Application of PIEZO1 gene mutation in selection of lambing number of Mancheng black goats

Abstract

The invention discloses application of an SNP (Single Nucleotide Polymorphism) marker influencing the lambing number of goats. The marker is located on a PIEZO1 gene on a capa circus 18 # chromosome, the specific SNP marker site is A / C base mutation at the 236bp position of SEQ ID NO: 1 in a sequence table, and the lambing number of CC genotype and AC genotype goat individuals at the site is remarkably higher than that of AA genotype individuals. According to the present invention, the Musheng black goats are adopted as the research object, the DNA sequence of the 33rd exon of the PIEZO1 gene is subjected to PCR amplification, the sequence has an SNP variation site, the influence of the site polymorphism on the 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 so as to provide the basis for the breeding of the Musheng black goats. 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] 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] Lamb number in goats is a key trait affecting the economic benefits of goat farming. Its heritability is low, and it is regulated by multiple genes with minor effects and environmental factors. Traditional breeding methods result in slow genetic progress and unstable selection effects. Molecular marker-assisted selection (MMR) allows for early selection with high efficiency and accuracy. Major genes in lamb number exist, and currently known major genes are mainly concentrated in hormone and follicle development pathways regulated by the TGF-β signaling pathway (such as BMPR1B and GDF9) and steroid hormone metabolism pathways (such as ESR1).

[0003] The PIEZO family is a key family of mechanosensitive cation channels in mammals, with PIEZO1 and PIEZO2 as its core members. Both possess a unique trilobed propeller-like trimer structure, which enables the conversion of mechanical stimuli into electrochemical signals, thereby mediating a wide range of mechanotransduction processes in the body. PIEZO2 is mainly enriched in sensory neurons and related tissues, with its core function focused on somatic sensory regulation, including the transduction of tactile, proprioceptive, and mechanoreceptive signals. It also participates in respiratory tract extension sensing, bladder fullness monitoring, and the construction of the blood pressure reflex arc. PIEZO1, on the other hand, participates in multiple physiological processes with mechanotransduction as its core function. In the cardiovascular system, it regulates vascular tone, blood pressure homeostasis, and angiogenesis by sensing blood flow shear forces, ensuring tissue blood supply. In the reproductive system, PIEZO1 is closely related to animal reproductive performance, regulating the proliferation and differentiation of placental trophoblast cells, maintaining uterine blood supply during pregnancy by regulating uterine artery vasodilation, and potentially influencing follicle dominance and ovulation efficiency by participating in the perception of the mechanomicroenvironment during follicle development. Therefore, PIEZO1 regulates reproduction from dominant follicle selection, ovulation, placental trophoblast fusion, uterine blood supply during pregnancy, and even the initiation of labor. Its physiological processes regulating reproduction may affect the number of lambs born. This invention discovered that a mutation site in the PIEZO1 gene is significantly correlated with the number of lambs born in goats, providing marker resources for marker-assisted selection breeding of the lambing 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 the 236th base of the nucleotide sequence shown in SEQ ID NO:1. Goat individuals with the CC and AC genotypes at this SNP marker site have significantly higher litter sizes than those with the AA 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 DNA fragment containing the SNP marker associated with the litter size trait in the tested goat. 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 AC genotypes at the SNP marker locus had significantly higher litter sizes than those with the AA genotype, indicating that the CC and AC 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 the genotype of the individual goat at the SNP marker locus. Individuals with the CC genotype at the SNP marker locus can be retained for breeding, while individuals with the AA genotype at the SNP marker locus can be culled. Alternatively, individuals with the CC genotype can be mated with individuals with the AC 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 AC genotype is significantly higher than that of individuals with AA genotype. (2) The SNP marker can be used for auxiliary selection of the lambing trait in Macheng Black Goats, screening out 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, AC, and AA 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 Hubei Jinyang (Macheng) Livestock Co., Ltd. has 101 adult Macheng black goat ewes with records of lambing from one to five litters, and their feeding, management and environmental conditions are the same.

[0012] 2. Genomic DNA extraction Blood was collected from the jugular vein of the above samples (5 mL / animal) 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 diluted to 100 mg / mL and stored at 4℃.

[0013] 3. Primer design Based on the sequence of the PIEZO1 gene on goat chromosome 18 (Ensembl database gene sequence number: ENSCHIG00000021797), 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 exon 33 of the PIEZO1 gene. The amplified product was 690 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×Taq PCR Master Mix, and 9.5 μL ddH2O. Amplification program: 95℃ pre-denaturation for 3 min; 94℃ denaturation for 25 s, 59℃ annealing for 25 s, 72℃ extension for 13 s, 32 cycles; 72℃ extension for 5 min.

[0015] (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 236 bp site of the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing, such as... Figure 1 As shown, the genotype of unimodal C is CC, the genotype of unimodal A is AA, and the genotype of bimodal C is AC. A mutation from base C to A changes the codon encoding arginine (Arg) to the codon encoding serine (Ser).

[0016] 5. Association analysis between PIEZO1 gene SNP markers and litter size 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: Y... ij =μ+G i +E ij 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.

[0017] 6. Significant difference in litter size among different genotypes of Macheng black goats The results of the litter size trait 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 AC genotypes was significantly higher than that with the AA genotype (p<0.05). This indicates that the CC and AC 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 for breeding, while individuals with the AA genotype can be culled. Alternatively, individuals with the CC genotype can be mated with those with the AC genotype to obtain more CC-type offspring, thereby gradually increasing the litter size of the Macheng Black Goat population.

[0018]

Claims

1. A method for selecting high-producing goats based on molecular markers associated with goat litter size, wherein the molecular marker is located in the nucleotide sequence SEQ ID NO:1, wherein the base at the 236bp site of the sequence has a C / A polymorphism, and the litter size of goat individuals with CC and AC genotypes at the molecular marker site is significantly higher than that of individuals with AA genotype; The method includes the following steps: (1) Extracting goat genomic DNA; (2) PCR amplification was performed using two specific primers to obtain a 690bp 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; The goat in question is the Macheng Black Goat.

2. The application of the molecular marker described in claim 1 in screening Macheng Black Goats with high lambing rates, wherein individuals with the CC genotype of the molecular marker are selected for breeding.

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