Application of LAMA5 gene SNP marker in goat weight selection

Abstract

The application discloses application of a SNP marker of an exon of a LAMA5 gene related to goat weight. The marker is located on an exon of a LAMA5 gene on a goat chromosome 13, and the specific SNP marker is a C / T base mutation at the 320th bp in SEQ ID NO: 1 in the sequence listing; the weight of a TT genotype is significantly higher than that of CT and CC types. The application takes Macheng black goats as research objects, amplifies DNA sequences of goat LAMA5 genes by using PCR, analyzes the influence of mutation site polymorphism on goat weight, and can be used for breeding the Macheng black goats, accelerates breeding of goat breeds taking the Macheng black goats as breeding materials, and provides a marker resource for marker-assisted selection breeding of goat 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 that affect the weight trait of goats in goat breeding. Background Technology

[0002] Body weight is a crucial trait in livestock breeding because it directly affects meat production, milk production, and reproductive performance. Single nucleotide polymorphisms (SNPs) provide information on the association between genotype and body weight, allowing breeders to screen for SNP markers in the genome to target body weight and achieve more precise breeding goals.

[0003] Laminin subunit alpha 5 (LAMA5) encodes the laminin α chain in vertebrates and participates in cell adhesion, differentiation, migration, signal transduction, neurite growth, and metastasis. LAMA5 is expressed in bone, muscle, ligaments, periosteum, trabeculae, and throughout cartilage, particularly in growth plate and articular chondrocytes. Laminins belong to the extracellular matrix glycoprotein family and control osteoblast differentiation, proliferation, migration, and survival, regulating bone formation. During early osteoblast differentiation, LAMA5 is significantly upregulated in the focal adhesion signaling pathway, while its absence negatively regulates actin cytoskeleton formation, vinculin localization, and WNT signaling. In exome sequencing of the human short stature family, LAMA5 is a strong candidate gene for human short stature. Enrichment analysis shows that LAMA5 is located in GO entries including skeletal system development and appendage development, and its functional groups are located in the Wnt signaling pathway. Furthermore, laminins are crucial early determinants of musculoskeletal development during sarcomere formation by controlling the balance between sarcomere cell proliferation and differentiation. In later fetal development, laminins are an indispensable part of the microenvironment of fibers and muscle stem cells, and are a determinant of normal muscle mass growth. Simultaneously, laminins play an equally important role in the development of the neuromuscular junction, and muscle stem cells located between the basement membrane and fibers are the primary source of adult muscle regeneration. As one of the laminins, a single nucleotide polymorphism (SNP) rs659822 in the human LAMA5 gene was found to be significantly associated with the body weight of subjects. In summary, LAMA5 participates in the regulation of biological processes in skeletal and muscle development, thereby influencing body size and weight in animals.

[0004] Macheng Black Goat is an excellent black goat meat breed in the Dabie Mountains region, characterized by its rapid growth and good fattening ability. Based on genome-wide selection signal analysis, Macheng Black Goats were divided into high-body recombinant and low-body recombinant groups. Positive selection signals were detected, and one positively selected SNP marker of the LAMA5 gene was found to be associated with goat body weight, providing marker resources for molecular marker-assisted selection breeding of body weight traits in Macheng Black Goats. Summary of the Invention

[0005] This invention provides a technical method for selective breeding of goat weight.

[0006] According to an embodiment of the present invention, the SNP marker is located at base 320 of the nucleotide sequence shown in SEQ ID NO:1. Individuals with the TT genotype of this SNP marker have significantly higher body weights than individuals with the CT and CC genotypes. By detecting the above-mentioned SNP marker in goats, the body weight of 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 breeding of superior goat breeds and improving the efficiency and accuracy of breeding.

[0007] This invention provides a primer pair for detecting the aforementioned 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 DNA fragment containing the aforementioned weight-related SNP marker in the goat being tested can be effectively amplified. Sequencing can then effectively detect this SNP marker, determine the genotype of the goat at the SNP marker locus, and thus effectively predict the weight of the goat. Specifically, goats with the TT genotype at this SNP locus have significantly higher weights than those with the CT and CC genotypes, and this can serve as an important criterion for judging goat weight. In goat breeding, individuals with the TT genotype at this SNP locus can be retained for breeding, while individuals with the CC genotype at this SNP marker 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 larger-weight goats, thereby gradually increasing the weight of the goat population.

[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. The weight of Macheng Black Goats with the TT genotype is significantly higher than that of Macheng Black Goats with the CT and CC genotypes; (2) The SNP marker can be used for auxiliary selection of the weight trait of Macheng Black Goats, and screen out Macheng Black Goats with large weight. It has important practical application value for further improving the weight 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 SNP markers TT, CT, and CC genotypes 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] 177 adult Macheng black goats from Hubei Jinyang (Macheng) Livestock Co., Ltd. were kept under the same feeding, management and environmental conditions. Blood samples of 5 ml were collected from blood collection tubes anticoagulated with dipotassium ethylenediaminetetraacetate (EDTA-2K). The samples were stored at -20℃, and their weight (unit: cm) in the morning fasting state was measured. The results were retained to one decimal place.

[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 LAMA5 gene (Ensembl database gene sequence number: ENSCHIG00000011048), 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 segment of the sequence containing exon 24 of the LAMA5 gene. The amplification product was 633 bp, and the nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing.

[0017] 4. Polymerase chain reaction (PCR) amplification and sequencing of the target sequence of the LAMA5 gene from Macheng black goats.

[0018] (1) PCR amplification system (20 μL): 1 μL DNA (50 mg / mL), 1 μL each of forward and reverse primers SEQ ID NO:2 and SEQ ID NO:3 (100 μM), 10 μL of 2×M5 HiPerplus Taq HiFi PCR mix (with blue dye) (purchased from 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 a final extension at 72℃ 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 320bp site of the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing. Figure 1 As shown, the genotype of unimodal C is CC, the genotype of unimodal T is TT, and the genotype of bimodal C is CT. A mutation from C to T changes the encoded proline (Pro) to leucine (Leu).

[0020] 5. Association analysis of LAMA5 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 +E ij

[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 analysis results of body weight of different genotypes in Macheng Black Goats are shown in Table 1. Table 1 shows that there are three genotypes at this locus. One-way ANOVA comparing the differences in body weight among different genotypes revealed that the body weight of Macheng Black Goats with the TT genotype was significantly higher than that with the CC and CT genotypes (p < 0.05). This indicates that this SNP locus can serve as an important criterion for judging the body weight of Macheng Black Goats. In the breeding of Macheng Black Goats, individuals with the TT genotype at this locus can be retained for breeding, while individuals with the CC genotype at this locus can be culled. Alternatively, TT individuals can be bred with CT individuals to obtain more TT offspring, thereby gradually improving the growth performance of the population.

[0026] Table 1. Correlation between different genotypes of LAMA5 gene mutation sites and body weight.

[0027]

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

Claims

1. A method for selecting a high weight goat according to the genotype of a SNP molecular marker site associated with goat weight, characterized in that, The SNP molecular marker site is located at 320 bp of SEQ ID NO:1 in the nucleotide sequence. This site indicates that the weight of goat individuals with the TT genotype is significantly higher than that of individuals with the CT and CC genotypes. The method includes the following steps: (1) Extracting goat genomic DNA; (2) PCR amplification was performed using two specific primers to obtain a 633 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 goat individual to be tested at the SNP molecular marker site based on the sequencing results; (5) Select goat individuals with the TT genotype at the above SNP molecular marker sites for breeding; The goat in question is the Macheng Black Goat.

2. To test the application of the SNP molecular marker locus genotype described in claim 1 in the breeding of a high-weight Macheng black goat population, and to select individuals of Macheng black goat with the TT genotype of the SNP molecular marker locus for breeding.

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