Application of PRICKLE1 gene mutation in goat body height selection

Abstract

The invention discloses application of an SNP (Single Nucleotide Polymorphism) marker highly related to a goat body. The marker is located on an exon of a PRICKLE1 gene of a goat chromosome 5, the specific SNP marker is C / T base mutation at the 305bp position of SEQ ID NO: 1 in a sequence table, and the body heights of TT and CT genotypes are significantly higher than those of CC type individuals. According to the present invention, the Musheng black goat is adopted as the research object, the PCR is adopted to amplify the DNA sequence of the goat PRICKLE1 gene, and the influence of the mutation site polymorphism on the goat body height is analyzed, such that the method can be used for breeding the Musheng black goat, and the Musheng black goat variety breeding using the Musheng black goat as the breeding material is accelerated; and a marker resource is provided for marker-assisted selection breeding of goat growth-related traits.

Description

Technical Field

[0001] The invention belongs to the technical field of animal molecular breeding, and relates to the application of a SNP marker affecting goat body height in goat breeding. Background Art

[0002] Body height is an important body size trait indicator in goat breeding. This trait is positively correlated with the body weight and carcass weight of animals and can be used to predict the body weight and carcass weight of goats. Body height is an important body size trait of Macheng black goats that has a strong correlation with body weight and is often used to evaluate their meat potential. Therefore, by selecting goats with larger body height for breeding, the growth rate and meat quality of the offspring can be indirectly increased, thereby improving the production efficiency of goats.

[0003] Prickle Planar Cell Polarity Protein 1 (PRICKLE1) encodes a cytoskeletal protein that plays a key role in the planar cell polarity pathway (PCP) signaling pathway of cell polarity, adhesion, and rearrangement. PRICKLE1 is involved in chondrocyte polarity during endochondral ossification and assists in the proximal and distal growth of endochondral elements. The PRICKLE1 gene is expressed in bone condensates, which differentiate into chondrocytes and subsequently form bones. The mutated PRICKLE1 gene changes the expression pattern of cell polarity pathway genes (including Wnt5a and Vangl2 genes in the PCP pathway), regulates cell polarity, and affects the expression of multiple factors, thereby hindering limb growth. Impaired PRICKLE1 function changes the expression of genes such as Bmp4, Fgf8, Vangl2, and Wnt5a, resulting in shortening and widening of limb bones and loss of finger joint segments. In summary, PRICKLE1 is involved in regulating the biological process of bone growth, which is beneficial to the growth and development of long bones and affects the height of animals. Its functional loss affects the maturation and directional division of chondrocytes, causing long bone growth defects.

[0004] Macheng Black Goat is an excellent black goat meat breed in the Dabie Mountains. It has the growth characteristics of fast growth rate and good fattening ability. Based on the whole genome selection signal analysis, a SNP marker associated with the body height of Macheng Black Goat was found in the PRICKLE1 gene, providing marker resources for molecular marker-assisted selection breeding of body shape-related traits of Macheng Black Goat. Summary of the invention

[0005] The invention aims to provide a technical method for breeding goats with high body height.

[0006] According to an embodiment of the present invention, the SNP marker is located at the 305th base of the nucleotide sequence shown in SEQ ID NO: 1. The body height of individuals with the TT and CT genotypes of the SNP marker is significantly higher than that of individuals with the CC genotype. By detecting the above-mentioned SNP marker of goats, the body height of goats can be evaluated according to the genotype of the SNP marker. Therefore, the SNP marker of the present invention is closely related to the body height of goats, and can be effectively used in molecular marker-assisted breeding of goats, and then goat breeding materials can be selected according to actual breeding needs, so that excellent goat varieties can be bred accurately and efficiently, thereby improving the efficiency and accuracy of breeding.

[0007] The present invention provides a primer pair for detecting a SNP marker as described in claim 1. 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 can effectively amplify the DNA fragment where the SNP marker related to the body height 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 goat individual to be tested at the SNP marker site can be determined, so as to effectively predict the body height of the goat to be tested. Specifically, the body height of individuals with TT and CT genotypes of the SNP site is significantly higher than that of individuals with CC genotype, which can be used as an important criterion for judging the body height of goats. In the goat breeding work, individuals with the genotype of the SNP site TT are retained, and individuals with the genotype of the SNP marker site CC are eliminated, so as to select goats with higher body height at low cost and high accuracy, indirectly increase the weight of goats, and gradually increase the body height and weight of 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 body height of Macheng black goats, and the body height of 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 body size traits of Macheng black goats to screen tall Macheng black goats, which has important practical application value for further improving the production performance of 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 in conjunction with 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 are described in detail below, and the present invention is further described in detail in conjunction with the embodiments. The embodiments herein are only used to illustrate the present invention and are not to be construed as limiting the present invention.

[0011] 1. Experimental samples A total of 217 adult Macheng black goats of Hubei Jinyang (Macheng) Animal Husbandry Co., Ltd. were raised and managed under the same environmental conditions. 5 ml of blood samples were collected in blood collection tubes anticoagulated with EDTA2K dipotassium edetate (EDTA-2K). The samples were stored at -20°C and their body height (unit: cm) in the morning in the fasting state was measured. The results were rounded to one decimal place.

[0012] 2. Genomic DNA Extraction Use disposable vacuum negative pressure blood collection tube (EDTA-K 2 The jugular vein blood (5 mL / goat) of the above-mentioned experimental sample was collected for anticoagulation, and the genomic DNA of the blood samples of Macheng black goats was extracted according to the instructions of the blood genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0013] 3. Primer Design According to the sequence of goat PRICKLE1 gene (Ensembl database gene sequence number: ENSCHIG00000012537), 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 Qingke Biotechnology Co., Ltd. and were used to amplify a sequence where the 9th exon of the PRICKLE1 gene is located. The amplified product is 544 bp, and the nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table.

[0014] 4. Polymerase chain reaction (PCR) amplification and sequencing of the target sequence of the PRICKLE1 gene of Macheng black goat (1) PCR amplification system (20 μL): 1 μL DNA (50 mg / mL), 1 μL each of the upstream and downstream primers SEQ ID NO: 2 and SEQ ID NO: 3 (100 uM), 10 μL 2×M5 HiPer plus Taq HiFi PCR mix (with blue dye) (purchased from Beijing Polymer Biotechnology Co., Ltd.), and 7 μL ddH2O. The amplification program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 55°C for 25 s, extension at 72°C for 8 s, 35 cycles, and final extension at 72°C for 5 min. (2) The PCR amplification product was 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 305 bp position of the nucleotide sequence shown in SEQ ID NO: 1 in the sequence list, such as Figure 1As shown, the genotype of the single peak T is TT, the genotype of the single peak C is CC, and the genotype of the double peak is CT. The base mutates from C to T, changing the encoded alanine (Ala) to valine (Val).

[0015] 5. Association analysis between PRICKLE1 gene SNP markers and body height in Macheng black goats One-way ANOVA in SPSS software was used to analyze the association between genotype and height. The specific linear analysis model is as follows: Y ij =μ+G i +E ij Where: Y ij is the individual phenotypic record; μ is the population mean; G i is the genotype effect of each site; E ij is a random error.

[0016] 6. Analysis of the significance of height differences among different genotypes of Macheng black goat The results of the analysis of the height of different genotypes of Macheng black goats are shown in Table 1. As can be seen from Table 1, there are three genotypes at this locus. The one-way ANOVA was used to compare the differences in average height between different genotypes. It was found that the height of Macheng black goats with TT and CT genotypes was significantly higher than that of CC genotype (p < 0.05), indicating that the TT and CT genotypes of this SNP locus can be used as an important criterion for judging the height of Macheng black goats. In the breeding of Macheng black goats, individuals with the genotype of TT at this locus can be retained and individuals with the genotype of CC at this locus can be eliminated. TT and CT type individuals can also be bred to obtain more TT type offspring individuals, thereby gradually increasing the height of the goat population and indirectly increasing the weight. Table 1 Correlation between different genotypes of PRICKLE1 gene mutation sites and body height Note: Different letters in the same column indicate significant differences (p<0.05).

Claims

1. A method for selecting goats based on a molecular marker associated with goat height, wherein the molecular marker is located in the nucleotide sequence SEQ ID NO: 1, wherein the height of goat individuals with TT and CT genotypes at the 305 bp position of the sequence is significantly higher than that of individuals with CC genotypes; The method comprises the following steps: (1) Extracting goat genomic DNA; (2) using two specific primers to perform PCR amplification to obtain a 544 bp 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 tested at the molecular marker according to the sequencing result; (5) selecting goat individuals with the TT genotype of the above molecular marker for seed preservation; (6) The goat is a Macheng black goat.

2. Application of the molecular marker according to claim 1 in the breeding of Macheng black goat population, selecting Macheng black goat individuals with TT genotype of the molecular marker for seed preservation.

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