SNP (Single Nucleotide Polymorphism) marker for increasing lambing number of goats and application of SNP marker
By detecting the SNP marker sites of the PITPNM1 gene, individual goats with CC or CT genotypes were screened for breeding, the problem of low heritability of goat lamb numbers was solved, efficient and accurate breeding selection was achieved, and the number of lambs in Macheng black goats was increased.
Patent Information
- Application Number
- CN202510449273.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-07-08
AI Technical Summary
The heritability of goat lamb number is low and affected by a variety of factors. The use of traditional breeding selection technology is slow, making it difficult to achieve early selection and precise selection.
The SNP marker of the PITPNM1 gene was discovered and used to detect the genotype of the marker site, provide primer pairs for PCR amplification and sequencing, determine the genotype of goat individuals, screen out CC or CT genotype individuals for breeding, eliminate TT genotype individuals, and achieve efficient and accurate breeding selection.
The number of lamb production of Macheng black goats has been significantly improved, the efficiency and accuracy of breeding have been improved, and the number of goats with a large number of lambs has been bred at low cost and high accuracy.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of animal molecular breeding, and relates to an SNP marker related to the litter size of Macheng black goats and its application in the breeding of Macheng black goats. Background Art
[0002] The reproductive performance of goats is an important indicator to measure their economic benefits, and the litter size is an important trait to measure the reproductive performance. The litter size of goats is affected by various factors, with low heritability and being controlled by polygenes with minor effects. Using traditional breeding selection techniques for breeding results in slow genetic progress. Selecting based on the genes controlling reproductive traits can achieve early selection and precise selection. In the present invention, whole-genome sequencing was performed on two individuals with different litter sizes of Macheng black goats, and it was found that a base mutation in PITPNM1 significantly affects the litter size.
[0003] The Phosphatidylinositol Transfer Protein Membrane-associated 1 (PITPNM1) gene encodes a phosphatidylinositol transfer protein that binds to the cytoplasmic side of the Golgi membrane. This protein is mainly distributed inside the cell, has the activity of phosphatidylinositol transfer protein, can bind metal ions at the same time, participates in the transport process of phosphatidylinositol in the cell, and plays a key role in the formation and maintenance of cell membranes. PITPNM1 also participates in physiological processes such as lipid metabolism, brain development, and light conduction. Lipid metabolism is an important physiological function of goats, providing energy for goats, especially a large amount of energy is required during follicle development and ovulation in the estrus period. During pregnancy and lactation, female goats also need energy to support the development of fetuses and the secretion of milk. Therefore, the nutritional status of female goats during the estrus period, pregnancy, and lactation has an important impact on the litter size. Only female goats with sufficient nutrition can produce more healthy lambs. The present invention found a new SNP mutation site in the PITPNM1 gene, and this mutation site affects the litter size 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 goat litter size.
[0005] The present invention provides an SNP marker that affects the goat litter size trait. This marker is located in the coding region of the goat PITPNM1 gene (sequence number: ENSCHIG00000000979) published in the Ensembl database. The C / T difference in the nucleotides at this marker site leads to different goat litter sizes.
[0006] The present invention provides a method for selecting goats for seed according to the genotype of the marker site. According to an embodiment of the present invention, the SNP marker is located at the 736th base of the nucleotide sequence shown in SEQ ID NO: 1, and the number of lambs of individuals with CC genotype and CT genotype of the SNP marker is significantly higher than that of individuals with TT genotype. By detecting the above SNP marker of Macheng black goat, the number of lambs 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 number of lambs of Macheng black goats, and can be effectively used for molecular marker-assisted breeding of Macheng black goats, and then the goat breeding materials can be selected according to the actual breeding needs, so that goat individuals with a large number of lambs can be accurately and efficiently selected, and the efficiency and accuracy of breeding can be improved.
[0007] The present invention provides a primer pair for detecting a SNP marker as claimed in claim 1. According to an embodiment of the present invention, the primers have nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, and 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 SNP marker can be effectively detected by sequencing, and the genotype of the individual 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.
[0008] Specifically, the number of lambs produced by individuals with CC genotype and CT genotype of the SNP site is significantly higher than that of individuals with TT genotype, which can be used as an important criterion for judging the number of lambs produced by goats. In goat breeding, the genomic sequence of the goat individual is amplified and sequenced using the primer pair of the SNP marker of the present invention to determine the genotype of the goat individual at the SNP marker site. The individuals with CC genotype of the SNP marker site can be retained for breeding, and the individuals with TT genotype of the SNP marker site can be eliminated. The CC genotype individuals can also be mated with the CT genotype individuals, and the CC genotype individuals can be selected from the hybrid offspring for seed preservation to obtain more CC genotype individuals, so as to achieve low-cost and high-accuracy breeding of goat individuals with a large number of lambs, thereby gradually increasing the number of lambs produced by the goat population.
[0009] 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 goats with CC genotype and CT genotype is significantly higher than that by goats with TT genotype; (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 number of lambs born 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
[0010] The above aspects of the present invention will be more easily understood in conjunction with the accompanying drawings in the description of the embodiments. Figure 1 Show the SNP marker CC, CT, TT genotype sequencing peak maps of the present invention. Specific embodiments
[0011] The embodiments of the present invention are described in detail below. 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 should not be construed as limiting the present invention.
[0012] 1. Experimental samples 178 adult Macheng black goats with lambing records of one to five litters from Hubei Jinyang (Macheng) Animal Husbandry Co., Ltd., with the same feeding management conditions and environmental conditions.
[0013] 2. Genomic DNA extraction Collect the blood samples (5 ml / animal) of the above samples with blood collection tubes anticoagulated with dipotassium ethylenediaminetetraacetate (EDTA2K dipotassium edetate, EDTA-2K), and store the samples at -20°C. Use the blood genomic DNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the genomic DNA from the blood samples of Macheng black goats, measure the DNA concentration of the samples and dilute it to 50 mg / mL, and store it at 4°C.
[0014] 3. Primer design According to the sequence of the goat PITPNM1 gene (gene sequence number in the Ensembl database: ENSCHIG00000000979), use Primer 6.0 to design a pair of specific primers SEQ ID NO:2 and SEQ ID NO:3. The primers are synthesized by Beijing Tsingke Biotechnology Co., Ltd. and are used to amplify a DNA sequence where the 13th exon of the PITPNM1 gene is located. The amplification product is 895 bp, and the nucleotide sequence is shown as SEQ ID NO:1 in the sequence listing.
[0015] 4. PCR amplification of the target sequence of the PITPNM1 gene of goat DNA and determination of genotype (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 μM), 10 μL of 2×M5 HiPer plus Taq HiFi PCR mix (Beijing Polymer Beauty Biotechnology Co., Ltd.), 7 μL of ddH2O. The amplification program is: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 25 s, annealing at 56°C for 25 s, extension at 72°C for 13 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 736bp site of the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing. As Figure 1 shown, the genotype of single peak C is CC, the genotype of single peak T is TT, and the genotype of double peak is CT. The base at the SNP marker site of 736bp in SEQ ID NO:1 of the sequence listing mutated from T to C, changing the codon encoding leucine (Leu) to the codon encoding proline (Pro).
[0016] 5. Association analysis of SNP markers of the PITPNM1 gene in Macheng black goats with litter size The single-factor variance analysis in SPSS software was used to conduct the association analysis between genotype and litter size. 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; E ij is the random error.
[0017] 6. Analysis of the significance 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. The single-factor variance analysis was used to compare the differences in average litter size among different genotypes. It was found that the litter size of Macheng black goats with the CC genotype and the CT genotype was significantly higher than that of the TT genotype (p < 0.05), indicating that the CC genotype and the CA genotype of this SNP 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 CC genotype at this locus can be retained for breeding, and individuals with the TT genotype at this locus can be eliminated. The CT type can also be mated with individuals with the CC genotype to obtain more offspring individuals with the CC genotype, thereby gradually increasing the litter size of the Macheng black goat population. Table 1 Correlation between different genotypes of the PITPNM1 gene mutation site in Macheng black goats and litter size Note: Different superscript letters in the same column indicate significant differences (p < 0.05).
Claims
1. A molecular marker related to the litter size of goats, the molecular marker is located in the nucleotide sequence SEQ ID NO: 1, where the 736th base of this sequence has C / T polymorphism, and the litter sizes of goat individuals with the CC genotype and CT genotype at this site are significantly higher than those of TT genotype individuals.
2. A method for selecting goats according to the molecular marker described in claim 1, the method comprising the following steps: (1) Extracting genomic DNA of goats; (2) Performing PCR amplification using two specific primers to obtain an 895bp 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 individual to be tested at the molecular marker according to the sequencing result; (5) Selecting goat individuals with the CC genotype of the above molecular marker for breeding; (6) The goat is Macheng Black Goat.
3. Application of the molecular marker described in claim 1 in screening Macheng Black Goats with high litter size, selecting goat individuals with the CC genotype of the molecular marker for breeding.