CN108753926B - Molecular marking method for thickness of pig back fat - Google Patents
Molecular marking method for thickness of pig back fat Download PDFInfo
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Abstract
The invention relates to a molecular marking method for pig back fat thickness. The operation steps are as follows: extracting porcine genomic DNA from porcine ear margin tissue; designing 1 pair of primers, namely an upstream primer and a downstream primer, from-1201 bp to-654 bp of the initial transcription site of the pig calbindin 2 (CALB 2) gene; obtaining 548bp long in-vitro amplification product between-1201 bp and-654 bp upstream of the transcription initiation site of the porcine CALB2 gene through polymerase chain reaction; carrying out restriction enzyme MspA1I enzyme digestion reaction on the in vitro amplification product; and (3) carrying out polymorphism (RLFP) detection on the enzyme digestion product to obtain a stripe AA type which is a genotype with excellent back fat progress, namely a mutant type, so that the piglet with the stripe AA type is selected for seed reservation, and the back fat thickness of the stripe AA type pig reaching 100kg of body weight is 0.95-1.27 mm thinner than that of the GG type individual.
Description
Technical Field
The invention belongs to the technical field of live pig feeding, and particularly relates to a molecular marking method for pig back fat thickness.
Background information
The thickness of the back fat of pigs is an important trait that affects the amount of lean meat produced by live pigs. The pig carcass lean meat percentage specified by the agricultural industry standard of the people's republic of China, "boar performance determination procedure" (NY/T822-2004) is that the left half carcass is divided into a front carcass, a middle carcass and a back carcass after lard and kidney are removed. The anterior and middle parts are cut perpendicularly at 6-7 intercostals, and the posterior part is cut perpendicularly from the lumbar and the sacral spine. The sebum, bones and lean meat of each trunk were separated and weighed separately. During separation, the muscle fat is not removed as lean meat and the skin muscle is not removed as fat meat, and then the ratio of the weight (kg) of the lean meat to the total weight of the sebum, the bone and the lean meat is used as the lean meat percentage. Therefore, thicker fat means less lean meat and thinner fat, more lean meat is produced, and consumers at home and abroad want to eat more lean meat and less fat, so that the breeding of live pigs want to produce more lean meat, i.e., the back fat is thinner, and better. The ' boar performance determination regulation ' of the agricultural industry standard of the people's republic of China (NY/T822-2004) stipulates that when a live pig grows to 85kg to 105kg of weight, the weight and the back fat thickness are determined one by one, the back fat thickness reaching 100kg of weight is calculated according to the determination result, the current day age is about 150-180 days, the breeding value of the back fat thickness reaching 100kg of weight is calculated according to the weight, and the selection is carried out according to the breeding value, so that the time, the labor and the fund are consumed.
Calbindin (Calbindin) is a generic term for several calcium-dependent binding proteins, originally found in the kidney and small intestine of chickens and mammals, and is classified into different subfamilies due to the fact that the hand-shaped domain (EF-hand) of Calbindin binds calcium in different amounts, and CALB2 is one of the calcium-binding proteins, and is mainly present in nervous tissues.
Calbindin 1 (CALB 1), was first found in the chicken small intestine and then in the mammalian kidney. It is also expressed in many neurons and endocrine cells, especially in the cerebellum. It is composed ofCALB1The gene encodes. CALB1 contains 4 active calcium binding domains and 2 modified domains that have lost calcium binding capacity. CALB1 acts as a calcium regulation and calcium ion sensor, and can bind 4 calcium ions in the hand-shaped domain (EF-hand). CALB1 was confirmed by high resolution nuclear magnetic resonance techniques to be one of the largest proteins identified at the time. CALB1 has only one strand, and the sequence has 263 residues. The sequence consists mainly of an alpha helix, containing 117 residues.CALB1In many tissues, especially the chicken small intestine, it is a vitamin D responsive gene with a distinct function in mediating calcium absorption, whereas in the brain its synthesis is independent of vitamin D. CALB1 and CALB3 have no homology except for the calcium binding site hand domain, CALB1 has 6 hand domains and CALB3 has 2.
Calbindin 2 (calbindin 2, CALB 2) is one of the members of the calbindin family, unlike CALB1 and CALB3, CALB2 is a member of the troponin C superfamily, which shares 58% homology with CALB2 proteinCALB2Gene coding, porcineCALB2The gene is located on chromosome 6, consists of 11 exons and codes 272 amino acids in total. PigCALB2The genes are expressed mainly in ovary, longissimus dorsi, psoas major, and adipose tissue of the back. The research data of CALB2 in pig is very little, and the results of human CALB2 research show that CALB2 has multiple cell functions, such as information targeting and buffering of intracellular calcium, and CALB2 can be used for diagnosing malignant mesothelioma, lung tumor and brain tumor. Calcium ion is the oldest and most widely used signal substance in the cell, and is involved in the regulation of almost all biological functions of the body, such as cardiac and muscle contraction, neural information transmission, learning and memory, embryogenesis and development, cell proliferation and apoptosis, cell division and differentiation, cellular energy metabolism, protein phosphorylation and dephosphorylation modification, gene expression and regulation, and the like[10]。
Disclosure of Invention
To achieve calcium binding protein 2: (CALB2) The invention provides a molecular marking method for pig back fat thickness, which is used for breeding pigs with thin back fat thickness by mutant detection at the upstream-1067 bp of a gene.
The method comprises the steps of extracting pig genome DNA and calcium binding protein 2: (CALB2) Amplification primers for gene upstream, in vitro amplification and genotype detection.
The operation steps of the molecular marker for the thickness of the pig back fat are as follows:
(1) extraction of genomic DNA from swine
On the day of birth of piglets, ear margin tissues of pigs are taken; extracting pig gene DNA from pig ear marginal tissue; the pig genome DNA is a nucleotide sequence with the total length of 548bp, wherein the nucleotide sequence comprises the initial transcription site of the pig calcium binding protein 2 (CALB 2) gene and is between-1201 bp and-654 bp;
(2) primer design
Designing 1 pair of primers, namely an upstream primer and a downstream primer, from-1201 bp to-654 bp of the initial transcription site of the pig calcium binding protein 2 (CALB 2) gene, wherein the sequences of the upstream primer and the downstream primer are as follows:
CALB2-F upstream primer: 5'-TGGTGTTCCTCTCTCCCCTG-3'
CALB2-R downstream primer: 5'-CCACTGTCCTCTCGTGTTGA-3', respectively;
(3) polymerase chain reaction
A. Preparation of polymerase chain reaction System
The polymerase chain reaction system comprises an upstream primer, a downstream primer, deoxynucleotide triphosphate, polymerase and porcine genomic DNA;
B. polymerase chain reaction
Performing polymerase chain reaction to obtain 548bp long in-vitro amplification product between-1201 bp and-654 bp upstream of the transcription initiation site of the pig CALB2 gene;
(4) enzyme digestion reaction
Carrying out restriction enzyme digestion reaction on the in vitro amplification product to obtain a digestion product;
(5) polymorphism (RLFP) detection
Carrying out polymorphism (RLFP) detection on the enzyme digestion product to obtain an AA type individual which cannot be cut and comprises a 548bp fragment; the genotype with excellent back fat thickness is a mutant type, namely AA type, individuals with AA type bands are selected for reserving seeds, and the back fat thickness of the AA type band pig reaching 100 kilograms of body weight is 0.95-1.27 mm thinner than that of the GG type individual.
The technical scheme for further limiting is as follows:
in the step (3), the operation of preparing the polymerase chain reaction system is as follows:
2ml of 4 XdNTP 2.5 mmol/l deoxynucleoside triphosphate, 0.5ml of each of 10 mmol/ml upstream primer and 10 mmol/ml downstream primer, 0.5ml of 10U/ml Taq polymerase, 1ml of a DNA genome template solution having a concentration of 100ng/ml, and 20 mmol/l magnesium oxide (MgCl)2) 2.0ml of 10 × Polymerase Chain Reaction (PCR) buffer solution and 13.5ml of pure water to a final volume of 20 ml; and mixed evenly to prepare 20 mul of polymerase chain reaction system.
In the step (3), the conditions of the polymerase chain reaction are as follows:
mu.l of Polymerase Chain Reaction (PCR) system was placed in a PCR apparatus under the following conditions:
firstly, pre-denaturation is carried out for 4 minutes at 95 ℃,
② denaturation at 95 ℃ for 30 seconds,
③ the annealing temperature is 58 ℃ for 30 seconds,
extension at 72 ℃ for 40 seconds,
fifthly, repeating the steps from the fourth step to the fourth step for 35 cycles,
sixthly, extending for 7 minutes at 72 ℃, and finally cooling to 4 ℃ for storage to obtain an in vitro amplification product with the length of 548bp between-1201 bp and-654 bp upstream of the transcription initiation site of the porcine CALB2 gene.
In the step (4), because the 1067 th base upstream of the transcription initiation site of the pig CALB2 gene is mutated from G to A, a restriction enzyme digestion polymorphic site of MspA1I restriction enzyme is caused, and the specific operation of the restriction enzyme digestion reaction is as follows: taking 5 mu l of in-vitro amplification product with the length of 548bp upstream of the transcription start site of the pig calcium-binding protein 2 (CALB 2) gene, respectively adding 0.5 mu l of MspA1I restriction enzyme and 1 mu l of MspA1I restriction enzyme buffer solution into a Polymerase Chain Reaction (PCR) tube, adding 3.5 mu l of water, uniformly mixing, and reacting at 37 ℃ for 3 hours to obtain 10 mu l of enzyme digestion product.
In the step (5), the detection operation of polymorphic site restriction fragment enzyme digestion polymorphism (RLFP) is as follows:
mu.l of the above digestion products were electrophoresed on 2.0% agarose gel for 1 hour under the following conditions: obtaining gel containing enzyme-cleaved products at normal temperature and 120V voltage; placing the gel containing the enzyme cutting products under a gel imaging system to observe the genotype, wherein the GG type individual is completely cut and comprises 415bp and 133bp fragments; the GA type individual is incompletely cut and comprises three fragments of 548bp, 415bp and 133 bp; the AA individual cannot be cut and comprises 548bp of a fragment; wherein the genotype with excellent back fat thickness is mutant type, namely AA type; the thickness of the back fat of the AA type pig strip when the weight of the AA type pig strip reaches 100 kilograms is 0.95-1.27 mm thinner than that of the GG type pig.
And selecting individuals with AA type genotypes for reservation, so that the breeding pigs with lower backfat thickness can be obtained, and the aim of improving the lean meat percentage is fulfilled.
The beneficial technical effects of the invention are embodied in the following aspects:
1. polymorphism exists at the upstream-1067 site of the transcription site of CALB2 gene for the first time, and is obviously related to the thickness of pig back fat. As the CALB2 gene has a regulation function in the aspects of signal transmission, embryonic development, cell proliferation, differentiation, gene expression and the like, and CALB2 is expressed abundantly in back adipose tissues, the mutation site of the CALB2 gene of a pig is analyzed, the mutation site (G is mutated into A) exists in an upstream regulation region-1067 bp of the 5' end of the CALB2 gene of the pig is firstly discovered, the restriction enzyme digestion polymorphism site of a restriction enzyme MspA1I is caused, therefore, population polymorphism is detected in 390 heads of Yorkshire pigs and 235 heads of Duroc pigs, the relevance of the polymorphism of the site and the back fat thickness of about 100kg body weight of the Yorkia and Duroc pigs is analyzed, the site is found to be obviously related to the back fat thickness of 100kg body weight, and the polymorphism can be used as a molecular marker of the back fat thickness of the pig.
2. The invention can collect ear tissues when the piglets are born, and then can judge whether each individual has a genotype with thinner back fat thickness through the steps of DNA extraction, polymerase chain reaction, enzyme digestion and the like, is used for the auxiliary selection of the back fat thickness in the breeding of the pigs, realizes the early breeding of the breeding pigs, and can accurately select and retain the breeding pigs even when the pigs are born. The gene marking method can fix the excellent genotype of the CALB2 gene only through one generation, namely, the excellent gene of the back fat thickness of the gene is homozygous through one generation breeding, while the conventional breeding method needs a large amount of performance measurement and descendant measurement, and can achieve the required effect through more than 5 generations of successive breeding, thereby greatly shortening the generation interval and accelerating the breeding process. The price of pure lean meat of the live pig is 10 yuan/kg higher than that of pure fat meat, the fat meat is difficult to sell, and the demand of people for the fat meat is quite small, so the breeding of the live pig is to select and breed towards the direction of thin back fat and high lean meat rate.
3. The method has the advantages of simple operation, low requirement on conditions in the polymerase chain reaction process, short amplified fragment length (548 bp), easy amplification, and improved amplification efficiency and genotype judgment accuracy.
4. The invention can obviously reduce the back fat thickness of pigs and improve the lean meat percentage of the pigs, and the back fat thicknesses of AA type individuals in about Kjeldahl pigs and Duroc pigs which reach 100kg of weight are respectively 0.95mm and 1.27mm thinner than those of GG type individuals.
Drawings
FIG. 1 shows the agarose gel electrophoresis of the CALB2 gene PCR amplification of the invention.
FIG. 2 is a restriction enzyme map of the CALB2 gene of the present invention with restriction enzyme (MspA 1I) located upstream of the gene for-1067 bp.
Detailed Description
The present invention will be further described with reference to the following examples.
A molecular marking method for pig back fat thickness comprises the following specific operation methods:
through the analysis of the relevance of CALB2 gene polymorphism and growth traits of the Yorkshire pigs and Duroc swinery, consistent results can be obtained, namely the AA individuals have thinner back fat and are excellent genotypes of the Yorkshire pigs and Duroc swinery, and two examples of the Yorkshire pigs and Duroc pigs are respectively described as follows:
example 1:
a total of 390 ear edge tissues from approximately Kechard pigs were collected and the backfat thickness was measured up to 100kg body weight. The genotype detection of the molecular marker CALB2 gene of the pig backfat thickness is respectively carried out, and the specific operation steps are as follows.
(1) Extracting genomic DNA of Yorkshire pig
The ear rim tissue was cut into pieces with scissors, and 600. mu.l of DNA tissue extract containing 40ml of 0.5M EDTA solution, 10ml of 1M Tris. Cl solution, 4ml of 5M NaCl solution, 20ml of 10% SDS solution, 30. mu.l of 10mg/ml proteinase K solution, and 125.07ml of double distilled water was poured into 200ml of DNA tissue extract. The extract is prepared as before. Then placing the mixture into a warm bath at 55 ℃ for 4 hours, adding 600ul of Tris saturated phenol, shaking up and down, uniformly mixing for 15 minutes, and centrifuging for 10 minutes by using a low-temperature high-speed centrifuge, wherein the rotating speed of the centrifuge requires 12000 r/min; taking out the supernatant by using a 1ml pipette, adding into a sterilized 1.5ml centrifuge tube, adding equal volume of Tris saturated phenol, shaking up and down, mixing uniformly for 15 minutes, and centrifuging for 10 minutes (12000 r/min); the supernatant was taken, mixed with an equal volume of Tris saturated phenol, chloroform, isoamyl alcohol (25:24:1) for 10 minutes, and centrifuged for 10 minutes (12000 rpm). The supernatant was added with an equal volume of chloroform isoamyl alcohol (24:1), mixed for 10 minutes, and centrifuged for 10 minutes (12000 rpm). The supernatant was added to 2 volumes of glacial ethanol (about 1ml), and then 1/10 volumes of about 60ml of NaAc (sodium acetate) were added and shaken horizontally, whereupon a flocculent, white DNA sample appeared. The samples were frozen at-20 ℃ for 30 minutes, and the DNA was removed or picked out or centrifuged (12000 rpm) for 10 minutes, with the DNA settled to the bottom of the tube. The DNA was washed with 75% ethanol, shaken and then centrifuged 5-10 (12000 rpm). Discarding 75% ethanol, naturally drying, adding appropriate amount (about 50ml) of TE to dissolve, and storing in-20 deg.C refrigerator.
(2) Primer preparation
Primers at two ends of the-1067 bp site at the upstream of the pig CALB2 gene are dissolved by double distilled water, and the concentration of the dissolved primers is 10 mmol/ml, and the dissolved primers are used for Polymerase Chain Reaction (PCR).
(3) Polymerase Chain Reaction (PCR)
20ml polymerase chain reaction system, consisting of 2ml of 4 x dNTP 2.5 mmol/l deoxyribonucleoside triphosphate, 0.5ml of each of 10 mmol/ml upstream primer and 10 mmol/ml downstream primer, 0.5ml of 10U/ml Taq polymerase, 1ml of DNA genome template solution with the concentration of 100ng/ml, and magnesium oxide (MgCl) containing 20 mmol/l2) 2.0ml of 10 × Polymerase Chain Reaction (PCR) buffer, adding 13.5ml of water to a final volume of 20 ml; and mixing uniformly.
Reaction conditions for polymerase chain reaction apparatus
Firstly, pre-denaturation is carried out for 4 minutes at 95 ℃;
② denaturation at 95 ℃ for 30 seconds;
③ annealing temperature is 58 ℃ for 30 seconds;
extension at 72 ℃ for 40 seconds;
fifthly, repeating the steps from the second step to the fourth step for 35 cycles;
sixthly, stretching for 7 minutes at 72 ℃, and finally cooling to 4 ℃ for storage.
The in vitro amplification product containing CALB2 gene upstream-1067 bp site is obtained through the reaction and detected by 2% agarose gel electrophoresis. The length of the CALB2 gene amplification fragment is 548 bp; see fig. 1. As shown in FIG. 1, the band is single, there is no non-specific amplification product, and the next analysis can be performed.
(4) Restriction fragment cleavage polymorphism (RLFP) detection of polymorphic sites
Taking 5ml of an amplification product of the gene, putting 0.5ml of MspA1I restriction endonuclease, 1ml of MspA1I restriction endonuclease buffer solution and 3.5ml of water into a Polymerase Chain Reaction (PCR) tube respectively, adding the mixture into the tube at a concentration of 10U/ml, uniformly mixing, putting the tube into an environment at 37 ℃ for reaction for 3 hours, then using 2.0% agarose gel, carrying out electrophoresis at normal temperature and 120V for 1 hour, putting the gel under a gel imaging system for observation, and observing the MspA1I restriction endonuclease digestion result, wherein the MspA1I restriction endonuclease digestion picture of the base of the position of 1067bp at the upstream of the CALB2 gene of the pig only contains a strip of 548bp, and is of GG type, and the Mspc 1I restriction endonuclease pictures of the positions of 3 and 7bp in the picture 2 are shown in lanes; the enzyme cutting map contains three bands of 548bp, 415bp and 133bp, which are GA type, and are shown as lanes 1, 2, 5, 6 and 8 in figure 2; the AA type of the DNA contains 415bp and 133bp bands, as shown in the 4 th lane of FIG. 2, and the DL 2000 Marker is shown in the M lane of FIG. 2.
The results of detecting 390 calcium binding protein 2 (CALB 2) gene polymorphisms of the Yorkshire pigs are shown in Table 1. The G allele frequency was 0.48 and the A allele frequency was 0.52, and the herd had abundant polymorphisms at this site, which was in Harder-Weinberg equilibrium in the Yorkshire pigs.
Correlation analysis of calcium binding protein 2 (CALB 2) gene polymorphism and backfat thickness of Yorkshire swinery
The back fat thickness at 5 cm from the dorsal midline between the 3 rd to last and 4 th ribs when the Yorkshire pigs grow to 100kg body weight, namely the back fat thickness reaching 100kg body weight, is measured by B-ultrasound. The genotype of CALB2 gene was also determined, and the thickness of the back fat at 100kg body weight in 390 subjects was subjected to least squares analysis using the General Linear Model (GLM) program of SPSS13.0 software, and the gene effect was analyzed, the results are shown in Table 2. The back fat thickness of GG type individuals which averagely reach 100kg body weight is 11.36mm at most and is 11.30mm thicker than that of GA type individuals, but the difference is not significant (P > 0.05), the back fat thickness of GG type individuals which averagely reach 100kg body weight is significantly thicker than that of AA type individuals by 10.41mm (P < 0.01), the back fat thickness of GA type individuals which averagely reach 100kg body weight is significantly larger than that of AA type (P < 0.05), so that mutant allele A is an excellent allele of the back fat thickness of the locus, and mutant AA is an excellent genotype of the back fat thickness of the locus. From gene effect analysis, the dominant effects are not completely dominant, and the G allele of the CALB2 gene shows positive additive effect on the back fat thickness; the average effect value of the G allele was 0.284, and the average effect values of the A alleles were-0.232, respectively; the average effect value of the A allele for replacing the G allele is-0.516, which indicates that the AA genotype in the Yorkshire pig group is an excellent genotype of the back fat thickness, the A allele is an excellent allele, the back fat thickness of the AA type individual is 0.95mm thinner than that of the GG type individual in the example, and the selection of the AA type pig can effectively reduce the back fat thickness of the Yorkshire pig, thereby improving the lean meat percentage and increasing more lean meat protein for human beings.
As can be seen from Table 2, the same row of shoulder marks with different letters (a and c) indicates that the difference is very significant: (P<0.01), marked ab and c, are significantly different (P<0.05); marked with the same letter to indicate that the difference is not significant (P)>0.05). In the table d indicates the dominant effect; a represents the additive effect of the gene; d represents the dominance degree; alpha is alpha1Represents the average effect of allele G; alpha is alpha2Represents the average effect of allele a; alpha represents the average effect value of the A allele in place of the G allele (i.e., alpha)2-α1）。
Example 2:
ear margin tissues of 235 duroc pigs were collected together with backfat thickness measured up to 100kg body weight. The genotype detection of the molecular marker CALB2 gene of the pig backfat thickness is respectively carried out, and the specific operation steps are as follows.
(1) Duroc pig genome DNA extraction
The ear rim tissue of Duroc pigs was cut into pieces with scissors, and 600. mu.l of DNA tissue extract containing 40ml of 0.5M EDTA solution, 10ml of 1M Tris. Cl solution, 4ml of 5M NaCl solution, 20ml of 10% SDS solution, 30. mu.l of 10mg/ml proteinase K solution, and 125.07ml of double distilled water was poured into 200ml of DNA tissue extract. The extract is prepared as before. Then placing the mixture into a warm bath at 55 ℃ for 4 hours, adding 600ul of Tris saturated phenol, shaking up and down, uniformly mixing for 15 minutes, and centrifuging for 10 minutes by using a low-temperature high-speed centrifuge, wherein the rotating speed of the centrifuge requires 12000 r/min; taking out the supernatant by using a 1ml pipette, adding into a sterilized 1.5ml centrifuge tube, adding equal volume of Tris saturated phenol, shaking up and down, mixing uniformly for 15 minutes, and centrifuging for 10 minutes (12000 r/min); the supernatant was taken, mixed with an equal volume of Tris saturated phenol, chloroform, isoamyl alcohol (25:24:1) for 10 minutes, and centrifuged for 10 minutes (12000 rpm). The supernatant was added with an equal volume of chloroform isoamyl alcohol (24:1), mixed for 10 minutes, and centrifuged for 10 minutes (12000 rpm). The supernatant was added to 2 volumes of glacial ethanol (about 1ml), and then 1/10 volumes of about 60ml of NaAc (sodium acetate) were added and shaken horizontally, whereupon a flocculent, white DNA sample appeared. The samples were frozen at-20 ℃ for 30 minutes, and the DNA was removed or picked out or centrifuged (12000 rpm) for 10 minutes, with the DNA settled to the bottom of the tube. The DNA was washed with 75% ethanol, shaken and then centrifuged 5-10 (12000 rpm). Discarding 75% ethanol, naturally drying, adding appropriate amount (about 50ml) of TE to dissolve, and storing in-20 deg.C refrigerator.
(2) Primer preparation
Primers at two ends of the-1067 bp site at the upstream of the pig CALB2 gene are dissolved by double distilled water, and the concentration of the dissolved primers is 10 mmol/ml, and the dissolved primers are used for Polymerase Chain Reaction (PCR).
(3) Polymerase Chain Reaction (PCR)
20ml polymerase chain reaction system, consisting of 2ml of 4 x dNTP 2.5 mmol/l deoxyribonucleoside triphosphate, 0.5ml of each of 10 mmol/ml upstream primer and 10 mmol/ml downstream primer, 0.5ml of 10U/ml Taq polymerase, 1ml of DNA genome template solution with the concentration of 100ng/ml, and magnesium oxide (MgCl) containing 20 mmol/l2) 2.0ml of 10 × Polymerase Chain Reaction (PCR) buffer, adding 13.5ml of water to a final volume of 20 ml; and mixing uniformly.
Reaction conditions for polymerase chain reaction apparatus
Firstly, pre-denaturation is carried out for 4 minutes at 95 ℃;
② denaturation at 95 ℃ for 30 seconds;
③ annealing temperature is 58 ℃ for 30 seconds;
extension at 72 ℃ for 40 seconds;
fifthly, repeating the steps from the second step to the fourth step for 35 cycles;
sixthly, stretching for 7 minutes at 72 ℃, and finally cooling to 4 ℃ for storage.
The in vitro amplification product containing CALB2 gene upstream-1067 bp site is obtained through the reaction and detected by 2% agarose gel electrophoresis. The length of the CALB2 gene amplification fragment is 548 bp; see fig. 1. As shown in FIG. 1, the band is single, there is no non-specific amplification product, and the next analysis can be performed.
(4) Restriction fragment cleavage polymorphism (RLFP) detection of polymorphic sites
Taking 5ml of an amplification product of the gene, putting 0.5ml of MspA1I restriction endonuclease, 1ml of MspA1I restriction endonuclease buffer solution and 3.5ml of water into a Polymerase Chain Reaction (PCR) tube respectively, adding the mixture into the tube at a concentration of 10U/ml, uniformly mixing, putting the tube into an environment at 37 ℃ for reaction for 3 hours, then using 2.0% agarose gel, carrying out electrophoresis at normal temperature and 120V for 1 hour, putting the gel under a gel imaging system for observation, and observing the MspA1I restriction endonuclease digestion result, wherein the MspA1I restriction endonuclease digestion picture of the base of the position of 1067bp at the upstream of the CALB2 gene of the pig only contains a strip of 548bp, and is of GG type, and the Mspc 1I restriction endonuclease pictures of the positions of 3 and 7bp in the picture 2 are shown in lanes; the enzyme cutting map contains three bands of 548bp, 415bp and 133bp, which are GA type, and are shown as lanes 1, 2, 5, 6 and 8 in figure 2; the AA type of the DNA contains 415bp and 133bp bands, as shown in the 4 th lane of FIG. 2, and the DL 2000 Marker is shown in the M lane of FIG. 2.
The results of detecting the polymorphism of the 235-head Duroc calcium binding protein 2 (CALB 2) gene are shown in Table 3. The frequency of the G allele is 0.43, the frequency of the A allele is 0.57, and the pig group has abundant polymorphism at the site.
Correlation analysis of calcium binding protein 2 (CALB 2) gene polymorphism of Duroc swinery and backfat thickness
The back fat thickness of the penultimate 3 rd and 4 th ribs at the position 5 cm away from the back midline between the ribs when the pig grows to 100kg body weight, namely the back fat thickness of 100kg body weight, is measured by B ultrasonic. The genotypes of the CALB2 genes were separately determined, and the back fat thickness at 100kg body weight of 235 subjects was subjected to least squares analysis using the General Linear Model (GLM) program of the SPSS13.0 software, and the gene effects were analyzed, with the results shown in Table 4. The back fat thickness of GG type individuals which averagely reach 100kg body weight is 10.56mm at most and is 10.25mm thicker than that of GA type individuals, but the difference is not significant (P > 0.05), and the back fat thickness of GG type individuals and GA type individuals which averagely reach 100kg body weight is extremely significantly thicker than that of AA type individuals (P < 0.01), so that mutant allele A is a good allele of the back fat thickness of the locus, and mutant AA is a good genotype of the back fat thickness of the locus. From gene effect analysis, the dominant effects are not completely dominant, and the G allele of the CALB2 gene shows positive additive effect on the back fat thickness; the average effect value of the G allele was 0.388, and the average effect values of the A alleles were-0.293, respectively; the average effect value of the A allele for replacing the G allele is-0.681, which indicates that the AA genotype in the Duroc pig group is the excellent genotype of the back fat thickness, the A allele is the excellent allele, in the example, the back fat thickness of the AA type individual of the Duroc pig is 1.27mm thinner than that of the GG type individual, and the AA type pig is selected and retained, so that the back fat thickness of the Duroc pig can be effectively reduced, the lean meat percentage is improved, and more lean meat protein is improved for human beings.
As can be seen from Table 4, the same row of shoulder marks with different letters (a and c) indicates that the difference is very significant: (P<0.01), marked with the same letter (P) indicates that the difference is not significant>0.05). In the table d indicates the dominant effect; a represents the additive effect of the gene; d represents the dominance degree; alpha is alpha1Represents the average effect of allele G; alpha is alpha2Represents the average effect of allele a; alpha represents the average effect value of the A allele in place of the G allele (i.e., alpha)2-α1）。
Claims (5)
1. A molecular marking method for pig back fat thickness is characterized by comprising the following operation steps:
(1) extraction of genomic DNA from swine
On the day of birth of piglets, ear margin tissues of pigs are taken; extracting pig gene DNA from pig ear marginal tissue; the total length of the pig genome DNA is 548bp including a nucleotide sequence between-1201 bp and-654 bp of the pig calcium-binding protein 2 gene initiation transcription site;
(2) primer design
Designing 1 pair of primers between-1201 bp and-654 bp of the initial transcription site of the pig calcium binding protein 2 gene, namely an upstream primer and a downstream primer, wherein the sequences of the upstream primer and the downstream primer are as follows:
CALB2-F upstream primer: 5'-TGGTGTTCCTCTCTCCCCTG-3'
CALB2-R downstream primer: 5'-CCACTGTCCTCTCGTGTTGA-3', respectively;
(3) polymerase chain reaction
A. Preparation of polymerase chain reaction System
The polymerase chain reaction system comprises a CALB2-F upstream primer, a CALB2-R downstream primer, deoxynucleotide triphosphates, polymerase and porcine genomic DNA;
B. polymerase chain reaction
Performing polymerase chain reaction to obtain 548bp long in-vitro amplification product between-1201 bp and-654 bp upstream of the transcription initiation site of the pig CALB2 gene;
(4) enzyme digestion reaction
Carrying out restriction enzyme digestion reaction on the in-vitro amplification product by using restriction enzyme MspA1I to obtain a digestion product;
(5) polymorphism detection
Carrying out polymorphism detection on the enzyme digestion product to obtain an AA type individual which cannot be cut and comprises a 548bp fragment; the genotype with excellent back fat thickness is a mutant type, namely AA type, individuals with AA type bands are selected for reserving seeds, and the back fat thickness of the AA type band pig reaching 100 kilograms of body weight is 0.95-1.27 mm thinner than that of the GG type individual.
2. The molecular marking method of the thickness of the back fat of the pig as claimed in claim 1, characterized in that: in the step (3), the operation of preparing the polymerase chain reaction system is as follows:
2ml of 4 XdNTP 2.5 mmol/l deoxynucleoside triphosphate, 0.5ml of each of 10 mmol/ml upstream primer and 10 mmol/ml downstream primer, 0.5ml of 10U/ml Taq polymerase, 1ml of DNA genome template solution with the concentration of 100ng/ml, 2.0ml of 10 Xpolymerase chain reaction buffer solution containing 20 mmol/l magnesium oxide and 13.5ml of pure water are sequentially added into a polymerase chain reaction tube to reach the final volume of 20 ml; and mixed evenly to prepare 20 mul of polymerase chain reaction system.
3. The molecular marking method of the thickness of the back fat of the pig as claimed in claim 1, characterized in that: in the step (3), the conditions of the polymerase chain reaction are as follows:
mu.l of the PCR system was placed in a PCR apparatus under the following conditions:
firstly, pre-denaturation is carried out for 4 minutes at 95 ℃,
② denaturation at 95 ℃ for 30 seconds,
③ the annealing temperature is 58 ℃ for 30 seconds,
extension at 72 ℃ for 40 seconds,
fifthly, repeating the steps from the fourth step to the fourth step for 35 cycles,
sixthly, extending for 7 minutes at 72 ℃, and finally cooling to 4 ℃ for storage to obtain an in vitro amplification product with the length of 548bp between-1201 bp and-654 bp upstream of the transcription initiation site of the porcine CALB2 gene.
4. The molecular marking method of the thickness of the back fat of the pig as claimed in claim 1, characterized in that: in the step (4), because the 1067 th base upstream of the transcription initiation site of the pig CALB2 gene is mutated from G to A, a restriction enzyme digestion polymorphic site of MspA1I restriction enzyme is caused, and the specific operation of the restriction enzyme digestion reaction is as follows: taking 5 mul of the in-vitro amplification product with the length of 548bp upstream of the transcription start site of the pig calcium-binding protein 2 gene, adding 0.5 mul of MspA1I restriction enzyme and 1 mul of MspA1I restriction enzyme buffer solution of 10U/mul into a polymerase chain reaction tube respectively, adding 3.5 mul of water, mixing uniformly, and reacting at 37 ℃ for 3 hours to obtain 10 mul of enzyme digestion product.
5. The molecular marking method of the thickness of the back fat of the pig as claimed in claim 1, characterized in that: in the step (5), the polymorphic detection operation of the polymorphic site restriction enzyme digestion is as follows:
mu.l of the above digestion products were electrophoresed on 2.0% agarose gel for 1 hour under the following conditions: obtaining gel containing enzyme-cleaved products at normal temperature and 120V voltage; placing the gel containing the enzyme cutting products under a gel imaging system to observe the genotype, wherein the GG type individual is completely cut and comprises 415bp and 133bp fragments; the GA type individual is incompletely cut and comprises three fragments of 548bp, 415bp and 133 bp; the AA individual cannot be cut and comprises 548bp of a fragment; wherein the genotype with excellent back fat thickness is a mutant type, namely AA type.
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