Structural variation site and application thereof in auxiliary detection of yak growth traits
The detection of structural mutation sites of yak EXOC4 gene through PCR amplification and electrophoresis solved the problem of insufficient research on yak growth traits, and achieved the improvement of yak breeding accuracy and efficiency.
Patent Information
- Application Number
- CN202510642469.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-19
- Publication Date
- 2025-08-08
AI Technical Summary
In the prior art, there are few studies on the structural variations of the whole genome of yaks and their impact on growth traits, and it is difficult to effectively assist yak breeding and breed improvement.
The structural variation sites at positions 24135756-24135950 on the yak EXOC4 gene were amplified by PCR, and three genotypes (II, ID, DD) were detected by agarose gel electrophoresis, and correlation analysis was performed to screen out molecular markers related to growth traits for early breeding.
Effective detection and early breeding of yak bust traits at 6 months old has been achieved, which improves the accuracy and efficiency of breeding and provides a basis for molecular breeding.
Smart Images
Figure CN120442813A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of molecular markers, and in particular relates to a structural variation site and its application in assisting the detection of yak growth traits. Background Art
[0002] Yaks (Bos grunniens), a unique cattle breed native to the Qinghai-Tibet Plateau, are a vital source of livelihood and income for herders on the plateau and an essential livestock species in the local animal husbandry economy. GWAS, as a key tool for genetic mapping, plays a crucial role in elucidating the genetic basis of important economic traits in livestock and poultry. In recent years, a growing number of studies have demonstrated that genomic structural variation plays a significant role in phenotypic variation in livestock and poultry. Structural variation (SV) generally refers to sequence and positional variations exceeding 50 bp in the genome, including but not limited to deletions, duplications, and more complex rearrangements such as large tandem duplications, inversions, retrotransposon insertions, and inversions. In livestock and poultry breeding, screening for variant loci closely associated with growth traits and analyzing their association with phenotypes can achieve early selection and improve the accuracy of breeding values. However, current research on SVs across the yak genome and their impact on growth traits is very limited.
[0003] EXOC4 is a component of the exosome complex and is involved in the docking of exosomes with the plasma membrane fusion site. EXOC4 regulates the histone modification proteins ATF2 and RNF20, while also influencing the expression of PAK1 and PAK2, thereby influencing cytoskeletal dynamics and cell migration. Furthermore, EXOC4 is a candidate gene for economic traits in livestock and poultry. Studies have shown that copy number variation in the EXOC4 gene is significantly correlated with duck breast muscle weight.
[0004] The present invention provides a method for structural variation site-assisted detection of yak growth traits and its application. By studying the association between the SV site of the EXOC4 gene in yaks and growth traits, sites that affect the growth and development traits of yaks are screened out, providing a basis for accelerating yak molecular breeding and variety improvement. Summary of the Invention
[0005] To address the challenges of existing technologies, the present invention provides a method for detecting yak growth traits using structural variation sites. Using yak genomic DNA as a template, the gene sequence containing the SV locus on the yak EXOC4 gene is amplified by PCR. The PCR amplification product is subjected to agarose gel electrophoresis to determine the genotype of the SV locus in the test sample. The SV molecular marker is located on chromosome 4 of the yak genome, Bosgru_v3.0, at positions 24135756-24135950.
[0006] Specifically include the following:
[0007] In a first aspect, the present invention provides an application of a reagent for detecting SV molecular markers in detecting the chest circumference trait of yaks at 6 months of age, wherein the SV molecular marker is located at positions 24135756-24135950 on chromosome 4 of the yak genome Bosgru_v3.0 version, Ensembl number: ENSBGRG0000000470; the SV molecular marker has three genotypes: II, ID, and DD; wherein, using the genomic DNA of the yak as a template, the SV molecular marker is amplified by PCR, a 714bp band is amplified, and the genotype is defined as a homozygous insertion type II genotype; two bands of 714bp and 519bp are amplified, and the genotype is defined as a heterozygous ID genotype; a 519bp band is amplified, and the genotype is defined as a homozygous deletion type DD genotype.
[0008] Preferably, the DD genotype is superior to the II and ID genotypes in the chest girth trait of the yak at 6 months of age.
[0009] Preferably, the reagents include a primer pair for amplifying a marker containing the SV molecule.
[0010] Preferably, the gene sequence containing the SV molecular marker is shown as SEQ ID NO.1.
[0011] Preferably, the primer pair is:
[0012] Upstream primer: 5′-CGAAGTCCTAATAATCCCCAGA-3′;
[0013] Downstream primer: 5′-TTTCTCTCATAACGTCGCCCT-3′.
[0014] In a second aspect, the present invention provides an application of a reagent for detecting SV molecular markers in early breeding of chest circumference traits in yaks at 6 months of age, wherein the SV molecular marker is located at positions 24135756-24135950 on chromosome 4 of the yak genome Bosgru_v3.0 version, Ensembl number: ENSBGRG0000000470; the SV molecular marker has three genotypes: II, ID and DD; wherein, using the genomic DNA of the yak as a template, the SV molecular marker is amplified by PCR, a 714bp band is amplified, and the genotype is defined as a homozygous insertion type II genotype; two bands of 714bp and 519bp are amplified, and the genotype is defined as a heterozygous ID genotype; a 519bp band is amplified, and the genotype is defined as a homozygous deletion type DD genotype.
[0015] Preferably, the DD genotype is selected for early selection of chest girth traits in yaks at 6 months of age.
[0016] Preferably, the reagents include a primer pair for amplifying a marker containing the SV molecule.
[0017] Preferably, the gene sequence containing the SV molecular marker is shown as SEQ ID NO.1.
[0018] Preferably, the primer pair is:
[0019] Upstream primer: 5′-CGAAGTCCTAATAATCCCCAGA-3′;
[0020] Downstream primer: 5′-TTTCTCTCATAACGTCGCCCT-3′.
[0021] In a third aspect, the present invention provides a kit for assisting in the detection of chest girth traits of yaks at 6 months of age using structural variation sites, the kit comprising a primer pair for amplifying the SV molecular marker:
[0022] Upstream primer: 5′-CGAAGTCCTAATAATCCCCAGA-3′;
[0023] Downstream primer: 5′-TTTCTCTCATAACGTCGCCCT-3′.
[0024] In a fourth aspect, the present invention provides the use of the kit described in the third aspect in assisting the detection of chest circumference traits of yaks at 6 months of age.
[0025] Preferably, the method for assisting in detecting the chest girth trait of yaks at 6 months of age is:
[0026] Using yak genomic DNA as a template, amplifying the gene sequence containing the SV molecular marker described in the first aspect by PCR;
[0027] According to the amplification results, a single band of 714 bp was amplified, and the genotype was defined as II genotype; two bands of 714 bp and 519 bp were amplified, and the genotype was defined as ID genotype; a single band of 519 bp was amplified, and the genotype was defined as DD genotype;
[0028] Among them, the chest circumference of 6-month-old yaks with DD genotype was better than that with II and ID genotypes.
[0029] In a fifth aspect, the present invention provides the use of the kit according to the third aspect in assisting the early selection of chest girth traits in yaks at 6 months of age.
[0030] Preferably, the method for assisting in detecting the chest girth trait of yaks at 6 months of age for early breeding is:
[0031] Using yak genomic DNA as a template, amplifying the gene sequence containing the SV molecular marker described in the first aspect by PCR;
[0032] According to the amplification results, a single band of 714 bp was amplified, and the genotype was defined as II genotype; two bands of 714 bp and 519 bp were amplified, and the genotype was defined as ID genotype; a single band of 519 bp was amplified, and the genotype was defined as DD genotype;
[0033] Among them, the chest circumference of 6-month-old yaks with DD genotype is better than that of II and ID genotypes; the DD genotype is selected for early selection of chest circumference traits in yaks at 6 months of age.
[0034] Combining all the above technical solutions, the advantages and positive effects of the present invention are as follows:
[0035] The present invention uses resequencing data to screen SV molecular markers for yak growth traits. The SV molecular marker is 195bp long (shown in SEQ ID NO.1) and is located at positions 24135756-24135950 on chromosome 4 of the yak genome Bosgru_v3.0. The present invention uses the genomic DNA of the yak individual to be tested as a template and uses PCR to amplify a partial fragment containing the SV gene. It is found that the SV molecular marker has three genotypes, II, ID and DD; the homozygous insertion type (II) is expressed as a 714bp band, the heterozygous ID type is expressed as two bands of 714bp and 519bp, and the homozygous deletion type (DD) is expressed as a 519bp band. According to the association analysis of the genotype of the above-mentioned SV site and the growth traits of yaks, it was found that the SV molecular marker was significantly correlated with the chest circumference of yaks at 6 months of age. The SV molecular marker obtained by the present invention has good repeatability among different yak individuals and stable amplification, and can be used as a molecular marker for growth trait selection in yak molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 Gel electrophoresis analysis of PCR products of yak SV molecular marker genes, where M, GL DNAmarker1000; II, homozygous insertion type (II type); ID, heterozygous type (ID type); DD, homozygous deletion type (DD type). DETAILED DESCRIPTION
[0037] The present invention will be described in detail below with reference to specific embodiments. Those skilled in the art will be able to determine the essential features of the present invention and, without departing from the spirit and scope of the present invention, may make various changes and modifications to the present invention. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.
[0038] The present invention utilizes PCR technology to detect the SV markers described herein and use them in molecular breeding, generally comprising the following steps:
[0039] (1) The upstream sequence of the EXOC4 gene was searched using the Ensembl database, and primer pairs containing the above-mentioned SV loci were designed using Primer5 software. PCR amplification and agarose gel electrophoresis were used to detect the typing of candidate SV loci in the yak population to be tested.
[0040] (2) SPSS 21.0 software was used to perform association analysis between the presence of three genotypes II, ID, and DD of the SV molecular marker and the growth traits of yaks;
[0041] (3) EXOC4 SV analysis was performed on all test samples to determine the dominant allele, and yaks with excellent growth traits were selected according to different genotypes.
[0042] Example 1 A method for SV locus-assisted detection of yak growth traits
[0043] 1. Yak blood sample collection
[0044] The experimental animals of the present invention are 240 yaks from Qinghai Datong cattle breeding farm. First, the growth traits of yaks at different age stages are measured, and then blood samples are collected by sampling blood from the jugular vein.
[0045] 2. Blood DNA Extraction
[0046] Yak blood genomic DNA was extracted using the TIANGEN medium-volume blood genomic extraction kit according to the kit instructions. The specific steps are as follows:
[0047] ① Thaw the frozen blood sample at room temperature, draw 1 mL of blood into a 15 mL centrifuge tube, add 200 μL of proteinase K solution, and mix well.
[0048] ② Add 2.4 mL of buffer GE to the centrifuge tube containing the blood sample and shake for 30 seconds to mix.
[0049] ③ Place in a 65℃ water bath for 10 minutes, shaking every 3 minutes to aid lysis.
[0050] ④ Add 2 mL of anhydrous ethanol to the sample and mix well. Flocculent precipitation may appear at this time.
[0051] ⑤ Transfer the solution and half of the flocculent precipitate from the previous step to an adsorption column CB5. Centrifuge at 3,000 rpm (~1,850 × g) for 3 minutes. Discard the waste liquid and return the adsorption column CB5 to the collection tube. Transfer the remaining solution from step 6 to the same adsorption column and repeat step 6.
[0052] ⑥ Add 2 mL of buffer GD to the adsorption column CB5, centrifuge at 5,000 rpm for 1 min, discard the waste liquid, and return the adsorption column CB5 to the collection tube.
[0053] ⑦ Add 2 mL of rinse solution PW to the adsorption column CB5, centrifuge at 5,000 rpm for 1 min, discard the waste liquid, and return the adsorption column CB5 to the collection tube.
[0054] ⑧ Add 2 mL of rinse solution PW to the adsorption column CB5, centrifuge at 5,000 rpm (~4,500×g) for 15 min, discard the collection tube, and place the adsorption column CB5 in a new 15 mL centrifuge tube.
[0055] 9. Add 200 μL of elution buffer TB dropwise to the center of the membrane. Allow to stand at room temperature for 5 minutes. Centrifuge at 5,000 rpm (~4,500 × g) for 2 minutes. Collect the solution into a centrifuge tube. Measure the DNA concentration and quality using a UV spectrophotometer. Store at -80°C.
[0056] Based on the results of whole-genome comparison of the resequencing data of the yak population to be tested and the reference genome, the present invention discovered an SV mutation site located in the upstream reference sequence of the yak EXOC4 gene sequence (positions 24135756-24135950 on chromosome 4 of the yak genome Bosgru_v3.0 version).
[0057] 3. Amplification of yak EXOC4 gene fragment
[0058] Based on the upstream sequence of the EXOC4 gene on chromosome 4 of the yak genome published in the Ensembl database (http: / / asia.ensembl.org / index.html) (shown as SEQ ID NO. 1) as a reference sequence, Primer5.0 was used to design PCR primer pairs containing the SV site (Table 1).
[0059] The PCR reaction program was as follows: 1) pre-denaturation at 95°C for 2 min; 2) denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min, for a total of 30 cycles; 3) extension at 72°C for 5 min.
[0060] Table 1 Target sequence PCR primers
[0061]
[0062] The amplified PCR products were detected by 2% agarose gel electrophoresis. Figure 1As shown, the homozygous insertion type II appears as a band of 714 bp; the heterozygous type ID appears as two bands of 714 bp and 519 bp; and the homozygous deletion type DD appears as a band of 519 bp.
[0063] 4. Association analysis of SV molecular markers with growth traits
[0064] The weight, height, body length, chest circumference and tube circumference of each yak to be tested were recorded at birth and at 6 months of age.
[0065] Association analysis model: First, a descriptive analysis of the data was performed to determine whether there were outliers, and then the data were corrected using least squares analysis. Based on the data characteristics, SPSS 21.0 software was used to associate the genotypes and growth traits of yaks. The linear model used in the analysis was as follows:
[0066] Yij=μ+Gij+Eij
[0067] Where Yij is the observed value of the growth trait, μ is the population mean, Gj is the fixed effect of genotype, and Eij is the random residual. All data are expressed as mean ± SE.
[0068] The results of association analysis between the SV molecular marker and yak growth traits are shown in Table 2. The SV molecular marker was significantly correlated with chest circumference at six months of age (P < 0.05), suggesting that it could be used as an auxiliary selection marker in yak molecular breeding. Six-month-old yaks with the DD genotype had superior chest circumference compared to those with the II and ID genotypes; the DD genotype is therefore a candidate for early selection for the six-month-old chest circumference trait.
[0069] Table 2 Results of association analysis between yak EXOC4 gene SV and growth traits
[0070]
[0071] Note: Different lowercase letters indicate significant differences (P<0.05).
[0072] 5. Application of the above SV molecular markers in cattle selection
[0073] This example uses the screened SV molecular markers as candidate molecular genetic markers and detects the genotypes of the SV molecular markers on the EXOC4 gene of different yak individuals to provide basic data for the application of marker-assisted selection of yak growth traits.
[0074] In summary, the present invention detected the SV molecular marker on the yak EXOC4 gene, and performed an association analysis on the SV molecular marker genotype of the gene and the growth traits of the yak. It was found that the SV molecular marker described in the present application was significantly correlated with the chest circumference of 6-month-old yaks (P<0.05), indicating that the molecular marker can be used as a candidate molecular genetic marker for the growth traits of yaks, and can be used for molecular-assisted breeding selection of yak growth traits, providing an effective method for the directional selection of yak growth traits.
[0075] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.
Claims
1. The use of a reagent for detecting SV molecular markers in detecting chest girth traits of yaks at 6 months of age, characterized in that: The SV molecular marker is located at positions 24135756-24135950 of chromosome 4 of the yak genome Bosgru_v3.0 version, EnsemblID: ENSBGRG00000004701; the SV molecular marker has three genotypes: II, ID and DD; among them, the SV molecular marker is amplified by PCR using the genomic DNA of the yak as a template, and a single band of 714 bp is amplified, and the genotype is defined as a homozygous insertion type II genotype; two bands of 714 bp and 519 bp are amplified, and the genotype is defined as a heterozygous ID genotype; and a single band of 519 bp is amplified, and the genotype is defined as a homozygous deletion type DD genotype.
2. The use according to claim 1, characterized in that The DD genotype is superior to the II and ID genotypes in the chest girth trait of the yaks at 6 months of age.
3. Application of a reagent for detecting SV molecular markers in early selection of chest girth traits in yaks at 6 months of age, characterized in that: The SV molecular marker is located at positions 24135756-24135950 of chromosome 4 of the yak genome Bosgru_v3.0 version, EnsemblID: ENSBGRG00000004701; the SV molecular marker has three genotypes: II, ID and DD; among them, the SV molecular marker is amplified by PCR using the genomic DNA of the yak as a template, and a single band of 714 bp is amplified, and the genotype is defined as a homozygous insertion type II genotype; two bands of 714 bp and 519 bp are amplified, and the genotype is defined as a heterozygous ID genotype; and a single band of 519 bp is amplified, and the genotype is defined as a homozygous deletion type DD genotype.
4. The use according to claim 3, characterized in that The DD genotype was selected for early selection of chest girth traits in yaks at 6 months of age.
5. The use according to any one of claims 1 to 4, characterized in that The reagents include a primer pair for amplifying a marker containing the SV molecule.
6. The use according to claim 5, characterized in that The gene sequence containing the SV molecular marker is shown in SEQ ID NO.
1.
7. The use according to claim 6, characterized in that The primer pairs are: Upstream primer: 5′-CGAAGTCCTAATAATCCCCAGA-3′; Downstream primer: 5′-TTTCTCTCATAACGTCGCCCT-3′.
8. A kit for detecting chest girth traits of yaks at 6 months of age using structural variation sites, characterized in that: The kit includes a primer pair for amplifying a SV molecular marker: Upstream primer: 5′-CGAAGTCCTAATAATCCCCAGA-3′; Downstream primer: 5′-TTTCTCTCATAACGTCGCCCT-3′.
9. Use of the kit as claimed in claim 8 in assisting the detection of chest girth traits in 6-month-old yaks.
10. Use of the kit according to claim 8 in assisting early selection of chest girth traits in yaks at 6 months of age.