Haplotype markers associated with pig fat deposition and their application

Abstract

The present invention provides a haplotype marker associated with pig fat deposition and its application. The haplotype marker comprises a nucleotide sequence of the porcine AACS gene, as shown in SEQ ID NO:1, wherein the polymorphism at bp 332 is A / C, the polymorphism at bp 408 is C / T, and the polymorphism at bp 427 is A / G. The haplotype marker provided by the present invention can be used for backfat thickness selection in pigs. The marker is not restricted by the age or sex of the pig and can be used for early pig selection, even enabling accurate selection at birth, significantly shortening the generation interval and accelerating the pig breeding process.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, in particular to a haplotype marker associated with pig fat deposition and an application thereof. Background Art

[0002] The amount of fat deposited in pigs directly affects the quality and flavor of pork. Studying the fat deposition mechanism in pigs and improving pork quality have always been the focus of animal husbandry experts' breeding work. Adipogenesis is a complex process regulated by multiple transcriptional events (Ghaben AL, Scherer PE. Adipogenesis and metabolic health. Nat Rev Mol Cell Biol 2019, 20(4):242-258). As a quantitative trait, it is also regulated by multiple genes. Compared with imported pigs, most local pig breeds in my country have a high fat content and are called fatty pigs. Excessive fat deposition leads to reduced economic benefits. Molecular marker-assisted selection technology has been widely used in breed selection and breeding. It is fast, effective, and not restricted by the environment. Exploring and finding molecular markers with clear functions and significant effects can promote the cultivation of high-quality breeds.

[0003] Dingyuan pigs are the most widely distributed and numerous pig breed in Anhui Province. They are large in size, weighing up to 89.69 kg at 10 months of age. They have thicker backfat and higher intramuscular fat content, both of which are higher than those of Large White and Landrace pigs, which weigh approximately 100 kg (Wu Yijing, Zhang Pan, Fu Yanru, Su Shiguang, Tian Guangyou, Zhang Hao, Li Qinggang. Determination and analysis of carcass and meat quality traits of Dingyuan pigs [J]. Pig Farming, 2019(05):78-80.). Tibetan pigs, as a plateau-type pig breed unique to my country, have the common characteristics of local pig breeds, with slow growth and high fat accumulation ability (Yang SL, Wang ZG, Liu B, Zhang GX, Zhao SH, Yu M, Fan B, Li MH, Xiong TA, Li K: Genetic variation and relationships of eighteen Chinese indigenous pig breeds. Genetics Selection Evolution 2003, 35(6):657-671.). Local pig breeds with strong fat deposition ability and lean meat pigs such as Large White and Landrace with relatively weak fat deposition ability are used as materials to screen genes and molecular marker sites related to fat deposition, and assist in pig breed selection.

[0004] Acetoacetyl-CoA synthetase, also known as acetoacetate-CoA (AACS), is a cytoplasmic ketone body (acetoacetate)-specific ligase found in a variety of adipogenic tissues (Ito M, Fukui T, Saito T, Tomita K: Acetoacetyl-CoA synthetase specific activity and concentration in rat tissues. Biochim Biophys Acta 1986, 876(2): 280-287). It can specifically activate acetoacetate to acetoacetyl-CoA, thereby synthesizing cholesterol and fatty acids. Studies have shown that a binding site for CCAAT / enhancer binding protein α (C / EBPα) was found in the AACS promoter region (Hasegawa S, Yamasaki M, Inage T, Takahashi N, Fukui T: Transcriptional regulation of ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, by C / EBP alpha during adipocyte differentiation. Bba-Gene RegulMech 2008, 1779(6-7):414-419).In humans, the AACS promoter is a peroxisome proliferator-activated receptor γ (PPARγ) target gene (Aguilo F, Camarero N, Relat J, Marrero PF, Haro D: Transcriptional regulation of the human acetoacetyl-CoA synthetase gene by PPARgamma. Biochem J 2010, 427(2): 255-264). AACS mRNA expression increased significantly on the 4th day after induced differentiation of 3T3-L1 cells. Its expression pattern during preadipocyte differentiation is very similar to that of acetyl-CoA carboxylase 1 (ACC-1), a key enzyme in fatty acid synthesis (Yamasaki M, Hasegawa S, Suzuki H, Hidai K, Saitoh Y, Fukui T: Acetoacetyl-CoA synthetase gene is abundant in rat adipose, and related with fatty acid synthesis in mature adipocytes. Biochem Bioph Res Co2005,335(1):215-219). Summary of the Invention

[0005] The purpose of the present invention is to provide a haplotype marker related to pig fat deposition and application thereof.

[0006] Studies have shown that the AACS gene may be an important gene for regulating fat deposition. The present invention performed transcriptomics on the subcutaneous adipose tissue of Dingyuan pigs with different backfat thicknesses, and the results showed that the gene was lowly expressed in the adipose tissue of individuals with high backfat thickness. In the adipose tissue of Tibetan pigs with stronger fat deposition ability, the expression level of this gene was also lower than that of lean pigs by about one gram. The screening and identification of the SNP sites of this gene provide theoretical support for pig genetic improvement. Sanger sequencing was used to screen the differential SNP sites between lean pigs and obese pigs, and the correlation between SNP sites and backfat thickness was analyzed within the breed to clarify the SNP sites and genotype effects that affect pig fat deposition.

[0007] To achieve the purpose of the present invention, in a first aspect, the present invention provides a haplotype marker associated with pig fat deposition, wherein the haplotype marker comprises a nucleotide sequence of the pig AACS gene as shown in SEQ ID NO: 1, wherein the polymorphism at the 332 bp is A / C, the polymorphism at the 408 bp is C / T, and the polymorphism at the 427 bp is A / G.

[0008] Furthermore, the genotype of the polymorphic site at bp 332 is AA or AC, and the corresponding pigs have a stronger fat deposition ability than the genotype CC;

[0009] The genotype of the polymorphic site at 408 bp is CC or CT, and the corresponding pigs have a stronger fat deposition ability than the genotype TT;

[0010] The genotype of the polymorphic site at 427bp is AA or AG, and the corresponding pigs have stronger fat deposition ability than the genotype GG.

[0011] In a second aspect, the present invention provides primers for amplifying the haplotype marker, comprising a forward primer as shown in SEQ ID NO: 2 and a reverse primer as shown in SEQ ID NO: 3.

[0012] In a third aspect, the present invention provides a detection reagent or kit containing the primers.

[0013] In a fourth aspect, the present invention provides a method for evaluating the fat deposition ability of pigs, comprising the following steps:

[0014] 1) Extracting genomic DNA from the pig to be tested;

[0015] 2) Using genomic DNA as a template, PCR amplification was performed using the primers shown in SEQ ID NOs: 2-3;

[0016] 3) Analyze the PCR amplification products.

[0017] Preferably, the PCR reaction system is: 10 μl of 2×PCR Mix, 100 ng of genomic DNA, 0.5 μl of 10 μmol / L forward and reverse primers, and the volume is filled up to 20 μl with dd H2O.

[0018] The PCR reaction program was as follows: pre-denaturation at 94-95°C for 5 min; denaturation at 94-95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 36 cycles; and extension at 72°C for 5 min.

[0019] Step 3) includes sequencing the amplified product, and judging according to the sequencing results as follows: if the genotype of the polymorphic site at the 332 bp position of the amplified product is CC, the genotype of the polymorphic site at the 408 bp position is TT, and the genotype of the polymorphic site at the 427 bp position is GG, then it is judged that the fat deposition ability of the tested pig is weak.

[0020] In a fifth aspect, the present invention provides any of the following uses of the haplotype marker, the primer, or the detection reagent or kit:

[0021] (1) Used to determine the fat deposition capacity of pigs;

[0022] (2) Used for early prediction of pig fat deposition capacity;

[0023] (3) Used for molecular marker-assisted breeding related to pig fat deposition ability.

[0024] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0025] (1) The haplotype markers provided by the present invention can be used for the selection of backfat thickness in pigs.

[0026] (2) The haplotype markers provided by the present invention are not restricted by the age, sex, etc. of the pigs and can be used for early selection and breeding of pigs. They can even be accurately selected when the pigs are just born, which can greatly shorten the generation interval and accelerate the pig breeding process.

[0027] (3) The detection method is fast and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The figure shows the mRNA expression of AACS genes in pig back fat and longissimus dorsi muscle in a preferred embodiment of the present invention.

[0029] Figure 2 This is a sequencing peak diagram of three linked sites in the 5' flanking region of the porcine AACS gene in a preferred embodiment of the present invention.

[0030] Figure 3 This is the amplification result of three linked sites in the 5' flanking region of the porcine AACS gene in a preferred embodiment of the present invention; the agarose gel concentration is 1.2%; the marker in the figure is DM2000; the fragment size in each lane in the figure is 631 bp.

[0031] Figure 4 The following is a sequence diagram of the three linked loci of the porcine AACS gene in a preferred embodiment of the present invention. The first row of peaks in the figure shows the genotypes of the three linked loci (1759 bp, 1683 bp, and 1664 bp), AA, CC, and AA, respectively. The second row of peaks shows the genotypes of AC, CT, and AG, respectively. The third row of peaks shows the genotypes of CC, TT, and GG, respectively. DETAILED DESCRIPTION

[0032] The present invention provides an identification method and application of haplotype markers associated with pig fat deposition, namely, using polymerase chain reaction (PCR) and PCR product sequencing to detect genotypes, and selecting pigs based on the genotypes, which can improve pork quality and accelerate the pig breeding process.

[0033] The present invention adopts the following technical solutions:

[0034] The present invention provides a haplotype genetic marker related to fat deposition traits in the porcine AACS gene screened by PCR sequencing method, the nucleotide sequence of which is shown in SEQ ID NO: 1.

[0035] In the sequence shown in SEQ ID NO: 1, there is an A-to-C mutation at bp 332 (i.e., n is a or c), a C-to-T mutation at bp 408 (i.e., n is c or t), and an A-to-G mutation at bp 427 (i.e., n is a or g). These three mutations are completely linked. These three sites are located 1759 bp, 1683 bp, and 1664 bp before the start codon ATG, respectively.

[0036] Wherein: the primer sequences for detecting the base mutation are shown in the sequence listing SEQ ID NO: 2 and SEQ ID NO: 3.

[0037] Forward primer F: 5'-AGGAGAAAGGAGCAGAGCCA-3'

[0038] Reverse primer R: 5'-TTGAGCAGGGAACGGAAT-3'

[0039] Identification and application of a haplotype marker associated with pig fat deposition, comprising the following steps:

[0040] Genomic DNA was extracted from pig ear tissue, and primers were designed based on the 5' flanking region of the porcine AACS gene. The resulting primer sequences were as follows: forward primer F: 5'-AGGAGAAAGGAGCAGAGCCA-3' (SEQ ID NO: 2), reverse primer F: 5'-TTGAGCAGGGAACGGAAT-3' (SEQ ID NO: 3). PCR amplification was performed on the porcine genomic DNA using the indicated primers. The PCR product was purified, cloned, and sequenced to obtain the nucleotide sequence set forth in SEQ ID NO: 1.

[0041] The present invention further provides an application of using PCR product sequencing to perform correlation analysis between individuals with different genotypes and pig fat deposition traits.

[0042] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0043] Example 1 Analysis of porcine AACS gene tissue expression

[0044] Dingyuan pigs (DY) were obtained from Ankang Agriculture and Animal Husbandry Company in Dingyuan County, Anhui Province, and were divided into a high back fat group (HBF) and a low back fat group (LBF) based on back fat thickness. Tibetan pigs (TP) and Yorkshire pigs (YH) were obtained from the teaching and practice ranch of Tibet Agriculture and Animal Husbandry College in Linzhi City, Tibet Autonomous Region. Dingyuan pigs were 300 days old, and the other pigs were 6 months old. Longissimus dorsi (LD) and back fat tissue (BF) were collected, and tissue RNA was extracted using the traditional Trizol method and reverse transcribed into cDNA.

[0045] The porcine AACS gene mRNA sequence was cloned by RACE assay. The following primers were designed using Primer Premier 5.0 software:

[0046] Forward primer F: 5'-AAGAGCATCCGCAACGCCAT-3'

[0047] Reverse primer R: 5'-CTTCTTGCCGTTGAGGGTGT-3'

[0048] The above primers were used to amplify cDNA from the back fat and longissimus dorsi muscle tissues of Dingyuan pigs, Tibetan pigs, and Yorkshire pigs using real-time fluorescence quantitative PCR. The PCR amplification used a 20 μl reaction system consisting of 10 μl 2×SuperReal PreMix Plus, 8 μl ddH2O, 0.5 μl each of the forward and reverse primers, and 1 μl of the cDNA template. The PCR amplification reaction system was pre-denatured at 95°C for 15 min and then cycled 40 times as follows: pre-denaturation at 95°C for 20 s, annealing at 55°C for 20 s, and extension at 72°C for 20 s. β-actin (upstream primer: 5'-GGACTTCGAGCAGGAGATGG-3'; downstream primer: 5'-AGGAAGGAGGGCTGGAAGAG-3') was used as an internal reference. –ΔΔCT The relative expression of AACS genes in different pig tissues was analyzed by Figure 1 ).

[0049] In adipose tissue, AACS gene expression levels in Tibetan pigs were significantly lower than in Yorkshire pigs, while no significant difference was observed in muscle tissue. Similar results were observed in Dingyuan pigs, which vary in backfat thickness. AACS gene expression levels in adipose tissue in the high-backfat group were significantly lower than in the low-backfat group, while no significant difference was observed in muscle tissue. This suggests that elevated AACS gene expression may negatively regulate back fat deposition in pigs.

[0050] Example 2 Screening of SNP sites in the porcine AACS gene

[0051] Ear tissue samples from the local Chinese breed "Tibetan pig" (collected from the teaching and practice ranch of Tibet Agricultural and Animal Husbandry College in Linzhi City, Tibet Autonomous Region) and the introduced breeds "Yorkshire pig" (collected from Anhui Kexin Pig Breeding Co., Ltd.) and "Landrace pig" (collected from Beijing Zhongshun Jingsheng Breeding Co., Ltd.) were selected, and genomic DNA from the tissues was extracted using the phenol / chloroform method.

[0052] The porcine AACS gene sequence (accession number NC_010456.5) was downloaded from NCBI. The following primers (SEQ ID NOs: 2-3) were designed using the software Primer Premier 5.0 based on the DNA sequence of the AACS gene downloaded from NCBI:

[0053] Forward primer F: 5'-AGGAGAAAGGAGCAGAGCCA-3'

[0054] Reverse primer R: 5'-TTGAGCAGGGAACGGAAT-3'

[0055] PCR amplification was performed using the above primers on genomic DNA from Tibetan pigs, Yorkshire pigs, and Landrace pigs. A 20 μl reaction system was typically used: 10 μl of 2× PCR Mix (half the volume of the PCR amplification reaction system), 100 ng of the pig genomic DNA to be tested, 0.5 μl of each 10 μmol / L forward and reverse primer, and finally, ddH2O was added to 20 μl and mixed thoroughly to obtain the PCR amplification reaction system. The PCR amplification reaction system was pre-denatured at 94-95°C for 5 minutes, followed by 36 cycles of pre-denaturation at 94-95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and extension at 72°C for 5 minutes to obtain the PCR product.

[0056] PCR products from the three pig breeds were purified using a Gel Extraction Kit (purchased from Shanghai Bioengineering Technology Co., Ltd., according to the kit instructions). Twenty individuals from each breed were selected for PCR amplification. 10 μl of the PCR product from each individual was pooled into a single sample for sequencing (BGI). The nucleotide sequence of the amplified product is shown in SEQ ID NO: 1.

[0057] Sequencing results were aligned using Chromas Pro software. In the sequence shown in SEQ ID NO: 1, there was an A-to-C mutation at 332 bp, a C-to-T mutation at 408 bp, and an A-to-G mutation at 427 bp. These three mutations were completely linked. These three sites were located at 1759 bp, 1683 bp, and 1664 bp upstream of the codon in the 5' flanking region, respectively. Figure 2 ).

[0058] Example 3 Establishment of a sequencing method for genotyping the porcine AACS gene A-1759C, C-1683T, and A-1664G sites

[0059] To quickly and conveniently detect the genotypes of the porcine AACS gene loci A-1759C, C-1683T, and A-1664G, PCR amplification was performed on porcine genomic DNA using the primers described in Example 1. The PCR reaction system consisted of a 20 μl system: 10 μl of 2×PCR Mix (half the volume of the PCR amplification reaction system), 100 ng of the porcine genomic DNA to be tested, 0.5 μl of each of the 10 μmol / L forward and reverse primers, and finally ddH2O to 20 μl. The mixture was mixed to obtain a PCR amplification reaction system. The PCR amplification reaction system was pre-denatured at 94-95°C for 5 minutes, followed by 36 cycles of denaturation at 94-95°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 minute, and extension at 72°C for 5 minutes to obtain a PCR product.

[0060] The PCR amplification product was electrophoresed on a 1.2% agarose gel with the addition of nucleic acid dye SYBR Green I. The PCR amplification effect was observed in a gel imaging system, and a clear band with a fragment size of 631 bp was observed ( Figure 3 ). Sequence the PCR product and determine the genotype by sequencing peak diagram ( Figure 4 ).

[0061] Example 4 Detection of genotype distribution of genetic markers in different pig populations

[0062] Ear tissue samples were collected from Tibetan pigs (collected from the teaching and practice ranch of Tibet Agriculture and Animal Husbandry College in Linzhi City, Tibet Autonomous Region), Yorkshire pigs (collected from Anhui Kexin Pig Breeding Co., Ltd.), and Landrace pigs (collected from Beijing Zhongshun Jingsheng Breeding Co., Ltd.), and genomic DNA samples of individual pigs were extracted using the phenol / chloroform method.

[0063] The genotypes of the A-1759C, C-1683T, and A-1664G linkage loci of the AACS gene in the three varieties were determined by amplified PCR product sequencing technology. The test results are shown in Table 1.

[0064] Table 1 Genotype distribution of porcine AACS gene A-1759C, C-1683T, A-1664G sites

[0065]

[0066] Tibetan pigs have a stronger ability to deposit fat than introduced breeds, making them a fatty pig. Table 1 shows that the three loci, A-1759C, C-1683T, and A-1664G, are completely linked, forming two haplotypes, ACA and CTG, and three genotypes, ACA / ACA, ACA / CTG, and CTG / CTG. The frequency of the ACA / ACA genotype of the AACS gene in Tibetan pigs is significantly higher than in Yorkshire and Landrace pigs, with ACA being the dominant haplotype. In contrast, the dominant haplotype at this locus in breeds with slower fat deposition (Yorkshire and Landrace pigs) is CTG. Based on this, it is preliminarily determined that the ACA / ACA genotype in the 5' flanking region indicates a strong fat deposition ability, while the CTG / CTG genotype indicates a weak fat deposition ability. These results indicate that the genotype and allele distribution of this gene at these three linked loci differ significantly between pig populations with different fat deposition abilities, potentially implicating the fat deposition trait.

[0067] Example 5 Analysis of the association between AACS genotype and pig backfat thickness in the new Dingyuan pig strain population

[0068] The new Dingyuan pig strain is a hybrid bred from the local Chinese Dingyuan pig breed and the imported Berkshire pig. Ear tissue samples were collected from 104 Dingyuan pigs (collected from the Ankang Agriculture and Animal Husbandry Company in Dingyuan County, Anhui Province). Corrected backfat thickness (Equation 1) and age at 70 kg body weight (Equation 2) were measured and calculated for all individuals. Backfat thickness was measured using B-ultrasound scanning at the third to fourth intercostal space, measured in millimeters. This thickness was then corrected using the corrected backfat thickness formula to obtain the corrected backfat thickness.

[0069] Formula 1: Corrected backfat thickness = measured backfat thickness × [A ÷ {A + [B × (measured body weight – 70)]}]

[0070] Among them, the values ​​of A and B are: boar A = 12.826, B = 0.11437; sow A = 13.983, B = 0.12601. Formula 2: Age at reaching 70 kg body weight = actual measured age - (actual measured weight - 70) / (actual measured weight / actual measured age * C)

[0071] Among them, boar: C = 1.826040; sow: C = 1.714615

[0072] The genotypes of the A-1759C, C-1683T, and A-1664G sites of the AACS gene of 104 new Dingyuan pig strains were determined using the PCR product individual sequencing method established in Example 4. The results are shown in Table 2.

[0073] Table 2 Association analysis between AACS gene A-1759C, C-1683T, A-1664G sites and backfat thickness and growth traits

[0074]

[0075] Note: ACA / ACA, ACA / CTG, and CTG / CTG represent three genotypes. n represents the number of samples. Alternating letters indicate extremely significant differences (P<0.01), and identical letters indicate no significant differences (P>0.05). Values ​​in the table are mean ± standard error.

[0076] Among the three genotypes present at this locus in the new Dingyuan pig strain, the ACA / ACA genotype had a frequency of 32.7%, the ACA / CTG genotype had a frequency of 55.8%, and the CTG / CTG genotype had a frequency of 11.5%. Correlation analysis between the genotypes at this locus and backfat thickness in this population showed that the ACA / ACA and ACA / CTG genotypes had backfat thickness greater than that of the CTG / CTG genotype by more than 3 mm, a highly significant difference. Association analysis with the age at which the three genotypes reached 70 kg body weight showed that the age at which the three genotypes reached 70 kg was similar, but the difference did not reach significance. This indicates that ACA / ACA and ACA / CTG are molecular markers for rapid fat deposition and can be used in molecular marker-assisted breeding in pigs.

[0077] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for evaluating the fat deposition capacity of pigs, characterized in that The following steps are involved: 1) Extracting genomic DNA from the pig to be tested; 2) Using genomic DNA as a template, PCR amplification was performed using the primers shown in SEQ ID NOs: 2-3; 3) Analyze PCR amplification products; Step 3) includes sequencing the amplified product, and judging according to the sequencing results as follows: if the genotype of the polymorphic site at the 332 bp position of the amplified product is CC, the genotype of the polymorphic site at the 408 bp position is TT, and the genotype of the polymorphic site at the 427 bp position is GG, then it is judged that the fat deposition ability of the tested pig is weak.

2. The method according to claim 1, characterized in that The PCR reaction system was as follows: 2× PCR Mix 10 μl, genomic DNA 100 ng, 10 μmol / L forward and reverse primers 0.5 μl each, and filled to 20 μl with dd H2O; The PCR reaction program was as follows: pre-denaturation at 94-95°C for 5 min; denaturation at 94-95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 36 cycles; and extension at 72°C for 5 min.

3. Any of the following uses of a haplotype marker associated with porcine fat deposition, a primer for amplifying the haplotype marker, or a detection reagent or kit containing the primer: (1) Used to determine the fat deposition capacity of pigs; (2) Used for early prediction of pig fat deposition capacity; (3) Molecular marker-assisted breeding related to pig fat deposition ability; The haplotype marker associated with pig fat deposition comprises a nucleotide sequence of the pig AACS gene, wherein the polymorphism at the 332 bp position is A / C, the polymorphism at the 408 bp position is C / T, and the polymorphism at the 427 bp position is A / G, as shown in SEQ ID NO: 1; The genotype of the polymorphic site at bp 332 is AA or AC, and the corresponding pigs have a stronger fat deposition ability than the genotype CC; The genotype of the polymorphic site at 408 bp is CC or CT, and the corresponding pigs have a stronger fat deposition ability than the genotype TT; The genotype of the polymorphic site at 427bp is AA or AG, and the corresponding pigs have stronger fat deposition ability than the genotype GG.

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