SNP (Single Nucleotide Polymorphism) molecular marker associated with correction of 100kg backfat thickness character of pig and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker associated with correction of a 100kg backfat thickness character of a pig and application of the SNP molecular marker. The SNP molecular marker is located on a pig chromosome 2 and contains six SNP molecular marker sites. Genetic typing is carried out by utilizing a gene chip technology, whole genome association analysis is carried out in combination with the 100kg backfat thickness correction character of the landrace, six SNP molecular markers obviously related to the character are screened out, and a new marker resource is provided for molecular breeding of the growth character of the boar.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular markers, and particularly relates to a SNP molecular marker associated with the pig backfat thickness trait of 100 kg and application. BACKGROUND

[0002] Backfat thickness (BFT) is an important indicator for measuring the fat deposition capacity and body composition structure of pigs, and is one of the economic traits with the highest attention in pig breeding. Since lean meat rate is difficult to measure directly and is significantly negatively correlated with BFT, BFT is often used as an indirect measurement indicator of lean meat rate. In addition, due to the high similarity between pigs and humans in physiological characteristics such as fat metabolism, research on the genetic basis of the backfat thickness trait not only helps to improve pork quality and lean meat rate, but also provides reliable animal model support for the mechanism research of human obesity and other complex traits.

[0003] In order to reveal the genetic basis of important economic traits of pigs, a large number of quantitative trait locus (QTL) mapping studies have been carried out in the past few decades. As of April 2021, the pig QTL database has included 33,143 QTLs, covering 699 different traits, of which 3,289 are related to fat deposition. In the study of backfat thickness traits, scientists have identified a number of key candidate genes, including IGF2 which regulates growth and development, MC4R which is related to appetite and energy metabolism, and LEPR which is involved in fat metabolism. These achievements provide an important basis for in-depth understanding of the genetic mechanism of backfat thickness, and also provide theoretical support for the development of related breeding strategies.

[0004] With the continuous progress of high-density single nucleotide polymorphism (SNP) chips and high-throughput genotyping technologies, genome-wide association analysis (GWAS) has gradually become an important means for analyzing the genetic basis of complex traits. Compared with traditional linkage analysis methods, GWAS has higher mapping accuracy and can efficiently identify genetic variations significantly associated with target traits in large-scale populations. In recent years, significant SNP sites discovered based on GWAS not only help to perfect the genetic map of backfat thickness traits, but also can be applied as molecular markers in molecular assisted selection (MAS) and genomic selection (GS), showing broad prospects in improving the efficiency of pig breeding.

[0005] Although many studies have made progress in the genetic basis analysis of pig backfat thickness traits, there is still a lack of stable and effective molecular marker resources for controlling the backfat thickness of Changbai pigs under the condition of 100 kg body weight, which to some extent restricts the precise breeding process of this trait. SUMMARY

[0006] The present application aims at overcoming the deficiencies of the prior art, and provides a SNP molecular marker associated with the 100kg backfat thickness trait of pigs and an application thereof.

[0007] To achieve the above-mentioned object, the technical scheme of the present application is as follows: The present application provides a SNP molecular marker associated with the 100kg backfat thickness trait of pigs, wherein the SNP molecular marker is located on chromosome 2 of pigs, and contains six SNP molecular marker sites. The first SNP molecular marker site 2:1280617 is located at the 1280617th base of chromosome 2 of pigs, and the polymorphic site is A or G; The second SNP molecular marker site 2:1280654 is located at the 1280654th base of chromosome 2 of pigs, and the polymorphic site is T or G; The third SNP molecular marker site 2:1283873 is located at the 1283873th base of chromosome 2 of pigs, and the polymorphic site is C or A; The fourth SNP molecular marker site 2:1283877 is located at the 1283877th base of chromosome 2 of pigs, and the polymorphic site is C or A; The fifth SNP molecular marker site 2:1284016 is located at the 1284016th base of chromosome 2 of pigs, and the polymorphic site is G or A; The sixth SNP molecular marker site 2:1284026 is located at the 1284026th base of chromosome 2 of pigs, and the polymorphic site is T or C.

[0008] The present application also provides an application of the SNP molecular marker in identifying the backfat thickness trait of pigs and screening pig breeds with low backfat thickness.

[0009] The present application also provides a primer pair for obtaining the SNP molecular marker, and the nucleotide sequence of the primer pair for obtaining the sequence containing the first SNP molecular marker site and the second SNP molecular marker site is as follows: Forward primer 1: CTGAGGCTGTGGGGGGGT; Reverse primer 1: GGACCTGGGGTATGGAGCC; The nucleotide sequence of the primer pair for obtaining the sequence containing the third SNP molecular marker site, the fourth SNP molecular marker site, the fifth SNP molecular marker site and the sixth SNP molecular marker site is as follows: Forward primer 2: TGGCCCAGGGCACACACC; Reverse primer 2: GCTGGGGGGCCACGACCT.

[0010] Further, the nucleotide sequence amplified by the primer pair Forward primer 1 and Reverse primer 1 is shown in SEQ ID NO: 1, and the first SNP molecular marker site is located at the 151th base in the sequence, and the second SNP molecular marker site is located at the 188th base in the sequence; The nucleotide sequence amplified by the primer pair Forward primer 2 and Reverse primer 2 is shown in SEQ ID NO: 2, and the third SNP molecular marker site is located at the 151th base in the sequence, the fourth SNP molecular marker site is located at the 155th base in the sequence, the fifth SNP molecular marker site is located at the 294th base in the sequence, and the sixth SNP molecular marker site is located at the 304th base in the sequence; When the first SNP molecular marker site is GG genotype, the second SNP molecular marker site is GG genotype, the third SNP molecular marker site is AA genotype, the fourth SNP molecular marker site is AA genotype, the fifth SNP molecular marker site is AA genotype, and the sixth SNP molecular marker site is CC genotype, the backfat thickness of the pig is lower than that of the pig with other genotypes.

[0011] The application also provides a detection method of the SNP molecular marker associated with the correction of the 100kg backfat thickness trait of a pig, which adopts the primer pair amplification, sequencing comparison and detection.

[0012] The application also provides the application of the primer pair in identifying the backfat thickness trait of a pig, screening a pig breed with low backfat thickness and the genetic breeding of the backfat thickness of a pig.

[0013] The application also provides a kit for detecting the SNP molecular marker, which comprises the primer pair.

[0014] The application also provides a method for identifying the backfat thickness trait of a pig by using the kit, which comprises the following steps: Detecting the SNP molecular marker site of the SNP molecular marker on pig chromosome 2, and determining the pig backfat thickness trait based on the SNP molecular marker site; When the first SNP molecular marker site is a GG genotype, the second SNP molecular marker site is a GG genotype, the third SNP molecular marker site is an AA genotype, the fourth SNP molecular marker site is an AA genotype, the fifth SNP molecular marker site is an AA genotype, and the sixth SNP molecular marker site is a CC genotype, the back fat thickness of the pig is lower than the back fat thickness of pigs with other genotypes.

[0015] Furthermore, the detection method comprises the following steps: (1) Extract DNA from the pig to be tested; (2) performing PCR amplification on the extracted DNA using the primer pairs in the kit; (3) Sequencing and analyzing the PCR amplification products to obtain sequencing results; (4) Based on the sequencing results, the genotype is obtained. When the first SNP molecular marker site is a GG genotype, the second SNP molecular marker site is a GG genotype, the third SNP molecular marker site is an AA genotype, the fourth SNP molecular marker site is an AA genotype, the fifth SNP molecular marker site is an AA genotype, and the sixth SNP molecular marker site is a CC genotype, the back fat thickness of the pig is lower than the back fat thickness of pigs with other genotypes.

[0016] The present invention also provides an application of the kit in identifying pig backfat thickness traits, screening pig breeds with low backfat thickness, and pig backfat thickness genetic breeding.

[0017] Beneficial effects of the present invention: Based on genotype data from an 80K high-density gene chip, this study conducted a genome-wide association study of backfat thickness in Landrace pigs at a corrected weight of 100 kg. The analysis identified six SNP molecular markers located in the LSP1 gene region on chromosome 2 that were significantly associated with this trait. These SNP molecular markers can be used as candidate markers for marker-assisted breeding of Landrace pigs for backfat thickness, providing a theoretical basis and technical support for genetic improvement of this breed.

[0018] Compared with the traditional phenotypic selection method, the application realizes efficient and accurate screening of individuals with thinner back fat through molecular marker technology, providing a reliable molecular breeding method for the breeding of high-quality breeding pigs. Breeding pigs with thinner back fat when corrected to 100 kg body weight means less fat deposition and higher lean meat rate, which meets the current efficient and cost-effective breeding goals. This feature is conducive to improving pork quality, optimizing feed utilization efficiency, and reducing breeding costs while ensuring yield. The technology has broad application prospects in breeding practice, not only helping to improve the economic benefits of pig breeding enterprises, but also helping to promote the progress of pig breeding technology in China and accelerate the breeding process of excellent germplasm. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a Manhattan plot for GWAS analysis; Figure 2 is a QQ plot for GWAS analysis; Figure 3 is a luciferase activity result plot for GG and AT haplotypes; Figure 4 is a luciferase activity result plot for AAAC and CCGT haplotypes. DETAILED DESCRIPTION

[0020] The application will be further described in detail below with specific examples, so that those skilled in the art can understand.

[0021] Example 1 Genotyping detection and data processing 1. DNA extraction (1) Collect the ear tissue of 7149 Landrace pigs, crush the collected ear tissue in a glass homogenizer, add an equal volume of cell lysis buffer prepared from 100 mmol / L Tris saturated phenol, 500 mmol / L disodium EDTA, 20 mmol / L sodium chloride (NaCL), 10% sodium dodecyl sulfate (SDS), and 20 μg / mL trypsin, mix well, and place in a 65℃ constant temperature water bath for 30 min; (2) Slowly shake the centrifuge tube containing the above solution for 15 min, centrifuge in a centrifuge at 12000 rpm for 5 min, and then take the supernatant into another centrifuge tube; (3) Add an equal volume of phenol-chloroform-isoamyl alcohol mixed solution (volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), shake well, centrifuge in a centrifuge at 12000 rpm for 5 min, and then take the supernatant into another centrifuge tube; (4) Add an equal volume of phenol chloroform isoamyl alcohol mixed solution (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), shake and mix, place in the centrifuge at 12000 rpm for 10 min, take the supernatant into another centrifuge tube; (5) Add 2 times the volume of pre-cooled anhydrous ethanol, stand until the ethanol evaporates, pick out the DNA precipitate and dissolve the DNA with ultrapure water; (6) Use the DNA concentration detector and agarose gel electrophoresis to detect the quality of the DNA, and obtain the DNA of all the Landrace pigs.

[0022] 2. Genotyping detection (1) According to the above extracted DNA, use pig 80k functional site gene chip (liquid chip) for genotyping, which contains more than 180000 SNP molecular marker sites; (2) Randomly select 140 samples from the above DNA for whole genome resequencing data, use fastpv0.23.2 software for quality control processing, use bwa v0.7.18 software for alignment, and finally use GATK v4.5 software for genotyping processing of sequencing data.

[0023] 3. Genotype filling Use Beagle v5.4 software, and use the above 140 Landrace pigs whole genome resequencing data as a reference group to fill the chip data, and select SNP molecular marker sites with filling accuracy higher than 0.8 for subsequent analysis.

[0024] Example 2 SNP molecular marker sites and Landrace pigs whole genome association analysis of corrected 100kg backfat thickness trait 1. Phenotype definition Corrected 100kg backfat thickness: The vertical distance from the skin of the back 5cm from the middle line between the 3rd and 4th ribs to the longest muscle membrane of the back of the pig is the actual value at the end of the measurement, which is calculated by the correction formula.

[0025] Reference NY_T 822-2019 Swine Production Performance Measurement Procedure (Industry Standard) for filtering of phenotype data: retain individuals with corrected 100kg backfat thickness phenotype value greater than 5mm at the end of the measurement.

[0026] 2. SNP molecular marker quality control According to the SNP molecular marker sites with filling accuracy higher than 0.8 screened out after filling of genotypes in Example 1, the SNP molecular marker sites after filling were respectively subjected to quality control using PLINK v1.9 software, and the SNP molecular marker sites with SNP missing genotype proportion <10%, individual missing genotype proportion <10%, and SNP minimum allele frequency (MAF) >5% were reserved, and finally 5923 samples and 13464392 SNP molecular marker sites were used for whole genome association analysis.

[0027] 3. Whole genome association analysis of Changbai pig corrected 100 kg backfat thickness trait The experimental pig population used in the whole genome association analysis of this example was Changbai pig, including 5923 pigs. According to the recorded information of the corrected 100 kg backfat thickness of the experimental pig population, the method based on mixed linear model was used, and the MLM analysis module in the rMVP software package was used to perform whole genome association analysis of SNP molecular markers and Changbai corrected 100 kg backfat thickness trait. The specific model is as follows: Y = Xβ + Sa + Zu + e; Wherein, Y: phenotype vector; X: fixed effect design matrix (including gender, determination season); β: fixed effect parameter vector; S: marker effect matrix (including SNP genotype data); a: marker effect parameter vector (corresponding to the effect of each SNP in the SNP genotype matrix S); Z: random effect design matrix (including kinship matrix); u: random effect parameter vector, u ~ N(0, Kσu2), N is a normal distribution, K is a kinship matrix, and σu2 is the variance of random effect; e: error vector, N~(0, Iσe2), wherein I is an identity matrix, and σe2 is the variance of residual.

[0028] The Manhattan plot and QQ plot were automatically generated by the rMVP software package according to the data, and the Bonferroni method was used to determine the association significance threshold, and the threshold was 0.05 / number of effective SNPs.

[0029] The results are shown in Figure 1 A total of 1564 SNPs exceeded the set significance threshold line. Among them, significant peaks appeared on chromosome 2 and chromosome 12. Based on this, 6 SNPs sites on chromosome 2 with stronger signal were selected as the target for subsequent analysis.

[0030] The results are shown in Figure 2As shown in the figure, by comparing the quantiles of the probability distribution of the actual -log(P) value and the expected -log(P) value, the two probability distributions are compared to further judge the reliability of the GWAS results. 10 The P-values ​​are consistent with expectations, with significant deviations only at high quantiles, suggesting a true association signal. The overall graph shows no systematic upward trend, indicating that the GWAS analysis results are reliable.

[0031] Table 1 Candidate SNPs for correcting 100 kg backfat thickness trait in Landrace pigs identified based on GWAS As can be seen from Table 1, this example screened out 6 SNP molecular marker sites that were significantly associated with the 100 kg backfat thickness trait of Changbai, all of which were on chromosome 2, namely 2:1280617, 2:1280654, 2:1283873, 2:1283877, 2:1284016 and 2:1284026.

[0032] The number of individuals with different genotypes at each SNP molecular marker site and the corresponding genotype-phenotype means were further counted.

[0033] Table 2 Genotype frequencies of candidate SNPs in Landrace pigs and genotype-corrected 100 kg backfat thickness phenotype means Note: The values ​​in brackets are the mean ± standard deviation of the backfat thickness adjusted for 100 kg for the genotype pigs.

[0034] The results are shown in Table 2. For SNP molecular marker site 2:1280617, when the genotype is GG, the Landrace pig's 100kg backfat thickness is thinner than that of individuals with other genotypes; for SNP molecular marker site 2:1280654, when the genotype is GG, the Landrace pig's 100kg backfat thickness is thinner than that of individuals with other genotypes; for SNP molecular marker site 2:1283873, when the genotype is AA, the Landrace pig's 100kg backfat thickness is thinner than that of individuals with other genotypes. thinner; for SNP molecular marker site 2:1283877, when the genotype is AA, the Landrace pig's corrected 100kg backfat thickness is thinner than that of individuals with other genotypes; for SNP molecular marker site 2:1284016, when the genotype is AA, the Landrace pig's corrected 100kg backfat thickness is thinner than that of individuals with other genotypes; for SNP molecular marker site 2:1284026, when the genotype is CC, the Landrace pig's corrected 100kg backfat thickness is thinner than that of individuals with other genotypes.

[0035] Example 3 Single marker analysis of SNP molecular marker associated with 100 kg backfat thickness trait of pig The above 6 molecular marker sites were associated with Changbai 100 kg backfat thickness trait using mixed linear model (MLM) in SAS software.

[0036] The specific model is as follows: Wherein, represents the 100 kg backfat thickness trait phenotype value of the i th individual; is the overall mean; is the effect of the i th genotype (fixed effect); is the seasonal effect (fixed effect); is the gender effect (fixed effect); is the random residual effect.

[0037] The significant difference of 100 kg backfat thickness trait was analyzed using significant T test, and the results are shown in Tables 3-9.

[0038] Table 3 Single marker association analysis level of SNP molecular marker site Table 4 Polymorphism comparison of 2:1280617 Table 5 Polymorphism comparison of 2:1280654 Table 6 Polymorphism comparison of 2:1283873 Table 7 Polymorphism comparison of 2:1283877 Table 8 Polymorphism comparison of 2:1284016 Table 9 Polymorphism comparison of 2:1284026 As shown in Table 3, the 6 SNP molecular marker sites are significantly related to 100 kg backfat thickness trait.

[0039] From Table 4 to Table 9, there is a significant difference between different genotypes of each SNP molecular marker site, indicating that the genetic variation of the SNP molecular marker site has a significant regulatory effect on the backfat thickness. Combined with the results of Table 2, for the 6 SNP sites 2:1280617, 2:1280654, 2:1283873, 2:1283877, 2:1284016 and 2:1284026, the genotype is GG, GG, AA, AA, AA, CC, respectively, and the corrected 100 kg backfat thickness of the long white pig is thinner than that of the individual with other genotypes.

[0040] Example 4 Dual-luciferase reporter assay verifies SNP molecular marker site 1. Plasmid vector construction (1) Since the 6 SNP molecular marker sites to be verified are close in distance and tightly linked, the 6 SNP molecular marker sites are divided into two groups to construct haplotypes for experimental verification. The first group is 2:1280617 and 2:1280654 (two groups of haplotypes are GG and AT, respectively), and the second group is 2:1283873, 2:1283877, 2:1284016 and 2:1284026 (two groups of haplotypes are CCGT and AAAC, respectively).

[0041] (2) According to the pig Sscrofa11.1 version reference genome in Ensembl database, the upstream and downstream of each group of haplotypes are expanded by 150 bp as insertion sequences.

[0042] In order to avoid introducing other variation sites by amplification, the corresponding haplotype sequence (only containing the target verification site) is directly synthesized to obtain the GG haplotype sequence, the AT haplotype sequence, the CCGT haplotype sequence and the AAAC haplotype sequence. The nucleotide sequences are shown in SEQ ID NO: 7~SEQ ID NO: 10.

[0043] (3) The GG haplotype sequence, the AT haplotype sequence, the CCGT haplotype sequence and the AAAC haplotype sequence are respectively recombined with the PGL3-promoter plasmid by double enzyme digestion method (double enzyme digestion site: NheI and XhoI) to obtain the recombinant plasmid PGL3-promoter-GG haplotype, the recombinant plasmid PGL3-promoter-AT haplotype, the recombinant plasmid PGL3-promoter-CCGT haplotype and the recombinant plasmid PGL3-promoter-AAAC haplotype.

[0044] 2. Transfection of dual-luciferase reporter gene recombinant plasmid into cells (1) PK15 cell culture: PK15 cells were cultured in DMEM medium containing 10% FBS and 1% P / S (penicillin and streptomycin).

[0045] (2) Cell plating: The cultured PK15 cells were inoculated into a 24-well plate, and the cells were cultured to a density of 70% to 80% for transfection experiments.

[0046] (3) Cell transfection: Transfection was performed using a jetPRIME® transfection kit, and according to the 24-well system provided in the kit instructions, 50 μl of jetPRIME® buffer was added to each well, 0.5 μg of recombinant plasmid and pRL-TK plasmid, and 1.2 μl of jetPRIME® reagent. After mixing all reagents, they were allowed to stand for ten minutes, and then the transfection complex was added to the transfection well and incubated in a 37°C incubator for 24 hours. The recombinant plasmid was recombinant plasmid PGL3-promoter-GG haplotype, recombinant plasmid PGL3-promoter-AT haplotype, recombinant plasmid PGL3-promoter-CCGT haplotype, or recombinant plasmid PGL3-promoter-AAAC haplotype.

[0047] 3. Dual luciferase reporter gene activity assay The dual luciferase activity detection was mainly performed using a dual luciferase reporter gene detection kit produced by Yeason Company, and according to the kit instructions, the specific operation was as follows: (1) The 24-well plate after transfection was taken out of the incubator, and the culture medium was removed, and the cells in the 24-well plate were washed twice with PBS.

[0048] (2) 200 μl of lysis solution was added to each well of cells, and lysis was performed on ice for 20 min.

[0049] (3) 20 μl of lysed solution was taken from each well and added to the enzyme-labeled plate.

[0050] (4) Preparation of firefly luciferase working solution and sea cucumber luciferase working solution: 50x firefly luciferase substrate was diluted to 1x using firefly luciferase buffer, and 50x sea cucumber luciferase substrate was also diluted to 1x using sea cucumber luciferase buffer. The firefly luciferase working solution and the sea cucumber luciferase working solution were prepared according to this preparation ratio.

[0051] (5) 100 μl luciferase working solution was added into each well of the enzyme-labeled plate in step (3), and the luminescence detection value of the firefly luciferase was measured using a multifunctional enzyme marker, and then 100 μl of renilla luciferase working solution was added, and the luminescence detection value of the renilla luciferase was measured using the multifunctional enzyme marker. At the same time, plasmid PGL3-promoter and pRL-TK plasmid were transfected and double luciferase reporter gene activity determination was carried out as a control group.

[0052] The experimental results were calculated, and the difference significance of the experimental results was analyzed using independent sample T test.

[0053] For the selected 6 SNPs, 4 haplotypes (GG, AT, AAAC, CCGT) were constructed for double luciferase reporter assay. According to the fluorescence value, a column chart was drawn, and whether there was a significant difference in the relative luciferase activity of the two groups of haplotypes was statistically analyzed. The results can verify whether the selected SNPs are located in the enhancer region, and whether the SNP site changes the enhancer activity. P value is obtained by independent sample T test, P≤0.05, P≤0.01, P≤0.001.

[0054] The results are shown in Table 2. Figure 3 The fluorescence activity of the two groups of haplotype vectors was significantly higher than that of PGL3-promoter, suggesting that the fragment was located in the enhancer sequence. The fluorescence activity of the GG haplotype vector was significantly higher than that of the AT haplotype (P<0.01), suggesting that the GG haplotype had stronger transcription promotion effect on the target gene than the AT haplotype.

[0055] The results are shown in Table 2. Figure 4 The fluorescence activity of the two groups of haplotype vectors was significantly higher than that of PGL3-promoter, suggesting that the fragment was located in the enhancer sequence. The fluorescence intensity of the AAAC haplotype was significantly higher than that of the CCGT haplotype (P<0.01), suggesting that the AAAC haplotype had stronger transcription promotion effect on the target gene than the CCGT haplotype.

[0056] According to the results in Table 2, the GG and AAAC haplotype individuals had significantly lower back fat thickness than the AT and CCGT haplotype individuals, indicating that the identified 6 SNP molecular marker sites might affect the activity of the cis-regulatory element of the target gene, thereby participating in the regulation of fat deposition process, and further affecting the back fat thickness trait of the Landrace pig.

[0057] Example 5 Obtaining SNP molecular markers associated with the correction of 100 kg back fat thickness trait in pigs 1. According to the pig Sscrofa11.1 version reference genome in Ensembl database, the nucleotide sequences upstream and downstream of the SNP molecular marker site are extracted, and the genomic DNA of the pig is amplified by PCR using the primer pair Forward primer 1 and Reverse primer 1, and the nucleotide sequences of the primer pair Forward primer 1 and Reverse primer 1 are shown in SEQ ID NO: 3~SEQ ID NO: 4: Forward primer 1: CTGAGGCTGTGGGGGGGT; Reverse primer 1: GGACCTGGGGTATGGAGCC; The sequence SNP-1 containing the first SNP molecular marker site 2:1280617 and the second SNP molecular marker site 2:1280654 is obtained, and the nucleotide sequence is shown in SEQ ID NO: 1, R is A / G, K is T / G, and the SNP molecular marker site 2:1280617 is located at the 151st base in the sequence, the polymorphic site is A / G, and the SNP molecular marker site 2:1280654 is located at the 188th base in the sequence, the polymorphic site is T / G.

[0058] 2. The genomic DNA of the pig is amplified by PCR using the primer pair Forward primer 2 and Reverse primer 2, and the nucleotide sequences of the primer pair Forward primer 2 and Reverse primer 2 are shown in SEQ ID NO: 5~SEQ ID NO: 6: Forward primer 2: TGGCCCAGGGCACACACC; Reverse primer 2: GCTGGGGGGCCACGACCT; obtained, which has the nucleotide sequence shown as SEQ ID NO: 2, wherein M is C / A, M is C / A, R is G / A, Y is T / C, SNP molecular marker site 2:1283873 is located at the 151st base in the sequence, the polymorphic site is C / A, SNP molecular marker site 2:1283877 is located at the 155th base in the sequence, the polymorphic site is C / A, SNP molecular marker site 2:1284016 is located at the 294th base in the sequence, the polymorphic site is G / A, and SNP molecular marker site 2:1284026 is located at the 304th base in the sequence, the polymorphic site is T / C.

[0059] The corresponding genes of the above six SNP molecular markers are LSP1.

[0060] Example 6 The present example provides a detection method of SNP molecular markers associated with the 100 kg backfat thickness trait of pigs. The detection is completed by amplification and sequencing comparison using the primer pairs Forward primer 1 and Reverse primer 1, Forward primer 2 and Reverse primer 2 in Example 5. The primer pair Forward primer 1 and Reverse primer 1 is used for sequence SNP-1 containing the first SNP molecular marker site 2:1280617 and the second SNP molecular marker site 2:1280654, and the primer pair Forward primer 2 and Reverse primer 2 is used for sequence SNP-2 containing the third SNP molecular marker site 2:1283873, the fourth SNP molecular marker site 2:1283877, the fifth SNP molecular marker site 2:1284016 and the sixth SNP molecular marker site 2:1284026.

[0061] Example 7 The present example provides a kit for detecting SNP molecular markers associated with the 100 kg backfat thickness trait of pigs, which comprises the primer pairs Forward primer 1 and Reverse primer 1, Forward primer 2 and Reverse primer 2 in Example 5.

[0062] The primer pair Forward primer 1 and Reverse primer 1 is used for the sequence SNP-1 containing the first SNP molecular marker site 2:1280617 and the second SNP molecular marker site 2:1280654, and the primer pair Forward primer 2 and Reverse primer 2 is used for the sequence SNP-2 containing the third SNP molecular marker site 2:1283873, the fourth SNP molecular marker site 2:1283877, the fifth SNP molecular marker site 2:1284016 and the sixth SNP molecular marker site 2:1284026.

[0063] Embodiment 8 The embodiment provides a method for identifying the back fat thickness trait of a pig by using the kit in Embodiment 6, and the method comprises the following steps: detecting SNP molecular marker sites of the SNP molecular markers associated with the pig corrected 100 kg back fat thickness trait on the chromosome 2 of the pig, and judging the back fat thickness trait of the pig according to the SNP molecular marker sites; When the first SNP molecular marker site is the GG genotype, the second SNP molecular marker site is the GG genotype, the third SNP molecular marker site is the AA genotype, the fourth SNP molecular marker site is the AA genotype, the fifth SNP molecular marker site is the AA genotype and the sixth SNP molecular marker site is the CC genotype, the back fat thickness of the pig is lower than that of the pig with other genotypes.

[0064] The detecting method comprises the following steps: (1) extracting the DNA of the pig to be detected; (2) performing PCR amplification on the extracted DNA by using the primer pair in the kit; (3) performing sequencing analysis on the PCR amplification product to obtain a sequencing result; (4) obtaining the genotype based on the sequencing result, and when the first SNP molecular marker site is the GG genotype, the second SNP molecular marker site is the GG genotype, the third SNP molecular marker site is the AA genotype, the fourth SNP molecular marker site is the AA genotype, the fifth SNP molecular marker site is the AA genotype and the sixth SNP molecular marker site is the CC genotype, the back fat thickness of the pig is lower than that of the pig with other genotypes.

[0065] The method for identifying the backfat trait of pigs according to the embodiment can accurately obtain the genotype of the SNP molecular marker site on the chromosome 2 of pigs, and the accuracy is high. The breeding pigs with thinner backfat at the weight of 100 kg means less fat deposition and higher lean meat rate, which meets the current breeding goal of high efficiency, cost reduction and benefit increase. This feature is conducive to improving pork quality, optimizing feed utilization efficiency, and reducing breeding cost while ensuring yield. The technology has broad application prospects in breeding practice of breeding pigs, which not only helps to improve the economic benefits of pig breeding enterprises, but also helps to promote the progress of pig breeding technology in China and speed up the breeding process of excellent germplasm.

[0066] Other parts not specifically described are prior art. Although the above embodiment describes the present application in detail, it is only a part of the embodiment of the present application, not all embodiments, and other embodiments can be obtained under the premise of no creativity according to the embodiment, which belongs to the protection scope of the present application.

Claims

1. A SNP molecular marker associated with the corrected 100 kg backfat thickness trait in pigs, characterized by: The SNP molecular marker is located on pig chromosome 2, which contains 6 SNP molecular marker sites; The first SNP molecular marker site 2: 1280617: located at base 1280617 of pig chromosome 2, the polymorphic site is A or G; The second SNP molecular marker site 2: 1280654: located at base 1280654 of pig chromosome 2, the polymorphic site is T or G; The third SNP molecular marker site 2: 1283873: located at base 1283873 of pig chromosome 2, the polymorphic site is C or A; The fourth SNP molecular marker site 2: 1283877: located at base 1283877 of pig chromosome 2, the polymorphic site is C or A; The fifth SNP molecular marker site 2: 1284016: located at base 1284016 of pig chromosome 2, the polymorphic site is G or A; The sixth SNP molecular marker site 2: 1284026: located at base 1284026 of pig chromosome 2, the polymorphic site is T or C.

2. Use of the SNP molecular marker according to claim 1 in identifying the pig backfat thickness trait and screening pig breeds with low backfat thickness.

3. A primer pair for obtaining the SNP molecular marker according to claim 1, characterized in that: The nucleotide sequence of the primer pair for obtaining the sequence containing the first SNP molecular marker site and the second SNP molecular marker site is as follows: Forward primer 1: CTGAGGCTGTGGGGGGGT; Reverse primer 1: GGACCTGGGGTATGGAGCC; The nucleotide sequence of the primer pair for obtaining the sequence containing the third SNP molecular marker site, the fourth SNP molecular marker site, the fifth SNP molecular marker site, and the sixth SNP molecular marker site is as follows: Forward primer 2: TGGCCCAGGGCACACACC; Reverse primer 2:GCTGGGGGGCCACGACCT.

4. The primer pair according to claim 3, wherein: The nucleotide sequence amplified by the primer pair Forward primer 1 and Reverse primer 1 is shown in SEQ ID NO: 1, and the first SNP molecular marker site is located at the 151st base in the sequence, and the second SNP molecular marker site is located at the 188th base in the sequence; The nucleotide sequence amplified by the primer pair Forward primer 2 and Reverse primer 2 is shown in SEQ ID NO: 2, and the third SNP molecular marker site is located at the 151st base in the sequence, the fourth SNP molecular marker site is located at the 155th base in the sequence, the fifth SNP molecular marker site is located at the 294th base in the sequence, and the sixth SNP molecular marker site is located at the 304th base in the sequence; When the first SNP molecular marker site is a GG genotype, the second SNP molecular marker site is a GG genotype, the third SNP molecular marker site is an AA genotype, the fourth SNP molecular marker site is an AA genotype, the fifth SNP molecular marker site is an AA genotype, and the sixth SNP molecular marker site is a CC genotype, the back fat thickness of the pig is lower than the back fat thickness of pigs with other genotypes.

5. A method for detecting a SNP molecular marker associated with the corrected 100kg backfat thickness trait in pigs, characterized by: The method uses the primer pair described in claim 3 for amplification and sequencing comparison to complete the detection.

6. Use of the primer pair according to claim 3 in identifying the pig backfat thickness trait, screening pig breeds with low backfat thickness, and pig backfat thickness genetic breeding.

7. A kit for detecting the SNP molecular marker according to claim 1, characterized in that: The kit comprises the primer pair according to claim 3.

8. A method for identifying pig backfat thickness using the kit according to claim 7, characterized in that: The following steps are involved: Detecting the SNP molecular marker site of the SNP molecular marker according to claim 1 on pig chromosome 2, and determining the pig backfat thickness trait based on the SNP molecular marker site; When the first SNP molecular marker site is a GG genotype, the second SNP molecular marker site is a GG genotype, the third SNP molecular marker site is an AA genotype, the fourth SNP molecular marker site is an AA genotype, the fifth SNP molecular marker site is an AA genotype, and the sixth SNP molecular marker site is a CC genotype, the back fat thickness of the pig is lower than the back fat thickness of pigs with other genotypes.

9. The method according to claim 8, characterized in that: The detection method comprises the following steps: (1) Extract DNA from the pig to be tested; (2) performing PCR amplification on the extracted DNA using the primer pairs in the kit according to claim 7; (3) Sequencing and analyzing the PCR amplification products to obtain sequencing results; (4) Based on the sequencing results, the genotype is obtained. When the first SNP molecular marker site is a GG genotype, the second SNP molecular marker site is a GG genotype, the third SNP molecular marker site is an AA genotype, the fourth SNP molecular marker site is an AA genotype, the fifth SNP molecular marker site is an AA genotype, and the sixth SNP molecular marker site is a CC genotype, the back fat thickness of the pig is lower than the back fat thickness of pigs with other genotypes.

10. Use of the kit according to claim 7 in identifying the pig backfat thickness trait, screening pig breeds with low backfat thickness, and genetic breeding of pig backfat thickness.

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