Method for evaluating backfat of large white pigs based on usp16 gene

Abstract

The application relates to the technical field of pig breeding improvement, and particularly relates to a method for evaluating backfat of large white pigs based on a USP16 gene, determining SNP molecular markers of the large white pigs, and judging whether the SNP molecular markers are one of the following SNP molecular markers, specifically, a g.192401821G>A site located in an intron region of the USP16 gene, a g.192407026G>A site located in a downstream regulation region of the USP16 gene, a g.192407237A>G site located in the downstream regulation region of the USP16 gene, a g.192408287T>C site located in the downstream regulation region of the USP16 gene, and a g.192408499C>T site located in the downstream regulation region of the USP16 gene. The evaluation method and the SNP molecular markers used in the method provide good theoretical support and effective guidance for breeding and improvement of growth traits of pigs.

Description

Technical Field

[0001] This invention relates to the field of pig breeding and improvement technology, and in particular to a method for evaluating backfat in Large White pigs based on the USP16 gene. Background Technology

[0002] Reproductive traits, meat quality traits, carcass traits, and growth traits are the main economic traits of pigs. Genetic improvement of these main economic traits directly affects the cost and efficiency of pig farming. Growth traits and carcass traits have always been the focus of genetic improvement. Pig growth traits are usually expressed as growth rate, feed conversion ratio, daily feed intake, and backfat thickness per 100 kg. These traits have moderate heritability and can be improved through genetic selection. Carcass traits are expressed as carcass weight, dressing percentage, carcass length, lean meat percentage, and fat percentage. These traits are mostly highly heritable. In the long process of breeding improvement, the drawbacks of traditional breeding methods have gradually become apparent. For example, for some traits with low heritability, improvement using traditional breeding methods yields minimal results. With the development of modern biotechnology, new genetic breeding improvement methods have begun to emerge.

[0003] Molecular marker-assisted selection using modern biotechnology is a commonly used breeding method, and effective molecular markers are a prerequisite and guarantee for molecular breeding. Detection of SNP molecular genetic markers for target trait functional genes can improve the accuracy of genomic selection during the breeding process. Therefore, identifying functional genes for important economic traits in pigs can provide more theoretical basis for subsequent practical work in pig breeding.

[0004] Currently, since the implementation of genome-wide association studies (GWAS) and high-throughput sequencing in pigs, many candidate genes and functional sites have been detected, but there are not many applicable, truly effective major genes and key causative mutations. Summary of the Invention

[0005] To address the technical problems mentioned in the background section, this invention provides a method for assessing backfat in Large White pigs based on the USP16 gene.

[0006] This invention employs the following technical solution: a method for assessing backfat in Large White pigs based on the USP16 gene, determining the SNP molecular markers of the Large White pigs, and judging whether the SNP molecular markers are one of the following SNP molecular markers, specifically:

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

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

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

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

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

[0012] Specifically, the genotypes of the Large White pigs with backfat ranging from large to small, located at the g.192401821G>A site in the intron region of the USP16 gene and with the dominant allele being G, are: GG genotype, GA genotype, and AA genotype.

[0013] The g.192407026G>A site, located in the downstream regulatory region of the USP16 gene, corresponds to the following genotypes of the Large White pigs, from largest to smallest backfat: AA genotype, GA genotype, and GG genotype.

[0014] Specifically, the g.192407237A>G site located in the downstream regulatory region of the USP16 gene corresponds to the following genotypes of the Large White pigs, from largest to smallest backfat: GG genotype, GA genotype, and AA genotype.

[0015] Specifically, the g.192408287T>C site located in the downstream regulatory region of the USP16 gene corresponds to the following genotypes of the Large White pigs, from largest to smallest backfat: 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 genotypes of the Large White pigs, from largest to smallest backfat, are ordered as follows: TT genotype, CT genotype, and CC genotype.

[0017] A primer pair combination for detecting SNP molecular markers in 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 its forward primer is shown in SEQ ID NO.1 of the sequence listing, and the nucleotide sequence of its reverse primer is shown in SEQ ID NO.2 of the sequence listing.

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

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

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

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

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

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This study used Large White and Landrace pigs as experimental populations to determine the phenotypes of three traits: age at 100 kg body weight, backfat thickness at 100 kg body weight, and eye muscle area at 100 kg body weight. Ear tissue was collected and genomic DNA was extracted. Primers were designed targeting the entire exon region of the USP16 gene and the upstream and downstream regulatory regions (2000-3000 bp). Pooled sequencing was used to screen for six SNP loci in the USP16 gene. Time-of-flight mass spectrometry was used to genotype the six selected SNP loci, and association analysis was performed between the SNP loci and the three traits of age at 100 kg body weight, backfat thickness at 100 kg body weight, and eye muscle area at 100 kg body weight, as well as pairwise comparisons between genotypes.

[0026] The evaluation methods and SNP molecular markers proposed in this scheme provide sound theoretical support and effective guidance for the breeding and improvement of growth traits in pigs. Attached Figure Description

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

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

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

[0030] Figure 4 This is a scatter plot of the genotyping results for the g.192376801C>A site in this embodiment of the invention.

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

[0032] Figure 6 This is a scatter plot of the genotyping results for the g.192401821G>A site in this embodiment of the invention.

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

[0034] Figure 8 This is a scatter plot of the genotyping results for the g.192407026G>A site in this embodiment of the invention.

[0035] Figure 9 This is a sequencing result diagram of the g.192407237G>A site in an embodiment of the present invention;

[0036] Figure 10 This is a scatter plot of the genotyping results for the g.192407237G>A site in this embodiment of the invention.

[0037] Figure 11 This is a sequencing result diagram of the g.192408287G>A site in an embodiment of the present invention;

[0038] Figure 12 This is a scatter plot of the genotyping results for the g.192408287G>A site in this embodiment of the invention.

[0039] Figure 13 This is a sequencing result diagram of the g.192408499G>A site in an embodiment of the present invention;

[0040] Figure 14 This is a scatter plot of the genotyping results for the g.192408499G>A site in an embodiment of the present invention. Detailed Implementation

[0041] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0042] Example:

[0043] The population used in this embodiment consisted of 238 Large White pigs and 220 Landrace pigs. The traits measured were age at 100 kg body weight, eye muscle area at 100 kg body weight, and backfat thickness at 100 kg body weight. The experimental population came from Bengbu Pengrui Agricultural and Animal Husbandry Industry Co., Ltd. in Anhui Province. All samples collected were ear samples, and the extracted DNA samples were stored at -80℃ for long-term preservation for subsequent experiments.

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

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

[0046] Main instruments and equipment: benchtop high-speed centrifuge, model: MIKRO22; 4-degree storage cabinet, model: YC-260L; horizontal low-temperature freezer, 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 system, model: DYCP-32P; ultrapure water system, model: labmini4uvf.

[0047] Key molecular biology software: Primer3plus: for primer design; Chromas: for analyzing sequencing peaks; Seqman: for sequencing result alignment and assembly; Plink: Hardy-Weinberg equilibrium test; Haploview: for linkage disequilibrium analysis between loci; SPSS: for significance calculation; R language: for association analysis between SNPs and traits; Endnote: for managing references.

[0048] Test methods

[0049] Step 1: DNA Extraction

[0050] (1) Take 20-30 mg of ear sample, about the size of a soybean, from the alcohol, cut it into small pieces with scissors, and place it in a 1.5 ml centrifuge tube. Add 200 μL of Buffer TL. To accelerate lysis, the finer the tissue is cut, the better.

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

[0052] (3) Place the lysis buffer in a centrifuge at ≥12000×g for 5 min, carefully remove the supernatant and transfer it to a sterile centrifuge tube;

[0053] (4) Add 220ul Buffer BL, vortex to mix, and let stand in a 70℃ water bath for 10min.

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

[0055] (6) Insert a DNA minicolumn into a 2ml collection tube. Transfer the solution obtained in the above steps, including a small amount of precipitate, into the adsorption column, centrifuge at 10000×g for 1 min, and discard the filtrate;

[0056] (7) Add 500ul BufferKB, centrifuge at 10000×g for 1min, and discard the filtrate;

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

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

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

[0060] Step 2, Primer Design

[0061] Therefore, the USP16 gene amplifies all its exon regions, and primers are designed for the 2-3000bp uncoding regions upstream and downstream of the gene. Then, pooled sequencing is performed to determine whether SNPs are present.

[0062] The primer design software used in this study was Primer3Plus. Before designing primers, the software settings should be configured according to general principles by clicking "General Settings". The commonly used primer length is 18-27 bp, and no more than 38 bp. Excessive length leads to extension temperatures exceeding 74℃, which is unsuitable for Taq DNA polymerase reactions; therefore, the primer length is set at 18-25 bp. Both excessively high and low GC content are detrimental to the reaction, and the GC content of the upstream and downstream primers should not differ too much; therefore, the GC content is generally set at 40-60%. In addition, other principles must be followed in primer design. For example, the 3' end of the primer should not terminate at the third codon position, as degeneracy at the third codon position is prone to occur, affecting the specificity and efficiency of amplification. The 3' end should not be A; when the 3' end is mismatched, the initiation efficiency varies greatly depending on the base. When the terminal strand is T, the initiation efficiency of the mismatch is greatly reduced. The initiation efficiency of G and C mismatches is between that of A and T, so T is the best choice for the 3' end. Bases should be randomly distributed, etc.

[0063] Following these primer design principles, paste the sequences for which you want to design primers into the dialog box, and click the green "Pickprimer" button in the upper right corner of the Primer3plus online software to design primers. Primer sequence information is shown in Table 2-1.

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

[0065]

[0066]

[0067] Step 3: PCR amplification

[0068] This experiment used 2×Taq-ASPCRMix(+Dye) from Jiangsu Yugong Life Science Technology Co., Ltd. The PCR reaction system was 50 μl. When using it, take 25 μl of 2×Taq-ASPCRMix(+Dye), add appropriate amounts of template and primers, and add ddH2O to make up the volume to achieve a 1× concentration before proceeding with 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 consisted of a mixture of DNA samples from 40 randomly selected Landrace and Large White pigs from different families, covering the entire pedigree, with a DNA concentration of 300-400. All qualified PCR amplification products were sent to BGI Genomics for pooled sequencing. PCR cycling parameters are shown in Table 2-3.

[0072] Table 2-3 PCR Cycling Parameters

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

[0074] The annealing temperature for each primer pair is referenced in the primer information table in Table 5.1. Steps 2 through 4 are set to 35 cycles.

[0075] Step 4: Detection of PCR amplification products

[0076] Prepare a 1% agarose gel. Using 5 μl of FY2000 DNA Marker as a reference, inject it into the first well of the gel. Then, inject 5 μl of the PCR amplification product into the remaining wells sequentially, followed by gel electrophoresis. After electrophoresis, observe whether the gel image is clear, bright, and consistent with the target fragment length. Qualified PCR amplification products are stored at -80℃ and sent to BGI Genomics for sequencing.

[0077] Step 5: SNP site detection

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

[0079] Step 6: Genotyping

[0080] Sequencing revealed six mutation sites in the USP16 gene. 493 ear DNA samples were sent to Beijing Compson Biotechnology Co., Ltd., where time-of-flight mass spectrometry was used to genotype 11 mutations.

[0081] First, the provided DNA sample was tested by electrophoresis to determine if it met the quality standards of the time-of-flight mass spectrometry method. A 2 μL sample of the original solution was taken using a loading buffer (TAKARA) with EB dye and 1 μL of TAE buffer agarose. The results showed that the overall sample quality was very high, meeting the quality standards of the time-of-flight mass spectrometry method. The DNA concentration was then adjusted to an appropriate level.

[0082] Next, based on the SNP site sequence information, the primer design software Assaydesign 3.1 from Sequenom was used to design and synthesize PCR reaction and single-base expansion 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 for PCR amplification, SAP digestion reaction and extension reaction.

[0086] (2) Dilute the reaction product (total 9 μL) 3 times and desalt it using resin. Spot the desalted sample onto the sample target, allow it to crystallize naturally, and then perform mass spectrometry analysis and collect data.

[0087] DNA extraction results from pig ear tissue

[0088] DNA was extracted from ear tissues of Large White and Landrace pigs using a tissue genomic DNA isolation kit. Randomly selected DNA samples were analyzed by 1% agarose gel electrophoresis. The results showed that the selected DNA samples exhibited clear, bright, and uniform bands without significant banding. Nucleic acid detection of all DNA samples revealed that the OD values ​​of all DNA samples were [not specified in the original text]. 260 / 280 The values ​​are all between 1.7 and 2.1, therefore they can be used for subsequent experimental operations, such as... Figure 1 As shown.

[0089] PCR amplification product detection results

[0090] 1. Detection results of USP16 gene PCR amplification products

[0091] Detection of PCR amplification products of the USP16 gene revealed one mutation site in the upstream regulatory region, one mutation site in the intron region, and four mutation sites in the downstream regulatory region. Some PCR amplification product detection results are shown below. Figure 2 As shown, the length of the amplified fragment is basically in line with expectations.

[0092] Population phenotypic analysis

[0093] Phenotypic values ​​of backfat thickness at 100 kg, eye muscle area at 100 kg, and age-related traits at 100 kg were analyzed in 458 pigs, including 238 Large White pigs and 220 Landrace pigs. The results are shown in Table 3-1. For the trait of backfat thickness at 100 kg, the mean value for Large White pigs was 9.06 mm, with a coefficient of variation of 18.15%; the mean value for Landrace pigs was 8.28 mm, with a coefficient of variation of 15.36%. For the trait of eye muscle area at 100 kg, the mean value for Large White pigs was 37.72 cm². 2 The coefficient of variation was 9.11%; the average length of the Landrace pig was 35.55 cm. 2 The coefficient of variation was 10.16%. For the age at 100 kg body weight trait, the mean value for Large White pigs was 166.84 days, with a coefficient of variation of 6.57%; the mean value for Landrace pigs was 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] USP16 gene polymorphism analysis

[0098] Using 22 primer pairs covering 2000-3000 bp of the upstream and downstream regulatory regions and 18 exons of the USP16 gene, DNA samples from 40 randomly selected pig ear tissues from the entire family were amplified by PCR and sequenced in a pooled manner. The resequencing sequences were compared with the original sequences, revealing a total of 6 mutation sites: one in the upstream regulatory region of the USP16 gene (g.192376801C>A), one in an intron region (g.192401821G>A), and four in the downstream regulatory region (g.192407026G>A, g.192407237A>G, g.192408287T>C, and g.192408499C>T).

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

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

[0101] Forty DNA samples randomly selected from the entire family lineage were subjected to pooled sequencing. The sequencing results were analyzed using Seqman and Chromas software to identify 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, which revealed three genotypes at this site: CC, CA, and AA. The sequencing results for the g.192376801C>A site are shown below. Figure 3 As shown in the figure, the scatter plot of the locus typing results is as follows: Figure 4 As shown.

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

[0103] Genotyping using time-of-flight mass spectrometry (TOF-MS) revealed the following results in a population of 237 Large White pigs: 23 individuals (10%) were genotype AA at the g.192376801C>A locus; 96 individuals (40%) were genotype CA; and 118 individuals (50%) were genotype CC. The frequency of allele A was 0.30, and the frequency of allele C was 0.70. In a population of 218 Landrace pigs, 28 individuals (13%) were genotype AA at the g.192376801C>A locus; 99 individuals (45%) were genotype CA; and 91 individuals (42%) were genotype CC. The frequency of allele A was 0.36, and the frequency of allele C was 0.64. Detailed results are shown in Table 3-2.

[0104] Table 3-2 Polymorphism of the 2g.192376801C>A site in the swine population

[0105]

[0106] Table 3-2 shows that the allele frequencies of A and C differ significantly between Large White and Landrace pig populations, with the A allele being more frequent in Landrace pigs than in Large White pigs. Hardy-Weinberg equilibrium calculations indicate that the g.192376801C>A locus conforms to Hardy-Weinberg equilibrium in both Large White and Landrace pig populations (P>0.05).

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

[0108] Polymorphism at the g.192376801C>A locus was detected in Large White and Landrace pigs, and the number of individuals with each of the three genotypes was counted. Association analyses of the g.192376801C>A locus polymorphism with backfat thickness at 100 kg, eye muscle area at 100 kg, and age at 100 kg body weight in both Large White and Landrace populations are shown in Tables 3-3 and 3-4.

[0109] Table 3-3 Association analysis of polymorphism at the C>A site in Large White pig g.192376801 with traits

[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, representing significance; ** indicates P < 0.01, representing highly significant.

[0112] Table 3-4 Association Analysis of Polymorphism at the C>A Locus in Landrace Pigs (g.192376801) with Traits

[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, representing significance; ** indicates P < 0.01, representing highly significant.

[0115] Association analysis of the g.192376801C>A locus gene polymorphism with the three traits showed that, in the Large White pig population, the association between the g.192376801C>A locus and age at 100kg body weight, backfat thickness at 100kg body weight, and eye muscle area at 100kg body weight was not significant; similarly, in the Landrace pig population, the association between the g.192376801C>A locus and age at 100kg body weight, backfat thickness at 100kg body weight, and eye muscle area at 100kg body weight was also not significant.

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

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

[0118] Forty DNA samples randomly selected from the entire family lineage were subjected to pooled sequencing. The sequencing results were analyzed using Seqman and Chromas software to identify 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, which revealed three genotypes at this site: GG, GA, and AA. The sequencing results for the g.192401821G>A site are shown below. Figure 5 As shown in the figure, the scatter plot of the locus typing results is as follows: Figure 6 As shown.

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

[0120] Genotyping using time-of-flight mass spectrometry (TOF-MS) revealed the following results in a population of 237 Large White pigs: 40 individuals (17%) were AA type at the g.192401821G>A locus; 103 individuals (43%) were GA type; and 94 individuals (40%) were GG type. The frequency of allele A was 0.39, and the frequency of allele G was 0.61. In a population of 220 Landrace pigs, the AA type at the g.192401821G>A locus was 82 individuals (37%); 88 individuals (40%) were GA type; and 50 individuals (23%) were GG type. The frequency of allele A was 0.57, and the frequency of allele C was 0.43. Detailed results are shown in Tables 3-5.

[0121] Table 3-5 Polymorphism of the g.192401821G>A site in the swine population

[0122]

[0123]

[0124] Table 3-5 shows that the allele frequencies of A and G differ significantly in the Large White pig population, but less so in the Landrace pig population. The frequency of the A allele is higher in Landrace pigs than in Large White pigs. Hardy-Weinberg equilibrium calculations indicate that the g.192401821G>A locus conforms to Hardy-Weinberg equilibrium in the Large White pig population (P>0.05), but deviates from Hardy-Weinberg equilibrium in the Landrace pig population (P<0.05).

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

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

[0127] Table 3-6 Association Analysis of 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, representing significance; ** indicates P < 0.01, representing highly significant.

[0130] Table 3-7 Association Analysis of G.192401821G>A Site 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, representing significance; ** indicates P < 0.01, representing highly significant.

[0133] Association analysis of the g.192401821G>A locus gene polymorphism with the three traits revealed the following: In the Large White pig population, the g.192401821G>A locus was not significantly associated with the trait of age at 100 kg body weight; however, the g.192401821G>A locus was significantly associated with the trait of backfat thickness at 100 kg body weight (P<0.05), with the dominant allele being G. The backfat thickness at 100 kg was significantly less in the GG and GA types than in the AA type (P<0.05); and the g.192401821G>A locus was significantly associated with the trait of eye muscle area at 100 kg body weight (P<0.05). In the Landrace pig population, the g.192401821G>A locus was not significantly associated with any of the three traits: age at 100 kg body weight, backfat thickness at 100 kg body weight, and eye muscle area at 100 kg body weight.

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

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

[0136] Forty DNA samples randomly selected from the entire family lineage were subjected to pooled sequencing. The sequencing results were analyzed using Seqman and Chromas software to identify the g.192407026G>A mutation site located downstream of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, which revealed three genotypes at this site: GG, GA, and AA. The sequencing results for the g.192407026G>A site are shown below. Figure 7 As shown, the scatter plot of the locus typing results Figure 8 As shown in the figure.

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

[0138] Genotyping using time-of-flight mass spectrometry (TOF-MS) revealed the following results in a population of 256 Large White pigs: 41 individuals (16%) were genotype AA at the g.192407026G>A locus; 102 individuals (40%) were genotype GA; and 113 individuals (44%) were genotype GG. The frequency of allele A was 0.36, and the frequency of allele G was 0.64. In a population of 235 Landrace pigs, the same genotype was found in a population of 235 Landrace pigs: 83 individuals (35%) were genotype AA at the g.192407026G>A locus; 95 individuals (40%) were genotype GA; and 57 individuals (24%) were genotype GG. The frequency of allele A was 0.56, and the frequency of allele C was 0.44. Detailed results are shown in Table 3-8.

[0139] Table 3-8 Polymorphism of the g.192407026G>A site in the swine population

[0140]

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

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

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

[0144] Table 3-9 Association Analysis of Polymorphism at the G.192407026G>A Site in Large White Pigs with Traits

[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, representing significance; ** indicates P < 0.01, representing highly significant.

[0147] Table 3-10 Association Analysis of Polymorphism at the G>A site in Landrace pigs (g.192407026) with Traits

[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, representing significance; ** indicates P < 0.01, representing highly significant.

[0150] Association analysis of the g.192407026G>A locus gene polymorphism with the three traits revealed the following: In the Large White pig population, the g.192407026G>A locus was not significantly associated with the trait of age at 100 kg body weight; the g.192407026G>A locus was significantly associated with the trait of backfat thickness at 100 kg body weight (P<0.05), with the dominant allele being G, and the backfat thickness at 100 kg in the GG and GA types was significantly smaller than that in the AA type (P<0.05); the g.192401821G>A locus was significantly associated with the trait of eye muscle area at 100 kg body weight (P<0.05). In the Landrace pig population, the g.192401821G>A locus was not significantly associated with any of the three traits: age at 100 kg body weight, backfat thickness at 100 kg body weight, and eye muscle area at 100 kg body weight.

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

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

[0153] Forty DNA samples randomly selected from the entire family lineage were subjected to pooled sequencing. The sequencing peaks were analyzed using Seqman and Chromas software to identify the g.192407237G>A mutation site located downstream of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, which revealed three genotypes at this site: GG, GA, and AA. The sequencing results for the g.192407237G>A site are shown below. Figure 9 As shown in the figure, the scatter plot of the locus typing results is as follows: Figure 10 As shown.

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

[0155] Genotyping using time-of-flight mass spectrometry (TOF-MS) revealed the following results in a population of 236 Large White pigs: 94 individuals (40%) were AA type at the g.192407237A>G locus; 102 individuals (43%) were GA type; and 40 individuals (17%) were GG type. The frequency of allele A was 0.61, and the frequency of allele G was 0.39. In a population of 220 Landrace pigs, the AA type at the g.192407237A>G locus was 51 individuals (23%); 87 individuals (40%) were GA type; and 82 individuals (37%) were GG type. The frequency of allele A was 0.43, and the frequency of allele C was 0.57. Detailed results are shown in Table 3-11.

[0156] Table 3-11 Polymorphism of the 192407237G>A site in the swine population

[0157]

[0158] Table 3-11 shows that the allele frequencies of A and G differ significantly in the Large White pig population but less so in the Landrace pig population. The frequency of the A allele is higher in Large White pigs than in Landrace pigs. Hardy-Weinberg equilibrium calculations indicate that the g.192407237A>G locus conforms to Hardy-Weinberg equilibrium in the Large White pig population (P>0.05) but deviates from Hardy-Weinberg equilibrium in the Landrace pig population (P<0.05).

[0159] 4.3 Association analysis of polymorphism at site g.192407237A>G with traits

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

[0161] Table 3-12 Association Analysis of Polymorphism at the G.192407237G>A Site in Large White Pigs with Traits

[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, representing significance; ** indicates P < 0.01, representing highly significant.

[0164] Table 3-13 Association Analysis of Polymorphism at the G>A Locus in Landrace Pigs (g.192407026) with Traits

[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, representing significance; ** indicates P < 0.01, representing highly significant.

[0167] Association analysis of the g.192407237A>G locus gene polymorphism with the three traits revealed the following: In the Large White pig population, the g.192407237A>G locus was not significantly associated with the trait of age at 100 kg body weight; however, the g.192407237A>G locus was significantly associated with the trait of backfat thickness at 100 kg body weight (P<0.05), with the dominant allele being A, and the backfat thickness at 100 kg for both the AA and GA types being significantly smaller than that for the GG type (P<0.05); the g.192407237A>G locus was also significantly associated with the trait of eye muscle area at 100 kg body weight (P<0.05). In the Landrace pig population, the g.192407237A>G locus was not significantly associated with any of the three traits: age at 100 kg body weight, backfat thickness at 100 kg body weight, and eye muscle area at 100 kg body weight.

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

[0169] 5.1 Sequencing and genotyping results analysis of the g.192408287T>C site

[0170] Forty DNA samples randomly selected from the entire family lineage were subjected to pooled sequencing. The sequencing results were analyzed using Seqman and Chromas software to identify the g.192408287T>C mutation site located downstream of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, which revealed three genotypes at this site: CC, CT, and TT. The sequencing results for the g.192408287T>C site are shown below. Figure 11 As shown in the figure, the scatter plot of the locus typing results is as follows: Figure 12 As shown.

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

[0172] Genotyping using time-of-flight mass spectrometry (TOF-MS) revealed the following results in a population of 238 Large White pigs: 40 individuals (17%) were classified as CC at the g.192408287T>C locus; 104 individuals (44%) as CT; and 94 individuals (39%) as TT. The frequency of allele C was 0.39, and the frequency of allele T was 0.61. In a population of 220 Landrace pigs, 82 individuals (37%) were classified as CC at the g.192408287T>C locus; 88 individuals (40%) as CT; and 50 individuals (23%) as TT. The frequency of allele C was 0.57, and the frequency of allele T was 0.43. Detailed results are shown in Table 3-14.

[0173] Table 3-14 Polymorphism of the G.192408287G>A site in the swine population

[0174]

[0175] Table 3-14 shows that the allele frequencies of C and T differ significantly in the Large White pig population but less so in the Landrace pig population, with the C allele being more frequent in Landrace pigs than in Large White pigs. Hardy-Weinberg equilibrium calculations indicate that the g.192408287T>C locus conforms to Hardy-Weinberg equilibrium in the Large White pig population (P>0.05) but deviates from Hardy-Weinberg equilibrium in the Landrace pig population (P<0.05).

[0176] 5.3 Association analysis of polymorphism at the g.192408287T>C site with traits

[0177] Polymorphism at the g.192408287T>C locus was detected in Large White and Landrace pig populations, and the number of individuals with each of the three genotypes was counted. Association analyses of the g.192408287T>C locus polymorphism with backfat thickness at 100 kg, eye muscle area at 100 kg, and age at 100 kg body weight in both Large White and Landrace pig populations are shown in Tables 3-15 and 3-16.

[0178] Table 3-15 Association Analysis of Polymorphism at the T>C Locus in Large White Pigs (g. 192408287) with Traits

[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, representing significance; ** indicates P < 0.01, representing highly significant.

[0181] Table 3-16 Association Analysis of Landrace Pig g.192408287T>C Locus Polymorphism with Traits

[0182]

[0183] Note: * indicates P < 0.05, representing significance; ** indicates P < 0.01, representing highly significant.

[0184] Association analysis of the g.192408287T>C locus gene polymorphism with the three traits revealed the following: In the Large White pig population, the g.192408287T>C locus was not significantly associated with the trait of age at 100 kg body weight; however, it was significantly associated with the trait of backfat thickness at 100 kg body weight (P<0.05), with the dominant allele being T, and the backfat thickness at 100 kg for both the TT and CT types being significantly smaller than that for the CC type (P<0.05); and it was also significantly associated with the trait of eye muscle area at 100 kg body weight (P<0.05). In the Landrace pig population, the g.192408287T>C locus was not significantly associated with any of the three traits: age at 100 kg body weight, backfat thickness at 100 kg body weight, and eye muscle area at 100 kg body weight.

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

[0186] 6.1 Sequencing and genotyping results analysis of the g.192408499C>T site

[0187] Forty DNA samples randomly selected from the entire family lineage were subjected to pooled sequencing. The sequencing results were analyzed using Seqman and Chromas software to identify the g.192408499C>T mutation site located downstream of the USP16 gene. All DNA samples were then sent to Beijing Compson Agricultural Technology Co., Ltd. for genotyping, which revealed three genotypes at this site: CC, CT, and TT. The sequencing results for the g.192408499C>T site are shown below. Figure 13 As shown in the figure, the scatter plot of the locus typing results is as follows: Figure 14 As shown.

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

[0189] Genotyping using time-of-flight mass spectrometry (TOF-MS) revealed the following results in a population of 238 Large White pigs: 94 individuals (39%) with the CC genotype at the g.192408499C>T locus; 102 individuals (43%) with the CT genotype; and 42 individuals (18%) with the TT genotype. The frequency of allele C was 0.61, and the frequency of allele T was 0.39. In a population of 220 Landrace pigs, the same genotype was also identified: 50 individuals (23%) with the CC genotype at the g.192408499C>T locus; 88 individuals (40%) with the CT genotype; and 82 individuals (37%) with the TT genotype. The frequency of allele C was 0.43, and the frequency of allele T was 0.57. Detailed results are shown in Table 3-17.

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

[0191]

[0192] Table 3-14 shows that the allele frequencies of C and T differ significantly in the Large White pig population but less so in the Landrace pig population, with the C allele being more frequent in Large White pigs than in Landrace pigs. Hardy-Weinberg equilibrium calculations indicate that the g.192408499C>T locus conforms to Hardy-Weinberg equilibrium in the Large White pig population (P>0.05) but deviates from Hardy-Weinberg equilibrium in the Landrace pig population (P<0.05).

[0193] 6.3 Association analysis of polymorphism at the g.192408499C>T site with traits

[0194] Polymorphism at the g.192408499C>T locus was detected in Large White and Landrace pig populations, and the number of individuals with each of the three genotypes was counted. Association analyses of the g.192408499C>T locus polymorphism with backfat thickness at 100 kg, eye muscle area at 100 kg, and age at 100 kg body weight in both Large White and Landrace pig populations are shown in Tables 3-18 and 3-19.

[0195] Table 3-18 Association Analysis of Polymorphism at the g.192408499C>T Site in Large White Pigs with Traits

[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, representing significance; ** indicates p < 0.01, representing highly significant.

[0198] Table 3-19 Association Analysis of Polymorphism at the g.192408499C>T Site in Landrace Pigs with Traits

[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, representing significance; ** indicates p < 0.01, representing highly significant.

[0201] Association analysis of the g.192408499C>T locus gene polymorphism with the three traits revealed the following: In the Large White pig population, the g.192408499C>T locus was not significantly associated with the trait of age at 100 kg body weight; however, it was significantly associated with the trait of backfat thickness at 100 kg body weight (p<0.05), with the dominant allele being C, and the backfat thickness at 100 kg for both CC and CT genotypes being significantly smaller than that for the TT genotype (p<0.05); and it was also significantly associated with the trait of eye muscle area at 100 kg body weight (p<0.05). In the Landrace pig population, the g.192408499C>T locus was not significantly associated with any of the three traits: age at 100 kg body weight, backfat thickness at 100 kg body weight, and eye muscle area at 100 kg body weight.

[0202] Association analysis of USP16 gene SNP sites with traits

[0203] By using pooled sequencing, the resequencing sequence was compared with the original sequence, and a total of 6 SNPs were found in the USP16 gene that may be related to related growth traits.

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

[0205] For the g.192376801C>A site located in the upstream regulatory region of the USP16 gene, statistical analysis of the three genotypes of this site and association analysis with traits revealed that the g.192376801C>A site was not significantly associated with any growth trait in 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 trait

[0207] For the g.192401821G>A locus located in the intron region of the USP16 gene, statistical analysis of the three genotypes of this locus and its association with traits revealed a significant association between the g.192401821G>A locus and the 100kg backfat thickness trait in Large White pigs (P<0.05), with the dominant allele being G. The GG and GA genotypes showed significantly less backfat thickness at 100kg than the AA genotype (P<0.05). Simultaneously, this locus was also significantly associated with the 100kg eye muscle area trait in Large White pigs (P<0.05). While the mean eye muscle area at 100kg was greater for the AA genotype than for the GA and GG genotypes, the difference was not statistically significant, possibly due to the limited sample size in this study. Further research with a larger sample size could validate this finding. This locus was not significantly associated with age at 100kg body weight. In the Landrace pig population, the g.192401821G>A locus was not significantly associated with any growth trait.

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

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

[0210] For the g.192407026G>A locus located downstream of the USP16 gene regulatory region, statistical analysis of the three genotypes of this locus and association analysis with traits revealed a significant association between the USP16 gene g.192407026G>A locus and the trait of backfat thickness at 100 kg in Large White pigs (P<0.05). The dominant allele was G, and the backfat thickness at 100 kg was significantly smaller in the GG and GA genotypes than in the AA genotype (P<0.05). Simultaneously, this locus was also significantly associated with the trait of eye muscle area at 100 kg in Large White pigs (P<0.05). While the mean eye muscle area at 100 kg was larger in the AA genotype than in the GA and GG genotypes, the difference was not statistically significant, possibly due to the limited sample size in this study. Further research with a larger sample size could validate this finding. This locus was not significantly associated with age at 100 kg body weight. In the Landrace pig population, the g.192407026G>A locus was not significantly associated with any growth trait.

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

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

[0213] For the g.192407237A>G locus located downstream of the USP16 gene regulatory region, statistical analysis of the three genotypes of this locus and its association with traits revealed a significant association between the g.192407237A>G locus and the trait of backfat thickness at 100 kg in Large White pigs (P<0.05). The dominant allele was A, and the backfat thickness at 100 kg was significantly smaller for the AA and GA genotypes than for the GG genotype (P<0.05). Simultaneously, this locus was also significantly associated with the trait of eye muscle area at 100 kg in Large White pigs (P<0.05). While the mean eye muscle area at 100 kg was larger for the GG genotype than for the AA and GA genotypes, the difference was not statistically significant, possibly due to the limited sample size in this study. Further research with a larger sample size could validate this finding. This locus was not significantly associated with age at 100 kg body weight. In the Landrace pig population, the g.192407237A>G site was not significantly associated with any growth trait.

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

[0215] Association analysis between g.192408287T>C site and trait

[0216] For the g.192408287T>C locus located downstream of the USP16 gene regulatory region, statistical analysis of the three genotypes of this locus and its association with traits revealed a significant association between the g.192408287T>C locus and the trait of backfat thickness at 100 kg in Large White pigs (P<0.05). The dominant allele was T, and the backfat thickness at 100 kg was significantly smaller in the TT and CT genotypes than in the CC genotype (P<0.05). Simultaneously, this locus was also significantly associated with the trait of eye muscle area at 100 kg in Large White pigs (P<0.05). While the mean eye muscle area at 100 kg was larger in the CC genotype than in the CT and TT genotypes, the difference was not statistically significant, possibly due to the limited sample size in this study. Further research with a larger sample size could validate this finding. The association between this locus and age at 100 kg body weight was not significant. In the Landrace pig population, the g.192408287T>C locus was not significantly associated with any growth trait.

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

[0218] Association analysis between g.192408499C>T site and trait

[0219] For the g.192408499C>T locus located downstream of the USP16 gene regulatory region, statistical analysis of the three genotypes of this locus and its association with traits revealed a significant association between the g.192408499C>T locus and the Large White pig population (P<0.05). The dominant allele was C, and the backfat thickness at 100kg was significantly smaller in the CC and CT genotypes than in the TT genotype (P<0.05). Simultaneously, this locus was also significantly associated with the trait of eye muscle area at 100kg in the Large White pig population (P<0.05). While the mean eye muscle area at 100kg was larger in the TT genotype than in the CC and CT genotypes, the difference was not statistically significant, possibly due to the limited sample size in this study. Further research with a larger sample size could validate this finding. This locus was not significantly associated with age at 100kg body weight. In the Landrace pig population, the g.192408499C>T site was not significantly associated with any growth trait.

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

[0221] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for assessing backfat in Large White pigs based on the USP16 gene, characterized in that, Identify the SNP molecular markers of the Large White pig and determine whether the SNP molecular markers are one of the following SNP molecular markers: The g.192401821G>A site is located in the intron region of the USP16 gene; the backfat thickness at 100kg is significantly less in the GG and GA types than in the AA type; The g.192407026G>A site is located in the downstream regulatory region of the USP16 gene; the backfat thickness at 100kg is significantly smaller for GG and GA types than for AA type; The g.192407237A>G site is located in the downstream regulatory region of the USP16 gene; the backfat thickness at 100kg is significantly smaller for both AA and GA types than for the GG type. The g.192408287T>C site is located in the downstream regulatory region of the USP16 gene; the backfat thickness at 100kg is significantly smaller for both the TT and CT types than for the CC type; The g.192408499C>T site is located in the downstream regulatory region of the USP16 gene; the backfat thickness at 100kg for both CC and CT types is significantly smaller than that for the TT type.

2. The application of primer pair combinations in identifying the backfat thickness trait of Large White pigs, characterized in that, The primer pair combination is used to detect SNP molecular markers in Large White pigs, wherein: The g.192401821G>A site located in the intron region of the USP16 gene was detected. The nucleotide sequence of its forward primer is shown in SEQ ID NO.1 of the sequence listing, and the nucleotide sequence of its reverse primer is shown in SEQ ID NO.2 of the sequence listing. The g.192407026G>A site located in the downstream regulatory region of the USP16 gene was detected. The nucleotide sequence of its forward primer is shown in SEQ ID NO.3 of the sequence listing, and the nucleotide sequence of its reverse primer is shown in SEQ ID NO.4 of the sequence listing. The g.192407237A>G site located in the downstream regulatory region of the USP16 gene was detected. The nucleotide sequence of its forward primer is shown in SEQ ID NO.5 of the sequence listing, and the nucleotide sequence of its reverse primer is shown in SEQ ID NO.6 of the sequence listing. The g.192408287T>C site located in the downstream regulatory region of the USP16 gene was detected. The nucleotide sequence of its forward primer is shown in SEQ ID NO.7 of the sequence listing, and the nucleotide sequence of its reverse primer is shown in SEQ ID NO.8 of the sequence listing. The g.192408499C>T site located in the downstream regulatory region of the USP16 gene was detected. The nucleotide sequence of its forward primer is shown in SEQ ID NO.9 of the sequence listing, and the nucleotide sequence of its reverse primer is shown in SEQ ID NO.10 of the sequence listing.

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