WO2018218857A1 - 改善猪肉质的myh4基因分子标记及在猪遗传改良中的应用 - Google Patents

改善猪肉质的myh4基因分子标记及在猪遗传改良中的应用 Download PDF

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WO2018218857A1
WO2018218857A1 PCT/CN2017/107606 CN2017107606W WO2018218857A1 WO 2018218857 A1 WO2018218857 A1 WO 2018218857A1 CN 2017107606 W CN2017107606 W CN 2017107606W WO 2018218857 A1 WO2018218857 A1 WO 2018218857A1
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pig
acid sequence
genotype
seq
nucleic acid
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黄路生
陈从英
熊信威
方绍明
宿英
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江西农业大学
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
    • A01K2217/056—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to mutation of coding region of the transgene (dominant negative)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • A01K2267/02—Animal zootechnically ameliorated
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the patent application requires application number CN201710406485.8, and the application date is June 2, 2017.
  • the invention name is the priority of the MYH4 gene molecular marker for improving pork quality and its application in pig genetic improvement.
  • the present application relates to a SNP marker for determining and/or genetically improving pork quality traits.
  • the "snow flesh" formed by the high intramuscular fat content is a fine meat.
  • the intramuscular fat content is closely related to the flavor and edible value of pork, which affects the tenderness and juiciness of pork.
  • Applicants used the large-scale Laiwu pig population to determine the intramuscular fat content of the longissimus dorsi of 316 individuals in all blood rims of Laiwu pigs.
  • the genome-wide association (GWAS) analysis detected the effect of chromosome 12 on the intramuscular fat content of pigs.
  • the main gene locus (QTL) the initial location of the interval is 650kb.
  • QTL interval was reduced to 525.99 kb using haplotype analysis and LDLA analysis.
  • the region was re-sequenced, the search and identification of polymorphic loci and its correlation with intramuscular fat content in pigs, gene expression analysis and eQTL mapping, and verification analysis of multiple populations were used to separate pork that affects intramuscular fat content.
  • the trait gene MYH4 is used to establish efficient and accurate gene breeding technology for the selection of pork traits such as intramuscular fat content.
  • one of the applications provides a SNP marker for a pig, the SNP marker comprising at least one of the following SNP markers:
  • nucleotide sequence I which is nucleotide Y at position 11031 from the 5' end of SEQ ID No. 1, said Y being selected from C or T; SEQ ID No. The 11031th position from the 5' end on 1 corresponds to the 58244116 site from the 5' end on chromosome 12 of the 10.2 version of the international pig genome;
  • SNP marker on nucleotide sequence II which is nucleotide Y at position 1780 from the 5' end on SEQ ID No. 2, said Y being selected from C or T; SEQ ID No. The 1780th position from the 5' end on 2 corresponds to the 58244116 site from the 5' end on chromosome 12 of the 10.2 version of the international pig genome;
  • the SNP marker at position 11031 from the 5' end of the nucleotide sequence is identical; wherein the nucleotide sequence III can be translated in the pig as shown in SEQ ID No. 3. Amino acid sequence having the same functional protein;
  • a SNP marker on nucleotide sequence VI which is a SNP marker with a degree of linkage disequilibrium r 2 ⁇ 0.8 with at least one of the SNPs of (I) to (III).
  • the above nucleotide sequence I is shown in SEQ ID No. 1; the above nucleotide sequence II is shown in SEQ ID No. 2.
  • the second application of the present application provides a nucleic acid sequence which is a nucleic acid sequence comprising a SNP marker as described in one of the claims, the nucleic acid sequence being selected from at least one of a DNA sequence, a cDNA sequence and an RNA sequence. .
  • the nucleic acid sequence is located on chromosome 12 of the 10.2 version of the International Pig Genome.
  • the nucleic acid sequence includes the SNP marker, regardless of its length, for example, its length may be 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 15 bp, 20 bp, 30 bp, 50 bp, 80 bp, 100 bp, 120 bp, 150 bp, 180 bp, 200 bp, 250 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 1000 bp, 1200 bp, 1500 bp, 2000 bp, etc., all of which are nucleic acid sequences claimed herein, but the nucleosides The acid sequence is not limited to the recited lengths.
  • the SNP marker is generally located at a central position or relatively close to the center of the selected nucleic acid sequence, for example, in a selected fragment of 20 bp, the SNP marker is generally in the 20 bp DNA fragment.
  • the SNP marker it is also possible to approach one of the two ends of the selected nucleic acid sequence, even at the first or last position.
  • the nucleic acid sequence has from 5 bp to 26560 bp.
  • the nucleic acid sequence may include all nucleotide sequences as shown in SEQ ID No. 1.
  • the nucleic acid sequence has from 5 bp to 10,000 bp.
  • the nucleic acid sequence may be a partial nucleotide sequence as shown in SEQ ID No. 1.
  • the nucleic acid sequence may also be a partial nucleotide sequence as shown in SEQ ID No. 2.
  • the nucleic acid sequence has from 5 bp to 5928 bp.
  • the nucleic acid sequence may be a partial nucleotide sequence as shown in SEQ ID No. 1.
  • the nucleic acid sequence may also be a partial nucleotide sequence as shown in SEQ ID No. 2.
  • nucleic acid sequence is selected from the group consisting of SEQ ID No. 1 and/or SEQ ID No. 2.
  • the nucleic acid sequence has from 5 bp to 1000 bp.
  • the nucleic acid sequence may be a partial nucleotide sequence as shown in SEQ ID No. 1.
  • the nucleic acid sequence may also be a partial nucleotide sequence as shown in SEQ ID No. 2.
  • the nucleic acid sequence has from 5 bp to 500 bp.
  • the nucleic acid sequence may be a partial nucleotide sequence as shown in SEQ ID No. 1.
  • the nucleic acid sequence may also be a partial nucleotide sequence as shown in SEQ ID No. 2.
  • the nucleic acid sequence has from 5 bp to 300 bp.
  • the nucleic acid sequence may be a partial nucleotide sequence as shown in SEQ ID No. 1.
  • the nucleic acid sequence may also be a partial nucleotide sequence as shown in SEQ ID No. 2.
  • the third application of the present application provides an amino acid sequence encoded by a nucleic acid sequence comprising a SNP marker as described in one of the present application, an amino acid X corresponding to the SNP marker, the X being selected from the group consisting of alanine Acid or proline; and when the SNP is labeled T, its corresponding amino acid is valine; when the SNP is labeled C, its corresponding amino acid is alanine; preferably, the nucleic acid sequence is capable of A nucleic acid sequence having the same functional protein as the amino acid sequence shown in SEQ ID No. 3 is translated in the pig; preferably, the amino acid sequence is SEQ ID No. 3; wherein, as shown in SEQ ID No. 3 The amino acid sequence, amino acid X, is located at position 576.
  • the fourth application of the present invention provides a SNP marker according to any one of the present application, a nucleic acid sequence according to the second application of the present application, and at least one of the amino acid sequences according to the third embodiment of the present invention for determining and/or genetically improving pork quality.
  • the pork quality trait includes at least one of intramuscular fat content, marbling, muscle redness, muscle yellowness, muscle brightness, muscle fiber type, and moisture content.
  • the accuracy of 80% or more can also be achieved by using at least one of the molecular markers of one of the applications (VI) to detect the quality of the pork traits, and these molecular markers are all causal mutation sites with, for example, (I). related.
  • the genetically improved pork trait pig is selected from the group consisting of Laiwu pig, Erhualian, Meishan pig, Luchuan pig, folk pig, rice pig, Huai pig, Hetao big ear pig, Yushan black pig, Minnan small ear pig. At least one of horse body pig, Vietnamese pig, eight eyebrow pig and blue pond pig.
  • a fifth aspect of the present invention provides a method for genetic improvement of a pig, the method comprising: determining a SNP marker of a breeding pig in a core population of a breeding pig as described in one of the present applications, and making a corresponding selection based on the SNP marker:
  • the breeding pig in the porcine core population selected from the 5' end of the SEQ ID No. 1 to the TT and TC genotypes is selected, and the elimination is CC at the site.
  • the genotype of the individual pig breeder increases the frequency of the allele T at the site one by one; preferably, the pig of the TT genotype at position 11031 from the 5' end on the SEQ ID No. 1 is Elimination of individual pigs at the locus of TC and CC genotypes, increasing the frequency of allele T at this locus from generation to generation;
  • the breeding pigs at the 1780th position from the 5' end on the SEQ ID No. 2 are selected as the TT and TC genotypes in the porcine core population, and the elimination is CC at the site.
  • the genotype of the individual pig breeder increases the frequency of the allele T at the site by generation; preferably, the 1780 locus from the 5' end on the SEQ ID No. 2 is a TT genotype of the breeding pig individual, Elimination of individual pigs at the locus of TC and CC genotypes, increasing the frequency of allele T at this locus from generation to generation;
  • a pig individual in which the SNP marker site on the nucleotide sequence III is a TT and TC genotype is selected among the pig core population, and the breeding pig individual at the site is a CC genotype, Increasing the frequency of the allele T at this site by generation; preferably, the SNP marker site on the nucleotide sequence III is a porcine individual of the TT genotype, and the TC and CC genotypes are eliminated at this site. Breeding individual, increasing the frequency of allele T at this locus from generation to generation;
  • a pig individual having a SNP marker site on the nucleotide sequence VI and a homologous genotype having at least one of the foregoing (I) to (III) is selected among the pig core populations, and eliminated.
  • the nucleic acid sequence of the breeding pig is analyzed to determine a SNP marker of the breeding pig, wherein the nucleic acid sequence is selected from the nucleic acid sequence of claim 2; Or determining the SNP marker of one of the breeding pigs of the breeding pig by analyzing the amino acid sequence of the breeding pig, wherein the amino acid sequence is selected from the amino acid sequence as described in the third of the present application.
  • the SNP marker in a nucleic acid sequence amplified by PCR can be determined by high fidelity PCR amplification, and subsequent sequencing analysis.
  • the primer pair used may be named SNP-F as shown in SEQ ID No.
  • the sixth application of the present application provides a method for determining the quality of a pork quality trait, the method comprising: determining a SNP marker of the pig according to one of the applications, and determining the pork quality trait according to the SNP marker :
  • the pork quality traits are superior to poor, and the genotypes of the 11031th position from the 5' end in the SEQ ID No. 1 are: TT genotype, TC genotype, and CC. genotype;
  • the pork quality traits are superior to poor, and the genotypes of the 1780th position from the 5' end in the SEQ ID No. 2 are: TT genotype, TC genotype, and CC. genotype;
  • the pork quality trait is superior to inferior, and the SNP genotype ranking on the nucleotide sequence III is: TT genotype, TC genotype and CC genotype;
  • the pork quality trait is superior or inferior, and the SNP genotype ranking on the nucleotide sequence VI is consistent with at least one of the (I) to (III) genotypes;
  • the pork quality trait includes at least one of intramuscular fat content, marble streak, muscle redness, muscle yellowness, muscle brightness, muscle fiber type, and moisture content, and the intramuscular fat content is superior, marbled Those with higher scores are better, those with higher muscle redness are better, those with higher muscle yellowness are better, those with higher muscle brightness values are better; those with more type I and IIa muscle fibers are better; The lower content is superior.
  • the nucleic acid sequence of the pig is analyzed to determine the SNP marker of the pig according to one of the applications, wherein the nucleic acid sequence is selected from the nucleic acid according to the second application of the present application. sequence.
  • a seventh aspect of the present invention provides a method for establishing a new pig line and/or a new pig variety for improving pork quality, comprising the steps of: for a pig having a SNP-labeled genotype of CC or TC as described in one of the applications, The CC genotype or TC genotype was mutated to the TT genotype by site-directed mutagenesis.
  • nucleotide C at position 11031 from the 5' end of SEQ ID No. 1 is mutated to T; for (II), from 5 at SEQ ID No. 2
  • the nucleotide C of the 1780th position of the terminus is mutated to T; for (III), the nucleotide C of the SNP marker site on nucleotide sequence III is mutated to T; for (VI), the nucleus is
  • the nucleotide mutation of the SNP tag site on the nucleotide sequence VI is a nucleotide which is identical to the mutation of at least one nucleotide of the above (I) to (III).
  • the mutation is carried out by means of a transgenic method or a method of gene editing.
  • mutation is performed using the CRISPR/Cas9 gene editing method.
  • the pig new line and/or the new pig variety of the target pork quality trait are obtained by cloning by performing a site-directed mutagenesis on the somatic cells of the pig.
  • commonly used methods for detecting SNP markers include: 1) based on a hybridization method, which is at least divided into a) a method using a ⁇ Tm method, b) a hybridization plus a fluorescent probe, and 2) an enzyme-based method, which is at least divided into a) DNA polymerase method, for example, PCR amplification using DNA polymerase, b) ligase method, c) restriction endonuclease method, such as searching for restriction enzyme sites around the causal mutation site for enzymatic cleavage, Thus, the causal mutation site genotype is obtained; d) exonuclease FEN method, e) RNase H method; 3) electrophoresis method, which is at least divided into SSCP single-strand conformation polymorphism and DGGE/TGGE denaturing gradient gel electrophoresis; Direct sequencing method. These methods can all be used to detect the SNP markers of the present application.
  • Pork quality traits are important economic traits, and intramuscular fat traits are important indicators of meat traits.
  • the causal mutation sites identified in this study that affect the intramuscular fat content can be directly applied to the genetic improvement and development of the meat quality traits of Chinese local pig breeds containing the causal mutation site.
  • genetic editing techniques can be used to construct new strains to solve the current situation of poor pork quality.
  • Figure 1 shows a genome-wide association (GWAS) analysis of the intramuscular fat content of Laiwu pigs; where: the abscissa indicates the chromosome number of the pig and the ordinate indicates the -logP value.
  • GWAS genome-wide association
  • Figure 2 shows the QTL fine positioning and haplotype sharing analysis of Laiwu pigs
  • the confidence interval contains two distinct haplotypes, the shared haplotype containing 8 SNPs, a total of 128.15 kb;
  • haplotype 1 The IMF content of haplotype 1 is extremely significantly higher than that of haplotype 2, indicating that haplotype 1 corresponds to Q;
  • Figure 3 shows the results of QTT and eQTL mapping of Laiwu pigs
  • T represents high IMF content
  • MYH4 gene expression was significantly associated with IMF content.
  • the X axis represents the expression level of MYH4, and the Y axis represents the IMF content;
  • (D) C represents a high expression level of MYH4 gene, and the Y axis represents the amount of MYH4 expression after correction of sex and batch.
  • Figure 4 shows the analysis of the longissimus muscle fiber types of different genotype individuals in Laiwu pigs.
  • the chimeric family in the present application refers to an Asian (Chinese) breeding pig (Bama Xiang pig, which utilizes four phenotypic differences in European and American pig breeds (Pitland, Duroc, Changbai and Dabai) and four phenotypes).
  • Laiwu pigs, Vietnamese pigs and Erhualian pigs have a chimeric population of domestic pigs produced by multiple generations of random mating.
  • the term ">" indicating a mutation refers to the mutual change of two single nucleotides before and after, for example, A>C or C>A means that A and C are mutually mutated at the position.
  • the above-mentioned PCR amplification was used to identify the g.58244116C>T mutation of MYH4, and the favorable allele genotype was selected for the individual to keep the seed, and the population was selected to improve the intramuscular fat content, marbled, muscle. The redness, yellowness and brightness reduce the moisture content to improve the meat quality.
  • the experimental pig population used in this application is Laiwu pig.
  • Laiwu pig is a local pig breed in China, and its origin is Laiwu City, Shandong province.
  • 316 progeny obtained by mating 12 Laiwu boars and 45 Laiwu sows were used as experimental animals, and slaughtering was carried out at 300 ⁇ 5 days old.
  • NPPC National Pig Production Association
  • a small piece of ear sample was collected from each of the above-mentioned Laiwu pigs, and the whole genome DNA was extracted by standard phenol-chloroform method. The concentration and quality were measured by Nanodrop-ND1000 spectrophotometer and uniformly diluted to 50 ng/ ⁇ l on the Illumina Beadstation platform according to the standard. The process was performed on a pig genome-wide 60K SNP chip (Illumina, USA) genotype determination.
  • the detection rate is less than 90%
  • the family Mendel error rate is higher than 0.1
  • the minimum allele frequency is less than 0.01
  • Hardy- Weinberg's equilibrium significance level is higher than the 10 -6 SNP
  • this application uses a linear mixed model single point regression analysis and combined with the GenABEL software package in the R program for GWAS analysis, which uses the similarity correction stratification effect of the inter-individual genome.
  • Significant levels of the genome were determined using the conservative Bonferroni correction method, ie the genomic significant level threshold was 1.01 x 10 -6 (0.05/49452).
  • the results of the GWAS analysis are shown in Figure 1.
  • the genome assembly version 10.2, referred to as the reference sequence is at 57.83 Mb of chromosome 12.
  • LOD the logarithm of the likelihood function ratio
  • the QTL confidence interval of the Laiwu pig population was determined to be 57.83-58.48 Mb, which is the main effect of affecting intramuscular fat content on pig chromosome 12 (SSC12).
  • the gene is located within this 650 kb long chromosomal region.
  • the applicant compared the DNA sequence obtained by re-sequencing 6 Laiwu pigs with the reference sequence, and selected 127 SNPs in the QTL region for mass spectrometry, so that the label density in the region reached 1 per 10Kb. Judging SNPs.
  • the obtained mass spectrometry results were subjected to quality control and combined with 60K quality control data for subsequent analysis.
  • this application conducted a haplotype sharing analysis in 316 Laiwu pigs.
  • the results are shown in Figure 2B, and the results indicate that most individuals share one of the two haplotypes.
  • the haplotype region has a size of 128.15 Kb and contains 8 SNPs.
  • the results are shown in Figure 2C, haplotype 1, haplotype 2, and all 316 Laiwu pigs.
  • the mean IMF phenotype values were: 11.80% ⁇ 0.24%, 7.01% ⁇ 0.30%, and 8.74% ⁇ 0.36%, respectively.
  • Resequencing 1 using L001-FP and L001-RP primer pairs the size of the fragment obtained by sequencing was 10259 bp; resequencing 2 using L003-FP and L003-RP primer pairs, the size of the fragment obtained by sequencing was 7947 bp; resequencing 3 using L004- FP and L004-RP primer pairs, the size of the fragment obtained by sequencing was 11606 bp; resequencing 4 using L006-FP and L006-RP primer pairs, the size of the fragment obtained by sequencing was 11898 bp; resequencing 5 using L011-FP and L011-RP primers The fragment size obtained by sequencing was 9824 bp; the re-sequencing 6 used L013-FP and L013-RP primer pair, the fragment size obtained by sequencing was 9847 bp; the re-sequencing 7 used L014-FP and L014-RP primer pair, and the fragment obtained by sequencing was obtained.
  • the size is 13314 bp; resequencing 8 uses L016-FP and L016-RP primer pairs, The size of the fragment obtained by sequencing was 9993 bp; the re-sequencing 9 used L017-FP and L017-RP primer pair, the size of the fragment obtained by sequencing was 10776 bp; the re-sequencing 10 used L019-FP and L019-RP primer pair, and the size of the fragment obtained by sequencing was 10337 bp; resequencing 11 using L020-FP and L020-RP primer pairs, the size of the fragment obtained by sequencing was 10370 bp; resequencing 12 using L021-FP and L021-RP primer pairs, the size of the fragment obtained by sequencing was 10367 bp; L023-FP and L023-RP primer pairs, the size of the fragment obtained by sequencing was 9702 bp; re-sequencing 14 using L024-FP and L024-RP primer pairs,
  • Re-sequencing 46 using L084-FP and L084-RP primer pairs the size of the fragment obtained by sequencing was 10053 bp; resequencing 47 using L086-FP and L086-RP primer pairs, the size of the fragment obtained by sequencing was 8675 bp; re-sequencing 48 using L087 -FP and L087-RP primer pairs, the size of the fragment obtained by sequencing was 6329 bp; re-sequencing 49 using L088-FP and L088-RP primer pairs, the size of the fragment obtained by sequencing was 10548 bp; re-sequencing 50 using L089-FP and L089-RP Primer pair, the size of the fragment obtained by sequencing is 11651 bp; resequencing 51 using L090-FP and L090-RP primer pairs, the size of the fragment obtained by sequencing was 5421 bp; resequencing 52 using L091-FP and L091-RP primer pairs, the size of the
  • PCR polymerase chain reaction
  • the PCR amplification conditions were: 94 ° C for 2 min; 98 ° C for 10 s, 68 ° C for 8 min, 30 cycles; and finally at 72 ° C for 10 min. After the PCR amplification products were uniformly mixed, they were entrusted to Novo Zhiyuan Company for sequencing and de nove assembly.
  • the untested sequences were sequenced by sanger, and 131 SNPs that fully met the QTL genotype were finally analyzed.
  • 131 SNPs 7 SNPs have been typed in the first stage of fine mapping of this application.
  • the SNP with the strongest intramuscular fat content (g.58244116C>T) was also found to be the strongest SNP affecting MYH4 gene expression. Based on the above results, the Applicant determined that the g.58244116C>T site is a causal mutation site that affects the intramuscular fat content of pigs.
  • g.58244116C>T is a causal mutation site
  • the laboratory collected 419 Lule pigs and 821 heads.
  • the muscle samples of the F6 group of the family and the 77 Yushan black pig ⁇ Duroc group were subjected to DNA extraction and determination of the intramuscular fat phenotype according to the above method.
  • the applicant further analyzed the effect of the g.58244116C>T locus on other indicators of Laiwu pigs.
  • the results are shown in Table 2.
  • the marble of the longest muscle of the TT type was found, and the redness, yellowness and brightness of the muscle were higher than those of the CC type, and the moisture content of the muscle was lowered.
  • the T (ie, Q) allele of g.58244116C>T has a significant effect on marble, muscle redness, yellowness, brightness and moisture content, but does not affect fat deposition and growth. Traits.
  • the phenotype data is calculated using the lsmeans method.
  • the experimental animals were used to use the Lule pig, the chimeric family F6 and the Yushan black pig ⁇ Duroc group.
  • 419 Lule pigs, 821 chimeric families F6 and 77 Yushan black pigs ⁇ Duroc pigs were collected and the intramuscular fat content of each individual was determined.
  • the genotypes of g.58244116C>T locus were determined by PCR amplification on 419 Lule pigs, 821 chimeric families F6 and 77 Yushan black pigs ⁇ Duroc pigs.
  • GenABEL software was then used to analyze the effect of genotype on phenotype.
  • MYH4 gene and its causal mutation site g.58244116C>T affect the longissimus dorsi muscle fiber type
  • g.58244116C>T not only increases intramuscular fat content but also affects muscle fiber type, which results in a significant reduction in type IIB muscle fibers in the longissimus dorsi muscle, while type I muscle fibers and type IIA muscle fibers are significantly increased.
  • Applicants used the LD heatmap software package in the R language to calculate the degree of linkage disequilibrium (r 2 ) between different markers in the Laiwu pig population.
  • r 2 ⁇ 0.8 as a threshold, a SNP marker around the mutation site g.58244116C>T of the MYH4 gene was obtained.
  • GWAS analysis confirmed that all molecular markers with r 2 ⁇ 0.8 were related to the intramuscular fat content reaching the genomic significant level, and their degree of phenotypic correlation depends on their gene causal mutation site g.58244116C>T The degree of tight linkage. From this, it can be seen that a molecular marker having a degree of linkage with the mutation site g.58244116C>T of MYH4, r 2 ⁇ 0.8, can also be used as an effective marker for meaty breeding.
  • the molecular marker of the causal mutation site g.58244116C>T of the major coding gene MYH4 is closely linked to the degree r 2 ⁇ 0.8 (that is, the molecular marker tends to be co-segregated in population genetics), both with marbled, muscular meat quality traits significant association redness, yellowness, brightness, and moisture content, and therefore all the MYH4 MYH4 causal mutation g.58244116C> T r site closely linked molecular markers 2 ⁇ 0.8 pig meat quality traits can be used as genetic improvement Molecular markers should be within the scope of this patent protection.

Abstract

提供了改善猪肉质的MYH4基因分子标记及其在猪遗传改良中的应用，所述分子标记包括如下至少一种：(I)SEQ ID No.1上的从5'端起的第11031位点的核苷酸Y；(II)SEQ ID No.2上的从5'端起的第1780位点的核苷酸Y，(III)与SEQ ID No.1和/或SEQ ID No.2具有90％以上的一致性的核苷酸序列III上的SNP标记，与SEQ ID No.1上的从5'端起的第11031位点的SNP标记具有一致性；其中，所述核苷酸序列III能够在所述猪中翻译出与如SEQ ID No.3具有相同功能蛋白质；(VI)与(I)至(III)中的至少一种中SNP的连锁不平衡程度r 2≥0.8的SNP标记；所述Y选自C或T。

Description

改善猪肉质的MYH4基因分子标记及在猪遗传改良中的应用
该专利申请要求申请号为CN201710406485.8，申请日为2017年6月2日，发明名称为改善猪肉质的MYH4基因分子标记及在猪遗传改良中的应用的优先权。
技术领域
本申请涉及一种用于测定和/或遗传改良猪肉质性状的SNP标记。
背景技术
随着人们生活水平的不断提高，对肉品质的要求越来越高。由于高肌内脂肪含量而形成的“雪花肉”是肉中精品。肌内脂肪含量与猪肉的风味和食用价值密切相关，它影响猪肉的嫩度和多汁性。肌内脂肪含量越高，肉的多汁性、嫩度、香味及总体可接受性越高。因此，研究猪高肌内脂肪含量形成的遗传学基础，对改善猪肉品质及生产有利于人体健康的优质肉具有重要意义。
发明内容
申请人利用大规模莱芜猪群体，测定了覆盖莱芜猪全部血缘316头个体的背最长肌肌内脂肪含量，通过全基因组关联(GWAS)分析检测到了12号染色体上影响猪肌内脂肪含量的主效基因位点(QTL)，初步定位的区间大小为650kb。通过增加QTL区间内的SNP标记密度，利用单倍型分析和LDLA分析，将QTL区间缩小到525.99kb。对该区域开展重测序，多态位点的搜寻、鉴别及其与猪肌内脂肪含量相关性研究，基因表达分析及eQTL定位，多个群体的验证分析等分离到影响肌内脂肪含量等猪肉质性状的因果基因MYH4，以建立高效准确的基因育种技术开展猪肌内脂肪含量等猪肉质性状的选育工作。
因此，本申请之一提供了一种猪的SNP标记，所述SNP标记包括如下SNP标记中的至少一种：
(I)核苷酸序列I上的SNP标记，为SEQ ID No.1上的从5’端起的第11031位点的核苷酸Y，所述Y选自C或T；SEQ ID No.1上的从5’端起的第11031位点对应于10.2版本国际猪基因组的12号染色体上的从5’端起的第58244116位点；
(II)核苷酸序列II上的SNP标记，为SEQ ID No.2上的从5’端起的第1780位点的核苷酸Y，所述Y选自C或T；SEQ ID No.2上的从5’端起的第1780位点对应于10.2版本国际猪基因组的12号染色体上的从5’端起的第58244116位点；
(III)与SEQ ID No.1和/或SEQ ID No.2所示的核苷酸序列具有90％以上的一致性的核苷酸序列III上的SNP标记，与SEQ ID No.1所示的核苷酸序列上的从5’端起的第11031位点的SNP标记具有一致性；其中，所述核苷酸序列III能够在所述猪中翻译出与如SEQ ID No.3所示的氨基酸序列具有相同功能蛋白质；
(VI)核苷酸序列VI上的SNP标记，其为与(I)至(III)中的至少一种SNP的连锁不平衡程度r2≥0.8的SNP标记。
上述核苷酸序列I如SEQ ID No.1所示；上述核苷酸序列II如SEQ ID No.2所示。
对于上述(III)的情况，本领域技术人员容易知道，生物体内的DNA序列具有自然突变或诱发突变的特点，因此，不同的猪品系、不同的猪品种，甚至是不同的猪个体内的DNA序列可能并不完全相同，而可能存在碱基颠换、转换、移码、缺失和插入的情况。当在其他猪品系、猪品种或猪个体中的对应于10.2版本国际猪基因组的12号染色体上MYH4基因中的SNP标记位点以外的碱基发生碱基颠换和/或转换时，不会影响所述SNP标记的位置；但是当在其他猪品系、猪品种或猪个体中的对应于10.2版本国际猪基因组的12号染色体上MYH4基因中的SNP标记位点以外的碱基发生移码、缺失和插入中的至少一种情况时，就可能导致所述SNP标记的位置，此时，可通过与所述SNP标记位点相邻的一个或多个碱基序列来确定所述SNP标记在这种情况下所处的位置。
本申请之二提供了一种核酸序列，所述核酸序列为包含如本申请之一所述的SNP标记的核酸序列，所述核酸序列选自DNA序列、cDNA序列和RNA序列中的至少一种。
所述核酸序列位于10.2版本国际猪基因组的12号染色体上。举例来说，所述核酸序列只要包括所述的SNP标记，不论其长度是多少bp，例如其长度可以是5bp、6bp、7bp、8bp、9bp、10bp、15bp、20bp、30bp、50bp、80bp、100bp、120bp、150bp、180bp、200bp、250bp、300bp、400bp、500bp、600bp、700bp、800bp、1000bp、1200bp、1500bp、2000bp等等，均是本申请所要求保护的核酸序列，但所述核苷酸序列并不限于所列举的长度。另外，所述SNP标记一般位于所选定的所述的核酸序列的中心位置或比较靠近中心的位置，例如，在20bp的选定片段中，所述SNP标记一般在这个20bp的DNA片段中的第7-14位中的一个位置；在1500bp的DNA片段中，所述SNP标记所处的位置的选择余地大大增大，其可以是在第100-1400位中的一个位置，优选在300-1200位中的一个位置，更优选在500-700位中的一个位置，这样设计的目的是有利于更加准确的检测所述的SNP标记；但是，在检测技术特别灵敏和/或特异性非常强的情况下，所述SNP标记也可以靠近所选定的核酸序列的两端中的一端，甚至是位于第一位或者末位。
在一个具体实施方式中，所述核酸序列具有5bp至26560bp。此时，所述核酸序列可以包括如SEQ ID No.1所示的全部核苷酸序列。
在一个具体实施方式中，所述核酸序列具有5bp至10000bp。此时，所述核酸序列可以为如SEQ ID No.1所示的部分核苷酸序列。所述核酸序列也可以为如SEQ ID No.2所示的部分核苷酸序列
在一个具体实施方式中，所述核酸序列具有5bp至5928bp。此时，所述核酸序列可以为如SEQ ID No.1所示的部分核苷酸序列。所述核酸序列也可以为如SEQ ID No.2所示的部分核苷酸序列。
在一个具体实施方式中，所述核酸序列选自SEQ ID No.1和/或SEQ ID No.2。
在一个具体实施方式中，所述核酸序列具有5bp至1000bp。此时，所述核酸序列可以为如SEQ ID No.1所示的部分核苷酸序列。所述核酸序列也可以为如SEQ ID No.2所示的部分核苷酸序列。
在一个具体实施方式中，所述核酸序列具有5bp至500bp。此时，所述核酸序列可以为如SEQ ID No.1所示的部分核苷酸序列。所述核酸序列也可以为如SEQ ID No.2所示的部分核苷酸序列。
在一个具体实施方式中，所述核酸序列具有5bp至300bp。此时，所述核酸序列可以为如SEQ ID No.1所示的部分核苷酸序列。所述核酸序列也可以为如SEQ ID No.2所示的部分核苷酸序列。
本申请之三提供了一种氨基酸序列，所述氨基酸序列由包含如本申请之一所述SNP标记的核酸序列编码得到，与所述SNP标记相对应的氨基酸X，所述X选自丙氨酸或缬氨酸；且当所述SNP标记为T时，其对应的氨基酸为缬氨酸；当所述SNP标记为C时，其对应的氨基酸为丙氨酸；优选所述核酸序列为能够在所述猪中翻译出与如SEQ ID No.3所示的氨基酸序列具有相同功能蛋白质的核酸序列；优选所述氨基酸序列为SEQ ID No.3；其中，在如SEQ ID No.3所示的氨基酸序列，氨基酸X位于第576位。
本申请之四提供了如本申请之一所述的SNP标记、本申请之二所述的核酸序列和如本申请之三所述的氨基酸序列中至少一种在测定和/或遗传改良猪肉质性状中的应用，所述猪肉质性状包括肌内脂肪含量、大理石纹、肌肉红度、肌肉黄度、肌肉亮度、肌肉纤维类型和水分含量中的至少一种。利用本申请之一(VI)的分子标记中的至少一种来检测猪肉质性状优劣时也能达到80％及以上的准确率，这些分子标记都是与例如(I)的因果突变位点相关的。
在一个具体实施方式中，遗传改良猪肉质性状的种猪选自莱芜猪、二花脸、梅山猪、陆川猪、民猪、米猪、淮猪、河套大耳猪、玉山黑猪、滇南小耳猪、马身猪、藏猪、八眉猪、蓝塘猪中的至少一种。
本申请之五提供了一种猪的遗传改良的方法，所述方法包括：确定种猪核心群中的种猪的如本申请之一所述的SNP标记，并根据所述SNP标记做出相应的选择：
对于(I)，在所述种猪核心群中选择在所述SEQ ID No.1上的从5’端起的第11031位点为TT和TC基因型的种猪个体，淘汰在该位点为CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；优选在所述SEQ ID No.1上的从5’端起的第11031位点为TT基因型的种猪个体，淘汰在该位点为TC和CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；
对于(II)，在所述种猪核心群中选择在所述SEQ ID No.2上的从5’端起的第1780位点为TT和TC基因型的种猪个体，淘汰在该位点为CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；优选在所述SEQ ID No.2上的从5’端起的第1780位点为TT基因型的种猪个体，淘汰在该位点为TC和CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；
对于(III)，在所述种猪核心群中选择在所述核苷酸序列III上的SNP标记位点为TT和TC基因型的种猪个体，淘汰在该位点为CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；优选在所述核苷酸序列III上的SNP标记位点为TT基因型的种猪个体，淘汰在该位点为TC和CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；
对于(VI)，在所述种猪核心群中选择在所述核苷酸序列VI上的SNP标记位点与前述(I)至(III)中的至少一种具有一致基因型的种猪个体，淘汰在该位点与前述(I)至(III)中的至少一种不具有一致基因型的种猪个体。
在一个具体实施方式中，利用分析所述种猪的核酸序列来确定所述种猪的如本申请之一所述的SNP标记，其中所述核酸序列选自如本申请之二所述的核酸序列；和/或利用分析所述种猪的氨基酸序列来确定所述种猪的如本申请之一所述的SNP标记，其中所述氨基酸序列选自如本申请之三所述的氨基酸序列。例如，可以通过高保真的PCR扩增，及之后的测序分析来确定通过PCR扩增出的核酸序列中的所述SNP标记。使用的引物对可以名称为SNP-F即如SEQ ID No.4(5’-CCTAGAAATGCTTTTGGTAAGTG-3’)和名称为SNP-R即SEQ ID No.5(5’-TCAGAGTTGGATTTTCCTATGCC-3)所示。或分析所述种猪的氨基酸序列来确定其属于本申请之三所述的氨基酸序列的哪一种，并通过该种猪的氨基酸序列来确定所述种猪的如本申请之一所述的SNP标记。
本申请之六提供了一种确定猪肉质性状优劣的方法，所述方法包括：确定所述猪的如本申请之一所述的SNP标记，并根据所述SNP标记确定所述猪肉质性状：
对于(I)，所述猪肉质性状从优到劣，以所述SEQ ID No.1上的从5’端起的第11031位点的基因型排序依次为：TT基因型、TC基因型和CC基因型；
对于(II)，所述猪肉质性状从优到劣，以所述SEQ ID No.2上的从5’端起的第1780位点的基因型排序依次为：TT基因型、TC基因型和CC基因型；
对于(III)，所述猪肉质性状从优到劣，所述核苷酸序列III上的所述SNP基因型排序依次为：TT基因型、TC基因型和CC基因型；
对于(VI)，所述猪肉质性状从优到劣，所述核苷酸序列VI上的所述SNP基因型排序与(I)至(III)中的至少一种基因型具有一致性；
所述猪肉质性状包括肌内脂肪含量、大理石纹、肌肉红度、肌肉黄度、肌肉亮度、肌肉纤维类型和水分含量中的至少一种，且肌内脂肪含量更高者为优，大理石纹评分更高者为优，肌肉红度数值更高者为优，肌肉黄度数值更高者为优，肌肉亮度数值更高者为优；I型和IIa型肌肉纤维更多者为优；水分含量更低者为优。
优选地，在一个具体实施方式中，利用分析所述猪的核酸序列来确定所述猪的如本申请之一所述的SNP标记，其中所述核酸序列选自如本申请之二所述的核酸序列。
本申请之七提供了一种改善猪肉品质的猪新品系和/或猪新品种建立的方法，其包括如下步骤：对于如本申请之一所述SNP标记的基因型为CC或TC的猪，通过定点突变将其中的CC基因型或TC基因型突变为TT基因型。
优选地，对于(I)，将SEQ ID No.1上的从5’端起的第11031位点的核苷酸C突变为T；对于(II)，将SEQ ID No.2上的从5’端起的第1780位点的核苷酸C突变为T；对于(III)，将核苷酸序列III上的SNP标记位点的核苷酸C突变为T；对于(VI)，将核苷酸序列VI上的SNP标记位点的核苷酸突变为与前述(I)至(III)中的至少一种核苷酸突变具有一致性的核苷酸。
优选地，利用转基因的方法或基因编辑的方法进行突变。
更优选地，利用CRISPR/Cas9的基因编辑方法进行突变。
在一个具体实施方式中，通过对所述猪的体细胞进行定点编辑突变后，通过克隆获得目标猪肉质性状的猪新品系和/或猪新品种。
此外，常用的检测SNP标记的方法包括：1)基于杂交方法，其至少分为a)利用ΔTm法，b)杂交加荧光探针的方法；2)基于酶的方法，其至少分为a)DNA聚合酶法，例如利用DNA聚合酶进行PCR扩增，b)连接酶法，c)限制性内切酶法，如寻找因果突变位点周围的限制性酶切位点进行酶切扩增，从而获知因果突变位点基因型；d)外切酶FEN法，e)RNase H法；3)电泳法，其至少分为SSCP单链构象多态性和DGGE/TGGE变性梯度凝胶电泳；4)直接测序法。这些方法都可以用来检测本申请的SNP标记。
本申请的有益效果：
猪肉质性状为重要的经济性状，而肌内脂肪性状又是肉质性状的重要指标。本研究所鉴别的影响肌内脂肪含量的因果突变位点，可以直接应用于含该因果突变位点的中国地方猪种肉质性状的遗传改良和开发利用。而对不含该因果突变位点的杜长大商品猪等，可以采用基因编辑技术，构建新的品系解决商品猪肉质较差的现状。在追求优良猪肉品质的今天，具有重大的商业价值，能产生巨大的经济效益。
附图说明
图1显示了莱芜猪肌内脂肪含量的全基因组关联(GWAS)分析图；其中：横坐标表示猪的染色体编号，纵坐标表示-logP值。
图2显示了莱芜猪QTL精细定位及单倍型共享分析图；
(A)莱芜猪IMF QTL区域内关联性分析。最高点用红色表示，与最高点不同的LD值区域用不同颜色表示，灰色线标注的区域是根据LOD值下降2和r2≥0.8确定的525.99-kb置信区间；
(B)置信区间内含两个明显的单倍型，该共享单倍型含8个SNPs，共128.15kb；
(C)单倍型1的IMF含量极显著高于单倍型2，表明单倍型1对应于Q；
(D)红框标注的为128.15kb区域内的基因。
图3显示了莱芜猪QTT及eQTL定位结果；
(A)IMF含量分布图(校正性别和批次的值)，T代表高IMF含量；
(B)MYH4基因表达与IMF含量显著相关。X轴代表MYH4的表达量，Y轴代表IMF含量；
(C)莱芜猪区域eQTL定位结果。X轴代表SNP的位置，Y轴代表-log10(P值)，最高点用红色表示；
(D)C代表MYH4基因表达量高，Y轴代表校正性别和批次后的MYH4表达量。
图4显示了莱芜猪不同基因型个体背最长肌肌纤维类型分析。
具体实施方式
以下通过优选的实施例的形式对本申请的上述内容再作进一步的详细说明，但不构成对本申请的限制。
本申请中的嵌合家系是指利用4个表型差异显著的欧美猪种(皮特兰，杜洛克，长白和大白)与4个表型显著差异的亚洲(中国)种猪(巴马香猪，莱芜猪，藏猪和二花脸猪)通过多世代随机交配产生的家猪嵌合群体。
除非特别说明，文中表示突变的“＞”是指前后两个单核苷酸互变，例如A＞C或C＞A均是指该位点上A与C互相突变。
针对种猪核心群个体，利用上述的PCR扩增鉴别MYH4的g.58244116C＞T突变，选择有利的等位基因型对个体留种，群体继代选育后提高肌内脂肪含量，大理石纹，肌肉的红度、黄度和亮度，减少水分含量，以此改善肉质。
实施例
1.实验动物
本申请使用的实验猪群体为莱芜猪。莱芜猪是中国地方猪种，原产地为山东省莱芜市。本申请使用以12头莱芜公猪和45头莱芜母猪交配获得的316头后代为实验动物，在300±5日龄进行屠宰测定。
2.实验方法
在猪屠宰后取11至14肋骨间背最长肌，用Minolta色度仪CM-2600d/2500d测定肌肉亮度(L*)、肌肉红度(a*)和肌肉黄度(b*)；用美国国家猪生产联合会(NPPC)制作的主观评分板判定猪肉的颜色(评分板共有6个值，分别为1-6；其中1＝苍白，6＝暗黑)和猪肉的大理石纹(评分板共有10个值，分别为1-10；其中1＝稀少，10＝极丰富)；用常规烘箱烘干法和索氏萃取法分别测定肉样的水分和肌内脂肪(IMF)含量；利用ATP酶法进行肌纤维染色。
3.猪全基因组60K SNP判型
采集上述莱芜猪每头个体一小块耳样，用标准苯酚-氯仿法提取全基因组DNA，经Nanodrop-ND1000分光光度计检测浓度和质量后统一稀释至50ng/μl，在Illumina Beadstation平台上根据标准流程进行猪全基因组60K SNP芯片(Illumina，美国)基因型判定。利用R语言GenABEL包中checkmarker对所有样本60K芯片扫描分型数据进行质量控制，剔除检出个体率低于90％、家系孟德尔错误率高于0.1、最小等位基因频率小于0.01，且哈代-温伯格平衡显著性水平高于10-6的SNP，最终得到49452个SNP的有效基因型数据。
4.全基因组关联(GWAS)分析
为了消除群体层化效应，本申请采用线性混合模型单点回归分析并结合R程序中的GenABEL软件包进行GWAS分析，分析模型中利用个体间基因组的相似度校正层化效应。基因组显著水平采用保守的Bonferroni校正方法确定，即基因组显著水平阈值为1.01×10-6(0.05/49452)。
GWAS分析结果如图1所示。从图中可知，在12号染色体鉴别到极显著影响肌内脂肪含量的基因位点，关联最强SNP为ALGA0067072(P＝1.75×10-12)，其位置对应于国际猪基因组参考序列(Sscrofa genome Assembly 10.2版本，简称参考序列)12号染色体的57.83Mb处。通过LOD(似然函数比值的对数)值下降2的方法，将莱芜猪群体的QTL置信区间确定为57.83-58.48Mb，即猪的12号染色体(SSC12)上影响肌内脂肪含量的主效基因定位在此650kb长的染色体区域内。
5.精细定位
为了进一步缩小QTL置信区间，申请人根据6头莱芜猪重测序获得的DNA序列与参考序列比对，在QTL区域选择了127个SNP进行质谱分型，使区域内标记密度达到了每10Kb含1个判型SNP。对得到的质谱分型结果进行质控后和60K质控后的数据进行合并，用于后续分析。
重新对增加标记密度后的基因型与IMF进行全基因组关联分析，结果表明与IMF关联最强SNP为rs49，位于12号染色体58.36Mb处，P值为4.81×10-15。最强关联SNP rs49解释的表型变异为34.7％，根据关联最强SNP LOD下降2和r2≥0.8确定的QTL置信区间为525.99kb(图2A)。即猪的12号染色体(SSC12)上影响肌内脂肪含量的主效基因精细定位在此525.99kb长的染色体区域内。
6.单倍型分析
针对这525.99Kb的QTL区域，本申请在316头莱芜猪中开展了单倍型共享分析。结果如图2B所示，结果表明，大部分个体共享了两种单倍型中的其中之一。该单倍型区域大小为128.15Kb，包含了8个SNP。我们计算了两种单倍型校正后的IMF表型值和所有316头莱芜猪校正后的平均表型值，结果如图2C，单倍型1、单倍型2和所有316头莱芜猪的平均IMF表型值分别为：11.80％±0.24％、7.01％±0.30％和8.74％±0.36％。携带单倍型1单倍型的莱芜猪肌内脂肪含量极显著高于携带单倍型2单倍型的莱芜猪，因此单倍型1对应的QTL基因型应该为Q，而单倍型2则为q。这128.15Kb的单倍型区域仅仅只有MYH4这一个基因(图2D)，由此确定肌球蛋白重链4(myosin heavy chain 4，MYH4)是一个强候选基因。
7.目标区域重测序
根据Ensembl网站(http：//asia.ensembl.org/index.html)上目标区域约526kb的猪基因组序列，用Primer3.0在线软件(http：//frodo.wi.mit.edu/)设计引物(SEQ ID No.6至SEQ ID No.121)，对实验莱芜猪群体中携带不同QTL基因型的25个个体进行重测序。重测序1使用L001-FP和L001-RP引物对，测序获得的片段大小为10259bp；重测序2使用L003-FP和L003-RP引物对，测序获得的片段大小为7947bp；重测序3使用L004-FP和L004-RP引物对，测序获得的片段大小为11606bp；重测序4使用L006-FP和L006-RP引物对，测序获得的片段大小为11898bp；重测序5使用L011-FP和L011-RP引物对，测序获得的片段大小为9824bp；重测序6使用L013-FP和L013-RP引物对，测序获得的片段大小为9847bp；重测序7使用L014-FP和L014-RP引物对，测序获得的片段大小为13314bp；重测序8使用L016-FP和L016-RP引物对， 测序获得的片段大小为9993bp；重测序9使用L017-FP和L017-RP引物对，测序获得的片段大小为10776bp；重测序10使用L019-FP和L019-RP引物对，测序获得的片段大小为10337bp；重测序11使用L020-FP和L020-RP引物对，测序获得的片段大小为10370bp；重测序12使用L021-FP和L021-RP引物对，测序获得的片段大小为10367bp；重测序13使用L023-FP和L023-RP引物对，测序获得的片段大小为9702bp；重测序14使用L024-FP和L024-RP引物对，测序获得的片段大小为9355bp；重测序15使用L025-FP和L025-RP引物对，测序获得的片段大小为10254bp；重测序16使用L028-FP和L028-RP引物对，测序获得的片段大小为13586bp；重测序17使用L030-FP和L030-RP引物对，测序获得的片段大小为11005bp；重测序18使用L031-FP和L031-RP引物对，测序获得的片段大小为9096bp；重测序19使用L034-FP和L034-RP引物对，测序获得的片段大小为10106bp；重测序20使用L038-FP和L038-RP引物对，测序获得的片段大小为10173bp；重测序21使用L043-FP和L043-RP引物对，测序获得的片段大小为12841bp；重测序22使用L044-FP和L044-RP引物对，测序获得的片段大小为10088bp；重测序23使用L045-FP和L045-RP引物对，测序获得的片段大小为10077p；重测序24使用L046-FP和L046-RP引物对，测序获得的片段大小为10040bpp；重测序25使用L047-FP和L047-RP引物对，测序获得的片段大小为13038bp；重测序26使用L048-FP和L048-RP引物对，测序获得的片段大小为10351bp；重测序27使用L049-FP和L049-RP引物对，测序获得的片段大小为6681bp；重测序28使用L050-FP和L050-RP引物对，测序获得的片段大小为8390bp；重测序29使用L051-FP和L051-RP引物对，测序获得的片段大小为7812bp；重测序30使用L053-FP和L053-RP引物对，测序获得的片段大小为9509bp；重测序31使用L055-FP和L055-RP引物对，测序获得的片段大小为8134bp；重测序32使用L056-FP和L056-RP引物对，测序获得的片段大小为7412bp；重测序33使用L058-FP和L058-RP引物对，测序获得的片段大小为6522bp；重测序34使用L059-FP和L059-RP引物对，测序获得的片段大小为6572bp；重测序35使用L060-FP和L060-RP引物对，测序获得的片段大小为7739bp；重测序36使用L065-FP和L065-RP引物对，测序获得的片段大小为8491bp；重测序37使用L069-FP和L069-RP引物对，测序获得的片段大小为13025bp；重测序38使用L070-FP和L070-RP引物对，测序获得的片段大小为10290bp；重测序39使用L071-FP和L071-RP引物对，测序获得的片段大小为10744bp；重测序40使用L073-FP和L073-RP引物对，测序获得的片段大小为7643bp；重测序41使用L075-FP和L075-RP引物对，测序获得的片段大小为9272bp；重测序42使用L077-FP和L077-RP引物对，测序获得的片段大小为6185bp；重测序43使用L078-FP和L078-RP引物对，测序获得的片段大小为8879bp；重测序44使用L080-FP和L080-RP引物对，测序获得的片段大小为9506bp；重测序45使用L082-FP和L082-RP引物对，测序获得的片段大小为9159bp；重测序46使用L084-FP和L084-RP引物对，测序获得的片段大小为10053bp；重测序47使用L086-FP和L086-RP引物对，测序获得的片段大小为8675bp；重测序48使用L087-FP和L087-RP引物对，测序获得的片段大小为6329bp；重测序49使用L088-FP和L088-RP引物对，测序获得的片段大小为10548bp；重测序50使用L089-FP和L089-RP引物对，测序获得的片段大小为 11651bp；重测序51使用L090-FP和L090-RP引物对，测序获得的片段大小为5421bp；重测序52使用L091-FP和L091-RP引物对，测序获得的片段大小为11376bp；重测序53使用L092-FP和L092-RP引物对，测序获得的片段大小为10603bp；重测序54使用L093-FP和L093-RP引物对，测序获得的片段大小为8701bp；重测序55使用L094-FP和L094-RP引物对，测序获得的片段大小为7599bp；重测序56使用L095-FP和L095-RP引物对，测序获得的片段大小为6061bp；重测序57使用L096-FP和L096-RP引物对，测序获得的片段大小为10897bp；重测序58使用L097-FP和L097-RP引物对，测序获得的片段大小为10868bp。
50μL的聚合酶链式反应(PCR)反应体系中，包括100ng猪基因组DNA，2.5mM MgCl2，0.4mM dNTP，使用上述设计的58对正反向引物各20pmol，2.5单位DNA聚合酶(La Taq酶)及1×La PCR buffer(缓冲液)(Takara公司)。PCR扩增条件为：94℃ 2min；98℃ 10s，68℃ 8min，30个循环；最后在72℃延伸10min。PCR扩增产物均匀混合后，委托诺禾致源公司进行测序和de nove组装，未测通的序列采用sanger测序，最终分析得到131个完全符合QTL基因型的SNP。在这131个SNP中，有7个SNP在本申请精细定位第一阶段已经分型，我们对剩下的124个SNP在316头莱芜猪中进行质谱分型，得到的基因分型数据整合至前面已有的基因型数据中，然后与肌内脂肪含量进行关联分析。结果表明12号染色体上影响肌内脂肪含量的最强SNP位点为g.58244116C＞T(P＝2.86×10-16)，其位置对应于国际猪基因组参考序列(Sscrofa genome Assembly 10.2版本)12号染色体的58.24Mb处。
8.因果基因MYH4的确定
8.1目标区域内候选基因
在上述525.99kb区域内包含了9个功能基因，分别是MYH13、MYH1、SCO1、ADPRM、TMEM220、MYH3、MYH2、MYH4和SHISA6。
8.2候选基因表达量与肌内脂肪含量的关联分析(QTT)及表达QTL(eQTL)定位
从135头莱芜猪背最长肌中提取总RNA，采用qRT-PCR技术检测肌细胞中上述9个候选基因的表达量，分析各基因的表达量与肌内脂肪含量的关联性。结果如图3所示，表明在上述9个候选基因中，MYH4基因的RNA转录水平与肌内脂肪含量之间有最强的相关性，相关系数为-0.39(P＝3.53×10-6)，因此确定MYH4是影响猪肌内脂肪含量的因果基因。结合135个个体的所有SNP基因型分型数据，通过关联分析发现与肌内脂肪含量关联性最强的SNP(g.58244116C＞T)也是影响MYH4基因表达最强的SNP。基于以上结果，申请人确定g.58244116C＞T位点是影响猪肌内脂肪含量的因果突变位点。
9.因果突变位点g.58244116C＞T在不同猪群体中的验证分析
为了进一步验证g.58244116C＞T为因果突变位点，即为了进一步验证g.58244116C＞T的因果突变在不同的猪群体中具有广泛性，本实验室采集了419头鲁莱猪、821头嵌合家系F6群体和77头玉山黑猪×杜洛克群体的肌肉样，按照上面的方法进行DNA的提取和肌内脂肪表型的测定。对于鲁莱猪群体和嵌合家系F6群体，对莱芜猪中所有符合QTL基因型的131个SNP用质谱分型，而玉山猪×杜洛克群体则只检测了g.58244116C＞T及周边几个位点 的基因型。结果显示，在三个群体中g.58244116C＞T均是关联最强位点(表1)。这进一步佐证了g.58244116C＞T是影响肌内脂肪含量的因果突变位点。因此，申请人进一步确证g.58244116C＞T是影响肌内脂肪含量的因果突变。
表1.g.58244116C＞T与不同群体肌内脂肪含量的关联分析
Figure PCTCN2017107606-appb-000001
10.MYH4基因及其因果突变位点g.58244116C＞T对莱芜猪肉质性状的影响分析
利用中国地方猪种(莱芜猪群体)为实验动物。316头莱芜猪饲养到300日龄后屠宰，分别测定每个个体的肉质性状。采用PCR扩增和测序对这316头莱芜猪个体开展g.58244116C＞T位点的基因型判定。然后利用GenABEL软件进行基因型对表型的影响效应分析。结果表明在莱芜群体中，g.58244116C＞T位点与肌内脂肪含量表型呈极显著相关性：TT基因型个体比CC基因型个体平均肌内脂肪含量值高6.5％。这说明该位点的效应与QTL导致肌内脂肪含量升高的效应相符。
申请人进一步分析了g.58244116C＞T位点对莱芜猪的其它指标的影响。结果如表2所示，发现TT型个体背最长肌的大理石纹，肌肉的红度、黄度和亮度均比CC型个体提高，且肌肉的水分含量降低。而对于脂肪沉积和生长等其他性状，并未检测到显著相关(P＞0.05)。因此，g.58244116C＞T的T(即Q)等位基因对大理石纹，肌肉的红度、黄度、亮度和水分含量等多个肉质指标有显著影响，但不影响脂肪沉积和生长等其他性状。
表2.g.58244116C＞T突变位点对肉质性状的影响效应。
Figure PCTCN2017107606-appb-000002
注：表型数据用lsmeans方法计算。
11.MYH4基因及其因果突变位点g.58244116C＞T显著提高鲁莱猪、嵌合家系F6和玉山黑猪×杜洛克猪肌内脂肪含量
利用鲁莱猪、嵌合家系F6和玉山黑猪×杜洛克群体为实验动物。采集了419头鲁莱猪、821头嵌合家系F6和77头玉山黑猪×杜洛克猪，分别测定每个个体的肌内脂肪含量。采用PCR扩增对这419头鲁莱猪、821头嵌合家系F6和77头玉山黑猪×杜洛克猪开展g.58244116C＞T位点的基因型判定。然后利用GenABEL软件进行基因型对表型的影响效应分析。结果表明在三个猪群体中，g.58244116C＞T位点与肌内脂肪含量呈极显著相关性(见表1)。在鲁莱群体中，TT基因型个体比CC基因型个体平均肌内脂肪含量值高5.73％；在嵌合家系F6群体中，TT基因型个体比CC基因型个体平均肌内脂肪含量值高2.22％；在玉山黑猪×杜洛克猪群体中，CT基因型个体比CC基因型个体平均肌内脂肪含量值高1.79％。这说明该位点的效应与莱芜群体QTL导致肌内脂肪含量升高的效应相符。
12.MYH4基因及其因果突变位点g.58244116C＞T影响背最长肌肌纤维类型
基于g.58244116C＞T基因型，我们选择了6头TT和5头CC基因型的莱芜猪背最长肌进行肌纤维染色(ATP酶法)。结果如图4所示，肌纤维类型分析发现TT型莱芜猪中I型肌纤维和IIA型肌纤维显著高于CC型个体，而IIB型肌纤维则比CC型莱芜猪显著减少。这表明，g.58244116C＞T不仅增加肌内脂肪含量还影响肌纤维类型，该突变会导致背最长肌中的IIB型肌纤维显著减少，而I型肌纤维和IIA型肌纤维显著增加。
13.连锁不平衡分析
申请人利用R语言中的LD heatmap软件包计算莱芜猪群体中不同标记之间的连锁不平衡程度(r2)。以r2≥0.8为阈值，获得MYH4基因的突变位点g.58244116C＞T周边的SNP标记。同时，GWAS分析证实r2≥0.8的所有分子标记均与肌内脂肪含量达到基因组显著水平以上的相关，而且它们与表型相关程度的高低取决于它们与基因因果突变位点g.58244116C＞T紧密连锁的程度。由此可知，与MYH4的突变位点g.58244116C＞T连锁程度r2≥0.8的分子标记也可以作为有效的肉质育种分子标记。
与主效编码基因MYH4的因果突变位点g.58244116C＞T紧密连锁程度r2≥0.8(即分子标记在群体遗传学上趋于共分离的状态)的分子标记，均与大理石纹，肌肉的红度、黄度、亮度和水分含量等肉质性状显著关联，所以MYH4及其凡是与MYH4因果突变g.58244116C＞T位点紧密连锁r2≥0.8的分子标记都可作为种猪肉质性状遗传改良的分子标记，均应在本专利保护的范围之内。
虽然本申请已经参照其优选地具体实施方式进行了描述，但是本领域的技术人员应该理解在没有脱离本申请的真正的精神和范围的情况下，可以进行的各种改变。例如，可以对本申请的主体、精神和范围进行多种改变以适应特定的情形、材料、材料组合物或方法步骤。所有的这些改变均包括在本申请明的权利要求的范围内。并且利用上述揭示的技术内容做出些许的变动或修饰均等同于等效实施案例，均属于技术方案范围内。

Claims (10)

  1. 一种猪的SNP标记，所述SNP标记包括如下SNP标记中的至少一种：
    (I)核苷酸序列I上的SNP标记，为SEQ ID No.1上的从5’端起的第11031位点的核苷酸Y，所述Y选自C或T；SEQ ID No.1上的从5’端起的第11031位点对应于10.2版本国际猪基因组的12号染色体上的从5’端起的第58244116位点；
    (II)核苷酸序列II上的SNP标记，为SEQ ID No.2上的从5’端起的第1780位点的核苷酸Y，所述Y选自C或T；SEQ ID No.2上的从5’端起的第1780位点对应于10.2版本国际猪基因组的12号染色体上的从5’端起的第58244116位点；
    (III)与SEQ ID No.1和/或SEQ ID No.2所示的核苷酸序列具有90％以上的一致性的核苷酸序列III上的SNP标记，与SEQ ID No.1所示的核苷酸序列上的从5’端起的第11031位点的SNP标记具有一致性；其中，所述核苷酸序列III能够在所述猪中翻译出与如SEQ ID No.3所示的氨基酸序列具有相同功能蛋白质；
    (VI)核苷酸序列VI上的SNP标记，其为与(I)至(III)中的至少一种SNP的连锁不平衡程度r2≥0.8的SNP标记。
  2. 一种核酸序列，所述核酸序列为包含如权利要求1所述的SNP标记的核酸序列，所述核酸序列选自DNA序列、cDNA序列和RNA序列中的至少一种。
  3. 根据权利要求2所述的核酸序列，其特征在于，所述核酸序列具有5bp至26560bp；优选所述核酸序列具有5bp至10000bp；优选所述核酸序列具有5bp至5928bp；优选所述核酸序列选自SEQ ID No.1和/或SEQ ID No.2；
    优选所述核酸序列具有5bp至1000bp；优选所述核酸序列具有5bp至500bp；优选所述核酸序列具有5bp至300bp。
  4. 一种氨基酸序列，所述氨基酸序列由包含如权利要求1所述SNP标记的核酸序列编码得到，与所述SNP标记相对应的氨基酸X，所述X选自丙氨酸或缬氨酸；且当所述SNP标记为T时，其对应的氨基酸为缬氨酸；当所述SNP标记为C时，其对应的氨基酸为丙氨酸；优选所述核酸序列为能够在所述猪中翻译出与如SEQ ID No.3所示的氨基酸序列具有相同功能蛋白质的核酸序列；优选所述氨基酸序列为SEQ ID No.3；其中，在如SEQ ID No.3所示的氨基酸序列，氨基酸X位于第576位。
  5. 如权利要求1所述的SNP标记、如权利要求2或3所述的核酸序列和如权利要求4所述的氨基酸序列中至少一种在测定和/或遗传改良猪肉质性状中的应用，所述猪肉质性状包括肌内脂肪含量、大理石纹、肌肉红度、肌肉黄度、肌肉亮度、肌肉纤维类型和水分含量中的至少一种；
    优选遗传改良猪肉质性状的种猪选自莱芜猪、二花脸、梅山猪、陆川猪、民猪、米猪、淮猪、河套大耳猪、玉山黑猪、滇南小耳猪、马身猪、藏猪、八眉猪、蓝塘猪中的至少一种。
  6. 一种猪的遗传改良的方法，所述方法包括：确定种猪核心群中的种猪的如权利要求1所述的SNP标记，并根据所述SNP标记做出相应的选择：
    对于(I)，在所述种猪核心群中选择在所述SEQ ID No.1上的从5’端起的第11031位点为TT和TC基因型的种猪个体，淘汰在该位点为CC基因型的种猪个体，以逐代提高该 位点的等位基因T的频率；优选在所述SEQ ID No.1上的从5’端起的第11031位点为TT基因型的种猪个体，淘汰在该位点为TC和CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；
    对于(II)，在所述种猪核心群中选择在所述SEQ ID No.2上的从5’端起的第1780位点为TT和TC基因型的种猪个体，淘汰在该位点为CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；优选在所述SEQ ID No.2上的从5’端起的第1780位点为TT基因型的种猪个体，淘汰在该位点为TC和CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；
    对于(III)，在所述种猪核心群中选择在所述核苷酸序列III上的SNP标记位点为TT和TC基因型的种猪个体，淘汰在该位点为CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；优选在所述核苷酸序列III上的SNP标记位点为TT基因型的种猪个体，淘汰在该位点为TC和CC基因型的种猪个体，以逐代提高该位点的等位基因T的频率；
    对于(VI)，在所述种猪核心群中选择在所述核苷酸序列VI上的SNP标记位点与前述(I)至(III)中的至少一种具有一致基因型的种猪个体，淘汰在该位点与前述(I)至(III)中的至少一种不具有一致基因型的种猪个体。
  7. 根据权利要求6所述的方法，其特征在于，利用分析所述种猪的核酸序列来确定所述种猪的如权利要求1所述的SNP标记，其中所述核酸序列选自如权利要求2或3所述的核酸序列；和/或
    利用分析所述种猪的氨基酸序列来确定所述种猪的如权利要求1所述的SNP标记，其中所述氨基酸序列选自如权利要求4所述的氨基酸序列。
  8. 一种确定猪肉质性状优劣的方法，所述方法包括：确定所述猪的如权利要求1所述的SNP标记，并根据所述SNP标记确定所述猪肉质性状：
    对于(I)，所述猪肉质性状从优到劣，以所述SEQ ID No.1上的从5’端起的第11031位点的基因型排序依次为：TT基因型、TC基因型和CC基因型；
    对于(II)，所述猪肉质性状从优到劣，以所述SEQ ID No.2上的从5’端起的第1780位点的基因型排序依次为：TT基因型、TC基因型和CC基因型；
    对于(III)，所述猪肉质性状从优到劣，所述核苷酸序列III上的所述SNP基因型排序依次为：TT基因型、TC基因型和CC基因型；
    对于(VI)，所述猪肉质性状从优到劣，所述核苷酸序列VI上的所述SNP基因型排序与(I)至(III)中的至少一种基因型具有一致性；
    所述猪肉质性状包括肌内脂肪含量、大理石纹、肌肉红度、肌肉黄度、肌肉亮度、肌肉纤维类型和水分含量中的至少一种，且肌内脂肪含量更高者为优，大理石纹评分更高者为优，肌肉红度数值更高者为优，肌肉黄度数值更高者为优，肌肉亮度数值更高者为优；I型和/或IIa型肌纤维类型更多者为优；水分含量更低者为优；
    优选利用分析所述猪的核酸序列来确定所述猪的如权利要求1所述的SNP标记，其中所述核酸序列选自如权利要求2或3所述的核酸序列。
  9. 一种改善猪肉品质的猪新品系和/或猪新品种建立的方法，其包括如下步骤：对于如权利要求1所述SNP标记的基因型为CC或TC的猪，通过定点突变将其中的CC基因型或TC基因型突变为TT基因型；
    优选对于(I)，将SEQ ID No.1上的从5’端起的第11031位点的核苷酸C突变为T；
    对于(II)，将SEQ ID No.2上的从5’端起的第1780位点的核苷酸C突变为T；
    对于(III)，将核苷酸序列III上的SNP标记位点的核苷酸C突变为T；
    对于(VI)，将核苷酸序列VI上的SNP标记位点的核苷酸突变为与前述(I)至(III)中的至少一种核苷酸突变具有一致性的核苷酸。
  10. 根据权利要求9所述的方法，其特征在于，利用转基因的方法或基因编辑的方法进行突变；更优选利用CRISPR/Cas9的基因编辑方法进行突变。
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