Application of SNP (Single Nucleotide Polymorphism) marker related to lambing number of goats

Abstract

The invention discloses application of an SNP (Single Nucleotide Polymorphism) marker influencing the lambing number of goats. The marker is located at the 6293801th basic group on the No.15 chromosome of a goat, the specific SNP marker site is C / G basic group mutation at the 392bp position of SEQ ID NO: 1 in a sequence table, and the lambing number of a CC genotype goat individual and a CG genotype goat individual at the site is remarkably higher than that of a GG genotype individual. The method comprises the following steps: by taking a Muncheng black goat as a research object, carrying out PCR (Polymerase Chain Reaction) amplification on a segment of ENSCHIG00000009743 gene, wherein the segment contains a goat 15 # chromosome g.6293801Cgt; according to the present invention, the DNA sequence of the G mutation site is analyzed, the influence of the specific genotype of the site on the lambing number of the Mancheng black goats is analyzed, and the individual breeding of the Mancheng black goats is performed according to the influence, such that the Mancheng black goat lambing number can be increased, and the breeding of the new variety (strain) using the Mancheng black goats as the breeding material can be accelerated; and a marker resource is provided for marker-assisted selective breeding of goat lambing number 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] The number of lambs born in goats is a key trait determining the economic benefits of goat farming. It is influenced by multiple genes and environmental factors, and its heritability is low, resulting in slow progress in genetic selection for this trait using traditional methods. Several major genes affecting the number of lambs born have been identified, including GDF9, BMP15, BMPR1B, KISS1, and KITLG.

[0003] This invention uses genome-wide association studies (GWAS) to discover that the g.6293801C>G site in the ENSCHIG00000009743 gene sequence on goat chromosome 15 is significantly associated with the number of lambs born in Macheng Black Goats, providing marker resources for marker-assisted selection breeding of the lambing number trait in Macheng Black Goats. Summary of the Invention

[0004] This invention provides a method for selecting goats for breeding based on the genotype of the marker locus.

[0005] According to an embodiment of the present invention, the SNP marker is located at base 392 of the nucleotide sequence shown in SEQ ID NO:1. Goat individuals with the CC and CG genotypes at this SNP marker site have significantly higher litter sizes than those with the GG genotype. By detecting the aforementioned SNP marker in Macheng Black Goats, the litter size of individual goats can be assessed based on the genotype at this SNP marker site. Therefore, the SNP marker of the present invention is closely related to the litter size 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, thereby enabling accurate and efficient breeding of goat individuals with high litter sizes, improving the efficiency and accuracy of selection during the breeding process.

[0006] This invention provides a primer pair for detecting the 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, 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 goat by PCR, and sequencing can effectively detect the SNP marker, determine the genotype of the tested goat at the SNP marker site, and thus effectively predict the litter size of the tested goat.

[0007] Specifically, individuals with the CC and CG genotypes at the SNP marker locus had significantly higher litter sizes than those with the GG genotype, indicating that the CC and CG genotypes at this SNP locus can serve as important criteria for determining the number of lambs produced in goats. In goat breeding, the genome sequence of individual goats is amplified and sequenced using the primer pairs for the SNP marker described in this invention to determine their genotype. Individuals with the CC genotype at the SNP marker locus can be retained for breeding, while individuals with the GG genotype at the same SNP marker locus can be culled. Alternatively, individuals with the CC genotype can be mated with individuals with the CG genotype to obtain more individuals with the CC genotype for breeding. This allows for low-cost and high-accuracy selection of goats with high litter sizes, thereby gradually increasing the number of lambs produced in 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 number of lambs born in Macheng Black Goats. The number of lambs born in Macheng Black Goats with CC genotype and CG genotype is significantly higher than that of individuals with GG genotype. (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. It has important practical application value for further improving the lambing number 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 from the above aspects of the invention in conjunction with the accompanying drawings. Figure 1 The sequencing peak diagrams of the CC, CG, and GG genotypes of the SNP marker sites of this invention are shown. 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 The 112 adult Macheng black goat ewes (with records of one to five litters) of Hubei Jinyang (Macheng) Livestock Co., Ltd. were kept under the same feeding, management and environmental conditions.

[0012] 2. Genomic DNA extraction Blood was collected from the jugular vein of the above samples (5 mL / goat) using disposable vacuum negative pressure blood collection tubes (EDTA-K2 anticoagulation). Genomic DNA was extracted from the blood samples of Macheng black goats using the Kangwei Genomics Blood Extraction Kit. The DNA concentration of the samples was measured and diluted to 100 mg / mL, and stored at 4℃.

[0013] 3. Primer design Based on the DNA sequence near the g.6293801C>G site on goat chromosome 15 (Ensembl database gene sequence number: ENSCHIG00000009743), a pair of specific primers, SEQ ID NO:2 and SEQ ID NO:3, were designed using Primer 6.0 software. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. These specific primers were used to amplify a DNA sequence containing the g.6293801C>G mutation site in the ENSCHIG00000009743 gene. The amplified product was 764 bp in length, and its nucleotide sequence is shown in SEQ ID NO:1 in the sequence listing.

[0014] 4. PCR amplification of the target sequence of sample DNA and determination of genotype (1) PCR amplification system (25 μL): 1 μL DNA (100 ng / μL), 1 μL each of primers SEQ ID NO:2 and SEQ ID NO:3 (10 uM), 12.5 μL 2×Rapid Taq Master Mix, and 9.5 μL ddH2O. Amplification program: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s, 55℃ annealing for 25 s, 72℃ extension for 15 s, 32 cycles; 72℃ final 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 392 bp site of the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing, as shown in the attached figure. Figure 1 As shown, the genotype of unimodal C is CC, the genotype of unimodal G is GG, and the genotype of bimodal C is CG. A mutation from base G to C changes the codon encoding glycine (Gly) to the codon encoding cysteine ​​(Cys).

[0015] 5. Association analysis between SNP markers and lambing number 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 was as follows: Y ij =μ+G i +E i : 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.

[0016] 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 CG genotypes was significantly higher than that with the GG genotype (p<0.05). This indicates that the CC and CG 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, while individuals with the GG and CG genotypes can be culled. Alternatively, individuals with the CC genotype can be bred with those with the CG 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 and litter size at the g.6293801C>G mutation site in Macheng Black Goats. Note: Different shoulder letters in the same column indicate significant differences (p < 0.05).

Claims

1. A method for selecting goats based on molecular markers associated with the number of lambs born, wherein the molecular markers are located in the nucleotide sequence SEQ ID NO:1, wherein goat individuals with the CC and CG genotypes at position 392bp of the sequence have a significantly higher number of lambs born than individuals with the GG genotype; The method includes the following steps: (1) Extracting goat genomic DNA; (2) PCR amplification was performed using two specific primers to obtain a 764bp 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; (6) The goat mentioned is the Macheng Black Goat.

2. The application of the molecular marker described in claim 1 in the breeding of lambing number traits in Macheng black goats, wherein goat individuals with the CC genotype of the molecular marker are selected for breeding.

Images