A molecular marker of the PPIL4 gene related to the residual feed intake trait of Large White pigs, as well as its detection method and application

Abstract

The present invention belongs to the field of molecular biology technology, and discloses a molecular marker of the PPIL4 gene related to the residual feed intake trait of Large White pigs, and a detection method and application thereof. The SNP marker is located on the nucleotide sequence of the peptidyl proline isomerase-like protein-4 gene (PPIL4) on pig chromosome 1, and the site is the g.16442886 nucleotide site on pig chromosome 1 of the international pig genome version 11.1 reference sequence, and has a C / T polymorphism. The SNP marker is significantly correlated with the residual feed intake trait of Large White pigs. The SNP marker provided by the present invention is related to the residual feed intake trait of pigs, and pig strains with low residual feed intake can be screened by identifying the SNP marker. The resulting pig strains with low feed-to-meat ratio and high growth rate have important economic benefits and social value.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to a PPIL4 gene molecular marker related to the residual feed intake trait of Large White pigs, a detection method and an application thereof. Background Art

[0002] The Large White, a widely bred commercial pig breed worldwide, holds a crucial position in modern animal husbandry due to its exceptional growth performance and reproductive capacity. Residual feed intake (RFI) is a key indicator of feed conversion efficiency. Low RFI generally reflects high feed efficiency, measured by measuring the difference between a pig's daily feed intake and its expected feed intake. RFI is significantly correlated with growth rate, feed conversion rate, and meat quality. Therefore, RFI is often selected as a primary trait in feed efficiency measurements, with differences in RFI indirectly predicting feed conversion performance.

[0003] With the continuous development of single nucleotide polymorphism (SNP) microarrays and genome sequencing technologies, genome-wide association analysis has become a powerful tool for genetic analysis of important economic traits in livestock and poultry. Residual feed intake is regulated by multiple, microgenes on multiple chromosomes, with a heritability of 0.12 to 0.52, making it a medium-heritability trait with promising selection potential. The peptidylproline isomerase-like protein-4 (PPIL4) gene encodes a peptidylproline isomerase-like enzyme, a member of the peptidylproline isomerase (PPIase) family. Members of this family regulate protein folding and function by catalyzing the cis-trans isomerization of proline residues in peptide chains. PPIL4 can enhance Wnt signaling by binding to JMJD6. The Wnt signaling pathway plays a key role in the proliferation and differentiation of muscle satellite cells. When muscle is damaged or growth is required, Wnt signaling drives satellite cells into differentiation by upregulating key transcription factors such as Myf5 and MyoD, ultimately leading to the formation of mature muscle fibers. Activation of the canonical Wnt / β-catenin pathway increases the accumulation of β-catenin in the nucleus, boosting the expression of cell cycle-related genes and promoting the transition of satellite cells from a quiescent state to a proliferative state. Furthermore, activation of this pathway inhibits adipogenesis. Mutations in the peptidylproline isomerase-like protein-4 gene significantly reduce the ability of its protein product to bind to JMJD6, thereby reducing the expression of the Wnt signaling pathway and ultimately affecting animal growth and development.

[0004] Therefore, by identifying the molecular marker sites of the peptidylproline isomerase-like protein-4 gene that affect residual feed intake and applying them to molecular marker-assisted selection, new pig breeds with high feed conversion rates can be bred, which is of great significance for reducing costs and increasing efficiency in pig production. Summary of the Invention

[0005] The purpose of the present invention is to address the shortcomings of existing technologies and the difficulty in predicting and breeding low feed-to-meat ratio pigs in advance, and to provide a PPIL4 gene molecular marker associated with low residual feed intake.

[0006] Another object of the present invention is to provide primers and detection methods for detecting the above-mentioned SNP markers and related applications.

[0007] The technical solution provided by the present invention is: a PPIL4 gene molecular marker related to the low residual feed intake trait, the SNP marker is located on the peptidyl proline isomerase-like protein-4 gene nucleotide sequence from 16400938 to 16446860bp on pig chromosome 1, the site of the SNP marker is the g.16442886 nucleotide site on pig chromosome 1 of the international pig genome version 11.1 reference sequence, and has a C / T polymorphism. The SNP marker is significantly correlated with the residual feed intake trait, and the residual feed intake of the CC genotype at this site is significantly lower than that of the TT genotype.

[0008] Preferably, the molecular marker sequence is as shown in SEQ ID NO: 1, and the SNP marker site is located at position 133 where a C / T polymorphism exists.

[0009] At the same time, the present invention also provides an amplification primer for amplifying the PPIL4 gene molecular marker related to the low residual feed intake trait, or for detecting the low residual feed intake trait of Large White pigs, which is designed based on the nucleotide sequence containing the SNP marker.

[0010] Preferably, the upstream primer sequence of the primer is shown as SEQ ID NO: 2, and the downstream primer sequence is shown as SEQ ID NO: 3.

[0011] The present invention also provides a method for detecting the PPIL4 gene molecular marker associated with the low residual feed intake trait, or for detecting the low residual feed intake trait of Large White pigs, the method comprising the following steps:

[0012] A sequence containing the SNP marker in the pig genome is amplified by PCR, and the amplified product is sequenced to determine the C / T polymorphism of the site.

[0013] The method specifically comprises the following steps:

[0014] (1) Take pig ear tissue samples and extract total DNA;

[0015] (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primers;

[0016] (3) The amplified product was sequenced, the sequencing results were analyzed, and the C / T polymorphism at position 133 of SEQ ID NO: 1 was interpreted.

[0017] In the method, preferably, the PCR amplification reaction system in step (2) is: 1.0 μL of DNA template, 1.0 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.125 μL of Taq enzyme reagent, and 17.38 μL of double-distilled water; wherein the concentration of the DNA template is 50 ng / μL, the concentration of the primers is 10 mol / L, and the Taq enzyme is a reagent from Dalian TaKaRa Biotechnology Co., Ltd.; the reaction procedure for PCR amplification is: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s; annealing at 60°C for 30 s, extension at 72°C for 30 s, 34 cycles; extension at 72°C for 5 min.

[0018] The present invention also provides the use of the SNP marker, the amplification primer, or the method in screening pig strains with low residual feed intake and low feed-to-meat ratio.

[0019] The application, wherein the screening of pig breeds with low residual feed intake and low feed-to-meat ratio is achieved by the following method: detecting the genotype of the piglet's g.16442886 nucleotide site, and breeding CC-type individuals with the g.16442886 nucleotide site as breeding pigs with low residual feed intake and low feed-to-meat ratio.

[0020] The present invention also provides a method for breeding a pig breed with low residual feed intake and low feed-to-meat ratio. The method comprises obtaining offspring piglets by self-pollination or hybridization, detecting the genotype of the piglets at the nucleotide site g.16442886 by using the method described in any one of claims 5 to 7, and selecting CC-type individuals at the nucleotide site g.16442886 as breeding pigs with low residual feed intake and low feed-to-meat ratio.

[0021] The present invention has the following beneficial effects:

[0022] The PPIL4 gene molecular marker provided by the present invention is associated with the low residual feed intake trait. Therefore, the low residual feed intake pig strain can be screened by identifying the SNP marker. The obtained low residual feed intake trait strain exhibits a low feed-to-meat ratio and has important economic benefits and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The electrophoresis diagram is the result of amplifying the peptidylproline isomerase-like protein-4 gene using the primers of the present invention.

[0024] Figure 2 This is the peak diagram of DNA sequencing results of different genotypes at the mutation site of the peptidylproline isomerase-like protein-4 gene. DETAILED DESCRIPTION

[0025] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0026] Example 1

[0027] 1. Source of experimental animals

[0028] The samples were collected from 200 Large White pigs of Shaanxi Huamu Xingnong Breeding Pig Technology Co., Ltd.

[0029] 2. Determination of residual feed intake

[0030] Average daily feed intake (ADFI), body weight (BW), and average daily gain (ADG) were collected from each pig during the fattening period from 90 to 160 days of age. Backfat thickness was measured using ultrasound when the pigs reached approximately 100 kg. After the performance measurements were completed, residual feed intake (RFI) was calculated using the following formula:

[0031] RFI=ADFI-(b1×ONBW+b2×OFFBW+b3×MWT+b4×ADG+b5×BF)

[0032] MWT=[(ONBW +OFFBW) / 2]^0.75

[0033] ADFI: average daily feed intake; ONBW: initial body weight; OFFBW: final body weight; MWT: mid-term body weight; ADG: average daily weight gain; BF: average backfat thickness; b1, b2, b3, b4, b5 corresponding trait regression coefficients.

[0034] 3. Genomic DNA Extraction

[0035] Pig ear tissue samples were collected, placed in a centrifuge tube filled with 70% alcohol, and stored in a -20°C refrigerator for later use.

[0036] Genomic DNA was extracted using an animal genomic DNA extraction kit. The required reagents include:

[0037] Proteinase K (Beijing Solaibao Technology Co., Ltd.)

[0038] Animal genomic DNA extraction kit: Sangon Biotech (Shanghai) Co., Ltd.

[0039] Rapid PCR premix: Bio-Rad Biotechnology (Beijing) Co., Ltd.

[0040] DNA Marker (DL1000): Shanghai Pudi Biotechnology Co., Ltd.

[0041] 10×Loading Buffer: Bio-Rad Biotechnology (Beijing) Co., Ltd.

[0042] Agarose: Sangon Biotech (Shanghai) Co., Ltd.

[0043] TBE (10×) solution: Shanghai Beyotime Biotechnology Co., Ltd.

[0044] The specific steps are as follows:

[0045] (1) Take 25 mg of left and right ear tissue from a centrifuge tube, add it to a 1.5 ml centrifuge tube, add 400 µl of Buffer Digestion, and vortex to mix. Incubate in a 65°C water bath for 1 h until the cells are completely lysed.

[0046] (2) Add 20 μL of Proteinase K solution, shake to mix, and place in a 56°C water bath for overnight digestion.

[0047] (3) Add 200 µl of Buffer PA, mix thoroughly by inversion, and place in a -20°C refrigerator for 5 min.

[0048] (4) Centrifuge at 10,000 rpm for 5 min at room temperature and transfer the supernatant (500-550 µl) to a new 1.5 ml centrifuge tube.

[0049] (5) Add an equal volume of isopropanol, invert 5-8 times to mix thoroughly, and let stand at room temperature for 2-3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant.

[0050] (6) Add 1 ml of 75% ethanol, rinse by inversion for 1-3 minutes, centrifuge at 10,000 rpm for 2 minutes, and discard the supernatant. (7) Repeat step 6.

[0051] (7) Open the lid and invert at room temperature for 5 to 10 minutes until the remaining ethanol is completely evaporated.

[0052] (8) Dissolve the obtained DNA in 50-100 µl of TE Buffer. The extracted DNA can be used immediately for the next step or stored at -20°C.

[0053] (9) Determine the concentration. After testing the mass and concentration using a spectrophotometer, dilute the sample to a uniform concentration of 50 ng / μL and store at -20°C for later use.

[0054] 4. Pig genome 60K SNP chip genotyping and genome-wide association analysis

[0055] The whole genome genotype of the pigs was determined according to the company's standard process. The "Zhongxin No. 1" pig 50K SNP chip was used to perform SNP genotyping on individual breeding pigs with residual feed intake measurements, and all SNP marker detection results were quality controlled. A genome-wide significant SNP site on chromosome 1 of the pig was detected using genome-wide association analysis. This site was significantly associated with the residual feed intake trait ( P =3.73E-04)( Figure 1 Comparison with the International Porcine Genome Reference Sequence, version 11.1, revealed that these SNPs are located within the coding region of the PPIL4 gene. Based on existing literature and functional annotation, the PPIL4 gene has the potential to regulate muscle growth in animals, and SNP variants within this gene may regulate and influence the residual feed intake phenotype in pigs.

[0056] Example 2

[0057] This example validates the SNP g.16442886C / T in the PPIL4 gene, found in Example 1, which is significantly associated with residual feed intake, in a Large White pig population. SEQ ID NO: 1 is the product of PCR amplification using primers SEQ ID NO: 2 and SEQ ID NO: 3. The SNP significantly associated with residual feed intake is mutation 133 of this sequence.

[0058] This example validates the SNP g.16442886C / T in the PPIL4 gene, found in Example 1, which is significantly associated with residual feed intake, in a Large White pig population. SEQ ID NO: 1 is the product of PCR amplification using primers SEQ ID NO: 2 and SEQ ID NO: 3. The SNP significantly associated with residual feed intake is mutation 133 of this sequence.

[0059] 1. Extraction of pig genomic DNA

[0060] Ear tissue samples from sows with a recorded residual feed intake trait were collected, placed in a centrifuge tube containing 70% alcohol, and stored at -20°C until use. Genomic DNA from pig ear tissue was extracted using the above method, and after quality and concentration testing, the DNA was diluted to 50 ng / μL and stored at -20°C until use.

[0061] 2. PCR amplification and sequencing of target fragments

[0062] PCR amplification was performed using the extracted porcine DNA as a template according to the designed specific primers: the PCR amplification reaction system was: 1.0 μL of DNA template, 1.0 μL each of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.125 μL of Taq enzyme, and 17.38 μL of double-distilled water; the PCR amplification reaction program was: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s; annealing at 60°C for 30 s, extension at 72°C for 30 s, 30 cycles; extension at 72°C for 5 min.

[0063] The PCR product was detected by electrophoresis in 1.2% agarose gel. The amplified target fragment was 442 bp in size. Figure 2 The remaining amplified products were sequenced, and the sequencing results were compared and analyzed with the pig PPIL4 gene fragment sequence in GenBank using DNAMAN software, and the g.16442886C / T genotype was interpreted. The chi-square test module of SAS software was used to perform single marker association analysis between SNP genotype and residual feed intake trait.

[0064] Table 1 shows the effect of the g.16442886C / T mutation in the PPIL4 gene on residual feed intake in a resource population. As shown in Table 1, individuals with the CC genotype at the g.16442886C / T SNP site have lower residual feed intake compared to those with the TT genotype. This suggests that in breeding stock, successive generations of individuals with the CC genotype at the g.16442886C / T locus can gradually reduce residual feed intake and improve feed-to-meat conversion and growth rate.

[0065] Table 1. Association analysis between the PPIL4 gene SNP site (g.16442886 C / T) and residual feed intake traits

[0066] .

Claims

1. A method for detecting low residual feed intake trait in Large White pigs, characterized in that: The steps include: PCR amplified a sequence containing a SNP marker in the pig genome, sequenced the amplified product, and determined the C / T polymorphism of the site; the SNP marker was located on the nucleotide sequence of the peptidyl proline isomerase-like protein-4 gene from 16400938 to 16446860 bp on pig chromosome 1, and the SNP marker site was the g.16442886 nucleotide site on pig chromosome 1 of the international pig genome version 11.1 reference sequence, which had a C / T polymorphism, wherein the residual feed intake of the CC genotype at the site was significantly lower than that of the TT genotype.

2. The method according to claim 1, wherein The following steps are involved: (1) Take pig ear tissue samples and extract total DNA; (2) using the extracted porcine genomic DNA as a template, and performing PCR amplification using primers, wherein the upstream primer sequence of the primers is shown in SEQ ID NO: 2, and the downstream primer sequence is shown in SEQ ID NO: 3; (3) Sequencing the amplified product, analyzing the sequencing results, and interpreting the C / T polymorphism of the site.

3. The method according to claim 2, wherein: The PCR amplification reaction system described in step (2) is: 1.0 μL of DNA template, 1.0 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, 0.125 μL of Taq enzyme reagent, and 17.38 μL of double-distilled water; wherein the concentration of the DNA template is 50 ng / μL, the concentration of the primers is 10 mol / L, and the Taq enzyme is a reagent from Dalian TaKaRa Biotechnology Co., Ltd.; the reaction procedure for PCR amplification is: pre-denaturation at 95°C for 1 min; denaturation at 95°C for 10 s; annealing at 60°C for 30 s, extension at 72°C for 30 s, 34 cycles; extension at 72°C for 5 min.

4. Use of a reagent for detecting a SNP marker in screening pig strains with low residual feed intake, wherein the SNP marker is located on the nucleotide sequence of the peptidylproline isomerase-like protein-4 gene from 16400938 to 16446860 bp on pig chromosome 1. The SNP marker site is the g.16442886 nucleotide site on pig chromosome 1 of the International Porcine Genome Version 11.1 reference sequence, and has a C / T polymorphism, wherein: The residual feed intake of the CC genotype at the locus was significantly lower than that of the TT genotype, and the pig strain was Large White pig.

5. The use according to claim 4, characterized in that The reagent is an amplification primer, the upstream primer sequence of the amplification primer is shown as SEQ ID NO: 2, and the downstream primer sequence is shown as SEQ ID NO:

3.

6. Use of the method according to any one of claims 1 to 3 in screening pig strains with low residual feed intake.

7. The use according to any one of claims 4 to 6, characterized in that The screening of low residual feed intake pig strains is achieved by the following method: detecting the genotype of the nucleotide site g.16442886 of the reserve pigs, and selecting CC type individuals of the nucleotide site g.16442886 as low residual feed intake breeding pigs.

8. A method for breeding a low residual feed intake pig strain, characterized in that: The offspring piglets of the breeding pigs are obtained by self-pollination or hybridization, the genotype of the nucleotide site g.16442886 of the reserve pigs is detected by the method described in any one of claims 1 to 3, and CC type individuals with the nucleotide site g.16442886 are selected as low residual feed intake breeding pigs, and the pig breed is Large White pig.

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