CN104450697B - SNP marker associated with oyster antiviral properties and application thereof - Google Patents

Abstract

The invention belongs to the fields of molecular biology and genetic breeding and in particular relates to an SNP marker associated with oyster antiviral properties and an application thereof. The SNP marker is positioned on a TLR2-1 (CGI_10005421) single exon gene in a Pacific oyster TLR immune pathway. The SNP marker and the application disclosed by the invention have the advantages that oyster parents can be subjected to genotype identification during young oyster breeding, and the oyster parents with the genotypes of A/A or A/C are screened, so that the antiviral properties of offspring groups are improved.

Description

A SNP marker associated with oyster antiviral properties and its application

technical field

The invention belongs to the field of molecular biology and genetic breeding, in particular to a SNP marker related to oyster anti-virus characteristics and its application.

Background technique

The long oyster belongs to the mollusk phylum and belongs to the clambranch/bivalves. Long oysters are native to China, Japan, and Korea along the Pacific Ocean, and were later introduced to the European and American continents. Due to its fast growth rate, delicious meat and strong environmental adaptability, the long oyster has high edible and economic value, and has gradually become an important cultured shellfish species in my country and even in the world.

So far, OsHV-1 has been detected in a variety of bivalve molluscs around the world, and OsHV-1 has been proved to be related to the large-scale death of larvae. huge economic losses.

The concept of PRR was first proposed by Charles Janeway in 1989, and the knowledge about the mechanism of cell recognition of microbial pathogens has exploded in the past decade. It is now well known that several classes of pattern recognition receptor family PRRs exist in the cellular microenvironment to recognize and infect viruses. The TLR family is one of the most well-studied PRR families. Herpes virus particles can be recognized by TLR2, which may recognize the glycoprotein of herpes virus, and then induce the expression of pro-inflammatory cytokines.

SNP is a single nucleotide polymorphism. As the latest generation of molecular markers, SNP has gradually shown great application potential in the fields of marine aquatic genetics and breeding. On average, there is one SNP every 20-30bp in the oyster genome. SNP analysis technology is mainly divided into two categories according to the nature of the research object: one is to analyze the unknown SNP, that is, to determine whether there is a SNP in a certain sequence, or to determine whether an unknown SNP is associated with a certain trait The second is to analyze known SNPs, that is, to analyze the genotypes of known SNP sites in different groups or individuals, to determine the genotype of each individual for a certain SNP one by one, and then to carry out subsequent gene positioning or association analysis and other research work. Contents of the invention

The purpose of the present invention is to provide a SNP marker related to oyster anti-virus traits and its application.

To achieve the above object, the technical solution adopted in the present invention is:

A SNP marker associated with the antiviral properties of oyster oyster. The SNP marker is located on the single exon gene of TLR2-1 (CGI_10005421) in the TLR immune pathway of oyster oyster.

The SNP marker is located on the leucine rich C-terminal recognition domain (Leucinerich repeat C-terminal domain) of the TLR2-1 gene protein molecule.

The base of the SNP marker located at the 858th base of the 5' end shown in SEQ ID NO.1 (the 286th amino acid of the amino acid sequence encoded by the base sequence shown in SEQ ID NO.1) is A or c.

The genotype of the SNP in the genomic DNA is A/A, A/C or C/C.

An application of the SNP marker related to the anti-virus characteristics of oyster oyster according to claim 1, the application of the SNP marker in identifying the anti-virus genotype of broodstock before seed breeding.

A method for identifying SNP markers related to the anti-virus traits of oyster oysters, comprising the following steps:

1) Use OsHV-1 (oyster herpes virus type 1) to artificially infect wild long oyster populations, and screen the 100 individuals who died first (TM group) and the 100 individuals who died last (TF group).

2) Extract the individual genomic DNA of two groups of oysters in step 1).

3) Take the 100 individual DNA obtained from the TM group in the above step 2), and mix the DNA of each individual in equal amounts to form a TM pool; take the 100 individual DNA obtained from the TF group, and mix the DNA of each individual in equal amounts to form a TF Mix pool.

4) The two gene pools obtained in step 3) are used for whole-genome resequencing using next-generation sequencing technology.

5) After the resequencing described in step 4), compare the sequences obtained by the two mixed pool sequencing with the oyster genome, and carry out the screening of the SNP sites in the whole genome;

6) Select oyster immune-related key pathway genes as candidate genes, design primers, and the specific primer sequence of the SNP site is (concentration 10uM):

Forward primer sequence F: 5'-GAATATTATCTTTTCACTACATACCTGACATG-3';

Reverse primer sequence R: 5'-GGATCAATATGTACTAGTTTGTTGAAGG-3'

The PCR in vitro amplification technology was used to amplify the target fragment of the candidate gene in vitro. PCR reaction program:

7) Entrust the amplified product obtained in step 6) to Shanghai Sunny Biotechnology Co., Ltd. for first-generation Sanger sequencing.

8) Manually analyze and count the sequencing peaks of the sequencing results described in step 7) using auxiliary software.

9) Use plink software to analyze the statistical results of step 8) and its antiviral traits, and mark the SNP.

Through the correlation analysis between the genotypes at the SNP site and the virus characteristic traits of the two populations of TM and TF, it was found that the TF genotype of the anti-virus population was more inclined to A/A or A/C, while the TM genotype of the virus-sensitive population was more biased. Prefer to C/C (P<0.05).

According to the above results, the results of identifying the marker SNP showed that individuals with genotype A/A or A/C at the above SNP loci were insensitive to the virus and had strong resistance to the virus; individuals with the genotype C/C were relatively resistant to the virus. Sensitive, less resistant to viruses.

A specific primer for detecting SNP markers related to the antiviral properties of oyster oysters, the specific primers are:

Forward primer sequence F: 5'-GAATATTATCTTTTCACTACATACCTGACATG-3';

Reverse primer sequence R: 5'-GGATCAATATGTACTAGTTTGTTGAAGG-3'.

A kit for detecting SNP markers associated with the antiviral properties of oyster oysters, the kit contains a forward primer sequence F: 5'-GAATATTATCTTTTCACTACATACCTGACATG-3';

Reverse primer sequence R: 5'-GGATCAATATGTACTAGTTTGTTGAAGG-3'

The advantages that the present invention has:

The SNP marker related to the anti-virus of oyster oyster is located at the 858th base of the ORF (Open reading frame, open reading frame) of the TLR2-1 gene of oyster oyster, the 286th amino acid of the amino acid sequence, and there are two types of this base, A and C Correspondingly, the SNP has two allelic forms, A and C, in the genomic DNA, and the individuals with the A genotype at the SNP site are more resistant to the OsHV-1 virus than the C genotype; the SNP in the genomic DNA The genotypes in are corresponding to three types: A/A, A/C, and C/C (the following genotypes refer to the genotype of the SNP in the genomic DNA); the amino acid codon corresponding to the SNP has a GAA valley GAC and GAC aspartic acid forms.

In the present invention, on the basis of resequencing the entire genomes of two virus-related populations and screening and checking the SNPs in the whole genome, PCR technology is used to amplify specific regions of specific candidate immune-related genes, and then Sanger sequencing is performed on the PCR products to check The SNP loci were verified, and the gene frequency of the SNP loci was correlated with anti-virus characteristics, so as to mark the SNP loci that were significantly related to the anti-virus characteristics of oysters. The SNP marker can be used for genotype identification of parent shellfish before seed breeding (oyster non-damage sampling to identify genotype), and by screening parent shellfish of A/A and A/C genotypes, the anti-virus characteristics of offspring populations can be improved. The SNP related to the oyster anti-virus characteristic marked by the present invention has a potential value for aquaculture breeding, and is beneficial to reduce the loss of oyster breeding industry.

Description of drawings

Fig. 1 is the electrophoresis detection diagram of the PCR product comprising SNP 10005421-858 provided by the embodiment of the present invention;

Figure 2 is a characteristic peak diagram of base sequencing of various genotypes provided by the embodiment of the present invention (the first base has a set of peaks, indicating that this site is C/T heterozygous), this figure does not represent the SNP described in the present invention Peak diagram, only the base peak diagram form is described here.

detailed description

The present invention is further illustrated below in conjunction with the examples, but the examples do not limit the present invention in any form, and the experimental materials used in the present invention are all purchased from the market.

Example 1

The above invention is further explained below with specific examples, and the specific steps are according to the SNP identification method in the description, namely:

1) Use OsHV-1 (oyster herpes virus type 1) to artificially infect wild long oyster populations, and screen the 100 individuals who died first (TM group) and the 100 individuals who died last (TF group). TM is the virus sensitive group, and TF is the antiviral group.

2) Extract the genomic DNA from the individual gill tissues of the two groups of long oysters in step 1).

3) Take the DNA of 100 individuals obtained from the TM group in the above step 2), mix the DNA of each individual in equal amounts to form a TM pool, take the DNA of 100 individuals in the TF group, and mix the DNA of each individual in equal amounts to form a TF pool. pool.

4) The two gene pools obtained in step 3) were entrusted to Shenzhen Huada Gene Technology Service Co., Ltd. to perform whole-genome resequencing using next-generation sequencing technology.

5) After the resequencing described in step 4), compare the sequences obtained by the two mixed pool sequencing with the oyster genome, and carry out the screening of the SNP sites in the whole genome;

6) Select oyster immune-related key pathway genes as candidate genes according to the oyster virus stress expression profile database, and use Primer Premier 5.0 software to design primers. The specific primer sequence for the SNP site is (concentration 10uM):

Forward primer sequence F: 5'-

GAATATTATTCTTTCACTACATACCTGACATG-3';

Reverse primer sequence R: 5'-GGATCAATATGTACTAGTTTGTTGAAGG-3'

The PCR in vitro amplification technology was used to amplify the target fragment of the candidate gene in vitro.

PCR amplification system:

PCR reaction program:

7) Entrust the amplified product obtained in step 6) to Shanghai Sunny Biotechnology Co., Ltd. to perform first-generation Sanger sequencing on it, and the sequencing primers are the PCR amplification primers described in step 6).

8) Use the auxiliary software seqscape v2.7 to sequence the peak map of the sequencing results described in step 7) (as shown in Figure 2, the sequencer will specifically identify four specific bases A, T, G, and C according to the detection results, Then give the sequencing peak map; if there is a set of peaks at the same site, it means that the genotype of this site is heterozygous for these two base types, and if a peak appears at this site, it means that the genotype of this site is that base type Homozygous) were manually counted and analyzed, and the statistical results of the genotype analysis of the two groups of individuals are shown in Table 1 (where 00 means missing information).

9) Use plink software to analyze the statistical results of step 8) and its association with antiviral traits, and mark the SNP, as shown in the 858th base of the 5' end as shown in SEQ ID NO.1 (shown in SEQ ID NO.1 The 286th amino acid of the amino acid sequence encoded by the base sequence).

Through association analysis, it was found that the allele frequency of A in SNP10005421-858 of the virus-resistant population TF was significantly higher than that of the virus-sensitive population TM, indicating that SNP10005421-858 was significantly related to the anti-virus characteristics of oysters, and could be used as an auxiliary selection for oyster anti-virus traits. marker, and allele A at SNP10005421-858 makes oysters more resistant to oyster herpesvirus type 1 (OsHV-1).

SEQ ID NO.1:

ATGATGCGGAAAGGAATAATGATGCTTTCTAGCCTATCTGTATTCTTAATGTTGATGTTTCTTGCTGGTTTATGTGACATGTGCCCCGTCTTACAGTGGGCAGGAAACAATGAAACAACTCTGAAATTTCTTACAAGGATGAAGATTGTTTCAAACGACACGTTTAGTAATTGCGACAAAAGTGTTCACCATCTCTTAATGGATGACTTAGGAATAGAAGAGGTTTCCCACAATGCATTAGAAGATTTCTATAACCTGAAATCTTTATCGTTATCAGGAAATTATATTTCCGCGCTGTCACCATTTTTACTGGAATACAAAAGAGGGTTGGAGTCTTTCAACATCGCAAATAATCGGCTCGAACGTATTGAGGACGGAACTTTTGAATATCTGGACAACGTAAAGGTAATTGAGCTCCAGTTTAACAACATTTCCTTTATCGGACCAGACGCTTTCTCAAGGAACCTCCGAGTTTTACAAACTGTAAACGTTTCCCATAATGCTCTAACATACGGAGAACCATGGCCATTTATACCGGAAGCGGACCCAACGGACGGCGAGGATTTGGTGTTTGACCTCGAATACAATCATATCTCGGAATTCCGGAACTCGCCTAACTGGACATACGATCTGGTTTCGCCCTTTGAGTACGACGTCAAGTTAAGTTTTAACAACATAACAAACATTCATATTGATGATGTTGTACATATATTCAATCCGGGTTTCCAAGGAAATACATTCGTAGAATACTTGTCTTTTAAAATCAATGCTTCTGAAAACCCTTTTTTCTGTGACTGTAATGTATATCCGTTTGCATCTTATTTGCGCGATTCGCTGTTTTATTTCTATAGAGTGGAAGAGTTCCGATACCGGTGTAACTCTCCTGATTCCTTGTCTCAAGAAGATTTCCTTCATGATATTCCCCTTGAGCAGTTTGTGTGTAACGTCACCGAAAACTGTCCCGACGGCTGTATCTGTCAGGAGCGGCCTTATTATGGAT ATCTTCACGTGAACTGTAGTTTCATGTACGGTTCTAACCAACAACCAATGAACACACTCCCTTTAACTCTTCCAACGTCCAAGAACGGCAAGATATCCTTACATCTGGATGGACACTACATCGCGTTACTAGAACAAAGGGGTTACCTTCCACAACTCGTAAATCTAACTCTCTCCTTCAACAAACTAGTACATATTGATCCAAAAGCTCTCACGTCCATGTCTGACAGACAATATCTAGATCTCTCACATAATTATCTCAAGTATGTGCCGAAAGAAATCCAGTATTTCAAGACGAATAAGGTTCGCATCGGTTCCAATCGCTTTGAGTGTAACTGCAACATGACTTGGATGGCCCAGTGGTTAAATCTGGATTCAAACGCTCCTGCTTACAATGCCCAGTGTGAATACGATGACGGCAGCGAGCGTTATTTTATACGCGACATCACCGATTCTTTGCTCAAATGCAGCTTTGAGAACCTCATCATTATAATCAGTGTCGTCGTTGGCATCCTCATCGCAATAATCATCGGGGCAGCCGTCACGGCCAGACGGTGTCCGTACGAGACAAAGGTTCTGATGTTTCGACTCTTCGGAGTGCACCCTGCCGACAAGTATCGCGTGGATTTGGAAGAACCACGTCAATACGACATCTATGTATCCCATTACGAACACGATTTACAGGCTAGACAATGGGTGAAATCAAAGTTCCTTCGCAAACTGGAAGAAAATCAAAAAAGAAAGTTCAAAGTCTTTTATTTCGAGCGAGATCTCAATGCTGGTACAGATATGTACGATGAATTAGTCACCCATATGAAACAATGCAGACGAATAGTGTTTATACTAACTAATGATTTTTTCAATGATGAAAAGAATATCTTTGAGGCAGATCAAGCAGAGATAGAACATCGAGCGAGCGACAATCTGCATGGACGTGTCATATATTTGTTATGGAACGAGAGCGTGCGAGAAAAAATAAAATTGGACCCATGGAAGTCTCG AATGGAGGGTAAACGCGTGCTCTGTCCAGACGACAAGTTTCTTCTGGTCTAAGATGAGATACGAACTGCCTTTCAAGGGGGTGCCTTAA

Length: 2088bp

Type: nucleic acid

Chain type: double chain

Topology: Linear

Molecule type: DNA

Feature name: cDNA

Source: Long Oyster

Table 1

Example 2

The components in the kit consist of the following:

Genomic DNA (15ng/ul) of individual gill tissue of Oyster oyster;

Specific primer F;

specific primer R;

Premix EXTaq polymerase Premix Taq ^TM (TaKaRa);

ddH ₂ O.

The amplification system used in the PCR reaction in the kit is calculated as 25 μl: 2ul long oyster individual gill tissue genomic DNA, 12.5ul premixed EXTaq polymerase Premix Taq ^TM (TaKaRa), 0.5ul forward primer F, 0.5ul reverse Primer R, the balance is double distilled water;

Forward primer sequence F: 5'-GAATATTATCTTTTCACTACATACCTGACATG-3';

Reverse primer sequence R: 5'-GGATCAATATGTACTAGTTTGTTGAAGG-3'

Wherein, the conditions of the PCR reaction are: 94° C. for 4 minutes; 94° C. for 10 seconds, 55° C. for 30 seconds, 72° C. for 2 seconds, 35 cycles; 72° C. for 10 minutes.

The product obtained from the reaction is the target sequence containing the SNP site, the sequence is shown in SEQ ID NO.1, and the base at the 2088bp site of the sequence is detected to determine the genotype of the sample.

Although, the present invention has been described in detail with general description, specific implementation and test above, but on the basis of the present invention, some modifications or improvements can be made to it, which will be obvious to those skilled in the art . Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (5)

1. A SNP marker related to the anti-virus characteristics of oyster oyster, characterized in that: the SNP marker is located on the TLR2-1 single exon gene in the TLR immune pathway of oyster oyster; The SNP marker is located at the 858th base at the 5' end shown in SEQ ID NO.1, and the base type is A or C. 2. the SNP marker associated with the long oyster anti-virus property according to claim 1, characterized in that: said SNP marker is located at the leucine rich C-terminal recognition domain (Leucine rich repeat) of the TLR2-1 gene protein molecule C-terminal domain). 3. The SNP marker related to the anti-virus property of oyster oyster according to claim 1, characterized in that: the genotype of the SNP in the genomic DNA is A/A, A/C or C/C. 4. A specific primer for detecting the SNP marker associated with the antiviral properties of oyster oyster, characterized in that: the specific primer is: Forward primer sequence F: 5'-GAATATTATCTTTTCACTACATACCTGACATG-3'; Reverse primer sequence R: 5'-GGATCAATATGTACTAGTTTGTTGAAGG-3'. 5. A test kit for detecting SNP markers associated with oyster anti-virus properties, characterized in that: the test kit contains Forward primer sequence F: 5'-GAATATTATCTTTTCACTACATACCTGACATG-3'; Reverse primer sequence R: 5'-GGATCAATATGTACTAGTTTGTTGAAGG-3'.