KR100960878B1 - Method for identification and parentage using microsatellite marker in Olive flounder - Google Patents

Abstract

The present invention relates to a method for analyzing genotype of each individual of the flounder, and more particularly, to a method for identifying and paternity using a composition capable of simultaneously amplifying eight flounder microsatellite markers. The individual identification method and paternity method using the composition of the present invention can reduce the analysis time and cost by analyzing the genotypes of eight flounder microsatellite markers simultaneously.

Olive flounder, microsatellite marker, multiple amplification polymerase chain reaction, individual identification, paternity

Description

Method for identification and parentage using microsatellite marker in Olive flounder}

The present invention relates to a method for analyzing genotype of each individual of the flounder, and more particularly, to a method for identifying and paternity using a composition capable of simultaneously amplifying eight flounder microsatellite markers.

1. Flounder Genotyping

Flounder ( Paralichthys olivaceus ) is a major aquaculture species that accounts for about half of Korea's total aquaculture production. Although the backbone of the industry, the recent decline in productivity has caused many fish prices. The decline in aquaculture productivity is pointed to not only external factors such as increase in production cost such as feed and labor costs, but also the problems of aquaculture flounder varieties, such as growth rate, disease resistance, increased malformation rate and decreased survival rate. These internal factors are closely related to the genetic composition of the halibut currently in farming, so it is urgent to improve the genotype analysis of the cultivated flounder population in Korea and to overcome the genetic degradation.

Much progress has been made in the molecular genetic studies of flounder using the recently developed molecular biological methods. In particular, the genetic markers of specific sequences within the genome are divided into the genetic characteristics of each individual. It can be applied to individual identification and paternity, and ultimately to breed improvement considering the genetic composition of population and individual.

2. Microsatellite markers

Microsatellite is a region where two to five bases are characteristically repeated among DNA nucleotide sequences in the genome, and various alleles exist according to the degree of repetition, and chromosomes are inherited from parents to offspring according to Mendel's genetic law. Used as markers for mapping (Maria RM et al ., Aquaculture 220: 203-218, 2003), or to define genetic characteristics of populations and individuals (Sekino M & Hara M, Mar Biotechnol 3: 572-589, 2001) and paternity (Hara M & Sekino M, Aquaculture 217: 107-114, 2003). These microsatellite marker specific sites can be amplified by polymerase chain reaction using primers of the upper and lower nucleotide sequences.

3. Multiple Amplification Polymerase Chain Reaction

Multiplex PCR is a method of amplifying two or more regions in a gene simultaneously in one reaction using respective primers. This method can simultaneously amplify several regions on DNA through one polymerase chain reaction, which is very economical in terms of time and cost compared to individual polymerase chain reactions, and is very efficient for genotyping an individual. The multi-amplification polymerase chain reaction is somewhat different from the general polymerase chain reaction, and various factors such as the nature and concentration of each primer reacted at the same time, the concentration and amount of each reagent added and the polymerase chain reaction conditions are mutually sensitive. And influence the outcome (Henegariu O et al ., Biotechniques 23: 504-511).

In this regard, the present inventors established a primer combination and an optimal multi-amplification reaction condition for analyzing eight flounder microsatellite markers at the same time, thereby genetic analysis, individual identification, and paternity of the flounder population using the genotype of the microsatellite marker allele of the flounder. The present invention was completed by confirming that confirmation is possible.

An object of the present invention is to develop a primer combination capable of analyzing multiple microsatellite markers having a DNA polymorphism at the same time, and to establish optimal multi-amplification reaction conditions, thereby enabling genetic individual recognition such as flounder identification and paternity. .

In order to achieve the above object, the present invention provides a pair of primers consisting of oligonucleotides having SEQ ID NOS: 9 and 10, which can specifically amplify the flounder microsatellite markers of SEQ ID NO: 1, the flounder microsatellite markers of SEQ ID NO: 2 A primer pair consisting of oligonucleotides having SEQ ID NOs 11 and 12 that can be specifically amplified, and oligonucleotides having SEQ ID NOs 13 and 14 that can specifically amplify the flounder microsatellite markers of SEQ ID NO: 3, respectively. A primer pair consisting of oligonucleotides having a primer pair, SEQ ID NOs: 15 and 16, respectively, capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 4, and specifically capable of amplifying the flounder microsatellite marker of SEQ ID NO: 5 Primer pair consisting of oligonucleotides having SEQ ID NOs: 17 and 18, respectively; Primer pair consisting of oligonucleotides having SEQ ID NOs: 19 and 20, respectively, capable of specifically amplifying satellite markers; oligos having SEQ ID NOs: 21, 22, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 7 Flounder microsatellites described in SEQ ID NOs: 1 to 8 comprising primer pairs consisting of nucleotides and primer pairs consisting of oligonucleotides having SEQ ID NOs: 23 and 24, respectively, capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 8 Provided are compositions that can specifically amplify a marker.

In addition, the present invention

1) extracting a DNA sample of the fish to be discriminated;

2) amplifying the DNA sample of step 1) using the composition; And

3) provides a flounder individual identification method comprising the step of analyzing the band pattern of the amplification product of step 2).

In addition, the present invention

1) extracting a DNA sample of the fish to be discriminated from the parent generation fish of the fish;

2) amplifying each of the DNA samples of step 1) using the composition;

3) analyzing the band patterns of the amplification products of step 2), respectively; And

4) Provides a paternity method comprising the step of comparing the band pattern of the fish to be discriminated against the band pattern of the parent generation fish of step 3).

In addition, the present invention provides a kit for identifying individuals and / or paternity of fish comprising the composition of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention can specifically amplify a pair of primers consisting of oligonucleotides having sequence numbers 9 and 10, respectively, capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 1, the flounder microsatellite marker of SEQ ID NO: 2 Primer pair consisting of oligonucleotides having SEQ ID NOs: 11 and 12, respectively; primer pair consisting of oligonucleotides having SEQ ID NOs: 13 and 14, respectively, capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 3, SEQ ID NO: 4 Primer pairs consisting of oligonucleotides having SEQ ID NOs: 15 and 16, respectively, capable of specifically amplifying the flounder microsatellite markers of SEQ ID NO: 17 and 18, which can specifically amplify the flounder microsatellite markers of SEQ ID NO: 5 Specific amplification of the flounder microsatellite marker of SEQ ID NO: 6, a pair of primers each consisting of oligonucleotides Primer pair consisting of oligonucleotides having SEQ ID NOs: 19 and 20, respectively; primer pair consisting of oligonucleotides having SEQ ID NOs: 21, 22, respectively, capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 7, SEQ ID NO: It is possible to specifically amplify the flounder microsatellite markers described in SEQ ID NOs: 1 to 8 comprising primer pairs consisting of oligonucleotides having SEQ ID NOs: 23 and 24, respectively, capable of specifically amplifying the flounder microsatellite markers of 8 It provides a composition.

Known microsatellite markers for flounder (Kim WJ et al., Mol Ecol Notes 3: 491-493, 2003; Sekino M & Hara M, Mol Ecol 9: 2155-2234, 2000; Coimbra MRM et al ., Fisheries Sci 67: 358-360, 2001; http://www.ncbi.nlm.nih.gov), considering the number of alleles, the size of the reaction product, the temperature of the amplification reaction, etc. K8 microsatellite markers were selected (see Table 1). SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22 and sequence capable of amplifying the marker Primer pairs consisting of two polynucleotides described as numbers 23 and 24 were designed (see Table 2).

At least one oligonucleotide of the primer pair may be labeled with a fluorescent material. The fluorescent material is not limited thereto, but may include 6-FAM, HEX, NED, ROX, and JOE. By using a variety of fluorescent materials in the composition and by combining different sizes of the amplification products using the composition, it can be confirmed whether each of the flounder microsatellite markers described in SEQ ID NOs: 1 to 8 is specifically amplified. Specifically, primer pairs consisting of two polynucleotides described in SEQ ID NOs: 9 and 10 can yield 78-138 bp amplification products by specifically amplifying the flounder microsatellite markers described in SEQ ID NO: 1. In the exemplary embodiment of the present invention, the polynucleotide represented by SEQ ID NO: 9 was labeled with 6-FAM fluorescent material so as to be distinguished from other amplification products by the size of the amplification product and the color of the fluorescent material. A primer pair consisting of two polynucleotides set forth in SEQ ID NOs: 11 and 12 can yield 136-180 bp amplification products by specifically amplifying the flounder microsatellite markers set forth in SEQ ID NO: 2. In the exemplary embodiment of the present invention, the polynucleotide represented by SEQ ID NO: 11 was labeled with 6-FAM fluorescent material to distinguish it from other amplification products by the size of the amplification product and the color of the fluorescent material. A primer pair consisting of two polynucleotides set forth in SEQ ID NOs: 13 and 14 can yield 192-242 bp amplification products by specifically amplifying the flounder microsatellite markers set forth in SEQ ID NO: 3. In the embodiment of the present invention, the polynucleotide represented by SEQ ID NO: 13 was labeled with 6-FAM fluorescent material so that the size of the amplification product and the color of the fluorescent material could be distinguished from other amplification products. Primer pairs consisting of two polynucleotides set forth in SEQ ID NOs: 15 and 16 can yield 78-162 bp amplification products by specifically amplifying the flounder microsatellite markers set forth in SEQ ID NO: 4. In the embodiment of the present invention, the polynucleotide represented by SEQ ID NO: 15 was labeled with HEX fluorescent material so that the size of the amplification product and the color of the fluorescent material could be distinguished from other amplification products. Primer pairs consisting of two polynucleotides set forth in SEQ ID NOs: 17 and 18 can yield amplification products of 163 to 235 bp by specifically amplifying the flounder microsatellite markers set forth in SEQ ID NO: 5. In the embodiment of the present invention, the polynucleotide represented by SEQ ID NO: 17 was labeled with HEX fluorescent material to distinguish it from other amplification products by the size of the amplification product and the color of the fluorescent material. Primer pairs consisting of two polynucleotides set forth in SEQ ID NOs: 19 and 20 can yield 107-125 bp of amplification products by specifically amplifying the flounder microsatellite markers set forth in SEQ ID NO: 6. In the embodiment of the present invention, the polynucleotide represented by SEQ ID NO: 19 was labeled with NED fluorescent material so that the size of the amplification product and the color of the fluorescent material could be distinguished from other amplification products. A primer pair consisting of two polynucleotides set forth in SEQ ID NOs: 21 and 22 can yield 125-169 bp of amplification products by specifically amplifying the flounder microsatellite markers set forth in SEQ ID NO: 7. In the embodiment of the present invention, the polynucleotide represented by SEQ ID NO: 21 was labeled with an NED fluorescent material to distinguish it from other amplification products by the size of the amplification product and the color of the fluorescent material. A primer pair consisting of two polynucleotides set forth in SEQ ID NOs: 23 and 24 can yield 178-212 bp amplification products by specifically amplifying the flounder microsatellite markers set forth in SEQ ID NO: 8. In the embodiment of the present invention, the polynucleotide represented by SEQ ID NO: 23 was labeled with an NED fluorescent material to distinguish it from other amplification products by the size of the amplification product and the color of the fluorescent material.

The primer pairs may each be included in the composition at a final concentration of 0.1-0.2 μM. The composition may be stored in a lyophilized state, or may further include a buffer solution for stably preserving the polynucleotide in an aqueous solution state.

In the present invention, SEQ ID NOS: 9 and 10, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, which can amplify the flounder microsatellite markers of SEQ ID NOs: 1 to 8, respectively A primer pair consisting of two polynucleotides of SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, and SEQ ID NOs: 23 and 24 was designed and amplified when PCR was performed simultaneously by mixing the primer pairs in one reaction solution. Since the size of the product is duplicated, in order to distinguish the preferred embodiment of the present invention, the fluorescent material of 6-FAM, HEX or NED was labeled on the forward primer (see Table 2 and FIG. 1). PCR was performed using the composition to confirm the amplification and the degree of amplification on a 1.5% agarose gel. As a result, it was confirmed that each marker was simultaneously amplified in all individuals of flounder mother and offspring (see FIG. 2). At this time, the DNA of the flounder mothers was extracted using phenol extraction method using TNES-Urea buffer solution, and the DNA of the flounder was used as a PCR template by the Killex method, and both cases showed good amplification results. However, if 8 markers are amplified simultaneously with the resolution of agarose gel, at least 8 to as many as 16 alleles cannot be distinguished by size. Therefore, the PCR amplification product can be classified into a genotype analyzer (ABI 3100 Genetic analyzer, USA). As a result of the analysis, genotypes of eight microsatellite markers of flounder were obtained simultaneously (see FIG. 3). In this case, alleles of the microsatellite markers represented by the respective peaks appear to overlap the size of eight markers, but may be distinguished with differently labeled fluorescent dyes. Blue peaks represent K1, K2 and K3 markers fluorescently labeled with 6-FAM, green peaks represent K4 and K5 markers labeled with HEX, and black peaks represent K6, K7 and K8 markers labeled with NED. Indicates. In addition, the size (bp) of each allele was determined using Genescan 400 HD ROX (ABI, USA), which is a size standard indicated by a red peak.

In addition, the present invention

1) extracting a DNA sample of the fish to be discriminated;

2) amplifying the DNA sample of step 1) using the composition; And

3) provides a flounder individual identification method comprising the step of analyzing the band pattern of the amplification product of step 2).

In step 1) the DNA sample can be extracted from the fin of the fish, the fish may preferably be a flounder.

The amplification reaction of step 2) can be carried out according to the multiple amplification polymerase chain reaction program. The PCR reaction can be made according to the program of initial denaturation, denaturation, annealing and stretching reactions 35 to 45 times, final stretching and cooling. The multi-amplification polymerase chain reaction program is characterized in that the cycle of denaturation, annealing and elongation reaction is divided into primary and secondary, and the annealing temperature of the secondary cycle is lower than that of the primary cycle. In this case, the number of repetitions of the second cycle is greater than that of the first cycle.

In a preferred embodiment of the present invention, the composition of the present invention is subjected to a multi-amplification polymerase chain reaction program in which the annealing temperature of the first cycle is 60 ° C., the number of repetitions is 20 and the annealing temperature of the secondary cycle is 55 ° C., and the number of repetitions is 25. As a result of performing a multi-amplification polymerase chain reaction on the parent generation of wild flounder and cultured flounder populations in Korea, 10 to 39 alleles were determined for each of the flounder microsatellite markers of SEQ ID NOs. The high expected heterozygosity of 0.710 to 0.920 was confirmed (see Table 3). In this case, the various allele numbers of the markers may indicate that the marker combination of the present invention is a very useful combination for analyzing gene diversity. In addition, the two alleles of the flounder population analyzed showed higher expected heterozygosity rates than the observed heterozygotes, which are calculated as the ratio of heterozygotes. When the population is maintained, the proportion of heterozygotes expected to be increased may be greater than that of the present. This is a result of calculating not only the number of alleles but also the frequency of each allele. When analyzed by the combination of the markers of SEQ ID NO: 1 to 8 has a large number of alleles, the frequency of each allele is a good population means that the genetic diversity is very high. In addition, as a result of genotyping of more than 10,000 genomes, not only the parental flounder but also the offspring of the offspring, no individuals with the same genotype were found. When the calculation is the same in all cases, since the probability of the same genotype is calculated to be 1 / 8.3 이하 10 ¹⁰ or less, the marker combination of SEQ ID NOs 1 to 8 of the present invention shows that the flounder can be identified by comparison of genotyping analysis ( See FIG. 4).

In addition, the present invention

1) extracting a DNA sample of the fish to be discriminated from the parent generation fish of the fish;

2) amplifying each of the DNA samples of step 1) using the composition;

3) analyzing the band patterns of the amplification products of step 2), respectively; And

4) Provides a paternity method comprising the step of comparing the band pattern of the fish to be discriminated against the band pattern of the parent generation fish of step 3).

Since the flounder microsatellite marker of the present invention is inherited to the offspring by the Mendel law, paternity can be identified by comparing the genotype of the offspring and the genotype of the parent population. Accordingly, in a preferred embodiment of the present invention, a multi-amplification polymerase chain reaction was carried out using the composition of the present invention for the parent and progeny flounder, and the parental identification of the progeny was performed by analyzing the band pattern of the amplification product. (See Figure 5). As a result of three parental checks with increasing number of offsprings, 99.9% of the individual genotypes were analyzed and 96.7% of the parents were identified (Table 4).

In other words, the marker combination and multiple amplification reaction conditions of SEQ ID NOs. 1 to 8 according to the present invention can be used very economically and efficiently for individual identification and paternity by halibut genotype profile analysis, As paternity is possible, it can be used for the development of excellent flounder varieties, the identification of producers and the establishment of tracking system for branding and protection.

In addition, the present invention provides a kit for identifying individuals and / or paternity of fish comprising the composition of the present invention.

The kit may further include a deoxynucleotide mixture (dNTP mixture) and a buffer as an amplification reaction element, and may further include a thermostable DNA polymerase such as Taq DNA polymerase. The kit for identifying and / or paternity of the present invention may further include fluorescent materials such as 6-FAM, HEX, NED, ROX, or JOE, agarose and electrophoresis buffer solution, etc. required to confirm PCR results. have. In addition, the kit of the present invention may be provided in the form of a premix of the amplification reaction element with the composition.

The individual identification method and paternity method using the composition of the present invention can reduce the analysis time and cost by analyzing the genotypes of eight flounder microsatellite markers simultaneously.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example 1 Extraction of Flounder Genomic DNA

<1-1> DNA Extraction for Long-term Storage

Part of pectoral fin tissue was excised for flounder genomic DNA extraction. The dorsal tissue is cut by about 1 cm of the softened area except for the ultra-lowest areas to facilitate regeneration after cutting, and TNES-Urea buffer solution (10 mM Tris-Cl; pH 7.5, 125 mM NaCl, 10 mM EDTA, 1% SDS, 6). M Urea,) was added to 600 µl, the proteinase K was treated at a final concentration of 100 ㎍ / ml, dissolved at 37 ° C. overnight, and then the same amount of phenol / chloroform / isopropanol was added and shaken at 12,000 rpm. After centrifugation for 1 minute, the upper layer was collected twice to remove cell debris and proteins and to purify DNA. Then, the precipitate was collected by centrifugation for 30 seconds at 12,000 rpm by adding twice the amount of 100% ethanol of the collected DNA solution, and washed with extra salt with 70% ethanol, followed by TE (10 mM Tris-Cl; pH 8.0, 1 mM EDTA) was dissolved in a buffer solution, refrigerated and used for analysis.

<1-2> Short-term Storage DNA Extraction

If no long-term storage is required after DNA extraction, a chelating resin method (Walsh PS et al ., Biotechniques 10: 506-518, 1991; Suenaga E & Nakamura H, J Chromatography B 820: 137-141, 2005), using the modified method.

Specifically, the pectoral fin tissue of the flounder was cut to 0.1 mg or less using a scalpel, and placed in a solution containing 200 μl of 5% Killex 100 (Bio-Rad, USA) and protease K at a final concentration of 100 μg / ml. Incubated for 15 minutes at 42 ℃, 15 minutes at 50 ℃. After boiling at 100 ° C. for 5 minutes, the cells were destroyed by vortexing to expose the DNA into the aqueous solution. At this time, the intracellular enzymes and cell debris contained in the extract are adsorbed onto the Killex resin (Bio-Rad, USA), and the supernatant is precipitated by centrifuging the extract containing the Killex resin at 6,000 rpm for 5 minutes. Was taken to obtain DNA.

Example 2 Preparation of Primer

<2-1> Microsatellite Marker Selection

Known microsatellite markers for flounder (Kim WJ et al., Mol Ecol Notes 3: 491-493, 2003; Sekino M & Hara M, Mol Ecol 9: 2155-2234, 2000; Coimbra MRM et al ., Fisheries Sci 67: 358-360, 2001; http://www.ncbi.nlm.nih.gov), microsatellite markers were selected in consideration of the number of alleles, the size of the reaction product, and the temperature of the amplification reaction.

As a result, markers K1 to K8 were selected, and the characteristics of the selected markers are shown in Table 1.

Characteristics of microsatellite markers Seat
(Locus)
SEQ ID NO: Repeat motif Repeated center size
(Size range of repeat motifs; bp) Genbank
accession no.
K1 One (CA) ₆ CG (CA) ₁₄ 42 AY328958 K2 2 (CT) ₁₄ AT (CT) ₄ 38 AY328977 K3 3 (TC) ₂₄ 48 AY328973 K4 4 (CT) ₅ CA (CT) ₁₄ 40 AY328982 K5 5 (GT) ₅ AT (GT) ₅ AA (GA) ₄ TT (GA) ₃ AA (GA) ₅ TT (GA) ₆ GTAA (GA) ₃ GT (GA) ₁₈ 114 AY328981 K6 6 (GACA) ₂ (CA) ₁₀ 28 AY328959 K7 7 (CT) ₆ CA (CT) ₁₄ (GT) ₆ 54 AY328976 K8 8 (AC) ₅ AA (CA) ₁₇ AT (AC) ₂ TC (TG) ₂ TTT (TG) ₉ 79 AY328965

<2-2> primer  making

Primer pairs for amplification of the markers K1 to K8 selected by the method of Example 2-1 were determined as shown in Table 2 as SEQ ID Nos. 9 to 24, respectively. When amplifying the markers using the primer pair, the size of the product was overlapped, so that the fluorescent primer of 6-FAM, HEX or NED was labeled on the forward primer during the biosynthesis process of the primer pair. 2 and FIG. 1).

Characteristics of primer pairs Marker SEQ ID NO: Primer sequence (5 '→ 3') Fluorescent label Annealing
Temperature (℃) Product size (bp) K1 9 F: GCG ACT CTC AAA AAT CCG TCA G 6-FAM 65 78-138 10 R: GGA CTT TGT GGG GTT TCA GGA - K2 11 F: GTG TAT GTG TTT CGT TTC CTG C 6-FAM 62 136-180 12 R: TGA AGG CTG TTG TTG CTG TAG - K3 13 F: GAA GGA TCT GGA CTC ATG GTG AC 6-FAM 67 192-242 14 R: CAG CAG CAA AGG CAG AAA GAG - K4 15 F: TCC TTC ACA CTA ATG GAG TCC A HEX 60 78-162 16 R: GTG TCC GAA AGG AAA ACT AGG - K5 17 F: ACA GTA AAA CAG TCC CTC CTG AAC HEX 60 163-235 18 R: AGG AGT CTG GAA CCA AAT GTC TG - K6 19 F: AGA GGA TAT CGA GGG GAG G NED 65 107-125 20 R: CAG CAG TAG CCG ATC TTA GTG - K7 21 F: TTG AAA CTG GAT GCC TCA C NED 60 125-169 22 R: TCT CAC GTT TCC ATG TCA G - K8 23 F: ATC AGG AAC GGG AGA GAC TC NED 63 178-212 24 R: GAG TTC GTC CTT CGT CTT CA -

Example 3 Genotyping

<3-1> Multiplex PCR

First, the DNA obtained from the flounder pectoral tissue by the method of Example 1 was used as template DNA, 5 μl of template DNA (50 ng), 1.5 μl of 10 ㅧ reaction buffer solution (Slogent, Korea), 1.5 μl of 5 Doctor Band Doctor (Slogent, Korea), 0.3 μl of 10 mM dNTP (Slogent, Korea), 0.3 μl of f-Taq (Slogent, Korea), and primers of SEQ ID NO: 9 to SEQ ID NO: 24 with final concentration of 0.1 to 0.2 μM A total of 15 μl of the reaction solution was obtained with the mixed primer set. At this time, the concentration of the primer set mixed at the same time is a primer pair consisting of oligonucleotides of SEQ ID NO: 9 and 10 which can specifically amplify the flounder microsatellite marker of SEQ ID NO: 1, respectively, the final concentration of 0.10 μM, SEQ ID NO: 2 A primer pair consisting of oligonucleotides of SEQ ID NOs: 11 and 12 capable of specifically amplifying flounder microsatellite markers, respectively, and a sequence number 13 capable of specifically amplifying flounder microsatellite markers having a final concentration of 0.13 μM and SEQ ID NO: 3, respectively. And a primer pair consisting of oligonucleotides of SEQ ID NOs: 15 and 16 capable of specifically amplifying a flounder microsatellite marker of SEQ ID NO: 4 and a final concentration of 0.10 μM, respectively, of a oligonucleotide of 14, respectively, and a final concentration of 0.13 μM, respectively. Ollie of SEQ ID NOS: 17 and 18, which can specifically amplify the flounder microsatellite markers of SEQ ID NO: 5 A primer pair consisting of oligonucleotides of SEQ ID NO: 19 and 20 capable of specifically amplifying a flounder microsatellite marker of SEQ ID NO: 6 and a final concentration of 0.20 μM and SEQ ID NO: 7, respectively, and a final concentration of 0.10 μM, SEQ ID NO: 7 A primer pair consisting of oligonucleotides of SEQ ID NO: 21 and 22 capable of specifically amplifying the flounder microsatellite markers of the final concentration 0.20 μM, respectively, SEQ ID NO: capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 8 Optimal amplification was obtained when primer pairs consisting of oligonucleotides of 23 and 24 were used, respectively, at a final concentration of 0.20 μM. After mixing well the total 15 μl PCR reaction solution, 95 ℃, 10 minutes; 95 ° C. 20 seconds, 60 ° C. 40 seconds, 72 ° C. 1 minute, 20 times; 95 ° C. 20 sec, 55 ° C. 40 sec, 72 ° C. 1 min, 25 times; PCR was performed using a Dyad PCR instrument (Bio-Rad, USA) at a condition of 72 ° C. for 10 minutes, and the final reaction product was confirmed by 1.5% (w / v) agarose gel electrophoresis.

As a result, as shown in FIG. 2, the PCR of the flounder mother DNA obtained by the phenol extraction method using the TNES-Urea buffer solution was carried out by PCR, and the DNA of the flounder offspring obtained by the killex was used as a template. When the PCR reaction was performed, it was confirmed that each marker was well amplified simultaneously in all subjects.

<3-2> Genotyping

The PCR reaction products obtained by the method of Example 3-1 were diluted 10 times, 30 times, 50 times, and 80 times with respect to four randomly extracted samples to determine the dilution rate for genotyping. Mix 1 μl, 0.2 μl of GeneScan 400HD ROX (ABI, USA), and 10 μl of HiDi formamide (ABI, USA) to increase the intensity of analytical peaks with a 3100 genetic analyzer (ABI, USA). The dilution concentration showing about 4000 rfu was confirmed, and the genotype was analyzed by the appropriate dilution at the ratio determined for the remaining samples.

As a result, as shown in Fig. 3, genotypes of eight microsatellite markers of flounder could be obtained at the same time. In this case, alleles of the microsatellite markers represented by the respective peaks appeared to overlap the size of eight markers, but could be distinguished with differently labeled fluorescent dyes (FIG. 3A). Blue peaks represent K1, K2 and K3 markers fluorescently labeled with 6-FAM, green peaks represent K4 and K5 markers labeled with HEX, and black peaks represent K6, K7 and K8 markers labeled with NED. Indicated. In addition, the size (bp) of each allele was determined using Genescan 400 HD ROX (ABI, USA), the size standard indicated by a red peak (FIG. 3B).

Example 4 Individual Identification

<4-1> Genotype Analysis of Parent Group

DNA was obtained from the method of Example 1 from 389 wild flounder and 735 cultured flounder in Korea. The genotype was analyzed by the method of Example 3 using the DNA as template DNA.

As a result, as shown in Table 3, the number of alleles of 10 to 39 alleles was confirmed for each marker, and the observed heterozygosity was found to be a high heterozygosity value of 0.677 to 0.902. In addition, the two alleles in the flounder population showed higher expected heterozygosity ratios of 0.710 to 0.920 compared to the observed heterozygotes, which were calculated as different heterozygote ratios.

Genotyping of the Flounder Population Locus K1 K2 K3 K4 K5 K6 K7 K8 Average Analysis number
(No. of fishes) 1124 1124 1124 1124 1123 1122 1102 1036 1124 Allele number
(No. of Alleles) 30 22 26 39 30 10 23 18 24.75 Allele size range
(Allele size range; bp) 78-138 136-180 192-242 78-162 163-235 107-125 125-169 178-212 - Observation heterojunction, Ho
Observed heterozygosity 0.853 0.890 0.881 0.880 0.902 0.677 0.844 0.788 0.839 Expected Release Amount, He
(Expected heterozygosity) 0.876 0.910 0.907 0.893 0.920 0.710 0.872 0.850 0.867 PIC ^* 0.865 0.903 0.900 0.884 0.914 0.656 0.861 0.835 0.852

Polymorphism information content (Botstein D et al . , American J Human Genetics 32: 314-331, 1980)

<4-2> Genotyping of Offspring

The genotype analysis of 10,000 progeny populations of the wild and cultured flounder populations of Example 4-1 and the degree of difference between genotypes were compared.

Specifically, DNA was obtained by the method of Example 1 from about 10,000 randomly selected 10,000 out of a population of progeny produced from artificial insemination from 210 wild flounder and 366 aquaculture flounder among the total 1124. It was. The genotype was analyzed by the method of Example 3 using the DNA as template DNA.

In addition, genotype comparisons were performed using the MSA (Microsatellite Analyzer; Dieringer, D & Schl ㆆ tterer, C, Mol Ecol Notes 3: 167-169, 2002) program. Standard genetic distance (Nei, M., Genetics 89: 538-590, 1978) matrix was analyzed.

In this case, when the same genotype is set to 0 and the genetic distance between the individuals represented by the value up to 1 when the genotypes are all different, the individuals having the same genotype between each individual were not found (see FIG. 4). This diversity is theoretically calculated when the alleles of the respective markers are the same, the probability of the same genotype is calculated as 1 / 8.3 ㅧ 10 ¹⁰ or less, so the marker combination of SEQ ID NOs 1 to 8 of the present invention Comparison of genotyping analysis shows that flounder can be identified.

Example 5 Paternity

In the results of Example 4, the genotype profile unique to each individual indicates the likelihood of paternity as compared to the genotype of the parent population as well as individual identification as these markers are inherited to the offspring by Mendel's law.

Genotyping and parental identification were performed on 244 offspring flounder produced from 221 flounder females and 200 males. Subsequently, a second analysis was performed on 802 animals by increasing the number of offsprings, and a third analysis was performed on 4300 animals, further increasing the number of offsprings.

DNA was obtained from the progeny flounder by the method of Example 1-2. The genotype was analyzed by the method of Example 3 using the DNA as template DNA. On the basis of the analysis results, paternity analysis was performed to finally identify the parent individuals by sequentially locating only the parent individuals corresponding to the allele types of markers of the offspring (see FIG. 5).

As a result, as shown in Table 4, 99.9% of the individual genotyping rates and 96.7% of the parents were identified.

Paternity Primary analysis Secondary analysis 3rd analysis system Analysis 244 803 4300 5347 Genotyping (%) 244 (100) 802 (99.9) 4300 (100) 5346 (99.9) Household confirmation (%) 244 (100) 757 (94.4) 4168 (96.9) 5169 (96.7)

1 is a diagram showing an allele size range and a fluorescent dye label for each marker.

Figure 2 is a diagram showing the results of electrophoresis on 1.5% agarose gel with the result of simultaneously amplifying the markers of SEQ ID NOs: 1 to 8 by multiple amplification reaction conditions using primer pairs described in SEQ ID NOs: 9 to 24. At this time, the DNA of the flounder mothers was extracted by phenol extraction, and the DNA of the flounders was extracted using the Killex method (M: 1Kb ladder; lanes 1-12: flounder mothers # 1-12; lanes 13-24). Halibut pups # 1-12).

Figure 3 shows the results of genotyping of the flounder, each peak showing the allele of the microsatellite marker:

a: As an example of genotyping results of three flounder individuals, eight markers overlapped simultaneously and appear as peaks [blue peak: K1, K2, K3 markers fluorescently labeled with 6-FAM; Green peak: K4, K5 marker labeled with HEX; Black peak: K6, K7, K8 marker labeled with NED; Red peak: size standard Genescan 400 HD ROX (ABI, USA); Number above peak: size of each gene (bp)]; And,

B: Genotype analysis of one flounder individual (gray bar above the peak: name and size range of each marker).

Figure 4 is a diagram showing the genetic distance between each individual. If the genotype of each individual is equal to 0, and if all genotypes are different, the genetic distance is represented as 1. Only the same object appears as a diagonal 0.

5 is a diagram showing a process of identifying the parental parent by comparing the genotype of the offspring with the genotype of the parent population.

<110> National Fisheries Research and Development Institute <120> Method for identification and parentage using microsatellite          marker in Olive flounder <130> 7P-10-68 <160> 24 <170> KopatentIn 1.71 <210> 1 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> K1 <400> 1 cacacacaca cacgcacaca cacacacaca cacacacaca ca 42 <210> 2 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> K2 <400> 2 ctctctctct ctctctctct ctctctctat ctctctct 38 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> K3 <400> 3 tctctctctc tctctctctc tctctctctc tctctctctc tctctctc 48 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> K4 <400> 4 ctctctctct cactctctct ctctctctct ctctctctct 40 <210> 5 <211> 114 <212> DNA <213> Artificial Sequence <220> <223> K5 <400> 5 gtgtgtgtgt atgtgtgtgt gtaagagaga gattgagaga aagagagaga gattgagaga 60 gagagagtaa gagagagtga gagagagaga gagagagaga gagagagaga gaga 114 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> K6 <400> 6 gacagacaca cacacacaca cacacaca 28 <210> 7 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> K7 <400> 7 ctctctctct ctcactctct ctctctctct ctctctctct ctgtgtgtgtgt gtgt 54 <210> 8 <211> 79 <212> DNA <213> Artificial Sequence <220> <223> K8 <400> 8 acacacacac aacacacaca cacacacaca cacacacaca cacacaatac actctgtgtt 60 ttgtgtgtgt gtgtgtgtg 79 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> K1 forward primer <400> 9 gcgactctca aaaatccgtc ag 22 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K1 reverse primer <400> 10 ggactttgtg gggtttcagg a 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> K2 forward primer <400> 11 gtgtatgtgt ttcgtttcct gc 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K2 reverse primer <400> 12 tgaaggctgt tgttgctgta g 21 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> K3 forward primer <400> 13 gaaggatctg gactcatggt gac 23 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K3 reverse primer <400> 14 cagcagcaaa ggcagaaaga g 21 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> K4 forward primer <400> 15 tccttcacac taatggagtc ca 22 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K4 reverse primer <400> 16 gtgtccgaaa ggaaaactag g 21 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> K5 forward primer <400> 17 acagtaaaac agtccctcct gaac 24 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> K5 reverse primer <400> 18 aggagtctgg aaccaaatgt ctg 23 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> K6 forward primer <400> 19 agaggatatc gaggggagg 19 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> K6 reverse primer <400> 20 cagcagtagc cgatcttagt g 21 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> K7 forward primer <400> 21 ttgaaactgg atgcctcac 19 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> K7 reverse primer <400> 22 tctcacgttt ccatgtcag 19 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> K8 forward primer <400> 23 atcaggaacg ggagagactc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> K8 reverse primer <400> 24 gagttcgtcc ttcgtcttca 20  

Claims (14)

Primer pair consisting of oligonucleotides having SEQ ID NOs: 9 and 10, respectively, capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 1, SEQ ID NO: 11 capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 2 And a pair of primers each consisting of oligonucleotides having 12, a pair of primers each consisting of oligonucleotides having SEQ ID NOs: 13 and 14 capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 3, and the flounder microsatellite of SEQ ID NO: 4 A primer pair consisting of oligonucleotides having SEQ ID NOs: 15 and 16 capable of specifically amplifying a marker, and oligonucleotides having SEQ ID NOs: 17 and 18, respectively capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 5 Primer pair consisting of, can specifically amplify the flounder microsatellite marker of SEQ ID NO: 6 A pair of primers consisting of oligonucleotides having SEQ ID NOs: 19 and 20, a pair of primers consisting of oligonucleotides having SEQ ID NOs: 21 and 22, respectively, capable of specifically amplifying the flounder microsatellite marker of SEQ ID NO: 7; A composition capable of specifically amplifying the flounder microsatellite markers set forth in SEQ ID NOs: 1 to 8 comprising primer pairs consisting of oligonucleotides having SEQ ID NOs: 23 and 24, respectively, capable of specifically amplifying flounder microsatellite markers. . The composition of claim 1, wherein at least one oligonucleotide of the primer pair is labeled with a fluorescent material. delete The composition of claim 1, wherein the primer pairs each comprise a final concentration of 0.1 to 0.2 μM. 1) extracting a DNA sample of the fish to be discriminated; 2) amplifying the DNA sample of step 1) using the composition of claim 1; And 3) A method for identifying a flounder, comprising analyzing a band pattern of the amplification product of step 2). 6. The method according to claim 5, wherein the amplification reaction of step 2) is performed according to a multiple amplification polymerase chain reaction program, characterized in that the cycle of denaturation, annealing and stretching reactions is carried out by dividing the cycle into primary and secondary. . delete 7. The method of claim 6, wherein the annealing of the primary cycle is carried out at a temperature 5 ° C higher than the annealing of the secondary cycle. 7. The method of claim 6, wherein the number of repetitions of the secondary cycle is five times greater than the number of repetitions of the primary cycle. 1) extracting a DNA sample of the fish to be discriminated from the parent generation fish of the fish; 2) amplifying each of the DNA samples of step 1) using the composition of claim 1; 3) analyzing the band patterns of the amplification products of step 2), respectively; And 4) paternity identification method comprising the step of comparing the band pattern of the fish to be discriminated against the band pattern of the parent generation fish of step 3). A kit for identifying an individual of a fish comprising the composition of claim 1. 12. The kit of claim 11, wherein the kit is provided in the form of a premix. Paternity kit of fish comprising the composition of claim 1. The kit of claim 13, wherein the kit is provided in the form of a premix.

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