KR101499695B1

KR101499695B1 - Genetic markers and methods for identifying Epinephelus septemfasciatus and Epinephelus akaara among family Serranidae

Info

Publication number: KR101499695B1
Application number: KR20140124486A
Authority: KR
Inventors: 안혜숙; 조재권; 안철민; 명정인
Original assignee: 대한민국
Priority date: 2014-09-18
Filing date: 2014-09-18
Publication date: 2015-03-16

Abstract

Provided in the present invention are species-specific genetic marker and method available of rapid and accurate identification of species of three species of serranidae. According to an aspect of the present invention, provided is a kit for identifying Epinephelus septemfasciatus including a primer set having the length of 15-50 nt, which is selected in a nucleic acid sequence represented by sequence number 1 or a compensating sequence of the same, and specifically amplifying polynucleotides including microsatellite marker consisting of the 275^th to 314^th nucleic acid of sequence number 1.

Description

[0001] The present invention relates to genetic markers and methods for distinguishing between the rapid identification of the locusts and the rhubarb,

More particularly, the present invention relates to a genetic marker for distinguishing a wild fish and / or a red barb among barley and fish and a method for distinguishing the same.

The family Serranidae is a tropical fish species in the North Pacific Ocean, which is not very high in production scale in Korea. It is included in the 10 major strategic items for the fisheries sector in response to the changes in the global fisheries market. It is the key species in question. In particular, China is emerging as a major consumer of high value-added goods, especially in the Chinese market. Taiwan's farming has dominated major markets such as China and Hong Kong. do. Korea has succeeded in producing artificial seedlings in 2003, but it is difficult to mass-produce domestic species as a tropical fish species. In the case of Javanese, which is called "Dachumbari" in dialect in Jeju Island, it is caught in small quantities within 10 days. It is very frequent that domestic or imported red barley or lanceolate which is sold and visually difficult to distinguish is turned into a red bean. Therefore, it is necessary to promptly and promptly distribute the fishes and red-bellied fish which are likely to be sold in the form of three species of fishes (jabari, redbari, and far-right fish) It is urgent to develop gene-based species discrimination technology that can discriminate accurately.

Korean Patent Laid-Open Publication No. 2014-0093889 discloses a method and a kit for distinguishing vaginally and lively fishes by using the D-loop site in the mitochondrial genomic sequence of Javanese and Lantus.

However, in the case of the above-mentioned patent, there is no way to distinguish between the lily and the red-billed fish among the barley and fish, which is related to the method of distinguishing between the bivalvia and the lively fish.

Disclosure of Invention Technical Problem [8] The present invention has been made to overcome the above-mentioned problems, and it is an object of the present invention to provide a species-specific gene marker for barnyardgrass and rhubarb. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, a polynucleotide comprising a microsatellite marker selected from the nucleic acid sequence set forth in SEQ ID NO: 1 or a complementary sequence thereof and consisting of the 275st to 314st nucleic acids of SEQ ID NO: 1 is specifically An Epinephelus septemfasciatus discrimination kit is provided comprising a primer set of 15 to 50 nt length capable of amplification.

According to another aspect of the present invention, there is provided a polynucleotide comprising a microsatellite marker selected from the nucleotide sequence represented by SEQ ID NO: 2 or a complementary sequence thereof and consisting of the 56th to 85th nucleotides of SEQ ID NO: 1, An Epinephelus akaara discrimination kit is provided comprising a set of primers 15 to 50 nt long capable of specifically amplifying adjacent polynucleotides of satellite markers.

In accordance with another aspect of the present invention, there is provided a multiplex PCR kit for the determination of the native fish and red sea bream in barley fish comprising the following primer set:

A 15 to 50 nt polynucleotide capable of specifically amplifying a polynucleotide comprising a microsatellite marker selected from the nucleotide sequence represented by SEQ ID NO: 1 or a complementary sequence thereof and consisting of the 275st to 314st nucleic acids of SEQ ID NO: Length primer set 1; And a microsatellite marker selected from the nucleotide sequence represented by SEQ ID NO: 2 or a complementary sequence thereof and consisting of the 56th to 85th nucleotides of SEQ ID NO: 1, or a polynucleotide comprising the adjacent polynucleotide of the microsatellite marker, A primer set of 15 to 50 nt long which can be amplified in a 2.

According to another aspect of the present invention, there is provided a DNA amplification method comprising: amplifying a genomic DNA of a fish object to be discriminated using the above-mentioned grouping kit; An electrophoresis step of electrophoresing the amplified DNA fragment; And analyzing a band pattern of the electrophoresed DNA fragment.

According to another aspect of the present invention, there is provided a DNA amplification step of amplifying a genomic DNA of a fish to be discriminated using the red-berry identification kit; An electrophoresis step of electrophoresing the amplified DNA fragment; And analyzing a band pattern of the electrophoresed DNA fragment.

According to another aspect of the present invention, there is provided a DNA amplification step of amplifying a genomic DNA of a fish object to be discriminated using the multiplex PCR kit for discrimination between three kinds of barley and fish; An electrophoresis step of electrophoresing the amplified DNA fragment; And analyzing a band pattern of the electrophoretic DNA fragment, wherein the method comprises the step of analyzing a band pattern of the electrophoretic DNA fragment.

According to one embodiment of the present invention as described above, it is possible to realize a fast and accurate species discrimination effect by using a species-specific gene marker for the wild fish and red-billed fish in barley and fish. Of course, the scope of the present invention is not limited by these effects.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view of an embodiment of the present invention comprising a microsatellite markers (green background) and a primer region (red letters on a yellow background) used to amplify the polynucleotide (332 bp) The invention provides a nucleic acid sequence (SEQ ID NO: 1) of a marker DNA according to an embodiment.
Figure 2 is a schematic diagram of an embodiment of the present invention comprising a red barley microsatellite marker (green background) and a primer site (red text on a yellow background) used to amplify polynucleotides associated therewith, in accordance with an embodiment of the present invention. (SEQ ID NO: 2) of the marker DNA according to the example.
FIG. 3 is a gel photograph showing species discrimination according to the size of a gene amplified using a multiplex PCR kit for identification in three species of barley and marine fishes according to an embodiment of the present invention.

Definition of Terms:

The term " Epinephelus akaara ", as used in this document, is a group of seaweed fish belonging to the Early River Ripper , which is 35 cm in length, with small yellow spots on a light brown background. It lives in the coastal reef area with high temperature and spawning around July to September, and Korea is distributed in Jeju Island, Central Japan, China and Taiwan.

The term " Epinephelus bruneu " as used in this document belongs to the early bass barbarians of Chrysomophyceae and is called " Dachumbari " in Jeju Island. It is one of high-priced high-priced fish species. However, This is very similar. The length of the body is about 60 cm. There are no thorns on the front gill cover, and there are seven black-brown patterns obliquely on a dark brown background. It lives in the rocky part of the coast, mainly at night, catches squid or small fish, and spawning occurs in August and October.

The term " Epinephelus septemfasciatus " as used herein refers to seawater having a body length of about 90 cm, belonging to the early river bass barb . The body has 7 dark brown patterns on the gray brown background, the ventral fin and the anal fin are black, the caudal fin is round, and the tail sack is high. Inhabits in coastal and deep rocky areas, catches crustaceans and fishes such as shrimp, crab, etc. and spawns in the coast from May to September. It lives in the southern sea including Jeju Island, and is distributed in the south of Hokkaido (Hokkaido), South China Sea and the Indian Ocean.

As used herein, "microsatellite" is also referred to as simple sequence repeat (SSR) or short tandem repeat (STR) and refers to a sequence of 2 to 6 basepairs at a particular locus in the genome It means repetitive genomic structure. Repetition frequency is often used for individual identification or population genetics analysis.

DETAILED DESCRIPTION OF THE INVENTION [

The primer set may be composed of a forward primer represented by a nucleic acid sequence represented by SEQ ID NO: 3 and a reverse primer represented by a nucleotide sequence represented by SEQ ID NO: 4.

In the kit for identifying red cells, the primer set may be composed of a forward primer represented by the nucleic acid sequence shown in SEQ ID NO: 5 and a reverse primer represented by the nucleotide sequence shown in SEQ ID NO: 6.

In the multiplex PCR kit, the primer set 1 may be composed of a forward primer represented by the nucleotide sequence shown in SEQ ID NO: 3 and a reverse primer represented by the nucleotide sequence shown in SEQ ID NO: 4, and the primer set 2 A forward primer represented by the nucleotide sequence shown in SEQ ID NO: 5 and a reverse primer represented by the nucleotide sequence shown in SEQ ID NO: 6.

In the multiplex PCR kit, the bivalve fish may be Epinephelus septemfasciatus , Epinephelus brunetus or Epinephelus akaara .

According to another aspect of the present invention, there is provided a DNA amplification step of amplifying a genomic DNA of a fish object to be discriminated using a multiplex PCR kit for discriminating between a fish and a red fish in the fish; An electrophoresis step of electrophoresing the amplified DNA fragment; And analyzing a band pattern of the electrophoretic DNA fragment, wherein the method comprises the step of analyzing a band pattern of the electrophoretic DNA fragment.

The present inventors have used a number of loci and combinations thereof to develop a genetic marker for the identification of the ladybird and redbud in barley. The two primers can increase economic efficiency and yield of data per reaction. In the present invention, the present invention is applied to a multiplex PCR method in order to obtain an economical and quick result in comparison with a single gene locus PCR reaction. The PCR technique can increase the accuracy of the analysis by increasing the types of primers included in the multiplex, but also can increase the inaccuracy if the sizes of the PCR products obtained from various gene loci are similar. Therefore, the number of primers included in the multiplex was selected so as to obtain reliable results with a minimum number of primer pairs.

According to the above-described method for distinguishing between the fishes and the red-billed fishes in the barley fish by the multiplex PCR, the genomic DNA of the barley and fish is obtained and amplified with the multiplex PCR kit to obtain PCR products (320 ~ 340 bp) is detected, it can be judged to be red-bellied if the PCR product (250 bp) amplified by the red-berry specific primer set appears.

The development of the species-specific marker of the present invention is carried out by analyzing through NGS (next generation sequencing), which is a method for mass analysis of base sequence fragments of barley and fish genomic DNA, and then screening the read (base sequence fragment) And primers were designed and confirmed by PCR to confirm stable amplification. Then, in the course of genotyping (microsatellite), we developed a species specific primer by selecting a read primer that has no allele and no amplification in the flexible species. In the present invention, the primers were designed with repeated sequences in between and the genotypes were analyzed through individual differences of the repeated sequences. However, in the case of the red barriers, the primers were designed for the adjacent sequences in the repeated sequences, We have developed a marker that shows specific amplification of the red barley species. When the primer is used to express the amplification of the lyso species, it is judged as a lyophil. Therefore, it has been proved that the primer set 1 and the set 2 can be effectively applied to the determination of the whitish and red-billed fish among the barley and the fish.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Preparation of experimental sample and DNA

The fishes used in the present invention were the fishes caught in the coastal waters off the coast of Geoje Island and five fish were used for each sample. All samples were stored in absolute ethanol at 20 ℃ until DNA extraction and TNES-Urea buffer method was used to isolate high molecular weight DNA for microsatellite isolation (Asahida et al . , &Lt; / RTI > Fish Sci Tokyo . 62: 727-730,1996). Total genomic Shotgun library was prepared from 2 ㎕ genomic DNA using GS DNA Library Preparation Kit (Roche Applied Science) and sequenced using Genome Sequencer FLX Titanium instrument (Roche Applied Science).

Example 2: Detection of a louse-specific marker

As a result of searching for a microsatellite on a lenticular language in the method of Example 1, the present inventors have found that a total of 2,500 microsatellites representing eight di-, tri- or tetra-nucleotide repetitive motifs Lt; RTI ID = 0.0 > sequence. &Lt; / RTI >

The micro-satellites selected the 40 candidate micro satellite sequences representing the micro-satellite array specific sequence adjacent (at least 100 bases) and jeoljeok a long (over 400 bp) was designed primers (An et al., Genes Genom , 34 : 681-688, 2012). Twenty - four of the 40 microsatellite loci were successfully amplified and further analysis of PCR amplification, reproducibility, and polymorphism level of these candidate markers revealed that 22 masturbations averaged to an average number of alleles of 6.41 The heterozygosity of the predicted heterozygosity was 0.53 and 0.57, respectively. The degree of deviation from the annual disequilibrium (linkage disequilibrium, LD) and the Hardi-Weinberg equilibrium (ARLEQUIN ver. 3.0. As a result, a significant association imbalance was detected in one pair after Bonferroni correction (P <0.05). Significant deviations from the Hardy-Weinberg equilibrium were found in four loci, which can be attributed to the presence of a null allele in these loci.

In addition, cross-amplification experiments were performed on eight of the 24 polymorphic microsatellite markers, eight for each of the two species of marshmallow and rhubarb. As a result, amplification did not occur at all in the case of 11 locusts, and cross - amplification occurred in both of the nine microsatellite markers in the jabari and red bari. In the case of two microsatellite markers, This happened. Therefore, it was found that the eleven microsatellite markers are specific for the lily fish, and are specific for the lily fish which can specifically discriminate the lily fish among the fishes.

However, in the case of the above 11 microsatellite markers, there is a case where the bass specific bands are unclear or the non-bass specific bands appear in the bass and red bass, and 11 out of the 11 bass specific bands are evident, (SEQ ID NO: 1, Fig. 1).

The above-mentioned grouper-specific primer sets are as shown in Table 1.

Example 3: Detection of red barley-specific markers

As in Example 2, the present inventors used a microsatellite analysis showing sequences (at least 100 bases) and a certain length (400 bp or more) adjacent to a non-specific sequence including a repeat sequence in a red barley microsatellite sequence Respectively. In the above experiment, it was confirmed that the specific primer set capable of specifically amplifying the site adjacent to the specific SSR did not produce the amplification product in other barley and fish such as lobster or Barbarie. Since the red barley specific primer set amplifies the adjacent polynucleotide instead of amplifying the polynucleotide containing the corresponding SSR (SEQ ID NO: 2, FIG. 2), it does not exhibit polymorphism in the length (250 bp). The red barley specific primer set is as shown in Table 1.

Example 3: Multiplex PCR

In accordance with one embodiment of the present invention, the primer pairs selected for the determination of each species were used to assess the level of PCR amplification, reproducibility and polymorphism.

Specifically, PCR amplification was performed using 0.25 U Extaq DNA polymerase (TaKaRa Biomedical Inc., Shiga, Japan), 1 × PCR buffer (PerkinElmer), 0.2 mM dNTP mix, 10 pmol forward or reverse primer The 5'-end of the forward primer was labeled with 6-FAM, NED and HEX dyes, which was performed using Applied Biosystems), 10 μl reaction solution containing 50 ng template DNA, and PTC 200 DNA Engine (MJ Research, Waltham, Mass., USA). The PCR reaction conditions were 95 ° C for 11 min and 95 ° C for 1 min, 94 ° C for 1 min, 58 ° C for 1 min and 72 ° C for 1 min. The final extension step lasted for 5 min at 72 ° C.

Analysis of microsatellite gene polymorphisms was performed using ABI PRISM 3100 automated DNA sequencer (Applied Biosystems). Alleles were analyzed using a molecular size marker (GENESCAN 400 HD [ROX]; Applied Biosystems) using GeneMapper v.4.1 (Applied Biosystems) PCR products were quantitated according to their relative PCR product sizes.

As a result, a gene marker was developed that exhibits stable amplification and species specific amplification in which no allele is found in the genotype analysis and no amplification occurs in the flexible species (FIG. 3). If the gene markers are used to identify the amplified lysine-specific amplification, it is identified as a lysosome. In conclusion, when multiplex PCR is performed using the combination of the red and the lyophilized primers, rapid and accurate species discrimination is possible because the gene is amplified only in the corresponding species and the amplified gene is not amplified.

primer The nucleic acid sequence (5 '-> 3') SEQ ID NO: Size (bp) KES F TGACATCACCTTTTCCCGAC 3 320-340
KES R TCACCGGGTGTTTATGAACC 4 KEA F TGCGCTGGTTCCAAAAAGTG 5 250
KEA R TGTACAGACACTCATGCGCA 6

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> Republic of Korea represented by National Fisheries Research & Development Institute <120> Genetic markers and methods for identifying three species belong to Serranidae <130> PD14-1498 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 501 <212> DNA <213> Epinephelus septemfasciatus <400> 1 ctgattggtg caatacgatt gtgacatcac cttttcccga ctgagccctg cccacttcca 60 atgaggaaga aaaggacaga gaaatacaca aatacagaaa tctgtcatgt gaaacaagct 120 gaagtgtgtt cacttaagtt agtgtgtcca tataggatcc tgtcactctt agaaagaagc 180 ttgttagcaa gatacttcat ctgggactaa tagtaaacga accaaacaac tgtgaaacag 240 tgaaaacaca ttagaacttt gttgaggacc tttattgatt gattgattga ttgattgatt 300 gattgattga ttgatttatt gactggattg attcatcagg ttcataaaca cccggtgaaa 360 aggcagtgac ttcaataaga tgccaatagt tataaataca gtattgcaca acatagaaat 420 gtgtatttag tctacaaagt gtgccaggta aaaccctgag cgcaggccat gtaaaatcat 480 acaattgata ttattatgtt a 501 <210> 2 <211> 978 <212> DNA <213> Epinephelus akaara <400> 2 ccccccgatt tgcatgaagg cgcgagggcc cgttcatcgc tgcttgcagc tttaattatt 60 attattatta ttattattat tattattcct ccaaatgaat tgcctttttg ggaggcttaa 120 catacccgaa aactcatgaa actttgcaca catctcagaa ctggtgaaaa aattgatagt 180 ttaagggtct catgcgctgg ttccaaaaag tgactcagta gcgcccccta caaaagtttg 240 aggaaacagc ccctgaagct agtttaacct acatgtatga aacttggcag gcttatgtaa 300 tgcttagaga catacaaaaa agcctcttgg aggcatcctc taaacccaac aggaagtcgg 360 ccattttgaa tttactgtgc aattttgatg catttttggc catttccagg ggtcctaaat 420 gtgtatgcgc atgagtgtct gtacataaat gtgtgaatgt gcagatacat acagggtctt 480 gcagatctgc attcatagca tgtgaatgtg ctgtacattt aatgtatgag gaaatgtacc 540 gcacatttta agtgtaatat attattctat tgagtttatt acgttctatt gatgccatat 600 atatatatga acacagccaa aattatcaag aaaattaagt gacaaaccaa taatgttctt 660 aatgaggcat ataggttaag aaaatgaggt caaacataaa tcactgactt gtattttcat 720 acagtttgtt tatattcatt tcaaatagta gagttaattt cacagagcta agcatcctct 780 tttgtccagc tgaaactaca ataagccctc agttgtctaa ataaggacac aacacataac 840 gttacacatt tgcattgtga attagctgtg ctctgccagc taacgttagc ttaaagcaaa 900 ggctaacata acaatctcac gttcagcgtt agtaagagac aggaccggtg gttaggtttt 960 acttctggaa agtctacg 978 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KES F <400> 3 tgacatcacc ttttcccgac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KES R <400> 4 tcaccgggtg tttatgaacc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KEA F <400> 5 tgcgctggtt ccaaaaagtg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KEA R <400> 6 tgtacagaca ctcatgcgca 20

Claims

A 15 to 50 nt polynucleotide capable of specifically amplifying a polynucleotide comprising a microsatellite marker selected from the nucleotide sequence represented by SEQ ID NO: 1 or a complementary sequence thereof and consisting of the 275st to 314st nucleic acids of SEQ ID NO: ( Epinephelus septemfasciatus ) discriminating kit, comprising a primer set of a predetermined length.

The method according to claim 1,
Wherein the primer set comprises a forward primer represented by a nucleic acid sequence represented by SEQ ID NO: 3 and a reverse primer represented by a nucleotide sequence represented by SEQ ID NO: 4.

A polynucleotide comprising a microsatellite marker selected from the nucleotide sequence represented by SEQ ID NO: 2 or a complementary sequence thereof and consisting of the 56th to 85st nucleic acids of SEQ ID NO: 1, or a polynucleotide comprising the adjacent polynucleotide of the microsatellite marker, containing 15 to 50 nt long primers to amplify, the reddish Bari (Epinephelus akaara) determination kit.

The method of claim 3,
Wherein the primer set comprises a forward primer represented by a nucleic acid sequence represented by SEQ ID NO: 5 and a reverse primer represented by a nucleotide sequence represented by SEQ ID NO: 6.

Multiplex PCR kit for the determination of the native fish and red bream in barley and fish, comprising the following primer sets:
A 15 to 50 nt polynucleotide capable of specifically amplifying a polynucleotide comprising a microsatellite marker selected from the nucleotide sequence represented by SEQ ID NO: 1 or a complementary sequence thereof and consisting of the 275st to 314st nucleic acids of SEQ ID NO: Length primer set 1; And
A polynucleotide comprising a microsatellite marker selected from the nucleotide sequence represented by SEQ ID NO: 2 or a complementary sequence thereof and consisting of the 56th to 85st nucleic acids of SEQ ID NO: 1, or a polynucleotide comprising the adjacent polynucleotide of the microsatellite marker, A primer set of 15 to 50 nt long which can be amplified by PCR.

6. The method of claim 5,
The primer set 1 comprises a forward primer represented by the nucleic acid sequence shown in SEQ ID NO: 3 and a reverse primer represented by the nucleic acid sequence shown in SEQ ID NO: 4.

6. The method of claim 5,
Wherein the primer set 2 comprises a forward primer represented by a nucleic acid sequence represented by SEQ ID NO: 5 and a reverse primer represented by a nucleotide sequence represented by SEQ ID NO: 6, wherein the primer set 2 is a reverse primer.

delete

A DNA amplification step of amplifying a genomic DNA of a fish object to be discriminated by the false-positive identification kit of claim 1 or 2;
An electrophoresis step of electrophoresing the amplified DNA fragment; And
And analyzing a band pattern of said electrophoretic DNA fragment.

A DNA amplification step of amplifying the genomic DNA of the fish to be discriminated by the red-berry identification kit of claim 3 or 4;
An electrophoresis step of electrophoresing the amplified DNA fragment; And
And analyzing a band pattern of said electrophoretic DNA fragment.

A DNA amplification step of amplifying the genomic DNA of the fish population to be discriminated by the multiplex PCR kit for discriminating between the fishes of the barley and the red barley according to claim 5 or 6;
An electrophoresis step of electrophoresing the amplified DNA fragment; And
And analyzing a band pattern of the electrophoretic DNA fragments.