Application of milk goat CTNND2 gene single nucleotide polymorphism marker in early selection of lambing traits

Abstract

The application discloses application of a milk goat CTNND2 gene single nucleotide polymorphism marker in early selection of lambing traits, and relates to the following technical scheme: a g.61357858G>A site of a milk goat CTNND2 gene screened through whole genome association analysis is subjected to large-group association analysis verification, and it is found that the site is a molecular marker that can be used for early selection of milk goat lambing traits, so that a method for detecting the milk goat CTNND2 gene single nucleotide polymorphism established by the application can be used for rapidly establishing a milk goat genetic resource group with excellent reproductive performance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and livestock breeding, and relates to the typing detection of single nucleotide polymorphism (SNP) in the CTNND2 gene of dairy goats and its application in molecular marker-assisted early selection breeding. Background Technology

[0002] Genetic mapping markers have evolved from the initial ABO blood group markers, restriction fragment length polymorphism (RFLP) markers, second-generation microsatellite markers, and short tandem repeats to the now widely used third-generation markers, namely SNP (single nucleotide polymorphism) markers. SNPs cover the genome at high density and have the advantage of low cost in large-scale genotyping.

[0003] With the completion of the sequencing and assembly of reference genomes for major species (pigs, cattle, sheep, etc.), genotyping technology and marker-assisted selection (MAS) are being applied more and more widely in animal husbandry and other fields, laying a foundation for the development of genetic breeding. In animal husbandry, traits such as lambing play a crucial role in the industry, but traditional breeding techniques require waiting until the ewe is pregnant before testing can be performed. However, by using marker-assisted selection technology, such as early selection using SNP markers that are highly specific, stable, and easy to analyze, it is possible to improve the production and reproductive performance of the herd, shorten the generation interval, and reduce breeding costs, thereby accelerating the genetic progress of breeding.

[0004] The CTNND2 (Catenin Delta 2) gene, located on chromosome 20 of goats, encodes a protein that, upon stimulation by hepatocyte growth factor, promotes the disruption of E-cadherin-based adhesion molecule junctions to facilitate cell proliferation. Studies have shown that the CTNND2 gene may play an important role in growth, development, and energy metabolism. Currently, there are no reports, either domestically or internationally, on the polymorphism of the CTNND2 gene in dairy goats or its genetic association with lambing traits.

[0005] The study "Functional Study of HSP 27 Gene in Apoptosis of Ovarian Granulosa Cells in Tibetan Sheep" points out that CTNND2 is a differentially expressed gene related to follicle development. However, the relationship between follicle development and lambing performance is not yet clear. Furthermore, lambing performance cannot be predicted through the biological function of the CTNND2 gene itself, making it difficult to implement early selection of high-producing individuals. The low lambing rate and low live lambing rate in some dairy goat populations still affect the high-quality and sustainable development of the dairy goat industry. Summary of the Invention

[0006] The purpose of this invention is to provide an application of the CTNND2 gene single nucleotide polymorphism marker in dairy goats for early selection of lambing traits. Based on the trait association results, it is possible to select dairy goat individuals with high lambing capacity at an early stage, thereby quickly establishing a dairy goat population with excellent reproductive performance.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for detecting single nucleotide polymorphisms in the CTNND2 gene of dairy goats includes the following steps:

[0009] Using the genomic DNA of the dairy goat to be tested (e.g., Xinnong Saanen dairy goat or an individual Saanen dairy goat) as a template, a partial fragment of the CTNND2 gene of dairy goats was amplified by PCR, and then the amplified product was directly sequenced. Based on the sequencing results, the genotype of the single nucleotide polymorphism site of the CTNND2 gene of the individual dairy goat was identified. The single nucleotide polymorphism site was selected from the CTNND2 gene mutation site CM048886.1:g.61357858G>A of dairy goats.

[0010] Preferably, the PCR amplification primers are:

[0011] Upstream primer: 5'-GGGACTTAGTGTATAGTACAGGGA-3'

[0012] Downstream primer: 5'-ACACAGTGAATCACAGCGCA-3'.

[0013] Preferably, the PCR reaction program is as follows: 95.0℃ pre-denaturation for 3 min; 94.0℃ denaturation for 15 s, 60.0℃ annealing for 15 s, for 34 cycles; 72.0℃ extension for 30-60 s; 72.0℃ final extension for 10 min.

[0014] Preferably, the genotypes of the single nucleotide polymorphism sites can be divided into three types based on sequencing results: GG, AG, and AA.

[0015] The above-mentioned method for detecting single nucleotide polymorphisms in the CTNND2 gene of dairy goats is applied in molecular marker-assisted selection breeding of dairy goats.

[0016] Application of single nucleotide polymorphism (SNP) sites in CTNND2 gene of dairy goats in molecular marker-assisted selection breeding of dairy goats, wherein the SNP sites are selected from the CTNND2 gene mutation site CM048886.1:g.61357858G>A.

[0017] Preferably, the AA genotype and mutant allele (base A) of the single nucleotide polymorphism site can both be used as DNA markers to improve the number of lambs born and / or the number of live lambs born in dairy goats.

[0018] Preferably, the dairy goat is a Saanen dairy goat or a Saanen dairy goat.

[0019] A detection kit for single nucleotide polymorphism (SNP) of the CTNND2 gene in dairy goats, comprising primer pairs for PCR amplification of SNP sites in the CTNND2 gene of dairy goats, wherein the SNP sites are selected from the mutation site CM048886.1:g.61357858G>A in the CTNND2 gene of dairy goats.

[0020] Preferably, the PCR amplification primers (i.e., the primer pairs mentioned above) are:

[0021] Upstream primer: 5'-GGGACTTAGTGTATAGTACAGGGA-3'

[0022] Downstream primer: 5'-ACACAGTGAATCACAGCGCA-3'.

[0023] The beneficial effects of this invention are reflected in:

[0024] Based on the association analysis of the SNP site (CM048886.1:g.61357858G>A) on the CTNND2 gene of dairy goats and the lambing trait of dairy goats, this invention discovered that the SNP site of the CTNND2 gene contains a DNA marker (specifically an SNP marker) that can improve the number of lambs born and the number of live lambs born in dairy goats. This marker can be applied in the early selection of lambing performance in dairy goats to quickly establish a genetic resource population of dairy goats with excellent reproductive performance.

[0025] This invention enables a simple and accurate detection of different genotypes of the SNP site (CM048886.1:g.61357858G>A) in the intron region of the CTNND2 gene in dairy goats within a dairy goat population through sequence amplification and direct sequencing. Attached Figure Description

[0026] Figure 1 Manhattan plot of genome-wide association analysis for the lambing number trait in dairy goats.

[0027] Figure 2 Manhattan plot of genome-wide association analysis for the number of live lambs produced in dairy goats.

[0028] Figure 3 Manhattan plot for genome-wide association analysis of mean birth weight trait in dairy goats.

[0029] Figure 4 Electrophoresis diagram of PCR amplification products of SNP loci (i.e., CM048886.1:g.61357858G>A) in different dairy goat individuals.

[0030] Figure 5 Sequencing peak diagram (reverse sequence) of the amplified product of the CTNND2 gene fragment (containing the target genotyping site CM048886.1:g.61357858G>A) in dairy goats: sequencing results of individuals with genotypes GG, AG, and AA. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments described are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0032] Based on the resequencing results of Saanen dairy goats with records of litter size, live lambs, and birth weight phenotypes, genome-wide association analysis (GWAS) was performed in conjunction with phenotypic data. The results ( Figure 1 , Figure 2 , Figure 3 The study showed that the number of lambs born was significantly associated with loci on chromosomes 7, 12, 20, and 24, and the number of live lambs born was significantly associated with loci on chromosomes 7, 20, and 27. However, these loci (including the mutation site on the CTNND2 gene on chromosome 20, i.e., the g.61357858G>A site in the dairy goat CTNND2 gene) were not significantly associated with the birth weight trait (the birth weight trait was only significantly associated with loci on chromosome 3).

[0033] Based on the above site screening results, this invention still attempted to verify them through large-scale population association analysis. Specifically, PCR amplification and direct sequencing methods were used to detect the potential polymorphisms of the initially screened sites (e.g., the g.61357858G>A site in the CTNND2 gene of dairy goats) in a dairy goat population. The different genotypes were then associated with the lambing and birth weight traits of dairy goats (birth weight can also be used to assess the growth and development performance of lambs born to ewes). This revealed that they can serve as molecular markers for molecular breeding-assisted selection in dairy goats, as detailed below.

[0034] 1. Laboratory animals

[0035] Using 341 Saanen dairy goats as the test subjects, blood samples were collected from full-term, normally parturient, healthy lactating ewes, and traits such as the number of lambs born, the number of live lambs born, and the birth weight were measured and recorded. The blood samples were collected in December 2022 and March 2023 (collection location: Saanen Goat Breeding Farm, Northwest A&F University, Yangling).

[0036] 2. Medicines and reagents

[0037] Blood DNA was extracted using a blood genomics column-based mini-extraction kit (0.1–1 mL), purchased from Kangwei Century Biotechnology Co., Ltd.

[0038] Reagents required for agarose gel electrophoresis: ① Plus DNA Marker, purchased from Beijing TransGen Biotech Co., Ltd.; ② 10×GelRed nucleic acid gel staining agent, purchased from Diyi Biotechnology Co., Ltd.; ③ Loading Buffer, purchased from Beijing Qingke Biotechnology Co., Ltd.; ④ Agarose, purchased from Beijing Jihua Biotechnology Service Co., Ltd.

[0039] Reagents required for PCR amplification: PrimeSTAR Max DNA Polymerase, 10×dNTPs, 2×GC buffer II (Mg 2+ All of them were purchased from TAKARA.

[0040] The reagent preparation method can refer to "Molecular Cloning: A Laboratory Manual" (2002) edited by Sambrook et al., and all reagents were prepared using deionized ultrapure water. The high-temperature sterilization conditions were 120°C for 30 minutes.

[0041] 3. Synthesize primers for the CTNND2 gene mutation site.

[0042] According to the NCBI, goats ( capra hircus Primers for amplifying the g.61357858G>A site of the CTNND2 gene in dairy goats were designed on chromosome 20 of the goat genome (GenBank accession number: GCA_026652205.1) and synthesized by Yangling Tianrun Aoke Biotechnology Co., Ltd. The specific sequences are as follows:

[0043] Upstream primer: 5'-GGGACTTAGTGTATAGTACAGGGA-3' (i.e., SEQ.ID.NO.1)

[0044] Downstream primer: 5'-ACACAGTGAATCACAGCGCA-3' (i.e. SEQ.ID.NO.2).

[0045] Using the primers described above, PCR amplification was performed. The amplified product (659bp) could be directly sent to the company for sequencing, thereby enabling genotyping of the goat CTNND2 gene g.61357858G>A site contained therein.

[0046] 4. Obtaining phenotypic values

[0047] 4.1 Methods for recording the number of lambs born, the number of live lambs born, and measuring birth weight

[0048] During the lambing season of 2022-2023, the number of lambs born to ewes and the number of live lambs born to ewes were recorded in detail. The birth weight of lambs was determined by weighing them after they had consumed colostrum, and the weight was measured in kilograms.

[0049] 4.2 Data Statistics

[0050] Record and organize the data, remove extreme values ​​beyond three standard deviations from the phenotypic data, and save the data for subsequent association analysis.

[0051] 5. PCR amplification of the target fragment of the CTNND2 gene in dairy goats.

[0052] 5.1 Blood Sample Collection from the Saanen Dairy Goat Experimental Population

[0053] When collecting blood samples from a total of 341 Saanen dairy goats, 5 mL of blood was drawn from each goat into an anticoagulant blood collection tube (EDTA), which was then quickly brought back to the laboratory and frozen at -80°C.

[0054] 5.2 Extraction and Detection of Genomic DNA from Blood Samples

[0055] Blood DNA extraction method: Refer to the instructions of Kangwei Century Biotechnology Co., Ltd. Blood Genomics Column Mini-Extraction Kit (0.1-1 mL), and store the extracted DNA sample at -80℃.

[0056] DNA samples were analyzed for concentration and absorbance at 260 nm and 280 nm using a UV spectrophotometer (NanoDrop1000, Thermo); integrity was assessed using 1% agarose gel electrophoresis.

[0057] 5.3 Genotyping of the CTNND2 gene in dairy goats at the g.61357858G>A locus

[0058] The specific reaction system for PCR amplification is shown in Table 1, and the reaction procedure is shown in Table 2. The concentration and specificity of the PCR products were detected using 1% agarose gel electrophoresis (120V, 30min). The results showed that the PCR products met the quality requirements. Figure 4 The sample was then sent to Yangling Tianrun Aoke Biotechnology Co., Ltd. for sequencing.

[0059] Table 1. PCR reaction system (20 μL)

[0060]

[0061] Table 2. PCR reaction procedure

[0062]

[0063] The returned sequencing results were analyzed using Chromas software. Figure 5It can directly perform genotyping and obtain genotype data of the g.61357858G>A locus of the CTNND2 gene in dairy goat samples.

[0064] 6. Genetic polymorphism indices at the g.61357858G>A locus in the CTNND2 gene of dairy goats.

[0065] Using the genotyping data of the CTNND2 gene at position g.61357858G>A in the Saanen dairy goat experimental population, the gene frequency, number of effective alleles (Ne), homozygosity (Ho), heterozygosity (He), and polymorphism information content (PIC) were calculated. Furthermore, the Hardy-Weinberg equilibrium was determined using the chi-square fitness test. The results show that the mutation site at position 61357858 (g.61357858G>A) of the CTNND2 gene in dairy goats is a SNP site, with its physical location on chromosome 20 (CM048886.1 equivalent to chromosome number) of 61357858 in the goat genome (GenBank accession number: GCA_026652205.1). Specific polymorphism information is shown in Table 3.

[0066] Table 3. Genetic polymorphism indicators of CTNND2 gene mutation sites in dairy goats

[0067]

[0068] 7. Genetic effect association analysis of the CTNND2 gene mutation site (CM048886.1:g.61357858G>A) in dairy goats.

[0069] In the association analysis between the g.61357858G>A site of the CTNND2 gene in dairy goats and lambing traits in Saanen dairy goats, genotype data were obtained from genotypes obtained by direct sequencing after PCR amplification. Lambing trait data for dairy goats included number of lambs born, number of live lambs born, and average birth weight (kg; if an individual had multiple lambs, the average birth weight was used for calculation).

[0070] The analysis was performed using the MIXED procedure in SAS 9.4 software. The results are displayed as mean ± standard deviation. The model formula is as follows:

[0071] y = 1μ + Xβ + Zα + e

[0072] In the formula: y is the original phenotypic value of lambing trait; μ is the population mean; X is the regression coefficient of fixed effects; β is all considered fixed effects (including SNP effects); Z is the correlation coefficient matrix of additive genetic effects; α is the individual random additive genetic effect. Let A be the additive genetic variance, A be the additive genetic correlation matrix, and e be the random residual effect.

[0073] The analysis results are shown in Table 4.

[0074] Table 4. Association analysis of CTNND2 gene mutation site polymorphism in dairy goats with lambing traits.

[0075]

[0076] Note: P-value indicates the degree of association between the g.61357858G>A site of the CTNND2 gene in dairy goats and the trait; P<0.05 indicates a significant difference, and different genotypes are indicated by lowercase superscript letters.

[0077] Table 4 shows that the CTNND2 gene g.61357858G>A locus in dairy goats was significantly correlated with the number of lambs born and the number of live lambs (P<0.05). Individuals with the AA genotype had the highest number of lambs born and the highest number of live lambs. There was no significant difference in the number of lambs born and the number of live lambs between individuals with the AG genotype and those with the AA genotype. Individuals with the AA and AG genotypes showed significantly higher lambing and live lambing traits than those with the GG genotype (P<0.05). Furthermore, individuals with the AA and AG genotypes showed a trend towards higher birth weight than those with the GG genotype (suggesting that individuals with the AA and AG genotypes have a stronger advantage in growth and development). Therefore, the mutation at the CTNND2 gene g.61357858G>A locus (G to A) in dairy goats has a significant genetic effect on lambing traits and can serve as a candidate marker for molecular marker-assisted selection, thus providing scientific data for breeding superior dairy goats.

[0078] In summary, given that different genotypes of the g.61357858G>A site in the CTNND2 gene of dairy goats contain molecular markers that can be used for early selection of lambing traits in dairy goats, the PCR-direct sequencing method for detecting single nucleotide polymorphisms in the CTNND2 gene of dairy goats established in this invention can be used to rapidly establish a population of dairy goats with excellent genetic resources, thereby facilitating the acceleration of the breeding process.

Claims

1. A type of dairy goat CTNND2 The application of single nucleotide polymorphism detection methods in marker-assisted selection breeding of dairy goats for litter size and / or live lamb count is characterized by: The dairy goat CTNND2 The method for detecting single nucleotide polymorphisms (SNPs) includes the following steps: Using dairy goat genomic DNA as a template, PCR amplification of dairy goat genomic DNA was performed. CTNND2 A partial gene fragment was obtained, and the amplified product was directly sequenced to identify dairy goats based on the sequencing results. CTNND2 The genotype of a single nucleotide polymorphism (SNP) site, wherein the SNP site is selected from dairy goats. CTNND2 Gene mutation site CM048886.1: g.61357858G>A; The AA genotype at the single nucleotide polymorphism site serves as a DNA marker to increase the number of lambs born and / or the number of live lambs in dairy goats. The dairy goat in question is a Saanen dairy goat.

2. The application according to claim 1, characterized in that: The amplification primers for the PCR are: Upstream primer: 5' GGGACTTAGTGTATAGTACAGGGA 3' Downstream primer: 5' ACACAGTGAATCACAGCGCA 3'.

3. The application according to claim 1, characterized in that: The PCR reaction procedure was as follows: pre-denaturation at 95.0℃ for 3 min; denaturation at 94.0℃ for 15 s; annealing at 60.0℃ for 15 s; for 34 cycles. Extend at 72.0℃ for 30–60 seconds; 72.0℃ for a final extension of 10 minutes.

4. The application according to claim 1, characterized in that: The genotypes of the single nucleotide polymorphism sites can be divided into three types based on sequencing results: GG, AG, and AA.

5. Dairy goats CTNND2 The application of a reagent for detecting single nucleotide polymorphisms (SNPs) in marker-assisted selection breeding of dairy goats for litter size and / or live lamb count is characterized by: The single nucleotide polymorphism site was selected from dairy goats. CTNND2 Gene mutation site CM048886.1: g.61357858G>A; The AA genotype at the single nucleotide polymorphism site serves as a DNA marker to increase the number of lambs born and / or the number of live lambs in dairy goats. The dairy goat in question is a Saanen dairy goat.

Images