Application of SUSD1 gene mutation in selection of number of lambs of Muncheng black goats

Abstract

The invention discloses application of an SNP (Single Nucleotide Polymorphism) marker in an SUSD1 gene influencing the lambing number of goats. The marker is located on an SUSD1 gene of a goat chromosome 8, the specific SNP marker is T / C base mutation at the 336bp position of SEQ ID NO: 1 in a sequence table, and the lambing number of TT genotype and CT genotype individuals is remarkably higher than that of CC genotype individuals. According to the present invention, the Musheng black goats are adopted as the research object, the DNA sequence of the 14th exon of the goat SUSD1 gene is subjected to PCR amplification, 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, such that the Musheng black goat lambing number can be increased, and the Musheng black goat lambing number can be improved. The breeding of a new variety (strain) taking the Muscheng black goat as a breeding material is accelerated, and a marker resource is provided for marker-assisted selective breeding of goat lambing number traits.

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 the 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 selection techniques for breeding, the genetic progress is slow. At present, major genes for the litter size of sheep have been discovered, and whole-genome sequencing technology provides a basis for finding major genes affecting the litter size. The present invention discovers that SUSD1 is an important gene affecting the litter size.

[0003] SUSD1 (Sushi Domain Containing 1) is a DNA methylation-driven gene, whose expression is regulated by DNA methylation and plays an important role in the formation of epigenetic characteristics. This gene encodes the precursor of Sushi domain protein 1. A typical Sushi domain contains about 60 amino acids and four cysteines, SUSD1. The Sushi domain (also known as the complement control protein domain) usually participates in protein-protein interactions. During reproduction, the immune balance between the mother and the embryo is crucial, which is necessary to avoid the mother's immune rejection of the embryo and also ensure the defense ability against pathogens. SUSD1 has an immune regulatory function and may indirectly affect the reproductive process through microenvironment immune regulation during embryo implantation, placenta formation, and maintenance of pregnancy. Fertilization and early embryo development require complex cell adhesion and signal transduction, including sperm-oocyte mutual recognition and blastocyst implantation into the endometrium and other links. The protein-protein interaction function of the Sushi domain contained in SUSD1 enables it to possibly participate in the mutual recognition or adhesion of cell surface molecules, and thus may play a certain auxiliary role in gamete binding, embryo implantation, and the formation of the uterine-embryo interface. The present invention discovers that a SNP marker in exon 14 of the SUSD1 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 the selection and breeding of the litter size of goats.

[0005] According to an embodiment of the present invention, the SNP marker is located at the 336th base of the nucleotide sequence shown in SEQ ID NO: 1, and the number of lambs of individuals with TT genotype and CT genotype of the SNP marker is significantly higher than that of individuals with CC genotype. By detecting the above SNP marker of Macheng black goat, the number of lambs can be effectively increased, thereby evaluating the number of lambs 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 for molecular marker-assisted breeding of Macheng black goats, and then can select goat breeding materials according to actual breeding needs, so that goat individuals with more lambs can be accurately and efficiently selected, and the efficiency and accuracy of breeding selection can be improved.

[0006] The present invention provides a primer pair for detecting the SNP marker. 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. The primer pair of the present invention can effectively amplify the DNA fragment where the SNP marker related to the number of lambs of the 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, thereby effectively predicting the number of lambs of the goat to be tested.

[0007] Specifically, the number of lambs born by individuals with the TT genotype and CT genotype of the above SNP locus is significantly higher than that of individuals with the CC genotype, which can be used as an important criterion for judging the number of lambs born by goats. In goat breeding, individuals with the genotype of the SNP locus TT can be retained for breeding, and individuals with the genotype of the SNP marker locus CT and CC can be eliminated. Individuals with the genotype of CT can also be mated with individuals with the genotype of TT to obtain more offspring with the genotype of TT, so as to achieve low-cost and high-accuracy breeding of excellent goat breeds, thereby gradually increasing the number of lambs born by 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 by Macheng black goats, and the number of lambs born by Macheng black goats with TT and CT genotypes is significantly higher than that of Macheng black goats with CC genotypes; (2) The SNP marker can be used for auxiliary selection of the number of lambs born by Macheng black goats to screen out Macheng black goats with high lamb numbers, 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 make the description of the embodiments more easily understood with reference 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, and the blue background area is the SNP site. Specific embodiments

[0010] The 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 lambing records of 1 to 5 litters) from Hubei Jinyang (Macheng) Animal Husbandry Co., Ltd. had 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 SUSD1 gene (gene sequence number in the Ensembl database: ENSCHIG00000004066), 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 14th exon of the SUSD1 gene is located. The amplification product is 947 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 SUSD1 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×M5HiPerplus 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 54.5°C for 25 s, extension at 72°C for 14 s, 35 cycles, and final extension at 72°C for 5 min. (2) The PCR amplification products were sent to Wuhan Kingfisher Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using SnapGene software to determine the genotype of the individual at the 336th 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 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 threonine (Thr) to methionine (Met).

[0015] 5. Association analysis of SNP markers of SUSD1 gene with litter size in Macheng black goats The single-factor variance analysis in SPSS software was used for the association analysis of genotype and litter size. The specific linear analysis model is 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 TT and CT genotypes is significantly higher than that of CC genotype (p < 0.05). This indicates that the TT genotype and CT genotype of this SNP marker locus can be used as important criteria 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 CT and CC genotypes at this locus can be eliminated. Or individuals with the CT genotype can be mated with TT-level individuals to obtain more offspring individuals with the TT genotype, so as to gradually increase the litter size of the goat population. Table 1 Correlation between different genotypes of SUSD1 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, wherein the molecular marker is located in nucleotide sequence SEQ ID NO: 1, and the kidding numbers of goat individuals with TT genotype and CT genotype at the 336th site of this sequence are significantly higher than those of individuals with CC genotype; The method comprises the following steps: (1) Extracting goat genomic DNA; (2) Performing PCR amplification using two specific primers to obtain a 947bp 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 to be tested at the molecular marker according to the sequencing result; (5) Selecting 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 breeding of Macheng Black Goat population, wherein goat individuals with the TT genotype of the molecular marker are selected for breeding.

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