CN106521000A - Molecular marker related to weaning weight and anti-diarrheal diseases of piglets and application of molecular marker in pig breeding - Google Patents

Abstract

The invention discloses a molecular marker related to the weaning weight and anti-diarrheal diseases of piglets and application of the molecular marker in pig breeding. Two SNP loci related to the weaning weight and anti-diarrheal diseases of the piglets are found in the IL6 gene by inventors, wherein one of the SNP loci related to the weaning weight and anti-diarrheal diseases of the piglets is 1753T/C which is located at the 1753th nucleotide in the IL6 gene sequence, and the other SNP locus related to the weaning weight of the piglets is 3789A/T which is located at the 3789th nucleotide of the IL6 gene sequence. Based on the SNP loci, the invention also provides a PCR-RFLP molecular marker containing the SNP loci. The piglets can be genotyped by the PCR-RFLP molecular marker technology, so that the piglets with the high weaning weight and anti-diarrheal disease ability are kept, and the breeding process of pigs is accelerated.

Description

To weaned piglet weight and the sick related molecular marker of diarrhea and its in pig breeding Using

Technical field

The invention belongs to molecular genetics field, and in particular to the molecular marker related to weaned piglet weight and diarrhea disease Preparation, detection method and application.

Background technology

Weaned piglet is the important indicator for weighing pig production performance again.There are some researches show, weaned piglet weight delivers day for sale with which Age and daily gain are significantly correlated, and with the increase of weaned piglet weight, live pig daily gain increases, marketing age declines.In live pig In modern intensive production, piglet diarrhea is one of modal problem during piglet produces, and diarrhoea can cause Piglet Development Slowly, or even dehydration is dead, the economic benefit of serious restriction pig industry.

Weaned piglet weight how is improved, the problem that piglet diarrhea sickness rate always perplexs pig industry is reduced.Although adopting Strengthen the measure such as feeding and management and vaccine immunity, weaned piglet weight can be improved to a certain extent, reduce piglet diarrhea sickness rate. But from hereditary angle, using GENERALIZATION OF MODERN BREEDING TECHNIQUE, select that diarrhea ability is strong, the piglet that weaning weight is high, Cai Nengcong Substantially solve problem.Traditional pig breeding method is by carrying out performance survey to purpose individuality itself, compatriot, ancestors or descendant Surely assessing individual breeding value, this system of selection accuracy is higher, achieves noticeable achievement, but exist high cost of determination, time-consuming effort, The shortcomings of early stage seed selection cannot be carried out.Now biotechnology is developed rapidly so that breeder can be developed for mesh Economic, quick, the accurate molecular marker of mark character, carries out molecular marker assisted selection, and then accelerates breeding work.

The premunition character of pig and weaning weight are all the complex characters by controlled by multiple genes, at present with regard to both character Molecule marking research has been achieved with certain achievement, but the number of labels for obtaining cannot still meet the needs of pig breeding, still needs to constantly Develop and develop new molecular marker.Interleukin-6 (Interleukin6, IL-6) is the cytokine with several functions, Research finds that IL-6 can participate in the generation evolution of various diseases by different signal paths.Physianthropy research finds IL-6 The risk of the diseases such as the hereditary variation of gene and inflammatory reaction syndrome, coronary heart disease, carcinoma of prostate is related.Nearest research is sent out Existing, IL-6 genes illustrate this except in addition to immunocyte, tissue, organ expression, also receiving the muscular tissue great expression of miniature pig in version Gene Swine muscle play important physiological function (king with etc., 2015).In consideration of it, the present invention is by finding pig IL-6 genes Hereditary variation, there is provided the molecular marker related to weaned piglet weight and diarrhea disease and its detection method, and it is applied to pig breeding.

The content of the invention

It is an object of the invention to provide to the molecule labelling method that piglet diarrhea is sick and weaning weight is related and being applied to In pig breeding.

In order to achieve the above object, following technological means be present invention employs：

The present invention has found two SNP sites in IL6 genes, wherein, the SNP related to weaned piglet weight and diarrhea disease Site is 1753T/C, at the 1753rd nucleotide of IL6 gene orders.With weaned piglet heavy phase close SNP site be 3789A/T, at the 3789th nucleotide of IL6 gene orders, the Gene bank accession number of described IL6 genes is NC_ 010451.3REGION:complement(101081042..101085451)GPC_000000591(https:// www.ncbi.nlm.nih.gov/nuccore/NC_010451.3？From=101081042＆to=101085451＆report =genbank＆strand=true), its nucleotide sequence is as shown in SEQ ID NO.1.

Further, the invention allows for the molecular marker developed based on described SNP site, it is preferred that described Molecular marker is PCR-RFLP molecular markers.

For described SNP site is converted into the primer pair of PCR-RFLP molecular markers also protection scope of the present invention it It is interior, it is wherein as follows for described 1753T/C SNP sites to be converted into the primer pair sequence of PCR-RFLP molecular markers：

IL6-3F：5'TGCGAATGGGTTCTGACT 3'；

IL6-3R：5'AATCGCTCTTCCCTCTGG 3'.

Primer pair sequence for 3789A/T SNP sites to be converted into PCR-RFLP molecular markers is as follows：

IL6-5F：5'CAAATGTGAACCTCCTCC 3',

IL6-5R：5'AACAACCTGGCTCTGAAA 3'.

Further, the invention allows for described SNP site, molecular marker and primer sequence are in pig breeding Application.

Further, the present invention proposes a kind of method for evaluating weaned piglet weight and diarrhea condition of disease power, and which includes Following steps：

(1) genomic DNA is extracted from pig ear tissue；

(2) PCR amplifications

With pig genomic DNA as template, performing PCR amplification is entered using following primer pair：

IL6-3F：5'TGCGAATGGGTTCTGACT 3'；

IL6-3R：5'AATCGCTCTTCCCTCTGG 3'；

Wherein, it is preferred that PCR amplification programs are as follows：94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 35s, 53 DEG C of annealing 35s, 72 DEG C of extension 40s, totally 35 circulations；Last 72 DEG C of extensions 8min；

(3) HpaII PCR-RFLP detections

The product that step (2) amplification is obtained is carried out into enzyme action with HpaII restricted enzyme, after enzyme action is finished, agar is used Sugared detected through gel electrophoresis enzyme action result, amplified production occur 3 kinds of banding patterns Jing after HpaII enzyme action, represent 3 kinds of different genes respectively Type：When amplified fragments cannot be recognized by HpaII, TT genotype is denoted as；When amplified fragments are two short-movie sections by HpaII enzyme action When, it is denoted as CC genotype；When amplified fragments are three short-movie sections by HpaII enzyme action, TC genotype is denoted as, in pig breeding, TT and TC genotype individuals are selected, CC genotype individuals is eliminated, average weaning weight and the diarrhea condition of disease power of swinery can be improved.

And a kind of method for evaluating weaned piglet weight, which comprises the following steps：

(1) genomic DNA is extracted from pig ear tissue；

(2) PCR amplifications

With pig genomic DNA as template, performing PCR amplification is entered using following primer pair：

IL6-5F：5'CAAATGTGAACCTCCTCC 3',

IL6-5R：5'AACAACCTGGCTCTGAAA 3'

Wherein, it is preferred that PCR amplification programs are as follows：94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 35s, 50 DEG C of annealing 35s, 72 DEG C of extension 40s, totally 35 circulations；Last 72 DEG C of extensions 8min；

(3) DraI PCR-RFLP detections

The product that step (2) amplification is obtained is carried out into enzyme action with DraI restricted enzyme, after enzyme action is finished, agarose is used Detected through gel electrophoresis enzyme action result, amplified production occur 3 kinds of banding patterns Jing after DraI enzyme action, represent 3 kinds of different genotype respectively： When amplified fragments are that two bar segments are other by DraI enzyme action, AA genotype is denoted as；When amplified fragments are three silvers by DraI enzyme action Duan Shi, is denoted as TT genotype；When amplified fragments are four short-movie sections by DraI enzyme action, AT genotype is denoted as, in pig breeding, AA and AT genotype individuals are selected, TT genotype individuals is eliminated, the average weaning weight of piglet group can be improved.

To sum up, the present invention has found two and diarrhea disease associated SNP positions heavy with weaned piglet in IL6 genes, wherein One SNP site is 1753T/C, and at the 1753rd nucleotide of IL6 gene orders, itself and weaned piglet are weighed and diarrhea Sick related, when the site is T, piglet has higher weaning weight and diarrhea ability, and cannot be known by HpaII Not, therefore by PCR-RFLP molecular marking techniques piglet can be divided into three kinds of genotype：TT genotype, TC genotype and CC Genotype.In pig breeding, select TT and TC genotype individuals, eliminate CC genotype individuals, can improve swinery weaning weight and Diarrhea condition of disease power, during pig breeding, reduces CC genotypic frequencies consciously, to improving the diarrhea disease of swinery, carrying It is a beneficial breeding measures for high weaned piglet weight.

Another SNP site is 3789A/T, at the 3789th nucleotide of IL6 gene orders, with weaned piglet weight Correlation, when the site is A, piglet has higher weaning weight, and cannot be recognized by DraI, therefore passes through PCR-RFLP Piglet can be divided into three kinds of genotype by molecular marking technique：AA genotype, AT genotype and TT genotype.In pig breeding, Site TT genotype individuals are eliminated, the average weaning weight of piglet group can be improved.During pig breeding, TT is reduced consciously Genotypic frequency, is a beneficial breeding measures for improving weaned piglet weight.

Description of the drawings

Fig. 1 is 1 pig IL6-3 primer amplified fragments agarose gel electrophoretograms of embodiment；

Wherein, M swimming lanes：DNA Marker DL2,000；Swimming lane 1 is primer amplified fragments；

Fig. 2 is 1 pig IL6-5 primer amplified fragments agarose gel electrophoretograms of embodiment；

Wherein, M swimming lanes：DNA Marker DL2,000；Swimming lane 1 is primer amplified fragments；

Fig. 3 is that pig IL6-3 primer extension increasing sequence comparison results of the present invention and SNP site show；

Fig. 4 is that pig IL6-5 primer extension increasing sequence comparison results of the present invention and SNP site show；

IL6 gene HpaII PCR-RFLP testing results of the Fig. 5 for embodiment 2；

In figure, M swimming lanes：DNA Marker DL2,000；Swimming lane 1 is CC genotype；Swimming lane 2 is TT genotype；Swimming lane 3 is TC genotype.

IL6 gene DraI PCR-RFLP testing results of the Fig. 6 for embodiment 3.

In figure, M swimming lanes：DNA Marker DL2,000；Swimming lane 1 is TT genotype；Swimming lane 2 is AA genotype；Swimming lane 3 is AT genotype.

Specific embodiment

Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from this In the case of bright spirit and essence, the modification made to the inventive method, step or condition or replacement belong to the scope of the present invention.

In case study on implementation of the present invention, variety source used derives from Lanxi County Heilongjiang Province kind pig farm.

Embodiment 1：The acquisition of the molecular marker related to piglet diarrhea disease and weaning weight:

1) design of primers of pig IL6 genes is expanded with partial dna sequence

Pig IL6 gene mRNA sequences (GeneBank accession number is downloaded from NCBI：) and genome sequence NM_214399.1 (GeneBank accession number：NC_010451.3REGION:complement(101081042..101085451)GPC_ Shown in 000000591, SEQ ID NO.1).Two sequences are compared, with download sequence as (NC_010451.3REGION: Shown in complement (101081042..101085451) GPC_000000591, SEQ ID NO.1) calligraphy or painting model sequence, utilize Primer5 software Design primers IL6-3 are used for expanding IL6 Gene Partials Inron2, Intron3 and complete Exon3 regions, if Meter primer I L6-5 is used for expanding IL6 Gene Partials Intron4 and part Exon5 regions, and all primers are by giving birth to work biological engineering (Shanghai) limited company synthesizes, and primer sequence is as shown in table 1 below：

1. pig IL6 gene amplification primers of table

2) pcr amplification reaction

Using Genomic DNA Kit test kits (purchased from Sangon Biotech (Shanghai) Co., Ltd.) from people pig and Genomic DNA is extracted in Landrace ear tissue, and concrete operation method is carried out with reference to Genomic DNA Kit kit specifications.

With people pig and Landrace genomic DNA as template, enter performing PCR amplification respectively using primer I L6-3 and IL6-5.PCR Reaction system is 25 μ L, and wherein template DNA is 1 μ L, each 0.5nmol/ μ L of upstream and downstream primer, 12.5 μ L of PCRmix (precious biological), Plus deionized water is to 25 μ L of cumulative volume.PCR programs are as follows：94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 35s, annealing 35s (IL6-3 is 53 DEG C, and IL6-5 is 50 DEG C), 72 DEG C of extension 40s, totally 35 circulations；Last 72 DEG C of extensions 8min.The amplification of PCR is produced The agarose gel of thing Jing 1.2% carries out electrophoresis detection.IL6-3 primers amplified fragments are 698bp (Fig. 1 and SEQ ID NO.2 It is shown), the amplified fragments of IL6-5 primers are 565bp (shown in Fig. 2 and SEQ ID NO.3).

3) PCR primer purification, sequencing and sequence alignment

IL6-3 with IL6-5 primer extension products cut purpose band Jing after 1.2% sepharose electrophoresis are separated, using agar Sugared gel DNA QIAquick Gel Extraction Kits (Beijing Tiangeng) purified pcr product, and send Sangon Biotech (Shanghai) Co., Ltd. to enter Row sequencing.Multiple sequence alignments are carried out using ClusterW softwares, the nucleoside in IL6-3 and IL6-5 primer amplified fragments is retrieved Acid mutation site.IL6-3 primers amplified fragments are 698bp, co-exist in coding mutation at 3, wherein at fragment 302bp T/C variations cause the change (Fig. 3) of HpaII restriction enzyme sites (C ↓ CGG) (at NC_010451.3 sequences 1753bp).IL6-5 Primer amplified fragments are 565bp, there is coding mutation at 1, A/T variation (the NC_010451.3 sequences at fragment 64bp At row 3789bp) cause the change (Fig. 4) of DraI restriction enzyme sites (TTT ↓ AAA).

Embodiment 2：The detection method in HpaII sites, its step are as follows:

1) primer sequence

Primer for PCR amplifications is IL6-3 primers, and primer sequence is as follows

IL6-3F:5'TGCGAATGGGTTCTGACT 3'；

IL6-3R:5'AATCGCTCTTCCCTCTGG 3'

2) PCR reaction conditions

Expanded in pig genomic DNA using IL6-3 primers, PCR reaction systems are 25 μ L, wherein template DNA is 1 μ L, each 0.5nmol/ μ L of upstream and downstream primer, 12.5 μ L of PCRmix (precious biological), plus deionized water is to 25 μ L of cumulative volume.PCR journeys Sequence is as follows：94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 35s, 53 DEG C of annealing 35s, 72 DEG C of extension 40s, totally 35 circulations；Finally 72 DEG C of extension 8min.

3) HpaII PCR-RFLP detections

PCR primer endonuclease reaction total system be 10 μ l, wherein 8.5 μ l of PCR primer, 0.5 μ l (10U/ μ l) HpaII inscribes Enzyme, 10 × buffer are 1 μ l.To be centrifuged after sample blending, 5h is put in 37 DEG C of water-baths, examined with 1.5% agarose gel electrophoresiies Enzyme action result is surveyed, genotype is recorded, take pictures under uviol lamp (Fig. 5).There is SNP mutation at sequence 302bp, the mutation is produced HpaII restriction enzyme sites.As shown in figure 5, IL6-3 primer extension products occur 3 kinds of banding patterns Jing after HpaII enzyme action, 3 kinds are represented respectively Different genotype.Wherein, 1 swimming lane is TC genotype (698bp, 397bp and 301bp)；2 swimming lanes are TT genotype (698bp)； 3 swimming lanes are CC genotype (397bp and 301bp), and 4 swimming lanes are DL2000Marker；

Embodiment 3：The detection method in DraI sites, its step are as follows：

1) primer sequence

Primer for PCR amplifications is IL6-5 primers, and primer sequence is as follows

IL6-5F:5'CAAATGTGAACCTCCTCC 3'

IL6-5R:5'AACAACCTGGCTCTGAAA 3'

2) PCR reactions

Expanded in pig genomic DNA using IL6-5 primers, PCR reaction systems are 25 μ L, wherein template DNA is 1 μ L, 12.5 μ L of each 0.5nmol/ μ L of upstream and downstream primer, PCRmix (precious biological), plus deionized water is to 25 μ L of cumulative volume.PCR programs are as follows：94 DEG C denaturation 4min；Then 94 DEG C of degeneration 35s, 50 DEG C of annealing 35s, 72 DEG C of extension 40s, totally 35 circulations；Last 72 DEG C of extensions 8min.

3) DraI PCR-RFLP detections

PCR primer endonuclease reaction total system be 10 μ l, wherein 8.5 μ l of PCR primer, 0.5 μ l (10U/ μ l) DraI restriction endonucleases, 10 × buffer is 1 μ l.To be centrifuged after sample blending, 5h is put in 37 DEG C of water-baths, detect enzyme with 1.5% agarose gel electrophoresiies Result is cut, genotype is recorded, take pictures under uviol lamp (Fig. 6).

There are two DraI restriction enzyme sites in the extension increasing sequence of IL6-5 primer 566bp, an intrinsic restriction enzyme site is located at At 413bp, another restriction enzyme site produced due to SNP mutation at 64bp.

As shown in fig. 6, IL6-5 primer extension products occur 3 kinds of banding patterns Jing after DraI enzyme action, represent respectively 3 kinds it is different Genotype.Wherein, 1 swimming lane is AT genotype (415bp, 351bp, 151bp and 64bp)；2 swimming lanes be AA genotype (415bp and 151bp)；3 swimming lanes are TT genotype (351bp, 151bp and 64bp), and 1 swimming lane is DL2000Marker.

Application of the molecular marker of 4 present invention of embodiment in colony's polymorphic detection：

The people pig of the implementation case and Landrace test colony from Lanxi County Heilongjiang Province kind pig farm.It is white in people pig and length In swinery body, using the gene frequency and genotypic frequency in HpaII PCR-RFLP technology for detection SNP (302C/T) sites；By table 2 is visible, and the site has three kinds of genotype in people's swinery body, and it is 68 that wherein TT types are individual, and it is 176 that TC types are individual, CC It is 37 that type is individual, and T gene frequencies are 0.56.Three kinds of genotype, wherein TT types are also detected that in Landrace colony Body is 8, and it is 50 that TC types are individual, and it is 47 that CC types are individual, and T gene frequencies are 0.31 (table 2), illustrate the inspection of the present invention Survey method can be used for population genetics analysis.

2 IL6 gene HpaII sites of table are in different pig kind genotypic frequencies and gene frequency

In people pig and Landrace colony, using the gene in DraI PCR-RFLP technology for detection SNP (64bp T/A) site Frequency and genotypic frequency；From table 3, there are three kinds of genotype in the site in people's swinery body, and it is 52 that wherein AA types are individual Individual, it is 129 that AT types are individual, and it is 32 that TT types are individual, and A gene frequencies are 0.55.Also detect that in Landrace colony It is 8 that three kinds of genotype, wherein AA types are individual, and it is 49 that AT types are individual, and it is 43 that TT types are individual, and A gene frequencies are 0.33 (table 3), illustrates that the detection method of the present invention can be used for population genetics analysis.

3 IL6 gene DraI sites of table are in different pig kind genotypic frequencies and gene frequency

Embodiment 5：Application of the molecular marker of the present invention in piglet diarrhea and weaning weight association analysiss

The test swinery of the implementation case is from Lanxi County Heilongjiang Province kind pig farm.With the people that the same period in autumn in 2013 is born Pig suckling pig is test material.Routinely production requirement feeding and management and the epidemic prevention under the same conditions of all experimental animals.It is young Pig is born to 35 ages in days and weans, and every morning and respectively once observes the defecation situation of piglet in each colony house afternoon, and by head inspection Anus whether there is piglet faecal condition in red and swollen and fecal pollution and pigsty, then carries out piglet diarrhea and scores and record, and scores Standard is as follows：Feces outward appearance is in strip or granular, is normal diarrhoea, scores as 0；Feces outward appearance is in soft stool, can shape, and is slight Diarrhoea, scores as 1；Feces outward appearance without segregation phenomenon, loose stool, is mild diarrhea, scores as 2 in thick shape liquid dung；Feces outward appearance is in Liquid, shapeless, liquid dung are separated, and Mucous Stool or pus just, are severe diarrhoea, score as 3.Scours index is that piglet birth starts extremely 35 age in days faecal conditions scoring sum.During testing, at the same weigh the birth weight of piglet, 7 age in days weights, 14 age in days weights, 21 days Age weight, 28 age in days weights and 35 age in days weaning weights, and recorded.

The HpaII PCR-RFLP technologies and DraI PCR-RFLP technologies set up using embodiment 1 by inventor exists respectively Carry out polymorphic detection in people's swinery body, and analyze the related of two kinds of molecular markers and piglet diarrhea index and some growth character Property.Using SAS statistical softwares, (8.0) GLM programs carry out single labelling variance analyses, institute for SAS Institute Inc, Version Adopt model for：

Yi=μ+Gi+ei

Wherein Yi is trait phenotypes value, and μ is meansigma methodss, and Gi is genotype effects；Ei is residual error effect.

In people's swinery body, HpaII sites different genotype is tied with the association analysiss of piglet diarrhea index and some growth character Fruit is shown in Table 4.As can be seen that when HpaII genotype is different, the individual Scours index of CC types is significantly higher than TT types individuality (P< 0.05), 7 individual age in days body weight of CC types, 14 age in days body weight, 21 age in days body weight, 28 age in days body weight and 35 age in days weanling weights are equal Substantially less than TC types individuality (P<0.05), 21 individual age in days body weight of CC types are also significantly lower than TT types individuality (P<0.05), therefore, CC genotype individuals are eliminated in breeding process can improve the average diarrhea ability of piglet colony and weanling weight.

In people's swinery body, DraI sites different genotype is tied with the association analysiss of piglet diarrhea index and some growth character Fruit is shown in Table 5.As can be seen that when DraI genotype is different, 14 individual age in days body weight of TT types are substantially less than AA types individuality (P< 0.05), 21 individual age in days body weight of TT types are substantially less than AA types and AT types individuality (P<0.05), 28 individual age in days body weight of TT types AT types individuality (P is substantially less than with 35 age in days body weight<0.05).Therefore, eliminating TT genotype individuals in breeding process can be with Improve the average weanling weight of piglet colony.

4 IL6 gene HpaII sites of table and people's porkling diarrhea of pigs index and the association analysiss table of the production traitss

5 IL6 gene DraI sites of table and people's porkling diarrhea of pigs index and the association analysiss table of the production traitss

Note：Above numerical value is least square mean value ± standard error, and when numerically target letter is different, capitalization represents poor Heteropole significantly (P<0.01), lowercase letter indication difference significantly (P<0.05).

Sequence table

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actcccagcc tccttatact atcagagcag ttgactctta atagatgctg tatggatggt 3420

tgagagggaa accagagaca agtaattagg gttagagggc cctgaagaac ttattaaaaa 3480

cggctttaag taaaagttct tcatttatct actgaattcc aactatgtgc caggcattcc 3540

ttaggataac agaaaacaag taaggtacta tggctgccca cggatgctca atggcctaac 3600

agagatgggc agcaaaggga tgtgtagagc tcagttgcta tgggagagct gcaggctggg 3660

tgggctcaga ggagaggcac ctccccctgt ctgggcgagt cacagcccca gttcacacct 3720

tcccccaaat gtgaacctcc tcccacaggc cagagcatcc ctccactgca gaggattcat 3780

tcaatattta aacaattatt tgtgttttct tttcccttca gggaaagaat ccagacaaag 3840

ccaccacccc taaccccacc acaaatgccg gcctgctgga taagctgcag tcacagaacg 3900

agtggatgaa gaacacaaag atcattctca tcctgcgcag ccttgaggat ttcctgcagt 3960

tcagcctgag ggccattcgg ataatgtagc tggggcacct gagattgatg ccgtccacgg 4020

gcattccctc ctctggtcag aaacctgtcc actgggcaca taacttatgt tgttctctat 4080

gaagaactaa aagtatgagc gttaggacac tattttaatt attttaattt attgatattt 4140

aaatatgtga tgtcgagtta atttatataa gtgatagata tttatatttt tattaagtgc 4200

cacttgaaat attttatgta tttgttttga aaaagtaacg taaaatggct acacggcttg 4260

aagatccttg ttgtttcaga gccaggttgt ttcttggagt gtgtaggctt acctcaaata 4320

aattgctaac ttatacgtat ttttaaaaga aatatttata ttgtatttat ataaagttta 4380

agttgttttt ataccaataa acaccttttt 4410

<210> 2

<211> 698

<212> DNA

<213> Sus scrofa

<400> 2

tgcgaatggg ttctgactcc acttttctta gagaacttcc tggccatggt agatcatgaa 60

tcagccctct agtggtgttc gttttgggag cacttatatg ataacagctc taatactctt 120

tcccagatgt gtgagaagta tgagaagtgt gaaaacagca aggaggtact ggcagaaaac 180

aacctgaacc ttccaaaaat ggcagaaaaa gacggatgct tccaatctgg gttcaatcag 240

gtatggatgc attacattca cttttagctg ctccccagtc caaagttctt cttcttataa 300

ccggtgtctg tacacatgta gaccaagcag gcatcaaaag taggggagaa atgtgaagaa 360

catcaggtaa atttcctgag gaggccaact taaatgttat taaaggcaac ttatttttga 420

atgacctggt cagaggtagc tcctcggtgt tgggattttc acagctgtca aaggggttag 480

aacagcccac aggtttagct agttcatcct gggtgtgatg gtccagggag gtttatcctc 540

cttggttgcc tgtagcagga tccagtccta atctccccac tgagaaagtt ctctgcaccc 600

tctccctcca gacagtgaga taacctgctt attccataaa acgaggtttc agtccaaccg 660

tggcaaaggc atgagggcag ccagagggaa gagcgatt 698

<210> 3

<211> 565

<212> DNA

<213> Sus scrofa

<400> 3

caaatgtgaa cctcctccca caggccagag catccctcca ctgcagagga ttcattcaat 60

attaaacaat tatttgtgtt ttcttttccc ttcagggaaa gaatccagac aaagccacca 120

cccctaaccc caccacaaat gccggcctgc tggataagct gcagtcacag aacgagtgga 180

tgaagaacac aaagatcatt ctcatcctgc gcagccttga ggatttcctg cagttcagcc 240

tgagggccat tcggataatg tagctggggc acctgagatt gatgccgtcc acgggcattc 300

cctcctctgg tcagaaacct gtccactggg cacataactt atgttgttct ctatgaagaa 360

ctaaaagtat gagcgttagg acactatttt aattatttta atttattgat atttaaatat 420

gtgatgtcga gttaatttat ataagtgata gatatttata tttttattaa gtgccacttg 480

aaatatttta tgtatttgtt ttgaaaaagt aacgtaaaat ggctacacgg cttgaagatc 540

cttgttgttt cagagccagg ttgtt 565

Claims (10)

1. to weaned piglet weight and the sick related SNP site of diarrhea, it is characterised in that described SNP site is 1753T/C, At the 1753rd nucleotide of IL6 gene orders, the nucleotide sequence of described IL6 genes is as shown in SEQ ID NO.1.

2. the SNP site closed with weaned piglet heavy phase, it is characterised in that described SNP site is 3789A/T, positioned at IL6 genes At 3789th nucleotide of sequence, the nucleotide sequence of described IL6 genes is as shown in SEQ ID NO.1.

3. the molecular marker developed based on the SNP site described in claim 1, it is preferred that described molecular marker is PCR- RFLP molecular markers.

4. the molecular marker developed based on the SNP site described in claim 2, it is preferred that described molecular marker is PCR- RFLP molecular markers.

5. for the SNP site described in claim 1 to be converted into the primer pair of PCR-RFLP molecular markers, it is characterised in that institute The primer sequence stated is as follows：

IL6-3F：5'TGCGAATGGGTTCTGACT 3'；

IL6-3R：5'AATCGCTCTTCCCTCTGG 3'.

6. for the SNP site described in claim 2 to be converted into the primer pair of PCR-RFLP molecular markers, it is characterised in that Described primer sequence is as follows：

IL6-5F：5'CAAATGTGAACCTCCTCC 3',

IL6-5R：5'AACAACCTGGCTCTGAAA 3'.

7. application of the SNP site described in claim 1 and/or 2 in pig breeding.

8. application of the molecular marker described in claim 3 or 4 in pig breeding.

9. it is a kind of to evaluate the heavy method with diarrhea condition of disease power of weaned piglet, it is characterised in that to comprise the following steps：

(1) genomic DNA is extracted from pig ear tissue；

(2) PCR amplifications

With pig genomic DNA as template, performing PCR amplification is entered using following primer pair：

IL6-3F：5'TGCGAATGGGTTCTGACT 3'；

IL6-3R：5'AATCGCTCTTCCCTCTGG 3'；

Preferably, PCR amplification programs are as follows：94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 35s, 53 DEG C of annealing 35s, 72 DEG C prolong 40s is stretched, totally 35 circulations；Last 72 DEG C of extensions 8min；

(3) HpaII PCR-RFLP detections

The product that step (2) amplification is obtained is carried out into enzyme action with HpaII restricted enzyme, it is after enzyme action is finished, solidifying with agarose Gel electrophoresis detect enzyme action result, and amplified production occurs 3 kinds of banding patterns Jing after HpaII enzyme action, represents 3 kinds of different genotype respectively： When amplified fragments cannot be recognized by HpaII, TT genotype is denoted as；When amplified fragments are two short-movie sections by HpaII enzyme action, It is denoted as CC genotype；When amplified fragments are three short-movie sections by HpaII enzyme action, TC genotype is denoted as, in pig breeding, is selected TT and TC genotype individuals, eliminate CC genotype individuals, can improve average weaning weight and the diarrhea condition of disease power of swinery.

10. it is a kind of to evaluate the heavy method of weaned piglet, it is characterised in that to comprise the following steps：

(1) genomic DNA is extracted from pig ear tissue；

(2) PCR amplifications

With pig genomic DNA as template, performing PCR amplification is entered using following primer pair：

IL6-5F：5'CAAATGTGAACCTCCTCC 3',

IL6-5R：5'AACAACCTGGCTCTGAAA 3'

Preferably, PCR amplification programs are as follows：94 DEG C of denaturations 4min；Then 94 DEG C of degeneration 35s, 50 DEG C of annealing 35s, 72 DEG C prolong 40s is stretched, totally 35 circulations；Last 72 DEG C of extensions 8min；

(3) DraI PCR-RFLP detections

The product that step (2) amplification is obtained is carried out into enzyme action with DraI restricted enzyme, after enzyme action is finished, agarose gel is used Electrophoresis detection enzyme action result, amplified production occur 3 kinds of banding patterns Jing after DraI enzyme action, represent 3 kinds of different genotype respectively：Work as expansion When increasing fragment is that two bar segments are other by DraI enzyme action, AA genotype is denoted as；When amplified fragments are three bar segments by DraI enzyme action, It is denoted as TT genotype；When amplified fragments are four short-movie sections by DraI enzyme action, AT genotype is denoted as, in pig breeding, is selected AA and AT genotype individuals, eliminate TT genotype individuals, can improve the average weaning weight of swinery.