CN112813175A - Method for quickly and auxiliarily detecting growth traits of cattle CHRDL1 gene CNV marker and application thereof - Google Patents
Method for quickly and auxiliarily detecting growth traits of cattle CHRDL1 gene CNV marker and application thereof Download PDFInfo
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Abstract
The invention discloses a method for quickly and auxiliarily detecting growth traits of cattle CHRDL1 gene CNV marker and application thereof: using the whole genome DNA of bovine blood as a template, respectively amplifying a CNV region of a bovine CHRDL1 gene and a segment of a reference gene BTF3 by using two pairs of primers P1 and P2 through a real-time fluorescent quantitative PCR technology according to the sequence 2 x 2‑ΔΔCtDetermining the copy number variation type of individual CHRDL1 gene as multi-copy type, deletion type or normal type. According to the correlation analysis result of the copy number variation of the CHRDL1 gene and the growth traits, the method can detect the CNV marker closely related to the growth traits of the cattle on the DNA level, can be used for marker-assisted selection of the growth traits of the cattle, and accelerates the process of fine breed breeding.
Description
Technical Field
The invention relates to the field of livestock molecular biology detection, in particular to a method for detecting a bovine CHRDL1 gene CNV marker based on a qPCR technology.
Background
At present, the research on beef cattle molecular breeding mainly focuses on the aspect of marker-assisted selection based on molecular markers. Molecular breeding, i.e., molecular marker-assisted selection (MAS), is to select genetic resources or breeding materials by means of DNA molecular markers, thereby improving the comprehensive properties 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 closely related to quantitative traits, so that greater genetic progress is obtained in the breeding of livestock and poultry.
Copy Number Variations (CNVs), which are structural Variations at the genomic sub-microscopic level, refer to the phenomenon of deletion or duplication of larger fragments in genomic DNA, involving fragment sizes between 50bp and Mb, including increased Copy Number gain and decreased Copy Number loss. The CNV regions cover more nucleotide content of the genome than Single Nucleotide Polymorphisms (SNPs), and the mechanism of association with phenotypic variation is most notably gene dose effects caused by variation in gene copy number or its associated regulatory elements.
The unknown copy number variation of the genome is studied by a comparative microarray-based genomic hybridization (CGH) method, by which the variation in the DNA copy number between the genome of a test sample and the genome of a control sample can be detected by simultaneously hybridizing samples (test sample and control sample) labeled with different fluorescein on one chip. The probes of the CGH chip cover the whole genome, and the CGH chip has the characteristics of high throughput, sensitivity, accuracy and resolution, and the test data has higher reliability. However, the resolution of CGH chips is at Mb level, and copy number variants of smaller fragments are not easily detected. Meanwhile, the detection operation is complicated, the time consumption is long, the cost is high, a large amount of template DNA is needed, and the large-scale popularization is not facilitated. The New Generation Sequencing (NGS) technology overcomes some inherent defects of the hybridization technology, and the application of pair sequencing can identify complex structural changes, improve the detection resolution, but 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 and MAPH. Among them, real-time fluorescent quantitative PCR (qPCR) is most commonly used. The method mainly includes two types, namely a fluorescent dye embedding method and a fluorescent hybridization probe method, according to different fluorescent chemical methods used by qPCR. The dye method has the advantages of low experimental cost, no need of designing and synthesizing probes,The method is convenient to use, and can detect the absolute copy number of the target fragment. For example, the amount of genomic DNA can be reflected by detecting the intensity of fluorescent signal by adding an excessive amount of SYBR Green dye molecules to the PCR reaction system, and the amount of genomic DNA can be reflected by relatively quantifying the target gene (with copy number variation) and the reference gene (without copy number variation) according to 2-ΔΔCtThe method statistics may detect the copy number of the candidate gene region of the sample.
Chord-like 1(CHRDL1) is a secreted glycoprotein with repeated cysteine-rich domains that binds to ligands of the BMPs family and is an antagonist of secreted Bone Morphogenetic Proteins (BMPs) expressed in mesenchymal tissues. The ability of CHRDL1 to enhance BMP-4 activity may be an important mechanism to elucidate the mechanisms of osteogenic differentiation and bone remodeling by human mesenchymal stem cells (hBMSCs). In the process of forming bone of hBMSCs, the function of BMP-4 is enhanced and depends on the regulation of CHRDL1 gene in BMP-SMAD signaling pathway.
At present, no literature report about the influence of CHRDL1 gene CNV on the growth traits of local cattle breeds is found.
Disclosure of Invention
The invention aims to provide a method for quickly and auxiliarily detecting growth traits of cattle CHRDL1 gene CNV markers and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting copy number variation of bovine CHRDL1 gene comprises the following steps:
using genome DNA of a cattle individual to be detected as a template, using a primer pair P1 and a primer pair P2 as primers, respectively amplifying a partial fragment of a copy number variation region of a CHRDL1 gene and a partial fragment of a BTF3 gene serving as an internal reference through real-time fluorescence quantitative PCR, and then identifying the copy number variation type of the CHRDL1 gene of the cattle individual according to a quantitative result;
the primer pair P1 is as follows:
the upstream primer F1: 5'-GCAGCCCATCTTCCACTTTT-3'
The downstream primer R1: 5'-AGCCTGAGATAAGAGGTCTCCA-3'
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 153bp, and the size of the PCR product fragment amplified based on the primer pair P2 is 166 bp.
Preferably, the copy number variation region of the CHRDL1 gene is located in 64369201bp-64378000bp of a bovine CHRDL1 gene reference genome sequence AC _ 000187.1.
Preferably, said copy number variation pattern is according to 2 x 2-ΔΔCtThe 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-ΔΔC＝2。
Preferably, the amplification system used for the real-time fluorescent quantitative PCR is as follows: 10 ng/. mu.L of template DNA 1. mu.L, 10. mu. mol/L of primer pair P1 or upstream and downstream primers corresponding to primer pair P2 are 0.5. mu.L, 2 XSSYBR Green QPCR Mix 5. mu.L and ddH respectively2O 3μL。
Preferably, the reaction procedure used for the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, and 39 cycles.
The method for detecting copy number variation of the bovine CHRDL1 gene is applied to bovine molecular marker-assisted selective breeding.
Preferably, in the Pinna parviflora, the individuals with the copy number variation type of deletion are obviously superior to the individuals with the normal type and the multiple copy type in the growth trait of the nojiri; in Qinchuan cattle, individuals with deletion copy number variation types are obviously superior to individuals with multiple copy types in the growth trait of chest depth; in southward summer cattle, individuals with the copy number variation type of normal type are obviously superior to individuals with deletion type and multiple copy type in the growth trait of weight; in the Yunnan cattle, the growth trait of waist width of an individual with a deletion type copy number variation is remarkably superior to that of a multi-copy type individual.
A kit for detecting copy number variation of bovine CHRDL1 gene comprises the primer pair P1 and the primer pair P2.
The invention has the beneficial effects that:
according to the invention, a cattle CHRDL1 gene reference region Chr X of 64369201bp-64378000bp is used as a locus, the copy number variation condition of the locus in a cattle group is detected by a real-time fluorescent quantitative PCR technology, and correlation analysis is carried out on important economic characters such as weight, waist width and the like; correlation analysis results show that the CNV locus of the cattle CHRDL1 gene is obviously related to important growth traits of cattle, and a DNA molecular marker (CNV marker) for marker-assisted selection of the growth traits of the cattle exists, so that the DNA molecular marker can be used for quickly establishing cattle populations with excellent genetic resources, and further, the improved breeding process is accelerated.
Compared with the prior art, the invention has the following advantages:
(1) the method for detecting copy number variation of the CHRDL1 gene of the cow is not limited by age, and can be used for early breeding of cows.
(2) The method for detecting the copy number variation of the CHRDL1 gene is accurate and reliable and is simple and convenient to operate.
(3) The detection of the copy number variation site of the CHRDL1 gene provides scientific basis for the auxiliary selection of the bovine molecular marker.
Drawings
FIG. 1 is an amplification curve drawn by qPCR (CHRDL1 gene) performed in the examples of the present invention.
FIG. 2 shows a melting curve obtained by qPCR (CHRDL1 gene) performed in the present example.
FIG. 3 shows the distribution of copy number variation of CHRDL1 gene in bovine populations, which was detected in the present example.
Detailed Description
The present invention will be described in further detail with reference to the following drawings and examples, which are provided for illustration only and are not intended to limit the scope of the present invention.
The invention aims at the copy number variation region Chr X of the cattle CHRDL1 gene (GenBank Accession No. AC-000187.1) screened in the re-sequencing, is 64369201bp-64378000bp, detects the copy number variation of the cattle CHRDL1 gene by utilizing qPCR, and reveals a CNV mark existing in the cattle CHRDL1 gene. The concrete description is as follows.
1. Sample collection and genomic DNA extraction
(1) Blood sample collection and data collection
TABLE 1 Experimental animal sample information
673 blood samples are collected together, 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 blood is brought back to a laboratory by an ice box and stored at-80 ℃; and (4) sampling, and simultaneously determining, collecting and inputting growth development basic data of corresponding individuals for later-stage correlation analysis.
(2) Extraction of DNA from blood sample (extraction of genomic DNA from sample by phenol-chloroform method)
Thawing frozen blood sample (mainly blood cells) at room temperature, sucking 2mL of blood into a 2.0mL centrifuge tube, and centrifuging at 12000rpm for 10min at 4 ℃; 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 for 10min at 4 deg.C, discarding liquid, and retaining precipitate.
② the precipitate is crushed into flocculent, 500 microliter of DNA extraction buffer solution and 6 microliter of protease K are added into a centrifuge tube, and the centrifuge tube is incubated overnight (about 16 hours) in a constant temperature water bath cabinet at 37 ℃ until the cell precipitate is completely digested, and the solution is clarified.
③ adding 1mL of Tris saturated phenol, placing on ice, gently shaking for 20min to fully mix, centrifuging at 4 ℃ and 12000r/min for 10min, 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; the upper aqueous phase was transferred to a 1.5mL centrifuge tube using a pipette.
Sixthly, adding 1mL of precooled absolute ethyl alcohol at (-20 ℃), shaking the mouth and the bottom for many times until DNA is separated out, and then placing the mixture for 30min at-20 ℃; centrifuging at 12000rpm for 10min at 4 deg.C, and discarding 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 standing at 60 deg.C for 30s to volatilize ethanol completely.
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 Gene and reference Gene
The sequence of the copy number variation region of the CHRDL1 gene (target gene) screened in the re-sequencing is found on NCBI, and primers (the amplified target fragment is 153bp) contained in the region are designed by using the sequence as a template and using Prime 5.0 software, and are compared in NCBI _ BLAST. The primer sequences are as follows (primer pair P1):
the upstream primer F1: 5'-GCAGCCCATCTTCCACTTTT-3'
The downstream primer R1: 5'-AGCCTGAGATAAGAGGTCTCCA-3' are provided.
Meanwhile, a primer for amplifying a specific fragment (166bp) in the BTF3 gene (internal reference gene) is designed by using a bovine BTF3 gene sequence (AC _000177.1) published by NCBI as a reference sequence and adopting the same method. The primer sequences are as follows (primer pair P2):
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3' are provided.
3. Real-time fluorescent quantitative PCR
The qPCR reaction system is shown in table 2.
TABLE 2.qPCR reaction System
The reaction procedure used for qPCR 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.
And determining that the primer is suitable for real-time fluorescent quantitative PCR analysis by drawing an amplification curve and a dissolution peak. 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 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).
Calculation of CNV type
Each sample was amplified with primers for the target and internal reference sequences (primer pair P1 and P2), respectively, and 3 replicates for each pair of primers. According to 2 x 2-ΔΔCtThe method performs an analysis of the copy number (FIG. 3). Wherein Δ Δ Ct ═ CT target gene-CT reference gene)Experimental group-(CT target gene-CT reference gene)Control group. The experimental group is a sample to be detected for the presence or absence of CNVs, the control group is a sample known to have no copy number variation, CTNamely 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. The homogeneity test of variance was then performed, and the differences between groups were statistically tested.
When the target sequence is a normal type sequence, a normalized value 2 x 2 is calculated-ΔΔCt2; when the target sequence is a deletion-type sequence, a normalization value 2 x 2 is calculated-ΔΔCt<2; when the target sequence is a multicopy sequence, a normalization value 2 x 2 is calculated-ΔΔCt>2。
5. Data analysis
Growth traits: height of body, height of cross, body slant length, chest circumference, abdominal circumference, tube circumference, chest width, chest depth, hip circumference, waist angle width, hip end width, head length, forehead width, nojiri length, body weight, etc.
The number of individuals of various CNV types (Loss, Median and Gain) in the detection population is counted, and the frequency of the various CNV types is counted. The calculation formula is as follows:
PC＝NC/N
wherein PC represents the frequency of a certain copy number variation type; NC represents the number of individuals in the population having this type of copy number variation; n represents the total number of detection populations.
The correlation analysis was performed using SPSS (25.0). In the data processing, according to different factors influencing growth 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:
Yijk＝μ+Gj+Eijk
wherein, Yijk(ii) recording the phenotype of the individual; μ is the population mean; gjThe copy number variation type of each site; eijkIs a random error. The results of the data processing are shown in tables 3 to 6.
TABLE 3 correlation analysis of CHRDL1 Gene CNV with growth traits of Pinus parviflora
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 3): individuals with the deletion type (Loss) copy number variation type of the CHRDL1 gene in the Pinus parviensis have better growth traits than those of other copy number variation types, and the copy number variation sites have a remarkable correlation with the growth traits of the nojiri (P < 0.05). Therefore, the Loss type of the CHRDL1 gene can be used as a candidate molecular genetic marker for improving the growth traits of the Pinus parviensis.
TABLE 4 correlation analysis of CHRDL1 gene CNV and Qinchuan cattle growth traits
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 4): individuals with deletion type (Loss) copy number variation types of the CHRDL1 gene in Qinchuan cattle have more copy types on growth traits, and the copy number variation sites have obvious correlation (P <0.05) with the growth trait of chest depth. Therefore, the Loss type of the CHRDL1 gene can be used as a candidate molecular genetic marker for improving the growth traits of Qinchuan cattle.
TABLE 5 correlation analysis of CHRDL1 gene CNV and Xianan cattle growth traits
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 5): individuals with the normal type (Median) copy number variation type of the CHRDL1 gene in southeast cattle are better in growth trait than individuals with other copy number variation types, and the copy number variation site has a remarkable correlation (P <0.05) with the weight of the growth trait. Therefore, the Median type of the CHRDL1 gene can be used as a candidate molecular genetic marker for improving the growth traits of the south-summer cattle.
TABLE 6 correlation analysis of CHRDL1 Gene CNV with growth traits of Yunnan cattle
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 deletion type (Loss) copy number variation types of the CHRDL1 gene in the Yunnan cattle are better than individuals with more copy types in the growth trait, and the copy number variation sites have obvious correlation with the growth trait of waist width (P < 0.05). Therefore, the Loss type of the CHRDL1 gene can be used as a candidate molecular genetic marker for improving the growth traits of the Yunnan cattle.
6. Application of cow CHRDL1 gene copy number variation type detection
The above results also indicate that CHRDL1 gene can be used as a candidate gene for improving the growth traits of cattle, and is presumed to have an effect on the development of traits such as cattle bones. Individuals with deletion types are selected from the cattle to be detected for seed reservation and propagation, and the advantages of the individuals on the phenotypes such as nojirima, chest depth and the like are utilized, so that the improvement of the excellent growth performance and the breeding process of cattle (such as Qinchuan cattle, Pinna cattle and other local cattle varieties) can be accelerated.
<110> northwest agriculture and forestry science and technology university
<120> method for rapidly detecting growth traits of cattle by CHRDL1 gene CNV marker in auxiliary mode and application thereof
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Claims (10)
1. A method for detecting copy number variation of bovine CHRDL1 gene, which is characterized by comprising the following steps: the method comprises the following steps:
by taking bovine genomic DNA as a template and a primer pair P1 and a primer pair P2 as primers, respectively amplifying a copy number variation region of the CHRDL1 gene and a partial fragment of the BTF3 gene serving as an internal reference by real-time fluorescent quantitative PCR, and then identifying the copy number variation type of the bovine CHRDL1 gene according to the quantitative result;
the primer pair P1 is:
the upstream primer F1: 5'-GCAGCCCATCTTCCACTTTT-3'
The downstream primer R1: 5'-AGCCTGAGATAAGAGGTCTCCA-3'
The primer pair P2 is:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3' are provided.
2. The method for detecting copy number variation of bovine CHRDL1 gene according to claim 1, wherein the method comprises the following steps: the copy number variation region of the CHRDL1 gene is located in 64369201bp-64378000bp of a bovine CHRDL1 gene reference genome sequence AC _ 000187.1.
3. The method for detecting copy number variation of bovine CHRDL1 gene according to claim 1, wherein the method comprises the following steps: said copy number variation type is according to 2 x 2-ΔΔCtThe 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。
4. The method for detecting copy number variation of bovine CHRDL1 gene according to claim 1, wherein the method comprises the following steps: the size of the amplification product fragment of the primer pair P1 is 153bp, and the size of the amplification product fragment of the primer pair P2 is 166 bp.
5. The method for detecting copy number variation of bovine CHRDL1 gene according to claim 1, wherein the method comprises the following steps: the real-time fluorescent quantitative PCR amplification system comprises 1 mu L of template DNA with the concentration of 10 ng/mu L and 0.5 mu L of upstream primer and downstream primer corresponding to the primer pair P1 or the primer pair P2 with the concentration of 10 mu mol/L respectively.
6. The method for detecting copy number variation of bovine CHRDL1 gene according to claim 1, wherein the method comprises the following steps: the reaction procedure of the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 20s, and 39 cycles.
7. Use of the method of any one of claims 1-6 for molecular marker assisted selection breeding in cattle.
8. Use according to claim 7, characterized in that: individuals with either the deletion or normal copy number variation types are superior in growth traits.
9. Use according to claim 8, characterized in that: the growth traits are one or more of nojiri length, chest depth, body weight and waist angle width.
10. A kit for detecting copy number variation of bovine CHRDL1 gene, which is characterized in that: the kit comprises a primer pair P1 and a primer pair P2 which are used for amplifying copy number variation regions of CHRDL1 gene and partial fragments of BTF3 gene serving as internal reference respectively through real-time fluorescent quantitative PCR;
the primer pair P1 is:
the upstream primer F1: 5'-GCAGCCCATCTTCCACTTTT-3'
The downstream primer R1: 5'-AGCCTGAGATAAGAGGTCTCCA-3'
The primer pair P2 is:
the upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3'
The downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG-3' are provided.
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| CN114657267A (en) * | 2022-04-28 | 2022-06-24 | 中国农业科学院兰州畜牧与兽药研究所 | Detection method and application of CNV marker of yak MICALL2 gene |
| CN116083596A (en) * | 2022-11-14 | 2023-05-09 | 宁夏大学 | SNP locus highly correlated with bovine primary organism and application thereof |
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| EP1263985A2 (en) * | 2000-01-22 | 2002-12-11 | The University Court Of The University Of Edinburgh | Methods for amplifying genetic material and uses thereof |
| CN109943647A (en) * | 2019-04-29 | 2019-06-28 | 西北农林科技大学 | A kind of method and its application of quick detection ox MLLT10 gene C NV label |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP1263985A2 (en) * | 2000-01-22 | 2002-12-11 | The University Court Of The University Of Edinburgh | Methods for amplifying genetic material and uses thereof |
| CN109943647A (en) * | 2019-04-29 | 2019-06-28 | 西北农林科技大学 | A kind of method and its application of quick detection ox MLLT10 gene C NV label |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114657267A (en) * | 2022-04-28 | 2022-06-24 | 中国农业科学院兰州畜牧与兽药研究所 | Detection method and application of CNV marker of yak MICALL2 gene |
| CN116083596A (en) * | 2022-11-14 | 2023-05-09 | 宁夏大学 | SNP locus highly correlated with bovine primary organism and application thereof |
| CN116083596B (en) * | 2022-11-14 | 2024-05-28 | 宁夏大学 | SNP locus highly correlated with bovine primary organism and application thereof |
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