AU2020101763A4 - SNP Genetic Marker of Fat Deposition Traits in Pigs and Its Application - Google Patents

Abstract

The invention belongs to the technical field of livestock genetic marker preparation. In particular, the invention relates to a SNP genetic marker for porcine fat deposition traits and its application. A gene fragment of porcine fat deposition traits used as a genetic marker is obtained from porcine OLR1 gene. Its nucleotide sequence is shown in SEQ ID NO: 1 and FIG. 4. There is a A/G base mutation at 181 bp of the sequence shown in FIG. 4, which leads to Pag I-RFLP polymorphism. The invention also discloses a method for preparing a genetic marker of pig fat deposition traits and an application of the prepared genetic marker in detection of polymorphism of pig fat deposition traits, providing a new marker for marker assisted selection of pigs. -1/4 BigWhite TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Landrace TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Qingping TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Meishan TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 BigWhite TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Landrace TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Qingping TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Meishan TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Big White GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Landrace GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Qingping GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Meishan GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 BigWhite GrGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Landrace GrGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Qingping ATGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Meishan ATGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Big White ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Landrace ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Qingping ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Meishan ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 BigWhite TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Landrace TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Qingping TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Meishan TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Fig. 1A - continued in Fig. lB

Description

-1/4

BigWhite TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Landrace TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Qingping TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Meishan TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60

BigWhite TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Landrace TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Qingping TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Meishan TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120

Big White GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Landrace GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Qingping GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Meishan GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180

BigWhite GrGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Landrace GrGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Qingping ATGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Meishan ATGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240

Big White ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Landrace ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Qingping ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Meishan ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300

BigWhite TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Landrace TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Qingping TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Meishan TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360

Fig. 1A - continued in Fig. lB

AUSTRALIA

PATENTS ACT 1990

PATENT SPECIFICATION FOR THE INVENTION ENTITLED:

SNP Genetic Marker of Fat Deposition Traits in Pigs and Its Application

The invention is described in the following statement:-

SNP Genetic Marker of Fat Deposition Traits in Pigs and Its Application

TECHNICAL FIELD

[0001] The invention belongs to the technical field of preparing molecular markers for

pigs, in particular to an SNP genetic marker for pig fat deposition traits and its

application. The genetic marker is obtained from porcine OLR1 gene.

BACKGROUND

[0002] With the development of molecular biology technology, molecular breeding

technologies such as molecular marker-assisted selection and molecular marker assisted

introgression have gradually emerged. The combination of these technologies and

conventional breeding technologies has greatly accelerated the process of pig breeding.

Molecular marker-assisted selection can quickly and accurately analyze the genetic

composition of individuals at the molecular level. So as to realize direct selection of

genotypes and carry out molecular breeding, which is an important application of genetic

engineering in modem livestock breeding. The molecular marker method can not only

shorten the breeding time limit, but also reduce the human and material resources. In

addition, the diversity of molecular markers has greatly improved the application

potential of molecular markers in animal breeding (Lu Shaoxiong, Wu Changxin., 2000).

Molecular markers used for assisted selection include protein markers, microsatellite

markers, single nucleotide polymorphism (SNP) markers, etc.

[0003] SNP marker refers to DNA sequence polymorphism caused by genomic single

nucleotide variation, including base conversion, transversion, single base insertion or deletion, etc., and which is recognized as the latest third generation DNA molecular markers. It is abundant in number and polymorphism. The detection methods include

PCR-SSCP, PCR-RFLP, sequencing and SNP chip. Restriction Fragment Length

Polymorphism, RFLP (RFLP) is based on the mutation of bases at the restriction sites of

the genomes of different varieties (individuals), or the insertion or deletion of bases

between the restriction sites, resulting in changes in the size of enzyme fragments. This

change can be detected by agarose electrophoresis, thus comparing the differences (i.e.

Polymorphism) in DNA levels of different varieties (individuals) (BeuzenN. D, et al.

2000).

[0004] Oxidized low-density lipoprotein receptor 1 ( oxidized low-density lipoprotein

receptors , OLR1) is a kind of receptors that can bind oxidized low-density lipoprotein

(oxLDL). Sawamura et al. (1997) first discovered the OLR1 gene in bovine endothelial

cells and successfully cloned the OLR1 gene in human endothelial cells. Subsequently,

the cDNA sequences of OLR1 genes in rats, mice and rabbits were successively cloned

(Miki et al., 1998; Kume et al., 1998). The amino acid sequences of OLR1 gene in these

species are similar. The porcine OLR1 gene has 5 introns and 6 exons, encoding 274

amino acids. The similarity between porcine OLR1 gene and human OLR1 gene is 79%,

and the similarity between porcine OLR1 gene and bovine OLR1 gene is the highest,

reaching 84% (Yamanaka et al., 1998; Ming et al., 2001). OLR1 gene is widely believed

to be closely related to the formation of cardiovascular diseases and plays an important

role in fat metabolism. Patricia et al. (2005) studied the role of OLR1 in adipocytes for

the first time, proving that OLR1 plays an important role in regulating lipid metabolism

and thus potentially regulating insulin resistance with the participation of PPAR and its related antidiabetic ligands. Juwon A et al. (2007) proved that extracellular signal regulated kinase (ERK) and c-Jun amino terminal kinase (JNK) pathways are important pathways for OLR1 to play a role in adipocytes. Studies by Sun Chao et al. (2009) have shown that OLRl gene plays a role through P38MAPK pathway during the differentiation of mouse precursor adipocytes and has certain effect on inhibiting adipocyte proliferation. Li Tuo (2013) analyzed the correlation between OLR1 gene genetic polymorphism and meat quality traits in Qinchuan cattle population, and found that there was a C > A mutation at g.10497 locus, and CC homozygous genotype was significantly correlated with loin eye area and loin eye depth of Qinchuan cattle. At present, the research of OLR1 gene mainly focuses on human, mouse, rat, cattle and other species, and there are few reports on the research of pig OLR1 gene. The applicant of this invention has carried out polymorphism research and association analysis on the gene in pigs, which provides important theoretical basis for genetic improvement of pigs.

SUMMARY

[0005] The object of the invention is to obtain SNP molecular markers related to fat

deposition characteristics and their applications. A specific DNA fragment was cloned

from porcine OLR1 gene, found SNP, and corresponding SNP detection methods were

established to analyze the relationship between SNP and porcine fat deposition traits, thus

providing a new molecular marker for marker-assisted selection in porcine.

The invention was performed by the following technical protocols:

The applicant discovered an SNP molecular marker related to pig fat deposition traits through isolation and cloning of pig OLRl gene, and its nucleotide sequence is as follows:

TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTT TAGTCTGGCCTGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGG TGGGTCTCTAGGCTCCAGCAGGCATGTCTATAAAACCATCCAGAAGATGGCA AAGATCCCTTTGGTTCTGTGGCTTCR (A/G) TGAATCTAATCTAAGATGTTCA CATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCTACAGGAATTCATCCAG CAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATCTCTGAGGAA ACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTTGT AAGTTTCCTATTCTTTTCCTAATCTGCTGGTGAAGTTACTGCCGCATTCCGCAG AGTGTCCTCTTCCTGCTGGAAAGAACCCTGGGAAATTTCTGAATCTCTCTCCT CAGCCCTCTCATGCAGAGGCTTGCCCCTTGACTCATTCAGACCTTCTCTTGAG TTTTCCTTCTTACATTTATTACTGATTTGAAGCTTTAAGACAAGGCCAAGTGC CTGACCCAAGGTAG

[0006]

[0007] R at 181bp in the above sequence is A or G, and this mutation leads to Pag I

RFLP polymorphism.

The Applicant of this invention designed a primer pair for amplifying the above

mentioned porcine OLR1 gene fragment (this primer pair is also a primer pair for

detecting the molecular marker of the present invention), the DNA sequence of which is

as follows:

Forward primer: 5 'TCCTCGGTGGAAGAGCAT3', corresponding to the sequence

shown in SEQ ID NO: 4.

Reverse primer: 5 'CTACCTTGGGTCAGGCAC3'; corresponding to the sequence of

SEQ ID NO: 5.

[0008] The invention establishes a method for screening molecular markers related to pig

fat deposition traits, which comprises the following specific steps:

Extracting porcine genomic DNA, according to the sequence information of porcine

OLRl gene published in NCBI, PCR amplification primer pair were designed (that

nucleotide sequence of which is shown in the sequence table SEQ ID NO: 4 and SEQ ID

NO: 5), PCR amplification is performed with this pair of primers, obtaining 589 bp

amplified fragment. The nucleotide sequence is shown in that sequence table SEQ ID

NO: 1 (where r at 181bp is A or G, Or see FIG. 4, Where r in the sequence shows the

position of the base mutation, That is, R at 181bp is A or G), SEQ ID NO: 2 (Meishan

pig: its mutant base at 18lbp is A) and SEQ ID NO: 3 (Large White pig: its mutant base

at 181bp is G), which lead to Pag I-RFLP polymorphism. Furthermore, the obtained PCR

product was digested by enzyme and applied to analyze the correlation between genotype

and pig fat deposition traits, providing a new molecular marker for molecular marker

assisted selection in pigs.

The molecular marker of the invention can be applied to the detection of pig fat

deposition traits.

The primer pair designed of the invention can also be applied to fat deposition detection.

More detailed technical solutions are described in the "Specific Embodiments".

BRIEF DESCRIPTION OF THE FIGURES

[0009] SEQ ID NO: 1 is the nucleotide sequence of the genetic marker prepared

according to the invention. 589 bp in length, the "r" shown at the 181bp of the sequence

is an allelic mutation, i.e. there is an A/G substitution. This mutation leads to a Pag I

RFLP polymorphism.

[0010] The sequence table SEQ ID NO: 2 is a partial nucleotide sequence amplified from

Chinese native pig breed "Meishan Pig". The length of the sequence is 589bp, of which

there is an allele mutation at the 18lbp of the sequence, namely base A.

[0011] The sequence table SEQ ID NO: 3 is a partial nucleotide sequence amplified from

foreign pig breed "Large White". The length of the sequence is 589bp, of which there is

an allele mutation at the 181bp of the sequence, namely base G.

[0012] SEQ ID NO: 4 and SEQ ID NO: 5 are that nucleotide sequence of the primer pair

design according to the invention.

[0013] Fig. 1: OLR1 sequence alignment results of large white pig, Landrace pig,

Meishan pig andQingpingpigrespectively. Bold English letters in the figure represent

SNP site.

[0014] FIG. 2 is the amplification result of intron 4 of pig OLR gene. The concentration

of agarose was 1.5%; Mark description in the figure: lane M: DL2000Marker; Lanes 1-4

were amplified fragments from Large White, Landrace, Meishan and Qingping pigs

respectively, with a fragment size of 589bp.

[0015] FIG. 3: Pigs OLR1 gene Pag I-RFLP detection results. The concentration of

agarose was 1.5%; The mark in the figure shows that lane M is DL2000Marker; GG

genotype, 589bp; AG genotype, 589bp, 179bp, 410bp; AA genotype, 179bp, 410bp.

[0016] FIG. 4 is a nucleotide sequence of intron 4 of porcine OLR1 gene amplified in

this invention, wherein r at 181bp is a A or G mutation, which leads to Pag I-RFLP

polymorphism.

[0017] Example 1 Amplification of Pig OLR1 Gene Fragment and Establishment of

Polymorphism Detection Method

[0018] The test pig breeds of the invention are large white pig, Landrace pig (foreign

blood pig breeds), Meishan pig and Qingping pig (Chinese local pig blood breeds), and

the samples are all provided by Hubei Key Laboratory of Animal Embryo and Molecular

Breeding, and Institute of Animal Husbandry and Veterinary, Hubei Academy of

Agricultural Sciences, which are commonly used breeds.

Extraction of Porcine Genomic DNA:

The genomic DNA kit produced by Beijing Baitaike Biotechnology Co., Ltd. (operated

according to the instructions of the kit) is used for extraction, and the specific steps are as

follows:

(1) Collect 20-50mg pig ear tissue, cut it into paste by ophthalmic surgery, put it into 2ml

centrifuge tube, add 200 ul lysate TL, and mix it well with pipette tips.

[0019] (2) Add 20 ul protease K (20mg/ml), turn it up and down, mix it completely, and

digest it overnight in a water bath at 55 °C.

(3) Add 200 ul of binding solution CB (the kit has its own), mix it completely up and

down, and leave it at 70 °C for 10min.

(4) After cooling, add 100 ul isopropanol, turn it violently and mix it well.

(5) Absorb the above mixture with 1mL gun head, add it to adsorption column AC,

centrifuge at 10000 rpm for 30s, and pour out the waste liquid in the collection tube.

6) Add 500p of inhibitor remove liquid IR (that kit is self-contained) , centrifuged at

12000 rpm for 30s, and waste liquid is discarded.

(7) Add 700 ul rinsing solution WB, centrifuged at 12000rpm for 30s, and the waste liquid was poured out.

(8) Repeat operation step 7.

(9) Put the adsorption column AC back into the collection tube, centrifuge at 12000 rpm

for 2min, and remove the rinsing solution as much as possible to prevent the residual

ethanol from inhibiting the downstream reaction.

[0020] Take out the adsorption column AC, put it into a clean centrifugal tube, add 50

100 ul elution buffer EB to the middle part of the adsorption membrane, place it at room

temperature for 3-5min, centrifuge at 12000 rpm for 1min, and collect the solution into

the centrifugal tube.

[0021] The concentration and quality of the extracted DNA were detected and then stored

at -20 °C for later use.

[0022] According to the genomic sequence of the OLR1 gene (Genbank accession

number: NC_010447.4, http://www.ncbi.nlm.nih.gov/nuccore/NC_010447.4), the

following primer pairs were designed:

A forward primer: 5'TCCTCGGTGGAAGAGCAT3', the sequence of which corresponds

to SEQ ID NO: 4;

Reverse primer: 5 'CTACCTTGGGTCAGGCAC3'; its sequence corresponds to SEQ ID

NO: 4.

PCR amplification was carried out in the genomic DNA of Large White, Landrace,

Meishan and Qingping pigs by using the above primers. The PCR reaction system was 25

ul, the concentration of each component in the system was 100 ng template DNA, 1 x

Taq buffer 2.5 mmol/L, MgCl2 1.5 mmol/L, dNTPs 2.5 mmol/L, the above primers were

0.5 mmol/L respectively, and 1U TaqDNA polymerase was used. The operation procedure of PCR was as follows: pre-denaturation 94 °C for 4 min; Then 94 °C 30 s, 58

°C 40 s, 72 °C 50 s, 34 cycles; Finally, the extension was continued at 72 °C for 10 min.

[0023] The PCR products of the above four pig breeds were purified by Gel Extraction

Kit (purchased from Shanghai Shenggong Bioengineering Technology Co., Ltd. And

operated according to the kit instructions), cloned and sequenced (sequenced by Shanghai

Shenggong Bioengineering Technology Co., Ltd.), and the size of the obtained fragment

was 589 bp, and its nucleotide sequence was shown in the sequence table SEQ ID NO: 2

(Meishan pig) and SEQ ID NO: 3 (Large White pig). A/G mutation at 181bp (246bp in

intron 4) of the fragment resulted in a digestion site change of restriction enzyme Pag I

(TCATGA).

Add 5.5 ul PCR products with 0.5 ul (1OU/ul) restriction enzyme, 10 x buffer lul, ddH20

3ul, and overnight at 37 °C. then, the PCR products were analyzed by 1.5% agarose gel

electrophoresis. The results of enzyme digestion were observed and recorded on gel

imaging system. When all the bases at 181bp were G, Pag I does not recognize this site,

which is marked as GG genotype (589bp), when all the bases at the mutant site were A,

Pag I enzyme recognized the site, which is marked as AA genotype (179bp + 41Obp), and

when both G and A were present, the site is marked as AG genotype (589bp + 179bp +

410bp). The PCR-Pag I-RFLP genotyping results of porcine OLR1 gene are shown in

FIG. 3.

[0024] Example 2 Verification of polymorphism distribution of this invention in

different pig breeds

[0050] The distribution frequency of PCR-Pag I-RFLP polymorphism in intron 4 of pig

OLR1 gene was detected in two Chinese local pig breeds (Meishan pig and Qingping pig) and two foreign pig breeds (Large White pig and Landrace pig). The detection results are shown in Table 1. The frequency of G allele is higher in large white pig and Landrace pig, AA allele is dominant in Meishan pig and Qingping pig, and A allele is higher, which indicates that there are significant differences between foreign pig breeds and

Chinese local pig breeds. As shown in Table 1.

[0025] Table 1 Genotype and allelic frequencies of porcine OLR1 gene intron 4

determined by PCR-Pag I-RFLP in different pig breeds

Breed Number Genotype frequencies Allele frequencies

AA AG GG A G

Large White 57 6 36 15 0.42 0.58

Landrace 46 0 8 38 0.09 0.91

Meishan 40 28 12 0 0.85 0.15

Qingping 50 30 18 2 0.78 0.22

[0026] Example 3 Association Analysis and Application of Molecular Markers Cloned

by the Invention and Pig Production Traits

[0027] In order to determine whether porcine OLR1 gene polymorphism is related to

porcine fat deposition traits, 331 pigs of Large white x Meishan resource family were

selected in this study(that hybridization method is commonly used in this field, Blood

samples were collected by the Key Laboratory of Pig Genetics and Breeding of Ministry

of Agriculture, Huazhong Agricultural University). The PCR-Pag I-RFLP method

established in Example 1 was used for polymorphism detection, and the GLM program of

SAS Institute Inc (Version 8.0) was used for analysis of association between different

PCR-Pag I-RFLP genotypes OLR1 gene with porcine fat deposition traits. The statistical

model was assumed to be:

[0028] Yijk=pi+Gi+Sj+Yk(+bijkXijk)+eijk

[0029] Yijk is the observed values of traits, p is the least square mean, Gi is the genotype

effect, , Sj and Yk are the fixed effects, which are respectively the sex and year effects,

bijk is the regression coefficient of slaughter weight or slaughter age, carcass traits are

covariant with slaughter weight, meat quality traits are covariant with slaughter age, and

eijk is the residual effect.

[0030] As it can be seen from Table 2, for carcass fat deposition traits, there are

significant differences in leaf fat weight, 6-7 rib fat thickness with different Pag I-RFLP

genotypes (P < 0.05), and the additive effect is significant. There were extremely

significant differences in thorax-waist fat thickness and average backfat thickness (P <

0.01), and the additive effect was extremely significant. G allele has the genetic effect of

significantly reducing fat deposition traits such as back fat thickness.

[0031] Table 2 Association analysis of porcine OLR gene PCR-PagI-RFLP genotypes

with fat deposition traits

Traits OLR1 PCR- Pag I -RFLP Genotype (t±SE) Effect (p±SE) AA AG GG Additive Dominance

Leaf fat weight (Kg) 0.861±0.030 a 0. 82 2 ±0.0 2 4 ab 0.770±0.036 0.045±0.023* -0.003±0.017 b

Caul fat weight (Kg) 1.218±0.033 1.172±0.027 1.182±0.040 0.017±0.026 0.014±0.019 Internal fat rate (%) 0.032±0.001 0.031±0.001 0.030±0.001 0.001±0.001 0.000±0.000 Shoulder fat thickness 3.783±0.084 3.593±0.068 3.570±0.100 0.106±0.066 0.041±0.047 (cm) 6-7 rib fat thickness (cm) 2.890±0.066a 2.866±0.053 a 2.670±0.078 0.110±0.051* -0.043±0.037 b

Thorax-waist fat thickness 2 .3 8 2 ±0.0 7 8 Aa 2.158±0.061b 1.925±0. 10 5 B 0.228±0.065** -0.002±0.045 (cm) Buttock fat thickness (cm) 2.104±0.082 1.973±0.066 1.891±0.098 0.106±0.064 0.012±0.046 Average backfat thickness 2 .7 4 2 {0 .0 6 1 A 2.621g0.050A 2 .5 0 3 ±0.0 7 4 B 0.120±0.048** 0.001±0.035 (cm) Fat percentage(%) 0.242±0.005 0.237±0.004 0.232±0.006 0.004±0.004 -0.000±0.002

[0032] Note: All the data in the table are least square means standard error. The same

letter in the same line means there is no significant difference. Significant differences

(within a trait) between the genotype classes are indicated with different lower case

superscripts are at P < 0.05, with different upper case superscripts at P < 0.01; Genotype

effect * indicated P < 0.05, * * indicated P < 0.01.

[0033] The main reference is described as below

Juwon A, Matthew K,Franklin, et al. Linoleic acid regulates the expression of oxidized low density lipoprotein receptor (OLR1) in 3T3-Ll adipocytes via ERK and JNK pathways [J]. FASEB Journal, 2007,21(5):A455 APR Patricia C, Hong-Ping Guan, Michael L. PPARy regulates adipocyte cholesterol metabolism via oxidized LDL receptor 1. Clin Invest, 2005, 115(8):2244-2256.

Sawamura T, Kume N, Aoyama T, et al. An endothelial receptor for oxidized low-density lipoprotein[J]. Nature 1997, 386: 73-77. Li tuo. Analysis of the association between the polymorphism of OLR1 gene and meat quality traits and its interaction with bta-miR-370 in Qinchuan cattle. Northwest Agricuture and Forest Science and Technology University, 2013.Sun Chao and Liu Chunwei, Effects of Fatty Acids on Proliferation and Differentiation of Mouse Preadipocytes and Expression of OLRl Gene [J] .Journal of Northwest A & F University, 2009, 37 (3): 1-6.

Claims (3)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A SNP genetic marker for pig fat deposition traits, the nucleotide sequence of which is as follows:

TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTC TGGCCTGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCT CTAGGCTCCAGCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCT TTGGTTCTGTGGCTTCR (A/G) TGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTC CCTACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATG GGGTTATCTCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTT TGATGCCCCACTTGTAAGTTTCCTATTCTTTTCCTAATCTGCTGGTGAAGTTACTG CCGCATTCCGCAGAGTGTCCTCTTCCTGCTGGAAAGAACCCTGGGAAATTTCTGA ATCTCTCTCCTCAGCCCTCTCATGCAGAGGCTTGCCCCTTGACTCATTCAGACCTT CTCTTGAGTTTTCCTTCTTACATTTATTACTGATTTGAAGCTTTAAGACAAGGCCA AGTGCCTGACCCAAGGTAG

The R at 181 bp in the above sequence is A or G, resulting in a Pag I-RFLP polymorphism

2. A method for screening SNP genetic markers of pig fat deposition trait, characterized in the following steps: extracting genomic DNA from pig ear tissue, designing primers according to pig OLR1 gene sequence, the DNA sequence of which is as SEQ sequence table ID NO: 4 and 5 shown. Executing PCR amplification, PCR product purification and clone sequencing in porcine genomic DNA with forward primer TCCTCGGTGGAAGAGCAT and reverse primer CTACCTTGGGTCAGGCAC to obtain nucleotide sequence as shown in sequence table SEQ ID NO: 1.

3. The Application of the genetic marker according to claim 1 in detection of pig fat deposition traits.

-1/4- 11 Aug 2020

Big White TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Landrace TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Qingping TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 Meishan TCCTCGGTGGAAGAGCATTTGTTCGAGATGCAGAGTAGGTACCTGTTTTTTAGTCTGGCC 60 ************************************************************ 2020101763

Big White TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Landrace TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Qingping TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120 Meishan TGGCCACTGATTACATGCATGTCCTTGGATGGACAGGGTATGGTGGGTCTCTAGGCTCCA 120

************************************************************ Big White GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Landrace GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Qingping GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 Meishan GCAGGCATGTCTATAAAACCATCCAGAAGATGGCAAAGATCCCTTTGGTTCTGTGGCTTC 180 ************************************************************ Big White GrGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Landrace GrGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Qingping ATGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 Meishan ATGAATCTAATCTAAGATGTTCACATCAACTCTTTGATCAATTCAGAAATGTTCCTCCCT 240 *********************************************************** Big White ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Landrace ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Qingping ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300 Meishan ACAGGAATTCATCCAGCAAACCATCGCCCATTCCAGTTTCCCATTCTGGATGGGGTTATC 300

Big White TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Landrace TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Qingping TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360 Meishan TCTGAGGAAACCCAACAACTCATGGCTCTGGGAGGACGGTACTCCTTTGATGCCCCACTT 360

************************************************************ Fig. 1A – continued in Fig. 1B

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Big White GTAAGTTTCCTATTCTTTTCCTAATCTGCTGGTGAAGTTACTGCCGCATTCCGCAGAGTG 420 Landrace GTAAGTTTCCTATTCTTTTCCTAATCTGCTGGTGAAGTTACTGCCGCATTCCGCAGAGTG 420 Qingping GTAAGTTTCCTATTCTTTTCCTAATCTGCTGGTGAAGTTACTGCCGCATTCCGCAGAGTG 420 Meishan GTAAGTTTCCTATTCTTTTCCTAATCTGCTGGTGAAGTTACTGCCGCATTCCGCAGAGTG 420

************************************************************ White Big 2020101763

Landrace

Qingping

Meishan

White Big Landrace Qingping

Meishan

White Big

Landrace

Qingping

Meishan

Fig. 1B (continued from Fig. 1A)

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Fig. 3 Fig. 2

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Fig. 4