A SNP marker affecting fatty acid composition in pig individuals

Abstract

The present invention provides a SNP marker that affects fatty acid composition in pig individuals. The SNP marker is located at the 301st position from the 5' end on SEQ ID No. 1, corresponding to the 111614768th position from the 5' end on chromosome 14 of the 11.1 version of the international pig genome, and is C or T.

Description

Technical Field

[0001] The present invention relates to the field of molecular markers, in particular to SNP markers that affect fatty acid composition in pig individuals. Background Art

[0002] As one of the important industries of animal husbandry, pig farming provides guarantee for human food demand. It is reported that the total consumption of pork in the world accounts for about one-third of all meat products. Pork is not only rich in high-quality protein, but also provides the human body with a variety of essential amino acids, fatty acids, rich trace elements and other nutrients.

[0003] Pork contains a large amount of adipose tissue, which is one of the important sources of energy for the body. In addition to participating in the body's immune mechanism and protecting organs, fat also participates in the body's growth and metabolism process. Adipose tissue is mainly composed of fatty acids and glycerol. Fatty acids are a class of long-chain carboxylic acid substances and are an important component of animal cells. Fatty acids include saturated fatty acids and polyunsaturated fatty acids (including monounsaturated fatty acids and polyunsaturated fatty acids), and the fatty acid composition is closely related to the nutritional value and taste of pork. In recent years, with the continuous improvement of living standards, people all over the world have an increasing demand for meat quality. However, saturated fatty acids C14:0, C16:0, etc. in pork are important factors leading to cardiovascular diseases such as coronary heart disease and atherosclerosis in humans. Therefore, these saturated fatty acids are defined as "bad" fatty acids.

[0004] Studies have shown that the monounsaturated fatty acid C18:1n9 is positively correlated with the flavor of pork and is beneficial to human health. In addition, C18:1n9 accounts for the highest proportion of fatty acids in pork, averaging about 45%. Among them, C18:1n9 in Spanish Iberian black pigs can reach about 55%, which is the best-flavored pork in the world. A pig ham can be sold for tens of thousands of yuan. C18:0 is a saturated fatty acid in pork. It has a certain content in pork and can be converted into the fatty acid C18:1n9, which is beneficial to the body. Domestic and foreign research teams have found that the heritability of the main fatty acid composition traits is between 0.3-0.7, which is a medium heritability trait. Because the determination of fatty acid traits is costly, time-consuming and labor-intensive, and requires sampling and determination after slaughter, it is not efficient to use traditional genetic breeding techniques based on family information to improve fatty acids.

[0005] Therefore, in order to better balance the fatty acid composition and produce healthy and high-quality pork, exploring the markers that affect the content of high-quality fatty acids such as C18:1n9 and improving the breeding population are of great significance to the production and economic benefits of the pig farming industry. Summary of the invention

[0006] One of the present inventions provides a SNP marker for pigs, which is located at the 301st position from the 5' end on SEQ ID No. 1, corresponding to the 111614768th position from the 5' end on chromosome 14 of the 11.1 version of the international pig genome, and is C or T.

[0007] The second aspect of the present invention provides the use of the SNP marker described in the first aspect of the present invention in determining the quality of fatty acid composition in pigs or pork.

[0008] In a specific embodiment, when the SNP marker is T, compared with when the SNP marker is C, the fatty acid produces at least one of the following traits:

[0009] 1) The content of C16:1n7 in the fatty acid is increased;

[0010] 2) The content of MUFA in the fatty acids is increased;

[0011] 3) the content of C18:1n9 in the fatty acid is increased;

[0012] 4) the content of C18:0 in the fatty acid is reduced;

[0013] 5) The content of SFA in the fatty acid is reduced;

[0014] or

[0015] When the SNP marker is C, compared with when the SNP marker is T, the fatty acid has at least one of the following effects:

[0016] 1) the content of C16:1n7 in the fatty acid is reduced;

[0017] II) the content of MUFA in the fatty acids is reduced;

[0018] III) the content of C18:1n9 in the fatty acid is reduced;

[0019] IV) the C18:0 content in the fatty acid is increased;

[0020] V) the content of SFA in the fatty acids is increased;

[0021] The third aspect of the present invention provides a method for genetic improvement of pigs, the method comprising: determining the SNP markers of the breeding pigs in the breeding pig core group as described in one of the present invention, and making corresponding selections according to the SNP markers:

[0022] In the core group of sows, sows with T / C and T / T genotypes at the 301st position from the 5' end on SEQ ID No. 1 are selected, and sows with C / C genotypes at this position are eliminated to increase the frequency of allele T at this position generation by generation.

[0023] In a specific embodiment, in the core group of sows, sow individuals with T / T genotype at the 301st position from the 5' end on the SEQ ID No. 1 are selected, and sow individuals with T / C and C / C genotypes at this position are eliminated to increase the frequency of allele T at this position generation by generation.

[0024] In a specific embodiment, the SNP marker of the sow according to one of the present invention is determined by analyzing the sequence of the nucleic acid of the sow, wherein the sequence of the nucleic acid is shown as SEQ ID No.1.

[0025] The fourth aspect of the present invention provides a method for determining the quality of pork meat, the method comprising: determining the SNP markers of the pig described in the first aspect of the present invention, and determining the pork meat quality traits according to the SNP markers:

[0026] The pork quality traits are arranged from best to worst according to the genotype of the 301st position from the 5' end on the SEQ ID No. 1: T / T genotype, T / C genotype and C / C genotype.

[0027] The fifth aspect of the present invention provides a method for establishing a new pig strain and / or a new pig variety with improved pork quality, which comprises the following steps: for a pig whose SNP marker genotype is T / C or C / C as described in one of the present invention, mutating the T / C or C / C genotype into a T / T genotype by site-directed mutagenesis.

[0028] In one embodiment, mutation is performed using a transgenic approach or a gene editing approach.

[0029] In one embodiment, mutation is performed using the CRISPR / Cas9 gene editing method.

[0030] Beneficial effects of the present invention:

[0031] The SNP marker 14_111614768 of the present invention is significantly correlated with the fatty acid composition trait, so the SNP marker of the present invention can be used to phenotype the site of the pig herd, and the SNP marker can be used to perform genetic improvement related to fatty acid composition on the pig herd.

[0032] By improving the 14_111614768 SNP site, increasing the frequency of the allele T at the site and reducing the frequency of the allele C, the content of C18:1n9 in fatty acids can be increased while reducing the content of C18:0, which shows that the site can affect the conversion of fatty acid C18:0 to C18:1n9. In addition, increasing the frequency of the allele T at the 14_111614768 site and reducing the frequency of the allele C can also increase the content of C16:1n7 and MUFA in fatty acids; and reduce the content of SFA in fatty acids. This is not only conducive to improving the flavor of meat, but also can reduce the risk of people suffering from cardiovascular and cerebrovascular diseases, which is beneficial to human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The Manhattan plot of C18:0 in the GWAS analysis of chimeric F6 is shown. The X-axis is the position of the SNP site on the chromosome, and the Y-axis is the -log 10 (P value).

[0034] Figure 2 The box plot (Box Plot) shows the percentage of different genotypes of the most significant site 111614768 in the fatty acid composition trait of the F6 population. Among them, the X-axis represents the genotype of the SNP site, the Y-axis represents the phenotypic value of the fatty acid composition of the individual, and the numbers above the box plot represent the number of individuals of each genotype in the pig herd (among which, C18:0 eliminates a phenotypic outlier. Cov0 represents covariates, including sex, slaughter age and slaughter batch). DETAILED DESCRIPTION

[0035] The present invention will be further described below in conjunction with examples, but the examples of the present invention are only exemplary descriptions, and the implementation methods do not constitute limitations of the present invention under any circumstances.

[0036] The chimeric family F6 pig group used in the present invention is the offspring of four Western commercial pig breeds (Duroc, Large White, Landrace, Pietrain) and four Chinese local pig breeds (Erhualian, Laiwu, Bamaxiang, Tibetan pig) after multiple generations of hybridization.

[0037] Example 1

[0038] 1. Acquisition, quality control and typing of whole genome resequencing data of pigs

[0039] 797 heads were randomly selected from the F6 population, and a small muscle tissue sample was collected from each individual. The genomic DNA of each individual was extracted using the standard phenol-chloroform method, and the extracted genomic DNA was dissolved in TE buffer. The quality of the extracted genomic DNA was tested using a Nanodrop-ND1000 spectrophotometer, and the quality standard was met when the A260 / 280 ratio was between 1.8 and 2.0 and the A260 / 230 ratio was around 1.7 and 1.9.

[0040] The concentration of the DNA samples that met the standards was diluted to 50 ng / μl, and each DNA sample was resequenced at a low depth using Illumina's HiSeqXTen sequencer platform (the average sequencing depth was about 7.8X). All the double-end reads obtained were aligned to the 11.1 version of the International Swine Genome using BWA software, and then the genotype data of each individual was obtained using SAMTools, Platypus, and Beagle software. Plink1.9 was used to perform quality control on the obtained genotype data, and individuals with minor allele frequency (MAF) < 0.03 and pedigree Mendelian error rate higher than 0.1 were eliminated. Finally, 29,441,528 SNPs were determined in the F6 population.

[0041] 2. GCMS quantitative determination of fatty acid content in pork

[0042] The fatty acid composition of pork is composed of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA).

[0043] Since the present invention focuses on determining the fatty acid composition phenotype in pork, based on the biochemical reaction of fatty acids and the easy separation of fats in polar solvents, fatty acid methyl esters, a fatty acid derivative obtained by saponification and methylation of fatty acids, are extracted, and the separated fatty acid methyl esters are qualitatively analyzed by gas chromatography (hereinafter referred to as "GCMS"), and the percentage of each fatty acid is obtained by converting the fatty acid-fatty acid methyl ester molecular weight coefficient according to the peak area normalization method. Specific inspection standards can be found in the "National Food Safety Standard Determination of Fatty Acids in Food GB5009.168-2016".

[0044] The detailed preparation method of the fatty acid extract of the longissimus dorsi muscle of 797 pigs in the F6 group can be found in the article "Folch, J., Lees, M., Sloane Stanley, GH, 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226 (1), 497-509." 10 g of pork was used to obtain 4 ml of the extract.

[0045] The fatty acid composition content (%) in the longissimus dorsi muscle extract of each individual (n=797) was detected by GCMS, and the descriptive statistical results of the five fatty acid composition phenotypes were obtained as follows, see Table 1. As can be seen from Table 1, SFA and MUFA in pork account for about 88.37% of the total, among which C18:1n9 accounts for the largest proportion in MUFA.

[0046] Table 1. Descriptive statistics of fatty acid composition phenotype of F6 population

[0047]

[0048] Note: MUFA (Monounsaturated Fatty Acids) are monounsaturated fatty acids, including C14:1n5, C16:1n7, C17:1n7, C18:1n9 and C20:1n9; SFA (Saturated Fatty Acid) are saturated fatty acids, including C14:0, C16:0, C17:0, C18:0 and C20:0.

[0049] 3. Genome-wide association study (GWAS)

[0050] The mixed linear model in GEMMA (Genome-wide Efficient Mixed Model Association algorithm, version 0.98.1) software was used to perform GWAS analysis on the SNP marker information of the F6 population obtained by the second-generation resequencing technology and the corresponding fatty acid composition traits of 797 individuals. The expression is as follows: y = Xa + Qb + u + e; u ~ MVNn (0, βt -1 K),e~MVNn(0,t -1 E). Where y represents the phenotypic value vector of all individuals, X represents the covariate matrix, a represents the corresponding coefficient vector including the intercept, Q represents the genotype vector of SNP, b represents the effect of SNP, u represents the random effect vector, e represents the error vector, β represents the ratio of the two variances, t -1represents the variance of the residual, K represents the affinity matrix, E represents the identity matrix, and MVNn represents the multivariate normal distribution.

[0051] The results of GWAS analysis of fatty acid C18:0 in the F6 population are shown in Figure 1 .Depend on Figure 1 It can be seen that the most significant site affecting the pig fatty acid C18:0 trait is located on chromosome 14.

[0052] The present invention only focuses on the case where the highest -log P value of chromosome 14 corresponds to physical position 111614768, and the SNP site is located at the 301st site from the 5' end on SEQ ID No. 1. The basic genetic parameter information of the fatty acid composition trait of the F6 population at this SNP site is shown in Table 2.

[0053] From the results in Table 2, we can see that 14_111614768SNP has the most significant effect on C18:0 and the largest effect value, and has the smallest effect on C18:1n9 and the smallest effect value.

[0054] Table 2. Basic genetic parameter information of SNP loci in F6 population

[0055]

[0056] The genotypes of each of the 797 individuals in the F6 population at the 14_111614768 SNP site were extracted from the sequencing file using PLINK software. After counting the number of individuals of each genotype, the genotypes of these individuals were matched one by one with their corresponding fatty acid compositions. Then, the multcomp package in the R language was used to statistically analyze the differences in phenotypic distribution under different genotypes. The results are shown in Figure 2 and Table 3. Among them, the P value is obtained from the variance test.

[0057] Depend on Figure 2 As shown in Table 3, compared with C, the T genotype can increase the content of C16:1n7, C18:1n9 and MUFA, and reduce the content of C18:0 and SFA. The above results show that the fatty acid composition in pork can be improved based on this site. While reducing C18:0 and SFA, the content of C16:1n7, C18:1n9 and MUFA can be increased, which can not only improve the flavor of pork, but also reduce the risk of cardiovascular and cerebrovascular diseases. Therefore, the three genotypes of this site are ranked as T / T>T / C>C / C for fatty acid composition.

[0058] Table 3. Effect of SNP site 14_111614768 on fatty acid composition of individual F6 pigs

[0059]

[0060] 4. The size of phenotypic variation that SNPs can explain

[0061] Heritability is the most important basic genetic parameter in quantitative genetics and can be divided into broad-sense heritability, narrow-sense heritability and realized heritability. In the breeding process, heritability generally refers to narrow-sense heritability (h 2 ), which refers to the ratio of the variance of the breeding value of a quantitative trait to the variance of the phenotypic trait. It is the additive effect after eliminating the dominant effect and epistatic effect, and can be stably inherited during the generation-to-generation transmission process.

[0062] Since the additive effect model is used in the present invention to perform GWAS analysis on fatty acid composition traits, the size of the phenotypic variance explained (PVE) by the SNP site is the h 2 The detailed information of PVE explained by this SNP site for the fatty acid composition traits of the F6 population is shown in Table 4.

[0063] As can be seen from Table 4, the most significant phenotype that can be explained by this site is C18:0.

[0064] Table 4. Phenotypic variation explained by the significant SNP site 14_111614768 on fatty acid composition traits

[0065]

[0066] Although the present invention has been described with reference to specific embodiments, it will be appreciated by those skilled in the art that various changes may be made without departing from the true spirit and scope of the present invention. In addition, the subject matter, spirit and scope of the present invention may be varied to accommodate specific situations, materials, material combinations and methods. All of these changes are included within the scope of the claims of the present invention.

Claims

1. Use of a pig SNP marker in determining the quality of fatty acid composition in pigs or pork, wherein the SNP marker is located at the 301st position from the 5' end on SEQ ID No. 1, corresponding to the 111614768th position from the 5' end on chromosome 14 of the 11.1 version of the international pig genome, and is C or T; When the SNP marker is T, compared with when the SNP marker is C, the fatty acid produces at least one of the following traits: 1) The content of C16:1n7 in the fatty acid is increased; 2) the content of MUFA in the fatty acids is increased; 3) The content of C18:1n9 in the fatty acid is increased; 4) the content of C18:0 in the fatty acid is reduced; 5) The content of SFA in the fatty acid is reduced; or When the SNP marker is C, compared with when the SNP marker is T, the fatty acid has at least one of the following effects: I) the content of C16:1n7 in the fatty acid is reduced; II) the content of MUFA in the fatty acids is reduced; III) the content of C18:1n9 in the fatty acid is reduced; IV) the C18:0 content in the fatty acid is increased; V) The SFA content in the fatty acids is increased.

2. A method for genetic improvement of pigs, the method comprising: Determine the SNP markers in the application of claim 1 for the sows in the sow core group, and make corresponding selections based on the SNP markers: Selecting sow individuals with T / C and T / T genotypes at the 301st position from the 5' end on the SEQ ID No. 1 in the sow core group, and eliminating sow individuals with C / C genotype at the position, so as to increase the frequency of allele T at the position generation by generation; The genetic improvement is to genetically improve at least one of the following fatty acid traits: 1) the content of C16:1n7 in fatty acids; 2) the content of MUFA in fatty acids; 3) the content of C18:1n9 in fatty acids; 4) the content of C18:0 in fatty acids; 5) the content of SFA in fatty acids.

3. The method according to claim 2, characterized in that In the core group of sows, sow individuals with T / T genotype at the 301st position from the 5' end on SEQ ID No. 1 are selected, and sow individuals with T / C and C / C genotypes at this position are eliminated to increase the frequency of allele T at this position generation by generation.

4. The method according to claim 2 or 3, characterized in that The SNP marker of the sow in the use of claim 1 is determined by analyzing the sequence of the nucleic acid of the sow, wherein the sequence of the nucleic acid is shown as SEQ ID No.

1.

5. A method for determining the quality of pork, the method comprising: Determine the SNP marker of the pig in the application of claim 1, and determine the pork quality trait based on the SNP marker: The pork quality traits are arranged from best to worst according to the genotype of the 301st position from the 5' end of the SEQ ID No. 1: T / T genotype, T / C genotype and C / C genotype; Among them, the pork quality traits are at least one of the following fatty acid traits: 1) the content of C16:1n7 in fatty acids; 2) the content of MUFA in fatty acids; 3) the content of C18:1n9 in fatty acids; 4) the content of C18:0 in fatty acids; 5) the content of SFA in fatty acids.

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