CN107151692B - Method for identifying pig living body backfat thickness and gene diagnosis kit - Google Patents
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Abstract
The invention discloses a gene diagnosis kit for identifying pig living body backfat thickness. The method for identifying the backfat thickness of the pig living body by using the gene diagnostic kit provided by the invention is as follows (1), (2), (3) or (4): (1) detecting whether the 460 th deoxyribonucleotide of MEG3 gene of a pig individual is T or A or T and A; (2) detecting whether the 1137 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual is A or G or A and G; (3) detecting whether the 1158 th deoxyribonucleotide of a second exon of the MEG3 gene of the pig individual is A, G or A and G; (4) whether the 51 st deoxyribonucleotide of the first intron of MEG3 gene of a pig individual is T or C or T and C is detected. Experiments prove that: the method of the invention is consistent with the actual measurement result of the back fat thickness of the live body with the weight of 100kg of the pig, and has important significance for selecting excellent pig breeds.
Description
Technical Field
The invention belongs to the field of genetic engineering, particularly relates to a gene diagnosis kit for identifying the back fat thickness of a pig living body, and particularly relates to a method for identifying or assisting in identifying the back fat thickness of the pig living body with the weight of 100kg based on MEG3 gene.
Background
The pig is the first of six livestock, and the pig raising industry plays an important role in the world, especially in the national economy of China. The specific gravity of pork in various meat products is always kept about one third, and the pork is one of the favorite non-staple foods. The life of people is continuously improved, and the traditional breeding technology can not meet the demand of people on pork. With the development of modern breeding technology, the production performance of pigs is remarkably improved, and the healthy breeding and fattening of the pigs are of great importance.
In recent years, the lean meat percentage and the growth rate of the pork are improved by introducing high lean meat percentage pig breeds in China, but the quality reduction and the flavor simplification of the pork have attracted attention of consumers. The quality and flavor of pork are also determined by the growth of skeletal muscle and fat deposition. In actual production, according to the national standard, the backfat thickness directly reflects the fat content of the pigs, and for the swinery corrected to the same weight standard, the larger the backfat thickness value is, the lower the lean meat percentage is, and the less the skeletal muscle content is. Therefore, the backfat thickness can be used as a judgment index for measuring the lean meat percentage of the pig. Therefore, how to effectively judge the back fat of the pigs in advance is a goal pursued by each pig farm operator and a breeder.
The development of molecular biotechnology has provided a new genetic marker based on DNA variation for pig breeding over the last decade. In particular, the emergence of molecular marker-assisted selection technology provides possibility for remarkably improving the quantitative traits of pigs.
Disclosure of Invention
The first purpose of the invention is to provide a method for identifying or assisting in identifying the backfat thickness of a live body of up to 100kg of weight of a pig.
The method for identifying or assisting in identifying the back fat thickness of the live body of the pig with the weight of 100kg comprises the following steps (1) and/or (2) and/or (3) and/or (4):
(1) detecting whether the genotype of a pig individual is TT genotype, AA genotype or TA genotype, and determining the back fat thickness of the pig individual in a living body with the weight of 100kg according to the genotype of the pig individual: the AA genotype of the pig individual reaches 100kg weight and the living backfat thickness is more than that of the TT genotype, and the TT genotype of the pig individual reaches 100kg weight and the living backfat thickness is more than that of the TA genotype;
the TT genotype is a homozygote of the 460 th deoxyribonucleotide of the second exon of the MEG3 gene which is T;
the TA genotype is a heterozygote of the 460 th deoxyribonucleotide of a second exon of the MEG3 gene, namely T and A;
the AA genotype is homozygote of 460 th deoxyribonucleotide of a second exon of MEG3 gene as A;
(2) detecting whether the genotype of a pig individual is an AA genotype, a GG genotype or an AG genotype, and determining the back fat thickness of the pig individual in a living body with the weight of 100kg according to the genotype of the pig individual: the AA genotype of the pig individual reaches 100kg weight and the living backfat thickness of the pig individual is more than that of the GG genotype, and the GG genotype of the pig individual reaches 100kg weight and the living backfat thickness of the pig individual is more than that of the AG genotype;
the AA genotype is a homozygote of the 847 th deoxyribonucleotide of the second exon of the MEG3 gene, wherein the A genotype is A;
the AG genotype is a heterozygote of the 847 th deoxyribonucleotide of the second exon of the MEG3 gene, namely A and G;
the GG genotype is a homozygote of the 847 th deoxyribonucleotide of the second exon of the MEG3 gene, namely G;
(3) detecting whether the genotype of a pig individual is an AA genotype, a GG genotype or an AG genotype, and determining the back fat thickness of the pig individual in a living body with the weight of 100kg according to the genotype of the pig individual: the AA genotype of the pig individual reaches 100kg weight and the living backfat thickness of the pig individual is more than that of the GG genotype, and the GG genotype of the pig individual reaches 100kg weight and the living backfat thickness of the pig individual is more than that of the AG genotype;
the AA genotype is a homozygote of 1158 th deoxyribonucleotide of a second exon of MEG3 gene, wherein A is the AA;
the AG genotype is a heterozygote of 1158 th deoxyribonucleotide of a second exon of MEG3 gene A and G;
the GG genotype is a homozygote of 1158 th deoxyribonucleotide of a second exon of MEG3 gene as G;
(4) detecting whether the genotype of a pig individual is TT genotype, CC genotype or TC genotype, and determining the back fat thickness of the pig individual in a living body with the weight of 100kg according to the genotype of the pig individual: the TT genotype of the pig individual reaches 100kg weight and the back fat thickness of the living body is more than that of the CC genotype of the pig individual, and the CC genotype of the pig individual reaches 100kg weight and the back fat thickness of the living body is more than that of the TC genotype of the pig individual;
the TT genotype is a homozygote of 51 th deoxyribonucleotide of a first intron of MEG3 gene, namely T;
the TC genotype is a hybrid of T and C at the 51 st deoxyribonucleotide of the first intron of the MEG3 gene;
the CC genotype is a homozygote of 51 th deoxyribonucleotide of a first intron of MEG3 gene, which is C;
the nucleotide sequence of the second exon of the MEG3 gene is shown as a sequence 1 in a sequence table;
the nucleotide sequence of the first intron of the MEG3 gene is shown as a sequence 2 in a sequence table.
In the above-mentioned method, the first step of the method,
in the step (1), the method for detecting whether the genotype of the pig individual is the AA genotype or the GG genotype or the AG genotype comprises the following steps A) or B):
A) direct sequencing;
B) sequencing the PCR amplification product containing 460 th deoxyribonucleotide of the second exon of MEG3 gene;
in the step (2), the method for detecting whether the genotype of the pig individual is the AA genotype or the GG genotype or the AG genotype is C) or D) as follows:
C) direct sequencing;
D) sequencing the PCR amplification product containing the 1137 th deoxyribonucleotide of the second exon of MEG3 gene;
in the step (3), the method for detecting whether the genotype of the pig individual is the AA genotype or the GG genotype or the AG genotype is as follows E) or F):
E) direct sequencing;
F) sequencing the PCR amplification product containing the 1158 th deoxyribonucleotide of the second exon of the MEG3 gene;
in the step (4), the method for detecting whether the genotype of the pig individual is TT genotype or CC genotype or TC genotype is G) or H as follows:
G) direct sequencing;
H) sequencing the PCR amplification product containing the 51 st deoxyribonucleotide of the first intron of the MEG3 gene;
the primers used for PCR amplification products in B), D) and F) are 1) or 2):
1) a primer pair A consisting of a single-stranded DNA molecule shown in a sequence 3 in the sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
2) a primer pair C consisting of a single-stranded DNA molecule shown in a sequence A and a single-stranded DNA molecule shown in a sequence B;
the sequence A is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 3 and has the same function with the sequence 3;
the sequence B is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 4 and has the same function with the sequence 4;
the primers used for the PCR amplification product in the H) are the following 3) or 4):
3) a primer pair B consisting of a single-stranded DNA molecule shown in a sequence 5 in the sequence table and a single-stranded DNA molecule shown in a sequence 6 in the sequence table;
4) a primer pair D consisting of a single-stranded DNA molecule shown in a sequence C and a single-stranded DNA molecule shown in a sequence D;
the sequence C is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 5 and has the same function as the sequence 5;
and the sequence D is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 6 and has the same function as the sequence 6.
The second object of the present invention is to provide a novel use of a substance for detecting the genotype of 460 th deoxyribonucleotide and/or 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of the second exon of MEG3 gene of a pig individual or a substance for detecting the genotype of 51 st deoxyribonucleotide of the first intron of MEG3 gene of a pig individual.
The invention provides an application of a substance for detecting the genotype of 460 th deoxyribonucleotide and/or 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual or a substance for detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of a pig individual in identifying or assisting in identifying the backfat thickness of a live body of a pig individual with the weight of 100 kg.
The invention also provides an application of a substance for detecting the genotype of 460 th deoxyribonucleotide and/or 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual or a substance for detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of a pig individual in preparing a product for identifying or assisting in identifying the back fat thickness of a pig individual in a living body with the weight of 100 kg.
The invention also provides an application of a substance for detecting the genotype of 460 th deoxyribonucleotide and/or 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual or a substance for detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of a pig individual in pig breeding.
The invention also provides an application of a substance for detecting the genotype of 460 th deoxyribonucleotide and/or 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual or a substance for detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of a pig individual in preparing a product for pig breeding.
The invention also provides an application of a substance for detecting the genotype of 460 th deoxyribonucleotide and/or 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual or a substance for detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of a pig individual in breeding of a pig with high lean meat percentage and/or high skeletal muscle content and/or short back fat thickness of 100kg weight living body of the pig.
The invention also provides an application of a substance for detecting the genotype of 460 th deoxyribonucleotide and/or 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual or a substance for detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of a pig individual in the preparation of products for breeding pigs with high lean meat percentage and/or high skeletal muscle content and/or short backfat thickness of 100kg weight living bodies of pigs.
The third purpose of the invention is to provide a product for identifying the back fat thickness of a live pig individual with the weight of 100 kg.
The product for identifying the back fat thickness of the live body of 100kg weight of the pig individual is a substance for detecting the genotype of 460 th deoxyribonucleotide and/or 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of a second exon of an MEG3 gene of the pig individual or a substance for detecting the genotype of 51 st deoxyribonucleotide of a first intron of an MEG3 gene of the pig individual.
In the above application or product, the substance for detecting the genotype of the 460 th deoxyribonucleotide and/or the 1137 th deoxyribonucleotide and/or the 1158 th deoxyribonucleotide of the second exon of the MEG3 gene of the pig individual or the substance for detecting the genotype of the 51 st deoxyribonucleotide of the first intron of the MEG3 gene of the pig individual is X1) -X6 as follows:
x1) is composed of a single-stranded DNA molecule shown in a sequence 3 in the sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
x2) is composed of a single-stranded DNA molecule shown in the sequence 5 in the sequence table and a single-stranded DNA molecule shown in the sequence 6 in the sequence table;
x3) is composed of a single-stranded DNA molecule shown in the sequence A and a single-stranded DNA molecule shown in the sequence B;
the sequence A is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 3 and has the same function with the sequence 3;
the sequence B is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 4 and has the same function with the sequence 4;
x4) is composed of a single-stranded DNA molecule shown in a sequence C and a single-stranded DNA molecule shown in a sequence D;
the sequence C is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 5 and has the same function as the sequence 5;
the sequence D is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 6 and has the same function with the sequence 6;
x5) contains the PCR reagents of X1) the primer pair A or X2) the primer pair B or X3) the primer pair C or X4) the primer pair D;
x6) contains X1) the primer pair A or X2) the primer pair B or X3) the primer pair C or X4) the primer pair D or X5) the PCR reagent kit.
The fourth purpose of the invention is to provide a method for breeding pigs with high lean meat percentage and/or high skeletal muscle content and/or short back fat thickness of 100kg weight of the pigs.
The method for breeding the boar with high lean meat percentage and/or high skeletal muscle content and/or short pig body backfat thickness of 100kg body weight comprises the steps of selecting the boar with TC genotype and/or TA genotype and/or AG genotype for breeding;
the TA genotype is a heterozygote of the 460 th deoxyribonucleotide of a second exon of the MEG3 gene, namely T and A;
the TC genotype is a hybrid of T and C at the 51 st deoxyribonucleotide of the first intron of the MEG3 gene;
the AG genotype is a heterozygote of 1137 th deoxyribonucleotide and/or 1158 th deoxyribonucleotide of a second exon of MEG3 gene, wherein A and G are/is used as the AG genotype;
the nucleotide sequence of the second exon of the MEG3 gene is shown as a sequence 1 in a sequence table;
the nucleotide sequence of the first intron of the MEG3 gene is shown as a sequence 2 in a sequence table.
The invention discovers that the T460A, A1137G and A1158G sites of the second exon of the pig MEG3 gene and the T51C site of the first intron of the MEG3 gene have a remarkable influence on the back fat thickness of 100kg weight of a pig in vivo, and provides a method for identifying or assisting in identifying the back fat thickness of 100kg weight of the pig in vivo based on the T51C, T460A, A1137G and A1158G sites. Experiments prove that: the method of the invention is consistent with the actual measurement result of the back fat thickness of the live body with the weight of 100kg of the pig, and has important significance for selecting excellent pig breeds.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The live backfat thickness of 100kg body weight of the pig in the following examples is the value of the live backfat thickness when the weight of the pig reaches 100 kg.
Example 1 method for assisting in identifying the backfat thickness of a live pig having a weight of up to 100kg
Screening of SNP sites of pig MEG3 gene
1. Extraction of DNA from a tissue sample of pig ear
Blood samples from 32 Changbai pigs from Hebei pig farms were collected, stored in 75% alcohol, and stored at-20 ℃. The method comprises the following steps of extracting the genome DNA of a blood sample by adopting a phenol-chloroform extraction method:
(1) preparation of ear tissue samples: cutting ear tissue sample with appropriate size, washing with normal saline to remove impurities and blood stain on surface, placing in 1.5ml centrifuge tube, cutting with ophthalmic scissors, and adding 500uL of tissue lysate (Beijing Baitai biological technology, Inc.; product number: AU 19011);
(2) adding 15-25 uL of proteinase K (SIGMA-ALDRICH; product number: SRE0005) into the centrifuge tube according to the size of the cut ear tissue sample;
(3) placing on a water bath shaker, digesting overnight at 55 ℃ to ensure that the ear tissue sample is completely digested to obtain completely digested ear tissue;
(4) placing the digested ear tissue sample at room temperature, adding equal volume of Tris-saturated phenol (Jiangsu Baolai Biotechnology Co., Ltd.), turning over, reversing and mixing for 5 min;
(5) when the mixture is centrifuged at 14000rpm/min for 10min, the centrifugal tube can generate a layering phenomenon, the upper layer is a water phase containing nucleic acid, and the lower layer is an organic phase containing other impurities.
(6) Carefully sucking the upper aqueous phase containing the nucleic acid by using a micropipette, placing the upper aqueous phase into a new 1.5ml centrifuge tube (preferably a blue pipette tip with a tip removed to avoid sucking up the lower organic phase), and discarding the lower organic phase;
(7) repeating the steps (4) to (6);
(8) to the centrifuge tube containing the aqueous phase was added an equal volume of saturated phenol: chloroform-1: 1, repeatedly reversing and uniformly mixing for 5min, centrifuging for 10min at 14000rpm/min, then sucking the upper layer liquid, and placing the upper layer liquid in a new 1.5mL centrifuge tube;
(9) repeating the step (8);
(10) to this was added saturated phenol: chloroform: the volume ratio of isoamyl alcohol is 25: 24: 1, turning over, reversing, uniformly mixing, and centrifuging at 14000rpm/min for 10 min;
(11) absorbing the upper layer liquid, adding 2 times of anhydrous ethanol (pre-cooled in a refrigerator at the temperature of minus 20 ℃) to precipitate DNA, and generating filiform or flocculent DNA precipitation in a centrifugal tube at the moment;
(12) carefully picking out filiform or flocculent DNA precipitate with a yellow gun head, placing in a new 1.5mL EP tube, adding 70% ethanol (-precooling at 20 deg.C) 500uL, washing DNA precipitate, gently inverting for 30s, and discarding 70% ethanol;
(13) repeating the step (12);
(14) placing the centrifugal tube containing the DNA precipitate in a room temperature environment, and drying for 20-30 min;
(15) the DNA precipitate was dissolved by adding 60 to 80uL of TE buffer or double distilled water (without vigorous shaking or centrifugation), and stored in a freezer at-20 ℃.
2. Amplification and sequencing of fragments of interest
(1) PCR amplification
And (3) performing PCR amplification by using the genomic DNA obtained in the step (1) as a template and respectively adopting a primer pair F1 and R1 and a primer pair F2 and R2 to obtain a PCR amplification product. The primer pair F1 and R1 were amplified to obtain the second exon (SEQ ID NO: 1) of MEG3 gene, and the primer pair F2 and R2 were amplified to obtain the first intron (SEQ ID NO: 2) of MEG3 gene. The primer sequences are as follows:
f1: ATGCCTGACGCAGTAACAA (SEQ ID NO: 3);
r1: CCGCTTCCTTCATCCCTC (SEQ ID NO: 4);
f2: AGCGACGTATTTAAGGT (SEQ ID NO: 5);
r2: TCTCCCACAGCACTCC (SEQ ID NO: 6).
PCR amplification System: 10 × LA PCR Buffer 2ul, 10mM dNTP Mix 1.6ul, upstream and downstream primers (10pmol/L) each 1ul, LA Taq DNA polymerase (5U/ul), genomic DNA 1ul, ddH2O 13.2.2 ul.
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C, annealing at 58 deg.C for 30s, extension at 72 deg.C for 10 min.
(2) Recovery and purification of PCR product
The PCR product was recovered and purified using an agarose gel purification recovery kit (Beijing Baitacg Biotechnology Co., Ltd.), and the specific procedures were in accordance with the instructions attached to the kit.
(3) Ligation reaction
The PCR amplification product recovered by the above purification was ligated with pMD18-T vector. The ligation reaction system was 5 μ L: PCR recycling product 2ul, pMD18-T carrier 0.5ul, Solution I2.5 ul, 4 ℃ connecting overnight, get the connecting product.
(4) Transformation of
The specific operation steps comprise taking 1 DH5 α competent cell (100 mu L) from an ultra-low temperature refrigerator at minus 80 ℃, rapidly placing on ice to melt the cell, adding the ligation product (volume of 5uL) into the competent cell, gently and repeatedly blowing and beating the cell by using a pipette to uniformly mix the cell, placing the cell on ice, standing for 30min, placing the cell in a water bath at 42 ℃, thermally shocking the cell for 90s, immediately carrying out ice bath for 2-3 min, adding 600 mu L of liquid LB culture medium (without ampicillin) into the cell, placing the cell in a constant temperature shaking table at 37 ℃, and culturing for 1h at the rotating speed of 170-200 r/min to recover the bacteria.
(5) Positive clone identification
Sucking 50-100 mu L of the recovered bacterial liquid by a pipette, uniformly coating the bacterial liquid on an agar plate containing ampicillin, putting the bacterial liquid in a constant-temperature incubator at 37 ℃ for 30min after the bacterial liquid is completely absorbed, so that the bacterial liquid is completely absorbed, then inversely placing the agar plate in the constant-temperature incubator, and culturing at 37 ℃ overnight.
And (4) selecting bacteria according to the growth condition of the colonies on the agar plate. Adding 1mL of liquid LB culture medium into 1.5mL of EP tubes, adding 2 μ L of ampicillin into each EP tube, picking 20 white colonies (selecting colonies with complete shapes and round dots) on agar plates by using a 10 μ L tip, respectively placing the white colonies into 1.5mL of EP tubes containing 1mL of liquid culture medium, placing the white colonies into a shaker at 37 ℃, carrying out amplification culture for 3-4 h at the rotating speed of 220R/min, and carrying out bacteria liquid PCR identification by adopting a primer pair F1 and R1 and a primer pair F2 and R2 when a turbidity phenomenon or white filamentous precipitates appear in the EP tubes.
The PCR reaction system is as follows: 10 × LA Buffer 1ul, dNTP Mix (2.5mM)0.8ul, upstream and downstream primers (pmol/L) 0.5ul each, LA polymerase 0.1ul, bacterial suspension 0.5ul, ddH2O 6.6.6 ul, and the total is 10 ul.
After the PCR reaction is finished, 1 microliter of PCR product is absorbed for 1.5 percent agarose gel electrophoresis detection, and the positive clone is preliminarily determined by taking a picture by an agarose gel imager.
(6) Sequencing validation
The bacterial liquid preliminarily identified as positive clone by PCR amplification is delivered to Shanghai Ying Jie Co., Ltd for sequencing.
(7) Acquisition of SNP sites (T51C, T460A, A1137G and A1158G)
According to the sequencing results, the 4 differential sites are obtained by comparing with DNAman software, namely T460A (460 th nucleotide of the sequence 1), A1137G (1137 th nucleotide of the sequence 1), A1158G (1158 th nucleotide of the sequence 1) and T51C (51 th nucleotide of the sequence 2).
The nucleotide at the T460A site of the second exon (sequence 1) of the pig MEG3 gene is T, the individual is homozygous, the genotype of the individual is named as homozygous TT genotype, the nucleotide at the T460A site of the second exon (sequence 1) of the pig MEG3 gene is A, the individual is homozygous, the genotype of the individual is named as homozygous AA genotype, the nucleotide at the T460A site of the second exon (sequence 1) of the pig MEG3 gene is T and A, the individual is heterozygous, and the genotype of the individual is named as heterozygous TA genotype.
The base of the A1137G site of the second exon (sequence 1) of the pig MEG3 gene is A, the individual is homozygous, the genotype of the individual is named as homozygous AA genotype, the base of the A1137G site of the second exon (sequence 1) of the pig MEG3 gene is G, the individual is homozygous, the genotype of the individual is named as homozygous GG genotype, the base of the A1137G site of the second exon (sequence 1) of the pig MEG3 gene is A and G, the individual is heterozygous, and the genotype of the individual is named as heterozygous AG genotype.
The base of the A1158G site of the second exon (sequence 1) of the pig MEG3 gene is A, the individual is homozygous, the genotype of the individual is named as homozygous AA genotype, the base of the A1158G site of the second exon (sequence 1) of the pig MEG3 gene is G, the individual is homozygous, the genotype of the individual is named as homozygous GG genotype, the base of the A1158G site of the second exon (sequence 1) of the pig MEG3 gene is A and G, the individual is heterozygous, and the genotype of the individual is named as heterozygous AG genotype.
The nucleotide at the T51C site of the first intron (sequence 2) of the pig MEG3 gene is T, the individual is homozygous and named as homozygous TT genotype, the nucleotide at the T51C site of the first intron (sequence 2) of the pig MEG3 gene is C, the individual is homozygous and named as homozygous CC genotype, the nucleotide at the T51C site of the first intron (sequence 2) of the pig MEG3 gene is T and C, the individual is heterozygous and named as heterozygous TC genotype.
Second, the correlation analysis of T51C, T460A, A1137G and A1158G of pig MEG3 gene and the back fat thickness of the live body of 100kg body weight of pig
Correlation analysis of T460A locus of second exon of (I) pig MEG3 gene and pig back fat thickness of 100kg body weight in vivo
In order to determine whether the T460A locus of the second exon of the pig MEG3 gene is related to the backfat thickness of 100kg weight of the live pig, 294 big white pigs in a Hebei pig farm are taken as experimental materials, the genotype of each pig individual is determined to be the TT genotype, the AA genotype or the TA genotype according to the method in the step one, and the backfat thickness of 100kg weight of the live pig is determined according to the genotype of the pig: the AA genotype of the pig individual reaches 100kg weight and the living backfat thickness is more than that of the TT genotype, and the TT genotype of the pig individual reaches 100kg weight and the living backfat thickness is more than that of the TA genotype.
1. Genotype(s)
The genotype detection result of 294 pig individuals shows that: the genotype of 288 pigs is TT genotype, the genotype of 2 pigs is AA genotype, and the genotype of 4 pigs is TA genotype. The results of detecting the genotype frequency and the allele frequency of the pig MEG3 gene in the swinery are shown in Table 1: as can be seen from table 1: the genotype frequency of TT homozygote is obviously higher than that of AA homozygote and TA heterozygote, and T allele is dominant gene.
TABLE 1 detection of genotype frequency and allele frequency of pig MEG3 gene in swinery
2. Correlation analysis of pig genotype and pig up to 100kg body weight in vivo backfat thickness
Statistical analysis of genotype and pig up to 100kg body weight in vivo backfat thickness was performed using SPSS 20.0 software, and multiple comparisons between samples were made.
The results are shown in table 2: the SNP (T460A locus) site has a significant influence on the pig body backfat thickness of 100kg, the pig body backfat thickness of 100kg with the AA genotype is more than that of the pig body with the TT genotype, the pig body backfat thickness of 100kg with the TT genotype is more than that of the pig body with the TA genotype, and the pig body backfat thickness of 100kg with the AA genotype is significantly more than that of the pig body with the TA genotype by about 7.17 mm.
TABLE 2 correlation analysis of single nucleic acid polymorphism of pig MEG3 gene with up to 100kg body weight in vivo backfat thickness
Note that significant differences are indicated in the table by different lower case letters (P <0.05), insignificant differences are indicated by the same letters (P >0.05), and values are expressed as least squares means. + -. standard error.
In summary, the nucleotide at the T460A locus of the second exon of the pig MEG3 gene can be determined to determine whether the pig individual is TT genotype, TA genotype or AA genotype, thereby assisting in identifying the back fat thickness of the live body of 100kg weight of the pig: the AA genotype of the pig has 100kg weight and more living backfat thickness than the TT genotype, and the TT genotype of the pig has 100kg weight and more living backfat thickness than the TA genotype;
the TT genotype is homozygote of nucleotide 460 of the second exon of the pig MEG3 gene as T;
the nucleotide of the AA genotype at 460 th site of the second exon of the pig MEG3 gene is homozygote of A;
the TA genotype is a heterozygote of the 460 th nucleotide of the second exon of the pig MEG3 gene as T and A;
the nucleotide sequence of the second exon of MEG3 gene is shown as sequence 1 in the sequence table.
Correlation analysis of A1137G locus of second exon of (II) pig MEG3 gene and pig back fat thickness of 100kg body weight in vivo
In order to determine whether the A1137G locus of the second exon of the pig MEG3 gene is related to the backfat thickness of 100kg weight of the living body of the pig, 294 big white pigs in a Hebei pig farm are taken as experimental materials, the genotype of each pig individual is determined to be the AA genotype, the GG genotype or the AG genotype according to the method in the step one, and the backfat thickness of 100kg weight of the living body of the pig is determined according to the genotype of the pig: the AA genotype of the pig individual reaches 100kg body weight and has more living backfat thickness than the GG genotype of the pig individual, and the GG genotype of the pig individual reaches 100kg body weight and has more living backfat thickness than the AG genotype of the pig individual.
1. Genotype(s)
The genotype detection result of 294 pig individuals shows that: the 26 pig genotype is AA genotype, the 164 pig genotype is GG genotype, and the 104 pig genotype is AG genotype. The results of detecting the genotype frequency and the allele frequency of the pig MEG3 gene in the swinery are shown in Table 3: as can be seen from table 3: the genotype frequency of GG homozygous type and AG heterozygous type is obviously higher than that of AA homozygous type and G allele which is a dominant gene.
TABLE 3 genotype frequency and allele frequency of pig MEG3 gene in swine herd
2. Correlation analysis of pig genotype and pig up to 100kg body weight in vivo backfat thickness
Statistical analysis of genotype and pig up to 100kg body weight in vivo backfat thickness was performed using SPSS 20.0 software, and multiple comparisons between samples were made.
The results are shown in Table 4: the SNP (A1137G site) site has a significant influence on the pig body backfat thickness of 100kg, the pig body backfat thickness of 100kg of weight of the AA genotype is more than that of the pig body with the GG genotype, the pig body backfat thickness of 100kg of weight of the TT genotype is more than that of the AG genotype, and the pig body backfat thickness of 100kg of weight of the AA genotype is significantly more than that of the AG genotype by about 1.23 mm.
TABLE 4 correlation analysis of pig MEG3 gene single nucleic acid polymorphism and up to 100kg body weight in vivo backfat thickness
Note that significant differences are indicated in the table by different lower case letters (P <0.05), insignificant differences are indicated by the same letters (P >0.05), and values are expressed as least squares means. + -. standard error.
In summary, the nucleotide at the A1137G locus of the second exon of the pig MEG3 gene can be determined to determine whether the pig individual is AA genotype, GG genotype or AG genotype, thereby assisting in identifying the back fat thickness of the pig in the living body with the weight of 100 kg: the AA genotype of the pig has 100kg body weight and more living backfat thickness than the GG genotype of the pig, and the TT genotype of the pig has 100kg body weight and more living backfat thickness than the AG genotype of the pig;
the nucleotide of the AA genotype at 1137 th site of the second exon of the pig MEG3 gene is homozygote of A;
the GG genotype is homozygote of nucleotide 1137 of second exon of pig MEG3 gene, and the nucleotide is G;
the AG genotype is a heterozygote of 1137 th nucleotide of a second exon of the pig MEG3 gene, A and G;
the nucleotide sequence of the second exon of MEG3 gene is shown as sequence 1 in the sequence table.
Correlation analysis of A1158G locus of second exon of (III) pig MEG3 gene and pig back fat thickness of 100kg body weight in vivo
In order to determine whether the A1158G locus of the second exon of the pig MEG3 gene is related to the backfat thickness of 100kg weight of the living body of the pig, 295 big white pigs in a Hebei pig farm are taken as experimental materials, the genotype of each pig individual is determined to be the AA genotype, the GG genotype or the AG genotype according to the method in the step one, and the backfat thickness of 100kg weight of the living body of the pig is determined according to the genotype of the pig: the AA genotype of the pig individual reaches 100kg body weight and has more living backfat thickness than the GG genotype of the pig individual, and the GG genotype of the pig individual reaches 100kg body weight and has more living backfat thickness than the AG genotype of the pig individual.
1. Genotype(s)
The genotype detection result of 295 pigs shows that: the genotype of 25 pigs is AA genotype, the genotype of 164 pigs is GG genotype, and the genotype of 106 pigs is AG genotype. The results of detecting the genotype frequency and the allele frequency of the pig MEG3 gene in the swinery are shown in Table 1: as can be seen from table 5: the genotype frequency of GG homozygous type and AG heterozygous type is obviously higher than that of AA homozygous type, and G allele is dominant gene.
TABLE 5 genotype frequency and allele frequency of pig MEG3 gene in swine herd
2. Correlation analysis of pig genotype and pig up to 100kg body weight in vivo backfat thickness
Statistical analysis of genotype and pig up to 100kg body weight in vivo backfat thickness was performed using SPSS 20.0 software, and multiple comparisons between samples were made.
The results are shown in Table 6: the SNP (A1158G site) site has a significant influence on the pig body backfat thickness of 100kg, the pig body backfat thickness of 100kg of the AA genotype is more than that of the pig body with the GG genotype, the pig body backfat thickness of 100kg of the GG genotype is more than that of the AG genotype, and the pig body backfat thickness of 100kg of the AA genotype is significantly more than that of the AG genotype by about 1.25 mm.
TABLE 6 correlation analysis of pig MEG3 Gene single nucleic acid polymorphism with Up to 100kg body weight in vivo backfat thickness
Note that significant differences are indicated in the table by different lower case letters (P <0.05), insignificant differences are indicated by the same letters (P >0.05), and values are expressed as least squares means. + -. standard error.
In summary, the nucleotide at the A1158G site of the second exon of the pig MEG3 gene can be determined to determine whether the pig individual is AA genotype, GG genotype or AG genotype, so as to assist in identifying the back fat thickness of the pig in the living body with the weight of 100 kg: the AA genotype of the pig has 100kg body weight and more living backfat thickness than the GG genotype of the pig individual, and the GG genotype of the pig has 100kg body weight and more living backfat thickness than the AG genotype of the pig individual;
the AA genotype is a homozygote of the 1158 th nucleotide of the second exon of the pig MEG3 gene, and the nucleotide is A;
the GG genotype is a homozygote of the 1158 th nucleotide of the second exon of the pig MEG3 gene, which is G;
the AG genotype is a heterozygote of the 1158 th nucleotide of the second exon of the pig MEG3 gene as A and G;
the nucleotide sequence of the second exon of MEG3 gene is shown as sequence 1 in the sequence table.
Correlation analysis of T51C locus of first intron of (fourth) pig MEG3 gene and pig back fat thickness of 100kg body weight in vivo
In order to determine whether the T51C locus of the first intron of the pig MEG3 gene is related to the backfat thickness of the pig in the living body with the weight of 100kg, 295 big white pigs in a Hebei pig farm are taken as experimental materials, the genotype of each pig individual is determined to be the TT genotype, the CC genotype or the TC genotype according to the method in the step one, and the backfat thickness of the pig in the living body with the weight of 100kg is determined according to the genotype of the pig: the TT genotype of the pig individual reaches 100kg weight and the back fat thickness of the living body is more than that of the CC genotype of the pig individual, and the CC genotype of the pig individual reaches 100kg weight and the back fat thickness of the living body is more than that of the TC genotype of the pig individual.
1. Genotype(s)
The genotype detection result of 295 pigs shows that: the 26 pig genotype is TT genotype, the 163 pig genotype is CC genotype, and the 106 pig genotype is TC genotype. The results of detecting the genotype frequency and the allele frequency of the pig MEG3 gene in the swinery are shown in Table 1: as can be seen from table 7: the genotype frequency of CC homozygous and CT heterozygous is obviously higher than that of TT homozygous, and C allele is a dominant gene.
TABLE 7 genotype frequency and allele frequency of pig MEG3 gene in swine herd
2. Correlation analysis of pig genotype and pig up to 100kg body weight in vivo backfat thickness
Statistical analysis of genotype and pig up to 100kg body weight in vivo backfat thickness was performed using SPSS 20.0 software, and multiple comparisons between samples were made.
The results are shown in Table 8: the SNP (T51C) site has a significant influence on the pig body backfat thickness of 100kg, the pig body with the TT genotype reaches 100kg, the body backfat thickness of the pig body is more than that of the pig individual with the CC genotype, the pig body with the CC genotype reaches 100kg, the body backfat thickness of the pig body is more than that of the pig individual with the TC genotype, and the pig body with the TT genotype reaches 100kg, the body backfat thickness of the pig body is more than that of the TC genotype by about 1.46 mm.
TABLE 8 correlation analysis of pig MEG3 gene single nucleic acid polymorphism and up to 100kg body weight in vivo backfat thickness
Note that significant differences are indicated in the table by different lower case letters (P <0.05), insignificant differences are indicated by the same letters (P >0.05), and values are expressed as least squares means. + -. standard error.
In summary, the nucleotide at the T51C site of the first intron of the pig MEG3 gene can be determined to determine whether the TT genotype, the CC genotype or the TC genotype of a pig individual, so as to assist in identifying the back fat thickness of a pig with 100kg weight in vivo: the TT genotype of the pig has 100kg weight and more living backfat thickness than the CC genotype, and the CC genotype of the pig has 100kg weight and more living backfat thickness than the TC genotype;
the TT genotype is homozygote of the 51 st nucleotide of the first intron of the pig MEG3 gene, namely T;
the CC genotype is homozygote of 51 th nucleotide of a first intron of the pig MEG3 gene, which is A;
the TC gene is a hybrid of the 51 st nucleotide of the first intron of the pig MEG3 gene, T and A;
the nucleotide sequence of the first intron of MEG3 gene is shown as sequence 2 in the sequence table.
Claims (6)
1. A method for identifying or assisting in identifying the backfat thickness of a live pig with the weight of 100kg comprises the following steps (1) and (2) and (3) and (4):
(1) detecting whether the genotype of a pig individual is TT genotype, AA genotype or TA genotype, and determining the back fat thickness of the pig individual in a living body with the weight of 100kg according to the genotype of the pig individual: the AA genotype of the pig individual reaches 100kg weight and the living backfat thickness is more than that of the TT genotype, and the TT genotype of the pig individual reaches 100kg weight and the living backfat thickness is more than that of the TA genotype;
the TT genotype is a homozygote of the 460 th deoxyribonucleotide of the second exon of the MEG3 gene which is T;
the TA genotype is a heterozygote of the 460 th deoxyribonucleotide of a second exon of the MEG3 gene, namely T and A;
the AA genotype is homozygote of 460 th deoxyribonucleotide of a second exon of MEG3 gene as A;
(2) detecting whether the genotype of a pig individual is an AA genotype, a GG genotype or an AG genotype, and determining the back fat thickness of the pig individual in a living body with the weight of 100kg according to the genotype of the pig individual: the AA genotype of the pig individual reaches 100kg weight and the living backfat thickness of the pig individual is more than that of the GG genotype, and the GG genotype of the pig individual reaches 100kg weight and the living backfat thickness of the pig individual is more than that of the AG genotype;
the AA genotype is a homozygote of 1137 th deoxyribonucleotide of a second exon of MEG3 gene, wherein A is the AA;
the AG genotype is a heterozygote of 1137 th deoxyribonucleotide of a second exon of MEG3 gene A and G;
the GG genotype is a homozygote of 1137 th deoxyribonucleotide of a second exon of MEG3 gene, namely G;
(3) detecting whether the genotype of a pig individual is an AA genotype, a GG genotype or an AG genotype, and determining the back fat thickness of the pig individual in a living body with the weight of 100kg according to the genotype of the pig individual: the AA genotype of the pig individual reaches 100kg weight and the living backfat thickness of the pig individual is more than that of the GG genotype, and the GG genotype of the pig individual reaches 100kg weight and the living backfat thickness of the pig individual is more than that of the AG genotype;
the AA genotype is a homozygote of 1158 th deoxyribonucleotide of a second exon of MEG3 gene, wherein A is the AA;
the AG genotype is a heterozygote of 1158 th deoxyribonucleotide of a second exon of MEG3 gene A and G;
the GG genotype is a homozygote of 1158 th deoxyribonucleotide of a second exon of MEG3 gene as G;
(4) detecting whether the genotype of a pig individual is TT genotype, CC genotype or TC genotype, and determining the back fat thickness of the pig individual in a living body with the weight of 100kg according to the genotype of the pig individual: the TT genotype of the pig individual reaches 100kg weight and the back fat thickness of the living body is more than that of the CC genotype of the pig individual, and the CC genotype of the pig individual reaches 100kg weight and the back fat thickness of the living body is more than that of the TC genotype of the pig individual;
the TT genotype is a homozygote of 51 th deoxyribonucleotide of a first intron of MEG3 gene, namely T;
the TC genotype is a hybrid of T and C at the 51 st deoxyribonucleotide of the first intron of the MEG3 gene;
the CC genotype is a homozygote of 51 th deoxyribonucleotide of a first intron of MEG3 gene, which is C.
2. The method of claim 1, wherein:
in the step (1), the method for detecting whether the genotype of the pig individual is TT genotype or AA genotype or TA genotype comprises the following steps A) or B):
A) direct sequencing;
B) sequencing the PCR amplification product containing 460 th deoxyribonucleotide of the second exon of MEG3 gene;
in the step (2), the method for detecting whether the genotype of the pig individual is the AA genotype or the GG genotype or the AG genotype is C) or D) as follows:
C) direct sequencing;
D) sequencing the PCR amplification product containing the 1137 th deoxyribonucleotide of the second exon of MEG3 gene;
in the step (3), the method for detecting whether the genotype of the pig individual is the AA genotype or the GG genotype or the AG genotype is as follows E) or F):
E) direct sequencing;
F) sequencing the PCR amplification product containing the 1158 th deoxyribonucleotide of the second exon of the MEG3 gene;
in the step (4), the method for detecting whether the genotype of the pig individual is TT genotype or CC genotype or TC genotype is G) or H as follows:
G) direct sequencing;
H) sequencing the PCR amplification product containing the 51 st deoxyribonucleotide of the first intron of the MEG3 gene;
the primers used for PCR amplification products in B), D) and F) are 1) or 2):
1) a primer pair A consisting of a single-stranded DNA molecule shown in a sequence 3 in the sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
2) a primer pair C consisting of a single-stranded DNA molecule shown in a sequence A and a single-stranded DNA molecule shown in a sequence B;
the sequence A is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 3 and has the same function with the sequence 3; the sequence B is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 4 and has the same function with the sequence 4;
the primers used for the PCR amplification product in the H) are the following 3) or 4):
3) a primer pair B consisting of a single-stranded DNA molecule shown in a sequence 5 in the sequence table and a single-stranded DNA molecule shown in a sequence 6 in the sequence table;
4) a primer pair D consisting of a single-stranded DNA molecule shown in a sequence C and a single-stranded DNA molecule shown in a sequence D;
the sequence C is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 5 and has the same function as the sequence 5; and the sequence D is a nucleotide which is obtained by deleting or adding or changing one or more nucleotides in the sequence 6 and has the same function as the sequence 6.
3. The application of the substance for detecting the genotypes of the 460 th deoxyribonucleotide, the 1137 th deoxyribonucleotide and the 1158 th deoxyribonucleotide of the second exon of the MEG3 gene of a pig individual and the substance for detecting the genotype of the 51 st deoxyribonucleotide of the first intron of the MEG3 gene of the pig individual in identifying or assisting in identifying the backfat thickness of a live pig body with the weight of 100 kg;
or detecting the genotype of 460 th deoxyribonucleotide, 1137 th deoxyribonucleotide and 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual and detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of the pig individual in preparing a product for identifying or assisting in identifying the backfat thickness of a live body of the pig individual reaching 100kg body weight.
4. The application of the substance for detecting the genotypes of the 460 th deoxyribonucleotide, the 1137 th deoxyribonucleotide and the 1158 th deoxyribonucleotide of the second exon of the MEG3 gene of a pig individual and the substance for detecting the genotype of the 51 st deoxyribonucleotide of the first intron of the MEG3 gene of the pig individual in pig breeding;
or detecting the genotype of 460 th deoxyribonucleotide, 1137 th deoxyribonucleotide and 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual and detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of the pig individual in preparing a product for pig breeding.
5. The application of the substance for detecting the genotypes of the 460 th deoxyribonucleotide, the 1137 th deoxyribonucleotide and the 1158 th deoxyribonucleotide of the second exon of the MEG3 gene of a pig individual and the substance for detecting the genotype of the 51 st deoxyribonucleotide of the first intron of the MEG3 gene of the pig individual in breeding of a pig with high lean meat percentage and/or high skeletal muscle content and/or short backfat of 100kg weight living body of the pig;
or detecting the genotype of 460 th deoxyribonucleotide, 1137 th deoxyribonucleotide and 1158 th deoxyribonucleotide of a second exon of MEG3 gene of a pig individual and detecting the genotype of 51 st deoxyribonucleotide of a first intron of MEG3 gene of the pig individual, in the preparation of products for breeding pigs with high lean meat percentage and/or high skeletal muscle content and/or short back fat thickness of 100kg weight living body of the pig.
6. A method for breeding boars with high lean meat percentage and/or high skeletal muscle content and/or short back fat thickness of 100kg weight living body comprises selecting the pigs with TC genotype, TA genotype and AG genotype for breeding;
the TA genotype is a heterozygote of the 460 th deoxyribonucleotide of a second exon of the MEG3 gene, namely T and A;
the TC genotype is a hybrid of T and C at the 51 st deoxyribonucleotide of the first intron of the MEG3 gene;
the AG genotype is a heterozygote of 1137 th deoxyribonucleotide and 1158 th deoxyribonucleotide of a second exon of MEG3 gene, wherein A and G are heterozygotes.
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