CN105296639B - SNP marker for tracing to the source and its application on No. 6 chromosomes of one pig - Google Patents
SNP marker for tracing to the source and its application on No. 6 chromosomes of one pig Download PDFInfo
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Abstract
The invention discloses the SNP marker for being used to trace to the source on No. 6 chromosomes of a pig and its applications, the SNP marker is obtained by ncbi database sequence alignment, the SNP marker is identified by HinfI restriction enzymes, total 570bp, there are one bases to replace at 403rd bit base, for 403T or 403G, sequence is as shown in SEQ ID NO.1.By analyzing SNP marker in the distribution situation for testing group's allelic in the experiment group 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 is close, rich polymorphism, gene frequency distributional difference is small between kind or strain, and heterozygosity is both greater than 0.3, which can be used as DNA source tracing of pork products.
Description
Technical field
The invention belongs to field of food safety, and in particular on No. 6 chromosomes of a pig for the SNP marker traced to the source and
It is applied.
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
Work, packaging, storage, transport and sale, there are the security risk of food, food-safety problem often has the links of food supply
Occur.Therefore need to carry out meat products can tracing management, establish the retrospect system of links from the place of production to dining table, to
The information that accurate and detailed article is provided for consumer is conducive 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 has been used as a kind of safety
Control measure are paid much attention to by multiple countries of 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 exist 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
Object tracing technology iris feature technology and DNA marker tracing technology (or DNA tracing technologies) etc., wherein DNA tracing technologies because
Detection means is simple, quickly, the features such as being difficult to forge become the widely applied quick tracing technology in countries in the world.With other labels
Method is compared, and DNA marker is put into superiority with peculiar：It directly occurs with DNA form, in each tissue, each of organism
Developmental stage can detect, and not influenced by environmental factor, and marker number is more, and organism whole gene group is spread.
SNP marker refers to the variation of single nucleotide acid on the same site of genome, normally behaves as two allele, suitable
High throughput automated analysis, so identifying molecular labeling of greatest concern as animal identification.
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, close 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 has accumulated a large amount of SNP marker, since single SNP labels of tracing to the source are nothings
Method is completed to trace to the source, and could realize and traces to the source therefore, it is necessary to one group of mutually independent SNP marker.With existing 13 known SNP points
Son label is as label combination of tracing to the source, in the pig groups introduced by height selection, when being traced to the source using the group echo, and meeting
There is the case where certain individuals cannot distinguish between.
In addition, in long-term breeding work, a large amount of SNP marker of accumulation often integrated distribution in certain chromosomes
On, and other part chromosome, such as No. 5, No. 6, No. 8, No. 14, No. 15 and No. 18 chromosome etc. then lack SNP marker, are easy to lead
Cause cascade phenomenon occur between molecular labeling, cannot be independent mutually between molecular labeling, the detection range for experiment of tracing to the source is reduced, is caused
Practical detectable range is less than expected detectable range.Therefore, the requirement that DNA traces to the source is carried out to pork product in order to meet, needed
The SNP marker for tracing to the source on other chromosomes is developed, trace to the source detection range and accuracy are improved.
Invention content
The purpose of the present invention is to provide the SNP marker for being used to trace to the source on No. 6 chromosomes of a pig and its applications, can
To be widely used in DNA source tracing of pork products, the safety for being especially used for pork product is traced to the source, and perfect existing label combines,
Raising is traced to the source accuracy in detection, and expansion is traced to the source scale.
To achieve the above object, technical scheme is as follows：
SNP marker on No. 6 chromosomes of pig for tracing to the source, the DNA sequence dna such as SEQ ID of the SNP marker
Shown in NO.1, total 570bp, and there are one bases to replace at the 403rd bit base, is 403T or 403G, DNA sequence dna such as SEQ ID
Shown in NO.1.
Further, the SNP marker is identified by HinfI restriction enzymes.
The SNP marker is obtained by ncbi database sequence alignment, and including 10 pig varieties or
In the experiment group (amounting to 245 individuals) of strain, the SNP marker is analyzed in the distribution feelings for testing group's allelic
Condition finds that gene frequency of the molecular labeling in different kinds and strain is close, 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 the described SNP marker traced to the source for pork DNA, using PCR-HinfI-RFLP methods into
Row, includes the following steps：
(1) the DNA sequence dna information of No. 6 chromosomes of pig, design forward and reverse primer separation No. 6 dyeing of pig are searched on NCBI
The DNA fragmentation of body；
(2) sequence alignment is carried out online using ncbi database, there are the sites that base substitutes for searching；
(3) foundation of PCR-HinfI-RFLP detection methods and the detection in replacement site；
(4) the ear tissue sample for acquiring 10 kinds and strain, amounts to 245 individuals；
(5) detection SNP marker is in the distribution of experiment group, statistical analysis, and further whether verification experimental verification is applicable in
In pork product is traced to the source.
Wherein, in the above-mentioned method for getting SNP marker, DNA fragmentation on described separation No. 6 chromosomes of pig
Forward and reverse primer sequence is as follows：
Forward primer：5 '-CCCTTGCCATTATCTCAC-3 ' (as shown in SEQ ID NO.2)；
Reverse primer：5 '-ATTTGTTCTTTACACTCAGCA-3 ' (as shown in SEQ ID NO.3).
During DNA traces to the source, the site as soon as often increase is traced to the source can further expand the number of detection sample, accurately
Property also with regard to higher, ideal SNP site should be generally evenly distributed on the different chromosome of pig, and in existing SNP site lack
Label 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 group including 10 pig varieties or strain, distribution of the SNP marker in experiment group allelic is analyzed
Situation finds that gene frequency of the molecular labeling in different kinds or strain is close, 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 traces to the source and the safety of pork product is traced to the source.
It is combined to improve existing SNP marker, improves the accuracy in detection for experiment of tracing to the source, expand scale of tracing to the source, it will
The Novel SNP molecular marker site that the present invention obtains on No. 6 chromosomes of pig and other existing published SNP marker sites
It is combined together and (amounts to 14 SNP sites) and carry out experiment of tracing to the source.
The present invention increases market pig source, 300 from different pig farms is picked altogether in slaughterhouse in experiment of tracing to the source
Individual selects 100 individuals at random from 300 individuals, detects the genotype of 14 SNP sites in 100 individuals, statistics
The genotype results of each individual find that 100 individuals possess respectively different genotype, can realize individual differentiation；Meanwhile
20 parts of muscle samples of picking in this 100 individuals at random, the genotype in 14 SNP marker sites of same detection statistics, so
100 individual genotype of heel are compared, and discovery can be found completely the same with 20 parts of muscle samples genotype
Individual finds the source individual of 20 parts of pig muscles by tracing to the source in 100 individuals, therefore, it is determined that the SNP points of the present invention
Son label 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 covering surface of label,
The accuracy traced to the source is improved, and expands the detection range for having molecular labeling.
Specific implementation mode
Technical scheme of the present invention is described in further detail below in conjunction with specific embodiment.
The lookup of 1 molecular labeling of embodiment
(1) design of primers
With the DNA sequence dna (Genbank of No. 6 chromosomes of pig：FN673773) it is template, the DNA pieces of design primer separation pig
Section (using the DNA of a Shen agriculture pig as the template of PCR amplification), primer is as follows：
Forward primer：5 '-CCCTTGCCATTATCTCAC-3 ' (as shown in SEQ ID NO.2)
Reverse primer：5 '-ATTTGTTCTTTACACTCAGCA-3 ' (as shown in SEQ ID NO.3)
PCR react total volume be 20 μ l, wherein pig genomic DNA about 100ng, (Promega companies) containing 1 × buffer,
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 amplification：94 DEG C of 4min, (94 DEG C of 30s, 59 DEG C of annealing 30s, 72 DEG C of 30s) cycle 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
It surveys.
(2) cloning and sequencing is analyzed
The DNA fragmentation of obtained No. 6 chromosome of pig is cloned as follows.
The purifying of PCR product：The gel containing target fragment is cut from Ago-Gel in the UV lamp, is put into 1.5ml
In Ependorff pipes, purified with PCR product purification kit (Tiangeng biochemical technology Co., Ltd).
Connection reaction：The PCR product of purifying is connect with pMD18-T carriers (Dalian treasured biotech firm), connection 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 the carrier T of 0.5 μ l
Department), the purified pcr product of 2.5 μ l, be eventually adding 2 μ l aqua sterilisas set 4 DEG C of water-baths stay overnight.
Conversion：Take 100-120 μ l competent cells (Tiangeng biochemical technology Co., Ltd) in 1.5ml under germ-free condition
In Ependorff pipes, mixing is added in the connection product of 5 μ l, places 30min on ice, 42 DEG C of heat shock 90s not shake therebetween
Dynamic Ependorff pipes, ice bath 3-4min after taking-up are added the LB liquid medium of 400 μ l antibiotic-frees, 37 DEG C keep flat 1h after fall
Set culture.
Bacterium colony PCR identifications：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, this is located on No. 6 chromosomes of pig, sequence such as SEQ ID through the spliced total 570bp of pig DNA sequence
Shown in NO.1.It finds that there are a bases to replace at its 403rd base through analysis, is 403T or 403G.Pass through molecular biology
Software is analyzed and ncbi database analysis, it is found that the replacement of the base at the 403rd base leads to HinfI-RFLP
(Restriction Fragment Length Polymorphism, the i.e. sites HinfI-RFLP) polymorphism.
(3) foundation of PCR-HinfI-RFLP detection methods and the detection in replacement site
10 μ l of endonuclease reaction total volume, wherein 1 × buffer 10 μ l, PCR product 3-5 μ l, restriction enzyme HinfI
For 0.5 μ l (5U), H is used2O supplies 10 μ l, 37 DEG C of water-bath 4h, weighs 0.6g agaroses and is dissolved in 15.75ml DEPC (Shanghai life works
Biotech firm) it handles in water, 37% formaldehyde of 5 × formaldehyde gel buffer solution and 4.25ml that 5ml is added after slightly cooling down is molten
Liquid, mixing glue detect digestion result after electrophoresis.As a result, it has been found that：A allele only has 570bp mono- in SEQ ID NO.1 sequences
A segment, G allele have two segments of 400bp and 170bp, the two allele constitute three kinds of genotype, GG, GT,
TT.And replaced in the 403rd bit base of the SEQ ID NO.1 sequences, base is T or G.
The distribution situation of 2 allele of embodiment
(1) design of group is tested
Test group：Acquire Pietrain (21), Shen Nong (17), great Bai (43), great Shen (52), long Shen (16),
The ear group of Du Shen (23), Pi great Shen (11), Shen of growing up (25), Du great Shen (17) and Du's (20) individuals of Pi great Shen
It knits, extracts DNA, amount to 245 DNA samples.
The purpose of experiment group is to detect distribution situation of the SNP marker in different cultivars.
(2) genetic test
Forward primer：5 '-CCCTTGCCATTATCTCAC-3 ' (as shown in SEQ ID NO.2)；
Reverse primer：5 '-ATTTGTTCTTTACACTCAGCA-3 ' (as shown in SEQ ID NO.3).
(condition such as embodiment 1) is expanded, it is all to detect experiment group with identical PCR-HinfI-RFLP methods
Idiotype.
(3) statistical analysis
All idiotypes of record experiment group, and gene frequency and heterozygosity are calculated, as a result such as the following table 1.
Table 1
As known from Table 1：Gene frequency of the SNP marker of the present invention in each kind is close, rich polymorphism；
Between kind or strain, the distributional difference of gene frequency A and G 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 traces to the source with safety.
3 SNP marker availability of embodiment is traced to the source verification experimental verification
The SNP marker of the present invention can be used for by tentative confirmation in traceability label, in order to ensure in label of tracing to the source
In availability, the present invention resamples in different location again, 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 label trace to the source verification experimental verification work to ratio.
In the ear tissue and each 100 parts of muscle sample for reviving slaughterhouse random acquisition pig individual, (each pig individual is adopted simultaneously
Collect ear tissue sample and muscle sample), ear tissue sample number E1-E100, muscle sample number M1-M100 extract muscle sample and ear respectively
The DNA of tissue sample is spare.
The HinfI 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 denoted as 1, two bands occurs and is denoted as 2, three bands are denoted as 3.Record sequence
It is ranked sequentially according to 2 site of table, the SNP marker site as II enzymes of Pvu of ADAMTS-1 genes identify is 1, genotype
Record result just makes number one, and the SNP marker site of I enzymes of the Eam1104 identification of ADD1 genes is 2, genotype note
Record result just comes second, and so on, the HinfISNP molecular labelings site on No. 6 chromosomes of pig of the application comes most
Afterwards, genotype results are indicated as the 14th number 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 the following table 3 and table 4.
In table 3, No. 82 individuals and No. 91 individual genotype are closely similar, and two individual preceding 13 tag values are equal
Unanimously, only difference is that the 14th label is different.It can be seen that tracing back merely with existing 13 SNP markers
When source, it may appear that the case where genotype repeats, the 14th is labeled as New SNP marker provided by the invention, illustrates that the present invention is added
SNP marker after, so that it may with smoothly by this, genotype distinguishes in two, 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 pork DNA with SNP marker.
The genotype results in 14 SNP marker sites of 20 parts of muscle samples with 100 individual genotype into
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 for finding 20 parts of pig muscles in 100 individuals by tracing to the source, realizes that meat sample is traced to the source to the accurate of individual.
Illustrate in conjunction with 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, can realize individual differentiation.
Table 3
| Number | Genotype | Number | Genotype | Number | Genotype |
| E1 | 33332332132311 | E35 | 33333232131332 | E68 | 23311222133231 |
| E2 | 21211233132123 | E36 | 21333321323232 | E69 | 23313333333222 |
| E3 | 33333232331121 | E37 | 22213233131323 | E70 | 21231233333213 |
| E4 | 23311232111312 | E38 | 31221211121113 | E71 | 21121333332233 |
| E5 | 23113332231332 | E39 | 22333231121223 | E72 | 21231122211121 |
| E6 | 23332221321321 | E40 | 33332223111332 | E73 | 31313113323222 |
| E7 | 33231232321331 | E41 | 21233113113322 | E74 | 23131113231122 |
| E8 | 23332222131123 | E42 | 33311312333121 | E75 | 31332213331111 |
| E9 | 21123123221123 | E43 | 21233112131323 | E76 | 23333333321312 |
| E10 | 31313211221111 | E44 | 33112113133133 | E77 | 23311232333321 |
| E11 | 21211332123113 | E45 | 21312233331232 | E78 | 22131232333332 |
| E12 | 12121313131112 | E46 | 33212323222112 | E79 | 12133233222323 |
| E13 | 33312232313213 | E47 | 31212333333312 | E80 | 31211212311133 |
| E14 | 32112212111323 | E48 | 21232312331233 | E81 | 21231222322323 |
| E15 | 23113132231112 | E49 | 33333232133222 | E82 | 21231233333332 |
| E16 | 23132311121123 | E50 | 31133211131321 | E83 | 21231233331331 |
| E17 | 23123232113213 | E51 | 33332333331231 | E84 | 33331332333322 |
| E18 | 32132231321323 | E52 | 21133232323223 | E85 | 31311222112211 |
| E19 | 21213132321112 | E53 | 21232132123112 | E86 | 12132112133223 |
| E20 | 21211212331332 | E54 | 21232332321113 | E87 | 23223212322221 |
| E21 | 23313332323313 | E55 | 21213232131212 | E88 | 21231233333113 |
| E22 | 33312233233211 | E56 | 33132122332313 | E89 | 23213331133213 |
| E23 | 23111212323113 | E57 | 33132312131133 | E90 | 23311222132222 |
| E24 | 13133331223211 | E58 | 31231221331212 | E91 | 21231333233333 |
| E25 | 32113232321112 | E59 | 31332333113311 | E92 | 31212212123111 |
| E26 | 21222223333332 | E60 | 21313232321131 | E93 | 33311232311133 |
| E27 | 33312232132113 | E61 | 31313133231333 | E94 | 32131332233332 |
| E28 | 32111222322221 | E62 | 32132211313332 | E95 | 23312332323113 |
| E29 | 32311213123113 | E63 | 33313233213331 | E96 | 13332223323112 |
| E30 | 32113213123223 | E64 | 21321332221313 | E97 | 21211132323331 |
| E31 | 33123311113312 | E65 | 32112213231222 | E98 | 21332212132213 |
| E32 | 11232131332233 | E66 | 31213133321131 | E99 | 32131331321112 |
| E33 | 33131232232113 | E67 | 32131133113323 | E100 | 23313231323331 |
| E34 | 31222212111133 |
Table 4
| Number | Genotype | Corresponding individual | Number | 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 (3)
1. the SNP marker on No. 6 chromosomes of pig for tracing to the source, the DNA sequence dna such as SEQ ID NO.1 of the SNP marker
Shown, total 570bp, there are one bases to replace at the 403rd bit base, is 403T or 403G, which is limited by HinfI
Property restriction endonuclease identification processed.
2. the primer pair for expanding the SNP marker on No. 6 chromosomes of pig described in claim 1 for tracing to the source, special
Sign is comprising forward primer, reverse primer, specific base sequence are as follows：
Forward primer：As shown in SEQ ID NO.2；
Reverse primer：As shown in SEQ ID NO.3.
3. application of the SNP marker as described in claim 1 in DNA source tracing of pork products, wherein utilize claim
Primer pair amplifies described in 2 go out the SNP marker sequence fragment.
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| 基于SNP标记的肉类溯源技术;张小波等;《肉类研究》;20110530;第25卷(第05期);第40-45页 * |
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