CN103320516B - Method and special product for assisted identification of swine backfat thickness character - Google Patents

Abstract

The invention discloses a method and a special product for the assisted identification of swine backfat thickness character. The method for the assisted identification of swine backfat thickness character comprises the following steps of: determining whether the deoxyribonucleotide 47033 of the GenBank Accession Number GU565976.1 of a peroxisome proliferator-activated receptor delta gene of a to be detected swine is T, or C, or T and C, so as to determine whether the genotype of the to be detected swine is TT or TC or CC, and determining the backfat thickness character according to the genotype of the to be detected swine, wherein the backfat thickness of the to-be-detected swine with the TT genotype is greater than that of the to be detected swine with the TC genotype, and the backfat thickness of the to be detected swine with the TC genotype is greater than that of the to be detected swine with the CC genotype. The method and the special product disclosed by the invention are used for breeding swine, so that the to be detected swine can be screened in the early stage, the problem of long time needed for selecting good breeding swine in the practical production can be effectively solved, the breeding cost is reduced, and the swine backfat thickness in the practical production is effectively reduced or increased.

Description

A method for auxiliary identification of pig backfat thickness traits and special products thereof

technical field

The invention relates to a method for assisting identification of pig backfat thickness traits and a special product thereof.

Background technique

One of the most famous characteristics of local pig breeds in my country is their excellent meat quality. These local breeds, breeds containing the blood of local breeds, and even Chinese and foreign hybrid commercial pork are widely welcomed by consumers because of their excellent meat quality, such as Beijing Black pork, Laiwu black pork, Heishan pork, Sutai pork, etc. are well received by consumers. Moreover, the price of these high-quality pork is 1.5-3 times that of ordinary pork, which fully reflects the commercial value of local breeds of pigs in my country at the current stage. However, most of these pig breeds are fat pigs, and their notable feature is the thick backfat. Excessive backfat deposition not only leads to slow growth, but also does not meet the requirements of the current situation for grain saving. As we all know, growing lean meat requires less energy than growing fat, and most local breeds of pigs in my country are fat pigs, and most of their feed-to-meat ratios are above 4:1. According to research, the genetic correlation between backfat thickness and feed-to-meat ratio can reach 0.55 (Hoque MA, Suzuki1K, Kadowaki H, Shibata T, Oikawa T. Genetic parameters for feed efficiency traits and their relationships with growth and carcass traits in Duroc pigs.J AnimBreed Genet.2007,124:108-116.), the genetic correlation with lean meat percentage can reach -0.58 (Sun Hua, Song Zhongxu, Li Lianghua, Peng Xianwen, Guo Wanzheng, Wu Huayu, Mei Shuqi. The main growth and Estimation of genetic parameters for carcass traits. Hubei Agricultural Sciences. 2009, 48, 3086-3089.). Therefore, the backfat thickness of local pig breeds and breeds containing local pig blood is more than 3 cm. The advantages of local pig breeds in my country have not been fully utilized. Compared with international commercial pig breeds, there is still great potential for improvement.

Based on the importance of the above-mentioned pig backfat thickness traits, carrying out breeding work for it is a focus of current breeding work. Due to the slow speed and low accuracy of traditional breeding, molecular breeding has become the main technical means of current breeding work due to its accuracy and rapidity.

Peroxisome proliferator-activated receptors (PPARs) are a class of ligand-activated nuclear transcription factors that belong to the steroid/thyroid/retinoic acid receptor superfamily and consist of three members: PPARα, PPARγ, and PPARδ (PPARD). , they can all mediate lipophilic small molecular compounds to regulate DNA transcription. PPARs can regulate the expression of many target genes involved in lipid metabolism inside and outside cells, especially genes encoding some important enzymes in the process of β-oxidation. In addition, PPARs are also involved in adipocyte (Evans RM, Barish GD, Wang YX. PPARs and the complexjourney to obesity. Nat Med. 2004, 10:355-361.).

Contents of the invention

The purpose of the invention is to provide a method for assisting identification of pig backfat thickness traits and a special product thereof.

The method for auxiliary identification and detection of pig backfat thickness provided by the present invention is to detect the GenBank Accession Number GU565976.1 of the peroxisome proliferator-activated receptor delta (PPARD) gene of the pig to be tested (updatedate is April 2010 26th) whether the 47033rd deoxyribonucleotide is T or C or T and C to determine whether the genotype of the pig to be tested is TT or TC or CC, and determine the backfat according to the genotype of the pig to be tested Thickness traits: the backfat thickness of the tested pigs of the TT genotype is higher than or candidately higher than that of the tested pigs of the TC genotype, and the backfat thickness of the tested pigs of the TC genotype is higher than or candidately higher than that of the CC genotype. Test pigs; the TT genotype is the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 (update date is April 26, 2010) of the peroxisome proliferator-activated receptor δ gene is T The CC genotype is C at position 47033 of the GenBank Accession Number GU565976.1 (updated on April 26, 2010) of the peroxisome proliferator-activated receptor δ gene The TC genotype is the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 (update date is April 26, 2010) of the peroxisome proliferator-activated receptor δ gene. Hybrid of C and T.

In the above method, the 47033rd deoxyribonucleotide of GenBank Accession Number GU565976.1 (update date is April 26, 2010) for detecting the peroxisome proliferator-activated receptor δ gene of the pig to be tested is T or C or T and C can specifically be T or C or T and C in the 95th position of sequence 3 or sequence 4 in the detection sequence list.

In the above method, the 47033rd deoxyribonucleotide of GenBank Accession Number GU565976.1 (update date is April 26, 2010) for detecting the peroxisome proliferator-activated receptor δ gene of the pig to be tested is The method of T or C or T and C can specifically be a sequencing analysis; the said sequencing analysis includes two steps of PCR amplification and sequencing the PCR amplification product; the primer pair used in the PCR amplification satisfies the following conditions: GenBank Accession Number GU565976.1 (update date is April 26, 2010) No. 47033 of the GenBank Accession Number GU565976.1 (update date is April 26, 2010) in the PCR amplification product containing the pig's genomic DNA as a template deoxyribonucleotides.

In the above method, the primer pair used for the PCR amplification may specifically be a primer pair composed of the single-stranded DNA shown in sequence 1 in the sequence listing and the single-stranded DNA shown in sequence 2 in the sequence listing.

A special product for assisting identification of pig backfat thickness provided by the invention is a reagent for assisting identification of pig backfat thickness.

The reagent provided by the present invention to assist in the identification of the pig's backfat thickness traits is GenBank Accession Number GU565976.1 (update date is April 26, 2010) for detecting the peroxisome proliferator-activated receptor δ gene of the pig to be tested. ) of the 47033rd deoxyribonucleotide is T or C or T and C substances.

Among the above reagents, the substance can specifically be a primer pair consisting of the single-stranded nucleotide shown in sequence 1 in the sequence listing and the single-stranded nucleotide shown in sequence 2 in the sequence listing. When the primer pair is used for PCR amplification, the amplified DNA fragment is the nucleotide shown in sequence 3 or the nucleotide shown in sequence 4 according to the difference of the genomic DNA of the pig to be tested.

Another special product provided by the present invention to assist in the identification of pig backfat traits is a kit containing the above-mentioned reagents for assisting in the identification of pig backfat traits.

In the above, to detect whether the 47033th deoxyribonucleotide of GenBank AccessionNumber GU565976.1 (update date is April 26, 2010) of the peroxisome proliferator-activated receptor δ gene of the pig to be tested is T or C Or the substances of T and C can be determined by at least one of the following methods in GenBank Accession Number GU565976.1 (update date is April 26, 2010) of the porcine peroxisome proliferator-activated receptor δ gene Reagents and/or instruments required for single nucleotide polymorphism at position 47033: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chip. Among them, SNP chips include chips based on nucleic acid hybridization reactions, chips based on single base extension reactions, chips based on allele-specific primer extension reactions, chips based on "one-step" reactions, chips based on primer ligation reactions, and chips based on Chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule-DNA binding reactions.

The application of the above-mentioned reagent or the above-mentioned kit in assisting in the identification of the pig's backfat traits and in the preparation of products for assisting in the identification of the pig's backfat traits all belong to the protection scope of the present invention.

The product can be a reagent or a kit, or a combination product of a reagent or a kit and an instrument, such as a combination product consisting of primers and a DNA sequencer, or a combination of PCR reagents, DNA sequencing reagents and a DNA sequencer product.

In the above, the backfat thickness may be the backfat thickness corresponding to the sixth and seventh ribs and/or the average backfat thickness.

In one embodiment of the present invention, the pig to be tested is a Damin hybrid pig.

The above method, reagent or kit can be used to breed pigs with thicker or thinner backfat traits, so as to be applied to pig breeding.

The invention also provides a method for cultivating thin backfat pigs.

The method for breeding thin backfat pigs provided by the present invention comprises selecting pigs of CC genotype or TC genotype for breeding; the CC genotype is the GenBankAccession of the peroxisome proliferator-activated receptor delta (PPARD) gene The 47033rd deoxyribonucleotide of Number GU565976.1 (update date is April 26, 2010) is homozygous for C, and the TC genotype is GenBank of the peroxisome proliferator-activated receptor δ gene The 47033rd deoxyribonucleotide of Accession Number GU565976.1 (update date is April 26, 2010) is a hybrid of C and T.

The experiment proved that the CC genotype population was 5.49 mm thinner and 5.60 mm thinner than the TT genotype population in six and seven rib backfat thickness and average backfat thickness (P<0.01), and the CC genotype population was thinner than the TC genotype population in six and seven ribs. Seven-rib backfat thickness and average backfat thickness were 3.12mm and 3.31mm thinner respectively (P<0.01). Therefore, the 47033rd position of the GenBank Accession Number GU565976.1 (update date is April 26, 2010) of the peroxisome proliferator-activated receptor δ gene can be used as a molecular breeding marker for pig backfat thickness traits.

Applying the invention to breeding pigs can carry out early screening of pigs to be selected, effectively alleviate the problem of long time for selecting good breeding pigs in actual production, reduce breeding costs, and effectively reduce or increase the backfat thickness of pigs in actual production. The detection method of the invention has the advantages of simple operation, low cost and high accuracy, and can realize automatic direct detection.

Description of drawings

Fig. 1 is a map of the sequencing results of sequence M1 and sequence M2 (sequencing map of g.47033C>T mutation site of porcine PPARD gene).

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The Damin hybrid pigs in the following examples are the F2 generation of large white pigs × Min pigs, obtained according to the methods in the literature: Luo W, Cheng D, Chen S, Wang L, Li Y, Ma X, Song X, Liu X, Li W,Liang J,Yan H,Zhao K,Wang C,Wang L,Zhang L.Genome-wide association analysis of meatquality traits in a porcine Large White×Minzhu intercross population.IntJ Biol Sci.2012,8:580-595 . The specific method is as follows: four Large White pigs were mated with 16 Min pigs to obtain F1 generation population, 9 boars in this F1 generation population were mated with 46 sows, and 575 F2 generation individuals were produced in three parities.

Embodiment 1, the backfat thickness trait of auxiliary identification pig

1. Determination of g.47033C>T polymorphism site of porcine PPARD gene

1. PCR amplification

According to the genome DNA sequence of pig PPARD gene (GenBank Accession Number GU565976.1) information, a pair of primers were designed as follows:

U (upstream primer): 5'-caaaacgcagtagcaggaaa-3' (sequence 1 of the sequence listing);

D (downstream primer): 5'-cccaacctctctagcacacc-3' (SEQ ID NO: 2 of the Sequence Listing).

Three Damin hybrid pigs were selected as experimental materials. Using the genomic DNA of each Damin hybrid pig as a template, PCR amplification was performed using primers U and D.

The amplification system is: 200ng of genomic DNA, 5μl of 10×PCR amplification buffer, 10mM of dNTPs, 50ng of upstream and downstream primers, 0.75U of Taq DNA polymerase, Mg ²⁺ 2.5mmol/L, and supplement the reaction system with ddH ₂ O to 50μl.

The PCR reaction conditions were: denaturation at 95°C for 5 min; then denaturation at 95°C for 20 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, a total of 35 cycles; finally, extension at 72°C for 10 min.

The PCR products were stored at -20°C after detection by agarose gel. The products amplified using the genomic DNA of three Damin hybrid pigs as templates were named product 1, product 2, and product 3, respectively.

2. Cloning sequencing and sequence analysis

The three PCR products were recovered and purified with agarose gel recovery kit (Tiangen Biochemical Technology Co., Ltd.), and the recovered DNA fragments were connected to the vector pGEM-T (Promega Company), and the ligated products were transformed into Escherichia coli DH5α competent cells (Tomorrow Baiao (Beijing) Technology Co., Ltd.), positive clones were screened according to the carbenicillin resistance marker on the vector, and recombinant plasmids containing recovered fragments were obtained. The T7 and SP6 promoter sequences on the recombinant plasmid vector were used as primers to determine its nucleotide sequence (English Weijieji (Shanghai) Trading Co., Ltd.). The sequencing results showed that product 1 was sequence M1, product 2 was a mixture of sequence M1 and sequence M2, and product 3 was sequence M2; both sequence M1 and sequence M2 were 212 bp in length, and there was only one deoxyribonucleotide difference (T /C) (see Figure 1), this deoxyribonucleotide is the 47033rd deoxyribonucleotide from the 5′ end of GenBank Accession Number GU565976.1 (update date is April 26, 2010), the single The nucleotide polymorphism is named g.47033C>T. The nucleotide sequence of the sequence M1 is shown in the sequence 3 of the sequence listing, and the nucleotide sequence of the sequence M2 is shown in the sequence 4 of the sequence listing. GenBank Accession Number GU565976.1 (update date: April 26, 2010) of the peroxisome proliferator-activated receptor δ gene is a homozygous genotype of T at the 47033rd deoxyribonucleotide from the 5′ end Named as TT, the pure DNA with C at the 47033rd deoxyribonucleotide from the 5′ end of the GenBank Accession Number GU565976.1 (update date is April 26, 2010) of the peroxisome proliferator-activated receptor δ gene The zygotic genotype is CC, and their heterozygous genotype is TC.

2. Correlation analysis of pig PPARD gene g.47033C>T polymorphism and pig backfat thickness and meat quality traits

In order to determine whether the g.47033C>T polymorphic locus is related to pig backfat thickness traits, 575 Damin hybrid pigs were used as experimental materials, and the following experiment was carried out: the genomic DNA of each pig was extracted, and the above-mentioned primers U and D were used for Perform PCR amplification and sequence analysis on the PCR amplification product of each pig to determine whether the genotype of each pig is TT, TC or CC, and the method is the same as step 1. Determine the traits of backfat thickness according to the genotype of the pig: the backfat thickness of the tested pigs with the TT genotype is higher than that of the tested pigs with the TC genotype, and the backfat thickness of the tested pigs with the TC genotype is higher than that of the tested pigs with the CC genotype Test pig.

Measure and record the backfat thickness of each pig at 240 days of age (the fat thickness at the thickest part of the shoulder, the backfat thickness corresponding to the sixth and seventh ribs (the backfat thickness at the sixth and seventh ribs), the fat thickness at the junction of the thorax and lumbar vertebrae, the thickness of the loin sacral joint fat thickness), intramuscular fat content (IMF) and other traits, and the least squares method was used to carry out the correlation analysis between the g.47033C>T locus and the traits. The model used is as follows:

Y=S+P+B+G+e

Among them, Y is the measured value of traits, S is the sex effect, P is the parity effect, B is the slaughter batch effect, G is the genotype effect, and e is the residual effect.

Table 1 Association of PPARD gene mutation site g.47033C>T genotype with fat deposition-related traits

genotype quantity Backfat thickness of six or seven ribs (mm) Average backfat thickness (mm) Intramuscular fat (%)

TT 161 40.01± ^0.77A 40.14± ^0.76A 3.14± ^0.18A TC 273 37.64±0.68 ^B 37.85± ^0.67B 3.11± ^0.16A CC 141 34.52± ^0.77C 34.54± ^0.76C 3.13± ^0.18A

Note: The same superscript letters in the same column indicate no significant difference (P>0.01), and different superscript letters in the same column indicate extremely significant difference (P<0.01).

Among them, fat thickness refers to the thickness of subcutaneous fat, excluding skin thickness. The average backfat thickness of the thickest part of the shoulder, the fat thickness of the six or seven ribs, the fat thickness of the thoracolumbar junction, and the fat thickness of the lumbar-sacral junction was taken as the average backfat thickness.

The results are shown in Table 1, which shows that in the traits of backfat thickness, the backfat thickness of TT genotype pigs is higher than that of TC genotype pigs, and the backfat thickness of TC genotype pigs is higher than that of CC genotype pigs; The backfat thickness and average backfat thickness of the TT genotype population at the corresponding position of the sixth and seventh ribs were 5.49 mm and 5.60 mm thinner (P<0.01), respectively, and the CC genotype population was lower than the backfat thickness of the TC genotype population at the corresponding position of the six and seven ribs. Fat thickness and average backfat thickness were 3.12mm and 3.31mm thinner, respectively (P<0.01). Description The present invention utilizes the single nucleotide polymorphism at position 47033 of GenBank Accession Number GU565976.1 (update date is April 26, 2010) of the peroxisome proliferator-activated receptor delta (PPARD) gene to identify pigs The method of backfat thickness traits is consistent with the actual measurement results of pig backfat thickness.

Therefore, in actual pig breeding, in order to obtain pigs with thinner backfat, it is better to select CC genotype pigs for breeding.

Claims (14)

1. A method for assisting identification of pig backfat thickness traits is to detect the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 of the peroxisome proliferator-activated receptor δ gene of the pig to be tested. T or C or T and C, to determine whether the genotype of the pig to be tested is TT or TC or CC, determine the backfat thickness trait according to the genotype of the pig to be tested: the backfat of the pig to be tested of the TT genotype Thickness is higher than or candidate is higher than the test pig of TC genotype, and the backfat thickness of the test pig of TC genotype is higher than or candidate is higher than the test pig of CC genotype; Described TT genotype is peroxidase The GenBank Accession Number GU565976.1 of the somatic proliferator-activated receptor δ gene is homozygous for T at the 47033rd deoxyribonucleotide from the 5′ end; the CC genotype is peroxisome proliferator-activated receptor δ The GenBank Accession Number GU565976.1 of the gene is homozygous for C at the 47033rd deoxyribonucleotide from the 5′ end, and the TC genotype is the GenBank Accession Number GU565976 of the peroxisome proliferator-activated receptor δ gene .1 The 47033rd deoxyribonucleotide from the 5' end is a hybrid of C and T. 2. The method according to claim 1, characterized in that: the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 of the peroxisome proliferator-activated receptor delta gene of the pig to be tested Whether it is T or C or the method of T and C is sequencing analysis; the sequencing analysis includes two steps of PCR amplification and sequencing the PCR amplification product; the primer pair used in the PCR amplification meets the following conditions: The pig's genomic DNA is used as a template for PCR amplification, and the product contains the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 of the pig's peroxisome proliferator-activated receptor delta gene. 3. The method according to claim 2, characterized in that: the primer pair used in the PCR amplification is composed of the single-stranded DNA shown in sequence 1 in the sequence listing and the single-stranded DNA shown in sequence 2 in the sequence listing primer pair. 4. The method according to any one of claims 1-3, characterized in that: the backfat thickness is the backfat thickness corresponding to six or seven ribs and/or the average backfat thickness. 5. A reagent for assisting the identification of pig backfat thickness, which is to detect the 47033rd deoxyribonucleotide of the GenBank Accession Number GU565976.1 of the peroxisome proliferator-activated receptor δ gene of the pig to be tested. T or C or a substance of T and C. 6. The reagent according to claim 5, characterized in that: the substance is a primer pair consisting of the DNA shown in sequence 1 in the sequence listing and the DNA shown in sequence 2 in the sequence listing. 7. The reagent according to claim 5 or 6, characterized in that: the backfat thickness is the backfat thickness corresponding to six or seven ribs and/or the average backfat thickness. 8. A kit for assisting identification of pig backfat thickness traits, comprising the reagent according to claim 5. 9. The kit according to claim 8, characterized in that: the kit for assisting in the identification of pig backfat thickness traits contains the reagent according to claim 6. 10. The kit according to claim 8 or 9, characterized in that: the backfat thickness is the backfat thickness corresponding to six or seven ribs and/or the average backfat thickness. 11. The reagent according to claim 5 or 6 or the application of the kit according to claim 8 or 9 in assisting the identification of the backfat traits of pigs, or in the preparation of products for assisting the identification of pigs' backfat thickness traits Applications. 12. The application according to claim 11, characterized in that: the backfat thickness is the backfat thickness corresponding to six or seven ribs and/or the average backfat thickness. 13. Application of the method according to any one of claims 1-4, the reagent according to any one of claims 5-7 or the kit according to any one of claims 8-10 in pig breeding. 14. A method for cultivating thin backfat pigs, comprising selecting pigs of CC genotype or TC genotype for breeding; said CC genotype is the autosome of GenBank Accession Number GU565976.1 of the peroxisome proliferator-activated receptor δ gene The 47033rd deoxyribonucleotide at the 5' end is homozygous for C, and the TC genotype is the 47033rd position at the 5' end of the GenBank Accession Number GU565976.1 of the peroxisome proliferator-activated receptor δ gene The deoxyribonucleotide is a hybrid of C and T.