Haploid molecular marker related to pig black / white hair color and application of haplotype molecular marker

Abstract

The invention belongs to the technical field of pig molecular markers, and particularly relates to haplotype molecular markers related to pig black / white hair color and application of the haplotype molecular markers. Four SNP loci obviously associated with black / white hair color characters are screened based on whole genome association analysis through whole genome high-depth sequencing in combination with re-sequencing data disclosed by an NCBI database, the four SNP loci are located in a No.6 chromosome and are completely linked, and the haplotype molecular marker composed of the four SNP loci can be used for auxiliary selection and breeding of pig black / white hair color.

Description

Technical Field

[0001] The invention belongs to the technical field of pig molecular markers, and in particular relates to haplotype molecular markers associated with pig black / white coat color and applications thereof. Background Art

[0002] Coat color is one of the most significant and obvious characteristics of mammals. Due to human preference and domestication of animals, as well as different habitats and climatic conditions, domestic pigs also show obvious differences in coat color. Black and white coat colors are more common in pigs and are typical phenotypic characteristics of multiple domestic pig breeds. Coat color is closely related to the economic traits and disease characteristics of pigs. Asian domestic pigs with all-black coats have the advantages of high fertility, tolerance to roughage, and good meat flavor. The commonly used maternal Large White pigs have white coats and have excellent characteristics of fast growth rate, high feed utilization rate, and high lean meat rate, but also have shortcomings such as macrocytic anemia and poor meat color (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): 825-42.). Coat color is a genetic marker that can be used for breed identification, purity testing, and judgment of kinship (Bai Xiaoqing, Wang Jinyong. Research progress in pig coat color genetics [J]. Animal Science and Veterinary Medicine, 2004, 21(8):32-33.), and is also one of the main breeding traits.

[0003] Genome-wide association study (GWAS) is a method for the overall association of common genetic variations across the entire genome, which can directly identify gene loci or marker loci that are closely related to phenotypic variation and have specific functions. GWAS has become a common method for marker-assisted breeding. The causes of coat color are complex. It is currently known that the black coat color of most pig breeds is caused by missense mutations in the MC1R gene (Lai F, Ren J, Ai H, et al. Chinese white Rongchang pig does not have the dominant white allele of KIT but has the dominant black allele of MC1R [J]. J Hered. 2007; 98 (1): 84-87.). Kijas et al. analyzed the sequence of MC1R gene of different coat color pigs and found that the coat color of wild boar is regulated by MC1R*1 allele, the dominant black of Meishan pig is regulated by MC1R*2 allele, the stripe of Hampshire pig is regulated by MC1R*3 allele, and the red of Duroc pig is regulated by MC1R*4 allele (KIJAS JM, WALES A, et al. Melanocortin receptor 1 (MC1R) mutations and coat color in pigs [J]. Genetics, 1998, 150 (3): 1177-1185.). Xu et al. found that Jinhua pigs, Shanggao Mengshan pigs, Dongshan pigs, and Ningxiang pigs have the MC1R*2 allele (Xu GL, Ren J, Ding NS, et al. Genetic analysis of the KIT and MC1R genes in Chinese indigenous pigs with belt-like coat color phenotypes [J]. Anim Genet. 2006; 37 (5): 518-519.). However, it has not yet been confirmed that all black / white hair pig breeds are caused by missense mutations in the MC1R gene.

[0004] The present invention performs GWAS through the LMM model of GEMMA software to screen out SNP molecular markers associated with black / white coat color. The correlation between the SNP molecular markers screened by the present invention and the black / white coat color of pigs reaches a significant level, providing a new molecular marker for gene-assisted selection of pig coat color. Summary of the invention

[0005] The purpose of the present invention is to provide a haplotype molecular marker related to the black / white coat color trait of pigs, obtain SNP typing data by aligning pig resequencing data to the pig reference genome, and use GWAS to screen SNPs related to pig coat color, thereby providing a SNP molecular marker combination related to pig black / white coat color based on whole genome resequencing screening.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The inventors integrated the self-sequencing resequencing data of Yunnan local pigs and the public resequencing data of the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ), including 366 black-haired pigs of 15 breeds and 265 white-haired pigs of 2 breeds, and aligned the resequencing data to the reference genome (genome version 11.1, Sscrofa11.1), obtained SNP typing, and screened out 4 SNP sites significantly associated with the black / white coat color trait of pigs through genome-wide association analysis. Referring to Ensembl, the nucleotide sequences of 50 bp upstream and downstream of each SNP site were obtained, as follows:

[0008] The nucleotide sequence of chr6:191762 is as shown in SEQ ID NO.1 or 2, the polymorphic site is located at the 51st position of the sequence, corresponding to the 191762nd position of chromosome 6 of the pig genome, and the polymorphic site is G or A;

[0009] The nucleotide sequence of chr6:195878 is as shown in SEQ ID NO.3 or 4, the polymorphic site is located at the 51st position of the sequence, and the polymorphic site corresponding to the 195878th position of chromosome 6 of the pig genome is C or T;

[0010] The nucleotide sequence of chr6:223834 is as shown in SEQ ID NO.5 or 6, the polymorphic site is located at the 51st position of the sequence, corresponding to the 223834th position of chromosome 6 of the pig genome, and the polymorphic site is A or C;

[0011] The nucleotide sequence of chr6:233254 is as shown in SEQ ID NO.7 or 8, the polymorphic site is located at the 51st position of the sequence, corresponding to the 233254th position of chromosome 6 of the pig genome, and the polymorphic site is A or G;

[0012] The reference genome version is Sus scrofa 11.1. The above four SNPs are completely linked to constitute haplotype molecular markers. Individuals with the homozygous genotype of haplotype ATCG have black coat color, and individuals with the homozygous genotype of haplotype GCAA have white coat color. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : The Manhattan plot provided in Example 2 of the present invention visualizes the results of the GWAS analysis.

[0014] Figure 2 : QQ graph provided by embodiment 2 of the present invention.

[0015] Figure 3 : Schematic diagram of SNP1-4 LD blocks and haplotype identification on chromosome 6 provided in Example 2 of the present invention.

[0016] Figure 4 : Schematic diagram of haplotype and haplotype combination frequency analysis consisting of 4 SNPs on chromosome 6 provided in Example 2 of the present invention; wherein A is the haplotype frequency analysis result, B is the all-black haplotype combination frequency analysis result, and C is the all-white haplotype combination frequency analysis result. DETAILED DESCRIPTION

[0017] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0018] Example 1 Genotyping Detection and Data Processing

[0019] (1) Sequencing and quality control

[0020] Ear samples of 185 black-haired Yunnan local pigs were collected and DNA was extracted. DNA quality was tested by DNA concentration meter and agarose gel electrophoresis. DNA sequencing library was constructed with qualified DNA. Whole genome resequencing was performed using DNBseq T10 platform (sequencing depth was 23.61×), and the raw data were filtered using the default parameters of Fastp v0.23.4 software. At the same time, the public resequencing data of 446 pigs obtained from the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ) were integrated, and a total of 631 pig resequencing data were obtained. BWA 0.7.17 software was used for alignment, the Sus scrofa 11.1 database was used as the reference genome (GCA_000003025.6), GATK v4.1.9.0 was used to separate SNPs and Indels, and the quality control filtering parameters were set as follows: QUAL < 30.0, QD < 2.0, MQ < 40.0, FS > 60.0, SOR > 3.0, MQRankSum < -12.5, and ReadPosRankSum < -8.0.

[0021] (2) Filtering and filling

[0022] PLINK v1.9 software was used to remove SNPs on sex chromosomes and unknown chromosomes, and the obtained SNPs were further filtered with the filtering parameters “-autosome-maf 0.05-geno 0.05”, and the Hardy-Weinberg equilibrium test was performed for single varieties with the parameter “-hwe 1e-6”. Beagle v5.2 default parameters were used for missing genotype filling, and finally 631 individuals and 9,220,408 SNPs were available for subsequent association analysis.

[0023] Example 2 Genome-wide association analysis of SNP molecular markers and black / white coat color traits in domestic pigs

[0024] (1) Phenotypic definition

[0025] According to the breed information corresponding to the individuals in the group, the coat color of each breed was counted by consulting the Chinese Livestock and Poultry Genetic Resources (Pig Genetic Resources) and related literature on pig genetic resources. The coat color was used as the phenotype, totaling 631 individuals of 17 breeds, including 2 white breeds (Large White Pig and Landrace Pig) with a total of 265 heads, and 15 black breeds (Bamei Pig, Debao Pig, Erhualian Pig, Enshi Black Pig, Neijiang Pig, Xiangxi Black Pig, Sichuan Tibetan Pig, Southern Anhui Black Pig, Southern Yunnan Small-eared Pig, Baoshan Pig, Mingguang Small-eared Pig, Gaoligong Mountain Pig, Saba Pig, Lijiang Pig, Diqing Tibetan Pig and other Chinese local pigs) with a total of 366 heads.

[0026] (2) Genome-wide association analysis

[0027] According to the SNP molecular markers selected after the above genotype filling, principal component analysis was performed using GCTA v1.94.1 software, with the white population as control and the black population as case, and genome-wide association analysis (GWAS) was performed using the LMM model in GEMMA v0.98.5 software to screen out SNP molecular markers that are significantly associated with the black / white coat color traits of pigs. The specific model is as follows:

[0028] y=Wα+Xβ+u+e;u~MVN n (0λτ -1 K); e~MVN n (0, τ -1 I n )

[0029] Where y represents the phenotypic value of the individual, α represents the fixed effect covariates including intercept and principal components, β is the SNP marker vector, μ is the random effect vector, and e represents the residual vector. W and X represent the association matrix of β and μ. MVN n represents n-dimensional multivariate normal distribution, τ- 1 is the residual variance 0, λ is the ratio between the two variance components, K is the kinship matrix, In is the identity matrix.

[0030] (3) Screening for SNP molecular markers associated with black / white coat color

[0031] The significance threshold was adjusted by Bonferroni, that is, P = 0.05 / 9220408. SNPs with a P value less than this were considered to be significantly associated with the trait. Based on the output results, the R language package CMplot was used to draw Manhattan plots and QQ plots ( Figure 1 and Figure 2 ). The SNPs associated with black / white coat color are concentrated on chromosomes 6 and 8, and are annotated to the MC1R gene and KIT gene. The MC1R gene is a key gene for regulating coat color, and the relationship between the MC1R gene and the white coat trait has not yet been clarified. Therefore, we focus on chromosome 6 and select four SNP molecular marker sites. The information of these sites is shown in Table 1.

[0032] Table 1 Candidate SNP loci for black / white coat color traits in domestic pigs identified based on GWAS

[0033]

[0034] (4) Linkage disequilibrium analysis and haplotype analysis

[0035] The population was typed for the screened SNP1-4, and linkage disequilibrium analysis was performed using the SHEsis online tool. The results showed that SNP1-4 were completely linked. Three haplotypes, H1 (GCAA), H2 (ATCG), and H3 (ATCA), were found in this experimental population, as shown in Table 2 and Figure 3 , 4 As shown. In the all-white population, the frequency of H1 haplotype is 100%; in the all-black population, the frequencies of H1, H2, and H3 haplotypes are 4.67%, 89.92%, and 5.41%; there are five haplotype combinations in the population, namely H1H1 (GCAA / GCAA), H1H2 (GCAA / ATCG), H2H2 (ATCG / ATCG), H2H3 (ATCG / ATCA), and H3H3 (ATCA / ATCA). Individuals with the H1H1 haplotype combination have white coat color, and individuals with the H2H2 haplotype combination have black coat color. The chi-square test of haplotype and coat color traits was performed using R, and the results showed that haplotype was significantly associated with coat color (χ 2 =1108.3, P < 2.2e-16), and further testing whether specific haplotypes were associated with coat color showed that haplotype H1 was significantly associated with all-white coat color (χ 2 =1104.4, P < 2.2e-16), haplotype H2 was significantly associated with all black coat color (χ 2=975.82, P < 2.2e-16), haplotype H3 was significantly associated with all black coat color (χ 2 =27.803,P=1.343e-07).

[0036] Table 2 Haplotype distribution and frequency of candidate SNP loci

[0037]

Claims

1. A haplotype molecular marker associated with the black / white coat color trait of pigs, characterized in that: The haplotype molecular marker consists of 4 SNP sites: SNP1 is located at position 191762 on chromosome 6, and the genotype of the polymorphic site is G or A; SNP2 is located at position 195878 on chromosome 6, and the genotype of the polymorphic site is C or T; SNP3 is located at position 223834 on chromosome 6, and the genotype of the polymorphic site is A or C; SNP4 is located at position 233254 on chromosome 6, and the genotype of the polymorphic site is A or G; The reference genome version was Sus scrofa 11.

1.

2. Use of the haplotype molecular marker described in claim 1 in the prediction or auxiliary selection of black / white coat color traits in pigs.

3. The use according to claim 2, characterized in that: The individuals with the homozygous genotype whose haplotype molecular marker is ATCG have black coat color, and the individuals with the homozygous genotype whose haplotype molecular marker is GCAA have white coat color.

4. The use according to claim 2 or 3, characterized in that: The pig breeds are Chinese local pigs, large white pigs and landrace pigs.

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