Method for predicting first-birth lambing number of Hu sheep and application thereof
By identifying the genotype of SNP marker loci in the EPAS1 gene, the problem of low lamb number in the plateau environment was solved, efficient screening and breeding were achieved, and the reproduction performance of lake sheep was significantly improved.
Patent Information
- Application Number
- CN202411534854.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-20
- Publication Date
- 2024-12-13
AI Technical Summary
In the plateau environment, the number of lamb production in lake sheep is affected by the low oxygen environment, resulting in a decline in breeding performance. It is difficult for the existing technology to effectively increase the number of lamb production in lake sheep.
By discovering SNP marker sites related to the number of lambs in the first birth of lake sheep in the EPAS1 gene, specific primer pairs were designed for PCR amplification and sequencing, TC and CC genotypes were identified to predict and screen individuals with high lambs. Molecular marker assisted breeding is then carried out.
It significantly improves the number of lambs born in the first child of the lake sheep, shortens the breeding cycle, improves the accuracy and efficiency of seed selection, is easy to operate and low-cost, and is suitable for large-scale screening.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of animal molecular breeding, and relates to the application of SNP markers related to the trait of litter size of Hu sheep in Hu sheep breeding. Background Art
[0002] The number of lambs born is a key indicator to measure the reproductive capacity of sheep and has a direct impact on the economic benefits of sheep farming. Hu sheep is a fine local sheep breed in China, famous for its high reproductive rate and strong adaptability. In the plateau environment, the low oxygen environment affects the physiological state of the ewes, especially during pregnancy, when ewes need a higher oxygen supply to maintain the normal development of themselves and multiple fetuses. Therefore, insufficient oxygen supply in the plateau environment may reduce the number of lambs born by Hu sheep, which poses new challenges and demands for maintaining and improving the reproductive performance of Hu sheep.
[0003] EPAS1, also known as hypoxia-inducible factor 2α (HIF-2α), is an important member of the hypoxia-inducible factor family. This protein is an oxygen-dependent transcription factor that can sense and respond to changes in intracellular oxygen concentration. Under normal oxygen levels, HIF-2α protein is regulated by proline hydroxylases (PHDs) and is ubiquitinated and degraded; under hypoxic conditions, HIF-2α is in a stable state, enters the cell nucleus, binds to HIF-1β, and activates the expression of multiple hypoxia response-related genes. EPAS1 plays a key role in physiological processes such as erythropoiesis, iron metabolism, and angiogenesis. At high altitudes, the oxygen level in the air is reduced. This protein can enhance blood oxygen levels and improve oxygen delivery efficiency, helping the body adapt to low oxygen environments.
[0004] In sheep production, the number of lambs born in the first litter directly affects the early income of the breeder. The present invention discovered SNP markers in the EPAS1 gene that are related to the number of lambs born in the first litter of Hu sheep, indicating that the EPAS1 gene is involved in the reproduction of Hu sheep under hypoxic conditions, and also provides genetic marker resources for marker-assisted selection of the number of lambs born in Hu sheep. Summary of the invention
[0005] The invention provides a method for solving the technical problem of the existing sheep breeding lamb number.
[0006] According to an embodiment of the present invention, the SNP marker is located at the 320th base of the nucleotide sequence shown in SEQ ID NO: 1, and the number of first-born lambs of Hu sheep individuals with TC and CC genotypes at the SNP marker site is significantly higher than that of individuals with TT genotypes. By detecting the above-mentioned SNP marker of Hu sheep, the number of first-born lambs can be effectively predicted, so that the first-born lambing performance of Hu sheep individuals can be evaluated according to the genotype of the SNP marker site. Therefore, the SNP marker of the present invention is closely related to the quantitative trait of the first-born lambs of Hu sheep, and can be effectively applied to the molecular marker-assisted breeding of Hu sheep, and then the sheep breeding materials can be selected according to the actual breeding needs, so as to accurately and efficiently screen out high-quality individuals with multiple lambs, and improve the efficiency and accuracy of breeding selection.
[0007] The present invention provides a primer pair for detecting the SNP marker of claim 1. According to an embodiment of the present invention, the primer pair has the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence table, and is used for detecting the SNP marker.
[0008] According to an embodiment of the present invention, the present invention provides a method for predicting the number of lambs produced in the first litter of sheep. The primer pair, amplification program and amplification system of the present invention can effectively amplify the fragment where the SNP marker related to the first litter number trait of the tested Hu sheep is located, and the detection of this SNP marker can be effectively achieved by sequencing, and the genotype of the SNP marker site of the tested Hu sheep can be determined, so as to effectively predict the number of lambs produced in the first litter of the tested Hu sheep. Specifically, the TC and CC genotypes of the SNP marker site can be used as an important criterion for judging the number of lambs produced in the first litter of Hu sheep. In the Hu sheep breeding work, sheep with the CC genotype of the SNP marker site genotype can be retained for breeding, and TC genotype individuals can also be mated with CC genotype individuals to obtain more CC type offspring individuals, and sheep with the genotype of the SNP marker site TT can be eliminated. Therefore, the present invention can be effectively applied to the molecular marker-assisted selection breeding of Hu sheep, so as to screen out Hu sheep individuals with a large number of lambs produced in the first litter at low cost and high accuracy.
[0009] The present invention has the following beneficial effects: (1) The SNP markers provided by the present invention are significantly correlated with the number of first-born lambs of Hu sheep, and the number of first-born lambs of sheep with TC and CC genotypes are significantly higher than that of sheep with TT genotypes. (2) By identifying the above-mentioned specific SNP markers, individuals with high yield potential can be screened out at an early stage, which can significantly shorten the breeding cycle, accelerate the breeding process of the first-born lambs of sheep, and improve the accuracy and efficiency of selection. (3) The detection method provided by the present invention is simple to operate, and the genotype of the target SNP site can be accurately detected by PCR amplification and conventional sequencing technology. This method is not only low-cost, but also suitable for large-scale screening and application, and has a good prospect for promotion and application. Specific embodiments
[0010] The embodiments of the present invention are described in detail below, and the present invention is further described in detail in conjunction with the embodiments. The embodiments herein are only used to illustrate the present invention and are not to be construed as limiting the present invention.
[0011] 1. Experimental samples The experimental samples in this embodiment were obtained from Hubei Zhiqinghe Agriculture and Animal Husbandry Co., Ltd., and blood samples were collected from 84 adult Hu sheep ewes, whose feeding and management conditions and environmental conditions were consistent. Jugular vein blood (5 mL / goat) was collected using a disposable vacuum negative pressure blood collection tube (EDTA-K2 anticoagulation), and the samples were stored at -20°C.
[0012] 2. Genomic DNA Extraction The genomic DNA from the blood samples of Hu sheep was extracted using the phenol-chloroform method. The extracted DNA was tested for quality using 1.0% agarose gel to ensure that the integrity and concentration of the DNA sample were suitable for subsequent experiments.
[0013] 3. Primer Design According to the sequence of sheep EPAS1 gene (Ensembl database gene sequence number: ENSOARG00000005722), a pair of specific primers SEQ ID NO: 2 and SEQ ID NO: 3 were designed using the NCBI online primer design tool. The primers were synthesized by Wuhan Jinkairui Biotechnology Co., Ltd. The specific primers were used to amplify a specific DNA sequence of the EPAS1 gene. The amplified product was 539 bp, and the DNA sequence was shown in SEQ ID NO: 1 in the sequence table.
[0014] 4. PCR amplification and sequencing of sheep EPAS1 gene target sequence (1) PCR amplification system (25 μL): DNA 1 μL, primers SEQ ID NO: 2 and SEQ ID NO: 3 (10 μM) 1 μL each, 2×Rapid Taq Master Mix 12.5 μL (Beijing Quanshijin Biotechnology Co., Ltd.), ddH2O 9.5 μL. Amplification program: 95°C pre-denaturation for 5 min, 95°C denaturation for 40 s, 60°C annealing for 40 s, 72°C extension for 5 min, 35 cycles. (2) The PCR amplification product was sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. The sequencing results were read using the SeqMan tool in DNASTAR to determine the genotype of the individual at the 320 bp position of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence list.
[0015] 5. Association analysis between EPAS1 gene SNP markers and litter size in Hu sheep The association analysis between genotype and litter size was analyzed using SAS8.1 software. The specific mixed linear model is as follows: Y ij =μ+G i +e ij where Y ij is the number of lambs born in the first litter; μ is the population mean; G j is the genotype effect; e ij is the random effect residual.
[0016] 6. Analysis of the significance of the difference in the number of lambs born among Hu sheep with different genotypes The results of the analysis of the number of lambs of different genotypes of Hu sheep are shown in Table 1. As can be seen from Table 1, there are three genotypes at this locus, and the difference in the number of lambs between different genotypes was compared by one-way analysis of variance. According to the analysis results, it was found that the number of first-born lambs of Hu sheep with TC and CC genotypes was significantly higher than that of TT genotype (p<0.05). This shows that the TC and CC genotypes of this SNP marker locus can be used as an important criterion for judging the high number of first-born lambs of Hu sheep. In the process of Hu sheep breeding, sheep with SNP markers of CC genotypes are selected and retained for breeding, and individuals with TT genotypes are eliminated. TC genotype individuals can also be bred with CC genotype individuals to obtain more CC genotype offspring individuals, thereby gradually increasing the number of first-born lambs of the Hu sheep population. Table 1. Correlation between the genotypes of different SNP loci of EPAS1 gene and the number of first litters of Hu sheep Note: Different lowercase letters in the same column indicate significant differences (P<0.05).
Claims
1. A method for selecting sheep based on a molecular marker associated with the lamb number trait of sheep, wherein the molecular marker is located in the nucleotide sequence SEQ ID NO: 1, wherein the number of first-born lambs of sheep individuals with TC and CC genotypes at the 320 bp position of the sequence is significantly higher than that of individuals with TT genotypes; The method comprises the following steps: (1) Extracting sheep genomic DNA; (2) using two specific primers to perform PCR amplification to obtain a 539 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 individual sheep to be tested at the molecular marker according to the sequencing results; (5) selecting sheep individuals with the CC genotype of the above molecular marker for breeding; (6) The sheep are Hu sheep.
2. Use of the SNP marker according to claim 1 in screening Hu sheep with high first-baby lamb production, and selecting sheep individuals with CC genotypes with the molecular marker for breeding.