Application of SNP marker for improving weaning weight of charollais sheep

Abstract

This invention discloses the application of a SNP marker in the EGFR gene that affects weaning weight in sheep. The marker is located in the EGFR gene on sheep chromosome 19, specifically at the T / C base mutation at 367 bp of SEQ ID NO:1 in the sequence listing. Sheep with the TT and TC genotypes at this site have significantly higher weaning weights (at 60 days of age) than those with the CC genotype. Using Charolais sheep as the research subject, this invention utilizes PCR amplification of SNP variations in exon 14 of the sheep EGFR gene to analyze the effect of specific genotypes at this variation site on weaning weight in Charolais sheep. Based on this analysis, selective breeding of Charolais sheep can be conducted to improve weaning weight, providing marker resources for marker-assisted selection breeding of sheep growth traits.

Description

Technical Field

[0001] This invention belongs to the field of animal molecular breeding technology and relates to the application of SNP markers related to weaning weight in Charolais sheep breeding. Background Technology

[0002] Growth rate directly affects mutton yield, making it a key trait in sheep breeding. Traits reflecting growth rate include birth weight, weaning weight, and adult weight. Weaning weight is a crucial indicator of sheep production performance; a higher weaning weight leads to higher lamb survival rates, faster later growth, shorter production cycles, and higher production efficiency. Molecular marker-assisted selection (MAG) technology enables early selection of individual livestock, offering high efficiency and accuracy. Therefore, utilizing this technology to select sheep for optimal weaning weight is of great significance.

[0003] Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein involved in physiological processes such as cell proliferation and differentiation, playing a crucial role in animal development, growth, and tissue regeneration. In pearl oysters, high expression of this gene is highly correlated with growth traits, while reduced gene expression decreases carcass and muscle traits in sheep. The Charolais sheep is a world-renowned meat sheep breed, an ideal sire breed, and an important subject for studying the molecular mechanisms of sheep growth traits. This invention discovers an association between a SNP marker in exon 14 of the EGFR gene and the weaning weight (60 days old) trait in Charolais sheep, providing a valuable marker resource for marker-assisted selection breeding of Charolais sheep growth traits. Summary of the Invention

[0004] This invention provides a technical method for selecting and breeding sheep with the correct weaning weight.

[0005] According to an embodiment of the present invention, the SNP marker is located at base 367 of the nucleotide sequence shown in SEQ ID NO:1. Charolais sheep individuals with the TT and TC genotypes at this SNP marker site have significantly higher weaning weights (at 60 days of age) than those with the CC genotype. By detecting the above-mentioned SNP marker in Charolais sheep, weaning weight can be effectively increased, thereby enabling the assessment of early growth rate of Charolais sheep individuals based on the genotype of this SNP marker site. Therefore, the SNP marker of the present invention can be effectively used for marker-assisted selection breeding of Charolais sheep, thereby enabling the selection of sheep breeding stock according to actual breeding needs, and thus accurately and efficiently selecting individuals with superior weaning weights, improving the efficiency and accuracy of breeding selection.

[0006] This invention provides a primer pair for detecting the SNP marker. According to an embodiment of the invention, the primers have the nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:3. Using this primer pair, the fragment containing the aforementioned SNP marker related to weaning weight in a Charlotte sheep can be effectively amplified. Sequencing can then effectively detect this SNP marker, determine the genotype of the Charlotte sheep at the SNP marker site, and thus effectively predict the weaning weight of the Charlotte sheep.

[0007] Specifically, the TT and TC genotypes at the aforementioned SNP marker loci can serve as important criteria for determining the weaning weight of Charolais sheep. In Charolais sheep breeding, individuals with the TT genotype at this SNP marker locus can be retained for breeding, while individuals with the CC genotype at this SNP marker locus can be culled. Alternatively, individuals with the TC genotype can be mated with individuals with the CC genotype to obtain more TT genotype offspring, thereby gradually increasing the weaning weight of the Charolais sheep population and achieving low-cost, high-accuracy selection of Charolais sheep with superior weaning weights.

[0008] The present invention has the following beneficial effects: (1) The SNP marker provided by the present invention is significantly correlated with the weaning weight (60 days old) of Charolais sheep, and the weaning weight of TT and TC genotype sheep is significantly higher than that of CC genotype sheep. (2) The SNP marker can be used for auxiliary selection of growth traits in Charolais sheep, screening out Charolais sheep with high weaning weight, which has important practical application value for further improving the growth rate of Charolais sheep and using Charolais sheep as a specific sire for breed selection. Attached Figure Description

[0009] The description of the embodiments will be more readily understood in conjunction with the above aspects of the invention and the accompanying drawings. Figure 1 The sequencing peak diagrams of the TT, TC, and CC genotypes of the SNP marker sites of this invention are shown, with the blue background area representing the SNP site. Specific Implementation

[0010] The embodiments of the present invention are described in detail below. The present invention will be further described in detail with reference to the embodiments. The embodiments are only used to illustrate the present invention and should not be construed as limiting the present invention.

[0011] 1. Experimental Samples

[0012] The 195 Charolais sheep were provided by Haofeng Sheep Breeding Cooperative in Zhangwu County, Fuxin City, Liaoning Province. They were growing and developing well and were healthy. Their feeding environment and conditions were consistent, and their birth weight and weaning weight were recorded.

[0013] 2. Genomic DNA extraction

[0014] Blood from the jugular vein of the above-mentioned sample (5 mL / animal) was collected using a disposable vacuum negative pressure blood collection tube (EDTA-K2 anticoagulant). Genomic DNA was extracted from the blood sample of Charolais sheep using the blood genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0015] 3. Primer design

[0016] Based on the sheep EGFR gene sequence (Ensembl database gene sequence number: ENSOARG00020016388), a pair of specific primers, SEQ ID NO:2 and SEQ ID NO:3, were designed using Primer 5.0 software. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. These specific primers were used to amplify a DNA sequence containing the fourteenth exon of the EGFR gene. The amplification product was 1137 bp, and the nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing.

[0017] 4. PCR amplification and sequencing of the target sequence of the sheep EGFR gene.

[0018] (1) PCR amplification system (25 μL): DNA 2 μL (20 ng / μL), primers SEQ ID NO:2 and SEQ ID NO:3 (10 μM) 1 μL each, 2×Rapid Taq Master Mix 12.5 μL (Nanjing Novizan Biotechnology Co., Ltd.), ddH2O 8.5 μL. Amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 17 s, 35 cycles; 72℃ extension for 5 min.

[0019] (2) The PCR amplification products were sent to Wuhan Jinkairui Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using SnapGene software to determine the genotype of the individual at the 367bp site of the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing. Figure 1 As shown, the genotype of unimodal T is TT, the genotype of unimodal C is CC, and the genotype of bimodal is TC.

[0020] 5. Association analysis between EGFR gene SNP markers and weaning weight at 60 days of age in Charolais sheep

[0021] One-way ANOVA was used in SPSS software to conduct an association analysis between genotype and weaning weight (60 days of age). The specific linear analysis model is as follows:

[0022] Y ij =μ+G i +E ij

[0023] Where: Yij For individual phenotypic records; μ is the population mean; G i Genotype effect; E ij This is random error.

[0024] 6. Significant difference in weaning weight among different genotypes of Charolais sheep

[0025] The weaning weight (60 days old) of Charolais sheep with different genotypes is shown in Table 1. Table 1 shows that there are three genotypes at this locus. One-way ANOVA comparing the differences in weaning weight among different genotypes revealed that the weaning weight of Charolais sheep with the TT and TC genotypes was significantly higher than that of the CC genotype (p<0.05). The weaning weight of Charolais sheep with the TC genotype was not different from that of sheep with the TT genotype. This indicates that the TT and TC genotypes at this SNP marker locus can serve as important criteria for judging high weaning weight in Charolais sheep. In the breeding of Charolais sheep, individuals with the TT genotype at this SNP marker locus can be retained for breeding, while individuals with the CC genotype can be culled. Alternatively, individuals with the TC and TT genotypes can be crossbred to obtain more TT offspring, thereby gradually increasing the weaning weight of the Charolais sheep population.

[0026] Table 1. Correlation between different genotypes of EGFR gene mutation sites in Charolais sheep and weaning weight at 60 days of age.

[0027]

[0028] Note: Different lowercase letters in the superscript of data in the same column indicate significant differences (P<0.05).

Claims

1. A method for selecting sheep with high weaning weight at 60 days of age based on the genotype of SNP molecular marker loci associated with weaning weight at 60 days of age, characterized in that, The SNP molecular marker site is located at 367bp of the nucleotide sequence SEQ ID NO:

1. This site indicates that the 60-day weaning weight of sheep individuals with TT and TC genotypes is significantly higher than that of individuals with CC genotypes. The method includes the following steps: (1) Extract sheep genomic DNA; (2) PCR amplification was performed using two specific primers to obtain an amplification product of 1137bp. The sequences of the two specific primers are SEQ ID NO:2 and SEQ ID NO:

3. (3) Sequencing the PCR amplification products to obtain sequencing results; (4) Determine the genotype of the sheep individual to be tested at the SNP molecular marker locus based on the sequencing results; (5) Select sheep individuals with the TT genotype at the above SNP molecular marker loci for breeding; The sheep in question are Charolais sheep.

2. To test the application of the genotype of the SNP molecular marker locus described in claim 1 in screening for high 60-day-old weaning weight Charolais sheep, and to select Charolais sheep individuals with the TT genotype of the SNP molecular marker locus for breeding.

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