SNP Molecular Marker Affecting Litter Interval Trait and Its Application

Abstract

The present invention discloses an SNP molecular marker affecting the litter interval trait. This SNP molecular marker is a C>A base mutation at the position of 29,375,835 bp on chromosome 7 of the international pig genome Ensemble Sscrofa11.1 version. The gene fragment of this SNP molecular marker locus is as shown in SEQ ID NO:1. The mutation site in the nucleotide sequence of SEQ ID NO:1 is the 161st base M, and M is selected from base C or A. This molecular marker is applied to the breeding of sow litter interval traits, the cultivation of high-fertility sow lines, and the improvement of the genetic traits of sow population fecundity. It can greatly shorten the litter interval, thereby significantly shortening the production process, reducing the risk of sow dystocia, reducing the harm to sows and improving the survival rate of piglets, improving the reproductive performance of sows / populations, and improving production efficiency.

Description

Technical Field

[0001] The present invention relates to the fields of molecular genetics and animal genetic breeding, and particularly relates to an SNP molecular marker affecting the litter interval trait and its application. Background Art

[0002] China is a large pig-raising country, and the market demand for pork production and quality is increasing day by day. Improving pork production and carcass quality has become an ongoing exploration by breeding scientists for a long time. Early breeding work mainly focused on phenotypic selection of pigs. With the continuous advancement of genomic work and the extensive development of genetic markers, molecular selection is gradually becoming a reliable and effective selection method.

[0003] Single nucleotide polymorphism (SNP) markers are the third-generation molecular markers, which refer to a polymorphism generated by a single-base mutation in the genomic DNA sequence. Such mutations include single-base transversions, transitions, insertions, and deletions. SNPs have the advantages of large quantity, high frequency, and low mutation rate, and are widely used in genomic analysis, automated bioinformatics detection, genetic research on simple and complex diseases, livestock breeding markers, and genetics research.

[0004] Genome-wide association studies (GWAS) are an important method for genetic improvement and mechanism analysis of livestock and poultry economic traits. The litter interval of pigs represents the length of the parturition process. The longer the litter interval, the higher the risk of dystocia and other problems in sows. This indicator is an important indicator reflecting the reproductive autonomy of sows. Conducting GWAS research on pig populations helps to quickly find meaningful molecular markers affecting the litter interval of pigs, providing a favorable theoretical basis for marker-assisted selection breeding of pigs. Summary of the Invention

[0005] The purpose of the present invention is to provide an SNP molecular marker affecting the litter interval trait and its application to solve the above problems.

[0006] According to one aspect of the present invention, there is provided an SNP molecular marker affecting the litter interval trait, and this SNP molecular marker is a C>A base mutation at the position of 29375835 bp on chromosome 7 of the international pig genome Ensemble Sscrofa 11.1 version.

[0007] In some embodiments, the gene fragment of this SNP molecular marker locus is as shown in SEQ ID NO.1, and the mutation site in the nucleotide sequence of SEQ ID NO:1 is the 161st base M, and M represents base C or A.

[0008] In certain embodiments, the primer sequences for detecting the molecular marker are as shown in SEQ ID NO:2 and SEQ ID NO:3.

[0009] The second aspect of the present invention provides the use of an SNP molecular marker affecting the litter interval trait in the breeding of sows for the litter interval trait.

[0010] In certain embodiments, the method for using the SNP molecular marker in the breeding of sows for the litter interval trait includes the following steps:

[0011] 1) Detect the C>A base mutation at the position of 29375835bp on chromosome 7 in replacement gilts;

[0012] 2) Select the individuals with the CC genotype of the allele detected in step 1) as the sows to be retained, which can shorten the litter interval of sows and improve the reproductive efficiency.

[0013] The third aspect of the present invention provides the use of an SNP molecular marker affecting the litter interval trait in the cultivation of high-fertility sow lines, where high fertility means a short litter interval during sow production.

[0014] In certain embodiments, the method for using the SNP molecular marker in the cultivation of high-fertility sow lines includes the following steps:

[0015] 1) Detect the C>A base mutation at the position of 29375835bp on chromosome 7 in replacement gilts;

[0016] 2) Select the individuals with the CC genotype of the allele detected in step 1) as the sows to be retained, and breed the retained sows;

[0017] 3) Detect the C>A base mutation at the position of 29375835bp on chromosome 7 in the sows born from the breeding in step 2), retain the individuals with the CC genotype, and conduct breeding to cultivate high-fertility sow lines.

[0018] The fourth aspect of the present invention provides the use of an SNP molecular marker affecting the litter interval trait in improving the genetic traits of sow population fertility.

[0019] In certain embodiments, the method for using the SNP molecular marker in improving the genetic traits of sow population fertility includes the following steps:

[0020] 1) Detect the C>A base mutation at the position of 29375835bp on chromosome 7 in replacement gilts;

[0021] 2) Select the individuals with the CC genotype of the allele detected in step 1) as the breeding sows, and breed the breeding sows.

[0022] 3) Detect the C>A base mutation at the 29375835bp position on chromosome 7 in the sows born from the breeding in step 2), retain the individuals with the CC genotype, and breed and select the CC individual sows again. Retain the individuals with the CC genotype among the offspring sows and eliminate other genotypes to gradually increase the frequency of the CC dominant allele genotype, thereby improving and enhancing the reproductive capacity of the offspring sow population.

[0023] Advantages of the present invention:

[0024] 1. By screening, the SNP molecular marker affecting the litter interval trait is obtained, thereby providing a basis for subsequent application in genetic improvement.

[0025] 2. Applying the SNP molecular marker affecting the litter interval trait to the breeding of the sow litter interval trait, individuals with a short litter interval can be selected through screening among the replacement sows and finally selected as breeding sows. Thereby, the production process can be shortened, the birth time interval of each piglet can be shortened by 12.95 minutes, the risk of dystocia in sows can be reduced, the harm to sows can be minimized, and the survival rate of piglets can be increased.

[0026] 3. Applying the SNP molecular marker affecting the litter interval trait to the cultivation of high-fertility sow lines, thereby obtaining a high-fertility sow line with a short litter interval, which can greatly shorten the production process, reduce the risk of dystocia in sows, minimize the harm to sows, increase the survival rate of piglets, improve the reproductive performance of sows, and improve production efficiency.

[0027] 4. Applying the SNP molecular marker affecting the litter interval trait to improving the genetic traits of the sow population's reproductive capacity, thereby shortening the litter interval of the sow population, shortening the production process, reducing the risk of dystocia in sows, minimizing the harm to sows, increasing the survival rate of piglets, improving the reproductive performance of sows, and improving production efficiency. Description of the Drawings

[0028] Figure 1 is the Manhattan plot of the GWAS results;

[0029] Figure 2 is the box plot of the correlation analysis between different genotypes of the SNP locus CNC10070635C>A of the molecular marker and the litter interval. Detailed Embodiments

[0030] The present invention will be further described in detail below in conjunction with the embodiments and the drawings, but the embodiments of the present invention are not limited thereto.

[0031] 1. Experimental animals

[0032] The experimental pig population used in this invention consists of 1,100 Landrace, Yorkshire, and Landrace×Yorkshire crossbred sows from Wen's Foodstuff Group Co., Ltd. They are the company's multiplication and production herds, and the herd pedigree records are detailed. The pigs are fed and watered ad libitum, and the entire feeding method, breeding conditions, etc. are always kept consistent, which is a conventional method.

[0033] 2. Phenotypic data collection

[0034] After the sows give birth, the piglets are delivered in a timely manner, recording the start time, end time of parturition, total number of piglets born, and the birth time of each piglet. The phenotypic value of the inter-birth interval is calculated by dividing the total parturition time by the total number of piglets born.

[0035] 3. Sample collection

[0036] Collect the tail docking and ear tissues of the above piglets and soak them in 75% ethanol, then store them in a -20°C refrigerator for later use.

[0037] 4. Detection of 50K SNP genotypes in the porcine whole genome

[0038] For each individual selected from the 1,100 sows in the above experimental pig population, ear tissues or tail docking tissues are collected, and genomic DNA is extracted using the standard phenol-chloroform method. The concentration and OD ratio (OD260 / 280, OD260 / 230) of the DNA in each sample are accurately measured using a Nanodrop2000 / 2000C nucleic acid and protein detector. For DNA samples that pass the detection by the NanoDrop2000 / 2000C nucleic acid and protein detector, the DNA is diluted to about 50 ng / μL according to the detected concentration. Then, 6 μL of the extracted DNA sample to be tested is mixed with 2 μL of Loading Buffer, loaded onto a 1% agarose gel, and electrophoresed at 150 V for 25 min. Observe and take pictures under an ultraviolet spectrophotometer and a gel imaging device to observe the integrity of the DNA.

[0039] The DNA samples are genotyped for the porcine whole genome 50K SNP chip (Illumina, USA) on the Illumina Beadstration platform according to the detection standard procedure. The Plink software is used to perform quality control on the 50K chip scanning genotyping data of all samples, excluding SNPs with a detected individual rate lower than 80%, a minor allele frequency less than 0.01, and a Hardy-Weinberg equilibrium significance level higher than 10 -5 ^-6^. Finally, effective genotype data of 39,163 SNPs are obtained.

[0040] 5. Genome-wide association (GWAS) analysis

[0041] To eliminate the population stratification effect, the present invention uses linear mixed model single-point regression analysis in combination with the GCTA software package for GWAS analysis. In the analysis model, the genomic similarity between individuals is used to correct the stratification effect. The Bonferroni method is used to determine the significance threshold for the association degree between SNPs and the litter interval trait. The genome-wide significant threshold is 0.05 divided by the number of effective SNP loci, that is, the genome-wide significant level threshold is 1.28×10 -6 , that is, 0.05 / 39163 (the number of effective SNPs); the chromosome-wide significant threshold is 1 divided by the number of effective SNP loci, that is, the chromosome-wide significant level threshold is 2.55×10 -5 , that is, 1 / 39163 (the number of effective SNPs).

[0042] The results of the GWAS analysis are as Figure 1 shown. As can be seen from Figure 1 , there are loci on chromosome 7 that significantly affect the litter interval in the experimental pig population. Among them, the most strongly associated SNP molecular marker is denoted as CNC10070635 C>A (P = 7.1E-7). This molecular marker is located at the position of 29375835bp on chromosome 7 of the international pig genome Ensemble Sscrofa 11.1 version, with a C>A mutation.

[0043] 6. Analysis of the association between different genotypes and the litter interval

[0044] According to Table 1 and Figure 2 , it can be seen that the SNP locus CNC10070635C>A of the molecular marker is significantly correlated with the litter interval (P<0.001), indicating that this molecular marker significantly affects the litter interval of pigs. By means of the assisted selection of this SNP locus in pigs, the litter interval of this population can be improved, and thus the breeding process can be accelerated.

[0045] In addition, according to Table 1 and Figure 2 shown, the litter interval of the CC genotype is shorter than that of the AC and AA genotypes, indicating that the homozygous CC is most favorable for the litter interval. The litter interval is an important indicator of the production performance of sows. A shorter litter interval indicates good reproductive performance of sows, meeting the requirements of modern production. Therefore, eliminating pigs with the AA genotype can bring more economic benefits. During the breeding process, we need to eliminate AA-type and AC-type breeding pigs and retain CC-type breeding pigs to gradually increase the frequency of allele C at this locus.

[0046] Table 1 Correlation between the SNP locus CNC10070635C>A of the molecular marker and the litter interval

[0047]

[0048] 7. Invention process of detecting SNP markers

[0049] (1) Primer design

[0050] The amplified target fragment containing the SNP locus significantly associated with the litter interval in sows is a 321-bp nucleotide sequence in chromosome 7. Primers were designed using the primer design software primerpremier6.0, and the upstream and downstream primer sequences for sequence amplification are as follows:

[0051] Upstream primer (SEQ ID NO:2): 5’-CTACAGCTCCGATTCGA CCC-3’, downstream primer (SEQ ID NO:3): 5’-TAATGCCCACGACACAGGAC-3’;

[0052] (2) PCR amplification

[0053] 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 and P002 to a 10-μL reaction system. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 2 min, then denaturation at 94°C for 30 s, annealing at 55°C for 20 s, extension at 72°C for 30 s, for 35 cycles, and finally extension at 72°C for 10 min.

[0054] (3) DNA sequence determination

[0055] Finally, the products after PCR amplification were sequenced. The sequence determination was completed by Sanger sequencing. The requirement for gene fragment sequencing was to sequence both strands through. The obtained sequence was compared with the NCBI genomic sequence to obtain the mutation of the corresponding SNP locus.

[0056] The sequencing results are shown in SEQ ID NO:1:

[0057]

[0058]

[0059] Note: M marked in the sequence list is the mutation site. M represents base C or A, which is underlined (the left side in the parentheses is the reference genome allele, and the right side is the mutated base, which is an allelic gene mutation). The primer sequence positions are shown in bold at the beginning and end of this sequence.

[0060] 8. Effect analysis of the CNC10070635C>A locus of the molecular marker on the litter interval

[0061] The present invention provides an SNP marker that can significantly reduce the farrowing interval of sows. By using this SNP for marker-assisted selection, the breeding process for selecting sows with a shorter farrowing interval can be greatly accelerated. If all AA-type individuals of the molecular marker affecting the piglet birth interval trait in the present invention are selected and bred into CC-type individuals, the birth interval of each piglet can be shortened by 12.95 minutes. The shorter the farrowing interval of pigs, the shorter the entire labor process, the less harm to sows, and the lower the mortality rate of piglets. This will greatly improve the reproductive performance of sows and create wealth for enterprises. Among the individuals of this SNP marker, by preferentially selecting the dominant allele (C) of this SNP in sows, the economic benefits of sows can be ultimately improved, thereby increasing the income of enterprises. The present invention detects the 161st base mutation site in the sequence of SEQ ID NO:1 and preliminarily applies the correlation analysis between its genotype and the farrowing interval trait of pigs, providing a new molecular marker for the marker-assisted selection of pigs.

[0062] 9. Application of the molecular marker CNC10070635C>A in the selection and breeding of sows with respect to the farrowing interval trait

[0063] 1) Detect the molecular marker of the CNC10070635C>A locus (at the 10070635bp position on chromosome 7) in gilts;

[0064] 2) Select and retain the individuals with the CC genotype of the allele detected in step 1) as breeding sows, which can shorten the farrowing interval of breeding sows and improve the reproductive efficiency.

[0065] 10. Application of the molecular marker CNC10070635C>A in the cultivation of sow lines with high fertility

[0066] 1) Detect the molecular marker of the CNC10070635C>A locus (at the 10070635bp position on chromosome 7) in gilts;

[0067] 2) Select and retain the individuals with the CC genotype of the allele detected in step 1) as breeding sows, and breed the breeding sows;

[0068] 3) Detect the molecular marker of the CNC10070635C>A locus (at the 10070635bp position on chromosome 7) in the sows born from the breeding in step 2), retain the CC genotype individuals, and conduct breeding to cultivate a sow line with high fertility.

[0069] 11. Application of the molecular marker CNC10070635C>A in improving the genetic traits of the reproductive capacity of the sow population

[0070] 1) Detect the molecular marker of the CNC10070635C>A locus (at the 10070635bp position on chromosome 7) in replacement gilts;

[0071] 2) Select and retain the individuals with the CC genotype of the allele detected in step 1) as the breeding gilts, and breed the breeding gilts;

[0072] 3) Detect the molecular marker of the CNC10070635C>A locus (at the 10070635bp position on chromosome 7) in the sows born from the breeding in step 2), retain the individuals with the CC genotype, and breed and select the CC individual sows again, retain the individuals with the CC genotype, and eliminate other genotypes to gradually increase the frequency of the CC dominant allele genotype, thereby improving and enhancing the reproductive performance of the offspring sow population.

[0073] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention. Sequence Listing <110> Wens Foodstuff Group Co., Ltd. <120> SNP Molecular Marker Affecting Litter Interval Trait and Its Application <130> 20220517 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 321 <212> DNA <213> Sus scrofa <400> 1 ggcctggatc cagcgttgct gtggctgtgg tataggccag cagctacagc tccgattcga 60 cccctagcct gggaacctcc atatgccgca ggagtggccc aagcaatgga aaaaagacca 120 aaaaaaaatt atcattcttt agttcccccc caccccacct mcttgagcaa tggtttatcc 180 actacatttc agtctcctga ccagaccttt gtcctgtgtc gtgggcatta acaacagtta 240 ttgaaacctg aggcaaaaaa agcaagccaa ggaaaacaga agagacatgg aatttttaaa 300 acatttccca atcatgattc a 321 <210> 2 <211> 20 <212> DNA <213> Sus scrofa <400> 2 ctacagctcc gattcgaccc 20 <210> 3 <211> 20 <212> DNA <213> Sus scrofa <400> 3 taatgcccac gacacaggac 20

Claims

1. Use of a product for detecting SNP molecular markers in the breeding of sow litter interval traits, wherein, The described SNP molecular marker is a C>A base mutation at the position of 29375835 bp on chromosome 7 of the international pig genome Ensembl Sscrofa 11.1 version.

2. The application according to claim 1, wherein The method of application includes the following steps: 1) Detect the described SNP molecular marker in gilts. 2) Select the individuals with the CC genotype of the allele detected in step 1) as the sows to be retained, which can shorten the farrowing interval of sows and improve the reproductive efficiency.

3. Use of a product for detecting SNP molecular markers in the cultivation of high-fertility sow lines, wherein, The described high fertility means that the farrowing interval of sows during production is short. The described SNP molecular marker is a C>A base mutation at the position of 29375835 bp on chromosome 7 of the international pig genome Ensembl Sscrofa 11.1 version.

4. The application according to claim 3, wherein The method of application includes the following steps: 1) Detect the described SNP molecular marker in gilts. 2) Select the individuals with the CC genotype of the allele detected in step 1) as the sows to be retained, and breed the retained sows. 3) Detect the described molecular marker in the sows born from the breeding in step 2), retain the individuals with the CC genotype, and conduct breeding to cultivate a high-fertility sow line.

5. Use of a product for detecting SNP molecular markers in improving the genetic traits of sow population fecundity, wherein, The described SNP molecular marker is a C>A base mutation at the position of 29375835 bp on chromosome 7 of the international pig genome Ensembl Sscrofa 11.1 version.

6. The application according to claim 5, wherein, The method of application includes the following steps: 1) Detect the described SNP molecular marker in gilts. 2) Select the individuals with the CC genotype of the allele detected in step 1) as the sows to be retained, and breed the retained sows. 3) Detect the described SNP molecular marker in the sows born from the breeding in step 2), retain the individuals with the CC genotype, and conduct breeding and selection again for the sows with the CC genotype. Retain the individuals with the CC genotype in the offspring sows and eliminate other genotypes to gradually increase the frequency of the CC dominant allele genotype, thereby improving and enhancing the fertility of the offspring sow population.

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