CN101603089B

CN101603089B - Molecular marker for identifying local pig anti-F4ac piglet diarrhea in China and breeding application

Info

Publication number: CN101603089B
Application number: CN2009101157165A
Authority: CN
Inventors: 黄路生; 任军; 晏学明; 艾华水
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2009-07-20
Filing date: 2009-07-20
Publication date: 2011-08-10
Anticipated expiration: 2029-07-20
Also published as: CN101603089A

Abstract

The invention discloses a molecular marker for identifying F4ac diarrhea affectability/resistance of newborn and weaned piglets of local pig breeds in China and application thereof in boar genetic improvement of the local pig breeds in China, wherein by cloning, a DNA segment as shown in a sequence table is obtained, a single nucleotide polymorphism (SNP) A183G is identified at a 183bp part of the sequence, and an SNaPshot method is adopted to perform genotyping on the SNP A183G. The invention also discloses a preparation method for obtaining the molecular marker and related primers and a method for performing diarrhea trait association analysis of local pigs in China by using the cloned molecular marker. The molecular marker and the methods can remarkably improve the ability of resisting diarrhea diseases of the weaned piglets of local pig breed population in China and greatly reduce the piglet mortality by using the modern molecular biology technique to detect polymorphic sites and using marker-assisted selection (MAS) to select advantageous genotype individuals having seed stocks.

Description

Differentiate the molecule marker and the seed selection application of the anti-F4ac grice diarrhoea of place of china pig

Technical field

The present invention relates to animal Protocols in Molecular Biology and breeding field, especially relate to a molecule marker and an application in the seed selection of Chinese native pig breed swine improvement thereof of differentiating Chinese native pig breed new life and weanling pig F4ac diarrhoea susceptibility/resistance.

Background technology

Grice diarrhoea is the common transmittable disease in the Swine Production, has caused the tremendous economic loss for world's pig industry.Investigate between large-scale of Denmark's pig industry and show: the newborn piglet of 6%-7% suffers from dysentery, makes the mortality ratio of the preceding piglet of wean reach 2.7%, accounts for 11.9% of whole mortality ratio during this.Even recover individuality, also can have a strong impact on and grow, the formation cad pig that has causes feeding cost to increase, and sales revenue descends.

Enterotoxigenic escherichia coli (Enterotoxigenic Escherichia coli, ETEC) F4 causes main pathogenic bacterium newborn and the preceding piglet diarrhoea of wean, with China and Denmark is example, and 35% and 40% the piglet diarrhoea of having an appointment respectively causes because of ETEC F4 infects.In China, its lethality rate is between 10%-30%, indivedual local even higher than this ratio.

ETEC F4 is divided into ab according to amynologic characteristic, three kinds of hypotypes of ac and ad, and wherein F4ac is topmost pathogenic former, in the U.S., Spain and Korea S, only is separated to the F4ac hypotype in the ETEC grice diarrhoea case.Behind the ETEC F4 invasion chitling road, be attached to the acceptor on intestinal epithelial cell surface by its specificity adhesin, a large amount of breedings produce enterotoxin, cause the intestinal epithelial cells secreting function hyperfunction, a large amount of power and waters are separated matter and are entered enteric cavity, cause a large amount of liquid accumulator in the enteric cavity, surpassed the intestines wall and weighed receptivity and cause diarrhoea.This shows that the piglet intestinal epithelial cell has or not ETEC F4 acceptor, particularly having or not the F4ac acceptor is the key whether piglet falls ill when being infected by ETEC F4.No acceptor pig shows as the opposing type, has the acceptor pig then to show as the susceptible type.Separate and differentiate pig ETEC F4ac acceptor gene and resistance site thereof thereby become the key of pig ETEC F4ac diarrhoea breeding for disease resistance.

Nineteen ninety-five, it is material that Sweden LeifAndersson group utilizes aper * Large White resource family colony, through the linkage analysis of three generations pedigree, first with ETEC F4ac receptor mapping in No. 13 karyomit(e)s, with Tf gene close linkage.2002, Switzerland Peter Vogeli group is a material with Large White and the white three generations's family of Da Bai * length, utilize the more microsatellite marker information of karyomit(e) No. 13, the F4ac acceptor further is positioned between S0068 and the Sw1030, with S0075 and Sw225 highly chain (θ=0.00) wherein.This result is Sweden LeifAndersson group and Denmark after 1 year

Group unites in the assignment of genes gene mapping research of carrying out and has obtained checking, and they also utilize FISH and radiation hybrid clone plate technique to disclose the F4ac acceptor to be positioned at No. 13 karyomit(e) q41 districts (SSC13q41), respectively with No. 3 chromosomal q28-29 of people and q21 district homology.Recently, Joller etc. further is positioned ETEC F4ac acceptor gene between q41 region S W207 and the S0283 by Fine Mapping.This research group by utilize extensive white Du Luoke * painted face in Beijing opera resource family colony on No. 13 karyomit(e)s, adopt the high-density microsatellite marker with F4ac acceptor gene Fine Mapping in the S0283 adjacent domain.

In addition, Grange etc. have done a series of detailed researchs to the character of F4 acceptor, be separated to two kinds of mucus type sialoglycoprotein (Intestine mucus glycoprotein at the chitterlings epithelial cell, IMTGP), molecular weight is respectively 210kDa (IMTGP-1) and 240kDa (IMTGP-2), and these two kinds of albumen are considered to pig to F4ac susceptibility and the important factor of determination of resistance.And studies show that expressing the proteic MUC4 gene of mucoitin is positioned at No. 13 karyomit(e) S0283 of pig adjacent domain, and analyze in conjunction with its biochemical functions, think that the MUC4 gene very likely is an important candidate gene of enterotoxic Escherichia coli F4ac acceptor.

In view of above background, the applicant has carried out search, the discriminating of pig MUC4 gene and adjacent domain polymorphism thereof in a deep going way and has influenced the research of piglet ETEC F4ac dysentery proterties, carries out the breeding for disease resistance work of diarrhea of weaned piglets disease in the hope of setting up the marker assisted selection technology.

Summary of the invention

First purpose of the present invention provides a molecule marker of differentiating Chinese native pig breed weanling pig F4ac diarrhoea susceptibility/resistance, detect this polymorphic site by modern molecular biology technique, utilize marker assisted selection (MAS) to select favourable genotype individuality to reserve seed for planting, can significantly improve the sick ability of diarrhea of Chinese native pig breed population weanling pig, significantly reduce the piglet mortality ratio.

Second purpose of the present invention is the application of this molecule marker in seed selection improvement Chinese native pig breed boar diarrhea characteristic of disease shape.

First purpose of the present invention is achieved in that

(1) the extensive polymorphic site of MUC4 gene adjacent domain (SNP) is searched:

Searching microsatellite marker S0283 on ENSEMBLE website (http://www.ensembl.org/index.html) is 94,369 at No. 13 chromosome positions of pig, 614bp-94,369,755bp.With S0283 is the center, and 5Mb is extended in front and back, promptly with chromosome 13:89, and 369,614bp-99,369,755bp is a query ID, is submitted on the ENSEMBLE website, downloads the dna sequence dna that obtains near the 10Mb of No. 13 karyomit(e) S0283 of pig.Simultaneously (National Center forBiotechnology Information, http://www.ncbi.nlm.nih.gov/) downloads to the partial dna sequence (sequence number: DQ848681) of the pig MUC4 gene of 29369bp from the NCBI website.With the SSC13 sequence that this 29369bp sequence grappling is downloaded, front and back are extended about 50kb more respectively, then obtain the dna sequence dna of one section 130kb, have included all DNA sequence of pig MUC4 gene.

Choose 8 ETEC F4ac of white Du Luoke and painted face in Beijing opera and stick the extreme individuality of phenotype, wherein 4 is strong adhesion type, and 4 are adhesion type not.In the dna fragmentation of acquired 130kb, design 34 pairs of primers altogether, wherein the every 5kb of the extragenic flanking sequence of MUC4 designs a pair of primer, every 2kb designs a pair of primer in the MUC4 gene, all above 8 individualities are carried out pcr amplification, adopt relatively sequencing discriminating pleomorphism site, with extensive search mononucleotide polymorphic mark (SNPs).

104 SNPs in the 130kb scope, have been differentiated altogether.Select wherein to be evenly distributed and 70 representative polymorphic sites, in 148 of 12 Chinese native pig breeds edge far away colony, carry out gene type, 64 SNP sites success somatotypes are arranged.Utilize the genotype data of somatotype success to carry out extensive SNP loci polymorphism and ETEC F4ac subsequently and stick single-point and multiple spot correlation analysis between the phenotype.Correlation analysis is the result show, the SNP A183G loci polymorphism of MUC4 gene adjacent domain and place of china pig ETEC F4ac stick phenotype cognation the most remarkable (P=2.68E-12).

(2) the concrete discrimination process of SNP A183G:

Utilize above-mentioned from including of ENSEMBLE website (http://www.ensembl.org/index.html) dna sequence dna of one section 130kb of pig MUC4 gene, use public Primer 5.0 software design primers F 1 and R1 (F1:5 ' G CTT AGA CAG TGA GAC ATC AACATC-3 ' and R1:5 '-AAT TAC AGG TGA CAC GCT TCC-3 ') amplification pig genomic dna (thymus nucleic acid).In the reaction system of the polymerase chain reaction of 25 μ l (PCR), comprise 25ng pig genomic dna, 1.0mM MgCl ₂, 100mM dNTP (four kinds of deoxynucleotide substrates of synthetic DNA), each 10pmol of forward and reverse primer, 2 unit archaeal dna polymerases (Taq enzyme) and 1 * PCR buffer (damping fluid) (Shanghai bio-engineering corporation, China).Amplification condition is: 94 ℃ of 4min; 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 60s, totally 34 circulations; Extend 10min at 72 ℃ at last.Amplified fragments adopts QIAquickPCR product purification test kit (QIAGEN, Germany) purifying, is checked order by order-checking center, China big gene Shanghai.Adopt the American National biotechnology (NCBI of information center, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) the public BLAST software in website, the dna sequence dna of order-checking back acquisition and the dna sequence dna of above-mentioned 130kb are carried out homology relatively, the result shows this sequence and announces sequence homology more than 99%, illustrates that thus this section sequence is an aim sequence.Choosing 8 ETEC F4ac then, to stick phenotype extremely individual, and wherein 4 is strong adhesion type, and 4 are adhesion type not.Adopt above-mentioned primers F 1 and R1 that above 8 individualities are carried out pcr amplification, adopt relatively sequencing order-checking, when using the SeqMan software analysis sequencing result in the public DNAStar software package, discovery exists a mononucleotide polymorphic site (SNPs), i.e. SNP A183G site at the 183rd Nucleotide place of this sequence.

(2) the portion gene sequence that be positioned at No. 13 karyomit(e) MUC4 of pig gene adjacent domain, comprises mutational site, 183bp place (SNPA183G) is as follows:

tgttagaaag?tccttgggct?ttaacccaaa?taacactgct?gtaactttaa?aggggacttt 60

tttttattca?gaattatgaa?tagatttgtt?tctaggactc?taagtcctat?acctgctccc 120

tcctcagtaa?agttcataaa?cttcgtttct?ctagctaagg?tttagattgg?aagacataca 180

ccrtgaaatt?attattaagc?ttatttcctc?ttcctaagaa?cataaataaa?agcaacgctc 240

tgtcttctgg?ctttgagtca?ccagatagaa?gctagttaca?tgccaaactt?actaataaac 300

ttattataga?ttcttggcaa?ttaatatact?tttggagcct?gcatcacctc?tgttaaaatc 360

aatattagca?gcaaaagttt?catgattgca?cagtccaagt?tttttaaagt?aaagatttgt 420

atttgtttgt?tcataaaagt?agtacagctc?tactcccagc?aatatggcag?aagagacaaa 480

cctttaacta?gagaacatgc?aaatgccaga?taatgtgtaa?taaatatttt?ctgaatgctg 540

agctgagatc?cgaagaaaga?aagggagacg?ctcaggagtg?aaggagttga?aaactaagat 600

gataagtaag?tgctctcata?caatttggag?atttctgaga?tctagagttt?ggcattttaa 660

tggctgcatg?gagaaaggaa?acaatcttag?atcaggacac?tgtagactga?atattttctg 720

cataaagctg?gaaacctcag?tgaaaaggtg?gtctagaaaa?atcaaaaaga?aaaaagaaaa 780

aggaggagtt?ccctggtggc?tcagtgggtt?aaggatccag?cgttttcact?gctggggctc 840

tggttactgc?tgag

(3) gene type of SNP A183G:

Use SNaPshot test kit (ABI company, the U.S.) to carry out gene type.Method is as follows:

1) design of primers

Amplification comprises the dna fragmentation and a SNaPshot that will detect SNP A183G site and extends primer (5 '-A AAA AAA AAA AAA GGA AAT AAG CTT AATAAT AAT TTC-3 ') the design primer to F2 and R2 (F2:5 '-ACG TTG GAT GCT TAG GAA GAGGAAATAAGC-3 ' and R2:5 '-ACG TTG GAT GCT TCG TTT CTC TAG CTAAGG-3 ').

2) pcr amplification condition and product purification

Use above-mentioned primer to F2 and R2 amplification pig genomic dna.In the PCR reaction system of 25 μ l, comprise 25ng pig genomic dna, 1.0mM MgCl ₂, 100mM dNTP, each 10pmol of forward and reverse primer, 2 Taq of unit enzymes and 1 * PCR buffer (Shanghai bio-engineering corporation, China).Amplification condition is: 94 ℃ of 4min; 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 30s, totally 35 circulations; Extend 10min at 72 ℃ at last.The PCR product detects with 2% agarose gel electrophoresis and takes pictures.Get 3 μ l PCR products, add 1 μ l ExoSAP-IT enzyme (Shanghai traditional Chinese medicines group company, China) and hatch 15min for 37 ℃, handle the 15min inactivator for 80 ℃ then.4 times of dilutions are standby behind the PCR product purification.

3) SNaPshot reaction and product purification

The SNaPshot reaction system is: SNaPshot reaction mixture (Multiplex Ready ReactionMix) 2 μ l; SNaPshot extends primer 0.5 μ l (the primer final concentration is 0.2 μ mol/L); Above-mentioned purifying dilution PCR product 1.5 μ l; Deionized water 1 μ l.The SNaPshot loop parameter is 96 ℃ of 10s, 50 ℃ of 5s, 60 ℃ of 30s, totally 25 circulations.Get that 5 μ l SNaPshot reaction product add 0.57 μ l 10 * buffer and 0.1 μ l CIP (SNaPshot reaction product purifying enzyme) is hatched 1h under 37 ℃, handle the 15min inactivators and reach the product purification effect for 75 ℃ then ,-20 ℃ of preservations are standby.

4) 3130XL genetic analyzer electrophoresis:

The electrophoresis mixed system comprises: 9.25 μ l Hi-Di formamide (methane amide denaturing agent); 0.5 the above-mentioned SNaPshot reaction product after purified of μ l; 0.25 μ l GeneScan 120LIZ size standard (electrophoresis intramolecularly mark).System mixing back places then immediately in 95 ℃ of sex change 5min handles 2min on ice.The centrifugal sample of going up.

Specifically declare the type method as shown in Figure 2.

Second purpose of the present invention is achieved in that the present invention utilizes this polymorphic site and flanking DNA sequence information, adopts Protocols in Molecular Biology to differentiate idiotype, at diarrhea characteristic of disease shape seed selection Chinese native pig breed boar.

The present invention has differentiated single nucleotide polymorphism (SNP) site that influences Chinese native pig breed weanling pig diarrhea characteristic of disease shape, detect this polymorphic site by modern molecular biology technique, utilize marker assisted selection (MAS) to select favourable genotype individuality to reserve seed for planting, can significantly improve the sick ability of diarrhea of Chinese native pig breed population weanling pig, significantly reduce the piglet mortality ratio.

Description of drawings

Fig. 1 is the sequence peak figure in the pig SNP A183G mutational site of the present invention's discriminating;

Fig. 2 declares type figure for the SNaPshot in SNP A183G site, and wherein a is the GG type; B is the AG type; C is the AA type;

Fig. 3 is the detected result of sticking of ETEC F4ac and brush border, (a) is adhesion type not (b) to be adhesion type.

Embodiment

Below in conjunction with embodiment and contrast accompanying drawing the present invention is described in further detail.

Select Chinese native pig breed boar core group individuality, utilize above-mentioned SNaPshot method to differentiate the genotype in No. 13 karyomit(e) MUC4 of pig gene adjacent domain SNP A183G site, select favourable genotype individuality (AA type) to reserve seed for planting, the anti-piglet diarrhoea performance of improvement Chinese native pig breed population after colony's subculture seed selection.

Embodiment:

Utilize 148 12 purebred piglets of local pig breed representing 6 ecotypes of China (comprising North China, south China, Central China, Jiang Hai, southwest, plateau type etc.) as laboratory animal, each pig kind is chosen at least 3 paternal familys, and each family is selected 3-4 1-2 monthly age individuality; After 148 purebred piglets are butchered, get small intestine jejunum part, extract the intestinal epithelial cell brush border, utilize E.coli F4ac bacterial strain to stick chitterlings epithelial cell brush border, by the result of sticking of phase microscope bacterial detection.The brush border of each sample detection more than 20, if have on the brush border more than 10% and have 2 bacterial adhesions at least, declaring this sample is adhesion type, if have the brush border of 2 bacterial adhesions at least less than 10%, but the brush border that 1-2 bacterial adhesion arranged is more than 10%, declaring this sample is weak adhesion type, otherwise is judged to not adhesion type.Stick design sketch as shown in Figure 3.

Use above-mentioned SNaPshot method that 148 purebred piglets are carried out SNP A183G genotype and judge, adopt the online common software of SHE to analyze this SNP loci polymorphism and an experiment pig ETEC F4ac and stick cognation (seeing Table 1) between the phenotypic character.The result shows that the susceptibility that infects of SNP A183G polymorphism and ETECF4ac is utmost point significant correlation (P=2.68E-12; LogP=11.57), G allelotrope is tumor susceptibility gene, and A allelotrope is resistant gene; Genotype GG and AG individuality are to the susceptible that infects of ETEC F4ac, and AA type individuality has resistivity (table 2) to infecting of ETEC F4ac.AA type individuality is the required genotype individuality of seed selection improvement of the sick type pig of diarrhea.In commercial boar core group, subculture seed selection AA type individuality can further be improved the diarrhea characteristic of disease shape of Chinese native pig breed population, reduces the mortality ratio of piglet.

Table 1SNP A183G polymorphism and ETEC F4ac stick the association analysis of phenotype

Table 2SNP A183G genotype and the ETEC F4ac of site in experiment pig colony sticks Phenotype Distribution

Claims

1. molecule marker of differentiating the anti-F4ac grice diarrhoea of place of china pig, it is characterized in that: the 183rd Nucleotide place of dna sequence dna as follows is the base mutation of A183-G183, comprises the situation of base A and bases G: