Application of LAMA5 gene SNP (Single Nucleotide Polymorphism) marker in goat weight selection

Abstract

The invention discloses an application of an SNP (Single Nucleotide Polymorphism) marker of an LAMA5 gene exon related to goat weight. The marker is located on an exon of an LAMA5 gene of a goat chromosome 13, the specific SNP marker is C / T base mutation at the 320bp position of SEQ ID NO: 1 in a sequence table, and the weight of a TT genotype is significantly higher than that of CT and CC type individuals. According to the present invention, the Musheng black goat is adopted as the research object, the PCR is adopted to amplify the DNA sequence of the goat LAMA5 gene, the influence of the mutation site polymorphism on the goat body weight is analyzed, the method can be used for breeding the Musheng black goat, the breeding of the goat variety using the Musheng black goat as the breeding material is accelerated, and the marker resource is provided for the marker-assisted selection breeding of the goat growth traits.

Description

Technical Field

[0001] The invention belongs to the technical field of animal molecular breeding, and relates to the application of a SNP marker affecting goat weight traits in goat breeding. Background Art

[0002] Weight traits are crucial in livestock breeding because they directly affect meat production, milk production, and reproductive performance. Single nucleotide polymorphisms (SNPs) provide information on the association between genotype and weight traits, and breeders can breed for weight traits through genomic screening of SNP markers to achieve more precise breeding goals.

[0003] Laminin subunit alpha 5 (LAMA5) encodes the vertebrate laminin alpha chain, which is involved in cell adhesion, differentiation, migration, signal transduction, neurite outgrowth and metastasis. LAMA5 is expressed in bones, muscles, ligaments, periosteum, trabeculae and whole cartilage, especially in growth plates and articular chondrocytes. Laminin belongs to the family of extracellular matrix glycoproteins, which controls the differentiation, proliferation, migration and survival of osteoblasts and regulates bone formation. In the early stage of osteoblast differentiation, LAMA5 in the focal adhesion signaling pathway genes is significantly upregulated, and the loss of LAMA5 negatively regulates the generation of actin cytoskeleton, vinculin localization and WNT signaling. In the exome sequencing of human short stature families, LAMA5 is one of the strong candidate genes for human short stature. Enrichment analysis shows that LAMA5 is located in GO terms including skeletal system development and appendage development, and the functional group of LAMA5 is located in the Wnt signaling pathway. In addition, laminin is a determinant of early musculoskeletal development during myometrium formation by controlling the balance between myotome cell proliferation and differentiation. In the later stages of fetal development, laminin is an indispensable part of the microenvironment of fibers and muscle stem cells and is a determinant of normal muscle mass growth. At the same time, laminin plays an equally important role in the development of neuromuscular junctions, and muscle stem cells located between the basement membrane and the fibers are the main source of adult muscle regeneration capacity. LAMA5, one of the laminins, was found to have a significant association between the single nucleotide polymorphism (SNP) rs659822 on the human LAMA5 gene and the weight of the subjects. In summary, LAMA5 is involved in the biological process of regulating bone and muscle development, thereby affecting the body shape and weight of animals.

[0004] Macheng Black Goat is an excellent black goat meat breed in the Dabie Mountains. It has the growth characteristics of fast growth rate and good fattening ability. Based on the whole genome selection signal analysis, Macheng Black Goat was used as the research object and divided into high body weight group and low body weight group. The positive selection signal was detected, and it was found that a positive selection SNP marker of the LAMA5 gene was related to the goat weight, which provided marker resources for the molecular marker-assisted selection breeding of the weight trait of Macheng Black Goat. Summary of the invention

[0005] The invention provides a technical method for goat weight breeding.

[0006] According to an embodiment of the present invention, the SNP marker is located at the 320th base of the nucleotide sequence shown in SEQ ID NO: 1. The weight of individuals with the TT genotype of the SNP marker is significantly higher than that of individuals with the CT and CC genotypes. By detecting the above-mentioned SNP marker of goats, the weight of goats can be evaluated according to the genotype of the SNP marker. Therefore, the SNP marker of the present invention is closely related to the weight of goats, and can be effectively used in molecular marker-assisted breeding of goats, and then goat breeding materials can be selected according to actual breeding needs, so that excellent goat varieties can be bred accurately and efficiently, and the efficiency and accuracy of breeding can be improved.

[0007] The present invention provides a primer pair for detecting the SNP marker. According to an embodiment of the present invention, the primer has a nucleotide sequence shown in SEQ ID NO:2 and SEQ ID NO:3. The primer pair can effectively amplify the DNA fragment where the SNP marker related to the weight of the goat to be tested is located. The detection of the SNP marker can be effectively achieved by sequencing, and the genotype of the goat individual to be tested at the SNP marker site can be determined, and the weight of the goat to be tested can be effectively predicted. Specifically, the weight of the goat with the TT genotype at the SNP site is significantly higher than that of the CT and CC genotype individuals, which can be used as an important criterion for judging the weight of the goat. In the goat breeding work, the individuals with the genotype of the SNP site TT can be retained for breeding, and the individuals with the genotype of the SNP marker site CC can be eliminated. The CT genotype individuals can also be mated with the TT genotype individuals to obtain more TT genotype offspring, so as to select goats with larger weight at low cost and high accuracy, thereby gradually improving 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, and the weight of Macheng black goats with TT genotype is significantly higher than that of Macheng black goats with CT and CC genotypes; (2) The SNP marker can be used for auxiliary selection of the weight trait of Macheng black goats to screen out Macheng black goats with large weight, which has important practical application value for further improving the weight of Macheng black goats and using Macheng black goats as materials for variety (or strain) breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The above aspects of the present invention will be more easily understood in conjunction with the accompanying drawings. Figure 1 The genotype sequencing peak diagram of the SNP markers TT, CT, and CC of the present invention is displayed, and the blue background area is the SNP site. Specific embodiments

[0010] The embodiments of the present invention are described in detail below, and the present invention is further described in detail in conjunction with the embodiments. The embodiments herein are only used to illustrate the present invention and are not to be construed as limiting the present invention.

[0011] 1. Experimental samples 177 adult Macheng black goats of Hubei Jinyang (Macheng) Animal Husbandry Co., Ltd. were raised and managed under the same environmental conditions. 5 ml of blood samples were collected in blood collection tubes anticoagulated with EDTA2K dipotassium edetate (EDTA-2K). The samples were stored at -20°C, and their body weight (unit: cm) in the morning in the fasting state was measured. The results were rounded to one decimal place.

[0012] 2. Genomic DNA Extraction The jugular vein blood (5 mL / goat) of the above-mentioned experimental sample was collected using a disposable vacuum negative pressure blood collection tube (EDTA-K2 anticoagulation), and the genomic DNA of the blood samples of Macheng black goats was extracted according to the instructions of the blood genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0013] 3. Primer Design According to the sequence of 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 used to amplify a sequence where the 24th exon of the LAMA5 gene is located. The amplified product is 633 bp, and the nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table.

[0014] 4. Polymerase chain reaction (PCR) amplification and sequencing of the target sequence of the LAMA5 gene of Macheng black goat (1) PCR amplification system (20 μL): 1 μL DNA (50 mg / mL), 1 μL each of the upstream and downstream primers SEQ ID NO: 2 and SEQ ID NO: 3 (100 uM), 10 μL 2×M5 HiPerplus Taq HiFi PCR mix (with blue dye) (purchased from Beijing Polymer Biotechnology Co., Ltd.), and 7 μL ddH2O. The amplification program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 55°C for 25 s, extension at 72°C for 10 s, 35 cycles, and final extension at 72°C for 5 min. (2) The PCR amplification product was 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 320 bp position of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence list, such as Figure 1 As shown, the genotype of single peak C is CC, the genotype of single peak T is TT, and the genotype of double peak is CT. The base mutates from C to T, changing the encoded proline (Pro) to leucine (Leu).

[0015] 5. Association analysis between LAMA5 gene SNP markers and body weight in Macheng black goats One-way ANOVA in SPSS software was used to analyze the association between genotype and body weight. The specific linear analysis model is as follows: Y ij =μ+G i +E ij Where: Y ij is the individual phenotypic record; μ is the population mean; G i is the genotype effect of each site; E ij is a random error.

[0016] 6. Analysis of the significance of weight differences among different genotypes of Macheng black goats The results of the analysis of the weight of different genotypes of Macheng black goats are shown in Table 1. As can be seen from Table 1, there are three genotypes at this locus. The one-way ANOVA was used to compare the differences in weight of different genotypes. It was found that the weight of Macheng black goats with TT genotype was significantly higher than that of CC and CT genotypes (p < 0.05). This shows that this SNP locus can be used as an important criterion for judging the weight of Macheng black goats. In the breeding of Macheng black goats, individuals with TT genotypes at this locus can be retained for breeding, and individuals with CC genotypes at this locus can be eliminated. TT and CT type individuals can also be mated to obtain more TT type offspring individuals, thereby gradually improving the growth performance of the group. Table 1 Correlation between different genotypes of LAMA5 gene mutation sites and body weight Note: Different letters in the same column indicate significant differences (p<0.05).

Claims

1. A method for selecting goats based on a molecular marker related to goat weight, wherein the molecular marker is located in the nucleotide sequence SEQ ID NO: 1, wherein the weight of goat individuals with TT genotype at the 320 bp position of the sequence is significantly higher than that of individuals with CT and CC genotypes; The method comprises the following steps: (1) Extracting goat genomic DNA; (2) using two specific primers to perform PCR amplification to obtain a 633 bp amplification product, wherein the sequences of the two specific primers are SEQ ID NO: 2 and SEQ ID NO: 3; (3) sequencing the PCR amplification product to obtain a sequencing result; (4) determining the genotype of the goat individual to be tested at the molecular marker according to the sequencing result; (5) selecting goat individuals with the TT genotype of the above molecular marker for seed preservation; (6) The goat is a Macheng black goat.

2. Application of the molecular marker according to claim 1 in the breeding of Macheng black goat population, selecting Macheng black goat individuals with TT genotype of the molecular marker for seed preservation.

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