SNP molecular markers on pig chromosome 17 related to pig abdominal circumference and their applications

Abstract

This invention belongs to the fields of molecular biology and molecular marker technology, specifically relating to a SNP molecular marker located on pig chromosome 17 that is associated with pig abdominal circumference and its application. The SNP site of this molecular marker on pig chromosome 17 corresponds to the T>C mutation at position 23660143 bp on chromosome 17 in the International Pig Reference Genome 11.1. This molecular marker was obtained through genome-wide association analysis and can significantly affect the pig abdominal circumference trait. This invention also provides a primer pair for identifying this molecular marker. Using this molecular marker and primer pair, an efficient and accurate molecular marker-assisted breeding technology can be established, which can be applied to the genetic improvement of pig abdominal circumference, thereby reducing pig abdominal circumference, increasing enterprise profits, and enhancing core competitiveness.

Description

Technical Field

[0001] This invention belongs to the fields of molecular biotechnology and molecular marker technology, specifically relating to a SNP molecular marker located on pig chromosome 17 that is related to pig abdominal circumference and its application. Background Technology

[0002] In recent years, breeding lean-type pigs has remained a major direction for pig farming development in my country, and abdominal circumference phenotype is an important indicator for selecting and retaining lean-type pigs. Abdominal circumference is negatively correlated with lean meat percentage; therefore, appropriately reducing abdominal circumference is beneficial for increasing carcass lean meat percentage and accelerating the breeding of lean-type pigs. Furthermore, pigs with full fore and hindquarters, small abdominal circumference, and a compact appearance score higher in live animal grading for lean-type pigs, resulting in better economic benefits. However, since abdominal circumference traits often only become relevant for selection after pigs have reached a certain age, traditional selection methods cannot determine whether to retain pigs for breeding in the early stages of growth, making the breeding process based on abdominal circumference traits relatively slow.

[0003] With the development of genotyping technologies such as gene chips and next-generation sequencing, breeders and scientists have discovered an increasing number of single nucleotide polymorphism (SNP) sites that are significantly associated with phenotypic traits through genome-wide association studies (GWAS). Based on this, marker-assisted selection (MAS) technology has been developed to shift from phenotypic selection to the identification of genes influencing trait genetic variation and functional genes, effectively promoting early selection of traits and significantly accelerating the breeding process. For example, if MAS technology can be used to improve abdominal girth in pigs, it will effectively accelerate the breeding process of this trait and improve production efficiency. Summary of the Invention

[0004] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide a SNP molecular marker located on chromosome 17 of pigs that is related to the abdominal circumference of pigs.

[0005] Another object of the present invention is to provide the application of the above-mentioned SNP molecular markers located on pig chromosome 17 that are related to pig abdominal circumference.

[0006] Another object of the present invention is to provide a primer pair for identifying the aforementioned SNP molecular markers located on pig chromosome 17 that are associated with pig abdominal circumference.

[0007] A fourth objective of this invention is to provide applications of the aforementioned primer pairs.

[0008] The fifth objective of this invention is to provide a method for genetic improvement of pigs.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A molecular marker of a SNP located on pig chromosome 17 that is associated with pig abdominal circumference, the SNP site of which corresponds to the T>C mutation at 23660143 bp on chromosome 17 in International Pig Reference Genome Version 11.1;

[0011] The nucleotide sequence of the SNP molecular marker located on pig chromosome 17 that is associated with pig abdominal girth is shown in SEQ ID NO.1, where M in the sequence is T or C, which leads to different abdominal girth traits;

[0012] The SNP site of the SNP molecular marker on pig chromosome 17 that is related to pig abdominal circumference is the nucleotide mutation of TC at position 263 of the sequence marked in SEQ ID NO:1 (a single base mutation of nucleic acid located at the 263 bp of this sequence fragment, named: g.23660143T>C);

[0013] The aforementioned SNP molecular markers located on pig chromosome 17 and associated with pig abdominal girth are used in the identification of abdominal girth traits and genetic breeding of Duroc pigs.

[0014] A method for detecting abdominal girth traits in pigs includes the following steps:

[0015] The above-mentioned SNP molecular markers related to pig abdominal circumference on pig chromosome 17 were detected. The SNP site of the SNP molecular marker is either T or C.

[0016] The preferred pigs are American Duroc strains and their synthetic lines;

[0017] A primer pair for identifying the aforementioned SNP molecular markers located on pig chromosome 17 that are associated with pig abdominal circumference, comprising primers primer-F and primer-R, has the following nucleotide sequence:

[0018] Upstream primer-F: 5'-CAACCCACTCCTTCCCCTTC-3';

[0019] Downstream primer primer-R: 5'-AATGAATGCTGGAGGGGGTG-3';

[0020] The application of the primer pairs described above in identifying the influence of abdominal girth traits in breeding pigs;

[0021] Application of the primer pairs in marker-assisted breeding of pigs;

[0022] Application of the primer pairs in reducing abdominal circumference in breeding pigs;

[0023] A method for genetic improvement of pigs, comprising the following steps:

[0024] Identify the aforementioned SNP molecular markers on chromosome 17 of pigs in the core breeding herd that are related to abdominal circumference, and make corresponding selections based on these molecular markers: select breeding pigs with the TT and TC genotypes at position 23660143bp on chromosome 17 according to the International Swine Reference Genome 11.1, and cull breeding pigs with the CC genotype at this position, in order to increase the frequency of the T allele at this locus in each generation, thereby reducing the abdominal circumference of the offspring pigs;

[0025] The preferred pigs are American Duroc strains and their synthetic lines;

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] (1) This invention studies and identifies the molecular markers that affect pig abdominal circumference and locate them on the nucleotide sequence of pig chromosome 17. It verifies their effect on the pig abdominal circumference trait and finally establishes an efficient and accurate molecular marker-assisted breeding technology. This technology is applied to the genetic improvement of reducing the abdominal circumference of breeding pigs, thereby increasing the lean meat rate of breeding pigs, reducing breeding losses, improving production efficiency, increasing enterprise economic profits, and increasing core competitiveness.

[0028] (2) This invention provides a primer pair for SNP molecular markers located on chromosome 17 of pigs that are related to pig abdominal circumference. Through this primer pair, an efficient and accurate molecular marker-assisted breeding technology can be established, and traits can be selected quickly and accurately, thus accelerating the breeding process. When applied to the genetic improvement of abdominal circumference traits in breeding pigs, the abdominal circumference of pigs can be reduced, thereby accelerating the progress of pig genetic improvement.

[0029] (3) By selecting the dominant allele of the molecular marker, the present invention provides a method for pig breeding, which can increase the frequency of the dominant allele generation by generation, reduce the abdominal circumference of the breeding pig, accelerate the progress of pig genetic improvement, and thus effectively improve the economic benefits of pig breeding. Attached Figure Description

[0030] Figure 1 This is a Manhattan plot of genome-wide association (GWAS) analysis of abdominal circumference in American Duroc pigs on chromosome 17; where: the horizontal axis represents the chromosome number of the pig; and the vertical axis represents the -logP value.

[0031] Figure 2 This is a graph showing the results of the analysis of the abdominal circumference phenotype ratios of pigs with different genotypes. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0033] Experimental pig population: A total of 2,206 American Duroc pigs were used in this experiment.

[0034] Example 1 specifically explains the measurement process of abdominal circumference in this invention.

[0035] The abdominal circumference phenotypes of all pigs were accurately measured using a soft tape measure according to a unified method, with a precision of centimeters. The pigs weighed between 105 and 150 kg during measurement. Before measurement, the pigs were fixed. When the pigs stood naturally, the soft tape measure was gently pressed against the body surface, avoiding excessive tension or relaxation. The measurement process was rapid and accurate. The measurement method of abdominal circumference was: the vertical circumference at the largest part of the abdomen.

[0036] The experimental pig population used in this invention consisted of 2,206 purebred American Duroc pigs from the Breeding Pig Branch of Guangdong Wenshi Food Group Co., Ltd., which was the core group of the breeding pig branch, and the pedigree records of the group were detailed. In this experiment, American Duroc breeding pigs were selected from this resource population and were fed and managed by professional staff according to a unified feeding standard, with free access to food and water.

[0037] Example 2 specifically explains the invention process of gene markers in this invention.

[0038] (1) Sample collection

[0039] The method for extracting DNA from ear tissue samples of American Duroc pigs referred to the standard phenol-chloroform method for extracting genomic DNA. The DNA quality and concentration of all samples were measured using a NanoDrop 2000 / 2000C nucleic acid and protein detector. The DNA quality detection standard was that the light absorption ratio A2_{60} / A2_{80} of the sample was between 1.8 and 2.0, and when the A2_{60} / A2_{30} ratio was between 1.8 and 2.1, the DNA sample was judged to be qualified. Finally, the qualified DNA samples were uniformly diluted to 50 ng / μL.

[0040] (2) Genotyping of 50K SNPs in the porcine whole genome

[0041] For the GeneSeek Genomic Profiler Porcine 50K SNP genotyping platform, chip hybridization and result scanning were performed according to the instructions and standard procedures of Illumina Infinium. Finally, genotype data were read using GenomeStudio software.

[0042] The genotype imputation website - SWIM (Swine Imputation Server) website (https: / / quantgenet.msu.edu / swim / ) was used to impute genotypes for 2,206 American Duroc pigs to obtain genotype data.

[0043] The genotypic data obtained after imputation were quality controlled using PLINK v1.90. Data with a rejection rate <99%, mimor allelic frequency (MAF) <1%, or deviation from Hardy-Weinberg equilibrium (HWE) were assessed with a p-value ≤10. -6 SNP markers were selected, excluding individuals with a detection rate <90%, a family Mendelian error rate >0.1, and SNPs located at unknown locations or on sex chromosomes. The remaining 281,798 SNP markers and 2,206 samples from the abdominal circumference quality control were used for subsequent data analysis.

[0044] (3) Genome-wide association analysis (GWAS)

[0045] To eliminate population stratification effects, this invention employs a linear mixed model with single-point regression analysis combined with GWAS analysis using the GenABEL software package in R. The analysis model utilizes inter-individual genomic similarity to correct for stratification effects. Referring to the human genome significance threshold, this invention sets the genome-level significance threshold for the association between SNPs and abdominal girth traits to 5 × 10⁻⁶. -8 The significance threshold at the chromosome level is 1×10⁻⁶. -6 .

[0046] GWAS analysis results are as follows Figure 1 As shown. From Figure 1 It is known that in Duroc, there is a locus on chromosome 17 that significantly affects abdominal girth, with the strongest association SNP being g.23660143T>C (P = 4.98 × 10⁻⁶). -7 (The 263rd nucleotide in SEQ NO.1 corresponds to the T>C mutation at 23660143 bp on chromosome 17 in International Pig Reference Genome Version 11.1).

[0047] (4) Association analysis between different genotypes and abdominal circumference phenotype in breeding pigs: According to Table 1, the SNP site g.23660143T>C of the molecular marker was significantly correlated with the abdominal circumference trait (P<0.01), indicating that this molecular marker significantly affects the abdominal circumference of pigs. Assisted selection at this SNP site in pigs can reduce the abdominal circumference of the population, thereby accelerating the breeding process of lean-type breeding pigs. Furthermore, according to Table 1 and... Figure 2 It was also found that the TT type had a lower abdominal circumference than the TC and CC types, indicating that homozygous CC was the most detrimental to the abdominal circumference of breeding pigs. Therefore, gradually eliminating CC-type breeding pigs in breeding to increase the frequency of the T allele at this locus in each generation can significantly reduce the abdominal circumference of the breeding pig population, bringing more economic benefits to breeding enterprises.

[0048] Table 1. Correlation between SNP sites of molecular markers (g. 23660143T>C) and traits.

[0049]

[0050] Note: Pigs weighing 120-150kg are relatively few in number and are not included in the statistics in the table above.

[0051] Example 3 explains in detail the invention process of detecting SNP markers.

[0052] (1) The DNA sequence of SEQ ID NO:1 on pig chromosome 17 was downloaded from the Ensembl website (http: / / asia.ensembl.org / index.html). Primers were designed using the primer design software Primer Premier 6.0. The DNA sequences of the designed primers are shown below:

[0053] Upstream primer-F: 5'-CAACCCACTCCTTCCCCTTC-3';

[0054] Downstream primer primer-R: 5'-AATGAATGCTGGAGGGGGTG-3'.

[0055] (2) PCR amplification system and conditions

[0056] Prepare a 10 μL system, including 1 μL DNA sample, 0.3 μL upstream primer, 0.3 μL downstream primer, 5 μL PCR mix, and 3.4 μL ddH2O. The PCR conditions are as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 64℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 35 cycles; and a final extension at 72℃ for 5 min.

[0057] (3) DNA Sequence Sequencing Identification: Sequencing was performed at BGI Genomics Co., Ltd. in Shenzhen, with two sequencing reactions for each gene fragment. The obtained sequences were compared with the NCBI genome sequence to identify mutations at corresponding SNP sites. The sequencing results are shown below:

[0058]

[0059]

[0060] Note: M marked in the sequence is the mutation site, indicated by an underline (the mutated base in parentheses represents the allele mutation). The beginning and end of the sequence are bolded to indicate the primer binding position.

[0061] Example 4: Analysis of the T>C effect of SNP sites g.23660143 on molecular markers

[0062] This invention provides a SNP molecular marker that can significantly reduce the abdominal circumference of Duroc breeding pigs. Using this SNP molecular marker for marker-assisted selection can greatly accelerate the abdominal circumference breeding process in Duroc pigs. If this invention selects all individuals with the CC genotype (affecting abdominal circumference) into TT genotype individuals, the abdominal circumference of each pig at a weight of 100-120 kg can be reduced by 2.38%. Therefore, by using marker-assisted selection to gradually cull pigs with the CC genotype within the population, the allele frequency of the T allele can be significantly increased, reducing the abdominal circumference of breeding sows, reducing breeding losses while improving animal welfare, accelerating the progress of pig genetic improvement, and thus effectively improving the economic benefits of pig breeding.

[0063] This invention provides a novel molecular marker for marker-assisted selection of pigs by detecting the mutation site at position 263 in the SEQ ID NO:1 sequence and conducting preliminary association analysis between its genotype and the abdominal circumference of the breeding pig.

[0064] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of a SNP molecular marker related to pig abdominal girth in the identification of pig abdominal girth traits and in the genetic breeding of pig abdominal girth traits, characterized in that: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1, where M in the sequence is T or C, resulting in different abdominal girth traits in pigs. The TT genotype has a lower abdominal girth than the TC and CC genotypes. The pigs mentioned are American Duroc strains and their synthetic lines.

2. A method for detecting abdominal girth traits in pigs, characterized in that... It includes the following steps: The SNP molecular marker described in claim 1 is detected, wherein the TT genotype has a lower abdominal circumference than the TC and CC genotypes; The pigs mentioned are American Duroc strains and their synthetic lines.

3. The application of a primer pair for identifying SNP molecular markers related to abdominal girth in pigs in the identification of abdominal girth traits, characterized in that: The primer pair comprises primers primer-F and primer-R, and their nucleotide sequences are as follows: Upstream primer-F: 5'-CAACCCACTCCTTCCCCTTC-3'; Downstream primer-R: 5'-AATGAATGCTGGAGGGGGTG-3'; The SNP molecular marker is the SNP molecular marker described in claim 1, wherein the TT genotype has a lower abdominal circumference than the TC and CC genotypes at the location of the molecular marker; The pigs mentioned are American Duroc strains and their synthetic lines.

4. The application of a primer pair for identifying SNP molecular markers related to pig abdominal girth in marker-assisted breeding of pig abdominal girth, characterized in that: The primer pair comprises primers primer-F and primer-R, and their nucleotide sequences are as follows: Upstream primer-F: 5'-CAACCCACTCCTTCCCCTTC-3'; Downstream primer primer-R: 5'-AATGAATGCTGGAGGGGGTG-3'; The SNP molecular marker is the SNP molecular marker described in claim 1, wherein the TT genotype has a lower abdominal circumference than the TC and CC genotypes at the location of the molecular marker; The pigs mentioned are American Duroc strains and their synthetic lines.

5. A method for genetic improvement of pigs, characterized in that... It includes the following steps: Identify the SNP molecular markers described in claim 1 for breeding pigs in the core breeding pig herd, and make corresponding selections based on the molecular markers: select breeding pigs with the SNP molecular markers described in claim 1 as TT genotype and TC genotype for successive generations, and eliminate breeding pigs with the CC genotype, so as to increase the frequency of the allele T at the SNP locus in each generation, thereby reducing the abdominal circumference of the offspring pigs. The pigs mentioned are American Duroc strains and their synthetic lines.

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