A snp molecular marker related to the body weight of large white pig at 120 kg body weight day age and application thereof

Abstract

This invention discloses a SNP molecular marker associated with the age at which Large White pigs reach 120 kg body weight and its application, belonging to the fields of biotechnology and animal genetics and breeding technology. The marker is located on chromosome 13 of the pig reference genome Sscrofa11.1, at chr13:206033545 (rs344256714), within the PDE9A gene coding region, with an allele of T / G, located at position 300 from the 5′ end of the nucleotide sequence shown in SEQ ID NO.1. Under the same feeding conditions, individuals with the TG genotype have a significantly lower D120 than those with the TT genotype, exhibiting a heterozygous overdominance effect. This invention, by detecting the genotype at this locus, enables early molecular prediction, hierarchical management, and marker-assisted selection of Large White pig growth rate. Based on the selection index and optimal contribution selection strategy constructed using this marker, genetic improvement of growth traits in Large White pig populations can be achieved.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and animal genetics and breeding technology. Specifically, it relates to a SNP molecular marker associated with the age of Large White pigs reaching 120kg body weight and its application. Background Technology

[0002] Pigs are an important livestock production animal in my country, and their production performance directly affects breeding efficiency and market supply. In large-scale pig farming, daily gain (ADG) and age at slaughter are key indicators for measuring economic value, especially the age at which pigs reach 120kg (D120), which is often used as an important reference standard for market-ready pigs. D120 not only affects feed utilization efficiency and the feeding cycle, but is also closely related to breeding costs and final economic returns. Groups with faster growth rates and who reach market weight at a shorter age typically have higher feed conversion ratios and market competitiveness; therefore, D120 is of great significance in genetic improvement and breed selection.

[0003] Marker-assisted selection (MAS) technology has been widely applied in pig breeding. Through methods such as GWAS, researchers have identified several SNP markers related to pig growth traits, such as the markers related to age at 100kg or 115kg body weight reported in patents CN107828895B and CN116287305B. However, existing technologies have obvious limitations: (1) Most studies focus on the 100kg or 115kg body weight standard, and there is insufficient research on specific molecular markers for slaughter standards of 120kg and above; (2) Pigs are in the peak period of fat deposition at the 100-120kg stage, and their energy metabolism differs from that in the early stage, so markers for early body weight standards are difficult to effectively predict later growth performance; (3) Existing markers mostly focus on homozygous dominance effects, ignoring heterozygous dominance phenomena that may exist at some loci.

[0004] However, although some SNP markers have been found to be associated with growth traits in pigs, their stability and reproducibility across different populations or environments remain problematic. Most existing markers have not been widely used in large-scale breeding practices; therefore, exploring reliable SNP markers associated with 120 kg daily weight gain in Large White pigs remains an urgent task. Summary of the Invention

[0005] The purpose of this invention is to provide a SNP molecular marker associated with the age of 120kg body weight in Large White pigs and its application, with a particular focus on the key economic trait of Large White pigs reaching 120kg body weight (D120). Specific SNP markers located in the PDE9A gene region have been identified, providing a new tool for the precise breeding of growth traits in Large White pigs.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a SNP molecular marker associated with the age of Large White pigs reaching 120 kg body weight. The allele of the SNP analysis marker is T or G, located at position 206033545 bp on chromosome 13 of the pig reference genome Sscrofa11.1, and is within the PDE9A gene coding region.

[0007] Furthermore, the SNP analysis marker is located at position 300 from the 5′ end in the nucleotide sequence shown in SEQ ID NO.1.

[0008] The present invention also provides the application of the SNP molecular marker in predicting the age at which Large White pigs reach a weight of 120 kg.

[0009] The present invention also provides the application of the SNP molecular marker in predicting the average daily weight gain of Large White pigs.

[0010] This invention also provides a method for determining the relative growth rate rank of Large White pigs based on the SNP molecular markers, comprising the following steps: (1) Collect biological samples of the large white pigs to be tested and extract genomic DNA; (2) Detect the genotype at the SNP molecular marker; (3) Determine the relative growth rate of the Large White pig based on the genotype at the SNP molecular marker detected in step (2).

[0011] Furthermore, the growth rate of Large White pigs with the TG genotype at the SNP molecular marker was relatively better than that of Large White pigs with the TT genotype. Beneficial effects

[0012] The SNP loci provided by this invention have clear coordinates and clear effect directions (TG is superior to TT), and can be detected and recognized across platforms with consistent results; it supports early molecular prediction and rapid screening, shortens the breeding cycle and reduces the cost of testing; it can be connected with GS / OCS to form a feasible improvement process of "marker + genomic selection + inbreeding control"; it is suitable for continuous rolling evaluation of the core population of enterprises, which facilitates the monitoring of G allele frequency and population D120 improvement progress, and improves breeding efficiency and economic benefits. Attached Figure Description

[0013] Figure 1 The phenotypic distribution and statistical characteristics of Large White pig D120.

[0014] Figure 2 GWAS Manhattan plot for growth traits at 120 kg (Sscrofa11.1; x-axis represents chromosomes, y-axis represents −log10 P).

[0015] Figure 3This is a differential distribution map of the TG and TT genotypes at the chr13:206033545 locus on the D120 phenotype (D120: TG). <TT)。 Detailed Implementation

[0016] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0017] To identify molecular markers associated with the growth rate of Large White pigs, this embodiment completed sample collection, genotyping, data filling and quality control, phenotypic statistics, and genome-wide association analysis (GWAS) under uniform feeding and testing conditions, and located the target loci.

[0018] 1. Experimental materials and phenotypic acquisition Ninety-seven healthy Large White pigs were selected, and ear tissue was collected. Basic information such as sex, date of birth, rearing environment, and litter number was recorded. Systematic feeding and management were implemented according to the company's SOPs to minimize environmental disturbances. During the fattening stage, individuals were weighed regularly until they reached 120 kg, and the age at which they reached 120 kg (D120) was recorded as the phenotypic data for analysis (Note: a smaller D120 is equivalent to a larger average daily gain [ADG]). Phenotypic data were included in the analysis after missing values, outliers, and logical checks to ensure accuracy and comparability.

[0019] 2. DNA extraction and microarray typing Ear tissue was processed using a standard animal genomic DNA extraction kit; DNA purity and concentration (A260 / A280 = 1.8–2.0) were determined using a spectrophotometer, and 50 ng / μL working solution was prepared, with integrity verified by agarose gel electrophoresis. All samples were genotyped using a Borredi 50K SNP chip (approximately 52,000 loci).

[0020] 3. Genotyping and Quality Control To improve marker coverage and data integrity, a reference panel based on multiple varieties / populations was constructed, and the microarray data was populated using MiniMac4. After population, PLINK was used for quality control: knockout site detection rate <95%, minor allele frequency (MAF) <0.01, and Hardy-Weinberg equilibrium test. Loci were identified; samples with low detection rates were removed at the individual level. A total of 2,630,497 high-quality SNPs were obtained for analysis.

[0021] 4. Phenotypic Distribution Statistics The D120 phenotypic data approximate a normal distribution. Figure 1 The coefficient of variation was 8.95%, which is consistent with the quantitative trait characteristics. Sex and strain (if stratified) had a significant impact on the relevant traits, and these were subsequently controlled for by fixed effects in the model.

[0022] 5. GWAS and Site Annotation The FarmCPU method from rMVP (R package) was used to detect the association between D120 and SNP. To control for population structure and batch effects, the following fixed effects were included in the model: principal component analysis (PCA), birth location, birth season, sex, final weight, and litter number; PCA score was included as a covariate in the model to reduce false positives. Bonferroni correction was used for significance thresholds; Manhattan plots and QQ plots were visualized using CMplot. Figure 2 Significant sites were annotated using bedtools based on the reference genome Sscrofa11.1.

[0023] The results showed that five sites significantly associated with D120 were detected on autosomes. Among them, the chr13:206033545 (rs344256714) site (located in the PDE9A gene coding region) at position 300 from the 5′ end of the nucleotide sequence shown in SEQ ID NO.1 was the most significant, with a P value of 8.2177 × 10⁻ 9 Therefore, chr13:206033545 was identified as a candidate molecular marker related to Large White pig D120 as described in this invention.

[0024] Specifically, SEQ ID NO.1 is:

[0025] This embodiment verifies the effect of the candidate SNPs identified in Example 1, clarifies their genotype-phenotype direction and statistical significance, and formulates executable judgment rules.

[0026] 1. Locus Information and Genotype Definition The target SNP was located at chr13:206033545 (PDE9A) of the reference genome Sscrofa11.1, with alleles of T / G, where T is the reference allele and G is the alternative allele, and the genotypes are TG and TT.

[0027] 2. Samples and Data Individuals from the same batch as in Example 1 that passed quality control were selected as validation samples. The phenotype was D120; individuals with missing phenotypes or genotypes were excluded to ensure a one-to-one correspondence.

[0028] 3. Statistical methods The impact of genotype on D120 was evaluated using a linear model: D120 = μ + Genotype + Xγ + ε, where Genotype is the genotyping factor (TG, TT), X is the fixed covariate (PCA, birth factory, birth season, gender, weight at measurement, litter number), and ε is the residual. The overall effect of Genotype was tested using analysis of variance (ANOVA), and post hoc multiple comparisons were performed; the significance threshold was corrected using Bonferroni. The results were presented as violin plots / box plots ( Figure 3 ).

[0029] 4. Results and decision rules Statistical results showed that there were extremely significant differences in D120 among different genotypes ( Figure 3 ), and the direction of the effect was stable: TG < TT, that is, individuals with the TG genotype reached 120 kg at a shorter age (grew faster, equivalent to a larger ADG), while individuals with the TT genotype required a longer age (equivalent to a smaller ADG). Based on this, a molecular decision rule was formed: under the same population and measurement conditions, the relative growth rate grade was determined as TG (excellent) > TT (inferior) according to the genotype of chr13:206033545.

[0030] 5. Breeding applications Marker-assisted selection (MAS) was implemented in the core herd: TG individuals were preferentially retained, and the proportion of TT individuals selected for breeding was reduced; in the annual rolling evaluation, the frequency of the G allele or TG genotype and the change in the mean D120 of the population were monitored, and it was used in combination with genomic selection (GS) or optimal contribution selection (OCS) to achieve the improvement goal of reducing D120 / increasing ADG while maintaining genetic diversity.

[0031] According to the marker rs344256714 screened and verified in Examples 1–2, by referring to NCBI (Sscrofa11.1), it is located at the position of chr13:206033545 bp on pig chromosome 13, which is a T / G variation. This SNP molecular marker is located at the 300th base from the 5′ end of the nucleotide sequence shown in SEQ ID NO.1; the base at this site can be represented by the IUPAC degenerate code K, and its alleles are G or T. This SNP molecular marker can be used for molecular prediction and hierarchical determination of the D120 / ADG traits of Large White pigs, and can be used for marker-assisted selection in the core herd to improve the growth rate of the population, shorten the slaughter age, and increase economic benefits.

[0032] Within the core group of breeding boars, ear tissue / blood samples were collected and genomic DNA was extracted. The chr13:206033545 locus was genotyped using methods such as microarray typing, sequencing typing, or KASP, ARMS-PCR, HRM, and TaqMan. Individuals with the TG genotype were prioritized for breeding; the proportion of individuals with the TT genotype was relatively reduced, or TT individuals were culled under the same conditions, to achieve early screening of faster-growing individuals (smaller D120 / larger ADG).

[0033] Genotyping at this locus was performed on replacement / candidate boars during weaning or early rearing, and the results were comprehensively evaluated in conjunction with phenotypic records such as stage weight, ADG (advanced growth factor), and backfat thickness. If an individual is of the TG genotype, a shorter age at which they will reach 120 kg can be predicted; if they are of the TT genotype, a relatively slower growth rate is predicted. This molecular information can be used to optimize resource input for measurement and boar station selection strategies, improving selection efficiency and shortening the breeding cycle.

[0034] In commercial pigs (such as two-way or three-way crossbred sows with Large White bloodlines), weaned piglets or early-stage fattening individuals can be genotyped at the chr13:206033545 locus, and the group can be graded and managed according to the rule that TG is superior to TT: TG individuals are given priority as breeding replacement sows / boars or as high-growth-potential fattening groups; TT individuals can be subject to differentiated feeding management or have their proportion used as breeding sources reduced, thereby increasing the average ADG and shortening the fattening cycle at the group level.

[0035] In the selection of replacement gilts, candidate gilts are genotyped at this locus, with priority given to retaining individuals with the TG genotype for entry into the core or propagation herds; under the same comprehensive index conditions, the proportion of individuals with the TT genotype is reduced. By continuously monitoring changes in the frequency of the G allele or TG genotype, and combining this with production performance records, rolling improvement of the growth traits of the maternal population can be achieved.

[0036] This method includes the following steps: S1, testing the rs344256714 genotype in replacement breeding pigs; S2, selecting individuals with a TG allele as breeding stock and mating them; S3, retesting the genotype at this locus in the offspring born from mating in step S2, prioritizing the selection of TG individuals for further breeding and participation in the next round of mating. Through continuous selection over multiple generations, the frequency of the G allele or TG genotype can be gradually increased, thereby cultivating a high-growth-rate strain with a smaller D120 and a larger ADG.

[0037] An improvement program targeting a decrease in D120 and an increase in ADG was established within the core population: Each generation, candidate individuals were genotyped and mated and selected based on the principle of prioritizing TG and reducing TT; D120, ADG, and other phenotypes were recorded simultaneously, and breeding values ​​were calculated. The genotyping results at this locus were incorporated into the selection index as a fixed effect or weighted information; inbreeding constraints were set during optimal contribution selection (OCS) to maintain genetic diversity. By continuously evaluating the changes in G allele frequency and population-average D120, the improvement progress can be quantified, and a replicable, enterprise-level process can be established.

[0038] A detection kit for detecting the genotype at the chr13:206033545 locus is provided, comprising at least: PCR amplification primers and / or allele-specific primers designed around the target locus, a fluorescent probe (such as a TaqMan probe or a KASP primer system), a reaction buffer and enzyme system, and instructions; the instructions specifying genotyping determination, positive / negative control settings, and genotype-phenotypic determination rules (D120:TG).<TT;ADG:TG> (TT). When using this product, follow the instructions to complete DNA extraction, amplification / detection, and result interpretation to achieve standardized, batch detection of this site.

Claims

1. A SNP molecular marker associated with Large White pigs body weight at 120 kg of age, characterized in that, The SNP analysis markers are T or G, located at position 206033545 bp on chromosome 13 of the pig reference genome Sscrofa11.1, and within the PDE9A gene coding region.

2. The SNP analysis marker of claim 1, wherein The SNP analysis marker is located at position 300 from the 5′ end in the nucleotide sequence shown in SEQ ID NO.

1.

3. The application of the SNP molecular marker of claim 1 in predicting the age at which Large White pigs reach 120 kg body weight.

4. The application of the SNP molecular marker of claim 1 in predicting the average daily weight gain of Large White pigs.

5. A method for determining the relative growth rate of Large White pigs based on the SNP molecular marker of claim 1, characterized in that, Includes the following steps: (1) Collect biological samples of the large white pigs to be tested and extract genomic DNA; (2) Detect the genotype at the SNP molecular marker; (3) Determine the relative growth rate of the Large White pig based on the genotype at the SNP molecular marker detected in step (2).

6. The method of claim 5, wherein, Large White pigs with the TG genotype have a relatively better growth rate than Large White pigs with the TT genotype.

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