SNP Molecular Markers Affecting the Trait of Weak Piglets and Their Applications

Abstract

The present invention discloses an SNP molecular marker affecting the trait of weak piglet number. This SNP molecular marker is a G>A base mutation at the position of 136569986 bp on chromosome 15 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 represents base G or A. This molecular marker is applied to the breeding of the trait of weak piglet number in sows, the cultivation of sow lines with high fertility, and the improvement of the genetic traits of the reproductive performance of the sow population, which can efficiently reduce the number of weak piglets at parturition, improve the reproductive performance of sows, and increase production efficiency.

Description

Technical Field

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

[0002] With the development of China's economy, the living standards of the people are constantly improving. China is a major pig-raising country and also a major pork-consuming country. However, with the progress of technology, the people have higher requirements for the quantity and quality of pork. Therefore, how to improve the efficiency of pig-raising has to mention the quality of piglets, as well as the number of live or healthy piglets per litter, which becomes particularly important.

[0003] The number of weak piglets in pigs is an important indicator reflecting the effective litter size of sows. The more weak piglets, the higher the mortality rate in the farrowing house. This indicator is an important one reflecting the reproductive level of sows. Conducting GWAS (Genome-wide association studies) on pig populations helps to quickly find meaningful molecular markers affecting the number of weak piglets in pigs, providing a favorable theoretical basis for marker-assisted selection breeding of pigs. Summary of the Invention

[0004] The object of the present invention is to provide an SNP molecular marker affecting the trait of weak piglet number to solve the above problems.

[0005] One aspect of the present invention provides an SNP molecular marker affecting the trait of weak piglet number, and this SNP molecular marker is a G>A base mutation at the position of 136569986bp on chromosome 15 of the international pig genome Ensemble Sscrofa 11.1 version.

[0006] 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, where M represents base G or A.

[0007] In some embodiments, the primer sequences for detecting this molecular marker are as shown in SEQ ID NO:2 and SEQ ID NO:3.

[0008] The second aspect of the present invention provides an application of an SNP molecular marker affecting the trait of weak piglet number in the breeding selection of the trait of weak piglet number in sows.

[0009] In some embodiments, the application method of this SNP molecular marker in the breeding selection of the trait of weak piglet number in sows includes the following steps:

[0010] 1) Detect the G>A base mutation at the position of 136569986 bp on chromosome 15 in replacement gilts;

[0011] 2) Select the individuals with the GG genotype detected in step 1) as the breeding gilts, which can effectively reduce the number of weak piglets born to sows and improve the reproductive efficiency.

[0012] The third aspect of the present invention provides an application of an SNP molecular marker affecting the weak piglet number trait in cultivating a high-fertility sow line, wherein the high fertility refers to a small number of weak piglets born during sow production.

[0013] In some embodiments, the application method of the SNP molecular marker in cultivating a high-fertility sow line includes the following steps:

[0014] 1) Detect the G>A base mutation at the position of 136569986 bp on chromosome 15 in replacement gilts;

[0015] 2) Select the individuals with the GG genotype detected in step 1) as the breeding gilts, and breed the breeding gilts;

[0016] 3) Detect the G>A base mutation at the position of 136569986 bp on chromosome 15 in the sows born from the breeding in step 2), retain the individuals with the GG genotype, and conduct breeding to cultivate a high-fertility sow line.

[0017] The fourth aspect of the present invention provides an application of an SNP molecular marker affecting the weak piglet number trait in improving the genetic traits of the reproductive capacity of the sow population.

[0018] In some embodiments, the application method of the SNP molecular marker in improving the genetic traits of the reproductive capacity of the sow population includes the following steps:

[0019] 1) Detect the G>A base mutation at the position of 136569986 bp on chromosome 15 in replacement gilts;

[0020] 2) Select the individuals with the GG genotype detected in step 1) as the breeding gilts, and breed the breeding gilts;

[0021] 3) Detect the G>A base mutation at the position of 136569986 bp on chromosome 15 in the sows born from the breeding in step 2), retain the individuals with the GG genotype, and conduct breeding and selection again for the GG individual sows, retain the individuals with the GG genotype in the offspring sows, and eliminate other genotypes to gradually increase the frequency of the GG dominant allele genotype, thereby improving and enhancing the reproductive capacity of the offspring sow population.

[0022] Advantages of the present invention:

[0023] 1. By screening, SNP molecular markers affecting the weak piglet number trait are obtained, thus providing a basis for subsequent applications in genetic improvement.

[0024] 2. When the SNP molecular markers affecting the weak piglet number trait are applied to the breeding of sows for the weak piglet number trait, individuals with fewer weak piglets can be selected through screening among replacement gilts, and finally selected as breeding sows. Thus, the average number of weak piglets per sow can be reduced by 0.06 heads. The fewer the weak piglets, the more beneficial it is to improve the reproductive performance of sows, and ultimately to increase the economic benefits of sows, thereby increasing the company's income.

[0025] 3. When the SNP molecular markers affecting the weak piglet number trait are applied to the cultivation of high-fertility sow lines, a high-fertility sow line with fewer weak piglets is obtained, which can reduce the number of weak piglets at farrowing, improve the reproductive performance of sows, and improve production efficiency.

[0026] 4. When the SNP molecular markers affecting the weak piglet number trait are applied to improving the genetic traits of the reproductive capacity of the sow population, the number of weak piglets at farrowing can be reduced, the reproductive performance of sows can be improved, and production efficiency can be improved. Brief Description of the Drawings

[0027] Figure 1 It is a Manhattan plot of GWAS results;

[0028] Figure 2 Box plot of the correlation analysis between different genotypes of the SNP locus CNC10152677 G>A of the molecular marker and the number of weak piglets. Detailed Embodiments

[0029] The present invention will be further described in detail below in conjunction with examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0030] 1. Experimental Animals

[0031] The experimental pig population used in the present invention is a total of 1439 Landrace, Yorkshire, and Landrace×Yorkshire crossbred sows of Wen's Food Group Co., Ltd., which are the company's multiplication and production herds, and the pedigree records of the herds are detailed. The pigs are fed ad libitum and provided with water, and the entire feeding method, feeding conditions, etc. are always kept consistent, which is a conventional method.

[0032] 2. Collection of Phenotypic Data

[0033] After the sows give birth, they are promptly assisted in parturition, and the total number of malformations and weak piglets is recorded as the phenotypic value of the weak piglet number.

[0034] 3. Sample Collection

[0035] Collect the above-mentioned piglet tail docking and ear tissues, soak them in 75% ethanol, and store them in a -20°C refrigerator for later use.

[0036] 4. Detection of 50K SNP genotypes of the porcine whole genome

[0037] For each individual among the 1439 sows selected from the above experimental pig population, collect ear tissues or tail docking tissues, extract the whole genome DNA using the standard phenol-chloroform method, and accurately measure the DNA concentration and OD ratios (OD260 / 280, OD260 / 230) of each sample using a Nanodrop2000 / 2000C nucleic acid and protein detector. For the DNA samples qualified by the NanoDrop2000 / 2000C nucleic acid and protein detector, dilute the DNA to about 50 ng / μL according to the detected concentration. Then mix 6 μL of the extracted DNA sample to be tested with 2 μL of Loading Buffer, load it onto a 1% agarose gel, and perform electrophoresis 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.

[0038] 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. Use the Plink software to perform quality control on the 50K chip scanning genotyping data of all samples, and eliminate SNPs with a detection individual rate lower than 80%, a minor allele frequency less than 0.01, and a Hardy-Weinberg equilibrium significance level higher than 10 -5 to finally obtain the effective genotype data of 39163 SNPs.

[0039] 5. Genome-wide association (GWAS) analysis

[0040] To eliminate the population stratification effect, the present invention uses a linear mixed model single-point regression analysis and combines with the GCTA software package for GWAS analysis. In the analysis model, the stratification effect is corrected using the genomic similarity between individuals. The Bonferroni method is used to determine the significance threshold for the association degree between SNPs and the weak piglet number 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).

[0041] The results of the GWAS analysis are as Figure 1 shown. From Figure 1It can be seen that there are loci on chromosome 15 in the experimental pig population that significantly affect the number of weak piglets. Among them, the most strongly associated SNP molecular marker is denoted as CNC10152677 G>A (P = 5.3E-7), which is a G>A mutation located at position 136569986 bp on chromosome 15 of the international pig genome Ensemble Sscrofa 11.1 version.

[0042] 6. Association analysis between different genotypes and the number of weak piglets

[0043] According to Table 1 and Figure 2 It can be seen that the SNP locus CNC10152677 G>A of the molecular marker is significantly correlated with the number of weak piglets (P<0.001), indicating that this molecular marker significantly affects the number of weak piglets in pigs. By means of assisted selection of this SNP locus in pigs, the number of weak piglets in this population can be improved, and thus the breeding process can be accelerated.

[0044] In addition, according to Table 1, it can also be seen that the number of weak piglets of the GG genotype is less than that of the AG and AA genotypes, indicating that the homozygous GG is the most beneficial to the number of weak piglets. The number of weak piglets is an important indicator of the production performance of sows. A short number of weak piglets indicates good reproductive performance of sows, which meets 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 breeding pigs of the AA and AG genotypes and retain breeding pigs of the GG genotype to gradually increase the frequency of allele C at this locus.

[0045] Table 1 Correlation between the SNP locus CNC10152677 G>A of the molecular marker and the number of weak piglets

[0046]

[0047] 7. Invention process of detecting SNP markers

[0048] (1) Primer design

[0049] The amplification target fragment containing the target fragment of the SNP locus significantly related to the number of weak piglets in sows is a 321-bp nucleotide sequence in chromosome 7. Primers are designed using the primer design software primer premier6.0, and the upstream and downstream primer sequences for sequence amplification are:

[0050] Upstream primer (SEQ ID NO:2): 5’-AGATGCTGTGACAACAAGGGA-3’;

[0051] Downstream primer (SEQ ID NO:3): 5’-TCCAAGGATAAAGATTCCTGATGA-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, followed by 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, sequence the product after PCR amplification. The sequence determination is completed using Sanger sequencing technology. The requirement for gene fragment sequencing is to sequence both strands through. Compare the obtained sequence with the NCBI genomic sequence to obtain the mutations at the corresponding SNP sites. After sequencing, its sequence is as shown in SEQ ID NO: 1:

[0056]

[0057] Note: In the sequence listing, M marked is the mutation site. M represents the base G or A, which is shown in bold and underlined (the left side in the parentheses is the reference sequence allele, and the right side is the mutated base), and the primer sequence positions are shown in bold at the beginning and end of this sequence.

[0058] 8. Effect analysis of the CNC10152677 G>A locus of the molecular marker on the number of weak piglets

[0059] The present invention provides an SNP marker that can significantly reduce the number of weak piglets. Using this SNP for marker-assisted selection can greatly accelerate the breeding process for selecting the number of weak piglets. If all AA-type individuals of the molecular marker affecting the number of weak piglets in the present invention are selected and bred into GG-type individuals, the number of weak piglets per sow will be reduced by 0.06 heads. The fewer the number of weak piglets, the more beneficial it will be to improve the reproductive performance of sows and create wealth for enterprises. Among the individuals of this SNP marker, by preferentially selecting the dominant allele (G) of this SNP in sows, the economic benefits of sows can be ultimately improved, thereby increasing the income of enterprises.

[0060] 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 number of weak piglets in pigs, providing a new molecular marker for marker-assisted selection in pigs.

[0061] 9. Application of the CNC10152677 G>A of the molecular marker in the breeding selection of the number of weak piglets in sows

[0062] 1) Detect the G>A base mutation at the CNC10152677 G>A locus (at position 136569986 bp on chromosome 15) in replacement gilts;

[0063] 2) Select the individuals with the GG genotype detected in step 1) as the gilts to be retained, which can effectively reduce the number of weak piglets born to sows and improve the reproductive efficiency.

[0064] 10. Application of the molecular marker CNC10152677 G>A in the cultivation of high-fertility sow lines

[0065] 1) Detect the G>A base mutation at the CNC10152677 G>A locus (at position 136569986 bp on chromosome 15) in replacement gilts;

[0066] 2) Select the individuals with the GG genotype detected in step 1) as the gilts to be retained, and breed the retained gilts;

[0067] 3) Detect the G>A base mutation at the CNC10152677 G>A locus (at position 136569986 bp on chromosome 15) in the sows born from the breeding in step 2), retain the individuals with the GG genotype, and conduct breeding to cultivate a high-fertility sow line.

[0068] 11. Application of the molecular marker CNC10152677 G>A in improving the genetic traits of the reproductive capacity of sow populations

[0069] 1) Detect the G>A base mutation at the CNC10152677 G>A locus (at position 136569986 bp on chromosome 15) in replacement gilts;

[0070] 2) Select the individuals with the GG genotype detected in step 1) as the gilts to be retained, and breed the retained gilts;

[0071] 3) Detect the G>A base mutation at the CNC10152677 G>A locus (at position 136569986 bp on chromosome 15) in the sows born from the breeding in step 2), retain the individuals with the GG genotype, and conduct breeding and selection again for the GG individual sows, retain the individuals with the GG genotype, and eliminate other genotypes to gradually increase the frequency of the GG dominant allele genotype, thereby improving and enhancing the reproductive capacity of the offspring sow population.

[0072] 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 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 the Trait of Weak Piglets and Its Application <130> 20220517 <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 321 <212> DNA <213> Sus scrofa <400> 1 gaatgagaaa aaaatcaaga tgctgtgaca acaagggata atgtccccaa agaactttac 60 tccatagcac actaaggact acaacacaga tctctgaacc tagtattctc actcgaaaac 120 agtcttgctt gatcctggaa tgcttgtaat tcattctgct mtaattttct agaatttagg 180 ccaggggtga ttacaagttc actcaaaaag ttcaagaaaa aagaaaataa aagaaacaaa 240 atatatcaat tcacatctgg aattcaggtt atcaagtaac atcatcagga atctttatcc 300 ttggaatcag aattctttcc t 321 <210> 2 <211> 21 <212> DNA <213> Sus scrofa <400> 2 agatgctgtg acaacaaggg a 21 <210> 3 <211> 24 <212> DNA <213> Sus scrofa <400> 3 tccaaggata aagattcctg atga 24

Claims

1. Application of a product for detecting SNP molecular markers in the breeding of the trait of the number of weak piglets born to sows, wherein, The SNP molecular marker is a G>A base mutation at the position of 136569986 bp on chromosome 15 of the international pig genome Ensembl Sscrofa 11.1 version.

2. The application according to claim 1, wherein, The method of application includes: 1) Detecting the said SNP molecular marker in gilts; 2) Selecting the individuals with the GG genotype of the allele detected in step 1) as the sows to be retained, which can effectively reduce the number of weak piglets born to 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 high fertility refers to the small number of weak piglets born during sow production. The SNP molecular marker is a G>A base mutation at the position of 136569986 bp on chromosome 15 of the international pig genome Ensembl Sscrofa 11.1 version.

4. The application according to claim 3, wherein The method of application includes: 1) Detecting the said SNP molecular marker in gilts; 2) Selecting the individuals with the GG genotype of the allele detected in step 1) as the sows to be retained, and breeding the sows to be retained; 3) Detecting the said SNP molecular marker in the sows born from the breeding in step 2), retaining the individuals with the GG genotype in the offspring sows, and breeding them 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 fertility genetic trait refers to the trait of the number of weak piglets born to sows. The SNP molecular marker is a G>A base mutation at the position of 136569986 bp on chromosome 15 of the international pig genome Ensembl Sscrofa 11.1 version.

6. The use according to claim 5, wherein: The method of application includes: 1) Detecting the said SNP molecular marker in gilts; 2) Selecting the individuals with the GG genotype of the allele detected in step 1) as the sows to be retained, and breeding the sows to be retained; 3) Detecting the said SNP molecular marker in the sows born from the breeding in step 2), retaining the individuals with the GG genotype, and breeding and selecting the sows with the GG genotype again, retaining the individuals with the GG genotype and eliminating other genotypes to gradually increase the frequency of the GG dominant allele genotype, thereby improving and enhancing the fertility of the offspring sow population.

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