CN112176070A - UCP3 gene related to pig intramuscular fat character, molecular marker and application thereof - Google Patents

Abstract

The invention discloses a UCP3 gene related to the intramuscular fat character of pigs, a molecular marker and application thereof, wherein the SNP molecular marker is positioned in a nucleotide sequence shown as SEQ ID NO: 6 at 2159bp, and at a genomic position 8387309 from the 5' end of chromosome 9 of porcine version 11.1. Firstly, identifying a variation site influencing the intramuscular fat content of pigs in 279 test swinery by utilizing a Fasttarget target region sequencing technology, and establishing a PCR-RsaI-RFLP gene rapid typing method aiming at the site; on the basis, the application effect of the site for improving the intramuscular fat in 500 test swineries is verified, the result shows that the intramuscular fat content of the swinery can be obviously improved by selecting the favorable genotype of the site, and the identified mutation site can be used for early, living and rapid marking auxiliary breeding of the intramuscular fat of the boar.

Description

UCP3 gene related to pig intramuscular fat character, molecular marker and application thereof

Technical Field

The invention belongs to the field of livestock molecular breeding, and mainly relates to screening of a molecular marker for pig intramuscular fat character marker-assisted selection and application thereof. Wherein the molecular marker is positioned in an intron region of a UCP3 gene, and the genome position of the molecular marker is positioned at the 8387309 site from the 5' end of the No. 9 chromosome of the 11.1 version of the pig.

Background

The pig industry is one of the mainstay industries of the livestock industry in China, pork is a main source of animal protein in the life of people, and the requirement on the pork quality is correspondingly improved along with the improvement of the consumption level of people. Therefore, genetic improvement of pork quality is a hot spot of research in the swine industry. The quality of meat (meat quality for short) can be evaluated by a plurality of technical indexes, such as meat color, back fat thickness, pH value, water retention capacity, intramuscular fat content, tenderness and the like. Intramuscular fat is an important index for evaluating pork quality, is closely related to meat quality characters such as tenderness and flavor of meat, and genetic improvement of intramuscular fat characters is always the focus of attention in the field of pig genetic breeding. However, the genetic improvement effect of the intramuscular fat character of the pig by using the conventional breeding technology is not ideal for a long time. Therefore, the identification of key genes influencing the intramuscular fat character and the development of valuable molecular markers have very important significance for the early and accurate breeding of the intramuscular fat character, the acceleration of the genetic improvement process of the intramuscular fat character of pigs and the improvement of pork quality.

In the aspect of meat quality traits, Halothane genes (Halothane, Hal), sour meat genes (RN) and Phosphorylase kinase gamma 1 (PHKG 1) are three main effective genes influencing the meat quality traits, and are widely applied to molecular marker-assisted breeding of pork color and drip loss traits. In 2019, Cho et al found that the pig MYH3 gene is a major gene affecting skeletal muscle fiber type and intramuscular fat deposition, and identified a causal mutation site in the promoter of the gene (Plos Genetics,15(10), 2019). However, the major genes identified to date affecting intramuscular fat are very limited and there is still a lack of effective molecular markers available for selection of intramuscular fat. Uncoupling protein 3(UCP3) is one of the members of uncoupling protein family, and research shows that it plays an important regulatory role in the processes of energy metabolism and glycolipid metabolism. In addition, the research shows that the UCP3 of the pig has SNPs sites related to fat deposition and backfat thickness meat quality traits. Nevertheless, whether UCP3 is related to intramuscular fat or not and whether a molecular marker which can be used for breeding the intramuscular fat of the pig exists in the gene or not is not clear.

Disclosure of Invention

In view of the above background, the applicant firstly clones the full-length cDNA sequence of the porcine UCP3 gene, analyzes the expression patterns of the porcine UCP3 gene in different tissues, different fiber types of skeletal muscle tissues and before and after differentiation of precursor adipocytes, preliminarily determines the relationship between UCP3 gene expression and intramuscular fat deposition, and lays a foundation for the future genetic improvement of pork quality traits by using the full-length sequence of the gene. On the basis, the invention utilizes the target region targeted sequencing technology to identify the genetic variation sites in the gene sequence, and performs correlation analysis on the polymorphism of the genetic variation sites and the production traits of the pigs in a pig resource population, and a molecular marker which can be used for breeding the intramuscular fat of the pigs is identified through analysis, so that the favorable genotype and the unfavorable genotype are determined, and the intramuscular fat content of the pig population can be obviously improved through the selection of the favorable genotype, thereby achieving the aim of improving the meat quality traits of the pigs.

In order to achieve the above object, the present invention is realized by the following techniques:

an SNP molecular marker related to the intramuscular fat trait of pigs, wherein the SNP molecular marker is positioned in a nucleotide sequence shown as SEQ ID NO: 6, and the variation site corresponds to 8387309 site from the 5' end of the 9 th chromosome of the 11.1 version of the pig, and can cause the RsaI-RFLP polymorphism.

A primer pair for amplifying the SNP molecular marker, which comprises a forward primer gUCP3-F and a reverse primer gUCP 3-R; the sequences of the forward primer gUCP3-F and the reverse primer gUCP3-R are shown as follows:

forward primer gUCP 3-F: GATGACCCTTTTCCTTTG

Reverse primer gUCP 3-R: GACTGAACTCCCTCCCTG are provided.

A kit for genotyping the G/T mutation site of the SNP molecular marker comprises the primer pair and the RsaI restriction endonuclease.

A pig intramuscular fat character molecular marker assisted breeding method comprises the steps of carrying out PCR amplification on a pig genome DNA to be detected by adopting the primer pair to obtain a gene fragment containing a G/T mutation site of the SNP molecular marker in claim 1, carrying out enzyme digestion on the gene fragment obtained by the PCR amplification by utilizing RsaI restriction endonuclease, and finally carrying out genotyping on the G/T mutation site of the SNP molecular marker through agarose gel electrophoresis; the gene fragment containing the G/T mutation site of the SNP molecular marker is subjected to restriction enzyme digestion by RsaI restriction enzyme to generate three genotypes, when the site is GG homozygous genotype, the site cannot be subjected to enzyme digestion by RsaI, and only one fragment is obtained after enzyme digestion reaction; when the locus is TT mutation homozygous genotype, the RsaI enzyme can cut the PCR product into two segments; when the locus is a GT heterozygous genotype, the RsaI enzyme can cut the PCR product into three segments; the intramuscular fat of the GT genotype individuals is obviously higher than that of the GG and TT genotype individuals.

The SNP molecular marker, the primer pair or the kit can be applied to the genetic improvement of the intramuscular fat traits of the pigs.

A UCP3 gene related to the intramuscular fat character of pigs, which has the nucleotide sequence shown in SEQ ID NO: 2, or a CDS region nucleotide sequence as set forth in figure 2. The full-length cDNA sequence of the gene is shown as SEQ ID NO:1 is shown.

The protein coded by the UCP3 gene related to the intramuscular fat character of the pig has the sequence shown in SEQ ID NO: 5.

The UCP3 gene related to the pig intramuscular fat character is applied to genetic improvement of the pig intramuscular fat character.

The technical scheme of the invention is obtained by adopting the following steps:

1. UCP3 gene clone related to pig intramuscular fat character and expression pattern analysis

The present invention first downloads PREDICTED Sus scrofa UCP3, mRNA sequence (NM-214049.1) as seed sequence from NCBI database. Respectively designing amplification primers of 5 'end and 3' end of UCP3 gene of pig and 5 '-RACE and 3' -RACE amplification primers according to the downloaded nucleotide sequence, and utilizing

RACE 5 '/3' Kit clones the 5 'and 3' untranslated region sequences of the UCP3 gene of the pig. According to the test result of 5' -RACE, the transcription initiation site of the gene is determined, and the full-length cDNA sequence of the porcine UCP3 gene is obtained by sequence alignment and splicing. The nucleotide sequence of the full-length cDNA of the porcine UCP3 gene is shown as SEQ ID NO 1; wherein the CDS nucleotide sequence is shown as SEQ ID NO 2; the nucleotide sequences of the 5 'untranslated region and the 3' untranslated region are respectively shown as SEQ ID NO 3 and SEQ ID NO 4; the coded amino acid sequence is shown as SEQ ID NO 5.

According to the obtained full-length cDNA sequence SEQ ID NO of the porcine UCP3 gene: 1, designing specific qRT-PCR quantitative primers, and detecting the expression modes of UCP3 genes in different tissues, different fiber types of skeletal muscle tissues and before and after differentiation of precursor adipocytes, wherein the gene quantitative detection primers are shown in Table 1. The expression pattern shows that the porcine UCP3 gene is closely related to fat deposition and is a functional gene which is potentially used for improving intramuscular fat of pigs.

2. Molecular marker identification related to intramuscular fat character of pig

The marker is positioned in a sequence table SEQ ID NO: 6 at 2159bp, with the variation site name SNV00065 in Table 2. Sequence listing SEQ ID NO: the 1 st site in 6 is the UCP3 transcription initiation site identified in the invention, and the position of the 1 st site on the UCP3 gene is defined as + 1. This mutation site corresponds to 8387309 from the 5' end of chromosome 9 of porcine version 11.1. The molecular marker is obviously related to the intramuscular fat character of the pig.

The invention discloses a method for preparing a gene fragment with UCP3 gene full-length cDNA sequence SEQ ID NO:1, extracting a nearly 3259bp sequence before ATG from an NCBI database, and then utilizing a Fasttarget target region targeted sequencing technology to comprehensively detect and analyze genetic variation sites in 279 (Pitland x Duroc) x (long white x large white) [ (Pilxdu) x (long x large) ] resource groups and 125 individuals from 6 different pig species including Duroc, large white, long white, floret, Meishan and Mi pigs, wherein the total number of the genetic variation sites in the region is 125, the specific sequencing primer sequences are shown in table 1, 122 genetic variation sites are identified (table 2), and the genotype frequency and the allele frequency of each genetic variation site among different pig species are statistically analyzed (table 3).

According to the genetic variation site genotyping result obtained by sequencing and the related trait records of (PixDu) x (Long x big) test population, the SAS software mixed linear model program (MLM) is utilized to perform correlation analysis on the genetic variation site polymorphism and the pig production trait, and finally 1 effective molecular marker site applicable to pig intramuscular fat trait breeding is determined, wherein the marker site is positioned in a sequence table SEQ ID NO: 6 at 2159bp (SNV00065) corresponding to position 8387309 of chromosome 9 of porcine version 11.1 from the 5' end.

Sequence listing SEQ ID NO:1 is the nucleotide sequence information of the full-length cDNA of the porcine UCP3 gene cloned by the invention.

Sequence listing SEQ ID NO: 2 is the nucleotide sequence information of CDS region of porcine UCP3 gene cloned by the invention.

Sequence listing SEQ ID NO:3 is the nucleotide sequence information of 5' UTR of the porcine UCP3 gene cloned by the invention.

Sequence listing SEQ ID NO: 4 is the nucleotide sequence information of the 3' UTR of the porcine UCP3 gene cloned by the invention.

Sequence listing SEQ ID NO: 5 is the amino acid sequence information encoded by CDS region of porcine UCP3 gene cloned by the present invention.

Sequence listing SEQ ID NO: 6 is a genomic sequence from the translation initiation site ATG to the transcription initiation site A of the UCP3 gene of the present invention.

3. Preparation and application of molecular marker related to porcine intramuscular fat

(1) According to the sequence table SEQ ID NO: 6, designing and amplifying a sequence comprising SEQ ID NO: 6, specific amplification primers gUCP3-F and gUCP3-R at 2159bp G/T variation site. The nucleotide sequences of the forward and reverse primers are shown below:

forward primer (gUCP 3-F): GATGACCCTTTTCCTTTG (see Table 1)

Reverse primer (gUCP 3-R): GACTGAACTCCCTCCCTG (see Table 1)

(2) Primer 5 software was used to screen for SEQ ID NO: 6 at 2159bp, and then PCR amplifying a restriction endonuclease containing a G/T variation site at a position of SEQ ID NO: 6 at 2159bp, carrying out enzyme cutting by using the selected RsaI restriction endonuclease at 37 ℃, and finally carrying out genotyping on the site by a 2% agarose gel electrophoresis technology to establish a gene fragment aiming at a G/T variation site of SEQ ID NO: 6 at 2159bp G/T variation site.

(3) Using the sequences for SEQ ID NO: the PCR-RsaI-RFLP genotyping method of G/T variant locus at 2159bp 6 carries out genotyping on 500 (Pipi X Du) X (long X big) quaternary commercial pigs, and utilizes SAS software to carry out correlation analysis on polymorphism of the locus and the determined meat quality traits by adopting a mixed linear model program (MLM), thereby determining favorable genotype to be applied to marker-assisted breeding of intramuscular fat traits of pigs.

The invention has the beneficial effects that:

the meat quality trait belongs to quantitative trait with low hereditary trait, is controlled by polygene, and the trait index can be determined only after slaughtering. Therefore, the conventional breeding method for genetic improvement has the problems of high cost, long period, difficult prediction of effect and the like. The invention identifies a variation site (G/T variation at 2159bp of SEQ ID NO: 6) which is obviously related to the porcine intramuscular fat in the UCP3 gene area, and the identified variation site can be used for early, living and rapid marking auxiliary breeding of the porcine intramuscular fat. In addition, the locus can be integrated into the existing genome breeding technology, so that the breeding accuracy of breeding pigs with high-quality pork is improved, and the important application value is achieved. Based on the influence of the UCP3 gene on the intramuscular fat character of the pig, the invention researches the expression mode of the UCP3 gene, proves that the UCP3 gene can be used as a target gene for improving the meat quality character of the pig, and can be modified by adopting the modern transgenic or gene editing technology and the like, thereby realizing the rapid genetic improvement of the high-quality pork pig.

Description of the drawings:

FIG. 1: is agarose gel electrophoresis picture of 5' RACE amplified fragment of porcine UCP3 gene. In the figure: m: DNA molecular weight Standard (DL2000 ladder).

FIG. 2: is agarose gel electrophoresis picture of 3' RACE amplified fragment of porcine UCP3 gene. In the figure: m: DNA molecular weight Standard (DL2000 ladder).

FIG. 3: is the expression pattern of the porcine UCP3 gene in different tissues.

FIG. 4: is the expression pattern of the porcine UCP3 gene in skeletal muscle tissues of different muscle fiber types.

FIG. 5: is the expression mode of the porcine UCP3 gene before and after the differentiation of the porcine fat precursor cells.

FIG. 6: is the result of the PCR-RsaI-RFLP genotyping of G/T variant sites at the position of G. +2159bp of the porcine UCP3 gene.

Detailed Description

The present invention will be described in detail with reference to specific examples.

Example 1: cloning of porcine UCP3 Gene full-Length cDNA clone

1.1 primer design

Using NCBI database (https://www.ncbi.nlm.nih.gov/) The predicted porcine UCP3 gene mRNA sequence (NM-214049.1) seed sequence as template to design amplification primers, wherein 5' -RACE specific primers (GSPs) should satisfy 23-28nt in length and 50-70% GC content, and Universal Primer Mix (G-PCR)

The universal primers designed by the RACE 5 '/3' Kit comprise two Long Primer and Short Primer) and have non-complementary 3 'ends, the two primers meeting the conditions are designed to be GSP1 and NGSP1 respectively, and GATTACGCCAAGCTT is added to the 5' end of the primers to facilitate the carrier connection and sequencing work in the subsequent test. The design of UCP3 gene 3 ' -RACE specific primers is similar to that of 5 ' -RACE specific primers, the designed primers are named as GSP2 and NGSP2, and GATTACGCCAAGCTT is added at the 5 ' end of the primer. Specific primer sequences are shown in table 1.

1.2 Generation of RACE-Ready cDNA

According to strict rules of

The RACE 5 '/3' Kit includes the steps of preparing 5 '-RACE and 3' -RACE cDNA reaction liquid separately, mixing successively at room temperature, and incubating in a thermal cycler at 42 deg.c for 90min and 70 deg.c for 10 min. After the reaction was completed, 90. mu.L of diluent Tricine-EDTA Buffer was added to dilute the first strand cDNA synthesis reaction 5 '/3' -RACE-Ready cDNA.

1.3, 5 '-RACE and 3' -RACE amplification

5' -RACE amplification: first round of amplification was performed using diluted 5 'RACE-Ready cDNA as template and Long primer and GSP1 as upstream and downstream primers in a PCR reaction system of 5' -RACE-Ready cDNA 2.5. mu.L, Long primer 5. mu.L, GSP 11.0. mu.L, Master Mix (premixed, containing 15.5. mu.L ddH2O, 25.0. mu.L 2 XSeqAmp Buffer, 1.0. mu.L SeqAmp DNA Polymerase). PCR reaction program (touchdown PCR): 30s at 94 ℃ and 3min at 72 ℃ for 5 cycles; 30s at 94 ℃, 30s at 70 ℃ and 30min at 72 ℃ for 5 cycles in total; 30s at 94 ℃, 30s at 68 ℃ and 3min at 72 ℃ for 20 cycles in total; finally, the mixture is stored at 4 ℃. The target fragments were not evident by electrophoresis, and the first round amplification product was diluted 50-fold based on this and used as a template for nested PCR. And (3) PCR reaction system: DNA template 2. mu.L, Short primer 0.5. mu.L, NGSP 10.5. mu.L, ddH₂O 7μL，I-5^TM 2×High-Fidelity Master Mix (Ongjingke organism) 10. mu.L. PCR reaction procedure: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 30s for 30 cycles; after 72 ℃ the extension was carried out for 7min and the cells were stored at 4 ℃. After electrophoresis of the amplified product, the band is obvious (figure 1), the target fragment is cut and recovered, the purified PCR product is connected with pClone007 Vector Kit (Optimus sp.) Vector, and the positive clone is sequenced after transformation to obtain the target sequence SEQ ID NO. 3.

3' -RACE amplification: using diluted first strand cDNA as template and Long primer and GSP2 as upstream and downstream primers, 2.5. mu.L of 3' -RACE-Ready cDNA, 5.0. mu.L of Long primer, 21.0. mu.L of GSP, Master Mix (premixed, including 15.5. mu.L of ddH)₂O, 25.0. mu.L of 2SeqAmp Buffer, 1.0. mu.L of SeqAmp DNA Polymerase) was used for the first round of amplification. PCR reaction program (touchdown PCR): 30s at 94 ℃ and 3min at 72 ℃ for 5 cycles; 30s at 94 ℃, 30s at 70 ℃ and 30min at 72 ℃ for 5 cycles in total; 30s at 94 ℃, 30s at 68 ℃ and 3min at 72 ℃ for 20 cycles in total; finally, the mixture is stored at 4 ℃. And (3) finding that the target fragment is not obvious by electrophoresis, diluting the first round amplification product by 50 times, and performing nested amplification by using the product as a template, wherein a PCR reaction system comprises: DNA template 2. mu.L, Short primer 0.5. mu.L, NGSP 20.5. mu.L, ddH₂O 7.0μL，I-5^TM2 × 10.0 μ L of High-Fidelity Master Mix (Scentillaceae). PCR reaction procedure: pre-denaturation at 98 ℃ for 2min, denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 30s for 30 cycles; after 72 ℃ the extension was carried out for 7min and the cells were stored at 4 ℃. After electrophoresis of the amplified product, the band is obvious (fig. 2), the target fragment is cut and recovered, the purified PCR product is connected with pClone007 Vector Kit (Optimus sp. Okagi) Vector, and the positive clone is sequenced after transformation, so that the target sequence SEQ ID NO: 4.

1.4 extraction of CDS region sequence of porcine UCP3 gene and full-length cDNA splicing

Firstly, extracting a predicted porcine UCP3 gene mRNA Sequence (NM-214049.1) from an NCBI database (https:// www.ncbi.nlm.nih.gov /), comparing and splicing an amplified 5 '-UTR and 3' -UTR Sequence with a UCP3 gene Coding Sequence (CDS), integrating overlapping regions, and finally obtaining the porcine UCP3 gene full-length cDNA Sequence, wherein the specific Sequence is shown as SEQ ID NO: 1. In addition, human (NM _003356.4) and mouse (NM _009464.3) UCP3 gene mRNA sequences were also downloaded from NCBI database (https:// www.ncbi.nlm.nih.gov /), and the CDS region nucleotide sequences of human and mouse UCP3 genes were extracted and aligned with the obtained full-length cDNA sequence of porcine UCP3 gene to determine the CDS nucleotide sequence of porcine UCP3 gene, the specific sequence being shown in SEQ ID NO: 2.

Example 2: analysis of expression pattern of porcine UCP3 Gene

In order to determine the relationship between UCP3 expression pattern and pig fat deposition, 3 Durock × Meishan pig binary hybrid pigs were randomly selected, and heart, liver, lung, spleen, kidney, fat, and biceps femoris muscles were collected, and skeletal muscle tissues of 5 test pigs of different muscle fiber types, biceps femoris (fast muscle, white muscle) and soleus muscle (slow muscle, red muscle) were collected. Total RNA extraction was performed using TRIzol reagent (Invitrogen Co.), and Prime Script was used^TMThe first strand cDNA was synthesized using the reagent (TaKaRa). The mRNA expression level of UCP3 gene was then determined by Real-time PCR. The cloned full-length cDNA nucleotide sequence SEQ ID NO:1 of the porcine UCP3 gene is taken as reference to design Real-time PCR quantitative primers, quantitative upstream and downstream primer sequences qUCP3-F and qUCP3-R are shown in table 1, all PCR reactions are carried out for 3 times, the relative expression level of the gene is calculated by adopting a delta Ct method, and the HPRT gene is taken as an internal reference gene to carry out the correction of the relative expression level of the gene. Charting was performed using GraphPad Prism 6.01 and significance test performed<0.05 indicated significant differences in gene expression,. about.P<0.01 indicates that the gene expression differed extremely significantly.

The detection result of the gene expression pattern shows that the porcine UCP3 gene is mainly expressed in high abundance in fat and skeletal muscle (figure 3), and the fact that the porcine UCP3 gene can regulate the pork quality by regulating the growth and fat deposition of the porcine skeletal muscle is suggested. The muscle fiber type difference is a key factor influencing the pork quality, and the expression level of the UCP3 gene in slow-muscle soleus is extremely lower than that of fast-muscle white muscle biceps femoris (P <0.01) (figure 4), which further suggests that the porcine UCP3 gene may be closely related to the pork quality. In addition, the intramuscular fat content of skeletal muscle tissues of different muscle fiber types is different, which also suggests that UCP3 gene is a potential functional gene affecting the formation of intramuscular fat. In addition, the expression level of UCP3 gene was significantly up-regulated after differentiation of pig adipose precursor cells into adipocytes, indicating that UCP3 gene has a function of promoting fat deposition (fig. 5).

Example 3: genetic variation site detection and intramuscular fat related molecular marker identification of porcine UCP3 gene

2.1 test pigs and determination of traits

The four-element 279 (skin X Du) X (length X big) commercial pigs related to the invention have the characteristics of body weight, backfat thickness and pH value_45min、pH_24h、L*_45min、a*_45min、b*_45min、L*_24h、a*_24h、b*_24hDrip loss_24hLoss of water_48hGlycogen content, glucose-6-phosphate content, lactic acid content, glycolytic potential, cooking loss, shear force. Finally, the thickness of the back fat of the rib is measured by an electronic vernier caliper, the pH value is measured by a portable pH meter (model: HI 9125portable pH, Australia), the boiling loss is calculated by the boiling loss rate for 10min, the shearing force is measured by a domestic instrument, the flesh colors L, a and b are measured by a portable color difference meter (model: CR-10, Japan), the dripping water loss is measured by a hanging method, the intramuscular fat content is measured by a Soxhlet extraction method, the lactic acid content, the glycogen content and the glucose content are measured by a Nanjing constructed kit (constructed, Nanjing), and the glucose-6 phosphate content is measured by a Sigma kit (product number: MAK 014).

2.2 identification of genetic variation sites of porcine UCP3 gene

The invention discloses a method for preparing a gene fragment with UCP3 gene full-length cDNA sequence SEQ ID NO:1, extracting a total 3259bp sequence (SEQ ID NO: 6) from the translation initiation site ATG to the transcription initiation site A from an NCBI database, and then utilizing a FastTarget target region targeted sequencing technology to comprehensively detect and analyze genetic variation sites in 279 Duroc x Changbai x Dangbai resource groups and 125 individuals which are derived from 6 different pig species and comprise Duroc, Dangbai, Changbai, Erhualian, Meishan and Mi pigs, wherein specific sequencing primer sequences are shown in Table 1, are compared and analyzed with a latest version Sscrofa11.1 reference sequence of a pig, 122 genetic variation sites are identified (Table 2), and the gene frequency statistical analysis of each genetic variation site among different pig species is carried out (Table 3).

2.3 trait Association analysis

On the basis of finishing 279 head (skin x Du) x (long x big) quaternary commercial pig gene sequencing typing, by utilizing test pig herd sex determination data, the invention adopts SAS statistical software (SAS Institute Inc, Version 8.0) Mixed Linear Model (MLM) program to perform correlation analysis on the identified genetic variation site polymorphism and character, and adopts Bonferroni method to perform P value correction. The statistical model is as follows:

Y_ijk＝μ+Genotype_i+Sex_j+Batch_k+b_ijkZ_ijk+e_ijk

Y_ijkis a character phenotype value; μ is the population mean; genotype_iFor genotype effects, Sex_j、Batch_kSex, slaughter batch effect respectively; b_ijklmIs the regression coefficient of carcass weight to meat quality character, Z_ijkFor carcass weight, e_ijkIs the residual effect.

Correlation analysis results showed that, in a 279 (pico × du) × (long × large) four-element commercial pig population, the sequence listing SEQ ID NO: 6 (G. +2159, SNV00065), which corresponds to 8387309 site from the 5' end of chromosome 9 of the 11.1 version of pig, and the polymorphism of the G/T variant site is obviously related to the intramuscular fat and flesh color traits of the pig. As shown in table 4, different genotypes at the g. +2159 locus were significantly associated with intramuscular fat, the intramuscular fat content in subjects with the GT genotype was significantly higher than that in subjects with the GG and TT genotypes, and the meat redness value a at 24 hours in subjects with the GT genotype was significantly higher than that in subjects with the GG genotype. Wherein the intramuscular fat content of the GT type individual can be improved by 11.02 percent compared with that of the GG type individual, and the intramuscular fat content of the GT type individual can be improved by 5.60 percent compared with that of the TT type individual.

Example 4: large group verification of influence of UCP3 gene g. +2159 variable locus on intramuscular fat

1.1 genotyping primer design and restriction enzyme screening

Firstly, according to the sequence table SEQ ID NO: 6, designing and amplifying a sequence comprising SEQ ID NO: 6, primers gUCP3-F and gUCP3-R are specifically amplified at the G/T variation site at 2159bp, and the sequences of the primers are shown in Table 1. Taking 2159bp site base as a core, extracting upstream and downstream 10bp sequences, and screening by using Primer 5 software to obtain a Primer sequence aiming at SEQ ID NO: 6 at 2159bp G/T variation site. Through analysis, the RsaI can be used for enzyme typing of the G/T variation site at 2159 bp.

1.2G. +2159bp G/T variant locus PCR-RsaI-RFLP genotyping method establishment

PCR amplification is carried out by using the gUCP3-F and gUCP3-R primers to obtain a primer containing the nucleotide sequence shown in SEQ ID NO: 6 at 2159bp, then utilizing the selected RsaI restriction endonuclease to carry out enzyme digestion at 37 ℃, and finally carrying out gene typing on the site by a 2% agarose gel electrophoresis technology. The PCR product amplified by the genetic variation locus is subjected to enzyme digestion by RsaI to generate three genotypes, when the locus is a GG homozygous genotype, the locus cannot be subjected to enzyme digestion by the RsaI, and only one fragment (G allele) is obtained after the enzyme digestion reaction; when the locus is a TT mutation homozygous genotype, the RsaI enzyme can cut the PCR product enzyme into two segments (T alleles); when the locus is a GT hybrid genotype, the RsaI enzyme can cleave the PCR product into three fragments (hybrid gene). The results of the RsaI-RFLP enzyme cutting and typing of the porcine UCP3 gene are shown in FIG. 6. The GG genotype has only one band; there were two bands for the TT genotype and three bands for the GT genotype (FIG. 6).

1.3 trait Association analysis

Using the sequences for SEQ ID NO: 6 PCR-RsaI-RFLP genotyping method of G/T variant locus at 2159bp carries out genotyping on 500 (Pipi X Du) X (long X big) quaternary commercial pigs,

the g. +2159 site (SNV00065) polymorphism was also analyzed in association with production traits using the mixed linear model of SAS software (MLM) program. The analysis result shows that different genotypes of the g. +2159 locus are obviously related to intramuscular fat, and the intramuscular fat content of the GT genotype individuals is obviously higher than that of the GG genotype individuals and the TT genotype individuals, wherein the intramuscular fat content of the GT genotype individuals can be improved by 3.79 percent compared with the GG genotype individuals, and the intramuscular fat content of the GT genotype individuals can be improved by 9.70 percent compared with the TT genotype individuals (Table 5). Therefore, the polymorphism of the g. +2159(SNV00065) locus has obvious correlation with the meat quality character of the porcine intramuscular fat, can be used as an important molecular auxiliary marker for genetic improvement of the porcine intramuscular fat, and has important significance for screening and breeding new high-quality pig breeds.

Although the invention has been described in detail, it will be appreciated by those skilled in the art that modifications will be apparent within the spirit and scope of the invention. For example, many modifications may be made to adapt a particular situation, material, composition of matter, or process step to the objective, spirit and scope of the present application. All such modifications are intended to be included within the scope of the claims of the present invention. Moreover, the technical content disclosed in the above description may be modified or changed equally to the equivalent embodiments, and all of the modifications and changes fall within the technical scope of the present invention.

Attached watch

TABLE 1 primers for targeted sequencing of the UCP3 gene region of pigs

The sequence of the lowercase letters is a linker sequence for RACE subsequent fragment vector connection and sequencing

TABLE 2 polymorphism of variation site of porcine UCP3 Gene region

Reference genome: ssicrofa 11.1

TABLE 3 statistics of genotype frequencies and allele frequencies of different pig species at variant sites of the UCP3 gene region of pig

Reference genome: sscrofa11.1

TABLE 4 Association analysis of porcine UCP3 gene G. +2159G/T variation site polymorphism and production traits (experimental group)

Glycogen; glucose; glucose-6-phosphate; residual glycogen & glucose: residual glycogen and glucose; lactic acid: lactic acid; glycolytic potential: glycolytic potential; backfat thickness: backfat thickness; pH 24 h: PH 24 hours after slaughtering; pH 45 min: slaughtering at 45min PH; l-24 h: the brightness of the meat color; a-24 h: redness of flesh; b-24 h: the yellowness of meat color; drip loss-24h (%): drip loss 24 hours after slaughter; drip loss-48h (%): water dripping loss is 48 hours after slaughtering; cooking loss (%); shear force (N): shearing force; intramuscul fat (%): intramuscular fat of meat-like wet weight

TABLE 5 Association analysis of porcine UCP3 gene G. +2159G/T variation site polymorphism and production traits (validation group)

Suet weight (kg): the weight of the leaf lard is heavy; backfat thickness: backfat thickness; l-24 h: the brightness of the meat color; a-24 h: redness of flesh; b-24 h: the yellowness of meat color; drop loss (%): drip loss 24 hours after slaughter; intramuscul fat (%): intramuscular fat on a meat-like dry weight basis.

Sequence listing

<110> Nanjing university of agriculture

CHANGZHOU FENGHUA ANIMAL HUSBANDRY Co.,Ltd.

<120> UCP3 gene related to pig intramuscular fat character, molecular marker and application thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2283

<212> DNA

<213> pig (Sus)

<400> 1

agagacggcc ggcacagggc actcgggtcc ctgctgccgc ctcctggggt ggagccctga 60

ggagcctcaa ggaggccctg cgcttccccc gccccggccg gactgggcag ggccagtccc 120

ctgctggtgc tgcgggagcc gggggaccgt cccagaagcc cgggctccgt ggctcggctc 180

ctgcagcctc ccccgcacgg aagcccaggc cgggcagctc tcctggaacc tccctgctgg 240

gccctggggg acctggcaga gcccctggga catggtgggc ctgaagcctc ccgaggtgcc 300

gcccacgacg gctgtgaagc ttctgggggg caccgcggcc tgcttcgccg acctcctcac 360

cttcccactg gacacggcca aggtccgcct gcaggtagat ccaggggaga accaggcggc 420

ccggagcgcc cagtaccgcg gggtgctggg caccattctc accatggtgc gcaacgaggg 480

cccccgcagc ccctacaacg ggctggtggc cggcctgcag cgccagatga gcttcgcctc 540

catccgcatc ggcctctacg actccgtcaa gcagctctac acgcccaagg gatcggacca 600

ctccagcatc accacccgga ttttggcggg ctgcaccacg ggggccatgg cagtgacctg 660

cgcccagccc acggacgtgg tgaaggttcg attccaggcc agcatacacg ccgggcccgg 720

gagcaacagg aagtacagcg ggacgatgga tgcctatagg accatcgcca gggaggaagg 780

ggtccggggc ctgtggaaag gaattctgcc caacatcacg aggaatgcca tcgtgaactg 840

tgccgagatg gtgacctatg atgtcatcaa ggagaaggtg ctagactacc acctgctcac 900

ggacaacctg ccctgccact ttgtctctgc cttcggagct ggcttctgtg ccaccgtggt 960

ggcctcccca gtggatgtgg tgaagacccg gtacatgaac tcgccccccg gccagtacca 1020

gaaccctctg gactgcatgc tgaagatggt gacccaggag ggccccacag ccttctataa 1080

aggattcaca ccttcctttt tgcgcttggg aagctggaac gtggtgatgt ttgtaagcta 1140

cgagcagctg aaacgggcct tgatgaaagt ccagatgctg cgggaatctc cgttttgagt 1200

aagacaaagc cacctggcgt ttcacgaagg caaaaccagc caaggggtga caggcgccgc 1260

cgtgggtccg cgcacataag gccacaggcc cacccgcatt tacacaacag tctacttgcc 1320

gccgactcgg aaataggagg aggagaactg tggtctccgg tgcttcacct tcaggacacg 1380

ttcgtgtggc actgacaaga tgctcaataa attatattaa tttgtgaaac ccactaggat 1440

ggatgcctaa catttaggca aaagaagata aaccagaatg gatccatatg gataaaacag 1500

acgtttgcaa acttccgtcc cctggaaaac gatcaggtgt caacagataa ggctgcaagc 1560

ctgggacagg attcagactc gatacacttg gaaatcagcc gagtctagag gggcactgcc 1620

aaagccacac aaggctttca acggcagcct tccgctaccc acggatcacc agccacattt 1680

gccatgtgtc tgcctggtag aacccaccag gattccggga ttcgggaaaa gaaaaagagc 1740

aaagccagct cctgtgacaa aggagccctg ctgggccggc tggggctgag gccgggatgc 1800

tcgggagacc cggagggtca ggagggcggg ctcggcctcc tctcccgtaa aatgggagca 1860

caaagccctc tctcctacct cccctggggt gtcacgagga tcaagcggga agctgggcaa 1920

ggaggcgctt tataaaagct ccatgaaatg tgtatataag gacgaggcct tgggaatgtt 1980

ttcgtccgtc agtgaggcag tgccggggaa gaagctttag aaagacacag atgtccagag 2040

gtcagaccaa aggaaccagg ggctggaggg gcagtggccg gagccccgcg cccatgcggc 2100

ctcccctccc tgctggactg aggcaccttg aagggtgtgg agtttgggga caaagaggta 2160

aatgctgggc ccagagcctc aagtaccagg actctgaatt tggggaaaag gattatgact 2220

caataaagga tgcgacacct tgggtgtggg aatgaaaaaa aaaaaaaaaa aaaaaaaaaa 2280

aaa 2283

<210> 2

<211> 927

<212> DNA

<213> pig (Sus)

<400> 2

atggtgggcc tgaagcctcc cgaggtgccg cccacgacgg ctgtgaagct tctggggggc 60

accgcggcct gcttcgccga cctcctcacc ttcccactgg acacggccaa ggtccgcctg 120

caggtagatc caggggagaa ccaggcggcc cggagcgccc agtaccgcgg ggtgctgggc 180

accattctca ccatggtgcg caacgagggc ccccgcagcc cctacaacgg gctggtggcc 240

ggcctgcagc gccagatgag cttcgcctcc atccgcatcg gcctctacga ctccgtcaag 300

cagctctaca cgcccaaggg atcggaccac tccagcatca ccacccggat tttggcgggc 360

tgcaccacgg gggccatggc agtgacctgc gcccagccca cggacgtggt gaaggttcga 420

ttccaggcca gcatacacgc cgggcccggg agcaacagga agtacagcgg gacgatggat 480

gcctatagga ccatcgccag ggaggaaggg gtccggggcc tgtggaaagg aattctgccc 540

aacatcacga ggaatgccat cgtgaactgt gccgagatgg tgacctatga tgtcatcaag 600

gagaaggtgc tagactacca cctgctcacg gacaacctgc cctgccactt tgtctctgcc 660

ttcggagctg gcttctgtgc caccgtggtg gcctccccag tggatgtggt gaagacccgg 720

tacatgaact cgccccccgg ccagtaccag aaccctctgg actgcatgct gaagatggtg 780

acccaggagg gccccacagc cttctataaa ggattcacac cttccttttt gcgcttggga 840

agctggaacg tggtgatgtt tgtaagctac gagcagctga aacgggcctt gatgaaagtc 900

cagatgctgc gggaatctcc gttttga 927

<210> 3

<211> 271

<212> DNA

<213> pig (Sus)

<400> 3

agagacggcc ggcacagggc actcgggtcc ctgctgccgc ctcctggggt ggagccctga 60

ggagcctcaa ggaggccctg cgcttccccc gccccggccg gactgggcag ggccagtccc 120

ctgctggtgc tgcgggagcc gggggaccgt cccagaagcc cgggctccgt ggctcggctc 180

ctgcagcctc ccccgcacgg aagcccaggc cgggcagctc tcctggaacc tccctgctgg 240

gccctggggg acctggcaga gcccctggga c 271

<210> 4

<211> 1085

<212> DNA

<213> pig (Sus)

<400> 4

gtaagacaaa gccacctggc gtttcacgaa ggcaaaacca gccaaggggt gacaggcgcc 60

gccgtgggtc cgcgcacata aggccacagg cccacccgca tttacacaac agtctacttg 120

ccgccgactc ggaaatagga ggaggagaac tgtggtctcc ggtgcttcac cttcaggaca 180

cgttcgtgtg gcactgacaa gatgctcaat aaattatatt aatttgtgaa acccactagg 240

atggatgcct aacatttagg caaaagaaga taaaccagaa tggatccata tggataaaac 300

agacgtttgc aaacttccgt cccctggaaa acgatcaggt gtcaacagat aaggctgcaa 360

gcctgggaca ggattcagac tcgatacact tggaaatcag ccgagtctag aggggcactg 420

ccaaagccac acaaggcttt caacggcagc cttccgctac ccacggatca ccagccacat 480

ttgccatgtg tctgcctggt agaacccacc aggattccgg gattcgggaa aagaaaaaga 540

gcaaagccag ctcctgtgac aaaggagccc tgctgggccg gctggggctg aggccgggat 600

gctcgggaga cccggagggt caggagggcg ggctcggcct cctctcccgt aaaatgggag 660

cacaaagccc tctctcctac ctcccctggg gtgtcacgag gatcaagcgg gaagctgggc 720

aaggaggcgc tttataaaag ctccatgaaa tgtgtatata aggacgaggc cttgggaatg 780

ttttcgtccg tcagtgaggc agtgccgggg aagaagcttt agaaagacac agatgtccag 840

aggtcagacc aaaggaacca ggggctggag gggcagtggc cggagccccg cgcccatgcg 900

gcctcccctc cctgctggac tgaggcacct tgaagggtgt ggagtttggg gacaaagagg 960

taaatgctgg gcccagagcc tcaagtacca ggactctgaa tttggggaaa aggattatga 1020

ctcaataaag gatgcgacac cttgggtgtg ggaatgaaaa aaaaaaaaaa aaaaaaaaaa 1080

aaaaa 1085

<210> 6

<211> 308

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Val Gly Leu Lys Pro Pro Glu Val Pro Pro Thr Thr Ala Val Lys

1 5 10 15

Leu Leu Gly Gly Thr Ala Ala Cys Phe Ala Asp Leu Leu Thr Phe Pro

20 25 30

Leu Asp Thr Ala Lys Val Arg Leu Gln Val Asp Pro Gly Glu Asn Gln

35 40 45

Ala Ala Arg Ser Ala Gln Tyr Arg Gly Val Leu Gly Thr Ile Leu Thr

50 55 60

Met Val Arg Asn Glu Gly Pro Arg Ser Pro Tyr Asn Gly Leu Val Ala

65 70 75 80

Gly Leu Gln Arg Gln Met Ser Phe Ala Ser Ile Arg Ile Gly Leu Tyr

85 90 95

Asp Ser Val Lys Gln Leu Tyr Thr Pro Lys Gly Ser Asp His Ser Ser

100 105 110

Ile Thr Thr Arg Ile Leu Ala Gly Cys Thr Thr Gly Ala Met Ala Val

115 120 125

Thr Cys Ala Gln Pro Thr Asp Val Val Lys Val Arg Phe Gln Ala Ser

130 135 140

Ile His Ala Gly Pro Gly Ser Asn Arg Lys Tyr Ser Gly Thr Met Asp

145 150 155 160

Ala Tyr Arg Thr Ile Ala Arg Glu Glu Gly Val Arg Gly Leu Trp Lys

165 170 175

Gly Ile Leu Pro Asn Ile Thr Arg Asn Ala Ile Val Asn Cys Ala Glu

180 185 190

Met Val Thr Tyr Asp Val Ile Lys Glu Lys Val Leu Asp Tyr His Leu

195 200 205

Leu Thr Asp Asn Leu Pro Cys His Phe Val Ser Ala Phe Gly Ala Gly

210 215 220

Phe Cys Ala Thr Val Val Ala Ser Pro Val Asp Val Val Lys Thr Arg

225 230 235 240

Tyr Met Asn Ser Pro Pro Gly Gln Tyr Gln Asn Pro Leu Asp Cys Met

245 250 255

Leu Lys Met Val Thr Gln Glu Gly Pro Thr Ala Phe Tyr Lys Gly Phe

260 265 270

Thr Pro Ser Phe Leu Arg Leu Gly Ser Trp Asn Val Val Met Phe Val

275 280 285

Ser Tyr Glu Gln Leu Lys Arg Ala Leu Met Lys Val Gln Met Leu Arg

290 295 300

Glu Ser Pro Phe

305

<210> 5

<211> 3259

<212> DNA

<213> pig (Sus)

<400> 5

agagacggcc ggcacagggc actcgggtcc ctgctgccgc ctcctggggt ggagccctga 60

ggagcctcaa ggaggccctg cgcttccccc gccccggccg gactggtaag accccgctcc 120

ccaccccgca gccccctccc ggcaggggtc cccggccacc gcaggacaga gcccctaggg 180

actgagatga gggaccggca gccctgaggg gggcactgca gtcccagctc ccgtgtctct 240

cagccaggac tcgcctgcgc ctggctctgc tggctgccgc tgctgtgatt ttgcagatcc 300

ccacaaataa aatgactttc tccttcgtct ggcccagggt ccccaggatg cctctggagc 360

ataaagccct tgccccacga ggaccccttt cctgagtggg gggtggcaga ccccccagct 420

ctgctcagac agcatcctca gcggggatgc cctggaggtt ggccaggagg gtgggctcgg 480

ggctgccccg acagacggag gacttgtttc taacgcagat tcccaggccc cggctcagac 540

ctgaatgggg acctcgggct gagggccaag aatctgctct gtaaacaggc agctccaggg 600

atcctgagca ctccctgaag tctgagactc tgggcagcgg aacaggcaca ggtgccagac 660

agacctgggc tccagtccca gctccgtcgc gttagcctta gccgaagcca ccaaagcccg 720

ggactcttgt tcatgaagca ggaacaacca cggccactgc ccgggagcct ttctataacg 780

gggttgaagg agtcaatgcc tgcgaagccc ccagtgcggt gcccggtgca ggaagcactc 840

agcagacatc cccggggcac cgctactcca ggccctgcag ggacgaaccc cctgagcccc 900

catagagagc agctggggtc ttccctctgc atgtctcctg tgccagtggc gaaggtctgc 960

cggctgctag ggccccccac caccgagtcc tggtcaggag agtgggagca ctggcctagg 1020

gcctcccccg accctgccag tctggagccc ccctcgcctg agcccttgtc tagccactgc 1080

gccaccgccc atggggctca gggctcagga cggccaccgc acaggtggct gctacagccc 1140

cggctgcacc agcccatcga cctcacaggt ccccgagcct ccagcagctc ccttccagct 1200

catccttgcc gccgccgccg ccggcaccga catgacccaa acaggcctga cgctgtcatt 1260

cctccctgag aaccaccaac ccggctggca ttctcactag atcaggtgca gccttggcaa 1320

tcgaggcccc ccacaaagaa caccacgggc accccagatc ttgttgccca gcaagcgggg 1380

tcccttggtc accagatcct ccctgggccc tcaggggagt gatagcatca gaatgacact 1440

cgcactctag ctagtcctca gccagaaaat ctccccctca tttgctgtcc aggtctaata 1500

ttccagaatc ggtatacaat tttattttat aggtcagtga ccatcaaagg cacaaatcat 1560

tatctgtaaa ttatatctca atttaaaaat ttttttaaat aaatataaaa tgttcagtgc 1620

aaccttgtgc taggaaagat caaaataaaa gcaccccctt ccttggggca ggactcaaaa 1680

ggtgtccttt ggataattct atacctgttt tcctgaaaat aagctccttt ggggcaaggg 1740

ccaagcatga ttcatcccgt atctttcctt ccagccataa aggcagtaaa atagctagtc 1800

attaagatag tcactcacac cttagcatga attagaacat tggcgccttc ttaacaacag 1860

acttgtctgg ggaaaggtga cagcaaacct aagagtccag ggagccatgt gggtgaagag 1920

acagcacagg gagaaccaga gttgtagaca gtggccctta aagaagcctc atgtgatgac 1980

ccttttcctt tggcagctaa tttcccactg gtcaacaggt tatccccatt tacccaagag 2040

aggttaaatg atatacccac ggtctcatca cccattagta agccatgaag acagggcagg 2100

agctcagggc tccgagcccc aggaccagag ttctcctgtt gctgtagaga agggggcgga 2160

cgtgctgccc ccttcacagc tctgtgggat gggccctggc tccaggggtg agaatgctga 2220

agggtccagg atccccgcac agggggaggg agtgttgtga cctggggtcc cagcgggtgt 2280

gacccactaa gaggtcagca ctacaggcct gatgcccagc agcccggggt tacaggaaca 2340

cagggctgcc cgggacaggt gtgccagcct cccaaagaaa gcagggctcc gccaggcccc 2400

tccggtaaac acagccctgc tccaccagca gcaagatgaa ggcaggcacg gtgcccacct 2460

aggggcaggg agggagttca gtccctccac caaggctgac cctcctctgt ctgagaagtt 2520

catccccttt ggctgtgaag atgcccgcag aggggtgagg aaggaaagat cacgcgctgg 2580

gtcagcggcg ggacccctct gccgccgccc cctgagccct ggggaggccc agcagcagca 2640

gggcccaggg cccgaccccc accgcgcctc ccaccaccgc agagcagcag cctgcgggtc 2700

ccacgctgcc gtcccagaag aatcgtcttt agtgacgaag aaggaagctt atgttttaca 2760

gaaataagag tatattcagg cagacttggg ggagcgaaaa cccttcaccc aagacagcga 2820

gcggcttaac tgcgccctga ggcgtgttga ttttgttgat ttgggtggaa ggacctcagg 2880

tgatgagaca gcagggcccc cccacacctc agggaacagc ctctccagca agccgctcac 2940

tgctggggaa ggagaatggg gctgggatgc agggtcagaa cctggtctgt cccaaccaga 3000

gccagtgtag accaagctct gggcagaggc tgggggagga tggcgagagc tacgggagga 3060

aagccccatt cttacacgtc gaatccacag ggcagggcca gtcccctgct ggtgctgcgg 3120

gagccggggg accgtcccag aagcccgggc tccgtggctc ggctcctgca gcctcccccg 3180

cacggaagcc caggccgggc agctctcctg gaacctccct gctgggccct gggggacctg 3240

gcagagcccc tgggacatg 3259

Claims (9)

1. An SNP molecular marker related to the intramuscular fat character of pigs, which is characterized in that the SNP molecular marker is positioned in a nucleotide sequence shown as SEQ ID NO: 6, and the variation site corresponds to 8387309 site from the 5' end of the 9 th chromosome of the 11.1 version of the pig, and can cause the RsaI-RFLP polymorphism.

2. A primer pair for amplifying the SNP molecular marker of claim 1, comprising a forward primer gcup 3-F and a reverse primer gcup 3-R; the sequences of the forward primer gUCP3-F and the reverse primer gUCP3-R are shown as follows:

forward primer gUCP 3-F: GATGACCCTTTTCCTTTG

Reverse primer gUCP 3-R: GACTGAACTCCCTCCCTG are provided.

3. A kit for genotyping the G/T mutation site of the SNP molecular marker of claim 1, comprising the primer pair of claim 2 and a RsaI restriction enzyme.

4. A molecular marker assisted breeding method for porcine intramuscular fat character is characterized in that a gene fragment containing the G/T mutation site of the SNP molecular marker in claim 1 is obtained by carrying out PCR amplification on the genome DNA of a pig to be detected by adopting the primer pair in claim 2, the gene fragment obtained by the PCR amplification is subjected to enzyme digestion by RsaI restriction endonuclease, and finally the G/T mutation site of the SNP molecular marker is subjected to genotyping by agarose gel electrophoresis; the gene fragment containing the G/T mutation site of the SNP molecular marker of claim 1 is cut by RsaI restriction endonuclease to generate three genotypes, when the site is GG homozygous genotype, the site can not be cut by RsaI, and only one fragment is generated after the enzyme cutting reaction; when the locus is TT mutation homozygous genotype, the RsaI enzyme can cut the PCR product enzyme into two segments; when the locus is a GT heterozygous genotype, the RsaI enzyme can cut the PCR product into three fragments; the intramuscular fat of the GT genotype individuals is obviously higher than that of the GG and TT genotype individuals.

5. Use of the SNP molecular marker of claim 1, the primer pair of claim 2 or the kit of claim 3 for genetic improvement of intramuscular fat traits in pigs.

6. The UCP3 gene related to the intramuscular fat character of the pig is characterized by having the sequence shown in SEQ ID NO: 2, or a CDS region nucleotide sequence as set forth in figure 2.

7. The UCP3 gene related to the pig intramuscular fat trait according to claim 1, wherein the full-length cDNA sequence of the gene is shown as SEQ ID NO:1 is shown.

8. The protein encoded by the gene UCP3 related to the porcine intramuscular fat trait of claim 1, wherein the protein has the amino acid sequence as shown in SEQ ID NO: 5.

9. Use of the UCP3 gene related to the porcine intramuscular fat trait of claim 6 or 7 in genetic improvement of the porcine intramuscular fat trait.

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