CN107022603B - Molecular marker for pig backfat thickness character and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of livestock molecular marker preparation, and particularly relates to a molecular marker for pig backfat thickness traits and application thereof. Two molecular markers related to the pig backfat thickness property are obtained by cloning. One of the molecular markers is ACOXL c.8C/T, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO: 1, an allelic mutation (C/T) occurs at base 290 of the sequence, which results in a threonine to isoleucine change. Another molecular marker is LMNA c.741G/C, an allelic mutation (G/C) occurs at base 341 of the sequence, which mutation causes a change from glutamate to aspartate. The molecular marker can be used for detecting polymorphism of pig backfat thickness related traits.
Description
Technical Field
The invention belongs to the technical field of livestock molecular marker preparation, and particularly relates to a molecular marker for pig backfat thickness traits and application thereof. The invention comprises a method for discovering and detecting ACOXL and LMNA gene variation sites related to the pig backfat thickness character and application thereof in the pig backfat thickness character.
Background
In recent years, the introduced breeding pigs including Duroc, Changbai and Dabai occupy the lean pork market in China, and the introduced varieties still need to be continuously measured and selected to keep the excellent characteristics of the pigs without degradation. Growth traits and carcass traits have been the target traits for breeding improvement, and development of early selection by molecular marker assisted selection has been achieved in recent years in addition to improvement by classical BLUP breeding methods. Candidate genes are screened based on the result of Genome-wide association analysis (GWAS), then the effect of the candidate genes is verified in different populations, and a foundation is laid for searching effective molecular markers in different populations and implementing molecular marker-assisted selection.
The backfat thickness is an important carcass trait, is negatively related to the lean meat percentage of the pig, has high genetic strength, is easy to measure and the like, and is often used as an indirect trait for improving the lean meat percentage to be used for genetic improvement of the lean meat pig. Meanwhile, the genetic basis of the pig meat production traits is discussed, and BFT reduction also becomes the research focus of pig carcass trait genetic improvement. According to data of the canadian pig improvement center (CCSI), BFT at 100kg body weight was 13.6mm in 1994 and 10.6mm in 2014, the total thinning was 22%, and the annual genetic progress was-0.2 mm/year from 1994 to 2014; the day-old of 100kg body weight was 166 days in 1994 and 147 days in 2014, which were shortened by 12% in total, and the annual genetic progress was-1 day/year.
acyl-CoA oxidase-like genes (ACOXL) encode acyl-CoA oxidases, which are members of the acyl-CoA oxidase family, and are first found in castor bean endosperm in Cooper, 1969 (Cooper T, Bevers H. beta. oxidation in microorganisms from castor bean chemistry, 1969,244: 3514-3520). Sandra et al, when studying type II diabetes in African Americans, discovered that ACOXL may be a candidate gene associated with type II diabetes by genome-wide scanning of susceptibility genes in 5 chromosomal regions of type II diabetes, and that ACOXL is involved in lipid metabolism (Hasstedt SJ, Highland HM, Elbein SC, Hanis CL, Das SK. five linkage region ear hardbuilding type 2diabetes genes in the African American subset of the GEN ID Study. journal of human genetics.2013,58: 378-383). Suneel et al performed genome-wide association analysis with 1433 big white pigs and found the region MARC0085867-ALGA0018683 on SSC3 associated with backfat thickness at the tenth rib and ACOXL as candidate genes for associated backfat thickness in this region (Onteru SK, Gorbach DM, Young JM, Garrrick DJ, Dekkers JC, Rothschild MF. wheel genome association expressed meal and related trails in the pig. plos one.2013,8: e 61756).
The LMNA gene encodes a nuclear Lamin type A (Lamin A/C), which is divided into two subunits, Lamin A and Lamin C, which are encoded by the same transcription unit, but which are selectively cleaved to generate 2 different transcripts which are then translated into two different subunits. A study by Prigogine et al on the Oji-Creer and Inuit population found that LMNA 1908C/T, a common variation of LMNA, is an important factor in the obesity-related trait associated with metabolic syndrome (Prigogine C, Richard P, Van den Bergh P, Groswasser J, deconck N. novel LMNAmutation presenting as segment genetic multiple, Pediatricneurology 2010. 43: 283) -286). Brors found by gene scanning of Pima Indian that the chromosomal lq21-23 region where the LMNA gene is located is abnormal and is associated with obesity and diabetes (Broers J, Ramaekers F, Bonne G, YaouRB, Hutchison C.Nucleams: vitamins and the same role in preprocessing. physical reviews.2006,86: 967-. Choi et al studied QTLs associated with fat deposition in the region of about 89cM of SSC4 in Korean pigs grown in white, F2 generation population, and compared pig and human maps in this region to use LMNA as potential candidates for backfat thickness (Choi BH, Lee JS, Lee SH, Kim SC, KimSW, Kim KS, Lee JH, Seong HH, Kim TH. Portine LMNA is a positional candidate gene associated with fatty growth and fat deposition. Asian-Australian j ournal of animal science.2012, 25: 1649).
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a molecular marker for cloning and detecting the pig backfat thickness character and application thereof. According to known sequences of ACOXL and LMNA genes, a mixed pool sequencing and PCR-RFLP method is utilized to find SNP sites in the two genes which are related to the backfat thickness trait at the tenth rib of the pig, and the SNP sites are subjected to correlation analysis with the target trait, so that two molecular markers related to the backfat thickness trait are obtained, and a new molecular marker resource is provided for marker-assisted selection of the pig.
The invention is realized by the following technical scheme:
firstly, re-sequencing data of 14 big white pigs and 4 long white pigs are utilized to analyze and obtain possible SNP sites of the ACOXL gene and the LMNA gene. Then cloning the exon sequence of the SNP, performing mixed pool sequencing and PCR-RFLP to perform enzyme cutting and typing, and finally obtaining the SNP. The ACOXL c.8C/T is positioned on the first exon, and the base mutation is shown in figure 2, so that the change from threonine to isoleucine is caused. LMNA c.741G/C, located on the fourth exon, and the base mutation, as shown in FIG. 3, caused a glutamic acid to aspartic acid change.
A molecular marker ACOXL c.8C/T for the pig backfat thickness property has a nucleotide sequence shown as follows:
AGGGATGTACTCACATCATATGGAAGTTCCCAGGCTAGGGGTCGAATCAGAGCTGCAGCTGCCAGCCTGCACAATAGCCACAGCAATGCAGGATCTGAGCCTTGTCTGTGACCTACACCACAGTTCTTTAACCCACTGAGTGAAGCCAGGGATCGAACCCACAACCTCATGGTTCCTAGTCAGATTTGTTTCCGCTGTGCCAAGATGGGAACTCCCTCAATCAAACATCTTAACACCCACTTTGTATCCATTCATCACAGGTATGATTTAAGTCTGGATGAAATGAGAAR(C/T)CTGACACTTCAGAGAGTGAAGTTTACCATGGGCCTACCTTTGTTAAAGCGTGCAATTCAGGAACAGGTAAGGTTCATTGT TATTTTGGTGTGTGTGAGTGAATGAGAGAGAGACAGAGACAGAGGGAGCAAGAAGATTTCGTTTTGCTTGTTTTTGTGACTTGTGGAAGGAAGCTCATGAATTTTGTCCTGTGGTGTGGGGGAGGGCCTCCTTCACCCTCCCTTTCTGCCCCCCCCAATATAATTGACATTCACTTTTCCCAAGCCCACAGGCTTAGGAATAAGGGAGTCAACAGAGGTAATGAAATATTTGTTCACAAAACAAATGTAATTTGGGGTATAAAATGCAAATATCTGAGAAATGTAATGACATCACTTTATAGGCTCTTCAAGAATTTTAAACTTGCCCC
r at base 290 of the above sequence is C or T (i.e., base C is replaced with base T), and this mutation results in a threonine to isoleucine change.
A molecular marker LMNA c.741G/C for the pig backfat thickness character has a nucleotide sequence shown as follows:
GTCAGAGCTGGGGCTGGGACATTGGCTGTGTGCAGAGCTCCCCTGCCTGACTCCCTTGTACTAGTGGATGGGGAGTTGGGTCTGGGGGGACGGGGAGTGGCCAGCCCTCAGGTTAAAGGGGGGCTCACAGTGGCTCCATTCGCGGTTAGGATTGGGTCGGGAGCTCAGCCACCTGCCTGGGTCCCATCCTCAGAGGACTAGTTCTGATTTTGGTTTCTGGGTCCAACCCTTCCAGGAGCTTCGGGAGACCAAGCGCCGCCATGAGACGCGGCTGGTGGAGATTGATAATGGGAAGCAGCGCGAGTTTGAGAGCCGGCCGGCTGGCAGATGCCCTGCAGGAR(G/C)
TGCGGGCCCAGCACGAGGACCAGGTGGTGCTCGCTCTCCTGTGGCTCCCTCGCTGCCTCTGACCCTGGCACCCCTCCCCCCACCTCTGCCACCCTGATACGTCCCTTGCGGGATCGGGTGGATGATAGCAGGAGCCCCGGGTGCCCAGGACCTGAGGCTGCAGCAGAGATGCCGTTCCCAGGTCCCTTCCGGCCCCTGCATCCCTAACCCCGCGTCTTCCCCTCCAG
the mutation, wherein R at base 341 of the above sequence is G or C (i.e., the base G is substituted for the base C), causes a change from glutamic acid to aspartic acid.
The applicant prepared a primer pair for amplifying the first exon sequence of ACOXL in which ACOXL c.8C/T is located and the fourth exon sequence of LMNA in which LMNAc.741G/C is located, and the nucleotide sequences of the primer pair are shown as follows:
(1) the first exon sequence of the ACOXL (also used as a primer for enzyme digestion) in which the ACOXL c.8C/T is positioned is amplified as follows: the amplified fragment is 501bp, the annealing temperature is 61 DEG C
A forward primer: CAGGATCTGAGCCTTGTCTGTG
Reverse primer: CCTCTGTTGACTCCCTTATTCCTA
(2) The primers for amplifying the fourth exon sequence of the LMNA in which the LMNA c.741G/C is located are as follows:
and (3) amplifying fragments: 855bp, annealing temperature: 60 deg.C
A forward primer: CCCCTGGACCTGTTTGTG
Reverse primer: CCTGGGAACGGCATCTCT
(3) LMNA c.741G/C the primers used in PCR-RFLP were as follows:
length of amplified fragment: 441bp, annealing temperature: 59 deg.C
A forward primer: TTCTCCTGAACGTGTCTGGATTACCACTGTTGAGAGCCGGCTGGCA
GATGCCCTGCAGCA
Reverse primer: CTGAGCTGGGCCGAGAGGCTGTCGATG
The molecular marker of the invention can be applied to the detection of the pig backfat thickness character
Drawings
FIG. 1: is a technical flow chart of the present invention.
FIG. 2: the ACOXL c.8C/T is located in the ACOXL first exon sequence. Mutations of this allele are shown in parentheses.
FIG. 3: results of mixed pool sequencing of ACOXL c.8C/T.
FIG. 4: ACOXL C.8C/T enzyme cutting typing glue picture.
FIG. 5: the fourth exon of LMNA where LMNA c.741G/C is located. Mutations of this allele are shown in parentheses.
FIG. 6: the result of mixed pool sequencing of LMNA c.741G/C.
FIG. 7: an enzyme digestion typing glue map of LMNA c.741G/C.
Detailed Description
Example 1
Screening of SNPs of ACOXL and LMNA genes
1. Primer design
Based on the possible SNP sites analyzed by the re-sequencing data, primers were designed to clone the first exon of the ACOXL gene (gene ID:100520554) and the fourth exon of the LMNA gene (gene ID:100126859) for mixed-pool sequencing, and the sequences of specific primers for amplifying the genes were as follows:
amplifying the ACOXL first exon sequence in which the ACOXL c.8C/T is positioned:
a forward primer: CAGGATCTGAGCCTTGTCTGTG
Reverse primer: CCTCTGTTGACTCCCTTATTCCTA
Amplifying the fourth exon sequence of the LMNA in which the LMNA c.741G/C is located:
a forward primer: CCCCTGGACCTGTTTGTG
Reverse primer: CCTGGGAACGGCATCTCT
PCR amplification conditions
The sample genes were obtained from DNA extracted from white pig ear-like and white pig semen (samples from laboratory pig farm of agriculture university in Huazhong, Wuhan city, Hubei province) by conventional methods, and the DNA samples used for the PCR procedure were each diluted to 20 ng/. mu.L. The primer concentration was 10uM, purchased from Wuhan Dynasty scientific and creative Biotech Co., Ltd. MIX was 2 XPCR MIX, purchased from Erdela biotech, Beijing, loading buffer, configured by itself (4.4 g EDTA, 0.25g xylenecyanine FF, 0.25g bromophenol blue was weighed out and dissolved in 200ml ddH2O, heated and stirred to dissolve, after addition of 180ml Glycerol, pH7.0 was adjusted with NaOH, ddH2O was brought to a constant volume of 500ml and stored at-20 ℃), at a concentration of 6 XO.
10 uL of ACOXL c.8C/T PCR system, 5 uL of 2 XPCR MIX, 3.6 uL of sterilized water, 10uMPF and PR0.2 uL, and 1 uL of 50 ng/. mu.L DNA sample. Pre-denaturation at 95 ℃ for 5 min, 35 cycles (95 ℃ 30s, 61 ℃ 30s, 72 ℃ 30s), extension at 72 ℃ for 5 min. The resulting product was 501bp in length and detected by 1.8% agarose gel electrophoresis.
LMNA c.741G/C10 uL PCR system, 5 uL 2 XPCR MIX, 3.6 uL sterilized water, 10uMPF and PR0.2 uL, and 50 ng/. mu.L DNA sample 1 uL. Pre-denaturation at 95 ℃ for 5 min, 35 cycles (95 ℃ 30s, 60 ℃ 52s, 72 ℃ 30s), extension at 72 ℃ for 5 min. The resulting product was 855bp in length and detected by electrophoresis on a 1.2% agarose gel.
Then, 10 samples were randomly selected and mixed in a block to be sent to a sequencing company for sequencing, namely, mixed pool sequencing. The sequencing results were viewed and edited with the software SeqMan.
ACOXL and LMNA Gene PCR-RFLP
(1) Primer sequence (design primer for enzyme cleavage typing)
ACOXL c.8C/T:
A forward primer: CAGGATCTGAGCCTTGTCTGTG
Reverse primer: CCTCTGTTGACTCCCTTATTCCTA
The primers used for the enzyme digestion of LMNA c.741G/C are as follows:
a forward primer: TTCTCCTGAACGTGTCTGGATTACCACTGTTGAGAGCCGGCTGGCA
GATGCCCTGCAGCA
Reverse primer: CTGAGCTGGGCCGAGAGGCTGTCGATG
(2) PCR amplification conditions
ACOXL c.8C/T: mu.L of PCR system, 5. mu.L of 2 XPCR MIX, 3.6. mu.L of sterilized water, 0.2. mu.L of 10uMPF and PR, and 1. mu.L of 50 ng/. mu.L DNA sample. Pre-denaturation at 95 ℃ for 5 min, 35 cycles (95 ℃ 30s, 61 ℃ 30s, 72 ℃ 30s), extension at 72 ℃ for 5 min. The length of the amplified product is 501 bp. Detection was performed by 1.8% agarose gel electrophoresis.
LMNA c.741G/C: mu.L of PCR system, 5. mu.L of 2 XPCR MIX, 3.6. mu.L of sterilized water, 0.2. mu.L of 10uMPF and PR, and 1. mu.L of 50 ng/. mu.L DNA sample. Pre-denaturation at 95 ℃ for 5 min, 35 cycles (95 ℃ 30s, 59 ℃ 30s, 72 ℃ 30s), extension at 72 ℃ for 5 min. The length of the amplified product was 441 bp. Detection was performed by 1.8% agarose gel electrophoresis.
(3) PCR-RFLP detection conditions
Enzyme digestion system: mu.L system, 0.1. mu.L endonuclease, 1. mu.L 10 Xbuffer, 3.9. mu.L sterilized water, and 5. mu.L PCR product. After mixing and centrifugation of the samples, they were digested overnight (about 12h) in an incubator at 37 ℃. After digestion, 5. mu.L of the product was electrophoresed on 1.8% agarose gel at low pressure for a long time (105V, 1h)
ACOXL C.8C/T three genotypes: CC type (501bp), CT type (501bp, 300bp, 201bp), TT type (300bp, 201 bp). The endonuclease used was EcoR I.
Three genotypes of LMNA c.741G/C: CC type (331bp, 110bp), CG type (331bp, 271bp, 110bp, 60bp), and GG type (271bp, 110bp, 60 bp). The endonuclease used was Pvu II.
(4) Correlation analysis of molecular marker and target trait
The test takes 369 big white pigs and 157 long white pigs as the research objects. Using mixed linear models
TABLE 1 results of association between C.8C/T site of ACOXL gene of large and white pig and 3 personality
Description of table 1: p < 0.05, p < 0.01; note: mean p < 0.05, mean p < 0.01.
LMNA c.741G/C was significantly associated with backfat thickness in both large white pigs and long white pigs (p < 0.05). Among them, there are 90 individuals of type CC, 184 individuals of type CG and 76 individuals of type GG in the large white pig. The CC type is obviously different from CG type and GG type (p is less than 0.05), and the CC type is the dominant genotype. In the long and white pigs, there were 90 individuals for type CC, 43 individuals for type CG and 8 individuals for type GG. The CC type is obviously different from CG type and GG type (p is less than 0.05), and the CG type is the dominant genotype.
TABLE 2 correlation results of LMNA gene c.741G/C site of large white pig and 3 personality
Description of table 2: p < 0.05, p < 0.01; note: mean p < 0.05, mean p < 0.01.
TABLE 3 correlation results of LMNA gene c.741G/C site of Changbai pig and 3 personality
Description of table 3: p < 0.05, p < 0.01; note: 0.05, 0.01.
Claims (1)
1. The application of the molecular marker in the detection of the pig backfat thickness trait is characterized in that the nucleotide sequence of the molecular marker is one of the following sequences:
AGGGATGTACTCACATCATATGGAAGTTCCCAGGCTAGGGGTCGAATCAGAGCTGCAGCTGCCAGCCTGCACAATAGCCACAGCAATGCAGGATCTGAGCCTTGTCTGTGACCTACACCACAGTTCTTTAACCCACTGAGTGAAGCCAGGGATCGAACCCACAACCTCATGGTTCCTAGTCAGATTTGTTTCCGCTGTGCCAAGATGGGAACTCCCTCAATCAAACATCTTAACACCCACTTTGTATCCATTCATCACAGGTATGATTTAAGTCTGGATGAAATGAGAAR(C/T)
TCTGACACTTCAGAGAGTGAAGTTTACCATGGGCCTACCTTTGTTAAAGCGTGCAATTCAGGAACAGGTAAGGTTCATTGTTATTTTGGTGTGTGTGAGTGAATGAGAGAGAGACAGAGACAGAGGGAGCAAGAAGATTTCGTTTTGCTTGTTTTTGTGACTTGTGGAAGGAAGCTCATGAATTTTGTCCTGTGGTGTGGGGGAGGGCCTCCTTCACCCTCCCTTTCTGCCCCCCCCAATATAATTGACATTCACTTTTCCCAAGCCCACAGGCTTAGGAATAAGGGAGTCAACAGAGGTAATGAAATATTTGTTCACAAAACAAATGTAATTTGGGGTATAAAATGCAAATATCTGAGAAATGTAATGACATCACTTTATAGGCTCTTCAAGAATTTTAAACTTGCCCC
r at base 290 of the sequence is C or T, the mutation causing a threonine to isoleucine change;
or
GTCAGAGCTGGGGCTGGGACATTGGCTGTGTGCAGAGCTCCCCTGCCTGACTCCCTTGTACTAGTGGATGGGGAGTTGGGTCTGGGGGGACGGGGAGTGGCCAGCCCTCAGGTTAAAGGGGGGCTCACAGTGGCTCCATTCGCGGTTAGGATTGGGTCGGGAGCTCAGCCACCTGCCTGGGTCCCATCCTCAGAGGACTAGTTCTGATTTTGGTTTCTGGGTCCAACCCTTCCAGGAGCTTCGGGAGACCAAGCGCCGCCATGAGACGCGGCTGGTGGAGATTGATAATGGGAAGCAGCGCGAGTTTGAGAGCCGGCCGGCTGGCAGATGCCCTGCAGGA R(G/C)
CTGCGGGCCCAGCACGAGGACCAGGTGGTGCTCGCTCTCCTGTGGCTCCCTCGCTGCCTCTGACCCTGGCACCCCTCCCCCCACCTCTGCCACCCTGATACGTCCCTTGCGGGATCGGGTGGATGATAGCAGGAGCCCCGGGTGCCCAGGACCTGAGGCTGCAGCAGAGATGCCGTTCCCAGGTCCCTTCCGGCCCCTGCATCCCTAACCCCGCGTCTTCCCCTCCAG
R at base 341 of the sequence is G or C, and the mutation results in a glutamic acid to aspartic acid change.
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