CN112813175B - Method for rapid auxiliary detection of growth traits by cattle CHRDL1 gene CNV markers and application thereof - Google Patents
Method for rapid auxiliary detection of growth traits by cattle CHRDL1 gene CNV markers and application thereof Download PDFInfo
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Abstract
The invention discloses a method for rapid auxiliary detection of growth traits by a cattle CHRDL1 gene CNV marker and application thereof: with bovine bloodThe whole genome DNA is used as a template, and two pairs of primers P1 and P2 are used for amplifying a CNV region of the bovine CHRDL1 gene and a segment region of an internal reference gene BTF3 respectively according to 2 x 2 by using a real-time fluorescence quantitative PCR technology ‑ΔΔCt The copy number variation type of the individual CHRDL1 gene is determined to be multicopy, deleted or normal. According to the correlation analysis result of CHRDL1 gene copy number variation and growth characteristics, the invention can detect CNV markers closely related to the cattle growth characteristics on the DNA level, can be used for marker auxiliary selection of the cattle growth characteristics, and accelerates the fine breed breeding process.
Description
Technical Field
The invention relates to the field of livestock molecular biology detection, in particular to a method for detecting a cow CHRDL1 gene CNV marker based on a qPCR technology.
Background
The current beef cattle molecular breeding research is mainly focused on marker-assisted selection based on molecular markers. Molecular breeding, namely molecular marker-assisted selection (MAS), is to select genetic resources or breeding materials by means of DNA molecular markers, so as to improve the variety of the comprehensive characters of livestock and poultry. In livestock breeding, the purposes of early seed selection and improvement of breeding value accuracy are achieved through the selection of DNA markers which are closely related to growth traits and closely related to quantitative traits, so that greater genetic progress is obtained in livestock breeding.
Copy number variation (Copy Number Variations, CNVs), which is a structural variation at the genomic sub-microscopic level, refers to the phenomenon of deletion or duplication of larger fragments in genomic DNA, involving fragments ranging in size from 50bp to a number Mb, including Copy number increase (Copy number gain) and Copy number decrease (Copy number loss). The CNV region covers more nucleotide content of the genome than does a Single Nucleotide Polymorphism (SNP), and the mechanism of association with phenotypic variation is most clearly the gene dose effect caused by variation in gene copy number or its associated regulatory elements.
The unknown copy number variation of genome was studied by microarray-based Comparative Genome Hybridization (CGH), and by simultaneously hybridizing samples labeled with different luciferins (test sample and control sample) on one chip, the variation of DNA copy number between the genome of the test sample and the genome of the control sample can be detected. The probes of the CGH chip cover the whole genome, and have the characteristics of high flux, sensitivity, accuracy and resolution, and the test data has higher credibility. However, CGH chip resolution is on the Mb level, and copy number variation fragments of smaller fragments are not easily detected. Meanwhile, the detection operation is complex, 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 of Sequencing (NGS) technology overcomes some of the disadvantages inherent in hybridization technology, and complex structural changes can be identified using paired sequencing, improving detection resolution, but still being costly.
For the detection of established CNV, methods based on PCR techniques 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 fluorescent chemical methods used in qPCR are mainly classified into two types, namely, fluorescent dye intercalation and fluorescent hybridization probe. The dye method has the advantages of low experimental cost, no need of designing synthetic probes, convenient use and capability of detecting the absolute copy number of the target fragment. For example, by adding an excessive amount of SYBR Green dye molecules to the PCR reaction system, the amount of genomic DNA can be reflected by detecting the intensity of fluorescent signal, by relatively quantifying the objective gene (having copy number variation) and the reference gene (having no copy number variation), and according to 2 -ΔΔCt Method statistics the copy number of the sample candidate gene region can be detected.
Chord-like 1 (CHRDL 1) is a secreted glycoprotein with repeated cysteine-rich domains that binds to BMPs family ligands and is a secreted Bone Morphogenic Protein (BMP) antagonist expressed in mesenchymal tissues. The ability of CHRDL1 to enhance BMP-4 activity may be an important mechanism for elucidating the mechanisms of osteogenic differentiation and bone remodeling of human bone marrow mesenchymal stem cells (hBMSCs). During the process of hBMSCs osteogenesis, BMP-4 function is enhanced and depends on the regulatory role of the CHRDL1 gene in BMP-SMAD signaling pathway.
At present, no literature report on the influence of CHRDL1 gene CNV on the growth traits of local cattle varieties is available.
Disclosure of Invention
The invention aims to provide a method for quickly assisting in detecting growth traits by using a cattle CHRDL1 gene CNV marker and application thereof.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a method for detecting bovine CHRDL1 gene copy number variation, comprising the steps of:
taking genome DNA of an individual cow to be detected as a template, taking a primer pair P1 and a primer pair P2 as primers, amplifying partial fragments of a copy number variation region of the CHRDL1 gene and partial fragments of a BTF3 gene serving as an internal reference respectively through real-time fluorescent quantitative PCR, and then identifying the copy number variation type of the CHRDL1 gene of the individual cow according to a quantitative result;
the primer pair P1 is as follows:
upstream primer F1:5'-GCAGCCCATCTTCCACTTTT-3'
Downstream primer R1:5'-AGCCTGAGATAAGAGGTCTCCA-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 153bp, and the PCR product fragment size amplified based on the primer pair P2 is 166bp.
Preferably, the copy number variation region of the CHRDL1 gene is 64369201bp-64378000bp of the reference genome sequence AC_000187.1 of the bovine CHRDL1 gene.
Preferably, the copy number variation is according to 2.multidot.2 -ΔΔCt The quantitative results were divided into three categories: multicopy (Gain), 2 x 2 -ΔΔCt >2; deletion (Loss), 2 x 2 -ΔΔCt <2; normal (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 the upstream and downstream primers corresponding to primer pair P2 are each 0.5. Mu.L, 2X SYBR Green QPCR Mix. Mu.L and ddH 2 O 3μL。
Preferably, the reaction procedure used for the real-time fluorescent quantitative PCR is as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 10s, annealing at 60℃for 20s,39 cycles.
The method for detecting bovine CHRDL1 gene copy number variation is applied to bovine molecular marker-assisted selective breeding.
Preferably, in the Pinanniu, the individuals with the copy number variation type are obviously superior to the individuals with the normal type and the multicopy type in the growth characteristic of long-length of the individual; in Qinchuan cattle, individuals with copy number variation types of deletion are obviously superior to individuals with multiple copy types in the growth characteristic of chest depth; in summer south cattle, individuals with normal copy number variation types are significantly better than those with deletion and multiple copies in weight, which is a growth trait; in the cloud cattle, the copy number variation type is obviously superior to the copy number variation type in the aspect of the growth character of the waist angle width.
A kit for detecting bovine CHRDL1 gene copy number variation comprises the primer pair P1 and the primer pair P2.
The beneficial effects of the invention are as follows:
according to the invention, the bovine CHRDL1 gene reference region ChrX of 64369201bp-64378000bp is taken as a site, the copy number variation condition of the site in a cattle group body is detected by a real-time fluorescent quantitative PCR technology, and the site is subjected to correlation analysis with important economic characters such as weight, waist angle width and the like; the correlation analysis result shows that the CNV locus of the cattle CHRDL1 gene is obviously related to the important growth traits of cattle, and DNA molecular markers (CNV markers) which are selected in an assisted manner by markers of the cattle growth traits exist, so that the cattle population with excellent genetic resources can be quickly established, and the fine breed breeding process is accelerated.
Compared with the prior art, the invention has the following advantages:
(1) The detection method for the bovine CHRDL1 gene copy number variation is not limited by age, and can be used for early breeding of cows.
(2) The method for detecting CHRDL1 gene copy number variation is accurate and reliable and is simple and convenient to operate.
(3) The detection of CHRDL1 gene copy number variation site provides scientific basis for bovine molecular marker assisted selection.
Drawings
FIG. 1 is an amplification curve drawn by qPCR (CHRDL 1 gene) performed in the example of the present invention.
FIG. 2 is a melting curve of qPCR (CHRDL 1 gene) performed in the example of the present invention.
FIG. 3 is a distribution of CHRDL1 gene copy number variation detected in the examples of the present invention in bovine populations.
Detailed Description
The present invention will now be described in further detail with reference to the accompanying drawings and examples which are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
The invention detects copy number variation of the bovine CHRDL1 gene by qPCR aiming at a copy number variation region ChrX: 64369201bp-64378000bp of the bovine CHRDL1 gene (GenBank Accession No. AC-000187.1) screened in the resequencing, and reveals a CNV marker existing in the bovine CHRDL1 gene. The specific description is as follows.
1. Sample collection and genomic DNA extraction
(1) Blood sample collection and data collection
TABLE 1 information on test animal samples
673 blood samples are collected in the invention, the samples are adult cows over 2 years old (24-36 months old), the blood collection method is that jugular vein blood is collected, and the adult cows are brought back to a laboratory by an ice chest and stored at-80 ℃; and (3) sampling and simultaneously measuring, collecting and recording growth development basic data of the corresponding individuals for later correlation analysis.
(2) Extraction of blood sample DNA (extraction of genomic DNA from a sample by phenol-chloroform method)
(1) Thawing frozen blood samples (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, reserving the precipitate, adding 1.5mL of PBS buffer, and stirring by vortex to suspend the precipitate, and gently shaking on ice for 15min; centrifuge at 12000rpm for 10min at 4℃and discard the liquid and leave the pellet.
(2) The pellet was triturated to a flocculent state, 500. Mu.L of DNA extraction buffer and 6. Mu.L of proteinase K were added to the centrifuge tube and incubated overnight (about 16 h) in a thermostatted water bath at 37℃until the cell pellet was completely digested and the solution was clear.
(3) 1mL of Tris saturated phenol is added, the mixture is placed on ice and gently shaken for 20min, the mixture is fully and uniformly mixed, the mixture is centrifuged for 10min at the temperature of 4 ℃ and the speed of 12000r/min, and the upper water phase is transferred into another sterilized 2.0mL centrifuge tube.
(4) 0.5mL of saturated phenol and 0.5mL of chloroform were added, and the mixture was left on ice with gentle shaking for 20min; the upper aqueous phase was transferred to another sterilized 2.0mL centrifuge tube.
(5) Adding 1mL of chloroform, placing on ice and gently shaking for 20min; centrifuging at 12000rpm at 4deg.C for 10min; the upper aqueous phase was transferred to a 1.5mL centrifuge tube using a pipette.
(6) Adding 1mL of pre-cooled absolute ethyl alcohol (-20 ℃), slightly shaking the mouth bottom for multiple times until DNA is separated out, and then standing at-20 ℃ for 30min; the mixture was centrifuged at 12000rpm at 4℃for 10min, and the ethanol was discarded.
(7) Adding 1mL of 70% ethanol, and gently shaking for 10min; centrifuging at 12000rpm for 10min at 4deg.C, discarding ethanol, and repeatedly rinsing once; standing at room temperature for 30min, and standing in oven at 60deg.C for 30s to volatilize ethanol.
(8) Adding 50 μl of ultrapure water, preserving at 4deg.C until DNA is completely dissolved, and preserving at-80deg.C after spectrophotometry to determine concentration.
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 was found on NCBI, and primers (153 bp for amplification of the target fragment) contained in this region were designed using Prime 5.0 software using this as a template, and aligned in NCBI_BLAST. The primer sequences are as follows (primer pair P1):
upstream primer F1:5'-GCAGCCCATCTTCCACTTTT-3'
Downstream primer R1:5'-AGCCTGAGATAAGAGGTCTCCA-3'.
Meanwhile, a primer for amplifying a specific fragment (166 bp) in a BTF3 gene (reference gene) is designed by using a bovine BTF3 gene sequence (AC_ 000177.1) published by NCBI as a reference sequence by adopting the same method. The primer sequences are as follows (primer pair P2):
the upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
Downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3'.
3. Real-time fluorescent quantitative PCR
The qPCR reaction system is shown in Table 2.
TABLE 2 reaction System for qPCR
The reaction procedure used for qPCR was:
(1) Pre-denaturation: 95 ℃ for 2min;
(2) Amplification reaction: denaturation at 95℃for 10s, annealing at 60℃for 20s,39 cycles.
The primers were determined by plotting the amplification curves and the dissolution peaks and were suitable for real-time fluorescent quantitative PCR analysis. The amplification curve is smooth, which shows that the qPCR reagent has good quality and the amplification system and conditions are proper (FIG. 1); the drawn dissolution curves, the curves of the samples are matched together, the curve trend is smooth, the peak height is high and sharp, and the primer dimer or the mixed peak caused by non-specific amplification does not exist, which shows that the primer mass is good (figure 2).
Calculation of CNV type
Each sample was amplified with primers (primer pair P1 and P2) for the target sequence and the internal reference sequence, respectively, and 3 replicates of each pair of primers. According to 2 x 2 -ΔΔCt The method performs copy number analysis (FIG. 3). Wherein delta is delta 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 whether CNVs exist or not, the control group is a sample without known copy number variation, C T Namely Cycle threshold, is the number of amplification cycles that pass when the fluorescent signal of the amplified product reaches a set threshold during PCR amplification. Then, variance alignment is carried outTesting, the differences between groups were statistically tested.
When the target sequence is a normal sequence, calculating a normalized value of 2 x 2 -ΔΔCt =2; when the target sequence is a deletion type sequence, calculating a normalized value of 2 x 2 -ΔΔCt <2; when the target sequence is a multicopy sequence, calculating a normalized value of 2 x 2 -ΔΔCt >2。
5. Data analysis
Growth traits: high, cross height, body diagonal length, chest circumference, abdomen circumference, tube circumference, chest width, chest depth, hip circumference, waist angle width, ischial end width, head length, forehead width, and length of the body and weight.
The number of individuals of each CNV type (Loss, median and Gain) in the detection population is counted, and the frequency of each CNV type 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 such copy number variation types; n represents the total number of detection populations.
Correlation analysis was performed using SPSS (25.0). In the data processing, according to the difference of factors influencing the growth character index, the environmental effect, the age, the genetic effect and the interaction effect are considered, the analysis is performed by adopting a fixed model, and meanwhile, the simplification is performed according to the actual situation. The complete model is as follows:
Y ijk ＝μ+G j +E ijk
wherein Y is ijk Recording the phenotype of the individual; mu is the population mean; g j Copy number variation type for each site; e (E) ijk Is a random error. The results of the data processing are shown in tables 3 to 6.
TABLE 3 analysis of the relationship between CNV and growth traits of Piano cattle by CHRDL1 Gene
Note that: the average shoulder marks have the same letters to indicate that the difference is not significant (P > 0.05), and the average shoulder marks have the letters to indicate that the difference is significant (P < 0.05); * P <0.05.
The correlation analysis results showed (see table 3): individuals with deletion (Loss) copy number variation in the CHRDL1 gene in the pinna cattle are superior to other copy number variation types in growth traits, and the copy number variation sites have a significant correlation (P < 0.05) with the growth trait of long-length. Therefore, the Loss type of CHRDL1 gene can be used as a candidate molecular genetic marker for improving the growth traits of the Pinans cattle.
TABLE 4 CHRDL1 Gene CNV Qinchuan cattle growth trait correlation analysis
Note that: the average shoulder marks have the same letters to indicate that the difference is not significant (P > 0.05), and the average shoulder marks have the letters to indicate that the difference is significant (P < 0.05); * P <0.05.
The correlation analysis results showed (see table 4): individuals with deletion (Loss) copy number variation types of CHRDL1 genes in Qinchuan cattle are more excellent in growth traits than individuals with more copy types, and the copy number variation sites have obvious correlation with the growth trait of chest depth (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 Qinchuan cattle.
TABLE 5 CHRDL1 Gene CNV analysis of growth traits of summer south cattle
Note that: the average shoulder marks have the same letters to indicate that the difference is not significant (P > 0.05), and the average shoulder marks have the letters to indicate that the difference is significant (P < 0.05); * P <0.05.
The correlation analysis results showed (see table 5): individuals with normal (Median) copy number variation types of the CHRDL1 gene in summer-south cattle are superior to other copy number variation types in growth traits, and the copy number variation sites have a significant correlation (P < 0.05) with the growth trait of body weight. Therefore, the Median type of CHRDL1 gene can be used as a candidate molecular genetic marker for improving the growth traits of the cattle in the south of summer.
TABLE 6 CHRDL1 Gene CNV and cloud Linked cattle growth trait correlation analysis
Note that: the average shoulder marks have the same letters to indicate that the difference is not significant (P > 0.05), and the average shoulder marks have the letters to indicate that the difference is significant (P < 0.05); * P <0.05.
The correlation analysis results showed (see table 6): individuals with deletion (Loss) copy number variation types of the CHRDL1 gene in the cloud cattle are more excellent in growth traits than individuals with more copy types, and the copy number variation sites have obvious correlation (P < 0.05) with the growth traits of waist angular width. Therefore, the Loss type of CHRDL1 gene can be used as a candidate molecular genetic marker for improving the growth traits of the cloud-linked cattle.
6. Application of bovine CHRDL1 gene copy number variation type detection
The results also show that the CHRDL1 gene can be used as a candidate gene for improving the growth traits of cattle, and is presumed to have influence on the development of the traits such as cattle bones. Selecting individual with deletion type from cattle to be tested, reserving seeds and propagating, and utilizing the advantages of the individual with deletion type on the phenotypes such as long jirime, deep chest and the like, thereby accelerating the excellent growth performance improvement and breeding process of cattle (for example, local cattle breeds such as Qinchuan cattle, pi Na cattle and the like).
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<120> method for rapid auxiliary detection of growth traits by cattle CHRDL1 gene CNV marker and application thereof
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Claims (5)
1. A method for detecting bovine CHRDL1 gene copy number variation is characterized in that: the method comprises the following steps:
using bovine genome DNA as a template, using a primer pair P1 and a primer pair P2 as primers, amplifying a copy number variation region of the CHRDL1 gene and a part of fragments of the BTF3 gene serving as an internal reference respectively through real-time fluorescent quantitative PCR, and then identifying the copy number variation type of the bovine CHRDL1 gene according to a quantitative result;
the primer pair P1 is as follows:
upstream primer F1:5'-GCAGCCCATCTTCCACTTTT-3'
Downstream primer R1:5'-AGCCTGAGATAAGAGGTCTCCA-3'
The primer pair P2 is as follows:
the upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
Downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3';
the copy number variation type is according to 2 x 2 -ΔΔCt The quantitative results were divided into three categories: multicopy, 2 x 2 -ΔΔCt >2; deletion type, 2 x 2 -ΔΔCt <2; normal, 2 x 2 -ΔΔCt ＝2；
In the Pinan cattle, the individuals with copy number variation types of deletion type are obviously superior to those of normal type and multicopy type in the aspect of growth property of long-length of the individual; in Qinchuan cattle, individuals with copy number variation types of deletion are obviously superior to individuals with multiple copy types in the growth characteristic of chest depth; in summer south cattle, individuals with normal copy number variation types are significantly better than those with deletion and multiple copies in weight, which is a growth trait; in the cloud cattle, the copy number variation type is obviously superior to the copy number variation type in the aspect of the growth character of the waist angle width.
2. The method for detecting bovine CHRDL1 gene copy number variation according to claim 1, wherein: the amplified product fragment size of the primer pair P1 is 153bp, and the amplified product fragment size of the primer pair P2 is 166bp.
3. The method for detecting bovine CHRDL1 gene copy number variation according to claim 1, wherein: the real-time fluorescence quantitative PCR amplification system comprises 10 ng/mu L of template DNA1 mu L and 10 mu mol/L of primer pair P1 or the upstream primer and the downstream primer corresponding to the primer pair P2 respectively, wherein the upstream primer and the downstream primer are 0.5 mu L.
4. The method for detecting bovine CHRDL1 gene copy number variation according to claim 1, wherein: the reaction program of the real-time fluorescence quantitative PCR is as follows: pre-denaturation at 95℃for 2min; denaturation at 95℃for 10s, annealing at 60℃for 20s,39 cycles.
5. Use of the method according to any one of claims 1-4 in molecular marker assisted selection breeding of cattle.
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| 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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