An SNP molecular marker related to pig body length, its amplification primer and application

Abstract

The present invention discloses an SNP molecular marker related to pig body length, its amplification primers and applications. This molecular marker is located at the -416th base and -288th base in the promoter region before the 5' UTR region of the pig HHEX gene (taking the first base before ATG as -1), with a C-T mutation and a G-A mutation respectively, and they are completely linked. The present invention for the first time discovers that a pair of completely linked SNP molecular markers in the pig HHEX gene are related to the pig body length trait. Therefore, it can be used for pig marker-assisted selection breeding, that is, it provides a new use of a pair of completely linked SNP molecular markers in the pig HHEX gene in marker-assisted breeding of pig body length traits, realizing the early screening of pig body length traits, and the screening method is simple and fast.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers, and particularly relates to an SNP molecular marker related to pig body length, an amplification primer thereof, and an application thereof. Background Art

[0002] China is the world's largest producer and consumer of pigs, and the proportion of pork in China's meat consumption has remained above 60% for a long time. Body length trait is one of the most important quantitative traits in the process of species evolution, and there are significant differences among species and different breeds within a species. Existing studies have shown that the heritability of body length trait is 0.29 - 0.55, which is a trait with medium or higher heritability. It is positively correlated with the growth, development, health, and diseases of organisms, and is a comprehensive and complex trait affected by multiple factors. In agricultural economic animals, the body length trait is closely related to the meat production of animals, so it has always been one of the important improvement traits in the livestock breeding industry. Developing rapid, simple, and effective molecular genetic markers for pig body length traits can be applied to the detection of large populations, for early selection and breeding, increasing the selection pressure for body length, and accelerating the process of genetic improvement work.

[0003] HHEX (Hematopoietically expressed homeobox protein) is a divergent homeoprotein transcription factor containing a DNA-binding homeodomain. Research shows that HHEX is a key transcription factor that forms a normal morphology by regulating cell proliferation and differentiation during the development of hematopoietic cells, vascular system, liver, thyroid, heart, pancreas, and skin. In chondrocytes, it has been found that HHEX can promote the expression of type X collagen (COL10), a chondrocyte marker factor, during the differentiation of mouse chondrocytes, thus affecting cartilage development. And cartilage development is directly related to the body length trait, indicating that the HHEX gene plays a key role in body length development. The functional research of HHEX and its family members in mouse models is relatively thorough, but there is still no report on its function in pigs. In addition, it is still unclear whether there are molecular markers in the pig HHEX gene that can be used for the selection and breeding of pig body length traits. Summary of the Invention

[0004] In view of this, the present invention provides an SNP molecular marker related to pig body length, an amplification primer thereof, and an application thereof.

[0005] The first object of the present invention is to provide an SNP molecular marker related to pig body length trait.

[0006] The second object of the present invention is to provide primers and kits for detecting the SNP molecular marker.

[0007] The third object of the present invention is to provide the application of the above SNP molecular markers, primers and kits in breeding long-body pigs.

[0008] The fourth object of the present invention is to provide a method for identifying pig body length using the said SNP molecular markers and a method for improving pig breeds.

[0009] The objects of the present invention are achieved by the following technical solutions:

[0010] First, using the genomes of 10 Wuzhishan pigs and 10 Large White pigs as templates, the partial base sequences of the promoter region of the pig HHEX gene were sequenced by PCR-based pooled sequencing. Through sequence analysis of a partial segment of the HHEX gene, the sequence results were analyzed using chromasPro v1.41 software to search for SNP mutation sites, and SNP sites affecting pig body length traits were screened out. It was found that there were C-T mutations and G-A mutations at the -416th base and -288th base (with the first base before ATG being -1) in the promoter region before the 5' UTR region of the HHEX gene, and they were completely linked.

[0011] Furthermore, through large-scale SNP genotyping work, using 24 Wuzhishan pigs (average body length), 22 Bama Xiang pigs (average body length), 24 Tibetan pigs (average body length), 34 Banna mini-pigs (average body length), 47 Jiangkou radish pigs (average body length), 37 Longlin pigs (average body length), 36 Min pigs (average body length), 14 Yanan black pigs (average body length), 35 Qianbei black pigs (average body length), 35 Saba pigs (average body length) as medium and short body length breed verification groups, and at the same time 30 Landrace pigs (average body length), 29 Large White pigs (average body length) as long body length breed verification groups (the body length data of the experimental pigs comes from "China Livestock and Poultry Genetic Resources Record - Pig Record" in May 2011), it was found that the average body length of the TT and AA genotype groups was significantly longer than that of the CC and GG genotype groups. Then, using the linear model of the GenABLE software package in R language for analysis, it was found that this SNP locus was significantly associated with the individual body length trait. Using Chinese local pig breeds such as Wuzhishan pigs, Bama Xiang pigs, Tibetan pigs, Banna mini-pigs, Jiangkou radish pigs, Longlin pigs, Min pigs, Yanan black pigs, Qianbei black pigs and Saba pigs as medium and short body length pig breeds and long body length Large White pigs and Landrace pigs for the SNP allele frequency analysis of these two SNP sites, it was found that the allele frequencies of the two SNP sites T and A were relatively low in Chinese local pig breeds, both lower than 0.30, while they were extremely high in Large White pigs and Landrace pigs, both higher than 0.90. Based on this situation, the above-mentioned association analysis view was further confirmed: these two SNP loci were significantly associated with pig body length traits, among which the alleles T and A were positively correlated with the long body length trait, and the alleles C and G were positively correlated with the medium and short body length trait.

[0012] Specifically, the present invention provides an SNP molecular marker related to pig body length, and the SNP molecular marker includes SNP1 locus and / or SNP2 locus; the SNP1 locus is a C / T mutation at the -416th base in the promoter region of the pig HHEX gene; the SNP2 is a G / A mutation at the -288th base in the promoter region of the pig HHEX gene; wherein, the first base before ATG is the -1st base.

[0013] Furthermore, the present invention provides primers for amplifying the SNP molecular marker.

[0014] Among them, the primers are characterized in that they include:

[0015] Upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO1-2, or upstream and downstream primers with nucleotide sequences as shown in SEQ ID NO3-4,

[0016] The present invention also provides a kit for identifying pig body length, including upstream and downstream primers shown in SEQ ID NO1-2, and EagI endonuclease.

[0017] The present invention also provides another kit for identifying pig body length, including upstream and downstream primers shown in SEQ ID NO2-3.

[0018] The present invention also provides the application of the SNP molecular marker, the primers, or the kit in breeding pigs with long body length; when the genotypes of SNP1 locus and SNP2 locus are CC and GG respectively, or CT and GA respectively, the pig to be tested is an individual with medium and short body length; when the genotypes of SNP1 locus and SNP2 locus are TT and AA respectively, the pig to be tested is an individual with long body length.

[0019] The present invention also provides a method for identifying pig body length using the SNP molecular marker, which is characterized by including the following steps:

[0020] Perform PCR amplification on the genomic DNA of the pig to be tested using the upstream and downstream primers shown in SEQ ID NO.1-2;

[0021] Digest the PCR product with EagI endonuclease, determine the genotypes of SNP1 locus and SNP2 locus according to the digestion result, and further determine the body length of the pig;

[0022] The method for determining the genotypes of SNP1 locus and SNP2 locus and determining the body length of the pig is: if the digestion product is 165bp, the genotypes of SNP1 locus and SNP2 locus are TT and AA respectively, and the pig to be tested is an individual with long body length;

[0023] If the enzyme digestion products are 108bp and 57bp, the genotypes of SNP1 and SNP2 are CC and GG respectively. If the enzyme digestion products are 165bp, 108bp and 57bp, the genotypes of SNP1 and SNP2 are CT and GA respectively. If the genotypes are CC and GG, or CT and GA, the pigs to be tested are medium and short body length individuals.

[0024] The present invention also provides a method for identifying the body length of pigs using the SNP molecular marker as described above, which is characterized by including the following steps:

[0025] Perform PCR amplification on the genomic DNA of the pigs to be tested using the upstream and downstream primers shown in SEQ ID NO.3-4, and detect the genotypes of SNP1 and SNP2 sites;

[0026] When the genotypes of SNP1 and SNP2 are CC and GG respectively, the pigs to be tested are medium and short body length individuals; when the genotypes of SNP1 and SNP2 are TT and AA respectively, the pigs to be tested are long body length individuals.

[0027] In the embodiment of the present invention, the reaction procedure of the above PCR amplification is: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s, 72°C for 15 s, for a total of 35 cycles; 72°C for 10 min.

[0028] The present invention also provides a method for improving pig breeds. Use the method for identifying the body length of pigs to identify the body length of the pigs to be tested, and use the long body length individuals as parents for breeding.

[0029] The effective effects of the present invention are as follows:

[0030] The present invention discloses for the first time SNP molecular markers related to pig body length traits, which are used to distinguish medium and short body length and long body length individuals, are applicable to Chinese pig breeds and foreign pig breeds, and have a wide range of applicable pig breeds. The method for detecting the genotypes of SNP molecular markers of the present invention can predict pig body length early, quickly, at low cost and effectively, has a broad application prospect in the improvement of pig breeds and strains, and can achieve excellent economic value. Description of the Drawings

[0031] Figure 1 For the sequence alignment of mutation positions of different pig breeds;

[0032] Figure 2 -A, Figure 2 -B are respectively the sequencing peak maps at the -416 position of the 5'UTR region pre-promoter region of the HHEX gene of Wuzhishan pigs and Large White pigs; Figure 2 -C, Figure 2Sequencing peak maps at the mutation position of the -288th base in the promoter region before the 5’UTR region of the HHEX gene in Wuzhishan pigs and Large White pigs, respectively;

[0033] Figure 3 Schematic diagram for analysis by creating the restriction enzyme EagI;

[0034] Figure 4 Agarose gel electrophoresis results after digestion with EagI for three genotypes. Specific implementation manners

[0035] The present invention provides an SNP molecular marker related to pig body length, its amplification primers and applications. Those skilled in the art can draw on the content of this article and appropriately improve 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 regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications 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.

[0036] In the embodiments of the present invention, the germplasm resources of various pig breeds are from the conservation farms in each origin area. For example, the Wuzhishan pigs in the embodiments are from the Wuzhishan Pig Resource Conservation Farm of the Hainan Academy of Agricultural Sciences, and the Large White pigs are from the Beijing Pig Breeding Center.

[0037] If not specifically specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0038] The following further elaborates the present invention in conjunction with embodiments:

[0039] Example 1 Obtaining of SNP molecular markers related to body length traits

[0040] Using the genomes of 10 Wuzhishan pigs and 10 Large White pigs as templates, for the above verification, the PCR technology was used to pool-sequence partial base sequences in the promoter region of the pig HHEX gene, and partial genomic sequences were supplemented.

[0041] Through sequence analysis of partial regions of the HHEX gene, the sequence results were analyzed using chromasProv1.41 software to find SNP mutation sites, and SNP sites affecting pig body length traits were screened out. It was found that there were C→T mutations and G→A mutations at the -416th base and -288th base (with the first base before ATG being -1) in the promoter region before the 5’UTR region of the HHEX gene, and they were completely linked.

[0042] According to the pig HHEX gene sequence (NM_001244579.1) published by NCBI, the HHEX gene is located on pig chromosome 14, consists of 4 exons, and is 1190 bp in length. Primers were designed to amplify the mRNA and promoter region sequence of the pig HHEX gene. The primer sequences in this example are shown in Table 1, and a 654-bp fragment of the pig HHEX gene promoter region can be amplified.

[0043] Table 1 Specific primers for SNP detection of HHEX gene

[0044]

[0045] Using the above primers, SNP screening was first carried out in the Wuzhishan pig and Large White pig populations with extremely significant differences in body length. Screening was carried out by combining various software (such as chromasPro v1.41, VectorNTI.Advance11, etc.) with alignment and association statistical analysis, and two suspected SNP loci affecting pig body length traits were found. At the -416th base and -288th base in the promoter region in front of the 5’UTR (with the first base before ATG being -1), there were respectively C→T mutations and G→A mutations (see Figure 1 ). According to the sequencing peak map, it was confirmed that there was indeed a G / A mutation at this position (see Figure 2 A- Figure 2 D).

[0046] Chinese indigenous pig breeds such as Wuzhishan pig, Bama Xiang pig, Tibetan pig, Banna mini-pig, Jiangkou radish pig, Longlin pig, Min pig, Yanan black pig, Qianbei black pig, and Saba pig were used as medium and short body length pig breeds, and the SNP allele frequency analysis was carried out with the long body length Large White pig and Landrace. It was found that the allele frequencies of the two SNP loci, T and A, were relatively low in Chinese indigenous pig breeds, both lower than 0.30, while they were extremely high in Large White pigs and Landrace, both higher than 0.90. This result further confirmed the above-mentioned association analysis view: The C→T mutation at the -416th base and G→A mutation at the -288th base in the promoter region in front of the 5’UTR of the pig HHEX gene (with the first base before ATG being -1) were significantly associated with pig body length traits. Among them, the alleles T and A were positively correlated with long body length traits, and the alleles C and G were positively correlated with medium and short body length traits.

[0047] Example 2 Establishment of a method for detecting SNP molecular markers related to pig body length traits

[0048] 1. Detection of SNP loci at the population level using RFLP restriction enzymes

[0049] Based on the research results of Example 1 and the characteristics of the sequences, the SNP molecular markers of Example 1 were detected at the population level using Restriction Fragment Length Polymorphism (RFLP). Since the two SNP loci are completely linked, when the base at the first locus (i.e., the -416th position in the promoter region before the 5' UTR region of the HHEX gene) is C, the base at the second locus (i.e., the -288th position in the promoter region before the 5' UTR region of the HHEX gene) is G, and when the base at the first locus is T, the base at the second locus is A. Therefore, a mutation was introduced using specific primers upstream of the mutation position to create an EagI endonuclease site (see Table 2). When the base at the first SNP locus is C, it can be digested by the EagI enzyme, and when it is T, it cannot be digested by the EagI enzyme, thereby being used for genotyping (such as Figure 3 ). PCR amplification was performed using the primers in Table 2.

[0050] Specifically, if the enzyme digestion product is 165 bp, the bases at the two mutation sites are TT and AA respectively. If the enzyme digestion products are 108 bp and 57 bp, the bases at the two mutation sites are CC and GG respectively. If the enzyme digestion products are 165 bp, 108 bp and 57 bp, the bases at the two mutation sites are CT and GA respectively.

[0051] Table 2 Specific primers for detecting G / A mutation using EagI digestion

[0052]

[0053]

[0054] Table 3 pstI digestion reaction system

[0055] Reagent Volume (ul) PCR Product 5 10* Buffer 2 EagI 0.5 <![CDATA[ddH2O]]> 12.5 Total Volume 20

[0056] After digestion, 20 μl of the digestion product was used for 4% agarose gel electrophoresis at 100 v for 50 min. Then, genotyping could be performed according to the positions of the electrophoresis bands, such as Figure 4 . It was found that the frequencies of the C and G genotypes at the two SNP loci were relatively high in Chinese local pig breeds, while the frequencies of the T and A genotypes were relatively high in foreign pig breeds such as Landrace and Large White pigs. The specific distribution frequencies are shown in Table 4 below:

[0057] Table 4 Distribution of genotype frequencies and allele frequencies of different pig breeds at the first SNP locus

[0058]

[0059]

[0060] 2. Population-level association analysis

[0061] The SNP genotyping data of the above 368 pigs were collated with the phenotypic data of body length traits (the body length data were from "Chinese Livestock and Poultry Genetic Resources Annals - Pig Annals" in May 2011), as shown in Table 5.

[0062] Table 5 Phenotypic statistics of body length traits in different pig populations

[0063]

[0064]

[0065] The results of population genotyping showed that the distribution frequencies of the two SNP alleles T and A in this invention were relatively high in long - bodied pig breeds such as Landrace and Large White pigs, significantly higher than those in Chinese medium - short - bodied pig breeds such as Wuzhishan pigs, Tibetan pigs, and Banna mini - pigs. Further, a linear regression model between the individual TA genotype and the body length phenotype was constructed. Finally, the linear regression model between the two was Y = 74.24X + 90.45, P = 0.040, indicating that the TA genotype in the SNP molecular marker of this invention was significantly positively correlated with the pig body length trait. In summary, the C→T and G→A loci were related to the pig body length trait and could be used as markers for the improvement work of breeding long - bodied populations.

[0066] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of primers for amplifying SNP molecular markers or a kit comprising the primers in breeding pigs with long body length; when the genotypes of SNP1 locus and SNP2 locus are CC and GG respectively, or CT and GA respectively, the pigs to be tested are individuals with medium and short body lengths; when the genotypes of SNP1 locus and SNP2 locus are TT and AA respectively, the pigs to be tested are individuals with long body lengths; The SNP molecular markers are SNP1 locus and SNP2 locus; the SNP1 locus is a C / T mutation at the -416th base in the promoter region of the porcine HHEX gene; the SNP2 is a G / A mutation at the -288th base in the promoter region of the porcine HHEX gene; wherein, The first base before ATG is designated as the -1 position; The accession number of the mRNA sequence of the porcine HHEX gene is NM_001244579.

1.

2. The use according to claim 1, characterized in that, The primer includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NOs. 1-2, or includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NOs. 3-4.

3. The use according to claim 1, characterized in that, The kit includes upstream and downstream primers shown in SEQ ID NOs. 1-2 and EagI endonuclease, or includes upstream and downstream primers with nucleotide sequences shown in SEQ ID NOs. 3-4.

4. A method for identifying the body length of pigs using the SNP molecular markers according to claim 1, characterized in that, It includes the following steps: Perform PCR amplification on the genomic DNA of the porcine sample to be tested using the upstream and downstream primers shown in SEQ ID NOs. 1-2; Digest the PCR product with EagI endonuclease, determine the genotypes of SNP1 and SNP2 sites according to the digestion results, and then determine the body length of the pig. The method for determining the genotypes of SNP1 and SNP2 sites and determining the body length of the pig is as follows: If the digestion product is 165 bp, the genotypes of SNP1 and SNP2 sites are TT and AA respectively, and the porcine sample to be tested is an individual with a long body length; If the digestion products are 108 bp and 57 bp, the genotypes of SNP1 and SNP2 sites are CC and GG respectively; if the digestion products are 165 bp, 108 bp and 57 bp, the genotypes of SNP1 and SNP2 sites are CT and GA respectively; if the genotypes are CC and GG, or CT and GA, the porcine sample to be tested is an individual with a medium or short body length.

5. A method for identifying the body length of pigs using the SNP molecular markers according to claim 1, characterized in that, It includes the following steps: Perform PCR amplification on the genomic DNA of the porcine sample to be tested using the upstream and downstream primers shown in SEQ ID NOs. 3-4 to detect the genotypes of SNP1 and SNP2 sites; When the genotypes of SNP1 and SNP2 sites are CC and GG respectively, the porcine sample to be tested is an individual with a medium or short body length; when the genotypes of SNP1 and SNP2 sites are TT and AA respectively, the porcine sample to be tested is an individual with a long body length.

6. A method for improving pig breeds in breeding, characterized in that, Use the method according to claim 4 or 5 to identify the body length of the porcine breed to be tested, and use the individuals with a long body length as parents for breeding.

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