Application of SNP marker related to body weight of goat

Abstract

The application discloses an application of a SNP marker of a JAK2 gene intron region affecting goat weight. The marker is located in a JAK2 gene on a goat chromosome 8, and the specific SNP marker is an A>G base mutation at the 635th bp of the sequence table SEQ ID NO:1; the body weight of a goat with a GG genotype is significantly higher than that of goats with AA and GA genotypes. The application takes Macheng black goats as research objects, uses PCR to amplify DNA sequence variation of the JAK2 gene of the goats, analyzes the influence of the mutation site polymorphism on the body weight of the goats, and carries out breeding of the goat individuals according to the same, so that the body weight of the Macheng black goats can be improved, the breeding of goat breeds using the Macheng black goats as breeding materials can be accelerated, and a marker resource is provided for marker-assisted selection breeding of growth traits of the goats.

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 goat weight traits in the breeding of Macheng Black Goats. Background Technology

[0002] Single nucleotide polymorphisms (SNPs) are a significant contributor to biological diversity, and whole-genome resequencing is a rapid and accurate method for identifying them. Human domestication of wild animals for meat, milk, and fur has led to changes in their genotypes and phenotypes. Under the combined influence of natural and artificial selection, the transition from wild to domestic goats has resulted in significant changes in body weight traits, leaving substantial selection imprints on the genome. Therefore, comparing the genomic data of wild and domestic goats can help identify SNP loci associated with body weight. Our analysis of whole-genome sequencing data from Macheng black goats and wild goats revealed a positively selected SNP locus located in the JAK2 gene, which may be a crucial factor influencing goat body weight.

[0003] Junas kinase 2 (JAK2) is a non-receptor tyrosine protein kinase belonging to the cytokine receptor-associated tyrosine kinase family. It can both bind to cytokine receptors for phosphorylation and catalyze the phosphorylation of downstream target proteins. JAK2 / STATs are a key pathway in cytokine and growth factor signaling, participating in many cellular activities and playing important biological functions in signal transduction, cell proliferation, and growth. Growth hormone (GH) is an important hormone regulating body weight, and JAK2 is crucial for GH signal transduction. The GH-activated JAK2 / GHR complex recruits various signaling proteins, thereby initiating multiple signaling pathways and cellular responses, including pathways and protein expression related to body weight regulation. JAK2's influence on body weight is reflected in skeletal development, as well as in the metabolism and energy homeostasis of muscle and adipocytes. The signaling protein SH2B1 recruited by the JAK2 / GHR complex activates JAK2 and enhances GH's regulation of the actin cytoskeleton. When this protein is recruited to the leptin receptor-JAK2 complex, it participates in body weight regulation, and its variation is associated with obesity. Upon activation, JAK2 induces the JAK2 / p42 / 44MAPK and JAK2 / PI3K cascades, thereby inducing growth hormone mRNA expression and leading to transactivation of the GH promoter, thus controlling growth and metabolism in mammals. Adipocytes can regulate body weight through JAK2; mice with JAK2 gene knockout have significantly higher body weight than their littermate control mice, with female mice showing particularly pronounced weight gain. In summary, JAK2 affects signal transduction and energy balance during growth and development, and may participate in the regulation of animal body weight. This invention uses Macheng Black Goats as a model to analyze the relationship between JAK2 gene polymorphism and body weight, discovering that a SNP site in this gene is associated with body weight. Individuals with the GG genotype at this site have significantly higher body weight, providing marker resources for marker-assisted selection breeding of body weight traits in Macheng Black Goats. Summary of the Invention

[0004] This invention provides a solution to the technical problems existing in the current breeding process of goat weight traits.

[0005] According to an embodiment of the present invention, the SNP marker is located at base 635 of the nucleotide sequence shown in SEQ ID NO:1. Individuals of the GG genotype Macheng Black Goat with this SNP marker have a significantly higher body weight than individuals with the GA and AA genotypes. By detecting the aforementioned SNP marker in goats, the body weight of the goats can be assessed based on the genotype of the SNP marker. Therefore, the SNP marker of the present invention is closely related to goat body weight and can be effectively used for marker-assisted breeding of goats. This allows for the selection of goat breeding stock according to actual breeding needs, thereby enabling accurate and efficient selection of superior individuals with high body weight, improving the efficiency and accuracy of breeding selection.

[0006] This invention provides a primer pair for detecting the SNP marker of claim 1. According to an embodiment of the invention, the primers are the nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:3, used for detecting the SNP marker.

[0007] According to embodiments of the present invention, the primer pairs of the present invention can effectively amplify the fragment containing the aforementioned weight-related SNP marker in the Macheng Black Goat under test by PCR. Sequencing can then effectively detect this SNP marker, determine the genotype of the individual goat at the SNP marker locus, and thus effectively predict the weight of the goat. Specifically, individuals with the GG genotype at this SNP marker locus have significantly higher weights than individuals with the GA and AA genotypes. Therefore, the GG genotype at this SNP marker locus can serve as an important standard for judging goat weight. In the breeding of Macheng Black Goats, individuals with the GG genotype at this SNP marker locus can be retained for breeding, while individuals with the AA and GA genotypes at this SNP marker locus can be culled, thereby gradually increasing the weight of the Macheng Black Goat population. Therefore, the primer pairs of the present invention for detecting the aforementioned SNP marker can be effectively used in molecular marker-assisted breeding of Macheng Black Goats, thereby enabling low-cost and high-accuracy selection of large-weight Macheng Black Goats.

[0008] The present invention has the following beneficial effects: (1) The SNP marker provided by the present invention is significantly correlated with the weight of Macheng Black Goats, and the weight of GG genotype goats is significantly higher than that of GA and AA genotype goats. (2) The SNP marker can be used for auxiliary selection of the weight trait of Macheng Black Goats, screening for Macheng Black Goats with high weight, which has important practical application value for further improving the growth performance of Macheng Black Goats and using Macheng Black Goats 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 GG, GA and AA 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

[0012] The fasting weight of 343 adult Macheng black goats of Hubei Jinyang (Macheng) Livestock Co., Ltd. was measured under the same feeding, management and environmental conditions.

[0013] 2. Genomic DNA extraction

[0014] Blood from the jugular vein of the above-mentioned experimental 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 Macheng black goat according to the instructions of the blood genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0015] 3. Primer design

[0016] Based on the sequence of the goat JAK2 gene (Ensembl database gene sequence number: ENSCHIG00000003903), a pair of specific primers, SEQ ID NO:2 and SEQ ID NO:3, were designed using NCBI's Primer-BLAST. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., and were used to amplify a DNA sequence containing the g.39273847A>G site of the JAK2 gene. The amplification product was 760 bp, and the DNA sequence is shown in the sequence listing SEQ ID NO:1.

[0017] 4. Polymerase chain reaction (PCR) amplification of the target sequence of the JAK2 gene from Macheng black goats and sequencing.

[0018] (1) PCR amplification system (20 μL): DNA 1 μL, primers SEQ ID NO:2 and SEQ ID NO:3 (10 μM) 1 μL each, 2×M5 HiPer plus Taq HiFi PCR mix (with blue dye) (Beijing Jumei Biotechnology Co., Ltd.) 12.5 μL, ddH2O 7 μL. Amplification program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 25 s, 55℃ annealing for 25 s, 72℃ extension for 12 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 at the 635bp site of the nucleotide sequence shown in SEQ ID NO:1. Figure 1 As shown, the genotype of unimodal G is GG, the genotype of unimodal A is AA, and the genotype of bimodal A is GA.

[0020] 5. Association analysis between JAK2 gene SNP markers and body weight in Macheng black goats.

[0021] One-way ANOVA in SPSS software was used to conduct an association analysis between genotype and body weight. The specific linear analysis model is as follows:

[0022] Y ij =μ+G i +Eij

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

[0024] 6. Significant Analysis of Weight Differences Among Different Genotypes of Macheng Black Goats

[0025] The results of the weight analysis of different genotypes of Macheng Black Goats are shown in Table 1. Table 1 shows that there are three genotypes at this locus. One-way ANOVA was used to compare the weight differences among the different genotypes. It was found that the weight of Macheng Black Goats with the GG genotype was significantly higher than that of those with the GA and AA genotypes (P < 0.05). There was no difference in weight between Macheng Black Goats with the AA genotype and those with the GA genotype. This indicates that the GG genotype at this SNP locus can be used as an important criterion for judging high weight in Macheng Black Goats. In the breeding of Macheng Black Goats, individuals with the GG genotype at this SNP marker locus can be retained for breeding, while individuals with the AA genotype can be culled. Alternatively, GG genotype individuals can be bred with GA genotype individuals to obtain more GG genotype individuals, until AA or GA genotype individuals are eliminated, thereby gradually increasing the weight of the Macheng Black Goat population.

[0026] Table 1. Body weight of individuals with different genotypes at the JAK2 gene mutation site in Macheng black goats.

[0027]

[0028] Note: Different shoulder letters in the same column indicate significant differences (p<0.05).

Claims

1. A method for selecting high-weight goats based on molecular markers associated with goat weight traits, wherein the molecular marker exhibits an A / G polymorphism at position 635 of the nucleotide sequence SEQ ID NO:1, wherein goat individuals with the GG genotype at position 635 of the sequence have significantly higher weights than individuals with the AA and GA genotypes; The method includes the following steps: (1) Extracting goat genomic DNA; (2) PCR amplification was performed using two specific primers to obtain a 760 bp amplification product. The two specific primer sequences are P1: SEQ ID NO:2 and P2: SEQ ID NO:

3. (3) Sequencing the PCR amplification products to obtain sequencing results; (4) Determine the genotype of the goat individual to be tested based on the sequencing results; (5) Select goat individuals with the GG genotype of the above molecular markers for breeding; The goat in question is the Macheng Black Goat.

2. The application of the molecular marker described in claim 1 in screening for high-weight Macheng black goats, wherein individuals with the GG genotype of the molecular marker are selected for breeding.

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