Use of snp molecular markers associated with pig girth and waist circumference

Abstract

The application discloses a SNP molecular marker related to the chest circumference and waist circumference of pigs and application thereof. The SNP molecular marker is located at the 62nd position from the 5' end of the nucleotide sequence shown in SEQ ID NO:1, corresponding to the position of 167553481bp on the chromosome 1 of the international pig reference genome 11.1 version 1, and a T>G mutation exists at the position. The SNP molecular marker provided by the application is significantly related to the chest circumference and waist circumference of pigs. By identifying the genotype of the SNP molecular marker, the identification of the chest circumference and / or waist circumference of pigs can be realized. By breeding pigs with the genotype GG of the SNP molecular marker, the breeding process of pigs can be accelerated, and the genetic improvement of pigs can be realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology, and relates to a SNP molecular marker related to the chest girth and waist girth of pigs and application thereof. BACKGROUND

[0002] Improvement of growth traits has been an important goal for raising pigs to improve economic efficiency and productivity. The chest girth and waist girth, as important phenotypic indicators for measuring the growth status of pigs, are closely related to key growth traits such as backfat thickness and body weight. The chest girth and waist girth of pigs not only reflect the body characteristics of pigs, but also indirectly reflect production traits such as meat production capacity and feed conversion efficiency of pigs. Therefore, optimization of the two traits is of great significance for improving the production performance and market competitiveness of pigs. Through traditional breeding methods, the chest girth and waist girth of pigs can be improved to a certain extent. However, as complex quantitative traits, the chest girth and waist girth are regulated by multiple genes. Therefore, through traditional breeding methods, the two traits are improved slowly and the effect is not obvious. SUMMARY

[0003] The application aims to provide a SNP molecular marker related to the chest girth and waist girth of pigs and application thereof.

[0004] According to one aspect of the application, a SNP molecular marker related to the chest girth and waist girth of pigs is provided, which is located at the 62nd position from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1, corresponding to the position of 167553481bp on chromosome 1 of the international pig reference genome version 11.1, and there is a T>G mutation at the position, and the nucleotide type is T or G, and the genotype of the SNP molecular marker is TT, TG or GG.

[0005] The SNP molecular marker provided by the application is significantly related to the chest girth and waist girth of pigs, mainly as follows: the chest girth and waist girth of pigs with genotype GG are greater than those of pigs with genotype TT and genotype TG, the chest girth of pigs with genotype TG is less than or equivalent to that of pigs with genotype TT, and the waist girth of pigs with genotype TG is greater than that of pigs with genotype TT. By identifying the SNP molecular marker single nucleotide polymorphism and / or the genotype of the SNP molecular marker, the chest girth and / or waist girth of pigs can be identified, and by breeding pigs with genotype GG of the SNP molecular marker, the breeding process of pigs can be accelerated, and genetic improvement of pigs can be realized.

[0006] Therefore, the SNP molecular marker related to the chest girth and waist girth of pigs provided by the application can be applied to:

[0007] (1) identifying the chest girth and / or waist girth of pigs;

[0008] (2) preparing products for identifying the chest girth and / or waist girth of pigs;

[0009] (3) Genetic improvement of pigs, based on the SNP molecular marker genotype GG of the selected pigs to achieve genetic improvement of pigs;

[0010] (4) Preparation of a product for assisting in the genetic improvement of pigs, based on the identification of the SNP molecular marker genotype of pigs to assist in the genetic improvement of pigs.

[0011] The chest circumference and waist circumference of pigs are indirect manifestations of the production traits of pigs, such as meat production capacity and feed conversion efficiency. The larger the chest circumference and waist circumference of pigs, the higher the meat production capacity and feed conversion efficiency of pigs. Therefore, by identifying the chest circumference and / or waist circumference traits of pigs, the meat production capacity and the feed conversion efficiency of pigs can be identified. Therefore, the SNP molecular marker related to the chest circumference and waist circumference of pigs provided by the present application can also be applied to assist in identifying the meat production capacity and / or the feed conversion efficiency of pigs, and to prepare a product for assisting in identifying the meat production capacity and / or the feed conversion efficiency of pigs.

[0012] In some embodiments, the chest circumference and / or waist circumference traits of pigs are the chest circumference and / or waist circumference of pigs at 100 kg body weight.

[0013] In some embodiments, the pigs are preferably Duroc and its synthetic lines.

[0014] According to another aspect of the present application, a primer pair is provided, which can specifically amplify an amplification fragment containing the single nucleotide polymorphism at position 62 from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 3.

[0015] The primer pair provided by the present application can amplify an amplification fragment containing the SNP molecular marker related to the chest circumference and waist circumference of pigs provided by the present application, and can be applied to identify whether the single nucleotide at position 62 from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1 is T or G. Therefore, the primer pair provided by the present application can be applied to identify the genotype of the SNP molecular marker related to the chest circumference and waist circumference of pigs and / or to prepare a product for identifying the genotype of the SNP molecular marker related to the chest circumference and waist circumference of pigs.

[0016] According to a third aspect of the present application, a kit is provided, and the composition of the kit comprises a primer pair with the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3.

[0017] The applications of the primer pair and the kit provided by the present application include but are not limited to:

[0018] (1) Identifying the genotype of the SNP molecular marker related to the chest circumference and waist circumference of pigs;

[0019] (2) preparing a product for identifying the genotype of the SNP molecular marker related to the pig's chest girth and waist girth;

[0020] (3) identifying the pig's chest girth and / or waist girth trait, and identifying the pig's chest girth and / or waist girth trait based on the identification of the genotype of the SNP molecular marker related to the pig's chest girth and waist girth;

[0021] (4) preparing a product for identifying the pig's chest girth and / or waist girth trait, and identifying the pig's chest girth and / or waist girth trait based on the identification of the genotype of the SNP molecular marker related to the pig's chest girth and waist girth;

[0022] (5) assisting in identifying the pig's meat production capacity, and assisting in identifying the pig's meat production capacity based on the identification of the pig's chest girth and / or waist girth trait;

[0023] (6) preparing a product for assisting in identifying the pig's meat production capacity, and assisting in identifying the pig's meat production capacity based on the identification of the pig's chest girth and / or waist girth trait;

[0024] (7) assisting in identifying the pig's feed conversion efficiency, and assisting in identifying the pig's feed conversion efficiency based on the identification of the pig's chest girth and / or waist girth trait;

[0025] (8) preparing a product for assisting in identifying the pig's feed conversion efficiency, and assisting in identifying the pig's feed conversion efficiency based on the identification of the pig's chest girth and / or waist girth trait;

[0026] (9) genetic improvement of pigs, and improving the genetics of pigs based on selecting pigs with the SNP molecular marker genotype of GG;

[0027] (10) preparing a product for assisting in the genetic improvement of pigs, and assisting in the genetic improvement of pigs based on identifying the genotype of the pig's SNP molecular marker.

[0028] In some embodiments, the kit provided by the present application can further comprise: dNTPs, DNA polymerase, Mg 2+ and other components of a conventional PCR reaction system.

[0029] According to a fourth aspect of the present application, a method for genetic improvement of pigs is provided, comprising the following steps:

[0030] (1) determining the genotype of the pig's SNP molecular marker related to the pig's chest girth and waist girth;

[0031] (2) selecting individuals with the SNP molecular marker genotype of GG, and eliminating individuals with the genotypes of TG and TT, thereby increasing the frequency of allele G at this locus from generation to generation, and improving the chest girth and waist girth of the offspring pigs and increasing the meat production of the offspring pigs.

[0032] In some embodiments, in step (1), the method for determining the genotype of the SNP molecular marker related to the chest girth and waist girth of the pig comprises the following steps:

[0033] Extracting the whole genome DNA of the pig, performing PCR amplification using a primer pair with nucleotide sequences as shown in SEQ ID NO: 2 and SEQ ID NO: 3, sequencing the amplification product, and determining the genotype of the SNP molecular marker related to the chest girth and waist girth of the pig based on the sequencing result.

[0034] In some embodiments, the pig is Duroc and its synthetic line. Duroc is widely used as the terminal sire of Duroc×Large White×Landrace commercial pigs, which is the most popular commercial pig breed in the world. Duroc directly affects the production performance of Duroc×Large White×Landrace commercial pigs, and the production performance of commercial pigs directly affects the economic benefits of the farm. Therefore, improving the chest girth and waist girth of the core group of Duroc pigs can greatly pass the advantageous genetics to the commercial pig offspring, enhance the production competitiveness of the commercial pigs, and thus improve the economic benefits of the farm.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] (1) The present application provides a SNP molecular marker on the nucleotide sequence of pig chromosome 1 related to the chest girth and waist girth of the pig, and verifies the effect of the SNP molecular marker on the chest girth and waist girth of the pig, which helps to establish a molecular marker assisted selection breeding technology for rapidly improving the chest girth and waist girth of the pig, and improves the breeding process of Duroc and its synthetic line.

[0037] (2) The present application provides a primer pair for identifying the SNP molecular marker related to the chest girth and waist girth of the pig. Through the primer pair, a high-efficiency and accurate molecular marker assisted breeding technology can be established to rapidly and accurately breed the traits and accelerate the breeding process. When applied to the genetic improvement of the chest girth and waist girth of the breeding pig, the chest girth and waist girth of the pig can be improved and the meat yield of the pig can be increased.

[0038] (3) The present application provides a method for genetic improvement of pigs by selecting the advantageous alleles of the SNP molecular marker related to the chest girth and waist girth of the pig, which can accelerate the genetic progress of Duroc pigs and shorten the time for Duroc improvement, thereby effectively improving the economic benefits of the breeding of breeding pigs. If all the TT type individuals of the SNP molecular marker affecting the chest girth and waist girth of the pig are selected to be GG type individuals, the chest girth and waist girth of each pig can be increased by 0.24 cm and 0.54 cm respectively at 100 kg body weight, and the increase of the chest girth and waist girth can increase the meat yield of the pig and improve the economic benefits of the commercial pig. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Figure for whole genome association (GWAS) analysis of Yorkshire pigs on chromosome 1 for 100 kg body weight on chest circumference;

[0040] Figure 2 Figure for whole genome association (GWAS) analysis of Yorkshire pigs on chromosome 1 for 100 kg body weight on chest circumference;

[0041] Figure 3 Figure for analysis of chest circumference results of pigs with different genotypes at 100 kg body weight;

[0042] Figure 4 Figure for analysis of waist circumference results of pigs with different genotypes at 100 kg body weight. DETAILED DESCRIPTION

[0043] The present application will be further described in conjunction with the embodiments. The embodiments are only used for explanation and do not limit the present application in any way. If no special description is made, the raw materials and reagents used in the embodiments are conventional products that can be commercially available; and the experimental methods not specified in the embodiments are usually carried out according to the conventional conditions in the art or according to the conditions recommended by the manufacturers.

[0044] Example 1: Identification and verification of SNP sites related to pig chest circumference and waist circumference

[0045] (1) Experimental pig population

[0046] The experimental pig population used in the present application is a purebred Yorkshire pig of the Pig Breeding Branch of Guangdong Wen's Food Group Co., Ltd., which is the core population of the Pig Breeding Branch and has detailed pedigree records. A total of 2080 Yorkshire pigs were selected from the resource population of the Pig Breeding Branch, and the pigs were fed under uniform feeding standards, free access to feed and water, and fed to 100±5 kg body weight.

[0047] (2) Phenotype measurement

[0048] The phenotypes of pig chest circumference and waist circumference were measured by soft tape according to the method, and the accuracy was cm. When the live weight of the pig reached 100±5 kg, the pig was fasted for 24 h. Before measurement, the pig was restrained, and the soft tape was gently attached to the body surface for measurement. The measurement method is as follows:

[0049] Chest circumference: the vertical circumference of the chest determined along the posterior margin of the scapula;

[0050] Waist circumference: the vertical circumference measured at the waist position (the anterior margin of the hind leg region).

[0051] (3) Pig genomic DNA extraction

[0052] The whole genome DNA of the Landrace pigs was extracted by the standard phenol-chloroform method after sampling the ears of the Landrace pigs, and the DNA of the purebred Landrace population was detected for quality and concentration by a Nanodrop-ND1000 spectrophotometer. The A260 / 280 ratio is 1.8-2.0, and the A260 / 230 ratio is 1.7-1.9 to determine the pass. Finally, the qualified DNA samples were uniformly diluted to 50 nanograms / microliter.

[0053] (4) 50K SNP genotype detection of the whole genome of pigs

[0054] Based on the GeneSeek Genomic Profiler Porcine 50K SNP typing platform, the chip hybridization and result scanning were carried out by using the instructions and standard process of Illumina Infinium. Finally, the genotype data was read by GenomeStudio software. The obtained genotype data was controlled for quality by PLINK v1.07, and the SNP markers with detection rate <99%, mimor allel frequency (MAF) <1% or Hardy-Weinberg Equilibrium (HWE) test P≤10 -6 were excluded. The individuals with detection rate <90% and high Mendelian error rate of family were excluded, and the SNPs located in unknown position and sex chromosome were excluded. Finally, 35755 effective genotype data of SNPs were obtained.

[0055] In order to improve the success rate of identifying key mutation sites of chest circumference and waist circumference traits, the SWIM website (http: / / 192.168.142.36:9088 / # / home) was used to increase the marker density of the Landrace pig whole genome 50K SNP chip in the application. Then, the 50K chip genotype data of all samples was controlled for quality by using PLINK software. In this process, the SNPs with detection individual rate lower than 90%, family Mendelian error rate higher than 0.1, minimum allel frequency less than 0.01 and Hardy-Weinberg balance significance level lower than 10 -6 were excluded. Finally, 11388559 effective genotype data of SNPs were obtained.

[0056] (5) Whole genome association (GWAS) analysis

[0057] In order to eliminate the population stratification effect, the linear mixed model single point regression analysis was used in the application, and the GWAS analysis was carried out by combining the R language GenABEL software package. The similarity of the genomes between individuals was used to correct the stratification effect in the analysis model. The genome level significant threshold is 10 -8 ; the chromosome level significant threshold is 10-5 .

[0058] The results of GWAS analysis are shown in Figure 1 and Figure 2 .

[0059] As shown in Figure 1 and Figure 2 , in Duroc, there is a SNP site on chromosome 1 that significantly affects 100 kg chest circumference and 100 kg waist circumference, and the most strongly associated SNP is g.62T>G (P=2.78x10 -7 ; P=1.15x10 -6 ), i.e., the 62nd nucleotide from the 5' end in SEQ NO. 1, corresponding to the T>G mutation at position 167553481 on chromosome 16 of the international pig reference genome version 11.1.

[0060] (6) Analysis of the association between different genotypes and the waist circumference and chest circumference phenotypes of pigs at 100 kg body weight to verify the effect of the SNP site on the waist circumference and chest circumference traits of pigs

[0061] The results are shown in Table 1, Figures 3-4 According to Table 1, Figures 3-4 , the SNP site g.62T>G of the molecular marker is extremely significantly associated with the waist circumference and chest circumference traits (P<0.01), indicating that the molecular marker significantly affects the waist circumference and chest circumference of pigs. Among them, pigs with the TT genotype of the molecular marker have smaller chest circumference than those with the GG genotype, and pigs with the TT genotype have smaller waist circumference than those with the GG and TG genotypes, indicating that the homozygote TT is unfavorable for the chest circumference and waist circumference of pigs. Chest circumference and waist circumference are important indicators for measuring the growth performance of pigs, and the larger the chest circumference and waist circumference, the better the growth performance of pigs. Therefore, the growth performance of pigs with the TT genotype is the worst, and in the process of breeding, pigs with the TT and TG genotypes need to be gradually eliminated, and pigs with the GG genotype need to be preserved to gradually increase the frequency of allele G at this site from generation to generation.

[0062] Table 1 Correlation analysis of SNP site g.26A>C and traits

[0063]

[0064] (7) Effect analysis

[0065] The present application provides a SNP molecular marker which is significantly related to the chest circumference and waist circumference traits of Duroc pigs, and using the SNP molecular marker for marker-assisted selection can accelerate the breeding process of the chest circumference and waist circumference of Duroc pigs. If all the TT type individuals of the molecular marker affecting the chest circumference and waist circumference traits of pigs are selected to GG type individuals, the chest circumference and waist circumference of each pig can be increased by 0.24 cm and 0.54 cm respectively at 100 kg body weight. Since the chest circumference and waist circumference are positively correlated with meat yield, breeders can increase the meat yield by increasing the chest circumference and waist circumference of pigs, and the body shape improvement of the chest circumference and waist circumference makes the pigs more beautiful, and the chest circumference and waist circumference of the appearance of the selected pigs can bring benefits to the pig breeding enterprises. Therefore, the potential of increasing the chest circumference and waist circumference to provide benefits for the pig industry is huge. In the SNP molecular marker individuals of the present application, by selecting the advantage allele (G) of the SNP of the Duroc pig, the economic benefits of the commercial pigs can be finally improved, thereby increasing the benefits of the enterprises.

[0066] Example 2 Genetic improvement method of pigs

[0067] The target fragment containing the SNP site which is significantly related to the 100 kg body weight waist circumference and chest circumference traits of the Duroc pig is a 157 bp nucleotide sequence in chromosome 1, and the specific sequence is shown as SEQ ID NO: 1, and the primer pair for PCR amplification is shown as SEQ ID NO: 2 and SEQ ID NO: 3.

[0068] SEQ ID NO: 1

[0069]

[0070]

[0071] The K marked in the sequence is a mutation site, which is T or G, and is an allele mutation; the bolded primer sequence binding position at the beginning and end of the sequence is shown.

[0072] Upstream primer primer-F: 5'-GATAGTGGGAACTGGAGGG-3' (SEQ ID NO: 2);

[0073] Downstream primer primer-R: 5'-GTAGTGAGAGAGACGGGTGG-3' (SEQ ID NO: 3).

[0074] The genetic improvement method of pigs comprises the following steps:

[0075] S1, determining the genotype of the SNP molecular marker related to the chest circumference and waist circumference of pigs

[0076] (1) Sampling from the ear of pig, extracting the whole genome DNA of pig according to the standard phenol-chloroform method, and then detecting the quality and concentration of the extracted DNA.

[0077] (2) PCR amplification

[0078] Prepare 10 μL system, which includes: 1 μL of DNA sample, 0.3 μL of upstream primer, 0.3 μL of downstream primer, 5 μL of PCR mix, 3.4 μL of ddH2O; the PCR mix includes dNTPs, DNA polymerase, Mg 2+ and the composition of the conventional PCR reaction system such as PCR reaction buffer.

[0079] PCR reaction program: 95 ℃ pre-denaturation for 5 min, 95 ℃ denaturation for 30 s, 64 ℃ annealing for 30 s, 72 ℃ extension for 30 s, a total of 35 cycles, and finally extension for 5 min at 72 ℃.

[0080] (3) DNA sequence sequencing identification

[0081] Sequencing the PCR amplification product, and sequencing the gene fragment in both positive and negative reactions, and determining the genotype of the to-be-tested pig SNP site g.26A>C according to the sequencing result.

[0082] S2, selecting the pig with the SNP site genotype of GG as the parent for breeding, and increasing the frequency of the allele G of the site generation by generation.

[0083] The above only describes some embodiments of the present application. For those skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the protection scope of the present application.

Claims

1. Use of a product for detecting a SNP molecular marker associated with pig girth and girth, characterized in that, The application comprises: (1) identifying pig girth and / or waist circumference traits; (2) preparing products for identifying pig girth and / or waist circumference traits; (3) pig genetic improvement, based on breeding pigs with SNP molecular marker genotypes of GG to improve the girth and / or waist circumference of pigs to achieve genetic improvement of pigs; (4) preparing products for assisting in pig genetic improvement, based on identifying pig SNP molecular marker genotypes to improve the girth and / or waist circumference of pigs to achieve genetic improvement of pigs; The SNP molecular marker site is located at position 62 from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1, corresponding to position 167553481 on chromosome 1 of the International Pig Reference Genome version 11.1, and the nucleotide type at this site is T or G, and the genotype of the SNP molecular marker is TT, TG or GG; wherein the girth and waist circumference of pigs with genotype GG are both greater than the girth and waist circumference of pigs with genotype TT and genotype TG, and the girth of pigs with genotype TG is less than or equivalent to the girth of pigs with genotype TT, and the waist circumference is greater than the waist circumference of pigs with genotype TT; The pig is a Yorkshire Duroc.

2. Use according to claim 1, characterized in that, The pig girth and / or waist circumference traits are the girth and / or waist circumference of the pig at 100 kg body weight.

3. Use according to claim 1 or 2, characterized in that, The products comprise at least one of the following: primer pairs and kits.

4. Use according to claim 3, characterized in that, The nucleotide sequence of the upstream primer of the primer pair is shown in SEQ ID NO: 2, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

3.

5. A method for genetic improvement of pigs, characterized in that, The method comprises the following steps: (1) determining the genotype of the pig's SNP molecular marker associated with pig girth and waist circumference; (2) breeding individuals with SNP molecular marker genotypes of GG, and eliminating individuals with genotypes of TG and TT to increase the frequency of allele G at this site from generation to generation; The SNP molecular marker associated with pig girth and waist circumference is located at position 62 from the 5' end of the nucleotide sequence shown in SEQ ID NO: 1, corresponding to position 167553481 on chromosome 1 of the International Pig Reference Genome version 11.1, and the nucleotide type at this site is T or G, and the genotype is TT, TG or GG; The pig is a Yorkshire Duroc.

6. The method of genetic improvement of swine according to claim 5, wherein, In step (1), the method for determining the genotype of the pig's SNP molecular marker associated with pig girth and waist circumference comprises the following steps: Extracting the whole genome DNA of the pig, performing PCR amplification using primer pairs with nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3, sequencing the amplification product, and determining the genotype of the pig's SNP molecular marker associated with pig girth and waist circumference based on the sequencing results.

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