CN108411003A - WNT2 genetic fragments are as the molecular labeling for influencing swine erythrocyte number character - Google Patents

Abstract

The invention belongs to the technical field of animal molecular marker screening, and specifically relates to a WNT2 gene fragment as a molecular marker affecting the number traits of pig red blood cells. The molecular marker of the invention is cloned from the WNT2 gene. The molecule is a fragment of the WNT2 gene. The gene is typed and screened by gene chip technology to obtain a molecular marker related to the number of pig red blood cells. The nucleotide sequence of the marker is shown in SEQ ID NO: 1. In the sequence There is a T/C allelic mutation at the 24th base of SEQ ID NO: 1, and when the 24th nucleotide on SEQ ID NO: 1 is C, it is determined to have a higher number of red blood cells. The invention provides a new SNP molecular marker for marker-assisted selection of pig immune traits, especially red blood cell number.

Description

WNT2 Gene Fragment as a Molecular Marker Affecting Pig Red Blood Cell Number Traits

technical field

The invention belongs to the technical field of animal molecular marker screening, and specifically relates to a WNT2 gene fragment as a molecular marker affecting the number traits of pig red blood cells. The molecular marker of the invention is cloned from the WNT2 gene. The molecular marker can be used for predicting the character of pig red blood cell number (RBC).

Background technique

my country has a large population and likes to eat pork, and the consumption of pork is large, accounting for half of the world's total (Liu Wentao et al., 2016). According to the data from the National Bureau of Statistics, in 2017, my country's pork output was 53.4 million tons, imported pork was 1.217 million tons, and the import value was as high as 15.06 billion yuan. Gradually transform into high-efficiency and low-cost large-scale farming, and the free-range farming model is gradually eliminated (Deng Qifeng et al., 2017). Large-scale farming pays attention to intensive feeding, with high stocking density and poor environmental conditions, the disease resistance of pigs is reduced and disease prevention and control is difficult, especially the outbreak of infectious diseases often causes devastating damage. Among them, viral infectious diseases are the most harmful. Common viral infectious diseases mainly include pseudorabies, porcine parvovirus disease, Japanese encephalitis, porcine reproductive and respiratory syndrome, and swine fever, which often lead to abortion of sows and death of piglets etc. (Wang Xuemin etc., 2004), bring huge loss to pig farm. Although drug therapy and vaccine prevention have played a key role, they have greatly increased production costs, and vaccines have high requirements for technical personnel. Improper use will affect the normal production of pigs, and even evolve into pathogens (Ge Caiyan, 2016), causing serious loss.

In recent years, the supervision of the breeding industry has been increasing day by day. Under the dual pressure of strict drug residue control and food safety, pig production has entered a bottleneck period, and disease-resistant breeding has been recognized as an important link in the comprehensive prevention and control of pig diseases (Wang Chao et al., 2014). Scholars at home and abroad attach great importance to disease-resistant breeding and have achieved a series of important results. In the long run, improving the disease-resistant performance of pigs on the basis of genetics and cultivating high-quality and high-yield pigs with disease-resistant performance are the fundamental solutions to pig production problems. way. It has been found that individuals with the AA genotype of the FUT1 gene M307 of the Sutai pig (Duroc×Meishan pig) have strong comprehensive disease resistance and good production performance without affecting carcass and meat quality traits (Bao et al., 2012), This achievement provides a good reference for disease resistance breeding. In recent years, food safety problems caused by drug residues have been plagued by agricultural and animal husbandry enterprises. Disease-resistant breeding can solve this problem and allow the cost of disease-resistant management to be shared equally.

The immune system is the natural barrier for animals to resist bacteria and viruses. The strength of the immune system directly determines the ability of animals to resist pathogenic microorganisms. The Cav1 gene of pigs plays a key role in preventing pathogenic microorganisms from invading the host (Liu et al., 2011). For porcine reproductive and respiratory syndrome (PRRS), porcine circovirus (PCVAD) and other diseases that attack the immune system and reduce pig disease resistance, resistance breeding for these two diseases is carried out by molecular means ( et al., 2014), which can improve the health of pig herds. In an intensive breeding environment, the disease spreads rapidly in pig herds, making disease prevention and control more difficult. Coupled with the variability and complexity of microbial infectious disease variants and serotypes, disease-resistant breeding will become an effective way to solve this problem.

Red blood cells are an important component of the body's immune system. The various immune molecules contained in them can regulate the innate and adaptive immune responses of white blood cells, and play an important role in the body's immunity (Guo Feng, 2004). The number of red blood cells in pigs and their disease resistance have a close connection. It contains a large number of immune molecules such as CR1, CR3, CD8, CD59, and SOD enzymes, which can recognize, adhere, concentrate, kill antigens, and clear immune complexes in the blood circulation, playing an important role in immunity (Guo Feng, 2002). Erythrocyte immunity itself has a complete self-regulatory system, the number is huge, and the chance of encountering immune complexes is thousands of times greater than that of white blood cells. It is the main way to clear circulating immune complexes in the body (Li Hongquan et al., 2002). The gene or molecular marker has important practical significance for improving the disease resistance of pigs. There have been relevant literature reports on the immune effect of red blood cells on pigs. At present, more studies are related to CR1-like factors. Its expression level is related to that of pigs. It is related to the level of erythrocyte immune adhesion (Zhang Jingjing et al., 2018). Blood is distributed throughout the body, and red blood cells have a wide range of immune functions, so it is of great significance to screen out molecular markers and genes that are significantly related to the number of red blood cells in pigs for disease-resistant breeding.

The correlation between the SNP found in the invention and the traits of red blood cell count (RBCs) of pigs has reached a very significant level, and provides new genetic resources for the research on immunity and growth traits of domestic pigs.

Contents of the invention

The purpose of the present invention is to overcome the defects in the prior art, improve the excavation of domestic pig disease-resistant breeding molecular marker resources, use gene chip technology to carry out typing of SNP, and use GWAS screening and injection of polymyocytes to determine the number of pig cells ( The SNPs associated with RBC) traits provide new molecular marker resources for pig disease resistance breeding.

Technical scheme of the present invention is as follows:

The applicant obtained the nucleotide sequence of the second intron of the WNT2 gene through gene chip technology and referring to Ensemble, the nucleotide of which is shown in SEQ ID NO:1. The sequence shown in SEQ ID NO: 1 is a WNT2 gene molecular marker that affects the number of red blood cells in pigs. The specific nucleotide sequence of the molecular marker is as follows:

CTTGCATTTGTGGAGATGATTCAR(T/C)ATTAAAGAAGGGGCCTTGGCTCTAGGAAAGAATTCTTCAAGGAGACTTTGTCTAGCATCAAAGTACGTCAGAGGCAGAACAAGGATTCATCAGCCATCGTGTGAAAGTTCCTCAGAAGGAGCAAATTCCAAACCGCTTTTAAGAAAAGCCTTTTTGTTACAGGTAGCCGGGAATCTGCCTTTGTGTACGCCATCTCCTCAGCTGGAGTTGTATTTGCCATCACCAGGGCCTGTAGCCAAGGAGAATTAAAGTCCTGTTCCTGTGACCCGAAGAAGAAGGGAACAGCCAAGGACAGCAAGGGCA，

The R at the 24th base of the above sequence is T or C, and the mutation leads to changes in red blood cell count traits, and when the 24th nucleotide on SEQ ID NO: 1 is C, the pig has a higher Red blood cell count.

The above sequence can be used as the SNP molecular marker for detecting the number traits of porcine red blood cells.

The applicant provides a method for screening SNP molecular markers associated with porcine erythrocyte number traits, the method comprising the steps of:

① Extract the genomic DNA from the pig ear tissue, and conduct DNA quality testing.

② Using gene chip technology for genotyping.

③A method based on a single-marker association regression model was used, with individual sex as a fixed effect, and the enABEL software package in the R statistical environment was used to conduct genome-wide association analysis (GWAS). The specific regression model is as follows: Y=Xb+Sa+Zu+e, where Y represents "the vector of phenotypes"; b represents the estimated value of "fixed effects (including gender) and the mean value of the phenotype μ "; α stands for "SNP replacement effect"; u stands for "random additive genetic effect"; obeys multidimensional normal distribution, u～N(0, Gσα ² ), G represents the genome similarity matrix (based on SNP markers), σα ² represents the polygenic additive variance (through which the heritability can be estimated); X, S, Z are the incidence matrices of b, α, and u respectively; e represents the "residual error vector" (a vector of residual errors ), obey the normal distribution, e～N(0,Iσe ² ), σe ² represents the residual variance.

The molecular markers screened by the invention can be used for non-diagnostic purposes in the association analysis between pig-related genes or genotypes and the number of red blood cells in pig immune traits, and provide a new molecular marker resource for molecular marker-assisted selection of pig red blood cell number traits .

Compared with the prior art, the present invention has the beneficial effects:

The present invention can detect the genotype of the pig by adopting gene chip technology in vitro, and evaluate the immune ability of the pig as a non-diagnostic purpose. Compared with current methods such as PCR-RFLP, ELISA, and flow cytometry, the present invention has simple, It has the outstanding advantages of quickness, high sensitivity and good specificity. For more detailed technical solutions, please refer to the examples in "Description of Drawings" and "Detailed Implementation Modes" of the specification.

Description of drawings

Sequence listing SEQ ID NO: 1 is the second intron fragment of the WNT2 gene cloned in the present invention. This fragment is the molecular marker screened in the present invention. The nucleotide sequence of the molecular marker is 327bp in length, and there is a T/C allele mutation in R at the 24th base of the sequence.

Fig. 1: Schematic diagram of the overall technical process of the present invention.

Figure 2: The intron sequence of the WNT2 gene cloned in the present invention, which is the molecular marker sequence of the present invention. Explanation of reference numerals: there is a T/C allele mutation at the 24th base of the sequence shown in Figure 2 (the English letter "R" at 24bp represents the mutation site).

Figure 3: Manhattan diagram of the present invention. Explanation of reference numerals: the molecular markers screened by the present invention are marked with black circles, and the markers are located in the intron region of the WNT2 gene on the No. 18 pig chromosome.

Detailed ways

Example 1: Genotyping Detection

(1) Using phenol extraction method to extract ear tissue DNA from Duroc×Erhualian F2 population

1) Grind the ear tissue samples of the Duroc × Erhualian F2 generation pig population (group samples provided by Guangdong Huanong Wens Animal Husbandry Co., Ltd.) in liquid nitrogen, add an equal volume of 1 × SET (1 mL), proteinase K ( 10ng/mL) to a final concentration of 200ug/mL, then add 10% sodium dodecyl sulfate (SDS) to a final concentration of 0.5%, and shake well. Incubate the digestion overnight in a 55 °C water bath.

2) Add an equal volume of Tris-saturated phenol to the digested tissue sample, slowly invert the centrifuge tube for 15 minutes, centrifuge in a low-temperature refrigerated centrifuge at 4°C and 11,000 rpm for 10 minutes, and carefully transfer the supernatant to another centrifuge tube. Mark the corresponding mark.

3) Add an equal volume of phenol/chloroform/isoamyl alcohol (volume ratio 25:24:1), slowly invert the centrifuge tube for 10 minutes, and centrifuge at 11,000 rpm for 10 minutes in a low-temperature (4°C) centrifuge, and carefully absorb the supernatant. Transfer to another clean centrifuge tube.

4) Add an equal volume of chloroform/isoamyl alcohol (volume ratio 24:1), slowly invert the centrifuge tube for 10 minutes, and store at low temperature (4°C)

In a refrigerated centrifuge, centrifuge at 11000rpm for 10min.

5) Aspirate the supernatant into a marked centrifuge tube, add 2.5 times the volume of pre-cooled absolute ethanol, and you can see white flocculent DNA.

6) Pick out the DNA precipitate with a pipette tip, place it in an EP tube with a corresponding number, let the ethanol evaporate at room temperature, and add an appropriate amount of ultrapure water (generally about 300ul) to dissolve the DNA.

7) Measure its concentration and purity on a DNA concentration analyzer, electrophoresis on 1% agarose gel at 80 volts for about 2 hours, and detect the quality of the extracted DNA under ultraviolet light.

(2) Determination of SNP genotype

The PorcineSNP60BeadChip genome-wide chip developed by Illumina Company contains more than 60,000 SNP sites, with an average step size of 40kb per marker, covering the pig genome. This chip integrates genetic differences of various pigs, including Duroc, Landrace, Pietrain and Large White, and can provide sufficient SNP density for genome-wide association analysis.

(3) Quality control

Use PLINK v1.07 software (from Shaun Purcell Company) to carry out quality control on the obtained genotype data, the elimination detection rate is <90%, the minor allele frequency (minor allele frequency, MAF) is <0.01, deviates from Hardy temperature Berg (Hardy-Weinberg Equilibrium, HWE) P≤10-5 SNP markers and individuals with a detection rate of <90%, finally 303 individuals and 49749 SNPs were used for GWAS research.

Example 2: Application of the WNT2 molecular marker typing method in the correlation analysis of pig immune traits

(1) Correlation analysis between WNT2 molecular marker typing results and immune traits

The experimental pigs used for the detection and analysis of the association between genotype and immune traits come from the F2 generation population (conventional breed) of Duroc×Erhualian crossbred by Guangdong Huanong Wen's Animal Husbandry Co., Ltd. The DNA used for genotyping is extracted from the ear tissue of the F2 generation of Duroc × Erhualian cross ("Duroc × Erhualian hybrid F2 generation" referred to as "pig" in the text of the manual and the table) is used for blood Blood samples for routine testing and flow cytometry were collected from 35-day-old piglets 4 hours after inoculation with polymyocytes (PolyI:C). Using the method based on the single-marker association regression model, using the gender of the individual as a fixed effect, the GWAS analysis was performed using the GenABEL software package in the R statistical environment. The specific regression analysis model is as follows: Y=Xb+Sa+Zu+e

Among them: Y stands for "vector of phenotypes" (the vector of phenotypes); b stands for "estimated value of fixed effects (including gender) and phenotype value mean μ"; α stands for "replacement effect of SNP"; u stands for "random Additive genetic effect"; subject to multidimensional normal distribution, u～N(0,Gσα ² ), G represents the genome similarity matrix (based on SNP markers), G represents the polygenic additive variance (the heritability is estimated through this variance ); X, S, and Z are the incidence matrix (incidence matrix) of b, σ, and u respectively; e stands for "residual error vector" (a vector of residual errors), obeying normal distribution, e～N(0,Iσe ² ) , which represents the residual variance.

The results of the association analysis are shown in Table 1.

Table 1 Effect of different genotypes of WNT2 gene intron fragment 24T/C polymorphism on red blood cell count in 35-day-old pigs

Table 1 shows: P<0.05 means significant difference; P<0.01 means extremely significant difference.

It can be seen from Table 1 that the number of red blood cells in individuals with genotype CC is significantly higher than that in TC individuals, so C is an allele that is beneficial to increase the number of red blood cells.

main reference

[1] Liu Wentao et al. Comparative Study on Pig Industry in China and the United States [J]. Chinese Journal of Animal Husbandry, 2016, 52(6): 3-7;

[2] Deng Qifeng, etc., the development status and challenges of my country's pig industry [J]. Guangdong Animal Husbandry and Veterinary Science and Technology, 2017, 42(4): 5-8;

[3] Wang Xuemin, etc., Infectious diseases causing reproductive disorders in pigs [J]. Animal Science and Veterinary Medicine, 2004, 21(2): 59-60; [4] Ge Caiyan, Scientific use of pig vaccines and attention Matters [J]. China Animal Health, 2016, 18(5): 30-31;

[4] Wang Chao et al. Related issues and research progress of pig disease-resistant breeding [J]. China Journal of Animal Husbandry, 2014, 50(22): 67-72;

[5]Bao W B, Ye L, Zhu J, et al.Evaluation of M307of FUT1gene as genetic marker for disease resistance breeding of Sutai pigs[J].Molecular Biology Reports,2012,39(4):4223-4228;

[6] Bertschinger, Bürgi, et al.[Inheritance and disease in the pig: possibilities of use for breeding].[J].Schweizer Archiv Für Tierheilkunde,2014,156(6):269-77;

[7] Liu X D, Chen H B, Tong Q, et al. Molecular characterization of caveolin-1in pigs infected with Haemophilus parasuis [J]. Journal of Immunology, 2011, 186(5): 3031-3046;

[8] Guo Feng, Erythrocyte Innate Immunity and Acquired Immunity [J]. Nature Magazine, 2004, 26(4): 194-199;

[9] Guo Feng, Research and Significance of Erythrocyte Immunity [J]. Nature Magazine, 2002, 24(5): 268-273;

[10] Li Hongquan et al., Overview of the research on erythrocyte immune function in animals [J]. Journal of Shanxi Agricultural University (Natural Science Edition), 2002, 22(3): 269-273;

[11] Zhang Jingjing et al. Study on immune adhesion function and CR1-like expression level of porcine erythrocytes [J]. Chinese Veterinary Science, 2018(05): 1-18.

sequence listing

<110> Huazhong Agricultural University

<120> WNT2 Gene Fragment as a Molecular Marker Affecting Pig Red Blood Cell Number Traits

<141> 2018-05-02

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 327

<212>DNA

<213> Pig (Sus scrofa)

<220>

<221> gene

<222> (1)..(327)

<220>

<221> mutation

<222> (24)..(24)

<400> 1

cttgcatttg tggagatgat tcacattaaa gaaggggcct tggctctagg aaagaattct 60

tcaaggagac tttgtctagc atcaaagtac gtcagaggca gaacaaggat tcatcagcca 120

tcgtgtgaaa gttcctcaga aggagcaaat tccaaaccgc ttttaagaaa agcctttttg 180

ttacaggtag ccgggaatct gcctttgtgt acgccatctc ctcagctgga gttgtatttg 240

ccatcaccag ggcctgtagc caaggagaat taaagtcctg ttcctgtgac ccgaagaaga 300

agggaacagc caaggacagc aagggca 327

Claims (2)

1. a kind of molecular marker that influences porcine erythrocyte count character cloned from WNT2 gene fragment, it is characterized in that, the nucleotide sequence of described molecular marker is as follows: CTTGCATTTGTGGAGATGATTCARATTAAAGAAGGGGCCTTGGCTCTAGGAAAGAATTCTTCAAGGAGACTTTGTCTAGCATCAAAGTACGTCAGAGGCAGAACAAGGATTCATCAGCCATCGTGTGAAAGTTCCTCAGAAGGAGCAAATTCCAAACCGCTTTTAAGAAAAGCCTTTTTGTTACAGGTAGCCGGGAATCTGCCTTTGTGTACGCCATCTCCTCAGCTGGAGTTGTATTTGCCATCACCAGGGCCTGTAGCCAAGGAGAATTAAAGTCCTGTTCCTGTGACCCGAAGAAGAAGGGAACAGCCAAGGACAGCAAGGGCA， The R at 24bp of the above sequence is T or C, resulting in a polymorphism of the WNT2 gene; and when the 24th nucleotide sequence of the above sequence is C, it is determined that the pig has a higher number of red blood cells. 2. It is required to apply the molecular marker described in 1 in marker-assisted selection of porcine erythrocyte number traits.