A molecular marker of the FRMD4A gene associated with the teat number 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 relates to a SNP marker of the FRMD4A gene associated with the teat number trait of Large White pigs, a detection method thereof, and an application thereof. The SNP marker is located on the nucleotide sequence of the FRMD4A gene on pig chromosome 10. The site of the SNP marker is the g.48019215 nucleotide site on pig chromosome 10 in the International Porcine Genome Version 11.1 reference sequence, and has a C / T polymorphism. The SNP marker is significantly associated with the teat number trait. The SNP marker provided by the present invention is associated with the teat number trait of pigs. By identifying the SNP marker, pig strains with a high teat number can be screened. The resulting high-fertility strains with a high teat number have important economic benefits and social value.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a FRMD4A gene molecular marker related to the teat number trait of Large White pigs, a detection method and an application thereof. Background Art

[0002] The Large White pig, a widely bred commercial pig breed worldwide, holds a crucial position in modern animal husbandry due to its exceptional growth performance and reproductive capacity. Nipple number is a key reproductive trait in pigs, directly impacting a sow's lactation ability and piglet survival rate. The greater the number of nipples, the more effective lactation space a sow can provide, which is crucial for providing adequate nutrition during lactation and promoting healthy piglet development. With significant improvements in litter size in recent years, the number of piglets in a litter sometimes exceeds the number of functional nipples a sow has, limiting piglet production. Improving sow lactation ability, increasing sow lactation space, and ultimately improving piglet nutritional intake and survival rates has become a top priority in the current livestock industry. The development and utilization of novel molecular breeding markers to increase nipple number has garnered significant attention.

[0003] In commercial pig breeding, teat number is a key indicator for assessing sow reproductive performance. Sows with a higher teat number are typically selected for breeding to improve overall herd reproductive efficiency. Teat number is regulated by multiple genes on different chromosomes, with a heritability of approximately 0.32 to 0.39, making it a medium-heritability trait. Traditional breeding methods based on pedigree and phenotypic records are costly and ineffective in improving reproductive traits. The FRMD4A (FERMdomain-containing4A) gene plays a crucial role in mammary gland development. Genome-wide association studies (GWAS) have identified FRMD4A as a key candidate gene regulating teat number in Large White pigs. Mammary gland and teat development depend on epithelial cell polarization. During teat formation, epithelial cells polarize to form distinct membrane domains, enabling cell differentiation and specific functions. Differentiation of the apical and basal membranes helps mammary ducts form tubular structures within the teat and maintains the functional characteristics of teat tissue. Arf6 is a core player in actin cytoskeleton dynamics and membrane trafficking. It is activated during the formation of primitive "punctate" adherens junctions and is essential for establishing epithelial polarity. Studies have shown that FRMD4A regulates Arf6 activation and influences epithelial polarization by linking specific guanine nucleotide exchange factors (GEFs) of the cytochrome family with Par-3 proteins. Deficiency of the FRMD4A gene can impair nipple development. Therefore, identifying FRMD4A genotypes and applying them to marker-assisted selection is crucial for developing new strains with increased nipple numbers, improving sow lactation capacity, and enhancing piglet nutrition. Summary of the Invention

[0004] The purpose of the present invention is to address the shortcomings of existing technologies and the difficulty in predicting and breeding pigs with multiple nipple numbers in advance, and to provide FRMD4A gene molecular markers related to the multiple nipple number trait and their breeding applications.

[0005] Another object of the present invention is to provide primers and detection methods for detecting the above-mentioned molecular markers and related applications.

[0006] The technical solution provided by the present invention is: a FRMD4A gene molecular marker related to the multiple nipple number trait, the SNP marker is located on the FRMD4A gene nucleotide sequence from 47384867 to 48048948 on pig chromosome 10, the SNP marker site is the g.48019215 nucleotide site on pig chromosome 10 of the international pig genome version 11.1 reference sequence, and has a C / T polymorphism. The SNP marker is significantly correlated with the nipple number trait, and the number of nipples of pigs with the CC genotype at this site is significantly higher than that of pigs with the TT genotype.

[0007] Preferably, the molecular marker sequence is as shown in SEQ ID NO: 1, and the SNP marker site is located at position 301 where a C / T polymorphism exists.

[0008] At the same time, the present invention also provides a FRMD4A gene molecular marker for amplifying the Large White pig teat number trait, or an amplification primer for detecting the Large White pig teat number trait, which is designed based on the nucleotide sequence containing the SNP marker.

[0009] 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.

[0010] The present invention also provides a method for detecting the FRMD4A gene molecular marker associated with the Large White pig teat number trait or detecting the Large White pig teat number trait, comprising the following steps:

[0011] 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:

[0013] (1) Take pig ear tissue samples and extract total DNA;

[0014] (2) using the extracted porcine genomic DNA as a template and performing PCR amplification using the primers described in claim 3 or 4;

[0015] (3) The amplified product was sequenced, the sequencing results were analyzed, and the C / T polymorphism at position 301 of SEQ ID NO: 1 was interpreted.

[0016] Furthermore, in the method described above, 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.

[0017] The present invention also provides the use of the SNP marker, the amplification primer, or the method in detecting the teat number trait of Large White pigs or screening pig breeds with a large teat number.

[0018] The application, wherein the screening of high nipple number pig strains is achieved by the following method: detecting the genotype of the piglet's g.48019215 nucleotide site, and selecting CC type individuals at the g.48019215 nucleotide site as high nipple number high-fertility breeding pigs.

[0019] The present invention also provides a method for breeding high-fertility sows with a large number of nipples. The method comprises obtaining offspring piglets by self-pollination or hybridization, detecting the genotype of the piglets at the nucleotide site g.48019215 using the method described in any one of claims 5 to 7, and selecting CC-type individuals at the nucleotide site g.48019215 as high-fertility sows with a large number of nipples.

[0020] The present invention has the following beneficial effects:

[0021] The FRMD4A gene molecular marker provided by the present invention is associated with the trait of multiple nipples. Therefore, pig breeds with multiple nipples can be screened by identifying the SNP marker. The resulting pig breeds with multiple nipples exhibit higher lactation performance and fertility, and have important economic benefits and social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The chromosome-level significant SNP marker results from the genome-wide association analysis for nipple number are shown. Information on the 18 autosomes and the X chromosome is indicated on the x-axis.

[0023] Figure 2 The electrophoresis diagram of FRMD4A gene amplified using the primers of the present invention is shown.

[0024] Figure 3 This is the peak diagram of DNA sequencing results for different genotypes of the molecular marker site of the FRMD4A 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. Phenotypic determination

[0030] When the sows reach 120 days of age, the effective nipples are counted and the non-functional nipples, such as blind nipples, inverted nipples and supernumerary nipples, are removed.

[0031] 3. Genomic DNA Extraction

[0032] 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.

[0033] Genomic DNA was extracted using an animal genomic DNA extraction kit. The required reagents include:

[0034] Proteinase K (Beijing Solaibao Technology Co., Ltd.)

[0035] Animal genomic DNA extraction kit: Sangon Biotech (Shanghai) Co., Ltd.

[0036] DNAMarker (DL1000): Shanghai Pudi Biotechnology Co., Ltd.

[0037] 10×LoadingBuffer: Bio-Tech (Beijing) Co., Ltd.

[0038] Agarose: Sangon Biotech (Shanghai) Co., Ltd.

[0039] TBE (10×) solution: Shanghai Beyotime Biotechnology Co., Ltd.

[0040] The specific steps are as follows:

[0041] (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 hour until the cells are completely lysed.

[0042] (2) Add 20 μL of Proteinase K solution, shake to mix, and place in a 56°C water bath for digestion overnight.

[0043] (3) Add 200µl Buffer PA, mix thoroughly by inversion, and place in a -20°C refrigerator for 5 minutes.

[0044] (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.

[0045] (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.

[0046] (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.

[0047] (7) Open the lid and invert at room temperature for 5 to 10 minutes until the remaining ethanol is completely evaporated.

[0048] (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.

[0049] (9) Determine the concentration. After testing the mass and concentration using a spectrophotometer, dilute the sample to 50 ng / μL and store at -20°C for later use.

[0050] 4. Pig genome 60K SNP chip genotyping and genome-wide association analysis

[0051] The whole genome genotype of pigs was determined according to the company's standard process. The "Zhongxin No. 1" pig 50KSNP chip was used to perform SNP genotyping on individual pigs with nipple number determination records and quality control of all SNP marker detection results was performed. Three genome-wide significant SNPs were detected using whole genome association analysis and concentrated on pig chromosome 10 ( Figure 1 ). By comparing with the reference sequence of the International Pig Genome Version 11.1, one of the SNPs significantly associated with nipple number ( P =4.63E-09) is located within the coding region of the FRMD4A gene. Based on existing literature and functional annotation, the FRMD4A gene is crucial for epithelial cell polarization. Its deficiency leads to impaired nipple development. SNP variation in the FRMD4A gene may regulate and influence the nipple number phenotype in pigs.

[0052] Example 2

[0053] This example validates the SNP g.48019215C / T on FRMD4A, identified in Example 1, which is significantly associated with teat number, 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 teat number is mutation 301 in this sequence.

[0054] 1. Extraction of pig genomic DNA

[0055] Ear tissue samples from sows with a recorded nipple number trait were collected, placed in a centrifuge tube filled with 70% ethanol, 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.

[0056] 2. PCR amplification and sequencing of target fragments

[0057] Using the extracted porcine DNA as a template, PCR amplification was performed according to the designed 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 reagent, and 17.38 μL of double-distilled water; the PCR amplification reaction procedure 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.

[0058] The PCR product was detected by electrophoresis in 1.2% agarose gel. The amplified target fragment was 412 bp in size. Figure 2 The remaining amplified products were sequenced, and the sequencing results were compared and analyzed with the porcine FRMD4A gene fragment sequence in GenBank using DNAMAN software. The g.48019215C / T genotype was interpreted, and the single marker association analysis between SNP genotype and teat number trait was performed using the chi-square test module of SAS9.4 software.

[0059] Table 1 shows the effect of the g.48019215C / T mutation on teat number in a resource population. As shown in Table 1, there is a significant difference in teat number between individuals with the CC genotype (g.48019215C / T) and those with the TT genotype (P < 0.05). This suggests that in sow selection, successive generations of individuals with the CC genotype at the g.48019215C / T locus can gradually increase teat number and improve sow lactation and fertility.

[0060] Table 1. Association analysis between SNP locus (g.48019215C / T) and nipple number trait

[0061] 。

Claims

1. A method for detecting the number of teats in Large White pigs, characterized in that: The steps include: A sequence containing a SNP marker in the pig genome is amplified by PCR, the amplified product is sequenced, and the C / T polymorphism of the site is determined; the SNP marker is located on the nucleotide sequence of the FRMD4A gene at positions 47384867 to 48048948 on pig chromosome 10, the SNP marker site is the g.48019215 nucleotide site on pig chromosome 10 of the International Swine Genome Version 11.1 reference sequence, and has a C / T polymorphism, wherein the number of nipples of pigs with the CC genotype at the site is significantly higher than that of pigs with the TT genotype; the nucleotide sequence of the SNP marker is shown in SEQ ID NO: 1, and the SNP marker site is located at position 301 where a C / T polymorphism exists.

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 using primers for PCR amplification; the primers are designed based on the nucleotide sequence containing the SNP marker as described in claim 1; (3) The amplified product was sequenced, the sequencing results were analyzed, and the C / T polymorphism at position 301 of SEQ ID NO: 1 was interpreted.

3. The method according to claim 2, wherein 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.

4. 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 each of the 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, and the concentration of the primers is 10 mol / L; 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.

5. Use of the primers in the method according to any one of claims 2 to 4, or the method according to any one of claims 1 to 4, in detecting the teat number trait of Large White pigs or screening Large White pig strains with a high teat number.

6. The use according to claim 5, characterized in that The screening of Large White pig strains with a high nipple number is achieved by the following method: detecting the genotype of the piglets at position 301 as shown in SEQ ID NO: 1, and selecting CC-type individuals at the nucleotide site as Large White pig breeders with a high nipple number and high fertility.

7. A method for breeding high-fertility pigs with a large number of nipples, characterized in that: The offspring piglets are obtained by self-pollination or hybridization, and the genotype of the nucleotide site g.48019215 of the piglets is detected by the method described in any one of claims 1 to 4, and CC type individuals with the nucleotide site g.48019215 are selected as high-fertility breeding pigs with multiple nipples, wherein the breeding pigs are Large White pigs.

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