A SNP marker affecting fatty acid composition in pig individuals

Abstract

The present invention provides a SNP marker that affects the fatty acid composition of pig individuals. The SNP marker is located at position 301 from the 5' end of SEQ ID No. 1, corresponding to position 112034471 from the 5' end of chromosome 8 in the 11.1 version of the international porcine genome, and is G or A.

Description

Technical Field

[0001] The present invention provides a SNP marker that affects fatty acid composition in pig individuals. Background Art

[0002] As a key sector of animal husbandry, pig farming ensures human food security. Global pork consumption reportedly accounts for approximately one-third of all meat products consumed. Pork is not only rich in high-quality protein but also provides the human body with a variety of essential amino acids, fatty acids, and a wealth of trace elements.

[0003] Pork contains a large amount of adipose tissue, which is one of the main sources of energy for the body. In addition to participating in the body's immune system and protecting organs, fat is also involved in the body's growth and metabolic processes. Adipose tissue is mainly composed of fatty acids and glycerol. Fatty acids are a class of long-chain carboxylic acids and are important components 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 around the world have increasingly demanded higher quality meat. However, saturated fatty acids such as C14:0 and C16:0 in pork are important factors that lead to cardiovascular diseases such as coronary heart disease and atherosclerosis in humans. Therefore, these saturated fatty acids are defined as "bad" fatty acids.

[0004] Research has shown that the monounsaturated fatty acid C18:1n9 is positively correlated with pork flavor and is beneficial to human health. Furthermore, C18:1n9 makes up the highest proportion of pork fatty acids, averaging approximately 45%. The C18:1n9 content in Spanish Iberian black pigs can reach approximately 55%, making them the most flavorful pork in the world, with a single pig ham fetching tens of thousands of yuan. C14:0 and C16:0, two fatty acids known to cause human disease, are present in certain concentrations in pork. Therefore, appropriately increasing the C18:1n9 content in pork fatty acids can enhance pork flavor and benefit human health. Reducing the levels of the "bad" fatty acids C14:0 and C16:0 can effectively mitigate the risk of various human diseases. Research groups at home and abroad have found that the heritability of major fatty acid composition traits ranges from 0.3 to 0.7, making them moderately heritable. Because fatty acid trait measurement is costly, time-consuming, and requires post-slaughter sampling, traditional genetic breeding techniques based on pedigree information are inefficient for improving fatty acid composition.

[0005] Therefore, in order to better balance the fatty acid composition and produce healthy and high-quality pork, exploring 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 pig SNP marker, which is located at the 301st position from the 5' end on SEQ ID No. 1, corresponding to the 112034471st position from the 5' end on chromosome 8 of the 11.1 version of the international pig genome, and is G or A.

[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 fatty acid composition of pigs or pork.

[0008] In one embodiment, when the SNP marker is A, compared with when the SNP marker is G, the fatty acid has an effect on at least one of the following traits:

[0009] 1) The content of C18:1n9 in the fatty acid is increased;

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

[0011] 3) the C18:1n9 / C16:1n7 ratio increased;

[0012] 4) the content of C14:0 in the fatty acid is reduced;

[0013] 5) the content of C16:0 in the fatty acid is reduced;

[0014] 6) the content of C16:1n7 in the fatty acid is reduced;

[0015] 7) The content of SFA in the fatty acids is reduced.

[0016] In one embodiment, when the SNP marker is G, compared with when the SNP marker is A, the fatty acid has at least one of the following effects:

[0017] 1) the content of C18:1n9 in the fatty acid is reduced;

[0018] II) the MUFA content in the fatty acids is reduced;

[0019] III) the C18:1n9 / C16:1n7 ratio decreased;

[0020] IV) the C14:0 content in the fatty acids is increased;

[0021] V) the C16:0 content in the fatty acids is increased;

[0022] VI) the content of C16:1n7 in the fatty acid is increased;

[0023] VII) The SFA content in the fatty acids is increased.

[0024] 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 the first aspect of the present invention, and making corresponding selections based on the SNP markers:

[0025] In the core group of sows, sows with G / A and A / A genotypes at the 301st site from the 5' end on SEQ ID No. 1 are selected, and sows with G / G genotypes at this site are eliminated to increase the frequency of allele A at this site generation by generation.

[0026] In one embodiment, in the core group of sows, sow individuals with the A / A genotype at the 301st site from the 5' end on SEQ ID No. 1 are selected, and sow individuals with the G / A and G / G genotypes at this site are eliminated to increase the frequency of allele A at this site generation by generation.

[0027] In a specific embodiment, the SNP marker of the sow as described in 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.

[0028] 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 as described in one aspect of the present invention, and determining the pork meat quality traits based on the SNP markers:

[0029] The pork quality traits are ranked from best to worst based on the genotype of the 301st position from the 5' end on SEQ ID No. 1: A / A genotype, G / A genotype and G / G genotype.

[0030] 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 the genotype of the SNP marker as described in one of the present inventions, the G / A or G / G genotype is mutated to the A / A genotype by site-directed mutagenesis.

[0031] In one embodiment, mutation is performed using a transgenic approach or a gene editing approach.

[0032] In one embodiment, mutation is performed using the CRISPR / Cas9 gene editing method.

[0033] Beneficial effects of the present invention:

[0034] The SNP marker 8_112034471 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.

[0035] By modifying the 8_112034471 SNP site, increasing the frequency of allele A and decreasing the frequency of allele G, the C18:1n9 and MUFA content of fatty acids can be increased; the C18:1n9 / C16:1n7 ratio can be increased; and the C14:0, C16:0, C16:1n7, and SFA content of fatty acids can be reduced. This not only improves the flavor of meat but also reduces the risk of cardiovascular disease, benefiting human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The Manhattan plot of the C18:1n9 / C16:1n7 ratio 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)).

[0037] Figure 2 A box plot shows the percentage of different genotypes of the most significant locus 112034471 in the fatty acid composition trait of the F6 population. The X-axis represents the genotype of the SNP locus, 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 herd (among which, one phenotypic outlier was excluded for the C18:1n9 / C16:1n7 ratio trait. Cov0 represents the covariate, including sex, age at slaughter, and slaughter batch). DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to examples. However, the examples are merely illustrative and do not limit the present invention in any way.

[0039] 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) through multiple generations of hybridization.

[0040] Example 1

[0041] 1. Acquisition, quality control and typing of pig whole genome resequencing data

[0042] A small muscle tissue sample was collected from 797 randomly selected F6 individuals. Genomic DNA was extracted from each individual using the standard phenol-chloroform method and dissolved in TE buffer. The extracted genomic DNA was tested for quality using a Nanodrop-ND1000 spectrophotometer. Quality standards were met when the A260 / 280 ratio was between 1.8 and 2.0, and the A260 / 230 ratio was between 1.7 and 1.9.

[0043] DNA samples meeting the criteria were diluted to 50 ng / μl and resequenced at a low depth (average sequencing depth of approximately 7.8X) using the Illumina HiSeqXTen sequencer. All paired-end reads were aligned to the International Porcine Genome (IPG) version 11.1 using BWA software. Genotype data for each individual were then generated using SAMTools, Platypus, and Beagle. Genotype data were quality-controlled using Plink 1.9, and individuals with minor allele frequencies (MAF) < 0.03 and pedigree Mendelian error rates (FMRs) above 0.1 were removed. A total of 29,441,528 SNPs were identified in the F6 population.

[0044] 2. GCMS quantitative determination of fatty acid content in pork

[0045] The fatty acid composition of pork is composed of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA).

[0046] Because the present invention focuses on determining the fatty acid composition phenotype in pork, based on the biochemical reactions of fatty acids and the ease of separation of oils in polar solvents, fatty acid methyl esters (FAMs), derived from fatty acid saponification and methylation reactions, are extracted. The separated FAMEs are then qualitatively analyzed using a gas chromatograph (GCMS). The percentage of each fatty acid is then calculated by converting the fatty acid-FAM molecular weight coefficient using peak area normalization. Specific testing standards are described in the National Food Safety Standard for the Determination of Fatty Acids in Food (GB 5009.168-2016).

[0047] The detailed method for preparing fatty acid extracts from the longissimus dorsi muscle of 797 pigs in the F6 population is described 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.

[0048] GCMS was used to analyze the fatty acid composition (%) of the longissimus dorsi muscle extract from each individual (n = 797). Descriptive statistics for seven fatty acid composition phenotypes were obtained and are shown in Table 1. As shown in Table 1, SFA and MUFA account for approximately 88.37% of the total content in pork, with C16:0 comprising the largest proportion of SFA and C18:1n9 comprising the largest proportion of MUFA.

[0049] Table 1. Descriptive statistics of fatty acid composition phenotypes of F6 population

[0050]

[0051] 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.

[0052] 3. Genome-wide association (GWAS) analysis

[0053] 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 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 the SNP, b represents the effect of the SNP, u represents the random effect vector, e represents the error vector, β represents the ratio of the two variances, t-1 represents the variance of the residual, K represents the kinship matrix, E represents the identity matrix, and MVNn represents the multivariate normal distribution.

[0054] The results of GWAS analysis of the high-quality fatty acid C18:1n9 / C16:1n7 ratio trait in the F6 population are shown in Figure 1 .Depend on Figure 1 It can be seen that the most significant locus affecting the ratio of high-quality fatty acids C18:1n9 / C16:1n7 in pigs is on chromosome 8.

[0055] The present invention focuses only on the case where the highest -log P value on chromosome 8 corresponds to physical position 112034471. This SNP is located at position 301 from the 5' end of SEQ ID No. 1. The basic genetic parameter information for the fatty acid composition trait of the F6 population at this SNP is shown in Table 2.

[0056] From the results in Table 2, we can see that the 8_112034471 SNP has the most significant effect on the C18:1n9 / C16:1n7 ratio and the largest effect value, and has the smallest effect on SFA and the smallest effect value.

[0057] Table 2. Basic genetic parameter information of SNP sites in F6 population

[0058]

[0059] The genotypes of each of the 797 individuals in the F6 population at the 8_112034471 SNP site were extracted from the sequencing files using PLINK software. After counting the number of individuals of each genotype, the genotypes of these individuals were matched one-to-one with their corresponding fatty acid compositions. The multcomp package in R was then used to statistically analyze the differences in phenotypic distributions under different genotypes. The results are shown in Figure 2 and Table 3, where the P values ​​are obtained from the variance test.

[0060] Depend on Figure 2As shown in Table 3, compared to G, genotype A increases C18:1n9, the C18:1n9 / C16:1n7 ratio, and MUFA content, while decreasing C14:0, C16:0, C16:1n7, and SFA content. The decrease in C16:1n7 and the increase in C18:1n9 result from the conversion of C16:1n7 to C18:1n9. These results suggest that this locus can be used to improve the fatty acid composition of pork. While decreasing C14:0, C16:0, C16:1n7, and SFA, it can also increase C18:1n9, the C18:1n9 / C16:1n7 ratio, and MUFA content. This not only improves pork flavor but also reduces the risk of cardiovascular and cerebrovascular diseases. Therefore, the order of superiority of the three genotypes at this locus for fatty acid composition is A / A > A / G > G / G.

[0061] Table 3. Effects of SNP 8_112034471 on fatty acid composition of F6 pigs

[0062]

[0063] 4. The amount of phenotypic variation that SNPs can explain

[0064] 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 proportion of the variance of the breeding value of a quantitative trait to the variance of the phenotypic trait. It is the additive effect part after eliminating the dominant effect and epistatic effect, and can be stably inherited during the generation-to-generation transmission process.

[0065] Since the present invention adopts the additive effect model to perform GWAS analysis on fatty acid composition traits, the phenotypic variance explained (PVE) size of the SNP site is the h 2 The detailed information on the PVE of the fatty acid composition traits of the F6 population explained by this SNP site is shown in Table 4.

[0066] As shown in Table 4, the most significant phenotype that can be explained by this locus is the C18:1n9 / C16:1n7 ratio.

[0067] Table 4. Phenotypic variation explained by significant SNP site 8_112034471 on fatty acid composition traits

[0068]

[0069] Although the present invention has been described with reference to specific embodiments, those skilled in the art will appreciate that various modifications may be made without departing from the true spirit and scope of the invention. Furthermore, the subject matter, spirit, and scope of the invention may be modified in various ways to adapt to specific circumstances, materials, combinations of materials, and methods. All such modifications are intended to be within the scope of the claims.

Claims

1. A porcine SNP marker for use in determining the fatty acid composition of pigs or pork. The nucleic acid sequence of the SNP marker is SEQ ID No.

1. Position 301 from the 5' end of SEQ ID No. 1 corresponds to position 112034471 from the 5' end of chromosome 8 in the 11.1 international porcine genome, and is either G or A. When the SNP marker is A, compared to when the SNP marker is G, the SNP marker has an effect on at least one of the following fatty acid traits: 1) The content of C18:1n9 in the fatty acid is increased; 2) the MUFA content in the fatty acids is increased; 3) Increased C18:1n9 / C16:1n7 ratio; 4) the C14:0 content in the fatty acid is reduced; 5) the C16:0 content in the fatty acid is reduced; 6) the content of C16:1n7 in the fatty acid is reduced; 7) The content of SFA in the fatty acids is reduced; or When the SNP marker is G, compared with when the SNP marker is A, the fatty acid has at least one of the following effects: 1) the content of C18:1n9 in the fatty acid is reduced; II) the MUFA content in the fatty acids is reduced; III) the C18:1n9 / C16:1n7 ratio decreased; IV) the C14:0 content in the fatty acids is increased; V) the C16:0 content in the fatty acids is increased; VI) the content of C16:1n7 in the fatty acid is increased; VII) The SFA content in the fatty acids is increased.

2. A method for genetic improvement of pigs, comprising: Determine a SNP marker for sows in a sow core group, where the nucleic acid sequence of the SNP marker is SEQ ID No. 1, located at position 301 from the 5' end of SEQ ID No. 1, corresponding to position 112034471 from the 5' end of chromosome 8 of the 11.1 version of the international porcine genome, and is G or A; make a corresponding selection based on the SNP marker: Selecting sow individuals with G / A and A / A genotypes at the 301st site from the 5' end of SEQ ID No. 1 from the sow core group, and eliminating sow individuals with the G / G genotype at this site to increase the frequency of allele A at this site generation by generation; When the SNP marker is A, compared to when the SNP marker is G, the fatty acid has at least one of the following effects: 1) The content of C18:1n9 in the fatty acid is increased; 2) the MUFA content in the fatty acids is increased; 3) Increased C18:1n9 / C16:1n7 ratio; 4) the C14:0 content in the fatty acid is reduced; 5) the C16:0 content in the fatty acid is reduced; 6) the content of C16:1n7 in the fatty acid is reduced; 7) The content of SFA in the fatty acids is reduced.

3. The method according to claim 2, wherein In the core group of sows, sow individuals with the A / A genotype at the 301st site from the 5' end on SEQ ID No. 1 are selected, and sow individuals with the G / A and G / G genotypes at this site are eliminated to increase the frequency of allele A at this site generation by generation.

4. The method according to claim 2 or 3, characterized in that The SNP marker of the sow according to 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 pork quality traits, the method comprising: Determine the pig SNP marker, where the nucleic acid sequence of the SNP marker is SEQ ID No. 1, located at position 301 from the 5' end of SEQ ID No. 1, corresponding to position 112034471 from the 5' end of chromosome 8 in the 11.1 version of the international porcine genome, and is G or A; determine the pork quality trait based on the SNP marker: The pork quality traits are ranked from best to worst based on the genotype of the 301st position from the 5' end of SEQ ID No. 1: A / A genotype, G / A genotype, and G / G genotype; When the SNP marker is A, compared to when the SNP marker is G, the fatty acid has at least one of the following effects: 1) The content of C18:1n9 in the fatty acid is increased; 2) the MUFA content in the fatty acids is increased; 3) Increased C18:1n9 / C16:1n7 ratio; 4) the C14:0 content in the fatty acid is reduced; 5) the C16:0 content in the fatty acid is reduced; 6) the content of C16:1n7 in the fatty acid is reduced; 7) The content of SFA in the fatty acids is reduced.

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