CN110592235B - Method for detecting USP16 gene CNV marker of dairy cow and application thereof - Google Patents
Method for detecting USP16 gene CNV marker of dairy cow and application thereof Download PDFInfo
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Abstract
The invention discloses a method for detecting a milk cow USP16 gene CNV marker and application thereof: using real-time fixesQuantitative PCR technology, using Chinese Holstein cow genome DNA as template, respectively using primer pair P1 and P2 to amplify CNV marking site of bovine USP16 gene and a section of region of internal reference gene BTF3, finally using 2 x 2 ‑ΔΔCt The method of (1) calculates and determines the copy number variation type of an individual, and classifies the result into a multicopy type, a deletion type and a normal type. The method can detect the CNV marker closely related to the lactation character of the Chinese Holstein cow on the DNA level, can accelerate the breeding process of the cow with excellent lactation performance, and is simple, rapid and convenient to popularize and apply.
Description
Technical Field
The invention relates to the field of livestock molecular biology detection, in particular to a method for detecting a CNV (CNV) marker of USP16 gene of a Chinese Holstein cow based on a real-time quantitative PCR (polymerase chain reaction) technology.
Background
Molecular marker-assisted selection (MAS) can utilize molecular markers related to quantitative traits, take marker information as auxiliary information, and accurately and quickly analyze genetic composition of individuals from a molecular level, thereby realizing selection of genotypes. The genetic marker, pedigree and phenotype information can be fully utilized, and compared with a conventional breeding method, the genetic marker, pedigree and phenotype information is rich in information amount and high in selection accuracy.
Copy Number Variations (CNV) refer primarily to structural variations in the genome of a DNA fragment deletion, duplication, insertion, or complex multi-site variation, ranging in size from 1kb to several Mb, compared to a reference genome. Along with the continuous improvement of the construction work of the reference genome of livestock and poultry, and based on the improvement, the group weight sequencing data is rich, so that a large amount of genome variation is found by people. The variation on the genome mainly includes two types, single nucleotide variation (SNP) and Copy Number Variation (CNV). The former has a large number and is widely used as a research and molecular breeding marker; in contrast, the number of CNVs found and validated to date is small, and there are few reports of breeding directly as molecular markers. The CNV has the characteristics of large fragment length, ubiquitous existence, wide genome coverage and the like, so that the CNV can obviously influence the livestock and poultry traits and has wide prospects in livestock and poultry genome research and breeding application.
Currently, dairy CNVs are of increasing interest. On one hand, the mature human genome CNVs detection technology has reference effect on researching the milk cow CNVs; on the other hand, the gradual perfection of the cow genome sequencing map has a promoting effect on the development of CNVs. The establishment and gradual improvement of the CNVs map have important significance for the research of the economic traits and the functional traits of the dairy cows and the promotion of the genetic improvement of the dairy cows.
Ubiquitin-specific protease 16(USP16) plays an important role in gene expression, cell cycle, cell self-renewal or senescence. Research shows that the gene is involved in the mitosis process of cell and plays an important role in separating chromosome. USP16 can also deubiquitinate histone H2A and can modulate the expression of cell division protein regulator 1(PRC 1). In addition, it was found that decreasing the expression level of USP16 in the breast increases tissue responsiveness to Wnt, resulting in upregulation of the downstream Wnt target Axin2, and expansion of the basal compartment and increased epithelial regeneration in vitro and in vivo. Therefore, the USP16 gene has certain influence on the development of the mammary gland of the dairy cow and the milk production.
At present, no literature report about the influence of the USP16 gene CNV on the lactation traits of cows (e.g., local cow breed: Chinese Holstein cows) is found.
Disclosure of Invention
The invention aims to provide a method for detecting a milk cow USP16 gene CNV marker 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 milk cow USP16 gene comprises the following steps:
a genomic DNA of a cow (for example, Holstein, China) is used as a template, a primer pair P1 and a primer pair P2 are used as primers, a partial fragment of a CNV region of a USP16 gene and a partial fragment of a BTF3 gene which is used as a reference are amplified through real-time quantitative PCR respectively, and then the copy number variation type of the USP16 gene of a cow individual is identified according to a quantitative result.
Preferably, the CNV region of the USP16 gene is located at position 6544001 to 6547600 of the reference genomic sequence NC _007299.6 of bovine USP16 gene.
Preferably, said copy number variation pattern is according to 2 x 2 -ΔΔCt The quantitative results were divided into three categories: multicopy (Gain), 2 x 2 -ΔΔCt >2; deletion form (Loss), 2 x 2 -ΔΔCt <2; normal type (Median), 2 x 2 -ΔΔCt ＝2。
Preferably, the primer pair P1 is:
the upstream primer F1: 5'-AGAACCCCCGGAAGTAGAGTA-3'
The downstream primer R1: 5'-CAAAGTGCTACCGCCATTCC-3', respectively;
the primer pair P2 is as follows:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3';
the size of the PCR product fragment amplified based on the primer pair P1 is 200bp, and the size of the PCR product fragment amplified based on the primer pair P2 is 166 bp.
Preferably, the real-time quantitative PCR amplification system comprises: mu.L of 10 ng/. mu.L template DNA, 1. mu.L, 10. mu. mol/L primer pair P1 or upstream and downstream primers corresponding to primer pair P2, 0.5. mu.L each, 2 XSSYBR Mix 5. mu.L and ddH 2 O 3μL。
Preferably, the reaction procedure of the real-time quantitative PCR is as follows: (1) pre-denaturation: 94 ℃ for 2 min; (2) and (3) amplification reaction: denaturation at 94 ℃ for 10s, annealing at 60 ℃ for 30s, 40 cycles.
The method for detecting the copy number variation of the USP16 gene of the dairy cattle is applied to the molecular marker-assisted selective breeding of the dairy cattle.
Preferably, the Chinese Holstein cow individual having a deletion type (Loss) copy number variation type is superior in the milk fat rate and the milk protein rate in the lactation trait, and the Chinese Holstein cow individual having a multiple copy type (Gain) copy number variation type is superior in the milk yield in the lactation trait.
The invention has the following beneficial effects:
the method for detecting the copy number variation of the USP16 gene disclosed by the invention detects the copy number variation condition of a cow (such as a Chinese Holstein cow) by a real-time quantitative PCR technology, and the detection result can be used for identifying candidate molecular genetic markers (CNV markers) related to the improvement of the lactation traits of the cow (related to important lactation traits such as milk yield, milk fat percentage, lactoprotein rate, somatic cell score and the like). Compared with a high-throughput sequencing method, a gene chip method and the like, the method is quick, simple and low in cost, can accurately identify the copy number type of an individual, provides molecular markers and practical basis for molecular breeding of Chinese Holstein dairy cow and other dairy cow varieties, and accelerates the breeding process of the dairy cows with excellent milk production performance.
Drawings
FIG. 1 is an amplification curve plotted by QPCR in an example of the present invention.
FIG. 2 is a solubility curve plotted by QPCR in an example of the present invention.
Detailed Description
The invention will now be described in detail with reference to the drawings and examples, which are given by way of illustration and not by way of limitation.
Based on the previous bovine genome re-sequencing research, the copy number variation from 6544001 to 6547600 of the bovine USP16 genome sequence NC-007299.6 is found, and based on the function of the USP16 gene and the regulation mechanism of CNV, the invention finds the relevance of the copy number variation of the USP16 gene and the lactation trait, and provides important practical basis for molecular breeding of dairy cows, and improvement of milk yield and milk quality.
The invention detects CNV marks based on QPCR technology and utilizes 2 x 2 data -ΔΔCt And (3) processing, wherein the copy number variation result is divided into three types: 2*2 -ΔΔCt >2 is a multicopy type; 2*2 -ΔΔCt <2 is deletion type, 2 x 2 -ΔΔCt Normal type 2.
1. Sample collection and genomic DNA extraction
(1) Blood sample collection and data collection
In the invention, 180 blood samples (collected in 8 months 2015) are collected from a grass beach farm dairy farm in Xian City of Shaanxi province, the samples are all adult Holstein cows, the blood collection method is jugular vein blood collection, and the blood samples are brought back to a laboratory by an ice box and stored at-80 ℃; during sampling, basic data (lactation amount, fat content, protein content and the like) of corresponding individuals are collected and recorded for later-stage correlation analysis.
(2) Extraction of DNA from blood samples
The sample was subjected to extraction of genomic DNA by the phenol-chloroform method.
Unfreezing a frozen blood sample (mainly blood cells) at room temperature, sucking 2mL of blood into a 2.0mL centrifuge tube, and centrifuging at 12000rpm at 4 ℃ for 10 min; discarding the liquid, keeping the precipitate, adding 1.5mL of PBS buffer solution, suspending the precipitate by vortex oscillation, and gently shaking on ice for 15 min; centrifuging at 12000rpm at 4 deg.C for 10min, discarding liquid, and retaining precipitate;
secondly, smashing the precipitate into flocculent, adding 500 mu L of DNA extraction buffer solution and 6 mu L of protease K into a centrifuge tube, and incubating overnight (about 16 h) in a constant-temperature water bath at 37 ℃ until the cell precipitate is completely digested, and clarifying the solution;
③ adding 1mL of Tris saturated phenol, placing the mixture on ice, gently shaking the mixture for 20min to fully mix the mixture, centrifuging the mixture for 10min at 12000r/min, and transferring the upper aqueous phase into another sterilized 2.0mL centrifuge tube.
Adding 0.5mL of saturated phenol and 0.5mL of chloroform, placing on ice and gently shaking for 20 min; the upper aqueous phase was transferred to another sterilized 2.0mL centrifuge tube.
Adding 1mL of chloroform, placing on ice and gently shaking for 20 min; centrifuging at 12000rpm for 10min at 4 deg.C; transferring the upper aqueous phase into a 1.5mL centrifuge tube by using a pipettor;
sixthly, adding 1mL of precooled absolute ethyl alcohol at (-20 ℃), gently buckling the bottom and shaking the bottom for multiple times until DNA is separated out, and then placing the mixture for 30min at-20 ℃; centrifuging at 4 deg.C and 12000rpm for 10min, and removing ethanol;
adding 1mL of 70% ethanol, and gently shaking for 10 min; centrifuging at 4 deg.C and 12000rpm for 10min, removing ethanol, and rinsing repeatedly; standing at room temperature for 30min, and oven drying at 60 deg.C for 30s to completely volatilize ethanol;
adding 50 mu L of ultrapure water, storing at 4 ℃ until DNA is completely dissolved, measuring the concentration by a spectrophotometer, and storing at-80 ℃.
2. Amplification of target and reference genes
The sequence of the copy number variation region screened out in the re-sequencing, namely 6544001 to 6547600 of the sequence of the USP16 gene (target gene) group, is found by taking a bovine USP16 gene (NC-007299.6) published by NCBI as a reference sequence. Primers contained in this region were designed using Prime 5.0 software and aligned in NCBI _ BLAST. The primer sequences are as follows (primer pair P1), and the size of the PCR product fragment is 200 bp:
the upstream primer F1: 5'-AGAACCCCCGGAAGTAGAGTA-3'
The downstream primer R1: 5'-CAAAGTGCTACCGCCATTCC-3', respectively;
meanwhile, a bovine BTF3 gene sequence (AC _000177.1) published by NCBI is used as a reference sequence, and a primer for amplifying a specific fragment (166bp) in the reference gene (BTF3 gene) is designed by the same method, wherein the primer sequence is as follows (primer pair P2):
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3', respectively;
the design completion time of the primers is as follows: year 2019, month 9.
3. Real-time fluorescence Quantification (QPCR)
Whether the primers are suitable for real-time quantitative PCR analysis is determined by plotting an amplification curve and a dissolution peak. The amplification curve is smooth, and the QPCR amplification system and conditions are appropriate (figure 1); the prepared dissolution curves were matched, and the curves were smooth, high and sharp, and had no primer dimer or hetero-peak due to non-specific amplification, indicating good primer quality (FIG. 2).
The QPCR reaction system is shown in Table 1.
TABLE 1 QPCR reaction System
The reaction procedure used for real-time quantitative PCR was:
(1) pre-denaturation: 94 ℃ for 2 min; (ii) a
(2) And (3) amplification reaction: denaturation at 94 ℃ for 10s, annealing at 60 ℃ for 30s, 40 cycles.
Calculation of CNV type
Each sample was amplified with primers for the gene sequence of interest and the reference gene sequence (primer pair P1 and P2), respectively, and 3 replicates for each pair of primers. According to 2 -ΔΔCt The method performs copy number analysis. Wherein Δ Δ Ct ═ C T target gene -C T reference gene ) Experimental group -(C T target gene -C T reference gene ) Control group . The experimental group is a sample to be detected for the presence of CNVs, and the control group is a sample known to have no copy number variation. 2 -ΔΔCt The copy number of the target gene sequence in the experimental group is expressed in terms of the fold of the control group. The expressed abundance of the gene was then logarithmically transformed (base 2 of 2) -ΔΔCt Logarithm of (d) to fit a normal distribution, and after performing a homogeneity test for variance, statistically testing the differences between groups. C T Namely 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 process.
When the amplified target gene sequence is a normal type sequence, 2 x 2 -ΔΔCt 2; when the amplified target gene sequence is a deletion type sequence, 2 x 2 -ΔΔCt <2; when the amplified target gene sequence is a multicopy sequence, 2 x 2 -ΔΔCt >2。
5. Data analysis
And counting the number of individuals of various copy number variation types in the detection population, and counting the frequency of various types. The calculation formula is as follows:
PC＝NC/N
wherein PC represents a frequency of a certain copy number type; NC represents the number of individuals in the population having a CNV type of C (Gain, media, or Loss); 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 lactation character indexes, considering environmental effect, age, 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:
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 type of each site; e ijk Is a random error.
The results of the data processing are shown in table 2.
TABLE 2 correlation analysis of USP16 Gene with different lactation traits of cows
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.
According to the statistical results of the genotypes (n 1, n2 and n3 in the table 2), the distribution of copy number variation sites from 6544001 to 6547600 of a bovine USP16 gene (NC-007299.6) group sequence in sampled Chinese Holstein cows is determined and accords with polymorphism.
As can be seen from table 2, the deletion type (Loss) of the cow at this site has a significant increase in the fat content of cow milk and a positive effect on the protein content of cow milk compared to the other two types, but at the same time, it has a significant down-regulation effect on the milk yield.
The results show that the CNV locus on the USP16 gene can be used as a candidate molecular genetic marker locus for improving the lactation character of Chinese Holstein cows, and the breeding process of the cows with excellent milk production performance is accelerated.
<110> northwest agriculture and forestry science and technology university
<120> method for detecting USP16 gene CNV marker of dairy cow and application thereof
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<213> Artificial Synthesis
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<212> DNA
<213> Artificial Synthesis
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<212> DNA
<213> Artificial Synthesis
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Claims (3)
1. Detect milk cowUSP16The application of the gene copy number variation method in the auxiliary selection breeding of the dairy cattle molecular marker is characterized in that: the detection cowUSP16A method of gene copy number variation comprising the steps of:
milk cow genome DNA is taken as a template, and real-time quantitative PCR amplification is respectively carried outUSP16Partial fragments of copy number variation regions of genes and their use as referenceBTF3Partial fragments of the gene, and then identifying the cow according to the quantitative resultUSP16Copy number variation type of gene;
the above-mentionedUSP16The amplification primer pair of the partial segment of the copy number variation region of the gene comprises:
the upstream primer F1: 5'-AGAACCCCCGGAAGTAGAGTA-3'
The downstream primer R1: 5'-CAAAGTGCTACCGCCATTCC-3', respectively;
the above-mentionedBTF3The amplification primer pairs of the partial segments of the genes are as follows:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3', respectively;
the milk cow is a Chinese Holstein milk cow, the milk cow is a Chinese Holstein milk cowUSP16The copy number variation region of the gene is located in cattleUSP166544001 to 6547600 of the gene reference genomic sequence NC _ 007299.6;
said copy number variation type 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 −ΔΔCt =2；
The above-mentionedUSP16The gene copy number variation is related to the lactation traits of Chinese Holstein cows;
the lactation trait is selected from milk yield or milk fat rate, the Chinese Holstein cow individual with deletion type copy number variation type has better milk fat rate in the lactation trait, and the Chinese Holstein cow individual with multi-copy type copy number variation type has better milk yield in the lactation trait.
2. The use of claim 1, wherein: the real-time quantitative PCR amplification system comprises 1 mu L of template DNA of 10 ng/mu L and 10 mu mol/LUSP160.5 mu L of each of upstream and downstream primers corresponding to the amplification primer pair of the partial segment of the gene copy number variation region; alternatively, the real-time quantitative PCR amplification system comprises 1. mu.L of 10 ng/. mu.L template DNA and 10. mu. mol/LBTF3The upstream and downstream primers of the amplification primer pair of the gene partial fragment were each 0.5. mu.L.
3. The use of claim 1, wherein: the reaction procedure of the real-time quantitative PCR is as follows: pre-denaturation: 94 ℃, 2 min; and (3) amplification reaction: denaturation at 94 ℃ for 10s, annealing at 60 ℃ for 30s, 40 cycles.
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