Application of TSC22D1 gene mutation in selection of number of lambs of Muncheng black goats

Abstract

The invention discloses an application of an SNP (Single Nucleotide Polymorphism) marker in a TSC22D1 gene capable of influencing the lambing number of goats. The marker is located on a TSC22D1 gene of a goat chromosome 12, the specific SNP marker is T / C base mutation at the 608bp position of SEQ ID NO: 1 in a sequence table, and the lambing number of a TT genotype goat individual is remarkably higher than that of a CT genotype individual and that of a CC genotype individual. According to the present invention, the Musheng black goats are adopted as the research object, PCR is adopted to amplify the DNA sequence of the first exon of the goat TSC22D1 gene, the effect of the specific genotype of the variation site on the Musheng black goat lambing number is analyzed, and the Musheng black goat individual breeding is selected according to the analysis result, such that the Musheng black goat lambing number can be increased, and the Musheng black goat lambing number can be improved. 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 goats and their application in goat breeding. Background Art

[0002] Reproductive performance is an important factor affecting the economic benefits of goats, and litter size is an important trait for measuring reproductive performance. The litter size of goats is affected by various factors, with low heritability and being controlled by minor polygenes. Using traditional breeding and selection techniques for breeding, the genetic progress is slow. 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 TSC22D1 is an important gene affecting litter size.

[0003] TSC22D1 belongs to the members of the "TSC-22 domain family", also known as TGFb1i4 and MGC17597. The TSC22D1 gene has high homology in different species such as rats, mice, and humans, and is a highly conserved gene. This gene contains two main transcripts: TSC22D1.1 and TSC22D1.2, encoding a long-chain protein and a short-chain protein respectively. These two transcripts have opposite functions in regulating cell growth, differentiation, and apoptosis, and are closely related to the proliferation and apoptosis of tumors. As a transcription factor, TSC22D1 can regulate the expression of multiple genes including C-type natriuretic peptide. It forms homo- or heterodimers with other transcription factors, repressors, or activators to inhibit or activate the transcription of target genes, thereby participating in important biological processes such as lipid metabolism regulation, induction of cell senescence, and remodeling of the extracellular matrix. TSC22D1 is expressed in reproductive organs such as the prostate, testis, and ovary. Since TSC22D1 participates in the regulation of cell growth, differentiation, and apoptosis as a transcription factor, it may play a certain role in the development and maturation of germ cells. The present invention discovers that a SNP marker in exon 1 of the TSC22D1 gene is associated with the litter size trait of Macheng black goats, providing 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 the litter size of goats.

[0005] According to an embodiment of the present invention, the SNP marker is located at the 608th base of the nucleotide sequence shown in SEQ ID NO: 1, and the number of lambs born 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 Macheng black goats, the number of lambs born can be effectively increased, thereby evaluating the number of lambs born of Macheng black goat individuals according to the genotype of the SNP marker site. Therefore, the SNP marker of the present invention can be effectively used in molecular marker-assisted breeding of Macheng black goats, and then goat breeding materials can be selected according to actual breeding needs, so that goat individuals with more lambs can be bred accurately and efficiently, thereby improving the efficiency and accuracy of breeding selection.

[0006] The present invention provides a primer pair for detecting the 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. The primer pair of the present invention can effectively amplify the DNA fragment containing the SNP marker associated with the litter size of the goat to be tested, effectively detect the SNP marker by sequencing, determine the genotype of the Macheng black goat individual to be tested at the SNP marker site, and thus effectively predict the litter size of the goat to be tested.

[0007] Specifically, individuals with the TT genotype at the aforementioned SNP locus have significantly higher litter sizes than individuals with the CT and CC genotypes, making this an important criterion for determining goat litter size. In goat breeding, individuals with the TT genotype at this SNP locus can be retained for breeding while those with the CT and CC genotypes are eliminated. Alternatively, individuals with the CT genotype can be mated with those with the TT genotype to obtain more TT genotype offspring. This allows for low-cost, high-accuracy selection of superior goat breeds, thereby gradually increasing the litter size of a 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 by Macheng black goats, and the number of lambs born by Macheng black goats with the TT genotype is significantly higher than that of Macheng black goats with the CT and CC genotypes; (2) The SNP marker can be used for auxiliary selection of the number of lambs born by Macheng black goats, and Macheng black goats with high lamb numbers can be screened out, which has important practical application value for further improving the number of lambs born by 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 by referring to the accompanying drawings. Figure 1 The genotype sequencing peak diagram of the SNP markers TT, CT, and CC of the present invention is displayed. Specific embodiments

[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 170 adult Macheng black goats (with lambing records of 1 to 5 litters) from Hubei Jinyang (Macheng) Animal Husbandry Co., Ltd. have the same feeding management conditions and environmental conditions.

[0012] 2. Genomic DNA extraction Blood samples (5 ml / animal) of the above samples were collected using vacuum negative pressure blood collection tubes anticoagulated with dipotassium edetate (EDTA-K2), and the samples were stored at -20°C. 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 TSC22D1 gene (gene sequence number in the Ensembl database: ENSCHIG00000012362), 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 1st exon of the TSC22D1 gene is located. The amplification product is 739 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 TSC22D1 gene and genotype determination (1) PCR amplification system (20 μL): 2 μL DNA (50 mg / mL), 0.5 μL each of 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 58°C for 25 s, extension at 72°C for 12 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 608th bp site of the nucleotide sequence shown as SEQ ID NO:1 in the sequence listing, as Figure 1As shown, the genotype of single-peak T is TT, the genotype of single-peak C is CC, and the genotype of double-peak is CT. The base mutation from C to T changes the encoded alanine (Ala) to valine (Val).

[0015] 5. Association analysis of SNP markers of the TSC22D1 gene and litter size in Macheng black goats Single-factor variance analysis in SPSS software was used for the association analysis of 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 was found that the litter size of Macheng black goats with the TT genotype was significantly higher than that of the CT and CC genotypes (p < 0.05). This indicates that the TT 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 the TT genotype at this locus can be retained, and individuals with the CC genotype at this locus can be eliminated. Also, individuals with the CT genotype can be mated with those with the TT genotype to obtain more offspring individuals with the TT genotype, thereby gradually increasing the litter size of the goat population. Table 1 Correlation between different genotypes of the TSC22D1 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 litter size of Macheng black goats, wherein the molecular markers are located in the nucleotide sequence SEQ ID NO: 1, and the litter size of goat individuals with the TT genotype at the 608th site of this sequence is significantly higher than that of individuals with the CT genotype and the CC genotype; The method includes the following steps: (1) Extract goat genomic DNA; (2) Perform PCR amplification using two specific primers to obtain a 739bp amplification product. The sequences of the two specific primers are SEQ ID NO:2 and SEQ ID NO:3; (3) Sequence the PCR amplification product to obtain a sequencing result; (4) Determine the genotype of the goat individual to be tested at the molecular marker according to the sequencing result; (5) Select goat individuals with the TT genotype of the above molecular marker for breeding; (6) The goat is Macheng Black Goat.

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

Images