CN113151489B - Molecular diagnosis method for evaluating growth traits based on cow ZNF146 gene CNV marker and application thereof - Google Patents

Molecular diagnosis method for evaluating growth traits based on cow ZNF146 gene CNV marker and application thereof Download PDF

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CN113151489B
CN113151489B CN202110218217.XA CN202110218217A CN113151489B CN 113151489 B CN113151489 B CN 113151489B CN 202110218217 A CN202110218217 A CN 202110218217A CN 113151489 B CN113151489 B CN 113151489B
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CN113151489A (en
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刘贤
黄永震
丁晓婷
蔡雯雯
徐美芳
丁涵
彭巍
黄殷琪
李欣淼
吴胜军
李志明
张子敬
王二耀
茹宝瑞
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Henan Animal Husbandry General Station
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Abstract

The invention discloses a molecular diagnosis method for evaluating growth traits based on a cow ZNF146 gene CNV marker and application thereof: based on real-time quantitative PCR, using bovine genome DNA to be detected as a template, respectively using two pairs of primers P1 and P2 to amplify the CNV region of ZNF146 gene and partial fragment of reference gene BTF3, and finally using 2 x 2 ‑ΔΔCt The method of (3) calculates and determines the type of copy number variation of the individual. The method can detect the CNV marker closely related to the growth traits of the cattle on the DNA level, and is simple and quick and convenient to popularize and apply.

Description

Molecular diagnosis method for evaluating growth traits based on CNV marker of cattle ZNF146 gene and application thereof
Technical Field
The invention belongs to the field of livestock molecular biology detection, and particularly relates to a method for detecting bovine ZNF146 gene copy number variation by utilizing qPCR (quantitative polymerase chain reaction), which can be based on 2 x 2 -ΔΔCt Determining the copy number variation type of the individual.
Background
Copy Number Variations (CNVs) refer to insertion or deletion Variations of genomic sequences greater than 50bp between two individuals of a species, a type of genomic structural variation. It can affect gene function and the phenotype of an individual by dose effects, position effects, blocking of functional genes, fusion of genes, exposure of 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.
The research on the molecular mechanism for regulating the growth and muscle development of the beef cattle can provide a theoretical basis for the development of the beef cattle industry. Research shows that some CNV sites are located inside functional gene and relevant to the normal growth and development of ox. Among the methods for detecting the known CNV, quantitative Real-Time PCR (qPCR) is a widely used technology, and has the advantages of simple operation, high sensitivity and high speed; 2 x 2 can be used by selecting a single copy gene of cattle, such as BTF3 gene verified by Liu and the like as an internal reference gene -ΔΔCt The method of (3) determines the type of copy number variation and the relative copy number of the individual.
The Zinc Finger Protein 146(Zinc Finger Protein 146, ZNF146) gene belongs to C2H2(Kruppel) type Zinc Finger Protein family, and the coded Zinc Finger Protein consists of 10 Zinc Finger motifs and lacks a trans-activation structural domain. The ZNF146 gene is located in human chromosome 19, and the encoded regulatory protein is involved in various cell activities, such as development, differentiation and the like. Evolutionary research analysis reveals that the expression of the bovine ZNF146 gene is located on bovine chromosome 18, and the encoded protein has 95% homology with human.
So far, no literature report about detecting the copy number variation of the ZNF146 gene of the cattle and the influence of the CNV of the ZNF146 gene on the growth traits of local cattle and other bred cattle varieties is found.
Disclosure of Invention
The invention aims to provide a molecular diagnosis method for evaluating growth traits based on a CNV marker of a cattle ZNF146 gene and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting copy number variation of a ZNF146 gene of a cow comprises the following steps: the method comprises the steps of taking genome DNA of a cattle individual to be detected as a template, taking a primer pair P1 and a primer pair P2 as primers, amplifying a copy number variation region of the ZNF146 gene and a partial fragment of the BTF3 gene serving as an internal reference through real-time quantitative PCR, and identifying the copy number variation type of the individual ZNF146 gene according to a quantitative result.
Preferably, the copy number variation region is located from position 47225201 to position 47229600 of the reference genomic sequence AC _000175.1 of the bovine ZNF146 gene.
Preferably, said copy number variation pattern is according to 2 x 2 -ΔΔCt The quantitative results were divided into three categories: multicopy type, 2 x 2 -ΔΔCt >2; deletion form, 2 x 2 -ΔΔCt <2; normal type, 2 x 2 -ΔΔC ＝2。
Preferably, the primer pair P1 is:
the upstream primer F1: 5'-CTCTCAGCGAACATCACTTAT-3'
The downstream primer R1: 5'-AAGGGTTGAGAACTGCGAGAA-3', respectively;
the primer pair P2 is as follows:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3', respectively;
the size of the PCR product fragment amplified based on the primer pair P1 is 187bp, and the size of the PCR product fragment amplified based on the primer pair P2 is 166 bp.
Preferably, the amplification system used for the real-time quantitative PCR comprises 1 muL of 10-50 ng/muL of template and 0.5 muL of each of the upstream primer and the downstream primer corresponding to 10 mumol/L of primer pair P1 or primer pair P2.
Preferably, the reaction procedure for real-time quantitative PCR is: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 20s for 39 cycles.
The method for detecting the copy number variation of the ZNF146 gene of the cattle is applied to the auxiliary selection breeding of the cattle molecular marker.
Preferably, in the case of the Yunling cattle, individuals having a deletion-type copy number variation pattern are superior in growth traits.
Preferably, individuals with normal copy number variation types are superior in growth traits among red cows, jianxian.
Preferably, the growth trait is selected from one or more of body height, oblique body length, cross height, chest circumference, waist width, nojiri length, abdominal circumference, canal circumference, chest width, chest depth, hip circumference, hip width, head length, forehead width, body weight and sacrum height.
Preferably, the copy number variation region is used as a copy number variation site, and has significant correlation with the chest circumference and the ischium width of a Yunnan cattle and significant correlation with the ischium width of a red cattle in Jiaxian county.
A kit for detecting copy number variation of a ZNF146 gene of a cow comprises the primer pair P1 and a primer pair P2.
The invention has the following beneficial effects:
according to the invention, the copy number variation condition of the ZNF146 gene in a cattle group is detected by a real-time quantitative PCR technology, and the correlation analysis result of the ZNF146 gene copy number variation and the important economic traits of cattle shows that the Copy Number Variation (CNV) locus of the ZNF146 gene has a molecular marker which obviously influences the growth trait advantages of the cow in Yunling and Jiaxian county. Therefore, by accurately detecting and identifying the copy number types of individuals, the cattle population with excellent genetic resources can be quickly established, and the breeding process of the excellent growth traits of the cattle is accelerated.
Compared with the prior art, the invention has the following advantages:
(1) the method for detecting the copy number variation of the ZNF146 gene of the cow is not limited by age, can be used for early breeding of cows, and can be selected even just after birth.
(2) The method for detecting the copy number variation of the ZNF146 gene is accurate and reliable and is simple and convenient to operate.
(3) The detection of the ZNF146 gene copy number variation site provides scientific basis for the auxiliary selection of the bovine molecular marker.
Drawings
FIG. 1 is an amplification curve plotted by qPCR (ZNF146 gene) performed in examples of the present invention.
FIG. 2 is a dissolution curve plotted by qPCR (ZNF146 gene) performed in the examples of the present invention.
FIG. 3 shows the distribution of ZNF146 gene copy number variation in bovine populations in accordance with the present invention.
Detailed Description
The invention is further 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 research of re-sequencing of bovine genome, the ZNF146 is found to generate copy number variation in a bovine genome reference region Chr 18:47225201bp-47229600bp, and the relevance of the copy number variation of the ZNF146 gene and the growth traits of cattle is further disclosed by combining the physiological action of the ZNF146 gene and the regulation mechanism of CNV, so that an important basis is provided for the molecular breeding of local cattle and related breeding varieties. The concrete description is as follows.
1. Sample Collection and genomic DNA extraction
(1) Collection of blood samples
TABLE 1 Experimental animal sample information
Figure BDA0002954788440000031
In the invention, 618 individual blood samples (table 1) are collected, wherein the samples are all adult cows more than 2 years old (24-36 months old), the blood collection method is jugular vein blood collection, the adult cows are brought back to a laboratory by an ice box and stored at-80 ℃; basic data collection and measurement were performed simultaneously: during sampling, basic data (growth traits such as body height, cross height, oblique body length, chest circumference, abdominal circumference, canal circumference, chest width, chest depth, hip circumference, waist angle width, hip end width, head length, forehead width, nojiri length, sacrum height and body weight) of corresponding individuals are collected and recorded for later-stage correlation analysis.
(2) Extraction of genomic DNA from blood samples
Unfreezing a frozen blood sample (mainly blood cells) at room temperature, sucking 500 mu L of the frozen blood sample into a 1.5mL centrifuge tube, adding Phosphate Buffer Solution (PBS) with the same volume, uniformly mixing, gently shaking, centrifuging at 4 ℃ at 12000r/min for 5min, and removing supernatant; repeating the above steps until the supernatant is transparent and the precipitate is transparent.
② adding 500 mu L of DNA extraction buffer solution into a centrifuge tube, gently blowing and beating to separate the blood cell sediment from the wall of the centrifuge tube, and carrying out water bath at 37 ℃ for 1 h.
③ adding protease K to 5 mu L (20mg/mL), mixing, digesting in 55 ℃ water bath overnight (about 16 h) until no flocculent precipitate is seen, clarifying the solution, adding 10 mu L protease K, mixing, and digesting continuously until the solution is clear.
Cooling the reaction solution to room temperature, adding 500 mu L of Tris saturated phenol, gently shaking for 15min to fully mix the Tris saturated phenol and the reaction solution, centrifuging the mixture for 10min at 4 ℃ at 12000r/min, and transferring the upper aqueous phase into another sterilized centrifuge tube; repeat step 1 time.
Fifthly, adding 500 mu L of chloroform, gently shaking for 20min to fully mix the mixture, centrifuging for 15min at 12000r/min at 4 ℃, and transferring the upper aqueous phase into another sterilized 1.5mL centrifuge tube.
Sixthly, adding 500mL of chloroform-isoamyl alcohol mixed solution (24:1), fully mixing for 20min, centrifuging for 10min at 4 ℃ at 12000r/min, and transferring the supernatant into another 1.5mL centrifuge tube.
Seventhly, adding 0.1 time volume of NaAc buffer solution and 2 times volume of ice-cold absolute ethyl alcohol, mixing and rotating the centrifugal tube until white flocculent precipitate is separated out.
Eighty percent (4 ℃) and 12000r/min are centrifuged for 10min, the supernatant is discarded, and the DNA sediment is rinsed for 2 times by 70 percent ice-cold ethanol.
Ninthly, centrifuging at 12000r/min for 10min at 4 ℃, removing supernatant, and volatilizing the ethanol at room temperature.
Adding 80-100 mu L of TE into the DNA solution after drying the red fluorescent powder, storing at 4 ℃ until the DNA is completely dissolved, detecting the quality by using an ultraviolet spectrophotometer, and storing at-80 ℃.
2. Design of specific primers for amplification of target gene and reference gene
The sequence of the copy number variation region screened out in the re-sequencing, namely 47225201 to 47229600 of the reference genome sequence of the ZNF146 gene (target gene) is searched by taking a bovine ZNF146 gene (AC _000175.1) published by NCBI as a reference sequence, primers contained in the region are designed by using Prime 5.0 software, and the primers are compared in NCBI _ BLAST. The primer sequences are as follows (primer pair P1), and the size of the amplified target fragment is 187 bp:
the upstream primer F1: 5'-CTCTCAGCGAACATCACTTAT-3'
The downstream primer R1: 5'-AAGGGTTGAGAACTGCGAGAA-3'
Meanwhile, a bovine BTF3 gene sequence (AC _000177.1) published by NCBI is used as a reference sequence, primers for amplifying a specific fragment (166bp) in a BTF3 gene (internal reference gene) are designed by the same method, and the primer sequences are as follows (primer pair P2):
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3'
The specificity of the primer pair P1 and P2 amplification products was verified by common PCR amplification and 1% agarose electrophoresis.
3. Real-time quantitative PCR
The qPCR amplification system is shown in table 2.
TABLE 2 amplification System for qPCR
Figure BDA0002954788440000051
The qPCR reaction procedure was:
(1) pre-denaturation: at 95 ℃ for 2 min;
(2) and (3) amplification reaction: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, and 39 cycles.
The amplification curve is smooth, which indicates that the QPCR reagent has good quality and the amplification system and conditions are appropriate (FIG. 1); the drawn dissolution curves are matched together, and the curves are smooth in trend, high and sharp in peak height, and free of hybrid peaks caused by primer dimers or nonspecific amplification, and show that the primer quality is good (figure 2). Primers were determined to be suitable for qPCR analysis by plotting amplification curves and melting peaks.
CNV type determination
Each sample was amplified with primers for the gene of interest and the reference gene (primer pair P1 and P2), respectively, and 3 replicates for each pair of primers. The experimental results adopted 2 x 2 -ΔΔCt The method carries out calculation, and the specific calculation method comprises the following steps:
ΔΔCt＝ΔCt (Experimental group) -ΔCt (reference group) ，ΔCt (Experimental group) ＝C T (Experimental group target gene) -C T (Experimental group internal reference gene) ，ΔCt (reference group) ＝C T (reference group target gene) -C T (reference group internal reference gene)
In the formula, the experimental group is an individual sample to be detected whether the copy number variation exists or not. The reference group is an individual sample with no known copy number variation, and the selected reference group of Qinchuan cattle individuals in the re-sequencing test can be adopted. C T Cycle threshold, which is the number of amplification cycles that pass when the fluorescence signal of the amplification product reaches a set threshold during the PCR amplification.
When the gene of interest is a normal (Median) sequence, a normalization value 2 x 2 is calculated -ΔΔCt 2. When the target gene is a deletion (Loss) sequence, a normalized value of 2 x 2 is calculated -ΔΔCt <2. When the gene of interest is a multicopy (Gain) sequence, a normalization value 2 x 2 is calculated -ΔΔCt >2。
5. Data processing
The number of individuals of various types (Gain, media and Loss) in the detection population is counted, and the frequencies of the various types are counted.
The correlation analysis was performed using SPSS (18.0). In the data processing, according to different factors influencing the body size property index, the environmental effect, the age, the gender, the genetic effect and the interaction effect are considered, a fixed model is adopted for analysis, and simplification is carried out according to the actual situation. The complete model is as follows:
Y ijk ＝μ+G j +E ijk
wherein, Y ijk (ii) recording the phenotype of the individual; μ is the population mean; g j The copy number variation type of each site; e ijk Is a random error.
The results of the data processing are shown in tables 3 to 7.
TABLE 3 correlation analysis of the CNV gene ZNF146 with different growth traits of Qinchuan cattle
Figure BDA0002954788440000061
Correlation analysis results show (see table 3): the deletion type (Loss) copy number variation type of the ZNF146 gene has certain promotion effect on the height, oblique length, cross height, chest circumference, chest width, chest depth, nojiri length, ischium end width, waist angle width and weight of Qinchuan cattle individuals.
TABLE 4 correlation analysis of the CNV gene ZNF146 with different growth traits of southeast cattle
Figure BDA0002954788440000071
Correlation analysis results show (see table 4): the deletion type (Loss) and the multiple copy (media) copy number variation type of the ZNF146 gene have no obvious effect on promoting the body height, oblique length, cross height, chest circumference, tube circumference and weight of the southeast summer cattle.
TABLE 5 correlation analysis of the ZNF146 gene CNV and different growth traits of Yunnan cattle
Figure BDA0002954788440000072
Figure BDA0002954788440000081
Note: the average shoulder marks with the same letter indicate no significant difference (P >0.05), the average shoulder marks with different letters indicate significant difference (P < 0.05); p <0.05, P < 0.01.
Correlation analysis results show (see table 5): individuals with deletion type (Loss) copy number variation types of the ZNF146 gene in the Yunnan cattle are superior in growth traits, and copy number variation sites have obvious relevance to two growth traits, namely chest circumference and ischial width (P < 0.05). Therefore, the Loss type of the ZNF146 gene can be used as a candidate molecular marker for improving the growth traits of the Yunnan cattle, and the breeding process of the excellent growth performance of the cattle is accelerated.
TABLE 6 correlation analysis of the different growth traits of ZNF146 Gene CNV with red cattle in Jiaxian county
Figure BDA0002954788440000082
Note: the average shoulder marks with the same letter indicate no significant difference (P >0.05), the average shoulder marks with different letters indicate significant difference (P < 0.05); p < 0.05.
Correlation analysis results show (see table 6): individuals with normal (media) copy number variation types of ZNF146 gene in red cattle in Jiaxian county are superior in growth trait, and the copy number variation sites have significant correlation with the growth trait of ischial width (P < 0.05). Therefore, the Loss type of ZNF146 gene can be used as a candidate molecular marker for improving the growth traits of red cattle in Jiaxian county, and the breeding process of excellent growth performance of yellow cattle is accelerated.
TABLE 7 correlation analysis of the CNV gene ZNF146 with different growth traits of Pinus parviflora
Figure BDA0002954788440000091
Correlation analysis results show (see table 7): the deletion type (Loss) and the multiple copy (media) copy number variation types of the ZNF146 gene have insignificant effects on promoting the body height, oblique body length, cross height, chest circumference, waist angle width and nojiri length of the Piano cattle.
In summary, the present invention shows copy number variation in bovine genomic sequences obtained by resequencingDesigning specific primers for the different regions, performing qPCR amplification by using bovine genomic DNA as a template, and performing qPCR amplification by using BTF3 gene as a reference gene and 2 x 2 -ΔΔCt The method calculates and judges the copy number type of the individual. By detecting the copy number variation condition of the ZNF146 gene of the cattle and according to the correlation analysis of different copy number variation types and growth traits, the copy number type with the dominant growth trait is found, so that basic data is provided for the molecular breeding of the cattle, and the improvement work of germplasm resources of the cattle is accelerated.
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Claims (3)

1. Detect oxZNF146Application of gene copy number variation method in bovine molecular marker-assisted selective breedingThe method is characterized in that: the detection cattleZNF146The method for gene copy number variation comprises the following steps:
taking the cattle genome DNA to be detected as a template, and respectively amplifying by real-time quantitative PCRZNF146Copy number variation region of gene and gene serving as internal referenceBTF3Partial fragment of gene, and then identifying cattle according to quantitative resultZNF146Copy number variation type of gene;
the copy number variation region is located inZNF14647225201 to 47229600 of the gene reference genomic sequence AC _ 000175.1;
said copy number variation pattern is according to 2 x 2 −ΔΔCt The quantitative results were divided into three categories: multicopy type, 2 x 2 −ΔΔCt >2; deletion type, 2 x 2 −ΔΔCt <2; normal type, 2 x 2 −ΔΔCt =2；
The amplification primer pair of the partial fragment of the copy number variation region is as follows:
the upstream primer F1: 5'-CTCTCAGCGAACATCACTTAT-3'
The downstream primer R1: 5'-AAGGGTTGAGAACTGCGAGAA-3';
saidBTF3The amplification primer pair of the partial segment of the gene is as follows:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3', respectively;
in the Yunnan cattle, individuals with deletion type copy number variation types are superior to individuals with multi-copy type and normal type copy number variation types in growth character bust and width of ischium;
in red cattle in Jiaxian county, individuals with the normal type of copy number variation were superior to individuals with the multicopy type or deletion type of copy number variation in the width of the sessile ends as a growth trait.
2. The use of claim 1, wherein: the real-time quantitative PCR amplification system comprises 1 mu L of template of 10-50 ng/mu L and 0.5 mu L of upstream primer and downstream primer corresponding to 10 mu mol/L amplification primer pair respectively.
3. The use of claim 1, wherein: the reaction procedure of the real-time quantitative PCR is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10s and annealing at 60 ℃ for 20s for 39 cycles.
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