) | Method for quickly detecting growth traits of cattle in auxiliary manner through PLA2G2A gene CNV markers and special kit | |
| CN105543352B (en) | Method for detecting copy number variation of Qinchuan cattle FGF13 gene and application thereof | |
| CN110894542A (en) | Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer | |
| CN111733218B (en) | Method for detecting growth characteristics of goat CCSER1 gene CNV marker and diagnostic kit thereof | |
| CN110029156B (en) | Method for detecting CNV marker of KAT6A gene of tea goat and application thereof | |
| CN112813175B (en) | Method for rapid auxiliary detection of growth traits by cattle CHRDL1 gene CNV markers and application thereof | |
| CN110760597B (en) | Method for detecting copy number variation of cattle NCSTN gene and application thereof | |
| CN111394474B (en) | Method for detecting copy number variation of GAL3ST1 gene of cattle and application thereof | |
| CN113151489B (en) | Molecular diagnosis method for evaluating growth traits based on cow ZNF146 gene CNV marker and application thereof | |
| CN110964790B (en) | Method for detecting tea-goat PIGY gene CNV marker and application thereof | |
| CN116200472B (en) | Application of cattle SCN9A gene InDel marker in early selection of meat quality traits | |
| CN113481303B (en) | Method for auxiliary detection of growth traits by cattle ACTR3 gene CNV markers and application thereof | |
| CN113604583B (en) | Method for auxiliary detection of growth traits by goat KCNJ15 gene CNV markers and special kit thereof | |
| CN113151501B (en) | Method for assisted detection of growth traits by cattle WBP1L gene CNV markers and application thereof | |
| CN110964839B (en) | Method for auxiliary detection of cattle growth traits through SERPINA3-1 gene CNV labeling and application thereof | |
| CN111321232B (en) | Method for rapidly detecting copy number variation of EIF4A2 gene of beef cattle and application thereof | |
| CN110964838B (en) | Method for rapidly detecting CNV marker of sheep LRRFIP1 gene and application thereof | |
| CN113151490B (en) | Growth character molecular marker auxiliary selection method based on cattle LOC107131166 gene CNV marker | |
| CN112410441A (en) | Method for identifying anti-cysticercosis trait of bee colony by using SNP marker KZ 288479.1-95621 | |
| CN110564829B (en) | Method for auxiliary detection of lactation traits of dairy cow NCAM2 gene CNV marker and special kit thereof | |
| CN112430675A (en) | Method for identifying anti-cysticercosis trait of bee colony by using SNP marker KZ 288474.1-322717 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220823 |