CN111088327B - Method for detecting cattle body size characters under assistance of SIKE1 gene CNV marker and application thereof - Google Patents
Method for detecting cattle body size characters under assistance of SIKE1 gene CNV marker and application thereof Download PDFInfo
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Abstract
The invention discloses a SIKA method for detecting cattle body size characters under the assistance of E1 gene CNV markers and application thereof are disclosed: using cattle blood whole genome DNA as a template, respectively amplifying partial fragments of a CNV region of a SIKE1 gene and an internal reference gene BTF3 by a real-time fluorescent quantitative PCR method, and calculating log according to a quantitative result 2 2 ‑ΔΔCt Identifying the copy number variation type of the individual SIKE1 gene. Based on the correlation between SIKE1 gene copy number variation and growth traits, the method provided by the invention can be used for quickly establishing genetic resource dominant populations of local cattle varieties such as Yunnan cattle and the like, is favorable for accelerating molecular marker-assisted selective breeding work of the Yunnan cattle and the like, and is simple, quick and convenient to popularize and apply.
Description
Technical Field
The invention relates to the field of livestock molecular biology detection, in particular to a QPCR (quantitative polymerase chain reaction) technology-based method for detecting a cattle SIKE1 gene CNV marker.
Background
Genomic DNA, as a carrier of genetic information carried by organisms, plays a crucial role in the development of individual phenotypes, while genomic variations are a major genetic source responsible for inter-individual and inter-species variation. Molecular marker-assisted selection (MAS), which is a technology for selecting genetic resources or breeding materials by means of DNA molecular markers to improve the comprehensive characters of livestock and poultry. In the breeding of livestock and poultry, the purposes of early seed selection and improvement of the accuracy of breeding values are achieved by selecting DNA markers which are closely related to growth traits and are closely related to quantitative traits, so that greater genetic progress is obtained in the breeding of livestock and poultry.
Copy Number Variations (CNVs) refer to the phenomenon of insertions, deletions, duplications and complex recombinations of fragments greater than 50bp in the genome, a structural variation at the genomic sub-microscopic level that can affect gene function and individual phenotype through dose effects, positional effects, blocking of functional genes, fusion genes, exposure of recessive alleles and potential transition effects. Research shows that some CNV sites are located inside functional genes and have correlation with the growth characteristics of livestock and poultry or coincide with QTL sites, which has certain influence on economic characteristics.
Currently, the detection methods commonly used for CNV are mainly classified into two types: one type is mainly used for detecting unknown CNV in the whole genome range, and comprises a genome chip and a high-throughput sequencing technology; the other class is mainly used for site-specific detection or validation of known CNVs.
The chip method mainly includes Comparative Genomic Hybridization (CGH) and SNP chips, and oligonucleotide probe chips in the Comparative genomic chips are widely used due to their characteristics of high precision, high sensitivity, small sample requirement and the like. The SNP chip does not need a control sample during detection, and is analyzed through the SNP signal intensity in a detected sample, so that the SNP chip has the main advantages of simultaneously providing copy number and genotype information and displaying loss of heterozygosity. However, the distribution of the probes on the SNP chip is not uniform in the genome, and the design of the probes in many complex regions is difficult. Therefore, the SNP chip has a certain limitation in detecting CNV. With the maturation of new generation sequencing technologies, direct re-sequencing to detect genomic structural variations has become the most effective detection means at present. Compared with hybridization technology, the sequencing technology for detecting CNV has many advantages, such as improving the resolution of CNV, determining the CNV boundary, detecting the absolute copy number of individual CNV, and having higher detection efficiency for CNV with complex structure change, but the method has higher cost.
For the detection of established CNVs, several methods based on PCR and hybridization techniques are generally employed. Such as QPCR, QMPSF, MLPA, FISH, Southern blotting, MAPH, and the like. Among them, real-time fluorescent quantitative PCR (QPCR, qRT-PCR) is most commonly used. The methods mainly include two types, i.e., a fluorescent dye intercalation method and a fluorescent hybridization probe method, depending on the chemical method of fluorescence used in QPCR. Excessive SYBR Green dye molecules are added into a PCR reaction system, so that DNA double strands can be specifically infiltrated and fluorescent signals can be emitted, free dye molecules only have low fluorescence background and hardly emit light, the increase of the signals and the increase of PCR products are synchronous, and the quantity of genome DNA can be reflected by detecting the intensity of the fluorescent signals. The copy number of the candidate gene of the sample is statistically detected according to the 2-delta Ct method by relatively quantifying the target gene (with copy number variation) and the reference gene (without copy number variation).
SIKE1(IKK inhibitor) is a small coiled-coil domain protein containing 207 amino acid residues, which is ubiquitous in most tissues including the heart. Regarding the function of the SIKE1 gene, related studies have shown that IKK inhibitors can act as negative regulators of the interferon pathway. However, no research report about the influence of copy number variation of the cattle SIKE1 gene on the growth and lactation traits of cattle is found at present.
Disclosure of Invention
The invention aims to provide a method for detecting cattle body size traits by using SIKE1 gene CNV marker assisted and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting a CNV marker of a cattle SIKE1 gene comprises the following steps:
the method comprises the steps of taking blood whole genome DNA extracted from a cattle to be detected (such as a Yunling cattle) as a template, taking a primer pair P1 and a primer pair P2 as primers, amplifying a copy number variation region of a SIKE1 gene and a partial fragment of a BTF3 gene as an internal reference through real-time fluorescence Quantitative PCR (QPCR), and identifying the copy number variation type of the SIKE1 gene of the cattle according to the quantitative result.
Preferably, the copy number variation region of the SIKE1 gene is located in the candidate region Chr3:28561571-28565970 of the bovine SIKE1 gene (reference genome sequence NC-037330.1).
Preferably, the copy number variation types are according to log 2 2 -ΔΔCt (i.e., - Δ Δ Ct) classifies the quantitative results into three categories: insertion type, Log 2 2 -ΔΔCt >0.5; deletion type, Log 2 2 -ΔΔCt <-0.5; normal type, -0.5 is less than or equal to Log 2 2 -ΔΔCt ≤0.5。
Preferably, the primer pair P1 is:
the upstream primer F1: 5'-TTCCACCCAAAGTGTGGATGTT-3'
The downstream primer R1: 5'-CTGCCTCCTGAATGGAACTGT-3', respectively;
the primer pair P2 is as follows:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3' are provided.
Preferably, the amplification system for real-time fluorescent quantitative PCR comprises: mu.L of 50 ng/. mu.L template DNA1 and 0.5. mu.L of each of the upstream and downstream primers corresponding to 10. mu. mol/L primer pair P1 or primer pair P2.
Preferably, the reaction procedure used for the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, for 40 cycles.
Preferably, the size of the PCR product fragment amplified based on the primer pair P1 is 125bp, and the size of the PCR product fragment amplified based on the primer pair P2 is 166 bp.
The method for detecting the cattle SIKE1 gene CNV marker is applied to cattle molecular marker-assisted selective breeding.
Preferably, in the cattle to be tested (e.g. the Yunnan cattle), individuals with deletion type copy number variation types are better than individuals with normal type and insertion type copy number variation types in growth traits (e.g. chest circumference, chest width, chest depth and nojiri length).
The invention has the beneficial effects that:
the invention utilizes genome DNA to carry out real-time fluorescence quantitative PCR, uses BTF3 gene as reference, can determine the type of copy number variation of the SIKE1 gene of cattle individuals according to the-delta-Ct value, and finds that the copy number variation of the SIKE1 gene can be used as a molecular marker. Compared with the prior art, the invention has the following advantages:
(1) the method for detecting copy number variation of the SIKE1 gene of the cattle is not limited by age, can be used for early breeding, and can be selected even when the individual is born;
(2) the method is quick and simple, has low cost, and can accurately and reliably identify the copy number type of the SIKE1 gene of the cattle individual;
(3) the invention provides scientific basis for molecular marker-assisted selection of the growth traits of the cattle to a certain extent, and can quickly establish cattle populations with excellent germplasm resources (such as the cattle population in the greenling), thereby accelerating the breeding process.
Drawings
FIG. 1 is a graph showing the amplification curve of QPCR (SIKE1 gene) performed in the present invention.
FIG. 2 is a graph showing the dissolution curve of QPCR (SIKE1 gene) performed in the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples, which are illustrative of the present invention and are not intended to limit the scope of the present invention.
The invention utilizes QPCR to detect copy number variation of SIKE1 gene of Yunnan cattle (candidate region Chr3:28561571-28565970 of cattle SIKE1 gene).
1. Yunling cattle sample collection
The collected Yunling cattle are from a small Yangxiang grassland animal science research institute (the collection time is 10 months in 2018) in Kunming city, Yunnan province, and are 173 cows in total, and the collection method is collecting blood from jugular veins. And recording the growth character data of the plants, such as body height, body weight, oblique body length, chest circumference, chest width, chest depth, nojiri length, ischium width, cross part height and the like, for subsequent correlation analysis.
2. Separation, extraction and purification of blood sample genome DNA
Reference is made to the method of Sambrock et al (2002).
3. Amplification of target gene and reference gene
A125 bp sequence in a copy number variation region (Chr3:28561571 and 28565970) of the SIKE1 gene is amplified by using a Primer 5.0 design Primer (Primer pair P1) as a reference sequence and a Primer (Primer pair P2) for amplifying a 166bp sequence in an internal reference gene (BTF3 gene) is designed by using the same method by using a bovine SIKE1 gene (target gene) sequence (GenBank Accession No. NC-037330.1) published by an NCBI database (http:// www.ncbi.nlm.nih.gov /) as a reference sequence and a bovine BTF3 gene sequence (AC-000177.1) published by the NCBI as a reference sequence. The sequence information of the primer set is shown in Table 1 (primer synthesis time: 11 months in 2019).
TABLE 1 primer information for real-time fluorescent quantitative PCR
Note: f represents an upstream primer, and R represents a downstream primer.
The amplification system used for QPCR was calculated at 10. mu.L as: mu.L of 50 ng/. mu.L template DNA 1. mu.L, 0.5. mu.L each of 10. mu. mol/L upstream and downstream primers, 5. mu.L of Bioeasy Master Mix (SYBR Green, no ROX), and 3. mu.L deionized water.
The reaction procedure for PCR amplification was:
(1) pre-denaturation: 1min at 95 ℃;
(2) and (3) amplification reaction: denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, 40 cycles.
Drawing a dissolution curve: 95 ℃ for 10s, from 65 ℃ to 95 ℃ and +0.5 ℃ for 5 s.
Primers were determined to be suitable for QPCR analysis by plotting amplification curves and melting peaks. The amplification curve is smooth, which indicates that the QPCR reagent has good quality and the 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).
4. Inference of copy number variation
Each sample was amplified with primers for the gene sequence of interest and the reference gene sequence, respectively (primer pair P1 and P2), and 3 repeats 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 existence of CNVs, the control group is a sample known to have no copy number variation, and a control group of cattle (Yunling cattle) individuals selected in a resequencing test can be adopted. 2 -ΔΔCt The copy number of the target sequence in the experimental group relative to the multiple of the control group, Ct is Cycle threshold, and is the number of amplification cycles that pass when the fluorescence signal of the amplification product reaches a set threshold value in the PCR amplification process. Logarithmic transformation of the expression abundance of the genes (base 2: 2) -ΔΔCt Logarithm of (d) to fit a normal distribution, and after performing a homogeneity test for variance, statistically testing the differences between groups.
When the target gene is a normal type (Median) sequence, according to Log 2 2 -ΔΔCt The normalized value (Log) is calculated to be about 0 2 2 -ΔΔCt Less than or equal to | plus or minus 0.5 |). Log when the gene of interest is a deletion (Loss) sequence 2 2 -ΔΔCt <-0.5. Log when the gene of interest is an insertion (Gain) sequence 2 2 -ΔΔCt >0.5。
5. Correlation analysis of CNV locus and growth traits of SIKE1 gene of Yunling cattle
And (3) association analysis model: firstly, performing description analysis on data to determine whether an outlier exists, and then correcting the data by using least square analysis; based on the data characteristics, the SPSS 23 software was used to analyze the effect of production traits between genotypes. A fixed model was used in the analysis of genotype effects:
Y ijk ＝μ+A i +CNV j +e ijk ，
wherein: y is ijk For trait observations, μ is the overall mean, A i Is age of the ith individual, CNV j As a fixed effect of the jth copy number variation type, e ijk Is a random error. Differences between each set of data were examined using multiple comparisons of LSDs and the results were expressed as means ± SE. The data processing results are shown in table 2.
TABLE 2 correlation analysis of copy number variation and growth traits of the SIKE1 gene of Yunnan cattle
Note: the values calculated in the table represent the mean ± standard error; the upper right corner of the numerical value is marked with a and b to represent the significant difference level P between the data in the same row is less than 0.05; the top right corner of the values is labeled A, B indicating a very significant level of P < 0.01 for the difference between the data in the same row.
Correlation analysis results showed (see table 2): the CNV locus of SIKE1 gene of a Yunnan cattle can obviously influence the chest circumference, the chest width, the chest depth and the nojiri length, and the dominant copy number variation type is deletion type, which indicates that the deletion type of the CNV locus of SIKE1 gene can be used as a candidate molecular genetic marker (CNV marker) for improving the growth traits of the Yunnan cattle, and for cows, the chest circumference, the chest width and the chest depth are also related to the improvement of the lactation traits.
6. Application of CNV marker in cattle breeding
The obtained candidate molecular genetic marker can be used for searching quantitative trait loci which are related to or closely linked with the candidate molecular genetic marker and influence the growth traits of the cattle. The method can also be used for molecular marker-assisted selection of cattle, namely, deletion type individuals are selected for reservation and propagation by detecting copy number variation types of CNV sites of SIKE1 genes of cattle, so that the breeding process of improvement of cattle varieties (such as Yunnan cattle) can be accelerated.
<110> northwest agriculture and forestry science and technology university
<120> method for auxiliary detection of cattle body size traits through SIKE1 gene CNV marker and application thereof
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<213> Artificial Synthesis
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ttccacccaa agtgtggatg tt 22
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<212> DNA
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ctgcctcctg aatggaactg t 21
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<213> Artificial Synthesis
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Claims (4)
1. Cattle detection methodSIKE1The application of the gene CNV marking method in the auxiliary selection breeding of the molecular marker of the cattle is characterized in that: the detected cattleSIKE1A method for marking gene CNV, comprising the following steps:
taking cattle genome DNA as a template and a primer pair P1 and a primer pair P2 as primers, and respectively carrying out real-time fluorescent quantitative PCR amplificationIncrease theSIKE1Copy number variation region of gene and gene serving as internal referenceBTF3Partial fragment of gene, and identifying cattle according to quantitative resultSIKE1Copy number variation type of gene;
saidSIKE1The copy number variation region of the gene is located in cattleSIKE1The gene candidate region Chr3: 28561571-28565970;
the copy number variation types are divided into three types according to-delta-Ct: insertion type, - Δ Δ Ct > 0.5; deletion type, -delta Ct < -0.5; normal type, -delta Ct is more than or equal to-0.5 and less than or equal to 0.5;
the primer pair P1 is as follows:
the upstream primer F1: 5'-TTCCACCCAAAGTGTGGATGTT-3'
The downstream primer R1: 5'-CTGCCTCCTGAATGGAACTGT-3'
The primer pair P2 is as follows:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3', respectively;
the cattle is a Yunling cattle; individuals with deletion type copy number variation types are superior to individuals with normal type and insertion type copy number variation types in growth traits;
the growth traits are one or more of bust, chest width, chest depth and nojiri length.
2. The use of claim 1, wherein: the real-time fluorescent quantitative PCR amplification system comprises: mu.L of 50 ng/. mu.L template DNA and 0.5. mu.L of each of the upstream and downstream primers corresponding to 10. mu. mol/L primer pair P1 or primer pair P2.
3. The use of claim 1, wherein: the reaction program of the real-time fluorescent quantitative PCR comprises the following steps: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 15s and annealing at 60 ℃ for 15s for 40 cycles.
4. The use of claim 1, wherein: the size of the PCR product fragment amplified based on the primer pair P1 is 125bp, and the size of the PCR product fragment amplified based on the primer pair P2 is 166 bp.
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