Application of CNTNAP5 gene mutation in selection of number of lambs of Mancheng black goats

Abstract

The invention discloses an application of an SNP (Single Nucleotide Polymorphism) marker in a CNTNAP5 gene capable of influencing the lambing number of goats. The marker is located on a CNTNAP5 gene of a goat chromosome 2, the specific SNP marker is C / G base mutation at the 308bp position of SEQ ID NO: 1 in a sequence table, and the lambing number of a CC genotype goat individual is remarkably higher than that of a GC genotype individual and a GG genotype individual. The method comprises the following steps: by taking the Mancheng black goats as a research object, amplifying a DNA sequence of a tenth exon of a goat CNTNAP5 gene by virtue of PCR, analyzing the effect of a specific genotype of a variation site on the lambing number of the Mancheng factor goats, and selecting individual Mancheng black goats for breeding according to the effect, so that the lambing number of the Mancheng black goats can be increased, and the aim of improving the lambing number of the Mancheng black goats is fulfilled. Cultivation of a new variety (strain) with the Muscheng black goats as breeding materials is accelerated, and a marker resource is provided for marker-assisted selective breeding of the number of goat lambs.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal molecular breeding, and relates to SNP markers related to the litter size of Macheng black goats and their application in goat breeding. Background Art

[0002] Reproductive performance is an important factor affecting the economic benefits of goats, and the litter size is an important trait for measuring reproductive performance. The molecular marker-assisted selection technology can achieve early selection of livestock individuals, with the characteristics of fast selection efficiency and high accuracy. The present invention discovers that a SNP marker of the CNTNAP5 gene significantly affects the litter size of goats.

[0003] Contactin Associated Protein 5 (CNTNAP5) is a neural transmembrane protein, mainly enriched in myelinated axons, belonging to the neurexin superfamily members, and is a class of cell adhesion molecules of the contactin family, mainly present in the brain, and plays a key role in neurite formation, neuron development, axonal domain organization, and axon guidance. Its deletion leads to axonal malformation and impaired neurotransmission function. The hypothalamic-pituitary-gonadal axis (HPG axis) controls key links such as gonadal development, ovulation cycle, and gamete formation in male and female animals through the interaction of hormone secretion and neural signals. CNTNAP5 is directly related to the structure and function of neural axons, may play a key role in the stability of neural networks, and thus produce multi-level "upstream regulation" on the reproductive system, possibly affecting hormone secretion, estrus cycle, and even the entire reproductive process. Existing studies have shown that CNTNAP5 is related to the body size of Sudanese goats and also participates in the adaptive evolution of sheep. The present invention discovers that a SNP marker in exon 10 of the CNTNAP5 gene is associated with the litter size trait of Macheng black goats, providing valuable marker resources for molecular marker-assisted selection breeding of the litter size trait of Macheng black goats. Summary of the Invention

[0004] The present invention aims to provide a technical method for selecting and breeding goats with high litter size.

[0005] According to an embodiment of the present invention, the SNP marker is located at the 308th base of the nucleotide sequence shown in SEQ ID NO:1. The litter size of individuals with the CC genotype of this SNP marker is significantly higher than that of individuals with the GC and GG genotypes. By detecting the above SNP marker in Macheng black goats, the litter size can be effectively increased, and thus the litter size of Macheng black goat individuals can be evaluated according to the genotype at this SNP marker locus. Therefore, the SNP marker of the present invention can be effectively used for molecular marker-assisted breeding of Macheng black goats, and then the breeding materials of goats can be selected according to actual breeding needs, so that goat individuals with more litter sizes can be accurately and efficiently selected, improving the efficiency and accuracy of breeding selection.

[0006] The present invention provides a primer pair for detecting the above SNP marker. According to an embodiment of the present invention, the primers have the nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:3. Using the primer pair of the present invention can effectively amplify the DNA fragment where the above SNP marker related to litter size in the goat to be tested is located. Through sequencing, the detection of this SNP marker can be effectively achieved, determining the genotype of the Macheng black goat individual to be tested at this SNP marker locus, and then effectively predicting the litter size of the goat to be tested.

[0007] Specifically, the litter size of individuals with the CC genotype at the above SNP locus is significantly higher than that of individuals with the GC and GG genotypes, which can be used as an important criterion for judging the litter size of goats. In the goat breeding work, individuals with the CC genotype at this SNP locus can be retained for breeding, and individuals with the GC and GG genotypes at this SNP marker locus can be eliminated. Or individuals with the GC genotype can be mated with individuals with the CC genotype to obtain more CC genotype offspring, realizing the selection of excellent goat varieties with low cost and high accuracy, and gradually increasing the litter size 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 litter size of Macheng black goats. The litter size of Macheng black goats with the CC genotype is significantly higher than that of Macheng black goats with the GC and GC genotypes; (2) This SNP marker can be used for the assisted selection of the litter size of Macheng black goats, screening out Macheng black goats with high litter sizes, which has important practical application value for further increasing the litter size 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 combination with the accompanying drawings for the description of the embodiments. Figure 1 Showing the sequencing peak maps of the CC, GC, and GG genotypes of the SNP marker of the present invention. SPECIFIC EXAMPLES

[0010] Embodiments of the present invention will be described in detail below. The present invention will be further described in detail in combination with the embodiments. The embodiments herein are only used to illustrate the present invention and should not be construed as a limitation to the present invention.

[0011] 1. Experimental samples 138 adult Macheng black goats (with records of the number of lambs born in 1 to 5 litters) from Hubei Jinyang (Macheng) Animal Husbandry Co., Ltd., with the same feeding management conditions and environmental conditions.

[0012] 2. Genomic DNA extraction Blood samples of the above samples (5 ml / animal) were collected using vacuum negative pressure blood collection tubes anticoagulated with dipotassium edetate (EDTA-K2), and the samples were stored at -20°C. The genomic DNA in the blood samples of Macheng black goats was extracted using a blood genomic DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. The DNA concentration of the samples was measured and diluted to 50 mg / mL, and stored at 4°C.

[0013] 3. Primer design According to the sequence of the goat CNTNAP5 gene (gene sequence number in the Ensembl database: ENSCHIG00000009353), 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 Tsingke Biotechnology Co., Ltd. and were used to amplify a DNA sequence where the 10th exon of the CNTNAP5 gene is located. The amplification product is 862 bp, and the nucleotide sequence is shown as SEQ ID NO:1 in the sequence listing.

[0014] 4. PCR amplification of the target sequence of the goat CNTNAP5 gene and genotype determination (1) PCR amplification system (20 μL): 2 μL DNA (50 mg / mL), 0.5 μL each of the upstream and downstream primers SEQ ID NO:2 and SEQ ID NO:3 (100 μM), 10 μL of 2×M5 HiPerplus Taq HiFi PCRmix (Beijing Polymer Beauty Biotechnology Co., Ltd.), 7 μL of ddH2O. The amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 57°C for 25 s, extension at 72°C for 13 s, 35 cycles, and final extension at 72°C for 5 min. (2) The PCR amplification products were sent to Wuhan Kingcare Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using SnapGene software to determine the genotype of the individual at the 308 bp site of the nucleotide sequence shown as SEQ ID NO:1 in the sequence listing. For example, Figure 1As shown, the genotype of single peak C is CC, the genotype of single peak G is GG, and the genotype of double peak is GC. The base mutation from G to C changes the encoded glycine (Gly) to alanine (Ala).

[0015] 5. Association analysis of SNP markers of CNTNAP5 gene and litter size in Macheng black goats The single-factor variance analysis in SPSS software was used to conduct the association analysis between genotype and litter size. The specific linear analysis model is described as follows: Yij = μ + Gi + Eij Where: Y ij is the individual phenotypic record; μ is the population mean; G i is the genotype effect; E ij is the random error.

[0016] 6. Significance analysis of differences in litter size among different genotypes of Macheng black goats The analysis results of litter size of different genotypes of Macheng black goats are shown in Table 1. It can be seen from Table 1 that there are three genotypes at this locus. By using single-factor variance analysis to compare the differences in average litter size among different genotypes, it is found that the litter size of Macheng black goats with CC genotype is significantly higher than that of GC and GG genotypes (p < 0.05). This indicates that the CC genotype of this SNP marker locus can be used as an important criterion for judging high litter size in Macheng black goats. In the breeding work of Macheng black goats, individuals with CC genotype at this locus can be retained, and individuals with GC and GG genotypes at this locus can be eliminated. Also, individuals with GC genotype can be mated with individuals with CC genotype to obtain more offspring individuals with CC genotype, thereby gradually increasing the litter size of the goat population. Table 1 Correlation between different genotypes of CNTNAP5 gene mutation site and litter size Note: Different superscript letters in the same column indicate significant differences (p < 0.05).

Claims

1. A method for selecting goats based on molecular markers related to the number of kids born to goats, said molecular markers being located in nucleotide sequence SEQ ID NO: 1, wherein the number of kids born to goat individuals with the CC genotype at the 308th site of this sequence is significantly higher than that of individuals with the GC and GG genotypes; The method comprises the following steps: (1) Extracting goat genomic DNA; (2) Performing PCR amplification using two specific primers to obtain an 862bp amplification product, and 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 CC genotype of the above molecular marker for breeding; (6) The goat is Macheng Black Goat.

2. Use of the molecular marker according to claim 1 in the cultivation of Macheng Black Goat population, and selecting goat individuals with the CC genotype of the molecular marker for breeding.

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