Haplotype molecular marker related to black / white coat color of pig and application thereof

Abstract

The application belongs to the technical field of pig molecular marker, and particularly relates to a haplotype molecular marker related to pig black / white hair color and application thereof. The application screens 4 SNP sites significantly related to the black / white hair color trait through whole genome high-depth sequencing in combination with resequencing data disclosed in the NCBI database, and based on whole genome association analysis. The 4 SNP sites are located on chromosome 6 and are completely linked, and the haplotype molecular marker composed of the 4 SNP sites can be used for pig black / white hair color assisted selection and breeding.

Description

Technical Field

[0001] The present 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 prominent 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 significant differences in coat color. Black and white coat colors are more common in pigs and are typical phenotypic characteristics of many 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 pig has a white coat and has the excellent characteristics of fast growth rate, high feed utilization rate, and high lean meat rate, but also has disadvantages 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 kinship judgment (Bai Xiaoqing, Wang Jinyong. Research progress on pig coat color inheritance [J]. Animal Science and Veterinary Medicine, 2004, 21(8):32-33.), and is also one of the main breeding traits.

[0003] Genome-wide association studies (GWAS) are a method for analyzing the overall association of common genetic variations across the entire genome. They can directly identify gene loci or markers that are closely associated with phenotypic variation and have specific functions. GWAS has become a common method for marker-assisted breeding. The causes of coat color are complex, and 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 the MC1R gene in different coat color pig breeds and found that the coat color of wild boar is regulated by the MC1R*1 allele, the dominant black color of Meishan pig is regulated by the MC1R*2 allele, the striped color of Hampshire pig is regulated by the MC1R*3 allele, and the red color of Duroc pig is regulated by the 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 pig breeds are caused by missense mutations in the MC1R gene.

[0004] The present invention uses the LMM model of GEMMA software to perform GWAS and 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 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 366 black pigs from 15 breeds and 265 white pigs from two breeds. The resequencing data were aligned to the reference genome (genome version 11.1, Sscrofa11.1) to obtain SNP typing. Through genome-wide association analysis, four SNP sites significantly associated with the black / white coat color trait of pigs were screened. The nucleotide sequences of the 50 bp upstream and downstream of each SNP site were obtained by referring to Ensembl, 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 position 51 of the sequence, corresponding to position 191762 of chromosome 6 of the porcine 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, and the polymorphic site is located at position 51 of the sequence, corresponding to the polymorphic site 195878 on chromosome 6 of the porcine genome, which 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 position 51 of the sequence, corresponding to position 223834 of chromosome 6 of the porcine 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 position 51 of the sequence, corresponding to position 233254 of chromosome 6 of the porcine 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 and constitute haplotype molecular markers. Individuals with the homozygous genotype of ATCG haplotype have black coat color, and individuals with the homozygous genotype of GCAA haplotype 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 the four 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 provided 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 are within the scope of the present invention.

[0018] Example 1 Genotyping Detection and Data Processing

[0019] (1) Sequencing and quality control

[0020] Ear samples were collected from 185 black Yunnan native pigs and DNA was extracted. DNA quality was tested using a DNA concentration meter and agarose gel electrophoresis. DNA sequencing libraries were constructed from qualified DNA. Whole-genome resequencing was performed using the DNBseq T10 platform (sequencing depth 23.61×). Raw data were filtered using Fastp v0.23.4 software with default parameters. Public resequencing data from 446 pigs obtained from the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ) were also integrated, resulting in a total of 631 pig resequencing data. BWA 0.7.17 software was used for alignment, the Sus scrofa 11.1 database was used as the reference genome (GCA_000003025.6), and GATK v4.1.9.0 was used to separate SNPs and Indels. 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. The resulting SNPs were further filtered with the filtering parameters “-autosome-maf 0.05-geno 0.05”, and the Hardy-Weinberg equilibrium test was performed for each variety with the parameter “-hwe 1e-6”. Missing genotypes were filled using the default parameters of Beagle v5.2. 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) Phenotype 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] Based on the SNP molecular markers selected after the above genotype filling, principal component analysis was performed using GCTA v1.94.1 software. With the white group as the control and the black group as the case, 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 trait 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 individual phenotypic value, α represents the fixed effect covariates including the 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 correlation matrix of β and μ. 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 using Bonferroni, i.e., 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 Manhattan plot and QQ plot were drawn using the R language package CMplot ( 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 regulating coat color, and the relationship between the MC1R gene and the white coat trait has not yet been clarified. Therefore, we focused on chromosome 6 and selected four SNP molecular marker sites. The information of these sites is shown in Table 1.

[0032] Table 1 Candidate SNP sites 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 SNPs 1-4, and linkage disequilibrium analysis was performed using the SHEsis online tool. The results showed that SNPs 1-4 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 the H1 haplotype was 100%; in the all-black population, the frequencies of the H1, H2, and H3 haplotypes were 4.67%, 89.92%, and 5.41%, respectively. There were five haplotype combinations in the population: H1H1 (GCAA / GCAA), H1H2 (GCAA / ATCG), H2H2 (ATCG / ATCG), H2H3 (ATCG / ATCA), and H3H3 (ATCA / ATCA). Individuals with the H1H1 haplotype combination had white coat color, while individuals with the H2H2 haplotype combination had black coat color. The chi-square test between haplotypes and coat color traits was performed using R, and the results showed that haplotypes were significantly associated with coat color (χ 2 =1108.3, P < 2.2e-16), and further testing of 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), and haplotype H2 was significantly associated with all-black coat color (χ 2=975.82, P < 2.2e-16), and 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 sites

[0037]

Claims

1. Application of haplotype molecular markers in the prediction or assisted selection of black / white coat color traits in 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 is Sus scrofa 11.1, and individuals with the homozygous genotype of the haplotype molecular marker ATCG have black coat color, and individuals with the homozygous genotype of the haplotype molecular marker GCAA have white coat color.

2. The use according to claim 1, characterized in that The pig breeds are 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, Large White pig and Landrace pig.

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