Application of SNP molecular marker related to pig birth weight traits

Abstract

The application belongs to the technical field of pig molecular marker screening, and particularly relates to a SNP molecular marker related to pig birth weight. The SNP molecular marker has a registration number of rs80876322, ear samples of Duroc pigs from a pig farm in southern China are collected, genomic DNA is extracted and quality detection is carried out, SNP detection in a whole genome range is carried out by using a GeneSeek Porcine 50K SNP high-density chip to obtain genotype information of individuals, and a SNP molecular marker related to pig birth weight is obtained through whole genome association analysis. The SNP molecular marker is located on chromosome 14, a nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO:1, an A / G allelic mutation (substitution) exists at the 51th base of the sequence, the mutation (substitution) causes nucleotide polymorphism of the sequence, and when the 51th nucleotide of the sequence shown in SEQ ID NO:1 is G, it is determined that the pig has greater birth weight.

Description

Technical Field

[0001] This invention belongs to the field of porcine molecular marker screening technology, specifically relating to the application of SNP molecular markers related to the birth weight trait in pigs. The molecular markers described in this invention can be used for the auxiliary prediction and selection of Duroc birth weight traits. Background Technology

[0002] Reproductive traits in pigs are among the most important economic traits in the breeding process, and their quality directly affects the economic benefits of pig production. Birth weight is one of the main indicators for measuring pig reproductive traits and is often used as a key factor in genetic improvement (Yang Yun, 2013). Duroc is an American pig breed, widely used as one of the terminal sires in modern commercial pig crossbreeding systems in my country due to its excellent characteristics such as fast growth rate, high lean meat percentage, and good meat quality (Lü Zhenghai, 2018). However, its poor reproductive performance and significantly low litter size have seriously hindered the process of new breed breeding (Huang Mingying, 2006). Therefore, improving the litter size of Duroc pigs plays an important role in accelerating pig genetic improvement and breeding new pig breeds that meet market demands.

[0003] Primary weight is a complex trait with low heritability, controlled by multiple genes with minor effects and environmental factors, with each gene having limited genetic efficacy. Currently, with the development of sequencing technology and the continuous improvement of species genomic information, genome-wide association studies (GWAS) have become an important tool for discovering genetic markers and candidate genes related to complex traits. GWAS involves screening for single nucleotide polymorphisms (SNPs) across the entire genome of a species, identifying SNPs associated with the corresponding traits, and predicting phenotypic / breeding values ​​based on the effects of these SNPs (Meuwissen et al. 2001). This achieves early selection, significantly shortens generation intervals, and improves the genetic progress of animal breeding (Xing et al. 2020).

[0004] The accuracy of genome-wide association studies (GWAS) is related to the size of the effective population, and both the quantity and quality of the effective population data have a significant impact on the assessment of genetic effects (Calus and Veerkamp 2007). Currently, due to the lack of standardized breeding records for Duroc breeds introduced to my country, the number of effective populations for GWAS is relatively small (Wu Changxin 2012; Ding Xiangdong et al. 2020), and most studies on birth weight traits remain at the level of phenotypic statistical analysis. Therefore, it is essential to conduct GWAS on pig birth weight by expanding the population size to identify key SNP loci associated with birth weight.

[0005] This invention collects ear tissue from 2258 Duroc pigs from a pig farm in southern China, extracts DNA from the ear tissue and performs quality testing. After obtaining the genotype information of each individual using the GeneSeek Porcine 50K SNP high-density chip, genome-wide association analysis is performed using the FarmCPU model (Liu X et al., 2016) with the phenotypic data of the corresponding individuals using the MVP package in R language. This identifies SNP molecular markers associated with the birth weight trait in Duroc pigs, providing new molecular markers for predicting the birth weight phenotype and breeding value in pigs, which is of great significance for improving pig reproductive traits.

[0006] The SNP molecular markers screened in this invention showed a significant correlation with the birth weight trait in Duroc pigs, providing a theoretical basis for the study of the genetic mechanism and genetic progress of the birth weight trait in pigs. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and screen for a SNP molecular marker associated with the birth weight trait in Duroc pigs. By using the GeneSeek Porcine 50K SNP high-density chip to perform genome-wide SNP detection on each individual to obtain the individual's genotype information, and then using GWAS to perform genome-wide association analysis to obtain SNPs that are significantly associated with the birth weight trait in Duroc pigs, a new molecular marker is provided for evaluating the birth weight phenotype and breeding value of pigs.

[0008] The technical solution of the present invention is as follows:

[0009] The applicant collected ear samples from Duroc pigs at a pig farm in southern China, extracted their genomic DNA and performed quality testing. Genotype information was obtained using a GeneSeek Porcine 50K SNP high-density microarray. Genome-wide association analysis was performed to identify SNPs associated with birth weight. The nucleotide sequence of the SNP locus with accession number rs80876322 was obtained from the Ensembl database. 50 bp samples were taken upstream and downstream of the SNP locus, and the sequence is shown in SEQ ID NO:1 of the sequence listing. The specific sequence is shown below:

[0010] ACACGCCTTGTGGAGAAATGCCAGCTTTGGGGATGAAGGGAGGGCTCAGR(A / G)GCCCAGGGCAAATGGAGGCCAGAGCCCCCACCTCCTCCCCTCTGCAGCCC

[0011] The R at the 51st base of the above sequence is an A / G allele (substitution) mutation, which causes the above sequence, namely the sequence described in SEQ ID NO:1, to produce nucleotide polymorphism.

[0012] This invention develops a molecular marker for predicting the birth weight of pigs using the above-mentioned fragment. When the nucleotide at position 51 of SEQ ID NO:1 is G, it is determined that the birth weight of Duroc pigs may be larger.

[0013] The above sequence can be used as a molecular marker for the selection of primary birth weight traits in Duroc pigs.

[0014] The applicant provides a method for screening SNP molecular markers associated with the birth weight trait in Duroc pigs, the method comprising the following steps:

[0015] The applicant identified a SNP molecular marker associated with the birth weight trait in Duroc pigs, and the nucleotide sequence of this molecular marker is shown below:

[0016] ACACGCCTTGTGGAGAAATGCCAGCTTTGGGGATGAAGGGAGGGCTCAGR(A / G)GCCCAGGGCAAATGGAGGCCAGAGCCCCCACCTCCTCCCCTCTGCAGCC

[0017] The R at position 51 of the above sequence is a substitution of A or G, which causes polymorphism in the above sequence.

[0018] Based on a SNP molecular marker associated with the birth weight trait of Duroc pigs, when the 51st nucleotide of the sequence shown in SEQ ID NO:1 is G, it is determined that the birth weight of the pig may be larger.

[0019] The SNP molecular markers of this invention can be used for the prediction and selection of pig birth weight.

[0020] The specific steps of this invention are as follows:

[0021] ① Ear samples were collected from Duroc pigs from a pig farm in southern China. Genomic DNA was extracted and quality tested. SNP genotyping data was obtained by genotyping using the GeneSeek Porcine 50K SNP high-density chip.

[0022] ②Statistical analysis was conducted on phenotypic data from 2,258 Duroc pigs from a pig farm in southern China, and the newborn pigs were reclassified as phenotypes.

[0023] ③ The FarmCPU model was used for genome-wide association analysis (GWAS) in the MVP package of R language. The model formula is as follows: yi =M i1 b1+M i2 b2+...+M it b t +S ij d j +e i (1) where y i M is the trait observation value of the i-th individual; i1 M i2 M it These are the genotypes of t potential associated loci added to the model, with this portion empty during the first iteration; b1, b2, ..., b t These are the corresponding effect values ​​of the possible associated sites added to the model; S ij It is the genotype of the j-th genetic marker in the i-th individual; d j It is S ij The corresponding effect value; e i It is the residual vector;

[0024] y i =u i +e i (2)y i It is the trait observation value of the i-th individual; u i It is the total genetic effect of the i-th individual; e i The residual vector model (2) generates possible associated sites as covariates and adds them to the model (1) for iterative calculation until it stops.

[0025] The molecular markers screened by this invention can be applied to association analysis of related genotypes or related traits of pig birth weight for non-diagnostic purposes, providing new molecular marker resources for marker-assisted selection of pig birth weight.

[0026] The advantages of this invention compared to the prior art are as follows:

[0027] This invention can detect the genotype of pigs in vitro using gene chip technology, thereby improving the detection and analysis of birth weight in Duroc pigs for non-diagnostic purposes. Compared with conventional methods such as PCR-RFLP, this invention has the advantages of being simple, fast, and highly sensitive.

[0028] For a more detailed technical solution, please refer to "Detailed Implementation". Attached Figure Description

[0029] Figure 1 : Schematic diagram of the technical process of this invention.

[0030] Figure 2This invention presents the 50bp upstream and downstream nucleotide sequences of the molecular marker rs80876322 screened in this invention, and the nucleotide sequence of the molecular marker of this invention. Figure annotations: Figure 2 There is an A / G allele mutation at the 51st base of the nucleotide marker shown (the letter "R" at 51bp is the mutation site).

[0031] Figure 3 Manhattan plot of genome-wide association analysis in this invention. Explanation of figure labels: The research object is the pig's birth weight trait; the markers indicated by the black circles and arrows are the molecular markers screened in this invention, located on pig chromosome 14. Detailed Implementation

[0032] Explanation of the sequence list:

[0033] SEQ ID NO:1 is the nucleotide sequence of the molecular marker associated with the birth weight trait of Duroc pigs screened in this invention. The sequence length is 101 bp. There is an allelic mutation (substitution) at the 51st bp of this sequence (i.e., A / G base substitution. For the convenience of making a nucleotide sequence listing, the base at the 51st bp of the sequence listing of this application is the mutated (substituted) base G as an example). The above substitution or mutation causes polymorphism in the nucleotide sequence shown in SEQ ID NO:1.

[0034] The sequence and genome-wide association analysis results in this invention are based on pig genome version 11.1.

[0035] Example 1: Genotyping and Detection

[0036] Ear samples were collected from Landrace pigs from a pig farm in southern China. Genomic DNA was extracted and quality tests were performed. Genotypic information of each individual was obtained by performing SNP detection across the entire genome using the GeneSeek Porcine 50K SNP high-density chip.

[0037] The raw data was converted to VCF format using Plink 1.9 software, and then the software was used to perform quality control on the VCF format files, specifically as follows:

[0038] 1) maf 0.01: Remove SNP sites with a minimum allele frequency of less than 1%;

[0039] 2) geno 0.1: Remove SNP sites with a genotype detection rate of less than 90%;

[0040] 3) mind 0.1: Remove individuals with a genotype deletion rate greater than 10%;

[0041] 4) hwe le-6: Excluding Hardy and Weinberg equilibrium test p-values ​​less than 10 -6 SNP sites,

[0042] 5) Use Beagle 4.1 software to impute missing genotypes in the quality-controlled microarray data. The command is as follows:

[0043] java-jar-Xmx8g beagle.jar gt=qc.file.vcf out=example.impute

[0044] (2) Genotype data were tested, and 2,258 individuals and 34,985 SNPs were used for genome-wide association analysis.

[0045] Example 2: Methods for genome-wide association analysis of primary birth weight traits in pigs

[0046] The birth weight phenotypes of Duroc pigs used for genotype association analysis were obtained from a pig farm in southern China, totaling 2258 individuals. The FarmCPU model was used, and genome-wide association analysis (GWAS) was performed using the FarmCPU model from the MVP package in R. The specific model formula is as follows: y i =M i1 b1+M i2 b2+...+M it b t +S ij d j +e i (1) where y i M is the trait observation value of the i-th individual; i1 M i2 M it These are the genotypes of t potential associated loci added to the model, with this portion empty during the first iteration; b1, b2, ..., b t These are the corresponding effect values ​​of the possible associated sites added to the model; S ij It is the genotype of the j-th genetic marker in the i-th individual; d j It is S ij The corresponding effect value; e i It is the residual vector; y i =u i +e i (2)y i It is the trait observation value of the i-th individual; u i It is the total genetic effect of the i-th individual; e i It is the residual vector.

[0047] Model (2) generates possible associated sites as covariates and adds them to model (1) for iterative calculation until it stops.

[0048] The significance levels of the genome-wide association analysis in this invention are shown in Table 1.

[0049] Table 1. Significance of genome-wide association analysis for rs80876322

[0050] H3GA0041735 14 101650509 8.75E-07

[0051] Table 1 Explanation: The significance level is defined as P-value < 0.05 / 34985 (Bonferroni corrected).

[0052] Example 3: Application of rs80876322 molecular marker genotyping method in association analysis of pig birth weight trait / Association analysis of rs80876322 molecular marker (SWQ ID NO:1 in sequence listing) with pig birth weight trait:

[0053] Association analysis between the rs80876322 molecular marker and the porcine birth weight trait was performed using a mixed linear model (MLM). The specific model is as follows:

[0054] y ijklmn =μ+G i +HYS j +Breed+ID m +ε ijklmn

[0055] Among them, y ijklmn G is the primary birth weight phenotypic value of the m-th individual; μ is the population mean; G i It is a genotype effect, HYS k It is the combined effect of birth location, birth year, and birth season (fixed effect); Breed is the variety effect (fixed effect); ID m It is an additive effect (random effect) on individuals, ε ijklmno The model residual effect was analyzed using the F-test to determine the significance of the differences in the primary weight trait among individuals with the three genotypes. The results are shown in Table 2.

[0056] Table 2 Polymorphism of the fragment rs80876322 (accession number rs80876322)

[0057]

[0058] Table 2 shows that P<0.05 indicates a significant difference; P<0.01 indicates a highly significant difference.

[0059] Table 2 shows that individuals with the GG genotype may have a higher birth weight, while individuals with the AA genotype may have a lower birth weight. In the genome-wide association analysis using the FarmCPU model, the rs80876322 marker reached genome-wide significance, indicating that this marker is significantly associated with the pig birth weight trait, and when this marker mutates to G, the piglets have a higher birth weight.

[0060] Main references:

[0061] [1] Lü Zhenghai. Introduction to introduced pig breeds and some local breeds in my country [J]. Modern Animal Husbandry Technology, 2018, 42(06):24.

[0062] [2] Yang Yun et al., Analysis of factors affecting sow reproductive performance [J]. Pig Farming, 2013, 01: 33-35.

[0063] [3] Huang Mingying et al. Statistical analysis of litter size of Landrace, Large White and Duroc sows [J]. China Animal Husbandry and Poultry Seed Industry, 2006, 12: 27.

[0064] [4] Xing Wenkai et al., Research progress in pig genome selection breeding. Chinese Journal of Animal Husbandry, 2020, 1-11.

[0065] [5] Wu Changxin. Breaking out of the vicious cycle of “introduction-degeneration-re-introduction-re-degeneration”. China Pig Industry, 2012, 7(04):10.

[0066] [6] Meuwissen THE, Hayes BJ, Goddard ME. Prediction of Total GeneticValue Using Genome-Wide Dense Marker Maps. Genetics (Austin), 2001, 157(4): 1819-1829.

[0067] [7] Calus MPL, Veerkamp RF. Accuracy of breeding values ​​when using andignoring the polygenic effect in genomic breeding value estimation with amarker density of one SNP per cM. Journal of Animal Breeding and Genetics, 2007, 124(6): 362-368.

[0068] [8]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。

Claims

1. The application of a molecular marker in marker-assisted selection of primary birth weight traits in pigs, wherein the pig breed is Duroc, characterized in that, The nucleotide sequence of the molecular marker is shown below: ACAGCGCTTGTGGAGAAATGCCAGCTTTGGGGATGAAGGGAGGGCTCAGR(A / G)GCCCAGGGCAAATGGAGGCCAGAGCCCCCACCTCCTCCCCTCTGCAGCCC The R at position 51 of the above sequence is a substitution of A or G, which causes nucleotide polymorphism in the sequence shown; individuals with the GG genotype have a higher birth weight; individuals with the AA genotype have a lower birth weight.

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