WO2020238218A1 - Snp marker combination and identification method for jiaxing black pig and raw meat product - Google Patents

Abstract

An SNP marker combination and an identification method for a Jiaxing black pig and a raw meat product. The method comprises: extracting a genome DNA of raw pig meat or a meat product, performing PCR amplification and then performing agarose gel electrophoresis and Sanger sequencing, and identifying a Jiaxing black pig and a meat product thereof according to an SNP genotype of a particular site in the sequencing result, wherein identification sites of the Sanger sequencing are sites pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200, and pig13-111071937 where specific mutations occur.

Description

SNP mark combination and identification method of Jiaxing black pig and raw meat products Technical field

The invention relates to the field of food safety monitoring, in particular to a SNP mark combination and identification method of Jiaxing black pig and raw meat products.

Background technique

The main local breeds of pigs in the Taihu Lake Basin are: Erhualian pigs, Meishan pigs, Fengjing pigs, Shawutou pigs, rice pigs, and Jiaxing black pigs. Meishan pigs are divided into Zhongmeishan pigs and Xiaomeishan pigs due to their body size. The local breeds of pigs in the Taihu Basin share the same roots and have their own characteristics. However, due to their similar body shapes, especially piglets, there is confusion in the production. The traditional body shape and appearance breed identification methods are used to identify the various breeds of the local pigs in the Taihu Basin. It is difficult to distinguish, which is not conducive to effective seed conservation and development and utilization. On the other hand, local varieties of pork in the Taihu Lake Basin are delicious and popular with consumers. Its economic value is higher than that of the hybrid offspring of Duroc, Landrace, and Great White pigs that are commonly used in the market. Therefore, there are fake Taihu offspring of the above hybrid pork on the market. The sales of local varieties of pork in the river basin, and the sales behavior of adulteration of the above-mentioned hybrid pork in local varieties of pork products (such as ham, minced meat, etc.) in the Taihu Basin. However, traditional methods cannot accurately slaughter, cut and processed meat products. Attribution judgment. Single nucleotide polymorphism (Single Nucleotide Polymorphism, SNP) mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level, which has abundant sites, wide distribution, high genetic stability, It is representative, convenient and quick to detect. Among them, within a certain breed (group), SNPs that only appear in one group are called breed-specific SNPs. Variety-specific SNP site screening and site combination method research is the key to the identification of varieties and products.

Compared with the previous two generations of molecular markers, the third-generation molecular marker SNP has the advantages of rich variation, low requirements for DNA samples, high stability, accurate determination, simple detection methods and high throughput. At present, the third-generation molecular marker SNP has been widely used in the fields of paternity identification, animal and plant species (line) identification, genetic breeding and other fields.

Summary of the invention

The purpose of the present invention is to address the shortcomings of the prior art and propose a SNP marker combination and identification method for identifying Jiaxing black pigs and raw meat products, using third-generation molecular marker identification and Sanger sequencing technology to identify Jiaxing black pigs and raw meat products. In the prior art, there is no problem with the identification method related to Jiaxing black pig and its meat products, and a method and related special primers for identifying Jiaxing black pig and its meat products with accurate results, simple operation and low price are provided.

The purpose of the present invention is achieved through the following technical solutions:

A combination of SNP markers for Jiaxing black pig and raw meat products, including: pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200 and pig13-111071937; said pig2-60239356 represents the 60239356th locus of pig chromosome 2 , Pig8-5778954 represents the 5778954 locus of pig chromosome 8, pig7-37079960 represents the 37079960 position of pig chromosome 7, pig13-16955200 represents the 16955200 position of pig chromosome 13, and pig13-111071937 represents pig 13 Chromosome 111071937 position.

Further, the Jiaxing black pig and its raw meat product identification method based on the combination of SNP markers first extract the genomic DNA of the raw pork or meat product to be identified, and then perform agarose gel electrophoresis and Sanger sequencing after PCR amplification to obtain SNPs The information of each detection site of the tag combination, when a specific mutation occurs at any three sites of the SNP tag combination, it can be identified as Jiaxing black pig and its meat products.

Further, in the PCR amplification, the sequences of the upstream and downstream primers at pig2-60239356 are shown in SEQ ID Nos. 1 to 2, and the sequences of the upstream and downstream primers at pig8-5778954 are shown in SEQ ID Nos. 3 to 4. As shown, the sequences of the upstream and downstream primers at pig7-37079960 are shown in SEQ ID Nos. 5-6, the sequences of the upstream and downstream primers at pig13-16955200 are shown in SEQ ID Nos. 7-8, and the upper and lower primers at pig13-111071937 The sequence of the downstream primer is shown in SEQ ID NO. 9-10.

Further, the comparison information of identification sites of sequencing products is shown in the following table:

SNP	REF	ALT
pig2-60239356	C	T
pig8-5778954	G	A
pig7-37079960	G	A
pig13-16955200	T	C
pig13-111071937	G	T

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

Further, the identification site information is: the mutation genotype and the reference genotype of each detection site. When the reference genotype appears at the test site of the pig to be tested, the site is considered to have no identification significance. When a mutant genotype occurs at a locus, the locus is considered to be of identification significance.

The beneficial effect of the present invention is that compared with the prior art, the present invention uses the unique SNP sites of Jiaxing black pig breeds as the identification basis, studies the method for identifying Jiaxing black pigs from the molecular level, and uses Sanger sequencing as the main molecular identification method. Can distinguish Jiaxing black pigs from other pig breeds (strains), and distinguish Jiaxing black pigs from common western pigs, such as: Xiaomeishan pig, Fengjing pig, Zhongmeishan pig, Erhualian pig, Mizu , Sauvignon head pig, Landrace pig, Large white pig, Duroc, Pietland, Berkshire, etc.

Detailed ways

The specific embodiments of the present invention are described in further detail below.

The present invention relates to a SNP marker of Jiaxing black pig and raw meat products, including: pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200 and pig13-111071937; said pig2-60239356 represents the 60239356th position of pig chromosome 2 Pig8-5778954 represents the 5778954 locus of pig chromosome 8, pig7-37079960 represents the 37799960 position of pig chromosome 7, pig13-16955200 represents the 16955200 position of pig chromosome 13, and pig13-111071937 represents pig Position 111071937 on chromosome 13.

The present invention provides a method for identifying Jiaxing black pigs and raw meat products based on the above-mentioned SNP marker combination. The genomic DNA of raw pork or meat products to be identified is first extracted, and then amplified by PCR and then subjected to agarose gel electrophoresis and Sanger sequencing , Get the information of each detection site of the SNP marker combination. When a specific mutation occurs at any three sites of the SNP marker combination, it can be identified as Jiaxing black pig and its meat products.

In the PCR amplification, the primers involved are shown in Table 1:

Table 1 Amplification site primer 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 amplified product.

For the PCR amplification, the reaction system is template DNA 1ng/uL, primer 1uL, H ₂ O 3.8uL, 2×Taq PCR Masrer Mix 50uL; and/or, the PCR reaction program is 95°C pre-denaturation 2min , 95°C pre-denaturation 30s, 60°C annealing temperature 30s, 72°C extension 1min, cycle times 30 times, 72°C extension 10min.

The mass concentration of the agarose gel is 2%.

For the gel electrophoresis, the band length requirements are as shown in Table 1.

The comparison information of identification sites of sequencing products is shown in Table 2:

Table 2 Comparison information of identification sites of sequencing products

SNP	REF	ALT
pig2-60239356	C	T
pig8-5778954	G	A
pig7-37079960	G	A
pig13-16955200	T	C
pig8-5778954	G	T

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

As shown in Table 2, the mutant genotypes and reference genotypes of each detection site. When the reference genotype appears at the test site of the pig to be tested, the site is considered to be of no identification significance. For example, if a pig A to be tested is located at pig2-60239356, if the sequencing data is C, it is considered that the pig A to be tested has no identification significance at pig2-60239356; if the sequencing data is T, it is considered that pig A to be tested has identification significance at pig2-60239356.

Since a single site is used as an identification basis, there is a false positive misjudgment, and the probability of a misjudgment is high, the present invention uses a combination of sites as an identification Marker.

The combined identification Marker information of each variety identification site is shown in Table 3.

Table 3 Identification mark combination information

As shown in Table 3, Marker1-10 are the identification marker combinations for Jiaxing black pigs. For example, when pig A to be tested has any one of the 10 Marker combinations, then pig A to be tested is considered Jiaxing black pig. .

The Jiaxing black pork products refer to Jiaxing black pig cut meat and pickled products and cooked food products processed and prepared using Jiaxing black pig as raw materials.

Randomly select 5 samples of pig ear tissues to be tested, and extract tissue DNA by SDS method.

The primers are used for PCR amplification of DNA samples.

The reaction system of the PCR reaction is a 10uL system: template DNA 1ng/uL, primers 1uL, H ₂ O 3.8uL, 2×Taq PCR Masrer Mix 50uL; and/or, the PCR reaction program is 95°C pre-denaturation 2min, 95°C Pre-denaturation for 30 seconds, annealing temperature at 60°C for 30 seconds, extension at 72°C for 1 min, 30 cycles, and extension at 72°C for 10 min.

Use 2% agarose gel, 1×TAE buffer as the medium electrophoresis to detect the amplification results, the gel electrophoresis conditions are current 10A, voltage 100V, time 40 minutes. The amplified products of different primers are compared with the standard product length in Table 1. If the product length is within the error range and consistent with the standard product length, the amplification result is considered qualified.

Perform Sanger sequencing on the PCR amplification products of qualified samples to obtain the information of each detection site, and analyze the sequencing results:

Table 4 SNP polymorphism analysis table of sample sequencing results

Sample/SNP	REF	ALT	1	2	3	4	5
pig2-60239356	C	T	√	To	To	√	To
pig8-5778954	G	A	√	To	√	To	To
pig7-37079960	G	A	√	To	√	To	To
pig13-16955200	T	C	√	To	√	√	√
pig13-111071937	G	T	√	To	√	To	To

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

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

Use the combination of markers to identify: Pig No. 1 detected mutations at pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200, and pig13-111071937, which are in line with Marker1, Marker2, Marker3, Marker4, Marker5 The identification information of Marker6, Marker7, Marker8, Marker9, Marker10 identified pig No. 1 as Jiaxing black pig; pig No. 2 had no mutation detected and did not meet the Marker information, and pig No. 2 was identified as Jiaxing black pig; Pig8-5778954, pig7-37079960, pig13-16955200, pig13-111071937 mutations were detected, in line with Marker4, Marker7, Marker9, Marker10, identified pig No. 3 as Jiaxing black pig; pig No. 4 in pig2-60239356, pig13-16955200 Mutations were detected at the locus, which did not meet the Marker information, and pig No. 4 was identified as Jiaxing black pig; pig No. 5 was detected with mutations at pig13-16955200 locus, which did not meet the Marker information, and pig No. 5 was identified as Jiaxing pig.

At present, there are no relevant patents on the identification of Jiaxing black pig breeds and their meat product varieties (strains) at home and abroad. The third-generation molecular markers are applied to the identification of Jiaxing black pigs and their meat product breeds (strains), filling the gap in the market and effectively Solve the problem of authenticating Jiaxing black pigs (strains). Compared with the existing patents that use the first-generation molecular markers (RFLP) and the second-generation molecular markers (SSR) for pig breed identification, the identification method has the advantages of simpler operation, more accurate results, fast and efficient. At the same time, the present invention uses the third-generation molecular marker SNP to overcome the shortcomings of fewer available sites for the first two generations of molecular markers, and simplifies the identification method compared with the identification technology of the first two generations of molecular markers.

The above specific implementations can be locally adjusted by those skilled in the art in different ways without departing from the principle and purpose of the present invention. The protection scope of the present invention is subject to the claims and is not limited by the above specific implementations. All implementation schemes within the scope are bound by the present invention.

Claims (5)

1. A SNP marker combination of Jiaxing black pig and raw meat products, characterized in that it comprises: pig2-60239356, pig8-5778954, pig7-37079960, pig13-16955200, and pig13-111071937; said pig2-60239356 represents pig chromosome number 2 60239356 locus, pig8-5778954 represents the 5778954 locus of pig chromosome 8, pig7-37079960 represents the 37799960 position of pig chromosome 7, pig13-16955200 represents the 16955200 position of pig chromosome 13, pig13-111071937 It represents the 111071937 locus of pig chromosome 13.
2. A Jiaxing black pig and its raw meat product identification method based on the SNP marker combination of claim 1, wherein the genomic DNA of the raw pork or meat product to be identified is first extracted, and then amplified by PCR and then subjected to agarose coagulation Gel electrophoresis and Sanger sequencing to obtain the information of each detection site of the SNP marker combination. When a specific mutation occurs at any three sites of the SNP marker combination, it can be identified as Jiaxing black pig and its meat products.
3. The method according to claim 2, characterized in that, in the PCR amplification, the sequence of the upstream and downstream primers at pig2-60239356 is as shown in SEQ ID Nos. 1-2, and the sequence of the upstream and downstream primers at pig8-5778954 The sequences are shown in SEQ ID NOs. 3 to 4, the sequences of the upstream and downstream primers at pig7-37079960 are shown in SEQ ID Nos. 5 to 6, and the sequences of the upstream and downstream primers at pig13-16955200 are shown in SEQ ID NOs. 7 to 8. As shown, the sequences of the upstream and downstream primers at pig13-111071937 are shown in SEQ ID NOs. 9-10.
4. The method according to claim 2, wherein the comparison information of the identified sites of the sequencing product is shown in the following table:

   SNP REF ALT pig2-60239356 C T pig8-5778954 G A pig7-37079960 G A pig13-16955200 T C pig13-111071937 G T

   In the table, REF represents the reference genotype, and ALT represents the mutant genotype.
5. The method according to claim 4, wherein the identification site information is: the mutant genotype and the reference genotype of each detection site, and when the reference genotype appears at the detection site of the pig to be tested, the site is considered The point has no identification significance. When a mutant genotype occurs at the detection site of the pig to be tested, the site is considered to have identification significance.