SNP molecular markers related to intramuscular fat traits in pigs, and their applications and detection methods

Abstract

The present invention relates to the field of molecular genetics, and particularly to SNP molecular markers related to intramuscular fat traits in pigs, their applications and detection methods. The present invention discovers a SNP locus (A-C) at the 12726th base in the intron region of the DKK2 gene or at the 114874954th position on porcine chromosome 8. The allele A is positively correlated with the low intramuscular fat trait, while the allele C is positively correlated with the high intramuscular fat trait. The present invention provides a method and a kit for detecting this SNP molecular marker. If the enzyme digestion product is two bands, the base at the mutation site is A; if the enzyme digestion product is one band, the base at the mutation site is C. The method of the present invention can predict the intramuscular fat content in pigs early, quickly, at low cost and effectively, and can be used as a molecular marker for genetic improvement of pig breeds. It has broad application and market prospects in pig breed improvement. The kit developed for this invention has considerable economic benefits and good social benefits.

Description

Technical Field

[0001] The present invention relates to the field of molecular genetics, and particularly to SNP molecular markers related to intramuscular fat traits in pigs, their applications and detection methods. Background Art

[0002] China is a major producer and consumer of pork, and pork occupies an important position in the national life. In 2021, the total meat production in the country was 88.870 million tons, of which the pork production was 52.960 million tons, accounting for 59.6% of the total meat production. The pig and pork industry is related to social stability and the fundamental livelihood of the people. At present, the domestic pork market mainly focuses on lean-type pigs, which have a fast growth rate and a high lean meat rate. However, meat quality was not included in the breeding improvement goals in the early pig breeding programs, resulting in uneven meat quality and flavor in the market. With the improvement of people's living standards, consumers will pay more attention to the meat quality of pork when purchasing. This demand not only promotes the transformation of the market but also drives the innovation of the industry. Producing pork with tender meat, unique flavor, and rich nutrition has become a new goal for the breeding industry and related workers.

[0003] The narrow definition of pork quality refers to the most intuitive sensory quality of pork for people, such as vision, smell, taste, and touch. Specific pork quality can be evaluated by the following indicators: meat color, muscle water - holding capacity, flavor, pH value, drip loss, marbling, water - holding capacity, tenderness, flavor, juiciness, etc. The broad - sense meat quality also includes the deep - processing quality, nutritional value, and hygienic quality of meat. A large number of literatures have reported that intramuscular fat content and its fatty acid composition are not only the reasons for the appearance of marbling in muscle but also highly correlated with meat quality traits such as pork juiciness, tenderness, and flavor. It is one of the main indicators that are recognized as the key concerns in production and research for affecting meat flavor and meat quality evaluation.

[0004] However, usually, meat quality indicators are measured after slaughter, which not only has a high detection cost but also is difficult to measure. In addition, meat quality traits are not only affected by their own genetic factors, but also environmental factors such as season, temperature, and pre - slaughter treatment will have an important impact on them. These factors may reduce the accuracy of genetic evaluation, and thus affect the genetic improvement of meat quality traits. Therefore, developing rapid, simple, and effective molecular genetic markers for pig intramuscular fat traits can be applied to the rapid detection of large populations, increase the selection pressure for intramuscular fat traits, and accelerate the process of genetic improvement work.

[0005] The protein encoded by the DKK2 (Dickkopf WNT Signaling Pathway Inhibitor 2) gene is a member of the Dickkopf family and is an inhibitor of the WNT signal. The WNT signaling pathway is a complex network of protein interactions, whose functions are most commonly seen in embryonic development and cancer, and also participate in the normal physiological processes of adult animals, etc. Existing research has shown that the WNT signal is involved in fat deposition and differentiation. Through the β-catenin pathway, it binds to the TCF / LEF transcription factor family to initiate transcription, and then binds to different transcriptional co-activators to promote the proliferation, maintenance or influence the differentiation of preadipocytes. Then, as an inhibitor of this pathway, DKK2 will also affect the proliferation or differentiation of adipocytes. Research on Qinchuan cattle has shown that the SNP (C29T) in the first exon of DKK2 is related to intramuscular fat content. Some researchers have also verified the role of the DKK2 gene in inhibiting adipogenesis using mouse 3T3-L1 embryonic fibroblasts and c3h / 10t1 / 2 mesenchymal stem cells. In addition, the DKK1 gene, which also belongs to the DKK family, has been studied more thoroughly and comprehensively at present. A number of studies have proven in humans and mice that the DKK1 gene can reduce and regulate fat production. However, from the perspective of previous research and literature review, the function of the DKK2 gene has not been verified in the pig species, its specific mechanism of action is not yet clear, nor is it known whether there are SNP sites related to porcine intramuscular fat traits and can thus be used as molecular markers in genetic breeding. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide SNP molecular markers related to porcine intramuscular fat traits, their applications and detection methods.

[0007] The present invention provides SNP molecular markers related to porcine intramuscular fat traits. The SNP molecular markers are located at the 12,726th base in the intron region of the porcine DKK2 gene or the 114,874,954th base on porcine chromosome 8, and the base at this site is A or C.

[0008] The present invention provides the application of the SNP molecular markers in detecting porcine intramuscular fat traits.

[0009] In the application of the present invention, the fat traits include intramuscular fat content. If the genotype of the SNP molecular marker is AA, the pig to be tested is of the low intramuscular fat type; if the genotype of the SNP molecular marker is CC, the pig to be tested is of the high intramuscular fat type; if the genotype of the SNP molecular marker is AC, the pig to be tested is of the intermediate intramuscular fat type.

[0010] The present invention also provides a primer pair for detecting the genotype of the SNP molecular marker. The primer pair is divided into an upstream primer and a downstream primer. The upstream primer has the nucleotide sequence shown in SEQ ID NO.1; the downstream primer has the nucleotide sequence shown in SEQ ID NO.2. The sequence of the upstream primer is AGGCTTCCTGCTTGTCA (SDEQ IDNO.1), and the primer is located at positions 114874697 - 114874714 bp on chromosome 8; the sequence of the downstream primer is TCTTATGTTTGTTGGCTGT (SEQ ID NO.2), and the primer is located at positions 114874686 - 114874705 bp on chromosome 8. The length of the product of this primer pair is 578 bp, and the annealing temperature is 59°C.

[0011] The present invention also provides a kit for detecting the genotype of the SNP molecular marker, and the kit includes the above-mentioned primer pair.

[0012] Furthermore, the kit provided by the present invention also includes Taq DNA polymerase and PCR buffer. This kit is convenient, fast, efficient, and has accurate results.

[0013] The present invention also provides a method for detecting the SNP molecular marker related to porcine intramuscular fat traits, which includes using the above-mentioned primer pair and / or the above-mentioned kit to detect the test substance.

[0014] In the method of the present invention, the method includes using the above-mentioned primer pair or the above-mentioned kit to perform PCR amplification on the genomic DNA of the test pig, digesting the PCR product with BspHI endonuclease. If the digestion products are 399 bp and 179 bp (two products), the base at the mutation site is A; if the digestion product is 578 bp (one product), the base at the mutation site is C.

[0015] In the method of the present invention, the PCR amplification reaction program is: 95°C for 5 min; 95°C for 30 s, 59°C for 30 s, 72°C for 35 s, for a total of 35 cycles; 72°C for 10 min; 4°C ∞; the digestion reaction program is: 37°C, overnight; 65°C for 20 min; 4°C ∞.

[0016] The present invention provides the application of any one of the following I to III in the improvement of porcine germplasm resources and / or the breeding of pigs with high or appropriate intramuscular fat:

[0017] I: the above-mentioned SNP molecular marker; II: the above-mentioned primer pair; III: the above-mentioned kit.

[0018] The present invention also provides a pig breeding method, which includes using the primer pair or the kit described above to detect the genotypes of parents or offspring. The method provided by the present invention distinguishes or predicts the intramuscular fat content of pigs based on the SNP molecular marker.

[0019] In the method of the present invention, the method specifically includes amplifying the BspHI endonuclease site created by the mutation using specific primers upstream of the 12726th base in the intron region of the pig DKK2 gene or the mutation position of the 114874954th base on pig chromosome 8, and detecting the SNP molecular marker genotype by the PCR-RFLP method.

[0020] The pigs of the present invention are divided into seven types: North China type, Central China type, South China type, Southwest type, Plateau type, Jianghai type and Exotic type; specifically including Min pigs, Zaozhuang black-covered pigs, Shaziling pigs, Daweizi pigs, Ningxiang pigs, Jinhua pigs, Xiaomeishan pigs, Lantang pigs, Putian pigs, Western Hubei pigs, Neijiang pigs, Tibetan pigs, Large White pigs, Berkshire pigs and Duroc pigs.

[0021] Taking Shaziling pigs as representatives of Chinese indigenous pig breeds, and Large White pigs and Berkshire pigs as representatives of exotic pig breeds, whole-genome resequencing was carried out. In previous phenotypic studies, it was found that as a Chinese indigenous pig breed, Shaziling pigs have better meat quality and meat flavor, especially in terms of higher intramuscular fat. To find the genomic data basis for the difference in intramuscular fat, the SNPs and selection signals of the whole-genome resequencing data of 41 pigs in 3 populations were analyzed and studied. Finally, after enrichment and annotation, it was found that the DKK2 gene is related to fat synthesis and differentiation. Combining the SNP Calling results, the loci affecting intramuscular fat in pigs were screened out, that is, the 12726th base in the intron region of the DKK2 gene or the 114874954th base on pig chromosome 8, and the base at this locus is A or C.

[0022] 299 pigs (15 groups in total) were used as verification materials (mainly Chinese local pig breeds, including 6 types, and individuals were not related, as shown in Table 1), and these pigs were typed using PCR-RFLP technology. Combined with the sequencing results, it was finally found that the A gene frequency of Duroc was dominant, and only AA and AC individuals were present, with a corresponding A gene frequency of 82.86%, and its intramuscular fat content was the lowest among the tested pig breeds; while the C gene frequency of Berkshire pigs (foreign fat-loving dual-purpose type) and all Chinese local pig breeds was dominant, and all were higher than 70% (except Berkshire, which may be due to the small number of samples). Large White pigs are an exception. As an imported pig breed, their intramuscular fat content is low. Logically, the A gene frequency should be dominant, but in fact, their typing is similar to that of some Chinese local pig breeds. It is speculated that this may be related to the hybridization and improvement of foreign Large White pig breeds with the domestically introduced Meishan pigs. On this basis, in the experiment, the A gene frequency was X and the intramuscular fat content was Y. The existing phenotypic data and typing results were used for regression analysis, and the final linear regression model between the two was Y=-1.973979263X+4.700316512, P=0.048694825, which reached a significant level. It can be seen that the A gene frequency of this SNP site is significantly negatively correlated with the intramuscular fat content.

[0023] The present invention provides a SNP molecular marker related to the intramuscular fat trait of pigs and its application and detection method. The present invention detects the exon region of the DKK2 gene of different pig breeds and finds that at the 12726th base in the intron region of the DKK2 gene or the 114874954th SNP site (AC) of the pig chromosome 8, allele A is positively correlated with the low intramuscular fat trait, while allele C is positively correlated with the high intramuscular fat trait. The present invention provides a method for detecting the SNP molecular marker, using a primer pair with a nucleotide sequence as shown in SEQ ID NO.1-2 to amplify the pig genomic DNA to be tested and digest it with BspHI. If the digestion products are 399bp and 179bp (two), the base at the mutation site is A, and if the digestion product is 578bp (one), the base at the mutation site is C. The method of the present invention can predict the intramuscular fat content of pigs early, quickly, at low cost and effectively, and can be used as a useful molecular marker in the genetic improvement of pig breeds to predict the intramuscular fat content of pigs quickly, early and at low cost; it has broad application prospects in pig breed improvement and a broad market, and the kit developed for this invention can generate considerable economic benefits and good social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings:

[0025] Figure 1 Showing relevant information such as the BspHI restriction site;

[0026] Figure 2 Showing the agarose gel electrophoresis results after BspHI digestion of three genotypes. Left: genotype AA; Middle: genotype AC; Right: genotype CC. Specific embodiments

[0027] The present invention provides an SNP molecular marker related to intramuscular fat traits in pigs, its application and detection method. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0028] Table 1: Validation population and its phenotypic data (Intramuscular fat content data of pigs are from "China's Livestock and Poultry Genetic Resources Annals - Pig Annals" in May 2011)

[0029]

[0030]

[0031] In Example 1 of the present invention, the DNA ear samples for resequencing data to screen SNPs related to intramuscular fat traits were sourced from the Xiangtan Livestock Breeding Station. The germplasm resources of various pig breeds used in Example 2 came from the conservation farms in their respective original regions. For example, the Tibetan pigs came from the Tibetan Pig Conservation Farm in Tibet, and the Duroc pigs in the example were sourced from Guangzhou Wens Group.

[0032] The following will describe the implementation schemes of the present invention in detail in combination with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0033] Obtaining of SNP Molecular Markers Related to Intramuscular Fat Traits in Pigs in Example 1

[0034] Based on the resequencing data of a total of 41 pigs including Shaziling pigs, Berkshire pigs, and Large White pigs (20 Shaziling pigs, 10 Berkshire pigs, and 11 Large Whites). First, the FastQC software and Trimmomatic software were used to perform quality control and screening on the reads data; the clean reads after quality control were aligned to the pig reference genome (version number: Sscrofa11.1-release100) using BWA, SAMtools, and GATK software to obtain a sorted and duplicate-marked bam file. Then, the HaplotypeCaller, SelectVariant, and VariantFiltration modules in the GATK software were used to perform variant detection on the bam file of each individual to obtain SNPs after quality control screening, and SnpEff was used to annotate the SNP information. The obtained SNPs were analyzed for selection signals using Fst&θπ, and GO and KEGG analyses were performed on the significantly gene regions in the population selection signal detection. According to the enrichment analysis information, after basic experimental function verification, the DKK2 gene related to intramuscular fat deposition and differentiation was screened. This gene is mainly related to embryonic development and is also related to the synthesis and differentiation of fat.

[0035] According to the sequencing data, the experimenter found a total of 34 differential SNPs in the DKK2 gene region. Among them, the 12726th base in the intron region of the pig DKK2 gene or the 114874954th base on pig chromosome 8 was suspected to be related to intramuscular fat traits because this locus is A in the Duroc (low intramuscular fat) reference genome and is basically all C (gene frequency 0.975) in Shaziling pigs (high intramuscular fat). Subsequently, the experimenter analyzed the SNP allele frequencies of 15 pig breeds with different intramuscular fat contents (including 299 pigs) and found that only Duroc had more AA types, with the A gene frequency being 82.26%. In other high intramuscular fat pig breeds, the C gene frequency was dominant and all were higher than 70% (except Berkshire). The verification results of this group further confirmed the conjecture that this SNP locus may be related to intramuscular fat content. Subsequently, the experimenter used the A gene frequency as X and the intramuscular fat content as Y, and performed a regression analysis in EXCEL using the existing phenotypic data and genotyping results. Finally, the linear regression model between the two was obtained as Y = -1.973979263X + 4.700316512, P = 0.048694825, reaching a significant level. The above results further confirmed the association between intramuscular fat content and this SNP locus: that is, this SNP locus is significantly associated with intramuscular fat content, its allele A is positively correlated with low intramuscular fat, and allele C is positively correlated with high intramuscular fat.

[0036] Example 2 Establishment of a Method for Detecting SNP Molecular Markers Related to Intramuscular Fat Traits

[0037] Based on the research results of Example 1 and the characteristics of the sequence, the SNP molecular markers of Example 1 were detected at the population level using Restriction Fragment Length Polymorphism (RFLP). A mutation was introduced upstream of the mutation site using specific primers to create a BspHI endonuclease site. When the base at this position is A, it can be digested by BspHI, and when it is C, it cannot be digested by BspHI, thereby being used for genotyping (such as Figure 1 ). PCR amplification was performed using the primers in Table 2, and then the PCR products were digested using the digestion system in Table 3.

[0038] Table 2: Specific Primers for Detecting A / C Mutation Using BspHI Digestion

[0039]

[0040] Table 3: Digestion Reaction System (30 μl)

[0041]

[0042] After overnight digestion, 5 μl of the digested product was used for 1.5% agarose gel electrophoresis at 150 v for 25 min. Then, genotyping could be performed according to the positions of the electrophoresis bands, such as Figure 2 . It was found that the frequency of the C genotype at this position was relatively high in Chinese local pig breeds, while the frequency of the A genotype was relatively high only in foreign pig breeds such as Duroc pigs. The specific genotyping results are shown in Table 4 below:

[0043] Table 4: Genotype Identification and Gene Frequency in Different Pig Breeds

[0044]

[0045] By performing an association analysis between the genotypes of individuals and their phenotypes, it was found that the mutation site of the SNP molecular markers of the present invention was significantly associated with the intramuscular fat content in pigs, and the distribution frequency of the A allele was relatively high in the Duroc lean-type pig breed, significantly higher than that in Chinese fat-type pig breeds such as Jinhua pigs and Neijiang pigs. Thus, it can be seen that the A / C locus is significantly related to intramuscular fat in pigs and can be used as a marker for population breeding and improvement work.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of SNP locus in detecting intramuscular fat trait of pigs; the SNP locus is located at the 12726th base in the intron region of the pig DKK2 gene, and the base at this locus is A or C; the fat trait is intramuscular fat content; base A is positively correlated with low intramuscular fat content, and base C is positively correlated with high intramuscular fat content.

2. Method for detecting SNP locus related to intramuscular fat content of pigs, characterized in that Including amplifying porcine genomic DNA, and then digesting the PCR product with the restriction enzyme BspHI; The amplification primer pair includes an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2; The SNP locus is located at the 12726th base in the intron region of the porcine DKK2 gene, and the base at this locus is A or C; if the digestion product is 399 bp and 179 bp, the SNP base is A; if the digestion product is 578 bp, the SNP base is C.

3. The method according to claim 2, characterized in that The reaction program for the amplification is: 95°C for 5 min; 95°C for 30 s, 59°C for 30 s, 72°C for 35 s, for a total of 35 cycles; 72°C for 10 min; 4°C ∞; the reaction program for the digestion is: 37°C, overnight; 65°C for 20 min; 4°C ∞.

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