A SNP molecular marker associated with pork color redness value and its application

Abstract

The present invention provides a SNP molecular marker associated with the redness value of pork color. The SNP molecular marker is located at the missense mutation site rs793627529 on the fourth exon (17:32631325) of the MRPS26 gene on chromosome 17 of the international pig genome version 11.1 reference sequence. The polymorphism of the molecular marker is G / A, and the pork color redness value of individuals with AA and AG genotypes is significantly higher than that of individuals with GG genotypes.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and relates to a SNP molecular marker related to pork color redness value trait and application thereof. Background Art

[0002] With the development of my country's economy, people are paying more and more attention to the quality of pork. High-quality pork products will be the trend of green, healthy and efficient development of pigs in the future. There are many evaluation indicators for pork quality, including meat color, intramuscular fat, pH value, tenderness and other indicators. Meat color is one of the important indicators in the pork quality evaluation system and can directly affect consumers' purchasing desire. In addition, the meat color index is closely related to important meat quality indicators such as intramuscular fat content and tenderness. The redness value (a*) of pork color is one of the most intuitive factors for consumers to judge the quality of pork. Therefore, the optimization of pig a* can effectively improve the quality of pork. Taking a* as a breeding target trait has become one of the strategies for innovation in the pig breeding industry.

[0003] The traditional method of meat color evaluation is mainly through subjective evaluation, that is, professionals observe the color of the longissimus dorsi muscle surface at the last rib and compare it with the standard colorimetric plate to determine the quality of pork color. Objective evaluation methods mainly include colorimeter method, chemical method and spectrophotometry. Compared with subjective evaluation methods, chemical method and spectrophotometry are more accurate and scientific, but their measurement steps are cumbersome and the sample processing time is too long. The colorimeter method is currently the most common objective evaluation method for measuring meat color traits. Most of the research based on meat color traits at home and abroad uses colorimeter for measurement. The colorimeter method mainly uses CIE Lab color space to evaluate meat color traits. It was proposed by the International Commission on Illumination (CIE). It is mainly divided into three parameters: L*, a* and b*. These three space parameters are used to construct a coordinate system to evaluate color, where L* represents brightness, a* represents redness, and b* represents yellowness.

[0004] The pig a* trait is a complex quantitative trait with a medium to low heritability ( h 2 = 0.25~0.6). Although traditional phenotypic selection and breeding methods based on pedigree information can help a* selection to a certain extent, progress is slow due to the difficulty and low accuracy of a* determination in vivo, the time-consuming and labor-intensive slaughter determination, and the long breeding cycle. Therefore, identifying molecular markers (Single nucleotide polymorphism, SNP) related to the a* trait, combined with marker-assisted selection (MAS) and genomic selection (GS) technology, can quickly select and improve the a* of pig herds to meet the market demand for high-quality pork.

[0005] For nearly half a century, the world's pig farming industry has been driven by the selection and breeding orientation of fast growth and high lean meat rate. Commercial pig production has been almost covered by lean pigs such as Duroc pigs, Landrace pigs, Large White pigs and their hybrid combinations. These breeds have contributed to meeting the human demand for pork quantity, but this has also led to a significant decline in pork quality, causing serious economic losses to pig slaughtering, pork retail and meat processing industries. Therefore, under the premise of ensuring pork supply, improving pork quality has become a hot topic in current research, and improving a* is a key link in improving pork quality. At present, the a* of foreign pig breeds and their hybrid combinations on the market is relatively low, and there is a large coefficient of variation. Therefore, it is urgent to improve the a* of these groups through breeding.

[0006] At present, many quantitative trait loci (QTLs) related to a* have been identified through various molecular experiments, gene chip typing, resequencing and other technologies, and have been summarized on the International Pig QTL Database website. As of now, there are 655 QTLs related to meat color recorded in the pig QTL database (version #53). Among them, there are 197 QTLs related to a* values, mainly distributed on chromosomes 6, 12 and 15; there are 126 QTLs related to b* values, mainly distributed on chromosomes 12, 14 and 15; there are 401 QTLs related to L* values, mainly distributed on chromosomes 2, 7 and 12. Despite this, the confidence interval of the QTLs affecting a* located at present is relatively large, and it is impossible to determine the true major effect gene and its key variation site, so it is difficult to directly apply it to the selection and improvement of breeding pigs. Summary of the invention

[0007] The purpose of the present invention is to identify a SNP marker related to the pork color redness value a* and develop it into a molecular marker for pig a* breeding in view of the shortcomings of using traditional breeding methods for pig a* trait, such as being time-consuming, labor-intensive, and having poor breeding results.

[0008] Another object of the present invention is to provide a primer pair for amplifying the above-mentioned SNP marker and a reagent or kit comprising the primer pair.

[0009] Another object of the present invention is to provide applications of the aforementioned SNP molecular marker or primer pair or the aforementioned reagent or kit, wherein the applications include but are not limited to (1) use for pig gene typing detection; (2) use for detection of pork color redness value; and (3) use for breeding of pork color redness trait.

[0010] The purpose of the present invention can be achieved through the following technical solutions:

[0011] In the first aspect, the present invention provides a SNP molecular marker related to the redness value of pork color. The SNP molecular marker is located at the missense mutation site rs793627529 on the fourth exon (17:32631325) of the MRPS26 gene on chromosome 17 of the international pig genome version 11.1 reference sequence. The polymorphism of the molecular marker is G / A, and the redness value of pork color of AA and AG genotype individuals is significantly higher than that of GG genotype individuals. Further, the SNP molecular marker contains the nucleotide sequence shown in SEQ ID NO: 1; the polymorphic site is located at the 401st position of the sequence shown in SEQ ID NO: 1.

[0012] The inventors found that the SNP molecular marker associated with the redness value of pork color is the missense mutation site rs793627529 on the fourth exon (17:32631325) of the MRPS26 gene on chromosome 17 of the international pig genome version 11.1 reference sequence, which has a G / A polymorphism and causes proline to mutate to leucine after mutation. The SNP molecular marker is significantly associated with the redness value (a*) of pork color ( P <0.05). AA and AG genotype individuals a* The values ​​were significantly higher than those of individuals with GG genotype; MRPS26 The gene expression level was significantly higher than that of individuals with GG genotype; a* Value and MRPS26 The gene expression levels were significantly positively correlated.

[0013] In a second aspect, the present invention provides a primer pair for amplifying the aforementioned SNP molecular marker, the primer pair comprising an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3.

[0014] In a third aspect, the present invention provides a reagent or a kit containing the aforementioned primer pair.

[0015] In a fourth aspect, the present invention provides any of the following uses of the aforementioned SNP molecular marker or primer pair or reagent or kit: (1) for pig genotyping detection; (2) for detection of pork color redness value; (3) for breeding of pork color redness trait.

[0016] In a fifth aspect, the present invention provides a method for detecting the redness value of pork color, the method comprising the following steps:

[0017] (1) extracting genomic DNA from pig tissue samples; (2) using the pig genomic DNA as a template, performing PCR amplification using an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3; (3) analyzing the genotype of the G / A polymorphic site of the aforementioned SNP molecular marker in the amplified product, and determining the redness value of pork color based on the genotype, wherein the redness value of pork color of individuals with AA and AG genotypes is significantly higher than that of individuals with GG genotypes.

[0018] In a sixth aspect, the present invention provides a method for breeding pork color redness traits, the method comprising using an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3 to perform PCR amplification on the genomic DNA of a breeding pig, analyzing and obtaining the genotype of the polymorphic site of the aforementioned SNP molecular marker, breeding AA and AG genotype individuals for breeding, and selecting high a* excellent breeding pigs. In some preferred embodiments, breeding AA genotype individuals for breeding.

[0019] In some specific embodiments, the present invention provides a method for developing a molecular marker associated with the redness value (a*) of pork color, wherein a primer pair is designed based on a nucleotide sequence containing a SNP significantly associated with pig a*, and PCR amplification is performed using pig genomic DNA as a template to convert the SNP site into a molecular marker; the SNP site significantly associated with pig a* is the international pig genome version 11.1 reference sequence pig chromosome 17 MRPS26 The missense mutation site rs793627529 on the fourth exon of the gene (17: 32631325) has a G / A polymorphism; the SNP marker polymorphism is significantly associated with a* in the Duroc × (Landrace × Large White) (DLY) three-way hybrid pig population ( P <0.05); the SNP is located at MRPS26 Gene exon 4 (17:32631325), detected by quantitative PCR MRPS26 The expression level of MRPS26 Gene expression is highly significantly correlated with a* ( R = 0.481, P <0.01); and the SNP rs793627529 is MRPS26 Gene regulation SNP sites, its AA type individuals MRPS26 The gene expression level was significantly higher than that of GG type individuals ( P<0.01); the upstream primer for genotype detection of the SNP site is shown in SEQ ID NO: 2, and the downstream primer is shown in SEQ ID NO: 3; the molecular marker sequence is shown in SEQ ID NO: 1, the SNP site is located at the 401st position of the nucleotide sequence shown in SEQ ID NO: 1, there is a G / A polymorphism, the a* of the AA genotype individual is significantly higher than that of the GG genotype, and the AA genotype individual MRPS26 The gene expression level was significantly higher than that of individuals with GG genotype.

[0020] The present invention has the following beneficial effects:

[0021] The SNP marker provided by the present invention is significantly correlated with the redness value of pork color, and the molecular marker and primer developed based on the SNP can be used for SNP genotyping detection. Therefore, the AA genotype individuals can be screened for breeding by identifying the SNP marker, and the obtained high-a* excellent breeding pigs can be used for the breeding of new varieties (matching lines), and can also be directly used for the production of commercial pigs, providing high-quality pork for the consumer market, and high-quality pork can bring additional economic benefits to breeding enterprises and slaughtering and processing enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Manhattan plot of genome-wide association analysis (GWAS) of meat redness value a*;

[0023] Figure 2 : Group analysis MRPS26 Correlation between gene expression and a*;

[0024] Figure 3 :rs793627529 site polymorphism MRPS26 Gene expression level;

[0025] Figure 4 :The effect of rs793627529 polymorphism on a*;

[0026] Figure 5 : MRPS26 Example of genotyping plot for gene rs793627529 locus. DETAILED DESCRIPTION

[0027] Example 1 Screening of SNP molecular markers associated with a* value

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0029] In this study, the redness (a*) value of the longissimus dorsi muscle of 140 Duroc × (Landrace × Large White) tri-hybrid pigs (DLY) was determined. DNA from the muscle tissue of this group was extracted according to the instructions of the tissue DNA extraction kit of Tiangen Biotechnology. The DNA that passed the quality inspection was genotyped using GeneSeek GGP Porcine 80 K SNP chip (Neogen). In order to obtain a higher density SNP data set, this study used the SWINE IMPUTATION website to fill the 80K chip data of this group. After filling, the plink software--indep-pairwise 50 10 0.5 parameters were used to perform LD pruning on the filled data set. Finally, 1496731 SNP sites remained for subsequent analysis.

[0030] This study is based on the use of GEMMA software to conduct a genome-wide association study (GWAS) of a* values ​​in this population. The GWAS statistical model is as follows:

[0031]

[0032] A vector representing the phenotype; represents the fixed effects vector (sex, slaughter batch, and age at slaughter); represents the corresponding fixed effect value matrix; represents the SNP marker vector; Represents the effect size matrix of SNP markers; represents the random effect value; represents the random error effect value; represents the variance of random error; Represents the ratio of two variance components; Represents the relationship matrix between individuals; represent n×n The identity matrix of order. represents the n-dimensional multivariate normal distribution. The chromosome significance level in this study is 1 / N, the genome significance level is 0.05 / N, and N is the number of SNPs used in GWAS analysis. Figure 1 As shown, the GWAS results show that the rs793627529 site located on chromosome 17 is the top SNP with a significant association a* value. MRPS26 The mutation is a missense mutation in the fourth exon of the gene (17:32631325). Mutations at this site can cause proline to mutate to leucine.

[0033] Subsequently, total RNA was extracted from the longissimus dorsi muscle samples of 140 DLY individuals using TRIzol reagent (Invitrogen, USA). Total RNA was quantified and quality controlled using Qubit 2.0 and Agilent 2100. MRPS26 The expression of genes was measured. In the 140 DLY groups, Figure 2 As shown, MRPS26 Pearson correlation analysis between gene expression and a* value showed that MRPS26 Gene expression is significantly positively correlated with a* ( R = 0.481, P <0.01).

[0034] Subsequently, the general linear model in IBM SPSS (v 20.0) software was used to statistically analyze the correlation between the three genotypes of the rs793627529 locus and the MRPS26 The correlation between gene expression (TPM inverse normalization) and a* value. The model is as follows:

[0035]

[0036] where Y ijklm is the meat redness value phenotype vector (or MRPS26 gene expression); μ is the population mean; G i , B j and S k are the fixed effects of gender, batch and SNP respectively; D l is the covariate of slaughter age; e ijklm The results showed that AA and AG genotypes MRPS26 Gene expression ( Figure 3 ) and a* values ​​were significantly higher than those of GG type ( Figure 4 ), where ** indicates P <0.01, * indicates P <0.05. Therefore, AA and AG types can be selected for seed use in the later stage.

[0037] The above-mentioned SNP molecular marker corresponds to the sequence shown in SEQ ID NO: 1, wherein the polymorphic site is located at the 401st position of the sequence shown in SEQ ID NO: 1, and the polymorphism is G / A.

[0038] SEQ ID NO: 1

[0039] gctcgtgcaa ccgctggttc tccgcctggt tccaagccat cagctcgcga tgctcagccg 60

[0040] cgtcctcctg cgccttgcgc tctgcctgga ccccggcccg ggcctcatgc agcttcttgc 120

[0041] gcacctcgga catgaactcc agcctgggcg gagacgcgcc caaaattggc actctcttcg 180

[0042] cccgcccgct cgctcgcccg ccccaacaca cacgccggac agcgacgacg gccgccgccc 240

[0043] cctccgtcgc acacacctga gcgcgcgcac cgtctggcga tactggcggt aacgctcggt 300

[0044] cagcacgaag aactccgtag gatccaccgc gggcggagtc gccacgcgcc cggctttgga 360

[0045] cttggccggc gggtcgtggc gggtcttgcg gccgcgcgtg r ggagcagaa gcggggccgg 420

[0046] gagccggccc gcaggccgcg cacccaggcc gcccagcacg cgcagcatgg cgaggcctcc 480

[0047] ccgcctccgg cgagccgcaa ggcttcatgg gtgccgggaa gtggcgcgcg aagcaccacg 540

[0048] ggacatgtag tccggcgact ctacccccac cctcaggagc ccgctgacac ctcaggctgc 600

[0049] tctcgcctgc tgtgtcctcc ctgctgggtc cagtcccgcc ccatgtcccc ggcccgcgtc 660

[0050] tctccagcca cacgcgggaa tggctgtcag taggggtgga agagagggga caaacgaaag 720

[0051] aagatgcaaa aactcaggaa acttcacacc tttattggag ggcagcgggg cgcagcggtc 780

[0052] gtggtcgggt ctgtgggagg a 801

[0053] Example 2 Verification of SNP molecular markers associated with a* value

[0054] 1. Source of experimental animals

[0055] The experimental pigs were sourced from Kangle Agriculture and Animal Husbandry Co., Ltd. in Changzhou City, Jiangsu Province, and the experimental pig herd was a three-way hybrid pig of Duroc × (Landrace × Large White).

[0056] 2. Extraction of porcine genomic DNA

[0057] Longissimus dorsi muscle tissues were collected from 280 pigs, with 4 tissue samples per pig, of which 1 was used for individual DNA extraction;

[0058] Refer to the instructions of the tissue DNA extraction kit of Tiangen Biotechnology Company, and the extraction is carried out in the following order:

[0059] ① First, add 68 mL and 200 mL of anhydrous ethanol to buffer GD and rinse solution PW respectively, and mix thoroughly.

[0060] ② Collect about 100 mg of tissue sample and place it in a 2 mL EP tube. After completely mincing, add 200 μL of buffer CT and vortex until completely suspended.

[0061] ③ Add 20 μL of proteinase K solution, mix well and digest overnight in a 56 ℃ metal bath until the ear sample tissue is dissolved. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0062] ④ Add 200 μL of buffer GB, mix thoroughly by inversion, and place in a 70 ℃ metal bath for 10 min. The solution should become clear. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0063] ⑤ Add 200 μL of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may appear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0064] ⑥ Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3, place the adsorption column in a collection tube, then centrifuge at 12,000 rpm for 30 s, pour out the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0065] ⑦ Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in the collection tube.

[0066] ⑧ Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0067] ⑨Repeat step ⑧.

[0068] ⑩ Place the adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm for 2 min, and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0069] ⑪ Transfer the adsorption column CB3 to a clean centrifuge tube, add 100 μL of elution buffer TE to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 min, centrifuge it at 12,000 rpm for 2 min, collect the solution in a centrifuge tube, add the solution obtained by centrifugation to the adsorption column CB3, let it stand at room temperature for 2 min, centrifuge it at 12,000 rpm for 2 min and collect the solution in a centrifuge tube.

[0070] After the mass and concentration were detected by Nanodrop-100 spectrophotometer, the concentration was diluted to 50 ng / μL and stored at -20 °C for future use.

[0071] 3. PCR amplification and sequencing of target fragments

[0072] Primer design:

[0073] Upstream primer: cggtcagcac gaagaactc 19 (SEQ ID NO: 2);

[0074] Downstream primer: acccctactg acagccattc 20 (SEQ ID NO: 3).

[0075] The extracted DNA was used as a template and PCR amplification was performed according to the designed primers: 1 μL of DNA template, 1 μL of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, and 22 μL of PCR Mix reagent (1.1× T3 SuperPCR Mix, Qingke Biotechnology); a PCR amplification system was set up.

[0076] PCR amplification procedure:

[0077] 30 cycles:

[0078] Pre-denaturation at 98°C for 2 min;

[0079] Denaturation at 98°C for 10 s;

[0080] Annealing at 60 °C for 10 s;

[0081] Extension at 72°C for 1 min;

[0082] Extension at 72°C for 2 min;

[0083] Store at 4°C.

[0084] The PCR product was detected by electrophoresis in 1.2% agarose gel. The amplified target fragment size was about 400 bp, which was consistent with the expected fragment size. The remaining amplified product was sequenced, and the sequencing results were compared and analyzed with the relevant gene fragment sequences of pigs in GenBank using SnapGene Viewer software to determine the genotype of the rs793627529 site ( Figure 5 ).

[0085] 4. Statistical Analysis

[0086] The general linear model of SAS software was used to analyze the effect of genotype on phenotype. The analysis model is:

[0087] Y ijnk = u i + G j + S n + W k + e ijnk

[0088] in: Y ijnk is a* of pig; u i represents the population phenotype mean; G j represents the genotype fixed effect of SNP; S n represents the gender fixed effects; W k is the covariate for carcass weight; e ijnk is the residual.

[0089] 5. Results

[0090] Table 1 shows the effect of the rs793627529 mutation site G / A on a* in the pig population.

[0091] Table 1 MRPS26 Association analysis between gene rs793627529 and pig a*

[0092]

[0093] Note: SD: standard deviation; a, b represent extremely significant differences between different letters in each row ( P <0.05)

[0094] As shown in Table 1, there are extremely significant differences in a* among the three genotypes at rs793627529 ( P <0.01, among which the a* of AA and AG genotypes was significantly higher than that of GG genotype ( P <0.05). It can be seen that in the pig population, the successive breeding of individuals with AA and AG genotypes at the rs793627529 locus can gradually improve the a* of the pig population and achieve the purpose of improving pork quality.

[0095] Example 3 Breeding a new pig breed with a high a* value using the screened SNP molecular markers

[0096] 1. Source of experimental animals

[0097] The use of rs793627529 locus for breeding and improving meat color redness value a* has been applied in two generations of Lujia black pig population. The experimental pigs came from the Science and Technology Ranch of Zhejiang Academy of Agricultural Sciences. The pig breed is Lujia black, which is a newly bred breed with ruddy meat color and obvious marbling characteristics. This study has measured the a* value of meat color of 407 Lujia black pigs.

[0098] 2. Extract the pig genomic DNA to be tested according to the method in Example 1.

[0099] 3. Using pig genomic DNA as a template, PCR amplification is performed using the primer pair shown in SEQ ID NO: 2 and SEQ ID NO: 3; the amplified product is sequenced, the sequencing results are analyzed, and the G / A polymorphism of the SNP molecular marker site related to the redness value of pork color is interpreted (the genotype of the SNP molecular marker site is determined). The PCR amplification system and PCR amplification procedure are the same as those in Example 1.

[0100] The results showed that after two generations of selection of AA and AG genotypes at the rs793627529 locus, the number of AA and AG genotype individuals increased significantly compared with the GG genotype, and the a* values ​​of AA and AG genotype individuals were significantly higher than those of the GG genotype. Therefore, this study found that selecting AA and AG genotype individuals for breeding can achieve the goal of improving the selection of new pig breeds with high meat redness values.

[0101] Table 2 MRPS26 Association analysis between gene rs793627529 and pig a*

[0102]

[0103] Note: SD: standard deviation; A, B represent extremely significant differences between different letters in each row ( P <0.01).

Claims

1. A method for detecting the redness value of pork color, characterized in that: The method comprises the following steps: (1) Take tissue samples from pigs to extract genomic DNA; (2) using porcine genomic DNA as a template, performing PCR amplification using an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3 to obtain an amplified product; (3) Analyze the SNP molecular marker associated with the pork color redness value in the amplification product described in step (2), wherein the SNP molecular marker contains the nucleotide sequence shown in SEQ ID NO: 1, the polymorphic site is located at the 401st position of the sequence shown in SEQ ID NO: 1, and the polymorphism of the molecular marker is G / A. The pork color redness value is determined according to the genotype. The pork color redness value of individuals with AA and AG genotypes is significantly higher than that of individuals with GG genotype.

2. A method for breeding pork redness trait, characterized in that: The method comprises: using an upstream primer as shown in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3 to perform PCR amplification on the genomic DNA of a breeding pig to obtain an amplified product, analyzing a SNP molecular marker related to the redness value of pork color in the amplified product, wherein the SNP molecular marker contains a nucleotide sequence as shown in SEQ ID NO: 1, the polymorphic site is located at the 401st position of the sequence as shown in SEQ ID NO: 1, the polymorphism of the molecular marker is G / A, and individuals with AA and AG genotypes are selected for breeding.

3. The breeding method according to claim 2, characterized in that: Select and breed individuals with AA genotype for breeding.

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