CN116656839A - Application of ITGB8 gene mutation in selection of lambing number of black goats in Macheng - Google Patents
Application of ITGB8 gene mutation in selection of lambing number of black goats in Macheng Download PDFInfo
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Abstract
The invention discloses application of SNP markers in ITGB8 genes for influencing goat lambing numbers. The SNP marker locus is C/A base mutation at 325bp of SEQ ID NO. 1 in a sequence table, the lambing number of the AA genotype goats at the locus is obviously higher than that of individuals with CA and CC genotypes, and AA is the dominant genotype of the black goats in the milch city. According to the invention, the black goats in the milch city are taken as research objects, SNP variation of the second exon of the ITGB8 gene of the goats is amplified by using PCR, the influence of the specific genotype of the variation site on the lambing number of the black goats in the milch city is analyzed, and accordingly, the seed reserving of the black goats in the milch city is carried out, so that the method can be used for improving the lambing number of the black goats in the milch city, and a marking resource is provided for marking-assisted selective breeding of the character of the lambing number of the goats.
Description
Technical Field
The invention belongs to the technical field of animal molecular breeding, and relates to application of SNP markers related to lambing number traits of black goats in the Macheng in breeding of black goats in the Macheng.
Background
The lambing number is an important index for evaluating the reproductive capacity of animals, and influences the reproductive benefit and economic benefit of goats. The lambing number is affected by a number of factors including fertilization, embryo implantation, embryo development, delivery, etc., wherein embryo implantation refers to the process of fertilized eggs developing into embryos on the endometrium, which is divided into three stages in sequence: adsorption, invasion and implantation. In the adsorption stage, the outer membrane surface of fertilized ovum is adhered to the epithelial cells of endometrium, and fixation is realized by the synergistic effect of various factors such as adhesion molecules, cell adhesion receptor, collagenase and the like. In the invasion phase, fertilized eggs invade the mesenchymal and basilar membrane layers under the endometrial epithelium with the aid of enzymes such as metalloproteinases and fatty acid oxidases. In the final stage of implantation, fertilized eggs are deeply implanted into the uterine wall by endocrine regulation and self-growth, and continue to develop into embryos. The process of embryo implantation is complex and requires a series of normal signaling pathways involved in embryo development, such as hormone secretion, expression of adhesion molecules, apoptosis, and the like.
Integrins (integrins) are adhesion molecules that mediate intrauterine blastocyst attachment and downstream signaling activation, consist of alpha and beta subunits, are the primary receptors for cell interactions with the extracellular matrix (ECM), and are involved in cell survival, proliferation and migration by binding to the extracellular matrix. Expression of various integrins (ITGAV, ITGA4, ITGA5, ITGB1 and ITGB 5) was detected in the luminal epithelium and trophoblast germ layers of sheep at early gestation, whereas expression of these integrins was not detected in non-gestating sheep. Upregulation of integrin beta 3 (ITGB 3) and integrin beta 5 (ITGB 5) in human utero may improve endometrial receptivity to embryos. The increased levels of integrin beta 8 (ITGB 8) mRNA and protein expression at the uterine implantation site of mice during embryo implantation also helped the mice establish endometrial receptivity. ITGB8 is highly conserved between species but differs greatly from other integrin beta subunits, suggesting that it may have unique functions. ITGB8 also can regulate the interaction between embryonic cells and uterine epithelial cells, helping to assist in the correct attachment of embryos to the endometrium, primarily by binding to ligands on the endometrium to provide the necessary signals for the implantation process. Mice knocked out ITGB8 develop defects in placenta and yolk sac vascular development, resulting in death of most embryos in mid-gestation, and the remaining embryos die shortly after birth due to intracerebral hemorrhage caused by abnormal vascular development. In the goat endometrial epithelial cells, miR-187 regulates the expression of ITGB8, the expression level of ITGB8 is increased in the uterine adhesion phase, and the miR-187/ITGB8 axis regulates the activity of Focal Adhesion Kinase (FAK) and proliferation of the goat endometrial epithelial cells, so that the goat reproductive rate can be influenced. ITGB8 affects embryo implantation and vascular development, is important for success or failure of pregnancy and embryo survival, and directly affects the survival rate of offspring, so that it is inferred that ITGB8 has a certain correlation with lambing number. At present, no research literature related to the lambing number of goats exists in ITGB8, and SNP markers related to the lambing number of the black goats in the Macheng can be found in the ITGB8 gene, so that the molecular mechanism related to ITGB8 reproduction can be further known, and marker resources are provided for marker-assisted selection of the lambing number of the black goats in the Macheng.
Disclosure of Invention
The invention provides a method for solving the technical problems existing in the prior art of breeding goats for lambing.
According to the embodiment of the invention, the SNP marker is positioned at the 325 th base of the nucleotide sequence shown in SEQ ID NO. 1, and the lambing number of the AA genotype milch goat individuals of the SNP marker locus is obviously higher than that of the CA and CC genotype individuals. By detecting the SNP markers of the black goats in the milch city, the lambing number of the black goats in the milch city can be effectively predicted, and therefore the lambing performance of the black goats in the milch city can be estimated according to the genotypes of the SNP marker loci. Therefore, the SNP marker is closely related to the lambing number character of the black goats in the milch city, can be effectively used for auxiliary selective breeding of the molecular marker of the black goats in the milch city, and further can select goat breeding materials according to actual breeding requirements, so that excellent individuals with a large lambing number can be accurately and efficiently bred, and the breeding selection efficiency and accuracy are improved.
The present invention provides a primer set for detecting the SNP marker according to claim 1. According to an embodiment of the present invention, the primer has the nucleotide sequences shown in SEQ ID NO. 2 and SEQ ID NO. 3 for detecting the SNP marker.
According to the embodiment of the invention, the primer pair, the amplification program and the amplification system can be used for effectively amplifying the fragments of the SNP markers related to the lambing number characters of the to-be-detected milch goat, the detection of the SNP markers can be effectively realized through sequencing, the genotype of the SNP marker sites of the to-be-detected milch goat can be determined, and the lambing number of the to-be-detected milch goat can be effectively predicted. Specifically, the AA genotype of the SNP marker locus can be used as an important standard for judging the lambing number of the black goats in the Macheng. In the breeding work of the black goats in the Macheng, the individuals with the genotype of the SNP marker loci of CA and CC can be eliminated by reserving the individuals with the genotype of the SNP marker loci of AA for breeding. Therefore, the molecular marker-assisted selective breeding method can be effectively used for molecular marker-assisted selective breeding of the black goats in the milch cities, and further can realize low-cost and high-accuracy selection of the black goats in the milch cities with a large lambing number.
The invention has the following beneficial effects: (1) The SNP marker provided by the invention is obviously related to the lambing number of the black goats in the milch city, and the lambing number of the goats with the AA genotype is obviously higher than that of the goats with the CA and CC genotypes. (2) The SNP marker can be used for auxiliary selection of lambing number characters of the black goats in the milch city, screens the black goats in the milch city with high lambing number, and has important practical application value for further improving the fertility of the black goats in the milch city and breeding varieties (or strains) by using specific black goats in the milch city as materials.
Drawings
The above aspects of the present invention will be more readily understood from the description of the examples with reference to the accompanying drawings, and FIG. 1 shows CC, CA and AA genotype sequencing peak diagrams of SNP marker loci of the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description of the invention, in conjunction with the examples, is provided to illustrate the invention and should not be construed as limiting the invention.
1. Experimental sample
The Hubei Jin (Macheng) livestock limited company has 147 adult Macheng black goat ewes recorded for three years (2020-2022), and the feeding management conditions and the environmental conditions are the same, and a disposable vacuum negative pressure blood collection tube (EDTA-K) 2 Anticoagulation) jugular blood (5 mL/min.) was collected.
2. Genomic DNA extraction
Genomic DNA of blood samples of the black goat in Macheng was extracted by referring to the instructions of the Tiangen Biochemical technology (Beijing) limited blood genomic DNA extraction kit.
3. Primer design
According to the sequence of goat ITGB8 gene (Ensembl database gene sequence number: ENSCHIG 00000019429), a pair of specific primers P1 (SEQ ID NO: 2) and P2 (SEQ ID NO: 3) are designed by using Primer 5.0 software, the primers are synthesized by Beijing qing department biotechnology Co., ltd, the specific primers are used for amplifying a segment of DNA sequence where the second exon of the ITGB8 gene is located, and the amplified product is 676bp, and the DNA sequence is shown as SEQ ID NO:1 in a sequence table.
PCR amplification of goat ITGB8 Gene target sequence and sequencing
(1) PCR amplification System (25. Mu.L): 1. Mu.L of DNA, 1. Mu.L of primers SEQ ID NO:2 and SEQ ID NO:3 (10. Mu.M) each, 2X Rapid Taq Master Mix 12.5.5. Mu.L (Nanjinouzan Biotech Co., ltd.) ddH 2 O9.5. Mu.L. Amplification procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 15s, annealing at 56℃for 15s, extension at 72℃for 10s, and cycling for 35 times; extending at 72℃for 5min.
(2) The PCR amplified products were sent to the Wohan Jin Kairui Biotechnology Co.Ltd for sequencing. The result of sequencing is analyzed by SnapGene software, and the genotype of the 325bp locus of the nucleotide sequence shown in SEQ ID NO:1 in the sequence table of the individual is judged, as shown in figure 1, the genotype of the unimodal C is CC, the genotype of the unimodal A is AA, and the genotype of the bimodal is CA. The mutation of the base from C to A changes the leucine (Leu) encoding the site to methionine (Met).
5. Correlation analysis of ITGB8 gene SNP markers and lambing number traits of black goats in Macheng
Correlation analysis between genotype and lambing traits was performed using one-way analysis of variance in SPSS software. The specific linear analytical model is as follows:
Y ij ＝μ+G i +E ij
wherein: y is Y ij Recording the phenotype of the individual; mu is the population mean; g i Genotype effect for each locus; e (E) ij Is a random error.
6. Analysis of significance of differences of lambing numbers among different genotypes of black goats in milch city
Results of analysis of the lambing number traits of different genotypes of the black goats in the milch city are shown in Table 1. As can be seen from Table 1, the site has three genotypes, the difference of the lambing numbers among different genotypes is compared by adopting single-factor analysis of variance, the lambing numbers of the MAC black goats with the AA genotype are found to be obviously higher than those of CA and CC genotypes (p < 0.05), and obvious differences exist among the three genotypes, so that the AA genotype of the SNP marker site can be used as an important standard for judging the high lambing number of the MAC black goats. In the breeding work of the black goats in the milch city, the individuals with the genotype of the SNP marker loci of AA can be reserved for breeding, the individuals with the genotypes of CA and CC are eliminated, and the proportion of the genotype of AA in the black goats in the milch city is increased, so that the lambing performance of the black goats in the milch city is gradually improved.
TABLE 1 correlation of different genotypes and lambing numbers at ITGB8 Gene mutation sites in Macheng Heishan sheep
Note that: different shoulder letters indicate significant differences (p < 0.05).
Claims (2)
1. A method for selecting goats according to molecular markers related to the lambing number characters of goats, wherein the nucleotide sequence of the molecular markers is SEQ ID NO. 1, and the lambing number of AA genotype goats individuals at the 325bp locus of the sequence is obviously higher than that of CA and CC genotype individuals; the method comprises the following steps:
(1) Extracting goat genome DNA;
(2) Performing PCR amplification by using two specific primers to obtain a 676bp 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 detected in the molecular marker according to the sequencing result;
(5) Selecting goat individuals with the AA genotype of the molecular marker for seed reservation;
(6) The goats are black goats in the city of hemp.
2. The use of the SNP marker of claim 1 for screening black goats in milch city with high lambing numbers, selecting AA genotype goats individuals with the molecular marker for seed retention.
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| CN117004743A (en) * | 2023-09-28 | 2023-11-07 | 云南省畜牧兽医科学院 | SNP (Single nucleotide polymorphism) marker related to multi-lamb character of black goats on cloud and application of SNP marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117004743A (en) * | 2023-09-28 | 2023-11-07 | 云南省畜牧兽医科学院 | SNP (Single nucleotide polymorphism) marker related to multi-lamb character of black goats on cloud and application of SNP marker |
| CN117004743B (en) * | 2023-09-28 | 2023-12-22 | 云南省畜牧兽医科学院 | SNP (Single nucleotide polymorphism) marker related to multi-lamb character of black goats on cloud and application of SNP marker |
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