Application of LTF gene mutation in colostrum yield selection of sheep

Abstract

The application discloses an application of a SNP marker in LTF gene for selection of colostrum yield traits of sheep. The marker is located in the LTF gene on chromosome 19 of sheep, and the specific SNP marker is a T / C base mutation at the 480th bp in SEQ ID NO: 1 in the sequence listing; the colostrum yield of a sheep individual with a TT genotype is significantly higher than that of an individual with a CC genotype. The application takes sheep as a research object, amplifies a DNA sequence of the 4th exon of the LTF gene of sheep by PCR, analyzes the effect of a specific genotype of the variation site on the colostrum yield of sheep, and accordingly selects and keeps sheep individuals for breeding, so as to rapidly screen excellent individuals with high colostrum yield, accelerate the breeding process of a population with high colostrum yield, and provide a marker resource for marker-assisted selection breeding of colostrum yield of 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 from ewes is rich in bioactive substances such as immunoglobulins, lactoferrin, and growth factors. It is a core nutrient source for newborn lambs to acquire passive immunity and maintain early growth and development. Its yield directly affects lamb survival rate, health status, and subsequent growth performance. Colostrum yield is a core quantitative trait for measuring the reproductive performance and production potential of ewes, and breeding sheep breeds (or populations) with high colostrum yield has become an important goal in current indoor sheep breeding. Molecular marker-assisted selection technology can achieve early selection of individual livestock, with the characteristics of fast selection efficiency and high accuracy. This invention discovers that the lactoferrin (LTF) gene is an important gene affecting sheep colostrum yield.

[0003] The lactoferrin (LTF) gene is a key functional gene of the animal's innate immune system. It encodes lactoferrin, which has multiple biological functions, including antibacterial, antiviral, and iron-binding and transport functions. LTF is widely distributed in neutrophil granules and secretions such as colostrum, milk, and saliva, and is closely related to lactation-related traits in animals. Polymorphisms in the bovine LTF gene (especially promoter region variations) are significantly associated with traits such as milk somatic cell number, lactoferrin concentration, milk yield, and disease resistance, making it an important marker resource for molecular breeding of lactation traits in dairy cows. Compared to cattle, there are no studies on the relationship between LTF gene polymorphisms and key lactation traits such as colostrum yield in sheep. The scarcity of relevant molecular marker resources limits the genetic improvement of colostrum yield in sheep. This invention discovers a SNP marker on exon 4 of the LTF gene that is significantly associated with colostrum yield in Hu sheep, providing an efficient and reliable genetic marker resource for marker-assisted selection breeding of colostrum yield in Hu sheep. This is of great significance for promoting the upgrading of sheep breeding technology and cultivating superior breeds with high colostrum yield. 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 the 480th base of the nucleotide sequence shown in SEQ ID NO:1. Individuals with the TT genotype 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, colostrum production 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 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 genotype at the aforementioned SNP locus have significantly higher colostrum production than those with the CC genotype, which can serve 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 TC and CC genotypes at this SNP locus can be culled. Alternatively, TC 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 production of sheep populations.

[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 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 TT, TC, and CC genotypes of the SNP marker sites 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 LTF gene sequence (Ensembl database gene sequence number: ENSOARG00020025074), 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 4 of the LTF gene. The amplification product was 968 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 LTF 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: 95℃ pre-denaturation for 3 min, 94℃ denaturation for 25 s, 55℃ annealing for 25 s, 72℃ extension for 10 s, 35 cycles, and 72℃ extension for 5 min. (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 480bp 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 TC. A mutation from T to C changes the encoded methionine (Met) to threonine (Thr).

[0015] 5. Association analysis of LTF 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.

[0016] 6. Significance analysis of differences in colostrum yield 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 comparing the differences in colostrum production among different genotypes revealed that sheep with the TT genotype 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 TC and CC genotypes at this locus can be culled. Alternatively, TC 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.

Claims

1. A method for selecting sheep with high colostrum production based on the genotype of a SNP molecular marker site associated with colostrum production, wherein the SNP molecular marker site is located at 480 bp of the nucleotide sequence SEQ ID NO:1, and the colostrum production of sheep individuals with the TT genotype is significantly higher than that of individuals with the CC genotype. The method includes the following steps: (1) Extract sheep genomic DNA; (2) PCR amplification was performed using two specific primers to obtain a 968 bp 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 SNP molecular marker locus genotype described in claim 1 in screening for high colostrum production in Hu sheep, and to select Hu sheep individuals with the TT genotype of the SNP molecular marker locus for breeding.

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