A molecular marker associated with pig carcass weight traits and application thereof

Abstract

The application discloses a SNP molecular marker associated with a pig carcass weight trait, wherein the SNP molecular marker is located at 97,786,583 bp of a No.1 chromosome in a 11.1 version reference sequence of a pig genome and a region closely linked in heredity thereof. The application also discloses a KASP primer group for detecting the SNP molecular marker. The application first discovers that the SNP molecular marker is significantly associated with the pig carcass weight trait, and the KASP primer group can be used to simply, accurately, efficiently and low-costly early screen genotypes of a large number of piglet individuals, reserve excellent genotype individuals and eliminate poor genotype individuals, so that the process of pig genetic improvement can be accelerated, and economic benefits in pig raising can be improved. The application provides a reliable molecular marker for pig genetic improvement and efficient breeding, and has important scientific significance and economic value.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to a molecular marker associated with the weight trait of pig carcasses and its application. Background Technology

[0002] Pig farming is one of the most important livestock industries in my country, with extremely high requirements for breeding costs. Carcass weight gain rate is one of the key factors affecting breeding costs. Given the same number of days of feeding and the same amount of feed, some pigs grow quickly and gain more weight, while others grow slowly and gain less. To save costs, early culling of slow-growing individuals and retaining fast-growing individuals is an important requirement for every farm. Besides physiological factors such as health at birth, genetic factors are also one of the important factors determining the growth rate of different pig individuals. The heritability of carcass-related traits in pigs is relatively high, approximately 0.55-0.60 (Wu Zhenfang and Chen Wenguang. Heritability and genetic correlation of carcass and meat quality traits in pigs [J]. North China Agricultural Journal, 2000, 15(Supplement): 46-50.), exhibiting a good genetic selection effect.

[0003] Molecular markers are a technique that utilizes the differences in genetic material among individuals to detect variations in DNA molecular sequences. This allows for the selection of superior genes from the genetic origin of different individuals. Theoretically, at the moment of piglet birth, superior individuals can be screened and identified based on molecular markers, retaining those with superior genes and culling those with inferior genes, thus saving breeding costs and maximizing breeding efficiency.

[0004] With the development of modern biotechnology, molecular marker technology has also made great progress. Older generation molecular marker technologies such as RFLP, RAPD, AFLP, and SSR have been replaced by single nucleotide diversity (SNP) detection technologies. Based on efficiency and cost considerations, commonly used SNP detection technologies include SNP microarrays and Kompetitive Allele Specific PCR (KASP). SNP microarray technology concentrates millions of SNP markers onto a small chip for simultaneous detection of SNPs across the entire genome of the same individual, offering advantages such as high efficiency and a wide detection range. KASP technology involves designing primers for a specific SNP locus for single-locus SNP detection, offering advantages such as simplicity, flexibility, speed, and low cost, making it ideal for rapid detection across large numbers of individuals. Therefore, in research, it is generally necessary to first use SNP microarrays to screen almost all genetic loci across the entire pig genome to identify one or more specific and effective genetic loci, and then use KASP technology to perform large-scale detection on all individuals to select those with favorable genotypes.

[0005] Genome-wide association study (GWAS) is a crucial technique for screening major genetic loci. It typically employs methods such as GeneSeek Porcine 50K SNP Chip or Illumina Porcine SNP60 BeadChip, which contain SNP loci evenly distributed across all pig chromosomes, to screen in a sampled pig population. Currently, in the Duroc-Landrace-Large White crossbred pig population, a number of important genetic loci related to pig growth rate have been identified, such as the 162,192,627 bp locus on chromosome 1 (Chinese Patent: CN110541038A), the 79,026,669 bp locus on chromosome 6 (Chinese Patent: CN108913779B), the 95,480,854 bp locus on chromosome 6 (Chinese Patent: CN114350820B), and the 114 bp locus on chromosome 6. The genetic locus of 632,184 bp on chromosome 7 (Chinese Patent: CN109371143B), the genetic locus of 7,990,990 bp on chromosome 7 (Chinese Patent: CN114182025B), the genetic locus of 27,045,611 bp on chromosome 7 (Chinese Patent: CN110106255B), and the genetic locus of 83,434,731 bp on chromosome 13 (Chinese Patent: CN109811063B) have made important contributions to improving the efficiency of the domestic pig farming industry. In recent research literature, Meng et al., through genome-wide association analysis, discovered that ARPP19 regulates the growth rate of Large White pigs by controlling mitosis and ONEUT1 by promoting cell division (Meng Qingli, Gao Hong, Huang Guoying, et al. Genome-wide association analysis of growth traits in American Large White pigs [J]. Pig Farming, 2018, (06): 57-61.). Xie et al. obtained 13 SNPs associated with the growth of Congjiang Xiang pigs through GWAS analysis, and the results of AS-PCR verification were consistent with the results of microarray detection (Xie Lingling. Polymorphism Study of SNPs Related to Growth Traits of Xiang Pigs [D]. Guizhou University, 2017).Meng et al. used GWAS analysis to study Yorkshire purebred pigs and found that PLCB1 and MAP2K6 are involved in the gonadotropin signaling pathway, and that these two genes contribute to growth at the onset of puberty (MENG Q, WANG K, LIU X, et al. Identification of growth trait related genes in a Yorkshire purebred pig population by genome-wide association studies[J]. Asian-Australasian journal of animal sciences, 2017, 30(4): 462-9.). Guo et al. conducted large-scale single-trait and multi-trait GWAS analysis on four different pig populations and meta-analysis on growth traits, screening out 44 genetic loci associated with 9 traits, including genetic loci located on pig chromosomes 4, 7 and X that are associated with pig growth rate (GUO Y, HUANG Y, HOU L, et al. Genome-wide detection of geneticmarkers associated with growth and fatness in four pig populations using four approaches[J]. Genetics, selection, evolution: GSE, 2017, 49(1):21.).

[0006] This invention uses Jinwu pigs as the experimental subject. Through genome-wide association analysis of 418 sampled pigs, a major genetic locus for carcass weight was identified. Based on the significant association between the SNP marker at this locus and the pig carcass weight trait, a KASP marker designed at this locus can be used for early screening of the future growth rate of newborn piglets, thereby improving breeding efficiency and achieving better farming benefits. These results have not been previously reported. Summary of the Invention

[0007] The purpose of this invention is to provide a major genetic locus that is significantly associated with the carcass weight trait of pigs, to provide the SNP molecular marker of this genetic locus, and to provide a KASP primer set for large-scale screening. This provides a new molecular marker for pig marker-assisted selection breeding, enabling early selection of pig growth rate, thereby shortening the breeding cycle, screening superior individuals, and improving breeding efficiency.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] The first aspect of the present invention provides a SNP molecular marker associated with the weight trait of pig carcasses, wherein the SNP molecular marker is located at 97,786,583 bp on chromosome 1 and within its closely linked region in the pig genome version 11.1 reference sequence.

[0010] Furthermore, chromosome 1 has a G / A polymorphism at 97,786,583bp, with three genotypes: GG, GA, and AA, among which the GG genotype is the preferred genotype.

[0011] The second aspect of this invention provides the application of the above-mentioned SNP molecular markers in marker-assisted selection breeding of pigs.

[0012] The third aspect of this invention provides the application of the above-mentioned SNP molecular markers in the identification of fast-growing pig breeds.

[0013] The fourth aspect of the present invention provides the application of the above-mentioned SNP molecular markers in detecting the SNP molecular marker genotypes.

[0014] The fifth aspect of the present invention provides a set of KASP primers for the above-mentioned SNP molecular markers, wherein the nucleotide sequences of the KASP primers are shown in SEQ ID NO.1 to SEQ ID NO.3.

[0015] The sixth aspect of this invention provides the application of the above-mentioned KASP primer set in marker-assisted selection breeding of pigs.

[0016] The seventh aspect of this invention provides the application of the above-mentioned KASP primer set in the identification of fast-growing pig breeds.

[0017] The eighth aspect of the present invention provides the application of the above-described KASP primer set in detecting the SNP molecular marker genotype.

[0018] The beneficial effects of this invention are:

[0019] This invention used the GeneSeek Porcine 50K SNP Chip to perform genome-wide association analysis on 418 Jinwu pig samples, identifying one major genetic locus affecting carcass weight, located at 97,786,583 bp on chromosome 1 in the pig genome version 11.1 reference sequence. This locus exhibits a G / A mutation, with individuals of the GG genotype showing a significantly additive additive effect (P < 0.01), where the average carcass weight was higher in individuals of the GA genotype than in those of the AA genotype.

[0020] Genotypic analysis was performed on 32 randomly sampled piglets using the KASP primer sets shown in SEQ ID NO:1-3. After 230 days of feeding, the average carcass weight of individuals with the GG genotype was found to be greater than that of individuals with the GA genotype, which was greater than that of individuals with the AA genotype, with a highly significant difference (P<0.01).

[0021] This invention is the first to discover a significant association between this SNP molecular marker and the carcass weight trait in pigs. Using the KASP primer set of this invention, early screening of a large number of individual piglets' genotypes can be performed simply, accurately, efficiently, and at low cost, retaining individuals with superior genotypes and culling those with inferior genotypes, thereby accelerating the process of pig genetic improvement and improving the economic efficiency of pig farming. This invention provides a reliable molecular marker for the genetic improvement and efficient breeding of pigs, and has significant scientific and economic value. Attached Figure Description

[0022] Figure 1 This is a KASP marker genotyping diagram of 32 sampled piglets and their parents in Example 2 of the present invention (FAM refers to the FAM fluorescent probe signal intensity, and HEX refers to the HEX fluorescent probe signal intensity).

[0023] Figure 2 This is a bar chart showing the carcass weight of 32 sampled piglets after 230 days of feeding according to genotype in Example 2 of the present invention (Pig genotype = pig genotype, Carcass Weight = carcass weight). Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention. Any modifications or substitutions made to the present invention without departing from its spirit and essence are within the scope of the present invention.

[0025] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, all reagents used in this invention are of analytical grade or higher.

[0026] Example 1: Obtaining SNP markers associated with major genetic loci in pig carcass weight

[0027] 1. Test materials

[0028] This experiment used Jinwu pigs (Jinwu pigs are a high-quality new breed of pig cultivated by the Zhejiang Academy of Agricultural Sciences. They are bred by crossbreeding Jinhua Liangtouwu pigs (a famous local breed in Zhejiang Province) with Duroc pigs and then fixed through crossbreeding and selection. They contain 37.5% Jinhua Liangtouwu pig bloodline and 62.5% Duroc pig bloodline) as the experimental subjects, with a number of 418 pigs. The entire experimental group was managed and fed by professionals at the Jinwu pig breeding base of Zhejiang Anji Anxing Animal Husbandry Co., Ltd., and all experimental pigs were in good health.

[0029] 2. Test methods

[0030] 2.1 Determination of carcass weight traits in pigs

[0031] Carcass weight data were measured from 418 individual pigs after standardized feeding for 230 days following weaning. After slaughter, the remaining body parts, including the fat and kidneys, after removing the head, hooves, tail, blood, hair, and internal organs, were weighed, and the obtained weight was the carcass weight of that pig.

[0032] 2.2 DNA Extraction

[0033] Spleen tissue was collected from each individual after slaughter for genomic DNA extraction. The DNA extraction kit used was the EasyPure Genomic DNA Kit (catalog number: EE101) manufactured by Beijing TransGen Biotechnology Co., Ltd., and the specific steps were followed according to the kit instructions. The extracted DNA was tested for concentration and quality and stored at -20℃ for later use.

[0034] 2.3 Genome-wide SNP typing using DNA microarrays

[0035] Pig genome-wide SNP genotyping was performed using the GeneSeek Porcine 50K SNP Chip. Both the chip and SNP genotyping technology services were provided by Neocare Biotech (Shanghai) Co., Ltd.

[0036] 2.4 Genome-wide association analysis

[0037] GWAS analysis was performed using the r.MVP package in R. The association analysis model used was the Farm CPU multi-site model. The Farm CPU multi-site model repeatedly used fixed-effects and random-effects models. Principal components, sex, and quantitative trait nucleotides (QTNs) were added as covariates in the fixed-effects model for association analysis (Liu X, Huang M, Fan B, et al. Iterative Usage of Fixed and RandomEffect Models for Powerful and Efficient Genome-Wide Association Studies[J]. PLoS Genetics, 2016, 12(2):e1005767).

[0038] The Farm CPU multi-site model is represented as follows:

[0039] y = Pbp + Mtbt + Sidj + e

[0040] Where y is the phenotypic (carcass weight) observation vector, P is the fixed effects matrix, including the first two principal components, sex and farm effect, Mt is used as the t genotype matrix of QTNs for the fixed effects, bp and bt are the correlation design matrices of P and Mt respectively, Si is the i-th marker to be tested, dj is the corresponding effect, and e is the residual effect vector.

[0041] 3. Results of genome-wide association analysis

[0042] Genome-wide association analysis of pig carcass weight revealed a major genetic locus significantly associated with this trait, located at 97,786,583 bp on chromosome 1 in the pig genome version 11.1 reference sequence. This locus exhibited G / A nucleotide diversity. In the tested sample population, individuals with the GG genotype had an average carcass weight of 88.1 kg, individuals with the GA genotype had an average carcass weight of 61.5 kg, and individuals with the AA genotype had an average carcass weight of 46.1 kg (Table 1). All differences were highly significant (P < 0.01).

[0043] Table 1: SNP marker genotype effects significantly associated with pig carcass weight trait

[0044]

[0045] The values ​​in the table are "mean ± standard deviation", and different superscript letters indicate that the difference is highly significant (P<0.01).

[0046] Example 2: Design and Application of KASP Markers

[0047] 1. Design of KASP primers

[0048] For the G / A nucleotide diversity site at positions 97,786,583 on chromosome 1 of the porcine genome version 11.1 reference sequence obtained in Example 1, a primer combination for detecting the nucleotide diversity at this site was designed, and its primer sequences are shown in SEQ ID NO.1 to SEQ ID NO.3. The upstream primers are SEQ ID NO.1 and SEQ ID NO.2, with the last bases being G and A, respectively, corresponding to the nucleotide diversity site. Bases 1 to 21 of SEQ ID NO.1 are adapters used for recognition by the HEX fluorescent probe in the kit, emitting a blue fluorescent signal upon detection; bases 1 to 21 of SEQ ID NO.2 are adapters used for recognition by the FAM fluorescent probe in the kit, emitting a red fluorescent signal upon detection. SEQ ID NO.3 is the downstream primer for this site, which is universal for both genotypes. Details are as follows:

[0049] SEQ ID NO.1: 5'-GAAGGTGACCAAGTTCATGCTTCCATAGATGAAGGACACG-3'

[0050] SEQ ID NO.2: 5'-GAAGGTCGGAGTCAACGGATTTCCATAGATGAAGGACACA-3'

[0051] SEQ ID NO.3: 5'-AAAGAGCTTCATGAGTGGTCCAT-3'

[0052] 2. DNA template

[0053] Approximately 25 mg of micro-tissue was excised from the ears of Duroc pig parents, Jinhua two-headed black pig parents, and 32 newborn Jinhua two-headed black pig samples for DNA template extraction. The DNA extraction method was performed according to the instructions in Section 2.2 of Example 1. After extraction and concentration determination, the DNA was diluted with double-distilled water to 10 ng / μL for use as DNA template in PCR reactions.

[0054] 3. Reaction system

[0055] In a 384-well plate, each sample was processed using a 5 μL reaction mixture, with 2.5 μL of DNA template, 2.5 μL of 2×KASP Master Mix, and 0.07 μL of KASP Assay Mix added sequentially. The latter two mixes were purchased from LGC Ltd., UK, catalog number KBS-1050-102 (www.lgcgroup.com / mastermixcheck).

[0056] 4. Reaction conditions

[0057] PCR reactions were performed in an IntelliQube machine (https: / / www.biosearchtech.com / products) in SNP genotyping inline mode (single membrane). The thermal cycling conditions were as follows: pre-denaturation 94℃ for 15 min, 1 cycle; denaturation 94℃ for 20 sec, annealing and extension 61℃~55℃ for 60 sec, 10 cycles, with the annealing and extension temperature decreasing by 0.6℃ per cycle; denaturation 94℃ for 20 sec, annealing and extension 55℃ for 60 sec, 28 cycles.

[0058] 5. Test Results

[0059] After the reaction, the genotype of each sample was read using KlusterCaller software (www.lgcgroup.com / software). In the amplification results, blue fluorescence signal represents the GG genotype, red fluorescence signal represents the AA genotype, and purple fluorescence signal represents the GA heterozygous genotype. The results are as follows: Figure 1 As shown ( Figure 1 The negative control was water instead of DNA template. Fifteen samples were of the same GG genotype as the Duroc parent, 15 samples were of the GA heterozygous genotype, and two samples were of the same AA genotype as the Jinhua two-headed black pig parent. Thirty-two piglets were raised for 230 days, and their carcass weights were measured at slaughter. The results are as follows: Figure 2 As shown, the mean carcass weight and standard deviation of individuals with the GG genotype were 89.9 ± 10.9 kg, those with the GA genotype were 59.3 ± 4.1 kg, and those with the AA genotype were 47.3 ± 5.9 kg. The differences among genotypes were highly significant (P < 0.01), indicating that this molecular marker has a good screening effect for superior individuals.

Claims

1. The application of a SNP molecular marker associated with pig carcass weight trait in marker-assisted selection breeding of Jinwu pigs for carcass weight trait, characterized in that, The SNP molecular marker is located at 97,786,583 bp on chromosome 1 in the pig genome version 11.1 reference sequence. This location exhibits G / A polymorphism, with three genotypes: GG, GA, and AA. Among them, the average carcass weight of individuals with the GG genotype is greater than that of individuals with the GA genotype, which in turn is greater than that of individuals with the AA genotype.

2. The application of a SNP molecular marker associated with pig carcass weight traits in the identification of Jinwu pig carcass weight traits, characterized in that, The SNP molecular marker is located at 97,786,583 bp on chromosome 1 in the pig genome version 11.1 reference sequence. This location exhibits G / A polymorphism, with three genotypes: GG, GA, and AA. Among them, the average carcass weight of individuals with the GG genotype is greater than that of individuals with the GA genotype, which in turn is greater than that of individuals with the AA genotype.

3. The application of a KASP primer set for detecting SNP molecular markers associated with pig carcass weight traits in marker-assisted selection breeding of Jinwu pigs for carcass weight traits, characterized in that... The SNP molecular marker is located at 97,786,583 bp on chromosome 1 in the pig genome version 11.1 reference sequence. This location exhibits G / A polymorphism, with three genotypes: GG, GA, and AA. The average carcass weight of individuals with the GG genotype is greater than that of individuals with the GA genotype, which in turn is greater than that of individuals with the AA genotype. The nucleotide sequences of the KASP primer set are shown in SEQ ID NO.1 to SEQ ID NO.

3.

4. The application of a KASP primer set for detecting SNP molecular markers associated with pig carcass weight traits in the identification of Jinwu pig carcass weight traits, characterized in that, The SNP molecular marker is located at 97,786,583 bp on chromosome 1 in the pig genome version 11.1 reference sequence. This location exhibits G / A polymorphism, with three genotypes: GG, GA, and AA. The average carcass weight of individuals with the GG genotype is greater than that of individuals with the GA genotype, which in turn is greater than that of individuals with the AA genotype. The nucleotide sequences of the KASP primer set are shown in SEQ ID NO.1 to SEQ ID NO.3.

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