WO2006076825A1 - Procede de detection de caracteristiques de qualite de porc et de caracteristiques de carcasse - Google Patents

Procede de detection de caracteristiques de qualite de porc et de caracteristiques de carcasse Download PDF

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WO2006076825A1
WO2006076825A1 PCT/CN2005/000074 CN2005000074W WO2006076825A1 WO 2006076825 A1 WO2006076825 A1 WO 2006076825A1 CN 2005000074 W CN2005000074 W CN 2005000074W WO 2006076825 A1 WO2006076825 A1 WO 2006076825A1
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traits
carcass
gene
seq
sequence
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PCT/CN2005/000074
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Chinese (zh)
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Kui Li
Shuhong Zhao
Zhengmao Zhu
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Kui Li
Shuhong Zhao
Zhengmao Zhu
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Priority to PCT/CN2005/000074 priority Critical patent/WO2006076825A1/fr
Priority to CN200580049090.2A priority patent/CN101142481B/zh
Publication of WO2006076825A1 publication Critical patent/WO2006076825A1/fr

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    • 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
    • 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
    • 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
    • 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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/124—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 present invention relates to a method for detecting pork quality and carcass traits.
  • China is a big pig country and the country with the most abundant pig resources. It has long since been domesticated as a domestic pig. After long-term selection, many local varieties with their own characteristics have been formed. There are only 48 records recorded in "Chinese Pig Breeds" (edited by Zhang Zhongge, China Local Pig Breeds, Shanghai Science and Technology Press, 1986). Compared with foreign commercial pigs, Chinese local varieties have many outstanding advantages: good meat quality, high litter size, good resistance to stress and adaptability, but they have two common shortcomings: low lean rate and increase Slow down. Therefore, increasing the carcass rate and increasing the growth rate has always been the main goal of the genetic improvement work of pigs in our country.
  • Carcass traits mainly include the following two aspects: On the one hand, the metrics of the components of the corpus callosum, such as the thickness of the dorsum, the area of the eye muscle, the length of the corpus callosum, the length of the small intestine, and various visceral weights; on the other hand, the weight percentage of each component Such as slaughter rate, leg-to-hip ratio, leg-to-hip bone rate, oil rate and lactose ratio.
  • Meat quality is a comprehensive trait that includes a range of evaluation indicators.
  • the definition of meat quality varies from country to country and is widely accepted as the definition of H 0 fm a rm (1994), ie meat quality should take into account sensory attributes (traits), technical factors, nutritional value, hygiene and toxicity or food safety. etc.
  • the main indicators for measuring meat quality include the following: PH value ( ⁇ 1, pH2), hydraulic capacity (water loss rate, drip loss, storage loss, cooked meat rate), intramuscular fat content, tenderness ( Shear force), marble, flesh, muscle fiber diameter, etc.
  • the growth hormone gene located on chromosome 12 of pig is neuroendocrine endogenous
  • the core gene regulating animal growth and development in the long axis, its product growth hormone has the functions of regulating metabolism, promoting growth and development, and is a major candidate gene related to pig growth and carcass traits.
  • Chinese and foreign scholars on its genotype and production traits The relationship has been extensively studied. Knorr et al. (Knorr C, Moser G, Muller E, Geldermann H. Associations of GH gene variants with performance traits in F2 generations of European wild boar, Pietrain and Meishan pigs.
  • Insulin-like growth factor I Insulin-like growth factor I was found in the resource population constructed by Changbai X Lantang pigs. Different genotypes increased daily weight after weaning, bone rate, carcass The traits such as lean meat and sebum rate have significant effects.
  • Liu Guilan et al Liu Guilan, Jiang Siwen, Xiong Yuanzhu, Zheng Yi, Qu Yanchun. Association analysis of IGF2 gene PCR-RFLP polymorphism and fat deposition related traits.
  • the cadaver was found to be associated with several carcass traits in different resource families.
  • Other candidate genes related to carcass traits include growth hormone releasing hormone (GHRH), thin Epiii, auxin (61 ⁇ 2re '/?), myogenin Myogenin, Somatostatin (5, Melanocortin-4, MC4R) and growth hormone receptor (GHR) (Xia, et al., 2003), MSTN (Jiang YL, Li N, Plastow G, Liu ZL, Hu XX, Wu CX. Identification of three SNPs in the porcine myostatin gene (MSTN) .
  • Anim Biotechnol, 2002, 13 : 173- 178) Isogenic.
  • Gene epth is considered to be a gene associated with growth and carcass traits (Sasaki S, Clutter AC, Pomp D. Assignment of the porcine obese (leptin) gene to chromosome 18 by linkage analysis of a new PCR - based polymorphism. Mamm Genome, 1996, 7: 471-472), but Jiang et al. (Jiang ZH, Gibson JP. Genetic polymorphisms in the leptin gene and their association with fatness in four pig breeds. Mamm Genome, 1999, 10: 191-193) did not A clear association was found.
  • Myogenin located on chromosome 9 of pigs, is a member of the gene family and its primary function is to regulate the differentiation of myoblasts into muscle fibers. Te Pas MF, Soumillion A, Harders FL, Verburg FJ, van den Bosch TJ, Galesloot P, Meu Giveaway TH. Influences of myogenin genotypes on birth weight, growth rate, carcass weight, backfat thickness, and lean weight of pigs Anim Sci, 1999, 77 : 2352-2356. ) The polymorphism of this gene was detected in two large white pigs. It was found that individuals with different genotypes had traits such as birth weight, growth rate and meat mass. Significant differences.
  • the mRNA expression levels of gene family members of the two selected muscles were compared. It was found that the mRNA expression levels of myogenin, myf-5, and MyoDl in the F-line (selective growth rate) were higher than those of the L-line (selected meat rate), in F- In the system, the thickness of the backfat is negatively correlated with the expression of myoblasts (Te Pas MF, Verburg FJ, Gerritsen CL, de Greef KH. Messenger ribonucleic acid expression of the MyoD gene family in muscle tissue at slaughter in relation to selection for porcine Growth rate. JAnim Sci, 2000, 78 : 69- 77).
  • Kim et al (Kim KS, Larsen N, Short T, Plastow G, Rothschild MF.
  • a missense variant of the porcine melanocortin-4 receptor (MC4R) gene is associated with fatness, growth and feed intake traits. Mamm Genome, 2000, 11 : 131 - 135) Genetic genetic variation detected in five pig commercial lines was significantly associated with backfat thickness.
  • Liu Guilan et al Liu Guilan, Jiang Siwen, Xiong Yuanzhu, Zheng Yi, Qu Yanchun. Scanning of MC4R Gene in Pig Resource Family and Its Relationship with Fat Traits Off analysis.
  • Single nucleotide polymorphism mainly refers to DNA sequence polymorphisms caused by variations in single nucleotides at the genome level. Polymorphisms expressed by SNPs involve only a single base variation, which can be caused by a single base transition or transversion, or by the insertion or deletion of a base. But the so-called SNP does not include the latter two cases. This variation may be a transition (C ⁇ T, GA on its complementary strand) or a transversion (C ⁇ A, G ⁇ T, CG, A ⁇ T). The incidence of conversion is always significantly higher than several other variations. SNPs with conversion variants account for about 2/3, and the incidence of other mutations is similar.
  • SNP detection methods often use some existing mature techniques, such as DNA sequencing, restriction fragment length polymorphism (RFLP), single-strand conformation polymorphism (SSCP), allele-specific oligonucleotides.
  • RFLP restriction fragment length polymorphism
  • SSCP single-strand conformation polymorphism
  • AS0 allele-specific oligonucleotides.
  • AS0 also employ microsequencing according to DNA array, dynamic allele-specific hybridization, oligonucleotide-specific ligation, DNA chip, and TaqMan system. Regardless of the method, the amplification of the target sequence must first be performed before other tests can be performed.
  • the method for detecting pork quality and carcass traits comprises using a pair of primers consisting of the nucleotide sequences of SEQ ID No : 1 and SEQ ID Na: 2 in the Sequence Listing to test the genomic DNA of the pig.
  • the PCR amplification is followed by single nucleotide polymorphism detection of the PCR amplification product, and it is determined whether the 177th base at the 5' end of SEQ ID NO: 3 in the sequence listing is A or G.
  • the single nucleotide polymorphism detection result of the PCR amplification product is the 177th base at the 5' end of SEQ ID NO: 3 in the sequence listing, that is, the 512th base at the 5' end of the sequence 4 in the sequence listing is G
  • the genotype of homozygote is GG
  • the 177th base that is, from the 5' end of sequence 4 of the sequence 4
  • the 512th base is A
  • the homozygous genotype is AA; their heterozygous genotype is AG.
  • the average backfat thickness of GG genotype individuals was significantly higher than that of AG genotype and AA genotype.
  • the ocular muscle surface of GG genotype pigs was significantly lower than that of AG genotype and AA genotype.
  • the alleles of this allele have strong interaction with gender, among which the water loss rate and pH trait of boars are not significant, but in different alleles, The variation of sow individuals with different allelic genotypes is significant, and the sows with homozygous G alleles have a slightly lower water loss rate than the sow individuals homozygous for the A allele, and The water loss rate of heterozygous sows is significantly higher than that of heterozygous boars.
  • pH traits the difference in pH between homozygous individuals of different sexes was not significant, while in hybrids, the pH of sows was significantly lower than that of boars, indicating that G alleles are Is a dominant effect.
  • the single nucleotide polymorphism can be detected by DNA sequencing, restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), allele-specific oligonucleotide hybridization ( AS0) is detected by microsequencing of DNA arrays, dynamic allele-specific hybridization, oligonucleotide-specific ligation, DNA chip, and TaqMan system.
  • RFLP restriction fragment length polymorphism
  • SSCP single strand conformation polymorphism
  • AS0 allele-specific oligonucleotide hybridization
  • a second object of the present invention is to provide a gene related to pork quality and carcass traits.
  • the gene for pork quality and carcass traits provided by the present invention which has a name of 7 ⁇ 7, has a nucleotide fragment consisting of the nucleotide sequence of SEQ ID NO:4 in the sequence listing.
  • Vectors, cell lines and host bacteria containing the genes for pork quality and carcass traits of the present invention are all within the scope of the present invention.
  • Figure lb is the physical map of pBluescript II SK (-)
  • Figure 2 is an electrophoresis map of PCR amplification products of porcine blood genomic DNA.
  • Figure 3 shows the sequencing peak of the PCR amplification product.
  • the PCR of the porcine X hamster hybrid cell DNA (purchased from the French Academy of Agricultural Sciences, Laboratoire de Gene ique Cellulaire, INRA) in the RH clone plate (INRA - Minnesota porcine radiation hybrid panel, ImpRH) was used as a template.
  • the amplification conditions were: 95. C denaturation 3 min; denaturation at 94 °C for 20 s, annealing at 65 °C for 30 s, extension at 72 °C for 30 s, 35 cycles. Finally extended at 72 ° C for 3 min.
  • the composition of the reaction system is shown in Table 1.
  • Template DNA C contains Mg PrimerL PrimerR dNTP Taq polymerase
  • homologous genes or homologous sequences of the gene were not found in the genome of the human genome, mouse genome and the like.
  • Example 2 Detection of single nucleotide polymorphisms of partial DNA sequences of MNTF1 1, primer design.
  • the UniGene number Ssc. 6303 of porcine MNTF1 was obtained using the cDNA sequence having the nucleotide sequence of the sequence 4 in the sequence listing as an information probe.
  • the DNAStar analysis tool was used to analyze the mutation sites in the exon region of the gene.
  • the amplification primers were designed based on the above cDNA sequence and the mutation site information obtained by the analysis. The sequence is as follows:
  • PCR amplification was performed using the total DNA of the blood genome of 129 pigs (30 Tongcheng pigs, 39 large white pigs, 30 Changbai pigs, 15 Dachangtong and 15 Changdatong ternary crosses) as templates.
  • the reaction system 10 X buffer 2 ⁇ 1 , Mg 2+ ( 15 niMol / L) 2 ⁇ 1, each primer 0. 2 ⁇ mol / L, ⁇ ⁇ / L dNTP mixture, 1. 5U Tag DNA polymerase, Template DNA 20ng.
  • 10 X buffer was from TaKaRa Taq kit (TaKaRa, Code No.: DR100A) Reaction procedure: pre-denaturation at 95 ° C for 5 min; denaturation at 94 ° C for 30 s, annealing at 65 ° C for 30 s, extension at 72 ° C for 30 s PCR amplification was carried out in 34 cycles at 72 ° C for 5 min.
  • the PCR amplification product was subjected to 2% agarose gel electrophoresis. The electrophoresis results are shown in Figure 2.
  • the obtained PCR product is about 333 bp in size, and the lanes 1-5 are Tongcheng pig, Dabai pig, Landrace pig, and Dachangtong.
  • the PCR amplification product of the total DNA of the pig blood genome of the ternary cross population of Changdatong, the M lane is the DNA molecular weight marker (100-1000 bp ladder). Purification, cloning and sequencing of the PCR product were then performed as follows:
  • (2) ligation reaction the purified PCR product is ligated to the pGEM-T easy vector, and the ligation reaction The total volume was 5 ⁇ l, which included 2.5 ⁇ 2 ⁇ buffer, 0.5 ⁇ of ⁇ vector, 1.5 ⁇ of purified PCR product, 0.5 ⁇ l of T 4 DNA ligase, and placed in a 16 ° C water bath overnight.
  • Miniprep preparation of plasmid Single colonies on the plate were picked, inoculated into 2-3 ml of LB, and cultured overnight at 37 V 300 r/min. The cells were collected by centrifugation for several seconds using a 1.5 ml EP tube at 12000 r/min. 100 ⁇ l of ice-cold solution I (50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0)) was added to each tube, and vortexed until the cells were sufficiently suspended.
  • ice-cold solution I 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0)
  • the sequencing result showed that the length of the PCR product was 333 bp, and there were 177 bases at the 5' end of SEQ ID NO: 3 from the sequence table, that is, there were G and A at the 512th base of the 5' end of sequence 4 in the sequence listing. Alleles ( Figure 3). In Figure 3, the arrows refer to polymorphic sites.
  • Example 3 detecting pig backfat thickness, eye muscle area, water loss rate and pH value
  • step 2 in Example 2 using the total DNA of the pig blood genome of the above 129 pigs as a template, in the primers 5'-GCGAGAAGCACCAGCCAGAA-3' (sequence 1) and 5'-TCAAGGCGGGAGTGAAGCAG-3' (PCR amplification was carried out under the guidance of sequence 2), and the amplified product was sequenced.
  • the homozygous genotype is GG; Since the 5'-end 177th base of SEQ ID NO: 3 in the sequence listing, that is, the 512th base at the 5' end of sequence 4 in the sequence listing is A, the homozygous genotype is AA; their hybrids The genotype is AG.
  • yi is the observed value of the trait, the overall mean
  • GENOTYPEi is the genotype effect
  • GROUPj is the effect of different hybrid combinations
  • GENOTYPEi X GROUP j is the interaction effect of the two, ⁇ "is a random error, assuming obedience (0, ⁇ 2 ) distribution.
  • the method for detecting pork quality and carcass traits of the present invention can be used for detecting pig backfat thickness, eye muscle area, water loss rate and pH value, and will play an important role in pig breeding.

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Abstract

L'invention concerne un procédé de détection de caractéristiques de qualité de porc et de caractéristiques de carcasse : on utilise une paire d'amorces à séquences SEQ ID Nos 1 et 2 pour amplifier l'ADN génomique des porcs par PCR, puis on détecte le produit de la PCR avec le polymorphisme nucléotidique unique pour déterminer si la base No 177 de l'extrémité 5' dans la séquence SEQ ID No 3 est A ou G.
PCT/CN2005/000074 2005-01-18 2005-01-18 Procede de detection de caracteristiques de qualite de porc et de caracteristiques de carcasse WO2006076825A1 (fr)

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PCT/CN2005/000074 WO2006076825A1 (fr) 2005-01-18 2005-01-18 Procede de detection de caracteristiques de qualite de porc et de caracteristiques de carcasse
CN200580049090.2A CN101142481B (zh) 2005-01-18 2005-01-18 一种检测猪肉质性状及胴体性状的方法

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CN100591692C (zh) * 2007-08-13 2010-02-24 中国农业科学院北京畜牧兽医研究所 一种猪脂肪沉积相关蛋白及其编码基因与应用
CN110157816A (zh) * 2019-06-06 2019-08-23 中国农业科学院北京畜牧兽医研究所 一种鉴定猪的肌纤维直径大小的方法及其所用引物对
CN110157818A (zh) * 2019-07-08 2019-08-23 云南东恒经贸集团猪育种有限公司 一种与猪眼肌高产性状相关的分子标记及其应用
CN110343772A (zh) * 2019-08-31 2019-10-18 贵州大学 一种鉴别香猪与大白猪的结构变异smc1a基因中sv320分子标记及其检测技术
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CN114369669A (zh) * 2022-01-20 2022-04-19 湖北省农业科学院畜牧兽医研究所 一种与猪肉质性状相关的分子标记及其应用
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CN1540350A (zh) * 2003-10-31 2004-10-27 中国农业科学院兰州兽医研究所 一种诊断猪瘟抗体的间接酶联免疫吸附测定方法

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100591692C (zh) * 2007-08-13 2010-02-24 中国农业科学院北京畜牧兽医研究所 一种猪脂肪沉积相关蛋白及其编码基因与应用
CN110157816B (zh) * 2019-06-06 2022-04-26 中国农业科学院北京畜牧兽医研究所 一种鉴定猪的肌纤维直径大小的方法及其所用引物对
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