Cnvr marker of bovine echdc3 gene and application thereof

Abstract

The application relates to a bovine ECHDC3 CNVR marker of a bovine gene and application thereof, and belongs to the technical field of molecular genetic breeding. The CNVR marker of the bovine ECHDC3 Gene of the application is located at chr13:12482697-12761334 in the international bovine reference genome sequence ARS-UCD1.2. The CNVR marker of the application has the advantages of high specificity and convenient detection, clear breeding application effect, rapid screening of excellent individuals with high C22:0 fatty acid content, significant acceleration of the genetic improvement process of fatty acid traits, effective improvement of beef quality, breeding and breeding economic benefits, and provision of technical support for high-quality development of the beef cattle industry.

Description

Technical Field

[0001] This invention relates to the field of molecular genetic breeding technology, and more particularly to cattle. ECHDC3 CNVR markers for genes and their applications. Background Technology

[0002] Beef cattle are a specialized breed developed by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences, through 43 years of crossbreeding, improvement, and continuous selection. They possess advantages such as rapid growth, high slaughter rate, and wide adaptability, and have become one of the core breeds in my country's beef cattle industry. Fatty acid composition is a core indicator for evaluating beef quality. C22:0 fatty acids (docosahexaenoic acid, behenic acid), as important long-chain saturated fatty acids, directly affect the flavor, juiciness, and nutritional value of beef. Research indicates that genetic improvement of C22:0 fatty acids is of great significance for enhancing the competitiveness of beef in the market.

[0003] Copy number variation markers (CNVRs), as important structural variations in the genome, influence gene expression through mechanisms such as gene dosage effects and regulatory element enhancement, playing a crucial role in the regulation of economic traits in livestock and poultry. Currently, research on fatty acid-related genetic markers in beef cattle mainly focuses on single nucleotide polymorphisms (SNPs), while the development of functional markers based on CNVRs is relatively scarce. Furthermore, existing technologies lack functional CNVR markers that can precisely regulate the C22:0 fatty acid content in beef cattle, thus limiting the efficiency of genetic improvement of fatty acid traits. Summary of the Invention

[0004] The purpose of this invention is to provide cattle ECHDC3 CNVR markers for genes and their applications are used to address the problem that the lack of CNVR markers related to beef fatty acids in existing technologies limits the efficiency of genetic improvement of beef fatty acid traits.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides cow ECHDC3 The CNVR marker of the gene is located at chr13:12482697~12761334 of the international bovine reference genome sequence ARS-UCD1.2.

[0006] Preferably, the leading SNP of the CNVR tag is BovineHD1300003563.

[0007] The present invention also provides the application of the CNVR marker in increasing the C22:0 fatty acid content of beef.

[0008] This invention also provides the application of the CNVR marker in the selection and / or assisted breeding of high-fat cattle.

[0009] The present invention also provides a primer set for detecting the CNVR marker, the nucleotide sequence of which is shown in SEQ ID NO. 1 to 4.

[0010] The present invention also provides a kit for detecting the CNVR marker, the kit comprising the primer set and the required enzyme and buffer.

[0011] This invention also provides a method for breeding high-fat cattle, comprising the following steps: (1) Extract genomic DNA from the cattle to be tested, perform PCR amplification using the primer set described above, and sequence the DNA to obtain the sequencing results; (2) Based on the sequencing results, select the cattle carrying the CNVR marker as breeding cattle and cull cattle that do not carry the marker. (3) Crossbreeding the breeding cattle to obtain high fatty acid cattle.

[0012] Preferably, the target sequence for sequencing in step (1) is a CNVR marker and / or a leader SNP.

[0013] Preferably, the sequencing method in step (1) is one or more of whole genome sequencing, targeted sequencing, and Sanger sequencing.

[0014] Preferably, in the breeding cattle described in step (2), the CNVR marker has CN=3; In step (2), the CNVR marker in the untested cow has CN=2.

[0015] The present invention has the following technical effects and advantages: This invention is the first to identify a CNVR marker in the chr13:12482697-12761334 region on chromosome 13 of beef cattle, clarifying that it works by upregulating... ECHDC3 The molecular mechanism by which gene expression levels regulate C22:0 fatty acid content was investigated. This CNVR marker has the advantages of high specificity and convenient detection, and its breeding application effect is clear. When applied to molecular marker-assisted breeding of beef cattle populations, it can quickly screen superior individuals with high C22:0 fatty acid content, significantly accelerate the genetic improvement process of fatty acid traits, effectively improve beef quality, breeding and breeding economic benefits, and provide technical support for the high-quality development of the beef cattle industry. The beef cattle screening method and related reagents obtained based on the CNVR markers of this invention can provide new molecular markers and technical means for improving fatty acid traits in beef cattle, which is of great practical significance for accelerating the process of molecular breeding of beef cattle. Attached Figure Description

[0016] Figure 1The LRR distribution and third-generation sequencing validation results of CNVR markers on chromosome 13 of beef cattle are shown in the figure above, where the LRR distribution results are shown in the figure below, and the third-generation sequencing validation results are shown in the figure below. Figure 2 Sanger sequencing results with CNVR-tagged breakpoints at both ends; Figure 3 The C22:0 fatty acid content of each individual beef cattle; Figure 4 Manhattan plot of genome-wide association analysis of C22:0 fatty acid content on chromosome 13 for each beef cattle, where the horizontal axis represents the chromosome number of the beef cattle and the vertical axis represents the -logP value; Figure 5 For beef cattle ECHDC3 Results of gene expression level measurement. Detailed Implementation

[0017] This invention provides cow ECHDC3 The CNVR marker of the gene is located at chr13:12482697~12761334 of the international bovine reference genome sequence ARS-UCD1.2.

[0018] In this invention, the leading SNP of the CNVR tag is BovineHD1300003563.

[0019] In this invention, the cattle are preferably beef cattle.

[0020] The present invention also provides the application of the CNVR marker in increasing the C22:0 fatty acid content of beef.

[0021] This invention also provides the application of the CNVR marker in the selection and / or assisted breeding of high-fat cattle.

[0022] The present invention also provides a primer set for detecting the CNVR marker, the nucleotide sequence of which is shown in SEQ ID NO. 1 to 4.

[0023] The present invention also provides a kit for detecting the CNVR marker, the kit comprising the primer set and the required enzyme and buffer.

[0024] This invention also provides a method for breeding high-fat cattle, comprising the following steps: (1) Extract genomic DNA from the cattle to be tested, perform PCR amplification using the primer set described above, and sequence the DNA to obtain the sequencing results; (2) Based on the sequencing results, select the cattle carrying the CNVR marker as breeding cattle and cull cattle that do not carry the marker. (3) Crossbreeding the breeding cattle to obtain high fatty acid cattle.

[0025] In this invention, the target sequence for sequencing in step (1) is a CNVR marker and / or a leader SNP.

[0026] In this invention, the sequencing method in step (1) is one or more of whole genome sequencing, targeted sequencing, and Sanger sequencing.

[0027] In this invention, in the breeding cattle described in step (2), CNVR is marked with CN=3; In step (2), the CNVR marker in the untested cow has CN=2.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] The test animals for this invention were a beef cattle resource group established by the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences, which came from the Ulagai Management Area of ​​Xilingol League, Inner Mongolia Autonomous Region. The feeding and management conditions were consistent, and the animals were fed according to the TMR standard until they were 24 months old and then slaughtered. In the reagents of this invention, the TIANamp blood DNA extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0030] Example 1: Feeding of test animals and sample collection After 1,100 beef cattle were fed according to the TMR standard until they reached 24 months of age, 5 mL of venous blood was collected from each head. Genomic DNA was extracted using the TIANamp blood DNA extraction kit. The purity of the genomic DNA was detected using a Nanodrop spectrophotometer, and the integrity of the genomic DNA was verified by agarose gel electrophoresis. The qualified samples were then diluted to 50 ng / μL.

[0031] Example 2: Filtering and Verification of CNVR Tags Initial screening of CNVR markers: Genotyping was performed using an Illumina BovineHD 770K SNP chip, and CNVR markers were detected using PennCNVR software. The quality control criteria were set as LRR standard deviation < 0.30, BAF drift < 0.01, and minimum SNP number ≥ 5. CNVR markers were obtained through initial screening. Third-generation sequencing validation: Two beef cattle individuals carrying candidate CNVR markers and two normal beef cattle individuals were selected for PacBio HiFi third-generation sequencing (coverage ≥30×). The repetition type and boundary accuracy of CNVR markers were visualized and verified using IGV software. Sanger sequencing validation: Specific primers were designed based on the CNVR marker boundary sequences and PCR amplification was performed (as shown in Table 1). The amplification program was set as follows: 98℃ pre-denaturation for 5 min → (98℃ denaturation for 10 s → 55℃ annealing for 5 s → 68℃ extension for 1 s) × 35 cycles → 68℃ final extension for 5 min. Sanger sequencing was performed on the PCR amplification products to determine the breakpoint locations and verify the authenticity of the CNVR markers. The results are as follows: Figures 1-2 As shown.

[0032] Table 1. Primers related to CNVR tags ; The results showed that a 278.6 kb CNVR marker existed in the chr13:12482697–12761334 region of chromosome 13, which contained… ECHDC3 Gene (chr13:12517107~12533297), ECHDC3 The first exon of the gene overlaps with the CNVR marker, and this region overlaps with the H3K4me3 and H3K27ac epigenetic modification peaks and ATAC-seq open signals in beef cattle fat, liver, and muscle tissue, indicating active transcriptional regulatory activity.

[0033] Example 3: Genome-wide association analysis of CNVR markers with C22:0 fatty acids Samples of the 12th-13th intercostal psoas major muscle from beef cattle, as described in Example 1, were collected. Total lipids were extracted and subjected to saponification and methylation treatment. The C22:0 fatty acid content was determined by gas chromatography (GC-2014 CAFsc). A general linear model was used to correct for factors such as sex, farm, and year. The association between the CNVR marker genotype and the C22:0 fatty acid content was analyzed. The results are as follows: Figures 3-4 As shown.

[0034] The results showed that the content of C22:0 fatty acids in the muscle tissue of beef cattle carrying the CNVR marker (CN=3) was significantly higher than that in beef cattle without the marker (CN=2). P =0.004).

[0035] Example 4: ECHDC3 Determination of gene expression levels qPCR technology was used to determine ECHDC3 Gene expression levels, and in terms of BTF3 The reference gene was used, and the relevant primer sequences are shown in Table 2. The results are as follows: Figure 5 As shown.

[0036] Table 2 qPCR related primers ; The results showed that beef cattle carrying the CNVR marker, ECHDC3 The gene expression level was 1.5 to 2.5 times higher than that of beef cattle without the CNVR marker, indicating that the CNVR marker is upregulated through a gene dose-response effect. ECHDC3 Gene expression enhances fatty acid β-oxidation efficiency, ultimately increasing C22:0 fatty acid content.

[0037] Example 5: Breeding of high-fat beef cattle (1) Genomic DNA was extracted from the venous blood of the beef cattle to be tested according to the method described in Example 1. PCR amplification was performed using the primer set in Table 1 and the PCR amplification program described in Example 2. Sequencing was performed on the CNVR marker to obtain the sequencing results. (2) Based on the sequencing results, select the cattle carrying the CNVR marker (CN=3) as breeding cattle and cull the cattle that do not carry the CNVR marker (CN=2); (3) Crossbreeding with breeding cattle and gradually increasing the frequency of CNVR markers in the beef cattle population to obtain high fatty acid cattle.

[0038] The results showed that the breeding method of this invention can increase the frequency of CNVR markers in beef cattle populations generation by generation, increasing the average C22:0 fatty acid content of beef cattle populations by 8.3%–12.5% ​​per generation, thereby significantly accelerating the genetic improvement process of fatty acid traits; the CNVR marker showed strong linkage disequilibrium with the leader SNP (BovineHD1300003563). r 2 =0.89).

[0039] As can be seen from the above embodiments, the present invention provides a cow ECHDC3 CNVR markers for genes and their applications. The CNVR markers of this invention have the advantages of high specificity and convenient detection, with clear breeding application effects. They can quickly screen superior individuals with high C22:0 fatty acid content, significantly accelerate the genetic improvement process of fatty acid traits, effectively improve beef quality, breeding and breeding economic benefits, and provide technical support for the high-quality development of the beef cattle industry.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Cow ECHDC3 CNVR markers for genes, characterized by, The CNVR marker is located at chr13:12482697~12761334 of the international bovine reference genome sequence ARS-UCD1.

2.

2. The CNVR mark according to claim 1, characterized in that, The leading SNP of the CNVR tag is BovineHD1300003563.

3. The application of the CNVR marker as described in claim 1 or 2 in increasing the C22:0 fatty acid content of beef.

4. The application of the CNVR marker as described in claim 1 or 2 in the selection and / or assisted breeding of high-fat cattle.

5. A primer set for detecting the CNVR marker as described in claim 1 or 2, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO.1 to 4.

6. A kit for detecting the CNVR marker as described in claim 1 or 2, characterized in that, The kit includes the primer set as described in claim 5, as well as the required enzymes and buffers.

7. A method for breeding high-fat cattle, characterized in that, Includes the following steps: (1) Extract genomic DNA from the cattle to be tested, perform PCR amplification and sequencing using the primer set described in claim 5, and obtain sequencing results; (2) Based on the sequencing results, select the cattle carrying the CNVR marker described in claim 1 or 2 as breeding cattle, and eliminate cattle that do not carry the marker. (3) Crossbreeding the breeding cattle to obtain high fatty acid cattle.

8. The breeding method according to claim 7, characterized in that, The target sequence for sequencing in step (1) is a CNVR marker and / or a leader SNP.

9. The breeding method according to claim 8, characterized in that, The sequencing method described in step (1) is one or more of whole genome sequencing, targeted sequencing, and Sanger sequencing.

10. The breeding method according to claim 9, characterized in that, In the breeding cattle described in step (2), CNVR is marked with CN=3; In step (2), the CNVR marker in the untested cow has CN=2.

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