Method for evaluating large white pig backfat based on USP16 gene

Abstract

The invention relates to the technical field of pig breeding improvement, in particular to a method for evaluating backfat of a large white pig based on a USP16 gene, which is used for determining an SNP (Single Nucleotide Polymorphism) molecular marker of the large white pig and judging whether the SNP molecular marker is one of the following SNP molecular markers: g.192401821Ggt located in an intron region of the USP16 gene; site A; the g.192407026Ggt is positioned in a downstream regulation and control region of the USP16 gene; site A; the g.192407237Agt is positioned in a downstream regulation and control region of the USP16 gene; a G site; the g.192408287Tgt is positioned in a downstream regulation and control region of the USP16 gene; a C site; the g.192408499Cgt is positioned in a downstream regulation and control region of the USP16 gene; and a T site. The evaluation method provided by the scheme and the used SNP molecular marker provide good theoretical support and effective guidance for breeding and improvement of growth traits of pigs.

Description

Technical Field

[0001] The invention relates to the technical field of pig breeding and improvement, and in particular to a method for evaluating the back fat of Large White pigs based on the USP16 gene. Background Art

[0002] Reproductive traits, meat quality traits, carcass traits and growth traits are the main economic traits of pigs. The genetic improvement of these main economic traits is directly related to the breeding cost and breeding efficiency of live pigs. Growth traits and ketone body traits have always been the focus of genetic improvement. Pig growth traits are usually expressed by growth rate, feed conversion rate, daily feed intake, 100kg backfat thickness, etc. The genetic coefficients of these traits are medium and can be improved through genetic selection. Carcass traits are expressed by carcass weight, slaughter rate, carcass length, carcass lean meat rate, fat rate, etc. Most of these traits are highly heritable traits. In the long process of breeding and improvement, the disadvantages of traditional breeding methods have gradually emerged. For example, some traits with low heritability have little effect on improvement based on traditional breeding methods. With the development of modern biotechnology, new genetic breeding and improvement methods have begun to emerge.

[0003] The use of modern biotechnology for molecular-assisted marker selection is currently a commonly used breeding method, and effective molecular markers are the premise and guarantee of molecular breeding. The detection of SNP molecular genetic markers of functional genes of target traits can improve the accuracy of genome selection in the breeding process. Therefore, the identification of functional genes of important economic traits of pigs can provide more theoretical basis for subsequent practical work in pig breeding.

[0004] At present, since the implementation of genome-wide association analysis (GWAS) and high-throughput sequencing of pigs, many candidate genes and functional sites have been detected, but there are not many major genes and key causal mutations that can be applied and are truly effective. Summary of the invention

[0005] In order to solve the technical problems raised in the background technology, the present invention provides a method for evaluating the back fat of Large White pigs based on the USP16 gene.

[0006] The present invention is implemented by the following technical scheme: a method for evaluating the back fat of Large White pigs based on the USP16 gene, determining the SNP molecular marker of the Large White pig, and judging whether the SNP molecular marker is one of the following SNP molecular markers, specifically:

[0007] The g.192401821G>A site located in the intronic region of the USP16 gene;

[0008] The g.192407026G>A site located in the downstream regulatory region of the USP16 gene;

[0009] The g.192407237A>G site located in the downstream regulatory region of the USP16 gene;

[0010] The g.192408287T>C site located in the downstream regulatory region of the USP16 gene;

[0011] The g.192408499C>T site located in the downstream regulatory region of the USP16 gene;

[0012] Specifically, the g.192401821G>A site located in the intron region of the USP16 gene, and the dominant allele is G, and the back fat of the Large White pig is ranked from large to small, and the corresponding genotypes are: GG genotype, GA genotype, AA genotype;

[0013] The g.192407026G>A site located in the downstream regulatory region of the USP16 gene, the back fat of the Large White pig is ranked from large to small, and the corresponding genotypes are: AA genotype, GA genotype, and GG genotype.

[0014] Specifically, the g.192407237A>G site located in the downstream regulatory region of the USP16 gene, the corresponding genotypes of the back fat of the Large White pig are ranked from large to small: GG genotype, GA genotype, and AA genotype.

[0015] Specifically, at the g.192408287T>C site located in the downstream regulatory region of the USP16 gene, the corresponding genotypes of the back fat of the Large White pig are ranked from large to small: CC genotype, CT genotype, and TT genotype.

[0016] Specifically, at the g.192408499C>T site located in the downstream regulatory region of the USP16 gene, the corresponding genotypes of the back fat of the Large White pig are ranked from large to small: TT genotype, CT genotype, and CC genotype.

[0017] A primer pair combination for detecting SNP molecular markers of Large White pigs, wherein:

[0018] The g.192401821G>A site located in the intron region of the USP16 gene was detected. The nucleotide sequence of the forward primer was shown in the sequence list as SEQ ID NO.1, and the nucleotide sequence of the reverse primer was shown in the sequence list as SEQ ID NO.2.

[0019] The g.192407026G>A site located in the downstream regulatory region of the USP16 gene is detected. The nucleotide sequence of the forward primer is shown in the sequence list as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in the sequence list as SEQ ID NO.4;

[0020] The g.192407237A>G site located in the downstream regulatory region of the USP16 gene is detected. The nucleotide sequence of the forward primer is shown in the sequence list as SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in the sequence list as SEQ ID NO.6;

[0021] The g.192408287T>C site located in the downstream regulatory region of the USP16 gene is detected. The nucleotide sequence of the forward primer is shown in the sequence list as SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown in the sequence list as SEQ ID NO.8;

[0022] The g.192408499C>T site located in the downstream regulatory region of the USP16 gene was detected. The nucleotide sequence of the forward primer was shown in the sequence list as SEQ ID NO.9, and the nucleotide sequence of the reverse primer was shown in the sequence list as SEQ ID NO.10.

[0023] A kit comprising the above primer pair combination.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This study uses Large White and Landrace pigs as the test population, measures the phenotypes of three traits: 100kg body weight age, 100kg back fat thickness, and 100kg eye muscle area, collects ear tissue and extracts genomic DNA, designs primers for the full coverage of the exon region of the USP16 gene, and the upstream and downstream regulatory regions of the gene 2000-3000bp, and screens out 6 SNP sites in the USP16 gene through mixed pool sequencing. The 6 selected SNP sites were genotyped using time-of-flight mass spectrometry, and association analysis was performed between the SNP sites and the three traits of 100kg body weight age, 100kg back fat thickness, and 100kg eye muscle area, as well as pairwise comparisons between genotypes.

[0026] The evaluation method proposed in this plan and the SNP molecular markers used provide good theoretical support and effective guidance for the breeding and improvement of pig growth traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the pig ear tissue DNA detection result in an embodiment of the present invention;

[0028] Figure 2 This is a diagram showing the detection results of a partial PCR amplification product of the USP16 gene in an embodiment of the present invention;

[0029] Figure 3 This is a graph showing the sequencing results of the g.192376801C>A site in an embodiment of the present invention;

[0030] Figure 4 It is a scatter plot of the typing results of the g.192376801C>A locus in the embodiment of the present invention;

[0031] Figure 5 This is a graph showing the sequencing results of the g.192401821G>A site in an embodiment of the present invention;

[0032] Figure 6 It is a scatter plot of the typing results of the g.192401821G>A locus in the embodiment of the present invention;

[0033] Figure 7 This is a graph showing the sequencing results of the g.192407026G>A site in an embodiment of the present invention;

[0034] Figure 8 It is a scatter plot of the typing results of the g.192407026G>A locus in the embodiment of the present invention;

[0035] Fig. 9 This is a graph showing the sequencing results of the g.192407237G>A site in an embodiment of the present invention;

[0036] Fig.10 It is a scatter plot of the typing results of the g.192407237G>A site in the embodiment of the present invention;

[0037] Fig.11 This is a graph showing the sequencing results of the g.192408287G>A site in an embodiment of the present invention;

[0038] Fig.12 It is a scatter plot of the typing results of the g.192408287G>A site in the embodiment of the present invention;

[0039] Fig.13 This is a graph showing the sequencing results of the g.192408499G>A site in an embodiment of the present invention;

[0040] Fig.14 It is a scatter plot of the g.192408499G>A site typing results in the embodiment of the present invention. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0042] Example:

[0043] The population used in this example is 238 Large White pigs and 220 Landrace pigs, and the measured traits are 100kg body weight age, 100kg eye muscle area and 100kg back fat thickness. The test population comes from Anhui Province Bengbu Pengrui Agriculture and Animal Husbandry Industry Co., Ltd., and the samples collected are ear samples. The extracted DNA samples are stored in a -80°C refrigerator for long-term storage for the next test.

[0044] Main reagents: Tissue genomic DNA isolation kit was purchased from Hangzhou Biomedical Technology Co., Ltd.; 2×

[0045] Taq-ASPCRMix (+Dye) was purchased from Jiangsu Yugong Life Science Co., Ltd.; FY2000 DNA Marker was purchased from Jiangsu Yugong Life Science Co., Ltd.; SuperRed nucleic acid dye was purchased from Beijing Lanjieke Technology Co., Ltd.; agarose was purchased from Jiangsu Yugong Life Science Co., Ltd.; primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.; genotyping data were provided by Beijing Compson Agricultural Technology Co., Ltd.

[0046] Main instruments and equipment: desktop high-speed centrifuge, model: MIKRO22; 4-degree storage cabinet, model: YC-260L; horizontal low-temperature refrigerator, model: MDF-25H; liquid nitrogen biological container, model: YDS-30-1; ice maker, model: IMS-40; fully automatic digital gel imaging system, model: JS-780; PCR instrument, model: K960; agarose horizontal electrophoresis instrument, model: DYCP-32P; ultrapure water machine, model: labmini4uvf.

[0047] Main molecular biology software: Primer3plus: used for primer design; Chromas: used for analyzing sequencing peak graphs; Seqman: sequencing result alignment and splicing; Plink: Hardy-Weinberg equilibrium test; Haploview: linkage disequilibrium analysis between sites; SPSS: significance calculation; R language: association analysis between SNPs and traits; Endnote: reference management.

[0048] Test methods

[0049] Step 1. DNA extraction

[0050] (1) Take 20-30 mg ear sample, about the size of a soybean, out of the alcohol, cut it into pieces with scissors and place it in a 1.5 ml centrifuge tube, and add 200 ul of BufferTL. In order to accelerate lysis, the tissue should be cut as finely as possible;

[0051] (2) Add 25ul of Proteinase K, vortex to mix, and place in a 50°C water bath. Vortex the sample once every 20-30 minutes until the sample is completely dissolved. The lysis time generally does not exceed 3 hours, and the lysate can also be left overnight;

[0052] (3) Centrifuge the lysate at ≥12,000 × g for 5 min, carefully remove the supernatant and transfer it to a sterile centrifuge tube;

[0053] (4) Add 220 μl of Buffer BL, vortex to mix, and place in a 70°C water bath for 10 min;

[0054] (5) Add 220 μl of anhydrous ethanol and vortex to mix;

[0055] (6) Insert a DNA minicolumn into a 2 ml collection tube. Transfer the solution obtained in the above step including a small amount of precipitate to the adsorption column, centrifuge at 10,000 × g for 1 min, and discard the filtrate;

[0056] (7) Add 500 μl Buffer KB, centrifuge at 10,000 × g for 1 min, and discard the filtrate;

[0057] (8) Add 600ul DNA Wash Buffer, centrifuge as above, and discard the filtrate. Repeat this step once;

[0058] (9) Reuse the 2 ml collection tube, open the lid and centrifuge at maximum speed (≥12000 × g) for 2 min to remove residual ethanol;

[0059] (10) Insert the DNA minicolumn into a sterile 1.5 ml centrifuge tube and add 100-150 ul of 65°C preheated Elution Buffer. Let stand at room temperature for 1-3 min, centrifuge at ≥13000×g for 1 min; the first elution will yield 60-70% DNA, and the eluted DNA will be re-loaded on the column for a second elution to yield another 20-30% DNA.

[0060] Step 2: Primer design

[0061] Therefore, all exon regions of the USP16 gene are amplified, and then primers are designed for the 2-3000bp non-coding regions upstream and downstream of the gene, and mixed pool sequencing is performed in the later stage to determine whether SNP exists.

[0062] The primer design software used in this study is primer3plus. Before designing primers, click Generalsetting to set the software according to the general principles. The commonly used primer length is 18-27bp, not more than 38bp. Because too long will cause its extension temperature to be greater than 74℃, which is not suitable for TaqDNA polymerase reaction, so the primer length is set to 18-25bp here. Too high or too low GC content is not conducive to the reaction, and the GC content of upstream and downstream primers cannot differ too much, so the GC content is generally set to 40-60%. In addition, there are some other principles to be followed in primer design, such as the 3′ end of the primer should not terminate at the third position of the codon, because the third position of the codon is prone to degeneracy, which will affect the specificity and efficiency of amplification; A cannot be selected at the 3′ end of the primer. When the 3′ end of the primer is mismatched, there is a great difference in the priming efficiency of different bases. When the last chain is T, the priming efficiency of the mismatch is greatly reduced. The priming efficiency of G and C mismatches is between A and T, so it is best to choose T at the 3′ end; the bases should be randomly distributed, etc.

[0063] Follow these primer design principles and paste the sequence for which primers need to be designed into the dialog box. Click the green button Pickprimer in the upper right corner of the primer3plus online software to design primers. The primer sequence information is shown in Table 2-1.

[0064] Table 2-1 Primer sequence information for mixed pool sequencing

[0065]

[0066]

[0067] Step 3: PCR amplification

[0068] The PCR amplification in this experiment uses 2×Taq-ASPCRMix (+Dye) from Jiangsu Yugong Life Science Co., Ltd. The PCR reaction system is 50μl. When using, take 25μl of 2×Taq-ASPCRMix (+Dye), add appropriate amount of template and primer, and add ddH2O to make up the volume, so that the concentration of the reaction system is 1×, and then you can carry out the PCR reaction. The PCR reaction system is shown in Table 2-2:

[0069] Table 2-2 PCR reaction system

[0070] Reagents Usage Final concentration 2×Taq-ASPCRMix(+Dye) 25μl 1× Forward primer (10 μM) 2μl 0.4μM Reverse primer (10 μM) 2μl 0.4μM Template DNA 3μl <![CDATA[ddH2O]]> 18μl

[0071] The template DNA is composed of a mixture of DNA samples from 40 randomly selected Landrace pigs and Large White pigs of different families, with a DNA concentration of 300-400. The qualified PCR amplification products are uniformly sent to BGI for mixed pool sequencing. The PCR cycle parameters are shown in Table 2-3:

[0072] Table 2-3 PCR cycle parameters

[0073] step Temperature(℃) time Pre-denaturation 94 3-5min transsexual 94 30sec annealing 55-65 30sec extend 72 20-40s / kb Final extension 72 5min

[0074] The annealing temperature of each pair of primers refers to the annealing temperature in the primer information table in Table 5.1, and 35 cycles are set from the second to the fourth step.

[0075] Step 4: PCR amplification product detection

[0076] Prepare a 1% agarose gel, use 5μl of FY2000DNAMarker as a reference and inject it into the first well of the gel, then draw 5μl of the PCR amplification product and inject it into the remaining gel wells in turn, and then perform gel electrophoresis. After the electrophoresis, observe whether the gel image is clear, bright, and consistent with the target fragment length. Qualified PCR amplification products are stored in a -80℃ refrigerator and sent to BGI for sequencing.

[0077] Step 5: SNP site detection

[0078] The nucleotide sequence of the sequencing results was compared with the original sequence using the NewPu Bio multiple sequence alignment online tool, the specific locations of the mutation sites were recorded, and then the sequencing peak graphs were analyzed using Seqman and Chromas software.

[0079] Step 6: Genotyping

[0080] After sequencing, 6 mutation sites were found in the USP16 gene. 493 ear DNA samples were sent to Beijing Compson Biotechnology Co., Ltd., and 11 mutations were typed using time-of-flight mass spectrometry.

[0081] First, the DNA sample provided was tested by electrophoresis to see if it met the quality standards of the time-of-flight mass spectrometry method. Using Loading buffer (TAKARA) EB dye and 1*TAE buffer agarose, 2ul of stock solution was taken for electrophoresis detection. The experimental results showed that the overall quality of the sample was very high and met the quality standards of the time-of-flight mass spectrometry method. Then the DNA was adjusted to an appropriate concentration.

[0082] Secondly, according to the SNP site sequence information, the primer design software Assaydesign3.1 of Sequenom was used to design and synthesize PCR reaction and single-base extension primers. The specific information of the primers is shown in Table 2-4.

[0083] Table 2-4 Primer sequence information for genotyping

[0084]

[0085] (1) Use the diluted DNA as a template to perform PCR amplification, SAP digestion reaction, and extension reaction.

[0086] (2) The reaction product (9 ul in total) was diluted 3 times and desalted using resin. The desalted sample was spotted on the sample target, and the natural crystal was put on the machine for mass spectrometry detection and data collection.

[0087] Results of DNA extraction from pig ear tissue

[0088] The ear tissue DNA of Large White and Landrace pigs was extracted using a tissue genomic DNA isolation kit. Some DNA was randomly selected and tested using 1% agarose gel electrophoresis. The results showed that the selected DNA sample bands were clear, bright, and neat, with no obvious dragging. All DNA samples were tested with a nucleic acid detector and found that the OD of all DNA samples was 260 / 280 The values ​​are all between 1.7 and 2.1, so they can be used for subsequent test operations, such as Figure 1 shown.

[0089] PCR amplification product detection results

[0090] 1. USP16 gene PCR amplification product detection results

[0091] The PCR amplification products of the USP16 gene were tested and it was found that there was one mutation site in the upstream regulatory region of the USP16 gene, one mutation site in the intron region, and four mutation sites in the downstream regulatory region. The test results of some PCR amplification products are as follows Figure 2 As shown, the length of the amplified fragment was basically consistent with expectations.

[0092] Population phenotypic value analysis

[0093] The phenotypic values ​​of 100kg backfat thickness, 100kg eye muscle area and 100kg body weight at age were analyzed for 458 pigs, including 238 Large White pigs and 220 Landrace pigs. The analysis results are shown in Table 3-1. For the trait of 100kg backfat thickness, the average value of Large White pigs was 9.06mm, with a coefficient of variation of 18.15%; the average value of Landrace pigs was 8.28mm, with a coefficient of variation of 15.36%. For the trait of 100kg eye muscle area, the average value of Large White pigs was 37.72cm 2 , the coefficient of variation is 9.11%; the average value of Landrace pig is 35.55cm 2 , with a coefficient of variation of 10.16%. For the trait of 100kg body weight per day, the average value of Large White pigs is 166.84 days, with a coefficient of variation of 6.57%; the average value of Landrace pigs is 162.60 days, with a coefficient of variation of 6.75%.

[0094] Table 3-1 Descriptive statistical analysis of pig phenotypic data

[0095]

[0096]

[0097] Analysis of USP16 gene polymorphism

[0098] 22 pairs of primers were used to amplify the DNA of 40 pig ear tissues randomly selected from the whole family for PCR, and then the resequenced sequences were compared with the original sequences. A total of 6 mutation sites were found, including 1 in the upstream regulatory region of USP16 gene (g.192376801C>A), 1 in the intron region (g.192401821G>A), and 4 in the downstream regulatory region (g.192407026G>A, g.192407237A>G, g.192408287T>C, and g.192408499C>T).

[0099] 1. Association analysis between g.192376801C>A locus and traits

[0100] 1.1. Sequencing and typing results analysis of the g.192376801C>A locus

[0101] 40 randomly selected DNA samples covering the entire family were pooled for sequencing, and the peak graphs of the sequencing results were analyzed using Seqman and Chromas software to screen out the g.192376801C>A mutation site located upstream of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, and it was found that there were three genotypes at this site: CC, CA, and AA. The sequencing results of the g.192376801C>A site are as follows Figure 3 As shown, the scatter plot of the site typing results is as follows Figure 4 shown.

[0102] 1.2 Gene frequency distribution and Hardy-Weinberg equilibrium test of the g.192376801C>A locus

[0103] Through time-of-flight mass spectrometry typing detection, among the 237 large white pigs, there were 23 AA-type individuals with g.192376801C>A locus, accounting for 10%; there were 96 CA-type individuals, accounting for 40%; and there were 118 CC-type individuals, accounting for 50%. The frequency of allele A was 0.30, and the frequency of allele C was 0.70. Among the 218 Landrace pigs, there were 28 AA-type individuals with g.192376801C>A locus, accounting for 13%; there were 99 CA-type individuals, accounting for 45%; and there were 91 CC-type individuals, accounting for 42%. The frequency of allele A was 0.36, and the frequency of allele C was 0.64. The specific results are shown in Table 3-2.

[0104] Table 3-2g.192376801C>A site polymorphism in pig populations

[0105]

[0106] As shown in Table 3-2, the allele frequencies of A and C in both the Large White and Landrace populations are quite different, and the frequency of A allele in Landrace is higher than that in Large White. According to the Hardy-Weinberg equilibrium test, the g.192376801C>A locus conforms to the Hardy-Weinberg equilibrium in both the Large White and Landrace populations (P>0.05).

[0107] 1.3. Association analysis between polymorphism of g.192376801C>A locus and traits

[0108] The polymorphism of g.192376801C>A locus was detected in Large White and Landrace pigs, and the number of individuals with three genotypes was counted. The association analysis of g.192376801C>A locus polymorphism with 100kg backfat thickness, 100kg eye muscle area and 100kg body weight at age in Large White and Landrace pig populations is shown in Table 3-3 and Table 3-4.

[0109] Table 3-3 Association analysis between g.192376801C>A site polymorphism and traits in Large White pigs

[0110] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area AA 23 166.54±11.60 9.40±1.11 38.67±3.59 CC 118 166.48±10.26 9.05±1.78 37.35±3.41 CA 96 167.22±11.76 8.98±1.58 37.93±3.43 P-value 0.827 0.657 0.068 AA-CA 0.791 0.272 0.357 AA-CC 0.981 0.354 0.095 CC-CA 0.625 0.75 0.223

[0111] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0112] Table 3-4 Association analysis between g.192376801C>A polymorphism and traits in Landrace pigs

[0113] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area CC 91 163.03±11.60 8.19±1.18 35.58±3.39 CA 99 161.96±10.86 8.29±1.35 35.24±3.87 P-value 0.998 0.247 0.634 AA-CA 0.438 0.488 0.187 AA-CC 0.748 0.305 0.377 CC-CA 0.506 0.614 0.525

[0114] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0115] After association analysis of the g.192376801C>A site gene polymorphism with the three traits, it can be seen that in the Large White pig population, the g.192376801C>A site is not significantly associated with 100kg body weight age, 100kg back fat thickness, and 100kg eye muscle area; in the Landrace pig population, the g.192376801C>A site is not significantly associated with 100kg body weight age, 100kg back fat thickness, and 100kg eye muscle area.

[0116] 2. Association analysis between the g.192401821G>A locus and traits

[0117] 2.1. Sequencing and typing results analysis of the g.192401821G>A site

[0118] 40 randomly selected DNA samples covering the entire family were pooled and sequenced. The peak graphs of the sequencing results were analyzed using Seqman and Chromas software to screen out the g.192401821G>A mutation site located in the intron region of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, and it was found that there were three genotypes at this site: GG, GA, and AA. The sequencing results of the g.192401821G>A site are as follows Figure 5 As shown, the scatter plot of the site typing results is as follows Figure 6 shown.

[0119] 2.2 Gene frequency distribution and Hardy-Weinberg equilibrium test of the g.192401821G>A locus

[0120] Through time-of-flight mass spectrometry typing detection, among the 237 large white pigs, there were 40 AA individuals with g.192401821G>A, accounting for 17%; there were 103 GA individuals, accounting for 43%; and there were 94 GG individuals, accounting for 40%. The frequency of allele A was 0.39, and the frequency of allele G was 0.61. Among the 220 Landrace pigs, there were 82 AA individuals with g.192401821G>A, accounting for 37%; there were 88 GA individuals, accounting for 40%; and there were 50 GG individuals, accounting for 23%. The frequency of allele A was 0.57, and the frequency of allele C was 0.43. The specific results are shown in Table 3-5.

[0121] Table 3-5g.192401821G>A site polymorphism in pig populations

[0122]

[0123]

[0124] As shown in Table 3-5, the allele frequencies of A and G differed greatly in the Large White pig population, but less in the Landrace pig population. The frequency of A allele in Landrace pig was higher than that in Large White pig. According to the Hardy-Weinberg equilibrium test, the g.192401821G>A locus conformed to the Hardy-Weinberg equilibrium in the Large White pig population (P>0.05), but deviated from the Hardy-Weinberg equilibrium in the Landrace pig population (P<0.05).

[0125] 2.3. Association analysis between g.192401821G>A site polymorphism and traits

[0126] The polymorphism of g.192401821G>A locus was detected in the Large White and Landrace pig populations, and the number of individuals with the three genotypes was counted. The association analysis of the g.192401821G>A locus polymorphism with 100kg backfat thickness, 100kg eye muscle area and 100kg body weight at age in the Large White and Landrace pig populations is shown in Tables 3-6 and 3-7.

[0127] Table 3-6 Association analysis between g.192401821G>A site polymorphism and traits in Large White pigs

[0128] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area AA 40 167.28±11.88 9.61±1.314 38.40±3.407 GG 94 166.75±10.34 8.92±1.834 37.22±3.320 GA 103 166.64±11.28 8.96±1.557 37.89±3.550 P-value 0.74 <![CDATA[0.031 * ]]> <![CDATA[0.042 * ]]> AA-GA 0.755 <![CDATA[0.033 * ]]> 0.431 AA-GG 0.798 <![CDATA[0.027 * ]]> 0.072 GG-GA 0.945 0.884 0.175

[0129] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0130] Table 3-7 Association analysis between g.192401821G>A polymorphism and traits in Landrace pigs

[0131] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area AA 82 162.34±9.991 8.28±1.316 35.75±3.495 GG 50 165.02±12.02 8.10±1.051 34.76±3.292 GA 88 161.45±11.18 8.38±1.354 35.81±3.878 P-value 0.21 0.58 0.18 AA-GA 0.593 0.634 0.907 AA-GG 0.175 0.436 0.128 GG-GA 0.067 0.230 0.101

[0132] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0133] After the association analysis of the g.192401821G>A site gene polymorphism with the three traits, it can be seen that in the large white pig population, the g.192401821G>A site is not significantly associated with the trait of 100kg body weight age; the g.192401821G>A site is significantly associated with the trait of 100kg back fat thickness (P<0.05), the dominant allele is G, and the 100kg back fat thickness of GG type and GA type is significantly smaller than that of AA type (P<0.05); the g.192401821G>A site is significantly associated with the trait of 100kg eye muscle area (P<0.05). In the Landrace pig population, the g.192401821G>A site is not significantly associated with the three traits of 100kg body weight age, 100kg back fat thickness, and 100kg eye muscle area.

[0134] 3. Association analysis between g.192407026G>A locus and traits

[0135] 3.1. Sequencing and typing results analysis of the g.192407026G>A site

[0136] 40 randomly selected DNA samples covering the entire family were pooled for sequencing, and the peak graphs of the sequencing results were analyzed using Seqman and Chromas software to screen out the g.192407026G>A mutation site located in the downstream region of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, and it was found that there were three genotypes at this site: GG, GA, and AA. The sequencing results of the g.192407026G>A site are as follows Figure 7 As shown, the site typing results are scattered Figure 8 As shown in the picture.

[0137] 3.2 Gene frequency distribution and Hardy-Weinberg equilibrium test of the g.192407026G>A locus

[0138] Through time-of-flight mass spectrometry typing detection, among the 256 large white pigs, there were 41 AA individuals with g.192407026G>A, accounting for 16%; there were 102 GA individuals, accounting for 40%; and there were 113 GG individuals, accounting for 44%. The frequency of allele A was 0.36, and the frequency of allele G was 0.64. Among the 235 Landrace pigs, there were 83 AA individuals with g.192407026G>A, accounting for 35%; there were 95 GA individuals, accounting for 40%; and there were 57 GG individuals, accounting for 24%. The frequency of allele A was 0.56, and the frequency of allele C was 0.44. The specific results are shown in Table 3-8.

[0139] Table 3-8g.192407026G>A site polymorphism in pig populations

[0140]

[0141] As shown in Table 3-8, the allele frequencies of A and G differ greatly in the Large White pig population, but less in the Landrace pig population. The frequency of A allele in Landrace pig is higher than that in Large White pig. According to the Hardy-Weinberg equilibrium test, the g.192407026G>A locus conforms to the Hardy-Weinberg equilibrium in the Large White pig population (P>0.05), but deviates from the Hardy-Weinberg equilibrium in the Landrace pig population (P<0.05).

[0142] 3.3. Association analysis between g.192407026G>A site polymorphism and traits

[0143] The polymorphism of g.192407026G>A locus was detected in the Large White and Landrace pig populations, and the number of individuals with the three genotypes was counted. The association analysis of the g.192407026G>A locus polymorphism with 100kg backfat thickness, 100kg eye muscle area and 100kg body weight at age in the Large White and Landrace pig populations is shown in Tables 3-9 and 3-10.

[0144] Table 3-9 Association analysis between g.192407026G>A site polymorphism and traits in Large White pigs

[0145] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area AA 40 167.28±11.88 9.61±1.314 38.40±3.407 GG 94 166.75±10.34 8.92±1.834 37.22±3.320 GA 102 166.44±11.16 8.95±1.565 37.87±3.561 P-value 0.773 <![CDATA[0.031 * ]]> <![CDATA[0.043 * ]]> AA-GA 0.683 <![CDATA[0.033 * ]]> 0.413 AA-GG 0.797 <![CDATA[0.027 * ]]> 0.072 GG-GA 0.847 0.888 0.191

[0146] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0147] Table 3-10 Association analysis between g.192407026G>A polymorphism and traits in Landrace pigs

[0148] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area AA 82 162.34±9.991 8.28±1.316 35.75±3.495 GG 50 165.02±12.02 8.10±1.051 34.76±3.292 GA 88 161.45±11.18 8.38±1.354 35.81±3.878 P-value 0.213 0.582 0.182 AA-GA 0.593 0.634 0.907 AA-GG 0.175 0.436 0.128 GG-GA 0.067 0.230 0.101

[0149] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0150] The association analysis of the g.192407026G>A site gene polymorphism with the three traits showed that in the large white pig population, the g.192407026G>A site was not significantly associated with the trait of 100kg body weight per day; the g.192407026G>A site was significantly associated with the trait of 100kg backfat thickness (P<0.05), the dominant allele was G, and the 100kg backfat thickness of GG type and GA type was significantly smaller than that of AA type (P<0.05); the g.192401821G>A site was significantly associated with the trait of 100kg eye muscle area (P<0.05). In the Landrace pig population, the g.192401821G>A site was not significantly associated with the three traits of 100kg body weight per day, 100kg backfat thickness, and 100kg eye muscle area.

[0151] 4. Association analysis between the g.192407237A>G locus and traits

[0152] 4.1. Sequencing and typing results analysis of the g.192407237G>A site

[0153] 40 randomly selected DNA samples covering the entire family were pooled for sequencing, and the peak graphs of the sequencing results were analyzed using Seqman and Chromas software to screen out the g.192407237G>A mutation site located in the downstream region of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, and it was found that there were three genotypes at this site: GG, GA, and AA. The sequencing results of the g.192407237G>A site are as follows Fig. 9 As shown, the scatter plot of the site typing results is as follows Fig.10 shown.

[0154] 4.2 Gene frequency distribution and Hardy-Weinberg equilibrium test of the g.192407237A>G locus

[0155] Through time-of-flight mass spectrometry typing detection, among the 236 large white pigs, there were 94 AA individuals with g.192407237A>G locus, accounting for 40%; there were 102 GA individuals, accounting for 43%; there were 40 GG individuals, accounting for 17%. The frequency of allele A was 0.61, and the frequency of allele G was 0.39. Among the 220 Landrace pigs, there were 51 AA individuals with g.192407237A>G locus, accounting for 23%; there were 87 GA individuals, accounting for 40%; there were 82 GG individuals, accounting for 37%. The frequency of allele A was 0.43, and the frequency of allele C was 0.57. The specific results are shown in Table 3-11.

[0156] Table 3-11g.192407237G>A site polymorphism in pig populations

[0157]

[0158] As shown in Table 3-11, the allele frequencies of A and G differ greatly in the Large White pig population, but less in the Landrace pig population. The frequency of the A allele in the Large White pig is higher than that in the Landrace pig. According to the Hardy-Weinberg equilibrium test, the g.192407237A>G locus conforms to the Hardy-Weinberg equilibrium in the Large White pig population (P>0.05), but deviates from the Hardy-Weinberg equilibrium in the Landrace pig population (P<0.05).

[0159] 4.3. Association analysis between polymorphism of g.192407237A>G locus and traits

[0160] The polymorphism of g.192407237A>G locus was detected in the Large White and Landrace pig populations, and the number of individuals with the three genotypes was counted. The association analysis of the g.192407237A>G locus polymorphism with 100kg backfat thickness, 100kg eye muscle area and 100kg body weight at age in the Large White and Landrace pig populations is shown in Tables 3-12 and 3-13.

[0161] Table 3-12 Association analysis between g.192407237G>A site polymorphism and traits in Large White pigs

[0162] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area AA 94 166.75±10.34 8.92±1.834 37.22±3.320 GG 40 167.28±11.88 9.61±1.314 38.40±3.407 GA 102 166.44±11.16 8.95±1.565 37.87±3.561 P-value 0.773 <![CDATA[0.031 * ]]> <![CDATA[0.043 * ]]> AA-GA 0.847 0.888 0.191 AA-GG 0.797 <![CDATA[0.027 * ]]> 0.072 GG-GA 0.683 <![CDATA[0.033 * ]]> 0.413

[0163] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0164] Table 3-13 Association analysis between g.192407026G>A polymorphism and traits in Landrace pigs

[0165] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area AA 51 164.90±11.93 8.13±1.062 34.88±3.384 GG 82 162.34±9.991 8.28±1.316 35.75±3.495 GA 87 161.47±11.24 8.36±1.355 35.75±3.855 P-value 0.24 0.606 0.22 AA-GA 0.077 0.311 0.178 AA-GG 0.192 0.512 0.183 GG-GA 0.605 0.688 0.997

[0166] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0167] The association analysis of the g.192407237A>G site gene polymorphism with the three traits showed that in the large white pig population, the g.192407237A>G site was not significantly associated with the trait of 100kg body weight per day; the g.192407237A>G site was significantly associated with the trait of 100kg backfat thickness (P<0.05), the dominant allele was A, and the 100kg backfat thickness of AA and GA types was significantly smaller than that of GG type (P<0.05); the g.192407237A>G site was significantly associated with the trait of 100kg eye muscle area (P<0.05). In the Landrace pig population, the g.192407237A>G site was not significantly associated with the three traits of 100kg body weight per day, 100kg backfat thickness, and 100kg eye muscle area.

[0168] 5. Association analysis between g.192408287T>C locus and traits

[0169] 5.1. Sequencing and typing results analysis of g.192408287T>C locus

[0170] 40 randomly selected DNA samples covering the entire family were pooled and sequenced. The peak graphs of the sequencing results were analyzed using Seqman and Chromas software to screen out the g.192408287T>C mutation site located in the downstream region of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, and it was found that there were three genotypes at this site: CC, CT, and TT. The sequencing results of the g.192408287T>C site are as follows Fig.11 As shown, the scatter plot of the site typing results is as follows Fig.12 shown.

[0171] 5.2 Gene frequency distribution and Hardy-Weinberg equilibrium test of g.192408287T>C locus

[0172] Through time-of-flight mass spectrometry typing detection, there are 40 CC-type individuals with g.192408287T>C locus in the 238 large white pigs, accounting for 17%; there are 104 CT-type individuals, accounting for 44%; there are 94 TT-type individuals, accounting for 39%. The frequency of allele C is 0.39, and the frequency of allele T is 0.61. In the 220 Landrace pigs, there are 82 CC-type individuals with g.192408287T>C locus, accounting for 37%; there are 88 CT-type individuals, accounting for 40%; there are 50 TT-type individuals, accounting for 23%. The frequency of allele C is 0.57, and the frequency of allele T is 0.43. The specific results are shown in Table 3-14.

[0173] Table 3-14g.192408287G>A site polymorphism in pig populations

[0174]

[0175] As shown in Table 3-14, the allele frequencies of C and T differ greatly in the Large White pig population, but less in the Landrace pig population. The frequency of the C allele in the Landrace pig is higher than that in the Large White pig. According to the Hardy-Weinberg equilibrium test, the g.192408287T>C locus conforms to the Hardy-Weinberg equilibrium in the Large White pig population (P>0.05), but deviates from the Hardy-Weinberg equilibrium in the Landrace pig population (P<0.05).

[0176] 5.3. Association analysis between g.192408287T>C polymorphism and traits

[0177] The polymorphism of g.192408287T>C locus was detected in the Large White and Landrace pig populations, and the number of individuals with the three genotypes was counted. The association analysis of the g.192408287T>C locus polymorphism with 100kg backfat thickness, 100kg eye muscle area and 100kg body weight at age in the Large White and Landrace pig populations is shown in Table 3-15 and Table 3-16.

[0178] Table 3-15 Association analysis between g.192408287T>C polymorphism and traits in Large White pigs

[0179] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area CC 238 167.28±11.88 9.61±1.31 38.40±3.40 CT 238 166.75±11.28 8.96±1.55 37.91±3.53 TT 238 166.75±10.34 8.92±1.83 37.22±3.32 P-value 0.732 <![CDATA[0.031 * ]]> <![CDATA[0.041 * ]]> CC-CT 0.795 <![CDATA[0.035 * ]]> 0.445 CC-TT 0.798 <![CDATA[0.027 * ]]> 0.072 TT-CT 0.999 0.853 0.163

[0180] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0181] Table 3-16 Association analysis between g.192408287T>C polymorphism and traits in Landrace pigs

[0182]

[0183] Note: * indicates P<0.05, which means significant; ** indicates P<0.01, which means extremely significant

[0184] The association analysis of the g.192408287T>C site gene polymorphism with the three traits showed that in the large white pig population, the g.192408287T>C site was not significantly associated with the trait of 100kg body weight per day; the g.192408287T>C site was significantly associated with the trait of 100kg backfat thickness (P<0.05), the dominant allele was T, and the 100kg backfat thickness of TT and CT types was significantly smaller than that of CC type (P<0.05); the g.192408287T>C site was significantly associated with the trait of 100kg eye muscle area (P<0.05). In the Landrace pig population, the g.192408287T>C site was not significantly associated with the three traits of 100kg body weight per day, 100kg backfat thickness, and 100kg eye muscle area.

[0185] 6. Association analysis between g.192408499C>T locus and traits

[0186] 6.1. Sequencing and typing results analysis of g.192408499C>T locus

[0187] 40 randomly selected DNA samples covering the entire family were pooled for sequencing, and the peak graphs of the sequencing results were analyzed using Seqman and Chromas software to screen out the g.192408499C>T mutation site located in the downstream region of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, and it was found that there were three genotypes of CC, CT, and TT at this site. The sequencing results of the g.192408499C>T site are as follows Fig.13 As shown, the scatter plot of the site typing results is as follows Fig.14 shown.

[0188] 6.2 Gene frequency distribution and Hardy-Weinberg equilibrium test of the g.192408499C>T locus

[0189] Through time-of-flight mass spectrometry typing detection, there are 94 CC-type individuals with g.192408499C>T locus in the 238 large white pigs, accounting for 39%; there are 102 CT-type individuals, accounting for 43%; there are 42 TT-type individuals, accounting for 18%;. The frequency of allele C is 0.61, and the frequency of allele T is 0.39. In the 220 Landrace pigs, there are 50 CC-type individuals with g.192408499C>T locus, accounting for 23%; there are 88 CT-type individuals, accounting for 40%; there are 82 TT-type individuals, accounting for 37%. The frequency of allele C is 0.43, and the frequency of allele T is 0.57. The specific results are shown in Table 3-17.

[0190] Tab.3-17Polymorphisms of g.192408499G>A locus in pig populations

[0191]

[0192] As shown in Table 3-14, the allele frequencies of C and T differ greatly in the large white pig population, but less in the landrace pig population. The frequency of C allele in large white pigs is higher than that in landrace pigs. According to the Hardy-Weinberg equilibrium test, the g.192408499C>T locus conforms to the Hardy-Weinberg equilibrium in the large white pig population (P>0.05), but deviates from the Hardy-Weinberg equilibrium in the landrace pig population (P<0.05).

[0193] 6.3. Association analysis between g.192408499C>T polymorphism and traits

[0194] The polymorphism of g.192408499C>T locus was detected in the Large White and Landrace pig populations, and the number of individuals with the three genotypes was counted. The association analysis of the g.192408499C>T locus polymorphism with 100kg backfat thickness, 100kg eye muscle area and 100kg body weight at age in the Large White and Landrace pig populations is shown in Table 3-18 and Table 3-19.

[0195] Table 3-18 Association analysis between g.192408499C>T polymorphism and traits in Large White pigs

[0196] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area CC 94 166.75±10.34 8.92±1.83 37.22±3.32 CT 102 166.44±11.16 8.95±1.56 37.87±3.56 TT 42 167.99±12.07 9.60±1.28 38.47±3.33 P-value 0.569 <![CDATA[0.031 * ]]> <![CDATA[0.034 * ]]> CC-CT 0.847 0.888 0.189 CC-TT 0.542 <![CDATA[0.025 * ]]> 0.052 CT-TT 0.443 <![CDATA[0.031 * ]]> 0.344

[0197] Note: * indicates p<0.05, which means significant; ** indicates p<0.01, which means extremely significant

[0198] Table 3-19 Association analysis between g.192408499C>T polymorphism and traits in Landrace pigs

[0199] genotype Number of individuals 100kg weight in days 100kg back fat thickness 100kg eye muscle area CC 238 165.02±12.02 8.10±1.05 34.76±3.29 CT 238 161.45±11.18 8.38±1.35 35.81±3.87 TT 238 162.34±9.991 8.28±1.31 35.75±3.49 P-value 0.213 0.582 0.182 CC-CT 0.067 0.23 0.101 CC-TT 0.175 0.436 0.128 TT-CT 0.593 0.634 0.907

[0200] Note: * indicates p<0.05, which means significant; ** indicates p<0.01, which means extremely significant

[0201] After the association analysis of the g.192408499C>T site gene polymorphism with the three traits, it can be seen that in the large white pig population, the g.192408499C>T site is not significantly associated with the trait of 100kg body weight age; the g.192408499C>T site is significantly associated with the trait of 100kg backfat thickness (p<0.05), the dominant allele is C, and the 100kg backfat thickness of CC type and CT type is significantly smaller than that of TT type (p<0.05); the g.192408499C>T site is significantly associated with the trait of 100kg eye muscle area (p<0.05). In the Landrace pig population, the g.192408499C>T site is not significantly associated with the three traits of 100kg body weight age, 100kg backfat thickness, and 100kg eye muscle area.

[0202] Analysis of association between USP16 gene SNP sites and traits

[0203] By pool sequencing, the resequenced sequences were compared with the original sequences, and a total of 6 SNPs were found on the USP16 gene that may be related to related growth traits.

[0204] Association analysis between g.192376801C>A locus and traits

[0205] For the g.192376801C>A site located in the upstream regulatory region of the USP16 gene, by statistically analyzing the three typing results of this site and analyzing the association with the traits, it was found that the g.192376801C>A site was not significantly associated with various growth traits in the Large White and Landrace pig populations. Therefore, this site cannot be used as an effective molecular marker.

[0206] Association analysis between g.192401821G>A locus and traits

[0207] For the g.192401821G>A site located in the intron region of the USP16 gene, by statistically analyzing the three typing results of the site and analyzing the association with the traits, it was found that the g.192401821G>A site of the USP16 gene was significantly associated with the 100kg backfat thickness trait in the large white pig population (P<0.05), and the dominant allele was G. The 100kg backfat thickness of the GG type and the GA type was significantly less than that of the AA type (P<0.05). At the same time, in the large white pig population, this site was also significantly associated with the trait of 100kg eye muscle area (P<0.05). From the mean values ​​of each genotype group, the 100kg eye muscle area of ​​the AA type was larger than that of the GA type and the GG type, but the difference was not significant, which may be related to the limited sample size of this study. The sample size can be expanded in the future to continue the research and verification. This site was not significantly associated with the age of 100kg body weight. In the Landrace pig population, the g.192401821G>A locus was not significantly associated with any growth traits.

[0208] Therefore, the g.192401821G>A locus is significantly associated with the 100kg backfat thickness trait of the Large White pig population and can be used as an effective molecular marker in molecular-assisted selection.

[0209] Association analysis between g.192407026G>A locus and traits

[0210] For the g.192407026G>A site located in the downstream regulatory region of the USP16 gene, by statistically analyzing the three typing results of the site and doing association analysis with the traits, it was found that the g.192407026G>A site of the USP16 gene was significantly associated with the trait of 100kg back fat thickness in the large white pig population (P<0.05), the dominant allele was G, and the 100kg back fat thickness of the GG type and the GA type was significantly smaller than that of the AA type (P<0.05). At the same time, in the large white pig population, this site was also significantly associated with the trait of 100kg eye muscle area (P<0.05). From the mean values ​​of each genotype group, the 100kg eye muscle area of ​​the AA type was larger than that of the GA type and the GG type, but the difference was not significant, which may be related to the limited sample size of this study. The sample size can be expanded in the future to continue the research and verification. This site was not significantly associated with the age of 100kg body weight. In the Landrace pig population, the g.192407026G>A locus was not significantly associated with any growth traits.

[0211] Therefore, the g.192407026G>A locus is significantly associated with the 100kg backfat thickness trait of the Large White pig population and can be used as an effective molecular marker in molecular-assisted selection.

[0212] Association analysis between g.192407237A>G locus and traits

[0213] For the g.192407237A>G site located in the downstream regulatory region of the USP16 gene, by statistically analyzing the three typing results of the site and analyzing the association with the traits, it was found that the USP16 gene g.192407237A>G site was significantly associated with the trait of 100kg backfat thickness in the large white pig population (P<0.05), the dominant allele was A, and the 100kg backfat thickness of the AA type and GA type was significantly smaller than that of the GG type (P<0.05). At the same time, in the large white pig population, this site was also significantly associated with the trait of 100kg eye muscle area (P<0.05). From the mean values ​​of each genotype group, the 100kg eye muscle area of ​​the GG type was larger than that of the AA type and GA type, but the difference was not significant, which may be related to the limited sample size of this study. The sample size can be expanded in the future to continue the research and verification. This site was not significantly associated with the age of 100kg body weight. In the Landrace pig population, the g.192407237A>G locus was not significantly associated with any growth traits.

[0214] Therefore, the g.192407237A>G locus is significantly associated with the 100kg backfat thickness trait of the Large White pig population and can be used as an effective molecular marker in molecular-assisted selection.

[0215] Association analysis between g.192408287T>C locus and traits

[0216] For the g.192408287T>C site located in the downstream regulatory region of the USP16 gene, by statistically analyzing the three typing results of the site and analyzing the association with the traits, it was found that the g.192408287T>C site of the USP16 gene was significantly associated with the trait of 100kg backfat thickness in the large white pig population (P<0.05), the dominant allele was T, and the 100kg backfat thickness of the TT type and CT type was significantly smaller than that of the CC type (P<0.05). At the same time, in the large white pig population, this site was also significantly associated with the trait of 100kg eye muscle area (P<0.05). From the mean values ​​of each genotype group, the 100kg eye muscle area of ​​the CC type was larger than that of the CT type and TT type, but the difference was not significant, which may be related to the limited sample size of this study. The sample size can be expanded in the future to continue the research and verification. This site was not significantly associated with the age of 100kg body weight. In the Landrace pig population, the g.192408287T>C locus was not significantly associated with any growth traits.

[0217] Therefore, the g.192408287T>C locus is significantly associated with the 100kg backfat thickness trait of the Large White pig population and can be used as an effective molecular marker in molecular-assisted selection.

[0218] Association analysis between g.192408499C>T locus and traits

[0219] For the g.192408499C>T site located in the downstream regulatory region of the USP16 gene, by statistically analyzing the three typing results of the site and analyzing the association with the traits, it was found that the g.192408499C>T site of the USP16 gene was significantly associated with the trait of 100kg backfat thickness in the large white pig population (P<0.05), the dominant allele was C, and the 100kg backfat thickness of the CC type and CT type was significantly smaller than that of the TT type (P<0.05). At the same time, in the large white pig population, this site was also significantly associated with the trait of 100kg eye muscle area (P<0.05). From the mean values ​​of each genotype group, the 100kg eye muscle area of ​​the TT type was larger than that of the CC type and CT type, but the difference was not significant, which may be related to the limited sample size of this study. The sample size can be expanded in the future to continue the research and verification. This site was not significantly associated with the age of 100kg body weight. In the Landrace pig population, the g.192408499C>T locus was not significantly associated with any growth traits.

[0220] Therefore, the g.192408499C>T locus is significantly associated with the 100kg backfat thickness trait of the Large White pig population and can be used as an effective molecular marker in molecular-assisted selection.

[0221] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for evaluating back fat of Large White pigs based on USP16 gene, characterized in that: Determine the SNP molecular marker of the Large White Pig, and judge whether the SNP molecular marker is one of the following SNP molecular markers, specifically: The g.192401821G>A site located in the intronic region of the USP16 gene; The g.192407026G>A site located in the downstream regulatory region of the USP16 gene; The g.192407237A>G site located in the downstream regulatory region of the USP16 gene; The g.192408287T>C site located in the downstream regulatory region of the USP16 gene; The g.192408499C>T site is located in the downstream regulatory region of the USP16 gene.

2. A method for evaluating backfat of Large White pigs based on USP16 gene according to claim 1, characterized in that: The g.192401821G>A site located in the intron region of the USP16 gene, the back fat thickness of the Large White pig is ranked from large to small, and the corresponding genotypes are: GG genotype, GA genotype, AA genotype; The g.192407026G>A site located in the downstream regulatory region of the USP16 gene, the back fat of the Large White pig is ranked from large to small, and the corresponding genotypes are: AA genotype, GA genotype, and GG genotype.

3. A method for evaluating backfat of Large White pigs based on USP16 gene according to claim 1, characterized in that: The g.192407237A>G site located in the downstream regulatory region of the USP16 gene, the back fat thickness of the Large White pig is ranked from large to small, and the corresponding genotypes are: GG genotype, GA genotype, and AA genotype.

4. A method for evaluating backfat of Large White pigs based on USP16 gene according to claim 1, characterized in that: The g.192408287T>C site located in the downstream regulatory region of the USP16 gene, the back fat thickness of the Large White pig is ranked from large to small, and the corresponding genotypes are: CC genotype, CT genotype, and TT genotype.

5. The method for evaluating backfat of Large White pigs based on USP16 gene according to claim 1, characterized in that: The g.192408499C>T site located in the downstream regulatory region of the USP16 gene, the back fat thickness of the Large White pig is ranked from large to small, and the corresponding genotypes are: TT genotype, CT genotype, and CC genotype.

6. A primer pair combination, characterized in that: Used to detect SNP molecular markers of Large White Pigs, including: The g.192401821G>A site located in the intron region of the USP16 gene was detected. The nucleotide sequence of the forward primer was shown in the sequence list as SEQ ID NO.1, and the nucleotide sequence of the reverse primer was shown in the sequence list as SEQ ID NO.

2. The g.192407026G>A site located in the downstream regulatory region of the USP16 gene is detected. The nucleotide sequence of the forward primer is shown in the sequence list as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in the sequence list as SEQ ID NO.4; The g.192407237A>G site located in the downstream regulatory region of the USP16 gene is detected. The nucleotide sequence of the forward primer is shown in the sequence list as SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in the sequence list as SEQ ID NO.6; The g.192408287T>C site located in the downstream regulatory region of the USP16 gene is detected. The nucleotide sequence of the forward primer is shown in the sequence list as SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown in the sequence list as SEQ ID NO.8; The g.192408499C>T site located in the downstream regulatory region of the USP16 gene was detected. The nucleotide sequence of the forward primer was shown in the sequence list as SEQ ID NO.9, and the nucleotide sequence of the reverse primer was shown in the sequence list as SEQ ID NO.

10.

7. A kit, characterized in that: It comprises the primer pair combination as claimed in claim 6.

8. Use of the primer pair combination as claimed in claim 6 in any of the following: identifying the trait of eye muscle area of ​​Large White pigs, increasing the eye muscle area of ​​Large White pigs, identifying the trait of back fat thickness of Large White pigs, increasing the trait of back fat thickness of Large White pigs, and genetic breeding of Large White pigs.

Images