A haplotype molecular marker associated with reddish-brown / black coat color in pigs and its application

Abstract

This invention belongs to the field of porcine molecular marker technology, specifically relating to a haplotype molecular marker associated with the reddish-brown / black coat color trait in pigs and its application. Based on high-depth whole-genome sequencing and integrating publicly available resequencing data from the NCBI database, this invention performs genome-wide association analysis to screen for four SNP loci significantly associated with reddish-brown / black coat color in pigs. These four SNP loci are located on chromosome 9 and are fully linked. The haplotype molecular marker formed by these loci can be used for marker-assisted selection of the reddish-brown / black coat color trait.

Description

Technical Field

[0001] This invention belongs to the field of porcine molecular marker technology, specifically relating to a haplotype molecular marker related to the reddish-brown / black coat color trait of pigs and its application. Background Technology

[0002] Coat color, as the most intuitive and varied phenotypic trait during animal domestication, is one of the important phenotypic characteristics of breed (Xu Jiguo, Cui Fangfang, Zhang Guosheng, et al. Research progress on the formation of diluted coat color phenotype in agricultural economic animals [J]. Guangdong Agricultural Sciences, 2024, 51(02): 1-15.). To date, researchers have identified multiple genetic loci and molecular markers related to pig coat color. In pig breeds such as Jinhua pig and Shaziling pig, the EDNRB gene is subject to strong selection (Ai H, Huang L, Ren J, et al. Genetic diversity, linkage disequilibrium and selection signatures in Chinese and Western pigs revealed by genome-wide SNP markers [J]. PLoS One, 2013, 8(2): e56001.). The dominant white coat color in domestic pigs is mainly due to the tandem duplication of a 450-Kb large fragment containing the entire KIT gene, and at the same time, a splicing mutation occurs in intron 17 of one copy of the KIT gene (Sun G, Liang X, Qin K, Qin Y, Shi X, Cong P, Mo D, Liu X, Chen Y, He Z. Functional analysis of KIT genestructural mutations causing the porcine dominant white phenotype using genome-edited mouse models[J]. Front Genetics, 2020, 11:138.). The MC1R gene regulates the ratio of pheomelanin to eumelanin production by modulating the cAMP signaling pathway in pigment cells, thereby affecting coat color (Lan J, Wang M, Qin K, Liu X, Shi X, Sun G, Liu X, Chen Y, He Z. Functional characterization of cAMP signaling of variant porcine MC1R alleles in PK15 cells[J]. Animal Genetics, 2022, 53(3):317-326.).

[0003] There may be certain associations between genes regulating coat color and other traits. The white coat color of Large White pigs is regulated by the KIT gene, and mutations in this gene also lead to lighter meat color in Large White pigs (Liang X, Lan J, Xu M, et al. Impact of KIT Editing on Coat Pigmentation and Fresh Meat Color in Yorkshire Pigs[J]. CRISPR J, 2022, 5(6): 825-842.). In pig breeding, coat color, as a usable genetic marker, is an important basis for identifying breeds and also has certain uses in determining hybrid combinations, breed purity and kinship, and evaluating product quality (Li Ming. Pig coat color and its application in breeding[J]. Journal of Animal Husbandry and Veterinary Medicine, 2017, 48(6): 123-130.). Therefore, coat color, as a key trait, deserves in-depth exploration in breeding research.

[0004] Coat color is a trait controlled by multiple genes and their complex interactions, and some coat color-related genes have not yet been fully identified. This invention integrates self-sequencing resequencing data of Yunnan local pigs and publicly available resequencing data from the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ), including 526 black-coated pigs from 22 breeds and 178 red-brown-coated pigs from 2 breeds, to screen for SNP molecular markers associated with red-brown / black coat color, providing a new molecular marker reference for marker-assisted selection and coat color prediction in pigs. Summary of the Invention

[0005] This invention integrates resequencing data of Yunnan local pigs obtained through self-sequencing with publicly available resequencing data from the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ), including 526 black-coated pigs from 22 breeds and 178 red-brown-coated pigs from 2 breeds. Genome-wide association analysis identified four SNP molecular markers significantly associated with the red-brown / black coat color trait in pigs. Based on the pig version 11.1 reference genome from the Ensembl database, the nucleotide sequences 50 bp upstream and downstream of each SNP were obtained, as follows:

[0006] The nucleotide sequence containing SNP1 is shown in SEQ ID NO.1 or 2. The SNP site is located at position 51 of the sequence, which corresponds to position 72588595 on chromosome 9 of pigs. The polymorphic site is G or A.

[0007] The nucleotide sequence of SNP2 is shown in SEQ ID NO.3 or 4. The SNP site is located at position 51, which corresponds to position 72714828 on chromosome 9 of pigs. The polymorphic site is T or G.

[0008] The nucleotide sequence of SNP3 is shown in SEQ ID NO.5 or 6. The SNP site is located at position 51, which corresponds to position 72785308 on chromosome 9 of pigs. The polymorphic site is T or C.

[0009] The nucleotide sequence of SNP4 is shown in SEQ ID NO.7 or 8. The SNP site is located at position 51, which corresponds to position 72809421 on chromosome 9 of pigs. The polymorphic site is G or A.

[0010] The above four SNP markers are completely linked and constitute haplotype molecular markers. Individuals with the homozygous genotype AGCA have black fur, while individuals with the homozygous genotype GTTG have reddish-brown fur. Attached Figure Description

[0011] Figure 1 The Manhattan plot drawn in Embodiment 2 of this invention visualizes the results of GWAS analysis.

[0012] Figure 2 The QQ graph drawn in Embodiment 2 of the present invention compares the two probability distributions by comparing the quantiles of the probability distributions of the actual -log(P) value and the expected -log(P) value, in order to further determine the reliability of the GWAS results.

[0013] Figure 3 The linkage disequilibrium analysis results of the four SNPs provided in Embodiment 2 of this invention are shown in the box. The larger the value, the stronger the correlation. Detailed Implementation

[0014] Example 1: Genotyping Detection and Data Processing

[0015] (1) Sequencing and quality control

[0016] Ear samples were collected from 196 black-haired Yunnan native pigs, and DNA was extracted. DNA quality was assessed using a DNA concentration analyzer and agarose gel electrophoresis. Sequencing libraries were then constructed from these DNA samples, and whole-genome resequencing was performed using the DNBseq T10 platform. The raw data was filtered using the default parameters of Fastp v0.23.4 software, with a sequencing depth of 23.61×. Simultaneously, publicly available resequencing data from 508 pigs obtained from the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ) were integrated, resulting in a total of 704 pigs' resequencing data. Alignment was performed using BWA 0.7.17 software with default parameters. The reference genome version was Sus scrofa 11.1 (GCA_000003025.6). SNPs were isolated using GATK v4.1.9.0 with the following hard filtering parameters: QUAL < 30.0, QD < 2.0, MQ < 40.0, FS > 60.0, SOR > 3.0, MQRankSum < -12.5, and ReadPosRankSum < -8.0.

[0017] (2) Data quality control and data entry

[0018] SNPs on sex chromosomes and unknown chromosomes were removed using PLINK v1.9 software. The obtained SNPs were further filtered with the quality control parameter "-autosome-maf 0.05-geno 0.05". For single-variety quality control, Harvey-Wen balance was performed with the parameter "-hwe1e-6". Deletion genotypes were filled using the default parameters of Beagle v5.2. Finally, 704 individuals and 9,053,744 SNPs were available for subsequent association analysis.

[0019] Example 2: Genome-wide association analysis of SNP molecular markers with black and reddish-brown coat traits in domestic pigs

[0020] (1) Phenotypic definition

[0021] Based on the breed information of individuals in the population, and by consulting the Chinese Livestock and Poultry Genetic Resources (Pig Records), relevant foreign pig breed websites, and related literature on pig genetic resources, the coat color of each breed was statistically analyzed, and coat color was used as a phenotype. A total of 704 individuals from 24 breeds were included. Among them, there were 178 individuals from 2 breeds that were red-brown (Duroc and Thomson pigs), and 526 individuals from 22 breeds that were black (Enshi Black Pig, Eight-Eyebrow Pig, Debao Pig, Erhualian Pig, Neijiang Pig, Tiegu Pig, Tibetan Pig, Southern Anhui Black Pig, Southern Yunnan Small-Eared Pig, Baoshan Pig, Mingguang Small-Eared Pig, Gaoligong Mountain Pig, Saba Pig, Lijiang Pig, Iberian Black Pig, Jeju Black Pig, Laiwu Black Pig, Lulai Black Pig, Min Pig, Basin Mountain Pig, Suhuai Pig, and Yanan Pig).

[0022] (2) Genome-wide association analysis

[0023] Based on the SNP molecular markers screened after genotyping, principal component analysis was performed using GCTAv1.94.1 software. Using the black population as the control and the red-brown population as the case, genome-wide association analysis was conducted using the LMM model in GEMMA0.98.5 software to screen out SNP molecular markers significantly associated with the red-brown / black coat color trait in pigs. The specific model is as follows:

[0024] y = Wα + Xβ + u + e; u ~ MVN n (0,λτ -1 K); e~MVN n (0,τ -1 I n )

[0025] Where y represents the individual's phenotypic value, α represents the fixed-effects covariates including the intercept and principal components, β is the SNP label vector, μ is the random-effects vector, and e represents the residual vector. W and X represent the correlation matrices of β and μ. MVN n Let τ represent an n-dimensional multivariate normal distribution. -1 Let the residual variance be 0, λ be the ratio between the two variance components, K be the kinship matrix, and I be the variance between the two components. n It is an identity matrix.

[0026] (3) Screening for SNP molecular markers associated with reddish-brown / black coat color

[0027] The significance threshold was adjusted using Bonferroni, i.e., P = 0.05 / 9053744. SNPs with a P value less than this were considered significantly associated with the trait. Based on the output, Manhattan plots and QQ plots were generated using the R package CMplot, as shown below. Figure 1 and Figure 2 As shown in Table 1, four SNP sites were selected.

[0028] Table 1. Candidate SNP loci for the reddish-brown / black coat color trait in domestic pigs identified based on GWAS.

[0029]

[0030] Table 2. Haplotype distribution and percentage of candidate SNP sites.

[0031]

[0032] As shown in Table 1, four SNP loci were identified that were significantly associated with the reddish-brown / black coat color trait in domestic pigs. Figure 3Table 2 shows that the four selected SNPs are completely linked, forming two haplotypes: AGCA and GTTG. When the haplotype is homozygous for AGCA, the coat color is black; when the haplotype is homozygous for GTTG, the coat color is reddish-brown.

Claims

1. Use of a haplotype molecular marker for the prediction of the red / brown / black color trait in pigs or for marker-assisted selection, characterized in that, The haplotype molecular marker is composed of 4 SNP sites: SNP1 is located at 72588595 of pig chromosome 9, the genotype of the polymorphic site is G or A; SNP2 is located at 72714828 of pig chromosome 9, the genotype of the polymorphic site is T or G; SNP3 is located at 72785308 of pig chromosome 9, the genotype of the polymorphic site is T or C; SNP4 is located at 72809421 of pig chromosome 9, the genotype of the polymorphic site is G or A; The reference genome version is Sus scrofa 11.1, the individual with homozygous genotype of the haplotype GTTG of the molecular marker is red-brown coat color, and the individual with homozygous genotype of the haplotype AGCA of the molecular marker is black coat color.

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