Beef cattle CIITA gene copy number variation detection method and application thereof

Abstract

The invention relates to the field of molecular genetic breeding, in particular to a beef cattle CIITA gene copy number variation detection method and application thereof.The detection method comprises the following steps that cattle genome DNA is used as a template, a primer pair P1 and a primer pair P2 are used as primers, and the primer pair P1 and the primer pair P2 are used as primers; the method comprises the following steps: respectively amplifying a copy number variation region of the CIITA gene and a part of fragments of an ACTB gene serving as an internal reference through real-time fluorescent quantitative PCR (Polymerase Chain Reaction), and then determining a copy number variation type of the CIITA gene according to a quantitative result; the primer pair P1 comprises an upstream primer F1 and a downstream primer R1, wherein the upstream primer F1 is 5 '-CTGGGTGGTGGTGGTGGAGTTG-3', and the downstream primer R1 is 5 '-GGAGGAGGTGAGAAGCAATGG-3'; the primer pair P2 comprises an upstream primer F2 and a downstream primer R2, wherein the upstream primer F2 is 5 '-CATCCTGACCCTAAGTA-3', and the downstream primer R2 is 5 '-CTCGTTGTAGAAGGTGTG-3'; the CIITA gene copy number variation detection method provided by the invention is not limited by individual age and sex, can be used for early breeding of cattle, and can quickly, simply and accurately identify individual copy number variation types at low cost and screen and establish a heat stress tolerant beef cattle group, so that the variety breeding or improvement time is shortened, and the breeding efficiency is improved.

Description

Technical Field

[0001] This invention relates to the field of molecular genetics breeding, and in particular to a method for detecting copy number variation in the CIITA gene of beef cattle and its application. Background Technology

[0002] Heat stress is a series of behavioral and physiological responses in animals caused by high temperatures. Under heat stress, livestock maintain a constant body temperature by increasing respiratory rate, sweating, and skin blood flow; however, prolonged exposure to high temperatures can severely impair bodily functions. Heat stress not only harms animal health and welfare but also reduces production performance, affects meat quality, and causes economic losses. With global warming, its adverse effects on livestock farming are intensifying, making the screening of heat-resistant genes and the development of adaptable beef cattle breeds of great importance.

[0003] Copy number variations (CNVs) are an important form of genomic structural variation, referring to the duplication or deletion of DNA segments, ranging from 1 kb to several Mb. CNVs can affect gene expression and biological function through gene dosage effects, gene breaks, gene fusions, and position effects, thus leading to phenotypic differences among individuals. Existing research has shown that CNVs are closely related to animal growth and development, disease resistance, and environmental adaptability; therefore, using CNVs as molecular markers for genetic modification of target traits has significant application potential.

[0004] Among existing CNV detection methods, real-time quantitative PCR is widely used due to its ease of operation, high sensitivity, and fast detection speed. This method can accurately determine the CNV type of an individual by comparing the relative quantitative results of the target gene and the internal reference gene, combined with –ΔΔCt analysis.

[0005] CIITA, a gene belonging to the NLR transcription factor family, is a key factor regulating MHC-II gene expression and plays an important role in antigen presentation, immune response, and cellular stress regulation. Previous cell experiments have shown that CIITA expression levels can affect the regulation of the pro-inflammatory cytokine IL-6, thereby participating in cell repair and muscle metabolism under stress.

[0006] However, there are currently no publicly available reports on the correlation between CIITA gene copy number variation and heat tolerance and growth traits in beef cattle. Therefore, there is an urgent need to establish a detection method based on CIITA gene CNV for rapid screening of individuals with heat tolerance advantages and excellent growth performance, providing a new tool for molecular marker-assisted selection and breeding of beef cattle. Summary of the Invention

[0007] To address the problems mentioned in the background art, the present invention aims to provide a method for detecting copy number variation (CNV) in the CIITA gene of beef cattle and its application in selective breeding for heat tolerance and growth traits. By identifying the CNV type of the CIITA gene in beef cattle, rapid detection of candidate molecular markers can be achieved at the DNA level, thereby establishing a population with heat tolerance potential and excellent growth performance, and accelerating the molecular breeding process of beef cattle.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for detecting copy number variation in the CIITA gene of beef cattle, comprising the following steps:

[0010] Genomic DNA was extracted from the cattle to be tested. Genotyping was then performed on the copy number variation region of the CIITA gene located at positions 9557000 to 9605500 on chromosome 25 of the bovine reference genome. This copy number variation was caused by multiple copies in this region. The identification was performed using real-time quantitative PCR, specifically including the following steps: using primer pairs P1 and P2 as primers, the copy number variation region of the CIITA gene and a partial fragment of the ACTB gene (used as an internal control) were amplified by real-time quantitative PCR. The copy number variation type of the CIITA gene was then determined based on the quantitative results. The genotype of the CIITA copy number variation region was classified as either a multiple copy type (CN=3) or a normal type (CN=2).

[0011] The primer pair P1 is:

[0012] Upstream primer F1: 5'-CTGGGTGGTGGTGGTGAGTTG-3'

[0013] Downstream primer R1: 5'-GGAGGAGGTGAGAAGAGCAATGG-3';

[0014] The primer pair P2 is:

[0015] Upstream primer F2: 5'-CATCCTGACCCTCAAGTA-3'

[0016] Downstream primer R2: 5'-CTCGTTGTAGAAGGTGTG-3'.

[0017] Preferably, the copy number variation type of the individual CIITA gene is divided into two categories based on the quantitative results of –ΔΔCt: Duplication type, –ΔΔCt>0.5; Normal type, –0.5≤–ΔΔCt≤0.5.

[0018] Preferably, the bovine reference genome is ARS-UCD2.0, and the copy number variation region of the CIITA gene covers exons 2 to 20 of the CIITA gene and the 3'UTR region.

[0019] Secondly, this invention provides the application of the above-mentioned method for detecting copy number variation of the CIITA gene in beef cattle in molecular marker-assisted selection breeding of beef cattle. It screens all individual beef cattle to be tested based on the copy number variation type, establishes a heat stress-tolerant beef cattle population, thereby shortening the breeding or improvement time and improving breeding efficiency.

[0020] Preferably, the normal type is a molecular marker associated with improving the heat tolerance potential and growth traits of cattle.

[0021] Preferably, the structural variation is identified through the following steps:

[0022] Growth performance and body temperature were measured in 202 West China cattle. Results showed that daily weight gain was significantly lower in July-September than in September-October (P<0.01). Rectal temperature under heat stress was significantly negatively correlated with relative daily weight gain (P<0.01, r=–0.378). Plasma metabolomics analysis of West China cattle in both high and low body temperature groups identified 30 heat stress plasma marker metabolites. GWAS analysis of these heat stress plasma marker metabolites and West China cattle CNVs identified 79 candidate genes. Notably, CNVs covering the class II trans-activator (CIITA) gene were associated with multiple carnitine metabolites. In the West China cattle population, the rectal temperature of the normal type (CN=2) individuals under heat stress was significantly lower than that of the multi-copy type (CN=3) individuals (P<0.05). At the same time, the 18-month weight (CN=2) of the normal type of CIITA gene was significantly (P<0.01) and significantly (P<0.05) better than that of the multi-copy type individuals.

[0023] Thirdly, the present invention provides a kit for detecting copy number variation of the CIITA gene in beef cattle. The kit includes: primer pair P1 for amplifying the copy number variation region of the CIITA gene in beef cattle, primer pair P2 for amplifying the internal reference gene ACTB, and real-time fluorescence quantitative PCR reaction system reagents required for amplification.

[0024] The primer pair P1 is:

[0025] Upstream primer F1: 5'-CTGGGTGGTGGTGGTGAGTTG-3'

[0026] Downstream primer R1: 5'-GGAGGAGGTGAGAAGAGCAATGG-3';

[0027] The primer pair P2 is:

[0028] Upstream primer F2: 5'-CATCCTGACCCTCAAGTA-3'

[0029] Downstream primer R2: 5'-CTCGTTGTAGAAGGTGTG-3'.

[0030] The beneficial effects of this invention are: 1. The CIITA gene copy number variation detection method provided by this invention is not limited by individual age and sex, and can be used for early selective breeding of cattle;

[0031] 2. This invention can quickly, simply, and cost-effectively identify the copy number variation type of an individual, screen and establish a heat stress-tolerant beef cattle population, thereby shortening the breeding or improvement time and improving breeding efficiency.

[0032] 3. This invention provides a scientific basis for molecular marker-assisted selection of heat resistance and growth traits in beef cattle to a certain extent. Attached Figure Description

[0033] Figure 1 This is a correlation analysis between rectal temperature and relative daily weight gain in the examples;

[0034] Figure 2 Volcano plots of differential metabolites in plasma metabolomics analysis of the hyperthermic and hypothermic groups in this example;

[0035] Figure 3 The Manhattan plot of GWAS performed in this example uses four acylcarnitine heat stress-labeled metabolites as phenotypes.

[0036] Figure 4 IGV visualization of the CIITA copy number variation regions identified by GWAS in the examples;

[0037] Figure 5 This refers to the coverage of multicopy genome reads in the examples;

[0038] Figure 6 The amplification curves for real-time quantitative PCR (CIITA gene) in the examples are shown.

[0039] Figure 7 The melting curve is shown in the example, which was plotted using real-time quantitative PCR (CIITA gene). Detailed Implementation

[0040] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art are within the protection scope of the present invention.

[0041] Example

[0042] This invention detects copy number variations (CNVs) in the CIITA gene and applies them to molecular breeding of beef cattle. The main steps include: referencing the bovine CIITA gene sequence in the NCBI database, designing primers using the Primer3 website, and employing real-time quantitative PCR to detect the copy number variations of candidate loci (CHR25:9557000–9605500) in the population; statistically analyzing the copy number variation types in relation to heat tolerance and growth development to screen for CNV markers related to heat tolerance and growth development; selecting individuals with excellent heat tolerance and good growth development based on the copy number variation types, and establishing a cattle herd for selective breeding. Detailed explanations are as follows.

[0043] 1. Sample Collection

[0044] This invention uses Huaxi cattle as the test subject, collects and uses blood samples from 202 individual Huaxi cattle, and records phenotypic data such as rectal temperature, weight at 18 months of age and 24 months of age.

[0045] 2. Extraction of genomic DNA from blood samples

[0046] Genomic DNA was extracted using the standard phenol-chloroform method. The extraction steps included cell lysis, protein removal, DNA precipitation, and purification. The obtained DNA was dissolved in TE buffer and stored at –80°C.

[0047] (1) Take 400 μL of whole blood sample and place it in a 1.5 mL centrifuge tube. Add 400 μL of STE buffer, 100 μL of 10% SDS and 40 μL of proteinase K respectively, and mix gently.

[0048] (2) Incubate overnight in a water bath at 56°C with shaking;

[0049] (3) Add 1000 μL of phenol:chloroform:isoamyl alcohol (25:24:1), gently invert and mix several times, and centrifuge at 4℃ and 12000 rpm for 10 minutes.

[0050] (4) Carefully aspirate the upper aqueous phase (containing DNA) into a new centrifuge tube, add an equal volume of chloroform:isoamyl alcohol (24:1), and mix by inverting again;

[0051] (5) Centrifuge at 4℃ and 12000rpm for 10 minutes, and transfer the supernatant again;

[0052] (6) Add an equal volume of pre-cooled isopropanol (-20℃) to the supernatant and gently invert to mix.

[0053] (7) Place it in a -20℃ refrigerator and let it stand for 30 minutes to 1 hour;

[0054] (8) Centrifuge at 4℃ and 12,000 rpm for 10 minutes, and discard the supernatant;

[0055] (9) Add 1 mL of 75% pre-cooled ethanol and mix gently to wash the DNA precipitate. After centrifugation again, discard the ethanol and air dry.

[0056] (10) Add 25 μL of LTE buffer to dissolve the DNA precipitate. After complete dissolution, store the DNA at -80°C.

[0057] 3. Design of target gene and internal reference gene-specific primers

[0058] Using the bovine CIITA gene sequence published in the NCBI database as a reference sequence, sequences of candidate regions for copy number variation in the CIITA gene (target gene) selected during resequencing (CHR25:9557000–9605500) were identified. Primers (primer pair P1) were designed using the Primer3 website to amplify a 135 bp sequence near the center of this region. Simultaneously, using the bovine ACTB gene sequence published in NCBI as a reference sequence, primers (primer pair P2) were designed using the same method to amplify a 91 bp sequence in the internal reference gene (ACTB gene). Primer pair sequence information is shown in Table 1.

[0059] Table 1 Primer information for real-time quantitative PCR

[0060]

[0061] Note: F1 or F2 is the upstream primer, and R1 or R2 is the downstream primer.

[0062] 4. Real-time quantitative PCR

[0063] The real-time quantitative PCR reaction system is shown in Table 2, with a total volume of 15 μL.

[0064] Table 2 Real-time quantitative PCR reaction system

[0065]

[0066] The amplification reaction procedure for real-time quantitative PCR is as follows:

[0067] (1) Pre-denature at 95℃ for 30s, then proceed with the amplification reaction as described in (2);

[0068] (2) 95℃ denaturation for 5s, 60℃ annealing for 30s, for a total of 40 cycles.

[0069] See Figure 6 and Figure 7 The primers were determined to be suitable for real-time quantitative PCR analysis by plotting amplification curves and melting peaks.

[0070] 5. CNV Type Determination

[0071] Calculate sample-ΔCt based on the qPCR results of the target gene and internal reference gene:

[0072] ΔΔCt=ΔCt (实验组) –ΔCt (参照组)

[0073] ΔCt (实验组) =Ct (实验组目的基因) –Ct (实验组内参基因) ,ΔCt (参照组) =Ct (参照组目的基因) –Ct (参照组内参基因 )

[0074] In this study, the experimental group consists of individual samples to be tested for copy number variations, while the control group consists of individual samples known to have no copy number variations. The control group, selected in the resequencing experiment, can be the Huaxi cattle individuals. Ct, or Cyclethreshold, refers to the number of amplification cycles required for the fluorescence signal of the amplification product to reach a set threshold during PCR amplification.

[0075] The ΔΔCt of each individual sample is calculated using the above formula, and the CNV type of the individual is identified according to the following criteria: –ΔΔCt>0.5 indicates multiple copy type (CN=3); –0.5≤–ΔΔCt≤0.5 indicates normal type (CN=2).

[0076] 6. Statistical analysis of CNV genotyping of the CIITA gene and its effects on heat resistance and growth traits

[0077] Physiological indicators of heat stress: rectal temperature;

[0078] Growth traits: Body weight at 18 months and 24 months of age.

[0079] CNV types: Normal and Duplication.

[0080] Statistical analysis: In data processing, unpaired t-tests were performed on traits such as copy number variation type, rectal temperature, body weight at 18 months, and body weight at 24 months. The statistical analysis results are shown in Table 3.

[0081] Table 3. Association analysis between CIITA gene copy number variation and heat stress physiological indicators and growth traits in West China cattle.

[0082]

[0083] Note: CN represents the copy number; * P<0.05, ** P < 0.01; n represents the number of individual samples with the same copy number.

[0084] Statistical analysis results showed that the copy number variation sites of the CIITA gene were significantly correlated with traits such as rectal temperature, body weight at 18 months of age, and body weight at 24 months of age in West China cattle. Therefore, the copy number variation site of the CIITA gene (CHR25:9557000–9605500) can serve as a candidate molecular genetic marker (CNV marker) site for effectively improving the heat tolerance and growth traits of beef cattle, thereby accelerating the selective breeding process.

[0085] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting copy number variation in the CIITA gene of beef cattle, characterized in that, Includes the following steps: Using bovine genomic DNA as a template and primer pair P1 and primer pair P2 as primers, the copy number variation region of the CIITA gene and a partial fragment of the ACTB gene, which served as an internal control, were amplified by real-time quantitative PCR. The copy number variation type of the CIITA gene was then determined based on the quantitative results. The primer pair P1 is: Upstream primer F1: 5'-CTGGGTGGTGGTGGTGAGTTG-3' Downstream primer R1: 5'-GGAGGAGGTGAGAAGAGCAATGG-3'; The primer pair P2 is: Upstream primer F2: 5'-CATCCTGACCCTCAAGTA-3' Downstream primer R2: 5'-CTCGTTGTAGAAGGTGTG-3'.

2. The method for detecting copy number variation in the CIITA gene of beef cattle according to claim 1, characterized in that, The copy number variation region of the CIITA gene is located in the candidate region of the bovine CIITA gene CHR25: 9557000–9605500.

3. The method for detecting copy number variation in the CIITA gene of beef cattle according to claim 1, characterized in that, The copy number variation type is divided into two categories based on –ΔΔCt: multicopy type, –ΔΔCt>0.5; normal type, –0.5≤–ΔΔCt≤0.

5.

4. The method for detecting copy number variation in the CIITA gene of beef cattle according to claim 1, characterized in that, The amplification reaction system of the real-time quantitative PCR includes 1 μL of template DNA (10-50 ng / μL) and 0.5 μL each of the upstream and downstream primers corresponding to primer pair P1 or primer pair P2 (10 μmol / L).

5. The method for detecting copy number variation in the CIITA gene of beef cattle according to claim 1, characterized in that, The real-time quantitative PCR reaction procedure includes the following steps: Pre-denaturation at 95℃ for 30 seconds; denaturation at 95℃ for 5 seconds; annealing at 60℃ for 30 seconds, for a total of 40 cycles.

6. The method for detecting copy number variation in the CIITA gene of beef cattle according to claim 1, characterized in that, The PCR product fragment size based on primer pair P1 is 135bp, and the PCR product fragment size based on primer pair P2 is 91bp.

7. The application of the method according to any one of claims 1-6 in molecular marker-assisted selection breeding of beef cattle.

8. The application according to claim 7, characterized in that, Individuals with normal copy number are superior to those with multiple copy number in terms of heat resistance and growth traits.

9. The application according to claim 8, characterized in that, The heat resistance is defined as rectal temperature, and the growth traits are body weight at 18 months and body weight at 24 months.

10. A kit for detecting copy number variation in the CIITA gene of beef cattle, characterized in that, include: Primer pair P1 for amplifying the copy number variation region of the CIITA gene in beef cattle, primer pair P2 for amplifying the internal reference gene ACTB, and reagents for the real-time quantitative PCR reaction system required for amplification. The primer pair P1 is: Upstream primer F1: 5'-CTGGGTGGTGGTGGTGAGTTG-3' Downstream primer R1: 5'-GGAGGAGGTGAGAAGAGCAATGG-3'; The primer pair P2 is: Upstream primer F2: 5'-CATCCTGACCCTCAAGTA-3' Downstream primer R2: 5'-CTCGTTGTAGAAGGTGTG-3'.

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