PPIL4 gene molecular marker related to residual feed intake character of large white pigs as well as detection method and application of PPIL4 gene molecular marker

Abstract

The invention belongs to the technical field of molecular biology, and discloses a large white pig residual feed intake character related PPIL4 gene molecular marker as well as a detection method and application thereof. The SNP marker is located on a nucleotide sequence of a peptidyl proline isomerase-like protein-4 gene (PPIL4) on a pig chromosome 1, the site is a g.16442886 nucleotide site on the pig chromosome 1 of a reference sequence of an international pig genome version 11.1, the SNP marker has C / T polymorphism, and the SNP marker is significantly related to the residual feed intake character of the large white pig. The SNP marker provided by the invention is related to the residual feed intake character of the pig, the pig strain with low residual feed intake can be screened by identifying the SNP marker, and the obtained pig strain with low feed conversion ratio and high growth speed has important economic benefits and social values.

Description

Technical Field

[0001] The 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] Large White pigs are widely raised as commercial pig breeds around the world. They occupy an important position in modern animal husbandry due to their excellent growth performance and reproductive capacity. The residual feed intake of pigs is an important indicator to measure the feed conversion efficiency of pigs. Low residual feed intake usually reflects that pigs have high feed utilization efficiency. It is obtained by measuring the difference between the daily feed intake and the expected feed intake of pigs. Residual feed intake is significantly correlated with the growth rate, feed conversion rate and meat quality traits of pigs. Therefore, residual feed intake is often used as the main trait in the determination of feed utilization efficiency, and the feed conversion performance of pigs is indirectly predicted by evaluating its difference.

[0003] With the continuous development of single nucleotide polymorphism (SNP) chips and genome sequencing technology, whole genome association analysis has become a powerful tool for genetic analysis of important economic traits in livestock and poultry. Residual feed intake is regulated by micro-effect polygenes on multiple chromosomes, with a heritability of 0.12-0.52, which is a medium heritability trait with good selection progress. The peptidylproline isomerase-like protein-4 (PPIL4) gene encodes a peptidylproline isomerase-like protein and is 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. Peptidylproline isomerase-like protein-4 can enhance Wnt signal transduction by binding to JMJD6, and the Wnt signaling pathway plays a key role in the proliferation and differentiation of muscle satellite cells: when muscles are damaged or need to grow, Wnt signals promote satellite cells to enter the differentiation stage by upregulating key transcription factors such as Myf5 and MyoD, and finally form mature muscle fibers. Activation of the classic Wnt / β-catenin pathway increases the accumulation of β-catenin in the nucleus, increases the expression of cell cycle-related genes, and promotes satellite cells to enter a proliferative state from a quiescent state. In addition, activation of this pathway also inhibits fat production. The mutant peptidylproline isomerase-like protein-4 gene significantly reduces the ability of its protein product to bind to JMJD6, thereby reducing the expression of the Wnt signaling pathway, 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 provide a PPIL4 gene molecular marker associated with low residual feed intake in view of the shortcomings of the prior art and the difficulty in predicting and breeding low feed-to-meat ratio pigs in advance.

[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 the 133rd position where the C / T polymorphism exists.

[0009] At the same time, the present invention also provides an amplification primer for amplifying 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, 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 related to 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: 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.

[0012] The method specifically comprises the following steps: (1) Taking large white pig ear tissue samples and extracting total DNA; (2) using the extracted porcine genomic DNA as a template and using the primers to perform PCR amplification; (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 determined.

[0013] 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.

[0014] 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.

[0015] The application, wherein the screening of pig strains with low residual feed intake and low feed-to-meat ratio is achieved by the following method: detecting the genotype of the piglet at the nucleotide site g.16442886, 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.

[0016] The present invention also provides a method for breeding a pig strain 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 nucleotide site g.16442886 of the piglets by 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.

[0017] The present invention has the following beneficial effects: The PPIL4 gene molecular marker provided by the present invention is associated with the low residual feed intake trait. Therefore, pig strains with low residual feed intake can be screened by identifying the SNP marker. The obtained low residual feed intake trait strains show low feed-to-meat ratio and have important economic benefits and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The electrophoresis diagram is the result of amplifying the peptidylproline isomerase-like protein-4 gene using the primers of the present invention.

[0019] Figure 2 This is the peak diagram of DNA sequencing results of different genotypes of the mutation site of the peptidylproline isomerase-like protein-4 gene. DETAILED DESCRIPTION

[0020] The following examples are used 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 all fall within the scope of the present invention.

[0021] Example 1

[0022] 1. Source of experimental animals Collected from 200 Large White pigs of Shaanxi Huamu Xingnong Breeding Pig Technology Co., Ltd.

[0023] 2. Determination of residual feed intake The average daily feed intake (ADFI), individual body weight (BW), and average daily gain (ADG) of each pig during the fattening period of 90 to 160 days of age were collected. When the pig weight reached about 100 kg, the back fat thickness was measured using ultrasound. After the production performance measurement was completed, the residual feed intake (RFI) formula was used to calculate: RFI=ADFI-(b 1 ×ONBW+b 2 ×OFFBW+b 3 ×MWT+b 4 ×ADG+b 5 ×BF) MWT = [(ONBW +OFFBW) / 2]^0.75 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 back fat thickness; b1, b 2 、b 3 、b 4 、b 5 Corresponding trait regression coefficients.

[0024] 3. Genomic DNA Extraction 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.

[0025] Use the animal genomic DNA extraction kit to extract genomic DNA. The required reagents include: Proteinase K (Beijing Solebow Technology Co., Ltd.) Animal genomic DNA extraction kit: Sangon Biotech (Shanghai) Co., Ltd. Rapid PCR premix: Bio-Rad Biotechnology (Beijing) Co., Ltd. DNA Marker (DL1000): Shanghai Pudi Biotechnology Co., Ltd. 10×Loading Buffer: Bio-Rad Biotechnology (Beijing) Co., Ltd. Agarose: Sangon Biotech (Shanghai) Co., Ltd. TBE (10×) solution: Shanghai Bio-Tech Biotechnology Co., Ltd.

[0026] The specific steps are as follows: (1) Take 25 mg of left and right ear tissues and place them in a centrifuge tube. Add them to a 1.5 ml centrifuge tube and add 400 µl of BufferDigestion. Mix by vortexing. Incubate at 65°C in a water bath for 1 h until the cells are completely lysed.

[0027] (2) Add 20 μL of Proteinase K solution, shake to mix, and place in a 56°C water bath to digest overnight.

[0028] (3) Add 200 µl of Buffer PA, mix thoroughly by inversion, and place in a -20°C refrigerator for 5 min.

[0029] (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.

[0030] (5) Add an equal volume of isopropanol, invert 5 to 8 times to mix thoroughly, and leave at room temperature for 2 to 3 minutes. Centrifuge at 10,000 rpm for 5 minutes at room temperature and discard the supernatant.

[0031] (6) Add 1 ml of 75% ethanol, invert and rinse for 1-3 min, centrifuge at 10,000 rpm for 2 min, and discard the supernatant. (7) Repeat step 6.

[0032] (7) Open the lid and invert at room temperature for 5 to 10 minutes until the remaining ethanol evaporates completely.

[0033] (8) Dissolve the obtained DNA in 50~100 µl TE Buffer. The extracted DNA can be used for the next step of the experiment immediately or stored at -20°C.

[0034] (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.

[0035] 4. Pig genome 60K SNP chip genotyping and genome-wide association analysis The whole genome genotype of pigs was determined according to the company's standard process. The "Zhongxin No. 1" pig 50K SNP chip was used to perform SNP genotype detection on individual breeding pigs with residual feed intake measurement records, and all SNP marker detection results were quality controlled. A genome-wide significant SNP site on chromosome 1 of pigs was detected using whole genome association analysis, which was significantly associated with the residual feed intake trait ( P =3.73E-04)( Figure 1 ). By comparing with the international pig genome version 11.1 reference sequence, the SNPs are located in the coding region of the PPIL4 gene. According to existing literature and functional annotations, the PPILL4 gene has the potential to regulate animal muscle growth, and the SNPs variation in this gene regulates the phenotype of pig residual feed intake.

[0036] Example 2

[0037] This example verifies the SNP site g.16442886C / T in the PPIL4 gene significantly associated with the residual feed intake trait obtained in Example 1 in a large white pig population. SEQ ID NO: 1 is the product sequence after PCR amplification of the designed primers SEQ ID NO: 2 and SEQ ID NO: 3, and the SNP site significantly associated with the residual feed intake is the 133rd mutation in the sequence.

[0038] This example verifies the SNP site g.16442886C / T in the PPIL4 gene significantly associated with the residual feed intake trait obtained in Example 1 in a large white pig population. SEQ ID NO: 1 is the product sequence after PCR amplification of the designed primers SEQ ID NO: 2 and SEQ ID NO: 3, and the SNP site significantly associated with the residual feed intake is the 133rd mutation in the sequence.

[0039] 1. Extraction of pig genomic DNA Ear tissue samples of sows with residual feed intake were collected and placed in a centrifuge tube filled with 70% alcohol and stored in a -20°C refrigerator for later use. Genomic DNA from pig ear tissue was extracted using the above method, and after quality and concentration testing, the concentration was diluted to 50 ng / μL and stored at -20°C for later use.

[0040] 2. PCR amplification and sequencing of target fragments Using the extracted pig DNA as a template, PCR amplification was performed according to the designed specific primers: the PCR amplification reaction system was: 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, and 17.38 μL of double distilled water; the reaction procedure for PCR amplification 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.

[0041] The PCR product was detected by electrophoresis in 1.2% agarose gel. The size of the amplified target fragment was 442 bp. 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.

[0042] Table 1 shows the effect of the PPIL4 gene g.16442886C / T mutation site on the residual feed intake trait in the resource population. As shown in Table 1, compared with the TT genotype individuals at the SNP site (g.16442886C / T), the CC genotype individuals have lower residual feed intake. This shows that in the selection of breeding pigs, the successive breeding of CC genotype individuals at the g.16442886C / T site can gradually reduce the residual feed intake of the pig herd and improve the feed-to-meat ratio and growth rate.

[0043] Table 1. Association analysis between PPIL4 gene SNP locus (g.16442886 C / T) and residual feed intake traits .

Claims

1. A PPIL4 gene molecular marker associated with residual feed intake trait, characterized in that: The SNP marker is located on the nucleotide sequence of the peptidyl proline 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 pig genome version 11.1 reference sequence, and has a C / T polymorphism, wherein the residual feed intake of the CC genotype at the site is significantly lower than that of the TT genotype.

2. The PPIL4 gene molecular marker associated with the low residual feed intake trait according to claim 1, characterized in that: The molecular marker sequence is shown in SEQ ID NO: 1, and the SNP marker site is located at the 133rd position where C / T polymorphism exists.

3. A primer for amplifying the PPIL4 gene molecular marker associated with the low residual feed intake trait as claimed in claim 1 or 2, or for detecting the low residual feed intake trait of Large White pigs, characterized in that: It is designed based on the nucleotide sequence containing the SNP marker as claimed in claim 1.

4. The amplification primer according to claim 3, characterized in that The upstream primer sequence of the primer is shown in SEQ ID NO:2, and the downstream primer sequence is shown in SEQ ID NO:

3.

5. A method for detecting the PPIL4 gene molecular marker associated with the low residual feed intake trait as claimed in claim 1 or 2, or for detecting the low residual feed intake trait of Large White pigs, characterized in that: The steps include: 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.

6. The method according to claim 5, characterized in that The following steps are involved: (1) Taking large white pig ear tissue samples and extracting total DNA; (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primers described in claim 3 or 4; (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 determined.

7. The method according to claim 6, characterized in that: 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 primer is 10 mol / L, and the Taq enzyme is a reagent from Dalian TaKaRa Biotechnology Co., Ltd.; the reaction procedure of 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.

8. Use of the SNP marker according to claim 1 or 2, the amplification primer according to claim 3 or 4, or the method according to any one of claims 5 to 7 in screening pig strains with low residual feed intake and low feed-to-meat ratio.

9. The use according to claim 8, characterized in that The screening of low residual feed intake and low feed-to-meat ratio pig strains is achieved by the following method: detecting the genotype of the g.16442886 nucleotide site of the reserve pigs, and selecting CC type individuals at the g.16442886 nucleotide site as low residual feed intake and low feed-to-meat ratio breeding pigs.

10. A method for breeding a pig strain with low residual feed intake and low feed-to-meat ratio, characterized in that: Obtain offspring piglets through self-pollination or hybridization, detect the genotype of the nucleotide site g.16442886 of the reserve pigs using the method described in any one of claims 5 to 7, and select CC type individuals at the nucleotide site g.16442886 as breeding pigs with low residual feed intake and low feed-to-meat ratio.

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