Application of ATP8A1 gene mutation in selection of number of lambs of Mancheng black goats
By using the SNP marker sites of the ATP8A1 gene in goats, especially the A/G mutation at 130 bp, screening and mating individuals with AA and GA genotypes, the problem of slow increase in lamb numbers in traditional breeding was solved, efficient molecular marker assisted breeding was achieved, and the number of lambs in Macheng black goats was significantly increased.
Patent Information
- Application Number
- CN202510435334.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-08
- Publication Date
- 2025-07-04
AI Technical Summary
The prior art is difficult to effectively increase the genetic progress of goat lamb production, the reproductive performance is slowly improved, and the traditional breeding selection efficiency is low.
Using the SNP marker sites of the ATP8A1 gene, especially the A/G mutation located at 130 bp, the goat genotype was determined by designing specific primers to amplify and sequencing, individuals with AA and GA genotypes were screened for breeding, individuals with GG genotypes were eliminated, and the proportion of AA genotypes in the population was gradually increased.
The number of lamb production in Macheng black goats has been significantly improved, efficient and accurate molecular marker assisted breeding has been achieved, and reproductive performance and breeding efficiency have been improved.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of animal molecular breeding, and relates to SNP markers related to the litter size trait of Macheng black goats and their application in the breeding of Macheng black goats. Background Art
[0002] Reproductive performance is an important factor affecting the economic benefits of goats, and litter size is an important trait for measuring reproductive performance. The litter size of goats is affected by various factors, with low heritability and being controlled by polygenes with minor effects. Using traditional breeding selection techniques for breeding results in slow genetic progress. Currently, a large number of candidate genes related to the multiple-litter trait of goats have been discovered and used for the improvement of local goat breeds and the cultivation of new breeds (matching lines). In the present invention, whole-genome sequencing was performed on two individuals selected for the litter size of Macheng black goats, and a base mutation in the ATP8A1 gene was found to be significantly correlated with the litter size.
[0003] P4-ATPase is a subfamily of P-type ATPases that can flip phospholipids across the membrane to generate lipid asymmetry, which is crucial for many cellular processes. This unidirectional transport generates phospholipid asymmetry across the cell membrane and is related to various key cellular processes, including cell and organelle shape determination, cell division, vesicle budding and transport, fertilization, phagocytosis, apoptosis, etc. Research has found that specifically assembling ATP8A1 with the liposome CDC50A forms a complex that can actively transport phosphatidylethanolamine (PE) across the lipid bilayer. During the maturation of eggs, PE may regulate intracellular signal transduction by affecting the fluidity and permeability of the cell membrane, thereby promoting meiosis and other physiological and biochemical processes of the eggs, enabling the eggs to have the ability to be fertilized. The present invention discovers a new SNP mutation site in the ATP8A1 gene, and this mutation site affects the litter size of Macheng black goats, providing marker resources for molecular marker-assisted selection breeding of the litter size trait of Macheng black goats. Summary of the Invention
[0004] The present invention provides a method to solve the technical problems existing in the current breeding of goat litter size.
[0005] The present invention provides an SNP marker that affects the litter size trait of goats. This marker is located in the coding region of the goat ATP8A1 gene (sequence number: ENSCHIG00000008265) published in the Ensembl database. The G / A difference in the nucleotides at this marker site results in different litter sizes of goats.
[0006] The present invention provides a method for selecting goats for breeding according to the genotypes of the marker loci. According to an embodiment of the present invention, the SNP marker is located at the 130th base of the nucleotide sequence shown in SEQ ID NO: 1. The litter size of individuals with the AA genotype and GA genotype at this SNP marker locus is significantly higher than that of individuals with the GG genotype. By detecting the above SNP marker of Macheng Black Goats, the litter size of goats can be evaluated according to the genotype of this SNP marker. Thus, the SNP marker of the present invention is closely related to the litter size trait of Macheng Black Goats and can be effectively used for molecular marker-assisted breeding of Macheng Black Goats. Furthermore, the breeding materials of goats can be selected according to the actual breeding needs, so as to accurately and efficiently select individuals with a large litter size and improve the efficiency and accuracy of breeding.
[0007] The present invention provides a primer pair for detecting the SNP marker recited in claim 1. According to an embodiment of the present invention, the primers have the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 and are used to detect the SNP marker. According to an embodiment of the present invention, the primer pair of the present invention can effectively amplify the DNA fragment where the above SNP marker related to litter size in the goat to be tested is located. Through sequencing, the detection of such SNP marker can be effectively achieved, the genotype of the goat to be tested at this SNP marker locus can be determined, and further the litter size of the goat to be tested can be effectively predicted.
[0008] Specifically, the litter size of individuals with the AA genotype and GA genotype at the SNP marker locus is significantly higher than that of individuals with the GG genotype, which can be used as an important criterion for judging the litter size of goats. In the goat breeding work, the genomic sequence of a goat individual is amplified with the primer pair of the SNP marker of the present invention and sequenced to determine the genotype of the goat individual at the SNP marker locus. Individuals with the AA genotype at this SNP locus can be retained for breeding, and individuals with the GG genotype at this SNP marker locus can be eliminated. Alternatively, individuals with the AA genotype can be mated with individuals with the CA genotype, and individuals with the AA genotype are selected for breeding in the hybrid offspring, gradually increasing the number of goat individuals with the AA genotype at the molecular marker locus in the population, thereby gradually increasing the litter size of the goat population and realizing molecular marker-assisted breeding for selecting goat individuals with a large litter size at low cost and high accuracy.
[0009] The present invention has the following beneficial effects: (1) The SNP marker provided by the present invention is significantly related to the litter size of Macheng Black Goats. The litter size of goats with the AA genotype and GA genotype is significantly higher than that of goats with the GG genotype. (2) This SNP marker can be used for the assisted selection of the litter size trait of Macheng Black Goats, screening Macheng Black Goats with a high litter size, which has important practical application value for further improving the fertility of Macheng Black Goats and using specific Macheng Black Goats as materials for variety (or strain) breeding. Brief Description of the Drawings
[0010] The above aspects of the present invention will be more easily understood in conjunction with the accompanying drawings for the description of the embodiments. Figure 1 To show the sequencing peak maps of AA, GA, and GG genotypes at the SNP marker locus of the present invention. Specific Embodiments
[0011] The embodiments of the present invention will be described in detail below. The present invention will be further described in detail in conjunction with the embodiments. The embodiments herein are only used to illustrate the present invention and should not be construed as a limitation to the present invention.
[0012] 1. Experimental Samples 141 adult female Macheng black goats from Hubei Jinyang (Macheng) Animal Husbandry Co., Ltd. (with lambing records of one to five litters) were used, and their feeding management conditions and environmental conditions were the same.
[0013] 2. Genomic DNA Extraction The jugular vein blood of the above samples (5 mL / animal) was collected using a disposable vacuum negative pressure blood collection tube (EDTA-K2 anticoagulant), and the genomic DNA of the Macheng black goat blood samples was extracted using a blood genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd.
[0014] 3. Primer Design According to the sequence of the goat ATP8A1 gene (gene sequence number in the Ensembl database: ENSCHIG00000008265), a pair of specific primers SEQ ID NO:2 and SEQ ID NO:3 were designed using Primer 6.0 software. The primers were synthesized by Beijing Tsingke Biotechnology Co., Ltd. and were used to amplify a DNA sequence where exon 25 of the ATP8A1 gene is located. The amplification product is 968 bp, and this DNA sequence is shown as SEQ ID NO:1 in the sequence listing.
[0015] 4. PCR Amplify the Target Sequence of the ATP8A1 Gene of the Sample Goat DNA and Determine the Genotype (1) PCR Amplification System (20 μL): 2 μL of DNA, 0.5 μL each of primers SEQ ID NO:2 and SEQ ID NO:3 (10 μM), 10 μL of 2×M5 HiPerplus Taq HiFi PCR mix (Beijing Polymer Beauty Biotechnology Co., Ltd.), 7 μL of ddH2O. Amplification program: Pre-denaturation at 95 °C for 3 min; denaturation at 94 °C for 25 s, annealing at 57.5 °C for 25 s, extension at 72 °C for 14 s, 35 cycles; extension at 72 °C for 5 min. (2) The PCR amplification products were sent to Wuhan Kingcare Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using SnapGene software to determine the genotype of the individual at the 130th bp site of the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing. As Figure 1 shown, the genotype of the single peak A is AA, the genotype of the single peak G is GG, and the genotype of the double peak is GA.
[0016] At the SNP marker site at the 130th bp of SEQ ID NO:1 in the sequence listing, the base G mutated to A, changing the codon encoding aspartic acid (Asp) to the codon encoding asparagine (Asn).
[0017] 5. Association analysis of the SNP marker of the ATP8A1 gene in Macheng black goats with litter size One-way analysis of variance in SPSS software was used to conduct an association analysis between the genotype and the litter size trait. The specific linear analysis model is as follows: Yij = μ + Gi + Eij Where: Y ij is the individual phenotypic record; μ is the population mean; G i is the genotype effect; E ij is the random error.
[0018] 6. Analysis of the significance of differences in litter size among different genotypes of Macheng black goats The analysis results of the litter size traits of different genotypes of Macheng black goats are shown in Table 1. It can be seen from Table 1 that there are three genotypes at this locus. One-way analysis of variance was used to compare the differences in litter size among different genotypes. It was found that the litter sizes of Macheng black goats with AA genotype and GA genotype were significantly higher than those with GG genotype (p < 0.05), indicating that the AA genotype and CA genotype of this SNP marker site can be used as important criteria for judging high litter size in Macheng black goats. In the breeding work of Macheng black goats, individuals with the AA genotype of this SNP marker site can be retained for breeding, and individuals with the GG genotype can be eliminated. The CA genotype can also be mated with individuals with the AA genotype to obtain more AA-type offspring individuals, increasing the proportion of individuals with the AA genotype in the Macheng black goat population, thereby gradually increasing the litter size of the Macheng black goat population. Table 1 Correlation between different genotypes of the ATP8A1 gene mutation site in Macheng black goats and litter size Note: Different superscript letters in the same column indicate significant differences (p < 0.05).
Claims
1. A molecular marker associated with the number of lambs born in a goat, the molecular marker being located at the nucleotide sequence SEQ ID NO: 1, wherein the 130 bp position of the sequence has a G / A polymorphism, and the number of lambs born in goats with AA genotype and GA genotype at the position is significantly higher than that of goats with GG genotype.
2. A method for selecting goats according to the molecular marker of claim 1, comprising the following steps: (1) Extracting goat genomic DNA; (2) using two specific primers to perform PCR amplification to obtain a 968 bp amplification product, wherein the sequences of the two specific primers are SEQ ID NO: 2 and SEQ ID NO: 3; (3) sequencing the PCR amplification product to obtain a sequencing result; (4) determining the genotype of the goat individual to be tested at the molecular marker according to the sequencing result; (5) selecting goat individuals with the AA genotype of the above molecular markers for seed preservation; (6) The goat is a Macheng black goat.
3. Use of the molecular marker according to claim 1 in screening Macheng black goats with high lamb production, and selecting goat individuals with the AA genotype of the molecular marker for breeding.