CN105255878B - It is used for SNP marker and its application traced to the source on No. 6 chromosomes of pig - Google Patents

It is used for SNP marker and its application traced to the source on No. 6 chromosomes of pig Download PDF

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CN105255878B
CN105255878B CN201510783471.9A CN201510783471A CN105255878B CN 105255878 B CN105255878 B CN 105255878B CN 201510783471 A CN201510783471 A CN 201510783471A CN 105255878 B CN105255878 B CN 105255878B
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snp marker
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pig
chromosomes
snp
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CN105255878A (en
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吴潇
唐雪明
吕贝贝
蒋玮
王金斌
武国干
李鹏
白蓝
刘华
赵凯
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Shanghai Academy of Agricultural Sciences
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Abstract

The invention discloses SNP marker and its application for being used to trace to the source on No. 6 chromosomes of pig, described SNP marker is obtained by ncbi database sequence alignment, the SNP marker is by TaiI restriction enzyme specific recognitions, common 550bp, there is a base to replace at 362nd bit base, for 362A or 362G, its sequence is as shown in SEQ ID NO.1.By analyzing distribution situation of the SNP marker in experiment colony allelic in the experiment colony including 10 pig varieties or strain, it was found that gene frequency of the SNP marker of the present invention in different kinds or strain approaches, rich polymorphism, gene frequency distributional difference is small between kind or strain, and heterozygosity is both greater than 0.3, the SNP marker can be used as DNA source tracing of pork products.

Description

It is used for SNP marker and its application traced to the source on No. 6 chromosomes of pig
Technical field
The invention belongs to field of food safety, be related to the SNP marker that is used for tracing to the source on No. 6 chromosomes of a pig and its Detection method.
Background technology
With the development of society, the link in agri-food supply chains is being continuously increased, from farm, pasture to food enterprise plus There is the potential safety hazard of food in work, packaging, storage, transport and sale, the links of food supply, food-safety problem often has Occur.Therefore need to carry out meat products can tracing management, establish the retrospect systems of the links from the place of production to dining table, so as to The information of accurate and detailed article is provided for consumer, is advantageous to operator and finds in time present in each link Hidden danger.Since the food security crisis of mid-term the 1990s, the retrospect of meat product is as a kind of safety Control measure, by the multiple national great attentions in the world.Meat product traceability system strengthens government department to meat product quality The ability to supervise of safety, therefore many countries establish meat product traceability system one after another and existed to reduce in meat product quality safety Risk.
Meat product tracing technology has label tracing technology, isotopic traceability technology, mineral element fingerprint tracing technology, organic Thing tracing technology iris feature technology and DNA marker tracing technology (or DNA tracing technologies) etc., wherein DNA tracing technologies because Detection means is simple, it is quick, be difficult to turn into the wide variety of quick tracing technology in countries in the world the features such as forgery.With other marks Method is compared, and DNA marker, which has, peculiar is put into superiority:It directly occurs with DNA form, each tissue in organism, each Developmental stage is detectable, is not influenceed by environmental factor, and marker number is more, throughout organism whole gene group.
SNP marker refers to the variation of single nucleotide acid on the same site of genome, normally behaves as two allele, very Suitable high throughput automated analysis, so having become animal identification identifies molecular labeling of greatest concern.
But the SNP marker traced to the source for meat product is different from general SNP marker, for single SNP marker, it must Must at least have following characteristics:(1) degree of variation is high, is approached in kind or strain allelic frequency;(2) equipotential base between kind Because distribution frequency difference is small;(3) heterozygosity is more than or equal to 0.3.
In long-term breeding work, researcher have accumulated substantial amounts of SNP marker, because single SNP marks of tracing to the source are nothings Method completes what is traced to the source, therefore, it is necessary to one group of separate SNP marker could be realized and traced to the source.With existing 13 known SNP points Son mark combines as mark of tracing to the source, in the swinery body introduced by height selection, when being traced to the source using the group echo, and meeting There are some undistinguishable situations of individual.
In addition, in long-term breeding work, the substantial amounts of SNP marker of accumulation often integrated distribution in some chromosomes On, and other part chromosome, such as No. 5, No. 6, No. 8, No. 14, No. 15 and No. 18 chromosomes etc. then lack SNP marker, easily lead Cause cascade phenomenon occur between molecular labeling, can not be independent mutually between molecular labeling, the detection range for experiment of tracing to the source is reduced, is caused Actual detectable scope is less than expected detectable scope.Therefore, in order to meet the requirement traced to the source pork product progress DNA, need The SNP marker for being used to trace to the source is developed on other chromosomes, improves trace to the source detection range and the degree of accuracy.
The content of the invention
, should it is an object of the invention to provide SNP marker and its application for being used to trace to the source on No. 6 chromosomes of a pig SNP marker can be widely applied to DNA source tracing of pork products, the particularly peace for pork product in market pig is cultivated Full property is traced to the source, perfect existing mark combination, and raising is traced to the source accuracy in detection, and expansion is traced to the source scale.
To achieve the above object, the technical solution adopted by the present invention is as follows:
It is used for the SNP marker traced to the source, the DNA sequence dna such as SEQ ID of the SNP marker on No. 6 chromosomes of one pig Shown in NO.1, common 550bp, and there is a base to replace at the 362nd bit base, it is 362A or 362G.
Further, described SNP marker is by TaiI restriction enzyme specific recognitions.
Described SNP marker is obtained by ncbi database sequence alignment, and including 10 pig varieties or In the experiment colony (amounting to 245 individuals) of strain, distribution feelings of the SNP marker in experiment colony allelic are analyzed Condition, it is found that gene frequency of the molecular labeling in different kinds and strain approaches, rich polymorphism, kind or strain Between gene frequency distributional difference it is small, the preliminary judgement SNP marker can be used as DNA source tracing of pork products.
The preparation method of one on described No. 6 chromosomes of the pig SNP marker that can be used for tracing to the source, using PCR- TaiI-RFLP methods are carried out, and are comprised the following steps:
1) the DNA sequence dna information of No. 6 chromosomes of pig is searched on NCBI, designs forward and reverse primer separation No. 6 chromosomes of pig DNA fragmentation;
2) sequence alignment is carried out online using ncbi database, find the site that base replacement be present;
3) PCR-TaiI-RFLP detection methods are established and site is replaced in detection;
4) the ear tissue sample of 10 kinds and strain is gathered, altogether 245 individuals;
5) distribution of the SNP marker in experiment colony, statistical analysis are detected, and further whether verification experimental verification is applied to During pork product is traced to the source.
Wherein, in the method for above-mentioned acquisition SNP marker, on described separation No. 6 chromosomes of pig DNA fragmentation just, Reverse primer sequences are as follows:
Forward primer:5 '-AAATGAAGAACAGGCAAGG-3 ' (as shown in SEQ ID NO.2);
Reverse primer:5 '-CTTCCCAGTAAACTTAGGAGATA-3 ' (as shown in SEQ ID NO.3).
During DNA traces to the source, often increase a site of tracing to the source, just can further expand the number of detection sample, accurately Property it is also just higher, preferable SNP site should be generally evenly distributed on the different chromosome of pig, and in existing SNP site lack Mark of tracing to the source on weary No. 6 chromosome.
The present invention detects the existing SNP marker on No. 6 chromosomes of pig by ncbi database sequence alignment, In experiment colony including 10 pig varieties or strain, distribution of the SNP marker in experiment colony allelic is analyzed Situation, it is found that gene frequency of the molecular labeling in different kinds or strain approaches, rich polymorphism, kind or product Gene frequency distributional difference is small between system, and heterozygosity is both greater than 0.3, and the preliminary judgement SNP marker can be used as pork production During product DNA is traced to the source and the security of pork product is traced to the source.
It can not complete to trace to the source because single SNP traces to the source to mark, therefore in simulated test of tracing to the source, the present invention is obtained No. 6 chromosomes of pig on Novel SNP molecular marker site be combined together with other existing published SNP marker sites (amounting to 14 SNP sites) carries out experiment of tracing to the source.The present invention increases market pig source, chosen altogether in slaughterhouse in experiment of tracing to the source 300 individuals from multiple different pig farms have been selected, have selected 100 individuals at random from 300 individuals, have detected this 100 The genotype of 14 SNP sites in body, the genotype results of each individual are counted, it is found that 100 individuals possess respective difference Genotype, can realize individual differentiation;Meanwhile 20 parts of muscle samples of picking, same detection statistics 14 in this 100 individuals at random The genotype in individual SNP marker site, be then compared with 100 individual genotype, discovery can find with The completely the same individual of 20 parts of muscle samples genotype, i.e., the source of 20 parts of pig muscles is found in 100 individuals by tracing to the source Individual, therefore, it is determined that the SNP marker of the present invention can be used for pork product and trace to the source.
Compared with prior art, beneficial effects of the present invention:
The SNP marker of the present invention is located on No. 6 chromosomes of pig, can further improve the detection coverage rate of mark, The accuracy traced to the source is improved, and expands the detection range of existing molecular labeling.
Embodiment
Technical scheme is described in further detail below in conjunction with specific embodiment.
The lookup of the molecular labeling of embodiment 1
(1) design of primers
With the DNA sequence dna (Genbank of No. 6 chromosomes of pig:FN675082 it is) template, the DNA pieces of design primer separation pig Section (template expanded using the DNA of a duroc as PCR), primer is as follows:
Forward primer:5 '-AAATGAAGAACAGGCAAGG-3 ' (as shown in SEQ ID NO.2)
Reverse primer:5 '-CTTCCCAGTAAACTTAGGAGATA-3 ' (as shown in SEQ ID NO.3)
PCR reaction cumulative volumes are 20 μ l, wherein pig genomic DNA about 100ng, containing 1 × buffer (Promega companies), 1.5mmol/L MgCl2, dNTP (Shanghai Sheng Gong biotech firms) final concentration of 150 μm of ol/L, the final concentration of 0.2 μm of ol/ of primer L, 2U Taq archaeal dna polymerases (Promega companies).
PCR is expanded:94 DEG C of 4min, (94 DEG C of 30s, 60 DEG C of annealing 30s, 72 DEG C of 30s) circulation 30 times, last 72 DEG C of extensions 10min。
PCR reaction products are detected with 1% agarose gel electrophoresis.PCR reaction products are examined with 1% agarose gel electrophoresis Survey.
(2) cloning and sequencing is analyzed
The DNA fragmentation of obtained No. 6 chromosome of pig is cloned as follows.
The purifying of PCR primer:The gel containing purpose fragment is cut from Ago-Gel under uviol lamp, is put into 1.5ml In Ependorff pipes, purified with PCR primer purification kit (Tiangeng biochemical technology Co., Ltd).
Coupled reaction:The PCR primer of purifying is connected with pMD18-T carriers (Dalian treasured biotech firm), coupled reaction is total Volume is 10 μ l, and including 5 μ l solution I (Dalian treasured biotech firm), (the precious biology in Dalian is public for 0.5 μ l carrier T Department), 2.5 μ l purified pcr product, it is eventually adding 2 μ l aqua sterilisas and puts 4 DEG C of water-baths and stay overnight.
Conversion:100-120 μ l competent cells (Tiangeng biochemical technology Co., Ltd) are taken under germ-free condition in 1.5ml In Ependorff pipes, 5 μ l connection product is added and mixed, 30min is placed on ice, 42 DEG C of heat shock 90s, not shaken therebetween Dynamic Ependorff pipes, ice bath 3-4min after taking-up add the LB fluid nutrient mediums of 400 μ l antibiotic-frees, 37 DEG C keep flat 1h after fall Put culture.
Bacterium colony PCR is identified:Bacterial strain after bacterium colony PCR identifications is in 37 DEG C of overnight incubations of LB culture mediums, and picking is multiple immediately Clone is sent to the sequencing of Shanghai life work biology Co., Ltd.
After tested, the common 550bp of DNA sequence dna through spliced No. 6 chromosomes of pig, sequence is as shown in SEQ ID NO.1. Find that a base at its 362nd base be present replaces through analysis, be 362A or 362G.By molecular biology software analysis and Ncbi database is analyzed, it is found that the base at the 362nd base is replaced causes TaiI-RFLP (Restriction Fragment LengthPolymorphism, i.e. TaiI-RFLP sites) polymorphism.
(3) foundation of PCR-TaiI-RFLP detection methods and the detection in replacement site
The μ l of endonuclease reaction cumulative volume 10, wherein 1 × buffer 10 μ l, PCR primer 3-5 μ l, restriction enzyme TaiI are 0.5 μ l (5U), use H2O supplies 10 μ l, 65 DEG C of water-bath 4h, weighs 0.6g agaroses and is dissolved in 15.75ml DEPC (Shanghai life work lifes Thing company) handle in water, 5ml 5 × formaldehyde gel buffer solution and 4.25ml 37% formalin are added after slightly cooling down, Glue is mixed, digestion result is detected after electrophoresis.
As a result find:A allele only has mono- fragment of 550bp in SEQ ID NO.1 sequences, and G allele has 363bp With two fragments of 187bp, the two allele can form three kinds of genotype, AA, AG, GG.And in the SEQ ID NO.1 sequences The 362nd bit base replace, A → G.
The distribution situation of the allele of embodiment 2
(1) design of colony is tested
Test group:Gather Pietrain (21), Shen Nong (17), great Bai (43), great Shen (52), long Shen (16), (20) Du Shen (23), Pi great Shen (11), Shen of growing up (25), Du great Shen (17) and Du Pi great Shen individual ear groups Knit, extract DNA, altogether 245 DNA samples.
The purpose of colony is tested in the distribution situation in detection SNP marker in different cultivars.
(2) genetic test
Forward primer:5 '-AAATGAAGAACAGGCAAGG-3 ' (as shown in SEQ ID NO.2);
Reverse primer:5 '-CTTCCCAGTAAACTTAGGAGATA-3 ' (as shown in SEQ ID NO.3).
(amplification condition is with embodiment 1) is expanded, experiment colony is detected with identical PCR-TaiI-RFLP methods and owns Idiotype.
(3) statistical analysis
All idiotypes of record experiment colony, and gene frequency and heterozygosity are calculated, as a result such as table 1 below.
Table 1
As known from Table 1:Gene frequency of the SNP marker of the present invention in each kind approaches, rich polymorphism; Between kind or strain, gene frequency A and G distributional difference are small;The heterozygosity H of all kinds or strain is all higher than 0.3, Therefore it was initially believed that the DNA that the SNP marker can be used for pork product traces to the source and traced to the source with security.
The SNP marker availability of embodiment 3 is traced to the source verification experimental verification
The SNP marker of the present invention can be used in traceability mark by tentative confirmation, in order to ensure in mark of tracing to the source In availability, the present invention again resampled in different location, with existing 13 SNP markers (as described in Table 2) It is used in combination (totally 14 SNP markers), carries out availability and trace to the source verification experimental verification, meanwhile, only to be divided with existing 13 SNP Son mark trace to the source verification experimental verification work to ratio.
In the individual ear tissue of recovery slaughterhouse random acquisition pig and each 100 parts of muscle sample, (each pig individual is adopted simultaneously Collect ear tissue sample and muscle sample), ear tissue sample numbering E1-E100, muscle sample numbering M1-M100, muscle sample and ear are extracted respectively The DNA of tissue sample is standby.
The TaiI of the pig of the present invention is detected in 20 muscle samples selected in 100 individual ear tissue samples and at random The genotype (amounting to 14 SNP marker sites) of SNP site and existing published 13 SNP sites, statistics is per each and every one The genotype results of body.According to digestion banding pattern, a band is designated as 1, two bands occurs and is designated as 2, three bands are designated as 3.Record order Arranged according to the site of table 2 order, the SNP marker site as the enzymes of Pvu II of ADAMTS-1 genes identify is 1, its genotype Record result just makes number one, and the SNP marker site of the enzymes of Eam1104 I identification of ADD1 genes is 2, its genotype note Record result just comes second, the like, the TaiI SNP markers site on No. 6 chromosomes of pig of the invention comes most Afterwards, its genotype results is indicated as the 14th numeral from left to right, and individual genotype can be expressed as: 21232322223311。
Table 2
The genotype in 14 SNP marker sites of each individual of statistics and meat sample, 100 individuals and 20 parts of muscle samples The genotype Statistical Comparison result of product is respectively such as table 3 below and table 4.
In table 3, No. 82 individuals and No. 91 individual genotype are closely similar, and preceding 13 tag values of two individual are equal Unanimously, only difference is that the 14th mark is different.As can be seen here, traced back merely with existing 13 SNP markers During source, it may appear that the situation that genotype repeats, the 14th is labeled as New SNP marker provided by the invention, illustrates to add the present invention SNP marker after, it is possible to smoothly by this in two genotype make a distinction, the addition of Novel SNP molecular marker of the invention The further perfect scale of tracing to the source for combination of tracing to the source, improves the accuracy traced to the source with SNP marker pork DNA.
The genotype results in 14 SNP marker sites of 20 parts of muscle samples are entered with 100 individual genotype Row compares analysis (comparison result is referring to table 4), and discovery can find the individual completely the same with 20 parts of muscle samples genotype, The source individual of 20 parts of pig muscles is found in 100 individuals by tracing to the source, realizes that meat sample is traced to the source to the accurate of individual.
Illustrate with reference to table 3 and table 4, experiment of tracing to the source, 100 individuals, 20 parts of muscle samples are carried out using 14 SNP markers Possess different genotype with 14 SNP marker detections, individual differentiation can be realized.
Table 3
Numbering Genotype Numbering Genotype Numbering Genotype
E1 33332332132312 E35 33333232131333 E68 23311222133232
E2 21211233132123 E36 21333321323232 E69 23313333333223
E3 33333232331122 E37 22213233131322 E70 21231233333212
E4 23311232111313 E38 31221211121113 E71 21121333332233
E5 23113332231331 E39 22333231121222 E72 21231122211123
E6 23332221321322 E40 33332223111331 E73 31313113323221
E7 33231232321332 E41 21233113113321 E74 23131113231122
E8 23332222131121 E42 33311312333123 E75 31332213331111
E9 31313211221111 E43 21233112131322 E76 23333333321313
E10 23333333111333 E44 33112113133133 E77 23311232333323
E11 21211332123113 E45 21312233331231 E78 22131232333331
E12 12121313131111 E46 33212323222112 E79 12133233222322
E13 33312232313212 E47 31212333333312 E80 31211212311132
E14 32112212111322 E48 21232312331231 E81 21231222322321
E15 23113132231113 E49 33333232133223 E82 21231233333332
E16 23132311121121 E50 31133211131322 E83 21211212111332
E17 23123232113213 E51 33332333331233 E84 33331332333321
E18 32132231321322 E52 21133232323222 E85 31311222112213
E19 21213132321111 E53 21232132123113 E86 12132112133222
E20 21211212331331 E54 21232332321112 E87 23223212322223
E21 23313332323312 E55 21213232131213 E88 23311312333111
E22 33312233233213 E56 33132122332311 E89 23213331133213
E23 23111212323113 E57 33132312131131 E90 23311222132221
E24 13133331223211 E58 31231221331212 E91 21231333233331
E25 32113232321113 E59 31332333113313 E92 31212212123113
E26 21222223333331 E60 21313232321132 E93 33311232311131
E27 33312232132111 E61 31313133231332 E94 32131332233331
E28 32111222322223 E62 32132211313331 E95 23312332323112
E29 32311213123112 E63 33313233213333 E96 13332223323112
E30 32113213123221 E64 21321332221312 E97 21211132323333
E31 33123311113312 E65 32112213231221 E98 21332212132212
E32 11232131332233 E66 31213133321131 E99 32131331321112
E33 33131232232113 E67 32131133113323 E100 23313231323332
E34 31222212111132
Table 4
Numbering Genotype Corresponding individual Numbering Genotype Corresponding individual
M1 31213133321131 E66 M11 32131331321112 E99
M2 21333321323232 E36 M12 31221211121113 E38
M3 23123232113213 E17 M13 11232131332233 E32
M4 21123123221123 E9 M14 33212323222112 E46
M5 33123311113312 E31 M15 31332213331111 E75
M6 13133331223211 E24 M16 32131133113323 E67
M7 21121333332233 E71 M17 23131113231122 E74
M8 33112113133133 E44 M18 23213331133213 E89
M9 31313211221111 E10 M19 31212333333312 E47
M10 31231221331212 E58 M20 33131232232113 E33

Claims (4)

1. it is used for the SNP marker traced to the source, the DNA sequence dna such as SEQ ID NO.1 of the SNP marker on No. 6 chromosomes of pig It is shown, common 550bp, and there is a base to replace at the 362nd bit base, it is 362A or 362G.
2. it is used for the SNP marker traced to the source on No. 6 chromosomes of pig according to claim 1, it is characterised in that the SNP Molecular labeling is by TaiI restriction enzyme specific recognitions.
3. the primer pair for expanding the SNP marker for being used for tracing to the source on No. 6 chromosomes of pig described in claim 1, it is special Sign is that it includes forward primer, reverse primer, and specific base sequence is as follows:
Forward primer:As shown in SEQ ID NO.2;
Reverse primer:As shown in SEQ ID NO.3.
4. the SNP marker for being used to trace to the source on No. 6 chromosomes of pig as claimed in claim 1 is in DNA source tracing of pork products Application.
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