CN112695106B - Method for quickly detecting growth traits of cattle in auxiliary manner through PLA2G2A gene CNV markers and special kit - Google Patents
Method for quickly detecting growth traits of cattle in auxiliary manner through PLA2G2A gene CNV markers and special kit Download PDFInfo
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Abstract
The invention discloses a method for quickly and auxiliarily detecting growth characters of cattle by using PLA2G2A gene CNV markers and a special kit, which are based on a real-time fluorescent quantitative PCR technology, take cattle genome DNA as a template, amplify partial fragments of PLA2G2A gene copy number variation regions, amplify partial fragments of cattle BTF3 genes as internal references, and finally utilize 2 ‑ΔΔCt The method calculates and determines the copy number variation type of the individual, and the result of the correlation analysis of the copy number variation type of the cattle PLA2G2A gene and the individual growth characteristics shows that the copy number variation region of the cattle PLA2G2A gene has CNV marks which can be used for representing the advantages of the individual growth characteristics such as deep chest, high chest circumference, abdomen circumference, high cross and the like, and the cattle population with excellent genetic resources can be quickly established through copy number variation typing detection, so that the auxiliary selection breeding process of the cattle molecular marks is quickened, the detection process is simple and quick, and the popularization and application are convenient.
Description
Technical Field
The invention belongs to the field of molecular genetic breeding, and relates to a method for detecting a cattle PLA2G2A gene CNV mark, in particular to a method for detecting a cattle PLA2G2A gene CNV mark by using a real-time fluorescent quantitative PCR (qPCR) technology and taking a BTF3 gene as a reference according to 2 -ΔΔCt Value determination of individual PLAType of copy number variation of 2G2A gene.
Background
In animal molecular breeding, the following two aspects are included: molecular marker assisted selection is a modern molecular breeding technique for improving animal varieties by using DNA markers or functional genes linked with important economic traits; transgenic breeding is a breeding technique for improving the productivity of animals by transferring genes or a group of genes related to functions, which have important functions, to recipient animals. The molecular marker assisted selection has improvement efficiency for characters with low genetic transmission, such as litter size, meat quality, carcass quality and the like, which is several times, even tens times, that of the traditional breeding method. If, in the course of breeding selection, the corresponding DNA markers or functional genes cover the entire genome and achieve a certain density and genetic effect, this is often also referred to as animal genome breeding. The molecular marker assisted selection cannot create variation and transfer of excellent functional genes among different species, and the transgenic technology can achieve the aim, so that the two breeding technologies have strong complementarity and are called molecular breeding technology. The molecular breeding can overcome the defects of the traditional hybridization breeding method, and has the characteristics of high efficiency and high speed. Molecular breeding particularly relates to almost all breeding fields such as protection, development and utilization of genetic resources, seed selection and matching, heterosis prediction, quantitative Trait Loci (QTL) detection and utilization and the like.
Copy number variation (Copy number variation, CNV) is a common genetic polymorphism, which is mainly represented by deletions and duplications at the sub-microscopic level, resulting from rearrangement of the genome, and generally refers to an increase or decrease in copy number of large fragments of the genome that are 1kb or more in length. The detection methods commonly used for CNV are mainly divided into two categories: the method is mainly used for detecting unknown CNV in the whole genome range, comprises genome chip technologies such as comparative genome hybridization chips (Comparative GenomicHybrid ization, CGH) and SNP chips and high-throughput sequencing technologies, and directly detecting genome structural variation through resequencing, and has become the most effective detection means at present, but has higher cost; the other is mainly used for site-specific detection or verification of known CNVs.
Type IIA phospholipase A2 (secretory phospholipase A group IIA, PLA2G 2A) is a phospholipase A2 (PLA 2) family member, and for type IIA phospholipase A2, it was originally discovered that it was resistant to bacteria, e.g., in 1995 it was discovered that a mouse strain with a frameshift mutation in the PLA2G2A gene was more susceptible to colorectal cancer than a mouse strain with a functional enzyme. Subsequently, transgenic or knockout studies of sPLA2 demonstrate an important role in various biological processes, such as digestion, host defense, and reproductive and skin homeostasis.
At present, no related research report about PLA2G2A genetic variation and cattle growth performance is found at home and abroad.
Disclosure of Invention
The invention aims to provide a method for quickly and auxiliarily detecting growth traits of cattle by using a PLA2G2A gene CNV marker and a special kit.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for detecting copy number variation of a cattle PLA2G2A gene, comprising the steps of:
taking genomic DNA of a cattle individual to be detected as a template, respectively using primer pairs P1 and P2, amplifying a copy number variation region of the PLA2G2A gene and a part of fragments of the BTF3 gene serving as an internal reference by real-time fluorescent quantitative PCR, and identifying the copy number variation type of the PLA2G2A gene of the cattle individual according to a quantitative result; the copy number variation region of the PLA2G2A gene is positioned in the PLA2G2A gene reference genome sequence Chr 2:133289295-133295334.
Preferably, the primer pair P1 is:
upstream primer F1:5'-TCCCAACAAGAGGACACAGC-3'
Downstream primer R1:5'-CCCGTCTCTCCAGCATCATC-3';
the primer pair P2 is as follows:
the upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
Downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3';
the PCR product fragment size amplified based on the primer pair P1 is 119bp, and the PCR product fragment size amplified based on the primer pair P2 is 166bp.
Preferably, the reaction system used for the real-time fluorescent quantitative PCR comprises 1. Mu.L of template (10 ng/. Mu.L of genomic DNA) and 0.5. Mu.L of each of the upstream and downstream primers (10 pmol/. Mu.L).
Preferably, the reaction procedure used for the real-time fluorescent quantitative PCR is: pre-denaturation at 95℃for 1min; denaturation at 95℃for 15s, annealing at 60℃for 15s, extension at 72℃for 30s, and 39-40 cycles; extending at 72℃for 10min.
Preferably, the copy number variation is according to 2 -ΔΔCt The quantitative results were divided into three categories: multicopy, 2 -ΔΔCt > 2; missing 0 is less than or equal to 2 -ΔΔCt <2; normal, 2 -ΔΔCt ＝2。
The method for detecting the copy number variation of the cattle PLA2G2A gene is applied to cattle molecular marker assisted selective breeding.
Preferably, the PLA2G2A gene copy number variation has obvious association with individual growth characters of cattle varieties such as Qinchuan cattle, original fixed cattle and the like, and in Qinchuan cattle groups, individuals with normal PLA2G2A gene copy number variation types are obviously superior to individuals with other two copy number variation types in terms of chest depth characters; in the original fixed cattle population, individuals with the copy number variation type of the PLA2G2A gene being deleted are obviously superior to individuals with the copy number variation type of multiple copy types and normal types in terms of growth characteristics (high, chest circumference, abdomen circumference and cross height).
A real-time fluorescent quantitative PCR kit for detecting copy number variation of a cattle PLA2G2A gene comprises the primer pair P1 and the primer pair P2.
The beneficial effects of the invention are as follows:
compared with the high-throughput sequencing method, the gene chip and other methods, the method and the kit for detecting the copy number variation of the cattle PLA2G2A gene disclosed by the invention are quick, simple and low in cost, can accurately identify the copy number variation types of a plurality of cattle variety individuals, and are convenient to popularize and apply. The invention can detect and obtain PLA2G2A gene copy number type closely related to cattle growth traits on the DNA level, can be used as an important candidate molecular marker (belonging to CNV markers) for marker-assisted selection of cattle growth traits, can quickly establish cattle population with excellent genetic resources, and can accelerate the promotion of cattle molecular marker-assisted selection breeding work.
Drawings
FIG. 1 is an electrophoresis chart of PCR amplification products of PLA2G2A gene CNV detection primer (primer pair P1); wherein the first lane on the right side is DNAmarker I, and the three lanes on the left side are PCR amplification products of 119bp target fragment of PLA2G2A gene.
Detailed Description
The present invention will now be described in detail with reference to the drawings and examples, which are only illustrative of the present invention and are not intended to limit the scope of the present invention.
In early-stage re-sequencing studies of the bovine genome, copy number variations were found that were located within the non-coding region Chr 2:133289295-133295334 of the bovine PLA2G2A gene. Therefore, the invention designs specific primers according to the region with copy number variation in the re-sequenced cattle PLA2G2A gene sequence, then uses 5 cattle varieties (Qinchuan cattle, summer south cattle, fixed cattle, cloud-line cattle and Pinan cattle) genome DNA as templates to carry out qPCR amplification, uses BTF3 gene as reference gene and uses 2 -ΔΔCt The method determines the copy number variation type of the individual. The invention detects and counts the type frequency of CNV type of the cattle PLA2G2A gene (copy number variation region of PLA2G2A gene), and carries out correlation analysis on the CNV type of the locus and the growth character of cattle. The result shows that the locus has obvious association with individual growth characters of cattle varieties such as Qinchuan cattle, original-fixed cattle and the like. The correlation analysis result of the cattle PLA2G2A gene copy number variation and the cattle important growth characteristics can provide a theoretical basis for cattle molecular breeding, facilitate the molecular marker auxiliary selection of cattle growth characteristics and quickly establish cattle populations with excellent genetic resources. The specific description is as follows.
1. Sample collection and genomic DNA extraction
(1) Blood sample collection (see table 1): qinchuan cattle (blood sample of cows of 24-48 months of age), solid cattle (blood sample of cows of 24-48 months of age), summer south cattle (blood sample of cows of 24-36 months of age), pinna cattle (blood sample of cows of 24-36 months of age), cloud-line cattle (blood sample of cows of 24-36 months of age), and recording growth trait data of individuals, such as height, body length, chest circumference, chest depth, abdominal circumference, long-jirime, ischial end width, cross height, etc., for subsequent association analysis.
TABLE 1 information on test animal samples
(2) Extraction of genomic DNA from blood samples (phenol-chloroform method)
(1) Frozen blood samples were thawed in a room temperature water bath and 2mL whole blood was transferred to a 2mL sterile centrifuge tube.
(2) Centrifuge at 12000r/min for 10min at 4deg.C, discard the liquid, leave the pellet, add 1.5mL PBS buffer, vortex to suspend the pellet, gently shake on ice for 15min.
(3) Centrifuging at 12000r/min at 4deg.C for 10min, discarding the liquid, and retaining the precipitate. Repeating the step (3) once.
(4) The mashed pellet was pelleted, 500. Mu.L of DNA extract and 6. Mu.L of proteinase k were added to the centrifuge tube, and incubated overnight (about 16 h) in a constant temperature water bath at 37℃until the pellet was digested and the solution clarified.
(5) 1mL of Tris-saturated phenol was added, gently shaken on ice for 20min, centrifuged at 12000r/min for 10min at 4℃and the upper aqueous phase was transferred to another 2mL centrifuge tube.
(6) 1mL of chloroform was added, gently shaken on ice for 20min, centrifuged at 12000r/min for 10min at 4℃and the upper aqueous phase was transferred to a new 1.5mL centrifuge tube.
(7) Adding 1mL of pre-cooled absolute ethanol (-20deg.C), shaking slightly to precipitate DNA, standing at-20deg.C for 30min, centrifuging at 4deg.C for 12000r/min for 10min, and discarding ethanol.
(8) 1mL of 70% ethanol was added, the mixture was gently shaken for 10min, centrifuged at 12000r/min at 4℃for 10min, and the ethanol was discarded. The washing is repeated once according to the step (8).
(9) Standing at room temperature for 30min, and oven drying at 60deg.C for 30s to evaporate ethanol.
Adding 50 μl of ultrapure water, preserving at 4deg.C until DNA is completely dissolved, and preserving at-80deg.C after spectrophotometry.
2. Specific primer design for amplifying target gene and reference gene
The sequence of the copy number variation region screened in resequencing, namely Chr 2:133289295-133295334, was found by using the PLA2G2A gene (NC_ 037329.1) published by NCBI as a reference sequence, and primers contained in this region were designed by using Prime 5.0 software. The primer sequences (primer pair P1) are as follows:
upstream primer F1:5'-TCCCAACAAGAGGACACAGC-3'
Downstream primer R1:5'-CCCGTCTCTCCAGCATCATC-3'
Referring to FIG. 1, primer pair P1 can specifically amplify a 119bp fragment of interest.
Meanwhile, the primer for amplifying a specific fragment (166 bp) in the reference gene (BTF 3 gene) is designed by adopting the same method by taking the bovine BTF3 gene (AC_ 000177.1) published by NCBI as a reference sequence, and the primer sequence is as follows (primer pair P2):
the upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
Downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3'
3. Real-time fluorescent quantitative PCR (qPCR)
The reaction system of qPCR is shown in table 2:
TABLE 2 reaction System for qPCR (10. Mu.L)
The reaction procedure for qPCR is shown in table 3:
TABLE 3 qPCR reaction procedure
4. Identification of individual CNV types
The abundance of gene expression was indexed according to the fold of the copy number of the experimental group relative to the control group (Log 2 2 -ΔΔCt ) And (5) carrying out variance homogeneity test, and counting the differences among the groups. The parting experiment result adopts 2 -ΔΔCt The method is used for calculation, and the specific calculation method comprises the following steps:
ΔΔCt＝ΔCt (Experimental group) -ΔCt (reference group)
Wherein, deltaCt (Experimental group) ＝Ct (Experimental group objective Gene) -Ct (reference Gene of Experimental group) ，ΔCt (reference group) ＝Ct (reference group target gene) -Ct (reference group internal reference Gene) The method comprises the steps of carrying out a first treatment on the surface of the The experimental group is an individual sample to be detected for copy number variation. The reference group is an individual sample known to have no copy number variation, and cattle individuals of the reference group selected in the resequencing test can be used. Ct is Cycle threshold, which is the number of amplification cycles that pass when the fluorescent signal of the amplified product reaches a set threshold during PCR amplification.
According to 2 -ΔΔCt The quantitative results were divided into three categories: multiple copies (duplicity), 2 -ΔΔCt > 2; missing (deletion), 0.ltoreq.2 -ΔΔCt <2; normal (Normal), 2 -ΔΔCt ＝2。
5. Data processing
Production data: high, cross portion high, body length, chest circumference, chest width, chest depth, jirimlength, ischial end width, waist angle width, body weight, body diagonal length, waistline, tube circumference, abdomen circumference, hip circumference, head length, forehead width.
The number of individuals of the three genotypes (Deletion, normal, duplication) in the detection population was counted.
Firstly, carrying out description analysis on data, determining whether outliers exist or not, and correcting the data by utilizing least square analysis; based on the data features, the GLM process using SPSS (20.0) software analyzes genotype and effects on individual traits in the vials. A simplified model was used in the analysis of genotype effects:
Y ik ＝μ+G k +e ik
wherein: y is Y ijkl As observed values of the characters, mu is the overall mean value, G k Is the fixed effect of the kth genotype, e ik Is a random error。
The correlation analysis results of the Qinchuan cattle PLA2G2A gene copy number variation and the growth characteristics are shown in Table 4:
TABLE 4 correlation of body size data with PLA2G2A Gene 3 copy number variation types in Qinchuan cattle populations
Note that: the lower case letters differ significantly (P < 0.05)
The result shows that the copy number variation type of the Qinchuan cattle PLA2G2A gene has obvious correlation with chest depth. Wherein the average value of individuals of the normal type is significantly higher than those of the multicopy type and the deletion type.
The correlation analysis results of the copy number variation and the growth traits of the summer south cattle PLA2G2A gene are shown in Table 5:
TABLE 5 correlation of body size data with PLA2G2A Gene 3 copy number variation types in the summer south cattle population
The results show that copy number variation of PLA2G2A gene has no significant effect on the growth trait development of summer-south cattle, but the phenotype of multicopy type individuals is inferior to normal and deleted individuals in terms of body weight, waist circumference, chest circumference and body diagonal length.
The correlation analysis results of Yun Lingniu PLA2G2A gene copy number variation and growth traits are shown in Table 6:
TABLE 6 correlation of body size data with 3 copy number variation types of PLA2G2A genes in the cloud Ling cattle population
The result shows that there is no obvious correlation between the copy number variation of the PLA2G2A gene and the growth traits of the cloud-plate cattle, but the average value of the multiple growth traits of the individual in the multicopy type is relatively high through data comparison.
The correlation analysis results of the copy number variation and the growth traits of the original-solid cattle PLA2G2A gene are shown in Table 7:
TABLE 7 correlation of body size data with PLA2G2A Gene 3 copy number variation types in the fixed cattle population
Note that: the lower case letters differ significantly (P < 0.05)
The result shows that the copy number variation of PLA2G2A gene has obvious influence on the height, chest circumference, abdomen circumference and cross part height of the original fixed cattle, and the average value of the height, chest circumference, abdomen circumference and cross part height of the missing individual is higher than that of the individual with other two copy number variation types, so that the method has obvious promotion effect on the growth characteristics.
The correlation analysis results of the copy number variation and the growth traits of the south-leather cattle PLA2G2A gene are shown in Table 8:
TABLE 8 correlation of body size data with PLA2G2A Gene 3 copy number variation types in Pinan cattle population
The results show that the copy number variation of the PLA2G2A gene has no significant effect on the growth traits of the Pinans cattle.
According to the results of the correlation analysis above: in the original fixed cattle from Ningxia region, PLA2G2A gene copy number is obviously related to body height, chest circumference, abdomen circumference and the like, and the missing individual body size data is better. Although in Qinchuan cattle, individuals with deletions exhibited lower chest depth traits than individuals with the other two copy number variation types. However, in general, the copy number variation site (Chr 2:133289295-133295334) of the cattle PLA2G2A gene is inversely related to growth traits. Based on the physiological action of PLA2G2A genes and the regulatory mechanism of CNV, a theoretical and practical basis is provided for molecular breeding of cattle.
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Claims (2)
1. Detection cattlePLA2G2AThe application of the gene copy number variation method in the cattle molecular marker assisted selective breeding is characterized in that:
the detection of cattlePLA2G2AA method of gene copy number variation comprising the steps of:
taking the genomic DNA of the cattle to be detected as a template, and respectively amplifying by using real-time fluorescent quantitative PCRPLA2G2ACopy number variation region of gene as internal referenceBTF3Partial fragments of the genes and then identifying cattle based on quantitative resultsPLA2G2AGene of geneCopy number variation type, thePLA2G2AThe copy number variation region of the gene is locatedPLA2G2AChr 2:133289295-133295334 of the gene reference genome sequence NC_ 037329.1; the cattle is Qinchuan cattle or original-fixed cattle;
the saidPLA2G2AThe amplification primer pair of partial fragments of the copy number variation region of the gene is as follows:
upstream primer F1:5'-TCCCAACAAGAGGACACAGC-3'
Downstream primer R1:5'-CCCGTCTCTCCAGCATCATC-3';
the saidBTF3The amplification primer pairs of the partial fragments of the genes are:
the upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
Downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3'
The copy number variation type is according to 2 -ΔΔCt The quantitative results were divided into three categories: multicopy, 2 -ΔΔCt > 2; missing 0 is less than or equal to 2 -ΔΔCt <2; normal, 2 -ΔΔCt =2；
In the Qinchuan cattle, the Chinese medicinal materials are prepared,PLA2G2Aindividuals with the copy number variation type of the gene being normal are superior to individuals with the copy number variation type of the deletion type and the multicopy type in terms of chest depth characters, and have obvious differences; in the case of a fixed-origin cattle,PLA2G2Aindividuals with the copy number variation type of the gene being deleted are superior to individuals with the copy number variation type of multiple copy types and normal types in high, chest, abdomen and cross high characters and have obvious differences.
2. The use according to claim 1, characterized in that: the reaction program of the real-time fluorescence quantitative PCR is as follows: pre-denaturation at 95℃for 1min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, and 39-40 cycles; extending at 72℃for 10min.
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