A SNP molecular marker related to the reproductive traits of Haimen goats and its application

Abstract

The present invention discloses a SNP molecular marker related to the reproductive traits of Haimen goats and its application, belonging to the field of biotechnology and livestock breeding. The SNP molecular marker contains a mutation site of Wnt10a g.28944633G>A. The present invention uses the blood genomic DNA of Haimen goats as a template and a method of direct sequencing of PCR products to determine the genotype. Among them, the AA genotype is significantly associated with the number of lambs born by Haimen goats, and can be used as an effective molecular marker to improve the reproductive traits of Haimen goats. The detection method of the present invention is simple, rapid, highly accurate and low-cost, can improve the breeding efficiency of Haimen goats, and has high application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and livestock breeding, and relates to a SNP molecular marker related to the reproductive traits of Haimen goats and an application thereof. Background Art

[0002] With the development of the national economy and the improvement of people's living standards, the market demand for mutton is getting higher and higher, but the production speed of sheep can no longer meet people's demand for mutton. Good breeds are the basis of animal husbandry production, so improving the degree of good breeds of sheep through scientific means is of great significance to the development of the breeding industry.

[0003] In modern large-scale breeding, the reproductive performance of ewes is an important factor affecting the economic benefits of farms. Reproductive traits are complex quantitative traits, involving multiple processes such as ovarian follicle development, oocyte maturation, ovulation, fertilization, and embryonic development, and are jointly regulated by multiple factors such as genetics, epigenetic modification, feeding management, hormones, and nutrition (Zhang Yanli, Guo Jiahe, Yao Xiaolei, & Wang Feng, 2022). In production, the reproductive traits of ewes are often improved by shortening the lambing interval or increasing the number of lambs per litter. The traditional breeding method is to infer and select by observing the phenotype, which has a long breeding cycle, a large workload, and poor results. In recent years, with the development of molecular biology, molecular marker-assisted selection technology (MAS) has become increasingly mature. The selection of target trait genotypes through molecular markers can improve breeding efficiency and provide strong support for the breeding of high-fertility trait goats.

[0004] SNP refers to a genomic variation caused by a single nucleotide change. It is the simplest and most common heritable variation in livestock and poultry, occurring at a frequency of about 1 / 1000bp in the entire genome. SNP has become the most widely used molecular marker due to its large number and easy detection (Lin Yuexia, Lv Yuhua, & Liao Rongrong, 2022).

[0005] The Wnt10a gene is a member of the Wnt gene family. The Wnt gene family consists of structurally related genes that encode secretory signaling proteins. The series of signal transduction pathways regulated by the Wnt gene are called the Wnt signaling pathway. This is a genetically extremely conserved signal transduction pathway that plays an important role in a variety of biological processes such as growth and development, metabolism, and stem cell maintenance. Wnt10a also plays a key role in wound healing, cancer growth, osteogenesis, and adipogenesis. Jia-He Zhang et al. (Jia-He Zhang et al., 2022) found that the estrus cycle of Wnt10a knockout mice was disrupted, the number of follicles was reduced, the endometrium was thinned, the serum estrogen level was reduced, and the testosterone and progesterone levels were increased; in neurobehavior, Wnt10a knockout mice showed spatial memory impairment and anxiety-like behavior.

[0006] The research on Wnt10a polymorphism is currently focused on human diseases. The study found that the Wnt10a variant site rs10177996 is associated with the sella calcification phenotype, and the probability of sella calcification in individuals with the TT genotype is significantly higher than that in individuals with the CT and TT genotypes ( Armando Brancher et al., 2023). Jiang Xinke et al. (Jiang Xinke, Yu Kang, Zhou Mengqi, Wu Yiqun, & Wang Feng, 2022) detected Wnt10a mutations in 6 patients with severe congenital tooth agenesis and detected a total of 5 mutation sites. They predicted the functional effects of the mutations on the protein and found that these mutations showed a certain degree of pathogenicity. MFAbid et al. (MFAbid et al., 2018) found that a nonsense heterozygous mutation in Wnt10a c.321C>A exon 2 may cause severe tooth dysplasia through the generation of premature termination codons. However, there are still no reports on the study of Wnt10a polymorphism in goat reproductive traits. Summary of the invention

[0007] In order to overcome the shortcomings of the prior art in the selection of Haimen goat reproductive traits, the object of the present invention is to provide a SNP molecular marker related to the reproductive traits of Haimen goats and an application thereof.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention claims the use of SNP molecular markers associated with the reproductive traits of Haimen goats or substances for detecting SNP molecular markers associated with the reproductive traits of Haimen goats in the screening and / or breeding of reproductive traits of Haimen goats:

[0010] The SNP molecular marker associated with the reproductive traits of Haimen goats is Wnt10a g.28944633G>A, located at 28944633bp of chromosome 2, which is a G>A mutation.

[0011] Furthermore, the reproductive trait is the number of lambs born.

[0012] Furthermore, the reproductive trait screening and / or breeding of Haimen goats is to use the above-mentioned Wnt10ag.28944633G>A molecular marker to identify the litter size trait of Haimen goats.

[0013] Furthermore, the reproductive trait screening and / or breeding of Haimen goats also includes using the above-mentioned Wnt10ag.28944633G>A molecular marker to regulate the number of lambs born by Haimen goats.

[0014] Furthermore, the substance contains (or is) a PCR primer for amplifying a Haimen goat genomic DNA fragment containing the SNP molecular marker.

[0015] In a specific embodiment of the present invention, the PCR primers include an upstream primer F1 and a downstream primer R1:

[0016] Upstream primer F1: 5′-TCTGTGGCGTGCCGTGTT-3′;

[0017] Downstream primer R1: 5′-CAGCCCATCGGAGCTGTCT-3′.

[0018] Furthermore, the genomic DNA of the blood of the Haimen goat to be tested is used as a template, PCR amplification is performed using the above-mentioned PCR primers, and the genotype of the SNP molecular marker is determined by direct sequencing of the PCR amplification product; the AA homozygous genotype individuals are retained, and the GG homozygous genotype individuals and the AG heterozygous genotype individuals are eliminated, thereby increasing the number of lambs born by Haimen goats generation by generation.

[0019] The different genotypes of Wnt10a g.28944633G>A had a significant effect on the number of lambs born in the third litter of Haimen goats. The number of lambs born in the third litter of ewes with AA genotype was significantly higher than that of ewes with GG genotype.

[0020] In a specific embodiment of the present invention, the Wnt10a g.28944633G>A genotype is determined based on the sequencing peak graph: when the peak graph shows a single peak A, the genotype is AA; when the peak graph shows a single peak G, the genotype is GG; when the peak graph shows a double peak of A and G, the genotype is AG.

[0021] In the second aspect, the present invention claims protection for a genetic breeding improvement method for the reproductive traits of Haimen goats, using the blood genomic DNA of the Haimen goat to be tested as a template, using the above-mentioned PCR primers to perform PCR amplification, and determining the genotype of the SNP molecular marker Wnt10a g.28944633G>A by direct sequencing of the PCR amplification product; retaining AA homozygous genotype individuals, eliminating GG homozygous genotype individuals and AG heterozygous genotype individuals, thereby increasing the number of lambs born by Haimen goats generation by generation.

[0022] Furthermore, in the above method, different genotypes of Wnt10a g.28944633G>A had a significant effect on the number of lambs born in the third litter of Haimen goats, and the number of lambs born in the third litter of ewes with AA genotype was significantly higher than that of ewes with GG genotype.

[0023] In a specific embodiment of the present invention, the PCR product is directly sequenced to determine the genotype.

[0024] In a specific embodiment of the present invention, the Wnt10a g.28944633G>A genotype is determined based on the sequencing peak graph: when the peak graph shows a single peak A, the genotype is AA; when the peak graph shows a single peak G, the genotype is GG; when the peak graph shows a double peak of A and G, the genotype is AG.

[0025] In a specific embodiment of the present invention, the reaction system of the PCR amplification is 40 μL, comprising: 2×Taq Plus Master MixⅡ20 μL; 1.6 μL of upstream and downstream primers; 2 μL of template DNA; 14.8 μL of deionized water; the reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 20 s, extension at 72°C for 30 s, for a total of 30 cycles; extension at 72°C for 5 min; and storage at 4°C after the completion of PCR.

[0026] In a specific embodiment of the present invention, selecting AA genotype individuals as parents in Haimen goat breeding can increase the number of lambs born in the third litter. The number of lambs born in the third litter of AA genotype individuals is 0.47 more than that of GG genotype individuals, and 0.28 more than that of AG genotype individuals.

[0027] The above-mentioned SNP molecular markers related to the reproductive traits of Haimen goats also fall within the protection scope of the present invention.

[0028] The above-mentioned substances used for detecting the SNP molecular markers related to the reproductive traits of Haimen goats also belong to the protection scope of the present invention.

[0029] Furthermore, the substance is a PCR primer for amplifying a Haimen goat genomic DNA fragment containing the SNP molecular marker, or a kit containing the PCR primer. The specific nucleotide sequence of the PCR primer is as described above.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention first discovered an effective SNP molecular marker in the Wnt10a gene that affects the litter size of Haimen goats. The kit provided by the present invention is used to detect the genotype of the molecular marker by PCR amplification and product direct sequencing. The method is simple, fast, highly accurate and inexpensive. The molecular marker of the present invention is used to screen Haimen goats, retain AA homozygous genotype individuals, eliminate GG homozygous genotype individuals and AG heterozygous genotype individuals, improve the reproductive performance of Haimen goats, accelerate the breeding progress of Haimen goats, and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the result of 2% agarose gel electrophoresis of the mixed PCR amplification products of the Wnt10a g.28944633G>A site (primer P1), where M represents DL2000 Plus DNA Marker and 1 represents Wnt10a g.28944633G>A.

[0033] Figure 2 This is the sequencing diagram of the PCR amplification product of Wnt10a g.28944633G>A site (primer is P1); the arrow indicates the mutation site.

[0034] Figure 3 This is the association analysis diagram between Wnt10a g.28944633G>A and the number of lambs born in the third litter of Haimen goats. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described in detail below in conjunction with the embodiments and related drawings. However, it is worth noting that the implementation of the present invention is not limited to this, and relevant personnel can implement it in other different forms without violating the purpose and spirit of the present invention.

[0036] Example 1 Typing and Identification of SNP Molecular Markers of Wnt10a Gene in Haimen Goats

[0037] 1. Experimental animals and sample collection

[0038] The Haimen goats used in this experiment were from Jiangsu Jinsheng Goat Breeding Technology Development Co., Ltd., with a total of 1,065 goats. All goats were healthy and grew up under the same breeding conditions and environment. 10 mL of blood from pregnant ewes was collected from the jugular vein, placed in an anticoagulant tube containing EDTA, and stored at -20°C. Phenotypic information such as the number of lambs born by ewes was collected for subsequent association analysis experiments.

[0039] 2. Main instruments

[0040] Pipette (Eppendorf), electronic balance (HENGJI), microwave oven (Galanz), refrigerator (Haier), handheld centrifuge (SCILOGEX, S1010E), vortexer (Dalong), digital constant temperature water bath (Changzhou Putian, HH-G2), high-speed refrigerated centrifuge (Eppendorf, 5424R), micro-spectrophotometer (NANODROP2000), PCR instrument (Applied Biosystems), electrophoresis instrument (Beijing Liuyi, DYY-6C), automatic digital gel imaging system (Tanon, Tanon-4100).

[0041] 3. Main Reagents

[0042] TIANGEN blood genomic DNA extraction kit (centrifugal column type), 50×TAE (Solarbio), agarose (BIOWEST), 2×Taq Plus Master MixⅡ (Dye Plus) (Vazyme, P213-03), 10000×TS-GelRed nucleic acid gel dye (TSINGKE, TSJ003), DL2000 Plus DNA Marker (Vazyme, MD102-02).

[0043] 4. Methods

[0044] 4.1 Extraction of genomic DNA from goat blood

[0045] Five hours in advance, the goat blood was taken out of the -20°C refrigerator and placed in a 4°C refrigerator for thawing. After the blood was completely thawed, DNA was extracted from the whole blood according to the instructions of the TIANGEN blood genomic DNA extraction kit.

[0046] DNA concentration and quality were measured using a micro-spectrophotometer. 260 / 280 Should be between 1.80 and 2.00, OD 260 / 230 It should be between 1.80 and 2.20. DNA samples that pass the test should be stored in a -20℃ refrigerator.

[0047] 4.2 Primer design for candidate SNP sites

[0048] The Ensembl database (https: / / asia.ensembl.org / index.html) was used to search for SNP loci related to the Wnt10a gene, and genotyping primers were designed using Primer Premier 5 software. The SNP locus information and primer information are shown in Tables 1 and 2.

[0049] Table 1 Wnt10a gene SNP site information

[0050]

[0051] Table 2 Wnt10a SNP genotyping primer sequences

[0052]

[0053] 4.3 Primer verification of candidate SNP sites

[0054] 4.3.1 Mixing pool production

[0055] Randomly select 50 DNA samples from 1065 Haimen goat samples, take 1 μL from each sample and add it to the same 1.5 mL centrifuge tube to make a DNA pool for subsequent candidate SNP site polymorphism and primer verification. Randomly select another 50 DNA samples and repeat the above operation to make 2 DNA pools.

[0056] 4.3.2 Pooled PCR amplification

[0057] (1) PCR amplification system (20 μL): 2× Taq Plus Master Mix II 10 μL; upstream and downstream primers 0.8 μL each; template DNA 1 μL; deionized water 7.4 μL.

[0058] (2) PCR amplification procedure: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s, for a total of 30 cycles; extension at 72°C for 5 min; storage at 4°C after PCR.

[0059] 4.3.3 Agarose gel electrophoresis of mixed PCR products

[0060] (1) Prepare 2% agarose gel: Measure 1 g agarose and 50 mL 1×TAE and pour into a conical flask. Heat in a microwave oven on high for 3 min until the solution is clear and transparent. Otherwise, extend the heating time appropriately. Let the solution stand until it is no longer hot, then add 5 μL of nucleic acid dye, mix thoroughly, pour onto a plate, and wait for it to solidify.

[0061] (2) After the agarose gel solidifies, add samples, including 6 μL of PCR product and 4 μL of DNA marker. Gently place the agarose gel in an electrophoresis tank filled with 1×TAE (the 1×TAE liquid level must cover the gel surface) and run electrophoresis at 140V for 35 minutes.

[0062] (3) After electrophoresis, transfer the gel to a fully automatic digital gel imaging system for observation.

[0063] The mixed pool DNA was subjected to agarose gel electrophoresis, and the results were as follows Figure 1 As shown in the figure, there is a clear band at the 220bp position of the Wnt10a SNP site, which is consistent with the expected fragment size and has no nonspecific miscellaneous bands, indicating that the two pairs of primers have good specificity and can be used for subsequent operations.

[0064] 4.4 Genotyping

[0065] 4.4.1 Sample PCR amplification

[0066] (1) PCR amplification system (40 μL): 2× Taq Plus Master Mix II 20 μL; upstream and downstream primers 1.6 μL each; template DNA 2 μL; deionized water 14.8 μL.

[0067] (2) PCR amplification procedure: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s, for a total of 30 cycles; extension at 72°C for 5 min; storage at 4°C after PCR.

[0068] 4.4.2 Sequencing of sample PCR products and result determination

[0069] The above PCR products and primer P1 were sent to Qingke Biotechnology for sequencing, and the sequencing results were compared and analyzed using SnapGene software.

[0070] The Wnt10a g.28944633G>A genotype was determined based on the sequencing peak graph: when the peak graph showed a single peak A, the genotype was AA; when the peak graph showed a single peak G, the genotype was GG; when the peak graph showed a double peak of A and G, the genotype was AG ( Figure 2 ).

[0071] The above results showed that the PCR amplification of Haimen goat genomic DNA using primer P1 and the interpretation of genotype by direct sequencing could quickly and accurately identify the SNP site of the Wnt10a gene in Haimen goats.

[0072] Example 2 Statistical analysis of Wnt10a gene SNP site polymorphism and its relationship with Haimen goat high fertility traits

[0073] 1. Statistical analysis of population genetic parameters at the Wnt10a g.28944633G>A locus

[0074] Genotyping of 1065 Haimen goats was performed according to the primers and methods designed in Example 1, and the population genetic parameters of this locus were calculated, including genotype frequency, allele frequency, homozygosity, heterozygosity, effective number of alleles, polymorphic information content, and Hardy-Weinberg equilibrium P value.

[0075] As shown in Table 3, there are three genotypes AA, AG, and GG at the Wnt10a g.28944633G>A locus in Haimen goats. Among them, the AG genotype is the dominant genotype, A is the dominant allele, and it is moderately polymorphic (0.25 < PIC < 0.5) and in a state of Hardy-Weinberg disequilibrium (P < 0.05).

[0076] Table 3 Population genetic parameters of the Wnt10a gene

[0077]

[0078] 2. Association between the Wnt10a g.28944633G>A locus and high fecundity traits in Haimen goats

[0079] Using SAS (8.0) software, the GLM procedure was used for least squares statistical analysis to analyze the association between different genotypes of Wnt10a g.28944633G>A and the number of lambs born in Haimen goats.

[0080] Taking genotype and season as fixed effects, the model is: Y ijk = μ + G i + S j + e ijk . In the formula, Y ijk is the phenotypic value of the number of lambs born per individual; μ is the population mean; G i is the genotype effect; S j is the season effect; e ijk is the random error.

[0081] 2.1 Association analysis between the Wnt10a g.28944633G>A locus and the number of lambs born in Haimen goats

[0082] Fixed effects that were not significantly associated with the individual phenotypic value except for the genotype effect were deleted, and the association analysis between the SNP locus of the Wnt10a gene and the number of lambs born in Haimen goats was performed. The results are expressed as "least square mean ± standard error".

[0083] The results are shown in Table 4 and Figure 3As shown, the Wnt10a g.28944633G>A locus was significantly associated with the number of third-parity lambs of Haimen goats, and the number of third-parity lambs of ewes with AA genotype was significantly higher than that of ewes with GG genotype.

[0084] Table 4 The association results between Wnt10a gene SNP loci and litter size in Haimen goats

[0085]

[0086] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0087] Example 3 Method for improving the reproductive traits of Haimen goats

[0088] The method for improving the reproductive traits of Haimen goats mainly includes the following steps:

[0089] 1. Collect blood from Haimen goats to be tested and extract blood genomic DNA.

[0090] 2. Using the primer P1 described in Example 1, PCR amplification was performed with blood genomic DNA as a template, and the genotype was determined by direct sequencing of the PCR amplification product.

[0091] in:

[0092] (1) PCR amplification system (40 μL): 2× Taq Plus Master Mix II 20 μL; upstream and downstream primers 1.6 μL each; template DNA 2 μL; deionized water 14.8 μL.

[0093] (2) PCR amplification procedure: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 20 s, and extension at 72°C for 30 s, for a total of 30 cycles; extension at 72°C for 5 min; storage at 4°C after PCR.

[0094] (3) Sequencing: The PCR product and primer P1 were sent to Qingke Biotechnology Co., Ltd. for direct sequencing.

[0095] (4) Genotype determination: SnapGene software was used to compare and analyze the sequencing peak graphs of the Wnt10a g.28944633G>A site. When the peak graph showed a single peak A, the genotype was AA; when the peak graph showed a single peak G, the genotype was GG; when the peak graph showed a double peak of A and G, the genotype was AG.

[0096] 3.The AA genotype of Wnt10a g.28944633G>A can be used as an effective molecular marker to improve the reproductive traits of Haimen goats.

[0097] Selecting AA genotype individuals as parents in Haimen goat breeding can increase the number of lambs born in the third litter. The number of lambs born in the third litter of AA genotype individuals is 0.47 more than that of GG genotype individuals and 0.28 more than that of AG genotype individuals.

[0098] By using the SNP molecular markers of the present invention to screen Haimen goats, retaining individuals with AA genotypes for genetic breeding, and eliminating individuals with GG and AG genotypes, the breeding goal of improving the reproductive performance of Haimen goats can be achieved.

Claims

1. Application of SNP molecular markers associated with the number of third-born lambs of Haimen goats or substances for detecting SNP molecular markers associated with the number of third-born lambs of Haimen goats in screening and / or breeding of the number of third-born lambs of Haimen goats; The SNP molecular marker associated with the number of third litters of Haimen goats is Wnt10a g.28944633 G>A, located at bp 28944633 of chromosome 2, is a G>A mutation.

2. The use according to claim 1, characterized in that: The substance contains or is a PCR primer for amplifying a Haimen goat genomic DNA fragment containing the SNP molecular marker.

3. The use according to claim 2, characterized in that: The PCR primers include an upstream primer F1 and a downstream primer R1; Upstream primer F1: 5′-TCTGTGGCGTGCCGTGTT-3′; Downstream primer R1: 5′-CAGCCCATCGGAGCTGTCT-3′.

4. The use according to claim 1, 2 or 3, characterized in that: Using the genomic DNA of the blood of the Haimen goat to be tested as a template, using the PCR primers described in claim 2 or 3 to perform PCR amplification, and using the method of direct sequencing of the PCR amplification product to determine the genotype of the SNP molecular marker; retaining the AA homozygous genotype individuals, eliminating the GG homozygous genotype individuals and the AG heterozygous genotype individuals, and increasing the number of lambs born in the Haimen goat generation by generation; Wnt10a g.28944633 G>A Different genotypes have a significant effect on the number of lambs born in the third litter of Haimen goats. The number of lambs born in the third litter of ewes with AA genotype is significantly higher than that of ewes with GG genotype.

5. The use according to claim 4, characterized in that: According to the sequencing peak diagram WNT10A g.28944633 G>A genotype determination: when the peak graph shows a single peak A, the genotype is AA; when the peak graph shows a single peak G, the genotype is GG; when the peak graph shows a double peak of A and G, the genotype is AG.

6. A method for improving the genetic breeding of Haimen goats' reproductive traits, characterized in that: Using the genomic DNA of the blood of the Haimen goat to be tested as a template, using the PCR primers described in claim 2 or 3 to perform PCR amplification, and determining the genotype of the SNP molecular marker described in claim 1 by direct sequencing of the PCR amplification product; retaining the AA homozygous genotype individuals, eliminating the GG homozygous genotype individuals and the AG heterozygous genotype individuals, and increasing the number of lambs born in the Haimen goat generation by generation; Wnt10a g.28944633 G>A Different genotypes have a significant effect on the number of lambs born in the third litter of Haimen goats. The number of lambs born in the third litter of ewes with AA genotype is significantly higher than that of ewes with GG genotype.

Images