KR102155967B1 - Genetic Marker Composition for Discrimination of Lebbeus Groenlandicus Species Originated Mitochondria Insertion Markers and Diagnosing Method using the Same - Google Patents

Abstract

The present invention relates to a genetic marker composition for discriminating species of Lebbeus groenlandicus derived from a mitochondrial insertion marker, a primer set for discriminating species of Lebbeus groenlandicus, a kit composition including the same, and a method for discriminating Lebbeus groenlandicus using the same. When using the genetic marker for discriminating the species of Lebbeus groenlandicus and the primer for amplifying the marker of the present invention, the Lebbeus groenlandicus of the present invention can be easily distinguished from other Dokdo shrimps caught in the waters around Dokdo, such as Pandalopsis japonica and Pandalus hypsinotus. In addition, it is possible to classify shrimp species in a faster, more accurate, and economical way.

Description

Genetic Marker Composition for Discrimination of Lebbeus Groenlandicus Species Originated Mitochondria Insertion Markers and Diagnosing Method using the Same}

The present invention relates to a genetic marker composition for discriminating species of prickly pear shrimp derived from a mitochondrial insertion marker and a method for discriminating prickly pear shrimp using the same.

Shrimp is a species belonging to Arthropoda, Malacostraca, and Decapoda. About 2,900 species are distributed worldwide, and about 90 species are known to inhabit the coast of Korea. Shrimp species are morphologically classified into the order of Dendrobranchiata, which is defined as'pawn,' and the order of Pleocyemata, which is defined as'shrimp'.

Shrimp is well known as a natural tonic food so that it improves the kidneys and blood circulation, and replenishes energy, and the effects of replenishing energy are recorded in ancient books such as Bonchogangmok, and it is expected to be more valuable as a health food.

Meanwhile, the Nagoya Protocol, announced in 2010, is an international agreement to share the benefits of utilizing biological resources, and the agreement is in effect in Korea from August 17, 2017. Korea is preparing for the Nagoya Protocol by discovering more than 100,000 species of domestic biological genetic resources and creating a database for resource use, centering on the National Institute of Biological Resources and the National Institute of Ecology established in 2012.

As the “Nagoya Protocol” comes into force, protecting and preserving our bio sovereignty is emerging as a very important issue. Regarding the preservation of our biological resources, the globular tree, the representative plant of Mt. Jirisan and Mt. Halla, was exported to the West in 1904 and used widely as a Christmas tree in foreign countries. The popular clove tree and the Korean native wheat known as Sorona are all biological resources of Korea exported overseas.

Therefore, various efforts are required to prevent the unauthorized export of Korean endemic organisms and secure biological resources. In particular, in the case of endemic species such as shrimp, which prepare for the Nagoya Protocol and have high economic value added, the development of species-specific markers for securing biological sovereignty is essential.

On the other hand, Lebbeus shrimp, well known as a kind of Dokdo shrimp groenlandicus ) is Korea's representative endemic species living in the sea near Dokdo, and is also called chicken shrimp. Prickly pear shrimp ( Litopenaeus) accounts for 99.9% of farmed shrimp in Korea. vannamei ) is an evolutionarily distant species. Particularly, since prickly pear shrimp has the meaning of providing an important link to the protection of Dokdo territorial sovereignty, it can contribute to the protection of Dokdo territorial sovereignty and securing Dokdo biodiversity through genome analysis and species identification of Prickly Pear shrimp.

In addition, species identification and classification according to morphological characteristics tends to cause variables according to growth stages or environmental factors, and species identification through classification according to morphological characteristics requires a lot of time, effort, and cost. It is only limited by researchers with expertise. However, due to the recent development of biotechnology, it has become possible to study the diversity of biological species at the DNA level, enabling rapid and accurate species identification through nucleic acid fingerprint analysis.

Currently, more than 100 species of shrimp mitochondrial genome sequences belonging to the Decahedron have been identified, but there is no mitochondrial sequence analysis of the Hippolytidae shrimp belonging to the Dokdo shrimp. Accordingly, there is no situation in the development of species-specific markers for classifying Dokdo shrimps caught in the waters around Dokdo, such as spiny pear shrimp, red lobster shrimp, and flaming shrimp.

Accordingly, the present inventors have made extensive research efforts to develop a species-specific marker that can distinguish one of the Dokdo shrimps from other shrimps. As a result, NAD3 (NADH dehydrogenase) in the mitochondrial genome sequence isolated from the muscle tissue of the Shrimp subunit 3) In the 4480th nucleotide sequence under the gene, it was confirmed that there is an insertion site of 39 bp in length unlike other shrimp species, and using primers prepared based on the insertion sequence By revealing that the shrimp can be easily distinguished from other shrimp species, the present invention was completed.

1. Korean Registered Patent Publication No. 10-1856539 “Microsatellite marker for determining the habitat of long-tailed leap side shrimp and a method for determining habitat using the same” (Announcement date: May 10, 2018) 2. Korean Registered Patent Publication No. 10-1913744 “Single nucleotide polymorphism (SNP) gene marker for determining the habitat of long-tailed leap side shrimp and its habitat identification method” (announcement date: November 1, 2018)

Accordingly, the main object of the present invention is to provide a mitochondrial marker composition for discriminating species of prickly pear shrimp and a primer set based thereon.

Another object of the present invention is to provide a method for discriminating a mitochondrial marker for discriminating the species of barbed shrimp and a primer set based thereon.

Another object of the present invention is to provide a kit for discriminating a mitochondrial marker and a primer set based on the mitochondrial marker for discriminating the species of barbed shrimp.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a genetic marker composition for discriminating species of thorny shrimp ( Lebbeus Groenlandicus ) consisting of the DNA sequence of SEQ ID NO: 2.

The present inventors performed genomic analysis of spiny shrimp to discover species-specific markers of spiny shrimp. As a result, attention was paid to the mitochondrial genome sequence of prickly pear shrimp. The mitochondrial genome of shrimp species varies from species to species with an average of 15.9Kb (range: 15,240bp to 16,899bp), while most of 13 protein-coding genes ( ND1, ND2, ND3, ND4, ND4L , ND5, ND6, COI , COII , COIII , ATP6 , ATP8 , CytB ), 22 tRNA genes, and 2 rRNA genes. The present inventors determined and analyzed the mitochondrial genome sequence of prickly pear shrimp, predicted the analyzed prickly pear mitochondrial gene, and then compared the gene sequence of 62 other shrimp mitochondria with the mitochondrial gene sequence of prickly pear Has discovered a conservative area. Afterwards, through the analysis of the phylogenetic tree and the phylogenetic tree of prawns, the closest prawns were found evolutionarily, and the mitochondrial genome sequence between the genetically preserved prawns was analyzed through an alignment method. of SEQ ID NO: 2 48bp length of the DNA sequence (5'-a gagtaagcaa ctaaaatttc ctgctcaata ataacgacac tgttaga-3 ') consisting of the visible fold shrimp (Lebbeus Groenlandicus ) gene markers for species discrimination were discovered.

The spiny pear shrimp of the present invention ( Lebbeus Groenlandicus ) The genetic marker for species discrimination is capable of complementary binding to the mitochondrial genome of Lebbeus Groenlandicus , so it can be used as a marker for discriminating species of Lebbeus Groenlandicus or a marker composition by itself. .

Prickly pear shrimp of the present invention ( Lebbeus Groenlandicus ) In the gene marker for species discrimination, the DNA sequence of SEQ ID NO: 2 is SEQ ID NO: 1 Prickly pear ( Lebbeus Groenlandicus ) It is characterized by being inserted under the 4,480th base of the NAD3 (NADH dehydrogenase subunit 3) gene of the mitochondrial full-length genome sequence. When comparing the DNA sequence of SEQ ID NO: 2 with the mitochondrial sequence of Chlorotocus crassicornis, which is a genetically close shrimp species, prickly pear shrimp is two nodes at the 4,480th nucleotide sequence corresponding to the lower part of the NADH dehydrogenase subunit 3 (NAD3) gene. It was found that there was an insertion of 39 base-pairs (bp) compared to the mitochondrial sequence of shrimp (Chlorotocus crassicornis).

In addition, when compared to the other 62 species of shrimp, from the 4,480th base to the 4,487th base 8bp primary insertion (5'-a gagtaag-3'); A conserved portion of 7 bp from base 4,488 to base 4,494 (5'-caa ctaa-3'); And a secondary insertion portion of 33 bp from base 4,495 to base 4,527 (5'-atttc ctgctcaata ataacgacac tgttaga -3'). Configuring this insertion portion is arranged well to the invention [4], prickly pear shrimp of the present invention due to the characteristics consisting of the DNA sequence of SEQ ID NO: 2 (Lebbeus Groenlandicus ) Species identification genetic markers can be used as a kind of insertion marker. The "insertion marker" or "Insertion marker" refers to a mutation in which some bases are inserted in the middle of the DNA sequence. The insertion marker searches for an insertion or deletion region through a method of comparing and analyzing genomic information between species, and based on the information, a primer used to amplify the "insertion marker" site is prepared. Can be used.

In the present invention, "marker" refers to a substance capable of analyzing species specificity between shrimps, and includes organic biomolecules such as nucleic acids (eg, DNA, mRNA, etc.) related to species identification of Lebbeus Groenlandicus . .

According to another aspect of the present invention, the present invention comprises a primer set capable of characteristically amplifying the DNA sequence of SEQ ID NO: 2 or a fragment thereof. Groenlandicus ) provides a primer set for species identification.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3'terminal hydroxyl group, which can form a complementary template and a base pair, and copy the template It refers to a short nucleic acid sequence that functions as a starting point for. The length and sequence of the primers should allow the synthesis of the extension product to start, and the specific length and sequence of the primers depend on the conditions of use such as temperature and ionic strength, as well as the complexity of the required DNA or RNA target. something to do. As a specific example, the specific length and sequence of the primer should be determined in consideration of various conditions such as guanine and cytosine contents (GC contents), GC arrangement, annealing temperature and ionic strength. Primers can initiate DNA synthesis in the presence of reagents for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates (dNTPs) at appropriate buffers and temperatures.

In the present invention, "forward primer" and "reverse primer" are each bound to the 3'-end and 5'-end of a specific site of a gene amplified by a gene amplification reaction to serve as the starting point of DNA synthesis. It means a primer that works.

Prickly pear shrimp of the present invention ( Lebbeus Groenlandicus ) In the primer set for species identification, the primer set may be used for amplification of the target sequence, and preferably may be used for amplification of the DNA sequence of SEQ ID NO: 2, which is an insertion marker for discriminating the species of P. .

Prickly pear shrimp of the present invention ( Lebbeus Groenlandicus ) In the species identification primer set, the primer set may be a primer set of SEQ ID NOs: 3 and 4.

In a specific embodiment of the present invention, the inventors of the present invention are based on the gene sequence of the insertion site of the mitochondrial genome, Lebbeus Groenlandicus ) It is possible to easily identify Lebbeus Groenlandicus by using a genetic marker for species identification, and by confirming the presence or absence of band formation, other shrimp species and Lebbeus Groenlandicus ) could be confirmed to be easily distinguishable, and when amplification using the primer sets of SEQ ID NOs: 3 and 4, thorny lobster ( Lebbeus Groenlandicus ) it was confirmed that a specific band of 244bp length of SEQ ID NO: 5 was amplified. Accordingly, the primer set of the present invention can be used as an insertion marker primer set for discriminating species of Lebbeus Groenlandicus .

According to another aspect of the present invention, the present invention is the spiny pear shrimp ( Lebbeus Groenlandicus ) Provides a kit for identifying species of spiny shrimp ( Lebbeus Groenlandicus ) including a primer set for species identification.

The "kit" of the present invention includes genomic DNA derived from a sample to be analyzed, the primer set of the present invention, an appropriate amount of DNA polymerase (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusfiliformis, Thermisflavus, Thermostable DNA polymerase obtained from Thermococcus literalis or Pyrococcus furiosus (Pfu)), dNTP mixture, PCR buffer, and water. The PCR buffer solution may contain KCl, Tris-HCl and MgCl ₂ . In addition, constituents necessary for performing electrophoresis capable of confirming the amplification of PCR products may be additionally included in the kit of the present invention. At this time, the concentration of MgCl ₂ greatly affects the specificity and quantity of amplification. In general, when Mg ^{2 +} ions are excessive, non-specific PCR amplification products increase, and when Mg ^{2 +} ions are insufficient, the yield of PCR products decreases. An appropriate amount of BSA may be additionally included in the PCR buffer solution.

According to another aspect of the present invention, the present invention comprises the steps of: (a) separating DNA from a target shrimp sample; (b) using the DNA isolated in step (a) as a template, and amplifying the target sequence using the primer set of claim 3 or 4; And (c) when the target sequence is amplified, Prickly Pear ( Lebbeus Groenlandicus ) The step of determining as the species; Provides a method for determining the species containing a spiny shrimp ( Lebbeus Groenlandicus ).

The spiny pear shrimp of the present invention ( Lebbeus Groenlandicus ) In the method for determining species, the target sequence is not limited thereto, but may be a DNA sequence of SEQ ID NO: 2.

In an embodiment of the present invention, when amplifying the gene sequence of the insertion site of the mitochondrial genome of SEQ ID NO: 2 of the present invention, the thorny lobster ( Lebbeus Groenlandicus ) was confirmed that a specific band of 244 bp length of SEQ ID NO: 5 was amplified, and it was found that no band was formed in Pandalus hypsinotus and Penaeus japonicas .

Thus, using the prickly pear shrimp (Lebbeus Groenlandicus) gene markers for species determination based on the gene sequence of the inserted (insertion) site of the mitochondrial genome of the present invention the visible fold shrimp (Lebbeus Groenlandicus ) can be easily identified, and other shrimp species and barbed shrimp ( Lebbeus Groenlandicus ) could be confirmed to be easily distinguishable.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a genetic marker composition for discriminating a species of prickly pear shrimp derived from a mitochondrial insertion marker, a primer set for discriminating a species of prickly pear ( Lebbeus Groenlandicus ), a kit composition comprising the same, and a method for discriminating prickly pear shrimp using the same do.

(b) In the case of using the genetic marker for discriminating the species of prickly pear shrimp of the present invention and the primer to amplify the marker, the prickly pear prawn of the present invention with other Dokdo shrimp caught in the waters around Dokdo, such as mulleung pear shrimp, It can be easily identified, and species classification of shrimp is possible in a faster, more accurate and economical way.

1 is a diagram schematically showing a genomic map of the mitochondria of prickly pear shrimp.
FIG. 2 is a diagram showing the results of analyzing the mitochondrial genome of 62 species of shrimp, and analyzing the sequence and sequence of each gene.
3 is a schematic diagram of analyzing and organizing the genetic location of spiny pear shrimp through evolutionary tree analysis.
FIG. 4 is a diagram showing a comparative analysis by aligning the specific insertion marker site of barbed shrimp with corresponding sequences of other shrimps.
Figure 5 is a spiny pear shrimp (LGR, Lebbeus Groenlandicus ) using the primer set of the present invention, dohwa shrimp (PHY, Pandalus hypsinotus ), and bovine shrimp (PJA, Penaeus japonicas ) after amplifying the genes, and taking the result of electrophoresis.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are for illustrative purposes only, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Analysis of mitochondrial genome of spiny shrimp

Mitochondrial genome analysis project of prickly pear shrimp was carried out.

First, the promethION long-read produced from muscle DNA was subjected to de novo assembly using a wtdbg2 (de novo sequence assembler) to obtain a mitochondrial sequence of 14,181 bp in length.

In order to correct the high error rate according to the long sequence, 3,372,627 short-reads were secured by aligning 500 gigabyte Illumina short-reads to the assembled prickly pear shrimp mitochondrial sequence with the minimap2 program (314,823 depth).

Based on the aligned single sequence, high-accuracy prickly lobster mitochondrial genome sequence was obtained through two polishing with the Pilon program, and a seed sequence for mitochondrial analysis was produced using the NOVOPlasty (ver. 3.0) program.

As a result of performing NOVOPlasty (ver. 3.0) assembly based on the 2.8 giga (G) (2,832,869,352) illumination sequence and the produced prickly pear seed sequence, the analysis of the mitochondrial genome structure of prickly pear consisting of 17,399 nucleotides was completed. I did.

The mitochondrial genes of spiny lobster were predicted using the MITOS (http://mitos.bioinf.uni-leipzig.de/index.py) program, through which 13 protein-coding genes, 22 tRNAs genes, and Then, two rRNAs genes were identified (see Table 1 and FIG. 1).

[Table 1] Mitochondrial locus information and annotations of spiny pear shrimp

Example 2. Search for mitochondrial markers specific to Prickly pear species

In order to compare and analyze the gene sequence and sequence of the mitochondrial genome of shrimps, 62 kinds of mitochondrial genome sequences (Table 2) were downloaded from the NCBI database and the gene sequence was compared and analyzed using MitoPhAST (Ver. 3.0) (Fig. 2). .

As a result, 58% (36 species) of the mitochondria of 62 species of shrimp were analyzed by placing it as the main structure, which is a structure in which most of the gene sequence of the Shrimp species of Decagonal order are in common, as shown in FIG. ), and some shrimps have been confirmed to have undergone modifications such as genus-specific translocation and inversion.

However, it was confirmed that the sequence of mitochondrial gene sequence of prickly pear shrimp was the same as the main structures found in other shrimp species. [Table 2] and [Table 3] show the grouping according to the mitochondrial genome information and gene sequence patterns of 62 species of cruciferous shrimp.

[Table 2]

[Table 3]

In order to find the evolutionary relationship between the 62 species of prawns and prickly pear prawns, a phylogenetic tree was created based on the maximum likelihood analysis by aligning the protein sequences of 62 mitochondria ( Fig. 3).

As a result, prickly pear shrimp was genetically bound to a single clade with the suborder shrimps, and among the shrimps whose mitochondrial genome sequence was identified, the lobster shrimp belonging to the Pandalidae family ( Pandalus hypsinotus ), and two segments. It was classified closest to shrimp ( Chlorotocus crassicornis ).

In order to investigate the polymorphism of the mitochondrial genome of prickly pear shrimp, the full-length mitochondrial genomes of 34 species of shrimp in the G1 group classified as having a gene sequence corresponding to the major structure were aligned using the clustalW program.

As a result, spiny pear shrimp is NADH dehydrogenase subunit 3 (NAD3) two words from the 4480th base sequence corresponding to the lower part of the shrimps gene (Chlorotocus crassicornis ) It was confirmed that there was an insertion of 39 base-pairs (bp) compared to the mitochondrial sequence (see FIG. 4). In addition, this sequence was found to be a sequence that was not found in 61 species of other shrimps, and it was identified as a specific sequence for the species of prickly pear, so that the gene sequence of the insertion site of the mitochondrial genome of SEQ ID NO: 2 was changed to Lebbeus. Groenlandicus ) was selected as a genetic marker for species discrimination.

Example 3. Preparation of primers specific to species of thorny embryo shrimp and species identification using primers

A primer set of SEQ ID NO: 3 and SEQ ID NO: 4 capable of amplifying the gene sequence of the mitochondrial genome insertion site of SEQ ID NO: 2, which is a sequence specific to the species of prickly pear species, was prepared and shown in [Table 4].

[Table 4]

For comparative experiments, CO1F (LCO1490) and CO1R (HC02198) primers were used as controls. The CO1F (LCO1490) and CO1R (HC02198) primers are used for species identification by amplifying the mitochondrial COI gene (mitochondrial cytochrome c oxidase subunit I) of metazoans of the invertebrate phyla through PCR reaction. It is used as a general purpose primer used.

In order to determine whether the species of prickly pear shrimp can be identified using the mitochondrial genome insertion marker selected in Example 2, the genomic DNA of prickly pear shrimp was isolated and the mitochondrial genome was inserted ( insertion) marker-based primer set was used to perform PCR.

Specifically, in order to isolate genomic DNA for genotyping, the spiny lobster ( Lebbeus Groenlandicus ) was pulverized and then frozen and powdered by treatment with liquid nitrogen. 700 µl of microprep buffer was added to the prickly pear shrimp powder, followed by incubation at 65° C. for 15 minutes and mixed at intervals of 5 minutes. Then, 700 µl of chloroform: isoamyl alcohol (chloroform: isoamylalcohol = 24:1) was added, mixed for 1 minute, and centrifuged at 11,000 rpm for 15 minutes to obtain a supernatant. About 60% volume of isopropanol was added to the separated supernatant, mixed until the DNA was condensed, and then centrifuged at 13,000 rpm for 5 minutes, and the supernatant was removed to obtain a DNA pellet. After washing and drying the obtained DNA pellet with 70% ethanol, 50 µl of 1xTE buffer to which 50 mg/L of RNaseA was added was added and left at 37° C. for 120 minutes to remove RNA contained in the DNA pellet, Was obtained. Then, in order to use as a template in the amplification reaction, the concentration of the spiny lobster DNA was measured and diluted to about 100 ng/µl.

Then, in order to amplify the insertion (insertion) marker of the mitochondrial genome discovered in Example 2, PCR was performed using the primer set of SEQ ID NO: 3 and SEQ ID NO: 4 using the obtained prickly pear DNA as a template. I did.

For the comparative experiment, the dohwa shrimp ( Pandalus hypsinotus ) and barley shrimp (P enaeus japonicas ) extracted using the same method as the DNA extraction method were used as controls.

For PCR, a PCR reaction product was configured with the reaction product composition shown in the following [Table 5], and PCR was performed under the conditions of the following [Table 6] to obtain an amplified marker product.

[Table 5]

[Table 6]

Then, the amplified Indel marker product was loaded on a 2.5% agarose gel and subjected to electrophoresis at 100V for 50-60 minutes to confirm the amplification band.

As a result, as shown in FIG. 5, in the control group using the CO1F (LCO1490) and CO1R (HC02198) primers of the comparative example, only bands of the same size were detected in all three types of shrimp, so that the metazoans of the conventional invertebrate phyla ( metazoan), it was confirmed that it was impossible to distinguish between species among shrimp animals when using the primer set used for species identification.

On the other hand, when the gene sequence of the mitochondrial genome insertion site of SEQ ID NO: 2 of the present invention is amplified using the above primer set, thorny shrimp ( Lebbeus Groenlandicus ) it was confirmed that the specific band of 244bp length of SEQ ID NO: 5 was amplified only. Contrary to this, it was found that no band was formed in Pandalus hypsinotus and Penaeus japonicas .

Based on the results as described above, the thorny embryo shrimp ( Lebbeus) based on the gene sequence of the insertion site of the mitochondrial genome of the present invention Groenlandicus ) It is possible to easily distinguish Lebbeus Groenlandicus using a genetic marker for species identification, and it is possible to easily distinguish between other shrimp species and Lebbeus Groenlandicus by confirming the presence or absence of band formation. I could confirm.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto for those of ordinary skill in the art. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Genome Research Foundation <120> Genetic Marker Composition for Discrimination of Lebbeus Groenlandicus Species Originated Mitochondria Insertion Markers and Diagnosing Method using the Same <130> PN190065AN <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 17399 <212> DNA <213> Unknown <220> <223> Lebbeus Groenlandicus <400> 1 tagataacgt cacggtgatt gttttcaaca aatcacaaag atattggcac tttatatttt 60 atttttgggg cttgagcggg tatagtaggt accgccttaa ggcttctaat ccgagcagaa 120 ttaggacaac ccggaagtct cattggaaac gaccaaattt ataatgtaat tgtcacagca 180 catgccttcg ttataatttt ctttatagtt atacccatta taatcggagg gtttggtaat 240 tggttagttc cccttatatt aggagctcct gatatggcct tcccccgtat gaacaacata 300 agattctggc ttcttccacc ttctttaact ttacttctat caaggggaat ggttgaaagg 360 ggagttggaa caggatgaac tgtctaccct cctttggcgg caggtattgc ccatgccggt 420 gcttctgtag acatggggat tttctcacta cacttggcgg gggtttcttc aatcttagga 480 gcagttaatt tcataacaac agtcattaat atacgatcta ccggaatgac aatagatcgc 540 atgcctttat ttgtttggtc agtattctta acagcaatct tattgcttct ctcactacca 600 gttcttgcag gagcaattac aatactcctt acagaccgaa atttaaacac ctctttcttt 660 gaccctgctg gaggagggga ccctatcctt taccaacatt tattttggtt cttcggccac 720 ccagaagttt atatcttaat cctacctgcc tttggcatga tttcccatat tattagacag 780 gagtcaggga aaaaagaaac attcggcact ctaggtataa tttatgctat aatagcaatt 840 ggcatcctag gttttgttgt atgggctcat catatattca cagtaggaat ggatgttgac 900 acccgtgctt attttacatc tgcaactatg attattgccg ttccaacagg aattaaaatc 960 ttcagttggt taggaactct acatgggacc caattcacct atagcccatc ccttctatgg 1020 gcgcttgggt ttgtgttctt attcaccgta ggaggtttaa ctggggttgt tttagctaac 1080 tcctcattag atatcatcct tcacgatact tattatgtgg tggcccattt tcactatgtt 1140 ctctcaatag gcgctgtttt tggtattttt gctggtattg cacattgatt ccccttgttc 1200 acaggaatca caataaaccc taagtggctc aaaattcact tcataacaat atttattggg 1260 gtaaatatta cctttttccc ccaacacttt ttaggactga atggaatacc tcgacgttac 1320 tcagactacc ctgacgctta tatcacatga aatgtagtct cctctattgg gtcaaccatt 1380 tccctattag cagttcttgg ctttgttata attgtgtgag aggccttcgt tagtctacga 1440 ccagtaatat tcactcttgc ccttccaact tctattgaat gacaacaaaa ccttccaccg 1500 gccgaccata gttttataga aatcccaata attgcaaaag gatatctagg gtggcagatc 1560 agtgccctga gtttaagcct caactaggta attatttatt acccctggaa aatggcaaca 1620 tgatccgcac taggcttcca agatagagtg tctcctttaa tggaacaatt aattttcttt 1680 cacgaccacg ctatattagt gttaatttta attactacct tagtagggta tataataggc 1740 tctttatttt ttaactctct tacaaaccgc taccttttag agaatcaaac aatcgaaatt 1800 atttgaacaa ttctacccgc ttttatttta atttttattg ccctaccgtc cctgcgccta 1860 ctttaccttt tagatgaagt aaatacccca agggtaacct tgaaagccat cggccaccaa 1920 tgatattgaa ggtatgaata ctctgatttc cttcaagtag agtttgacgc ctatatagtt 1980 ccttcggcag acttaccaga ctcaggattc cggcttttag atgtagacaa ccgaaccgtc 2040 ttaccaataa acacccaaat ccgaaccctt atctctgcag cagatgtaat tcactcctga 2100 acagtacctg cattaggggt aaaagctgac gcgatccctg gccgactaaa ccaaatcaga 2160 ttttacataa accgggcggg gttatttttt ggtcaatgtt ctgaaatttg cggcgcaaat 2220 cacagattta tacctattgt tattgaaaga gtttcaatta atgctttctt agattgagtt 2280 gtagcaacta gagacgacgc cttatcagat gactgaagtg taagttcagg tcttttaacc 2340 ctgctatagt agaaacaact acttctgata accttaaaat tagttaaata ataacatagg 2400 cttgtcaagc ctaaattatc aaactttgat attttaaaca ctaatgcccc aaatagcccc 2460 ccttctttgg ttacctttat ttattttttt ctctgctatc tttttaatat ttactgcttt 2520 taactactct attaaaaccc caattaaaat ttctaatgta tcaagaacaa ccttaagcca 2580 caaattaacc tggaaatgat aactaattta ttttctagtt ttgaccctat gtcaagagtt 2640 ttcaacctct cacttaactg actctctgct cttttaggac taaggtttct accttattta 2700 tactgggcta tacaaacccg ctgatcccta ctgtgggcta ataccactat tgcaatccac 2760 aaagaattta aagttttatt aggcccgact gactcagggg cctctatctt atttgttagt 2820 ttatttagct ttatcgtatt taataatttt ttaggcttac taccttatgt tttcacaaga 2880 tctagccacc tcaccataac actagctctt gcccttcctt tatggttatc ttttataacc 2940 tacggaatta ttgtacacac ccaacatata ttagcccact tagtcccctc tgggaccccg 3000 ggcgccttaa taccttttat agttttaatt gaaacagtaa gtattataat ccgcccatga 3060 accctagccg tgcgattagc cgctaatata atcgcaggac acctcttact aaccctctta 3120 ggaaacactg gaacctctct ttcctttaca ctcctatccg tccttatttt agcccaaatt 3180 ctccttctaa tcttggaagc agccgttgct gttattcaat cctatgtgtt tgctgttctt 3240 aggactctct acgccagaga agttaactaa tgtcaacaca cggacaccac acataccact 3300 tagtagatat aagcccttga ccccttacag ggtctattag ggccataata ttaacaactg 3360 gattagttaa atgatttcac caatttaaca tagacctcct tattttaggg tttttagcca 3420 cattattaac aataattcaa tgatgacgcg atatcacacg agagggtacc taccaaggcc 3480 ttcacacaaa ccgtgtggtt ataggattac aatggggtat aatcttattt atcgcctctg 3540 aagtcttgtt tttcttctcc tttttttggg ccttcttcca tagaagctta gcccctacct 3600 cagagttggg gaattgctgg ccccctattg gaattcaacc ttttgacccc ttccaaattc 3660 cccttctaaa tacagccatt cttctagcct cgggagctac tgttacctgg gcccatcacg 3720 cccttctaga atcaaatcac acacaagccc tccaaaggtt agccttaaca gtaattctag 3780 gaatttattt ttcagcactt caagccgtag aatatattga ggctcccttc tctattgcag 3840 attctgttta tggagcaacc ttttttgtag ccacagggtt ccatgggtta cacgtgttaa 3900 ttggatcttc ttttttatta gtttgcctaa tccggatgtt tatgtgccat ttctcagaaa 3960 cccaccactt tggatttgaa gccgccgcat gatactggca ctttgttgat gtagtatgac 4020 tgttcttata tatttccatt tactgatgag gtggttaaaa agaaaatatt tttttagtat 4080 aattagtatt attgacttcc aatcaatagg cctggggttc ccagaaaaaa taattttttt 4140 acttataact ataactttaa tcatccttat attagctcta gtagtaataa taatttcctc 4200 tctcctctct aaaaaaataa ttacagaccg agaaaaaaac tcgccgtttg agtgtggatt 4260 tgacccgaaa ggttccgccc gtttgccatt ttccctccga ttttttttga ttgctgttat 4320 tttcttaatt tttgatgtag agatcacact tctcttaccc ttagcctcaa tttataatac 4380 gagtagaatc ttctcttgaa cagttacagg aatatttttt cttattattt tactagcggg 4440 gctttaccac gaatgaaacc aaggagccct taattgaaaa gagtaagcaa ctaaaatttc 4500 ctgctcaata ataacgacac tgttagaagc tatagtcaac aatgattcct gatttgcaat 4560 taggaggtgc tctattgagc tggcttcaag ccaagaagcg aacgtcgctt ttaggttcga 4620 tctaaactcc ggaggtcctt ctcccttggc tattaattaa agccaaaaag cggcctatca 4680 ctgttaatga taaaattgaa agcgtctttc taattaaaga gaaatgtaaa tgctacaaga 4740 aaaggtttct aaccttagct tttgcggcct aagccccgct ctttctccgt cattatagtg 4800 taaataacac actatacttt cattgtagag ataaagggac tccttttaaa gatacctagg 4860 ggcagaatgc cacttctaca gcttcaatgt aacgcccttt tattaaacta cccaagtagg 4920 acccttactt atagaaaact atccttctat aaaaaccatt aaagcaacta accatataac 4980 taaagaaatc atataaattt taaccctatt atcctgagct acctgcaaat aattagaaga 5040 tttaattaaa gttgaataag ccccctgacc accataaaac tcccttcacc ctatatcccc 5100 tacccccgaa taaaacttac caaccttcaa aatacttaaa ctaacccccc gtgtagaaat 5160 taagggtaaa aaccatatag accccctaaa tacaactaaa ctataataac gcagagattt 5220 taaagaataa ttcaccctta taatatttaa taaataaccc aataaagctc cagcccctct 5280 cactaataaa gctaataatt ttatactcct tggcaagcaa attatataag gggcgggaaa 5340 aataagtcaa gctaaagccg cgccgccaaa aaccgagcct cctcctaata ataatattgg 5400 ccaagttata acgggattgt cgtccctcac ccctctttcc gcactaagat taaaatctct 5460 cactaaccta taaaaaacca aacgaaaagt ataacacact gttaaaccag tggccactac 5520 taaaagccaa aagcaaacct cgtttaaggg agatgaaaag gcgacctcta aaattaaatc 5580 cttagaataa aaccctgcta aaaaaggtgt tccacataga gctaagttag caagattcac 5640 ccttgctact ctaagcggta taaagctaac agtacaccct atgacacgaa tatcttggta 5700 atctccaaca ctatggataa tagaacctgc acatataaat aaaagggcct taaagagagc 5760 atgagctaac aaatgaaaaa atgccaactc ggcccatcct atagctaaaa ttgttattat 5820 aactcctaat tgactaagtg tcgacaaagc aataattttt ttaagatccg tttcaaaatt 5880 agcacctaat ccagccataa atatagttaa cctagctaat aataggagac aactttgctc 5940 tgaagttccc tctaacgcag gactaaaccg aattaaaagg taaacccccg ccgtaacaag 6000 agtagaagag tgaactaatg ctgaaactgg agttggggca gctattgccg caggaagtca 6060 cgctgaaaaa ggaatttgag cccttttagt tatagcagct actacaataa aagctactaa 6120 ccccaattgg gcccccatcg cttccctccc aacataaaaa ataaaatttc aacccccaag 6180 actaaatatt aatgcaatac taagtaggat acaaacatct cccacccgat tggaaagtgc 6240 agttaacata cccgcgttag cagacttttc attttgataa tagataacca gtgcataaga 6300 aacaagccct aatccatctc agcccaaaag aatcctaatt atattaggac taataatcaa 6360 caaccctatt gataaaacaa aacctaaaac caagtatata aaacgattaa tattaggatc 6420 tctagccata taactccccc tataaaataa aactatagaa gagataaacc tcacaaaagc 6480 taaaaatatt atagaaaccc aatcaataat taaccttata cccaaataag acgaatttaa 6540 agttacaatt tctcactcaa taaagaaaga attgccggat aaaagcaaaa aaagggaaac 6600 ccttaagccc cttaaagcta ctatagctag gaaaactatt ctaataacat ttatttttaa 6660 accccataac actgttcaaa atactttatc catatatctt tggccccaca aaccaacatt 6720 tttattaaac tatttaaact aaattaaatt taaaaccaat cttcttttta aaattaatag 6780 atttaagggt agtcaatgaa gcaccaaaat taaaaactca cgcaccttac cagaacaaca 6840 cgaaaataaa gaattaacaa acttgccatg ttggcttatt gaatatatat ataaagtgta 6900 ggccgccctg aaaaaagata aagctgctac ccttaccaaa gcaaacttag atcaacaacc 6960 tactcttata ataagtctaa tctcacctaa aagattaagg gaaggagggg ctgccatatt 7020 acaagccctt aaaacaaatc accataaagc catcctaggt ataaaactaa gcaagccctt 7080 cctaatgaaa aggcttcgac tcccaatccg ttcataagct atattagcaa tacaaaaaag 7140 gccagaagaa caaagaccgt gcccaactat aacagaaact gctcctccta acccccatca 7200 cctaaacaat ataaggccac acaacactaa acctatgtga gcaacagagg aataagcaat 7260 taaagattta acatccgcct ggcgcatgca aattaaacta acaattgcac cccctgtggc 7320 accaaatcta atccaaactc aagtaaatct taaagactgt tctttaaata aaggcagcac 7380 ccgtaaaagc ccataacccc ctaactttaa taaaacacct gctaaaatca ttgaccccgc 7440 tacgggagct tctacatgag ccttaggcaa ccataaatga actataaaaa caggaagttt 7500 aacaatgaaa gcaaagaccg aacaaaaata tcaaattaac cttgtagaag ttgaagaggt 7560 taacccgtct accagcccaa taaccaaagt tcctccttgg cctttaaacc taagtaaaga 7620 aactaataag ggtagtgagg caaatagtgt ataaaataat atatacaccc ccgcttgaag 7680 acggtcgggt tggtaccccc aacctaaaat taaaataaaa gtaggaatta aagacctctc 7740 aaaacaaaca taaaataata aataatccat gcaagaaaat gttaaaacaa gacttaataa 7800 caaaagaaca tttaccttta aaaacaacct aaaatagttt cctaagtcat aaatcttctg 7860 tctccttcca actacaagag cggccaccca aaaacttaac aaaattaaga catacctaat 7920 cgagtcaatt ccaaatataa cccctaagcc cgcaatgtga gagccaactc ctgcaaataa 7980 actaaataaa accctaacta aaagcaacga acaaaaaaga gtagttcact ccctacaaat 8040 tataagtatc gataatacaa aaaaaacaaa ctttaacatt gaagtgctct aaaactatta 8100 aaattatcat tcccgtgtct gcgaaccact gaaacaagca aagctaaacc tagtgcccct 8160 tcgcaagccg ctaaagttaa aaaaaataaa acaaaaaaat tatctcctcc taaagtaaca 8220 atacttaaac ttataaccca aaaaactcta agtataataa actcaagcct taataaagaa 8280 cttaataaat gtttacgaac cctgacaaaa gaacccactc cacacaataa tataacaact 8340 ggaatggtgt attgaaataa aagaactaac attagcctta atagtttata taaaactccg 8400 gtcttgtaaa ccgaaaaaga aattattttt cttaaagcat cagaggagag aatgcccttc 8460 ccatctttag tctccaaaac taatattcta attaaactat cgccctgctc tgaaatttct 8520 cttacactta acataatatc tttgatctta accacctctc ttattttttc acaaataacg 8580 cagccccttt ccataggatt aataattgtg gttcaaacca tcttaattgc atttgttgcc 8640 ggagttggaa gtaactctac ttgattctca tatatcctat tcctagtttt cttaggagcg 8700 atgctcgtgt tgttcctcta tgttgcctcc ttagccccaa atgaagcctt tccaatctca 8760 tctactataa ttgcaatttt attaatcgga attctaagcc ccctcccact ttttctctta 8820 gatcaaatag ccgtcccact taaaataaca attgaaggaa cttcttttag agcagaacaa 8880 attataattt cagccaaata taaactaggg ctaatatata gcttaccttc tataaaccta 8940 acgatatata tcatcatata tttactacta acacttattg ttgtagtaaa aattacaaac 9000 accttttttg ggccacttcg tttaacctaa tgacaatacc cttacgtaaa tcccatcccc 9060 tcttcagaat tgctaacgga gccttagtag acctgccgac gccggcaaat atctcaatcc 9120 tttgaaactt tggatccctc ttagggctat gtttaatcac ccaaatcgcc acggggcttt 9180 tccttgctat acattacaca gcagatatta gactagcttt ttcaagtgtt gctcatattt 9240 gccgagacgt aaattatgga tgattacttc gaactttaca cgccaatgga gggtccttct 9300 tcttcatttg cctttttata cacattgggc gaggtattta ttatggctca tttatgttca 9360 tacatacttg aatagtcggt gtcgtaatcc tattcttaac tataggaact gcgttccttg 9420 gatatgtgtt accttgagga caaatatcct tctgaggagc aacagttatt actaacttag 9480 cctcagccat cccttatatc ggaactaatt tagtccagtg aatctgaggg gggtttgcag 9540 ttgataatgc cactcttaca cgctttttta cttttcactt tttattccct tttatagttg 9600 ctgcagcaac tatagtccat attctattcc tacaccaaac aggatctaat aacccacttg 9660 gaatttctag ccaagtagat aaaattccct tccaccctta cttctcgtat aaagatattg 9720 taggttttat tgtaataatc gcaggacttg ttattttaac acttttaaac ccttacctat 9780 taggagatcc cgataacttt atccctgcca atcctttagt aacaccggcc catatccaac 9840 cagaatgata ctttttattt gcttatgcca tcctacgatc catccccaat aaattaggag 9900 gagttattgc cttagtgctt tctgttgcta tcttgtttat ccttcctttt acccacaagg 9960 caaaattccg aagtcttgca ttttaccctt taaaccaatt cttattctga tccatagtag 10020 ttattgttat tttactaacc tgaattggag cccgacctgt tgaagacccc tatgttttaa 10080 ctgggcaaat tctaaccgtt ttatactttg cctattatat tgtcaaccct ctcagattaa 10140 ttttatggga taaaatacta gattgagaag ttaactttat ggaaagttta tgctttgaaa 10200 gcatactaaa agagttcaat tcccttactt cttccctaat tctaacttta ttacaaagtt 10260 atattaatat cacctccaat aaaaccttta ttcccatata aaatacacaa aaatttaagg 10320 ctacaggcaa aaaccttttt caagccaaat atattaactt atcataacga aagcgcggta 10380 gcgttccacg cacccataca aaactaaacg caacaaaaag ggcttttaaa taaaaaacga 10440 accttattaa acttccccct ataaaaataa cactaaaaag aacccttata aaaagaatac 10500 tagcatactc tgctataaaa ataagagcaa agccgcctgc cctgtactca gtattaaaac 10560 cagaaactaa ctctgattca ccttctgcaa aatcaaaagg agtccgatta gtctcagcta 10620 aacaagatgc aaatcaaaca agagccaagg ggcccataaa aactctgaac caaagatacc 10680 gctgataatc tctaaacaaa ctcagcctaa acccacccac taaaaataaa gtacccaata 10740 aaattaaagc caaccttacc tcataagaaa tagtttgagc tacagcacgt aaacacccaa 10800 gcaaggcata tttagagtta gaggaccaac ccgctcctat tgtagtgtac actcctaaac 10860 tagtacaaca taaaaaaaac aaaaccctta tcttaaaact taacaaacca aactcatagg 10920 gcattctaca ccaaactaat aaagctacaa atagcctaaa aacaggagat acataataag 10980 gaataaaatt agatattgta gggagagttt gctccttagt gaataatttt actgcgtctg 11040 aaaaaggctg aagaatccct ataaacccta ctttatttgg ccctttccgg atttgaatat 11100 aacctaaaat ttttcgctca agaagagtta aaaaagcgac tgccaccaac acaaaaataa 11160 ctaataaaat aaccctaact agcttagata actctataat aaactttaca aaagtttgtt 11220 atttatagaa cttgcctata tatttaggtc ctaagcctaa cgcactactc tgccaaaata 11280 acaaaattac tattcatcaa aatctaacag atttgctggc ccttttgatc ctttcgtact 11340 aaaaagggct tcttttacct gagagataga aaccaacctg gctcacaccg gtttgaactc 11400 aaatcatgta aagttttaat ggtcgaacag accaactaac taagctgcta cacttaaaag 11460 atactttaat tcaacatcga ggtcgcaact aatttcatcg ataggaactc tcagaaaaaa 11520 ttacgctgtt atccctaaag taacttaatc ttttaatctt tattaaagga tcaactaact 11580 aaataaaact attcctttca cttacagtta ttaaaaatta tataatagtc gccccaacct 11640 aacaataata catctcactc aatttaaaca aaaatttatg aagcagaaat aaattgtaaa 11700 gttttatagg gtcttatcgt cccctcagaa tatttaagcc ttttcactta aaagtaaaat 11760 tcaattttta aggaggagac agcttccttc tcgtgcagcc attcattcca gcctccaatt 11820 aagagactat tgattatgct accttcgcac ggtcagaata ccgcggccct ttagcacaca 11880 gccagtgggc agactaaatt gtttatataa accaacaaaa atgtttttga taaacagccg 11940 gaagtctaat ttgccgagtt aattctcctt aacaaaaata tattataatt taatcaaatt 12000 ttaattccat aaaacacaat aaacactcta ctaatacaac cactttaact atatttttta 12060 tattaaaaca cactttctct taacttaatt caaacaaata aaaatatata tttattcacc 12120 acattgatta taacaccctc taaaattagc taattttaag cctattccaa tgaaaatata 12180 acttatttaa ccttaaacta aactaactaa cgagctcatg ccccctaaaa tctcagccaa 12240 cctaactaac aattgacgct aaattaaatt tatttcagaa aaaattagat atagaaagac 12300 gactagcata tcactcctaa tctttaattt ctaatacctt taccacggta attagtttta 12360 aacattagct cctttccctt cgaaaataaa taaaatttta tagccaaata ggaagaccct 12420 gatacaaaag gtacaaatat ttctttttat taattcacaa agttccccta cagtactttt 12480 aacctttaac caacgacact aatttcaggc gacttactat taacatatta ttcccattac 12540 tctgaccaat ataacaattt aaccttatta atcaattatt tatcctttta gagcacgtcc 12600 ttattagacg atcacttcat tgtaagtgaa gcactttatt agctatcctc tatataaaat 12660 tccaggggcc ccttccggca cccctactat gttacgactt atctcatttt aagatgagag 12720 cgacgggcga tgtgtacata tcacagagct aaaatcaaat taccttaata aaataatatt 12780 acaattaaat ccaattttta aaagtatttc acccttttag ccaataactt aattaactgt 12840 aacccactta cactttttta taagctgcac cttgacctaa tatatacatt caataaataa 12900 ttccaatagt tttctctcga aacaatctaa taatggcggt atacaaactg atacacgaaa 12960 aaaagtaagg tatttcgggg attatcgttt atgaagcagg ttcctctaag aagaatgtga 13020 taccgccaag ttctttgggt ttcaagacag taactattac tacccaggag cttattaatt 13080 tcaaaatagt atagcagggt atctaatcct attttaaccc tatctcttac gggcttcaat 13140 gttaaacgaa atgctctatt ctacacaaat attaaatttc acttttttat ttttaaaata 13200 acacatcaat caataaacaa tcaccttacc ccacacctaa aacctcctct tatttaatca 13260 gtctaaccgc ggctgctggc acaagtttag cctcaattaa aaagttctct agttcaagct 13320 atcgcccatg tcccaaatct ttacgctgag atataataac cctaatttaa taaataacga 13380 agtttaagca cctacttaca tacgcaacta cctaacaaag gaaattatag ccagaatcaa 13440 actaatatat tatattaata atgggggggc tgccataatt aaaaacaatc aataataatt 13500 aaggtataca aatttaacta cttataacaa acaaatattt accgctgtaa aaaacaaccc 13560 aatctaaaaa aagggccaaa ctaatcccat ttcccccttc ttttccattt acgaccacct 13620 tttatcataa attaacttct ctgttgaaaa cgtatatttt taccccctca ctctaacttt 13680 aaccactgta ctaattttta ttttttatcc aaaaaagttg tgttgatatt ataattcttt 13740 tcgattttaa taatagtgat atgagactaa tttgttagtc tcatctcaca taaattaatt 13800 gtatacaatt aataargaat taatagttag gtaattagag caagatttgg aaacttttat 13860 aaattgagaa ataaggacat aaagcaagtg taagttttta tcgttaaaga gagattacac 13920 tatatgatga gaatataagg acaaaaaaga tcttataaat taagctatgg ggacaaaaaa 13980 gatcttataa attaagctat ggagacaaaa aagaccttat aaattaagct atgaaggaca 14040 aaaaagggat tataaattaa gctataggga cataaagcaa gtgtaagttc ttatcgttaa 14100 agagagatta cactatatga taagaaccct aacgactagg aagattttat aattaaagaa 14160 atgaagccat aaaacaagtg aaagttctta tcgttaaaga ctgatcacaa tatatactaa 14220 gaatataagg acaaaaaaga ttttataaac tgagctataa gggacaaaaa agatattata 14280 aattaagcta taaggacata aagcaagtgt aagtttttat cgttaaagag agattacact 14340 atatgatgag aatataagga caaaaaaggt tttataaatt aagatataga gacaaaaaag 14400 attttataaa ttaagctata aaggacaaaa aagggattat aaattaagct atggggacat 14460 aaagcaagtg taagttctta tcgctaaaga gagattacac tatatgataa gaaccctaac 14520 gaccaggaag gctttataat taaagaaatg aagccataaa acaagtgaaa gttcttatcg 14580 ttaaagactg attagaatat aggctaagaa taacccatca agttaattaa ataattataa 14640 ttgaaggtta tatatattaa attaaatgtc tacgatataa gtagtaaata gaccctttag 14700 gaaaattact tttattccgg aggaagtgta attttctgtt ttaaagggtc tatttactta 14760 tacaattcat attttaatta tactatacgt gtaatattaa agaatattta attttacaga 14820 aagggtatag atatataaac taaactttaa tattaaatat ttaattcaga cttaaataga 14880 tgtgtaaaat caatcaaaga gtataaatcg ataaatgtgt taggtcatta cttaaatatt 14940 aatcgttccc tttctagaat tcctttatgt ttcccattat gggaaacgga attctacctt 15000 taaagggaac tctaattcta gactaaaata actatttaca gagaattctg taaatagata 15060 ttaatctaaa ttttttataa ttacttaacc ttatataagc tatttcaaat taaagcttat 15120 aaatcgagaa caagattctt tatacacttt aaataattat aataaatcac gtttacgtgt 15180 ttgaaagata aaacactatc aacttctata aataactata aaaattagca attaattaaa 15240 aaaaaaatca tacatatatt gtatgtctct gacccccaca gaaaaaactc cgttttacta 15300 tgaatggtag tgtgcttgat aaaaagatcg ctttgataga gcggaatatg ggagatacct 15360 cctcactatt gaccatctta tcggaacctt ataatatgtg agttaagagc ttgccgtccc 15420 tgacgataaa atctaacgta attattcctt agatcaacta aaattggtct accataatta 15480 caaaaacccc tgggccacct cccagcaacc aatgtaatgt gagctaaaac tcaccgatta 15540 atgaataaat taacttaaaa ccccccccgt ggttaaacac gcaatccgaa aaataaagat 15600 ttgccgtgga aatcacaccc tggaacctta taatatgtga gttaagagct tgccgtccct 15660 gacgatttaa gctaactgta ctcttcccta aggcaactaa aattgccgtc cggaataaaa 15720 cctttagcta aatcccagca accaatgtag gtgagctaaa aatcaccgat tggtgatcaa 15780 attagcccac aataaccact taaagcaaat ccataaactt actgtaatag tcaacccaag 15840 caaatcacca aatcaataaa ttagttttaa cggcttacaa aacccaattt ttcactccga 15900 ttgactcaaa ctctgtataa aacaccttta acttacttaa taataaacgg ccttagccct 15960 gtcctatcaa tagctggcaa catagttaaa aaccttaggg gaccaaactt aacaataaat 16020 cttgaccctg ccattatgaa ttatgggatt ccaccatctc cttaaagatc aaaacttcac 16080 gtgctaaaca ccaaaacata aattacctgg ccaactaggc ctacgtgctg aaagataagc 16140 taaacttaag cttacgggtt cataccccgc ctatgaaatt attattctcc ttcaaatttt 16200 ttttctaaac ccagcccgta tcctcttcct aacaactctc acattagggg ccctgcttgc 16260 agccagaaga acctcttgat taaccgcctg gtttggatta gagttaaacc ttctttcatt 16320 catcccgcta atcgcgtcta cctctaacat ctactcctct gagagagcct taaaatattt 16380 cctaattcaa gcgctaggct ctgccttaat cttagctagg gcccctggaa ttttattgtt 16440 aaaaaattta actttaaccc caattatttt ggcgctttta attaaaatag gagccgcgcc 16500 cttccacttt tgatttccct ctgtaataca aggaatccaa tgaaaccaag cccttatttt 16560 aataacaatt caaaaaatag ccccattatt tgtattaacc tatgcccttt ccaggggaag 16620 aataatgccc tactttatcg tcaaaatatc ttctatcctc tcatcgattg ttggtagatt 16680 ggggggttta aaccaaacgc tccttcgtaa aattatagct tactcctcca ttaaccattt 16740 agcttggata ttagccgctc tgtctttcca aaaatcctta ttaacgcatt acttcgcagt 16800 ttatgttatt gtagtagcct cagttacctt tatttttata tcctaccaaa tttttcattt 16860 taaccaaata tttcaaattg ataattcaaa cacacttgta aaagtagggt tattttccac 16920 tttattctca ataggaggcc tgcctccctt tctaggattt atccctaaat taatagtagt 16980 gcaaggctta gctttgacag gacaaacact ctggctagct ttcctacttt taacagcagt 17040 ggtaacacta attttctacg tccgactcct tttaatagga atagttctaa cagccgtaaa 17100 accccgccct actctcacta aaaaagacta cctacccctg ctaattttaa cagctctcaa 17160 cctaactcct cttatcgccc ctttatcaat cttcatgcct ttttagggct ttaagttaaa 17220 aaaactacca gccttcaaag cttgcaacga ggatgttgcc cctcaggccc ttaagcttag 17280 gttaaaactc cttcagaatt gcagtctggt atgcacattt acactactaa gccgtcagag 17340 aaatataaat ttcgtctaca gatttacagt ctatcgccta accctcagcc acctaacac 17399 <210> 2 <211> 48 <212> DNA <213> Unknown <220> <223> Lebbeus Groenlandicus <400> 2 agagtaagca actaaaattt cctgctcaat aataacgaca ctgttaga 48 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5'PCR primer <400> 3 cccgtttgcc attttccctc 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 3'PCR primer <400> 4 agtgtcgtta ttattgagca gga 23 <210> 5 <211> 244 <212> DNA <213> Artificial Sequence <220> <223> PCR band sequence <400> 5 cccgtttgcc attttccctc cgattttttt tgattgctgt tattttctta atttttgatg 60 tagagatcac acttctctta cccttagcct caatttataa tacgagtaga atcttctctt 120 gaacagttac aggaatattt tttcttatta ttttactagc ggggctttac cacgaatgaa 180 accaaggagc ccttaattga aaagagtaag caactaaaat ttcctgctca ataataacga 240 cact 244

Claims (7)

Prickly pear shrimp consisting of the DNA sequence of SEQ ID NO: 2 ( Lebbeus Groenlandicus ) Gene marker composition for species identification. The method of claim 1,
DNA sequence of SEQ ID NO: 2, SEQ ID NO: 1, prickly pear shrimp (Lebbeus Groenlandicus) mitochondrial full-length genome of SEQ ID NAD3 (NADH dehydrogenase subunit 3) prickly pear shrimp (Lebbeus Groenlandicus, characterized in that the insert under 4480th bases of the gene of ) Genetic marker composition for species identification. A primer set for discriminating species of thorny shrimp ( Lebbeus Groenlandicus ) comprising a primer set capable of characteristically amplifying the DNA sequence of SEQ ID NO: 2 or a fragment thereof. The method of claim 3,
The primer set capable of characteristically amplifying the DNA sequence of SEQ ID NO: 2 or a fragment thereof is a primer set of SEQ ID NOs: 3 and 4 ( Lebbeus Groenlandicus ) Species identification primer set. Prickly pear shrimp according to claim 3 or 4 ( Lebbeus Groenlandicus ) Prickly pear shrimp ( Lebbeus Groenlandicus ) species identification kit containing a primer set for species identification. (a) separating DNA from the target shrimp sample;
(b) using the DNA isolated in step (a) as a template, and amplifying the target sequence using the primer set of claim 3 or 4; And
(c) When the target sequence is amplified, Prickly Pear ( Lebbeus Groenlandicus ) the step of determining the species; Prickly pear shrimp ( Lebbeus Groenlandicus ) containing the species determination method. The method of claim 6,
The target sequence, characterized in that consisting of the DNA sequence of SEQ ID NO: 2 Prickly pear shrimp ( Lebbeus Groenlandicus ) species identification method.

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