CN107151691B - Method for identifying day age and eye muscle area of 100kg body weight of pig based on BMP7 gene - Google Patents
Method for identifying day age and eye muscle area of 100kg body weight of pig based on BMP7 gene Download PDFInfo
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Abstract
The invention discloses a method for identifying the day age and the eye muscle area of a pig with the weight of 100 kg. The method of the invention is (1) or (2) as follows: (1) detecting whether the 422 th deoxyribonucleotide of the fourth intron of the BMP7 gene of the pig individual is T or C or T and C; (2) and detecting whether the 241 th deoxyribonucleotide of the fifth intron of the BMP7 gene of the pig individual is T or C or T and C. The test proves that: the method for identifying the day age of 100kg body weight and the eye muscle area of the pig by using the 422 th single nucleotide polymorphism of the fourth intron and the 241 th single nucleotide polymorphism of the fifth intron of the BMP7 gene is consistent with the actual measurement result of the day age of 100kg body weight and the eye muscle area of the pig, and has important significance for selecting excellent pig breeds.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a method for identifying the day age and the eye muscle area of a pig with the weight of 100kg based on a BMP7 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.
The day-old pig with the weight of 100kg is an important index for evaluating the growth speed of the pig, and the utilization rate of the feed can be indirectly reflected. A large amount of data show that the day age of the pigs with the weight of 100kg is shortened, the growth speed is higher, the production cost of a pig farm can be reduced, and the economic effect of the pig farm can be obviously improved. The heritability is an important parameter of quantitative character, and the genetic progress and the individual breeding value can be predicted according to the heritability. Therefore, how to effectively improve the growth speed of the pigs, shorten the weight day age of 100kg, and make judgment in advance is a target pursued by each pig farm operator and breeder.
The pig backfat thickness and the eye muscle area are directly related to the pig lean meat percentage and are important index parameters in genetic breeding and performance identification of pigs, and the larger the eye muscle area of the pig is, the higher the lean meat percentage is. The heritability is an important parameter of quantitative character, and the genetic progress and the individual breeding value can be predicted according to the heritability. Therefore, how to increase the eye muscle area and increase the lean meat percentage and make a judgment in advance is a goal pursued by each pig farm operator and 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 day age of the pig with the weight of 100kg and/or the eye muscle area character of the pig with the weight of 100 kg.
The method for identifying or assisting in identifying the day-old pig with the weight of 100kg and/or the eye muscle area character of the pig with the weight of 100kg comprises the following steps (1) and/or (2) and/or (3):
(1) detecting whether the genotype of a pig individual is TT genotype, CC genotype or TC genotype, determining the day age of the pig individual with the weight of 100kg according to the genotype of the pig individual: the pig individual with the TC gene type reaches 100kg, the weight of the pig individual with the day age is more than that of the pig individual with the CC gene type, and the pig individual with the CC gene type reaches 100kg, the weight of the pig individual with the day age is more than that of the pig individual with the TT gene type;
(2) detecting whether the genotype of a pig individual is TT genotype, CC genotype or TC genotype, and determining the eye muscle area of the pig individual with the weight of 100kg according to the genotype of the pig individual: the CC gene type pig individual reaches 100kg weight, the eye muscle area of which is more than that of the TT gene type pig individual, and the TT gene type pig individual reaches 100kg weight, the eye muscle area of which is more than that of the TC gene type pig individual;
(3) detecting whether the genotype of a pig individual is TT genotype, CC genotype or TC genotype, determining the day age of the pig individual with the weight of 100kg according to the genotype of the pig individual: the CC genotype pig individual reaches 100kg weight day age and is more than the TC genotype, and the TC genotype pig individual reaches 100kg weight day age and is more than the TT genotype;
in the 1) and 2), the TT genotype is a homozygote of the 422 th deoxyribonucleotide of the fourth intron of the BMP7 gene, namely T; the CC genotype is homozygote of 422 th deoxyribonucleotide of a fourth intron of a BMP7 gene, which is C; the TC genotype is a heterozygote of a 422 th deoxyribonucleotide of a fourth intron of a BMP7 gene, wherein the 422 th deoxyribonucleotide is T and C;
in the step 3), the TT genotype is a homozygote of the 241 th deoxyribonucleotide of the fifth intron of the BMP7 gene, namely T; the CC genotype is a homozygote of 241 th deoxyribonucleotide of a fifth intron of a BMP7 gene, which is C; the TC genotype is a heterozygote of 241 th deoxyribonucleotide of a fifth intron of a BMP7 gene, namely T and C.
In the above method, the method for detecting whether the genotype of the pig individual is TT genotype, CC genotype or TC genotype comprises the following steps A) or B):
A) direct sequencing;
B) sequencing a PCR amplification product A containing 422 th deoxyribonucleotide of the fourth intron of the pig BMP7 gene or sequencing a PCR amplification product B containing 241 th deoxyribonucleotide of the fifth intron of the pig BMP7 gene;
the primers used for the PCR amplification product A 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 B 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 purpose of the invention is to provide a new application of the substance for detecting the 422 th deoxyribonucleotide of the fourth intron of BMP7 gene of a pig individual or the substance for detecting the 241 th deoxyribonucleotide of the fifth intron of BMP7 gene of a pig individual.
The invention provides an application of a substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of a BMP7 gene of a pig individual or a substance for detecting the 241 th deoxyribonucleotide genotype of the fifth intron of a BMP7 gene of the pig individual in identification or auxiliary identification of the day age of 100kg weight of a pig and/or the eye muscle area character of 100kg weight of the pig.
The invention also provides an application of a substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of a pig individual or a substance for detecting the 241 th deoxyribonucleotide genotype of the fifth intron of the BMP7 gene of the pig individual in preparing a product for identifying or assisting in identifying the daily age of the pig with the weight of 100kg and/or the eye muscle area character of the pig with the weight of 100 kg.
The invention also provides application of the substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of a pig individual or the substance for detecting the 241 th deoxyribonucleotide genotype of the fifth intron of the BMP7 gene of the pig individual in pig breeding.
The invention also provides an application of the substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of a pig individual or the substance for detecting the 241 th deoxyribonucleotide genotype of the fifth intron of the BMP7 gene of the pig individual in preparing a product for pig breeding.
The invention also provides application of the substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of a pig individual or the substance for detecting the 241 th deoxyribonucleotide genotype of the fifth intron of the BMP7 gene of the pig individual in breeding of a pig with high growth speed and/or 100kg weight, short day-old pig and/or high lean meat percentage and/or 100kg weight and large eye muscle area.
The invention also provides application of a substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of a pig individual or a substance for detecting the 241 th deoxyribonucleotide genotype of the fifth intron of the BMP7 gene of the pig individual in preparing products for breeding pigs with high growth speed, 100kg weight day-old pigs and/or high lean meat percentage and/or 100kg weight eye muscle area.
The third purpose of the invention is to provide a product for identifying or assisting in identifying the daily age of the pig with the weight of 100kg and/or the eye muscle area character of the pig with the weight of 100 kg.
The product provided by the invention is a substance for detecting the genotype of 422 th deoxyribonucleotide of the fourth intron of BMP7 gene of a pig individual or a substance for detecting the genotype of 241 th deoxyribonucleotide of the fifth intron of BMP7 gene of a pig individual.
In the above application or the above product, the substance for detecting the genotype of the 422 nd deoxyribonucleotide of the fourth intron of the BMP7 gene of the pig individual or the substance for detecting the genotype of the 241 nd deoxyribonucleotide of the fifth intron of the BMP7 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 the boar with high growth speed and/or short weight day of 100 kg.
The method for breeding the boar with high growth speed and/or short weight day age of 100kg comprises the steps of selecting the pig with TT genotype for breeding;
the TT genotype is a homozygote of the 422 th deoxyribonucleotide of the fourth intron of the BMP7 gene as T or a homozygote of the 241 th deoxyribonucleotide of the fifth intron of the BMP7 gene as T;
the nucleotide sequence of the fourth intron of the BMP7 gene is shown as a sequence 1 in a sequence table;
the nucleotide sequence of the fifth intron of the BMP7 gene is shown as a sequence 2 in the sequence table.
The fifth purpose of the invention is to provide a method for breeding boars with high lean meat percentage and/or large eye muscle area of 100kg body weight.
The method for breeding the boar with high lean meat percentage and/or large eye muscle area of 100kg body weight comprises the steps of selecting the pig with the TC genotype for breeding;
the TC genotype is a heterozygote of a 422 th deoxyribonucleotide of a fourth intron of a BMP7 gene, namely T and C;
the nucleotide sequence of the fourth intron of the BMP7 gene is shown as a sequence 1 in a sequence table.
In the above method or the above application or the above product, the 422 nd deoxyribonucleotide of the fourth intron of the BMP7 gene is also the 9136 th nucleotide of the pig BMP7 genome (NC — 010459.4); the 241 th deoxyribonucleotide of the fifth intron of the BMP7 gene is also the 7679 th nucleotide of the pig BMP7 genome (NC-010459.4).
The invention discovers that the T422C site and the T241C site of the fourth intron of the pig BMP7 gene have obvious influence on the day age and the eye muscle area of 100kg weight of pigs, and provides a method for identifying or assisting in identifying the day age and the eye muscle area of 100kg weight of pigs based on the SNP sites. The test proves that: the method for identifying the day age of 100kg body weight and the eye muscle area of the pig by using the 422 th single nucleotide polymorphism of the fourth intron and the 241 th single nucleotide polymorphism of the fifth intron of the BMP7 gene is consistent with the actual measurement result of the day age of 100kg body weight and the eye muscle area 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 day-to-day age of 100kg body weight of the pigs in the following examples is the time from the birth date of the pigs to 100kg body weight of the pigs.
The eye muscle area of 100kg body weight of the pig in the following examples is the eye muscle area of the living body when the weight of the pig reaches 100 kg.
Example 1 method for identifying pigs at 100kg body weight day of age and eye muscle area based on BMP7 Gene
Screening of SNP sites of pig BMP7 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 fourth intron (sequence 1) of the BMP7 gene is obtained by amplifying the primer pair F1 and R1, and the fifth intron (sequence 2) of the BMP7 gene is obtained by amplifying the primer pair F2 and R2. The primer sequences are as follows:
f1: 5'-GGGGTAGACAGGGCAGAGCAA-3' (SEQ ID NO: 3);
r1: 5'-TGGCAGGTGAGTGGAGGTGG-3' (SEQ ID NO: 4);
f2: 5'-GTGGCGTTCATGTACGAGTTGAGTGG-3' (SEQ ID NO: 5);
r2: 5'-CACGAAAGGCTCTGCTGCCTGTTT-3' (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 13.2ul。
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 site (T422C, T241C)
According to the sequencing results, DNAman software is used for comparison to obtain 2 differential sites: a T422C site and a T241C site, wherein the T422C site is 422 th nucleotide of a fourth intron (sequence 1) of the pig BMP7 gene, namely 9136 th nucleotide of the pig BMP7 genome (NC _ 010459.4); the T241C site is 241 th nucleotide of the fifth intron (sequence 2) of the pig BMP7 gene, namely 7679 th nucleotide of the pig BMP7 genome (NC-010459.4).
The basic groups at the T422C locus of the fourth intron (sequence 1) of the pig BMP7 gene are all T individuals, the individuals are homozygous individuals, and the genotype of the individuals is named as homozygous TT genotype; the basic groups of the T422C site of the fourth intron (sequence 1) of the pig BMP7 gene are all C individuals, the individuals are homozygous individuals, and the genotype of the individuals is named as homozygous CC genotype; the pig BMP7 gene fourth intron (sequence 1) has T422C base and C individual, the individual is heterozygous individual, the individual's genotype is named as heterozygous TC genotype.
The basic groups at the T241C locus of the fifth intron (sequence 2) of the pig BMP7 gene are all T individuals, the individuals are homozygous individuals, and the genotype of the individuals is named as homozygous TT genotype; the basic groups of the T241C site of the fifth intron (sequence 2) of the pig BMP7 gene are all C individuals, the individuals are homozygous individuals, and the genotype of the individuals is named as homozygous CC genotype; the pig BMP7 gene fifth intron (sequence 2) has T241C base of T and C individual, the individual is heterozygous individual, the individual's genotype is named as heterozygous TC genotype.
II, correlation analysis of T422C and T241C sites of the pig BMP7 gene with day-old pig body weight of 100kg and eye muscle area
Correlation analysis of T422C locus of pig BMP7 gene and pig weight day age of 100kg and eye muscle area
In order to determine whether the T422C locus of the BMP7 gene of the pig is related to the weight day age and the eye muscle area of the pig of 100kg, 382 big white pigs in the 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 weight day age and the eye muscle area of the pig of 100kg are determined according to the genotype of the pig: the pig individual with the TC gene type reaches 100kg, the weight of the pig individual with the day age is more than that of the pig individual with the CC gene type, and the pig individual with the CC gene type reaches 100kg, the weight of the pig individual with the day age is more than that of the pig individual with the TT gene type; the CC genotype pig individual 100kg weight eye muscle area is more than that of the TT genotype pig individual, and the TT genotype pig individual reaches 100kg weight eye muscle area which is more than that of the TC genotype pig individual;
1. genotype(s)
The genotype detection result of the 382 pig individuals shows that: the genotype of 13 pigs is TT genotype, the genotype of 121 pigs is TC genotype, and the genotype of 248 pigs is CC genotype. The results of detecting the genotype frequency and the allele frequency of the pig BMP7 gene in the swinery are shown in Table 1: as can be seen from table 1: the genotype frequency of CC homozygote and TC heterozygote is higher than that of TT homozygote, and C allele is a dominant gene.
TABLE 1 detection of genotype frequencies and allele frequencies of the porcine BMP7 Gene in swinery
2. Correlation analysis of pig genotype with pig weight day age and eye muscle area of up to 100kg
Statistical analysis of genotype and day age of pigs to 100kg body weight and eye muscle area was performed using SPSS 20.0 software and multiple comparisons between samples were made.
The results are shown in table 2: the SNP (T422C) site has obvious influence on the pig body weight day age and eye muscle area of 100kg, the TT type day age of 100kg is obviously lower than that of the TT type and the CC type, and the pig individual with the TC genotype reaches the pig individual with the body weight day age of 100kg more than that of the CC genotype, so that the TT genotype pig has higher growth speed in the actual pig breeding. The CC genotype reaches 100kg body weight and the eye muscle area is higher than the TC genotype and the TT genotype, and the TT genotype reaches 100kg body weight and the eye muscle area is higher than the TC genotype.
TABLE 2 correlation analysis of the mononucleotide polymorphism of the fourth intron of the porcine BMP7 gene with up to 100kg body weight day of age and eye muscle area
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 of the T422C site of the fourth intron of the pig BMP7 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 100kg weight day age and eye muscle area of the pig: the pig individual with the TC gene type reaches 100kg, the weight of the pig individual with the day age is more than that of the pig individual with the CC gene type, and the pig individual with the CC gene type reaches 100kg, the weight of the pig individual with the day age is more than that of the pig individual with the TT gene type; the CC genotype pig individual 100kg weight eye muscle area is more than that of the TT genotype pig individual, and the TT genotype pig individual reaches 100kg weight eye muscle area which is more than that of the TC genotype pig individual;
the 422 th nucleotide of the fourth intron of the TT genotype of the pig BMP7 gene is a homozygote of T;
the 422 th nucleotide of the fourth intron of the CC genotype of the pig BMP7 gene is homozygote of C;
the TC genotype is a heterozygote of the 422 th nucleotide of the fourth intron of the pig BMP7 gene, namely T and C;
the nucleotide sequence of the fourth intron of the BMP7 gene is shown as a sequence 1 in a sequence table.
Correlation analysis of T241C site of (II) pig BMP7 gene and day age of up to 100kg body weight of pig
In order to determine whether the T241C locus of the BMP7 gene of the pig is related to the day age of 100kg weight of the pig, 295-head white pigs in a hogpen 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 first step, and the day age of 100kg weight of the pig and the eye muscle area of 100kg of the pig are determined according to the genotype of the pig: the CC genotype pig individual reaches 100kg weight day age and is more than the TC genotype, and the TC genotype pig individual reaches 100kg weight day age and is more than the TT genotype;
1. genotype(s)
The genotype detection result of 295 pigs shows that: the genotype of 41 pigs is TT genotype, the genotype of 114 pigs is CC genotype, and the genotype of 140 pigs is TC genotype. The results of detecting the genotype frequency and the allele frequency of the pig BMP7 gene in the swinery are shown in Table 3: as can be seen from table 3: the genotype frequency of TC heterozygous and CC homozygous is obviously higher than that of TT homozygous, and C allele is a dominant gene.
TABLE 3 genotype frequency and allele frequency of the porcine BMP7 Gene in the test Swine herd
2. Correlation analysis of pig genotype and day age of 100kg weight of pig
Statistical analysis was performed on genotypes and day-old pigs up to 100kg body weight using SPSS 20.0 software, and multiple comparisons between samples were made.
The results are shown in Table 4: the SNP (T241C) site has obvious influence on the pig with the weight of 100kg per day, the TT type is obviously lower than the TC type and the CC type when the pig with the weight of 100kg per day is aged, the TT type is 100kg per day and is lower than the TC type, and the TC type is 100kg per day and is lower than the CC type, so that the TT genotype pig has higher growth speed in the actual pig breeding.
TABLE 4 analysis of association between single nucleotide polymorphism of the fifth intron of the porcine BMP7 gene and day-old up to 100kg body weight
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 of the T241C site of the fifth intron of the pig BMP7 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 weight day age of 100kg of pigs: the CC genotype pig individual reaches 100kg weight day age and is more than the TC genotype, and the TC genotype pig individual reaches 100kg weight day age and is more than the TT genotype;
the nucleotide at the 241 th site of the fifth intron of the porcine BMP7 gene in the TT genotype is a homozygote of T;
the CC genotype is homozygote of the 241 th nucleotide of the fifth intron of the pig BMP7 gene, which is C;
the TC genotype is a heterozygote of the 241 th nucleotide of the fifth intron of the pig BMP7 gene, namely T and C.
The nucleotide sequence of the fifth intron of the BMP7 gene is shown as a sequence 2 in the sequence table.
Claims (9)
1. A method for identifying or assisting in identifying the day-old pig with the weight of 100kg and/or the eye muscle area trait of the pig with the weight of 100kg is as follows (1) and/or (2):
(1) detecting whether the genotype of a pig individual is TT genotype, CC genotype or TC genotype, determining the day age of the pig individual with the weight of 100kg according to the genotype of the pig individual: the pig individual with the TC gene type reaches 100kg, the weight of the pig individual with the day age is more than that of the pig individual with the CC gene type, and the pig individual with the CC gene type reaches 100kg, the weight of the pig individual with the day age is more than that of the pig individual with the TT gene type;
(2) detecting whether the genotype of a pig individual is TT genotype, CC genotype or TC genotype, determining the eye muscle area of the pig individual with the weight of 100kg according to the genotype of the pig individual: the CC gene type pig individual reaches 100kg weight of pig individual with eye muscle area larger than the TT gene type, and the TT gene type pig individual reaches 100kg weight of pig individual with eye muscle area larger than the TC gene type;
in the (1) and (2), the TT genotype is a homozygote of the 422 th deoxyribonucleotide of the fourth intron of the BMP7 gene, namely T; the CC genotype is homozygote of 422 th deoxyribonucleotide of a fourth intron of a BMP7 gene, which is C; the TC genotype is a heterozygote of a 422 th deoxyribonucleotide of a fourth intron of a BMP7 gene, wherein the 422 th deoxyribonucleotide is T and C;
the pig is a big white pig.
2. In order to detect whether the genotype of a pig individual is TT genotype, CC genotype or TC genotype, the method for identifying or assisting in identifying the day-age character of the pig with the weight of 100kg comprises the following steps of: the CC genotype pig individual reaches 100kg weight day age and is more than the TC genotype, and the TC genotype pig individual reaches 100kg weight day age and is more than the TT genotype; the TT genotype is a homozygote of 241 th position deoxyribonucleotide of a fifth intron of a BMP7 gene, namely T; the CC genotype is a homozygote of 241 th deoxyribonucleotide of a fifth intron of a BMP7 gene, which is C; the TC genotype is a heterozygote of 241 th deoxyribonucleotide of a fifth intron of a BMP7 gene, namely T and C;
the pig is a big white pig.
3. The method according to claim 1 or 2, characterized in that: the method for detecting whether the genotype of the pig individual is the TT genotype, the CC genotype or the TC genotype comprises the following steps A) or B):
A) direct sequencing;
B) sequencing a PCR amplification product A containing 422 th deoxyribonucleotide of the fourth intron of the pig BMP7 gene or sequencing a PCR amplification product B containing 241 th deoxyribonucleotide of the fifth intron of the pig BMP7 gene;
the primer used by the PCR amplification product A is a primer pair A consisting of a single-stranded DNA molecule shown in a sequence 3 in a sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
the primer used by the PCR amplification product B is a primer pair B consisting of a single-stranded DNA molecule shown in a sequence 5 in a sequence table and a single-stranded DNA molecule shown in a sequence 6 in the sequence table.
4. The application of the substance for detecting the genotype of the 422 th deoxyribonucleotide of the fourth intron of the BMP7 gene of a pig individual in identifying or assisting in identifying the day age of 100kg weight of the pig and/or the eye muscle area character of 100kg weight of the pig;
or detecting the genotype of the 422 th deoxyribonucleotide of the fourth intron of the BMP7 gene of a pig individual in preparing a product for identifying or assisting in identifying the daily age of the pig with 100kg body weight and/or the eye muscle area character of the pig with 100kg body weight;
the substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of the pig individual is a primer pair A consisting of a single-stranded DNA molecule shown in a sequence 3 in a sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
the pig is a big white pig.
5. The application of the substance for detecting the genotype of 241 th deoxyribonucleotide of the fifth intron of BMP7 gene of a pig individual in the identification or the auxiliary identification of the day-old character of 100kg body weight of the pig;
or detecting the genotype of 241 th deoxyribonucleotide of the fifth intron of BMP7 gene of a pig individual in preparing a product for identifying or assisting in identifying the daily age trait of 100kg body weight of the pig;
the substance for detecting the genotype of the 241 th deoxyribonucleotide of the fifth intron of the BMP7 gene of the pig individual is a primer pair B consisting of a single-stranded DNA molecule shown in a sequence 5 in a sequence table and a single-stranded DNA molecule shown in a sequence 6 in the sequence table;
the pig is a big white pig.
6. The application of the substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of a pig individual or the substance for detecting the 241 th deoxyribonucleotide genotype of the fifth intron of the BMP7 gene of the pig individual in breeding of a boar with a high growth speed;
or detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of the pig individual or detecting the 241 th deoxyribonucleotide genotype of the fifth intron of the BMP7 gene of the pig individual in preparing products for breeding pigs with high growth speed;
the substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of the pig individual is a primer pair A consisting of a single-stranded DNA molecule shown in a sequence 3 in a sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
the substance for detecting the genotype of the 241 th deoxyribonucleotide of the fifth intron of the BMP7 gene of the pig individual is a primer pair B consisting of a single-stranded DNA molecule shown in a sequence 5 in a sequence table and a single-stranded DNA molecule shown in a sequence 6 in the sequence table;
the pig is a big white pig.
7. The application of the substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of a pig individual in breeding a boar with high lean meat percentage;
or detecting the application of the substance of the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of a pig individual in the preparation of products for breeding pigs with high lean meat percentage;
the substance for detecting the 422 th deoxyribonucleotide genotype of the fourth intron of the BMP7 gene of the pig individual is a primer pair A consisting of a single-stranded DNA molecule shown in a sequence 3 in a sequence table and a single-stranded DNA molecule shown in a sequence 4 in the sequence table;
the pig is a big white pig.
8. A method for breeding boar with fast growth speed comprises selecting pig with TT genotype for breeding;
the TT genotype is a homozygote of the 422 th deoxyribonucleotide of the fourth intron of the BMP7 gene as T or a homozygote of the 241 th deoxyribonucleotide of the fifth intron of the BMP7 gene as T;
the pig is a big white pig.
9. A method for breeding boars with high lean meat percentage comprises selecting the boars with CC genotype for breeding;
the CC genotype is homozygote of 422 th deoxyribonucleotide of a fourth intron of a BMP7 gene, which is C;
the pig is a big white pig.
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