CN110106262B

CN110106262B - SNP marker combination and identification method of Zhongmeishan pigs and raw meat products

Info

Publication number: CN110106262B
Application number: CN201910463209.4A
Authority: CN
Inventors: 潘玉春; 王起山; 岳阳
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2020-11-24
Anticipated expiration: 2039-05-30
Also published as: CN110106262A

Abstract

The invention discloses a SNP marker combination and identification method of Meishan pigs and raw meat products, which comprises the steps of extracting genome DNA of raw pork or meat products, carrying out agarose gel electrophoresis and Sanger sequencing after PCR amplification, and identifying the Meishan pigs and the meat products thereof according to SNP genotypes of characteristic sites of sequencing results; the Sanger sequencing has the following identification sites: specific mutations appear at the positions of pig11-7097640, pig15-53346339, pig1-8851878, pig5-2060225 and pig 6-8539623; the invention solves the problem that the prior art has no identification method related to the Zhongmeishan pigs and meat products thereof.

Description

SNP marker combination and identification method of Zhongmeishan pigs and raw meat products

Technical Field

The invention relates to the field of food safety monitoring, in particular to a SNP marker combination and an identification method of Zhongmeishan pigs and raw meat products.

Background

Local pig breeds in the Taihu lake basin mainly comprise: the pig feed comprises Erhualian pigs, Meishan pigs, Fengjing pigs, Sanwu pigs, rice pigs and Jiaxing black pigs, wherein the Meishan pigs are divided into medium Meishan pigs and small Meishan pigs according to body types. The local variety pigs in the Taihu river basin have the same common source and have various characteristics, but because the body types and the appearances of the local variety pigs are similar, particularly piglets are confused in production, and the traditional body type and appearance variety identification method is difficult to distinguish the local variety pigs in the Taihu river basin, so that the work of breed conservation, development and utilization is not facilitated to be effectively carried out. On the other hand, the pork of the local variety of the Taihu river basin is delicious and popular with consumers, and the economic value of the Taihu river basin local variety pork is higher than that of the pork of the hybrid offspring such as Duroc, Changbai and Dabai pigs which are common in the market, so that the situation that the pork of the local variety of the Taihu lake river basin is counterfeited by the pork of the hybrid offspring and the selling behavior of the pork product (such as ham, minced meat and the like) of the local variety of the Taihu river basin with the hybrid pork adulterated in the market occur, but the traditional method cannot accurately judge the attribution of the killed and cut pork and the processed meat product. Single Nucleotide Polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by Single nucleotide variation on genome level, and has the characteristics of abundant sites, wide distribution, high genetic stability, representativeness, convenient and fast detection and the like. Among them, SNPs appearing in only one population in a certain range of breeds (population) are called breed-specific SNPs. The screening of variety specific SNP sites and the research of a site combination method are the key points for developing variety and product identification.

Compared with the first two generations of molecular markers, the third generation of molecular marker SNP has the advantages of rich variation, low requirement on DNA samples, high stability, accurate determination, simple and convenient detection method, high flux and the like. At present, the third generation molecular marker SNP has been widely applied to the fields of paternity test, animal and plant variety (strain) identification, genetic breeding and the like.

Disclosure of Invention

The invention aims to provide an SNP marker combination and an identification method for identifying the Zhongmeishan pigs and the raw meat products aiming at the defects of the prior art, and the third generation molecular marker identification and Sanger sequencing technology are utilized to identify the Zhongmeishan pigs and the raw meat products, so that the problem that no identification method related to the Zhongmeishan pigs and the meat products thereof exists in the prior art is solved, and the method for identifying the Zhongmeishan pigs and the meat products thereof and related special primers are provided, wherein the method has accurate results, simple operation and low cost.

The purpose of the invention is realized by the following technical scheme:

an SNP marker combination of Zhongmeishan pigs and raw meat products comprises: pig11-7097640, pig15-53346339, pig1-8851878, pig5-2060225 and pig6-8539623, wherein the pig11-7097640 represents the 7097640 th site of the pig11 chromosome, the pig15-53346339 represents the 53346339 th site of the pig15 chromosome, the pig1-8851878 represents the 8851878 th site of the pig1 chromosome, the pig5-2060225 represents the 2060225 th site of the pig5 chromosome, and the pig6-8539623 represents the 8539623 th site of the pig6 chromosome.

Further, the identification method of the Zhongmeishan pig and the raw meat products thereof based on the SNP marker combination comprises the steps of firstly extracting the genome DNA of the raw pork or meat products to be identified, then carrying out agarose gel electrophoresis and Sanger sequencing after PCR amplification to obtain the information of each detection site of the SNP marker combination, and when specific mutation occurs at any three sites of the SNP marker combination, identifying the Zhongmeishan pig and the meat products thereof.

Further, in the PCR amplification, the sequences of the upstream and downstream primers at the pig11-7097640 are shown as SEQ ID NO. 1-2, the sequences of the upstream and downstream primers at the pig15-53346339 are shown as SEQ ID NO. 3-4, the sequences of the upstream and downstream primers at the pig1-8851878 are shown as SEQ ID NO. 5-6, the sequences of the upstream and downstream primers at the pig5-2060225 are shown as SEQ ID NO. 7-8, and the sequences of the upstream and downstream primers at the pig6-8539623 are shown as SEQ ID NO. 9-10.

Further, the sequencing product identification site alignment information is shown in the following table:

SNP	REF	ALT
			pig15-53346339	A	T
pig5-2060225	G	A
			pig6-8539623	T	C
pig1-8851878	C	T
			pig11-7097640	C	T

in the table, REF represents a reference genotype and ALT represents a mutant genotype.

Further, the identification site information is: and when the mutant genotype of the detection site of the pig to be detected appears, the site is considered to have identification significance.

The invention has the beneficial effects that: compared with the prior art, the method for identifying the Zhongmeishan pigs is researched and identified from a molecular level by taking the special SNP locus of the Zhongmeishan pig variety as an identification basis and taking Sanger sequencing as a main molecular identification method, so that the Zhongmeishan pigs and other pig varieties (strains) can be identified and distinguished mutually, and the Zhongmeishan pigs and common western pig varieties can be identified and distinguished, for example: small Meishan pig, Fengjing pig, rice pig, Erhualian pig, Jiaxing black pig, Shakusneh pig, Changbai pig, Dabai pig, Duroc, Petland and Bakka, etc.

Detailed Description

The following further describes the embodiments of the present invention in detail.

The invention relates to an SNP marker of Zhongmeishan pigs and raw meat products, which comprises the following components: pig11-7097640, pig15-53346339, pig1-8851878, pig5-2060225 and pig6-8539623, wherein the pig11-7097640 represents the 7097640 th site of the pig11 chromosome, the pig15-53346339 represents the 53346339 th site of the pig15 chromosome, the pig1-8851878 represents the 8851878 th site of the pig1 chromosome, the pig5-2060225 represents the 2060225 th site of the pig5 chromosome, and the pig6-8539623 represents the 8539623 th site of the pig6 chromosome.

The invention provides a method for identifying Zhongmeishan pigs and raw meat products based on the SNP marker combination, which comprises the steps of firstly extracting genome DNA of raw pork or meat products to be identified, then carrying out agarose gel electrophoresis and Sanger sequencing after PCR amplification to obtain information of each detection site of the SNP marker combination, and identifying the Zhongmeishan pigs and the meat products thereof when specific mutation occurs at any three sites of the SNP marker combination.

The PCR amplification is carried out, wherein the related primers are shown in a table 1:

TABLE 1 amplification site primers and product information

In the table, F represents the upstream primer, R represents the downstream primer, length represents the length of the standard product, and F' -position represents the position of the SNP site in the amplification product.

The PCR amplification comprises a reaction system of 1ng/uL template DNA, 1uL primer and H₂O3.8 uL, 2 XTaq PCR Masrer Mix 50 uL; and/or the reaction program of the PCR reaction comprises pre-denaturation at 95 ℃ for 2min, pre-denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, cycle times for 30 times and re-extension at 72 ℃ for 10 min.

The mass concentration of the agarose gel is 2%.

The length of the band of the gel electrophoresis is required to be as shown in Table 1.

The sequencing product identification site alignment information is shown in table 2:

TABLE 2 sequencing products identification site alignment information

As shown in table 2, the mutation genotype and the reference genotype of each detection site, when the reference genotype appears in the detection site of the pig to be detected, the site is considered to have no identification significance, and when the mutation genotype appears in the detection site of the pig to be detected, the site is considered to have identification significance, for example, one pig a to be detected is at the pig position of pig15-53346339, and if the sequencing data is A, the pig a to be detected is considered to have no identification significance at the pig position of pig 15-53346339; and if the sequencing data is T, the pig A to be detected is considered to have identification significance at the pig sites of pig 15-53346339.

Because the single locus is used as the identification basis, false positive misjudgment exists, and the misjudgment probability is higher, the invention utilizes the locus combination as the identification Marker.

The information of the Marker for identifying each variety identifying site combination is shown in table 3.

TABLE 3 authentication tag combination information

As shown in table 3, Marker1-10 is a medium meishan pig identification Marker combination, for example, when the pig a to be detected has any one of the site mutation combinations in 10 Marker combinations, the pig a to be detected is considered to be a medium meishan pig.

The meat product of Zhongmei shan pig is cut meat of Zhongmei shan pig, and pickled product and cooked food product prepared from Zhongmei shan pig as raw material.

Randomly selecting 5 parts of the porcine ear tissue samples to be detected, and extracting tissue DNA by adopting an SDS method.

And carrying out PCR reaction amplification on the DNA sample by using the primer.

The reaction system of the PCR reaction is a 10uL system: template DNA 1ng/uL, primer 1uL, H₂O3.8 uL, 2 XTaq PCR Masrer Mix 50 uL; and/or the reaction program of the PCR reaction is pre-denaturation at 95 ℃ for 2min, pre-denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 1min, cycle times for 30 times and re-extension at 72 ℃ for 10 min.

The amplification result was detected by electrophoresis in 2% agarose gel and 1 XTAE buffer as medium under the conditions of current 10A, voltage 100V and time 40 min. Comparing the lengths of the amplification products of different primers with the standard products in the table 1, and determining that the amplification result is qualified if the product length is within the error range and is consistent with the length of the standard product.

Performing Sanger sequencing on qualified sample PCR amplification products to obtain information of each detection site, and analyzing a sequencing result:

TABLE 4 sample sequencing results SNP polymorphism analysis Table

sample/SNP

REF

ALT

1

2

3

4

5

pig15-53346339

A

T

√

pig5-2060225

G

A

√

pig6-8539623

T

C

√

pig1-8851878

C

T

√

pig11-7097640

C

T

√

In the table, REF represents a reference genotype, ALT represents a mutant genotype, and V represents a detected mutant genotype.

As shown in Table 4, from the sequencing data, if only a single locus is used as the identification basis, one pig to be tested belongs to multiple breeds at the same time.

The identification is carried out by using a site combination Marker mode: the No.1 pig detects mutations at positions of pig5-2060225, pig6-8539623, pig1-8851878 and pig11-7097640, meets identification information of Marker3, Marker6, Marker8 and Marker10, and is identified as a Zhongmeishan pig; the No. 2 pig detects mutations at the pig sites of pig15-53346339 and pig6-8539623, the mutations at the two sites do not accord with Marker information, and the No. 2 pig is identified as not a Zhongmeishan pig; the 3-pig detects mutations at the positions of pig15-53346339, pig5-2060225, pig6-8539623, pig1-8851878 and pig11-7097640, and meets the conditions of Marker1, Marker2, Marker3, Marker4, Marker5, Marker6, Marker7, Marker8, Marker9 and Marker10, and the 3-pig is identified as a Meishan pig; no. 4 pig detects mutation at pig sites of pig15-53346339, pig6-8539623 and pig11-7097640, meets marker1, and is identified as Zhongmeishan pig; no.5 pig detected mutation at the pig1-8851878 site, and did not accord with Marker information, and the No.5 pig was identified as not a Zhongmeishan pig.

At present, no relevant patent about identification of the Zhongmeishan pig species and the meat product variety (strain) thereof exists at home and abroad, and the third generation molecular marker is applied to identification of the Zhongmeishan pig species and the meat product variety (strain) thereof, so that the market vacancy is filled, and the counterfeit identification problem of the Zhongmeishan pig (strain) is effectively solved. Compared with the existing patent for identifying pig breeds by utilizing the first generation molecular marker (RFLP) and the second generation molecular marker (SSR), the identification method has the advantages of simpler operation, more accurate result, rapidness and high efficiency. Meanwhile, the invention utilizes the third generation molecular marker SNP to overcome the defect that the first two generations of molecular markers can use less sites, and compared with the identification technology of the first two generations of molecular markers, the identification method is simplified.

The foregoing embodiments may be modified in many different ways by those skilled in the art without departing from the spirit and scope of the invention, which is defined by the appended claims and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

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Claims

1. A method for identifying Zhongmeishan pigs and raw meat products thereof based on SNP marker combination comprises the following steps: pig11-7097640, pig15-53346339, pig1-8851878, pig5-2060225 and pig6-8539623, wherein the pig11-7097640 represents the 7097640 th site of the pig11 chromosome, the pig15-53346339 represents the 53346339 th site of the pig15 chromosome, the pig1-8851878 represents the 8851878 th site of the pig1 chromosome, the pig5-2060225 represents the 2060225 th site of the pig5 chromosome, and the pig6-8539623 represents the 8539623 th site of the pig6 chromosome, and the method is characterized in that genomic DNA of raw pork or meat products to be identified is firstly extracted, then agarose gel electrophoresis and Sanger sequencing are carried out after PCR amplification to obtain information of each detection site of the SNP marker combination, and when specific mutations occur at any three sites of the SNP marker combination, the pig with the Meishan pig and meat products thereof can be identified.

In the PCR amplification, the sequences of the upstream and downstream primers at the pig11-7097640 are shown as SEQ ID NO. 1-2, the sequences of the upstream and downstream primers at the pig15-53346339 are shown as SEQ ID NO. 3-4, the sequences of the upstream and downstream primers at the pig1-8851878 are shown as SEQ ID NO. 5-6, the sequences of the upstream and downstream primers at the pig5-2060225 are shown as SEQ ID NO. 7-8, and the sequences of the upstream and downstream primers at the pig6-8539623 are shown as SEQ ID NO. 9-10.

The sequencing product identification site alignment information is shown in the following table: