SNP (Single Nucleotide Polymorphism) molecular marker related to influence on stillbirth number of sows on pig chromosome 1 and application of SNP molecular marker

Abstract

The invention discloses a related SNP (Single Nucleotide Polymorphism) molecular marker influencing the dead birth number of sows on a pig chromosome 1. An SNP site of the molecular marker corresponds to a 230578111 site Cgt on a chromosome 1 of a reference sequence of an international pig genome version 11.1; the polymorphism of the basic group at the site leads to the difference of the number of stillbirth birth of pigs. By verifying the influence effect of the molecular marker on the stillbirth number of the sow, an efficient and accurate molecular marker assisted breeding technology is finally established, and is applied to genetic improvement of reduction of the stillbirth number of boars, so that the reproductive performance of offspring pigs is improved, and the economic profit of enterprises is increased. Accordingly, a corresponding genetic improvement method is established, and by optimizing the dominant alleles of the SNP, the frequency of the dominant alleles can be increased generation by generation, the number of stillbirth of sows is reduced, and the progress of genetic improvement of pigs is accelerated, so that the economic benefit of breeding of the boars is effectively improved. Besides, a primer pair for amplifying a nucleotide sequence of the SNP molecular marker is further researched and designed, breeding traits can be rapidly and accurately bred through the primer pair, and the breeding process is accelerated.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biotechnology and molecular marker technology, and particularly relates to an SNP molecular marker related to the number of stillborn piglets in sows on porcine chromosome 1 and its application. Background Art

[0002] China is the largest producer and consumer of live pigs in the world. However, the generally low reproductive performance of sows remains an important problem restricting the development of the pig industry. Therefore, the breeding of maternal pigs with high reproductive performance has become a current research hotspot. In recent years, with the implementation of the pig genetic improvement policy in China, significant progress has been made in the breeding of sow reproductive performance, and traits such as litter size, number of live piglets, and number of teats have been greatly improved. However, due to the certain positive genetic and phenotypic correlation between total litter size and the proportion of weak piglets and the number of stillborn piglets, the increase in litter size is often accompanied by an increase in the number of weak and stillborn piglets. With the continuous optimization of breeding goals, breeders no longer simply pursue an increase in litter size in the breeding of sow reproductive performance, but focus on the genetic improvement of key traits such as reducing the number of stillborn piglets and increasing the survival rate of live piglets to maximize economic benefits.

[0003] A stillborn piglet refers to a fetus that dies in the uterus during pregnancy and is delivered in a dead state during parturition. Stillbirth can occur at any stage of pregnancy, and it is usually more common during the parturition process, mainly because the parturition process of the sow is too long and the piglet stays in the birth canal for too long, resulting in death due to hypoxia. Among reproductive traits, the number of stillborn piglets produced by a sow is also one of the important indicators to measure the reproductive ability of the sow. The fewer the number of stillborn piglets, the more the number of live and healthy piglets, indicating that the sow has excellent reproductive performance to a certain extent and has the potential to create higher economic value. Therefore, the key point of pig farm breeding is to select and retain such sows with high reproductive performance and optimize reproductive management to maintain their high-yield performance. However, the traditional breeding method of sows mainly relies on phenotypic and pedigree information, and relevant data for breeding can usually be obtained only after the sow gives birth. This method results in a long genetic improvement cycle, making it difficult to optimize reproductive performance in a timely manner and limiting the efficiency of genetic progress.

[0004] With the development of molecular genetics and genomic selection technology, it has become possible to conduct genetic evaluations and early selections using genomic information, which can not only improve breeding accuracy but also accelerate genetic progress, providing a more precise improvement strategy for enhancing the reproductive capacity of sows. Genome-wide association study (GWAS), as one of the common methods for screening important molecular markers, has been widely applied in genetic research of species such as humans, cattle, sheep, and pigs. By using GWAS to identify major genes and key molecular markers affecting the number of stillbirths in pigs, the genetic mechanism of sow reproductive performance can be deeply analyzed. Applying these key genes and molecular markers to genomic selection (GS) breeding technology can not only significantly accelerate the genetic progress of the number of stillbirths in piglets but also effectively improve the overall reproductive capacity of sows, thereby enhancing the production efficiency and economic benefits of the pig industry. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an SNP molecular marker related to the number of stillbirths in sows on porcine chromosome 1 and its application.

[0006] To solve the above technical problem, the present invention adopts the following technical solutions:

[0007] An SNP molecular marker related to the number of stillbirths in sows on porcine chromosome 1, wherein the SNP locus corresponds to a C>T mutation at position 230578111 on chromosome 1 of the international porcine genome version 11.1 reference sequence.

[0008] The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, where M in the sequence is C or T.

[0009] A primer pair for amplifying the nucleotide sequence of the SNP molecular marker, including primer P001-F and primer P002-R, whose base sequences are SEQ ID NO.2 and SEQ ID NO.3.

[0010] The application of the SNP molecular marker related to the number of stillbirths in sows on porcine chromosome 1 and its application or the primer pair in pig assisted breeding.

[0011] Pig assisted breeding is a genetic improvement to reduce the number of stillbirths in breeding pigs.

[0012] A genetic improvement method for pigs, detecting the SNP molecular marker, eliminating individuals with TC genotype and TT genotype at the SNP locus, and retaining individuals with CC genotype at the SNP locus as breeding pigs, and gradually increasing the frequency of the dominant allele C at this locus, thereby reducing the number of stillbirths in offspring pigs.

[0013] The pig is a purebred Duroc.

[0014] In view of the problems existing in the current genetic improvement technology for the number of stillborn piglets in sows, the inventors discovered an SNP molecular marker related to the number of stillborn piglets in sows on Sus scrofa chromosome 1. The SNP locus corresponds to the C>T mutation at position 230578111 on chromosome 1 of the international pig genome version 11.1 reference sequence. The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, where M in the sequence is C or T (i.e., the nucleotide mutation of C266-T266 at the 266th position from the 5' end). The polymorphism of the base at this site results in differences in the number of stillborn piglets in pigs. By verifying its effect on the number of stillborn piglets in sows, the inventors finally established an efficient and accurate molecular marker-assisted breeding technology and applied it to the genetic improvement of reducing the number of stillborn piglets in breeding pigs, thereby improving the reproductive performance of offspring pigs and increasing the economic profit of enterprises. Accordingly, the inventors established a corresponding genetic improvement method for pigs. By selecting the advantageous allele of this SNP, the frequency of the advantageous allele can be increased generation by generation, the number of stillborn piglets in sows can be reduced, and the progress of pig genetic improvement can be accelerated, thus effectively improving the economic benefits of breeding pig breeding. In addition, the inventors also designed a primer pair for amplifying the nucleotide sequence of the SNP molecular marker. Through this primer pair, the reproductive traits can be selected quickly and accurately, and the breeding process can be accelerated. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the Manhattan plot of the genome-wide association analysis of the stillborn trait on chromosome 1 in Duroc pigs. In the figure: the abscissa represents the chromosome number of pigs, and the ordinate represents the -logP value.

[0016] Figure 2 is the sequencing result diagram of the target DNA sequence. DETAILED DESCRIPTION OF THE INVENTION

[0017] Example 1

[0018] (1) Experimental animals

[0019] The experimental pig population used in this invention was 100 purebred Duroc breeding sows from the breeding pig branch of Guangxi Yangxiang Group Co., Ltd. The pigs were fed and watered ad libitum, and the entire feeding method and breeding conditions were conventional methods and remained consistent throughout.

[0020] (2) Sample collection

[0021] The ear tissues of the above-mentioned Duroc sows were collected and soaked in an ethanol solution with a volume fraction of 75% and stored in a -20°C refrigerator for later use.

[0022] (3) Genotyping of 80K SNPs in the pig genome

[0023] Ear tissues were collected from each of the 100 Duroc breeding pigs selected from the above-mentioned population, and genomic DNA was extracted using the standard phenol-chloroform method. The concentration and OD ratios (OD260 / 280, OD260 / 230) of the DNA in each sample were accurately measured using a Nanodrop2000 / 2000C nucleic acid and protein detector. The DNA samples that passed the detection by the NanoDrop2000 / 2000C nucleic acid and protein detector were diluted to approximately 50 ng / μL according to the detected concentration. Then, 6 μL of the extracted DNA sample to be tested was mixed with 2 μL of Loading Buffer and loaded onto a 1% agarose gel by mass / volume ratio, and electrophoresed at 150 V for 25 min. Observation and photography were carried out under an ultraviolet spectrophotometer and a gel imaging device to observe the integrity of the DNA.

[0024] The DNA samples were sent to Wuhan Shadow Gene Technology Co., Ltd. for genotyping determination of the porcine whole-genome 80K functional locus chip according to the company's standard procedures. The checkmarker in the GenABEL package of R language was used to perform quality control on the 80K chip scanning genotyping data of all samples, and SNPs with a detected individual rate lower than 90%, a pedigree Mendelian error rate higher than 0.1, a minor allele frequency less than 0.05, and a Hardy-Weinberg equilibrium significance level higher than 10 -6 were excluded, and finally, effective genotype data of 100,881 SNPs were obtained.

[0025] (4) Genome-wide association (GWAS) analysis

[0026] To eliminate the population stratification effect, the present invention adopted a single-point regression analysis using a linear mixed model and combined with the GenABEL software package of R language for GWAS analysis. The stratification effect was corrected using the genomic similarity between individuals in the analysis model. The Bonferrini method was used to determine the significance threshold for the association degree between SNPs and the stillbirth number trait. The chromosome-level significant threshold was 1 divided by the number of effective SNP loci, that is, the genome-wide significant level threshold was 9.91E-06, that is, 1 / 100,881 (the number of effective SNPs).

[0027] (5) Association analysis between different genotypes of this locus (rs319330613) and the stillbirth number

[0028] Table 1 Correlation analysis between the SNP locus g.266C>T of the molecular marker and the stillbirth number

[0029]

[0030] Example 2 Amplification and sequencing of the target DNA sequence

[0031] (1) Primer design

[0032] Download the DNA sequence of SEQ ID NO:1 on Sus scrofa chromosome 1 through the Ensembl website (http: / / asia.ensembl.org / index.html). And use the primer design software primer premier 6.0 to design primers. The DNA sequences of the designed primers are as follows:

[0033] P001-F: 5'-ACCTTTGCCATAGCCTCCAAA-3',

[0034] P002-R: 5'-GGGGAATTCATGGCTTTCAGC-3';

[0035] (2) PCR amplification

[0036] Add 1 μL of DNA template, 3.4 μL of double-distilled water, 5 μL of 2×Tag PCR StanMixwithLoading Dye, and 0.3 μL each of primers P001-F and P002-R to a 10 μL reaction system. The PCR reaction conditions are as follows: After pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for 35 cycles, and finally extension at 72°C for 5 min.

[0037] (3) DNA sequence determination

[0038] DNA sequence sequencing identification: Conducted at BGI-Shenzhen. The gene fragment is sequenced in both forward and reverse reactions. Compare the obtained sequence with the NCBI genomic sequence to obtain the mutations at the corresponding SNP sites. The sequencing results are as Figure 2 shown. Among them, M marked in the sequence list is the mutation site (the mutated base is in parentheses, which is an allelic gene mutation), and the primer sequence positions are underlined and bolded at the beginning and end of this sequence.

[0039] Example 3 Analysis of the effect of SNP site g.266C>T of molecular marker

[0040] According to Table 1, for the number of stillbirths, the effect of the dominant allele genotype (CC) of SNP site g.266C>T is significantly reduced by 1.34 compared to the phenotypic average of the inferior allele (TT) type. Therefore, through marker-assisted selection, gradually eliminate the pigs with genotype TT within the population, which can significantly increase the allele frequency of allele C, reduce the probability of stillbirth piglets in sow breeding, reduce breeding losses while improving animal welfare, and accelerate the improvement progress of pig genetic defects, thereby effectively improving the economic benefits of pig breeding.

[0041] The present invention detects the 266th base mutation site in the SEQ ID NO.1 sequence and preliminarily applies the correlation analysis between its genotype and the number of stillborn piglets in sows, providing a new molecular marker for molecular marker-assisted selection of pigs.

Claims

1. A SNP molecular marker related to the number of stillbirths in sows on Sus scrofa chromosome 1, characterized in that Its SNP locus corresponds to the C>T mutation at position 230578111 on chromosome 1 of the reference sequence of the 11.1 version of the international pig genome.

2. The SNP molecular marker related to the number of stillbirths in sows on Sus scrofa chromosome 1 according to claim 1, characterized in that: The nucleotide sequence of the SNP molecular marker is SEQ ID NO.1, where M in the sequence is C or T.

3. A primer pair for amplifying the nucleotide sequence of the SNP molecular marker according to claim 2, characterized in that It includes primer P001-F and primer P002-R, and their base sequences are SEQ ID NO.2 and SEQ ID NO.

3.

4. Application of the SNP molecular marker related to the number of stillborn piglets in sows on pig chromosome 1 described in claim 1 or 2 or the primer pair described in claim 3 in pig assisted breeding.

5. The application according to claim 4, characterized in that: The pig assisted breeding is a genetic improvement to reduce the number of stillborn piglets in breeding pigs.

6. A method for genetic improvement of pigs, characterized in that: Detect the SNP molecular marker described in claim 1 or 2, eliminate individuals with the TC genotype and TT genotype at the SNP locus, and retain individuals with the CC genotype at the SNP locus as breeding pigs. Gradually increase the frequency of the dominant allele C at this locus, thereby reducing the number of stillborn piglets in the offspring pigs.

7. The method for genetic improvement of pigs according to claim 6, wherein: The pig is a purebred Duroc.

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