SNP (Single Nucleotide Polymorphism) molecular marker related to character of pig corrected to 100kg day-age and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to the character of the corrected 100 kg day age of a pig and application, major genes and functional mutation related to the corrected 100 kg day age of the pig are obtained through screening and are verified in a group, and a new molecular marker is provided for auxiliary selection and prediction improvement of the corrected 100 kg day age of the pig. The method has important significance on auxiliary screening of pigs.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biotechnology and molecular marker technology, and specifically relates to an SNP molecular marker related to the trait of corrected age at 100 kg in pigs and its application. Background Art

[0002] Pigs are important economic animals in livestock production. As one of the earliest domesticated animals by humans, domestic pigs originated from wild boars in East Asia (Sus scrofa). Since 10,000 years ago, pigs have been domesticated in many places in Eurasia (Groenen MA, Archibald AL, Uenishi H, Tuggle CK, Takeuchi Y, Rothschild MF, et al. Analyses of pig genomes provide insight into porcine demography and evolution. Nature. 2012; 491(7424): 393 - 8). The domestication of wild boars and subsequent intensive artificial selection have led to significant phenotypic changes in many traits of domestic pigs, including behavior, body composition, reproduction, and coat color (Rubin CJ, Megens HJ, Martinez Barrio A, Maqbool K, Sayyab S, Schwochow D, et al. Strong signatures of selection in the domestic pig genome. Proc Natl Acad Sci U S A. 2012; 109(48): 19529 - 36). Due to the independent domestication process, there are distinct germplasm characteristics between European pig breeds and Asian pig breeds. For example, European pig breeds have a higher lean meat percentage and larger body size, while Asian pig breeds have a higher fat content and the advantages of high litter size and early maturity.

[0003] The corrected age at 100 kg in pigs is an important target trait for measuring the growth rate and production performance of pigs (Wang K, Liu D, Hernandez - Sanchez J, et al. Genome Wide Association Analysis Reveals New Production Trait Genes in a Male Duroc Population. PLoS ONE, 2017, 10(9)). Therefore, exploring and discovering molecular markers related to the genetic mechanism of corrected age at 100 kg is helpful for better carrying out pig breeding research.

[0004] Genome-wide association study (GWAS), as an association analysis method, has the advantages of better effect and higher statistical power than linkage analysis in the related research of complex traits. It was first proposed by Risch in 1996 (Risch N, Merikangas K. The future of genetic studies of complex human diseases. [J]. Science, 1996, 273(3): 350-354.). GWAS is a powerful tool for detecting the association between genotyped (or imputed) common single nucleotide polymorphism markers and unknown causal variants through LD (linkage disequilibrium). Genome-wide association analysis has been widely applied to various studies, including various complex human diseases and important economic traits of livestock, etc. (Visscher P M, Brown M A, McCarthy M I, et al. Five years of GWAS discovery [J]. The American Journal of Human Genetics, 2012, 90(1): 7-24.).

[0005] Most genetic variations are located in non-coding regulatory regions, and correctly identifying their target genes requires reconstructing chromatin interactions of cis-regulatory elements in physiologically relevant cell or tissue types. BL-HiChIP (in situ bridge-linked Hi-C followed by chromatin immunoprecipitation) is a protein-centered chromatin conformation method. Compared with ChIA-PET, BL-HiChIP increases the yield of conformational information reading by more than 10-fold and reduces the input requirement by more than 100-fold (Mumbach MR, Rubin AJ, Flynn RA, Dai C, Khavari PA, Greenleaf WJ, et al. HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nature Methods. 2016;13(11):919-22.). It can be used to identify the interactions between enhancers associated with diseases and distal target genes (Mumbach MR, Satpathy AT, Boyle EA, Dai C, Gowen BG, Cho SW, et al. Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements. Nature Genetics. 2017;49(11):1602-12.), while linking regulatory elements to target genes and revealing the potential mechanisms of gene expression dysregulation in diseases (Song M, Yang X, Ren X, Maliskova L, Li B, Jones IR, et al. Mapping cis-regulatory chromatin contacts in neural cells links neuropsychiatric disorder risk variants to target genes. Nat Genet. 2019;51(8):1252-62.).

[0006] Therefore, new methods are needed to explore SNP molecular markers related to the 100-kg corrected age trait in pigs, providing valuable references for pig breeding. Summary of the Invention

[0007] The object of the present invention is to explore new SNP molecular markers related to the age at 100 kg corrected in pigs, and to provide valuable references for the research on the trait of the age at 100 kg corrected in pigs and pig breeding.

[0008] In view of this, the solution of the present invention is as follows:

[0009] In the first aspect of the present invention, new SNP molecular markers related to the age at 100 kg corrected in pigs are proposed, including SNP001 to SNP834, which respectively correspond to the following positions and mutations on the pig reference genome version Sscrofa11.1:

[0010]

[0011]

[0012]

[0013]

[0014]

[0015]

[0016] In the second aspect of the present invention, the application of the SNP molecular markers in the research related to the trait of the age at 100 kg corrected in pigs and pig breeding is proposed.

[0017] Furthermore, the research on the trait related to the age at 100 kg corrected in pigs is to identify the traits related to the age at 100 kg corrected in pigs by using the pig SNP molecular markers described in the first aspect.

[0018] Furthermore, the pig breeding is molecular marker-assisted breeding of pigs.

[0019] Furthermore, the SNP molecular markers are preferably at least one of SNP136, SNP143, SNP147, SNP150, SNP153, SNP155, SNP156, SNP159, SNP161, and SNP164.

[0020] In the third aspect of the present invention, a method for detecting the trait of the age at 100 kg corrected in pigs is proposed. The steps include detecting the nucleotide mutation types of the pig SNP molecular markers described in the first aspect on the pig chromosome; specifically, it can be achieved by designing detection primers for each SNP locus.

[0021] In the fourth aspect of the present invention, a method for genetic improvement of pigs is proposed. By determining the SNP molecular markers described in the first aspect of the breeding pigs in the core population of breeding pigs, and making corresponding selections according to one of the SNP molecular markers: selecting the genotype with a short corrected age at 100 kg on the chromosome of the reference genome Sscrofa11.1 version of the successive breeding pigs of breeding pigs, and eliminating the genotype with a long corrected age at 100 kg.

[0022] Further, the SNP molecular markers are at least one of SNP136, SNP143, SNP147, SNP150, SNP153, SNP155, SNP156, SNP159, SNP161, and SNP164.

[0023] Preferably, the breeding process is as follows:

[0024] For the molecular marker SNP136, select individuals with the CC or CA genotype at 159729800 bp on chromosome 1, and eliminate individuals with the AA genotype at this point;

[0025] For the molecular marker SNP143, select individuals with the TT or TG genotype at 160044355 bp on chromosome 1, and eliminate individuals with the GG genotype at this point;

[0026] For the molecular marker SNP147, select individuals with the TT genotype at 160347188 bp on chromosome 1, and eliminate individuals with the TC and CC genotypes at this point;

[0027] For the molecular marker SNP150, select individuals with the AA or CA genotype at 160614440 bp on chromosome 1, and eliminate individuals with the CC genotype at this point;

[0028] For the molecular marker SNP153, select individuals with the AA or GA genotype at 161037225 bp on chromosome 1, and eliminate individuals with the GG genotype at this point;

[0029] For the molecular marker SNP155, select individuals with the AA or AG genotype at 161282331 bp on chromosome 1, and eliminate individuals with the GG genotype at this point;

[0030] For the molecular marker SNP156, select individuals with the AA or AG genotype at 161355252 bp on chromosome 1, and eliminate individuals with the GG genotype at this point;

[0031] For the molecular marker SNP159, select the AA or AG at 161686878 bp on chromosome 1, and eliminate the GG genotype at this point;

[0032] For the molecular marker SNP161, select individuals with the GG or AG genotype at position 161987727 bp on chromosome 1, and eliminate individuals with the AA genotype at this position;

[0033] For the molecular marker SNP164, select individuals with the AA or GA genotype at position 162241690 bp on chromosome 1, and eliminate individuals with the AA genotype at this position.

[0034] Furthermore, the successive selection is carried out generation by generation to gradually increase the frequency of the dominant allele genotype at the SNP locus, thereby improving and enhancing the reproductive traits of the offspring pigs.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention has screened out the major genes and functional mutations (SNPs) related to the corrected age at 100 kg in pigs and verified them within the population, providing new molecular markers for the assisted selection and predictive improvement of the corrected age at 100 kg in pigs, which is of great significance for the assisted screening of pigs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic flow chart of the screening process of the SNP molecular marker related to the trait of corrected age at 100 kg in pigs according to the present invention.

[0038] Figure 2 It is a Manhattan plot of SNP loci significantly related to the corrected age at 100 kg in pigs at the whole genome level in Example 1.

[0039] Figure 3-12 They are successively box plots corresponding to the genotypes and phenotypes of 10 SNP loci, namely SNP136, SNP143, SNP147, SNP150, SNP153, SNP155, SNP156, SNP159, SNP161, and SNP164 in Example 2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] The technical solutions of the present invention will be clearly and completely described below in conjunction with the preferred embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0041] In one embodiment, by integrating H3K27ac BL-HiChIP and cis-regulatory element annotation, a three-dimensional enhancer-promoter interaction network across the pig genome was constructed. Major genes and functional mutations (SNPs) related to the corrected age at 100 kg in pigs were screened out and verified within the population, providing new molecular markers for the assisted selection and predictive improvement of the corrected age at 100 kg in pigs, which is of great significance for the assisted screening of pigs.

[0042] The applicant integrated the re-sequencing data of 66,161 pigs of 58 breeds publicly available in the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ) and the European Bioinformatics Institute (EMBL-EBI, https: / / www.ebi.ac.uk / ), and aligned the pig re-sequencing data to the pig reference genome (genome version 11.1, Sscrofa11.1) to obtain SNP genotyping data. A total of 834 SNP loci related to the trait of corrected age at 100 kg in pigs were screened out using genome-wide association study (GWAS), and their physical location information is shown in the following table.

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[0045]

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[0047]

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[0049] The above SNPs from SNP0011 to SNP834 reached a significant level of correlation with the trait of corrected age at 100 kg in pigs, providing new genetic resources for the research on the trait of corrected age at 100 kg in pigs.

[0050] In one embodiment, a method for screening SNP molecular markers related to pig growth traits is provided, and the method includes the following steps:

[0051] The applicant provides a method for screening SNP molecular markers related to the trait of corrected age at 100 kg in pigs, and the method includes the following steps:

[0052] 1) Integrate the resequencing data of 66,161 pigs of 58 breeds publicly available in the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ) and the European Bioinformatics Institute (EMBL-EBI, https: / / www.ebi.ac.uk / ), and align the pig resequencing data to the pig reference genome (genome version 11.1, Sscrofa11.1) to obtain SNP genotyping data;

[0053] 2) According to the GWAS study in the literature (Yang R, Guo X, Zhu D, Tan C, Bian C, Ren J, et al. Accelerated deciphering of the genetic architecture of agricultural economic traits in pigs using a low-coverage whole-genome sequencing strategy. Gigascience. 2021;10(7).), obtain the major QTL at the physical positions of 30,242,768 - 30,522,596 on porcine chromosome 7, and extract all 1,036 SNP sites within these QTLs for subsequent epigenetics screening.

[0054] 3) Construct a porcine whole-genome enhancer-promoter three-dimensional interaction network through H3K27ac BL-HiChIP, and screen the above SNP sites, only retaining SNPs that meet all of the following conditions simultaneously:

[0055] ① Located within the loop anchor of BL-HiChIP; ② Located within cis-regulatory elements (enhancer or promoter); ③ Located within chromatin open regions or transcription factor footprints; ④ The allele frequency difference between 4 commercial lean pig breeds (Large White, Landrace, Duroc, and Pietrain) and 6 Chinese indigenous pig breeds (Meishan, Bamei, Jinhua, Tongcheng, Rongchang, and Erhualian pigs) is greater than 0.5; ⑤ The SNP is predicted by the motifbreakR software to have a strong effect on transcription factor binding.

[0056] 4) According to the genotypes and phenotypic values of the population, verify the effects of the above-filtered SNP sites on the age at 100 kg, and detect whether there are differences in the age at 100 kg among pig populations with different alleles.

[0057] It is understandable that using the allele frequencies of multiple pig breeds greater than 0.5 as the screening criteria is to ensure that the population used has sufficient gene pool richness, and the SNP loci screened are meaningful. It is applicable to all breeds of pigs in the population used. The molecular markers obtained by screening can be applied to the genotype analysis of genes related to the age at 100 kg corrected in pigs or the association analysis of traits related to the age at 100 kg corrected in pigs for non-diagnostic purposes, providing new molecular marker resources for the molecular marker-assisted selection of the age at 100 kg corrected in pigs.

[0058] Screening of SNP Molecular Markers Related to the Trait of the Corrected 100-kg Body Weight Age in Pigs in Example 1

[0059] Integrate and download the resequencing data of 66,161 pigs of 58 breeds publicly available in the NCBI database (SRA, http: / / www.ncbi.nlm.nih.gov / sra / ) and the European Bioinformatics Institute (EMBL-EBI, https: / / www.ebi.ac.uk / ); use SRAToolkit (V2.8.2) to convert the original data into fastq files, and then use Trimmomatic (V0.36) software to perform preliminary quality control on the fastq files. After this step, use the Burrows-Wheeler Aligner 0.7.17 (BWA) software to align the remaining high-quality reads with the pig reference genome (version 11.1), and select the uniquely aligned sequences for genotyping. To obtain high-quality variant data, use GATK (V4.0.3.0) software to perform quality control through the 'QUAL < 30.0 || QD < 2.0 || FS > 60.0 || MQ < 40.0 || SOR > 4.0 || ReadPosRankSum < -8.0' command to obtain genotype data available for GWAS analysis.

[0060] Application of the Molecular Marker Genotyping Method in the Detection of the Trait of the Corrected 100-kg Body Weight Age in Pigs in Example 2

[0061] Association analysis of molecular markers with the age at 100 kg corrected trait: Used for the detection of the association analysis between genotype and the age at 100 kg corrected trait. Adopt the method of Mann-Whitney-U one-tailed test and use the scipy.stats module in the Python statistical environment for calculation.

[0062] Such as Figure 3-12As shown, the box plots of genotypes and phenotypes corresponding to the 10 SNP loci with the smallest P values in the order of chromosomal positions are SNP136, SNP143, SNP147, SNP150, SNP153, SNP155, SNP156, SNP159, SNP161, and SNP164. The figure legends are explained as follows: 0 represents wild-type homozygosity. 1 represents heterozygosity. 2 represents mutant homozygosity. The asterisk represents the degree of significant difference between the two genotypes. Figures 3 to 12 The SNP locus is a site mutation that has been verified to have a significant difference in the age at which pigs are corrected to 100 kg.

[0063] For the physical position chr1: 159729800, i.e., SNP136. Genotype 0: CC (wild type), genotype 1: CA (heterozygous mutant), genotype 2: AA (homozygous mutant), P value: 1.4×10 -48 . Figure 3 It shows that for individuals with the genotype CA or AA (mutant), their age at which they are corrected to 100 kg is higher than that of CC-type (wild type) individuals. Under the Mann-Whitney-U one-tailed test, the SNP136 marker reached the genome-wide significant level (P<0.05), indicating that this marker is not only significantly associated with the trait of the age at which pigs are corrected to 100 kg, but also when this marker mutates from C to A, it is not conducive to reducing the age at which pigs are corrected to 100 kg. Therefore, for the molecular marker SNP136, select CC-type or CA-type individuals at 159729800 bp on chromosome 1, and eliminate AA-type individuals at this point; preferably retain CC-type individuals.

[0064] For the physical position chr1: 160044355, genotype 0: TT (wild type), genotype 1: TG (heterozygous mutant), genotype 2: GG (homozygous mutant), P value: 1.3×10 -52 . From Figure 4 it can be seen that for the molecular marker SNP143, select TT-type or TG-type individuals at 160044355 bp on chromosome 1, and eliminate GG-type individuals at this point; preferably retain TT-type individuals.

[0065] From Figure 5 it can be seen that for the molecular marker SNP147, select TT-type individuals at 160347188 bp on chromosome 1, and eliminate TC-type and CC-type individuals at this point; preferably retain TT-type individuals.

[0066] From Figure 6 it can be seen that for the molecular marker SNP150, select AA-type or CA-type individuals at 160614440 bp on chromosome 1, and eliminate CC-type individuals at this point; preferably retain AA-type individuals.

[0067] It can be seen from Figure 7 that for the molecular marker SNP153, individuals with the AA or GA genotype at position 161037225 bp on chromosome 1 are selected, and individuals with the GG genotype at this point are eliminated.

[0068] It can be seen from Figure 8 that for the molecular marker SNP155, individuals with the AA or AG genotype at position 161282331 bp on chromosome 1 are selected, and individuals with the GG genotype at this point are eliminated.

[0069] It can be seen from Figure 9 that for the molecular marker SNP156, individuals with the AA or AG genotype at position 161355252 bp on chromosome 1 are selected, and individuals with the GG genotype at this point are eliminated.

[0070] It can be seen from Figure 10 that for the molecular marker SNP159, the AA or AG genotype at position 161686878 bp on chromosome 1 is selected, and individuals with the GG genotype at this point are eliminated.

[0071] It can be seen from Figure 11 that for the molecular marker SNP161, individuals with the GG or AG genotype at position 161987727 bp on chromosome 1 are selected, and individuals with the AA genotype at this point are eliminated.

[0072] It can be seen from Figure 12 that for the molecular marker SNP164, individuals with the AA or GA genotype at position 162241690 bp on chromosome 1 are selected, and individuals with the AA genotype at this point are eliminated.

[0073] By preferentially selecting the advantageous genotypes of the above SNP loci, the corrected age at 100 kg of pigs can be significantly shortened, the growth efficiency can be improved, and the purpose of ultimately improving the economic benefits of commercial pigs can be achieved.

[0074] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A SNP molecular marker associated with the trait of pigs corrected to 100 kg per day, characterized in that: The SNP sites of the SNP molecular markers include SNP001 to SNP834, which correspond to the following positions and mutations on the pig reference genome version Sscrofa11.1:

2. Application of the SNP molecular marker described in claim 1 in research related to pig corrected weight up to 100 kg and in pig breeding.

3. The use according to claim 2, characterized in that: The research related to the trait of pigs corrected to 100 kg age is to use the pig SNP molecular markers described in claim 1 to identify the main effect genes and functional mutations related to the trait of pigs corrected to 100 kg age.

4. The use according to claim 2, characterized in that: The pig breeding is molecular marker assisted breeding of pigs.

5. The use according to claim 2, characterized in that: The SNP molecular marker is at least one of SNP136, SNP143, SNP147, SNP150, SNP153, SNP155, SNP156, SNP159, SNP161, and SNP164.

6. A method for detecting the 100 kg corrected age trait of a pig, characterized in that: The steps include detecting the nucleotide mutation type of the pig SNP molecular marker described in claim 1 on the pig chromosome.

7. A method for genetic improvement of pigs, characterized in that: Determine the SNP molecular markers described in claim 1 of the breeding pigs in the breeding pig core group, and make corresponding selections based on one of the SNP molecular markers: correct the genotypes that are short in age up to 100 kg on the chromosomes of the reference genome Sscrofa11.1 version of the breeding pigs, and eliminate the genotypes that are corrected to be long in age up to 100 kg.

8. The use according to claim 7, characterized in that: The SNP molecular marker is at least one of SNP136, SNP143, SNP147, SNP150, SNP153, SNP155, SNP156, SNP159, SNP161, and SNP164.

9. The use according to claim 8, characterized in that: The breeding process is as follows: For molecular marker SNP136, CC or CA individuals at 159729800bp on chromosome 1 were selected, and AA individuals at this point were eliminated; For the molecular marker SNP143, select the TT or TG type individuals at 160044355bp on chromosome 1 and eliminate the GG type individuals at this point; For molecular marker SNP147, TT-type individuals at 160347188bp on chromosome 1 were selected, and TC-type and CC-type individuals at this point were eliminated; For the molecular marker SNP150, the AA or CA type individuals at 160614440 bp on chromosome 1 were selected, and the CC type individuals at this point were eliminated; For molecular marker SNP153, select AA or GA individuals at bp 161037225 of chromosome 1 and eliminate GG individuals at this point; For molecular marker SNP155, select AA or AG individuals at bp 161282331 of chromosome 1 and eliminate GG individuals at this point; For molecular marker SNP156, select AA or AG individuals at bp 161355252 of chromosome 1 and eliminate GG individuals at this point; For molecular marker SNP159, select AA or AG type at bp 161686878 of chromosome 1 and eliminate GG type individuals at this point; For the molecular marker SNP161, GG or AG individuals at bp 161987727 of chromosome 1 were selected, and AA individuals at this point were eliminated; For the molecular marker SNP164, AA or GA individuals at 162241690bp of chromosome 1 were selected, and AA individuals at this point were eliminated.

10. The use according to claim 7, characterized in that: The successive breeding is carried out generation by generation to gradually increase the frequency of the dominant allele type of the SNP locus, thereby improving the correction 100kg day-old trait of the offspring pigs.

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