Application of PTPRK gene mutation in sheep colostrum yield selection

Abstract

The invention discloses application of an SNP (Single Nucleotide Polymorphism) marker influencing the colostrum yield of sheep in a PTPRK gene. The marker is located on a PTPRK gene of a sheep chromosome 8, the specific SNP marker site is C / T base mutation at the 488 bp position of SEQ ID NO: 1 in a sequence table, and the colostrum yield of TT and CT genotype sheep individuals at the site is remarkably higher than that of CC genotype individuals. Hu sheep is used as a research object, PCR is used for amplifying a DNA sequence of an eighth exon of a sheep PTPRK gene, the sequence has an SNP variation site, the colostrum yield of a specific genotype Hu sheep individual at the variation site is analyzed, and the Hu sheep individual is selected and reserved for breeding according to the colostrum yield, so that excellent individuals with high colostrum yield are rapidly screened, the breeding process of a high colostrum yield population is accelerated, and the breeding efficiency of the sheep PTPRK gene is improved. A marker resource is provided for marker-assisted selection breeding of the colostrum yield of the sheep.

Description

Technical Field

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

[0002] Colostrum is the core source of nutrition and passive immune protection for newborn lambs. Its yield directly determines the early survival rate, health level, and later production performance of lambs, and is a key factor affecting the large-scale development and economic benefits of the sheep farming industry. Therefore, breeding sheep breeds with high colostrum yield has become an important research direction in the field of intensive sheep breeding. Colostrum yield is a typical quantitative trait, regulated by multiple genes. Traditional phenotypic selection breeding has drawbacks such as long cycle, limited accuracy, and low selection efficiency, making it difficult to meet the needs of industrial upgrading of intensive sheep breeding. Molecular marker-assisted selection (MAS) technology, based on the correlation between the genetic polymorphism of functional genes and target traits, can achieve early and precise screening of individual livestock, significantly shortening the breeding cycle and improving the efficiency of genetic improvement, providing an effective way to solve the bottlenecks of traditional breeding.

[0003] The PTPRK gene is an important member of the protein tyrosine phosphatase (PTP) family. Its encoded protein participates in a series of key biological processes, including cell proliferation, differentiation, adhesion, and signal transduction, by regulating intracellular tyrosine phosphorylation levels. The PTPRK gene is widely expressed in neural and epithelial tissues; downregulation of its expression significantly enhances cell proliferation and invasion. Furthermore, the protein expressed by this gene inhibits excessive keratinocyte proliferation in response to the TGF-β1 signaling pathway. Current research on the PTPRK gene mainly focuses on cell biology and human diseases, with no reported applications in livestock breeding. This invention discovers that a SNP marker on exon 8 of the PTPRK gene is significantly correlated with colostrum production in Hu sheep, providing a new genetic marker resource for marker-assisted selection of colostrum production in Hu sheep. This has significant practical implications for promoting innovation in sheep breeding technology and accelerating the development of superior breeds with high colostrum production. Summary of the Invention

[0004] This invention aims to provide a technical method for breeding sheep colostrum production.

[0005] According to an embodiment of the present invention, the SNP marker is located at base 488 of the nucleotide sequence shown in SEQ ID NO:1. Individuals with the TT and CT genotypes of this SNP marker have significantly higher colostrum production than individuals with the CC genotype. By detecting the above-mentioned SNP marker in Hu sheep, the colostrum production of sheep can be effectively increased, thereby assessing the colostrum production of individual Hu sheep based on the genotype of the SNP marker locus. Therefore, the SNP marker of the present invention can be effectively used for molecular marker-assisted breeding of Hu sheep, thereby enabling the selection of sheep breeding materials according to actual breeding needs, and thus accurately and efficiently selecting sheep individuals with higher colostrum production, 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 the primer pair of this invention, the DNA fragment containing the aforementioned colostrum-related SNP marker in the sheep to be tested can be effectively amplified. Sequencing can then effectively detect this SNP marker, determine the genotype of the sheep at the SNP marker site, and thus effectively predict the colostrum yield of the sheep.

[0007] Specifically, individuals with the TT and CT genotypes at the aforementioned SNP locus produced significantly higher colostrum yields than those with the CC genotype, serving as an important criterion for judging sheep colostrum production. In sheep breeding, individuals with the TT genotype at this SNP locus can be retained for breeding, while individuals with the CT and CC genotypes at this SNP locus can be culled. Alternatively, CT genotype individuals can be mated with TT genotype individuals to obtain more TT genotype offspring, achieving low-cost and high-accuracy selection of superior sheep breeds, thereby gradually increasing the colostrum yield of the sheep population.

[0008] The present invention has the following beneficial effects: (1) The SNP marker provided by the present invention is significantly correlated with the colostrum production of Hu sheep, and the colostrum production of TT and CT genotype sheep is significantly higher than that of CC genotype sheep; (2) The SNP marker can be used for auxiliary selection of sheep colostrum production, screening out Hu sheep with high colostrum production, which has important practical application value for further improving the colostrum production of Hu sheep and using Hu sheep as material for breed (or strain) 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 SNP markers TT, CT, and CC genotypes of this invention are shown. 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 Hubei Zhiqinghe Livestock Co., Ltd. provided its breeding ewes with standardized feed and free access to water from July to August 2023, and ensured they received routine vaccinations. The ewes were completely isolated from their lambs throughout the postpartum period. Ewes were allowed to nurse five times daily, and lamb weight was recorded before and after each feeding. The weight difference before and after each feeding was counted as the colostrum yield per feeding. Colostrum was manually collected from both mammary glands every 12 hours and the yield was measured using an electronic balance. The daily colostrum yield per ewe was the sum of the weight difference before and after five lamb feedings and the colostrum yield from two milkings. The average colostrum yield on days 2 and 3 was taken as the colostrum yield per ewe.

[0012] 2. Genomic DNA extraction Blood samples (5 mL / sheep) were collected from the above-mentioned ewes using vacuum negative pressure blood collection tubes anticoagulated with dipotassium edetate (EDTA-K2) and stored at -20℃. Genomic DNA was extracted from the sheep blood samples using the blood genomic extraction kit from Kangwei Century Biotechnology Co., Ltd. The DNA concentration was determined and diluted to 50 mg / mL, then stored at 4℃.

[0013] 3. Primer design Based on the sheep PTPRK gene sequence (Ensembl database gene sequence number: ENSOARG00020004343), a pair of specific primers, SEQ ID NO:2 and SEQ ID NO:3, were designed using Primer 6.0. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and were used to amplify a DNA sequence containing exon 8 of the PTPRK gene. The amplification product was 948 bp, and the nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing.

[0014] 4. PCR amplification of the target sequence of the sheep PTPRK gene and determination of genotype. (1) PCR amplification system (20 μL): 2 μL DNA (50 mg / mL), 0.5 μL each of forward and reverse primers SEQ ID NO:2 and SEQ ID NO:3 (100 uM), 10 μL of 2×M5 HiPer plus Taq HiFi PCR mix (Beijing Jumei Biotechnology Co., Ltd.), and 7 μL of ddH2O. The amplification program was as follows: 95℃ pre-denaturation for 3 min, 94℃ denaturation for 25 s, 55℃ annealing for 25 s, 72℃ extension for 10 s, 35 cycles, and a final extension at 72℃ for 5 min.

[0015] (2) The PCR amplification products were sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using SnapGene software to determine the genotype of the individual at the 488bp site of the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing, such as... Figure 1 As shown, the genotype for unimodal T is TT, the genotype for unimodal C is CC, and the genotype for bimodal T is CT. A mutation from C to T changes the encoded arginine (Arg) to histidine (His).

[0016] 5. Association analysis between PTPRK gene SNP markers and colostrum yield in Hu sheep One-way ANOVA in SPSS software was used to conduct an association analysis between genotype and colostrum yield. The specific linear analysis model is as follows: Yij=μ+Gi+Eij Where: Yij is the individual phenotypic record; μ is the population mean; Gi is the genotype effect; and Eij is the random error.

[0017] 6. Significance analysis of differences in colostrum production among different genotypes of Hu sheep The analysis results of colostrum production by different genotypes in Hu sheep are shown in Table 1. Table 1 shows that there are three genotypes at this locus. One-way ANOVA was used to compare the differences in average colostrum production among different genotypes, revealing that sheep with the TT and CT genotypes had significantly higher colostrum production than those with the CC genotype (p < 0.05). This indicates that the TT genotype at this SNP marker locus can serve as an important criterion for judging colostrum production in sheep. In Hu sheep breeding, individuals with the TT genotype at this locus can be retained, while individuals with the CT and CC genotypes can be culled. Alternatively, CT genotype individuals can be mated with TT genotype individuals to obtain more TT genotype offspring, thereby gradually increasing the colostrum production of the sheep population.

[0018]

Claims

1. A method for selecting sheep with high colostrum production based on the genotype of SNP molecular marker sites associated with colostrum production, wherein the SNP molecular marker sites are located at 488bp of the nucleotide sequence SEQ ID NO:1, and the colostrum production of sheep individuals with TT and CT 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 a 948bp amplification product. 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 Hu sheep.

2. To test the application of the genotype of the SNP molecular marker locus described in claim 1 in screening Hu sheep with high colostrum production, and to select Hu sheep individuals with the TT genotype of the SNP molecular marker locus for breeding.

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