CN110982908B - Molecular marking method for fat deposition and meat quality characters of lean type fixed-distance pig strain - Google Patents
Molecular marking method for fat deposition and meat quality characters of lean type fixed-distance pig strain Download PDFInfo
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Abstract
The invention discloses a molecular marker for fat deposition and meat quality character of a lean type fixed-distance pig strain, which has a nucleotide sequence shown as SEQ ID NO:1, the 398 th base in the sequence is a base mutation site, and the base mutation site is G or C. The invention discloses a primer pair for detecting the molecular marker. The invention discloses a molecular marking method for fat deposition and meat quality character of a lean type fixed-distance pig strain, which comprises the following steps: extracting the genomic DNA containing the molecular marker from the pig; performing polymerase chain reaction by using the primer pair, the genome DNA and the PCR reaction buffer solution to obtain an in vitro amplification product; carrying out enzyme digestion reaction on the in-vitro amplification product by using restriction enzyme to obtain an enzyme digestion product; and (4) carrying out polymorphism detection on the enzyme digestion product to obtain the genotype of each individual. The invention discloses application of the molecular marker and/or the primer pair in breeding of lean-type remote pig strain live pigs.
Description
Technical Field
The invention relates to the technical field of livestock molecular markers, in particular to a molecular marker for fat deposition and meat quality traits of a lean type remote pig strain, and primers, a method and application thereof.
Background
The lean type fixed-distance pig strain is a new product line obtained by breeding after crossing Bakkaia and fixed-distance pigs, the blood systems of the lean type fixed-distance pig strain containing Bakkaia and the fixed-distance pigs are both 50 percent, the hair is completely black, and the body size is between the Bakkaia and the fixed-distance pigs. The index variation coefficient of lean meat percentage, backfat thickness, intramuscular fat content, flesh color, marbling and the like in the unseeded colony is larger (> 20%), so that the characters such as lean meat percentage, backfat thickness, intramuscular fat, flesh color, marbling and the like can be fixed and stably inherited only by breeding for 5 generations or more. However, in the traditional breeding process, intramuscular fat, flesh color and marbling of the breeding pigs cannot be bred, because the characters cannot be measured in a living body, the meat quality of the slaughtered pigs must be obtained after being analyzed, and the slaughtered pigs cannot be used as the breeding pigs for breeding the next generation, so that a marking method for breeding the breeding pigs must be found to assist in selection.
The back fat thickness and the intramuscular fat are inversely related in the conventional breeding, namely, the intramuscular fat content of the pigs with thinned back fat thickness is reduced by breeding, so that the intramuscular fat content is reduced along with the reduction of the back fat thickness by the traditional back fat thickness selection, and the intramuscular fat content is an important index for evaluating the flavor, the taste and the tenderness of the pork and is the most important index for the pork quality. In the breeding process of live pigs, pig breeds with thin backfat and high intramuscular fat content are expected to be obtained, and the breeds with both the thin backfat and the high intramuscular fat content can not be obtained by the traditional breeding method, so that the method for seeking related molecular markers is an effective auxiliary selection method. The pork color and the marble veins are also important indexes about the pork quality, the pork color and the marble veins are the first impression when consumers buy the pork, so the pork price is directly influenced by the quality of the pork color and the marble veins, the two characters cannot be obtained by directly measuring the living bodies of the breeding pigs, the pork sample is taken after slaughter and then measured, the breeding pigs are used as breeding pigs, the breeding pigs cannot be used after slaughter, so the two characters belong to indirect characters, the selection of the indirect characters in breeding needs to be carried out according to the characters or marks related to the breeding pigs, the breeding targets can also be obtained, and the breeding is carried out through the related marks.
While phosphotriesterase related gene (PTER) is essential for normal development of cells, its inactivation will lead to canceration of cells. Scientists research on human PTER gene finds that the gene is highly expressed in adipose tissue, plays an important role in the accumulation process of fat, and has a certain correlation with the degree of obesity, and the single nucleotide polymorphism (rs 10508503) of human PTER gene has an obvious correlation with obesity. In addition, PTER can cause the change of leptin concentration in vivo, further promote the occurrence of obesity.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a molecular marker for fat deposition and meat quality character of a lean-type remote pig strain, and a primer, a method and application thereof.
A molecular marker for fat deposition and meat quality traits of a lean-type farrowing pig strain has a nucleotide sequence shown as SEQ ID NO:1, the 398 th base in the sequence is a base mutation site, and the base mutation site is G or C.
A primer pair for detecting the molecular marker has the following nucleotide sequences:
a forward primer: 5 'GTCAAGTTCATAAGTTTTGGGCAC-3',
reverse primer: 5 'TAGGGCAAGCAGGAAGAGTCAGC-3'.
A molecular marking method for fat deposition and meat quality characters of a lean type fixed-distance pig strain comprises the following steps:
(1) Extracting the genomic DNA containing the molecular marker from the pig;
(2) Performing polymerase chain reaction on the primer pair, the genomic DNA obtained in the step (1) and a PCR reaction buffer solution to obtain an in vitro amplification product;
(3) Carrying out enzyme digestion reaction on the in-vitro amplification product obtained in the step (2) by using restriction enzyme to obtain an enzyme digestion product;
(4) And (4) carrying out polymorphism detection on the enzyme digestion product obtained in the step (3) to obtain the genotype of each individual.
Preferably, in the step (1), on the day of birth of the lean-type fixed-distance pig breeding line piglet, the ear margin tissue of the pig is taken; extracting genomic DNA from ear margin tissue; the genome DNA is a nucleotide sequence containing swine phosphotriesterase related gene (PTER) chr10:45384562 locus, and the total length is 719bp;
preferably, in step (2), the polymerase chain reaction system comprises: mu.L of 10mmol/mL forward primer, 0.5. Mu.L of 10mmol/mL reverse primer, 1. Mu.L of DNA genome template solution with a concentration of 100 ng/. Mu.L, 10. Mu.L of PCR reaction buffer, and 8. Mu.L of pure water, and the final volume is 20. Mu.L.
Preferably, the polymerase chain reaction in step (2) is specifically performed as follows: 20 mul of the polymerase chain reaction system is put into a polymerase chain reaction instrument, and the reaction conditions are as follows:
(1) pre-denaturation at 95 ℃ for 4min,
(2) the denaturation is carried out for 30s at the temperature of 95 ℃,
(3) annealing at the temperature of 52.9 ℃ for 30s,
(4) the extension is carried out for 45s at the temperature of 72 ℃,
(5) repeating the 35 cycles of the steps (2) to (4),
(6) extending for 5min at 72 ℃, and finally cooling to 10 ℃ for storage to obtain an in vitro amplification product.
Preferably, in step (3), the enzyme digestion reaction is specifically performed as follows: and (3) adding 0.5 mu L of 10U/mu L MspI restriction enzyme and 1 mu L MspI restriction enzyme buffer solution into 5 mu L of the in-vitro amplification product obtained in the step (2), adding 3.5 mu L of water, uniformly mixing, and reacting at 37 ℃ for 3h to obtain 10 mu L of enzyme digestion product.
Preferably, in step (4), the polymorphism (RLFP) detection operation is as follows: and (3) carrying out electrophoresis on 5 mu L of the enzyme digestion product obtained in the step (3) for 1h by adopting agarose gel with the mass fraction of 1.5%, wherein the electrophoresis conditions are as follows: normal temperature and 120V voltage; obtaining gel containing enzyme cutting products, and placing the gel containing the enzyme cutting products in a gel imaging system to observe the genotype.
Preferably, the genotypes in step (4) are as follows:
GG individuals are completely non-cleavable and comprise two fragments of 185bp and 534 bp;
the GC type individual is not completely cut and comprises four fragments of 185bp, 534bp,321bp and 213 bp;
the CC individuals are completely cut and comprise three fragments of 185bp, 321bp and 213 bp.
The excellent genotype with thin back fat thickness, high intramuscular fat content, high flesh color score and high marbling score is a mutation type, namely CC type, and the pig with thin back fat thickness, high intramuscular fat content, high flesh color score and high marbling score can be obtained by selecting the individual with the CC type strip for reservation.
Genotype detection and slaughter meat quality determination of lean type farrowing pig strains show that when the weight of a CC type pig with the strips reaches 90kg, the back fat thickness is 2.34mm thinner than that of a GG type individual, the intramuscular fat content of the CC type pig is 0.92 percent higher than that of the GG type individual, and the CC type pork and marbling scores are respectively 0.45 and 0.42 higher than that of the GG type individual, so that the significant levels are achieved.
And selecting individuals with CC genotypes for reservation, so that the breeding pigs with thin backfat thickness, high intramuscular fat content, high flesh color score and marble mark score can be obtained, and the aims of increasing lean meat percentage, intramuscular fat and meat quality are fulfilled.
The PTER gene of the pig is positioned on a No. 10 chromosome and consists of 5 exons, the applicant finds that one mutation exists in an intron 3 of the gene in the whole genome sequencing of a research remote pig, the mutation is from G to C (a 45384562 site of the chromosome 10), and the endonuclease MspI can be identified, so the applicant designs primers aiming at the front and the back of the site, amplifies a segment containing the site, cuts the amplified product by using the MspI endonuclease, carries out electrophoresis detection on the enzyme cut product, judges the genotype, finds that the site has polymorphism in a lean-type remote pig line pig group (a hybrid generation of Bakka and the remote pig, and a later generation produced by utilizing the hybrid generation male and the breeding of a sow is called a lean-type remote pig line, namely an F2 generation), the polymorphism of the site in the first generation (F2) of the lean type remote pig line is found to be rich, the correlation among backfat thickness, intramuscular fat content, flesh color and marbling when the polymorphism of 86F 2 generation groups and the body weight measured after slaughtering of the site reach 90kg is analyzed, and the site is found to be opposite to the back fat deposition and the intramuscular fat deposition, namely, the back fat deposition of the same allele is more, the intramuscular fat deposition is less, the back fat deposition is less, the intramuscular fat deposition is more, which is exactly the same as the pursuit of backfat thickness and intramuscular fat content in the breeding process of the live pigs, so that the auxiliary selection is carried out according to the site, and the breeding process of the lean type remote pig line with the lean meat percentage and the high intramuscular fat content can be accelerated; meanwhile, the locus is found to be obviously related to the meat color and marbling, and the selection by utilizing the locus can improve the meat quality of the swinery.
An application of the molecular marker in the breeding of lean-type farrowing pig strain live pigs.
An application of the primer pair in the breeding of lean-type farrowing pig strain live pigs.
The invention has the following beneficial technical effects:
1. polymorphism exists in chr10:45384562 site of PTER gene for the first time, and the polymorphism is obviously related to pig back fat thickness, intramuscular fat content, flesh color and marbling. Therefore, the applicant analyzes the mutation site of the PTER gene of the lean type remote pig, firstly discovers that the mutation site (G is mutated into C) exists at the chr10:45384562 position of the pig PTER gene, and the restriction enzyme polymorphism site of the restriction enzyme MspI is caused, so that the colony polymorphism is detected in 87 lean type remote pig strains, and the relevance of the polymorphism of the site and the back fat thickness, the intramuscular fat content, the flesh color and the large marble texture of the lean type remote pig is analyzed when the body weight of the lean type remote pig reaches 90kg, and the significant relevance of the back fat thickness, the intramuscular fat content, the flesh color and the marble texture of the site and the body weight of the lean type remote pig is found, and the site can be used as a molecular marker of the fat deposition and the meat quality traits 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, higher intramuscular fat content, better flesh color and marble texture 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, the intramuscular fat content, the flesh color and the marble texture in the breeding of the pigs, realizes the early breeding of the breeding pigs, and can accurately select and retain the pigs even when the pigs are born. The excellent gene type of the PTER gene can be fixed by using the gene marking method only through one generation, and the conventional breeding method can achieve the required effect through a large amount of performance measurement and descendant measurement and over 5 generations of successive breeding, so the molecular marking method can greatly shorten the generation interval and accelerate the breeding process.
3. The method has the advantages of simple operation, low requirement on conditions in the polymerase chain reaction process, easy amplification, and improved amplification efficiency and genotype judgment accuracy.
4. The invention can obviously reduce the back fat thickness of the lean type fixed-distance pig strain, improve the lean meat rate of the pig, improve the intramuscular fat content in the lean meat and improve the meat quality and marbling. When the weight of the lean type fixed-distance pig strain reaches 90kg, the thickness of the back fat of the CC type individual is 2.34mm thinner than that of the GG type individual; the content of intramuscular fat of the CC type is 0.92 percent higher than that of the GG type; the CC type is 0.45 minutes higher than the GG type; CC type is 0.43 minutes higher than GG type marble.
Drawings
FIG. 1 is the agarose gel electrophoresis picture of the PCR amplification of PTER gene of the invention.
FIG. 2 is a restriction enzyme (MspI) cleavage map of PTER gene of the present invention.
FIG. 3 shows the nucleic acid sequence of the molecular marker obtained in the present invention, in which the box is marked with S, i.e., SNP site, and G is mutated into C as the mutation site of the base.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
Example 1
A molecular marking method for fat deposition and meat quality characters of a lean type fixed-distance pig strain comprises the following steps:
(1) Scoring lean type fixed-distance pig strain
Feeding the 87 lean type fixed-distance pig strain to 90kg under the same feeding environment for slaughtering, and measuring the back fat thicknesses at the 3 rd and 4 th ribs of the penultimate pig by using a vernier caliper after slaughtering; taking 100 g of longissimus dorsi at the last 3 and 4 ribs to measure the content of intramuscular fat; scoring the longissimus dorsi at the 3 rd to last and 4 th ribs for flesh color and marbling;
(2) Extraction of genomic DNA
Extracting genome DNA from ear marginal tissues of 87 lean type fixed-distance pig strains; the concentration of each genomic DNA was measured and diluted with pure water to a concentration of 100 ng/. Mu.L;
(3) Primer pair Synthesis
1 pair of primers, namely an upstream primer and a downstream primer, is designed between chr10:45384165 to chr10:45384883 of the PTER gene of the pig, and the sequences of the upstream primer and the downstream primer are as follows:
PTER-F upstream primer: 5' GTCAAGTTCATAAGTTTTTGGCAC-3
PTER-R downstream primer: 5 'TAGGGCAAGCAGGAAGAGTCAGC-3';
submitting the sequences of the upstream primer and the downstream primer to a primer synthesis company for primer synthesis; respectively diluting the synthesized upstream primer and downstream primer with pure water according to the synthesis information to make the final concentration be 10mmol/mL;
(4) Polymerase chain reaction
A. Preparation of polymerase chain reaction System
The polymerase chain reaction system comprises an upstream primer, a downstream primer, a PCR premix and pig genome DNA, wherein the pig genome DNA is 719bp of the full length of a nucleotide sequence between chr10:45384165 and chr10:45384883 of a PTER gene;
the specific operation for preparing the polymerase chain reaction system is as follows: adding 10 mul PCR premix, 0.5 mul 10mmol/mL upstream primer, 0.5 mul 10mmol/mL downstream primer, 1 mul pig genome DNA solution with concentration of 100 ng/mul and 8 mul pure water into a polymerase chain reaction tube in sequence to obtain a final volume of 20 mul; mixing uniformly to prepare 20 mu L of polymerase chain reaction system;
B. polymerase chain reaction
Performing polymerase chain reaction, putting 20 μ L of polymerase chain reaction system into a polymerase chain reaction instrument, wherein the reaction conditions are as follows:
(1) pre-denaturation at 95 ℃ for 4min,
(2) the denaturation is carried out for 30s at the temperature of 95 ℃,
(3) annealing at the temperature of 52.9 ℃ for 30s,
(4) the extension is carried out for 45s at the temperature of 72 ℃,
(5) repeating the 35 cycles of the steps (2) to (4),
(6) extending for 5min at 72 ℃, finally cooling to 10 ℃ for storage,
after the reaction is finished, obtaining an in vitro amplification product of 719bp length between chr10:45384165 and chr10:45384883 of the pig PTER gene; mu.L of the in vitro amplification product was electrophoresed for 1 hour with 1.5% agarose gel under the following conditions: at room temperature and 120V, a gel of the amplified product of PTER was obtained, as shown in fig. 1, showing that: the in vitro amplification product has a single band and can be used for the next restriction reaction.
(5) Enzyme digestion reaction
Carrying out restriction enzyme digestion reaction on the in vitro amplification product, wherein a basic group G at chr10:45384562 of the pig PTER gene is mutated into C, so that an MspI restriction enzyme digestion site is formed;
the specific operation steps of the enzyme digestion reaction are as follows: taking 5 mu L of the in vitro amplification product of the swine PTER gene 719bp long, respectively adding 0.5 mu L of MspI restriction enzyme with the concentration of 10U/mu L and 1 mu L of MspI restriction enzyme buffer solution, adding 3.5 mu L of water, uniformly mixing, and reacting at 37 ℃ for 3h to obtain 10 mu L of enzyme digestion product.
(6) Polymorphism (RLFP) detection
The enzyme products were tested for polymorphisms (RLFP), and 5. Mu.L of the enzyme products were electrophoresed for 1h on 2.0% agarose gel, 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 specific steps are as follows:
the completely non-cleavable GG-type DNA fragment comprises 185bp and 534bp fragments;
the incompletely cut fragment is GC type, and comprises four fragments of 185bp, 534bp,321bp and 213 bp;
the CC type individual is completely cut and comprises three fragments of 185bp, 321bp and 213 bp;
the PTER gene polymorphism detection results of 87 lean fixed-distance pigs are shown in the following table. The frequency of the G allele was 0.517 and the frequency of the C allele was 0.483, and this herd had abundant polymorphism at this site.
The 90kg backfat thickness, intramuscular fat content, flesh color and marbling scores of the 87 lean type remote pig lines were each subjected to a least squares analysis using the General Linear Model (GLM) program of the SPSS13.0 software, and the gene effects were analyzed, the results of which are shown in the following table.
Note: the same row of shoulder marks with different letters (a and c) indicates that the difference is extremely significant (P)<0.01 Marked ab represents significant difference from c (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 alpha 1 Represents the average effect of allele G; alpha is alpha 2 Indicating the average effect of allele C; alpha represents the average effect value of the C allele in place of the G allele (i.e., alpha) 2 -α 1 )。
From the above table, it can be seen that:
the 90kg backfat thickness of GG type individuals is extremely thicker than that of CC type (P < 0.01), and the 90kg backfat thickness difference between GG type individuals and GC type individuals is not significant (P > 0.05); 90kg backfat thickness for GC type individuals was significantly thicker than for CC type (P < 0.05); thus, the elite genotype at 90kg backfat thickness was CC, and the average effect value of the elite allele was-1.185 for the C, C allele instead of G.
The intramuscular fat content is highest in CC type, and is extremely higher than that of GC type and GG type individuals (P < 0.01), the difference between GC type and GG type is not significant (P > 0.05), therefore, the CC type is favorable for intramuscular fat deposition, CC is a good allele type for intramuscular fat deposition of lean type remote pig strains, and the average effect of C and C in place of G is 0.443.
The flesh color score is 3.50 points at the maximum according to CC type, and is remarkably higher than GC and GG type (P < 0.05), the difference between GC and GG type is not remarkable (P > 0.05), so that CC type is a good genotype of flesh color trait, C allele is a good allele of flesh color trait, and the average effect value of C instead of G is 0.220.
The marbling score is 3.26 points at the maximum of CC type, which is remarkably higher than GC and GG type (P < 0.05), and the difference between GC and GG type is not remarkable (P > 0.05), so that CC type is a good genotype of marbling traits, C allele is a good allele of marbling traits, and the average effect value of C instead of G is 0.211.
In conclusion, for 4 traits of 90kg backfat thickness, intramuscular fat content, flesh color and marbling, the excellent alleles at the site are all C, and the excellent genotypes are all CC types, so that individuals with the genotypes being CC types can be selected for reservation, and the frequency of the allele C is increased, thereby obtaining high-quality pork with thinner backfat, higher intramuscular fat content and better flesh color and marbling.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and equivalent substitutions or changes according to the technical solution and the inventive concept of the present invention should be covered by the scope of the present invention.
Sequence listing
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Claims (8)
1. A molecular marker for fat deposition and meat quality traits of a lean type farrowing pig strain is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO:1, the 398 th base in the sequence is a base mutation site, and the base mutation site is G or C.
2. A primer pair for detecting the molecular marker of claim 1, wherein the nucleotide sequence is as follows:
a forward primer: 5 'GTCAAGTTCATAAGTTTTGGGCAC-3',
reverse primer: 5 'TAGGGCAAGCAGGAAGAGTCAGC-3'.
3. A molecular marking method for fat deposition and meat quality characters of a lean type fixed-distance pig strain is characterized by comprising the following steps:
(1) Extracting genomic DNA from a pig comprising the molecular marker of claim 1;
(2) Performing polymerase chain reaction by using the primer pair of claim 2, the genomic DNA obtained in the step (1) and a PCR reaction buffer solution to obtain an in vitro amplification product;
(3) Carrying out enzyme digestion reaction on the in-vitro amplification product obtained in the step (2) by using restriction enzyme to obtain an enzyme digestion product; the specific operation of the enzyme digestion reaction is as follows: adding 0.5 mu L of 10U/mu L MspI restriction endonuclease and 1 mu L MspI restriction endonuclease buffer solution into 5 mu L of in-vitro amplification product obtained in the step (2), adding 3.5 mu L of water, uniformly mixing, and reacting at 37 ℃ for 3h to obtain 10 mu L of enzyme digestion product;
(4) Carrying out polymorphism detection on the enzyme digestion product obtained in the step (3) to obtain the genotype of each individual, wherein each genotype is as follows;
GG individuals are completely non-cleavable and comprise two fragments of 185bp and 534 bp;
the GC type individual is not completely cut and comprises four fragments of 185bp, 534bp,321bp and 213 bp;
the CC type individual is completely cut and comprises three fragments of 185bp, 321bp and 213 bp;
the CC type individual backfat is thinner, the intramuscular fat content is higher, and the flesh color and marbling of the high-quality pork are better, so that the excellent genotype is the CC type.
4. The method of claim 3, wherein in step (2), the polymerase chain reaction system comprises: mu.L of 10mmol/mL forward primer, 0.5. Mu.L of 10mmol/mL reverse primer, 1. Mu.L of DNA genome template solution with a concentration of 100 ng/. Mu.L, 10. Mu.L of PCR reaction buffer, and 8. Mu.L of pure water, and the final volume is 20. Mu.L.
5. The method of claim 3, wherein the polymerase chain reaction in step (2) is specifically performed as follows: 20 mul of the polymerase chain reaction system is put into a polymerase chain reaction instrument, and the reaction conditions are as follows:
(1) pre-denaturation at 95 ℃ for 4min,
(2) the denaturation is carried out for 30s at the temperature of 95 ℃,
(3) annealing the blank at the temperature of 52.9 ℃ for 30s,
(4) the extension is carried out for 45s at the temperature of 72 ℃,
(5) repeating the 35 cycles of the steps (2) to (4),
(6) extending for 5min at 72 ℃, and finally cooling to 10 ℃ for storage to obtain an in vitro amplification product.
6. The method of claim 3, wherein the polymorphism (RLFP) detection operation in step (4) is performed by: and (3) carrying out electrophoresis on 5 mu L of the enzyme digestion product obtained in the step (3) for 1h by using agarose gel with the mass fraction of 1.5%, wherein the electrophoresis conditions are as follows: normal temperature and 120V voltage; obtaining gel containing the enzyme cutting product, and placing the gel containing the enzyme cutting product under a gel imaging system to observe the genotype.
7. Use of the molecular marker of claim 1 in the breeding of lean-type remote pig strain live pigs.
8. Use of the primer pair of claim 2 in breeding a lean-type remote pig strain live pig.
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