CN110564829B - Method for auxiliary detection of lactation traits of dairy cow NCAM2 gene CNV marker and special kit thereof - Google Patents
Method for auxiliary detection of lactation traits of dairy cow NCAM2 gene CNV marker and special kit thereof Download PDFInfo
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Abstract
The invention discloses a method for auxiliary detection of lactation traits of a milk cow NCAM2 gene CNV marker and a special kit thereof: using real-time quantitative PCR technology, using Chinese Holstein cow genome DNA as template, respectively using two pairs of primers P1 and P2 to amplify CNV marker locus of cow NCAM2 gene and a section of region of internal reference gene BTF3, finally using 2*2 ‑ΔΔCt The method of (1) calculates and determines the copy number variation type of an individual, and classifies the result into a multicopy type, a deletion type and a normal type. The invention can detect the CNV mark closely related to the lactation character of Chinese Holstein cows on the DNA level, and can accelerate the breeding process of the cows with excellent lactation performance. The method is simple and rapid, and is convenient for popularization and application.
Description
Technical Field
The invention relates to the field of livestock molecular biology detection, in particular to a method for detecting a CNV (CNV) marker of a NCAM2 gene of a Chinese Holstein cow based on a real-time quantitative PCR (polymerase chain reaction) technology.
Background
Molecular marker-assisted selection (MAS) can utilize molecular markers related to quantitative traits, and marker information is used as auxiliary information to accurately and rapidly analyze genetic composition of an individual from a molecular level, thereby realizing selection of genotypes. The genetic marker, pedigree and phenotype information can be fully utilized, and compared with a conventional breeding method, the genetic marker, pedigree and phenotype information is rich in information amount and high in selection accuracy.
Molecular DNA markers (DNA molecular markers) are DNA fragments that reflect the genomic specificity of each individual and inter-population. Molecular markers include Restriction Fragment Length Polymorphism markers (RFLP), random Amplified genomic DNA Polymorphism markers (RAPD), amplified Fragment Length Polymorphism markers (AFLP), microsatellite DNA (MS), and Single Nucleotide Polymorphism markers (SNP), among others.
In contrast, copy Number Variations (CNV) are a new form of variation, which refers to mutations in the copy number of a DNA fragment of 1kb to Mb in length, including amplification, deletion, insertion, inversion, etc. of a single DNA fragment, and also including various combinations of complicated chromosomal amplification, deletion, and insertion, and thus have a greater genetic effect than SNPs and Indels because they cover a wider range.
Along with the continuous perfection of molecular breeding technology, more and more variations are known by people, in recent years, the research on SNP is relatively more, the research on CNV is relatively less, and the molecular breeding marker has the advantages of large fragment length, ubiquitous existence, wide genome coverage and the like, so that the molecular breeding marker can obviously influence the livestock and poultry properties and has wide application prospect as the molecular breeding marker.
At present, the research on the CNVs of cows is increasing. On one hand, the mature human genome CNVs detection technology has reference effect on researching the milk cow CNVs; on the other hand, the gradual perfection of the cow genome sequencing map has a promoting effect on the development of CNVs. The establishment and gradual improvement of the CNVs map have important significance for the research of the economic traits and the functional traits of the dairy cows and the promotion of the genetic improvement of the dairy cows.
The expression product of the NCAM2 gene is a nerve adhesion molecule 2 which is closely related to the nervous system, and the related process of lactation is regulated by nerves, but at present, only the NCAM2 gene is presumed to be related to lactation, and the influence of the copy number variation of the NCAM2 gene on the lactation traits of dairy cows (such as Chinese Holstein dairy cows) is not reported.
Disclosure of Invention
The invention aims to provide a method for auxiliary detection of lactation traits of a dairy cow NCAM2 gene CNV marker and a special kit thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting the copy number variation of the NCAM2 gene of a dairy cow comprises the following steps:
using the genomic DNA of a cow (for example, holstein, china) as a template, using a primer pair P1 and a primer pair P2 as primers, respectively amplifying a CNV region partial fragment of the NCAM2 gene and a reference BTF3 gene partial fragment by real-time quantitative PCR, and then identifying the copy number variation type of the NCAM2 gene of the cow individual according to the quantitative result.
Preferably, the CNV region of the NCAM2 gene is located from 14873201 to 14876400 of the bovine NCAM2 gene reference genomic sequence AC _ 000158.1.
Preferably, said copy numberThe type of variation is according to 2*2 -ΔΔCt The quantitative results were divided into three categories: multicopy (Gain), 2*2 -ΔΔCt >2; deletion type (Loss), 2*2 -ΔΔCt <2; normal type (Median), 2*2 -ΔΔCt ＝2。
Preferably, the primer pair P1 is:
an upstream primer F1:5'-TGGGCAATAGGTGATCCATCC-3'
A downstream primer R1:5'-TCAGCAGGAAAACACAGGCTAT-3';
the primer pair P2 is as follows:
an upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
A downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3';
the size of the PCR product fragment amplified based on the primer pair P1 is 130bp, and the size of the PCR product fragment amplified based on the primer pair P2 is 166bp.
Preferably, the real-time quantitative PCR amplification system comprises: mu.L of 10 ng/. Mu.L template DNA 1.25. Mu.L, 10. Mu. Mol/L primer pair P1 or upstream and downstream primers corresponding to primer pair P2 each 0.5. Mu.L, 2 XSSYBR Mix 6.25. Mu.L and ddH 2 O 3.75μL。
Preferably, the reaction procedure of the real-time quantitative PCR is: (1) pre-denaturation: 94 ℃ for 2min; (2) amplification reaction: denaturation at 94 ℃ for 10s, annealing at 60 ℃ for 30s,40 cycles.
Preferably, the method for detecting the copy number variation of the NCAM2 gene of the dairy cattle is applied to the molecular marker-assisted selective breeding of the dairy cattle.
Preferably, chinese Holstein cow individuals with multiple copy number (Gain) variation types are superior in lactation traits, and the copy number variation sites have significant correlation with the milk fat rate (P < 0.05).
A detection kit for the CNV marker of the NCAM2 gene of the dairy cow comprises the primer pair P1 and P2.
The invention has the beneficial effects that:
the method for detecting the copy number variation of the NCAM2 gene disclosed by the invention detects the copy number variation condition of a cow (such as a Chinese Holstein cow) by a real-time quantitative PCR technology, and the detection result can be used for identifying candidate molecular genetic markers (CNV markers) related to the improvement of the lactation traits of the cow (related to important lactation traits such as milk yield, milk fat percentage, lactoprotein rate, somatic cell score and the like). Compared with a high-throughput sequencing method, a gene chip method and the like, the method is quick, simple and low in cost, can accurately identify the copy number type of an individual, provides molecular markers and practical basis for molecular breeding of Chinese Holstein dairy cow and other dairy cow varieties, and accelerates the breeding process of the dairy cows with excellent milk production performance.
Drawings
FIG. 1 is an amplification curve plotted by QPCR in an example of the present invention.
FIG. 2 is a solubility curve plotted by QPCR in an example of the present invention.
FIG. 3 is the distribution of the copy number variation of the NCAM2 gene in the cow population in the example of the present invention.
Detailed Description
The invention will now be described in detail with reference to the drawings and examples, which are given by way of illustration and not by way of limitation.
Based on the previous cattle genome re-sequencing research, the copy number variation from 14873201 to 14876400 of the cattle NCAM2 genome sequence AC _000158.1 is found, and based on the function of the NCAM2 gene and the regulation mechanism of CNV, the invention finds the relevance between the copy number variation of the NCAM2 gene and the lactation character, and provides important practical basis for the molecular breeding of dairy cattle, the improvement of milk yield and milk quality.
The invention detects CNV marks based on QPCR technology and utilizes 2*2 to data -ΔΔCt And (3) processing, and dividing the copy number variation result into three types: 2*2 -ΔΔCt >2 is a multicopy type; 2*2 -ΔΔCt <2 is deletion, 2*2 -ΔΔCt =2 is normal type.
1. Sample collection and genomic DNA extraction
(1) Blood sample collection and data collection
In the invention, 310 blood samples (collected in 8 months in 2015) are collected from a grass beach farm dairy cow farm in Xian city, shaanxi province, all the samples are adult Chinese Holstein cow cows, the blood collection method is jugular vein blood collection, and the blood is brought back to a laboratory by an ice box and stored at-80 ℃; during sampling, basic data determination (lactation amount, fat content, protein content and the like) acquisition and recording are carried out on corresponding individuals, so that the relevant analysis at the later stage is realized.
(2) Extraction of DNA from blood samples
The sample was subjected to extraction of genomic DNA by the phenol-chloroform method.
(1) Thawing frozen blood sample (mainly blood cells) at room temperature, sucking 2mL blood into a 2.0mL centrifuge tube, and centrifuging at 12000rpm at 4 ℃ for 10min; 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 15min; centrifuging at 12000rpm at 4 deg.C for 10min, discarding liquid, and retaining precipitate;
(2) smashing the precipitate into flocculent, adding 500 mu L of DNA extraction buffer solution and 6 mu L of protease K into a centrifuge tube, and incubating overnight (about 16 h) in a constant-temperature water bath at 37 ℃ until the cell precipitate is completely digested, and clarifying the solution;
(3) adding 1mL of Tris saturated phenol, placing on ice, gently shaking for 20min to fully mix, centrifuging at 12000r/min for 10min at 4 ℃, and transferring the upper aqueous phase into another sterilized 2.0mL centrifuge tube.
(4) Adding 0.5mL of saturated phenol and 0.5mL of chloroform, placing on ice, and gently 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 for 10min at 4 deg.C; transferring the upper aqueous phase into a 1.5mL centrifuge tube by using a pipettor;
(6) adding 1mL of precooled absolute ethyl alcohol (-20 ℃), gently bottoming and shaking for multiple times until DNA is separated out, and then placing for 30min at-20 ℃; centrifuging at 4 deg.C and 12000rpm for 10min, and removing ethanol;
(7) adding 1mL of 70% ethanol, and gently shaking for 10min; centrifuging at 4 deg.C and 12000rpm for 10min, removing ethanol, and rinsing repeatedly; standing at room temperature for 30min, oven drying at 60 deg.C for 30s to volatilize ethanol;
(8) add ultra pure water 50 u L,4 degrees C storage until DNA completely dissolved, spectrophotometer determination concentration, the-80 degrees C storage.
2. Amplification of target and reference genes
The sequences of the copy number variation regions screened out in the re-sequencing, namely 14873201 to 14876400 of the sequence of the NCAM2 gene (target gene) group, are found by taking a bovine NCAM2 gene (AC _ 000158.1) published by NCBI as a reference sequence. Primers contained in this region were designed using Prime 5.0 software and aligned in NCBI _ BLAST. The primer sequences are as follows (primer pair P1), the size of the PCR product fragment is 130bp:
an upstream primer F1:5'-TGGGCAATAGGTGATCCATCC-3'
A downstream primer R1:5'-TCAGCAGGAAAACACAGGCTAT-3';
meanwhile, a bovine BTF3 gene sequence (AC _ 000177.1) published by NCBI is used as a reference sequence, and the same method is adopted to design a primer for amplifying a specific fragment (166 bp) in the reference gene (BTF 3 gene), wherein the primer sequence is as follows (primer pair P2):
an upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
A downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3'
The design completion time of the primers is as follows: year 2018, month 5.
3. Real-time fluorescence Quantification (QPCR)
Whether the primers are suitable for real-time quantitative PCR analysis is determined by plotting an amplification curve and a dissolution peak. The amplification curve is smooth, and the QPCR amplification system and conditions are appropriate (figure 1); the drawn dissolution curves are matched together, and the curves are smooth in trend, high and sharp in peak height, and free of hybrid peaks caused by primer dimers or nonspecific amplification, and show that the primer quality is good (figure 2).
The QPCR amplification system is shown in Table 1.
TABLE 1 QPCR reaction System
The reaction procedure of real-time quantitative PCR was:
(1) Pre-denaturation: 94 ℃ for 2min;
(2) And (3) amplification reaction: denaturation at 94 ℃ for 10s, annealing at 60 ℃ for 30s,40 cycles.
Calculation of CNV type
Each sample was amplified with primers for the gene sequence of interest and the reference gene sequence (primer pair P1 and P2), respectively, and 3 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 presence of CNVs, and the control group is a sample known to have no copy number variation. 2 -ΔΔCt The copy number of the target gene sequence in the experimental group is expressed in multiples relative to the control group. The expressed abundance of the gene was then logarithmically transformed (base 2 of 2) -ΔΔCt Logarithm of (d) to fit a normal distribution, and after performing a homogeneity test for variance, statistically testing the differences between groups. C T Namely Cycle threshold, which is the number of amplification cycles that pass when the fluorescence signal of the amplification product reaches a set threshold during the PCR amplification process.
When the amplified target gene sequence is a normal sequence, 2*2 -ΔΔCt =2; when the amplified target gene sequence is a deletion sequence, 2*2 -ΔΔCt <2; when the amplified target gene sequence is a multicopy sequence, 2*2 -ΔΔCt >2。
5. Data analysis
And counting the number of individuals of various copy number variation types in the detection population, and counting the frequency of various types. The calculation formula is as follows:
PC＝NC/N
wherein PC represents a frequency of a certain copy number type; NC represents the number of individuals in the population having a CNV type of C (Gain, media, or Loss); n represents the total number of detection populations.
The correlation analysis was performed using SPSS (18.0). In the data processing, according to different factors influencing lactation character indexes, environmental effects, age, genetic effects and interaction effects thereof are considered, a fixed model is adopted for analysis, and simplification is carried out according to actual conditions. The complete model is as follows:
Y ijk ＝μ+G j +E ijk
wherein Y is ijk (ii) recording the phenotype of the individual; μ is the population mean; g j The copy number type of each site; e ijk Is a random error.
The results of the data processing are shown in table 2.
TABLE 2 correlation analysis of NCAM2 Gene with different lactation traits in cows
Note: the average shoulder marks with the same letter indicate no significant difference (P > 0.05), the average shoulder marks with different letters indicate significant difference (P < 0.05); * P <0.05.
According to the statistic result of the genotype (figure 3, and N1, N2 and N3 in the table 2), the distribution of the copy number variation sites from 14873201 to 14876400 of the group sequence of the bovine NCAM2 gene (AC _ 000158.1) in the sampled Chinese Holstein cows is determined, which accords with the polymorphism.
Correlation analysis results show (see table 2): compared with other two types, the multi-copy type (Gain) of the dairy cow at the locus has the advantages that the fat content in the cow milk is obviously increased, and certain positive effects on the protein content in the cow milk and the yield of the cow milk are achieved.
The results show that the CNV locus on the NCAM2 gene can be used as a candidate molecular genetic marker locus for improving the lactation character of Chinese Holstein cows, and the breeding process of the cows with excellent lactation performance is accelerated.
<110> northwest agriculture and forestry science and technology university
<120> method for auxiliary detection of lactation traits of dairy cow NCAM2 gene CNV marker and special kit thereof
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Claims (4)
1. Detect milk cowNCAM2A method for marking gene CNV, comprising the following steps:
milk cow genome DNA is taken as a template, and real-time quantitative PCR amplification is respectively carried outNCAM2Partial fragments of the CNV region of a gene and referenceBTF3Partial fragments of the gene, and then identifying the cow according to the quantitative resultNCAM2Copy number variation type of gene;
saidNCAM2The CNV region of the gene is located in cattleNCAM214873201 to 14876400 of the gene reference genomic sequence AC _ 000158.1;
the copy number variation type is according to 2*2 −ΔΔCt The quantitative results were divided into three categories: multicopy type, 2*2 −ΔΔCt >2; deletion type, 2*2 −ΔΔCt <2; normal, 2*2 −ΔΔCt =2；
SaidNCAM2The amplification primer pair of the CNV region partial segment of the gene is as follows:
an upstream primer F1:5'-TGGGCAATAGGTGATCCATCC-3'
A downstream primer R1:5'-TCAGCAGGAAAACACAGGCTAT-3';
saidBTF3The amplification primer pair of the partial segment of the gene is as follows:
an upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
A downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3';
the dairy cows are selected from Chinese Holstein dairy cow individuals;
chinese Holstein cow individuals with multi-copy number variation types have better lactation character milk fat rate.
2. The method of claim 1 for testing dairy cowsNCAM2A method for marking gene CNV, which is characterized in that: the real-time quantitative PCR amplification system comprises 1.25 muL of 10 ng/muL template DNA and 0.5 muL of upstream primer and downstream primer corresponding to 10 mumol/L amplification primer pair respectively.
3. The method of claim 1 for testing dairy cowsNCAM2A method for marking gene CNV, which is characterized in that: the reaction procedure of the real-time quantitative PCR is as follows: pre-denaturation: 94. 2min at the temperature; and (3) amplification reaction: 94. denaturation at 10s, annealing at 60 ℃ for 30s,40 cycles.
4. Milk cowNCAM2A detection kit for a gene copy number variation marker, characterized in that: including use in real-time quantitative PCR amplificationNCAM2Primer pair of copy number variation marker sites of genes, wherein the copy number variation marker sites are positioned in cattleNCAM2The gene reference genomic sequence AC _000158.1 is within 14873201 to 14876400;
NCAM2the amplification primer pair of the partial segment of the copy number variation region of the gene comprises:
an upstream primer F1:5'-TGGGCAATAGGTGATCCATCC-3'
A downstream primer R1:5'-TCAGCAGGAAAACACAGGCTAT-3';
BTF3the amplification primer pair of the partial segment of the gene is as follows:
an upstream primer F2:5'-AACCAGGAGAAACTCGCCAA-3'
A downstream primer R2:5'-TTCGGTGAAATGCCCTCTCG-3';
the copy number variation type is according to 2*2 −ΔΔCt The quantitative results were divided into three categories: multicopy type, 2*2 −ΔΔCt >2; deletion form, 2*2 −ΔΔCt <2; normal, 2*2 −ΔΔCt =2；
The dairy cows are selected from Chinese Holstein dairy cow individuals;
chinese Holstein cow individuals with multi-copy number variation types have better lactation character milk fat rate.
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