SNP (Single Nucleotide Polymorphism) molecular marker influencing total nipple number of large white pig and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker influencing the total nipple number of a large white pig and an application, and aims to solve the technical problem that gene research on related characters of the total nipple number of a sow is lacked at present. According to the molecular marker, a pig whole genome sequence and high-density SNP chip data are utilized, the molecular marker remarkably related to the total nipple number of the large white pig is detected on the basis of a pig whole genome association analysis strategy, and the molecular marker has C / G mutation on the 10512725th site of the 13th chromosome of a reference sequence of an international pig genome 11.1 version. By screening the dominant alleles of the SNP site generation by generation, the frequency of the dominant alleles can be improved, the total papilla number of pigs is increased, the genetic improvement progress of the pigs is accelerated, and the economic benefit of pig breeding is effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of marker-assisted selection (MAS) in pigs, and specifically to a SNP molecular marker affecting the total nipple number of Large White pigs and its application. Background Art

[0002] Large White pigs, also known as Yorkshire pigs, are native to Yorkshire and northern England. Large White pigs are excellent lean meat pigs introduced into my country. Large White pigs introduced into my country have strong reproductive capacity, with an average litter size of more than 14 pigs, but their trunks are generally not long, and the average number of nipples is less than seven pairs.

[0003] With the development of pig farming technology and pig breeding, the number of sows born has increased year by year, but the number of sows' nipples is relatively fixed, so the effective number of sows' nipples cannot meet the needs of lactation. Compared with other reproductive traits, the number of nipples is more susceptible to the influence of congenital inheritance. Lactating piglets have the physiological characteristics of fixed nipples. If the effective number of nipples cannot meet the needs of piglets, the piglets will be malnourished and even compete for nipples. Therefore, it is increasingly critical to increase the number of sows' nipples through breeding.

[0004] The heritability of the number of nipples in pigs is low (about 0.3), and the SNP molecular marker technology that affects the total number of nipples in large white pigs is an innovative breakthrough in the genetic direction of pig reproductive traits. Compared with traditional breeding improvement technology, this technology has a more significant genetic effect and a faster trait improvement speed. This technology provides a new idea for the genetic improvement of pig reproductive traits and also promotes the development of pig farming.

[0005] The information disclosed in this background technology section is only used to deepen the understanding of the background technology of the present disclosure, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art known to those skilled in the art. Summary of the invention

[0006] The purpose of the present invention is to provide a SNP molecular marker that affects the total teat number of Large White pigs, and to apply it to genotype identification, genetic breeding, etc., in order to solve the current technical problem of lack of genetic research on traits related to the total teat number of sows.

[0007] Using the pig whole genome sequence and high-density SNP (single nucleotide polymorphism) chip data, a molecular marker significantly associated with the total nipple number of Large White pigs was detected based on the pig whole genome association study (GWAS). The SNP molecular marker was a C / G mutation at base 10512725 of chromosome 13 based on the international pig genome version 11.1 reference sequence. If the SNP molecular marker is used in molecular marker-assisted selection and genomic selection, it can promote the genetic improvement of pigs and improve the economic benefits of breeding.

[0008] The first aspect disclosed in the present application relates to a SNP molecular marker that affects the total nipple number of Large White pigs, based on the presence of a C / G conversion type single nucleotide polymorphism variation at base 10512725 of chromosome 13 of the international porcine genome version 11.1 reference sequence.

[0009] The second aspect disclosed in the present application relates to a method for selecting / identifying a trait related to the total teat number of Large White pigs using a SNP molecular marker that affects the total teat number of Large White pigs, comprising the following steps: (1) Take tissue samples from the pigs to be tested and extract whole genome DNA; (2) scanning the whole genome DNA using pig whole genome resequencing; (3) Obtain resequencing data, check the genotype of locus 10512725 on chromosome 13 based on the international porcine genome version 11.1 reference sequence, interpret the C / G polymorphism of this locus, and select / identify the total teat number trait of Large White pigs based on the characteristic that the total teat number of genotypes CG and GG is higher than the total teat number of genotype CC.

[0010] According to a third aspect disclosed in the present application, SNP molecular markers that affect the total nipple number of Large White pigs are used in reagents for selecting / assisting in the selection of Large White pig breeds / lines with traits related to the total nipple number.

[0011] According to a fourth aspect disclosed in the present application, SNP molecular markers that affect the total nipple number of Large White pigs are used in the selection of varieties / lines related to the trait of the total nipple number of Large White pigs.

[0012] The present application discloses a fifth aspect, a breeding method for increasing the total number of nipples of pigs, comprising the following steps: (1) Based on the international pig genome version 11.1 reference sequence, the genotype of chromosome 13, locus 10512725, of the pig population was determined; (2) Select individual sows with CG or GG genotype at the 10512725th locus and eliminate those with CC genotype at the locus, so as to increase the frequency of the G allele at the locus from generation to generation, thereby increasing the total number of nipples in the offspring.

[0013] In some embodiments of the present disclosure, the pig breed is Large White pig and its synthetic strain.

[0014] One or more technical solutions provided in the embodiments of the present application have at least any of the following technical effects or advantages: The present application determines that a molecular marker related to the total nipple number of Large White pigs is located on chromosome 13 of the pig and has a single nucleotide polymorphism variation of the C / G conversion type at the 10512725th base of the nucleotide sequence, and verifies its effect on the total nipple number trait. By optimizing the dominant allele of the molecular marker, a breeding method for increasing the total nipple number of pigs is proposed, and an accurate and efficient molecular marker-assisted selection method is established, which can genetically improve the quantitative trait of the total nipple number of Large White pigs, improve the reproductive performance of offspring pigs, shorten the improvement time, and effectively improve the economic benefits of the pig farming industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a Manhattan plot of the genome-wide association analysis (GWAS) of the total nipple number of Large White pigs on chromosome 13 in one embodiment of the present application; wherein the horizontal axis represents the pig chromosome number, and the vertical axis represents the -logP value. DETAILED DESCRIPTION

[0016] The specific implementation modes of the present application are described below in conjunction with examples. However, the following examples are only used to illustrate the present invention in detail and do not limit the scope of the present invention in any way.

[0017] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; unless otherwise specified, the biological reagents and raw materials involved are all commercially available conventional products; unless otherwise specified, the experimental methods involved are all conventional methods.

[0018] Example: SNP marker site determination and development 1. Source of experimental animals In this case, a group of 1151 purebred Large White pigs from a pig farm in Henan Province was used as the experimental group. The pigs were allowed to eat and drink freely, and the entire feeding method and feeding conditions were kept consistent, all of which were conventional methods.

[0019] 2. Sample collection and phenotypic recording The ear tissues of the above pigs were collected, stored in 2 mL centrifuge tubes containing 75% ethanol, stored in a -20°C refrigerator, and the total number of nipples was recorded by counting.

[0020] 3. Extraction and detection of genomic DNA All sample DNA in this case was extracted in the laboratory according to the instructions of the DNA extraction kit: (1) Take 10-20 mg of ear tissue the size of a mung bean, absorb the alcohol on the tissue with a clean paper towel, put it into a 1.5 ml centrifuge tube, and cut it into small pieces as much as possible.

[0021] (2) Add 400 μL ACL Solution, shake and mix for 1 min, add 20 μL Proteinase K, and place in a 55°C water bath for 20 min-3 h. Take out and mix several times during this period, and lyse until the sample is clear and transparent.

[0022] (3) Add 100 μL of NPB solution, shake vigorously, and place in a 70°C water bath for 10 min.

[0023] (4) Cool to room temperature, add 250 μL of anhydrous ethanol, mix well. If precipitation occurs, blow and mix well with a blue pipette tip and add to the adsorption column.

[0024] (5) Centrifuge at 8000 rpm for 1 min, remove the GenClean Column, and discard the waste liquid in the collection tube.

[0025] (6) Place the GenClean Column back into the collection tube, add 500 μL of AW1 Solution, centrifuge at 8,000 rpm at room temperature for 1 min, and discard the waste liquid.

[0026] (7) Place the GenClean Column back into the collection tube, add 500 μL AW2 Solution, centrifuge at 8,000 rpm at room temperature for 1 min, and discard the waste liquid.

[0027] (8) Repeat step (7) once.

[0028] (9) Remove the GenClean column, discard the waste liquid in the collection tube, put the GenClean column back into the collection tube, and centrifuge at 12,000 rpm at room temperature for 1 min to remove the residual AW2 Solution. Open the lid for 2 min.

[0029] (10) Place the GenClean column in a new clean 1.5 ml centrifuge tube, add 50 μL of AE Buffer to the center of the column, leave at room temperature for 2 min, then centrifuge at 12,000 rpm for 1 min at room temperature. The liquid in the centrifuge tube is the DNA. Dissolve it in 40-50 μL of TE solution and store at -20°C.

[0030] DNA concentration was tested by UV spectrophotometer, and DNA quality was tested by 1% agarose gel electrophoresis. DNA of qualified quality was placed in a 4 ℃ refrigerator for standby use or stored at -20 ℃ for long term storage.

[0031] The qualified DNA was diluted to about 50ng / μL and sent to Neogen Biotech (Shanghai) Co., Ltd. for 50K SNP chip genotyping of pigs. PLINK V1.9 was used to perform quality control on the SNP chip test results. The quality control standards are as follows: (1) Delete the sites that do not correspond to the positions of the pig reference genome version 11.1 (Sus scrofa11.1) and the sites on the Y chromosome; (2) Deleting sites with SNP detection rates less than 90%; (3) Delete the data that do not meet the Hardy-Weinberg equilibrium test (P < 10 -6 ) site; (4) Delete sites with a minimum allele frequency of less than 5%; (5) Delete samples with a detection rate less than 90%.

[0032] 4. Genome-wide association analysis The univariate mixed linear model of GEMMA software was used to perform genome-wide association analysis on the constructed haplotypes. The model is as follows: , Among them, y represents the phenotypic value vector of the trait, n samples represent n traits, w represents the fixed effect matrix; α represents the c vector of corresponding coefficients including the intercept; x represents the n vector of marker genotypes; β represents the effect size of the marker; u represents the n vector of random effects; ε represents the n vector of errors; t-1 represents the residual variance; λ represents the ratio of two variance components; k represents the known n×n correlation matrix, In represents the n×n identity matrix; MVNn represents the n-dimensional multivariate normal distribution.

[0033] The lm function in R was used to determine the impact of the effect. The significance threshold at the genome level was 0.05 / N, and the significance threshold at the chromosome level was 1 / N, where N is the number of haplotypes used for analysis. The R package rMVP was used for visualization of the results.

[0034] GWAS analysis results Figure 1 As shown, there is a locus in chromosome 13 of the Large White pig population that significantly affects the total nipple number of Large White pigs.

[0035] 5. Correlation analysis between different genotypes and total teat number phenotypes of sows From the above analysis results, it can be seen that the SNP site of the molecular marker corresponds to the 10512725th mutation of chromosome 13 of the international pig genome version 11.1 reference sequence and is extremely significantly correlated with the total number of teats in Large White pigs (P<0.01), indicating that the molecular marker significantly affects the total number of teats in Large White pigs; further statistical analysis of experimental data shows that the selection of the GG genotype at the SNP site (Chr13:10512725) can increase the number of teats by 0.1 on average (see Table 1); therefore, the breeding pigs with the GG genotype at the 10512725th site are selected, and the breeding pigs with the CG genotype at this site are eliminated, so as to increase the frequency of the allele G at this site from generation to generation, thereby increasing the total number of teats in the offspring sows. And then accelerate the breeding process.

[0036] Table 1 Correlation between molecular marker SNP site chr13:10512725C>G and papilla number .

[0037] Although some preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0038] Obviously, those skilled in the art can make various changes and modifications to the present application without departing from the spirit and scope of the inventive concept. Thus, if these modifications and variations to the present application fall within the scope of the claims of the present application and their equivalents, the present application is also intended to include these modifications and variations.

Claims

1. A SNP molecular marker affecting the total nipple number of Large White pigs, characterized in that: Based on the international pig genome version 11.1 reference sequence, a single nucleotide polymorphism variation of the C / G transition type exists at base 10512725 of chromosome 13.

2. A method for selecting / identifying the trait of total teat number of Large White pigs using the SNP molecular markers of claim 1, characterized in that: The following steps are involved: (1) Take tissue samples from the pigs to be tested and extract whole genome DNA; (2) scanning the whole genome DNA using pig whole genome resequencing; (3) Obtain resequencing data, check the genotype of locus 10512725 on chromosome 13 based on the international porcine genome version 11.1 reference sequence, interpret the C / G polymorphism of this locus, and select / identify the total teat number trait of Large White pigs based on the characteristic that the total teat number of genotypes CG and GG is higher than the total teat number of genotype CC.

3. Use of the SNP molecular marker affecting the total nipple number of Large White pigs as described in claim 1 in the preparation of reagents for breeding / identifying Large White pig varieties / lines related to the total nipple number trait.

4. Use of the SNP molecular marker affecting the total nipple number of Large White pigs as described in claim 1 in breeding varieties / strains related to the trait of the total nipple number of Large White pigs.

5. A breeding method for increasing the total number of nipples in pigs, characterized in that: The steps include: (1) Based on the international pig genome version 11.1 reference sequence, the genotype of chromosome 13, locus 10512725, of the pig population was determined; (2) Select individual sows with CG or GG genotype at the 10512725th locus and eliminate those with CC genotype at the locus, so as to increase the frequency of the G allele at the locus from generation to generation, thereby increasing the total number of nipples in the offspring.

6. The breeding method according to claim 5, characterized in that: The breeding pigs are Large White pigs and their synthetic strains.

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