KR102110100B1 - SNP primer set for golden flat fish and identification method of golden flat fish same - Google Patents

Abstract

The present invention provides a golden flatfish discrimination primer set comprising a primer set consisting of SEQ ID NO: 3 and SEQ ID NO: 4, and a method for discriminating golden flatfish using the same. The present invention allows accurate discrimination of the golden flatfish genotype of a BCO gene using seeds or the young of flatfish, and thus can predict stabilization of the supply and demand of golden flatfish by developing an accurate species discrimination technique. Also, when the seeds are delivered to a farm and nurtured to a size that can be sold, it is possible to pursue national interest by increasing fishery income and exports.

Description

Molecular marker for discrimination of golden halibut and method for discriminating golden halibut using the same {SNP primer set for golden flat fish and identification method of golden flat fish same}

The present invention relates to a molecular marker for discriminating golden halibut showing a golden body color, and a method for discriminating a golden halibut showing a golden body color using the same. It relates to a diagnostic method using this.

Flounder ( Paralichthys olivaceus_ is one of the major fish farmed in Asian countries including Japan and China. It is a cultured fish that accounts for more than 45% of Korea's aquaculture production as of 2018. It is a saltwater fish belonging to the flounder flounder that inhabits in Canopia.

The body shape is a flat shape adapted to the environment under the sea, and the body color has a yellowish-brown protective color that is indistinguishable from the sandy bottom. Body color is brown or dark on the eye side and white on the opposite side. The eye flocking to one side, which is characteristic of flounder, does not appear during the period of the dent, but as it grows, the right eye gradually moves to the left. It usually lives on sandy bottoms of less than 200m in depth, and depending on the season, it moves from place to place to feed or lay eggs. The flounder inhabiting the west coast of Korea spends the winter by flocking to the south between autumn and winter, and when it comes again in spring, it moves north and mates and spawns.

In recent years, economic damage has been steadily increasing and increasing in aquaculture fish farming halibut due to environmental effects such as decrease in halibut consumption, high water temperature and red tide. The decrease in consumption, which is the main cause of this phenomenon, can be confirmed through statistical data showing the rapid increase in consumption of salmon on behalf of halibut. Therefore, the development of new high value-added varieties targeting Japan and China is actively being pursued to pioneer new markets for halibut.

In particular, golden halibut is a new breed with a yellowish-yellow body color, and is rarely found in nature with a probability of millions of times. It is a high value-added variety that is traded at a price that is about 3-4 times higher than that of ordinary halibut. In addition, golden halibut boasts a superior texture with a sticky meat compared to regular halibut, and the golden color of halibut is preferred as a good ingredient to produce a luxurious and special look.

Aquaculture Golden flounder is the first live fish to receive K-FISH certification, an integrated export brand from the Ministry of Oceans and Fisheries earlier this year. Export contracts were signed with Canada, China, the Philippines and Singapore in 2017, and exports are expected to expand due to the high interest of countries in the Greater China and Southeast Asia. It is expected that mass production will be carried out by screening four mutant golden flounder formed with melanin pigment abnormalities and breeding them with the next 2,000 second-generation flounder.

Although various genetic and environmental factors act as a cause of having a golden body color, the main cause is accumulation of beta-carotene, which is a major yellow color, in the body. Beta-carotene is a natural yellow pigment present in various plants, fruits, and microalgae.

The structure of beta-carotene is an isoprenoid that is biochemically synthesized from 8 isoprenes, and has beta-rings at both ends of 40 carbons. It is metabolized to retinoic acid through retinal by beta-carotene oxygenase 1, and synthesis of rosafluene and beta-ionone by BCO2 and strong antioxidant action are toxic and carcinogenic Ease the substance. That is, harmful (active) oxygen plays an important role in protecting cells by preventing damage to cells in the body. It is associated with a very important role in inhibiting mitochondrial oxidative stress.

Recently, it has been revealed that a mutation in the BCO2 gene induces beta-carotene accumulation in the body and changes the body color to yellow, and a study has been conducted on the correlation between gene variation and body color expression of BCO2 in other tibial fish. Accordingly, the present invention is to provide a primer set and a discrimination method capable of identifying and amplifying a golden halibut specific SNP by performing a single nucleotide polymorphism (SNP) analysis of the halibut BCO2 gene.

In Korean Patent Registration No. 10-2018-0088956, a primer set for discriminating male and female flounder including a primer set of SEQ ID NOs: 5 and 6, a primer set of SEQ ID NOs: 7 and 8, and a primer set of SEQ ID NOs: 9 and 10 and the same Disclosed is a method for discriminating male and female flounder. Korean Patent Registration No. 10-2010-0089645 relates to a set of primers for discrimination of halibut females and a method of discriminating male and female halibuts using the same. The method of discrimination of halibut sex in the present invention includes PCR aromatase primer set or FEF1α for halibut sex determination with base sequences of FArom-F3 (5 '-TGGCTACAGGGTACCAAAGG-3') and FArom-R3 (5 '-GAACCGAATGGCTGGAAGTA-3') PCR Elongation Factor-1 alpha primer set for discrimination of flounder with base sequence of -F1 (5 '-GCAGCTCATTGTTGGAGTCA-3') and F-EF1α-R1 (5 '-ACACTTGCAGGGTTGTAGCC-3') amplifying the DNA extracted from the flounder through PCR using any one of the primer sets), and measuring the aromatase gene expression of each of the amplified products, and then measuring the aromatase gene expression between the products. Disclosed is a method for discriminating male and female halibut, which includes the step of determining the sex of halibut fish by relative comparison. Republic of Korea Patent No. 10-2011-0040665 relates to the use of the Fig α or Scp3 gene as a marker composition for discrimination of halibut sex, a marker composition for discrimination of halibut using Fig α or Scp3 gene, a composition for discriminating halibut sex using the sex Disclosed is a discriminative diagnostic method. Republic of Korea Patent Registration No. 10-2014-0124525 discloses a SNP marker that can genetically discriminate the male and female of halibut seedlings by comparing and analyzing the halibut male and female genomes.

In the present invention, since the signaling mechanism and specific gene related to the body color of the golden halibut are not known, research on the golden body color of the halibut is still in an unsatisfactory level, and it is difficult to collect a seedling having a golden body color gene. In order to solve the problem of additional cost required for production because the expression of the golden body color of the produced seedling is not confirmed, it is intended to provide a gene marker capable of identifying the specific genotype of the golden flounder.

In order to achieve the above object, the golden halibut discrimination single nucleotide polymorphism (SNP) primer set according to an embodiment of the present invention may be composed of SEQ ID NO: 3 and SEQ ID NO: 4. In addition, the kit for discriminating golden flounder of the present invention may include the primer set.

Method for determining the golden halibut according to another embodiment of the present invention comprises the steps of extracting the DNA of the target halibut sample; Generating a PCR product by performing a polymerase chain reaction (PCR) using the DNA extracted in the step as a template and using the primer pairs described in SEQ ID NOs: 3 to 4; And analyzing whether to amplify the PCR product.

While the existing sequencing analysis method takes about 2-3 days to derive the final result, the present invention can easily and rapidly amplify based on PCR to derive the flounder with the golden body color gene and confirm the result with a small amount of DNA. It is possible.

In addition, by using the primer set for discrimination of golden halibut according to the present invention and the method of discriminating golden halibut using the same, it is possible to accurately discriminate the golden halibut genotype of the BCO gene by using the halibut of the seedling or pomace stage, and by developing the accurate species discrimination technology. Not only can the stabilization of the supply and demand of golden flounder be predicted, but when the seedlings are delivered to the farm and nurtured to a size that can be sold, they can pursue national interest by increasing fishery income and exports.

Figure 1 shows the flounder showing a general body color and the golden flounder of the present invention.
Figure 2 shows a part of the BCO2 gene sequence of the Genbank flounder reference genome sequence.
3 shows the first SNP site of the BCO gene.
4 shows the second SNP site of the BCO gene.
Figure 5 shows a primer capable of amplifying the SNP genotype of a regular flounder.
6 shows a primer capable of amplifying the SNP genotype of golden flounder.
7 is a result of PCR electrophoresis using a general flounder specific marker including SNP1 and SNP2.
8 is a result of PCR electrophoresis using golden flounder specific markers including SNP1 and SNP2.

The term referred to in the present invention, flounder ( Paralichthys olivaceus ) is a saltwater fish belonging to the flounder family of flounder, which lives mainly in the nearby waters of Korea, China, and Japan.

Figure 1 shows the flounder showing a general body color and the golden flounder of the present invention. As shown in FIG. 1, the flounder having a general body color has brown and olive colors on the side with eyes, while the golden flounder has prominent gold and yellow colors.

Accordingly, the present invention is to provide a primer set represented by SEQ ID NOs: 3 to 4 capable of discriminating the golden flounder.

The term, nucleotide, referred to in the present invention is a deoxyribonucleotide or a ribonucleotide present in a single-stranded or double-stranded form, and may include analogs of nucleotides that are not specifically mentioned otherwise.

The term referred to in the present invention, polymorphism (polymorphism) means a case where two or more alleles exist in a single locus, and a polymorphic site means a locus in which the allele exists. Single Nucleotide Polymorphism (SNP) of the present invention refers to the difference in one base of a specific region of a DNA sequence between alleles in a population or individual.

The allele of the present invention means several types of a gene that exist at the same locus of a homologous chromosome. Alleles are also used to indicate polymorphism, for example, SNPs have two types of alleles.

In the present invention, a marker (marker) refers to a nucleotide sequence used as a reference point when identifying genetically unspecified associated loci. The term also applies to nucleic acid sequences complementary to a marker sequence, such as a nucleic acid used as a primer set capable of amplifying a marker sequence. The location of a molecular marker on a genetic map is called a genetic locus.

The present invention provides a golden flounder discrimination kit comprising a primer set comprising SEQ ID NOs: 3 to 4 and a reagent for performing a DNA amplification reaction. The kit may include DNA polymerase, dNTPs, buffers, etc. as reagents for performing the amplification reaction. In addition, the kit of the present invention may further include a user guide describing optimal conditions for performing the reaction. As a guide for the kit, in more detail, it includes a brochure or leaflet in the form of a brochure, leaflet attached to the kit, and a description on the surface of the package including the kit. can do. In addition, the handbook may include information disclosed or provided through an electric medium such as the Internet.

The term referred to in the present invention, the primer is a nucleic acid sequence having a short free 3-terminal hydroxyl group (free 3'-hydroxyl group) can form a complementary template (template) and base pair (base pair) and start for copying the template A short nucleic acid sequence that functions as a point.

In addition, primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures and four different nucleoside triphosphates. PCR conditions, sense and antisense primer lengths can be modified based on those known in the art. These oligonucleotide primers can be easily prepared and used by those skilled in the art according to methods known in the art.

Hereinafter, an example of an experiment related to a molecular marker for discriminating a golden halibut showing a golden body color of the present invention and a method for discriminating a golden halibut using the same will be described in detail with reference to the accompanying drawings.

Experimental Example 1. Extraction of genomic DNA from halibut

All golden halibut samples according to the experimental examples of the present invention were collected from Haeyeon Co., Ltd., and 15 natural halibut samples and 10 aquaculture animals were used as general halibut samples. In order to extract 25 genomic DNA from common flounder and golden flounder, the tail fin fixed to 99.9% alcohol was cut into 10 mg, placed in a 1.5 ml tube containing distilled water, and washed with water 5 times to remove alcohol.

The tissue was completely crushed using a homogenizer, and genomic DNA was extracted and purified using a commercially available DNA extraction kit (DNeasy Blood & Tissue kit, Qiagen). Thereafter, the concentration and purity of the extracted genomic DNA was confirmed using an absorbance photometer.

Experimental Example 2. Sequence analysis of BCO2 gene and development of single nucleotide polymorphism (SNP) marker

In order to analyze a part of the base sequence of the BCO2 gene of the flounder, PCR was performed using primers for sequencing. Figure 2 shows a part of the BCO2 gene sequence of the Genbank flounder reference genome sequence. The BCO2 gene was found using Genbank's halibut reference genome sequence, and 5 halibut and 5 halibut were used for the analysis.

To amplify the BCO2 gene region, a PCR reaction solution was prepared using the AccuPower PCR masterMix (Bioneer, Korea) as shown in Table 1 below, and PCR was performed using the primers in Table 2. PCR amplification conditions are shown in Table 3.

PCR reaction solution conditions reagent Reagent amount per 20 μl reaction solution 2xPCR Master Mix 10 μl Forward Primer (10 pmole / μl) 1 μl Reverse Primer (10 pmole / μl) 1 μl Template DNA 2 μl D.W 7 μl Total volume 20 μl

Primer sequence for sequencing Base sequence (5 '-> 3') Product size (bp) BCO2_F: 5'-AGACTGGCTGTGATGTACTCT-3 ' 199 BCO2_R: 5'-AGGTTGTTCCCACACCTTTCA-3 '

PCR conditions process Temperature ( ^o C) time Number of reactions Initial denaturation reaction 95 5 minutes One Denaturation reaction 95 30 seconds 30 Binding reaction 58 30 seconds Kidney reaction 72 30 seconds Final kidney reaction 72 1 minute One

After securing the nucleotide sequence of the completed PCR amplification product, a golden flounder specific SNP was developed using a multiple sequence alignment program, and FIG. 3 shows the first SNP site of the BCO gene, and FIG. 4 shows the second SNP site.

Experimental Example  3. Plain flounder and Golden flounder  allele specific PCR primer  Production and PCR  analysis

Usually, the annealing temperature of the primer is determined based on the Tm value after calculating the calculation formula of {4 * (G + C) + 2 * (A + T)}, and the accuracy of the primer induced by raising the annealing temperature is This is actually a very low level because it is generated by a single difference in SNP sequence.

Therefore, the phenomenon caused by arranging one mismatch sequence around the SNP sequence can significantly improve the accuracy of the primer because two sequence differences occur if the SNP location does not have the correct sequence.

Accordingly, the allele specific primer according to the present invention was prepared by mismatching the nucleotide sequence of -1 or -2 in the SNP at the 3 'end with TG or CA to perform the allele specific PCR method, and Table 4 below. Denotes a primer capable of amplifying the SNP sites of the common flounder and golden flounder prepared through the above method. In addition, Figures 5 and 6 show the SNP sites of the common and golden flounder to be amplified with the primer sets represented by SEQ ID NOs: 1 to 4.

The method of changing the -2 nucleotide sequence of the SNP site gives specificity to the last 3 'base sequence of the primer, and the -2 nucleotide sequence of the 3' SNP is changed from A to G, and SNP2 is changed from T to G. By changing it, the general halibut SNP identification primers (O_AS_F, _R) and the golden halibut SNP identification primers (G_AS_F, _R) were designed.

Although sequencing of SNPs through existing primer preparation and method is useful, the existing method is labor intensive and requires a high cost procedure. Accordingly, the allele-specific PCR using the modified primer according to the experimental example of the present invention has an effect suitable for multi-sample or multi-location SNP sequencing (genotyping) that does not require separate equipment, and is low cost and simple to implement. It is easy to shorten the time.

allele specific primer Discriminant species Discrimination SNP Base sequence (5 '-> 3') Product size (bp) Sequence number Normal SNP1 O_AS_F: 5'-AGAGTTATACTGTAGTATGTCGGG-3 ' 100 One SNP2 O_AS_R: 5'-GCTGTTTTCTTGTACAGTCTTGAGTT-3 ' 2 Gold SNP1 G_AS_F: 5'-AGAGTTATACTGTAGTATGTCGGC-3 ' 102 3 SNP2 G_AS_R: 5'-CTGTTTTCTTGTACAGTCTTGAGTC-3 ' 4

Reagent composition for PCR using the primer is shown in Table 5, PCR reaction conditions are shown in Table 6. PCR products were confirmed by electrophoresis.

PCR reaction solution conditions reagent Reagent amount per 20 μl reaction solution 2xPCR Master Mix 10 μl Forward Primer (10 pmole / μl) 1 μl Reverse Primer (10 pmole / μl) 1 μl Template DNA 2 μl D.W 7 μl Total volume 20 μl

PCR conditions process Temperature ( ^o C) time Number of reactions Initial denaturation reaction 95 5 minutes One Denaturation reaction 95 30 seconds 30 Binding reaction 59 30 seconds Kidney reaction 72 30 seconds Final kidney reaction 72 1 minute One

7 shows the results of PCR electrophoresis using general halibut specific markers including SNP1 and SNP2. 8 is a result of PCR electrophoresis using golden flounder specific markers including SNP1 and SNP2.

According to the above results, in the case of O_AS primer for SNP identification of normal halibut, it was well amplified in normal halibut of 25 mils. In addition, the golden halibut SNP identification G_AS primer was amplified only in the golden halibut but not in the normal halibut.

As a result of the analysis using the SNP marker, the SNP genotype does not distinguish between the homotype (homozygote) and the heterotype (heterozygote), and it is possible to confirm the genotype of the golden flounder by simultaneously confirming two SNP genotypes with one PCR analysis.

If PCR amplification is performed using a golden flounder primer, it can be determined that the BCO2 gene has a golden flounder genotype, which is very effective in early identification of golden flounder, a high value-added variety.

By developing a technology for discrimination of halibut with a golden body color gene, it is possible to quickly select seeds with a genotype, which is economical with cost reduction, and mass production continues if golden halibut is produced in aquaculture through seed selection. It is expected to bring economic benefits, such as increasing income from fish and securing national profits from exports, making it industrially available.

<110> BLUGEN KOREA <120> SNP primer set for golden flat fish and identification method of          golden flat fish same <130> p20-0070316 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> G_AS_Forward primer <400> 1 agagttatac tgtagtatgt cggg 24 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> G_AS_Reverse primer <400> 2 gctgttttct tgtacagtct tgagtt 26 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> G_AS_Forward primer <400> 3 agagttatac tgtagtatgt cggc 24 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> G_AS_Reverse primer <400> 4 ctgttttctt gtacagtctt gagtc 25

Claims (3)

Single nucleotide polymorphism (SNP) primer set for golden halibut discrimination consisting of the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4
Single nucleotide polymorphism (SNP) composition for discrimination of golden flounder comprising the primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4
Extracting the DNA of the discrimination target flounder sample; Polymerase chain reaction (PCR) using the extracted DNA as a template and using a single nucleotide polymorphism (SNP) primer set for golden halibut discrimination consisting of the nucleotide sequences shown in SEQ ID NOs: 3 and 4. Generating a PCR product by performing; And analyzing the amplification of the PCR product.

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