WO2006076825A1 - A method for detecting the pork quality traits and carcass traits - Google Patents

A method for detecting the pork quality traits and carcass traits 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 CN200580049090.2A priority Critical patent/CN101142481B/en
Priority to PCT/CN2005/000074 priority patent/WO2006076825A1/en
Publication of WO2006076825A1 publication Critical patent/WO2006076825A1/en

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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.

Abstract

A method for detecting the pork quality traits and carcass traits is disclosed here. The method according to this invention uses a pair of primers with the sequences of SEQ IN No. 1 and 2 to amplify the genome DNA of pigs by PCR, then detects the product of PCR with the single nucleotide polymorphism test to determine whether the No. 177 base of the 5' end in SEQ ID No.3 is A or G.

Description

一种检测猪肉质性状及胴体性状的方法 技术领域  Method for detecting pork quality traits and carcass traits
本发明涉及一种检测猪肉质性状及胴体性状的方法。  The present invention relates to a method for detecting pork quality and carcass traits.
背景技术 Background technique
中国是养猪大国， 也是猪种资源最丰富的国家， 很早就开始将野猪驯化 为家猪。 经过长期选择， 形成了许多各具特色的地方品种， 仅《中国猪品种 记》 （张仲葛主编， 中国地方猪品种志， 上海科技出版社， 1986 )记载的就 有 48个。与国外的商品猪相比，中国地方品种具有许多突出的优点： 肉质好， 产仔数高， 抗逆性好和适应性强等， 但却有着两个共同的缺点： 瘦肉率低和 增重慢。 因此， 增加胴体痩肉率和提高生长速度一直是我国家猪的遗传改良 工作的主要目标。  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.
胴体性状主要包括以下两个方面： 一方面是胴体各组分的度量值， 如背 膘厚度、 眼肌面积、胴体长、 小肠长度以及各种内脏重等；另一方面是各组成 的重量百分比， 如屠宰率、 腿臀比率、 腿臀肉骨率、 板油率和内脂率等。  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.
肉质是一个综合性状， 它包括一系列的评价指标。 关于肉质的定义在各 个国家有所差异，广泛接受的是 H0fmarm (1994)的定义，即肉质应考虑感官属 性 （性状） 、 技术因素、 营养价值、 卫生和毒性或食品的安全性等方面。 在 猪的育种中， 度量肉质性状的主要指标包括以下内容： PH值（ρΗ1、 pH2 ) 、 系水力（失水率、滴水损失、贮存损失、 熟肉率）、肌内脂肪含量、嫩度（剪 切力） 、 大理石紋、 肉色、 肌纤维直径等。 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. In pig breeding, the main indicators for measuring meat quality include the following: PH value (ρΗ1, 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.
许多胴体性状属于数量性状，由多基因控制、并存在主基因（major gene) 效应。 由于胴体性状表现晚且不便活体测量， 采用常规育种方法对其进行选 择周期长， 收效慢。 分子生物学技术的发展， 使人们可以在 DNA水平寻找控 制胴体性状的主基因或与其紧密连锁的分子标记， 在育种过程中用于标记辅 助选择 (marker assisted selection, MAS) , 以便提高选择进展， 更好地改 善胴体品质， 满足人们的需要， 同时获得较大的经济效益。  Many carcass traits are quantitative traits, controlled by multiple genes, and have a major gene effect. Due to the late performance of the corpus callosum and inconvenient in vivo measurement, the selection period is long and the effect is slow by conventional breeding methods. The development of molecular biology technology has enabled people to search for the main gene controlling the carcass trait or the molecular markers closely linked to it at the DNA level, and to use marker assisted selection (MAS) in the breeding process in order to improve the selection progress. Better improve the quality of the carcass, meet people's needs, and at the same time obtain greater economic benefits.
目前， 通过候选基因法， 已经找到了一些影响猪的胴体品质的基因。 位 于猪 12号染色体上的生长激素基因 （Growth Hormon, G )是神经分泌内生 长轴中调控动物生长发育的核心基因， 其产物生长激素具有调节新陈代谢、 促进生长发育等作用， 是与猪的生长和胴体性状相关的主要候选基因， 中外 学者对它的基因型与生产性状的关系进行了广泛的研究。 Knorr等（ 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. Anim Genet, 1997, 28 : 124- 128. )发现梅山猪与皮特兰 猪杂交的 F2代群体中， 不同的 ^基因型与 8个胴体性状显著相关。 李加琪 等（李加琪， 陈赞谋， 刘德武， 刘小红， 孙宝丽， 凌飞， 张豪， 陈瑶生 . IFG-1 基因对长白 X蓝塘猪资源群生产性能的遗传效应分析。 遗传学报， At present, some genes affecting the carcass quality of pigs have been found by the candidate gene method. The growth hormone gene (Growth Hormon, G) 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. Anim Genet, 1997, 28 : 124- 128. ) In the F2 population of pigs and Pietrain pigs, different genotypes were significantly associated with 8 carcass traits. Li Jiaqi et al (Li Jiaqi, Chen Zhanmou, Liu Dewu, Liu Xiaohong, Sun Baoli, Ling Fei, Zhang Hao, Chen Yaosheng. Genetic effects of IFG-1 gene on the production performance of Changbai X Lantang pig population.
2003, 30 (9)： 835-839 )发现在长白 X蓝塘猪构建的资源群体中类胰岛素生长 因子 1 (Insulin -like growth factor I， 不同基因型对断奶后日增重、 骨率、胴体瘦肉量和皮脂率等性状有显著影响。刘桂兰等（刘桂兰， 蒋思文， 熊远著， 郑嵘， 屈彦纯. IGF2基因 PCR- RFLP多态性与脂肪沉积相关性状的 关联分析。 遗传学报， 2003， 30 (12) : 1107-1112) 分析了类胰岛素生长因子 2 (Insulin -like growth factor II， Ji)基因第 8内含子两个 l酶 切位点的多态性在大白猪 X梅山猪构成的 F2代中的多态性分布情况， 发现 B 位点 基因型的个体显著比 B2B2基因型的个体背膘薄 18. 28 % (P<0. 01 ) ， 瘦肉率高 8. 71 % (P<0. 01 )。垂体转录因子（Pituitan transcription factor Ι, ΡΙΤΙ ) 是生长激素、 催乳素和促甲状腺素重要的调节因子， ¥11等（ Yu TP, Tuggle CK, Schmitz CB， Rothschild MF. Association of PIT1 polymorphisms with growth and carcass traits in pigs. J Anim Sci, 1995， 73 : 1282-1288) 曾报道尸 777与平均背膘厚存在显著相关， 近年来 1(11171等（ Kuryl J， Pierzchala M. Association of POUIFI/Rsal genotypes with carcass traits in pigs. J Appl Genet, 2001, 42 : 309 - 316· )和 Brunsch等（ Brunsch C， Sternstein I， Reinecke P， Bieniek J. Analysis of associations of PIT1 genotypes with growth, meat quality and carcass composition traits in pigs. J Appl Genet, 2002， 43 : 85- 91)分别在不同的资源家系中检测到尸 与数种胴体性状相关。 其它与胴体性状相关的候选基因还包括生长激素释放 素 i GHRH)、瘦素 eptiii、生长素（ 6½re '/?)、肌细胞生成素 Myogenin 、 生长激素抑制素（somatostatin, 5 、黑素皮质激素受体 4 (Melanocortin-4, MC4R)和生长素受体（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)等基因。 位于 18号染色体上的瘦素基因 epth 被认 为是与生长、 胴体性状相关的一个基因 ( 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) , 但 Jiang等 (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)的研究却没有发现明显的关联。 位于猪 9 号染色体上的肌细胞生成素 Myogenin)是 基因家族的成员， 主要功能 是调节成肌细胞分化为肌纤维。 Te Pas等（ Te Pas MF, Soumillion A, Harders FL, Verburg FJ, van den Bosch TJ, Galesloot P, Meuwissen 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. )在两个大白猪群中检测了该基因的多态性，分析发现不同基因型 的个体在出生重、 生长速度和痩肉量等性状上有显著差异。 对两个选择系肌 肉的 基因家族成员 mRNA表达水平比较发现 F-系 （选择生长速度） 中 myogenin, myf-5, MyoDl的 mRNA表达水平比 L一系（选择痩肉率）高， 在 F - 系中，背膘厚与成肌细胞的表达成负相关（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等（ 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)在 5个猪商品 系中检测到 基因遗传变异与背膘厚显著相关。刘桂兰等（刘桂兰， 蒋思 文， 熊远著， 郑嵘， 屈彦纯.猪资源家系 MC4R基因扫描及其与脂肪性状的相 关分析。 遗传学报， 2002, 29 (6) : 497-501 )对大白 X梅山的 F2代 174个体 检测发现 基因多态性与猪胸腰椎间膘厚（Ρ〈0. 05 )、臀部膘厚 (Ρ<0. 02)、 平均背膘厚 (Ρ<0. 04)、眼肌宽度 (Ρ〈0. 003)、眼肌面积 (Ρ〈0. 05)、皮率 (Ρ〈0. 02) 呈显著相关。 2003, 30 (9): 835-839 ) 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. Chinese Journal of Genetics, 2003 , 30 (12) : 1107-1112) Analysis of the polymorphism of two l-inscribed sites of the 8th intron of the insulin-like growth factor II (JI) gene in the Great White Pig X Meishan pig The polymorphism distribution in the F2 generation was found to be significantly lower than that of the B 2 B 2 genotype by 18.28% (P<0.01), and the lean meat rate was 8 71 % (P < 0.01). Pituitan transcription factor (ΡΙΤΙ) is an important regulator of growth hormone, prolactin and thyrotropin, ¥11, etc. (Yu TP, Tuggle CK, Schmitz CB, Rothschild MF. Association of PIT1 polymorphisms with growth and ca Racs traits in pigs. J Anim Sci, 1995, 73 : 1282-1288) It has been reported that corpse 777 is significantly associated with average backfat thickness, in recent years 1 (11171 et al. ( Kuryl J, Pierzchala M. Association of POUIFI/Rsal genotypes with Carcass traits in pigs. J Appl Genet, 2001, 42 : 309 - 316· ) and Brunsch et al (Brunsch C, Sternstein I, Reinecke P, Bieniek J. Analysis of associations of PIT1 genotypes with growth, meat quality and carcass composition traits in Jgsl. J Appl Genet, 2002, 43 : 85- 91) 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. Leptin located on chromosome 18. 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, Meuwissen 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. Acta Genetica Sinica, 2002, 29 (6) : 497-501 ) The genetic polymorphism of the F2 generation of 174 individuals in Dabai X Meishan was found to be thick and thick in the thoracic and lumbar intervertebral joints (Ρ<0.05) and thick in the buttocks (Ρ< 0. 02), average backfat thickness (Ρ<0. 04), eye muscle width (Ρ<0. 003), eye muscle area (Ρ<0. 05), skin rate (Ρ<0. 02) are significant Related.
采用基因组扫描法， 研究者们还定位了一些影响胴体性状的数量性状位 点（QTL)。影响背膘厚的主基因被定位于第 2和 7号染色体上（de Koning DJ, Janss LL, Rat t ink AP， van Oers PA, de Vries BJ, Groenen MA, van der Poel JJ, et al. Detection of quantitative trait loci for backfat thickness and intramuscular fat content in pigs (Sus scrofa) . Genetics, 1999， 152 : 1679-1690)。 Paszek等（ Paszek AA， Wilkie PJ， Flickinger GH, Miller LM, Louis CF, Rohrer GA, Alexander LJ, Beattie CW, Schook LB. Interval mapping of carcass and meat quality traits in a divergent swine cross. Anim Biotechnol, 2001, 12 : 155- 165)利用 119个分子标记对 116 个 F2个体的胴体和肉质性状进行分析后发现在猪 12号染色体上存在影响胴 体长、第 10肋骨背膘厚、 平均背膘厚、板油率、 眼肌面积和肌内脂肪含量等 多个性状的 QTL。 最近， Clop等（Clop A， Ovilo C, Perez- Enciso M， Cercos A, Tomas A, Fernandez A, Coll A, et al. Detection of QTL affecting fatty acid composition in the pig. Ma腦 Genome, 2003, 14 : 650- 656)对伊比 利亚猪与长白猪构建的 F2进行基因组扫描， 发现 12号染色体上存在影响不 饱和脂肪酸 (亚麻酸)含量的 QTL。 Yue (Yue G, Schroffel JR， Moser G， Bartenschlager H, Reiner G， Geldermann H. Linkage and QTL mapping for Sus scrofa chromosome 12. Journal of Animal Breeding and Genetics, 2003. 120 : 95-102)等以野猪、 梅山猪和皮特兰构成的三个 F2群体为研究对象， 在 12号染色体上发现了数个影响 13- 14肋骨间的背膘厚、 瘦肉切割率等性状的 QTL。据统计， 除了 SSC16和 SSC17外， 猪的所有染色体上均发现了影响背膘 厚的 QTL (Bidanel JP, Rothschild MF. Current status of quantitative trait locus mapping in pigs, pig news and information, 2002, 23 : 39N-53N)。  Using genomic scanning, the researchers also identified quantitative trait loci (QTL) that affect carcass traits. The major genes affecting the backfat are located on chromosomes 2 and 7 (de Koning DJ, Janss LL, Rat t ink AP, van Oers PA, de Vries BJ, Groenen MA, van der Poel JJ, et al. Of quantitative trait loci for backfat thickness and intramuscular fat content in pigs (Sus scrofa) . Genetics, 1999, 152 : 1679-1690). Paszek et al (Paszek AA, Wilkie PJ, Flickinger GH, Miller LM, Louis CF, Rohrer GA, Alexander LJ, Beattie CW, Schook LB. Interval mapping of carcass and meat quality traits in a divergent swine cross. Anim Biotechnol, 2001, 12 : 155- 165) Using 119 molecular markers to analyze the carcass and meat quality traits of 116 F2 individuals, it was found that there was an effect on the chromosome 12 of pigs, the length of the carcass, the thickness of the back 10th rib, the average backfat thickness, the oil rate, QTL for multiple traits such as eye muscle area and intramuscular fat content. Recently, Clop et al. (Clop A, Ovilo C, Perez- Enciso M, Cercos A, Tomas A, Fernandez A, Coll A, et al. Detection of QTL affecting fatty acid composition in the pig. Ma Brain Genome, 2003, 14: 650- 656) A genome scan of F2 constructed in Iberian pigs and Landrace pigs revealed a QTL affecting the content of unsaturated fatty acids (linolenic acid) on chromosome 12. Yue (Yue G, Schroffel JR, Moser G, Bartenschlager H, Reiner G, Geldermann H. Linkage and QTL mapping for Sus scrofa chromosome 12. Journal of Animal Breeding and Genetics, 2003. 120 : 95-102) Waiting for wild boar, Meishan The three F2 populations consisting of pigs and Pietrain were studied, and several QTLs affecting the traits of backfat thickness and lean meat cutting rate between 13-14 ribs were found on chromosome 12. According to statistics, except for SSC16 and SSC17, QTLs affecting backfat were found on all chromosomes of pigs (Bidanel JP, Rothschild MF. Current status of quantitative trait locus mapping in pigs, pig news and information, 2002, 23 : 39N -53N).
单核苷酸多态性（single nucleotide polymorphism, SNP) , 主要是指在 基因组水平上由单个核苷酸的变异所引起的 DNA序列多态性。 SNP所表现的多 态性只涉及到单个碱基的变异， 这种变异可由单个碱基的转换 (transition) 或颠换 (transversion)所引起， 也可由碱基的插入或缺失所致。 但通常所说 的 SNP并不包括后两种情况。这种变异可能是转换 (C→T， 在其互补链上则为 G-A) , 也可能是颠换 (C→A， G→T, C-G, A→T)。 转换的发生率总是明显高 于其它几种变异，具有转换型变异的 SNP约占 2/3，其它几种变异的发生几率 相似。 Single nucleotide polymorphism (SNP), 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检测方法常釆用一些已有的成熟技术， 如 DNA测序、 限制性酶切片 段长度多态性 (RFLP)、 单链构象多态性 (SSCP)、 等位基因特异的寡聚核苷酸 杂交 (AS0)等， 也采用根据 DNA列阵的微测序法、 动态等位基因特异的杂交、 寡聚核苷酸特异的连接、 DNA芯片以及 TaqMan系统等。 但不管哪一种方法， 首先必须进行靶序列的扩增， 然后才能进行其它检测。  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. Hybridization (AS0) and the like 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.
发明公开 Invention disclosure
本发明的目的是提供一种检测猪肉质性状及胴体性状的方法。  It is an object of the present invention to provide a method for detecting pork quality and carcass traits.
本发明所提供的检测猪肉质性状及胴体性状的方法， 是用由具有序列表 中 SEQ ID No : 1和 SEQ ID Na: 2的核苷酸序列组成的一对引物对待测猪的 基因组 DNA进行 PCR扩增， 然后对 PCR扩增产物进行单核苷酸多态性检测， 确定自序列表中 SEQ ID NO: 3的 5' 端第 177位碱基为 A还是 G。 The method for detecting pork quality and carcass traits provided by the present invention 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.
如果 PCR扩增产物的单核苷酸多态性检测结果是自序列表中 SEQ ID NO: 3的 5' 端第 177位碱基，即自序列表中序列 4的 5 ' 端第 512位碱基为 G时， 其纯合体的基因型为 GG; 自序列表中 SEQ ID NO: 3的 5 ' 端第 177位碱基， 即自序列表中序列 4的 5 ' 端第 512位碱基为 A时，其纯合体的基因型为 AA; 它们的杂合体基因型为 AG。  If 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; from the 5' end of SEQ ID NO: 3, 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.
其中， GG基因型个体的平均背膘厚度极显著地高于 AG基因型和 AA 基因型的个体， GG基因型猪的眼肌面积极显著地低于 AG基因型和 AA基 因型的个体。 在失水率和 pH性状中， 该等位基因不同等位基因型与性别 有较强的互作效应， 其中在不同等位基因中， 公猪的失水率和 pH性状变 化不显著， 而不同等位基因基因型的母猪个体之间变化很显著， 表现为 G 等位基因纯合的母猪个体失水率稍高于 A等位基因纯合的母猪个体，而且 杂合子母猪的失水率显著的高于杂合子公猪的失水率。 而在 pH值性状中， 不同性别的纯合个体的 pH值之间差异不显著， 而在杂合体中， 母猪的 pH值 显著的低于公猪的 pH值， 表明 G等位基因体现的是显性效应。 Among them, 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. In the water loss rate and pH trait, 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. In 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.
所述单核苷酸多态性检测可采用 DNA测序、 限制性酶切片段长度多态性 (RFLP)、 单链构象多态性 (SSCP)、等位基因特异的寡聚核苷酸杂交 (AS0)、根 据 DNA列阵的微测序法、动态等位基因特异的杂交、寡聚核苷酸特异的连接、 DNA芯片以及 TaqMan系统等进行检测。  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.
本发明的第二个目的是提供一种猪肉质性状及胴体性状相关基因。  A second object of the present invention is to provide a gene related to pork quality and carcass traits.
本发明所提供的猪肉质性状及胴体性状相关基因， 名称为 7^7， 具有 由序列表中序列 4的核苷酸序列组成的核苷酸片段。  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.
含有本发明的猪肉质性状及胴体性状相关基因的载体、 细胞系及宿主菌 均属于本发明的保护范围。  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.
附图说明 DRAWINGS
图 la为 Sfi I A和 Sfi I B接头序列  Figure la is the Sfi I A and Sfi I B linker sequences
图 lb为 pBluescript II SK (-)的物理图谱  Figure lb is the physical map of pBluescript II SK (-)
图 2为猪血液基因组 DNA的 PCR扩增产物电泳图谱  Figure 2 is an electrophoresis map of PCR amplification products of porcine blood genomic DNA.
图 3为 PCR扩增产物的测序峰图  Figure 3 shows the sequencing peak of the PCR amplification product.
实施发明的最佳方式 The best way to implement the invention
实施例 1、 MVTFI的获得  Example 1. Acquisition of MVTFI
利用 Clontech公司的 SMART cDNA Library Construction Kit和改造的 pBluescript II SK克隆载体（pBluescript II SK的改造载体， 具体为将 pBluescript II SK (-) 的 EcoR I和 Not I之间的序列改造为 Sfi I A和 Sfi I B接头序列， 利用 Sfi I A和 Sfi I B酶切后可定向插入 cDNA片断（Sfil A →Sfi I B) 。 Sfi I A和 Sfi I B接头序列和 pBluescript II SK (-)的物理 图谱分别如图 la和图 lb所示），用猪胚胎发育第 55天的骨骼肌组织的 mRNA， 按照常规方法构建了全长 cDNA文库。通过对文库克隆子的测序获得了大量的 在骨骼肌组织中表达的基因。  Using the SMART cDNA Library Construction Kit from Clontech and the engineered pBluescript II SK cloning vector (transformed vector of pBluescript II SK, specifically transforming the sequence between EcoR I and Not I of pBluescript II SK (-) into Sfi IA and Sfi The IB linker sequence was digested with Sfi IA and Sfi IB and inserted into the cDNA fragment (Sfil A → Sfi IB). The physical maps of the Sfi IA and Sfi IB linker sequences and pBluescript II SK (-) are shown in Figure la and Figure lb, respectively. As shown in the figure, a full-length cDNA library was constructed according to a conventional method using mRNA of skeletal muscle tissue on the 55th day of porcine embryo development. A large number of genes expressed in skeletal muscle tissues were obtained by sequencing library clones.
利用比较功能基因组学原理和方法，利用 BLAST在 NCBI的数据库中进行 比对没有发现在人鼠以及其它物种存在同源基因， 仅在牛物种中存在同源 EST, 它具有由序列表中 SEQ ID Na: 4的核苷酸序列 （cDNA)组成的核苷酸 片段， 将该基因命名为猪》 基因。 应用 cDNA macroarray分析发现该基因在不同的发育阶段具有差异表达， 应用 Northern 杂交试验验证了该基因是一个在不同发育时期差异表达的基 因。 Using comparative functional genomics principles and methods, comparisons using BLAST in the NCBI database did not reveal the presence of homologous genes in human and other species, only homologous ESTs in bovine species, which have the SEQ ID in the sequence listing. A nucleotide fragment consisting of a nucleotide sequence (cDNA) of Na : 4, which is named as a porcine gene. The cDNA macroarray analysis showed that the gene was differentially expressed at different developmental stages. Northern hybridization assay was used to verify that the gene is a differentially expressed gene at different developmental stages.
根据猪 cDNA序列设计一对引物：  Design a pair of primers based on the pig cDNA sequence:
5， - GCGAGAAGCACCAGCCAGAA -3， (PrimerL)，  5, - GCGAGAAGCACCAGCCAGAA -3, (PrimerL),
5， - TCAAGGCGGGAGTGAAGCAG _3， (PrimerR)  5, - TCAAGGCGGGAGTGAAGCAG _3, (PrimerR)
以 RH克隆板 ( INRA - Minnesota porcine radiation hybrid panel , ImpRH)中的猪 X仓鼠杂种体细胞的 DNA (购自法国农业科学院， Laboratoire de Genet ique Cellulaire, INRA)为模板进行 PCR扩增。 扩增条件为： 95。C 变性 3min; 94°C变性 20s， 65°C退火 30s， 72°C 延伸 30 s， 35个循环。 最 后在 72°C延伸 3min。 其中的反应体系组成如表 1所示。  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.
表 1. PCR的反应体系  Table 1. Reaction system of PCR
总体 ddH20 10 X Buffer lOmM/L lOmM/L lOmM/L 5υ/ μ 1 模板 DNA 积 C含 Mg PrimerL PrimerR dNTP Taq聚合酶Overall ddH 2 0 10 X Buffer lOmM/L lOmM/L lOmM/L 5υ/ μ 1 Template DNA C contains Mg PrimerL PrimerR dNTP Taq polymerase
0 μ 1 5. 75 μ 1 1 μ 1 0. 75 μ 1 0. 75 μ 1 0. 5 μ 1 0. 25 μ 1 1 μ 1 (20 ng) 扩增片段经 2%的琼脂糖凝胶电泳进行 PCR扩增片段分型。 其中， 阳性结 果的为 1， 阴性结果的为 0。 结果为： 0 μ 1 5. 75 μ 1 1 μ 1 0. 75 μ 1 0. 75 μ 1 0. 5 μ 1 0. 25 μ 1 1 μ 1 (20 ng) The amplified fragment was subjected to 2% agarose gel electrophoresis. PCR amplification fragment typing was performed. Among them, the positive result is 1, and the negative result is 0. The result is:
001000001010110000100100001000000101100000011000100000000011100000 1110 100000001001101000111101000001100001000101000000, 将该结果提交 至 Ij IMpRH数据统计分析服务器  001000001010110000100100001000000101100000011000100000000011100000 1110 100000001001101000111101000001100001000101000000, submit the result to Ij IMpRH data statistical analysis server
( http： //www. toulouse. inra. f r/l gc/p i g/RH/IMpRH. html / ) 上进 行分析， 得到如表 2的结果：  ( http: //www. toulouse. inra. f r/l gc/p i g/RH/IMpRH. html / ) Perform the analysis and get the results as shown in Table 2:
表 2. PCR扩增片段分型的分析结果  Table 2. Analysis results of PCR amplified fragment typing
基因 存留率(％)染色体连锁的标记 断裂频率 RH 图距 LOD值  Gene retention rate (%) chromosome-linked marker fragmentation frequency RH map distance LOD value
MNTF1 29 7 SSC12B09 0.6 0.92 3.47 MNTF1 29 7 SSC12B09 0.6 0.92 3.47
经过生物信息学分析和同源性比较， 在人基因组、 小鼠基因组等物种的 组中没有发现该基因的同源基因或者同源序列。  After bioinformatics analysis and homology comparison, homologous genes or homologous sequences of the gene were not found in the genome of the human genome, mouse genome and the like.
实施例 2、 MNTF1的部分 DNA序列的单核苷酸多态性的检测 1、 引物设计 . Example 2. Detection of single nucleotide polymorphisms of partial DNA sequences of MNTF1 1, primer design.
用由具有序列表中序列 4核苷酸序列的 cDNA序列为信息探针， 获得猪 MNTF1的 UniGene编号 Ssc. 6303。 利用 DNAStar分析工具分析在该基因的外 显子区域中的突变位点。根据上述 cDNA序列以及分析获得的突变位点信息设 计扩增引物。 序列如下：  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:
5' - GCGAGAAGCACCAGCCAGAA -3，（正向） （序列 1 )，  5' - GCGAGAAGCACCAGCCAGAA -3, (forward) (sequence 1),
5， - TCAAGGCGGGAGTGAAGCAG -3' (反向） （序列 2 )  5, - TCAAGGCGGGAGTGAAGCAG -3' (reverse) (sequence 2)
2、 PCR扩增及其产物的纯化、 克隆和测序  2. PCR amplification and purification, cloning and sequencing of its products
分别以 129头猪（30头通城猪， 39头大白猪， 30头长白猪， 15头大长 通和 15头长大通三元杂交群体）血液基因组的总 DNA为模板进行 PCR扩增。 其中， 反应体系： 10 X缓冲液 2 μ 1 , Mg2+ ( 15niMol/L) 2 μ 1， 每种引物 0. 2 μ mol/L, ΙΟΟμιηοΙ/L dNTP混合物， 1. 5U Tag DNA聚合酶， 模板 DNA 20ng。 其中， 10 X缓冲液来自于 TaKaRa Taq试剂盒（TaKaRa公司， Code No.： DR100A) 反应程序：95°C预变性 5min; 94°C变性 30s， 65°C退火 30 s， 72°C延伸 30s，34 个循环， 72°C延伸 5min下进行 PCR扩增。 PCR扩增产物进行 2 %琼脂糖凝胶 电泳， 电泳结果如图 2所示， 表明获得的 PCR产物大小约为 333bp， 1-5泳道 分别为通城猪， 大白猪， 长白猪， 大长通和长大通三元杂交群体猪血液基因 组的总 DNA的 PCR扩增产物， M泳道为 DNA分子量标记 （100— 1000 bp ladder) 。 然后按照如下方法进行 PCR产物的纯化、 克隆和测序： 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. Wherein, the reaction system: 10 X buffer 2 μ 1 , Mg 2+ ( 15 niMol / L) 2 μ 1, each primer 0. 2 μ mol / L, ΙΟΟ μιηοΙ / L dNTP mixture, 1. 5U Tag DNA polymerase, Template DNA 20ng. Among them, 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. It indicates that the obtained PCR product is about 333 bp in size, and the lanes 1-5 are Tongcheng pig, Dabai pig, Landrace pig, and Dachangtong. And 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:
( l ) PCR产物的纯化： 在紫外灯下从琼脂糖凝胶上切下含目的片段的凝 胶， 放入 1. 5 ml Ependorff管中， 于 70 °C温育至凝胶完全融化， 然后用 PCR 产物纯化试剂盒（Promega)纯化 PCR产物， 按照试剂盒说明书操作， 具体步 骤是在每 300 μ ΐ 融化的凝胶中加入 1 ml树脂（Resin) ， 混匀 20 s， 将 Resin/DNA混合物装入注射器， 使浆液通过微柱（Minicolumn)挤出。再在注 射器中加入 80%的异丙醇 2 ml ， 轻推活塞使异丙醇通过 Minicolumn挤出， 取下 Minicolumn装入 1. 5 ml Ependorff管中， 10， 000g离心 2 min以干燥 Resin, 将 Minicolumn装入另一个干净的 1. 5 ml Ependorff管中， 加入 30-50 μ ΐ无菌水，静置 Imin, 10, 000g离心 20s， 以洗脱 DNA存于 Ependorff 管中。  ( l) Purification of the PCR product: The gel containing the desired fragment was cut from the agarose gel under UV light, placed in a 1.5 ml Ependorff tube, and incubated at 70 ° C until the gel completely melted, then Purify the PCR product using the PCR Product Purification Kit (Promega) and follow the kit instructions by adding 1 ml of resin (Resin) to each 300 μM melted gel and mixing for 20 s. Resin/DNA mixture The syringe was loaded and the slurry was extruded through a microcolumn (Minicolumn). Then add 80 ml of 80% isopropanol to the syringe, gently push the piston to extrude the isopropanol through Minicolumn, remove the Minicolumn into a 1.5 ml Ependorff tube, centrifuge at 10,000 g for 2 min to dry Resin, Minicolumn was placed in a clean 1.5 ml Ependorff tube, 30-50 μM sterile water was added, Imin was allowed to stand, and 10 000 g was centrifuged for 20 s to elute the DNA in an Ependorff tube.
( 2 )连接反应： 将纯化的 PCR产物与 pGEM- T easy载体连接， 连接反应 总体积是 5 μ1， 其中包括 2.5 μΐ 2Χ缓冲液， 0.5 μΐ的 Τ载体， 1.5 μΐ 的纯化 PCR产物， 0.5 μ 1 的 T4 DNA连接酶， 置 16°C水浴过夜。 (2) ligation reaction: the purified PCR product is ligated to the pGEM-T easy vector, and the ligation reaction The total volume was 5 μl, which included 2.5 μΐ 2Χ buffer, 0.5 μΐ of Τ vector, 1.5 μΐ of purified PCR product, 0.5 μl of T 4 DNA ligase, and placed in a 16 ° C water bath overnight.
(3)感受态细胞的制备： 从 37°C培养了 16- 20 h的新鲜平板上挑取一 个 DH5a 单菌落接种于 2ml LB中， 于 37°C振荡培养 3h， 转接 1 ml菌液于含 有 30 ml LB的瓶中， 继续在 37°C振荡培养约 4h， 待 OD600达到 0.3- 0.4时 将瓶从摇床取出置冰浴冷却 10-15min， 然后将菌液转入离心管中于 4Ό 4，000g离心 10 min 以收集细胞， 将离心管倒置以弃净培养液， 用 10 ml冰 预冷的 0.1 mol/L 的 CaCl2重悬沉淀， 冰浴 30 min, 重复 4°C 4， 000g离心 10 min一次， 用 4 ml冰预冷的 0.1 mol/L的 CaCl2重悬沉淀， 置 4Ό保存备 用。 (3) Preparation of competent cells: A single DH5a colony was picked from a fresh plate cultured at 37 ° C for 16-20 h, inoculated into 2 ml of LB, shaken at 37 ° C for 3 h, and transferred to 1 ml of bacterial solution. In a bottle containing 30 ml LB, continue to shake culture at 37 ° C for about 4 h. When the OD600 reaches 0.3-0.4, remove the bottle from the shaker and cool it for 10-15 min. Then transfer the bacterial solution to the centrifuge tube at 4 Ό. Centrifuge at 4,000 g for 10 min to collect the cells, invert the tube to discard the culture solution, resuspend the pellet with 10 ml of ice-cold 0.1 mol/L CaCl 2 , ice bath for 30 min, repeat 4 ° C 4 000 g After centrifugation for 10 min, the pellet was resuspended in 4 ml of ice-cold 0.1 mol/L CaCl 2 and stored for 4 备用.
(4)转化： 无菌状态下取 100-120 μ 1感受态细胞于 1.5 ml Ependorff 管中， 将 5 μΐ的连接产物加入混匀， 在冰上放置 30 min， 42°C 热激 90 s， 其间不要摇动 Ependorff管， 取出后冰浴 3-4 min, 加入 400 μ 1无抗生素的 LB液体培养基， 37°C振荡培养 45 min。 取 100 μ 1涂布于已提前 4 h涂布了 IPTG( Isopropylthio-β -D-galactoside, 异丙基硫代一 -D—半乳糖苷）和 X- gal的琼脂平板上， 37°C平放 1 h后倒置培养。  (4) Transformation: 100-120 μl of competent cells were aseptically placed in a 1.5 ml Ependorff tube, and 5 μΐ of the ligation product was added to the mixture, placed on ice for 30 min, and heat shocked at 42 ° C for 90 s. Do not shake the Ependorff tube during this time. Remove the ice bath for 3-4 min, add 400 μl of antibiotic-free LB liquid medium, and incubate at 37 °C for 45 min. 100 μl was applied to agar plates coated with IPTG (Isopropylthio-β-D-galactoside, isopropylthio-D-galactoside) and X-gal 4 h earlier, at 37 ° C After 1 h, the culture was inverted.
(5)质粒的小量制备： 挑取平板上的单菌落， 接种于 2-3ml LB中， 37 V 300r/min培养过夜。用 1.5ml EP管 12000r/min离心数秒收集菌体。每管 加入 100 μ 1用冰预冷的溶液 I (50 mM葡萄糖， 25mM Tris- HC1 (pH8.0)， 10mM EDTA(pH8.0) )，涡旋振荡至菌体充分悬浮。加入新配制的溶液 II [0.2M NaOH, l%SDS]200 l, 快速颠倒混匀， 冰浴 5min， 然后加入预冷的溶液 III (5M乙酸 钾， 冰乙酸 11.5ml， H20 28.5ml ) 150μ 1, 混匀后冰浴 5min， 12000r/min 离心 5rain， 将上清转至另一 EP管中， 加入苯酚： 氯仿： 异戊醇 500μ1， 涡 旋振荡， 离心后小心吸取上层水相， 加入 2倍体积的无水乙醇， - 2CTC沉淀 30min, 12000r/min离心 5rain， 沉淀用 70%乙醇洗涤 2次， 抽干， 加入含有 RNA酶的 TE 20 μ 1。  (5) 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 μl 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. Add the newly prepared solution II [0.2M NaOH, l%SDS] 200 l, mix quickly by inversion, ice bath for 5 min, then add pre-cooled solution III (5M potassium acetate, glacial acetic acid 11.5ml, H20 28.5ml) 150μ 1 After mixing, ice bath for 5 min, centrifugation at 12000 r/min for 5 min, transfer the supernatant to another EP tube, add phenol: chloroform: isoamyl alcohol 500 μl, vortex, centrifuge carefully, then pipet the upper aqueous phase, add 2 times The volume of absolute ethanol, - 2CTC was precipitated for 30 min, centrifuged at 12000 r/min for 5 min, the precipitate was washed twice with 70% ethanol, drained, and TE 20 μl containing RNase was added.
(6)重组质粒的酶切鉴定： 取 3μ1质粒 DNA与双蒸水混匀， 使其总体 积为 10 μ 1, 加入 5U限制性内酶 EcoRl及 1 μ 1相应的 10X限制性内切酶反 应缓冲液，轻弹管壁混匀并离心，置 37°C水浴 1- 2小时，取 2- 3μ1反应液于 琼脂糖凝胶电泳检测， 得到酶切结果与预计完全相同的目的重组质粒。 ( 7 )测序： 重组质粒釆用双脱氧末端终止法在 DNA自动测序仪上进行测 序， 序列测定由上海博亚生物技术有限公司完成。 测序结果表明该 PCR产物 的长度为 333bp,自序列表中 SEQ ID NO: 3的 5 ' 端第 177位碱基， 即自序列 表中序列 4的 5 ' 端第 512位碱基处存在 G、 A两个等位基因 （图 3 ) 。 图 3 中， 箭头所指的是多态位点。 (6) Identification of the recombinant plasmid by digestion: Mix 3μ1 of plasmid DNA with double distilled water to make the total volume of 10 μ 1, add 5U restriction enzyme EcoRl and 1 μ 1 corresponding 10X restriction endonuclease reaction Buffer, lightly bombard the tube wall and mix and centrifuge, set the water bath at 37 ° C for 1-2 hours, take 2- 3 μ1 reaction solution on agarose gel electrophoresis, and obtain the recombinant plasmid of the same purpose as the expected digestion. (7) Sequencing: The recombinant plasmid was sequenced on a DNA automatic sequencer by the dideoxy termination method, and the sequence was determined by Shanghai Boya Biotechnology Co., Ltd. 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.
实施例 3、 检测猪背膘厚度， 眼肌面积， 失水率和 pH值  Example 3, detecting pig backfat thickness, eye muscle area, water loss rate and pH value
以 62头纯繁通城猪、 22头纯繁长白猪、 24头长白 (大白 X通城） 早和 21头大白 (长白 X通城） 早三元杂交组合猪， 共 129个个体为实验 对象进行性状关联分析， 具体方法如下：  62 heads of pure prosperous city pigs, 22 heads of pure prolific white pigs, 24 heads of Changbai (Dabai X Tongcheng) and 21 heads of white (Changbai X Tongcheng) early ternary cross-breeding pigs, a total of 129 individuals Conduct trait correlation analysis, the specific method is as follows:
1、按照实施例 2中步骤 2的方法， 分别以上述 129头猪的猪血液基因组 的总 DNA为模板， 在引物 5' - GCGAGAAGCACCAGCCAGAA -3' (序列 1)和 5' - TCAAGGCGGGAGTGAAGCAG -3' (序列 2)的引导下进行 PCR扩增， 对扩增产物进 行测序。 如测序结果表明自序列表中 SEQ ID NO: 3的 5 ' 端第 177位碱基， 即自序列表中序列 4的 5 ' 端第 512位碱基为 G时，其纯合体的基因型为 GG; 自序列表中 SEQ ID NO: 3的 5 ' 端第 177位碱基， 即自序列表中序列 4的 5 ' 端第 512位碱基为 A时， 其纯合体的基因型为 AA; 它们的杂合体基因型为 AG。  1. According to the method of 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. If the sequencing result indicates that the 177th base of the 5' end of SEQ ID NO: 3 in the sequence list, that is, the 512th base of the 5' end of the sequence 4 in the sequence list is G, 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.
2、 用如下最小二乘模型分析（屠宰体重， 胴体重， 屠宰率， 瘦肉重， 脂 肪重， 骨重， 平均膘厚， 眼肌面积）和肉质性状 （肌间脂肪含量， pH值， 失 水率， 大理石纹评分， 肉色， 滴水损失） ：  2. Analysis by the following least squares model (slaughter weight, body weight, slaughter rate, lean meat weight, fat weight, bone weight, average thickness, eye muscle area) and meat quality traits (intermuscular fat content, pH value, loss) Water rate, marbling score, flesh color, drip loss):
Ylj= μ + GENOTYPEi + GR0UPj+ GENOTYPEi X GR0UPj+ ε u, Ylj = μ + GENOTYPEi + GR0UPj+ GENOTYPEi X GR0UPj+ ε u ,
其中， yi」是性状观察值， 为总体均数， GENOTYPEi为基因型效应， GROUPj 为不同杂交组合的效应， GENOTYPEi X GROUP j为这两者的互作效应， ε "为随机 误差， 假定服从 Ν (0， σ 2)分布。 Where 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, ε "is a random error, assuming obedience (0, σ 2 ) distribution.
结果表明在所检测的群体中， GG基因型的个体有 89个， ΑΑ基因型有 9 个个体， ΤΤ基因型有 31个个体。不同基因型之间性状的性状显著差异的结果 (最小二乘均数和标准误分析）如表 3、表 4、表 5所示， 其它性状在不同的 基因型之间没有显著的差异。 表 3表明， GG基因型的平均背膘厚度均极显著 地高于 AG基因型和 ΑΑ基因型的猪 （P〈0. 01 ) 。 相反， GG基因型猪的眼肌面 积均极显著地低于 AG基因型和 AA基因型的猪（P〈0. 01 ) 。 说明 G等位基因 是影响背膘厚的增效基因， 而 A等位基因是影响眼肌面积的增效基因。 表 3.不同基因型与背膘厚性状和眼肌面积性状的关联分析 The results showed that among the tested populations, there were 89 individuals with GG genotype, 9 individuals with genotype, and 31 individuals with genotype. The results of significant differences in traits between different genotypes (least squares means and standard error analysis) are shown in Tables 3, 4, and 5, and other traits did not differ significantly between different genotypes. Table 3 shows that the average backfat thickness of the GG genotype was significantly higher than that of the AG genotype and the genotype genotype (P < 0.01). In contrast, the ocular muscle area of GG genotype pigs was significantly lower than that of AG genotype and AA genotype (P < 0.01). G allele It is a synergistic gene that affects the backfat, and the A allele is a synergistic gene that affects the area of the eye muscle. Table 3. Association analysis between different genotypes and backfat traits and ocular muscle area traits
Figure imgf000013_0001
Figure imgf000013_0001
注： **表示在 0.01水平上差异极显著。 表 4表明该等位基因不同等位基因型与性别有较强的互作效应， G等位 基因纯合的母猪个体失水率稍高于 G等位基因纯合的公猪个体。 两者差异不 显著， 同样 A等位基因纯合的不同性别的个体的失水率之间差异不显著，但 G 等位基因纯合个体的失水率大于 A等位基因纯合个体的失水率， 而在杂合个 体中， 母猪的失水率显著的高于公猪的失水率。 表 4.不同基因型与失水率的关联分析  Note: ** indicates that the difference is extremely significant at the 0.01 level. Table 4 shows that the different alleles of this allele have a strong interaction with gender. The sows homozygous for the G allele have a slightly lower water loss rate than the homozygous boar individuals. The difference between the two is not significant. The difference in the water loss rate of individuals with homozygous A alleles is not significant, but the G-allele homozygous individuals have a higher water loss rate than the A allele homozygous individuals. Water rate, and in heterozygous individuals, the sow's water loss rate is significantly higher than the boar's water loss rate. Table 4. Association analysis between different genotypes and water loss rate
Figure imgf000013_0002
Figure imgf000013_0002
注： *表示在 0.05水平上差异显著。 表 5表明在 pH值性状中，不同性别的纯合个体的 pH值之间差异不显著， 但 G等位基因的纯合个体的 pH值低于 A等位基因纯合个体的 pH值， 而在杂 合体中， 母猪的 pH值显著低于公猪的 PH值， 表明 G等位基因体现的是显性 效应。 表 5.不同基因型与 pH值的关联分析 Note: * indicates a significant difference at the 0.05 level. Table 5 shows that in pH traits, the difference in pH between homozygous individuals of different sexes is not significant, but the pH of homozygous individuals of G allele is lower than that of homozygous individuals of A allele, and In the hybrid, the pH of the sow is significantly lower than the P H value of the boar, indicating that the G allele exhibits a dominant effect. Table 5. Association analysis between different genotypes and pH
Figure imgf000014_0001
Figure imgf000014_0001
注： *表示在 0.05水平上差异极显著。  Note: * indicates that the difference is extremely significant at the 0.05 level.
工业应用  Industrial application
本发明的检测猪肉质性状及胴体性状的方法可用于检测猪背膘厚度， 眼 肌面积， 失水率和 pH值， 将在猪的育种中发挥重要作用。  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.

Claims

权利要求 Rights request
1、 检测猪肉质性状及胴体性状的方法， 是用由具有序列表中 SEQ ID Ns: 1和 SEQ ID No : 2的核苷酸序列组成的一对引物对待测猪的基因组 DNA进行 PCR扩增， 然后对 PCR扩增产物进行单核苷酸多态性检测， 确定自序列表中 SEQ ID NO: 3的 5 ' 端第 177位碱基为 A还是 G。 1. A method for detecting pork traits and carcass traits by PCR amplification of genomic DNA of a test pig using a pair of primers consisting of the nucleotide sequences of SEQ ID Ns: 1 and SEQ ID No : 2 in the sequence listing Then, the PCR amplification product is subjected to single nucleotide polymorphism detection, 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.
2、猪肉质性状及胴体性状相关基因，具有由序列表中序列 4的核苷酸序 列组成的核苷酸片段。  2. Pork quality traits and carcass trait related genes having a nucleotide fragment consisting of the nucleotide sequence of SEQ ID NO: 4 in the sequence listing.
3、 含有权利要求 2所述的猪肉质性状及胴体性状相关基因的载体。 A carrier comprising the pork-like trait and the steroid-related gene according to claim 2.
4、 含有权利要求 2所述的猪肉质性状及胴体性状相关基因的细胞系。A cell line comprising the pork-like trait and the steroid-related gene of claim 2.
5、 含有权利要求 2所述的猪肉质性状及胴体性状相关基因的宿主菌。 A host strain comprising the gene for pork quality and carcass trait according to claim 2.
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