Application of HTATSF1 gene in regulation and control of cattle muscle growth and development and breeding

Abstract

According to the application of the HTATSF1 gene in regulation and control of cattle muscle growth and development and breeding, the influence of the HTATSF1 gene on muscle growth and development and the correlation between HTATSF1-CNV and cattle growth traits are verified, a reliable molecular marker is provided for cattle seed selection and breeding, and help is provided for cattle variety improvement. A foundation is laid for association between copy number variation and growth traits of the cattle HTATSF1 gene, cattle molecular marker-assisted selection breeding work can be accelerated, the method is simple, rapid and convenient to popularize and apply, and a complete solution from fundamental research to industrial application is provided for improvement of cattle germplasm resources.

Description

Technical Field

[0001] This invention belongs to the field of molecular genetics research technology, specifically the application of the HTATSF1 gene in regulating muscle growth and development in cattle and in breeding. Background Technology

[0002] With technological advancements and improved living standards, beef is playing an increasingly important role in the food structure; however, the country still heavily relies on imports. Therefore, efforts are underway to improve both the quality and quantity of beef to drive the industrialization of the cattle industry and meet consumer demand. Currently, molecular marker-assisted selection (MAS) is primarily used for livestock breed improvement.

[0003] Current marker-assisted selection focuses on single nucleotide polymorphisms (SNPs) and insertions / deletions (InDels). In recent years, copy number variations (CNVs) have received considerable attention. Copy number variations refer to the insertion and deletion of 50 bp genomic sequences between two individuals of the same species, providing complementary genomic structural information to SNP data. Compared to SNPs, CNVs affect higher genomic sequences and have a greater impact. These include changes in gene structure and dosage, changes in gene regulation, and exposure to recessive alleles. DNA copy number variations are associated with specific chromosomal rearrangements and genomic diseases and are ubiquitous in mammalian genomes.

[0004] Currently, there are four main methods for verifying copy number variations (CNVs): CNVplex is a new technology for detecting CNVs, improved upon multiplex ligation probe amplification technology. FISH uses a fluorescence detection system to perform qualitative and quantitative analysis of target DNA molecules. ddPCR determines the initial copy number of the target DNA molecule based on the Poisson distribution principle and the number and proportion of positive droplets. qPCR is a technique that uses changes in fluorescence signal to monitor the amount of product in each cycle of PCR amplification in real time. It can perform large-scale CNV detection daily, making it a commonly used verification method in laboratories.

[0005] HTATSF1 is an important regulatory gene that encodes a heterogeneous ribonucleoprotein, a unique protein molecule located in the cell nucleus. Proteins in the nucleus participate in various biochemical processes, including RNA processing, gene stabilization, nucleotide transport, and the regulation of DNA expression. Research has revealed that the HTATSF1 gene also plays a crucial role in regulating RNA metabolism, making it a current research hotspot.

[0006] However, to date, the use of the HTATSF1 gene CNV marker in the auxiliary detection of growth traits has not been observed. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides the application of the HTATSF1 gene in regulating muscle growth and development in cattle and in breeding, verifies the effect of the HTATSF1 gene on muscle growth and development and the correlation between HTATSF1-CNV and growth traits in cattle, and provides a reliable molecular marker for cattle selection and breeding, in order to help improve the breed of cattle.

[0008] To achieve the above objectives, this invention provides the application of the HTATSF1 gene in regulating muscle growth and development in cattle.

[0009] Furthermore, the HTATSF1 gene can promote the differentiation of bovine myoblasts.

[0010] This invention also provides the application of HTATSF1 gene copy number variation in molecular breeding of cattle.

[0011] Furthermore, the sequence of the copy number variation region of the HTATSF1 gene is ChrX:20216801-20218800.

[0012] Furthermore, the specific breed of cattle mentioned is the Xia'nan cattle, and the copy number variation of the multi-copy HTATSF1 gene is used as a molecular marker for early selection of growth traits in Xia'nan cattle.

[0013] Furthermore, the growth traits are body height, cross-shaped height, body oblique length, and chest circumference.

[0014] Furthermore, the specific breed of cattle mentioned is the Qaidam Yellow Cattle, and the copy number variation of the normal type HTATSF1 gene is used as a molecular marker for early selection of growth traits in Qaidam Yellow Cattle.

[0015] Furthermore, the growth trait is body height.

[0016] Furthermore, using the genomic DNA of the cattle to be tested as a template, the copy number variation region of the HTATSF1 gene and a partial fragment of the BTF3 gene as an internal control were amplified by real-time quantitative PCR. Then, the copy number variation type of the cattle ZNF133 gene was identified based on the quantitative results.

[0017] Furthermore, the amplification primer pair for the copy number variation region of the HTATSF1 gene is as follows:

[0018] Upstream primer F1: 5'-CTTTTTCTCCAGCTTTTCTGCCC-3'

[0019] Downstream primer R1: 5'-CATTTCAGTTGCGGCTGCTT-3'.

[0020] Furthermore, the fragment size of the copy number variation region product of the HTATSF1 gene amplified by real-time quantitative PCR was 172 bp, and the fragment size of the partial fragment product of the BTF3 gene amplified by real-time quantitative PCR was 166 bp.

[0021] Furthermore, the reaction procedure used for real-time quantitative PCR is as follows: (1) pre-deformation at 95℃ for 30s; (2) denaturation at 95℃ for 10s, annealing at 60℃ for 30s, treatment at 72℃ for 30s, for a total of 40 cycles.

[0022] Furthermore, the amplification system used for real-time quantitative PCR includes: 1 μL of 10 ng / μL template DNA, and 0.2 μL each of the upstream and downstream primers corresponding to primer pair P1 or primer pair P2 (10 μmol / L). PreMix Ex TaqTMII 5μL and ddH2O3.6μL.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] In this invention, the HTATSF1 gene plays a clear regulatory role in the muscle growth and development of cattle, inhibiting the proliferation of bovine myoblasts while promoting their differentiation. This characteristic reveals the crucial role of genes in muscle development, providing a new perspective and entry point for a deeper understanding of the molecular mechanisms of bovine muscle growth. By studying how the HTATSF1 gene regulates the proliferation and differentiation of myoblasts, researchers can gain a more comprehensive understanding of the intrinsic laws governing bovine muscle growth, laying a solid theoretical foundation for subsequent breeding work. Secondly, based on the influence of the HTATSF1 gene on the proliferation and differentiation of bovine myoblasts, potential molecular marker reference sites can be provided for the selection and breeding of beef cattle, as well as insights into regulating the growth and development of myoblasts. For example, by regulating the expression level of the HTATSF1 gene, the growth rate and quality of bovine muscle can be affected to a certain extent, thereby breeding bovine breeds with better muscle growth and meat quality, which is of great significance for improving the economic benefits of cattle farming.

[0025] Using HTATSF1 gene copy number variation (CNV) as a molecular marker in the molecular breeding of cattle is highly specific, allowing for precise differentiation of different CNV types. For example, in Xia'nan and Qaidam cattle breeds, multi-copy or normal CNVs can serve as early selection criteria for growth traits, making breeding more targeted, avoiding indiscriminate selection, and improving efficiency. HTATSF1 CNV provides a rapid and effective approach for molecular breeding of cattle. By analyzing the correlation between CNV and growth traits, individuals with superior traits can be screened, accelerating the breeding process, shortening the breeding cycle, and reducing breeding costs. Furthermore, different copy number types are suitable as markers for different breeds, which helps in selecting superior populations and improving germplasm resources. Attached Figure Description

[0026] Figure 1 Tissue expression profile of the HTATSF1 gene;

[0027] Figure 2 For si-HTATSF1 fragment interference efficiency;

[0028] Figure 3 The expression of proliferation-related marker genes at the mRNA and protein levels is shown in Figure 1. (A) Expression of proliferation marker genes at the mRNA level; (B) Expression of proliferation marker genes at the protein level.

[0029] Figure 4 This shows the expression of differentiation marker genes at the mRNA level.

[0030] Figure 5 To identify regions of copy number variation in the HTATSF1 gene and design primer locations;

[0031] Figure 6 The copy number distribution of the HTATSF1 gene in seven cattle breeds;

[0032] Figure 7 The copy number ratio of the HTATSF1 gene in seven cattle breeds. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0034] The HTATSF1 gene is involved in rRNA and protein synthesis, and muscle is a rich protein storage reservoir. This invention focuses on the HTATSF1 gene. First, siRNA was used to reduce the expression of the HTATSF1 gene. si-HTATSF1 was transfected into bovine myoblasts, and the expression of proliferation and differentiation marker genes was detected to verify the regulatory role of the HTATSF1 gene in bovine myoblast development. Second, based on whole-genome resequencing results, a CNV region was found in this gene. Real-time quantitative PCR was used to verify the distribution of HTATSF1-CNV in seven different local cattle breeds and its association with growth traits, exploring the effect of HTATSF1-CNV on cattle growth, thereby selecting potential genetic markers for marker-assisted breeding of cattle. Specifically:

[0035] I. Verify the regulatory role of the HTATSF1 gene in bovine myoblast development.

[0036] 1. Materials and Methods

[0037] 1.1 Test Materials

[0038] Bovine primary myoblasts were isolated and cultured; the RNA samples used for tissue expression of the HTATSF1 gene were from six tissues of three fetal cattle: heart, liver, spleen, lung, kidney, and muscle.

[0039] 1.2 Test Reagents

[0040] Trizol reagent (main component is phenol) (Sangon Biotech Co., Ltd.), real-time PCR mix, reverse transcription kit, and protein marker were purchased from Genstar. Other items included loading buffer, electrophoresis buffer, skim milk powder, nitrocellulose membrane, and antibodies.

[0041] 1.3 Synthesis of interference fragments in the CDS region of the HTATSF1 gene

[0042] The interference fragment of the HTATSF1 gene CDS region used in this invention was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0043] 1.4 Isolation and Culture of Primary Bovine Myocytes

[0044] Bovine primary myoblasts were isolated and cultured using collagenase digestion. Culture conditions: suitable humidity, 37°C, 5% carbon dioxide, aseptic culture in proliferation medium.

[0045] 1.5 Cell resuscitation

[0046] Activating frozen cells requires rapid thawing to prevent cell death due to ice crystal damage. After thawing, a suitable environment and sufficient nutrition are needed to restore normal cell activity.

[0047] 1.6 Cell Culture and Cell Transfection

[0048] Under normal circumstances, the culture medium should be changed the day after successful cell resuscitation. When the cell density reaches 60%-80%, transfection experiments can be performed using liposomes. The bovine HTATSF1 gene interference fragment and the liposome complex are transfected into bovine primary myoblasts using liposomes. A control group without the interference fragment is also set up. Each treatment is performed in triplicate to improve the reliability of the experimental results.

[0049] 1.7 Cell proliferation and differentiation culture after HTATSF1 gene interference

[0050] After transfection, cells were cultured at 37°C in a 5% CO2 incubator for 24 hours, after which they were harvested for further analysis. Following the 24-hour culture, bovine primary myoblasts were induced to differentiate by culturing them in differentiation medium containing 2% horse serum. Differentiation was induced through starvation for 5 days, with the medium changed every other day. Cell morphology was then observed under a microscope. Once myotubes appeared, the differentiated myoblasts were harvested for subsequent experiments.

[0051] 1.8. RNA extraction and cDNA synthesis

[0052] cellular RNA was extracted using the Trizol method. cDNA synthesis was performed using a reverse transcription kit; specific reaction systems are shown in Tables 1 and 2.

[0053] Table 1 shows the reaction system for removing genomic DNA.

[0054]

[0055] Reaction conditions: 42℃, 2 min; store at 4℃.

[0056] Table 2 shows the reverse transcription reaction.

[0057]

[0058] Reaction conditions: 37℃, 15 min; 85℃, 5 sec; 4℃, store.

[0059] Dilute the cDNA and store at -20°C.

[0060] 1.9 RT-qPCR detection of marker genes for cell proliferation and differentiation

[0061] RT-qPCR was used to detect the expression of myoblast proliferation and differentiation marker genes at the mRNA level, and to investigate the role of the HTATSF1 gene in myoblast proliferation and differentiation. β-actin was used as an internal control, and the expression was calculated using a 2... -ΔΔCt The RT-qPCR reaction system is shown in Table 3, the reaction procedure is shown in Table 4, and the primers are shown in Table 5.

[0062] Table 3 shows the RT-qPCR reaction system.

[0063]

[0064]

[0065] Table 4 shows the RT-qPCR reaction procedure.

[0066]

[0067] Table 5 lists the primers for the relevant genes.

[0068]

[0069] 1.10 Protein Extraction and Western Blot Assay

[0070] Total protein was extracted from cells for Western blotting assay.

[0071] 1.11 Statistical Analysis

[0072] SPSS 20.0 was used for statistical analysis, and the results were plotted using GraphPad Prism 8. The data were analyzed for significance using t-tests. *P<0.05 indicates a significant difference, and **P<0.01 indicates an extremely significant difference.

[0073] 2. Test Results

[0074] 2.1, such as Figure 1 The figure shows the expression profile of HATSF1 in different tissues of cattle. This invention uses real-time quantitative PCR to detect the expression of the HATSF1 gene in different tissues (heart, liver, spleen, lung, kidney, and muscle) of Qinchuan cattle fetuses. The results show that the tissue with the highest expression level of HATSF1 gene is muscle, followed by heart. This expression profile provides a research basis for further study on the function of HATSF1 gene.

[0075] 2.2. Interference efficiency of the HTATSF1 gene

[0076] To investigate the function of the HTATSF1 gene, we transfected bovine myoblasts with siRNA and assessed the interference efficiency. Using cDNA derived from the reverse transcription of RNA extracted from the cells as a template, we performed real-time quantitative PCR. Figure 2 The interference efficiency of the si-HTATSF1 fragment is shown. The results indicate that the interference fragment can significantly reduce the expression of the HTATSF1 gene and can be used for subsequent experimental treatments.

[0077] 2.3 Effects of HTATSF1 gene interference on myoblast proliferation

[0078] The expression of this gene was detected at the RNA and protein levels using real-time quantitative PCR and Western blotting, respectively. The real-time quantitative results are as follows: Figure 3 As shown in Figure A, the results indicate that HTATSF1 gene interference increased the expression of PCNA, CDK2, Cyclin D, and Cyclin E, with Cyclin E expression showing a highly significant increase; the results of Western blotting experiments are as follows. Figure 3 Figure B shows that the expression level of Cyclin E increased after interfering with the HTATSF1 gene, indicating that the HTATSF1 gene has an inhibitory effect on myoblast proliferation.

[0079] 2.4 Effects of HTATSF1 gene interference on myoblast differentiation

[0080] The expression levels of differentiation-related marker genes MyoD, MyoG, and MyHC at the mRNA level were detected using real-time quantitative PCR. The results are as follows: Figure 4 As shown, the expression levels of differentiation marker genes MyoD, MyoG, and MyHC decreased after HTATSF1 gene interference, with MyHC expression decreasing significantly, indicating that the HTATSF1 gene promotes myoblast differentiation.

[0081] 3. Analysis of Experimental Results

[0082] The experimental results show that the HTATSF1 gene is expressed at a certain level in the muscle tissue of beef cattle, which may play a regulatory role in muscle growth and development; and the HTATSF1 gene inhibits the proliferation of bovine myoblasts and promotes their differentiation.

[0083] II. Investigating the effects of HTATSF1-CNV on the growth of cattle

[0084] Based on our previous sequencing of the cattle genome, we discovered a CNV region in the HTATSF1 gene, with the sequence ChrX: 20216801-20218800. To investigate its role in the growth and development of cattle, this invention analyzed the copy number distribution of the HTATSF1 gene in seven cattle breeds and performed correlation analysis with growth traits such as body length, body height, and cross height, aiming to provide a reference for molecular breeding. The specific steps are as follows:

[0085] 1. Materials and Methods

[0086] 1.1 Experimental Animals

[0087] This invention uses Xia'nan cattle, Qinchuan cattle, Pinan cattle, Jiaxian red cattle, Guyuan yellow cattle, Ji'an yellow cattle, and Qaidam yellow cattle as research subjects, and collects blood samples from each. All individuals are adults, and their growth trait data, such as body height, cross-shaped height, body length, chest circumference, and cannon bone circumference, are recorded for subsequent correlation analysis.

[0088] 1.2 Test Methods

[0089] (1) Sample collection

[0090] Blood samples were collected from the jugular vein of cattle using the ACD anticoagulation method. The samples were stored in ice packs and transported back to be frozen in an ultra-low temperature freezer at -80°C.

[0091] (2) Extraction of bovine whole blood genomic DNA

[0092] Blood DNA was extracted using the phenol-chloroform method.

[0093] (3) Primer design

[0094] Based on the bovine HTATSF1 gene sequence (sequence number: AC_000187.1) published in the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), primers for detecting the DNA copy number of the HTATSF1 gene in the bovine ChrX: 20216801-20218800 region were designed using Primer 5.0 software (e.g., Figure 5 (as shown in the figure), and the BTF3 gene, which is known to have no copy number variation, was used as an internal reference. Primer information is shown in Table 6. All primers were synthesized by Shanghai Sangon Biotech.

[0095] Table 6 shows the primer information for the HTATSF1 and BTF3 genes.

[0096]

[0097] (4) RT-qPCR detection

[0098] Using genomic DNA as a template, copy number variation was quantitatively detected by RT-qPCR. Since the BTF3 gene exhibits no copy number variation, it was used as a control. Each sample was tested in triplicate to ensure accurate and valid data.

[0099] The RT-qPCR reaction system and procedure are the same as described in step 1.9.

[0100] (5) Data Processing and Analysis

[0101] Experimental results were obtained using 2 -△△Ct The calculation is performed using the following method:

[0102] ΔΔCt=ΔCt (实验组) -ΔCt (参照组)

[0103] ΔCt (实验组) =Ct (实验组目的基因) -Ct (实验组内参基因)

[0104] ΔCt (参照组) =Ct (参照组目的基因) -Ct (参照组内参基因)

[0105] In the formula, the experimental group consists of individual samples to be tested for copy number variations. The control group consists of individual samples known to have no copy number variations, which can be the cattle individuals selected as the control group in the resequencing experiment. C t Cyclethreshold, or CycleThreshold, is the number of amplification cycles required for the fluorescence signal of the amplification product to reach a set threshold during PCR amplification.

[0106] Based on -ΔΔCt, quantitative results are divided into three categories: Duplication (-ΔΔCt > 0.5), Deletion (-ΔΔCt < -0.5), and Normal (-0.5 ≤ -ΔΔCt ≤ 0.5).

[0107] Using SPSS 20.0, a software for biostatistical data analysis, we conducted an association analysis between different copy number variation types (deletion type, normal type, and multiple copy type) and growth traits of cattle.

[0108] During data processing using SPSS, considering the multiple factors affecting body size traits, such as environmental effects, age, breed, genetic effects, and their interactions, a fixed model was used for analysis, while also being simplified based on actual circumstances. The simplified model is as follows:

[0109] Yijk=μ+Bj+CNVk+eijk

[0110] Where Yijk represents the numerical record of individual phenotypic traits; μ represents the mean of each trait in the population; CNVk represents the influence of the CNV type of the HTATSF1 gene in the k-th individual; and eijk represents random error. One-way LSD ANOVA was used, and the results were plotted using GraphPad Prism8. *P<0.05 indicates significant difference, and **P<0.01 indicates extremely significant difference.

[0111] 2. Experimental Results

[0112] 2.1 Distribution of Copy Number Types in Seven Cattle Breeds

[0113] In RT-qPCR results data ( Figure 6 , Figure 7 In the study, the Ct values ​​of the HTATSF1 and BTF3 genes in the sample were detected, and the relative copy number of the HTATSF1 gene was calculated, as shown in Table 7.

[0114] Table 7 shows the distribution of copy number types in the seven cattle breeds.

[0115]

[0116] 2.2 Association Analysis of HTATS F1 Copy Number Variation and Growth Traits in Yellow Cattle

[0117] (1) Association analysis between HTATSF1-CNV and growth traits of Xia'nan cattle

[0118] The association analysis results of different copy numbers of the HTATSF1 gene with growth traits in adult Xia'nan cattle are shown in Table 8. The association analysis showed that the multiple copy (duplication) variant of the HTATSF1 gene had a certain promoting effect on the growth traits of Xia'nan cattle, with a significant effect on body height (P < 0.05) and a highly significant effect on cross-shaped height, body length, and chest circumference (P < 0.01). Among them, the growth trait data of individuals with CN = 4 and CN ≥ 5 were significantly increased compared with CN = 3.

[0119] Table 8. Association analysis of Xia'nan cattle HTATSF1-CNV with growth traits

[0120]

[0121] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05), while identical lowercase letters indicate no significant differences; different uppercase letters in the same line indicate extremely significant differences (P < 0.01), while identical uppercase letters indicate no significant differences. (The same applies below, omitted.)

[0122] 2) Association analysis between HTATSF1-CNV and growth traits of Qinchuan cattle

[0123] The association analysis between HTATSF1-CNV and the growth traits of Qinchuan cattle is shown in Table 9. There was no significant correlation between the two, but the deletion copy number variation had a certain promoting effect.

[0124] Table 8. Association analysis of Qin Chuan cattle HTATSF1-CNV with growth traits

[0125]

[0126]

[0127] (3) Association analysis between HTATSF1-CNV and growth traits of Pinan cattle

[0128] As shown in Table 9, there was no significant correlation between this locus and growth traits in Pinan cattle, but the body size data of normal individuals were better than those of the deletion and multiple copy types.

[0129] Table 9. Association Analysis of HTATS F1-CNV and Growth Traits in Pinan Cattle

[0130]

[0131] (4) Correlation analysis between HTATSF1-CNV and growth traits of Guyuan Yellow Cattle

[0132] As shown in Table 10, this locus was not significantly associated with the growth traits of Guyuan cattle, but the height of the crosshead, body length, and chest circumference were all higher in the missing type than in the normal type and the multicopy type.

[0133] Table 10 shows the correlation analysis between Guyuan Yellow Cattle HTATSF1-CNV and growth traits.

[0134]

[0135] (5) Correlation analysis between HTATSF1-CNV and growth traits of Jiaxian Red Cattle

[0136] The association analysis between HTATSF1-CNV and growth traits of Jiaxian Red Cattle is shown in Table 11. There was no significant correlation, but some body size traits, such as cross height, sac height, and chest depth, showed a trend of missing and normal types being higher than the multicopy type.

[0137] Table 11 shows the correlation analysis between Jiaxian Red Cattle HTATSF1-CNV and growth traits.

[0138]

[0139]

[0140] (6) Correlation analysis between HTATSF1-CNV and growth traits of Ji'an Yellow Cattle

[0141] As shown in Table 12, there was no significant correlation between HTATSF1-CNV and the growth traits of Ji'an Yellow Cattle. Body height, body length, cross height, and chest circumference showed that the missing type was larger than the normal type, while cannon bone circumference showed that the normal type was slightly larger than the missing type.

[0142] Table 12 shows the correlation analysis between HTATSF1-CNV and growth traits of Ji'an Yellow Cattle.

[0143]

[0144] (7) Association analysis between HTATSF1-CNV and growth traits of Qaidam cattle

[0145] As shown in Table 13, there is a significant correlation between HTATSF1-CNV and the body height trait of Qaidam cattle. The normal type is significantly higher than the multicopy type. In other traits, the normal type is more advantageous than the multicopy type.

[0146] Table 13 shows the association analysis between HTATSF1-CNV and growth traits in Qaidam cattle.

[0147]

[0148]

[0149] Note: If the same letter is on the average value, the difference is not significant (P>0.05); if the letters on the average value are different, the difference is significant (P<0.05). * P<0.05.

[0150] 3. Analysis of Experimental Results

[0151] This invention analyzed the correlation between CNVs and growth traits in various cattle breeds. Data showed that CNVs significantly affected body height, body length, cross hump height, and chest girth. It is speculated that this is because the CNV region of this gene encodes a protein that binds to RNA and influences the bovine phenotype. Using copy number variations in the HTATSF1 gene as molecular markers, CNVs highly correlated with target traits can be selected. CNV genotyping allows for the screening of animals exhibiting superior phenotypic performance, thereby accelerating the breeding process, shortening the breeding cycle, and providing a more effective method for breeding work.

[0152] The distribution of HTATSF1 gene copy number variation varied significantly among the seven cattle breeds, with a large proportion of individuals exhibiting gene deletion or multiple copy types. Furthermore, the HTATSF1 gene significantly impacted the growth traits of Xia'nan and Qaidam cattle. It had a significant effect on body height in Xia'nan cattle, and a highly significant effect on body length, cross-shaped height, and chest girth, with the multiple copy type showing a clear advantage. In Qaidam cattle, it significantly affected body height, with the normal type being superior to the multiple copy type. Therefore, partial copy number variations of the HTATSF1 gene can serve as molecular markers for early selection of growth traits in cattle, accelerating the breeding of superior cattle populations and the improvement of Chinese cattle germplasm resources.

[0153] In summary, the presence of a CNV region in the HTATSF1 gene of Chinese Yellow Cattle has been detected. Studies have shown that copy number variation of this gene plays an important role in the growth and development of cattle. This provides a preliminary reference for future research on the role of CNV in the HTATSF1 gene in larger and different Chinese Yellow Cattle populations, and is expected to be used in molecular marker-assisted selection breeding of cattle, thus realizing the great potential of CNV.

[0154] This invention provides the application of the HTATSF1 gene in regulating muscle growth and development in cattle and in breeding. It verifies the influence of the HTATSF1 gene on muscle growth and development and the correlation between HTATSF1-CNV and growth traits in cattle, providing a reliable molecular marker for cattle selection and breeding, aiming to assist in cattle breed improvement. This invention lays the foundation for the association between copy number variation of the HTATSF1 gene and growth traits in cattle, helping to accelerate marker-assisted selection breeding work in cattle. This method is simple, rapid, and easy to promote and apply, providing a complete solution from basic research to industrial application for cattle germplasm resource improvement.

[0155] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the HTATSF1 gene in regulating muscle growth and development in cattle.

2. The application according to claim 1, characterized in that, The HTATSF1 gene can promote the differentiation of bovine myoblasts.

3. Application of HTATSF1 gene copy number variation in molecular breeding of cattle.

4. The application according to claim 3, characterized in that, The sequence of the copy number variation region of the HTATSF1 gene is ChrX:20216801-20218800.

5. The application according to claim 3, characterized in that, The specific breed of cattle mentioned is the Xia'nan cattle, and the copy number variation of the multi-copy HTATSF1 gene is used as a molecular marker for early selection of growth traits in Xia'nan cattle.

6. The application according to claim 5, characterized in that, The growth traits are body height, cross-shaped height, body length, and chest circumference.

7. The application according to claim 3, characterized in that, The specific breed of cattle mentioned is the Qaidam Yellow Cattle, and the copy number variation of the normal type HTATSF1 gene is used as a molecular marker for early selection of growth traits in Qaidam Yellow Cattle.

8. The application according to claim 7, characterized in that, The growth trait is body height.

9. The application according to claim 3, characterized in that, Using the genomic DNA of the cattle to be tested as a template, the copy number variation region of the HTATSF1 gene and a partial fragment of the BTF3 gene, which served as an internal control, were amplified by real-time quantitative PCR. The copy number variation type of the cattle ZNF133 gene was then identified based on the quantitative results.

10. The application according to claim 9, characterized in that, The primer pair for amplifying the copy number variation region of the HTATSF1 gene is as follows: Upstream primer F1: 5'-CTTTTTCTCCAGCTTTTCTGCCC-3' Downstream primer R1: 5'-CATTTCAGTTGCGGCTGCTT-3'.

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