Application of CABP4 gene mutation in selection of number of lambs of Muncheng black goats

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) marker of a CABP4 gene influencing the lambing number of goats and application of the SNP marker. A site of the SNP marker is C / T base mutation at the 467bp of SEQ ID NO: 1 in a sequence table, the lambing number of a CC genotype goat and a CT genotype goat at the site is remarkably higher than that of a TT genotype individual, and CC is a dominant genotype of a Musheng black goat. According to the present invention, the Musheng black goats are adopted as the research object, the DNA sequence of the first exon of the goat CABP4 gene is subjected to PCR amplification, the sequence has an SNP variation site, the influence of the specific genotype of the variation site on the Musheng black goat lambing number is analyzed, the individual Musheng black goat breeding is performed according to the influence, and the method can be used for improving the Musheng black goat lambing number, and a marker resource is provided for marker-assisted selective breeding of goat lambing number traits.

Description

Technical Field

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

[0002] Reproductive performance is an important factor affecting the economic benefits of goats, among which the number of lambs is an important trait to measure reproductive performance. The number of lambs in goats is affected by many factors. Its heritability is low and it is controlled by micro-effect polygenes. The use of traditional breeding selection technology for breeding has slow genetic progress. It has been found that there are major effect genes in the number of lambs in goats. Whole genome sequencing technology provides a basis for finding major effect genes affecting the number of lambs. We selected two individuals with the number of lambs in goats for whole genome sequencing and found that a base mutation in CABP4 was significantly correlated with the number of lambs in goats.

[0003] The calcium binding protein family (CABPs) is a class of proteins related to calcium ion binding, including CaBP1, CaBP2, CaBP4 and other members. They all have EF-hand calcium binding domains and can regulate signal transduction and synaptic transmission in neurons by binding to calcium ions, and mainly play a role in the nervous system. Among them, CABP4 is expressed in the brain, ganglia, lungs and testes of mammals. The function of CABP4 that has been studied more clearly is that it is expressed in the mammalian retina, especially in the synaptic terminals of photoreceptor cells, and regulates the synaptic transmission of photoreceptor cells by interacting with calcium ion channels (such as CaV1.4). Compared with other CABP family members, CABP4 has different binding sites with calmodulin. This difference enables CABP4 to provide additional regulatory effects in addition to the regulation of calmodulin. Therefore, the protein can enhance the opening frequency of calcium ion channels, thereby increasing the influx of calcium ions, which is more conducive to the release of neurotransmitters and signal transmission. In mouse models, mutations in the CABP4 gene lead to changes in protein expression levels and changes in neuronal firing frequency. Mutations in the CABP4 gene are associated with a variety of diseases, including autosomal recessive congenital stationary night blindness, and some mutations in CABP4 are also associated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The present invention, through whole genome sequencing and association analysis, found that there is a new SNP site in the CABP4 gene, and the mutation site is significantly correlated with the number of goats born, providing marker resources for molecular marker-assisted selection breeding of the number of goats born in Macheng black goats. Summary of the invention

[0004] The invention provides a solution to the technical problems existing in the existing goat lambing selection and breeding process.

[0005] The invention provides a SNP marker affecting the trait of goat litter size, which corresponds to the coding region of the goat CABP4 gene (sequence number: ENSCHIG00000012267) published in the Ensembl database, and the C / T difference of the nucleotide at the marker site leads to the difference in goat litter size.

[0006] The present invention provides a method for selecting goats for seed according to the marker genotype. According to the implementation of the present invention, the SNP marker is located at the 467th base of the nucleotide sequence shown in SEQ ID NO: 1, and the number of lambs produced by CC genotype and CT genotype goats at the SNP marker site is significantly higher than that of TT genotype individuals. By detecting the above-mentioned SNP marker of Macheng black goat, its number of lambs can be effectively predicted, so that the lambing performance of Macheng black goat individuals can be evaluated according to the genotype of the SNP marker site. Therefore, the SNP marker of the present invention is closely related to the number of lambs trait of Macheng black goat, and can be effectively used for molecular marker-assisted selection breeding of Macheng black goat, and then the goat breeding material can be selected according to the actual breeding needs, so that excellent individuals with a large number of lambs can be accurately and efficiently selected, and the efficiency and accuracy of breeding selection can be improved.

[0007] The present invention provides a primer pair for detecting the SNP marker of claim 1. 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, and is used to detect the SNP marker. According to an embodiment of the present invention, the primer pair of the present invention can effectively perform PCR amplification on the fragment where the SNP marker related to the lamb number trait of the Macheng black goat to be tested is located, and the detection of the SNP marker can be effectively achieved through sequencing, and the genotype of the individual Macheng black goat to be tested at the SNP marker site can be determined, so as to effectively predict the lamb number of the Macheng black goat to be tested.

[0008] Specifically, the number of lambs born by goats with CC genotype and CT genotype at the SNP marker site is significantly higher than that of individuals with TT genotype, indicating that the CC genotype at the SNP site can be used as an important criterion for judging the number of lambs born by Macheng black goats. In the breeding of Macheng black goats, the genome sequence of individual goats is amplified and sequenced using the primers of the SNP marker of the present invention to determine the genotype of the individual goats. Individuals with CC genotype at the SNP marker site can be retained for breeding, and CC genotype individuals can be mated with CT genotype individuals, and CC genotype individuals can be selected from the hybrid offspring for seed preservation, and individuals with CC genotype at the SNP marker site can be eliminated, so as to gradually increase the number of lambs born by the goat group, and then goats with a large number of lambs can be selected at low cost and high accuracy, realizing molecular marker-assisted breeding of goat lambs.

[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 of Macheng black goats, and the number of lambs of goats with CC genotype and CT genotype is significantly higher than that of individuals with TT genotype. (2) The SNP marker can be used for auxiliary selection of the number of lambs trait of Macheng black goats, and Macheng black goats with high number of lambs can be screened, which has important practical application value for further improving the reproductive capacity of Macheng black goats and using specific Macheng black goats as materials for variety (or strain) breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The above aspects of the present invention will be more easily understood in conjunction with the accompanying drawings. Figure 1 The CC, CT and TT genotype sequencing peak diagrams of the SNP marker sites of the present invention are displayed. Specific embodiments

[0011] 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.

[0012] 1. Experimental samples The 129 adult Macheng black goat ewes of Hubei Jinyang (Macheng) Animal Husbandry Co., Ltd. have a record of one to five litters of lambs, and their feeding management conditions and environmental conditions are the same.

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

[0014] 3. Primer Design According to the sequence of 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. The specific primers were used to amplify a DNA sequence where the mutation site of the first exon of the CABP4 gene was located. The amplified 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 goat CABP4 gene and sequencing to determine the genotype (1) PCR amplification system (20 μL): 2 μL DNA, 0.5 μL primers SEQ ID NO: 2 and SEQ ID NO: 3 (10 μM), 10 μL 2× Rapid Taq Master Mix (Nanjing Novogene Biotech Co., Ltd.), ddH 2 O 7μL. Amplification program: 95℃ pre-denaturation for 3min; 94℃ denaturation for 25s, 61℃ annealing for 25s, 72℃ extension for 14s, 35 cycles; 72℃ extension for 5min. (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 467 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 of the SNP marker site at 467 bp of SEQ ID NO: 1 in the sequence list mutates from C to T, changing the codon encoding tryptophan (Ser) to the codon encoding leucine (Leu).

[0016] 5. Association analysis between SNP markers of CABP4 gene and litter size in Macheng black goats One-way ANOVA in SPSS software was used to conduct the 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 is the individual phenotypic record; μ is the population mean; G i is the genotype effect; E ij is a random error.

[0017] 6. Analysis on the significance of differences in the number of lambs born among different genotypes of Macheng black goats The results of the analysis of the number of lambs 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 difference in the number of lambs between different genotypes was compared by one-way analysis of variance. It was found that the number of lambs of Macheng black goats with CC genotype and CT genotype was significantly higher than that of TT genotype (p<0.05), indicating that the CC genotype and CT genotype of this SNP marker locus can be used as an important criterion for judging the high number of lambs of Macheng black goats. In the breeding of Macheng black goats, individuals with CC genotypes at this SNP marker locus can be retained for breeding, and TT genotypes can be eliminated. Individuals with CT genotypes can also be mated with CC genotypes to obtain more CC-type offspring individuals, thereby gradually increasing the number of lambs of the Macheng black goat population. Table 1 Correlation between different genotypes of CABP4 gene mutation sites and litter size in Macheng black goats Note: Different letters in the same column indicate significant differences (p<0.05).

Claims

1. A molecular marker associated with goat litter size, the molecular marker being located in the first exon region of the goat CABP4 gene, the specific nucleotide sequence being SEQ ID NO: 1, wherein the 467th base of the sequence has a C / T polymorphism, and the number of litters born by goat individuals with CC and CT genotypes at this site is significantly higher than that by individuals with TT genotypes.

2. A method for selecting goats according to the molecular marker of claim 1, comprising the following steps: (1) Extracting goat genomic DNA; (2) using two specific primers to perform PCR amplification to obtain a 672 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 CC genotype of the above molecular markers for breeding; (6) The goat is a Macheng black goat.

3. Use of the molecular markers described in claim 1 and claim 2 in screening Macheng black goats with high lamb production, and selecting goat individuals with the CC genotype of the molecular markers for breeding.

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