Application of method for detecting cattle TEX10 gene copy number variation in cattle molecular marker-assisted selective breeding and kit

Abstract

The invention discloses application of a method for detecting cattle TEX10 gene copy number variation in cattle molecular marker-assisted selective breeding and a kit, and belongs to the technical field of molecular biological detection. Based on a fluorescent quantitative PCR (Polymerase Chain Reaction) technology, cattle blood sample whole genome DNA (Deoxyribose Nucleic Acid) is used as a template, a copy number variation region of cattle TEX10 genes is amplified, cattle BTF3 genes are amplified to be used as contrast, then a 2 * 2-Ct method is used for calculating copy number variation types of individuals, correlation analysis is carried out according to TEX10 gene copy number variation and growth traits, and the cattle TEX10 gene copy number variation and growth traits are identified. The method lays a foundation for establishing the association between the cattle TEX10 gene copy number and the growth traits, is beneficial to accelerating the molecular marker-assisted selective breeding work of cattle, so that an excellent cattle population is established, and has the characteristics of simplicity, rapidness and convenience in popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetics research, and specifically relates to a method for detecting yellow cattle TEX10 Application of gene copy number variation method in molecular marker-assisted selection breeding of cattle and kit. Background Art

[0002] Heredity, the cornerstone of species stability, ensures that genetic information from parents is accurately and correctly transmitted to offspring, maintaining the species' fundamental characteristics and stability. Variation, on the other hand, is the core driving force of biological evolution, providing unlimited possibilities for species to adapt to complex and changing environments and prompting organisms to continuously evolve and differentiate to adapt to different ecological niches. Copy number variations (CNVs) are a common and widespread structural variation found in the genome. They refer to complex structural rearrangements of chromosomes compared to the reference genome, resulting in deletions or duplications of DNA segments typically ranging from 50 bases to megabases in length, causing individual genomes to differ from the normal diploid state. This form of variation is widely distributed throughout the genome, and its presence not only enriches the genetic diversity of the genome but can also have profound effects on gene function and expression, thereby playing a role in multiple aspects of biological growth and development, physiological function, and disease susceptibility. Currently, array-based comparative genome hybridization (aCGH) technology, SNP chip technology, and whole-genome sequencing (WGS) technology based on next-generation sequencing (NGS) have become the main means of detecting CNV.

[0003] In recent years, many studies have used qPCR technology to verify different CNVs in various livestock and poultry, and found that these genetic variations can affect the phenotype of animals. For example, studies have confirmed that mouse skeletal muscle growth and fiber composition are regulated by transcription factors. TEX10 There are also correlation analysis showing that APOL3 The CNV of the gene is associated with the body size of Yunling cattle and Pinan cattle. This shows that the qPCR method for verifying CNV is desirable and widely used. At the same time, other experiments have shown that KLF6 Gene expression affects the relevant growth traits of the cattle population through the negative correlation between DNA copy number and gene expression level, which shows that the gain and loss of gene copies in the genome are related to gene dosage, and CNV affects gene expression, which in turn can affect the growth and development of livestock.

[0004] TEX10As key genes encoding transcription factor proteins, the TEX gene family belongs to the TEX gene family. The TEX gene family plays a vital role in cell biology and is widely involved in multiple core processes such as cell proliferation, differentiation, DNA replication, DNA repair, and gene transcription. TEX10 Genes in mammalian muscle cells regulate muscle growth and development, playing a crucial role in processes such as proliferation, apoptosis, and differentiation. However, while research on the TEX10 gene has been conducted in other species, no studies have yet reported on CNVs in the important livestock breed of cattle. Yunling cattle, a yellow cattle breed with significant economic and genetic value in my country, have growth traits directly linked to breeding efficiency and breed improvement. In-depth research on copy number variation in the TEX10 gene in Yunling cattle, and analysis of its association with key growth traits, is of great scientific and practical significance.

[0005] Therefore, it is crucial to study the copy number variation of this gene and analyze its association with the important growth traits of Yunling cattle. This can provide a theoretical basis for the molecular breeding of yellow cattle in my country, facilitate marker-assisted selection of growth traits of yellow cattle, and quickly establish a yellow cattle population with excellent genetic resources. Summary of the Invention

[0006] The existing methods for detecting CNV in cattle genes have problems of insufficient accuracy and result deviation, and the existing detection technology is not suitable for cattle. TEX10 The sensitivity of gene CNV is poor, it is difficult to capture subtle variations, and breeding lacks a strong targeted molecular marker technology. The purpose of this invention is to provide a method for detecting cattle TEX10 Application of gene copy number variation method in molecular marker-assisted selection breeding of cattle and kit.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a method for detecting yellow cattle TEX10 Application of gene copy number variation method in molecular marker-assisted selection breeding of cattle.

[0008] The genomic DNA of cattle to be tested was used as a template to amplify the TEX10 Gene copy number variation regions and controls BTF3 Part of the gene fragment, and then identify the cattle based on the quantitative results TEX10 Type of gene copy number variation; The TEX10 The copy number variation region of the gene is located at 66043201bp-66045200bp on chromosome Chr8.

[0009] The copy number variation type is based on CN=2×2 -△△Ct The quantitative results were divided into three categories: multi-copy type, i.e., copy number CN>2; normal type, i.e., copy number CN=2; and deletion type, i.e., copy number CN<2.

[0010] The amplification TEX10 The primer pair sequences for the copy number variation region of the gene are shown as SEIQ NO.1~SEIQ NO.2.

[0011] Further, the expansion TEX10 The primer pair for the copy number variation region of the gene (primer pair P1) is: Upstream primer F1: 5'-GAGCTGTAAAGAGTCTGTGCCTTT -3' (as shown in SEIQ NO.1) Downstream primer R1: 5'-GCATCTTCTGGTATGTCCCAAT-3' (as shown in SEIQ NO.2) The amplification BTF3 The primer pair sequences of the genes are shown as SEIQ NO.3~SEIQ NO.4.

[0012] Further, the expansion BTF3 The sequence of the primer pair for the gene (primer pair P2) is: Upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA -3' (as shown in SEIQ NO.3); Downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG -3' (as shown in SEIQ NO.4).

[0013] Furthermore, based on amplification TEX10 The size of the PCR product amplified by the primer pair in the copy number variation region of the gene is 75 bp. BTF3 The PCR product amplified by the primer pair of the gene was 166 bp in size.

[0014] The amplification system used in the real-time quantitative PCR includes: 1 μL of 10 ng / μL template DNA, 0.2 μL each of the upstream and downstream primers corresponding to 10 μmol / L primer pair P1 or primer pair P2, 5 μL of 2×qPCR mixture, and 3.6 μL of sterile double-distilled water.

[0015] The reaction procedure of the real-time quantitative PCR was as follows: (1) pre-denaturation at 95°C for 10 min; (2) denaturation at 95°C for 15 s, annealing at 60°C for 60 s, for a total of 39 cycles.

[0016] Among the copy number variation types, individuals with multiple copies are superior to individuals with deletion and normal copy number variation types in growth traits.

[0017] The growth traits are chest girth and ischium width.

[0018] The present invention provides a method for determining the growth characteristics of yellow cattle. TEX10 A real-time quantitative PCR detection kit for gene copy number variation, comprising an amplification TEX10 Gene copy number variation regions and reference genes BTF3 The primer pair of the gene TEX10 The copy number variation region of the gene is located at 66043201bp-66045200bp of chromosome Chr8, and the primer pair sequences are shown as SEIQ NO.1~SEIQ NO.4.

[0019] Compared with the prior art, the present invention achieves the following technical effects: The present invention provides a method for detecting yellow cattle TEX10 The application of gene copy number variation methods in molecular marker-assisted selection breeding of cattle allows for the direct correlation of TEX10 gene copy number variation (CNV) detection with growth traits (such as muscle development and body conformation) in cattle, enabling early selection. Compared to traditional phenotypic selection, this technology can significantly shorten the breeding cycle and improve selection accuracy. Quantitative analysis of CNV markers allows for precise assessment of the genetic potential of individual cattle, avoiding blind selection and improving resource utilization efficiency.

[0020] Furthermore, the detection method disclosed in the present invention TEX10 Compared with high-throughput sequencing methods, gene chips and other methods, the gene copy number variation method is fast, simple, low-cost, and can accurately identify the copy number type of an individual.

[0021] Furthermore, the present invention is effective for Yunling cattle TEX10 Gene( TEX10 The CNV types at the locus (a copy number variation region of the gene) were detected and their frequencies were analyzed. Association analysis was also conducted between this locus and growth traits in Yunling cattle. The results showed that the frequency of multiple copies of this locus was the highest, and that a CNV of 3 at this locus had a significant positive effect on ischium width and chest circumference in Yunling cattle.

[0022] The present invention provides a method related to the growth traits of yellow cattle TEX10 A real-time quantitative PCR detection kit for gene copy number variation was designed, with specific primers designed for the copy number variation (CNV) region of the TEX10 gene, combined with an internal reference gene (such as BTF3 Genes) for standardized analysis, enabling precise quantification TEX10The sensitivity of this assay for detecting copy number variation in the gene is significantly improved compared to traditional methods, enabling the capture of subtle changes in gene dosage and reducing bias in results. The kit is easy to use and low-cost, making it suitable for large-scale breeding applications. It can quickly and cost-effectively detect CNVs in the TEX10 gene, promoting the widespread use of molecular breeding technology in cattle breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 for TEX10 Electrophoresis of PCR amplification products of gene CNV detection primers; Lane 1 is DNA marker I, Lane 2 is BTF3 The PCR product of gene-specific primers (primer pair P2) has a fragment size of 166 bp; lane 3 is TEX10 The PCR product of gene-specific primers (primer pair P1) had a fragment size of 75 bp; Figure 2 is the melting curve detection diagram of the primer of the present invention; wherein A is BTF3 Primer melting curve; B is TEX10 - Melting curve of CNV primers; Figure 3 represents the copy number types and their proportions in the five different cattle breeds of the present invention, QC represents Qinchuan cattle, PN represents Pinan cattle, YL represents Yunling cattle, XN represents Xianan cattle, and JX represents Jiaxian red cattle; Figure 4 is the frequency of occurrence of CNV types in five different cattle breeds of the present invention, QC represents Qinchuan cattle, PN represents Pinan cattle, YL represents Yunling cattle, XN represents Xianan cattle and JX represents Jiaxian red cattle. DETAILED DESCRIPTION

[0024] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0025] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0026] The following examples utilize conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art.

[0027] The present invention is based on the cattle recorded in the NCBI database TEX10 The gene sequence (NC_037335.1) was used as the benchmark to screen out the resequencing data. TEX10 The candidate interval of the copy number variation of the gene (target gene) was precisely located at the 66043201-66045200 position of the Chr8 chromosome. TEX10 The physiological role of genes and the regulatory mechanism of CNV, research TEX10 The correlation between gene copy number variation and growth traits is very necessary and can provide an important theoretical basis for molecular breeding of cattle.

[0028] The present invention is based on the cattle obtained by resequencing TEX10 The specific primers were designed based on the region where the copy number variation occurred in the genome sequence. Then, qPCR amplification was performed using bovine genomic DNA as a template. BTF3 The gene was used as the internal reference gene, and 2×2 -ΔΔCt Method to determine the copy number type of an individual.

[0029] The present invention utilizes qPCR technology to detect TEX10 The copy number variation of genes is analyzed, and different copy number variation types are associated with growth traits to find copy number types with advantageous growth traits, thereby providing basic data for molecular breeding of cattle and accelerating the improvement of Chinese cattle germplasm resources. The specific instructions are as follows.

[0030] 1. Sample Collection and Genomic DNA Extraction (1) Blood sample collection Table 1: Sample sources and numbers

[0031] As shown in Table 1, this study selected five representative breeds: Qinchuan cattle (QC), Pinan cattle (PN), Yunling cattle (YL), Xianan cattle (XN), and Jiaxian Red cattle (JX). Blood samples were systematically collected from 756 individuals, all 24 months old, via jugular vein sampling. The samples were analyzed for key growth traits, including cross-section height, body height, weight, body length, chest circumference, chest width, rump length, abdominal circumference, and ischium width, to further explore potential associations between copy number variation and various growth traits.

[0032] (2) Extraction of DNA from blood samples Frozen blood samples (mainly blood cells) were thawed at room temperature. 500 μL of blood was drawn into a 1.5 mL centrifuge tube. An equal volume of phosphate buffered saline (PBS) was added and mixed. The tube was gently shaken and centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was discarded and the above steps were repeated until the supernatant was transparent and the precipitate was transparent.

[0033] Add 500 μL of DNA extraction buffer to the centrifuge tube, gently pipette to separate the blood cell pellet from the tube wall, and place in a 37°C water bath for 1 h.

[0034] Add proteinase K to 5 μL (20 mg / mL) and mix well. Digest in a 55°C water bath overnight (about 16 h) until the flocculent precipitate disappears and the solution becomes clear. If it is still not clear, add 10 μL of proteinase K, mix well, and continue digesting until it becomes clear.

[0035] The reaction solution was cooled to room temperature, 500 μL of Tris-saturated phenol was added, and the mixture was gently shaken for 15 min to mix thoroughly. The mixture was centrifuged at 12,000 rpm for 10 min at 4°C. The upper aqueous phase was transferred to another sterile centrifuge tube and the above steps were repeated once.

[0036] Add 500 mL of chloroform and shake gently for 20 min to mix thoroughly. Centrifuge at 12,000 rpm at 4°C for 15 min and transfer the upper aqueous phase to another sterilized 1.5 mL centrifuge tube.

[0037] Add 500 mL of a mixture of chloroform and isoamyl alcohol (24:1), mix thoroughly for 20 min, centrifuge at 12,000 rpm at 4°C for 10 min, and transfer the supernatant to another 1.5 mL centrifuge tube.

[0038] Add 0.1 volumes of NaAc buffer and 2 volumes of ice-cold anhydrous ethanol, mix and rotate the centrifuge tube until a white flocculent precipitate is precipitated.

[0039] Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, and rinse the DNA pellet twice with 70% ice-cold ethanol.

[0040] Centrifuge at 4°C, 12,000 rpm for 10 min, discard the supernatant, and allow the ethanol to evaporate completely at room temperature.

[0041] Add 80-100 μL of TE to the dried DNA solution and dissolve it. Store it at 4°C until the DNA is completely dissolved. Check its quality using a UV spectrophotometer and store it at -80°C.

[0042] 2. Design of specific primers for amplification of target genes and internal reference genes According to the cattle published by NCBI TEX10 The gene column (NC_037335.1) is the reference sequence, and the sequence of the copy number variation region screened out in the resequencing is found, that is, TEX10 The target gene sequence is located in the interval 66043201bp-66045200bp of chromosome Chr8. Primers encompassing this region were designed using Prime 5.0 software and compared using NCBI_BLAST. The primer sequences are as follows (primer pair P1): Upstream primer F1: 5'-GAGCTGTAAAGAGTCTGTGCCTTT-3' (SEQ.ID.NO.1) Downstream primer R1: 5'-GCATCTTCTGGTATGTCCCAAT-3' (SEQ.ID.NO.2) At the same time, the cattle published by NCBI BTF3 The gene sequence (AC_000177.1) was used as the reference sequence, and the same method was used to design and amplify the internal reference gene ( BTF3 The primers for a specific fragment (166 bp) in the gene sequence are as follows (primer pair P2): Upstream primer F2: 5'-AACCAGGAGAAACTCGCCAA-3' (SEQ.ID.NO.3) Downstream primer R2: 5'-TTCGGTGAAATGCCCTCTCG -3' (SEQ.ID.NO.4) The specificity of the primers for the amplification products of P1 and P2 was verified by conventional PCR amplification and 2% agarose electrophoresis, for example, see Figure 1 .

[0043] 3. Real-time Quantitative PCR The qPCR reaction system is shown in Table 2 below.

[0044] Table 2: qPCR reaction system

[0045] The PCR reaction procedure was as follows: (1) pre-denaturation at 95 °C for 10 min, followed by amplification according to (2); (2) denaturation at 95 °C for 15 s, annealing at 60 °C for 60 s, for a total of 39 cycles.

[0046] In this study, real-time quantitative polymerase chain reaction (qPCR) technology was used to TEX10- CNV primers and BTF3The specificity of the primers was verified and tested, and the results were as follows Figure 2 shown. BTF3 Primer melting curve, TEX10 The melting curves of the two primers, A, B, and C, showed high consistency, with a single, sharp peak without any bends or twists. The amplification curves of the other two primers showed a smooth and distinct "S"-shaped trend, and the slopes of the curves remained consistent, indicating comparable amplification efficiencies. The cycle threshold (Ct value) fell steadily within the normal range of 20 to 30, further confirming that the genomic DNA amplification process was in the expected normal state. This result indicates that the amplified product is highly specific and meets the characteristics of the expected target product.

[0047] 4. Individual CNV Type Determination and Data Analysis This experiment was designed to set up three sets of parallel replicates for each sample to enhance the accuracy and credibility of the qPCR experimental results. -△△Ct Method: Classify the individual genotypes in the test population. The specific calculation method is: ΔΔCt = ΔCt-ΔCt (中位数) , ΔCt = Ct (目的基因) -Ct (内参基因) , The target genome is the individual sample to be tested for copy number variation amplified by the P1 primer pair. The reference genome is the individual sample amplified by the P2 primer pair. (中位数) The ΔCt is the median in the group.

[0048] According to the formula, the CN of each individual to be tested is calculated as 2×2 -△△Ct , and according to the CNV type determination criteria: multiple copy type, copy number CN>2; deletion type, copy number CN<2; normal type, copy number CN=2, determine the copy number type of the tested cattle individual.

[0049] Association analysis model: SPSS 22 software was used to analyze the production trait effects among genotypes. A fixed model was used to analyze the genotype effects: Y ijk = μ +A i +CNV j + e ijk Where: Y ijk is the observed value of the trait, μ is the overall mean, A i is the age of the i-th individual, CNV j is the fixed effect of the jth copy number variation type, e ijkThe differences between the data groups were tested using LSD multiple comparisons, and the test results were expressed as Mean ± SE.

[0050] 5. TEX10 Distribution of gene CNVs By analyzing samples from 756 adult cows, we systematically studied the copy number variation (CNV) distribution of the TEX10 gene in five different cattle breeds. The results are shown in the Appendix. Figures 3 and 4 And shown in Table 3.

[0051] By the attached Figure 3 The data show that the copy number variation types of Pinan cattle (PN) and Xianan cattle (XN) are mainly concentrated in the deletion type; in contrast, Yunling cattle (YL) and Jiaxian red cattle (JX) show more multi-copy types; and for Qinchuan cattle (QC), the distribution of its CNV types is relatively discrete.

[0052] Table 3: Frequency of CNV types

[0053] By the attached Figure 4 As shown in Table 3, different types TEX10 - The distribution of CNVs in these five cattle breeds was significantly different, and this finding is important for further revealing TEX10 The genetic variation patterns and potential functions of genes in different cattle breeds are of great significance.

[0054] 6. TEX10 Association analysis between gene CNV loci and growth traits of five Chinese cattle breeds (1) Analysis of the relationship between copy number variation types and growth traits in Qinchuan cattle In the Qinchuan cattle population, 199 individuals were TEX10 -CNV was associated with 10 growth traits. According to the results shown in Table 4, TEX10 -CNV has a significant effect on the body length trait of Qinchuan cattle (P<0.05). Among them, the average body length of individuals with multiple copies of the variant type can reach 139±12.29cm, which is significantly longer than the other two types. Body length is an important trait affecting the growth, development and production performance of Qinchuan cattle. Therefore, individuals with multiple copies should be given priority when selecting Qinchuan cattle. At the same time, we can see from the table that although the copy number variation type is not significantly correlated with growth traits other than body length (P>0.05), body height, cross height, chest circumference, chest width, and rump length of the multiple copies have a trend of good development. This suggests that when selecting and breeding Qinchuan cattle, we should pay more attention to individuals with multiple copies.

[0055] Table 4: Qinchuan cattle TEX10Correlation analysis between genes and growth traits

[0056] Note: The values ​​in the same row are marked with a, b and *, indicating significant differences ( P <0.05).

[0057] (2) Analysis of the relationship between copy number variation types and growth traits in Pinan cattle The experimental group consisted of 206 Pi Nan cattle. The six growth traits measured were compared with TEX10 -CNV association analysis was performed and the results shown in Table 5 were obtained. TEX10 Different types of gene copy number variation have a very significant effect on the chest circumference of Pinan cattle ( P <0.01), and the normal genotype is the dominant genotype, with an average chest circumference of 176.03 ± 14.68 cm, showing a significant advantage over the multi-copy genotype. Individuals with the normal genotype tend to develop well in all growth traits, making the normal genotype a suitable molecular marker for Pinan cattle breeding.

[0058] Table 5: Pinan cattle TEX10 Correlation analysis between genes and growth traits

[0059] Note: The values ​​in the same row are marked with A, B and **, indicating extremely significant differences ( P <0.01).

[0060] (3) Analysis of the relationship between copy number variation types and growth traits in Yunling cattle Association analysis of 15 growth traits was performed on 196 individuals in the Yunling cattle population. The results (see Table 6) showed a significant correlation between TEX10-CNV and chest circumference in Yunling cattle (P < 0.05), with the multi-copy variant being the dominant genotype for this trait, with a mean value of 197.86 ± 10.08 cm, significantly superior to the deletion and normal genotypes. Furthermore, TEX10-CNV was significantly correlated with ischium width in Yunling cattle (P < 0.01). The multi-copy variant was also the dominant genotype, with a mean value of 22.52 ± 2.06 cm, showing a highly significant advantage over the deletion variant. Therefore, when selecting Yunling cattle, consideration should be given to retaining individuals with the multi-copy variant to optimize the genetic structure of the Yunling cattle population and improve breeding outcomes.

[0061] Table 6: Yunling cattle TEX10 Correlation analysis between genes and growth traits

[0062] Note: The values ​​in the same row are marked with a, b and *, indicating significant differences ( P <0.05); A, B, and ** in the same row indicate extremely significant differences ( P <0.01).

[0063] Yunling cattle TEX10 The gene copy number variation site is significantly associated with the growth traits of chest circumference and ischium width. In terms of ischium width, the multi-copy type (CN≥3) performs best, significantly better than the normal type and the deletion type. Ischium width is an important indicator affecting development and meat production performance, which is very important for Yunling cattle breeding. In future breeding, the multi-copy type can be given priority. The average chest circumference of the CN≥3 group is higher than that of other types ( P <0.05). Therefore, TEX10 The CN≥3 copy number type of the gene can be used as a molecular marker for early selection of body length and chest girth traits in Yunling cattle.

[0064] (4) Analysis of the relationship between copy number variation types and growth traits in Xianan cattle Copy number variation was detected in 106 Xia Nan cattle, and association analysis with seven growth traits and the TEX10-CNV region was performed. The results are shown in Table 7. The analysis showed that the different copy number variation types of the TEX10 gene had no significant effect on the growth traits of the Xia Nan cattle population (P>0.05). TEX10-CNV had no statistically significant effect on waist and abdominal circumference or weight. However, individuals with the normal type had a significant advantage in two important traits: chest circumference (mean 195.46±9.02 cm) and weight (mean 558.62±63.79 kg). Individuals with the other two types had only slight advantages in certain traits. Therefore, individuals with the normal type have the potential to become the dominant genotype.

[0065] Table 7: Xianan cattle TEX10 Correlation analysis between genes and growth traits

[0066] (5) Analysis of the relationship between copy number variation types and growth traits in Jiaxian Red Cattle In the study of 48 individuals in the Jiaxian Red Cattle population, as shown in Table 8, no TEX10 - There is a statistically significant association between CNV and growth traits ( P >0.05). Individuals with multiple copies showed a trend of better development in growth traits such as body length, cross height, and weight, although these differences did not reach statistical significance. This observation suggests that TEX10-CNV has potential positive effects on these growth traits in Jiaxian Red Cattle, worthy of further exploration in future studies. Due to the small number of Jiaxian Red Cattle tested, more accurate and specific effects and mechanisms of action require further research and verification.

[0067] Table 8: Jiaxian Red Bull TEX10 Correlation analysis between genes and growth traits

[0068] Note: The values ​​in the same row marked with a, b and * indicate significant differences (P<0.05).

[0069] (1) The deletion, normal, and multi-copy types of TEX10-CNV occurred in all five cattle populations, with the multi-copy and deletion types accounting for a larger proportion.

[0070] (2) TEX10-CNV was significantly correlated with chest circumference and ischium width of Yunling cattle, body length of Qinchuan cattle, and chest circumference of Pinan cattle.

[0071] 6. Cow TEX10 Application of gene copy number variation detection The above results also show that TEX10 This gene could be a candidate for improving growth traits in cattle, and is hypothesized to influence the development of traits such as bones. Individuals with multiple copies of this gene could be selected for breeding and multiplication, leveraging their phenotypic advantages in chest girth and ischium width. This could accelerate the improvement and selection of superior growth performance in cattle (e.g., local yellow cattle breeds such as Yunling and Qinchuan).

[0072] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. Application of a method for detecting copy number variation of the cattle TEX10 gene in cattle molecular marker-assisted selection breeding.

2. The use according to claim 1, characterized in that Using cattle genomic DNA as a template, the copy number variation region of the TEX10 gene and a partial fragment of the BTF3 gene as a control were amplified by real-time quantitative PCR. The copy number variation type of the cattle TEX10 gene was then identified based on the quantitative results. The copy number variation region of the TEX10 gene is located at 66043201bp-66045200bp of Chr8 chromosome.

3. The use according to claim 2, characterized in that The copy number variation types are divided into three categories based on the quantitative results of CN=2×2-△△Ct: multiple copy type, that is, the copy number CN>2; normal type, that is, the copy number CN=2; and deletion type, that is, the copy number CN<2.

4. The use according to claim 2, characterized in that The primer pair sequences for amplifying the copy number variation region of the TEX10 gene are shown as SEIQ NO.1 to SEIQ NO.

2.

5. The use according to claim 2, characterized in that The primer pair sequences for amplifying the BTF3 gene are shown as SEIQ NO.3 to SEIQ NO.

4.

6. The use according to claim 2, characterized in that The amplification system used in the real-time quantitative PCR includes: 1 μL of 10 ng / μL template DNA, 0.2 μL each of the upstream and downstream primers corresponding to 10 μmol / L primer pair P1 or primer pair P2, 5 μL of 2×qPCR mixture, and 3.6 μL of sterile double-distilled water.

7. The use according to claim 2, characterized in that The reaction procedure of the real-time quantitative PCR was as follows: (1) pre-denaturation at 95°C for 2 min; (2) denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for a total of 35 cycles.

8. The use according to claim 2, characterized in that Among the copy number variation types, individuals with multiple copies are superior to individuals with deletion and normal copy number variation types in growth traits.

9. The use according to claim 8, characterized in that The growth traits are chest girth and ischium width.

10. A real-time quantitative PCR detection kit for TEX10 gene copy number variation related to cattle growth traits, characterized in that: The kit includes a primer pair for amplifying the copy number variation region of the TEX10 gene and the internal reference gene BTF3 gene. The copy number variation region of the TEX10 gene is located at 66043201bp-66045200bp of the Chr8 chromosome, and the primer pair sequence is shown as SEIQ NO.1 to SEIQ NO.4.

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