An SNP molecular marker for detecting backfat thickness in pigs, primers containing this molecular marker, and their applications.

Abstract

This invention relates to a SNP molecular marker for detecting backfat thickness in pigs, primers containing this molecular marker, and their applications, belonging to the field of molecular biotechnology. This invention provides intron variant sites in the COL22A1 gene and their application in detecting backfat thickness in pigs. These variant sites are located at introns 4184306 and 4184654 in the COL22A1 gene, both being polymorphic SNP sites. The genotype of site g.4184306 is CC, CT, or TT, used to determine backfat thickness at the thoracolumbar junction and average backfat thickness in pigs. The genotype of site g.4184654 is AA, AG, or GG, used to determine backfat thickness at the shoulder. The beneficial effects of this invention are: by selecting the dominant alleles of these two SNP molecular markers, it provides a reference for SNP molecular marker-assisted breeding related to backfat thickness in pigs, which is of great significance for improving pig carcass and meat quality.

Description

Technical Field

[0001] This invention relates to an SNP molecular marker for detecting backfat thickness in pigs, primers containing the molecular marker, and their applications, belonging to the field of molecular biotechnology. Background Technology

[0002] Studying and scientifically utilizing the molecular mechanisms underlying different economic traits in pigs has always been a primary task for scientists and breeders. Backfat, located between the dermis and the longissimus dorsi muscle, is mainly composed of water, collagen, and lipids. Its thickness can be used to estimate carcass grading and lean meat content, and is one of the main indicators for individual pig carcass grading. In slaughter assessments, it is generally evaluated by the backfat thickness at the shoulder, the thoracolumbar junction, and the loin-sacral junction, as well as the average backfat thickness of these three areas. If an individual has excessive backfat, i.e., excessive subcutaneous fat deposition, it will significantly affect the individual's appearance value, prolong the feeding cycle, and reduce the lean meat percentage, thereby greatly reducing the economic benefits of pork products. Therefore, effectively reducing backfat thickness has always been a major task in improving carcass quality in the pig industry. Given that backfat thickness is a highly heritable trait, identifying genetic variations related to pig backfat thickness and then using molecular or genomic marker-assisted selection to breed strains with lower backfat thickness that meet market demands has very high application value. In recent years, genes and markers related to backfat thickness in pigs have been continuously discovered, such as insulin-like growth factor 2 (IGF2), collagen type XXI (alpha 1, COL21A1), leptin receptor (LEPR), and melanocortin 4 receptor (MC4R). Genetic variations in these genes can significantly affect backfat thickness in different pig breeds, laying the foundation for further exploration of the genetic mechanisms of subcutaneous fat formation in pigs, and also providing new ways to accelerate the improvement of pig carcass quality.

[0003] Collagen type XXII alpha 1 chain (COL22A1) belongs to the collagen superfamily and is a fibril-associated collagen subgroup with a discontinuous triple helix. Located in the collagen-containing extracellular matrix, it binds to collagen fibers through its C-terminal collagen domain and mediates protein-protein interactions through its N-terminal non-collagen domain. It is expressed in connective tissue, muscle, and subcutaneous adipose tissue, playing important roles in angiogenesis, endothelial cell morphogenesis, and extracellular matrix organization. Collagen XXII is the first specific extracellular matrix protein found only at tissue junctions. Since extracellular matrix integrity and structural remodeling processes can affect adipocyte growth, differentiation, and metabolism, and are related to the regulation of adipogenesis during adipose tissue expansion, the role of the COL22A1 gene in the extracellular matrix may influence adipocyte formation. Currently, COL22A1 has been identified as a core gene associated with type 2 diabetes and high-fat diets in humans, particularly in astrocytes and endothelial cells. Transcriptome sequencing has revealed high expression of the COL22A1 gene in high-marbled beef cattle populations. Furthermore, since cells involved in adipogenesis and fibrosis originate from the same progenitor cell pool, the COL22A1 gene may also indirectly regulate lipid accumulation. It is clear from this that the COL22A1 gene can influence adipogenesis. However, to date, there are no functional studies of the COL22A1 gene in pigs, nor are there any studies on the association between COL22A1 gene variations and economic traits in pigs. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing an SNP molecular marker for detecting backfat thickness in pigs, primers containing this molecular marker, and their applications, thereby providing marker resources and a basis for improving subcutaneous fat traits and carcass quality in pigs.

[0005] This invention solves the technical problem through the following technical solution: First, it provides an SNP molecular marker for detecting the thorax-lobe junction and average backfat thickness in pigs, wherein the SNP molecular marker is located on chromosome 4. COL22A1 At base 4184306 of intron 21 of the gene, the genotype of the SNP molecular marker site is CC, CT, or TT. Therefore, this SNP is named g.4184306 T>C.

[0006] A SNP molecular marker for detecting backfat thickness in the shoulder region of pigs, the SNP molecular marker being located on chromosome 4. COL22A1 At base 4184654 of intron 21 of the gene, the genotype of the SNP molecular marker site is AA, AG, or GG. Therefore, this SNP is named g.4184654 A>G.

[0007] pig COL22A1See Chromosome 4, NC_010446.5 (4069045..4313198) for gene sequence.

[0008] The present invention further provides a primer pair for detecting the above-mentioned SNP molecular markers, wherein the sequences of the primer pair are as follows:

[0009] COL22A1-3F: 5'-GCTGTGGTCAAGGGGAAAAG-3' SEQ ID NO: 5

[0010] COL22A1-3R: 5'-GGATGGAGATGTGGGACTGG-3'. SEQ ID NO: 6

[0011] The amplified sequences of the primer pairs are shown in SEQ ID NO. 1-4. In SEQ ID NO: 1, the bases at positions 4184306 and 4184654 are T and A, respectively; in SEQ ID NO: 2, the bases at positions 4184306 and 4184654 are C and A, respectively; in SEQ ID NO: 3, the bases at positions 4184306 and 4184654 are T and G, respectively; and in SEQ ID NO: 4, the bases at positions 4184306 and 4184654 are C and G, respectively. Position 4184306 corresponds to position 264 in the sequence, and position 4184654 corresponds to position 612 in the sequence, which is the molecular marker site.

[0012] This invention further provides the application of the above-mentioned SNP molecular markers and primer pairs in detecting backfat thickness in pigs. Genotyping of the C / T mutation site of the SNP molecular marker at position 4184306 shows that the backfat thickness at the thorax and loin junction and the average thickness of pork samples with the TT genotype are lower than those with the CC genotype. Genotyping of the A / G mutation site of the SNP molecular marker at position 4184654 shows that the backfat thickness at the shoulder of pork samples with the AA genotype is also lower than that with the GG genotype.

[0013] This invention further provides a method for genetically improving the low backfat thickness trait in pigs through assisted breeding. The method involves sequencing and genotyping the C / T mutation site of the SNP molecular marker at position 4184306. The detection method includes the following steps:

[0014] Step 1: Extract DNA from the longissimus dorsi muscle tissue of pigs. Use primers with SNP molecular markers to...

[0015] Perform PCR amplification to obtain the amplification product;

[0016] The second step is to detect and sequence the amplified products by agarose gel electrophoresis.

[0017] The third step is to determine the mutation sites at positions 4184306 and 4184654 based on the sequencing map.

[0018] genotype.

[0019] The PCR reaction system in the first step is as follows:

[0020] T3 Mix 22µl

[0021] Primer-F 1µl

[0022] Primer-R 1µl

[0023] DNA 1µl

[0024] Total 25µl;

[0025] The reaction conditions were: 98℃ for 3 min; 98℃ for 10 s, 65℃ for 10 s, 72℃ for 10 s, 34 cycles; 72℃ for 3 min; and stored at 4℃.

[0026] The third criterion is that the backfat thickness and average backfat thickness at the chest-waist junction of individual pigs with the TT genotype at position 4184306 are lower than those of individuals with the CC genotype; the A / G mutation sites of the SNP molecular marker at position 4184654 are sequenced and genotyped, and the backfat thickness at the shoulder of individual pigs with the AA genotype is lower than that of individuals with the GG genotype.

[0027] This invention analyzes the correlation between the specific extracellular matrix protein COL22A1 at tissue junctions and the backfat thickness trait in pigs. Two SNP loci associated with backfat thickness were screened in the intron region, and genes and molecular genetic markers related to backfat thickness were obtained. By selecting the dominant alleles of the two SNP molecular markers, the beneficial effects are: it can provide a reference for SNP molecular marker-assisted breeding related to backfat thickness in pigs, and has important significance for the improvement of pig carcass and meat quality. Attached Figure Description

[0028] Figure 1 Sequencing maps of different genotypes at five mutation sites in the COL22A1 gene. A. Sequencing map of different genotypes at the G>A site (g.4184257); B. Sequencing map of different genotypes at the T>C site (g.4184306); C. Sequencing map of different genotypes at the A>G site (g.4184592); D. Sequencing map of different genotypes at the A>T site (g.4184598); E. Sequencing map of different genotypes at the A>G site (g.4184654).

[0029] Figure 2 Linkage disequilibrium analysis plot of COL22A1 gene mutation sites. The degree of linkage disequilibrium between each pair of SNP sites is expressed as r.2 The value is used to measure this, and is represented in the box as a numerical value multiplied by 100, r. 2 A value >0.8 indicates that the two SNP sites are not in linkage equilibrium. Detailed Implementation

[0030] Example

[0031] The present invention will be further described below with reference to the embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally carried out in accordance with known means in the art.

[0032] experimental animals

[0033] This study used 302 Duroc-Landrace-Large White crossbred pigs. The experimental animals were obtained from Jiangsu Huaiyin Wenshi Livestock Co., Ltd. and were raised under the same conditions. The longissimus dorsi muscle tissue was collected for DNA extraction.

[0034] Test Procedure

[0035] Back fat thickness measurement

[0036] Using vernier calipers, the backfat thickness of each pig was measured at three locations: the thickest part of the shoulder on the left half of the carcass, the junction of the thoracic and lumbar vertebrae, and the junction of the lumbar and sacral vertebrae. The average backfat thickness at these three locations was calculated as the average backfat thickness of the individual.

[0037] Muscle DNA Extraction and Concentration Determination

[0038] (1) Cut off about 100mg of sample, chop it, and put it into a 2.0ml EP tube;

[0039] (2) Add 1 ml of lysis buffer and 50 µl of proteinase K to the EP tube and mix thoroughly. Digest in a water bath at 55 °C for 12 h to overnight. The next day, add 900 µl of Tris saturated phenol to a 2 ml tube, mix on a shaker for 15 min, and centrifuge at 12000 rpm for 15 min at 4 °C.

[0040] (3) Take 1000µl of the upper layer liquid into a 2ml EP tube, add 500µl of chloroform / isoamyl alcohol (24:1, v:v) and 500µl of saturated phenol, mix well on a shaker for 15min, centrifuge at 12000rpm for 15min at 4℃.

[0041] (4) Take 850µl of the upper layer liquid into a 2ml EP tube, add 850µl of chloroform / isoamyl alcohol (24:1, v:v), mix well on a shaker for 15min, and centrifuge at 12000rpm for 15min at 4℃.

[0042] (5) Take 700µl of the upper layer liquid and put it into a 2ml EP tube, add 700µl of chloroform, mix well on a shaker for 15min, and centrifuge at 12000rpm for 15min at 4℃.

[0043] (6) Take 500µl of the upper layer liquid into a 1.5ml EP tube, add 2 times the volume of anhydrous ethanol, shake gently, and centrifuge at 10000rpm for 10min at 4℃.

[0044] (7) Discard the supernatant, add 1 ml of 70% anhydrous ethanol, shake gently, and centrifuge at 10,000 rpm for 5 min at 4°C.

[0045] (8) Pour off the upper layer of liquid, remove the remaining liquid in the tube, and blow dry in the clean bench until there are no water droplets in the EP tube. The drying time is about 25 minutes.

[0046] (9) Add 50µl of 55℃ deionized water to the sample to completely dissolve the DNA, perform sample quality testing, and finally store it in a -20℃ refrigerator.

[0047] (10) The DNA concentration of all porcine longissimus dorsi muscle samples was determined using a NanoDrop spectrophotometer, and the OD260 / OD280 ratio was calculated to determine the DNA extraction quality. If the OD260 / OD280 ratio of all samples should be 1.8-2.0, then the DNA extraction quality is qualified.

[0048] Primer design

[0049] Based on pig gene sequences from the Ensembl database COL22A1 (Chromosome 4, NC_010446.5 (4069045..4313198)) The following primers were designed to amplify a fragment of 807 bp.

[0050] COL22A1-3F: 5'-GCTGTGGTCAAGGGGAAAAG-3'

[0051] COL22A1-3R: 5'-GGATGGAGATGTGGGACTGG-3'.

[0052] PCR amplification

[0053] The PCR reaction system used in this experiment is shown in Table 1:

[0054] Table 1 Sequencing PCR Reaction System

[0055] Components Volume T3 Mix 22µl Primer-F 1µl Primer-R 1µl DNA 1µl Total 25µl

[0056] The PCR reaction program was as follows: 98℃, 3 min; 98℃, 10 s, 65℃, 10 s, 72℃, 10 s, 34 cycles; 72℃, 3 min; store at 4℃.

[0057] Agarose gel electrophoresis detection

[0058] The PCR amplification products were electrophoresed on a 1.5% agarose gel at a voltage of approximately 120V for about 30 minutes, and then observed using a gel imaging system.

[0059] 2.6 Sequencing of PCR products

[0060] The amplified products were subjected to agarose gel electrophoresis. Single band samples that met the target fragment length and had high abundance were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0061] 2.7 Sequence Alignment and Linkage Disequilibrium Analysis

[0062] The sequencing files were viewed and compared using DNAstar 7.1 software to analyze the genotype information of each individual. Linkage disequilibrium between multiple loci was analyzed using HaploView 4.2 software.

[0063] 2.8 Statistical Analysis

[0064] Using SPSS 25.0 software to... COL22A1 The association between gene mutation and backfat thickness was analyzed, and the linear model is as follows: Y ijklm = μ + W i + G j + S k + M l + F m + e ijklm . Y ijklm This refers to the thickness of the back fat. μ The population mean; W i Let carcass weight be a covariate; G j For genotype effect, S k For gender, M l Male livestock G j , S k andM l Set as a fixed effect; F m For female animals, the effect is set as random. e ijk To account for residual effects, the Bonferroni multiple comparison method was used to compare differences between different genotypes, and the results are expressed as least squares mean ± standard error.

[0065] Test results

[0066] 3.1 SNP genotyping

[0067] In pig populations, chromosome 4 COL22A1 The intron region of the gene was amplified to obtain an 807 bp fragment. The PCR product was then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. After sequencing, a total of [number missing] fragments were obtained. COL22A1 Five SNP sites were found in the gene amplification region: g.4184257 G>A, g.4184306 T>C, g.4184592 A>G, g.4184598 A>T, and g.4184654 A>G. Figure 1 AE).

[0068] 3.2 Linkage Disequilibrium Analysis of SNP Sites

[0069] Given that tagged SNPs are a small number of SNP sites identified through linkage disequilibrium that can represent all SNPs within a region of interest and are as independent of each other as possible, linkage disequilibrium analysis was performed on the above 5 SNP sites. This revealed that the r values ​​of the g.4184257 G>A and g.4184306 T>C sites were... 2 The value is 0.99, indicating almost complete linkage disequilibrium, while the r values ​​at the two sites g.4184598 A>T and g.4184654 A>G are... 2 The value is 0.81, which is close to a state of chain disequilibrium. Figure 2 Since a single SNP in a linkage disequilibrium state can represent all SNPs within that linkage disequilibrium region, three SNPs, g.4184306 T>C, g.4184592 A>G, and g.4184654 A>G, were selected as tag SNP sites for subsequent association analysis.

[0070] 3.3 Correlation analysis between SNP sites and backfat thickness

[0071] Association analysis to find COL22A1The correlation between gene intron variation and backfat thickness in pigs was investigated, and the results showed that the g.4184306T>C site was significantly associated with the thoracolumbar junction and average backfat thickness in pigs. P <0.05, among which the TT genotype individuals had significantly lower backfat thickness at the thorax-lobe junction and average backfat thickness than the CC genotype individuals (Table 2), while the mutation g.4184654 A>G was significantly associated with backfat thickness at the shoulder of pigs ( P <0.05), and the backfat thickness of the shoulders of pigs with the AA genotype was also significantly lower than that of the GG genotype individuals (Table 2).

[0072] Table 2. Correlation analysis of the g.4184306 T>C, g.4184592 A>G, and g.4184654 A>G sites with backfat thickness at different locations. Note: Data are expressed as least squares mean ± standard error. Different lowercase letters in different genotype populations at the same locus indicate significant differences. P <0.05).

[0073] Results Analysis

[0074] The two mutation sites screened in this invention are significantly correlated with the backfat thickness trait in pigs and can be developed into SNP molecular markers for detecting backfat thickness in pigs. For COL22A1 At the g.4184306 T>C locus, individuals with the TT genotype had significantly lower back fat thickness at the chest-waist junction and lower average back fat thickness than individuals with the CC genotype; at the g.4184654 G>A locus, individuals with the AA genotype had significantly lower shoulder back fat thickness than individuals with the GG genotype.

[0075] This invention screened a SNP locus associated with the thorax-lobe junction and average backfat thickness in pigs, as well as a SNP locus associated with backfat thickness in the shoulder area. This led to the acquisition of functional genes and molecular genetic markers related to backfat thickness. By optimizing the dominant alleles of these SNP molecular markers, a reference basis can be provided for SNP molecular marker-assisted breeding related to backfat thickness in pigs, and it is also of great significance for improving pig carcass traits.

[0076] In addition to the above-described embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. The application of a SNP molecular marker related to the backfat thickness trait in pigs, characterized in that: The backfat thickness of the pig includes the backfat thickness at the thoracolumbar junction, the average backfat thickness, and the shoulder backfat thickness; the SNP molecular markers for the backfat thickness at the thoracolumbar junction and the average backfat thickness are located at position 4184306 on chromosome 4 of the 11.1 version of the reference genome, and the SNP molecular marker for the shoulder backfat thickness is located at position 4184654 on chromosome 4 of the 11.1 version of the reference genome. The genotype of the SNP molecular marker at position 4184306 is CC, CT, or TT, and the genotype of the SNP molecular marker at position 4184654 is AA, AG, or GG. The nucleotide sequences of the SNP molecular markers are shown in SEQ ID NO: 1-4. The molecular detection method for measuring backfat thickness in pigs includes the following steps. Step 1: Extract DNA from the longissimus dorsi muscle tissue of pigs. Perform PCR amplification using primers with SNP molecular markers to obtain the amplification product. The second step is to detect and sequence the amplified products by agarose gel electrophoresis. The third step involves determining the genotypes of mutation sites at positions 4184306 and 4184654 based on the sequencing map. The average backfat thickness at the shoulder, thoracolumbar junction, and lumbosacral junction is used as the average backfat thickness of an individual. The criteria for determination are that pigs with genotype CC have higher backfat thickness at the thoracolumbar junction and average backfat thickness than pigs with genotype TT; pigs with genotype GG have higher shoulder backfat thickness than pigs with genotype AA; and the pigs in question are Pidgeon-Duchy-Landrace-Landrace-Landrace-Great Wall four-way crossbred pigs.

2. The application of the SNP molecular markers related to the backfat thickness trait in pigs according to claim 1, characterized in that: The nucleotide sequences of the primer pairs are shown in SEQ ID NO: 5 and SEQ ID NO:

6.

3. The application of the SNP molecular markers related to the backfat thickness trait in pigs according to claim 1, characterized in that: In the first step, the PCR reaction system is as follows: T3 Mix 22µl Primer-F 1µl Primer-R 1µl DNA 1µl Total 25µl; The reaction conditions were: 98℃ for 3 min; 98℃ for 10 s, 65℃ for 10 s, 72℃ for 10 s, 34 cycles; 72℃ for 3 min; and stored at 4℃.

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