NL2031807B1 - Grass carp reovirus gcrv-wl1609 and use thereof - Google Patents

Abstract

The present disclosure belongs to the field of virus detection, and specifically sets forth a grass carp reovirus (GCRV) WLl609, RT—PCR detection primers, and use thereof. The inventors first isolate a low virulent strain of GCRV from grass carp, which is a 5 natural virulent strain with null genotype. The GCRV is deposited at CCTCC under accession number V201978. The virus has a 15 bp nucleotide deletion at l,043—l,057 bp of a coding region of S9 segment (encoding VP6 protein). According to a sequence of the virus, primers are designed as: Fl: 5'—GAGGCCATAGTCTCGGAAGC—3'; lO F2: 5'—GCGTACAATGCTGATGGACG—3'; and R: 5'-CCAGTACGTGTCAATAGCGT-3'. The primers are capable of detecting the GCRV, with excellent specificity and high sensitivity.

Description

P1346/NLpd

GRASS CARP REOVIRUS GCRV-WL1609 AND USE THEREOF

TECHNICAL FIELD

The present disclosure belongs to the field of molecular biological detection of fish viruses, and specifically relates to a grass carp reovirus (GCRV) WL1609, RT-PCR detection primers, and use thereof.

BACKGROUND ART

Grass carp hemorrhagic disease is a viral disease that does serious harm to healthy grass carp rearing, with a mortality of at least 90%. The economic loss caused by the disease can be up to over one billion yuan. The pathogen of the grass carp hemorrhagic disease is GCRV. At present, there are more than 50 GCRV isolates, which can be divided into three genotypes according to the gene sequence variation. Because GCRV is a segmented double-stranded

RNA virus and the genomic characteristics of the virus determine easy recombination among different segments, it is speculated that the attenuation mechanism of three disclosed low virulent strains of type II GCRV (GCRV-II) may be caused by fragment recombination.

However, the attenuation mechanism of this low virulent strain of

GCRV-II, GCRV-WL1609, isolated and identified by the applicants may be caused by fragment deletion. In nature, recombinant viruses are relatively universal, but deletion strains are relatively rare.

SUMMARY

An objective of the present disclosure is to provide a grass carp reovirus WL1609 (GCRV-WL1609). The virus has been submitted to and deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China on October 30, 2019 under accession number V201978, with a taxonomic name of GCRV-WL1609 strain.

Compared with the prior art, the present disclosure has the following advantages:

1. The method provided by the present disclosure is fast and convenient: a reverse transcription-polymerase chain reaction (RT-

PCR) assay constructed by the present disclosure requires only a few hours, and substantially improves detection efficiency compared with conventional viral isolation methods that require several days. 2. The method provided by the present disclosure is strongly specific: the method adopts a nested PCR assay using two primer pairs and two-round nucleic acid amplification, and a specific sequence region of a target sequence is recognized by the two primer pairs to guarantee high specificity of the PCR assay in amplification.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Example 1: culture and characteristics of GCRV-WL1609: 1) The applicants periodically detected healthy-looking grass carps according to degenerate primers designed by a conserved sequence at the S2 segment of GCRV, and finally a healthy-looking grass carp sample could be amplified to obtain a fragment. The liver, brain, spleen, and kidneys were eviscerated from the grass carp sample. The culture medium was diluted 100-fold and centrifuged at 2,000 rpm to collect a supernatant. The supernatant was filtered, inoculated into a single layer of grass carp liver cells L8824, and cultured at 25°C for 7 days. There was no significant cytopathy, namely, the virulent strain could not enable the grass carp liver cells to produce a cytopathic effect (CPE).

Culture medium: M199, 10% fetal bovine serum (FBS), pH 7.0- 7.2 2) Inoculated cells were collected and morphologically identified under transmission electron microscope (TEM). 3) Viruses were collected and subjected to whole genome sequencing. GCRV whole genome sequences S1 to S11 are shown in SEQ

ID NO: 1 to SEQ ID NO: 11, respectively, which are GCRV-II. It was found by whole-genome alignment (WGA) that the virulent strain belonged to GCRV-II; however, compared with known GCRV-II isolates, the virus had a 15 bp nucleotide deletion at 1,043-1,057 bp of a coding region of S9 segment (encoding VP6 protein) (where the VP6 protein encoded by the S9 segment had 5 amino acid deletions in the in the corresponding position), and other 10 genome segments were highly homologous to the GCRV-II (with a similarity of >98%).

Morphological characteristics of GCRV: Virion has a double- layered capsid without envelope, and is a 5:3:2 stereosymmetric regular icosahedron with a diameter of approximately 50-70 nm, which is similar to a typical GCRV particle. The virus has been submitted to and deposited at the China Center for Type Culture

Collection (CCTCC), Wuhan University, Wuhan, China on October 30, 2019 under accession number V201978, with a taxonomic name of

GCRV-WL1609 strain.

Example 2:

Primers designed based on the difference between the S9 segment (shown in SEQ ID NO: 9) of GCRV-WL1609 and the complete sequence of other virus:

The present disclosure exemplifies a common nested PCR assay.

The detection of GCRV-WL1609 can be completed based on other primers designed by the differential sequence, fluorescent quantitative primers, LAMP primers, digital PCR primers, and the like.

RT-PCR primers for a GCRV-WL1609 include: Fl: 5'-

GAGGCCATAGTCTCGGAAGC-3'; F2: L'-GCGTACAATGCTGATGGACG-3'; and R: 5'-CCAGTACGTGTCAATAGCGT-3"'.

Example 3:

The GCRV-WL1609 was detected by using PCR primers for GCRV-

WL1609 according to the following method: 1. Extraction of total RNA from grass carp tissue or infected cells and synthesis of cDNA: Total RNA was extracted from an unknown sample. The reverse transcription system was prepared as follows: 3 pL of total RNA, 1 pL of Oligo (dT)18 primer, and 8 uL of sterile double distilled water were mixed well at 65°C for 5 min and immediately placed on ice bath; 4 pL of 5xReaction Buffer, 1 pL of RNase Inhibitor, 2 pL of 5 mM dNTPs, and 1 pL of M-MLV

Reverse Transcriptase were added to the reaction mixture on ice bath, mixed well, and held at 42°C for 1 h and 95°C for 5 min; the resulting CDNA template was stored at -20°C for later use. 2. Nested PCR amplification: (1) First-round PCR amplification

A 25 |L reaction system was used: The first-round PCR primer combination was F1/R; 0.5 pL each of 20 uM upstream and downstream primers Fl and R were added to a PCR tube, respectively; 2 pL of 2.5 mM dNTPs, 1.5 pL of 25 mM MgCl.,, 0.5 pL of 5 U Tag DNA

Polymerase, 1 uL of template cDNA, and 2.5 pL of 10xThermoPol

Reaction Buffer were added, respectively, and two tubes of the reaction system were diluted to 25 pL with sterile double distilled water. The reaction program was as follows: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 48°C for 30 s, and extension at 72°C for 30 =; extension at 72°C for 10 min and storage at 4°C. (2) Second-round PCR amplification

The first-round PCR product was diluted 10- to 1,000-fold; 1 pL of diluted PCR product was used as a template for the second- round PCR amplification, and the primer combination for the second-round PCR amplification was F2/R; the reaction system and program were the same as those in the first-round PCR amplification.

Fl: 5'-GAGGCCATAGTCTCGGAAGC-3'; F2: 5'-GCGTACAATGCTGATGGACG- 3'; and R: 5'"-CCAGTACGTGTCAATAGCGT-3"'. 3. Analysis of nested PCR product:

After nested PCR amplification, 5 pL of final reaction product was added to 2% agarose gel containing 0.5 pg/mL ethidium bromide (EB) and run at a voltage of 100 V for 30 min, and the electrophoretogram of the PCR product was observed on a gel imaging system. If Fl and R can amplify a 477 bp characteristic band and F2 and R can amplify a 279 bp characteristic band, the sample will be positive; if there is no band, the sample will be negative.

Example 4:

Specificity test for RT-PCR primers for GCRV

Unknown virus samples in Table 1 were detected by using the method of Example 3, and a negative control group (double distilled water) and a positive control group (GCRV-WL1609) were set up. Specifically, the unknown virus samples were: type I grass carp reovirus 873 (GCRV-873), type II grass carp reovirus 106 (GCRV-106}, type III grass carp reovirus HGDRV, spring viremia of 5 carp virus (8VCV), and cyprinid herpesvirus 2 (CyHV-2).

Table 1 Specificity tests for GCRV RT-PCR assay kits

Test No. ~~ Name Amplified result

IO GCcRv-wnleoo + 2 GCRV-873 - 3 GCRV-106 - 4 HGDRV - 5 SVCV - 6 CyHV-2 -

The results are shown in Table 1. Characteristic bands of 477 bp and 279 bp were only detectable in the positive control group (GCRV-WL1609), and no characteristic band was detected in other groups, indicating that RT-PCR primers for GCRV-WL1609 had strong specificity.

Example 5:

Sensitivity of RT-PCR primers for GCRV

The cDNA template of GCRV-WL1609 with a concentration of 1,650 ng/uL was diluted 10-fold; the limit of detection (LOD) of

PRC was detected by using the primers provided by the present disclosure. The detection result showed that the target band could also be detected by a 10°-fold dilution, namely, at least 16.5 pg/uL cDNA template could be detected.

Example 6:

Use of RT-PCR primers for GCRV-WL1609 in preparing a GCRV assay kit: 1. Extraction of total RNA from grass carp tissue and synthesis of cDNA: Total RNA was extracted from liver, spleen, and kidney tissues of the GCRV-WL1609-infected grass carp. The reverse transcription system was prepared as follows: 3 uL of total RNA, 1 pL of Oligo (dT)18 primer, and 8 pL of sterile double distilled water were mixed well at 65°C for 5 min and immediately placed on ice bath; 4 uL of 5SxReaction Buffer, 1 uL of RNase Inhibitor, 2 uL of 5 mM dNTPs, and 1 pL of M-MLV Reverse Transcriptase were added to the reaction mixture on ice bath, mixed well, and held at 42°C for 1 h and 95°C for 5 min; the resulting cDNA template was stored at -20°C for later use. 2. Nested PCR amplification:

A 25 |L reaction system was used: 0.5 |L each of two primer pairs, F1/R or F2/R, with a concentration of 20 uM, were added to two PCR tubes, respectively; 2 pL of 2.5 mM dNTPs, 1.5 pL of 25 mM

MgCl:, 0.5 pL of 5 U Tag DNA Polymerase, 1 pL of template cDNA, and 2.5 pL of 10xThermoPol Reaction Buffer were added, respectively, and two tubes of the reaction system were diluted to 25 pL with sterile double distilled water. The reaction program was as follows: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 48°C for 30 s, and extension at 72°C for 30 s; extension at 72°C for 10 min and storage at 4°C.

Fl: 5'-GAGGCCATAGTCTCGGAAGC-3",

F2: 5'-GCGTACAATGCTGATGGACG-3', and

R: 5 '-CCAGTACGTGTCAATAGCGT-3T. 3. Analysis of nested PCR product:

After nested PCR amplification, 5 pL of final reaction product was added to 2% agarose gel containing 0.5 pg/mL ethidium bromide (EB) and run at a voltage of 100 V for 30 min, and the electrophoretogram of the PCR product was observed on a gel imaging system. If the electrophoregram shows characteristic bands of 477 bp and 279 bp, the result will be positive; if there is no band, the result will be negative.

The result illustrated that the positive control (GCRV-

WL1609) showed characteristic bands of 477 bp and 279 bp, while the negative control (water) showed no obvious band; the tissue sample of the GCRV-WL1609-infected grass carp also showed characteristic bands of 477 bp and 279 bp, indicating that the detected tissue sample was GCRV-WL1609-positive, which was in accordance with practical situation.

Example 7:

Infectivity experiment of GCRV-WL1609:

The GCRV-WL1609 strain cultured by grass carp liver cells was serially diluted to five concentration gradients in the range of

10% to 10° copies/uL with M199 medium, which were used for the infectivity experiment of grass carps (body length 10-15 cm), respectively. The infectivity experiment was divided into an experimental group and a control group. Each group contained 30 grass carps. In the experimental group, each grass carp was injected intraperitoneally with 0.1 mL of cytotoxin of different concentration gradients in the range of 10: to 10° copies/uL; in the control group, each grass carp was injected intraperitoneally with 0.1 mL of phosphate-buffered saline (PBS). During the experiment, the water temperature was controlled at 28°C, infection symptoms of fish bodies were observed, and deaths were counted. Experimental results showed that neither death nor obvious morbidity was observed in experimental grass carps injected with five concentration gradients in the range of 10° to 10° copies/uL and control grass carps injected with PBS, indicating that different concentrations of cytotoxin of GCRV-WL1609 had no pathogenicity to all grass carps. The preliminary study shows that the virulent strain may be a naturally mutant low virulent strain, which has potential as an attenuated vaccine for use in the immune control of grass carp hemorrhagic disease.

The virus has not been publicly reported yet, and there is no effective assay.

SEQUENCE LISTING

<110> Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences <120> GRASS CARP REOVIRUS GCRV-WL1609 AND USE THEREOF <130> HKJP20220400203 <160> 14 <170> Patentin version 3.5 <210> 1 «211» 3927 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of Si <400> 1 gtaattttgg gacaagatgg cattgtttgg attgagacta tcggagaaac ttaacgcacc 60 cacggaacaa caaattataa aacgaaagac atatggcgac atcatccgaa tattatccgg 120 caaaacatct gaacctggct ggcggatagt gcaacacgac aaaaagagcg gagttgggac 180 gattcgactt tacccaccat tcaatggatg gacaccccgt atattatctg gtgcgcaaga 240 ctggacaata tcgacatggg tccgatggct agagaatagg gcgcaaaagt tttgcgagta 300 tetactgtcg caacatacac tacagggata tgcttggcgc atagtgaacg ggatagttct 360 agctgacatg ctgggttctt tcttgaatgg tgagtcttgt cttgccctga tgecactget 420 actactggac aacgaggaca gcccattgct tggcagactt aagggtagga catttccgtg 480 gtcaacgacg atggaaggga cggcagatta cgtgctaaca gtagctggtg tcaaatacat 540 tegcgcacta cctgctatcg gagacagtat gggctacgag tggacgtgca cattcggcaa 600 acacattcct gcatatgcta aagtggctta ctatgatacg gaatttagtc gcaactccgec 660 catacgcgat cattcgacat cegtctegct gctcgagcat tatgatacac catcgatggc 720 accccacatc gtgatccecgt cacgcgtgte actagccact gggtttggca gcctaatcte 780 tgtggatgag ttattattgc ttgaaagcgc gatagacggg gcgatgcaga atgcaaatgc 840 tggtaggttt caggctgtat tgcgaaggga tgaaacgtac tgggtggtta actcgaagat 900 atcaacacgt gacttgagtt tgcagtctag acttcggcaa ctgggatgga tgatagccaa 960 cggtttctca tttgagcggc aatggccgac gcaggcaaca gagactatgg tcaaggaact 1020 gatcggtgat ttgatgggac ccggggagcc atttaacgcg atgactgctg gcatcagcta 1080 catacctcta ttcegcgatt ccccatcagt gatggacacg gtcaacggac gtcgctacaa 1140 gcggacacag atgaggagcg gtcgcccatc gggcagtaca cccgcgcaaa aggattccga 1200 cgtaccctta cgatggcaac cacaatggag cgatattagc actcaaacac tggatgatga 1260 cgatgattac cagcatgcgc tagtcaaggg gaaagtatac aaactaggcg teggcttcag 1320 cggtggcgca ttcattagga catggtctaa cagtagtggc ggaatggtga tccgtgatga 1380 cgacatgcct gagttgcctt tagaatattt cggtgtgcag caacagcaca gacggtctat 1440 attctcccaa ctgaggaaga cgggcgacaa atctttgctt aaggatgcgg cagtgctaca 1500 gttegtatca accgttatgg acccacatgc tggcgaacct gtactgaagg atgattttte 1560 tatgttgtac cttggggcat ctaatatcca tacctcttcc aacgagcccc tcattcttga 1620 tcggttgcgc aaaggcactg tccccggtgt acccggcaca acacacatca cgcagatggg 1680 atatgaagtg gtgcatggac cagtgataga tgctaggaag ccaataccaa cgggcacgta 1740 cacgctggtg tatagtgacg tggaccaaat tgtgaatggt ggtgaggaca tggtagcagc 1800 gacagagaca gctaaacgtg tcaccctcgt agcgctaagt tgcaccactc ttgggggcat 1860 taccgtcgtg aaaatcaatt tcccgaatcg acgctggtgg acagtgtttt tcaacacatt 1920 cagacaatct gcgcgcgact acgctctggt caagccgatg atcgtcaaca atgttgaggt 19880 gtacctggta atgatgcatc gaaccatgtc agggaactta accccgactg ctaacttaag 2040 gctattcctc ggtcaactga tgacgcgtta cagagagatt gatacggtca tgcaggccat 2100 tccgacccca aatcagtcag attcggacac ggcegttgatc ggtatgagca ccattcagct 2160 atttggcact gaagccgtcg tgaaaacgtg caatcggggt aaccgcgcta taaccgtatt 2220 ggcaaaccgt actggtggca cgcagttgac gttcggaatt gagaattttg gtggctctaa 2280 ctcacttacg ttgtgtggtce gccggtcact actatcagat agacgtctac ggagactaaa 2340 agagctacct atgacagacc tgtcctcact gagtttccaa catagaaccg ccccattcgt 2400 taccccatca ttcttttttg atcgtecage agacggtttc agtgtgcttg ctgcggggta 2460 caacgccatt caacgctcaa tggattggac tggtttgaaa gtactggacc taggaacggg 2520 ccccgaatgc cgatgcctat cactctttcc agcggataca cccgtcgaaa tgattgacat 2580 ccgtcccgcc gttgagcgaa tggacatttg ggaaactgat acaacttatc acgaggcgga 2640 cttcttgcta cccaccatat ggaacatgac aactgcgacc gcgtgtacag ctattttcte 2700 attaggaaat gcegctggtt tecgcaatac gcctctactt actgttctgc agaacgtcat 2760 ggacgaatgc aagcgaaagg gcattgagaa gatgttgttc caactgaact gccccctcac 2820 ctctaaacgt ctgggtcgtg attggttgca aattaatact aagaagaaaa ccttcacctt 2880 ccctacactt tcacgagtcg aaccgtacag cacggcagac gaggtggaga aagcgatcag 2940 aaataccttt cccaacgcag cctactcgtg gattacacta tctgacactc tggggtggat 3000 tgccacggcg actctggggg gaatgcagct agacatgagt attettgatg atgtattatt 3060 actgagcgag tacttacccc tgatgctgat agatacacgc acaacgggca ttaccgcaac 3120 aggccaactt gtggttaatc aacaaacaca gctcactatt cccgtccccg gtcgcgacgc 3180 tagcatcaga atgctgctgc aggacgtttt ggttgcagaa ggtggcggac tcaaattatg 3240 gtctcaggac ggaacgctga cccaacagta tcaggactca ccatcggcat atatatggac 3300 ttggactcca tccactgtgg gtgtgcattc cataaacgta ggtgacatag gacatgggca 3360 agttttatge ggcaccgtca atgttggage tccegatgca catacggtcg tgacttacgg 3420 agatacaatt gactataccg aggcaggaaa tgtcgcgacg atcgctgtgg attcatacta 3480 tcgcgtagac gtcttctatc atgatggcga ggagtataaa ctagtgaatc cctcccgggc 3540 ttgcgtgaag tacaataatg gagcaccgga gttaaacttc atcgcggact tctcrtaacac 3600 agcggtagtg atgtttcttt gcgatgtcac ggatggcegtt atcggccaac aagtgtttgt 3660 gagacttaat aagctacaca cgttacaatt ccctactacc gaaccactca ttatttctga 3720 cgtcttgcca aacgagtcat ggatcgtacg cgaatctggc accccaatat gcacactccc 3780 cttatctgcc ggtctgccac aggattggtc tgagtgcgac ctgaactacg ccgtccatgc 3840 atatggtcat tcctacatag ttccccctgg gtcgtatgag ttagtccgtg tggtctcata 3900 gatgctcatc accccatgct attcatc 3927

<210> 2 <211> 3867 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of S2 <400> 2 gtaacttttc tgatgatgga ccatgtgtac caaggcctct ctcaatcact acgcgatttc 60 atacccttte tcagaggcga gaaagacata aatgacaatg atttttcaca agcatcggca 120 gattggcacg gtcatcaacg gtcttatgct acatccctat tatccaaaat accgttcact 180 acgtgcatac gggtatccca tgaaatcttt gtcagacgcg actggcgaaa atattacaga 240 atagacgagt caggccgtgt tcgtcgagta cccgtaatcg atgacgaaga cgtctttgte 300 cccaatgccg atatccgtcc acttctgatt ccaatgaggt cgcagccaga ttacggcatc 360 ctacatccag atattcaatc aggcgcggac gcgggagttg ggagcgatcg tatggcagca 420 gcgttcttca agacagcgtc atcccaagta cggcagctaa agatggacgt cagtcggcca 480 ctactagcac tactactggc gtggtgtagt gaagggtcaa caagccgagt gattactgat 540 atctecacct ccagagacat agcaacatgt ccacctetac acgctctcca gcaggtattg 600 tcatattaca cgaaagacaa cgtaccactc ctgccaacca tgattgtcag gtcaggcccc 660 ctttggatag tgccgccgac gggcaagtcc atcccatcac atataccact gctgatcgcc 720 gatttggtca acttgtccat actgggttat gtgtgcaata ttgacgacga gttattgget 780 tgcggtgtgg aggtattcct agcagctget cgctctgagt cctactcaca tacactactc 840 gcatgcaagg ctttgtttcc cgcactcagt ttgcacgcgc tatacagaga tggcttcatt 900 ggcggtaagg tgccggtgat ggaatgggct gagccacgaa acaaatacac atttaaatgg 960 gtagggtcga gagtgattag cgcggatatc ctcaatatgc caccagttga ctatacagac 1020 gtggacagaa tatgtgaaag gtatggcctg gggattatca agacacgaat tggtaagcat 1080 gtatcggctt gcaatatgca caaccatgca agtatgaagt tagtacgtga cgtaatggct 1140 ttaacgtcgg gcacgttcat cgtceggtcc gctaccgaat ctgtcctaaa agagtatgcg 1200 caaccaccat tgatcaaaca acctattcga gaaagtgact ggtcaggtta tattggtaac 1260 gttegctact tgaaggcgac tgctcctggt ccggctcaat acctgtggtc gagatggcgc 1320 gacgcggcta tgcggttact ggcatcaccg agacttgacg accccctcte ccaagcaatc 1380 ttacgcactc aatatgtcac agccagaggt ggatcaggag cgaacatgcg agatgcattg 1440 atctcagctg gtgcgaaact gcccaactat gccggtgtgg gggtgaagat gtctacaaaa 1500 atcgtgcaag tcgcgcaaat cgctgatctg ccattcgagt tgcaagcagc gggtgtagat 1560 gcagccgtag ccatggggtc gcgcaatcaa gtgcaaagac ggaccagagt catcatgccg 1620 ttaagcccaa ttcagcagaa cgtctcagct atacacacca taaccgccga tgtgataaac 1680 aaggcgatga acctttcgac cacatcecggg tctgctgtgc atgagaaggt ggtgcctctg 1740 ttaatgtacg cttccatccc ccccaatcgt gtcataaaca tcgacatcaa ggcgtgcgac 1800 gcttccatca cgcccgacta cttcttaagc gtaatatgcg gtgctattca tcatggttta 1860 tcgaacgaag ccggatactc accattcatg ggcgtccctc ccgctgatgt aattgatgcc 1920 acgttcccaa cgaaccccgg taggagacgg attacaggta agcagaacat ggtacagcat 1980 trtggcacgtc tatacaagaa gccttttcaa tacgacgtaa atgatccatt ctcacccgga 2040 aacaaatttc aatttgacac cactgtcttt ccctctggtt ccacagcaac gtccacggag 2100 cataccgcta acaatagcac catgtttgac tacttcctaa ctcactacgt accacagaac 2160 gctcaatcac cgacgcttaa gcacatcgtg cgtggtatgt cgattcagcg gaactacgta 2220 tgtcaaggag atgatggcat atgtatattg gaccattatg gcgggaggcg cgtgagtaat 2280 gaggacataa acgagttcat caaactcctc atagactacg gtggactatt cggctggcgg 2340 tatgatatag acttccacgg taatgccgaa ttccttaaat tatgttgect acttgggtgt 2400 cgcgtaccaa atgctagtcg tcacgcacct gtcggccgtg agcatgcaac tgccgatgcg 2460 agcgaagcct ggceccggaat gatagacata gttatcggga tgtacgtcaa cggtattagc 2520 gacgcctatg actggcggaa gtggttacga ttcacttggg ccttactatg cttegtttcg 2580 cgagcattca tcaatcaact tgaccgacgc attaccgttc agttccccgt gtggactttc 2640 gtatacctag gagtcccacc tatatccgeg ttcggatcecg ggccatacat gttttccagc 2700 tacatgcctc gaggcgattt gggtatttac tcgttgatta ccttgcgtaa ggaatggatt 2760 atgcaacgct gcegtcgatct caattactca acaactcatg tgtcccgagc gttcggtgac 2820 tgcgatgtga gaaaattatt tgctgggttg ggaatatacg caggatacta cgctgctcag 2880 atgccgcgaa ccccaccacg acagcgtcag tctaccaccc ccgaagtgag agaggagttc 2940 atcggtgcte tgaaccgctt cctgttttat gacagggagc taacacgtcg tgtggaacgt 3000 ggacgcatgc tatgggataa gttrtgggcag acaatcgccg gcaaatacat aaggcgggtt 3060 ccttcactac gtgatgtgcc atcgaagtgg ttctctgacg cgcgagaagc ggacactgct 3120 acgcagggcg acatattccg tttagcatcg gatttagagc gccgcttcaa attgcaccca 3180 caacattttt ctgatttact cgaggcttac cttegtgteg agtgggatgc gggagacgtg 3240 gtaccaccag caattgaccc agatgttcct gtegtggctg gtatcgactt gacaaacgat 3300 gacctgttect tcaaattagt ctcagtcggg ccattgatgc agtetgtccg cegttacttt 3360 cagcaaacat tgtttgtggg caaaactgtc tccggtatgg atgttgagca tctcgacgcc 3420 acgctgctac gactgaaaat acttggcgeg ccacctgagg catttaaggg agttctgcaa 3480 atggctggct tttccgacac acaggcgaac gaaatctata gtaaagtcgt catcgctgac 3540 gcccgtttgg ttcagattgc ccgcgtegte aacctacacg tacctgacaa atggatgcte 3600 cttaattttg attacctatt gcgcgatgte atctcacatc agcaattgct cataactgat 3660 aggaactcta agttaccagc tcggcgtcca tggatgcgcg caatcctcaa atttcttggc 3720 gcaggcgtag tgatgacaca ggtaggcccg gtgcgataca tgtatgtcaa gcaaatcaag 3780 ggtggecggeg ccgcactttc aaacctctgec gagatgtgga tgcgaaaagt gtagtgatca 3840 ggectgtccc catcggaaat tttcatc 3867

<210> 3

<2ll> 3753 <212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S53

<400> 3 gtaatttttg tgccaatgca tcgtcataac agaacacgtg gtgaaaaacg tcatgaagac 60 cccaaagaca atgatactga aaaagagaca acgactaaga caaatacttc caagaaagag 120 acgtccactt ctaatgatga catggtgcca gttgattcgec gcaatcctaa catcgatcag 180 acgtcggtgg agattagcaa agagaaagct gaagctggtg acgatactag catacggccc 240 gacgaaagtc ctgaaaatac gaagacgcgt gctagtgctt cgacggccgt gagctcagat 300 ggcggactca aaaggcagga gtcatcaaca ggcgacccgc ccgttgtcaa tcctaaaaac 360 atcatgccta tagcgccccc aacagtgaag ccggcaccaa cgggacatgt ggcgaagtat 420 ctatgcaagg catgtccacc agagttcgac agcccggaca agccgcaaca tcatcaggct 480 ccggcgcaca gcgttgcgga tgctcaatct ttcacatcaa cggcagtcaa cgaggcgatt 540 gccacattca caaagtcttg ggacggtctg cgagctagtt ccagcagcga taacgttgea 600 atcatcaaag aatacgtgaa gtcgttatcc gacgagcagc cgctgctcat agtggaagat 660 gagggattga gctcctcttt catactgact gagatggtga acagtttacc catcgtgaac 720 gagattatag gatatcagtg gttcgaccga tcttatgaca ttccagcatc tctaccgacc 780 ggagcgatca atcgtatcgt atgtgtgacg gggtgggctg aatccgtatc tcattctcgt 840 ggcegcgaag tgcaaacgat gccaccgacg cacttgaacg tggacattta ccgacggaca 900 ccaagcgcgc agacaacgaa ctctcagcag tggaatccca atgctggcag agccaatgtt 960 ctaatcatgt tgctaaggct cgctctggcc aactgtcgca tcaacaaatc gacagctttt 1020 cgcaacgacg caaccgtcat gtccactggt agactactac gctcattcaa gaaggacgac 1080 ctactgttga aggcatatcc atctcgagtg ttgacagatc aggtgagcaa gaataatctg 1140 tttctgagac ggcagatcgc tgataggatc ggacgtgaca tgaaggccca ggtgatgatg 1200 gggatgatcg ctgcttacaa tgatgtctat gaaacatcag acacattgac catctacata 1260 cgcgaatgct acctactgct cctgcgccaa ttgcatctac caccgactca actgtcgata 1320 gccatcagcc aatgcgcaca cgggttcatc acgttcatca gcgcgtccgc agccaaccca 1380 atgatcatat gcccatggcg cgcaaccagt gaagataggc gtttggcagc agtgtttgac 1440 atcatgcgtc tgatcgatag ctcaagttcg atgctaccag tgctgcagga attagcgaat 1500 gcactacacg cttegtctct actgaaaata gaccccgagg tgattggcca ggccttacgc 1560 ggagtgagcg agtcagccgt gcagaatgtg tcaccggteg ctctgatcac aaaactctta 1620 cgaccaacag ggaatgactt catggcattc tgggttaccc tagcatcctg ggcgtataat 1680 ggcatctgta caaccgtgat agccgacgac agcttcccag ccattggctec gaatgtggtg 1740 acaatcgaag cgctcatgca agccatgatc gtggctatcg ctttgccgtt gacaactgac 1800 cctaatggac cgatgaaatg cttcatgctt gtagctaacc tattggcggc agatgagatt 1860 gtggagargg gagtgcgagg tctataccaa acaacgccag cccaggcatt taaccaccca 1920 acgttgtggt ctagagcttt catcgatccg actaccatcg acagacataa cagtccttgg 1980 ctgtacgcat gggcaacact gatacatcag ttctggccaa atccatcgca ggttaatttec 2040 ggagcccctg atatactggg ttcagcaaac ctcttcaccg ctccaggcgt attgcaccta 2100 ccactggaca gacgcccacc agacatgctg gctgtcaggc ctacgtatga cgctgaaatg 2160 ttegtgtgga tcaaggaggt catcgggttt ttcattcagc tgaccaatca gggtttegte 2220 acacgcacaa taggacgacc aacattggag tctacacgtg gtgtactaga gcagctacga 2280 cgtgtgccat caatgacccc cgcatggcta atgggcecttet tcccegagga gttagetgte 2340 ttagcgtcag tacaaaaggt gcagccaatc caaatcccat acgcgcgtac tgctatcaat 2400 gacattgtca ccaacctagg tgtctcgaga caggatccaa tcgacagggg tgatatgctt 2460 ggctacatgg cccagactgg aacgactatt ggtgtgacga tccctgtgag tgccgaggcg 2520 ttagtagtgt gtctacttga tggcgttgtg gaccctgaca ttgtacctac ccaacattac 2580 gcccgaatgt atgccgcatt gtttgaaggc aacgccatgt tcgcatcagc ccagcgtgce 2640 gttgtactac gtgaggccta tgtctgtgcce agagatgtga tatcccaatg cgctaccacg 2700 tcatttcctg ctaggcaaga tatgcagtgg atacgtcaac tgaatacgta tgctagtaca 2760 gcagcggaga tttgcaagga aatcgacgac gactttaaag aggcttttga catctcagga 2820 gatctgttgt ccccaattta taacgagtat gacccccgcg tatcccgatt aggcgtggct 2880 tatcgcacgt acggtggtga agttgtgtet gaagtgattg agccaggtgc ctcggctatt 2040 gacagagcct ttggaatggt gattgagact ttccaacatg agtataactt attcaactac 3000 gccaagggcg atatcatact tggacgcgtt gtgacaacgg ccatacataa ttccctcgac 3060 ccaccccctg gatatgtgtt tgatgccaca acacctggtg tccacgtcgc cggctctcge 3120 cttttattte aagcgcatat gggeggacgt cccgcaatgc tgctegacga acacgacatc 3180 gctcagagct tggatggaga ctggattatg actttgaaca ctctacaggc tggacgcgca 3240 ggtttcctca cccacatgtg gcctegtata gcctcaggta acacacgagt aagagtgatc 3300 atcggagctt tccctttegt cctgaactat catccecctgc aagaagacta cagcgcctat 3360 gaattgtcga ctgcctggtt agagaccatg actgtaggcg gcgttggacc cgtgccette 3420 gcattgacta tccccagtcc gtatgaggga ttcacaggca tcttccatta trtatatctgc 3480 gccactgaaa cgaacaccaa tggaatgttc tctaccaatg ctgcattccc cgccacacgc 3540 accggtacag acgtgccaat tgacgagtct cgatggggag ccctaattga ccctgcatac 3600 gaccccgatt tagtgcaact gccegtgaag attgacttgt attccaaaca tcgccgttac 3660 aactacgcgt atccaggtgt cgctgctttg ggaactatgg cgggagtaga gtaaccacgt 3720 tctggaccac gcacatgggg cactgtattc atc 3753

<210> 4 <2ll> 2263 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of 54 <400> 4 gtattttgag ctactcctgg caacatggeg atgecgtecgt cgtcggttgg ttcaagatcg 60 tttcgatatc aatacatgaa ctctccaact catggaaaat ctcaacgcag tctaccaaaa 120 ttgaaccttt ctgataccca gctaattcgc gatgtaccca aaatgctaac aagtggcagc 180 caaacttcaa aaattaccct gacactggge gacaagcagt tttctgacgt cctactgect 240 tcccgtcaaa aatggaatga tcacactgat ctgttgagca gatacatctc aatgcgcgct 300 gatggaacag agcgtgatgt gcactccttc caagaattgg tccatctaat ttcaaccaaa 360 atgaagcagg atggctttga ctggtcttcc aacatacccg aatttgctga ttatgttage 420 actttgactt ctaatgagcc tcagcctcta gacacgattt ccttgacctc ttcttteget 480 caactgcctt ctgagtctecc ggcacaatca gtcattgtta gtgagcgcte ctatcggcca 540 actcgacaac gtgctgctga ggtgaggcgt gagccccaat ctgatactag cgatagtgat 600 tcccgatgtc caccaggtcg cgagccccca tatggcaaag gccgcatgga tccggagacg 660 tataacatcg aaaggaaggc ctacctgcte ggtgacccat acgctgtccc cccacatgag 720 aactcaagcg cttacttcgc tgcattctgc ctcactcaat tgcgcgcaca cgccacgggc 780 tttttctcta attctgctge cggcacactg ctagccacta gagatgagca actcattcte 840 ctatctccct tgtacagcge cgtggagcat tctgaatctg gcgaatcctt ctatcacatc 900 tacgaccggg tgaagcattc gtctgaccca ctcgccacat tcacgattct gtacgtggac 960 cagctgctat ctgggacgac gaactcaggg cccttgetgc gcgcaataga tgggaaactg 1020 ctaatcaaca tcactcccac cgcactagtg tggctatgcg aacataatct gtctgatggc 1080 trtaatgtcct gtcaagcccg tcgcatcata ctcggagtcg accacttaat gtcacgactce 1140 gactegaccg ctgctttgct agcttgcctg ctaatggatc acaagctttc cttcctacgt 1200 cacgctccgce tcatggtgtt ccgcttcctg atgactgagc taacctcgac gcatcgtgtec 1260 gctatgctca aatcctttat cgctcacgcc gctagactcc ccaaccttca tgccttctat 1320 ggcecccgatc gcttcacacg gctaatagat ttcatgtcca tctetgcccc agcagcacca 1380 gatccaacac ccctgcatga tgctgaatca cacctggccg agatacagaa actcatggcc 1440 ggtatcaaac tgcagacttc atcctgtgat gcagccatac gcgacatcca actctctcga 1500 tegatgtttg agacagcgct tgccacttta cgcgacagat cctcegcttc tgccaatctt 1560 gacacttcte tgcttgacac atatctcaag gagcacacct gcattaatgt caaagagggt 1620 ccgectgectge ttacgctcgg catcgcgaac gataaagtca atggcatgct agcttcacga 1680 cacgcacgtt tggatgaatt caaggctcta tgctcatcca agtccggtaa tgctgacgca 1740 acgatggccc tgaaggcgca gatagacgaa ttgcggactg cgaacgagca cctgaccgct 1800 gagaacgtct cgacaattgg cgaactggcec ttggcgcata aggagatcat cagtaaagat 1860 gagaccattg ctgcattgag ggatgaaatg cgegctttag aaacaagagt cactgcctta 1920 actgctgagc atgagatgga ccagcatacc atcgcccgce tctcetcttt ggctaagctc 1980 gagcattctc ctaactactc ccaccaccgt cctccgccgc ccactttctc tgatctgacc 2040 gctcaaatgt ctggtgcatc cgctgccatc actcagtctg aacgttctct catgtccgcc 2100 tcatgcatcg gagcttctgc agctaatcta gacaaggtat ccatcaacga tttgttctac 2160 gactcaggcc attaatacct catttatgtc tctcgctcac tacgcttgat ctgccgccca 2220 ccgcgcacgt gacacttcgc atgccgcegec tcacgctttec atc 2263

<210> 5 <2ll> 2229 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of S5 <400> 5 gtaatttagc gatgttactc attctgccca cgtacgatgg agcgecgcgat gacgacttcc 60 tctccaccta caatatcgaa ttcacaagca aaatccactc cgatccaaac gtgcaggtgt 120 tagcaatgcc atggctgatc acacagctga ctcgattatt agacatcaac gacatcgcca 180 ctttgacccg ctgcctggac gaaatcacac cgcgacaccc cggggtattt gtettactec 240 ccaaatggcg atcattcgct cgcgtgtgcce tgactaattc tcaccttaat atatggaaaa 300 tacctaggtc cgttctaact gttacggcca atggtgaacg cacggagaag aagattagcg 360 attacattga cggtctggtg acatcatcgt cggacgtgtt gactggegtt aagcgtgtac 420 agaacggtgc cgtatgctac acgacgacgt taatggtatt aggcgcacca atacgaaaat 480 ggtgtcaaga cgacgcttac tctggggctt acgctegtgc cgacattaag gctttacaca 540 gacccaattg gccegtteceg cccaaagtaa caacccgaat tctgatcact aatctaccac 600 taggcgtgtc tgaaaacgct atcatgcteg accgcgctta ctggccacct gatgtgacgce 660 atggcaccct attattggag agatatgacg cgttegtgtc tactgccegc atatgcgact 720 tgactgcagt ggccatgttc cttggtgtcg aaaaacggct acctcgtaaa gtggatgagt 780 cataccgtag tgcacttcgc atgttaagac tgccatttecg tgatcacgcg caatcagtga 840 cgtctggtac cgccacgggg accatcacat gcgatatccg agtccgactg acgtgcaaaa 900 attcaaatag cccgaaggtg gctgcacgta ctgtgactat tctcagcgta ctaacatcat 960 tgctgtctat atacaatgtg gacatggacg tggactaccc attgcgacag gaaactggcg 1020 agatggtcga atggatactt atcgtcatca tgctgagtag tggactacgt actagcggag 1080 gccaggcagt gtgttgggat gcagcattta caatctcaac cccatttgac acgatcactg 1140 tgatgcatcg tagcccggat aagtataaaa tatcgccgte atatctatcc gacactgccc 1200 atgtgtatac aattgccatg gccaaggggt cctttaaatc caccattctt aaagccctat 1260 catcatggtt tggggacaaa cgtatatttg actctgctgc agctttcgat tcagatgacg 1320 cgggcaacac ggtatcaccc acctttgagg agctactatt gactgagtgg aggaagttag 1380 gtacggtttt gtgcgatgaa gccgcaatgg ccctggtgac aggaactgaa gggtgcgcga 1440 catccctagc ggatctgtac gatgcctttg tgactaccta tgcctccacc gtcactccag 1500 taatcagaga cacaataggt ccactgctgc gtegtgcaat atcctgcctc ttcgtacatt 1560 gegacactga ggtgcagaac gctaacatat ctacccatac cttccgatgc catatcccaa 1620 tggtgacgca gagcaatatc ttgatcagga ctggtecgctg cggaggcgtt actttccaag 1680 tcatcctgga ttactgctat cgatgtcteg ctgccggtgc cgacgaaatg tggtttggtg 1740 acacagtacg tcctttactg gatccttggt tacgtgettc ctcactggtt gggtatggat 1800 ggttggctgt aggtataggc gtcccaggta cgtcacttgc taaggctgga tggggagtat 1860 cgaccgacat catttaccga gccacatact gttatatata cgcaccggtc acgcggtttg 1920 atgcgataca gttcatggca gagcgccggt tccgaggtcg tatgaattta ggcccactcg 1980 cagcaacaga tcgacgggat gtgagcaaat atgaggttgg tttctcctgg ttgcctcaac 2040 ttegtcgtga tagctteggt ccggacgttt tgatgtccat ctegtctatc tcttatatcg 2100 atctttectgt cggatgtact gtgggtgagg agttaagcac gtegegccct ctcactacgt 2160 ggcctgaggt accacttcca catggggatt aatggattac cccatgccgt ggtatatcgc 2220 atattcatc 2229

<210> © <211> 2030 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of 356 <400> 6 gtaatttaag catagcgtgg agaccgactt acggccacta tcatgggaaa cgtccagacg 60 aacaacacta cagtcaatat caatggcgac ggtaatagct tcggtgccga aagcggcatg 120 cggtegacag ctgcgcctga aatcatgtta tcacccggaa aacttaaccc gaatggactc 180 gcatggatga aagtcgcagg atcaggtatg ggaccaggtt cactgcaaat tgtacacgca 240 acagacgggt caccgtattg ttacatccct ccggacgcca tgtctagtat ggaaaaatca 300 gcaggtgccg ttaatttgtg ggaacctcta ttcgccgcta tattcgaaac ctgctctaca 360 gtgaacgtgt ccgactttac tgatgagttc agtgcctacg ttggcgtcag tgaacccgag 420 gccctcaaga agtacatcga gaaaggagtc ttcatgtcaa catcacaagc gaaaaatttc 480 tttgggacac tcgggcagag gatggcgagg ataaagggtt ggtctgaaga tatacggacg 540 gcggctgcta tgataccagt agacacacct catggctcgg tcacgtgcga ttggaagagc 600 ttacttagat tttgggacga aaatctacca gtggataatg tatgcaaaca ataccaaaat 660 gctgtatatg aggtagcatt gaagatatac cctgcgttac gggccggaaa tagtggatca 720 ettctctctg atgtcgttgg cgtattagct ggtggtgecgg caggaactac agcgtctgga 780 gtgagtatgg tcgaaggtgt gacacgtaat gagcaagtgt ctcgaattct gacatcagca 840 gacgttgata ttttgacgtc gtcagcgcca gtgcctgtct cagctttgag aactccacca 900 gtggaattgt cctatcaacc cagcgccctg agtgccaaga ccaccccatg gttggttega 960 tatcccggga ctactgccat agagaagaca ttcgacgtcg ggacaacatc caagacgacg 1020 tattacctga gcatgggtaa ctccggtggt ggagatttga tgattgatct gaagcgactc 1080 ccagcatgcg gacttgaata ctccctgecgt ggtatcccga tcatatacga taccaatctg 1140 accgcggcga agctggctaa ggttactcct gcattgctga tgctgcagac ggctaaaccc 1200 cttagtgcgg agatcaccgc cgcagatatt caagctatca cacctcttgt agtagggact 1260 gataagctca acactttggt caccactggt tttggcaata ttcgcaacat taccgacttc 1320 tcaatgtctg caatttggga accagagaca gtcagcgcgg caggcaatta ctatctatgg 1380 ccgactgtaa tcggtgatgc atcaatgaca tcagactggg ggacaattag cacatctcta 1440 gctaatggca ggctccgtgt cgcacctctg gacctgacte atgcactcca caaaggcaat 1500 gtegtggagt caatcgtacc atctgatgtg ctaggtaatg cctcaccaga ggaaatgcgt 1560 teegcegttac cagcagatgt gctgacagct ttcaaagcca agctcacaac agtggcttct 1620 gtagttggcc gtgccttaaa ccccaacgac agtgcgcatg caccatcctec cggcaccgte 1680 cteggcccgc ttgcaattga aaacaaggcc caatcgaaac ctaaacccgt atcagatctg 1740 tggatagctg ctcggcgtgg tgtgaatcta ttcgtagcct ctccaatcgc gagtctgcaa 1800 acaggcgtgc cggtcatggg ggacagcagc gtgttgacaa gcttgatggg tggtgcaact 1860 acegcgttga atactggcga tatgggtacc ccaagcctgg aggccacagc gaaacgggca 1920 gctagagttg ctggaggact gctacgacag agagtcatgg acaaattaac tcgatactgg 1980 cctcctccgt cttaggtcgt gtacaccgac cccegctttg cattttcatc 2030

<210> 7

<211> 1604

<212> DNA <213> Artificial Sequence

<220>

<223> DNA sequence of S7

<400> 7 gtaatttaac tgccaagatg gccactcgtg acagccgcat gactacagag cacgttatcg 60 ccctaattct cgcatactcc acaactttag atgaagcaaa gatacacgaa gagattacga 120 atctgcagtc ggcagtggecg caactgacag cgcgattgac agagttgact gatgctgtga 180 ctagctttga tgcacgctta acccaagtag aacagcaggt tagtaaattyg tacgacaaga 240 ttgggatact atctaacagg ataggcgcca acaccgccag tatgaatttc ttggctggca 300 tagtggatgc gacgagaata gatgcagtgg atgtaccact atacatatat atagtcgatg 360 accagaggeg cctcgcaata gccacgggag aagggttatt tgtgaaagat cagaagttaa 420 acgggtacga tgtgcgtagc tttccaccta tagccgtcgc taaatataat aacatattat 480 cgttctcgct gagctcagcc cccccactgg acatagttga tggtaagttg gctgtcagca 540 taactagcag gctattcatc accagtggaa aattagacac taattcatat accggttcat 600 cgagtgtgga tgtatctgga gcgacggcag aaaagacggt gagcgttaga acgattaacc 660 ccatcattgc aggtcaggat ggcttgtcce tatcactaag cggtatcctg gaggtcggcg 720 ctggcttatt actatcaggt aggtcttgge cccttacatt ggatctacgt gtgagtaccc 780 cattagcata tgaccatacc ggaggattat ccgttgtaac acggtatccc ttagatgtga 840 agtctgctgg catgggtgtc aacatgcagg tgcccctacg gctaaacggt gtcgacttag 900 gattggcata caatacgcag gatttttcga tagttgacgg atatttgact ttgaacaggt 960 cccaacgaaa attggaggaa ctcggtgagg tggttgacat gaacgctact ctagtcgact 1020 taaatgacgg tggattgcag gttctagaga cagatctaag cttggagaag cctctraacg 1080 ctatgcaatg cctatttgag catgagggta catggcgttg aagtttggtg tgcgaatgtc 1140 cacggaaccg acgattacaa ctgtccgtgt taatgttcac agcacttggt ctgtctctgt 1200 ccaggaatca atagcaatct gcgcgataac caccactacg gctggagctg gtaaaggaca 1260 ggggccccgg gtgactattc ctttcagaag agctgctctc accgatgata tgatgcgtgc 1320 aggcttcgat gtgtggaaga gcgattatat cagggaggaa agcatttgta ggtacgttac 1380 gtggccteta ccgacgatgg tccgacaagc atctacttac ggaattctac gcacacggcc 1440 gtetctatcc aactaaggat ggcatcgagt tgattttaac acctaggtct acggatgacg 1500 agtatgagtg ggctggtctg cgtaatgtgc attggacggt agtttgctgt aagtaaagtg 1560 cgtgctggct ccagtgaggt accgcagttg gcagtatatt catc 1604 <210> 8

<211> 1556

<212> DNA

<213> Artificial Sequence <220>

<223> DNA sequence of S38

<400> 8 gtaatttaga cctttcctat cacccgcggt atcaaccagt ctttaacatg tatctggaac 60 tgttcatagc tatgatgtgg cacttcatgc tcgctttctg cgcgtatgtg tggtacatac 120 atggtcctac ccttegacgc ctctaccaac gcctgactgc tactactgct aacgctatct 180 gccaaacgga ctctacttac gtctgcactg caactgtttc caccatgaca gatgatctga 240 ctaacgctte ctettecgcc tccacctcca gtactgcttec ctceggctacg accagttttg 300 tagctctgcc taggctgteg cttgatgatt tgaagaccca ttccaaattc cgctggctgt 360 ggttttccaa Laacccattg accattgact tatccactga cgaactcaca actcccttca 420 atagtcctca gcttcatgat gtcttgttca tgcctcaata tactctcgca gcttatccet 480 atatgcatcg ctctctccat ttggcgcttt actttegctt cggctataac atgcgctacc 540 tgctacgcga acatcctggc attccaccgc ttegegtcac ctgcccatct acgcgtgtgc 600 tattcatgga caaacagcgc aataaatcgg aagagcctgt ctgttctatt tgtegcctgt 660 ggattgtgga cggccctcag cttcccatcg gtaactgtgg cagaaagtac aaagtccatg 720 aaatattcaa cgctttcagt ctcgagcctc accacattgt aaatgctttt gacgtcattc 780 caatcgtcte tctcctcaaa tgcctgcaac tggatgtttc tatgctggcc ggcttgtcte 840 tcaccgcact gacgcagctt tcagaattat caagagatcc agacgttaca tccgctggct 900 trtcttgcectgc tattcaattc gatggtcaca cttccaccgt ggattgccgt ggcgategtt 960 tgtatccaca tcctatgtta cttatcttgt ttgctttgtt ttgetaccgc cgccgttccec 1020 gtgtttattt ccgtgaatgc tacttgcgcg accaccgceg cttgacttca tccctacccg 1080 atatggttcc ctactcccgc accccaggca aggcaaaggc taagagcccg taaatgtaat 1140 ggcaaggtct agtgtaaata tgtatataaa tgttgaatat cgttacgctt tgcgatgctc 1200 agagtataat aaaaatgaaa aactacataa aaacttaaaa acaagatatg tgtataacca 1260 agctgtatat gttcgctgaa aaaccaaaaa gatcaagctt cgcaaaacta tgttgtttat 1320 cgctgtatat gtacgcataa aaaccaaaaa gaacaaatgt actcatcacg cttcatgtta 1380 atacactggc attccatatt ccagcgcagt cactgtaaag atgtataaca cggacgatat 1440 taacgccttc attgtatgtg cagcacagat gcaatagaga ggactgtttg atagcgggtg 1500 gcgtgttttt gacagcctgc gggtggcaat gcatgtacag agggtctatt tgcatc 1556 <210> 9 <211> 1284 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of S9 <400> 9 tatggagcga tccacttaca atatctgcac cccaggcttt ttcggcgcga atgttccgcec 60 ttttaaaaca atagacatac aacgatctac gacgggtggt aatacgcttt ggaatgcgcg 120 cggtcacgat gctttcagga ctcaccctaa agtggtatct catgagaagg atttccetet 180 aatttataca gagcagttca cattcaactt gcttattgge gcetgcctcc agcaacccct 240 actgcagaac tcgattgacc gacagtggag gggtatgatt tggacatctg atcggctcag 300 ctcgttgcgt atagtgccgc cgaattcacg tgctgctgat ctgcctcgtg catatcgtac 360 gttggacctt gcgaattacc cactatgggg aactacacgt gctgccctac agacactatg 420 gatggacagt tgtctgatga cattagagag tttatctgcg agaggacctt ttctttacct 480 ceggcatcct caagctcgac cagacgggaa tgttttacga gccttacggc agcatatttc 540 caagccaatg gaggccatag tctcggaagc ctaccaatca atagctatgg gacctttgac 600 attgcaaggc gtacaatgct gatggacgtc agtgaccacc ctcatctatc tagcctctct 660 gaccggtegt ctgggccctg accgtacata ctatggcttc tacgtccaat tccctaagaa 720 acggaaattc gaggatcteg gatactttgc gtacaatgct gatggacgta acgtcgctgt 780 cctgcaatcc attaacgcct acatctactg tgcctcacct gattggcagt acagctgtge 840 cctctactac ttgcatgtcc ttteggccct atcgcetctcc tggactgatc cagttggaat 900 gataaatggc ttctcgtgtg tcaatcaatt cacggacgtt cctggctggt caaccacaaa 960 ccgtgctttg cacacgcata gcttcaactg gttcaaccta ctggaggacg ctattgacac 1020 gtactggacc aatgccgagg gacaggccat gcggcaggaa tggacggcgg cgcgagacag 1080 gtggcgaatg atcatggacg caacccgtga tgaagatgac ttggtagttt ttcgtacgcc 1140 agacgattgt cgtagaaggc tcaaacctta tggggacaat aactggacgc gtgcttacga 1200 tactgccgat gtggtgcggg tgttggatcg cttattccct taggacctaa tccacccgac 1260 gggaggcaag actcgatttt catc 1284

<210> 10 <211> 1124 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of 510 <400> 10 gtaatttagc tctgtactat caacatggeg ggtgtgtcte tcaacatcaa tcgcaacatc 60 tcaaactcgg catcaacgat ctttctcgaa gatattccat tactgtcatg ttcagtgcgg 120 tgtgaacccyg gtaaaggacg cgaattacca aaatttaaca tgagctgccc tgctattaac 180 gcgatgggtc gatgtcttaa tccaatgaag ttcattgctg agcactgggt ccccaacagt 240 ccaagtcgta aaccatccag acagcattgg cgtaatgttt tgaatggact ggaattcagt 300 aatggtcgtg gattcgatgt gctgagttte tcaccagcgg gcatggctgt tcttegtgac 360 atcctgacag aagatagcgt gaaatattgc tttgacgaga gtaacacttg cagtttgtte 420 accttgctgt acactctgtg ttgcgatgcc gcaggcgtag aaccgatgga cttggattca 480 cgtcagacgg acgccagtgc ccggatggtg agctaccaag atcgcgctat cgtgctgacc 540 tctaatgagg caggagacag aattgagccg tggaatgttg agctcgacaa ggagtttgga 600 aatccagatc tgctcagccg tctgaacatc tcatatggcg tgcaacgata tggcgactcec 660 aaagccagca cagacactct gaccttgget gatgccccag agaggtccaa gcctgctctg 720 attactgtgc aacccttatt ggtggctatg tgcatcaaac agtctttgga tggcttgctg 780 gctttatctg atttgcgcct gagattcgat cagtatcctg gatacgcaaa tgctctcatg 840 aatgctatgg ccatgtacgce ttgcctagat cgtgacttga tgcgttttct gctccgctta 900 gaaatgactc acgcgagcac ggtgtctgaa gtggctgagt gctggaggaa ctctcgcaac 960 tctegcgatg caacaggttg tcatattgte ccacgtcaag gtttgctcat catcgtttcc 1020 ggagatgtcg aggtaagacg tatatttgcg cagatgctgt agacactgcc gtgtggtcgg 1080 cgataacggt actcatcgtc gactaaccca gagacgtatt catc 1124

<210> 11 <211> 1107 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of 511 <400> 11 gtaatttttg tgettgcacc aattatcggt aagtatggaa ccagcaaaac cattgacgtt 60 tetggatctec acccgactca atgaggcaat ctgctctcgg gectctettet ttgataatga 120 taacaactgc tggtctgtgt ctccaatcgc cccaaggcaa gccaaattcc atgactcagt 180 tgtctgtatt cgatgtggag cgcctattga caaagttcat gcaatgtcaa ttccaccacc 240 ccctgtgcat ggctgcatce caatgctgga tcatagccaa tgggaagatt tgtacgagtt 300 ggctgatgat atgggccgct gtatttggtg ggctaaaaag caactggtca tctggatgga 360 gggaatagtg aatctgaagg ctggtaaggt gtataatgat aatgtgagta accgcagtga 420 atggccagat gaggtgtggg atgaaacatg caaaatcttc tgtaaatggg caacgcaaaa 480 tecgtgtggcg agccgttgga tccagtcacc atcacgtgtg tacaagtttc ttcgtgacca 540 ggaaagtaag atgaacatcg atgctctgga gccatccaat catcagattt ttcaggcacc 600 accgcaatgg cctgagatag ctatggctgt cccgcactgg tccccgtccg tgcatgagat 660 gctaaatggt cacaaaatgg tgatgattgt gcceecgettg tccatgccag tcatatttga 720 tcccgccaac ggttacgtcg ccccaatcta caccgcggcc atgatcagtc tcccatccca 780 atggtgggtg tcacaatatg taaaagttca tggaagccac atggtgccac gtctgtatgg 840 tgatgacgtc ccaaccttac gttccecgcet gcggaatgct tetaccacaa cctcccactc 900 tcatacgcaa ctcctgatgc tgcctgaagc tcaatcgacc ttcaaacctg agatccaggg 960 acagtaaagg aaagtaagat gaacatcgat gctctggagc tatccaacca tcagattttt 1020 caggcacctg agatccaggg acagtaatga ctgattccat accacatgcg cagcgcgttt 1080 ggccgtaggt gagcacactt tttcatc 1107

<210> 12 <2l1> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer Fl <400> 12 gaggccatag tctcggaagc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer F2 <400> 13 gcgtacaatyg ctgatggacg 20

<210> 14 <2ll> 20 <212> DNA <213> Artificial Sequence

<220> <223> Primer R

<400> 14 ccagtacgtg tcaatagcgt 20

SEQUENCE LISTING

<110> Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences <120> GRASS CARP REOVIRUS GCRV-WL1609 AND USE THEREOF <130> HKJIP20220400203 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 3927 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of S1 <400> 1 gtaattttgg gacaagatgg cattgtttgg attgagacta tcggagaaac ttaacgcacc 60 cacggaacaa caaattataa aacgaaagac atatggcgac atcatccgaa tattatccgg 120 caaaacatct gaacctggct ggcggatagt gcaacacgac aaaaagagcg gagttgggac 180 gattcgactt tacccaccat tcaatggatg gacaccccgt atattatctg gtgcgcaaga 240 ctggacaata tcgacatggg tccgatggct agagaatagg gcgcaaaagt tttgcgagta 300 tctactgtcg caacatacac tacagggata tgcttggcgc atagtgaacg ggatagttct 360 agctgacatg ctgggttctt tcttgaatgg tgagtcttgt cttgccctga tgccactgct 420 actactggac aacgaggaca gcccattgct tggcagactt aagggtagga catttccgtg 480 gtcaacgacg atggaaggga cggcagatta cgtgctaaca gtagctggtg tcaaatacat 540 tcgcgcacta cctgctatcg gagacagtat gggctacgag tggacgtgca cattcggcaa 600 acacattcct gcatatgcta aagtggctta ctatgatacg gaatttagtc gcaactccgc 660 catacgcgat cattcgacat ccgtctcgct gctcgagcat tatgatacac catcgatggc 720 accccacatc gtgatcccgt cacgcgtgtc actagccact gggtttggca gcctaatctc 780 tgtggatgag ttattattgc ttgaaagcgc gatagacggg gcgatgcaga atgcaaatgc 840 tggtaggttt caggctgtat tgcgaaggga tgaaacgtac tgggtggtta actcgaagat 900 atcaacacgt gacttgagtt tgcagtctag acttcggcaa ctgggatgga tgatagccaa 960 cggtttctca tttgagcggc aatggccgac gcaggcaaca gagactatgg tcaaggaact 1020 gatcggtgat ttgatgggac ccggggagcc atttaacgcg atgactgctg gcatcagcta 1080 catacctcta ttccgcgatt ccccatcagt gatggacacg gtcaacggac gtcgctacaa 1140 gcggacacag atgaggagcg gtcgcccatc gggcagtaca cccgcgcaaa aggattccga 1200 cgtaccctta cgatggcaac cacaatggag cgatattagc actcaaacac tggatgatga 1260 cgatgattac cagcatgcgc tagtcaaggg gaaagtatac aaactaggcg tcggcttcag 1320 cggtggcgca ttcattagga catggtctaa cagtagtggc ggaatggtga tccgtgatga 1380 cgacatgcct gagttgcctt tagaatattt cggtgtgcag caacagcaca gacggtctat 14409 attctcccaa ctgaggaaga cgggcgacaa atctttgctt aaggatgcgg cagtgctaca 1500 gttcgtatca accgttatgg acccacatgc tggcgaacct gtactgaagg atgatttttc 1560 tatgttgtac cttggggcat ctaatatcca tacctcttcc aacgagcccc tcattcttga 1620 tcggttgcgc aaaggcactg tccccggtgt acccggcaca acacacatca cgcagatggg 1680 atatgaagtg gtgcatggac cagtgataga tgctaggaag ccaataccaa cgggcacgta 1740 cacgctggtg tatagtgacg tggaccaaat tgtgaatggt ggtgaggaca tggtagcagc 1800 gacagagaca gctaaacgtg tcaccctcgt agcgctaagt tgcaccactc ttgggggcat 1860 taccgtcgtg aaaatcaatt tcccgaatcg acgctggtgg acagtgtttt tcaacacatt 1920 cagacaatct gcgcgcgact acgctctggt caagccgatg atcgtcaaca atgttgaggt 1980 gtacctggta atgatgcatc gaaccatgtc agggaactta accccgactg ctaacttaag 2040 gctattcctc ggtcaactga tgacgcgtta cagagagatt gatacggtca tgcaggccat 2100 tccgacccca aatcagtcag attcggacac ggcgttgatc ggtatgagca ccattcagct 2160 atttggcact gaagccgtcg tgaaaacgtg caatcggggt aaccgcgcta taaccgtatt 2220 ggcaaaccgt actggtggca cgcagttgac gttcggaatt gagaattttg gtggctctaa 2280 ctcacttacg ttgtgtggtc gccggtcact actatcagat agacgtctac ggagactaaa 2340 agagctacct atgacagacc tgtcctcact gagtttccaa catagaaccg ccccattcgt 2400 taccccatca ttcttttttg atcgtccagc agacggtttc agtgtgcttg ctgcggggta 2460 caacgccatt caacgctcaa tggattggac tggtttgaaa gtactggacc taggaacggg 2520 ccccgaatgc cgatgcctat cactctttcc agcggataca cccgtcgaaa tgattgacat 2580 ccgtcccgcc gttgagcgaa tggacatttg ggaaactgat acaacttatc acgaggcgga 2640 cttcttgcta cccaccatat ggaacatgac aactgcgacc gcgtgtacag ctattttctc 2700 attaggaaat gccgctggtt tccgcaatac gcctctactt actgttctgc agaacgtcat 2760 ggacgaatgc aagcgaaagg gcattgagaa gatgttgttc caactgaact gccccctcac 2820 ctctaaacgt ctgggtcgtg attggttgca aattaatact aagaagaaaa ccttcacctt 2880 ccctacactt tcacgagtcg aaccgtacag cacggcagac gaggtggaga aagcgatcag 2940 aaataccttt cccaacgcag cctactcgtg gattacacta tctgacactc tggggtggat 3000 tgccacggcg actctggggg gaatgcagct agacatgagt attcttgatg atgtattatt 3060 actgagcgag tacttacccc tgatgctgat agatacacgc acaacgggca ttaccgcaac 3120 aggccaactt gtggttaatc aacaaacaca gctcactatt cccgtccccg gtcgcgacgc 3180 tagcatcaga atgctgctgc aggacgtttt ggttgcagaa ggtggcggac tcaaattatg 3240 gtctcaggac ggaacgctga cccaacagta tcaggactca ccatcggcat atatatggac 3300 ttggactcca tccactgtgg gtgtgcattc cataaacgta ggtgacatag gacatgggca 3360 agttttatgc ggcaccgtca atgttggagc tcccgatgca catacggtcg tgacttacgg 3420 agatacaatt gactataccg aggcaggaaa tgtcgcgacg atcgctgtgg attcatacta 3480 tcgcgtagac gtcttctatc atgatggcga ggagtataaa ctagtgaatc cctcccgggc 3540 ttgcgtgaag tacaataatg gagcaccgga gttaaacttc atcgcggact tctctaacac 3600 agcggtagtg atgtttcttt gcgatgtcac ggatggcgtt atcggccaac aagtgtttgt 3660 gagacttaat aagctacaca cgttacaatt ccctactacc gaaccactca ttatttctga 3720 cgtcttgcca aacgagtcat ggatcgtacg cgaatctggc accccaatat gcacactccc 3780 cttatctgcc ggtctgccac aggattggtc tgagtgcgac ctgaactacg ccgtccatgc 3840 atatggtcat tcctacatag ttccccctgg gtcgtatgag ttagtccgtg tggtctcata 3900 gatgctcatc accccatgct attcatc 3927 <210> 2

<211> 3867

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S2

<400> 2 gtaacttttc tgatgatgga ccatgtgtac caaggcctct ctcaatcact acgcgatttc 60 ataccctttc tcagaggcga gaaagacata aatgacaatg atttttcaca agcatcggca 120 gattggcacg gtcatcaacg gtcttatgct acatccctat tatccaaaat accgttcact 180 acgtgcatac gggtatccca tgaaatcttt gtcagacgcg actggcgaaa atattacaga 240 atagacgagt caggccgtgt tcgtcgagta cccgtaatcg atgacgaaga cgtctttgtc 300 cccaatgccg atatccgtcc acttctgatt ccaatgaggt cgcagccaga ttacggcatc 360 ctacatccag atattcaatc aggcgcggac gcgggagttg ggagcgatcg tatggcagca 420 gcgttcttca agacagcgtc atcccaagta cggcagctaa agatggacgt cagtcggcca 480 ctactagcac tactactggc gtggtgtagt gaagggtcaa caagccgagt gattactgat 540 atctccacct ccagagacat agcaacatgt ccacctctac acgctctcca gcaggtattg 600 tcatattaca cgaaagacaa cgtaccactc ctgccaacca tgattgtcag gtcaggcccc 660 ctttggatag tgccgccgac gggcaagtcc atcccatcac atataccact gctgatcgcc 720 gatttggtca acttgtccat actgggttat gtgtgcaata ttgacgacga gttattggct 780 tgcggtgtgg aggtattcct agcagctgct cgctctgagt cctactcaca tacactactc 840 gcatgcaagg ctttgtttcc cgcactcagt ttgcacgcgc tatacagaga tggcttcatt 900 ggcggtaagg tgccggtgat ggaatgggct gagccacgaa acaaatacac atttaaatgg 960 gtagggtcga gagtgattag cgcggatatc ctcaatatgc caccagttga ctatacagac 1020 gtggacagaa tatgtgaaag gtatggcctg gggattatca agacacgaat tggtaagcat 1080 gtatcggctt gcaatatgca caaccatgca agtatgaagt tagtacgtga cgtaatggct 1140 ttaacgtcgg gcacgttcat cgtccggtcc gctaccgaat ctgtcctaaa agagtatgcg 1200 caaccaccat tgatcaaaca acctattcga gaaagtgact ggtcaggtta tattggtaac 1260 gttcgctact tgaaggcgac tgctcctggt ccggctcaat acctgtggtc gagatggcgc 1320 gacgcggcta tgcggttact ggcatcaccg agacttgacg accccctctc ccaagcaatc 1380 ttacgcactc aatatgtcac agccagaggt ggatcaggag cgaacatgcg agatgcattg 14409 atctcagctg gtgcgaaact gcccaactat gccggtgtgg gggtgaagat gtctacaaaa 1500 atcgtgcaag tcgcgcaaat cgctgatctg ccattcgagt tgcaagcagc gggtgtagat 1560 gcagccgtag ccatggggtc gcgcaatcaa gtgcaaagac ggaccagagt catcatgccg 1620 ttaagcccaa ttcagcagaa cgtctcagct atacacacca taaccgccga tgtgataaac 1680 aaggcgatga acctttcgac cacatccggg tctgctgtgc atgagaaggt ggtgcctctg 1740 ttaatgtacg cttccatccc ccccaatcgt gtcataaaca tcgacatcaa ggcgtgcgac 1800 gcttccatca cgcccgacta cttcttaagc gtaatatgcg gtgctattca tcatggttta 1860 tcgaacgaag ccggatactc accattcatg ggcgtccctc ccgctgatgt aattgatgcc 1920 acgttcccaa cgaaccccgg taggagacgg attacaggta agcagaacat ggtacagcat 1980 ttggcacgtc tatacaagaa gccttttcaa tacgacgtaa atgatccatt ctcacccgga 2040 aacaaatttc aatttgacac cactgtcttt ccctctggtt ccacagcaac gtccacggag 2100 cataccgcta acaatagcac catgtttgac tacttcctaa ctcactacgt accacagaac 2160 gctcaatcac cgacgcttaa gcacatcgtg cgtggtatgt cgattcagcg gaactacgta 2220 tgtcaaggag atgatggcat atgtatattg gaccattatg gcgggaggcg cgtgagtaat 2280 gaggacataa acgagttcat caaactcctc atagactacg gtggactatt cggctggcgg 2340 tatgatatag acttccacgg taatgccgaa ttccttaaat tatgttgcct acttgggtgt 2400 cgcgtaccaa atgctagtcg tcacgcacct gtcggccgtg agcatgcaac tgccgatgcg 2460 agcgaagcct ggcccggaat gatagacata gttatcggga tgtacgtcaa cggtattagc 2520 gacgcctatg actggcggaa gtggttacga ttcacttggg ccttactatg cttcgtttcg 2580 cgagcattca tcaatcaact tgaccgacgc attaccgttc agttccccgt gtggactttc 2640 gtatacctag gagtcccacc tatatccgcg ttcggatccg ggccatacat gttttccagc 2700 tacatgcctc gaggcgattt gggtatttac tcgttgatta ccttgcgtaa ggaatggatt 2760 atgcaacgct gcgtcgatct caattactca acaactcatg tgtcccgagc gttcggtgac 2820 tgcgatgtga gaaaattatt tgctgggttg ggaatatacg caggatacta cgctgctcag 2880 atgccgcgaa ccccaccacg acagcgtcag tctaccaccc ccgaagtgag agaggagttc 2940 atcggtgctc tgaaccgctt cctgttttat gacagggagc taacacgtcg tgtggaacgt 3000 ggacgcatgc tatgggataa gtttgggcag acaatcgccg gcaaatacat aaggcgggtt 3060 ccttcactac gtgatgtgcc atcgaagtgg ttctctgacg cgcgagaagc ggacactgct 3120 acgcagggcg acatattccg tttagcatcg gatttagagc gccgcttcaa attgcaccca 3180 caacattttt ctgatttact cgaggcttac cttcgtgtcg agtgggatgc gggagacgtg 3240 gtaccaccag caattgaccc agatgttcct gtcgtggctg gtatcgactt gacaaacgat 3300 gacctgttct tcaaattagt ctcagtcggg ccattgatgc agtctgtccg ccgttacttt 3360 cagcaaacat tgtttgtggg caaaactgtc tccggtatgg atgttgagca tctcgacgcc 3420 acgctgctac gactgaaaat acttggcgcg ccacctgagg catttaaggg agttctgcaa 3480 atggctggct tttccgacac acaggcgaac gaaatctata gtaaagtcgt catcgctgac 3540 gcccgtttgg ttcagattgc ccgcgtcgtc aacctacacg tacctgacaa atggatgctc 3600 cttaattttg attacctatt gcgcgatgtc atctcacatc agcaattgct cataactgat 3660 aggaactcta agttaccagc tcggcgtcca tggatgcgcg caatcctcaa atttcttggc 3720 gcaggcgtag tgatgacaca ggtaggcccg gtgcgataca tgtatgtcaa gcaaatcaag 3780 getggcggcg ccgcactttc aaacctctgc gagatgtgga tgcgaaaagt gtagtgatca 3840 ggcctgtccc catcggaaat tttcatc 3867 <210> 3

<211> 3753

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S3

<400> 3 gtaatttttg tgccaatgca tcgtcataac agaacacgtg gtgaaaaacg tcatgaagac 60 cccaaagaca atgatactga aaaagagaca acgactaaga caaatacttc caagaaagag 120 acgtccactt ctaatgatga catggtgcca gttgattcgc gcaatcctaa catcgatcag 180 acgtcggtgg agattagcaa agagaaagct gaagctggtg acgatactag catacggccc 240 gacgaaagtc ctgaaaatac gaagacgcgt gctagtgctt cgacggccgt gagctcagat 300 ggcggactca aaaggcagga gtcatcaaca ggcgacccgc ccgttgtcaa tcctaaaaac 360 atcatgccta tagcgccccc aacagtgaag ccggcaccaa cgggacatgt ggcgaagtat 420 ctatgcaagg catgtccacc agagttcgac agcccggaca agccgcaaca tcatcaggct 480 ccggcgcaca gcgttgcgga tgctcaatct ttcacatcaa cggcagtcaa cgaggcgatt 540 gccacattca caaagtcttg ggacggtctg cgagctagtt ccagcagcga taacgttgca 600 atcatcaaag aatacgtgaa gtcgttatcc gacgagcagc cgctgctcat agtggaagat 660 gagggattga gctcctcttt catactgact gagatggtga acagtttacc catcgtgaac 720 gagattatag gatatcagtg gttcgaccga tcttatgaca ttccagcatc tctaccgacc 780 ggagcgatca atcgtatcgt atgtgtgacg gggtgggctg aatccgtatc tcattctcgt 840 ggccgcgaag tgcaaacgat gccaccgacg cacttgaacg tggacattta ccgacggaca 900 cCaagcgcgc agacaacgaa ctctcagcag tggaatccca atgctggcag agccaatgtt 960 ctaatcatgt tgctaaggct cgctctggcc aactgtcgca tcaacaaatc gacagctttt 1020 cgcaacgacg caaccgtcat gtccactggt agactactac gctcattcaa gaaggacgac 1080 ctactgttga aggcatatcc atctcgagtg ttgacagatc aggtgagcaa gaataatctg 1140 tttctgagac ggcagatcgc tgataggatc ggacgtgaca tgaaggccca ggtgatgatg 1200 gggatgatcg ctgcttacaa tgatgtctat gaaacatcag acacattgac catctacata 1260 cgcgaatgct acctactgct cctgcgccaa ttgcatctac caccgactca actgtcgata 1320 gccatcagcc aatgcgcaca cgggttcatc acgttcatca gcgcgtccgc agccaaccca 1380 atgatcatat gcccatggcg cgcaaccagt gaagataggc gtttggcagc agtgtttgac 14409 atcatgcgtc tgatcgatag ctcaagttcg atgctaccag tgctgcagga attagcgaat 1500 gcactacacg cttcgtctct actgaaaata gaccccgagg tgattggcca ggccttacgc 1560 ggagtgagcg agtcagccgt gcagaatgtg tcaccggtcg ctctgatcac aaaactctta 1620 cgaccaacag ggaatgactt catggcattc tgggttaccc tagcatcctg ggcgtataat 1680 ggcatctgta caaccgtgat agccgacgac agcttcccag ccattggctc gaatgtggtg 1740 acaatcgaag cgctcatgca agccatgatc gtggctatcg ctttgccgtt gacaactgac 1800 cctaatggac cgatgaaatg cttcatgctt gtagctaacc tattggcggc agatgagatt 1860 gtggagatgg gagtgcgagg tctataccaa acaacgccag cccaggcatt taaccaccca 1920 acgttgtggt ctagagcttt catcgatccg actaccatcg acagacataa cagtccttgg 1980 ctgtacgcat gggcaacact gatacatcag ttctggccaa atccatcgca ggttaatttc 2040 ggagcccctg atatactggg ttcagcaaac ctcttcaccg ctccaggcgt attgcaccta 2100 ccactggaca gacgcccacc agacatgctg gctgtcaggc ctacgtatga cgctgaaatg 2160 ttcgtgtgga tcaaggaggt catcgggttt ttcattcagc tgaccaatca gggtttcgtc 2220 acacgcacaa taggacgacc aacattggag tctacacgtg gtgtactaga gcagctacga 2280 cgtgtgccat caatgacccc cgcatggcta atgggcttct tccccgagga gttagctgtc 2340 ttagcgtcag tacaaaaggt gcagccaatc caaatcccat acgcgcgtac tgctatcaat 2400 gacattgtca ccaacctagg tgtctcgaga caggatccaa tcgacagggg tgatatgctt 2460 ggctacatgg cccagactgg aacgactatt ggtgtgacga tccctgtgag tgccgaggcg 2520 ttagtagtgt gtctacttga tggcgttgtg gaccctgaca ttgtacctac ccaacattac 2580 gcccgaatgt atgccgcatt gtttgaaggc aacgccatgt tcgcatcagc ccagcgtgcc 2640 gttgtactac gtgaggccta tgtctgtgcc agagatgtga tatcccaatg cgctaccacg 2700 tcatttcctg ctaggcaaga tatgcagtgg atacgtcaac tgaatacgta tgctagtaca 2760 gcagcggaga tttgcaagga aatcgacgac gactttaaag aggcttttga catctcagga 2820 gatctgttgt ccccaattta taacgagtat gacccccgcg tatcccgatt aggcgtggct 2880 tatcgcacgt acggtggtga agttgtgtct gaagtgattg agccaggtgc ctcggctatt 2940 gacagagcct ttggaatggt gattgagact ttccaacatg agtataactt attcaactac 3000 gccaagggcg atatcatact tggacgcgtt gtgacaacgg ccatacataa ttccctcgac 3060 ccaccccctg gatatgtgtt tgatgccaca acacctggtg tccacgtcgc cggctctcgc 3120 cttttatttc aagcgcatat gggcggacgt cccgcaatgc tgctcgacga acacgacatc 3180 gctcagagct tggatggaga ctggattatg actttgaaca ctctacaggc tggacgcgca 3240 ggtttcctca cccacatgtg gcctcgtata gcctcaggta acacacgagt aagagtgatc 3300 atcggagctt tccctttegt cctgaactat catcccctgc aagaagacta cagcgcctat 3360 gaattgtcga ctgcctggtt agagaccatg actgtaggcg gcgttggacc cgtgcccttc 3420 gcattgacta tccccagtcc gtatgaggga ttcacaggca tcttccatta ttatatctgc 3480 gccactgaaa cgaacaccaa tggaatgttc tctaccaatg ctgcattccc cgccacacgc 3540 accggtacag acgtgccaat tgacgagtct cgatggggag ccctaattga ccctgcatac 3600 gaccccgatt tagtgcaact gcccgtgaag attgacttgt attccaaaca tcgccgttac 3660 aactacgcgt atccaggtgt cgctgctttg ggaactatgg cgggagtaga gtaaccacgt 3720 tctggaccac gcacatgggg cactgtattc atc 3753 <210> 4

<211> 2263

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S4

<400> 4 gtattttgag ctactcctgg caacatggcg atgcgtccgt cgtcggttgg ttcaagatcg 60 tttcgatatc aatacatgaa ctctccaact catggaaaat ctcaacgcag tctaccaaaa 120 ttgaaccttt ctgataccca gctaattcgc gatgtaccca aaatgctaac aagtggcagc 180 caaacttcaa aaattaccct gacactgggc gacaagcagt tttctgacgt cctactgcct 240 tcccgtcaaa aatggaatga tcacactgat ctgttgagca gatacatctc aatgcgcgct 300 gatggaacag agcgtgatgt gcactccttc caagaattgg tccatctaat ttcaaccaaa 360 atgaagcagg atggctttga ctggtcttcc aacatacccg aatttgctga ttatgttagc 420 actttgactt ctaatgagcc tcagcctcta gacacgattt ccttgacctc ttctttcgct 480 caactgcctt ctgagtctcc ggcacaatca gtcattgtta gtgagcgctc ctatcggcca 540 actcgacaac gtgctgctga ggtgaggcgt gagccccaat ctgatactag cgatagtgat 600 tcccgatgtc caccaggtcg cgagccccca tatggcaaag gccgcatgga tccggagacg 660 tataacatcg aaaggaaggc ctacctgctc ggtgacccat acgctgtccc cccacatgag 720 aactcaagcg cttacttcgc tgcattctgc ctcactcaat tgcgcgcaca cgccacgggc 780 tttttctcta attctgctgc cggcacactg ctagccacta gagatgagca actcattctc 840 ctatctccct tgtacagcgc cgtggagcat tctgaatctg gcgaatcctt ctatcacatc 900 tacgaccggg tgaagcattc gtctgaccca ctcgccacat tcacgattct gtacgtggac 960 cagctgctat ctgggacgac gaactcaggg cccttgctgc gcgcaataga tgggaaactg 1020 ctaatcaaca tcactcccac cgcactagtg tggctatgcg aacataatct gtctgatggc 1080 ttaatgtcct gtcaagcccg tcgcatcata ctcggagtcg accacttaat gtcacgactc 1140 gactcgaccg ctgctttgct agcttgcctg ctaatggatc acaagctttc cttcctacgt 1200 cacgctccgc tcatggtgtt ccgcttcctg atgactgagc taacctcgac gcatcgtgtc 1260 gctatgctca aatcctttat cgctcacgcc gctagactcc ccaaccttca tgccttctat 1320 ggccccgatc gcttcacacg gctaatagat ttcatgtcca tctctgcccc agcagcacca 1380 gatccaacac ccctgcatga tgctgaatca cacctggccg agatacagaa actcatggcc 14409 ggtatcaaac tgcagacttc atcctgtgat gcagccatac gcgacatcca actctctcga 1500 tcgatgtttg agacagcgct tgccacttta cgcgacagat cctccgcttc tgccaatctt 1560 gacacttctc tgcttgacac atatctcaag gagcacacct gcattaatgt caaagagggt 1620 ccgctgctgc ttacgctcgg catcgcgaac gataaagtca atggcatgct agcttcacga 1680 cacgcacgtt tggatgaatt caaggctcta tgctcatcca agtccggtaa tgctgacgca 1740 acgatggccc tgaaggcgca gatagacgaa ttgcggactg cgaacgagca cctgaccgct 1800 gagaacgtct cgacaattgg cgaactggcc ttggcgcata aggagatcat cagtaaagat 1860 gagaccattg ctgcattgag ggatgaaatg cgcgctttag aaacaagagt cactgcctta 1920 actgctgagc atgagatgga ccagcatacc atcgcccgcc tctcctcttt ggctaagctc 1980 gagcattctc ctaactactc ccaccaccgt cctcegccgc ccactttctc tgatctgacc 2040 gctcaaatgt ctggtgcatc cgctgccatc actcagtctg aacgttctct catgtccgcc 2100 tcatgcatcg gagcttctgc agctaatcta gacaaggtat ccatcaacga tttgttctac 2160 gactcaggcc attaatacct catttatgtc tctcgctcac tacgcttgat ctgccgccca 2220 ccgcgcacgt gacacttcgc atgccgccgc tcacgctttc atc 2263 <210> 5

<211> 2229

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S5

<400> 5 gtaatttagc gatgttactc attctgccca cgtacgatgg agcgcgcgat gacgacttcc 60 tctccaccta caatatcgaa ttcacaagca aaatccactc cgatccaaac gtgcaggtgt 120 tagcaatgcc atggctgatc acacagctga ctcgattatt agacatcaac gacatcgcca 180 ctttgacccg ctgcctggac gaaatcacac cgcgacaccc cggggtattt gtcttactcc 240 ccaaatggcg atcattcgct cgcgtgtgcc tgactaattc tcaccttaat atatggaaaa 300 tacctaggtc cgttctaact gttacggcca atggtgaacg cacggagaag aagattagcg 360 attacattga cggtctggtg acatcatcgt cggacgtgtt gactggcgtt aagcgtgtac 420 agaacggtgc cgtatgctac acgacgacgt taatggtatt aggcgcacca atacgaaaat 480 ggtgtcaaga cgacgcttac tctggggctt acgctcgtgc cgacattaag gctttacaca 540 gacccaattg gcccgttccg cccaaagtaa caacccgaat tctgatcact aatctaccac 600 taggcgtgtc tgaaaacgct atcatgctcg accgcgctta ctggccacct gatgtgacgc 660 atggcaccct attattggag agatatgacg cgttcgtgtc tactgcccgc atatgcgact 720 tgactgcagt ggccatgttc cttggtgtcg aaaaacggct acctcgtaaa gtggatgagt 780 cataccgtag tgcacttcgc atgttaagac tgccatttcg tgatcacgcg caatcagtga 840 cgtctggtac cgccacgggg accatcacat gcgatatccg agtccgactg acgtgcaaaa 900 attcaaatag cccgaaggtg gctgcacgta ctgtgactat tctcagcgta ctaacatcat 960 tgctgtctat atacaatgtg gacatggacg tggactaccc attgcgacag gaaactggcg 1020 agatggtcga atggatactt atcgtcatca tgctgagtag tggactacgt actagcggag 1080 gccaggcagt gtgttgggat gcagcattta caatctcaac cccatttgac acgatcactg 1140 tgatgcatcg tagcccggat aagtataaaa tatcgccgtc atatctatcc gacactgccc 1200 atgtgtatac aattgccatg gccaaggggt cctttaaatc caccattctt aaagccctat 1260 catcatggtt tggggacaaa cgtatatttg actctgctgc agctttcgat tcagatgacg 1320 cgggcaacac ggtatcaccc acctttgagg agctactatt gactgagtgg aggaagttag 1380 gtacggtttt gtgcgatgaa gccgcaatgg ccctggtgac aggaactgaa gggtgcgcga 14409 catccctagc ggatctgtac gatgcctttg tgactaccta tgcctccacc gtcactccag 1500 taatcagaga cacaataggt ccactgctgc gtcgtgcaat atcctgcctc ttcgtacatt 1560 gcgacactga ggtgcagaac gctaacatat ctacccatac cttccgatgc catatcccaa 1620 tggtgacgca gagcaatatc ttgatcagga ctggtcgctg cggaggcgtt actttccaag 1680 tcatcctgga ttactgctat cgatgtctcg ctgccggtgc cgacgaaatg tggtttggtg 1740 acacagtacg tcctttactg gatccttggt tacgtgcttc ctcactggtt gggtatggat 1800 gettggctgt aggtataggc gtcccaggta cgtcacttgc taaggctgga tggggagtat 1860 cgaccgacat catttaccga gccacatact gttatatata cgcaccggtc acgcggtttg 1920 atgcgataca gttcatggca gagcgccggt tccgaggtcg tatgaattta ggcccactcg 1980 cagcaacaga tcgacgggat gtgagcaaat atgaggttgg tttctcctgg ttgcctcaac 2040 ttegtcgtga tagcttcggt ccggacgttt tgatgtccat ctcgtctatc tcttatatcg 2100 atctttctgt cggatgtact gtgggtgagg agttaagcac gtcgcgccct ctcactacgt 2160 ggcctgaggt accacttcca catggggatt aatggattac cccatgccgt ggtatatcgc 2220 atattcatc 2229 <210> 6

<211> 2030

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S6

<400> 6 gtaatttaag catagcgtgg agaccgactt acggccacta tcatgggaaa cgtccagacg 60 aacaacacta cagtcaatat caatggcgac ggtaatagct tcggtgccga aagcggcatg 120 cggtcgacag ctgcgcctga aatcatgtta tcacccggaa aacttaaccc gaatggactc 180 gcatggatga aagtcgcagg atcaggtatg ggaccaggtt cactgcaaat tgtacacgca 240 acagacgggt caccgtattg ttacatccct ccggacgcca tgtctagtat ggaaaaatca 300 gcaggtgccg ttaatttgtg ggaacctcta ttcgccgcta tattcgaaac ctgctctaca 360 gtgaacgtgt ccgactttac tgatgagttc agtgcctacg ttggcgtcag tgaacccgag 420 gccctcaaga agtacatcga gaaaggagtc ttcatgtcaa catcacaagc gaaaaatttc 480 tttgggacac tcgggcagag gatggcgagg ataaagggtt ggtctgaaga tatacggacg 540 gcggctgcta tgataccagt agacacacct catggctcgg tcacgtgcga ttggaagagc 600 ttacttagat tttgggacga aaatctacca gtggataatg tatgcaaaca ataccaaaat 660 gctgtatatg aggtagcatt gaagatatac cctgcgttac gggccggaaa tagtggatca 720 cttctctctg atgtcgttgg cgtattagct ggtggtgcgg caggaactac agcgtctgga 780 gtgagtatgg tcgaaggtgt gacacgtaat gagcaagtgt ctcgaattct gacatcagca 840 gacgttgata ttttgacgtc gtcagcgcca gtgcctgtct cagctttgag aactccacca 900 gtggaattgt cctatcaacc cagcgccctg agtgccaaga ccaccccatg gttggttcga 960 tatcccggga ctactgccat agagaagaca ttcgacgtcg ggacaacatc caagacgacg 1020 tattacctga gcatgggtaa ctccggtggt ggagatttga tgattgatct gaagcgactc 1080 ccagcatgcg gacttgaata ctccctgcgt ggtatcccga tcatatacga taccaatctg 1140 accgcggcga agctggctaa ggttactcct gcattgctga tgctgcagac ggctaaaccc 1200 cttagtgcgg agatcaccgc cgcagatatt caagctatca cacctcttgt agtagggact 1260 gataagctca acactttggt caccactggt tttggcaata ttcgcaacat taccgacttc 1320 tcaatgtctg caatttggga accagagaca gtcagcgcgg caggcaatta ctatctatgg 1380 ccgactgtaa tcggtgatgc atcaatgaca tcagactggg ggacaattag cacatctcta 14409 gctaatggca ggctccgtgt cgcacctctg gacctgactc atgcactcca caaaggcaat 1500 gtcgtggagt caatcgtacc atctgatgtg ctaggtaatg cctcaccaga ggaaatgcgt 1560 tccgcgttac cagcagatgt gctgacagct ttcaaagcca agctcacaac agtggcttct 1620 gtagttggcc gtgccttaaa ccccaacgac agtgcgcatg caccatcctc cggcaccgtc 1680 ctcggcccgc ttgcaattga aaacaaggcc caatcgaaac ctaaacccgt atcagatctg 1740 tggatagctg ctcggcgtgg tgtgaatcta ttcgtagcct ctccaatcgc gagtctgcaa 1800 acaggcgtgc cggtcatggg ggacagcagc gtgttgacaa gcttgatggg tggtgcaact 1860 accgcgttga atactggcga tatgggtacc ccaagcctgg aggccacagc gaaacgggca 1920 gctagagttg ctggaggact gctacgacag agagtcatgg acaaattaac tcgatactgg 1980 cctcctccgt cttaggtcgt gtacaccgac ccccgctttg cattttcatc 2030 <210> 7

<211> 1604

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S7

<400> 7 gtaatttaac tgccaagatg gccactcgtg acagccgcat gactacagag cacgttatcg 60 ccctaattct cgcatactcc acaactttag atgaagcaaa gatacacgaa gagattacga 120 atctgcagtc ggcagtggcg caactgacag cgcgattgac agagttgact gatgctgtga 180 ctagctttga tgcacgctta acccaagtag aacagcaggt tagtaaattg tacgacaaga 240 ttgggatact atctaacagg ataggcgcca acaccgccag tatgaatttc ttggctggca 300 tagtggatgc gacgagaata gatgcagtgg atgtaccact atacatatat atagtcgatg 360 accagaggcg cctcgcaata gccacgggag aagggttatt tgtgaaagat cagaagttaa 420 acgggtacga tgtgcgtagc tttccaccta tagccgtcgc taaatataat aacatattat 480 cgttctcgct gagctcagcc cccccactgg acatagttga tggtaagttg gctgtcagca 540 taactagcag gctattcatc accagtggaa aattagacac taattcatat accggttcat 600 cgagtgtgga tgtatctgga gcgacggcag aaaagacggt gagcgttaga acgattaacc 660 ccatcattgc aggtcaggat ggcttgtccc tatcactaag cggtatcctg gaggtcggcg 720 ctggcttatt actatcaggt aggtcttggc cccttacatt ggatctacgt gtgagtaccc 780 cattagcata tgaccatacc ggaggattat ccgttgtaac acggtatccc ttagatgtga 840 agtctgctgg catgggtgtc aacatgcagg tgcccctacg gctaaacggt gtcgacttag 900 gattggcata caatacgcag gatttttcga tagttgacgg atatttgact ttgaacaggt 960 cccaacgaaa attggaggaa ctcggtgagg tggttgacat gaacgctact ctagtcgact 1020 taaatgacgg tggattgcag gttctagaga cagatctaag cttggagaag cctcttaacg 1080 ctatgcaatg cctatttgag catgagggta catggcgttg aagtttggtg tgcgaatgtc 1140 cacggaaccg acgattacaa ctgtccgtgt taatgttcac agcacttggt ctgtctctgt 1200 ccaggaatca atagcaatct gcgcgataac caccactacg gctggagctg gtaaaggaca 1260 geggccccgg gtgactattc ctttcagaag agctgctctc accgatgata tgatgcgtgc 1320 aggcttcgat gtgtggaaga gcgattatat cagggaggaa agcatttgta ggtacgttac 1380 gtggcctcta ccgacgatgg tccgacaagc atctacttac ggaattctac gcacacggcc 14409 gtctctatcc aactaaggat ggcatcgagt tgattttaac acctaggtct acggatgacg 1500 agtatgagtg ggctggtctg cgtaatgtgc attggacggt agtttgctgt aagtaaagtg 1560 cgtgctggct ccagtgaggt accgcagttg gcagtatatt catc 1604 <210> 8

<211> 1556

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S8

<400> 8 gtaatttaga cctttcctat cacccgcggt atcaaccagt ctttaacatg tatctggaac 60 tgttcatagc tatgatgtgg cacttcatgc tcgctttctg cgcgtatgtg tggtacatac 120 atggtcctac ccttcgacgc ctctaccaac gcctgactgc tactactgct aacgctatct 180 gccaaacgga ctctacttac gtctgcactg caactgtttc caccatgaca gatgatctga 240 ctaacgcttc ctcttcegcc tccacctcca gtactgcttc ctcggctacg accagttttg 300 tagctctgcc taggctgtcg cttgatgatt tgaagaccca ttccaaattc cgctggctgt 360 ggttttccaa taacccattg accattgact tatccactga cgaactcaca actcccttca 420 atagtcctca gcttcatgat gtcttgttca tgcctcaata tactctcgca gcttatccct 480 atatgcatcg ctctctccat ttggcgcttt actttcgctt cggctataac atgcgctacc 540 tgctacgcga acatcctggc attccaccgc ttcgcgtcac ctgcccatct acgcgtgtgc 600 tattcatgga caaacagcgc aataaatcgg aagagcctgt ctgttctatt tgtcgcctgt 660 ggattgtgga cggccctcag cttcccatcg gtaactgtgg cagaaagtac aaagtccatg 720 aaatattcaa cgctttcagt ctcgagcctc accacattgt aaatgctttt gacgtcattc 780 caatcgtctc tctcctcaaa tgcctgcaac tggatgtttc tatgctggcc ggcttgtctc 840 tcaccgcact gacgcagctt tcagaattat caagagatcc agacgttaca tccgctggct 900 ttcttgectge tattcaattc gatggtcaca cttccaccgt ggattgccgt ggcgatcgtt 960 tgtatccaca tcctatgtta cttatcttgt ttgctttgtt ttgctaccgc cgccgttccc 1020 gtgtttattt ccgtgaatgc tacttgcgcg accaccgccg cttgacttca tccctacccg 1080 atatggttcc ctactcccgc accccaggca aggcaaaggc taagagcccg taaatgtaat 1140 ggcaaggtct agtgtaaata tgtatataaa tgttgaatat cgttacgctt tgcgatgctc 1200 agagtataat aaaaatgaaa aactacataa aaacttaaaa acaagatatg tgtataacca 1260 agctgtatat gttcgctgaa aaaccaaaaa gatcaagctt cgcaaaacta tgttgtttat 1320 cgctgtatat gtacgcataa aaaccaaaaa gaacaaatgt actcatcacg cttcatgtta 1380 atacactggc attccatatt ccagcgcagt cactgtaaag atgtataaca cggacgatat 14409 taacgccttc attgtatgtg cagcacagat gcaatagaga ggactgtttg atagcgggtg 1500 gcgtgttttt gacagcctgc gggtggcaat gcatgtacag agggtctatt tgcatc 1556 <210> 9

<211> 1284

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S9

<400> 9 tatggagcga tccacttaca atatctgcac cccaggcttt ttcggcgcga atgttccgcc 60 ttttaaaaca atagacatac aacgatctac gacgggtggt aatacgcttt ggaatgcgcg 120 cggtcacgat gctttcagga ctcaccctaa agtggtatct catgagaagg atttccctct 180 aatttataca gagcagttca cattcaactt gcttattggc gcctgcctcc agcaacccct 240 actgcagaac tcgattgacc gacagtggag gggtatgatt tggacatctg atcggctcag 300 ctcgttgcgt atagtgccgc cgaattcacg tgctgctgat ctgcctcgtg catatcgtac 360 gttggacctt gcgaattacc cactatgggg aactacacgt gctgccctac agacactatg 420 gatggacagt tgtctgatga cattagagag tttatctgcg agaggacctt ttctttacct 480 ccggcatcct caagctcgac cagacgggaa tgttttacga gccttacggc agcatatttc 540 caagccaatg gaggccatag tctcggaagc ctaccaatca atagctatgg gacctttgac 600 attgcaaggc gtacaatgct gatggacgtc agtgaccacc ctcatctatc tagcctctct 660 gaccggtcgt ctgggccctg accgtacata ctatggcttc tacgtccaat tccctaagaa 720 acggaaattc gaggatctcg gatactttgc gtacaatgct gatggacgta acgtcgctgt 780 cctgcaatcc attaacgcct acatctactg tgcctcacct gattggcagt acagctgtgc 840 cctctactac ttgcatgtcc tttcggccct atcgctctcc tggactgatc cagttggaat 900 gataaatggc ttctcgtgtg tcaatcaatt cacggacgtt cctggctggt caaccacaaa 960 ccgtgctttg cacacgcata gcttcaactg gttcaaccta ctggaggacg ctattgacac 1020 gtactggacc aatgccgagg gacaggccat gCggcaggaa tggacggegg Cgcgagacag 1080 gtggcgaatg atcatggacg caacccgtga tgaagatgac ttggtagttt ttcgtacgcc 1140 agacgattgt cgtagaaggc tcaaacctta tggggacaat aactggacgc gtgcttacga 1200 tactgccgat gtggtgcggg tgttggatcg cttattccct taggacctaa tccacccgac 1260 gggaggcaag actcgatttt catc 1284 <210> 10

<211> 1124

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S10

<400> 10 gtaatttagc tctgtactat caacatggcg ggtgtgtctc tcaacatcaa tcgcaacatc 60 tcaaactcgg catcaacgat ctttctcgaa gatattccat tactgtcatg ttcagtgcgg 120 tgtgaacccg gtaaaggacg cgaattacca aaatttaaca tgagctgccc tgctattaac 180 gcgatgggtc gatgtcttaa tccaatgaag ttcattgctg agcactgggt ccccaacagt 240 ccaagtcgta aaccatccag acagcattgg cgtaatgttt tgaatggact ggaattcagt 300 aatggtcgtg gattcgatgt gctgagtttc tcaccagcgg gcatggctgt tcttcgtgac 360 atcctgacag aagatagcgt gaaatattgc tttgacgaga gtaacacttg cagtttgttc 420 accttgctgt acactctgtg ttgcgatgcc gcaggcgtag aaccgatgga cttggattca 480 cgtcagacgg acgccagtgc ccggatggtg agctaccaag atcgcgctat cgtgctgacc 540 tctaatgagg caggagacag aattgagccg tggaatgttg agctcgacaa ggagtttgga 600 aatccagatc tgctcagccg tctgaacatc tcatatggcg tgcaacgata tggcgactcc 660 aaagccagca cagacactct gaccttggct gatgccccag agaggtccaa gcctgctctg 720 attactgtgc aacccttatt ggtggctatg tgcatcaaac agtctttgga tggcttgctg 780 gctttatctg atttgcgcct gagattcgat cagtatcctg gatacgcaaa tgctctcatg 840 aatgctatgg ccatgtacgc ttgcctagat cgtgacttga tgcgttttct gctccgctta 900 gaaatgactc acgcgagcac ggtgtctgaa gtggctgagt gctggaggaa ctctcgcaac 960 tctcgcgatg caacaggttg tcatattgtc ccacgtcaag gtttgctcat catcgtttcc 1020 ggagatgtcg aggtaagacg tatatttgcg cagatgctgt agacactgcc gtgtggtcgg 1080 cgataacggt actcatcgtc gactaaccca gagacgtatt catc 1124 <210> 11

<211> 1107

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of S11

<400> 11 gtaatttttg tgcttgcacc aattatcggt aagtatggaa ccagcaaaac cattgacgtt 60 tctggatctc acccgactca atgaggcaat ctgctctcgg gctctcttct ttgataatga 120 taacaactgc tggtctgtgt ctccaatcgc cccaaggcaa gccaaattcc atgactcagt 180 tgtctgtatt cgatgtggag cgcctattga caaagttcat gcaatgtcaa ttccaccacc 240 ccctgtgcat ggctgcatcc caatgctgga tcatagccaa tgggaagatt tgtacgagtt 300 ggctgatgat atgggccgct gtatttggtg ggctaaaaag caactggtca tctggatgga 360 gggaatagtg aatctgaagg ctggtaaggt gtataatgat aatgtgagta accgcagtga 420 atggccagat gaggtgtggg atgaaacatg caaaatcttc tgtaaatggg caacgcaaaa 480 tcgtgtggcg agccgttgga tccagtcacc atcacgtgtg tacaagtttc ttcgtgacca 540 ggaaagtaag atgaacatcg atgctctgga gccatccaat catcagattt ttcaggcacc 600 accgcaatgg cctgagatag ctatggctgt cccgcactgg tccccgtccg tgcatgagat 660 gctaaatggt cacaaaatgg tgatgattgt gccccgcttg tccatgccag tcatatttga 720 tcccgccaac ggttacgtcg ccccaatcta caccgcggcc atgatcagtc tcccatccca 780 atggtgggtg tcacaatatg taaaagttca tggaagccac atggtgccac gtctgtatgg 840 tgatgacgtc ccaaccttac gttcccgcct gcggaatgct tctaccacaa cctcccactc 900 tcatacgcaa ctcctgatgc tgcctgaagc tcaatcgacc ttcaaacctg agatccaggg 960 acagtaaagg aaagtaagat gaacatcgat gctctggagc tatccaacca tcagattttt 1020 caggcacctg agatccaggg acagtaatga ctgattccat accacatgcg cagcgcgttt 1080 ggccgtaggt gagcacactt tttcatc 1107 <210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer F1

<400> 12 gaggccatag tctcggaagc 20 <210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer F2

<400> 13 gcgtacaatg ctgatggacg 20 <210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer R

<400> 14 ccagtacgtg tcaatagcgt 20

Claims (6)

CONCLUSIONS 1. Isolated grass carp reovirus GCRV-WL1609, the GCRV having been deposited with CCTCC under accession number V201978. The specific gene of the GCRV according to claim 1, wherein the gene is shown in SEQ ID NO: 9. 3. Detection primers for the specific gene according to claim 2. Detection primers for the GCRV according to claim 1, wherein the detection primers comprise: F1: 5'-GAGGCCATAGTCTCGGAAGC-3'; F2: 5'-GCGTACAATGCTGATGGACG-3'; and R: 5'-CCAGTACGTGTCAATAGCGT-3'. Use of the GCRV WL1609 according to claim 1 in the preparation of a grass carp reovirus vaccine. Use of the primers according to claim 3 in the preparation of a GCRV test kit.