Application of a CABP4 gene mutation in the selection of lambing numbers in Macheng black goats

Abstract

This invention discloses the application of a SNP marker affecting the number of lambs born in goats. The SNP marker site is a C / T base mutation at position 467 bp of SEQ ID NO:1 in the sequence listing. Goats with the CC and CT genotypes at this site have a significantly higher lambing rate than those with the TT genotype. Using Macheng Black Goats as the research subject, this invention utilizes PCR to amplify the DNA sequence of the first exon of the goat CABP4 gene. This sequence contains a SNP variant site. The influence of a specific genotype at this variant site on the number of lambs born in Macheng Black Goats is analyzed, and based on this analysis, Macheng Black Goat individuals can be selected for breeding. This method can be used to increase the number of lambs born in Macheng Black Goats and provides marker resources for marker-assisted selection breeding of goat lambing traits.

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 the lambing number trait of Macheng Black Goats in the breeding of Macheng Black Goats. Background Technology

[0002] Reproductive performance is a crucial factor affecting the economic benefits of goats, with litter size being a key trait for measuring reproductive performance. Litter size in goats is influenced by multiple factors, has low heritability, and is controlled by numerous minor genes. Traditional breeding selection techniques result in slow genetic progress. Currently, major genes affecting litter size have been identified, and whole-genome sequencing technology provides a foundation for identifying these genes. We performed whole-genome sequencing on two goat individuals with high litter size and found that a single base mutation in CABP4 was significantly correlated with litter size.

[0003] The calcium-binding protein family (CABPs) is a class of proteins involved in calcium ion binding, including several members such as CaBP1, CaBP2, and CaBP4. All members possess an EF-hand calcium-binding domain and regulate signal transduction and synaptic transmission in neurons by binding to calcium ions, primarily functioning in the nervous system. CABP4 is expressed in the mammalian brain, ganglia, lungs, and testes. Its most clearly understood function is its expression in the mammalian retina, particularly at the synaptic terminals of photoreceptor cells, where it regulates synaptic transmission by interacting with calcium ion channels (such as CaV1.4). Compared to other CABP family members, CABP4 has a different binding site with calmodulin. This difference allows CABP4 to provide additional regulatory functions beyond calmodulin regulation. Therefore, this protein enhances the opening frequency of calcium ion channels, thereby increasing calcium ion influx and facilitating neurotransmitter release and signal transduction. In mouse models, mutations in the CABP4 gene lead to changes in protein expression levels and altered neuronal firing frequency. Mutations in the CABP4 gene are associated with a variety of diseases, including autosomal recessive congenital static night blindness. Some CABP4 mutations are also associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). This invention, through whole-genome sequencing and association analysis, discovered a novel SNP site in the CABP4 gene. This mutation site is significantly correlated with litter size in goats, providing marker resources for marker-assisted selection breeding of litter size in Macheng Black Goats. Summary of the Invention

[0004] This invention provides a solution to the technical problems existing in the current breeding process for goat litter size.

[0005] This invention provides an SNP marker that affects the number of lambs born in goats. The marker corresponds to the coding region of the goat CABP4 gene (sequence number: ENSCHIG00000012267) published in the Ensembl database. The difference in the C / T ratio of nucleotides at this marker site leads to different numbers of lambs born in goats.

[0006] This invention provides a method for selecting goats for breeding based on a marker genotype. According to an embodiment of the invention, the SNP marker is located at base 467 of the nucleotide sequence shown in SEQ ID NO:1. Goats with the CC and CT genotypes at this SNP marker site have significantly higher lambing numbers than those with the TT genotype. By detecting the aforementioned SNP marker in Macheng Black Goats, their lambing numbers can be effectively predicted, thereby enabling the assessment of lambing performance of Macheng Black Goats based on the genotype at this SNP marker site. Therefore, the SNP marker of this invention is closely related to the lambing number trait of Macheng Black Goats and can be effectively used for marker-assisted selection breeding of Macheng Black Goats. This allows for the selection of goat breeding stock according to actual breeding needs, enabling accurate and efficient selection of superior individuals with high lambing numbers, thus improving the efficiency and accuracy of breeding selection.

[0007] This invention provides a primer pair for detecting the SNP marker described in claim 1. According to an embodiment of the invention, the primers have the nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:3, and are used to detect the SNP marker. According to an embodiment of the invention, the primer pair can effectively amplify the fragment containing the SNP marker related to the litter size trait in the tested Macheng Black Goat by PCR. Sequencing can effectively detect this SNP marker, determine the genotype of the tested Macheng Black Goat at the SNP marker site, and thus effectively predict the litter size of the tested Macheng Black Goat.

[0008] Specifically, the number of lambs born to goats with the CC and CT genotypes at the SNP marker locus was significantly higher than that of individuals with the TT genotype, indicating that the CC genotype at this SNP locus can serve as an important criterion for determining the number of lambs born to Macheng Black Goats. In the breeding of Macheng Black Goats, the genome sequence of individual goats is amplified and sequenced using the primers for the SNP marker described in this invention to determine the genotype of the individual goats. Individuals with the CC genotype at the SNP marker locus can be retained for breeding, or individuals with the CC genotype can be mated with individuals with the CT genotype. In the hybrid offspring, individuals with the CC genotype are selected for breeding, while individuals with the CC genotype at the SNP marker locus are culled. This gradually increases the number of lambs born to the goat population, enabling the low-cost and high-accuracy selection of goat individuals with a high number of lambs, thus realizing molecular marker-assisted breeding based on the number of lambs born to goats.

[0009] The present invention has the following beneficial effects: (1) The SNP marker provided by the present invention is significantly correlated with the number of lambs born in Macheng Black Goats, and the number of lambs born in CC and CT genotype goats is significantly higher than that of TT genotype individuals. (2) The SNP marker can be used for auxiliary selection of the lambing trait in Macheng Black Goats, screening for Macheng Black Goats with high lambing numbers, which has important practical application value for further improving the reproductive capacity of Macheng Black Goats and using specific Macheng Black Goats as material for breed (or strain) selection. Attached Figure Description

[0010] 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 CC, CT, and TT genotypes of the SNP molecular marker sites of this invention are shown. Specific Implementation

[0011] 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.

[0012] 1. Experimental Samples The 129 adult Macheng black goat ewes of Hubei Jinyang (Macheng) Livestock Co., Ltd., which have records of one to five litters, are kept under the same feeding, management and environmental conditions.

[0013] 2. Genomic DNA extraction Blood from the jugular vein of the above-mentioned sample (5 mL / animal) was collected using a disposable vacuum negative pressure blood collection tube (EDTA-K2 anticoagulation). Genomic DNA was extracted from the blood sample of Macheng black goat using the blood genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd.

[0014] 3. Primer design Based on the sequence of the goat CABP4 gene (Ensembl database gene sequence number: ENSCHIG00000012267), a pair of specific primers, SEQ ID NO:2 and SEQ ID NO:3, were designed using Primer 5.0 software. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. These specific primers were used to amplify a DNA sequence containing the mutation site in the first exon of the CABP4 gene. The amplification product was 672 bp, and the nucleotide sequence is shown in the sequence listing SEQ ID NO:1.

[0015] 4. PCR amplification of the target sequence of the goat CABP4 gene and sequencing to determine the genotype. (1) PCR amplification system (20 μL): DNA 2 μL, primers SEQ ID NO:2 and SEQ ID NO:3 (10 μM) 0.5 μL each, 2×Rapid Taq Master Mix 10 μL (Nanjing Novizan Biotechnology Co., Ltd.), ddH2O 7 μL. Amplification program: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s, 61℃ annealing for 25 s, 72℃ extension for 14 s, 35 cycles; 72℃ extension for 5 min. (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 467bp site of the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing. 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. In the sequence listing, the SNP marker site at 467bp of SEQ ID NO: 1 is mutated from C to T, changing the codon encoding tryptophan (Ser) to the codon encoding leucine (Leu).

[0016] 5. Association analysis of CABP4 gene SNP markers and litter size in Macheng black goats One-way ANOVA in SPSS software was used to conduct an association analysis between genotype and litter size. The specific linear analysis model is as follows: Y ij =μ+G i +E ij Where: Y ij For individual phenotypic records; μ is the population mean; G i Genotype effect; E ij This is random error.

[0017] 6. Significant difference in litter size among different genotypes of Macheng black goats The results of the litter size analysis 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 litter size among different genotypes revealed that the litter size of Macheng Black Goats with the CC and CT genotypes was significantly higher than that with the TT genotype (p<0.05). This indicates that the CC and CT genotypes at this SNP marker locus can serve as important criteria for judging high litter size in Macheng Black Goats. In the breeding of Macheng Black Goats, individuals with the CC genotype at this SNP marker locus can be retained for breeding, while those with the TT genotype can be culled. Alternatively, individuals with the CT genotype can be mated with those with the CC genotype to obtain more CC-type offspring, thereby gradually increasing the litter size of the Macheng Black Goat population. Table 1. Correlation between different genotypes of the CABP4 gene mutation site in Macheng black goats and the number of lambs born. Note: Different shoulder letters in the same column indicate significant differences (p<0.05).

Claims

1. A method for selecting high-producing goats using molecular markers associated with goat litter size, wherein the nucleotide sequence of the molecular marker is SEQ ID NO:1, wherein the 467th base of the sequence has a C / T polymorphism, and the litter size of goat individuals with CC and CT genotypes at this site is significantly higher than that of individuals with TT genotypes; The method includes the following steps: (1) Extracting goat genomic DNA; (2) PCR amplification was performed using two specific primers to obtain a 672 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 based on the sequencing results; (5) Select goat individuals with the CC 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-producing Macheng black goats, wherein goat individuals with the CC genotype of the molecular marker are selected for breeding.

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