A snp molecular marker related to total litter size of pigs and application thereof

Abstract

The application discloses a SNP (Single Nucleotide Polymorphism) molecular marker related to total piglet number of pigs and application thereof. Therefore, through application of the molecular marker, a high-efficiency and accurate molecular marker assisted breeding technology can be quickly established, and the molecular marker assisted breeding technology is applied to genetic improvement of improvement of total piglet number of breeding pigs, so that the reproductive performance of a pig population is improved, economic profit of an enterprise is improved, and core competitiveness is increased.

Description

Technical Field

[0001] This invention relates to the field of molecular markers and animal genetics breeding, and in particular to a SNP molecular marker related to the total number of piglets born and its application. Background Technology

[0002] In hog production, litter size is an important economic indicator and a key factor affecting the economic benefits of hog production. Traditional breeding methods for litter size are mainly based on the observation and evaluation of reproductive traits, but this method has certain limitations, such as long breeding cycles and high evaluation difficulty. Total litter size is a complex trait controlled by multiple genes and has low heritability. Therefore, using molecular markers for assisted breeding is a more effective selection breeding method.

[0003] Thanks to the development and widespread adoption of high-throughput sequencing technology, the study of genomic data has provided a wealth of information for pig breeding. Genome-wide association studies (GWAS) are used to identify gene loci associated with litter size traits in pigs, leading to a better understanding of the genetic mechanisms underlying total litter size. This not only provides a scientific basis for improving the total litter size of pig breeds but also helps breeders select pigs with higher total litter sizes, thereby improving the economic benefits for breeding companies. Summary of the Invention

[0004] The purpose of this invention is to provide an SNP molecular marker related to the total number of piglets born and its application, in order to solve the above-mentioned problems.

[0005] According to a first aspect of the present invention, a SNP molecular marker associated with total litter size in pigs is provided, wherein the SNP molecular marker is an A>C base mutation located at position 107777216 bp on chromosome 1 of the international pig genome Sscrofa 11.1 version (this molecular marker can be abbreviated as g.141A>C hereinafter). Therefore, this molecular marker enables the rapid establishment of efficient and accurate marker-assisted breeding technology, which can be applied to the genetic improvement of total litter size in breeding pigs, thereby improving the reproductive performance of pig populations, increasing enterprise economic profits, and enhancing core competitiveness.

[0006] In some embodiments, the upstream and downstream gene sequences of the SNP molecular marker site are shown in SEQ NO.1, and the SNP molecular marker is located at position 141 of the sequence shown in SEQ ID No:1, where M represents an A>C base mutation (this molecular marker can be abbreviated as g.141A>C in the following text).

[0007] According to a second aspect of the present invention, an application of a SNP molecular marker in the selection of total litter size in pigs is provided. This SNP molecular marker is an A>C base mutation located at position 107777216 bp on chromosome 1 of the international pig genome Sscrofa 11.1 version, or an A>C base mutation represented by M at position 141 of the sequence shown in SEQ ID No:1. Therefore, by applying this SNP molecular marker, the total litter size of a sow population can be increased, significantly improving pig reproductive performance, enhancing the core competitiveness of enterprises, and increasing the economic value of sows.

[0008] In some implementations, the application includes the following steps:

[0009] 1) Detect the molecular marker g.141A>C in replacement breeding pigs;

[0010] 2) Select individuals with the AA or AC genotype obtained in step 1) as breeding pigs, and cull individuals with the CC genotype to breed pigs with high total litter size.

[0011] According to a third aspect of the present invention, an application of an SNP molecular marker in breeding high total litter size pig breeds is provided. This SNP molecular marker is an A>C base mutation located at position 107777216 bp on chromosome 1 of the international pig genome Sscrofa 11.1 version, or an A>C base mutation represented by M located at position 141 of the sequence shown in SEQ ID No:1. Therefore, by breeding high total litter size pig breeds, the gilts bred from these breeds all possess the high total litter size trait, thereby improving the reproductive performance of gilts and achieving the economic goal of producing more piglets.

[0012] In some implementations, the application includes the following steps:

[0013] 1) Detect the molecular marker g.141A>C in replacement breeding pigs;

[0014] 2) Select individuals with the AA or AC genotype obtained in step 1) as breeding pigs, and mate these breeding boars and sows.

[0015] 3) Detect the genotype of the molecular marker g.141A>C in the pigs born from mating in step 2), retain individuals with AA or AC genotypes, eliminate individuals with CC genotypes, and breed them to cultivate high reproductive performance pig breeds with high total litter size.

[0016] According to a fourth aspect of the present invention, a method for genetic improvement of pigs is provided, the method comprising the following steps:

[0017] 1) Detect the molecular marker g.141A>C in replacement breeding pigs;

[0018] 2) Select breeding pigs with the molecular marker genotype AA or AC from step 1), and cull individuals with the CC genotype;

[0019] 3) Use the individuals selected in step 2) as breeding pigs for breeding. In the offspring, continue to select breeding pigs with molecular marker genotypes of AA or AC from step 1) and eliminate individuals with AA genotype; in order to increase the frequency of allele A in the offspring pig population generation by generation, thereby increasing the total number of piglets born in the offspring pigs.

[0020] Therefore, this method can improve the frequency of allele A in a pig population, thereby increasing the total number of piglets born in offspring.

[0021] According to a fifth aspect of the present invention, an application of an SNP molecular marker in screening piglets for the genetic trait of high total litter size is provided. This SNP molecular marker is an A>C base mutation located at position 107777216 bp on chromosome 1 of the international pig genome Sscrofa 11.1 version, or an A>C base mutation represented by M at position 141 of the sequence shown in SEQ ID No:1. Therefore, piglets with high total litter size can be selected as replacement gilts at the piglet stage, maximizing savings on replacement gilt breeding costs and improving enterprise competitiveness.

[0022] In some implementations, the application includes the following steps:

[0023] 1) Detect the molecular marker g.141A>C in the piglets to be screened;

[0024] 2) When the genotype of the molecular marker detected in step 1) is AA or AC, the piglet to be screened has the genetic trait of high total litter size and is retained; when the genotype is CC, the piglet to be screened has the genetic trait of low total litter size and is culled.

[0025] According to a sixth aspect of the present invention, a primer pair is provided for detecting SNP molecular markers associated with total pig litter size, the nucleotide sequence of which is shown below:

[0026] P001-F: 5'-ACCACCCCATGAGTCAAGAACCC-3',

[0027] P002-R: 5'-AGACAATGCCAAGACTCCCTAGAGG-3'.

[0028] Therefore, this primer pair can efficiently detect the genotypes of SNP molecular markers related to total litter size in pigs and screen for dominant genotypes. This primer pair can be used to quickly establish an efficient and accurate molecular marker-assisted breeding technology, and to rapidly and accurately improve the breeding of pigs with high total litter size, thereby accelerating the breeding progress.

[0029] According to a seventh aspect of the present invention, a kit is provided for detecting SNP molecular markers associated with total piglet birth rate, the kit containing primer pairs with the following nucleotide sequences:

[0030] P001-F: 5'-ACCACCCCATGAGTCAAGAACCC-3',

[0031] P002-R: 5'-AGACAATGCCAAGACTCCCTAGAGG-3'.

[0032] According to an eighth aspect of the present invention, an application is provided for the use of an SNP molecular marker related to total litter size in pigs, or a primer pair capable of detecting the molecular marker, or a kit containing the primer pair, in screening for total litter size traits in pigs, identifying total litter size traits in pigs, breeding pig breeds with high total litter size traits, improving total litter size traits in pigs, and genetically improving reproductive traits in pigs.

[0033] Therefore, by using the above molecular markers, primer pairs, or kits, the genotypes of SNP molecular markers related to the total number of piglets born can be efficiently detected, and dominant genotypes can be screened out. This molecular marker, primer pair, or kit can be used to quickly establish an efficient and accurate molecular marker-assisted breeding technology, and to rapidly and accurately improve the breeding of pigs with high total number of piglets born, thereby accelerating the breeding progress.

[0034] The beneficial effects of this invention are:

[0035] 1. A SNP molecular marker related to the total number of piglets born has been disclosed. This molecular marker can be used to quickly establish an efficient and accurate molecular marker-assisted breeding technology, which can be applied to the genetic improvement of the total number of piglets born, thereby improving the reproductive performance of the pig population, increasing the economic profits of enterprises, and enhancing their core competitiveness.

[0036] 2. An application of an SNP molecular marker in the selection of total litter size in pigs is disclosed. By applying this SNP molecular marker, the total litter size of the sow population can be increased, which can greatly improve the reproductive performance of pigs, enhance the core competitiveness of enterprises, and increase the economic value of sows.

[0037] 3. An application of SNP molecular markers in breeding high total litter count pig breeds is disclosed. By breeding high total litter count pig breeds, the gilts bred from these breeds all possess the high total litter count trait, thereby improving the reproductive performance of gilts and achieving the economic goal of producing more piglets.

[0038] 4. A genetic improvement method for pigs is disclosed, which can improve the frequency of allele A in a pig population, thereby increasing the total number of piglets born in offspring.

[0039] 5. An application of SNP molecular markers in screening piglets with high total litter size is disclosed. This allows piglets with high total litter size to be selected as replacement gilts at the piglet stage, which can minimize the breeding costs of replacement gilts and improve the competitiveness of enterprises.

[0040] 6. A primer pair is disclosed that can detect SNP molecular markers related to total litter size in pigs. This primer pair can efficiently detect the genotypes of SNP molecular markers related to total litter size in pigs and screen for dominant genotypes. This primer pair can be used to quickly establish an efficient and accurate molecular marker-assisted breeding technology, and quickly and accurately select and improve breeding pigs to increase total litter size, thereby accelerating breeding progress.

[0041] 7. A kit for detecting SNP molecular markers related to total litter size in pigs is disclosed. This kit can efficiently detect the genotypes of SNP molecular markers related to total litter size in pigs and screen for dominant genotypes. This primer pair can be used to quickly establish an efficient and accurate molecular marker-assisted breeding technology, and to rapidly and accurately improve the breeding of high-yielding pigs by selective breeding, thereby accelerating the breeding progress.

[0042] 8. This paper discloses the application of a SNP molecular marker related to total litter size in pigs, or a primer pair capable of detecting this molecular marker, or a kit containing this primer pair, in screening for total litter size traits in pigs, identifying total litter size traits in pigs, breeding pigs with high total litter size traits, improving total litter size traits in pigs, and genetically improving reproductive traits in pigs. This allows for the rapid establishment of efficient and accurate marker-assisted breeding technology, enabling rapid and accurate selection and improvement of breeding pigs to achieve high total litter size, thus accelerating breeding progress. Attached Figure Description

[0043] Figure 1 A GWAS Manhattan plot for genome-wide association analysis of total litter size on chromosome 1 in Large White, Landrace, and Duroc pigs: where the horizontal axis represents the chromosome position of the pigs; the vertical axis represents the -logP value. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0045] Example 1

[0046] (1) Laboratory animals

[0047] The experimental pig herd used in this invention consisted of 535 purebred Large White pigs from Guangdong Guangken Livestock Group Co., Ltd., which was the company's core herd. The pigs had free access to feed and water, and the feeding methods and conditions were kept consistent throughout the process, which was a conventional method.

[0048] (2) Sample collection

[0049] The collected sow ear tissue was soaked in a 75% ethanol solution and stored at -20°C for later use.

[0050] (3) Pig genome 50K SNP genotyping

[0051] Ear tissue was collected from each of the 535 Large White pigs in the aforementioned resource population. Whole-genome DNA was extracted using the standard phenol-chloroform method. The concentration and OD ratio (OD260 / 280, OD260 / 230) of each sample were accurately determined using a NanoDrop 2000 / 2000C nucleic acid and protein analyzer. DNA samples that passed the NanoDrop 2000 / 2000C nucleic acid and protein analyzer test were diluted to approximately 50 ng / μL. 6 μL of the extracted DNA sample was then mixed with 2 μL of loading buffer and loaded onto a 1% (w / v) agarose gel. Electrophoresis was performed at 150 V for 25 min. The DNA integrity was observed and photographed using a UV spectrophotometer and gel imaging device.

[0052] DNA samples were sent to Beijing Compson Biotechnology Co., Ltd. for genotyping using a 50k SNP microarray (Zhongxin No. 1 breeding microarray, Beijing Compson Biotechnology Co., Ltd.). The genotyping data from all samples were quality controlled using the `checkmarker` function in the `GenABEL` package of the R language. Individuals with a detection rate below 90%, a family Mendelian error rate above 0.1, a minimum allele frequency below 0.05, and a Hardy-Weinberg equilibrium significance level above 10 were excluded. -6 The SNPs were analyzed, and finally, 27,843 valid genotype data of SNPs were obtained.

[0053] (4) Genome-wide association analysis (GWAS)

[0054] To eliminate population stratification effects, this invention uses GCTA software to calculate principal component characteristics of all individuals based on whole-genome sequence information, and uses the first five principal components as covariates to correct for the influence of potential population stratification on the results. This invention uses a linear mixture model in GEMMA software for GWAS analysis of the total litter size trait. The Bonferrini method is used to determine the significance threshold of the association between SNPs and the total litter size trait. The genomic significance threshold is 0.05 divided by the number of effective SNP loci, i.e., the genomic significance threshold is 1.80E-06, or 0.05 / 27843 (number of effective SNPs); the chromosomal significance threshold is 1 divided by the number of effective SNP loci, i.e., the chromosomal significance threshold is 3.59E-05, or 1 / 27843 (number of effective SNPs).

[0055] GWAS analysis results are as follows Figure 1 As shown. From Figure 1 It was found that a locus significantly affecting total litter size exists on chromosome 1 of Large White, Landrace, and Duroc pigs (this locus is located at position 107777216 bp of chromosome 1 in the international pig genome Sscrofa 11.1, representing an A>C mutation). The gene sequences upstream and downstream of this significantly associated SNP locus are shown in SEQ ID No:1. The SNP locus is located at position 141 of the sequence shown in SEQ ID No:1, where M represents an A>C mutation. This molecular marker can be abbreviated as g.141A>C in this text (P-value 3.46e-07). The gene sequences upstream and downstream of this SNP are shown in SEQ ID No:1.

[0056]

[0057] Note: M marked in the sequence listing is the mutation site. M site is an A>C mutation, which is displayed in bold font (the mutated base in parentheses is the allele mutation). The positions of the designed primer sequences are indicated by underscores and italics at the beginning and end of the sequence.

[0058] (5) Association analysis between different genotypes and total litter size phenotype

[0059] Table 1 shows that the molecular marker g.141A>C is highly significantly correlated with the total litter size trait (P<0.001), indicating that this molecular marker significantly affects the total litter size of pigs. Selecting pigs at this SNP locus can increase the total litter size of the population, thereby accelerating the breeding process. Furthermore, Table 1 shows that individuals with the AA genotype have a higher total litter size than those with the CC and AC genotypes. The phenotypic mean of AA individuals is 1.40 and 0.89 higher than that of CC and AC individuals, respectively. Additionally, the analysis of variance based on the two phenotypes shows that the distribution of the total litter size phenotype among the three genotypes is highly significant (P<0.01). Therefore, gradually retaining AA genotype breeding pigs in breeding to increase the frequency of allele A at this locus through successive generations can significantly increase the total litter size of breeding pigs, bringing greater economic benefits to pig farming enterprises.

[0060] Table 1. Statistical analysis of the relationship between the mutation site at chromosome 107777216 on molecular markers and the total litter size trait.

[0061]

[0062] Note: *** indicates a highly significant difference.

[0063] Example 2: Target DNA Sequence Amplification and Sequencing

[0064] (1) Primer design

[0065] The DNA sequence of SEQ ID NO:1 on pig chromosome 1 was downloaded from the Ensembl website (http: / / asia.ensembl.org / index.html), and primers were designed using the primer design software Oligo 7. The DNA sequences of the designed primers are shown below:

[0066] P001-F: 5'-ACCACCCCATGAGTCAAGAACCC-3' (SEQ ID NO: 2), P002-R: 5'-AGACAATGCCAAGACTCCCTAGAGG-3' (SEQ ID NO: 3);

[0067] (2) PCR amplification

[0068] To a 10 μL reaction mixture, add 1 μL of DNA template, 3.4 μL of double-distilled water, 5 μL of 2×Tag PCR StanMix with Loading Dye, and 0.3 μL each of primers P001-F and P002-R. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, followed by 35 cycles of 94℃ denaturation for 30 s, 57.6℃ annealing for 30 s, and 72℃ extension for 45 s, with a final extension at 72℃ for 5 min.

[0069] (3) DNA sequencing

[0070] DNA sequence sequencing and identification: Performed at BGI Genomics Co., Ltd. in Shenzhen, the gene fragments were sequenced using both forward and reverse reactions. The obtained sequences were compared with the NCBI genome sequence to identify mutations at corresponding SNP sites. The sequencing results were consistent with the SEQ ID NO:1 sequence.

[0071] Example 3: Analysis of the effect of molecular marker g.141A>C

[0072] Table 1 shows that the dominant allele AA, associated with the molecular marker g.141A>C, increases the total litter size by 1.40 compared to the inferior allele CC. Therefore, by using marker-assisted selection or genomic selection to gradually retain AA-type pigs within a population, the frequency of the dominant allele A can be significantly increased, thereby improving the total litter size of breeding pigs, accelerating the breeding improvement process, and ultimately effectively enhancing the economic benefits of pig breeding.

[0073] Example 4: Application of the molecular marker g.141A>C in the breeding of pigs with the total litter size trait.

[0074] 1) Detect the molecular marker g.141A>C in replacement breeding pigs;

[0075] 2) Select individuals with the AA or AC genotype obtained in step 1) as breeding pigs, and cull individuals with the CC genotype to breed pigs with high total litter size.

[0076] Example 5: Application of the molecular marker g.141A>C in the breeding of high total litter size pig breeds

[0077] 1) Detect the molecular marker g.141A>C in replacement breeding pigs;

[0078] 2) Select individuals with the AA or AC genotype obtained in step 1) as breeding pigs, and mate these breeding boars and sows.

[0079] 3) Detect the molecular marker g.141A>C in the pigs born from mating in step 2), retain individuals with AA or AC genotypes, eliminate individuals with CC genotypes, and breed them to cultivate high-fertility pig breeds with high total litter size.

[0080] Example 6: A method for genetic improvement of pigs

[0081] 1) Detect the molecular marker g.141A>C in replacement breeding pigs;

[0082] 2) Select breeding pigs with the molecular marker genotype AA or AC from step 1), and cull individuals with the CC genotype;

[0083] 3) Use the individuals selected in step 2) as breeding pigs for breeding. In the offspring, continue to select breeding pigs with molecular marker genotypes of AA or AC from step 1) and eliminate individuals with genotype CC. This will increase the frequency of allele A in the offspring pig population generation by generation, thereby increasing the total number of piglets born in the offspring pigs.

[0084] Example 7: Application of the molecular marker g.141A>C in screening piglets for the genetic trait of high total litter size.

[0085] 1) Detect the molecular marker g.141A>C in the piglets to be screened;

[0086] 2) When the genotype of the molecular marker detected in step 1) is AA or AC, the piglet to be screened has the genetic trait of high total litter size and is retained; when the genotype is CC, the piglet to be screened has the genetic trait of low total litter size and is culled.

[0087] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and all such modifications and improvements fall within the scope of protection of the invention.

Claims

1. The application of a SNP molecular marker in the selection of pigs for the total litter size trait, wherein, The upstream and downstream gene sequences of the SNP molecular marker site are shown in SEQ ID No:

1. The SNP molecular marker is located at position 141 of the sequence shown in SEQ ID No:1, where M represents an A>C base mutation. The application includes the following steps: 1) Detect the SNP molecular markers in replacement breeding pigs; 2) Select individuals with the AA or AC genotype obtained in step 1) as breeding pigs, and cull individuals with the CC genotype to breed pigs with high total litter size.

2. Application of a SNP molecular marker in breeding high total litter size pig breeds, wherein, The upstream and downstream gene sequences of the SNP molecular marker site are shown in SEQ ID No:

1. The SNP molecular marker is located at position 141 of the sequence shown in SEQ ID No:1, where M represents an A>C base mutation. The application includes the following steps: 1) Detect the SNP molecular markers in replacement breeding pigs; 2) Select individuals with the AA or AC genotype obtained in step 1) as breeding pigs, and mate these breeding boars and sows. 3) Test the molecular markers described above on the pigs born from mating in step 2), retain individuals with AA or AC genotypes, eliminate individuals with CC genotypes, and breed them to cultivate high-fertility pig breeds with high total litter size.

3. A method for genetic improvement of pigs, wherein, The method includes the following steps: 1) Detect SNP molecular markers in replacement breeding pigs, wherein the upstream and downstream gene sequences of the SNP molecular marker site are shown in SEQ ID No:1, and the SNP molecular marker is located at position 141 of the sequence shown in SEQ ID No:1, where M represents an A>C base mutation; 2) Select breeding pigs with the molecular marker genotype AA or AC from step 1), and cull individuals with the CC genotype; 3) Use the individuals selected in step 2) as breeding pigs for breeding. In the offspring, continue to select breeding pigs with molecular marker genotypes of AA or AC from step 1) and eliminate individuals with genotype CC. This is to increase the frequency of allele A in the offspring pig population generation by generation, thereby increasing the total number of piglets born in the offspring pigs.

4. Application of a SNP molecular marker in screening piglets for the genetic trait of high total litter size, wherein, The upstream and downstream gene sequences of the SNP molecular marker site are shown in SEQ ID No:

1. The SNP molecular marker is located at position 141 of the sequence shown in SEQ ID No:1, where M represents an A>C base mutation. The application includes the following steps: 1) Detect the SNP molecular markers described above in the piglets to be screened; 2) When the genotype of the SNP molecular marker detected in step 1) is AA or AC, the piglet to be screened has the genetic trait of high total litter size and is retained; when the genotype is CC, the piglet to be screened has the genetic trait of low total litter size and is culled.

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