CN105349674A - Detection method of CNV mark related to qinchuan cattlegrowth and application - Google Patents
Detection method of CNV mark related to qinchuan cattlegrowth and application Download PDFInfo
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Abstract
The invention discloses a detection method of a CNV mark related to qinchuan cattlegrowth and application. The CNV mark refers to copy number variation of Chr 4: 118264951-118267170 in a cattle SHH gene candidate region. The detection method includes the steps that a genome DNA of a qinchuan cattle serves as a template, a primer pair P1 and a primer pair P2 serve as primers, a qinchuan cattle SHH gene CNV region and a reference gene are amplified through real-time fluorescence quantification PCR, and the result is divided into an inserting type, a deletion type and a normal type according to log<2>2<delt delt Ct>, so that the copy number variation of the qinchuan cattle SHH gene is authenticated. By means of the detection method, the CNV mark closely relates to the production traits of the qinchuan cattle can be detected on the DNA level, and the CNV mark can serve as an important candidate molecular mark of assistant mark selection of the qinchuan cattlegrowth traits.
Description
Technical field
The present invention relates to domestic animal molecular Biological Detection field, be specifically related to a kind of detection method and the application that detect the SHH gene C NV mark that Qinchuan Cattle growth traits is correlated with.
Background technology
Copy number variation (CopyNumberVariations, CNVs) refers in genomic dna compared with the disappearance of large fragment or polyisomenism.CNVs is the structure variation of a kind of sub-microscopic level on genome, and the clip size related to is between 1kb to multiple Mb, and comprising copy number increases (Copynumbergain) and copy number minimizing (Copynumberloss).The variation of some copy number belongs to polymorphism category, does not impact vegeto-animal phenotype, and the variation of some copy number then affects genetic expression by upsetting gene order and changing gene content, thus causes phenotypic difference and phenotypic adaptation.
The technology being applied to copy number variation detection at present mainly contains: (1) comparative genome hybridization (CGH): CGH can on whole karyomit(e) or karyomit(e) subzone level, the copy number of DNA sequence dna between different genes group is detected, thus finds copy number variation.But this technology resolving power is in Mb level, more the copy number fragment of small segment then not easily detects.This technological operation is loaded down with trivial details simultaneously, low, the consuming time length of flux and cost intensive, needs comparatively a large amount of template DNAs, is unfavorable for large-scale popularization.(2) multiplex ligation-dependent probe amplification (MLPA): MLPA is a kind of copy number detection method grown up for 2002.This technology has relative quantification function more accurately, but the method probe preparation is comparatively complicated, simultaneously complex operation step, length consuming time.And adopt capillary electrophoresis as analysis means, flux is lower, cost is higher and belong to open-sky technique, is easy to the pollution causing PCR primer.(3) high-resolution fusion curve analysis (HRM): HRM was in invention in 2003, it is controlled by accurate Cooling rate and the instruction of DNA saturable dye, achieves by the melting temperature (Tm) of research PCR primer sequence and identifies PCR primer.The advantages such as this technology has fast, cheapness, high-throughput, have following deficiency simultaneously: the prerequisite that the method realizes is mutational site must be heterozygosis, thus adds the cost of experiment and the difficulty of design, and reduces the flux of detection.Further, the difference of single core thuja acid is less for the impact of melting curve, and even some difference affects the skew of melting curve hardly, causes detection sensitivity lower.(4) real-time fluorescence quantitative PCR (qPCR): according to the difference of the fluorescence chemical method that qRT-PCR uses, is mainly divided into fluorescence dye embedding inlay technique and fluorogenic hybridization probe method two class.Excessive SYBRGreen dye molecule is added in PCR reaction system, can DNA double chain be infiltrated specifically and launch fluorescent signal, free dye molecule then only has very low autofluorescent background luminous hardly, thus guarantee that the increase of signal is synchronous with the increase of PCR primer, the intensity by detecting fluorescent signal reflects the quantity of genomic dna.By carrying out relative quantification, according to 2 to goal gene (there is copy number variation) and with reference to gene (without copy number variation)
-Δ Δ Ctmethod statistic detects the copy number of sample candidate gene.The advantage of dye method be experimental cost low, without the need to design and synthesis probe, easy to use, can the absolute copy number of testing goal fragment, but be not suitable for the high throughput testing of large sample.
Molecular breeding, i.e. molecular marker assisted selection (molecularmark-assistselection, MAS), this technology selects genetic resources or breeding material by DNA molecular marker, carries out breed improvement to the Comprehensive Traits of livestock and poultry.In Animal Breeding, by closely related to growth traits, and the selection of DNA marker with quantitative character tight association, reach early stage seed selection and improve the object of breeding value accuracy, thus in Animal Breeding, obtain larger genetic progress.
SHH gene is the important regulatory factor of fetal development and Various Tissues orga-nogenesis, can promote that mescenchymal stem cell is to one-tenth flesh or osteoblast differentiation, and suppress Adipose Differentiation, and regulate and control the growth of four limbs, nerve and Digestive tract, thus affect growing of economic animal.High-flux sequence finds copy number variation (CNVs) that there is 2220bp in SHH gene, contain this Gene Partial exon, intron and 3 ' UTR critical function region, the Function of this gene and relevant target gene thereof may be changed, and then have influence on growing of body.At present, there is not yet the bibliographical information about this CNVs of SHH gene affects Qinchuan Cattle growth traits.Therefore, study this gene copy number variation and itself and the association analysis of Qinchuan Cattle important growth traits is most important, theoretical foundation can be provided for China's Qinchuan Cattle molecular breeding, be convenient to the marker assisted selection of Chinese Qinchuan Cattle growth traits, set up the Qinchuan Cattle population that genetic resources is excellent fast.
Summary of the invention
The object of the present invention is to provide and a kind ofly grow the detection method and application that relevant CNV marks to Qinchuan Cattle.
To achieve these goals, present invention employs following technical scheme:
A kind of detection method growing relevant CNV to Qinchuan Cattle and mark; comprise the following steps: with Qinchuan Cattle genomic dna for template; with primer pair P1 and primer pair P2 for primer; to be increased respectively Qinchuan Cattle SHH gene C NV region and reference gene BTF3 by real-time fluorescence quantitative PCR, according to the copy number variation of quantitative result qualification Qinchuan Cattle SHH gene.
Described CNV mark refers to the copy number variation of ox SHH gene candidate region Chr4:118264951-118267170.
Described copy number variation is according to log
22
-Δ Δ Ctquantitative result is divided three classes: insert type, Log
22
-Δ Δ Ct>0.5; Absence type, Log
22
-Δ Δ Ct<-0.5; Normal type, Log
22
-Δ Δ Ct≤ | ± 0.5|.
Described primer pair P1 is:
Upstream primer F1:5 '-CGCACGCAATGAGACTTTA-3 ',
Downstream primer R1:5 '-AATAGCCAGGAGAGGTGAA-3 ';
Described primer pair P2 is:
Upstream primer F2:5 '-AACCAGGAGAAACTCGCCAA-3 ',
Downstream primer R2:5 '-TTCGGTGAAATGCCCTCTCG-3 '.
Described real-time fluorescence quantitative PCR amplification system used is counted with 20 μ L: 50ng/ μ L template DNA 1 μ L, the primer pair P1 of 10pmol/L or each 1 μ L of the upstream and downstream primer corresponding to primer pair P2,2 × SYBRGreenqPCRMix10 μ L, and deionized water 7 μ L.
Described real-time fluorescence quantitative PCR response procedures used is: (1) denaturation 95 DEG C of 30s; (2) amplified reaction: 95 DEG C of sex change 10s, 60 DEG C of annealing 30s, 39 circulations.
The above-mentioned application of detection method in Qinchuan Cattle molecular breeding growing relevant CNV to Qinchuan Cattle and mark.
The described CNV tag application Qinchuan Cattle kind that molecular marker assisted selection growth traits is outstanding in early days.
The significant correlation that grows of the different copy number in described SHH gene C NV region and Qinchuan Cattle, wherein the growth traits of insert type individuality is significantly higher than absence type individuality.
The present invention makes a variation according to the copy number of ox SHH gene candidate region Chr4:118264951-118267170, particularly, described CNV mark is positioned at the 7776bp-9995bp region of ox SHH gene (GenBankAccessionNo.AC_000161) sequence, with the genomic dna of ox to be measured for masterplate, real-time fluorescence quantitative PCR is utilized to detect genome C NVs, according to log
22
-Δ Δ Ctresult is divided three classes: Log
22
-Δ Δ Ct>0.5 is then insert type; Log
22
-Δ Δ Ct<-0.5 is then absence type, Log
22
-Δ Δ Ct≤ | ± 0.5| is then normal type.According to the detected result of cow genome group CNVs, if copy number is insert type, then ox phenotype to be measured is comparatively excellent; If copy number is absence type, then test individual phenotype is poor.According to embodiments of the invention, with ox SHH gene candidate region Chr4:118264951-118267170 for candidate locus, detect the copy number variation situation of this site in Qinchuan Cattle colony of colony by Real-Time Fluorescent Quantitative PCR Technique, and carry out association analysis with the important economical trait such as body weight and chest measurement; If the copy number variation type detecting SHH gene candidate site is insert type, then ox phenotype to be measured is more excellent; If copy number variation type is absence type, then test individual phenotype is poor.
Compared with prior art, the present invention has following advantage:
(1) Qinchuan Cattle SHH gene copy number variation detection method provided by the invention, not by the restriction at age, can be used for the early stage seed selection of cow, even just can select when just birth;
(2) detect the method for ox SHH gene copy number variation accurately and reliably, easy and simple to handle;
(3) the detecting of ox SHH gene copy number variation site, the molecular marker assisted selection of growing for ox provides scientific basis.
Accompanying drawing explanation
Fig. 1 is the amplification curve carrying out qPCR drafting in the present invention;
Fig. 2 is the solubility curve carrying out qPCR drafting in the present invention.
Embodiment
Below in conjunction with drawings and Examples, the present invention is elaborated.
The present invention utilizes the copy number variation of real-time fluorescence quantitative PCR to Qinchuan Cattle SHH gene detect and for molecular breeding, generally include following steps:
(1) adopt the copy number variation situation of real-time fluorescence quantitative PCR (qPCR) technology for detection candidate locus in colony, screen the CNV relevant to Qinchuan Cattle growth traits and mark;
(2) utilize SPSS19.0 software that copy number variation type and ox growth traits etc. are carried out association analysis;
(3) the Qinchuan Cattle seed selection of growth traits excellence is carried out according to copy number variation type.
1, Qinchuan Cattle sample collection
The present invention is specifically using place of china Qinchuan Cattle kind as detected object, and the blood sample sample collection of 208 Qinchuan Cattles is from Qinchuan Cattle stock breeding center, Shaanxi Province.
2, the separation of genomic dna, extraction, purifying
Reference Sambrocketal (2002) method.
3, the amplification of target sequence and reference sequences
The ox SHH gene order (GenBankAccessionNo.AC_000161) announced with ncbi database (http://www.ncbi.nlm.nih.gov/) is for reference sequences, utilize Primer5.0 to design real-time fluorescence quantitative PCR primer pair and detect SHH gene copy number variation, its primer pair sequence information is as shown in table 1.Determine whether primer is applicable to qPCR and analyzes by drawing amplification curve (Fig. 1) and dissolving peak.According to the solubility curve drawn, together, and curve tendency is level and smooth for each sample curves, peak height and sharp, without the assorted peak (Fig. 2) that primer dimer or non-specific amplification cause.
Internal reference sequence is the known sequence that there is not copy number variation, the sequence of one section of 166bp namely in BTF3 gene.
The primer information of table 1 real-time fluorescence quantitative PCR
Wherein, carry out real-time fluorescence quantitative PCR amplification system used to count with 20 μ L: 50ng/ μ L template DNA (extracting the genomic dna from blood sample sample) 1 μ L, each 1 μ L, 2 × SYBRGreenqPCRMix10 μ L, the deionized water 7 μ L of upstream and downstream primer of 10pmol/L.
The response procedures of pcr amplification is: (1) denaturation: 95 DEG C of 30s; (2) amplified reaction: 95 DEG C of sex change 10s, 60 DEG C of annealing 30s, 39 circulations; (3) solubility curve is drawn: 95 DEG C of 5s ,-0.01 DEG C/s, 65 DEG C of 1min.
4, the deduction of copy number variation
Each sample increases with the primer of target sequence and reference sequences respectively, and 3 repetitions of often pair of primer.According to 2
-Δ Δ Ctmethod carries out the analysis of copy number.Wherein Δ Δ Ct=(C
t goal gene-C
t reference gene)
experimental group-(C
t goal gene-C
t reference gene)
control group.Experimental group is the sample with or without CNVs to be detected, and control group is the known sample without copy number variation.2
-Δ Δ Ctrepresent be the copy number of experimental group target sequence relatively and the multiple of control group.Then the gene expression abundance of gene is carried out logarithmic transformed (with 2 for the end 2
-Δ Δ Ctlogarithm) make it to meet normal distribution, after carrying out homogeneity test of variance, statistical test respectively organize between difference.
When target sequence is normal type sequence, according to Log
22
-Δ Δ Ctcalculate normalized value about 0 (Log
2
2
-ΔΔCt≤|±0.5|)。When target sequence is absence type sequence, Log
22
-Δ Δ Ctcalculate normalized value Log
22
-Δ Δ Ct<-0.5.When target sequence is insert type sequence, Log
22
-Δ Δ Ctcalculate normalized value Log
22
-Δ Δ Ct>0.5.
5, the association analysis of Qinchuan Cattle SHH gene C NV site and growth traits
The association analysis of table 2.1SHH gene C NV and Qinchuan Cattle kind growth traits
The association analysis of table 2.1SHH gene C NV and Qinchuan Cattle kind growth traits
Production data: body length, height, body weight, chest measurement, chest breadth, chest depth and hip cross are high.
Relation analysis model: be first described analysis to data, determines whether there is outlier, and recycling Least square analysis is to Data correction; According to data characteristics, the production traits effect between each genotype of application SPSS19 software analysis.Fixed model is have employed when analyzing genotype effects:
Y
ijk＝μ+A
i+CNV
j+e
ijk
Wherein: Y
ijkfor character observation value, μ is population mean, A
ibe i-th individual age, CNV
jfor the fixed effect of a jth copy number variation type, e
ijkfor random error.Otherness between each group of data adopts LSD multiple comparisons to test, and test-results represents with Mean ± SE form.
Association analysis result shows (see table 2): Qinchuan Cattle SHH gene C NV site can the height at remarkably influenced 24 monthly age, body length, body weight, hip cross high, and the body length at 30 monthly ages, chest measurement, body weight, chest breadth and chest depth.Further, in two age levels, advantage copy number variation type is insert type, illustrates that the candidate molecules genetic marker of Qinchuan Cattle growth traits can be improved in this CNV site of SHH gene as one.
6, above-mentioned CNV is marked at the application in Qinchuan Cattle seed selection
The CNV obtained can be used as candidate molecules genetic marker, finds or the closely linked quantitative trait locus that affect ox growth traits relevant to it, to carry out molecular marker assisted selection to Qinchuan Cattle, thus accelerates the seed selection process of Qinchuan Cattle breed improvement.
Claims (9)
1. one kind grows to Qinchuan Cattle the detection method that relevant CNV marks; 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; to be increased respectively Qinchuan Cattle SHH gene C NV region and reference gene BTF3 by real-time fluorescence quantitative PCR, according to the copy number variation of quantitative result qualification Qinchuan Cattle SHH gene.
2. a kind of detection method growing relevant CNV to Qinchuan Cattle and mark as claimed in claim, is characterized in that: described CNV mark refers to the copy number variation of ox SHH gene candidate region Chr4:118264951-118267170.
3. a kind of detection method growing relevant CNV to Qinchuan Cattle and mark as claimed in claim 1, is characterized in that: described copy number variation is according to log
22
-Δ Δ Ctquantitative result is divided three classes: insert type, Log
22
-Δ Δ Ct>0.5; Absence type, Log
22
-Δ Δ Ct<-0.5; Normal type, Log
22
-Δ Δ Ct≤ | ± 0.5|.
4. a kind of detection method growing relevant CNV to Qinchuan Cattle and mark as claimed in claim 1, is characterized in that: described primer pair P1 is:
Upstream primer F1:5 '-CGCACGCAATGAGACTTTA-3 ',
Downstream primer R1:5 '-AATAGCCAGGAGAGGTGAA-3 ';
Described primer pair P2 is:
Upstream primer F2:5 '-AACCAGGAGAAACTCGCCAA-3 ',
Downstream primer R2:5 '-TTCGGTGAAATGCCCTCTCG-3 '.
5. a kind of detection method growing relevant CNV to Qinchuan Cattle and mark as claimed in claim 1, it is characterized in that: described real-time fluorescence quantitative PCR amplification system used is counted with 20 μ L: 50ng/ μ L template DNA 1 μ L, the primer pair P1 of 10pmol/L or each 1 μ L of the upstream and downstream primer corresponding to primer pair P2,2 × SYBRGreenqPCRMix10 μ L, and deionized water 7 μ L.
6. a kind of detection method growing relevant CNV to Qinchuan Cattle and mark as claimed in claim 1, is characterized in that: described real-time fluorescence quantitative PCR response procedures used is: (1) denaturation 95 DEG C of 30s; (2) amplified reaction: 95 DEG C of sex change 10s, 60 DEG C of annealing 30s, 39 circulations.
7. as the application of the method in claim 1-6 as described in any one claim in Qinchuan Cattle molecular breeding.
8. apply as claimed in claim 7, it is characterized in that: the described CNV tag application Qinchuan Cattle kind that molecular marker assisted selection growth traits is outstanding in early days.
9. apply as claimed in claim 7, it is characterized in that: the significant correlation that grows of the different copy number in described SHH gene C NV region and Qinchuan Cattle, wherein the growth traits of insert type individuality is significantly higher than absence type individuality.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755444A (en) * | 2016-12-31 | 2017-05-31 | 东北农业大学 | A kind of soybean gene copy number analysis of variance method |
| CN107119114A (en) * | 2017-04-12 | 2017-09-01 | 武汉科技大学 | A kind of detection method of type ii diabetes related gene KCNIP1 copies number variation and application |
| 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 |
| 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 |
| CN110964839A (en) * | 2020-01-03 | 2020-04-07 | 西北农林科技大学 | Method for detecting growth traits of cattle under assistance of SERPINA3-1 gene CNV marker and application thereof |
| CN114657267A (en) * | 2022-04-28 | 2022-06-24 | 中国农业科学院兰州畜牧与兽药研究所 | Detection method and application of CNV marker of yak MICALL2 gene |
| CN114686602A (en) * | 2022-04-28 | 2022-07-01 | 中国农业科学院兰州畜牧与兽药研究所 | Detection method and application of yak HSF1 gene CNV marker |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525662A (en) * | 2009-04-02 | 2009-09-09 | 西北农林科技大学 | Molecular marking method for indicating and identifying size of Qinchuan cattle body by GDF5 gene |
| CN101899527A (en) * | 2010-08-30 | 2010-12-01 | 西北农林科技大学 | Molecular marking method of A-FABP gene predicted Qinchuan cattle meat quality |
| CN102363803A (en) * | 2011-05-09 | 2012-02-29 | 西北农林科技大学 | Method for detecting linked mutation of AMPD1 gene of Qinchuan cattle |
| CN103031376A (en) * | 2012-12-10 | 2013-04-10 | 西北农林科技大学 | Genetic diagnosis method for detecting different cattle groups at home and abroad |
| CN103233001A (en) * | 2012-06-25 | 2013-08-07 | 西北农林科技大学 | Qinchuan cattle FoxO1 gene mononucleotide polymorphism molecular marker detection method and application |
| CN103320429A (en) * | 2013-05-20 | 2013-09-25 | 西北农林科技大学 | Method for detecting Qinchuan cattle Wnt7a gene single nucleotide polymorphism, and application thereof |
| CN103695416A (en) * | 2013-09-29 | 2014-04-02 | 西北农林科技大学 | Mononucleotide polymorphism detection method of Qinchuan cattle CFL2 gene and application thereof |
| CN103740830A (en) * | 2014-01-13 | 2014-04-23 | 西北农林科技大学 | Molecular marking method for indicating Qinchuan cattle meat tenderness and water-holding capacity by utilizing THRSP (thyroid hormone responsive spot) genes |
-
2015
- 2015-11-30 CN CN201510867756.0A patent/CN105349674B/en not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101525662A (en) * | 2009-04-02 | 2009-09-09 | 西北农林科技大学 | Molecular marking method for indicating and identifying size of Qinchuan cattle body by GDF5 gene |
| CN101899527A (en) * | 2010-08-30 | 2010-12-01 | 西北农林科技大学 | Molecular marking method of A-FABP gene predicted Qinchuan cattle meat quality |
| CN102363803A (en) * | 2011-05-09 | 2012-02-29 | 西北农林科技大学 | Method for detecting linked mutation of AMPD1 gene of Qinchuan cattle |
| CN103233001A (en) * | 2012-06-25 | 2013-08-07 | 西北农林科技大学 | Qinchuan cattle FoxO1 gene mononucleotide polymorphism molecular marker detection method and application |
| CN103031376A (en) * | 2012-12-10 | 2013-04-10 | 西北农林科技大学 | Genetic diagnosis method for detecting different cattle groups at home and abroad |
| CN103320429A (en) * | 2013-05-20 | 2013-09-25 | 西北农林科技大学 | Method for detecting Qinchuan cattle Wnt7a gene single nucleotide polymorphism, and application thereof |
| CN103695416A (en) * | 2013-09-29 | 2014-04-02 | 西北农林科技大学 | Mononucleotide polymorphism detection method of Qinchuan cattle CFL2 gene and application thereof |
| CN103740830A (en) * | 2014-01-13 | 2014-04-23 | 西北农林科技大学 | Molecular marking method for indicating Qinchuan cattle meat tenderness and water-holding capacity by utilizing THRSP (thyroid hormone responsive spot) genes |
Non-Patent Citations (2)
| Title |
|---|
| 滑留帅: "牛SHH基因通过PPARg通路调控脂肪生成", 《中国优秀博士学文论文》 * |
| 马云等: "秦川牛分子标记研究进展", 《中国牛业科学》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755444A (en) * | 2016-12-31 | 2017-05-31 | 东北农业大学 | A kind of soybean gene copy number analysis of variance method |
| CN107119114A (en) * | 2017-04-12 | 2017-09-01 | 武汉科技大学 | A kind of detection method of type ii diabetes related gene KCNIP1 copies number variation and application |
| 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 |
| 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 |
| CN107523643B (en) * | 2017-10-20 | 2020-08-04 | 西北农林科技大学 | Method for auxiliary detection of growth traits of cattle KCNJ12 gene CNV marker and special kit thereof |
| CN110964839A (en) * | 2020-01-03 | 2020-04-07 | 西北农林科技大学 | Method for detecting growth traits of cattle under assistance of SERPINA3-1 gene CNV marker and application thereof |
| CN114657267A (en) * | 2022-04-28 | 2022-06-24 | 中国农业科学院兰州畜牧与兽药研究所 | Detection method and application of CNV marker of yak MICALL2 gene |
| CN114686602A (en) * | 2022-04-28 | 2022-07-01 | 中国农业科学院兰州畜牧与兽药研究所 | Detection method and application of yak HSF1 gene CNV marker |
| CN114657267B (en) * | 2022-04-28 | 2023-07-14 | 中国农业科学院兰州畜牧与兽药研究所 | Detection method and application of yak MICALL2 gene CNV marker |
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