KR20100132321A - Molecular markers and method for selection of the backfat thickness trait in pig using single nucleotide polymorphism of lepr gene - Google Patents

Abstract

PURPOSE: A method for selecting backfat thickness of a pig is provided to measure accurate genetic ability and to raise pig with high quality. CONSTITUTION: A SNP of leptin receptor gene(LEPR) is used as a molecular marker for evaluating phenotype of a pig. The SNPs are -200, -124, -25 or +161 loci mutant based on transcription initiation base(+1). The phenotype is backfat thickness of pig. A microarray for selecting the backfat thickness contains the molecular marker. A method for selecting backfat thickness of pig comprises: a step of obtaining a biological sample having a LEPR gene; a step of amplifying the LEPT gene by PCR; a step of detecting SNP from the PCR product; and a step of selecting SNP having significance in backfat thickness.

Description

Molecular markers and method for selection of the backfat thickness trait in pig using single nucleotide polymorphism of LEPR gene}

The present invention relates to a method for screening porcine backfat thickness, and more specifically, to select a single nucleotide polymorphism from the pig leptin receptor (LEPR) gene, and among them, a single nucleotide polymorphism having a significant correlation with the backfat thickness of the pig. It relates to a method for screening the thickness of the pig back fat using.

Information at the DNA level provides important information not only for producers, but also for breeders, in selecting specific key variants. DNA information can be used for selection of quantitative traits called marker help selection (MAS). Molecular markers can be used to improve the accuracy of selection, to select sex-limited traits, and to be useful for carcass traits such as meat quality. To date, several genes or markers have been used in the pig industry.

<Gene or markers currently used in pig industry>

Gene or Marker Related traits or fields of use Remarks Paternity - Non-exclusive use HAL Flesh ESR, PRLR, RBP4 Mountain embroidery Exclusive Use (PIC) KIT White hair MC1R Red / black hair MC4R Growth, obesity FUT1 Edema (E. Coli F18) Exclusive Use (PIC / ITH Switzerland) RN Flesh Non-exclusive use (Uppsala, INRA, Kiel) AFABP, HFABP Muscle fat Non Exclusive (IPG) PRKAG3 Flesh Exclusive (PIC) CAST year IGF2 Conductor composition Exclusive Use (Seghers)

Conventional methods for selecting species have been selected only by selection indexes based on phenotypes. Recently, however, it has been confirmed that marker help selection (MAS) can be effectively used in traits regulated by quantitative locus as well as traits regulated by a single gene.

As described above, many markers are also used for the improvement of pigs in important positions as livestock, and in particular, the backfat thickness has been recognized as one of the important factors for evaluating the quality of pigs.

Under the above technical background, the present inventors analyzed genes and gene mutations that regulate the quantitative trait loci (QTL) associated with intramuscular fat content of porcine chromosome 6 to identify biomarkers associated with intramuscular fat content. For the development, we selected the leptin receptor (LEPR) gene present in the quantitative locus as a candidate gene, and compared the DNA sequences corresponding to the transcriptional control region of the gene to find a single nucleotide polymorphism (SNP) and found the single nucleotide polymorphism. As a result of analyzing the association with the fat morphology, we discovered molecular weight factors by discovering the backfat thickness and the significant monobasic polymorphism.

Accordingly, it is an object of the present invention to provide a single base polymorph that can be used for screening the backfat thickness of a pig, a method for screening the backfat thickness of a pig using the same, and polynucleotides, microarrays and kits that can be used therein.

In order to achieve the above object, the present invention provides a molecular marker for evaluating the expression traits of pigs using Single Nucleotide Polymorphism (SNPs) of leptin receptor gene (LEPR).

In the molecular marker according to an embodiment of the present invention, the single nucleotide polymorphism (SNPs) is at least one selected from the group consisting of -200, -124, -25 and +161 based on the transcription start base (+1) It may be characterized by a mutation of the left position.

In the molecular marker according to an embodiment of the present invention, the phenotype of the animal may be characterized in that the back fat thickness of the pig.

According to another aspect of the invention, the polynucleotide constituting a part of the leptin receptor (LEPR) gene of the pig, 8 or more including the -200th position from the transcription start base (+1) of the leptin receptor (LEPR) gene Consecutive bases, at least 8 contiguous bases containing the -124 th position, at least 8 contiguous bases containing the -25 th position and at least 8 contiguous bases comprising the +161 th position and bases complementary to the bases; One or more polynucleotides selected from the group consisting of can be provided.

According to another aspect of the present invention, a microarray for porcine back fat thickness screening may be provided comprising the polynucleotide comprising the polynucleotide.

According to another aspect of the invention, there may be provided a pig back fat thickness screening kit comprising the polynucleotide.

According to another aspect of the invention, the step of obtaining a biological sample material comprising a leptin receptor (LEPR) gene from a pig; Amplifying the leptin receptor (LEPR) gene obtained from the sample; Searching for a single nucleotide polymorphism (SNP) from the amplification product of the leptin receptor (LEPR) gene; Selecting a single base polymorph having a statistically significant difference with the thickness of the pig back fat out of the single base polymorphs detected in the step; And it may be provided a method for screening the back fat thickness of the pig comprising the step of selecting the back fat thickness of the pig using the statistically significant single base polymorphism.

In the method for screening porcine backfat thickness according to an embodiment of the present invention, the single nucleotide polymorphism searched is -200th (A / G) from the start point of transcription of leptin receptor (LEPR) gene (+1),- And the 124th (C / T), -25th (A / C), and + 161th (G / C) digits.

In the method for screening porcine backfat thickness according to an embodiment of the present invention, one or more primers of SEQ ID NO: 1 to SEQ ID NO: 10 may be used for amplifying the leptin receptor (LEPR) gene. .

According to another aspect of the invention, alleles of the single nucleotide polymorphism from the start of transcription (+1) of the pig leptin receptor (LEPR) gene are -200, -124, -25 and +161 Methods may be provided for screening the backfat thickness of a pig using one or more single base polymorphisms selected from the group consisting of sites.

In the method for screening porcine back fat thickness according to an embodiment of the present invention, the -200th position of the single nucleotide polymorphism is G / G, the -124th position is T / T, and the -25th position is C / C and the + 161th position may be characterized as being C / C to be the pig with the thinnest back fat thickness.

According to the present invention, it is possible to measure the genetic ability 25-35% more accurately than the selection effect by the phenotypic evaluation, which can drastically increase the genetic capacity improvement of the sows, and the back fat thickness is one of the important factors for assessing meat quality. Therefore, it can greatly contribute to domestic fostering of excellent sows.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 PCR Amplification of Porcine LEPR Gene

Unlike the humans, the pig LEPR gene has separate exons 1 and 2, and the amplicon region of 2.072 kb, including exons 1 and 2 and the transcriptional regulatory region, can be amplified by 5 amplicons. Primers were prepared as shown in Table 1.

Primer set

Primer Sequence
Amplification Product Size (bp)
Annealing temperature
(℃) LEPRpro1
(SEQ ID NOs 1 and 2) Fw: 5'-GGATTCTATTGTGTTGTTTTACCTCA-3 '
Rv: 5'-AATGTCTCCCTCCCTGCTTC-3 ' 500 60 LEPRpro2
(SEQ ID NOs 3, 4) Fw: 5'-GATCGAACCCACAACCTCAT-3 '
Rv: 5'-CGTCCTTGAAGAGTCTAACTCCA-3 ' 528 60 LEPRpro3
(SEQ ID NOs 5, 6) Fw: 5'-TCTGCACACAGTAGGTCCTCA-3 '
Rv: 5'-CGGTCCTTTACCCAGCTGTA-3 ' 514 60 LEPRpro4
(SEQ ID NOs 7, 8) Fw: 5'-AGCATTTTGCACACCTGATG-3 '
Rv: 5'-ACCTTGGCAAAGGCAGAGAT-3 ' 562 60 LEPRpro5
(SEQ ID NOs: 9, 10) Fw: 5'-GGCCCTCCCTTCCTTTTATT-3 '
Rv: 5'-GTCGCGGTCCCTTTAGTTTC-3 ' 578 60

Four varieties with five traits (daily weight gain, feed requirement, back fat thickness, meat growth rate, selection index) obtained from the 2nd pig capacity test center belonging to the Korean Swine Association in order to confirm the genetic variation of the transcription control region. (Landrace, Duroc, Yorkshire, Berkshire) Genomic DNA was used from each of 40 heads of blood using the Wizard Genomic DNA Purification Kit (Promega, CA, USA). 2.5 μl of PCR reaction buffer (500 mM KCl; 100 mM Tris-Cl, pH8.2; 15 mM MgCl ₂ ; 0.1% Triton X-100, 5% DMSO), 2 μl of 2.5 mM dNTPs, 1 μl of 10 pmole primer , 25-50ng of genomic DNA, 0.2U of Hot-start Taq DNA polymerase (Genetbio) was added to the final volume of 25μ filled with sterile distilled water. PCR amplification reaction was 94 ℃ 10 minutes denaturation reaction after 94 ℃ 30 seconds, 60 ℃ 30 seconds, 72 ℃ 30 seconds amplification for 40 rotations and then performed the final extension reaction at 72 ℃ 10 minutes. After the PCR, 5 μl of the PCR product was subjected to electrophoresis on 1.5% agarose gel and stained with ethidium bromide to confirm amplification.

Example 2 Detection of Monobasic Polymorphism (SNP) from PCR Product of Porcine LEPR Gene

In order to discover a single base polymorphism in the transcriptional control region of the leptin receptor (LEPR) gene, the product amplified by PCR reaction was purified using a gene amplification product purification kit (PCR Purification Kit, CoreBio PPHTS-30), and then BigDye Terminator. Direct sequencing was performed using the Ver3.1 Cycle Sequencing Kit (Applied Biosystems) and ABI 3730XL DNA sequencer (Applied Biosystems).

The nucleotide sequence determined by individual was searched for single nucleotide polymorphism (SNP) in the DNA sequence through multi-alignment using SeqMan program (DNASTAR). Through this search, a total of 11 SNPs were identified.

Variance analysis on the association with the backfat thickness trait among the 11 monobasic polymorphisms was performed as -200A / G, -124C / T, -25A / C and Four monobasic polymorphs, +161 G / C, were selected. Table 3 below shows the results of analysis of the variance of the association between the pig leptin receptor (LEPR) gene transcription control region SNP and the backfat thickness trait using 160 heads of four varieties (L, D, Y, B).

Mean Square (Pr> F) df -200 A / G -124T / C -25A / C +161 G / C Breed 3 0.045 (0.1572) 0.052 (0.1218) 0.075 (0.0421) 0.066 (0.0574) Sex One 0.539 (<.0001) 0.541 (<.0001) 0.487 (<.0001) 0.349 (0.0004) SNP effect 2 0.174 ( 0.0016 ) 0.085 ( 0.0444 ) 0.104 ( 0.0231 ) 0.099 ( 0.0246 ) Test days One 0.027 (0.3058) 0.015 (0.4543) 0.028 (0.3068) 0.005 (0.6466)

Definitions and meanings of terms used in [Table 2] are as follows.

df (degree of freedom): The degree of freedom is calculated as “degree of freedom = number of levels of factor effects -1”. In the case of breed, the four degrees of freedom are investigated.

-Pr (Probability of Significance), F (F-Distribution Probability)

  : The probability value by Pr> F is based on the F-distribution analysis (ANOVA) in the statistical hypothesis test. Judging from the results of the survey, the results were statistically significant.

-SNP effect (Single Nucleotide Polymorphism)

 : The probability (Pr> F) of the distribution of the trait (backfat thickness) by the variation of the single base is statistically equal to that of the general variation (Pr> F). Judging from the difference in back fat thickness.

As shown in [Table 3], when the transcription initiation base was used as a reference (+1), -200A / G, -124C / T, -25A / C, and + 161G / C showed a single base polymorphism and statistically significant backfat thickness. Significance (P <0.05) was acknowledged.

In addition, as can be seen in [Table 4], in the case of GG at -200A / G, TT at -124C / T, CC at -25A / C and CC type at + 161G / C. Low backfat thickness trait. Table 4 shows the minimum donor mean and standard error of the backfat thickness for the transcriptional regulatory region SNP genotype of the pig leptin receptor (LEPR) gene.

-200 A / G -124T / C -25A / C +161 G / C AA 1.30 ± 0.033 ^b CC 1.31 ± 0.020 ^a AA 1.36 ± 0.027 ^a CC 1.26 ± 0.026 ^b AG 1.38 ± 0.023 ^a TC 1.36 ± 0.024 ^b AC 1.34 ± 0.022 ^a CG 1.36 ± 0.026 ^a GG 1.27 ± 0.021 ^b TT 1.26 ± 0.038 ^a CC 1.25 ± 0.029 ^b GG 1.33 ± 0.024 ^a

Allele frequencies for four varieties of Landrace, Duroc, Yorkshire, and Barkshire were examined. Table 4 shows the frequency of alleles by the single base polymorphism and variety identified in the transcriptional regulatory region of the pig leptin receptor (LEPR) gene.

-200 A / G -124C / T -25A / C +161 G / C Breed Genotype
Allele frequency Genotype
Allele frequency Genotype
Allele frequency Genotype
Allele frequency No AA AG GG A G No CC CT TT C T No AA AC CC A C No GG GC CC G C Landrace 37 4 12 21 0.27 0.73 36 21 12 3 0.75 0.25 36 11 11 14 0.46 0.54 37 13 12 12 0.51 0.49 Duroc 35 2 11 22 0.21 0.79 35 22 11 2 0.79 0.21 35 2 11 22 0.21 0.79 39 12 2 25 0.33 0.67 Yorkshire 39 11 22 6 0.56 0.44 39 16 17 6 0.63 0.37 39 12 22 5 0.59 0.41 36 17 12 7 0.64 0.36 Berkshire 40 8 16 16 0.40 0.60 40 16 15 9 0.59 0.41 39 18 19 2 0.71 0.29 39 8 24 7 0.51 0.49 Total 151 25 61 65 0.37 0.63 150 75 55 20 0.68 0.32 149 43 63 43 0.50 0.50 151 50 50 51 0.50 0.50

Of the four SNPs identified in the transcriptional regulatory region of the leptin receptor (LEPR) gene, the correlation between genotype and backfat thickness trait was analyzed for the four SNPs shown in FIG. Through the process, the model I was analyzed using the equation.

Model I: Y _{ijkl =} μ ₊ Breed _{i +} Sex _{j +} SNP _{k +} bx _{ijkl +} e _ijkl

Where Y _ijkl is the dorsal fat thickness of the first SNP genotype of the th sex of the th breed, μ is the overall mean, Breed _i is the breeding effect set as a fixed effect, and Sex _j is the sex effect (j = 1, 2) , SNP _k is the SNP genotyping effect, b is the regression coefficient for the test age, x _ijkl is the test date set to covariation, and e _ijkl means random error.

The LEPR gene, a receptor for leptin, known as the obesity gene in humans, is considered to be closely related to fat traits. Dogs have been found, and the single nucleotide polymorphisms found are effective as genetic DNA markers for assisted selection techniques for backfat thickness control.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a diagram showing the nucleotide sequence of the transcriptional regulatory region of the leptin receptor (LEPR) gene and the single nucleotide polymorphism (SNP) site.

<110> RURAL DEVELOPMENT ADMINISTRATION <120> Molecular markers and method for selection of the backfat          thickness trait in pig using single nucleotide polymorphism of          LEPR gene <130> NPF-14837 <160> 11 <170> KopatentIn 1.71 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro1 Forward Primer <400> 1 ggattctatt gtgttgtttt acctca 26 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro1 Reverse Primer <400> 2 aatgtctccc tccctgcttc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro2 Forward Primer <400> 3 gatcgaaccc acaacctcat 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro2 Reverse Primer <400> 4 cgtccttgaa gagtctaact cca 23 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro3 Forward Primer <400> 5 tctgcacaca gtaggtcctc a 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro3 Reverse Primer <400> 6 cggtccttta cccagctgta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro4 Forward Primer <400> 7 agcattttgc acacctgatg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro4 Reverse Primer <400> 8 accttggcaa aggcagagat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro5 Forward Primer <400> 9 ggccctccct tccttttatt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LEPRpro5 Reverse Primer <400> 10 gtcgcggtcc ctttagtttc 20 <210> 11 <211> 2072 <212> DNA <213> LEPR gene of Sus scrofa domesticus <400> 11 ggattctatt gtgttgtttt acctcacaat aacttgagga aattagtatt tctactccaa 60 ttctttgttt ccatccagtg ctggatggaa acaaagaatc ttgtctccag taaagtgaca 120 ctggccctgg gaggatactc ctttagggaa aaagttccat ttgctttttt tttttgtttt 180 gtttttttat cttttctagg gccacacccg tggcatatgg aggttcccag gctaggggtc 240 taattggagc tacagctgcc agcctatgcc acagccacag caacaccaga tccgagcctc 300 gtctgcaacc tacaccacag ctcacggcaa cgctggatcc ttatcccact gagtgaggcc 360 agggatcgaa cccacaacct catggttcct agtgggattc gttaacaact tagccacgat 420 cagaactccg aaaaagtacc atttgtacag ctccacaggt tggccaattg gctgggttgg 480 gaagcaggga gggagacatt gcttctgccc tgcccatctc tgggctacct tctctttatg 540 tctttgatgc ttcttcgcat aaggatgttg ggcaaattca tttttagaga cctttaaaag 600 ttaaccatcc acccaatcta gagcggtcca ctccccttta ttcttttttc cttaccggat 660 tggttccctt catatcaccc acaacaatgg ataattacat gaatatttgc ttgtcataac 720 accaggttca aggaacataa taaccacacc tagcatattg tctgcacaca gtaggtcctc 780 aataaaaatg tataaataaa tacacacata gataaatgaa tattttgagt agagagcaaa 840 aaaaaaataa tgcttactac tttgtctttg gagttagact cttcaaggac ggattttttt 900 ttttttttta acttactacg tcaaacgcct ttgaatttcc agattcaaag agaagtggtc 960 atttagttca acccttcatt agaagcatga gtgccttctg cagctttcct gacaagcatg 1020 acccaaaatg ctcggctggg tgaaacatcc ttggtgacag gaaactcact acctcactac 1080 ttctggtctt tttcatttag ctttccttcc cactcccggt tattgcattc tgtattgcag 1140 cattttgcac acctgatgca gcaggagcct ggaggtctag aagtgggagg cgcagaggga 1200 acccaatgcc cctcctaggt ctgagactcc ggctcagggt gggcgcgcct cgcctacagc 1260 tgggtaaagg accggcgcca ccttccctct gggttcgcct ttattctcgg cttccctgcg 1320 gacaaggggt cctccttccc aaacgcctcg catttcctcc cccaggcccg ctgccacgcg 1380 cagccgcatc cctgccgccg ccccagtcac ctgcgtggcc acccggcggc tcaggggccg 1440 ctccttccgc cccgggagtg gtggcggcgg gcgcggggcg cgcgggaggc cacccggccc 1500 tcccttcctt ttattctctc ccctccgctc cccctccgcc cctcctccca cactctgccg 1560 ccttcgctct cctcagagtt gcctggcacc ttccgcgacg agtttggagc cgccccaccg 1620 cagagcccac ggcccgccga gcacgagcga cgcaggtgcc cgagcccccg cgccgccgcc 1680 atctctgcct ttgccaaggt gggaccccgg cctcaaggtg ggacccagct ctccccgggg 1740 ccccttactt cccccttttc ccgctcccgg ccacgcgagg agccgaacgc cgcggctgga 1800 gcccgcgggc cacctgggga ccaggaggcc ggggccgctt gggcggcgtc accggagggg 1860 cccgtgcctg gcgggagggg cggcgggggc tctcggctcc ccggagccag gctctcggga 1920 ggcccccggg gcgaggagca tcagggcgcg cggtccaggg ccgccagcct agccttggcg 1980 cgcacctgcc gggcgccccc gaaaggacgt cccatctggc caacttcttt tccaggtctt 2040 cttgctactt tggaaactaa agggaccgcg ac 2072  

Claims (11)

Molecular marker that evaluates the expression traits of pigs using Single Nucleotide Polymorphism (SNPs) of leptin receptor gene (LEPR). The method of claim 1, The single nucleotide polymorphisms (SNPs) is a molecular marker, characterized in that the mutation of one or more loci selected from the group consisting of -200, -124, -25 and +161 based on the transcription start base (+1). The method of claim 1, The phenotype of the animal is a molecular marker, characterized in that the back fat thickness of the pig. Polynucleotide constituting a part of the leptin receptor (LEPR) gene of the pig, 8 or more consecutive bases, including the -200 position from the transcription start base (+1) of the leptin receptor (LEPR) gene, the -124 position One or two selected from the group consisting of at least 8 consecutive bases comprising, at least 8 consecutive bases comprising the -25th site and at least 8 consecutive bases comprising the + 161th site and bases complementary to the bases Or more polynucleotides. Pig back fat thickness screening microarray comprising the polynucleotide comprising the polynucleotide according to claim 4. Pig back fat thickness screening kit comprising the polynucleotide according to claim 4. Obtaining a biological sample material comprising leptin receptor (LEPR) gene from a pig; Amplifying the leptin receptor (LEPR) gene obtained from the sample; Searching for a single nucleotide polymorphism (SNP) from the amplification product of the leptin receptor (LEPR) gene; Selecting a single nucleotide polymorphism having a statistical significance with the pig isofat thickness among the single nucleotide polymorphisms detected in the step; And Screening the thickness of porcine back fat using the statistically significant single base polymorphism Pig back fat thickness screening method comprising a. The method of claim 7, wherein the single nucleotide polymorphism searched is -200th (A / G), -124th (C / T), -25th (from transcription initiation point (+1) of the leptin receptor (LEPR) gene) A / C) and + 161st (G / C) positions for screening porcine back fat thickness. The method of claim 7, wherein at least one primer of SEQ ID NO: 1 to SEQ ID NO: 10 is used for amplification of the leptin receptor (LEPR) gene. One or more single alleles of the single base polymorphism from the start of transcription (+1) of the pig leptin receptor (LEPR) gene are selected from the group consisting of -200, -124, -25 and +161 A method of screening the thickness of the back fat of a pig using a base polymorphism. 11. The method of claim 10, wherein the -200th position of the single nucleotide polymorphism is G / G, the -124th position is T / T, -25th position is C / C, +161 is the C / C A method for screening the thickness of the back fat of a pig, characterized in that it is determined that the back fat is the thinnest pig.

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