KR102491088B1 - microsatellite marker for identification of geographical origin of Pagrus major and its use - Google Patents

Abstract

The present invention relates to the development of a microsatellite marker for identifying the country of origin of Japanese and domestic Pagrus majors distributed in Korea and a method for determining the country of origin using the same. The present invention relates to the method for identifying Japanese and domestic Pagrus majors, which performs genotyping for each Pagrus major population using a combination of newly developed microsatellite markers based on a genotyping by sequencing method so as to effectively determine the origin of Pagrus majors. The present invention includes the microsatellite markers and a method for analyzing population affinity by country of origin. Therefore, the present invention provides nine new microsatellite markers which can determine the origin of domestic and Japanese Pagrus majors distributed in Korea and the method for determining the origin of domestic and Japanese Pagrus majors using the same. Therefore, the present invention increases public confidence in the management of the origin of marine products.

Description

Microsatellite marker for identification of geographical origin of red snapper and its use {microsatellite marker for identification of geographical origin of Pagrus major and its use}

The present invention relates to a microsatellite marker capable of distinguishing domestic and Japanese red sea bream ( Pagrus major ) and its use.

Red sea bream ( Pagrus major ) is a fish species belonging to the sea bream family ( Sparidae ) of sea bass ( Perciformes ) and lives in the water depth of 10-200m along the Northwest Pacific coast of Korea, Japan, China, Taiwan and Vietnam. In addition, red sea bream is a migratory fish that migrates to the coast of China and the west coast of Korea in the spring after wintering in the southern waters of Jeju Island in Korea. The back is red and the belly is white. Red sea bream is a high-quality food ingredient and is consumed as raw fish, grilled, steamed, etc. in Korea, Japan, and China, and is a fish species with high economic value.

Korea recorded the highest production, recording 11,090 tons in 2009 from 1,398 tons in 2000. The latest production in 2019 was recorded at 7,712 tonnes. In terms of production amount, it recorded the maximum production amount of about 95 billion won in 2009 and about 80.3 billion won in 2019, accounting for 2.7% of the total fish production amount (National Statistical Office).

Currently, imported red sea bream in Korea is steadily imported from Japan in the amount of 2,302 tons in 2017, 3,449 tons in 2018, and 2,504 tons in 2019 (Ministry of Food and Drug Safety). However, at present, the rate of close inspection for all edible imported aquatic product varieties is very low at 6%, and domestic and Japanese red sea bream cannot be distinguished with the naked eye, raising concerns about Japanese fish in Korea and demand for safe aquatic food. It is increasing. In addition, since some unscrupulous distributors try to distribute Japanese products by disguising them as domestic products, there are cases in which objective identification standards and techniques for identifying the country of origin are needed in the process of import, quarantine, and distribution to prevent illegal distribution.

Microsatellite (MS) marker, restriction fragment length polymorphism (RFLP), allozyme, mitochondrial DNA, random amplified Polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and single nucleotide polymorphism (SNP) have been developed.

In this study, microsatellite markers were used to systematically distinguish the country of origin of domestic and Japanese red sea bream using DNA genetic information.

Microsatellite markers are molecular markers that have been widely used for genetic relationship analysis in genetics since the 1990s. Simple sequence repeat (SSR) is a locus on DNA in which a short base pair (bp) sequence of 1 to 6 appears repeatedly. ) is also called Since microsatellite markers follow Mendel's laws of genetics and have high polymorphism and reproductivity, they have been used for lineage, group genetic relationship analysis, and paternity for various organisms including mammals and plants since the 1990s. , It is used as a genotyping analysis technique for individual identification, and since 2000, it is a useful molecular marker applied to breed identification and country of origin in animals such as cattle, pigs, and salmon.

The present inventors developed 9 new microsatellite markers using GBS information as molecular markers to discriminate domestic and Japanese red sea bream. Develop a method of comparison to provide a method to efficiently determine the country of origin. In addition, considering the economical aspect, 5 (PmMS32, PmMS54, PmMS78, PmMS37, PmMS97) or 3 (PmMS32, PmMS54) out of 9 markers (PmMS28, PmMS31, PmMS32, PmMS37, PmMS53, PmMS54, PmMS76, PmMS78, PmMS97) , PmMS97), the present invention was completed by confirming that the country of origin can be determined sufficiently in the same way when using microsatellite.

KR 20150089655 A

The present invention is characterized by providing a new microsatellite marker for distinguishing domestic red sea bream and Japanese red sea bream distributed in Korea and a method for effectively determining the country of origin through genotype analysis using the same.

In order to achieve the above object, the present invention provides microsatellite markers for determining the origin of at least one red snapper selected from the group consisting of PmMS28, PmMS31, PmMS32, PmMS37, PmMS53, PmMS54, PmMS76, PmMS78 and PmMS97.

In one embodiment of the present invention, the "microsatellite marker" is that PmMS28 is represented by SEQ ID NO: 19, PmMS31 is represented by SEQ ID NO: 20, PmMS32 is represented by SEQ ID NO: 21, and PmMS37 is represented by SEQ ID NO: 22, PmMS53 is represented by SEQ ID NO: 23, PmMS54 is represented by SEQ ID NO: 24, PmMS76 is represented by SEQ ID NO: 25, PmMS78 is represented by SEQ ID NO: 26, and PmMS97 is represented by SEQ ID NO: It is characterized by being represented by 27.

In addition, the present invention is a primer set represented by SEQ ID NO: 1 to 2 capable of specifically amplifying the PmMS28 microsatellite marker; Primer sets represented by SEQ ID NOs: 3 to 4 capable of specifically amplifying the PmMS31 microsatellite marker; Primer sets represented by SEQ ID NOs: 5 to 6 capable of specifically amplifying the PmMS32 microsatellite marker; Primer sets represented by SEQ ID NOs: 7 to 8 capable of specifically amplifying the PmMS37 microsatellite marker; Primer sets represented by SEQ ID NOs: 9 to 10 capable of specifically amplifying the PmMS53 microsatellite marker; Primer sets represented by SEQ ID NOs: 11 to 12 capable of specifically amplifying the PmMS54 microsatellite marker; Primer sets represented by SEQ ID NOs: 13 to 14 capable of specifically amplifying the PmMS76 microsatellite marker; Primer sets represented by SEQ ID NOs: 15 to 16 capable of specifically amplifying the PmMS78 microsatellite marker; and primer sets represented by SEQ ID NOs: 17 to 18 capable of specifically amplifying the PmMS97 microsatellite marker.

In addition, the present invention provides a kit for determining the country of origin of red sea bream containing the homologous primer set.

In one embodiment of the present invention, the "kit for determining the country of origin of red sea bream" may include a buffer solution and a DNA polymerase, but is not limited thereto.

In addition, the present invention comprises the steps of PCR amplifying the nucleic acid sample of the red sea bream to be analyzed using a homologous primer set; It provides a method for determining the origin of red sea bream, comprising the step of analyzing the genetic diversity of red sea bream by analyzing the alleles of each locus of the product amplified in the above step.

In one embodiment of the present invention, the "method for determining the origin of red sea bream" is the number of alleles per locus of the red sea bream sample in the step of analyzing genetic diversity, the observed heterozygosity rate, the expected heterozygosity rate, the polymorphism information index, and the allele It may be characterized by analyzing one or more selected from among abundance and inbreeding coefficient, but is not limited thereto.

In addition, the present invention comprises the steps of PCR amplifying the nucleic acid sample of the red sea bream to be analyzed using a homologous primer set; It provides a method for determining the origin of red sea bream, characterized in that it comprises the step of comparing the genetic relationship by country of origin of red sea bream by analyzing the alleles of each locus of the product amplified in the above step.

In one embodiment of the present invention, "method for determining origin of red sea bream" is characterized by comparing one or more selected from Pairwise Fst values and genotype likelihood matrix values in the step of comparing genetic relatedness by origin of red sea bream It may be, but is not limited thereto.

The development of microsatellites provided by the present invention and the method for determining origin using the microsatellite is an effective and economical method using a small amount of fin tissue to perform genetic distance and group analysis of Japanese and domestic red sea bream, indicating that it is possible to determine the country of origin. indicate Therefore, by using the present invention, it is possible not only to crack down on illegal acts occurring in the distribution process, but also to help increase the public's trust in the management of the origin of aquatic products and increase fishery income.

1 is a diagram for explaining a method for discriminating between domestic red sea bream and Japanese red sea bream using a microsatellite marker.
Figure 2 is to confirm the suitability of 100 newly developed microsatellite markers through GBS data, PCR reaction was performed using the genomic DNA of each domestic and Japanese red sea bream as a template, and the product was subjected to agarose gel electrophoresis This is a diagram of the results confirmed by the method.
3 is a diagram showing the results of amplifying each gene locus in domestic and Japanese red sea bream using the nine microsatellite markers selected in Example 3 (B) and analyzing the DNA fragment size of the PCR product.
Figure 4 is a diagram of the results showing the allele frequencies of the genotypes of domestic red sea bream and Japanese red sea bream for each of the 9 microsatellite marker loci analyzed through DNA fragment capillary electrophoresis.
5 is a diagram of PCoA analysis results showing the distribution and relationship of genetic variation of domestic red sea bream and Japanese red sea bream for each of nine microsatellites.
Figure 6 is a diagram showing the results of genotype likelihood calculations and PCoA analysis results showing the genetic relatedness of domestic red sea bream and Japanese red sea bream through Arlequin based on the analysis results of 9 microsatellite markers.
Figure 7 is a genotype likelihood calculation showing the genetic relatedness of Korean and Japanese red sea bream through Arlequin based on the analysis results of five microsatellite marker combinations (PmMS32, PmMS54, PmMS78, PmMS37, and PmMS97) with the highest Fst values. It is a diagram of values and pCoA analysis results.
Figure 8 shows the genotype likelihood calculation value and PCoA analysis results showing the genetic relatedness of domestic and Japanese red sea bream through Arlequin based on the analysis results of three selected microsatellite marker combinations (PmMS32, PmMS54, and PmMS97). It is also

An object of the present invention is to provide a microsatellite marker for determining the origin of at least one red snapper selected from the group consisting of PmMS28, PmMS31, PmMS32, PmMS37, PmMS53, PmMS54, PmMS76, PmMS78, and PmMS97.

The term 'microsatellite marker' of the present invention is a molecular marker widely used for genetic relationship analysis in genetics, and is a simple sequence on DNA where a short base pair (bp) sequence of 1 to 6 appears repeatedly. Also called repeat (SSR). Because of its high polymorphism and reproductivity, it is used as a genotyping analysis technology for pedigree, group genetic relationship analysis, paternity confirmation, and individual identification for various organisms, and since 2000, cattle, It is a useful molecular marker applied to breed identification and country of origin in animals such as pigs and salmon.

The present invention provides forward primers of SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18 specific for microsatellite markers. And an object of the present invention is to provide at least one pair of primers for determining the country of origin of red sea bream selected from the group consisting of reverse primer pairs.

The term 'primer' of the present invention is a single-stranded nucleic acid sequence composed of short nucleotide sequences, which plays a role of providing a starting point by forming complementary hydrogen bonds with template strands in PCR or DNA synthesis.

In addition, it is characterized by analyzing the genotype of domestic and Japanese red sea bream by determining the size of PCR products targeting the nine microsatellite markers through capillary electrophoresis.

A method for analyzing genotypes and determining the origin of red sea bream using the nine microsatellite markers includes primers of SEQ ID NOs 1 and 2 to amplify PmMS28, primers of SEQ ID NOs 3 and 4 to amplify PmMS31, and PmMS32 to amplify PmMS32. Primers of SEQ ID NOs: 5 and 6, primers of SEQ ID NOs: 7 and 8 to amplify PmMS37, primers of SEQ ID NOs: 9 and 10 to amplify PmMS53, primers of SEQ ID NOs: 11 and 12 to amplify PmMS54, SEQ ID NOs to amplify PmMS76 Primers 13 and 14, primers SEQ ID NOs 15 and 16 to amplify PmMS78, and primers SEQ ID NOs 17 and 18 to amplify PmMS97 are included.

In addition, the primers of the present invention may be labeled with a fluorescent material, and primers of SEQ ID NOs: 1 to 18 may be added or excluded.

The present invention provides forward primers of SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18 specific for microsatellite markers. And an object of the present invention is to provide a kit for determining the origin of red sea bream, including at least one pair of primers for determining the origin of red sea bream selected from the group consisting of pairs of reverse primers.

The present invention is specific to the microsatellite marker composition consisting of PmMS28, PmMS31, PmMS32, PmMS37, PmMS53, PmMS54, PmMS76, PmMS78, PmMS97 nucleic acid samples of red sea bream to be analyzed, and to the nucleotide sequence represented by SEQ ID NOs: 1 to 18 PCR amplification using the primers formed; An object of the present invention is to provide a method for determining the origin of red sea bream, comprising the step of analyzing the genetic diversity of red sea bream by analyzing the alleles of each locus of the product amplified in the above step.

Finally, the present invention is specific to the microsatellite marker composition consisting of PmMS28, PmMS31, PmMS32, PmMS37, PmMS53, PmMS54, PmMS76, PmMS78, and PmMS97 nucleic acid samples of red sea bream to be analyzed, and bases represented by SEQ ID NOs: 1 to 18 PCR amplification using primers consisting of the sequence; The purpose is to provide a method for determining the origin of red sea bream, characterized in that it comprises the step of comparing the genetic relationship by country of origin of red sea bream by analyzing the alleles of each locus of the product amplified in the above step.

That is, it includes a method of analyzing the genetic polymorphism index based on nine microsatellites and a method of determining the country of origin by comparing the genetic distance through a genetic distance calculation program.

In addition, considering the economical aspect among the 9 microsatellite markers, a combination of 5 microsatellite markers (PmMS32, PmMS54, PmMS78, PmMS37, PmMS97) and 3 microsatellite markers (PmMS32, PmMS54, PmMS97) were used. genotype analysis.

The method for determining the country of origin of red snapper using the five microsatellite markers includes primers of SEQ ID NOs: 5 and 6 to amplify PmMS32, primers of SEQ ID NOs: 7 and 8 to amplify PmMS37, and SEQ ID NOs: 11 and 12 to amplify PmMS54. Primers of SEQ ID NOs: 15 and 16 to amplify PmMS78, and primers to SEQ ID NOs: 17 and 18 to amplify PmMS97 are included.

The method of determining the country of origin of red sea bream using three markers includes primers of SEQ ID NOs: 5 and 6 to amplify PmMS32, primers of SEQ ID NOs: 11 and 12 to amplify PmMS54, and primers of SEQ ID NOs: 17 and 18 to amplify PmMS97. do.

In addition, it includes a method of determining the country of origin through a method of analyzing a genetic polymorphism index based on a combination of the five and three microsatellite markers and a method of comparatively analyzing genetic relationships through a genetic distance calculation program.

Hereinafter, the present invention will be described in detail by embodiments of the present invention with reference to the accompanying drawings. However, the following examples are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the description of the claims described later and equivalent scopes interpreted therefrom.

Example 1. Development of a method for determining the country of origin of domestic and Japanese red sea bream using microsatellite markers

The method for determining the country of origin of domestic and Japanese red sea bream using microsatellite markers according to the present invention is to secure red sea bream samples and isolate genomic DNA (A), select microsatellite genetic loci for determining the origin of red sea bream through genotyping by sequencing (B), After fragment analysis using the 9 microsatellite markers selected in (B) above, genotype analysis by individual red sea bream (C), genetic polymorphism index analysis by country of origin of red sea bream based on the information obtained in (C) above (D ), comparison of genetic relatedness by country of origin of red sea bream (E), and discrimination of origin of domestic and Japanese red sea bream (F) (FIG. 1).

Example 2. Red snapper sample acquisition and genomic DNA isolation (A)

90 domestic red sea bream used for genotyping analysis to determine the country of origin were purchased from Jagalchi, Tongyeong, and Pohang fish markets in Busan, and 90 Japanese red sea bream were purchased from a specialized seafood importer. Fin or muscle tissue of each individual of red sea bream was sampled and stored at -80 °C until analysis. AccuPrep® Genomic DNA Extraction kit (Bioneer, Korea) was used to extract and purify genomic DNA from fins or muscles of about 0.5 cm, and diluted to a constant concentration of 10 ng/μl before use. did

Example 3. Selection of microsatellite genetic loci for determining origin of red sea bream (B)

To secure a new microsatellite marker candidate group through GBS (Genotyping by sequencing) library construction, GCA_002897255.1 (https://www.ncbi. nlm.nih.gov/assembly/GCA_002897255.1/) assembly was used as a reference to analyze 96 individuals (48 Korean red sea bream, 48 Japanese red sea bream). 1974 markers were obtained from the database obtained from GBS reads, and among them, 100 candidate microsatellite markers were selected to discriminate domestic and Japanese red sea bream. In order to confirm the suitability of the selected 100 microsatellite markers, a primary confirmation was performed through PCR.

As specific PCR reaction conditions, 1 μl of template genomic DNA (10ng/μl), 1 μl of forward primer (10 pmol), 1 μl of reverse primer (10 pmol), and 10 μl of Prime Taq Premix (2X) from Genetbio were mixed. Distilled water was added to adjust the final volume to 20 μl. The PCR device used SimpliAmp™ Thermal Cycler (Applied biosystems), and the PCR reaction temperature conditions were 95 °C for 5 minutes, 94 °C for 30 seconds, 60 °C for 30 seconds, and 72 °C for 30 seconds. The reaction condition consisting of 1 minute was set to 1 cycle and repeated 40 times. Then, after extension reaction at 72 ° C for 7 minutes, 2 μl of the PCR product was confirmed by electrophoresis on a 2% agarose gel (FIG. 2). Nine microsatellite loci were selected for which the size difference of PCR products was confirmed through electrophoresis results, and they were used for fragment analysis (C), and information on each primer and marker was summarized in Tables 1 and 2.

Locus sequence number primer sequence PMMS28 One F: 5'- TGGAAACTGGGAGAAACGGG 2 R: 5'- CATGCAGATTAAACAAAGAGAAGAAAT PMMS31 3 F: 5'- TCCACTGAGTTAAAAGGGTCCA 4 R: 5'- ACAAGGCGGATCTAATGCGT PMMS32 5 F: 5'- CAGCCTGACAGCTCTCACAT 6 R: 5'-ATTGGGAAATGGCAGCGCAT PMMS37 7 F: 5'-ACTTTGAACCTGAGCCACA 8 R: 5'- TCTTCAATGTGTGAGTGGGCT PMMS53 9 F: 5'-TGAGATCATCCTCCTCCCAGA 10 R: 5'- AGGTTCTCATCATTTCCCTGGG PmMS54 11 F: 5'-GCGGCAGCAACAACGATTTT 12 R: 5'-TGCCTGTTTTTCGTTTGATTTTGT PMMS76 13 F: 5'-ACGGCTCACCTTGGATTTGA 14 R: 5'-CCACGAGTGAATGTGTTCCC PMMS78 15 F: 5'- TGCATGTACCTCTCTGTGCC 16 R: 5'- TGCAGGATTCAGAACCAGATGT PMMS97 17 F: 5'- GCCAGGACAGAAAATGAAGCA 18 R: 5'- GCAGCATCTTCAAAGGCACC

Locus ID Repeat motif sequence number base sequence Size (bp) Tm Ta PMMS28 Seq_104664974 (AC) ₂₀ 19 TGGGAGAAACGGGTACCCTGGGTCCGTCTG (AC)n ATATATATATACTGTATATGTATTTCTTCT 95-121 57.36 60 PMMS31 Seq_21359525 (TAA) ₈ 20 CAACTTTTCACCCACTAAGTCATAATCCTC (TAA) _n TATATGACGCATTAGATCCGCCTTGTTCCA 100-145 59.82 60 PMMS32 Seq_16597448 (AC) ₆ tcacgcgt(AC) ₆ 21 CCCCGTGCCATGAAGCCACGCACATACTGT (AC) _n TCACGCGT(AC) _n GCAAATGCGCTGCCATTTCCCAATGCCAGA 96-106 61.33 60 PMMS37 Seq_67108515 (TG) ₆ 22 ATTTTGCTGTTTTATGTGTGTGTGAGTTTA (TG) _n TCGCCAGCCCACTCACACATTGAAGAGGTT 96-102 59.58 60 PMMS53 Seq_101487188 (GA) ₁₀ 23 GTTTGAGATCATCCTCCTCCCAGAGCCTCT (GA) _n AAAAAACAGAATCCAATAAATTGGCTGCAA 92-101 59.76 60 PmMS54 Seq_30817975 (CAA) ₇ 24 AGATGCTTTGGTACGCTACAGAGAACACCT (CAA) _n CAGAAAGAAAACAAAATCAAACGAAAAACA 123-129 59.07 60 PMMS76 Seq_81269849 (AC) ₁₀ 25 TCCCCAGGCCGCTTTGTCCTGAACACGAAA (AC) _n AGTTAGAGAAACTGGGAACACATTCACTCG 120-146 58.84 60 PMMS78 Seq_29115048 (AC) ₈ 26 TCTCTGTGCCTCACAGACACACAGACACAG (AC) _n ATTCCAACACACACGTGCTGACAGCCATGA 92-123 59.69 60 PMMS97 Seq_79278127 (GT) ₈ 27 CAGGACAGAAAATGAAGCATGCCAGAGAAA (GT) _n GGAAAGTGAATGAGTAACCTCCTCTCGCCA 109-123 60.12 60

Example 4. Genotype analysis through fragment analysis (C)

Using the 9 microsatellite markers selected in Example 3, fragment analysis was performed to confirm the genetic diversity of each group of 90 domestic red sea bream and 90 Japanese red sea bream. Primers labeled with 5'-Fam (6-FAM) specific for each marker were synthesized and used for PCR. The PCR products were genotyped using an ABI3730xL Genetic Analyzer (Applied Biosystems, USA) equipped with a capillary array, and GeneMapper The size of alleles was calculated using software 5 (Applied Biosystems, CA, USA). The amplified product was separated by size using capillary electrophoresis to determine the size of the DNA fragment (FIG. 3). The genotype of each individual was determined and compared according to the size of the amplified product, and the determined genotype was allele frequency for 9 loci for each group of domestic (KR, n = 90) and Japanese (JP, n = 90) ) is shown in FIG. 4 as a bar graph.

Example 5. Genetic polymorphism index analysis by country of origin of red sea bream (D)

Number of alleles per locus (Na), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphic information content (PIC) were Cervus version 3.0. 7 (Marshall et al., 1998). Allelic richness (Ar) and inbreeding coefficient (Fis) were measured using the FSTAT 2.9.4 program (Goudet 1995). The p-value (pHWD) for the Hardy-Weinberg equilibrium test expected value was calculated using Arlequin 3.5.2.2 (Exocoffier et al., 2005) (Table 3).

When a total of 180 domestic (KR, n=90) and Japanese (JP, n=90) individuals were set as one population and genetic variability was analyzed using 9 microsatellite markers, a total of 70 alleles were detected. The total average number of alleles per locus ranged from 4 to 11, with an average of 7.8. By locus, PmMS54 and PmMS37 had the fewest number of alleles, 4 and 5, respectively, and PmMS31 showed the highest number of alleles with 13. Likewise, the overall average of allele abundance (Ar) was 7.322, and PmMS31 (Ar: 12.03) showed the highest value, and PmMS54 (Ar: 3.999) and PmMS37 (Ar: 3.978) showed the lowest value.

The He value for each group represents the expected heterozygosity assuming that the group is in genetic equilibrium, and the Ho value represents the heterozygosity of individuals actually in the observed heterozygous state. The overall average He value for the 9 locus of 180 individuals analyzed in this study was 0.694, and the overall average Ho was 0.617, and there was no significant difference between Ho and He values in all groups. In addition, when viewed by locus, Ho (0.85) and He (0.865) values appear to be the highest in all analyzed red snapper individuals when analyzed with PmMS28, and Ho (0.378) and He (0.431) have the lowest values when analyzed with PmMS76. appeared to have

The PIC value was between 0.848 (PmMS28) and 0.411 (PmMS76) within the two populations. Deviations from Hardy-Weinberg equilibrium were observed at 4 loci (PmMS28, PmMS53, PmMS54, PmMS78) in the Japanese population among 9 loci in the Korean and Japanese populations (p<0.0001). Fis was 0.112 on average, and Fis for all 9 loci was 0.054 for KR and 0.063 for JP, suggesting that some inbreeding occurred in the group by origin of red snapper. Overall, the genetic diversity index using the nine markers did not show a significant difference between the two origin groups.

Populations PMMS28 PMMS31 PMMS32 PMMS37 PMMS53 PmMS54 PMMS76 PMMS78 PMMS97 Mean KR
(n=90) Na 11 13 3 4 6 4 7 9 6 7 Ar 11 13 3 3.989 6 4 6.989 9 5.989 6.996 Ho 0.889 0.787 0.522 0.722 0.667 0.478 0.467 0.697 0.756 0.665 He 0.875 0.821 0.557 0.658 0.730 0.669 0.464 0.790 0.761 0.703 PIC 0.856 0.799 0.492 0.583 0.685 0.595 0.436 0.754 0.717 0.657 HWE 0.134 0.015 0.439 0.507 0.543 0.001 0.061 0.084 0.110 0.210 Fis -0.016 0.042 0.062 -0.098 0.087 0.287 -0.005 0.119 0.008 0.054 JP
(n=90) Na 9 9 5 4 5 4 5 7 5 5.9 Ar 9 8.978 4.978 3.978 4.977 4 5 7 5 5.879 Ho 0.811 0.822 0.667 0.667 0.622 0.333 0.289 0.307 0.600 0.569 He 0.824 0.758 0.528 0.524 0.578 0.567 0.388 0.628 0.643 0.604 PIC 0.796 0.725 0.465 0.457 0.514 0.507 0.364 0.597 0.575 0.556 HWE 0.000 0.335 0.013 0.003 0.000 0.000 0.001 0.000 0.420 0.086 Fis 0.016 -0.085 -0.264 -0.275 -0.077 0.413 0.256 0.513 0.067 0.063 All populations
(n=180) Na 11 13 7 5 7 4 7 10 6 7.8 Ar 10.721 12.030 6.489 3.978 6.322 3.999 6.930 9.492 5.933 7.322 Ho 0.850 0.804 0.594 0.694 0.644 0.406 0.378 0.503 0.678 0.617 He 0.865 0.797 0.769 0.607 0.666 0.649 0.431 0.741 0.722 0.694 PIC 0.848 0.775 0.732 0.538 0.619 0.584 0.411 0.711 0.677 0.655

Example 6. Comparison of genetic relatedness by country of origin of red sea bream (E)

Principal Coordinate (PCoA) analysis was performed using the GenAlEx program (Peakall and Smouse 2006) to represent the geometric relationship by origin group in two dimensions using the genetic variation distribution of domestic and Japanese red sea bream. In addition, in order to analyze the genetic relatedness between groups using 9 loci, Arlequin 3.5.2.2 (Exocoffier et al., 2005) was used to calculate pairwise Fst values and genotype likelihood matrix values, and OriginPro 8.5 software (OriginLab , USA).

The Fst values among the groups of origin were high in the order of PmMS32, PmMS54, PmMS78, PmMS37, PmMS97, PmMS53, PmMS28, PmMS76, and PmMS31, and there were significant differences between the domestic and Japanese groups in all markers except for PmMS53 (p=0.02703). appeared (p<0.00001) (Table 4). When all 9 microsatellite markers used in this analysis were applied, the Fst value was 0.11078 (Table 5), and the 5 microsatellite markers with the highest Fst value (PmMS32, PmMS54, PmMS78, PmMS37, PmMS97) When applied, an Fst value of 0.1711 (p<0.00001) was recorded (Table 6). When three microsatellite markers (PmMS32, PmMS54, PmMS97) were applied, the Fst value was 0.22956 (p <0.00001), which was higher (Table 7).

As a result of performing PCoA analysis for each of the 9 markers, in the case of PmMS32, which had the highest Fst value, clusters were formed for each individual of domestic (K) and Japanese (J) red sea bream, showing a degree of variability of 84.80% (FIG. 5).

Locus Fst P value PMMS28 0.03595 p<0.00001 PMMS31 0.01848 p<0.00001 PMMS32 0.45457 p<0.00001 PMMS37 0.05258 p<0.00001 PMMS53 0.0365 p<0.00001 PmMS54 0.09181 p<0.00001 PMMS76 0.02095 0.02703 PMMS78 0.08199 p<0.00001 PMMS97 0.05441 p<0.00001

Locus Fst P value Total 9 markers 0.11078 p<0.00001

Locus Fst P value 5 markers
(PmMS32, PmMS54, PmMS78, PmMS37, PmMS97) 0.1711 p<0.00001

Locus Fst P value 3 markers
(PmMS32, PmMS54, PmMS97) 0.22956 p<0.00001

Example 7. Determination of origin of domestic and Japanese red sea bream (F)

Based on the analysis results of the nine microsatellite markers used in the analysis, the genotype likelihood calculation results were tabulated through Arlequin as shown in FIG. In the PCoA results, some individuals were vague, but overall, they showed a mutation rate of 29.31% (by component: 14.69%, 8.2%, 6.42%).

Similarly, when 5 microsatellite markers (PmMS32, PmMS54, PmMS78, PmMS37, PmMS97) with high Fst values were applied, the PCoA result (total mutation rate of 43.5%, by component: 22.5%, 12.08%, 8.92%) was Clear differences by country of origin can be seen in genotype likelihood comparison (Fig. 7). Similarly, when three markers (PmMS32, PmMS54, and PmMS97) were used, a clearer difference was observed in the genotype likelihood analysis result, and the highest mutation rate was 58.56% in PCoA analysis (28.61%, 16.85%, 13.11%) (FIG. 8).

From the above results, based on the pairwise Fst values, PCoA results, and genotype likelihood matrix analysis of domestic and Japanese red sea bream using nine newly developed markers through genotyping by sequencing data, variations between groups of origin can be effectively grouped. there was. When the same analysis is performed using 5 (PmMS32, PmMS54, PmMS78, PmMS37, PmMS97) to 3 (PmMS32, PmMS54, PmMS97) markers considering the economic aspect, comparison of genotype likelihood matrix and PCoA by country of origin It was confirmed that domestic and Japanese red sea bream could be sufficiently discriminated using the analysis.

<110> Pukyong National University Industry-University Cooperation Foundation <120> microsatellite marker for identification of geographical origin of Pagrus major and its use <130> PN2106-299 <160> 27 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> artificial sequence <220> <223> PmMS28_primer_F <400> 1 tggaaactgg gagaaacggg 20 <210> 2 <211> 27 <212> DNA <213> artificial sequence <220> <223> PmMS28_primer_R <400> 2 catgcagatt aaacaaagag aagaaat 27 <210> 3 <211> 21 <212> DNA <213> artificial sequence <220> <223> PmMS31_primer_F <400> 3 tccactgagt taaagggtcc a 21 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> PmMS31_primer_R <400> 4 acaaggcgga tctaatgcgt 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> PmMS32_primer_F <400> 5 cagcctgaca gctctcacat 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> PmMS32_primer_R <400> 6 attgggaaat ggcagcgcat 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> PmMS37_primer_F <400> 7 actttgaacc ctgagccaca 20 <210> 8 <211> 21 <212> DNA <213> artificial sequence <220> <223> PmMS37_primer_R <400> 8 tcttcaatgt gtgagtgggc t 21 <210> 9 <211> 21 <212> DNA <213> artificial sequence <220> <223> PmMS53_primer_F <400> 9 tgagatcatc ctcctcccag a 21 <210> 10 <211> 22 <212> DNA <213> artificial sequence <220> <223> PmMS53_primer_R <400> 10 aggttctcat catttccctg gg 22 <210> 11 <211> 20 <212> DNA <213> artificial sequence <220> <223> PMMS54_primer_F <400> 11 gcggcagcaa caacgatttt 20 <210> 12 <211> 24 <212> DNA <213> artificial sequence <220> <223> PmMS54_primer_R <400> 12 tgcctgtttt tcgtttgatt ttgt 24 <210> 13 <211> 20 <212> DNA <213> artificial sequence <220> <223> PMMS76_primer_F <400> 13 acggctcacc ttggatttga 20 <210> 14 <211> 20 <212> DNA <213> artificial sequence <220> <223> PMMS76_primer_R <400> 14 ccacgagtga atgtgttccc 20 <210> 15 <211> 20 <212> DNA <213> artificial sequence <220> <223> PmMS78_primer_F <400> 15 tgcatgtacc tctctgtgcc 20 <210> 16 <211> 22 <212> DNA <213> artificial sequence <220> <223> PMMS78_primer_R <400> 16 tgcaggattc agaaccagat gt 22 <210> 17 <211> 21 <212> DNA <213> artificial sequence <220> <223> PMMS97_primer_F <400> 17 gccaggacag aaaatgaagc a 21 <210> 18 <211> 20 <212> DNA <213> artificial sequence <220> <223> PMMS97_primer_R <400> 18 gcagcatctt caaaggcacc 20 <210> 19 <211> 100 <212> DNA <213> artificial sequence <220> <223> PMMS28 <400> 19 tgggagaaac gggtaccctg ggtccgtctg acacacacac acacacacac acacacacac 60 acacacacac atatatatat actgtatatg tatttcttct 100 <210> 20 <211> 84 <212> DNA <213> artificial sequence <220> <223> PMMS31 <400> 20 caacttttca cccactaagt cataatcctc taataataat aataataata ataatatatg 60 acgcattaga tccgccttgt tcca 84 <210> 21 <211> 92 <212> DNA <213> artificial sequence <220> <223> PMMS32 <400> 21 ccccgtgcca tgaagccacg cacatactgt acacacacac actcacgcgt acacacacac 60 acgcaaatgc gctgccattt cccaatgcca ga 92 <210> 22 <211> 72 <212> DNA <213> artificial sequence <220> <223> PMMS37 <400> 22 attttgctgt tttatgtgtg tgtgagttta tgtgtgtgtg tgtcgccagc ccactcacac 60 attgaagagg tt 72 <210> 23 <211> 80 <212> DNA <213> artificial sequence <220> <223> PMMS53 <400> 23 gtttgagatc atcctcctcc cagagcctct gagagagaga gagagagaga aaaaaacaga 60 atccaataaa ttggctgcaa 80 <210> 24 <211> 81 <212> DNA <213> artificial sequence <220> <223> PMMS54 <400> 24 agatgctttg gtacgctaca gagaaccct caacaacaac aacaacaaca acagaaagaa 60 aacaaaatca aacgaaaaac a 81 <210> 25 <211> 80 <212> DNA <213> artificial sequence <220> <223> PMMS76 <400> 25 tccccaggcc gctttgtcct gaacacgaaa acacacacac acacacacac agttagagaa 60 actgggaaca cattcactcg 80 <210> 26 <211> 76 <212> DNA <213> artificial sequence <220> <223> PMMS78 <400> 26 tctctgtgcc tcacagacac acagacacag acacacacac acacacattc caacacacac 60 gtgctgacag ccatga 76 <210> 27 <211> 76 <212> DNA <213> artificial sequence <220> <223> PMMS97 <400> 27 caggacagaa aatgaagcat gccagagaaa gtgtgtgtgt gtgtgtgggaa agtgaatgag 60 taacctcctc tcgcca 76

Claims (9)

A set of microsatellite markers for distinguishing domestic and Japanese red sea bream consisting of PmMS28, PmMS31, PmMS32, PmMS37, PmMS53, PmMS54, PmMS76, PmMS78 and PmMS97. According to claim 1,
The PmMS28 is represented by SEQ ID NO: 19, PmMS31 is represented by SEQ ID NO: 20, PmMS32 is represented by SEQ ID NO: 21, PmMS37 is represented by SEQ ID NO: 22, and PmMS53 is represented by SEQ ID NO: 23 , PmMS54 is represented by SEQ ID NO: 24, PmMS76 is represented by SEQ ID NO: 25, PmMS78 is represented by SEQ ID NO: 26, and PmMS97 is represented by SEQ ID NO: 27, characterized in that domestic and Japanese red sea bream discrimination A set of microsatellite markers for use. Primer pairs represented by SEQ ID NOs: 1 to 2 capable of specifically amplifying the PmMS28 microsatellite marker;
Primer pairs represented by SEQ ID NOs: 3 to 4 capable of specifically amplifying the PmMS31 microsatellite marker;
Primer pairs represented by SEQ ID NOs: 5 to 6 capable of specifically amplifying the PmMS32 microsatellite marker;
Primer pairs represented by SEQ ID NOs: 7 to 8 capable of specifically amplifying the PmMS37 microsatellite marker;
Primer pairs represented by SEQ ID NOs: 9 to 10 capable of specifically amplifying the PmMS53 microsatellite marker;
Primer pairs represented by SEQ ID NOs: 11 to 12 capable of specifically amplifying the PmMS54 microsatellite marker;
Primer pairs represented by SEQ ID NOs: 13 to 14 capable of specifically amplifying the PmMS76 microsatellite marker;
Primer pairs represented by SEQ ID NOs: 15 to 16 capable of specifically amplifying the PmMS78 microsatellite marker; and
A primer set for distinguishing domestic and Japanese red sea bream consisting of primer pairs represented by SEQ ID NOs: 17 to 18 capable of specifically amplifying the PmMS97 microsatellite marker. A kit for distinguishing domestic and Japanese red sea bream containing the primer set of claim 3. According to claim 4,
Wherein the kit comprises a buffer solution and a DNA polymerase. (a) PCR amplifying a nucleic acid sample of red sea bream to be analyzed using the primer set of claim 3;
(b) analyzing the genetic diversity of red sea bream by analyzing the alleles of each locus of the product amplified in step (a). According to claim 6,
The step of analyzing the genetic diversity is to analyze one or more selected from the number of alleles per locus of the red sea bream sample, the observed heterozygosity rate, the expected heterozygosity rate, the polymorphic information index, the allele abundance, and the inbreeding coefficient. How to distinguish domestic and Japanese red sea bream. (a) PCR amplifying a nucleic acid sample of red sea bream to be analyzed using the primer set of claim 3;
(b) analyzing the alleles of each locus of the product amplified in step (a) to compare the genetic relationship by country of origin of red sea bream. Method for distinguishing domestic and Japanese red sea bream. According to claim 8,
The step of comparing the genetic affinity by country of origin is a domestic and Japanese red sea bream discrimination method, characterized in that for comparing one or more selected from Pairwise Fst values and genotype likelihood matrix values.

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