CN115786527A - SNP molecular marker related to pig intramuscular fat character and application and detection method thereof - Google Patents
SNP molecular marker related to pig intramuscular fat character and application and detection method thereof Download PDFInfo
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Abstract
The invention relates to the field of molecular genetics, in particular to an SNP molecular marker related to porcine intramuscular fat traits and an application and a detection method thereof. The invention discovers that the SNP locus (A-C) of 12726 th base of an intron region of a DKK2 gene or 114874954 th chromosome eight of a pig has positive correlation between allele A and a low intramuscular fat character and positive correlation between allele C and a high intramuscular fat character. The invention provides a method and a kit for detecting the SNP molecular marker, if the number of enzyme digestion products is two, the base at the mutation position is A; if the enzyme digestion product is one, the base at the mutation position is C. The method can be used for predicting the intramuscular fat content of the pig in an early, rapid, low-cost and effective manner, and can be used as a molecular marker to be applied to genetic improvement of pig breeds. The kit developed by the invention has considerable economic benefit and good social benefit.
Description
Technical Field
The invention relates to the field of molecular genetics, in particular to an SNP molecular marker related to porcine intramuscular fat traits and an application and a detection method thereof.
Background
China is a big country for pork production and consumption, and pork plays an important role in national life. In 2021, the meat yield of the whole country is 8887.0 ten thousand tons, wherein the pork yield is 5296.0 ten thousand tons, which accounts for 59.6 percent of the total meat yield, and the live pig and pork industry is related to the fundamental of the social stability and the livelihood. At present, the domestic pork market mainly comprises lean pork, the growth speed of the lean pork is high, the lean meat rate is high, however, the meat quality is not brought into the goal of breed selection improvement in early pig breeding programs, so that the pork in the market is good in meat quality and flavor. With the improvement of the living standard of people, consumers pay more attention to the meat quality of pork during purchasing. This need has driven industry innovation while driving market changes. The production of pork which is fresh and tender in meat quality, unique in flavor and rich in nutrition becomes a new target of breeding industry and related workers.
The pork quality is narrowly defined as the most intuitive sensory quality of pork, such as vision, smell, taste, touch, etc., and the specific pork quality can be evaluated by the following indexes: flesh color, muscle water retention, flavor, pH, drip loss, marbling, water retention, tenderness, flavor, juiciness, and the like. The meat quality in a broad sense also includes the deep processing quality, nutritional value, sanitary quality and the like of the meat. A great deal of documents report that the intramuscular fat content and the fatty acid composition thereof are not only the reason for marbling of muscles, but also highly related to meat quality traits such as juiciness, tenderness, flavor and the like of pork, and are one of the main indexes which are mainly concerned in production and research and influence the meat quality and flavor evaluation.
However, the meat quality index is usually determined after slaughter, which not only has high detection cost, but also is difficult to determine. In addition, meat quality traits are influenced by self genetic factors, environmental factors such as seasons, temperature, pre-slaughter treatment and the like can cause important influence on the meat quality traits, and the factors can reduce the accuracy of genetic evaluation and further influence the genetic improvement of the meat quality traits. Therefore, the molecular genetic marker for the intramuscular fat character of the pig is developed quickly, conveniently and effectively, can be applied to the quick detection of large groups, increases the selective pressure of the intramuscular fat character and accelerates the process of genetic improvement work.
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 WNT Signaling. The WNT signaling pathway is a complex network of protein action, the function of which is most common in embryonic development and cancer, and is also involved in normal physiological processes in adult animals, among others. The WNT signal is shown to be combined with TCF/LEF transcription factor family to start transcription through a beta-catenin channel by participating in fat deposition and differentiation, and further combined with different transcription co-activators to promote the proliferation, maintenance or influence differentiation of preadipocytes. DKK2, acting as an inhibitor of this pathway, will also affect the proliferation or differentiation of adipocytes. Studies on qinchuan cattle showed that the SNP (C29T) present in the first exon of DKK2 is associated with intramuscular fat content. Researchers also verify that the DKK2 gene has the effect of inhibiting adipogenesis by using mouse 3T3-L1 embryonic fibroblasts and c3h/10T1/2 mesenchymal stem cells. In addition, the DKK1 gene belonging to the DKK family is more thoroughly and comprehensively studied at present, and several studies have demonstrated that the DKK1 gene can reduce and regulate the generation of fat in humans and mice. However, from the previous research and the reference of literature data, the DKK2 gene function has not been verified on pig species, the specific action mechanism is not clear, and whether SNP sites exist or not are related to the intramuscular fat character of pigs is not clear, so that the DKK2 gene function can be used as a molecular marker for genetic breeding.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide an SNP molecular marker related to the intramuscular fat character of pigs, and an application and a detection method thereof.
The invention provides an SNP molecular marker related to the porcine intramuscular fat character, wherein the SNP molecular marker is positioned at the 12726 th base of an intron region of a porcine DKK2 gene or the 114874954 th base of a porcine chromosome eight, and the base of the site is A or C.
The invention provides application of the SNP molecular marker in detecting the intramuscular fat character of pigs.
In the application of the invention, the fat character comprises intramuscular fat content. The genotype of the SNP molecular marker is AA, and the pig to be detected is intramuscular fat low type; the genotype of the SNP molecular marker is CC, and the pig to be detected is intramuscular fat high type; and if the genotype of the SNP molecular marker is AC, the pig to be detected is the intramuscular fat intermediate type.
The invention also provides a primer pair for detecting the SNP molecular marker genotype, wherein the primer pair is divided into an upstream primer and a downstream primer, and the upstream primer has a nucleotide sequence shown as SEQ ID NO. 1; the downstream primer has a nucleotide sequence shown as SEQ ID NO. 2. The sequence of the upstream primer is AGGCTTCCTGCTTGTCTCA (SDEQ ID NO. 1), and the primer is on chromosome 8 from 114874697 to 114874714bp; the sequence of the downstream primer is TCTTATGTTTGTTGGCTGT (SEQ ID NO. 2), and the primer is from chromosome 8, 114686 to 114874705bp. The length of the primer pair product is 578bp, and the annealing temperature is 59 ℃.
The invention also provides a kit for detecting the genotype of the SNP molecular marker, and the kit comprises the primer pair.
Furthermore, the kit provided by the invention also comprises Taq DNA polymerase and PCR buffer solution, and the kit is convenient, quick and efficient to use and accurate in result.
The invention also provides a method for detecting the SNP molecular marker related to the porcine intramuscular fat character, which comprises the step of detecting an object to be detected by using the primer pair and/or the kit.
In the method, the primer pair or the kit is used for PCR amplification of the genome DNA of the pig to be detected, bspHI endonuclease is used for enzyme digestion of a PCR product, and if the enzyme digestion product is 399bp and 179bp, the base of the mutation position of the two products is A; if the enzyme cutting product is 578bp, (one product) the base at the mutation position is C.
In the method of the present invention, the PCR amplification reaction procedure is: 5min at 95 ℃; 30s at 95 deg.C, 30s at 59 deg.C, and 35s at 72 deg.C for 35 cycles; 10min at 72 ℃; infinity at 4 ℃; the enzyme digestion reaction program is as follows: at 37 ℃ overnight; 20min at 65 ℃; infinity at 4 ℃.
The invention provides an application of any one of the following I-III in pig germplasm resource improvement and/or breeding of high or suitable intramuscular fat pig breeds:
i: the SNP molecular marker; II: the primer pair; III: the kit is provided.
The invention also provides a breeding method of pigs, which comprises the step of detecting the genotype of parents or filial generations by using the primer pair or the kit. The invention provides a method for distinguishing or predicting intramuscular fat content of pigs according to the SNP molecular marker.
In the method, the method specifically comprises the steps of amplifying mutation-created BspHI endonuclease sites at 12726 th base of an intron region of a pig DKK2 gene or upstream of a mutation position of 114874954 th base of pig chromosome eight by using a specific primer, and detecting the SNP molecular marker genotype by using a PCR-RFLP method.
The pigs of the invention are divided into seven types: north China type, china-Medium type, south China type, southwest type, plateau type, river-sea type and exotic type; the pig feed comprises Min pigs, zaozhuang black-covered pigs, sand ridge pigs, daweizi pigs, ningxiang pigs, jinhua pigs, small plum mountain pigs, lantang pigs, pu Tian pigs, huxi pigs, nenjiang pigs, tibetan pigs, big white pigs, bake summer pigs and Duroc pigs.
And (3) performing whole genome re-sequencing by taking the sand-ridge pigs as local pig breeds in China and the white pigs and the Baker pigs as external pig breeds. In early phenotypic studies, sand ridge pigs serving as local pig breeds in China are found to have good meat quality and meat flavor, and particularly have high intramuscular fat. To find the genomic data basis for intramuscular fat differences, SNPs and selection signals were analyzed and studied for genome-wide re-testing data of a total of 41 pigs in 3 cohorts. After final enrichment and annotation, the DKK2 gene was found to be involved in fat synthesis and differentiation. Combining the SNP Calling result, a site influencing the intramuscular fat of the pig, namely 12726 th base of an intron region of a DKK2 gene or 114874954 th base of a pig eight chromosome is screened, and the base of the site is A or C.
299 pigs (15 groups in total) are taken as verification materials (mainly local pig breeds in China are taken as main materials, including 6 types, and the individuals have no genetic relationship, as shown in table 1), and the pigs are typed by utilizing a PCR-RFLP technology. Finally, combining with a sequencing result, the frequency of the A gene of Duroc is dominant, and only AA and AC individuals exist, the frequency of the corresponding A gene is 82.86%, and the intramuscular fat content of the A gene is the lowest in the pig species to be tested; the C gene frequency of the pigs including the Baker summer (externally fat meat and fat dual-purpose type) and all the pig breeds in China is dominant and is higher than 70 percent (except for the Baker summer, the number of samples is probably less). The white pig as an exception has the characteristic that the intramuscular fat content is lower as an externally introduced pig breed, the A gene frequency is considered to be dominant, but the actual typing is similar to that of a part of local pig breeds in China, and the white pig breed is suspected to be related to the hybridization improvement of the foreign white pig breed and the domestic introduced Meishan pig. On the basis, in the test, the A gene frequency is X, the intramuscular fat content is Y, the existing phenotype data and the typing result are utilized to carry out regression analysis, and finally the linear regression model between the A gene frequency and the typing result is Y = -1.97397363X +4.700316512, and P = -0.048694825 reaches a significant level, so that the A gene frequency of the SNP site is in a significant negative correlation with the intramuscular fat content.
The invention provides an SNP molecular marker related to the intramuscular fat character of pigs and an application and a detection method thereof. The invention detects the exon regions of DKK2 genes of different pig varieties, and discovers that the allele A of an 12726 base of the intron region of the DKK2 gene or an SNP site (A-C) of 114874954 of a pig chromosome eight, is positively correlated with the low intramuscular fat character, and the allele C is positively correlated with the high intramuscular fat character. The invention provides a method for detecting the SNP molecular marker, which comprises the steps of amplifying the genome DNA of a pig to be detected by utilizing a primer pair with a nucleotide sequence shown as SEQ ID NO. 1-2, and carrying out enzyme digestion by using BspHI, wherein if the enzyme digestion products are 399bp and 179bp (two), the base at the mutation position is A, and if the enzyme digestion product is 578bp (one), the base at the mutation position is C. The method can predict the content of the intramuscular fat of the pigs at early stage, quickly and effectively with low cost, can be used as a useful molecular marker to be applied to genetic improvement of pig breeds, and can predict the intramuscular fat condition of the pigs at early stage and low cost; the kit developed by the invention can generate considerable economic benefit and good social benefit.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts based on the drawings:
FIG. 1 shows the information about BspHI cleavage sites;
FIG. 2 shows the agarose electrophoresis results after digestion with BspHI for the three genotypes, left: genotype AA; the method comprises the following steps: genotype AC; and (3) right: genotype CC.
Detailed Description
The invention provides SNP molecular markers related to the intramuscular fat traits of pigs and an application and a detection method thereof, and a person skilled in the art can realize the SNP molecular markers by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Table 1: the verification population and the phenotype data thereof (the intramuscular fat content data of the pigs comes from Chinese livestock genetic resource Zhi-pig Zhi 2011 5 months)
Example 1 of the present invention DNA ear samples used to screen resequencing data for SNPs associated with intramuscular fat traits were sourced from livestock breeding station in quan, hunan Tan. The breeder pig germplasm resources used in example 2 were from the conservation site in various regions of origin, e.g., the Tibetan pigs were from the Tibetan pig conservation site, and the Duroc pigs in the examples were from the Guangzhou Wenshi group.
Embodiments of the present invention will be described in detail below with reference to examples, and it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
Example 1 acquisition of SNP molecular marker associated with intramuscular fat trait in pig
Based on the re-sequencing data of 41 pigs (20 pigs in sand, 10 pigs in Barkha and 11 pigs in white) in total of sand pigs, barkha pigs and white pigs. Firstly, performing quality control and screening on reads data by using FastQC software and Trimmomatic software; the controlled clean reads were aligned to the porcine reference genome (version number: sscrofa11.1-release 100) using BWA, SAMtools and GATK software to obtain a sorted and repeat-tagged bam file. And then, carrying out mutation detection on the bam file of each individual by using a haplotypecall, selectVariant and VariantFiltration module in the GATK software to obtain SNPs after quality control screening, and annotating the information condition of the SNPs by using SnpEff. The obtained SNPs are subjected to selection signal analysis by using Fst & theta pi, and GO and KEGG analysis are performed on gene regions which are significant in population selection signal detection. According to the enrichment analysis information, DKK2 gene related to intramuscular fat deposition and differentiation, which is shown to be mainly related to embryonic development and also related to fat synthesis and differentiation, was screened after the basic experimental function verification.
Based on sequencing data, the experimenter found a total of 34 differential SNPs in the DKK2 gene region, of which 12726 th base in the intron region of the pig DKK2 gene or 114874954 th base of pig chromosome eight were suspected to be associated with the intramuscular fat trait, since this site was a in the duroc (low intramuscular fat) reference genome and was essentially all C in the sand ridge pig (high intramuscular fat) (gene frequency 0.975). Subsequently, the experimenter analyzed the SNP allele frequencies of 15 pig breeds (including 299 head pigs) with different intramuscular fat contents, and found that only Duroc had more AA types, wherein the frequency of the A gene was 82.26%, while other high intramuscular fat pig breeds all had the advantage of the C gene frequency, and all had higher than 70% (except for Packxia). The group verification result further confirms the hypothesis that the SNP locus is possibly related to the intramuscular fat content, then, an experimenter performs regression analysis in EXCEL by using the existing phenotypic data and typing results with the A gene frequency as X and the intramuscular fat content as Y, and finally obtains a linear regression model between the A gene frequency and the Y = -1.97395663X +4.700316512, and the P = -0.048694825 to reach a significant level, and the result further confirms the relevance of the intramuscular fat content and the SNP locus: namely, the SNP locus is obviously related to the content of the intramuscular fat, the allele A of the SNP locus is positively related to the low intramuscular fat, and the allele C of the SNP locus is positively related to the high intramuscular fat.
Example 2 establishment of method for detecting SNP molecular markers associated with intramuscular fat trait
The SNP molecular markers of example 1 were population-level detected using Restriction Fragment Length Polymorphism (RFLP) according to the study results of example 1 and the selection of sequence characteristics. BspHI endonuclease sites were created by introducing mutations upstream of the mutation site using specific primers, which were cleaved by BspHI for the base A and not by BspHI for the base C, and were used for typing (see FIG. 1). PCR amplification was performed using the primers of Table 2, and then the PCR product was digested with the digestion system of Table 3.
Table 2: specific primer for detecting A/C mutation by BspHI enzyme digestion
Table 3: enzyme digestion reaction system (30. Mu.l)
After overnight digestion, 5. Mu.l of the digested product was subjected to 1.5% agarose gel electrophoresis 150v,25min, and then typed according to the position of the electrophoresis band as shown in FIG. 2. The result shows that the genotype at the position C is more frequent in local pig breeds in China, while the genotype A is only more frequent in foreign pig breeds such as Duroc pig breeds, and the specific typing conditions are shown in the following table 4:
table 4: genotype identification and gene frequency situation of different pig species
Correlation analysis is carried out between the genotype and the phenotype of an individual, and the mutation site of the SNP molecular marker disclosed by the invention is found to be obviously related to the intramuscular fat content of pigs, and the distribution frequency of the A allele in Duroc lean-type pig breeds is relatively higher and is obviously higher than that of Chinese fatty type pig breeds such as Jinhua pigs, neijiang pigs and the like. Therefore, the A/C locus is obviously related to the intramuscular fat of the pig and can be used as a mark for population breeding improvement work.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The SNP molecular marker related to the porcine intramuscular fat character is characterized in that the SNP molecular marker is positioned at the 12726 th base of the intron region of the porcine DKK2 gene or the 114874954 th base of the porcine chromosome eight, and the base of the site is A or C.
2. The application of the SNP molecular marker of claim 1 in detecting intramuscular fat traits of pigs.
3. The use of claim 2, wherein the fat trait comprises intramuscular fat content.
4. The primer pair for detecting the SNP molecular marker genotype of claim 1, which is characterized in that the primer pair is divided into an upstream primer and a downstream primer, wherein the upstream primer has a nucleotide sequence shown as SEQ ID NO. 1; the downstream primer has a nucleotide sequence shown as SEQ ID NO. 2.
5. A kit for detecting the genotype of the SNP molecular marker according to claim 1, wherein the kit comprises the primer set according to claim 4.
6. The method for detecting the SNP molecular marker related to the intramuscular fat trait of the pig is characterized by comprising the step of detecting a test object by using the primer pair of claim 4 and/or the kit of claim 5.
7. The method according to claim 6, which comprises using the primer pair of claim 4 or the kit of claim 5, PCR amplifying the genomic DNA of the pig to be detected, and performing enzyme digestion on the PCR product by BspHI endonuclease, wherein if the enzyme digestion product is 399bp and 179bp, the base at the mutation position is A; if the enzyme cutting product is 578bp, the base at the mutation position is C.
8. The method of claim 7, wherein the PCR amplification reaction procedure is: 5min at 95 ℃; 35 cycles of 95 ℃ 30s,59 ℃ 30s,72 ℃ 35 s; 10min at 72 ℃; infinity at 4 ℃; the enzyme digestion reaction program is as follows: at 37 ℃ overnight; 20min at 65 ℃: infinity at 4 ℃.
9. The application of any one of the following I to III in improving the pig germplasm resources and/or breeding high or suitable intramuscular fat pig breeds:
i: the SNP molecular marker of claim 1;
II: the primer pair of claim 4;
III: the kit of claim 5.
10. A method of breeding pigs, comprising detecting the genotype of the parent or progeny using the primer pair of claim 4 or the kit of claim 5.
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| PCT/CN2022/112041 WO2024021172A1 (en) | 2022-07-25 | 2022-08-12 | Snp molecular marker associated with intramuscular fat traits in pigs, use thereof, and method for detecting same |
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| WO2024021172A1 (en) * | 2022-07-25 | 2024-02-01 | 中国农业大学 | Snp molecular marker associated with intramuscular fat traits in pigs, use thereof, and method for detecting same |
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| CN105255870B (en) * | 2015-10-30 | 2018-04-03 | 中国农业大学 | A kind of SNP marker and its detection method related to fat thickness at back of pig character |
| CN105624310B (en) * | 2016-02-29 | 2017-07-14 | 华南农业大学 | A kind of molecular labeling for influenceing pig intramuscular fat character and application |
| CN108315433B (en) * | 2018-03-06 | 2018-12-21 | 华南农业大学 | It is a kind of influence Duroc boar intramuscular fat content molecular labeling and application |
| CN110106263B (en) * | 2019-05-30 | 2020-11-17 | 浙江大学 | SNP marker combination and identification method of Pudong white pig and raw meat product |
| CN112176070B (en) * | 2020-08-03 | 2022-04-19 | 南京农业大学 | UCP3 gene related to pig intramuscular fat character, molecular marker and application thereof |
| CN112322753B (en) * | 2020-11-27 | 2023-09-26 | 广西扬翔股份有限公司 | SNP molecular marker related to intramuscular fat of pork and application thereof |
| CN115786527A (en) * | 2022-07-25 | 2023-03-14 | 三亚中国农业大学研究院 | SNP molecular marker related to pig intramuscular fat character and application and detection method thereof |
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