KR20190053351A - Method for identification and parentage using marker in Ephinephelus akaara - Google Patents

Abstract

The present invention relates to a microsatellite marker for analyzing the genotype of each individual of Epinephelus akaara, and a method for identifying individuals and determining parentage of Epinephelus akaara using the same. Specifically, the present invention, ten Epinephelus akaara microsatellite marker and a set of each pair of primers for amplifying the same, includes a method of identifying individuals and determining parentage, as a method of amplifying and comparing and analyzing a DNA sample of Epinephelus akaara to be discriminated by using the primer pair. The microsatellite marker of the present invention can provide a highly efficient marker for identifying individuals and determining parentage of broodstock group of Epinephelus akaara, and can be effectively used for identifying individuals and determining parentage.

Description

[0001] The present invention relates to a microsatellite marker for identification and parental identification of red-

The present invention relates to a microsatellite marker for analyzing genotypes of each individual of red ruby, and a method for identification and identification of red ruby using the same. More specifically, the present invention relates to a method for identifying 10 red ruby microsatellite markers, And a method of amplifying and comparing and analyzing a DNA sample of a red barbarie to be discriminated by using the pair of primers, thereby identifying an individual and confirming paternity.

Ephinephelus akaara is an aquatic fish of the sea anemone that lives in the coastal reef area and is hiding in a rock hole or rock gaps due to its coastal anchoring. Spawning season is from June to August, and it is mainly caught in Jeju University area in Korea. It is said that white fish is cheaper, cleaner and chewy, and is treated as a fine species among barley and fish, but its number is very small.

There is a small amount of catch compared to the value as an edible resource of red barley, and there is a high dependence on aquaculture. Therefore, in order to improve aquaculture productivity, there is a continuing need to develop a good seed having high growth rate, disease resistance, malformation incidence and survival rate, and to establish a mass production system. The discrimination of the superior seeds is closely related to the genetic composition of the redbari and can be based on genotyping of the redbari group which is captured or cultivated in our country.

However, at present, the determination of the red barley seeds has no method other than the visual confirmation method for the living body, and it is impossible to read the naked eyes after the prototype has changed for the dish or the dish. In the meantime, it has been reported that the DNA reading technique is capable of reading the source species even in living tissue, post-incision tissue, or cooked food. Thus, studies using DNA-based PCR techniques have been reported to identify animal source species, At present, there is no report of genetic diagnosis for identification and paternity identification.

The microsatellite is a region in which 2 to 5 bases are repeatedly characterized in the DNA base sequence in the genome. Various alleles exist depending on the degree of repetition. Because they are inherited from the parent to offspring according to the genetic code of Mendel, (Maria RM et al., Aquaculture 220: 203-218, 2003), defining the genetic characteristics of populations and individuals (Sekino M & Hara M, Mar Biotechnol 3: 572-589, 2001) and paternity identification (Hara M & Sekino M, Aquaculture 217: 107-114, 2003). These microsatellite marker specific sites can be amplified by polymerase chain reaction using the primers of the up and down base sequences including them.

Therefore, the present inventors have found that by combining a combination of pliers capable of analyzing 10 red barley microsatellite markers and establishing optimum PCR conditions, it is possible to identify genetic analysis, identification and identification of red barley populations using microsatellite marker alleles of the red barley Confirming that paternity identification is possible.

Korea Patent Publication No. 10-2009-0069898 Korean Patent Publication No. 10-2016-0110594

It is an object of the present invention to develop a primer pair combination capable of analyzing a microsatellite marker having DNA polymorphism and to establish conditions for analysis through multiplex amplification, Thereby enabling object recognition.

In order to solve the above-mentioned problems, the inventors of the present invention provide the microsatellite markers for identification and paternity identification of SEQ ID NOs: 1-10.

Also, as respective primer pairs capable of specifically amplifying the microsatellite markers of SEQ ID NOS: 1 to 10, SEQ ID NOS: 11 and 12, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: 1 A pair of primers consisting of an oligonucleotide having an amino acid sequence of SEQ ID NO: 2, an oligonucleotide having SEQ ID NOs: 13 and 14 capable of specifically amplifying a red barbit satellite marker of SEQ ID NO: 2, A primer pair consisting of an oligonucleotide having SEQ ID NOS: 15 and 16, respectively, capable of amplification, and an oligonucleotide having SEQ ID NOS: 17 and 18, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: SEQ ID NOS: 19 and 20, which can specifically amplify the primer pair, the red barbit microsatellite marker of SEQ ID NO: 5, A pair of primers consisting of oligonucleotides each having SEQ ID NOS: 21 and 22 capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: 6, a pair of primers consisting of oligonucleotides of SEQ ID NO: A pair of primers consisting of an oligonucleotide having SEQ ID NOS: 23 and 24, respectively, which can be specifically amplified, an oligonucleotide having SEQ ID NOS: 25 and 26, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: A pair of primers consisting of an oligonucleotide having SEQ ID NOS: 27 and 28, respectively, capable of specifically amplifying the red barbit satellite marker of SEQ ID NO: 9, and a red barbit microsatellite marker of SEQ ID NO: 10, A primer consisting of an oligonucleotide having SEQ ID NOs: 29 and 30, respectively, It provides a set of primers comprising a.

In order to accomplish another object of the present invention, the inventors of the present invention have found that 1) a step of extracting a reddish-purple DNA sample to be discriminated, 2) amplifying the DNA sample, and 3) a band pattern of the amplified product And analyzing the plurality of red barriers.

In order to accomplish still another object of the present invention, the inventors of the present invention have found that 1) a step of extracting a DNA sample of a red barley and a parental generation red barley to be discriminated, 2) And 3) comparing and analyzing the band pattern of the amplified product.

The present invention relates to a microsatellite marker for analyzing the genotypes of each individual of the red barley, a pair of primers capable of amplifying the same, and a red barley individual identification and paternity identification method using the same. More specifically, The present invention relates to individual identification and paternity identification methods using primer pairs for microsatellite markers, respectively. The microsatellite markers can provide highly efficient markers for individual identification and paternity identification for the red-billed parent group.

FIG. 1 shows the result of amplification (PCR) of a DNA sequence of an actual rhabdomyote using a primer pair set of the present invention as a template.
Fig. 2 shows the result of determination of microsatellite marker genotypes of 26 red rats using the primer pair set of the present invention.
FIG. 3 shows the result of the identity test and the parentage test using the CERVUS program using the determined genotypes. As a result, it was confirmed that F1 produced in Bbr05M (male) and Bbr03F (male) F2.

The present invention is directed to the microsatellite markers of SEQ ID NOS: 1 to 10 present on the red barley DNA for individual identification and paternity identification.

Microsatellite (MS) DNA markers are DNA fragments that are present on nuclear DNA, with about 2 to 10 nucleotides forming repeat units. Repeating units have a variable number of repeats in the DNA sequence, and the genotype is different. The polymorphism of microsatellite markers shows various genotypes in the same species and is used as a genetic marker for object tracking and paternity identification. The identification system of Hanwoo is used for tracking the production history, and short tandem repeat (STR) markers on human Y chromosome are used as markers for sub-identification

In order to select the microsatellite markers, the present inventors determined the genomic sequence of the red barley through the next generation sequencing method, and found that among the microsatellite markers showing serial repetition in the determined sequence, the genotype is 3 We selected microsatellite markers that are more than two branches. Of the selected markers, 10 were selected for the frequency of high polymorphism and the distribution of alleles in the red barriers group. The selected markers were BBr_2nt_02 (SEQ ID NO: 1), BBr_2nt_12 (SEQ ID NO: 2), BBr_2nt_15 (SEQ ID NO: 3), BBr_2nt_16 (SEQ ID NO: 4), BBr_2nt_24 7), BBr_2nt_31 (SEQ ID NO: 8), BBr_2nt_32 (SEQ ID NO: 9), and BBr_2nt_34 (SEQ ID NO: 10).

The 10 types of microsatellite markers selected in this way have very high polymorphic information content (PIC) of 0.7 or more, and observed heterozygosity (HObs) and expected heterozygosity (HExp) 0.5 or more. As a result, it can be seen that the red barley microsatellite micoker selected in the present invention is highly efficient for individual identification or paternity identification as a whole.

In addition, the present inventors devised a pair of primers of SEQ ID NOs: 11 to 30 capable of amplifying the red barbit microsatellite marker. These are each primer pairs capable of specifically amplifying the microsatellite markers of SEQ ID NOS: 1 to 10, respectively, and having SEQ ID NOS: 11 and 12, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: 1 A pair of primers consisting of an oligonucleotide having an amino acid sequence of SEQ ID NO: 2, an oligonucleotide having SEQ ID NOs: 13 and 14 capable of specifically amplifying a red barbit satellite marker of SEQ ID NO: 2, A primer pair consisting of an oligonucleotide having SEQ ID NOS: 15 and 16, respectively, capable of amplification, and an oligonucleotide having SEQ ID NOS: 17 and 18, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: SEQ ID NOs: 19 and 20 which can specifically amplify the red barbithe satellite marker of SEQ ID NO: 5 A pair of primers consisting of oligonucleotides having respective oligonucleotides, a pair of primers consisting of oligonucleotides having SEQ ID NOS: 21 and 22, respectively, capable of specifically amplifying the red barbit satellite marker of SEQ ID NO: 6, SEQ ID NOS: 23 and 24, respectively, capable of specifically amplifying the oligonucleotide of SEQ ID NO: 8, oligonucleotides each having SEQ ID NOS: 25 and 26 capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: A primer pair consisting of an oligonucleotide having SEQ ID NOS: 27 and 28, respectively, capable of specifically amplifying a red barbit microsatellite marker of SEQ ID NO: 9, and a red barbit microsatellite marker of SEQ ID NO: 10, A primer consisting of an oligonucleotide having amplified SEQ ID Nos. 29 and 30, respectively It is a pair.

The present invention provides a composition comprising the designed primer pair and capable of specifically amplifying the red barbit satellite markers of SEQ ID NOS: 1 to 10. That is, in order to identify an individual of the red barley, identification of the individual of the red barley and identification of the paternity can be performed by confirming whether the microsatellite marker region is amplified in the DNA separated from the red barley using the designed primer pairs .

Accordingly, the present invention provides a primer pair consisting of oligonucleotides having SEQ ID NOs: 11 and 12, respectively, capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: 1, a red barbit microsatellite marker of SEQ ID NO: 2, A primer pair consisting of an oligonucleotide having SEQ. ID. NO. 13 and SEQ. ID. NO. 14, respectively, which can be amplified with a primer pair, and a primer pair consisting of an oligonucleotide having SEQ ID NO. 15 and 16 respectively capable of specifically amplifying the red barbit satellite marker of SEQ ID NO. A primer pair consisting of an oligonucleotide having SEQ ID NOS: 17 and 18, respectively, capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: 4, a pair of primers consisting of an oligonucleotide having the amino acid sequence of SEQ ID NO: A primer pair consisting of an oligonucleotide having SEQ ID NOS: 19 and 20 respectively, a red barley microsphere of SEQ ID NO: 6 A primer pair consisting of oligonucleotides each having SEQ ID NOS: 21 and 22 capable of specifically amplifying the marker, an oligonucleotide having SEQ ID NOS: 23 and 24, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: A pair of primers consisting of an oligonucleotide having SEQ ID NOS: 25 and 26, respectively, capable of specifically amplifying a red barbithe satellite marker of SEQ ID NO: 8, a pair of primers consisting of an oligonucleotide having a nucleotide sequence of SEQ ID NO: A primer pair consisting of an oligonucleotide having SEQ. ID. NO. 27 and SEQ. ID. NO. 28, respectively, amplifiable by primers, and a primer pair consisting of an oligonucleotide having SEQ ID NO. 29 and 30 respectively capable of specifically amplifying a red barbit microsatellite marker of SEQ ID NO: 1 to 10, wherein the red barbit microsatellite marker is selected from the group consisting of ≪ / RTI >

The primer pair may be labeled with at least one oligonucleotide fluorescent substance. The fluorescent material may include, but is not limited to, FAM, HEX, NED, ROX and JOE, preferably FAM, HEX, NED. 1 to 10 can be specifically amplified by using various fluorescent materials in the above composition and different sizes of amplification products using the above composition.

The present invention relates to a method for preparing a DNA sample, comprising the steps of 1) extracting a DNA sample of a red barbarie to be discriminated, 2) amplifying the sample of the first step using the composition, And 3) analyzing the band pattern of the amplification product of step 2.

In the above step 1), the amplification reaction may be performed according to a multiplex amplification polymerase chain reaction program. The PCR reaction can be performed according to a program of 35 to 45 cycles of final denaturation, denaturation, annealing and extension reactions, final elongation and cooling. The PCR reaction of the present invention is not limited thereto, but preferably a Mastercycler Gradient (Eppendorf, Germany) or a similar thermal cycle PCR system is used.

The present invention also provides a method for detecting a fish, comprising the steps of: 1) extracting a DNA sample of a redbarie and a parental fish of the fish to be discriminated; 2) amplifying each of the DNA samples of the first step using the composition; 3) analyzing the band patterns of the amplification products of the two stages respectively, and 4) comparing the band patterns of the red-berry band of the parental generation with the band patterns of the red-billed band of the three- to provide.

The microsatellite markers of the flounder of the present invention are inherited to the offspring by the Mendel's law, so that parent identification can be possible by comparing the genotype of the offspring with the genotype of the parental group. Thus, in a preferred embodiment of the present invention, a multi-amplification polymerase chain reaction was performed using the composition of the present invention in a parent and a progeny flounder, and the parental identification of the offspring generation was performed by analyzing the band pattern of the amplification product . In one embodiment of the present invention, parental identification was performed using the CERVUS program to perform an identity test and a parentage test, from which it was possible to establish a pedigree that identifies individuals and confirms their parental status.

The marker combination and multiple amplification reaction conditions of SEQ ID NOS: 1 to 8 according to the present invention described above can be used economically and efficiently for identification and paternity identification by analysis of a red barley genotype profile, , It can be used for the identification of producers and construction of tracking system for the development, branding and protection of flounder varieties.

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

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

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1> DNA extraction of red barley genome

Total DNA extraction of red barley was done by cutting tail fin. The fin tissue was cut into 0.5cm × 0.5cm, and placed in a 1.6ml microcentrifuge tube and transferred to the laboratory. Separation of DNA from caudal fin was performed using DNeasy Blood / Tissue kit (Qiagen, Germany). The separated DNA fragments were dissolved in TE buffer (10 mM Tris-Cl; pH 8.0, 1 mM EDTA) buffer solution and stored in a refrigerator and used as a template for polymerase chain reaction (PCR).

< Example  2> primer  making

1. Fine satellites Marker  selection

Epinephelus akaara 5 whole genome sequences were determined by next generation sequencing (NGS), and microsatellite markers representing serial repeats in the determined sequence were classified into three or more genotypes. . Among the selected markers, 10 microsatellite markers showing high frequency of polymorphism and distribution of alleles were selected as a genetic marker system for identification and paternity identification in the red barriers group.

In the genome sequence determined by NGS, 10 repeats of microsatellites and individual genotypes Repeat unit No. of genotypes Individual ID 0615 0616 0617 0618 0619 BBr _ 2nt _02 ct 3 30 26 26 28 28 BBr _ 2nt _12 tg 3 28 32 34 32 34 BBr _ 2nt _15 ta 3 20 18 28 18 18 BBr _ 2nt _16 ca 3 32 32 42 26 42 BBr _ 2nt _24 ac 4 42 40 32 42 44 BBr _ 2nt _26 ca 4 46 50 46 34 48 BBr _ 2nt _30 tg 4 22 38 36 36 37 BBr _ 2nt _31 ac 4 46 44 42 42 26 BBr _ 2nt _32 ac 4 52 36 48 40 48 Bbr _ 2nt _34 ga 5 50 44 40 32 42

The 10 types of microsatellite markers selected by the above method have very high polymorphic information content (PIC) of 0.7 or more, and observed heterozygosity (HObs) and expected heterozygosity (HExp) Lt; RTI ID = 0.0 &gt; 0.5 &lt; / RTI &gt;

A group analysis of red barley using 10 combinations of microsatellite markers Locus k N HObs HExp PIC NE-1P NE-2P NE-PP NE-I NE-SI BBr _ 2nt _02 13 26 0.846 0.847 0.812 0.493 0.325 0.146 0.047 0.347 BBr _ 2nt _12 6 25 0.640 0.818 0.775 0.565 0.387 0.204 0.066 0.366 BBr _ 2nt _15 10 26 0.692 0.740 0.704 0.647 0.458 0.247 0.097 0.411 BBr _ 2nt _16 9 26 0.808 0.824 0.781 0.550 0.375 0.193 0.063 0.362 BBr _ 2nt _24 10 26 0.885 0.865 0.831 0.466 0.301 0.131 0.040 0.336 BBr _ 2nt _26 9 26 0.615 0.787 0.748 0.594 0.411 0.214 0.076 0.383 BBr _ 2nt _30 12 26 0.885 0.863 0.831 0.461 0.297 0.124 0.039 0.337 BBr _ 2nt _31 11 26 0.885 0.859 0.826 0.471 0.305 0.131 0.041 0.339 BBr _ 2nt _32 12 26 0.885 0.878 0.846 0.435 0.276 0.113 0.034 0.328 Bbr _ 2nt _34 8 26 0.500 0.784 0.735 0.617 0.439 0.252 0.087 0.387

Informational power of 10 kinds of microsatellite markers evaluated in a group of 26 redbari Statistic content Measured value Number of individuals 26 Number of loci 10 Mean number of alleles per locus 10 Mean proportion of individuals typed 0.9962 Mean expected heterozygosity 0.8264 Mean polymorphic information content (PIC) 0.7889 Combined non-exclusion probability (first parent) (NE-1P) 0.00159773 Combined non-exclusion probability (second parent) (NE-2P) 0.00002937 Combined non-exclusion probability (parent pair) (NE-PP) 0.00000002 Combined non-exclusion probability (identity) (NE-I) 2.83 × 10 ^-13 Combined non-exclusion probability (sib identity) (NE-SI) 0.0000352

As shown in Table 3 above, the average number of alleles was 10.0 on a total of 10 loci, and the average HExp was 0.8264 and the average PIC was 0.7889. In the paternity test using 10 selected microsatellite markers, the parental negation rate (NE-1P) for the first parent is 0.001597, but if there is information about the first parent, The NE-2P and NE-PP were all below 0.0005, the recommended level of the International Society of Animal Genetics. The rate of occurrence of the same species (NE-I) and the same species in the same species (NE-SI) was also low. Overall, it is considered to be a highly efficient marker system for individual identification and paternity identification.

2. primer  making

Primers (forward F, reverse R) were designed for each marker to amplify 10 types of microsatellite markers by polymerase chain reaction (PCR). The designed sequences are shown in the following table.

Primer sequences designed for analysis of 10 species of microsatellite markers Locus Forward primer sequence Reverse primer sequence BBr _ 2nt _02 CTCCCTCCCTTTCTCCATTC CGACCGTCTGAGGTCTAAGG BBr _ 2nt _12 CACCGCCATCAAACTAGACA GTGTCGCTAATGTGGGGACT BBr _ 2nt _15 CCTGTGAGCTCTGTCCCTGT TTGAATTTCTGGGGCAGTTC BBr _ 2nt _16 GGCTAAAAGAGGGGTGGAGT CCATGCCAAAGGCAAAATTA BBr _ 2nt _24 CAGACTGCCTCCACACTTGA ACAGCCTCTGACCACAGCTT BBr _ 2nt _26 TTTCAAATCCACCTGCCAAT CAGCACTGGCAGAGAAAACA BBr _ 2nt _30 TTACCCAGCTGTGCTGAATG CACCAGTACTGACGCTCTGG BBr _ 2nt _31 GCTGGATGAGCTTTCCTCTG GTGAGCAGAGTCGGATGTGA BBr _ 2nt _32 TGCTACTGCATCCACACAAA AAGCGAGCAAACAAACACAG Bbr _ 2nt _34 CTTGGAGGGATGAATCGAGA ACAGAGTCTGCAGCATCCAA

< Example  3> Genotype analysis

One. PCR  reaction

(50 ng / mu l), 1 mu l of 10 x reaction buffer solution (GenetBio, Korea) and 1 mu l of the template DNA (50 ng / mu l) (5 units / μl) of Taq DNA polymerase (GenetBio, Korea) and 10 μM dNTP (GenetBio, Korea) and 0.2 μl (5 units / μl) of forward primer and reverse primer corresponding to the locus of the final concentration of 0.1 μM And mixed to prepare a final reaction solution of 10 μl. The prepared PCR reaction solution is denatured at 95 ° C for 10 minutes, followed by 35 cycles of 95 ° C for 30 seconds, -58 ° C for 30 seconds, and 72 ° C for 45 minutes, followed by holding at 72 ° C for 5 minutes. PCR is performed using a Mastercycler Gradient (Eppendorf, Germany) or a similar thermal cycle PCR system.

As a result, it was confirmed that the markers of the present invention were simultaneously amplified from the PCR analysis results of all the red rhesus species analyzed. (Fig. 1)

2. Genotype analysis

The PCR reaction product amplified by the method of Example 3-1 was randomly diluted 10 times with TE buffer solution for genotyping and 1 μl thereof and 0.2 μl of size standard GeneScan 400HD ROX (ABI, USA) , And 10 μl of HiDi formamide (ABI, USA) were analyzed by genotype analyzer (3100 genetic analyzer; ABI, USA). In addition, gene fragments amplified after labeling three fluorescent particles such as FAM, HEX and NED in the forward primer among the respective fine satellite marker primers were developed using a DNA sequencer ABI 3130XL.

As a result, as shown in FIG. 2, at the same time, genotypes of 10 microsatellite markers of red ruby were obtained. In this case, alleles of the microsatellite markers represented by the respective peaks are classified into differently labeled fluorescent dyes. The blue peaks represent the 6-FAM fluorescence-labeled marker, the green is the position of the allele of the marker labeled with HEX, and the black indicates the position of the marker labeled with NED.

In addition, the sizes (bp) of the alleles were measured as follows by comparison with Genescan 400 HD ROX (ABI, USA) in which the size of the corresponding allele was orange. This is the result of determining the microsatellite marker genotypes of 26 individuals of the red barley using the above primers.

&Lt; Example 4 > Object identification and paternity identification

Using the genotypes determined, identity tests and parentage tests were performed using the CERVUS program. The same individuals did not appear in the result of the identity test, and the family diagrams of F1 and F2 produced from Bbr05M (male) and Bbr03F (male) produced based on the result of the paternity test are shown in FIG.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Jeju National University <120> Method for identification and parentage using marker in          Ephinephelus akaara <130> PN1711-412 <160> 30 <170> KoPatentin 3.0 <210> 1 <211> 509 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 1 cttacctact cattgacgct ttctctgcaa cccccccacc accaccacca ccacctccac 60 ccccccatca acactcggct gttgacgtga gccgtgtaac aatcctccct gtttccatga 120 caaccacctg ctgctgtcct cccctcatct gtctctccct ccctcccttt ctccattccc 180 tctctctctc tctctctctc tctctctctc cccccttttc cccctctcag ttgtctccct 240 tctctccgca cactgttggt atttcaaggt caaagagggt cacagccaga tagtgagata 300 gagacagaga taggggtgga aagtggggaa aggagagcga taacaatgcg agagaagaca 360 cacagaggga ggctggagga caaaaaaatc ccccagacgt tgttattgtt agcaccttag 420 acctcagacg gtcgccttat tcaacctagc ttggctcgct gtcctccctg cacttatcat 480 aacctcgttt tgtgtcaacc ttgagccaa 509 <210> 2 <211> 543 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 2 ttcacaatga ttttcgacca tagtgtgccc ccttggatca ccaggagctg tatgctcagt 60 taaatgtgat ttatttagtg tggactggac tgattctcaa atgtcagctg aaacttaatg 120 ttacattttg ctccagttat gtctttttta tacatcagta gctcttacat ggacaaccct 180 catagctttt atctttcatc agcacaacct tttgtttccg ctggtttaac aatagtgcca 240 tgctgcagat taagcctaac tccaccgcca tcaaactaga caccattgtg tttgtgtgtt 300 tgtgtgtgtg tgtgtgtgtg tgtgtgtgcg cgccaggtta gaggggtgtg tataaagtgc 360 agtccccaca ttagcgacac cattcacaca gttttttatt atgtttggat ccaatgttga 420 tctgacaaag atttatgttg ggacaaaagt ttgctttctg gtattgtgtt tgctctatat 480 ttgttttgga gagaccgtgc acatgcagaa cttaaagagc ccagcgtttc agccccccct 540 ccc 543 <210> 3 <211> 620 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 3 tatttcttcc ttccatcttg tctgcacact tgtttcccag tgttcccaca tgctcagcca 60 tcagcactga aaactcacgt attttcctgc catcctgcac ttctctggaa tatgtatgtg 120 aatttatgca tttgttttgg acctgccaga aggaaagcag gtttccaaaa gcaaaaatct 180 gctctcaaca ccagctttct tacatttaca ctgaaataat taaagccaac ttgtgattca 240 agggcagtgt atattacaat gaaaccctgt gagctctgtc cctgtccacg gagtattctt 300 tatatatata tatatatata tgtacacaca aaacgcccac agacgcaatc accaagaact 360 gccccagaaa ttcaagcgaa ggttatagaa attctctttg tagtggcaaa caaaatatcc 420 ctgagctttg aaaatcaccc tgcattattt ccgcagcatc cttggtgctg ctctgtgttg 480 cttattcttt cacacagcaa atgcctctct tagtcacgtt aggcagcttc tccatcaagg 540 ctgttttaat tcttctccac accagtgagt ttttagctgc atttgattgt aaactgcacg 600 cagaaccaat acacacacaa 620 <210> 4 <211> 503 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 4 tttttaatat aattctgatt catttgtgaa tagactacag tggaattaac cgcagccctt 60 caaaacaaat cccctttatc caccctctgc ctccttctct ctctctgggg ctaaactgga 120 ctcggcctgg ctaaaagagg ggtggagtag tctaattcat cagaaacagg tcacacacac 180 acacacacac acacacacac acatgcctgg ctacgaaatc atttctgcaa ccacaaaatc 240 actttaatat attcccagtg ctacttttga atattaataa aatatcaatt attaattttg 300 cctttggcat ggcaagatat acaagtttta attagtgtac tgtactcttt cacaattaaa 360 aaaataattg atgtccatac atgcgggcat tttgctgtga tggtctttct tgttgttgtc 420 aacagaggag gcggtgagac ggtgcgggca ctctcgcttc ttcctcttgt ccgcagttca 480 gatgcacttc aggcctgagg cta 503 <210> 5 <211> 642 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 5 cattagctgt actgattgtc acttgcttgg tttgtagggg tgtgtgtgtg tgtgtggtgt 60 ggtgatgggg tcaggaaggg gagggggggc gcagagagag ggagagaggg ggacagagga 120 ggtagagaca gaaggaaaga gcaaggaaaa gagagaaaga aagcaagcgc tgtctcagtc 180 gt; cactctccca gactgcctcc acacttgacc gctcaagctg agccacacat gcgcacgcat 300 acacacacac acacacacac acacacacac gcaacaaaca aacaaaccaa gctgtggtca gaggctgtgg ccaagtgtcc ttgcatagac 420 agcacctttt agttcagcca ctgacccaat caaaaagcca ccacagctct tcagcacgct 480 atttcattca atggaggagc ccagcagcta tagcagtgaa tcccactgga tcactaagcc 540 ttgataggaa gacaacacta cacaaaaatg tttcagagtt gccaaatggc ccctaccaat 600 cttctgctgc caccaccacc tccaccagcg cctctgccaa aa 642 <210> 6 <211> 596 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 6 tctctctccc tctctctctc tccctctctc tctctcactc actcactcac tcactcactc 60 acacacacag aatcacacac acagagtcac aaattcacac attcaaacac acacaccggt 120 ccggatgctg aatacacact cccagtgcgg agcagttcag ctcagcatgt tgaaacagac 180 accaggctgc tctttgtttt aattattgta cggaagcttg tttcaaatcc acctgccaat 240 ctgtctctct cacacacaca cacacacaca cacacacaca cacacacaca cacacaccct 300 ccaccctgcc actccctctc tgtattatct gcagataaaa cccttccagt gaggcgttgc 360 agatttcata tgaatgtgta aattccaatt aggattagct tgttttctct gccagtgctg 420 atagcggctt tagcatgtcg ctggaggaag tgtcggccta ctggaactca tgatagacaa 480 gctgatgagg ccagattagt tcctttcagc tgtgtgtgtt tgtgtgcggg tgtgtgtggg 540 tgatgctctt catagctgaa actaattagc aggttgtctc tgctgtggag gggacg 596 <210> 7 <211> 622 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 7 aatttctctt acattttctc taattttcta tctcgagaaa acttgagaga agccagcccc 60 agatgcacca atatttctct accatccaaa tctgccctta cccagctgtg ctgaatgatg 120 aaccccactt catcacaagt aacctattag catgggtaac accttgtaaa cactgtataa 180 cagcaggtgt tgtgctgtgt gcaaaaactc tggcacatac ccaagaattg gagtagtggc 240 accaaaacag gctgtaagaa gaagaagctc tgcagtagtg aaattatttt tgtttttttt 300 tgtgtgtgtg tgtgtgtgtg tgaaataaac ttaaacaacg ttaacatgtg agcttccaga 360 gcgtcagtac tggtgttaca ggcaagtcaa aaacacagca atggaaaaat atttgtgatg 420 ctgtaaatac tgcagcagtg gtggaggaag tattcagacc cttgacttaa tgaaaaagta 480 ctagaaccac actgcgaaaa tactctgtaa ctagtagaac cctgcattta atatattact 540 taagtaaaag catgtaagtg caatcaggaa attgtattta ggtattaaaa gtaaaggtag 600 tcaatgcgga aaaacttaaa ta 622 <210> 8 <211> 646 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 8 gttcagggct gcttcatgtc attctttctt gctcctctca gaggcaggta aagtaacaga 60 gggatctggg aggacacggc gggaacagtg ggctctgcct gggccagctg ctccctctgt 120 tggagctaaa gacactgtac tgtactggat acttagagta gcatcgctcg acataatggg 180 gttataatct agaattccct gaaaatgctg taaaatgaca actaatctag gtggcacagg 240 tgtggctgga tgagctttcc tctgtgtgat tgcactgtgc tgtgggttgc tcgctctttt 300 acacacacac acacacacac acacacacac acacacacac acacacgctc atacaataaa 360 tcacatccga ctctgctcac caagcggccc agcctatccc ggggtcatct caaaatcacc 420 atttaatgtt taacggcaaa gctggctttg atatcagcgg cagataaagt ggaatttctt 480 gattgcctcg gtcaaagagg ctccgctcgc tgagaaaatg atttattgga cacggggaag 540 tcggtggaga tcaaaggaga cggacgagcc tccaactcac accgcgggcc gggagcaggc 600 tggggctcgt ccaatcactc gcaccagcgg ggcccctgtc actgct 646 <210> 9 <211> 652 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 9 acacttggct cagttttgga ccttagggat ttgggttgac aatccagata gcaacatttg 60 ggctaactgt gggccatgat cagcttactg tattaatgca taacatttac cacaatgctg 120 acatatgacc agcaggagtc ctggacttgg caactgtgtt tgtgtttata gcagaaaaaa 180 attcacaagt tgttttagac aggagactct gataaacagg ctgaacattc actgtggcag 240 gtattacctt taaaatccat ctgctactgc atccacacaa acaatacctt acatgagcta 300 acacacacac acacacacac acacacacac aggttcatat gtctgtagag ctgttagatg tttaagtttc atcaaacaca tgcaggttct 420 ctggttctgg ggccgctggc aaagagatta gcaaacacac acactgtgtt tgtttgctcg 480 cttatgagtt tgtcactgca acatacagta tagagcatgc tttcatttcc agtaaagtgg 540 caaacacacg ttagtgtgtg aatggcagct cacccagaac catcagcaca cacagagttt 600 ggcatcaacg aacccgacct cgcttatgtt cagcctggct ctgctcgact gg 652 <210> 10 <211> 650 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 10 attcacatgt gtgcatttgc aattttgaga gaggggcaca tgcaggggtc acgagagaaa 60 gagacaaccg tcaatttagt tcccctcgcc ttcatcaaac acataccctc ggccaaatgt 120 gcttgcattg gggcatttat gtttgcgagc ggtctgtata gataagcttc aaatttatgc 180 cagcaggagg aagataatga cctggtggat ttgtccttgg agggatgaat cgagaaactg 240 aggggaaagg gaggacgatg gcggagatgg gggagattat gcaggagatg gaatagcaag 300 gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga ggtggtttaa 360 aagttagaaa tgctgaatgt catatgagtg ggagagggta cagagcataa aatcacacag 420 gaaggcactt gttaatgcaa cagttggatg ctgcagactc tgtagaatat tggtctgaca 480 ctgtgggcct cccctcagac aaaatcgaga ccatttggca aattgccaag agtaaaagta 540 tgccaaatta gctattagca gctctgatct gcactgtact cacggatgta cagacatcca 600 tgattggatt cctttgatac caaagaaaac ttaatgattc tcgggcaagt 650 <210> 11 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 11 ctccctccct ttctccattc 20 <210> 12 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 12 cgaccgtctg aggtctaagg 20 <210> 13 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 13 caccgccatc aaactagaca 20 <210> 14 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 14 gtgtcgctaa tgtggggact 20 <210> 15 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 15 cctgtgagct ctgtccctgt 20 <210> 16 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 16 ttgaatttct ggggcagttc 20 <210> 17 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 17 ggctaaaaga ggggtggagt 20 <210> 18 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 18 ccatgccaaa ggcaaaatta 20 <210> 19 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 19 cagactgcct ccacacttga 20 <210> 20 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 20 acagcctctg accacagctt 20 <210> 21 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 21 tttcaaatcc acctgccaat 20 <210> 22 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 22 cagcactggc agagaaaaca 20 <210> 23 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 23 ttacccagct gtgctgaatg 20 <210> 24 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 24 caccagtact gacgctctgg 20 <210> 25 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 25 gctggatgag ctttcctctg 20 <210> 26 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 26 gtgagcagag tcggatgtga 20 <210> 27 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 27 tgctactgca tccacacaaa 20 <210> 28 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 28 aagcgagcaa acaaacacaj 20 <210> 29 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 29 cttggaggga tgaatcgaga 20 <210> 30 <211> 20 <212> DNA <213> Unknown <220> <223> Epinephelus akaara <400> 30 acagagtctg cagcatccaa 20

Claims (9)

SEQ ID NO: 1 to SEQ ID NO: 10. The microsatellite marker according to claim 1, wherein the microsatellite marker comprises a primer pair consisting of an oligonucleotide having SEQ ID NOs: 11 and 12, respectively, capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: 1, A primer pair consisting of oligonucleotides each having SEQ ID NOS: 13 and 14 capable of specifically amplifying the satellite marker, a primer pair consisting of SEQ ID NOS: 15 and 16, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: A primer pair consisting of an oligonucleotide having an amino acid sequence of SEQ ID NO: 4, an oligonucleotide having SEQ ID NOs: 17 and 18 capable of specifically amplifying a red barbit satellite marker of SEQ ID NO: 4, A pair of primers consisting of oligonucleotides each having SEQ ID NOS: 19 and 20, A primer pair consisting of oligonucleotides each having SEQ ID NOS: 21 and 22 capable of specifically amplifying the red barbit microsatellite marker, SEQ ID NOS: 23 and 24 capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: A pair of primers consisting of an oligonucleotide having SEQ ID NOS: 25 and 26, respectively, capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: 8, a pair of primers consisting of an oligonucleotide of SEQ ID NO: A pair of primers consisting of oligonucleotides each having SEQ ID NOS: 27 and 28 capable of specifically amplifying the marker, and an oligonucleotide having SEQ ID NOS: 29 and 30, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: A pair of primers selected from the group of primer pairs comprising a pair of primers consisting of nucleotides Which is amplifiable by a microsatellite marker for identification or paternity identification. A primer pair consisting of oligonucleotides having SEQ ID NOS: 11 and 12, respectively, capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: 1, a sequence capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: 2 A pair of primers consisting of oligonucleotides each having SEQ ID NOS: 13 and 14, a pair of primers consisting of oligonucleotides having SEQ ID NOS: 15 and 16 respectively capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: 3, A primer pair consisting of oligonucleotides each having SEQ ID NOS: 17 and 18 capable of specifically amplifying the red barbit microsatellite marker, SEQ ID NOS: 19 and 20 capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: , A pair of primers consisting of an oligonucleotide having an amino acid sequence of SEQ ID NO: A pair of primers consisting of oligonucleotides having SEQ ID NOS: 21 and 22, respectively, a pair of primers consisting of oligonucleotides having SEQ ID NOS: 23 and 24, respectively, capable of specifically amplifying the red barbithe satellite marker of SEQ ID NO: 7, A primer pair consisting of oligonucleotides having SEQ ID NOS: 25 and 26, respectively, capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: 8, a sequence capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: A primer pair consisting of oligonucleotides having numbers 27 and 28 respectively, and a pair of primers consisting of oligonucleotides having SEQ ID NOs: 29 and 30, respectively, capable of specifically amplifying the red barbit microsatellite marker of SEQ ID NO: A composition capable of specifically amplifying the red barbit microsatellite marker described as 1 to 10 . 4. The composition of claim 3, wherein at least one oligonucleotide of the primer pair is labeled with a fluorescent material. 5. The composition according to claim 4, wherein the fluorescent material is selected from the group consisting of FAM, HEX, NED, ROX and JOE. 1) extracting a DNA sample of a red barbarie to be discriminated;
2) amplifying the DNA sample of step 1) using the composition of claim 3; And
3) analyzing the 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 multiplex amplification polymerase chain reaction program. 1) extracting a DNA sample of a fish to be discriminated and a fish of a parent household of the fish;
2) amplifying each of the DNA samples of step 1) using the composition of claim 1;
3) analyzing each band pattern of the amplification product of step 2); And
4) comparing the band pattern of the parent household fish of step 3) with the band pattern of the fish to be discriminated. A kit for the identification and / or paternity of fish comprising the composition of claim 3.

Images