CN112342301B - Method for detecting CNV (CNV) mark of NSMF (NSMF) gene of Hu sheep and application of CNV mark - Google Patents

Method for detecting CNV (CNV) mark of NSMF (NSMF) gene of Hu sheep and application of CNV mark Download PDF

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CN112342301B
CN112342301B CN202011259217.6A CN202011259217A CN112342301B CN 112342301 B CN112342301 B CN 112342301B CN 202011259217 A CN202011259217 A CN 202011259217A CN 112342301 B CN112342301 B CN 112342301B
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孙伟
曹修凯
袁泽湖
王善禾
王珊
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Abstract

The invention provides a method for detecting Hu sheepNSMFGene CNV (carbon nanotubes) marking method and application thereof, wherein CNV marking refers to Hu sheepNSMFCopy number variation in gene candidate region chr3:545787-597706, using Hu sheep genome DNA as template, using normal double copy geneANKRD1As reference genes, hu sheep were amplified by fluorescent quantitative PCR, respectivelyNSMFPartial fragments of the genes and reference genes according to 2 x 2 −ΔCt Quantitative results the Hu sheep individuals were classified into an increased type, a deleted type and a normal type. The invention detects CNV markers closely related to the Hu sheep body length character on the DNA level and is applied to molecular marker assisted selection of the Hu sheep growth character, thereby accelerating the genetic breeding process of the Hu sheep.

Description

Detection Hu sheepNSMFGene CNV marking method and application thereof
Technical Field
The invention relates to a method for detecting Hu sheep based on fluorescent quantitative PCR technologyNSMFA method for marking gene CNV, which belongs to the technical field of livestock molecular biology detection.
Background
The applicant knows that sheep germplasm resources in China are rich, but the problems of lack of high-quality mutton sheep germplasm resources, delay of breeding progress, excessive dependence of mutton sheep fine breed on import and the like exist, and the sustainable healthy development of sheep germplasm resources protection and mutton sheep industry in China is seriously influenced. Hu sheep is a famous lamb skin sheep variety in China, and has the advantages of strong adaptability, high lambing rate, fast growth, good meat quality and the like. The genetic breeding development of the Hu sheep is quickened, and the method has great significance for the germplasm innovation and industrialization of sheep in China.
Molecular breeding mainly refers to DNA molecular marker assisted breeding, and is a technology for genetically improving biological populations by using molecular markers (or DNA markers) on the DNA level. The marker assisted breeding is a modern breeding technology for improving economic traits of livestock by utilizing key genes of important economic traits or DNA markers linked with quantitative trait loci, is a combination of a molecular biology technology and a traditional breeding technology, and can achieve the purposes of early seed selection and improvement of accuracy of breeding values by typing individual DNA markers, so that the genetic breeding progress of the livestock is accelerated.
Copy number variation (copy number variation, CNV) refers to the duplication or deletion of larger fragments in genomic DNA, typically 50 bp to several Mb in fragment size, which is a structural variation at the genomic sub-microscopic level. Compared with the DNA genetic markers (SNP and Indel) which are widely applied at present, the genome region covered by the CNV is larger, the change of gene expression (mRNA and protein) is more obvious, and researches prove that the CNV has obvious association with the phenotype of certain complex characters of human beings and livestock and poultry. CNV can therefore be used as a novel DNA marker for marker-assisted selection.
The current CNV identification methods are mainly divided into two categories: one class is mainly used for detecting unknown CNV over the whole genome, including genomic chips (comparative genomic hybridization chip and SNP chip) and high throughput sequencing technologies (genomic re-sequencing and high throughput chromatin conformation capture sequencing); the other class is mainly used for specific site CNV detection or validation (hybridization-based detection and PCR-based detection). The CNV identification method in the prior art has the following characteristics:
(1) Unknown CNV is detected based on genome chip, but the method has the advantages of long time consumption, poor accuracy, difficult detection of small-fragment CNV and the like, although the method has the advantages of high flux and low price; (2) With the maturation of new generation sequencing technology, detecting the structural variation of the genome directly by re-sequencing has become the most effective detection means at present, and the method overcomes the defects of genome chips, does not need complex chip design work, and can more accurately detect the structural variation of the genome by applying deep sequencing; (3) The methods for detecting or verifying the CNV at the specific site based on hybridization mainly comprise QMPSF, MLPA, FISH, southern blotting, MAPH and the like, and the methods have the defects of complicated operation steps, long time consumption, radioactivity and the like; (4) At present, fluorescent quantitative PCR is the most commonly used method for detecting or verifying known CNV, and the method has the advantages of simple and convenient operation, rapidness, safety, low cost and reliable result. The fluorescence quantitative PCR reaction system is added with excessive SYBR Green dye molecules, which can specifically permeate into DNA double chains and emit fluorescent signals, the free dye molecules have low fluorescence background and hardly emit light,thus, the increase of the signal is synchronized with the increase of the fluorescent quantitative PCR product, and the amount of genomic DNA can be reflected by detecting the intensity of the fluorescent signal. By relative quantification of the target gene (with copy number variation) and the reference gene (normal two copies, no variation), according to 2.multidot.2 −ΔCt The method is used for counting and detecting the copy number of the sample candidate genes.
NSMFGene (NMDA Receptor Synaptonuclear Signaling And Neuronal Migration Factor) also known asNELF(Nasal Embryonic Luteinizing Hormone-Releasing Hormone Factor) the gene is closely related to olfactory nerve development and luteinizing hormone secretion, and abnormal expression of the gene leads to abnormal gonadal and olfactory development.
The heavy sequencing of the Hu sheep genome shows that,NSMFthe gene candidate region (chr 3:545787-597706, oar_v4.0) had a 51920 bp copy number variation, which may affect the function of the gene. But at present, no relatedNSMFThe influence of the gene CNV on the growth traits of Hu sheep is reported in the literature.
Disclosure of Invention
The invention aims to solve the technical problems of overcoming the defects of the prior art and providing a method for detecting Hu sheepNSMFA method for marking gene CNV and application thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
detection Hu sheepNSMFA method for gene CNV labelling comprising the steps of:
respectively amplifying Hu sheep by using genomic DNA of Hu sheep to be detected as a template and utilizing fluorescent quantitative PCRNSMFGene copy number variation region and reference geneANKRD1Is used for judging Hu sheep according to quantitative resultsNSMFCopy number variation type of gene.
The Hu sheepNSMFThe gene copy number variation region is located inNSMFGene candidate region chr3:545787-597706.
The copy number variation type is to make the quantitative result according to 2.multidot.2 −ΔCt The Hu sheep individuals are classified by the method: increase type, 2 x 2 −ΔCt Not less than 2.5; deletion type, 2 x 2 −ΔCt <1.5; normal, 1.5.ltoreq.2.times.2 −ΔCt <2.5。
The Hu sheepNSMFThe amplification primer pair P1 of the gene copy number variation region is as follows:
upstream primer F1: 5'-GGCTACATCTGGGACCATCTT-3' the number of the individual pieces of the plastic,
downstream primer R1: 5'-CGAGCCGCCCAATTTTATGGT-3';
the reference geneANKRD1The amplification primer pair P2 of the partial fragment is as follows:
the upstream primer F2: 5'-AAGACCCCCGAAATGCTACC-3' the number of the individual pieces of the plastic,
downstream primer R2: 5'-GCCGACCTCAACGTCAAGAA-3'.
The amplification system used for the fluorescent quantitative PCR included 10 ng/. Mu.L of genomic template DNA 1.0. Mu.L, 10 mM of the upstream and downstream primers corresponding to the primer pair P1 or P2 of 0.5. Mu.L each, 2 XSYBR Green Mix (Takara) 6.5. Mu.L, and deionized water 4.5. Mu.L.
The reaction program of the fluorescent quantitative PCR is as follows: 95. pre-denaturing at a temperature of 10min; 95. denaturation at 15℃for 30 s at 60℃for 40 cycles.
The fragment size of the fluorescent quantitative PCR amplification product based on the primer pair P1 was 137 bp, and the fragment size of the fluorescent quantitative PCR amplification product based on the primer pair P2 was 128 bp.
Above Hu sheep detectionNSMFThe gene CNV labeling method is applied to molecular marker-assisted selective breeding of Hu sheep.
Individuals with the copy number variation type being normal are superior in growth traits to individuals with both deletion and addition copy number variation types.
The growth characters are primary weight, 6 month-old body height and 6 month-old body length.
In the invention, sheep is used asNSMFThe gene candidate region chr3 is 545787-597706 as a CNV detection candidate region, and the copy number variation condition of the locus in a Hu sheep population is detected by a fluorescent quantitative PCR technology and is related to important economic characters such as weight, body size and the like; individuals with normal copy number variation types develop traits (birth weight, 6 month oldHigh, 6 month old) is superior to individuals with both deletion and addition copy number variation types. The gene CNV is researched and is subjected to association analysis with important economic characters of Hu sheep, and the gene CNV can be used for molecular marker assisted selection of the growth characters of the Hu sheep, so that the genetic breeding process of the Hu sheep is quickened, and the Hu sheep population with excellent genetic resources is quickly established.
Compared with the prior art, the invention has the following advantages:
(1) The Hu sheep provided by the inventionNSMFThe gene CNV detection method is not limited by the age of the Hu sheep, can be used for early breeding of the ewes, and can be selected even just after birth;
(2) CheckingNSMFThe method for marking the gene CNV is accurate and reliable, simple to operate and low in cost;
（3）NSMFthe identification of the gene CNV marker can provide scientific basis for auxiliary selection of the Hu sheep molecular marker.
Drawings
FIG. 1 shows amplification curves of fluorescent quantitative PCR according to the present invention.
FIG. 2 shows melting curves of fluorescence quantitative PCR according to the present invention.
FIG. 3 is a agarose gel electrophoresis chart of a fluorescent quantitative PCR amplification product according to the present invention.
Detailed Description
The following describes the technical scheme of the present invention in further detail by combining examples: the embodiment is implemented on the premise of the technical scheme of the invention, and a detailed implementation mode and a specific operation process are provided, but the protection authority of the invention is not limited to the implementation. The reagents and materials used in this example are all commercially available and are not listed here.
The CNV mark based on fluorescent quantitative PCR technology related by the invention refers to Hu sheepNSMFCopy number variation in gene candidate region chr3:545787-597706 (Hu sheep genome version oar_v4.0) with Hu sheep genomic DNA as template and normal double copy geneANKRD1As reference genes, hu sheep were amplified by fluorescent quantitative PCR, respectivelyNSMFPartial fragments of the gene and the reference gene are determined based on quantitative resultsHu sheepNSMFCopy number variation type of gene. Specifically, the copy number variation type is to quantify the result according to 2 x 2 −ΔCt The Hu sheep individuals are classified by the method: increase type, 2 x 2 −ΔCt Not less than 2.5; deletion type, 2 x 2 −ΔCt <1.5; normal, 1.5.ltoreq.2.times.2 −ΔCt <2.5, selecting according to the parting resultNSMFThe normal individuals of the gene CNV are taken as dominant individuals, and the seeds are reserved for cultivation.
1. Hu sheep sample collection
Using hu sheep as a detection object, 432 hu sheep jugular vein blood samples were collected from Jiang Susheng Xuzhou su sheep industry limited.
2. Isolation, extraction and purification of genomic DNA
Reference is made to the method of Sambrock et al (2002).
3. Amplification of target sequences and reference sequences
Hu sheep published with NCBI database (http:// www.ncbi.nlm.nih.gov /)NSMFGene sequence (GenBank oar_v4.0) as reference sequence was amplified by Primer 5.0 designNSMFFluorescent quantitative PCR primer (namely primer pair P1, the gene sequence of which is shown as sequence 1 and sequence 2 in a sequence table) of the gene copy number variation region, wherein the internal reference sequence is a sequence without copy number variation, namelyANKRD1The sequence of 143 bp in the gene was amplified using Primer 5.0 designANKRD1The fluorescent quantitative PCR primer of the sequence of the gene segment (namely primer pair P2, the gene sequence of which is shown as sequence 3 and sequence 4 in a sequence table). Primer pair sequence information is shown in table 1.
TABLE 1 fluorescent quantitative PCR primer information
Gene Primer pair Primer sequence (5 '-3') Amplified fragment
NSMF P1 F1：GGCTACATCTGGGACCATCTT 137 bp
R1：CGAGCCGCCCAATTTTATGGT
ANKRD1 P2 F2：AAGACCCCCGAAATGCTACC 128 bp
R2：GCCGACCTCAACGTCAAGAA
The amplification system used for fluorescent quantitative PCR was 13. Mu.L: 10 ng/. Mu.L of template DNA (genomic DNA extracted from blood sample), 0.5. Mu.L of each of the upstream and downstream primers corresponding to the primer pair P1 or P2 of 10 mM, 6.5. Mu.L of 2 XSYBR Green Mix (Takara), and 4.5. Mu.L of deionized water.
The reaction procedure used for performing fluorescent quantitative PCR was: (1) Pre-denaturation: 95. 10min at the temperature; (2) amplification reaction: 95. denaturation at 15℃for 30 s at 60℃for 40 cycles; (3) drawing a dissolution curve: 95. 10 ℃ C., s, -0.01 ℃/s,65 ℃ C., 1 min.
The primers were determined to be suitable for fluorescent quantitative PCR analysis by drawing amplification curves, dissolution curves and agarose gel electrophoresis. The amplification curve is smooth, which indicates that the fluorescent quantitative PCR amplification system and conditions are proper (see FIG. 1); the dissolution curves of all samples have obvious single peak values at the same positions on the abscissa, which indicates that the amplified products are single (see FIG. 2); agarose gel electrophoresis showed that the size of the fluorescent quantitative PCR product was as expected and the amplified fragment was correct (see FIG. 3).
4. Inference of copy number variation
Each individual was amplified with primers for the target sequence and the internal reference sequence, respectively, and 3 technical replicates were set for each individual. According to 2 x 2 −ΔCt The method performs copy number analysis. Wherein Δct= (C T target sequence - C T internal reference sequence ). When the Hu sheep individual to be detected is normal, the value of 1.5 is less than or equal to 2 x 2 −ΔCt <2.5; when the Hu sheep individual to be detected is absent, 2 x 2 −ΔCt <1.5. When the Hu sheep individual to be detected is increased, 2 x 2 −ΔCt ≥ 2.5。
5. Hu sheepNSMFCorrelation analysis of gene CNV locus and growth character
Production data: birth weight, 6 month-old body height, and 6 month-old body length.
Correlation analysis model: analysis with SPSS 18 software using the following fixed modelNSMFPhenotypic effects of gene CNV.
Y ijk = μ +A i +S j +CNV k + e ijk
Wherein:Y ijk in order to obtain the observed value of the character,μas a global average value of the values,A i is the firstiThe age of the Hu sheep alone,S j is the firstjThe sex of the individual in the head,CNV k is the firstkThe fixed effect of the individual copy number variation types,e ijk is a random error.
The correlation analysis results showed (see table 2): in the group of Hu sheep,NSMFthe gene CNV locus can obviously influence the growth and development of individuals, and the normal individuals are better than the deletion type individuals and the addition type individuals, which shows thatNSMFThe CNV locus of the gene can be used as a molecular marker for improving the growth characteristics of Hu sheep.
TABLE 2NSMFCorrelation analysis of gene CNV and Hu sheep growth character
6. Application of CNV marker in Hu sheep breeding
By using the obtained candidate molecular genetic markers, molecular marker assisted selection can be carried out on characters such as Hu sheep body length and the like, so that the breeding process of hu sheep variety improvement is quickened.
In a word, the invention detects CNV markers closely related to Hu sheep body length traits on the DNA level and is applied to molecular marker assisted selection of Hu sheep growth traits, thereby accelerating the genetic breeding process of Hu sheep.
The foregoing is merely illustrative of the embodiments of the present invention, and the scope of the present invention is not limited thereto, and any person skilled in the art will appreciate that modifications and substitutions are within the scope of the present invention, and the scope of the present invention is defined by the appended claims.
Sequence listing
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gccgacctca acgtcaagaa 20

Claims (8)

1. Detection Hu sheepNSMFA method for marking a gene CNV, comprising the steps of:
respectively amplifying Hu sheep by using genomic DNA of Hu sheep to be detected as a template and utilizing fluorescent quantitative PCRNSMFGene copy number variation region and reference geneANKRD1Is used for judging Hu sheep according to quantitative resultsNSMFCopy number variation type of the gene; the Hu sheepNSMFThe gene copy number variation region is located inNSMFGene candidate region chr3, 545787-597706, the Hu sheep genome version is oar_v4.0;
the copy number variation type is to make the quantitative result according to 2.multidot.2 −ΔCt The Hu sheep individuals are classified by the method: increase type, 2 x 2 −ΔCt Not less than 2.5; deletion type, 2 x 2 −ΔCt <1.5; normal type, 1.5-1 2*2 −ΔCt <2.5。
2. A detection method according to claim 1NSMFThe method for marking the gene CNV is characterized in thatNSMFThe amplification primer pair P1 of the gene copy number variation region is as follows:
upstream primer F1: 5'-GGCTACATCTGGGACCATCTT-3' the number of the individual pieces of the plastic,
downstream primer R1: 5'-CGAGCCGCCCAATTTTATGGT-3';
the reference geneANKRD1The amplification primer pair P2 of the partial fragment is as follows:
the upstream primer F2: 5'-AAGACCCCCGAAATGCTACC-3' the number of the individual pieces of the plastic,
downstream primer R2: 5'-GCCGACCTCAACGTCAAGAA-3'.
3. A detection method according to claim 1NSMFA method for marking gene CNV, characterized in that: the amplification system used for the fluorescent quantitative PCR comprises 10 ng/. Mu.L of template DNA 1.0. Mu.L, 0.5. Mu.L of upstream and downstream primers corresponding to the primer pair P1 or P2 of 10 mM, 6.5. Mu.L of 2 XSYBR Green Mix and 4.5. Mu.L of deionized water.
4. A detection method according to claim 1NSMFThe method for marking the gene CNV is characterized in that the reaction program of the fluorescent quantitative PCR is as follows: 95. pre-denaturing at a temperature of 10min; 95. denaturation at 15℃for 30 s at 60℃for 40 cycles.
5. A detection method according to claim 2NSMFA method for marking gene CNV, characterized in that: the fragment size of the fluorescent quantitative PCR amplification product based on the primer pair P1 was 137 bp, and the fragment size of the fluorescent quantitative PCR amplification product based on the primer pair P2 was 128 bp.
6. Use of the method according to any one of claims 1 to 5 in CNV marker-assisted selection breeding of hu sheep.
7. The use according to claim 6, wherein: individuals with the copy number variation type being normal are superior in growth traits to individuals with both deletion and addition copy number variation types.
8. The use according to claim 7, wherein: the growth characters are primary weight, 6 month-old body height and 6 month-old body length.
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CN106498080A (en) * 2016-12-14 2017-03-15 兰州大学 Method and its application using PCR SSCP quick detection sheep NELF gene mononucleotide polymorphisms
CN110029156A (en) * 2019-05-08 2019-07-19 西北农林科技大学 A kind of method and its application of detection tea card sheep KAT6A gene C NV label
CN110079610A (en) * 2019-05-08 2019-08-02 西北农林科技大学 A kind of method and its application of detection tea card sheep BAG4 gene C NV label
CN110079615A (en) * 2019-06-10 2019-08-02 西北农林科技大学 A kind of method and its application of detection tea card sheep KMT2D gene C NV label

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Publication number Priority date Publication date Assignee Title
CN106498080A (en) * 2016-12-14 2017-03-15 兰州大学 Method and its application using PCR SSCP quick detection sheep NELF gene mononucleotide polymorphisms
CN110029156A (en) * 2019-05-08 2019-07-19 西北农林科技大学 A kind of method and its application of detection tea card sheep KAT6A gene C NV label
CN110079610A (en) * 2019-05-08 2019-08-02 西北农林科技大学 A kind of method and its application of detection tea card sheep BAG4 gene C NV label
CN110079615A (en) * 2019-06-10 2019-08-02 西北农林科技大学 A kind of method and its application of detection tea card sheep KMT2D gene C NV label

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