CN109280700A - The method of accurate measurement Eriocheir sinensis mitochondria whole genome sequence - Google Patents

Abstract

The present invention relates to the methods of precise determination Eriocheir sinensis mitochondria whole genome sequence and the whole genome sequence obtained by this method.The method comprising the steps of: (1) extracting the complete genome DNA in Eriocheir sinensis musculature, construct gene library, be sequenced using second generation sequencing technologies；(2) sequencing read is screened with reference to nearly edge species mitochondria whole genome sequence, except stoning DNA fragmentation, then carries out the splicing of mitochondrial genomes, then prediction annotation is carried out to encoding egg white gene, rRNA and the tRNA of the mitochondrial genomes of splicing；(3) by assembled good mitochondrial genomes sequence and nearly edge edge species mitochondrial genomes sequence alignment, according to conserved region, referring again to the mitochondria whole genome sequence design primer of the nearly edge species, PCR amplification is carried out to corresponding second generation sequencing sample, pcr amplification product is sequenced using first generation sequencing technologies, corrects the mitochondrial genomes sequence of splicing.This method has the advantages that fast and accurately.

Description

The method of accurate measurement Eriocheir sinensis mitochondria whole genome sequence

Technical field

The invention belongs to field of biotechnology, specifically, being related to a kind of accurate measurement full base of Eriocheir sinensis mitochondria Because of group sequence method and the Eriocheir sinensis mitochondria whole genome sequence measured by this method.

Background technique

Mitochondria has and uniquely has " chromosome in double membrane structure and unique DNA molecular and zooblast The semiautonomous organelle of genome outside ", while it also has the ability of entire genetic information transmitting and expression system.In recent years To come, mitochondrial genomes have become very characteristic molecular labeling, because it is with some unique properties, such as: point The smaller, structure of son amount is simple, matrilinear inheritance, intronless, evolution rate are high and without genetic recombination etc..Due to structure of mitochondria Function and heredity importance and particularity, mitochondrial DNA phylogenetic systematics, molecular evolution and in terms of obtain It is widely applied.

Currently, carrying out sequencing to mtdna sequence is broadly divided into three categories method: first generation sequencing, the second generation are sequenced It is sequenced with the third generation.First generation sequencing technologies are mainly characterized by sequencing reading length up to 1000bp, and accuracy is up to 99.999%, But the shortcomings that aspects such as its sequencing cost is high, and flux is low, has seriously affected its really large-scale application.Thus first generation sequencing Technology is not optimal sequencing approach.Second generation sequencing technologies (also known as " high throughput sequencing technologies ") greatly reduce sequencing Cost, and sequencing speed greatly improved, but read segment (200bp -500bp) and its application is allowed to be limited, and is accurate Property is also lower than generation sequencing technologies, and main cause is just derived from the replacement of base.Third generation sequencing technologies compared with preceding two generation, Their maximum features are exactly single-molecule sequencing, and speed is sequenced quickly, readable 10 dNTP per second, but its sequencing error rate ratio It is higher, reach 15%, this is almost the common fault of current single-molecule sequencing technology.

When mitochondrial genomes are sequenced, 1. if using first generation sequencing technologies, it is excessively high to will cause sequencing cost, The problems such as workload is too big has seriously affected its really large-scale application, thus first generation sequencing technologies are not most ideal Sequencing approach；2. if will cause the accurate of its sequencing using second generation sequencing technologies (also known as " high throughput sequencing technologies ") Property reduce, and influence final result.3. third generation sequencing technologies are compared with preceding two generation, it is relatively high that there is also sequencing error rates Disadvantage.As it can be seen that these sequencing technologies cannot all obtain mitochondrial genomes ideally.

Currently, Eriocheir, which is sorted in academia still, very big dispute, Accurate Determining is carried out to the mitochondrial DNA of Eriocheir It is necessary so that its classification is accurately positioned.For this purpose, developing a kind of fast and accurately high sequencing approach of property is also that have very much must It wants.

Summary of the invention

For the above-mentioned technical problems in the prior art, it is an object of the present invention to provide in a kind of precise determination The method of magnificent Eriocheir mitochondria whole genome sequence, this method are based on high-flux sequence, have quickly, high excellent of accuracy Point.

It is a further object of the present invention to provide a kind of Eriocheir sinensis mitochondria complete genome DNA sequences, according to the sequence The biological classification of Eriocheir sinensis can be accurately positioned.

As the first aspect of the present invention, the present invention provides a kind of accurate measurement Eriocheir sinensis mitochondria full-length genomes The method of sequence, this method are based on high-flux sequence, comprising the following steps:

(1) complete genome DNA in the Eriocheir sinensis musculature is extracted, gene library is constructed, utilizes the second generation Sequencing technologies (high throughput sequencing technologies) are sequenced；

(2) it is screened, is removed by reference to sequencing read of the nearly edge species mitochondria whole genome sequence to high quality Then core DNA fragmentation carries out the splicing of mitochondrial genomes, chooses optimal splicing result, then to the mitochondria base spliced Because the encoding egg white gene, rRNA and tRNA of group carry out prediction annotation；

(3) by assembled good mitochondrial genomes sequence and nearly edge species mitochondrial genomes sequence alignment, according to conservative Area carries out PCR to corresponding second generation sequencing sample referring again to the nearly edge species mitochondria whole genome sequence design primer Amplification sequencing, PCR product are carried out using first generation sequencing technologies, are finally corrected to the mitochondrial genomes sequence of splicing, It can be obtained Eriocheir sinensis mitochondria whole genome sequence.

In a preferred embodiment of the invention, it is extracted in the Eriocheir sinensis musculature in step (1) Complete genome DNA uses phenol/chloroform method.

In a preferred embodiment of the invention, nearly edge species described in step (2) are eriocheir japonicus The closely square crab (Hemigrapsus sanguineus) of (Eriocheir japonica), meat ball, Helice wuana (Helicana ) and Eriocheir sinensis (Eriocheir sinensis) wuana.

In a preferred embodiment of the invention, the splicing of step (2) Mitochondria genome is to utilize NOVOPlasty splicing software carries out the splicing of more Kmer parameters.

In a preferred embodiment of the invention, in step (2) to the encoding egg white gene of mitochondrial genomes, RRNA and tRNA carries out prediction annotation, and to be utilized respectively blast software, Barrnap 0.4.2 and tRNAscan-SE v1.3.1 soft Part.

In a preferred embodiment of the invention, PCR amplification the primer described in step (3) includes following 19 pairs Primer:

1st pair of primer: F:SEQ ID NO.1 and R:SEQ ID NO.2,

2nd pair of primer: F:SEQ ID NO.3 and R:SEQ ID NO.4,

3rd pair of primer: F:SEQ ID NO.5 and R:SEQ ID NO.6,

4th pair of primer: F:SEQ ID NO.7 and R:SEQ ID NO.8,

5th pair of primer: F:SEQ ID NO.9 and R:SEQ ID NO.10,

6th pair of primer: F:SEQ ID NO.11 and R:SEQ ID NO.12,

7th pair of primer: F:SEQ ID NO.13 and R:SEQ ID NO.14,

8th pair of primer: F:SEQ ID NO.15 and R:SEQ ID NO.16,

9th pair of primer: F:SEQ ID NO.17 and R:SEQ ID NO.18,

10th pair of primer: F:SEQ ID NO.19 and R:SEQ ID NO.20,

11st pair of primer: F:SEQ ID NO.21 and R:SEQ ID NO.22,

12nd pair of primer: F:SEQ ID NO.23 and R:SEQ ID NO.24,

13rd pair of primer: F:SEQ ID NO.25 and R:SEQ ID NO.26,

14th pair of primer: F:SEQ ID NO.27 and R:SEQ ID NO.28,

15th pair of primer: F:SEQ ID NO.29 and R:SEQ ID NO.30,

16th pair of primer: F:SEQ ID NO.31 and R:SEQ ID NO.32,

17th pair of primer: F:SEQ ID NO.33 and R:SEQ ID NO.34,

18th pair of primer: F:SEQ ID NO.35 and R:SEQ ID NO.36,

19th pair of primer: F:SEQ ID NO.37 and R:SEQ ID NO.38.

In a preferred embodiment of the invention, the system total volume of PCR amplification described in step (3) is 50 μ L, Including each 2 μ L of forward and reverse primer, 2 μ L of DNA profiling (concentration is 50ng/ μ L), DNase-Free deionized water 19 μ L, Taq PCR MasterMix 25μL。

In a preferred embodiment of the invention, the response procedures of PCR amplification described in step (3) are as follows: 94 DEG C pre- Denaturation 5 minutes；94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute, and totally 35 recycle；After circulation terminates, 72 DEG C Extending 10 minutes, 10 DEG C of termination reactions.

As a second aspect of the invention, the present invention provides a kind of Eriocheir sinensis mitochondria complete genome DNA sequence, As shown in SEQ ID NO.58.

The present invention also provides a kind of for expanding the primer of Eriocheir sinensis mitochondria complete genome DNA, the primer packet Include following 19 pairs of primers:

1st pair of primer: F:SEQ ID NO.1 and R:SEQ ID NO.2,

2nd pair of primer: F:SEQ ID NO.3 and R:SEQ ID NO.4,

3rd pair of primer: F:SEQ ID NO.5 and R:SEQ ID NO.6,

4th pair of primer: F:SEQ ID NO.7 and R:SEQ ID NO.8,

5th pair of primer: F:SEQ ID NO.9 and R:SEQ ID NO.10,

6th pair of primer: F:SEQ ID NO.11 and R:SEQ ID NO.12,

7th pair of primer: F:SEQ ID NO.13 and R:SEQ ID NO.14,

8th pair of primer: F:SEQ ID NO.15 and R:SEQ ID NO.16,

9th pair of primer: F:SEQ ID NO.17 and R:SEQ ID NO.18,

10th pair of primer: F:SEQ ID NO.19 and R:SEQ ID NO.20,

11st pair of primer: F:SEQ ID NO.21 and R:SEQ ID NO.22,

12nd pair of primer: F:SEQ ID NO.23 and R:SEQ ID NO.24,

13rd pair of primer: F:SEQ ID NO.25 and R:SEQ ID NO.26,

14th pair of primer: F:SEQ ID NO.27 and R:SEQ ID NO.28,

15th pair of primer: F:SEQ ID NO.29 and R:SEQ ID NO.30,

16th pair of primer: F:SEQ ID NO.31 and R:SEQ ID NO.32,

17th pair of primer: F:SEQ ID NO.33 and R:SEQ ID NO.34,

18th pair of primer: F:SEQ ID NO.35 and R:SEQ ID NO.36,

19th pair of primer: F:SEQ ID NO.37 and R:SEQ ID NO.38.

Method of the invention is quickly to measure Eriocheir sinensis mitochondrial genomes on the basis of second generation sequencing.It will Assembled good mitochondria whole genome sequence and reference species (that is, nearly edge species mitochondria whole genome sequence) full base of mitochondria Because of a group sequence alignment, then unusual sequences segment is corrected using first generation sequencing technologies, finally obtains point-device acquisition Eriocheir sinensis mitochondrial genomes sequence.

Method of the invention combines the advantages of first generation and second generation sequencing technologies, and the method overcome first generation sequencings The heavy workload of technology, it is at high cost, time-consuming the defects of, and the reading segment for overcoming second generation sequencing technologies is limited, surveys The defects of sequence error rate is high solves its defect that cannot really apply on a large scale.The advantages of using first generation sequencing technologies Abnormal Eriocheir sinensis mitochondrial genomes sequence is pointedly corrected, the accuracy rate of sequencing is significantly improved.

Compared with prior art, the present invention has the advantage that

The sequence accuracy of Eriocheir sinensis mitochondria complete genome DNA of the invention is high, carries out for Eriocheir sinensis quasi- True biological classification is laid a good foundation；

Required chondriogen segment can be accurately positioned using primer of the invention, to correct mitochondrial genomes sequence Column, overcome overlap, the gap and faulty sequence that can often generate using second generation sequencing technologies；

Reduce tedious steps and the design costs such as design of primers；

It is sequenced compared to the first generation, the method for measurement Eriocheir sinensis mitochondria whole genome sequence of the invention eliminates A large amount of human resources and material resources, and also save a large amount of time；

It is sequenced compared to the second generation, the method for measurement Eriocheir sinensis mitochondria whole genome sequence of the invention is sequenced quasi- True rate is higher, has reached the accuracy rate of first generation sequencing, and can be accurately positioned and correct mistake sequence, and scientific research result is more It is accurate credible.

Detailed description of the invention

Fig. 1 is Eriocheir sinensis complete genome DNA ring-type figure of the invention.

Fig. 2 is initial data base composition distribution example diagram of the present invention using the sequencing of Illumina HiSeq TM platform.

Fig. 3 is initial data base Mass Distribution example diagram of the present invention using the sequencing of Illumina HiSeq TM platform.

Specific embodiment

Present inventor is during obtaining the DNA sequence dna, first with second generation sequencing technologies to the China of building The gene library of Eriocheir complete genome DNA is sequenced, then by reference to nearly edge species mitochondria whole genome sequence pair The sequencing read of high quality is screened, and except the splicing for carrying out mitochondrial genomes after stoning DNA fragmentation, chooses optimal splicing As a result, carrying out prediction annotation to the encoding egg white gene, rRNA and tRNA of the mitochondrial genomes spliced again；It finally will be assembled Good mitochondrial genomes sequence and nearly edge species mitochondrial genomes sequence alignment, according to conserved region, referring again to the nearly edge The mitochondria whole genome sequence design primer of species expands corresponding second generation sequencing sample, and PCR product utilizes the Generation sequencing technologies are sequenced, and are finally corrected to the mitochondrial genomes sequence of splicing.Method of the invention can quickly, Eriocheir sinensis mitochondrial genomes sequence is accurately obtained, is laid a good foundation for its biological classification is accurately positioned.

The sequence of Eriocheir sinensis mitochondria complete genome DNA is as shown in SEQ ID NO.58.

TTACATGCAACGGAATTGCACCGTTTCTTAAAAGATCAAAACTTCTTATGCTCTTTACATCAGCATAT AAATTATTTTCAAACCCTAGTGATTTACACATCTTTAAACTTGCAATTTAATATTATTTTTATACTATAGGGCCTA ATAAAGGAATCTCATTCGTCTACAGATTTACAATCTGCCGCTTACAACTCAGCCACTTTATTAATTTTTAAATAAA TAGTATACTCGATGACTGAAAGGTAAGTATAGATCTTTTAAATCTAATATAGTAATTTAACATTTACTTCGAGTAA GGAATTAGTTAAAATATAATAACATTTGCTTGTCATGCAAAAGTCATCTTTAAGATACTCCTTAAATTTCAGTTTT ATTTGTTTTATGGTGTAGATAACACATTGTATTTTCGTTACAAAACCGAAACTAATGTTTCTAGAACATTATAATA CTTTGCCCACAGATAATATTTATCTCTTTTGCAGCTTCAATGCAATATTCTATATAAATTATATAAGCTACTTATA TTAATCTAGCTTTACGTTATACGCCATTAACTAAATTTAAATTTAAAATTAACATATTTAAAGGAAATCAATGTAA AAATAGAACTAAATATTCTCGAACTTTTCCTGACCTACAAGAATATAAACTACTGACGAAAATCCCATGCTGACTT AAACTATATATGTATAAAGTATAAGCTGCTCTGAAAAAAGAAAGAAATATAATTCCCATTACTGTAAATGTCCTTC ACCTTATTAAACTAATAATCAACCTAATCTCCCCGAATAGATTTAAAGTAGGGGGAGCAGCCATATTACCCACTCT AAATAAAAATCATCACAAACCTATCCTAGGTATAAAATTTAATAAACCCTTACTAACTAAAAGACTACGCCTACCA ACTCGCTCGTATACTATATTAGCCAAACAAAATAATCCAGAAGAACATAACCCGTGTCCGACTATTACTACAACAG CTCCTATTAGTCCTCATCATCTAAAAATCATTAGACCACATAGTACTATTCTCATATGAACAACCGAAGAATAAGC AATCAAGGACTTTATATCTACCTGGCGTAGACACAAAAGACTCACTACTACTCCCCCTACTAAACTAATTCTAACC CATAACGACGAAAAATTCAAGCTAATTTTCTGAAATATCAATAACACCCGAATCAAACCATACCCGCCTAACTTTA ATAAAACTCCTGCTAAAATTATAGAACCAGCAACCGGGGCTTCTACGTGAGCTTTTGGCAACCATAAATGAAATAT ATATATCGGTAATTTAACTAAAAAAGCCATCACCCTTCTAAAATATCAAATACTGTTTGCCCAGCTAACTTCTAAA ATAAGATTTCTTAATATTATAACTAACCTATATTCTTTTATATAAATATACAATAAAGACCCCAATAAAGGTAAAG ATAGTGACAAAGTATAAAAAAGTATATAAACTCCAGCTTGAATTCGTTCAGGTTGATATCCCCATCCAAGAATCAA AATTAAAGTGGGAATTAACGATATTTCAAATATAATATAAAACAACAAATAATTTAATGAAGAAAATGTTACTAAA AGCCTAAATAAAAGTACTAAATTTACCAAGATAAACATACCCCTAAAATTATTTATTATTTTTACTCGCTCCCTCG AAATAACAATAAGAAATATAATTCAAACACTTAAATAAACTATAATATAACTGACATAATCTATTCCTAAATTAAA TCCCAAACTATATATATATATATTATGAAAACATCTTAAACCAACTAAAAATCTTAATAATCCTAATCCTATCTGA GCTGACTTTCAGTCATCCTTTAATTTTATCAATAGAACTACAGGTAACAAAAACTTTAACATTCCAGAAGATTAAA TCTTTTAAAATAATCATTTCCATGCCTACGAACAATAAGAACTAATAAAGATAAACCAAGAGCTCCTTCACATACA GCTAAAGTTAAAAAAAATAAAGAAATGTAAATTTCCCTACCAACTATAGACAACTGTAACCTTAATAATCAAAAAA CTCCAAGCATTATAAATTCTAATCTTAACAACGAATTTAATAAATGTTTATTATAAGAAATAAATCTCCATAAACC ACAAAAAATTATAAATACCGCCCCAAACACTCTCAAAAATCTAAAATTTATCATTAGTTTTAATAGTTAATTTAAA AAACTAAAACATTGGTGTTAAAATCCTAAACATGAAATTTTTTCTTAAAACTCAAGAGAATTATTATCTCTATTAT TTTTATATTAACAATCCCCTTATTAATTGTTTCTTCCTTCTTTTTTATACAACTCTCTCACCCTTTGGCAATAGGC CTTATATTATTTATTCAAACAGTCTTAATTTCAATTACAATTGGAGTTTCCAACTTTTCTTTTTGATTTTCTTATA TTCTGTTTTTAGTGTTTTTAGGAGGAATACTAGTTTTATTCATTTATATTGCTTCTTTAGCTTCAAATGAATCATT TTCTTTTTCTTTAATTACTTTTTTCTTTGCTATTATTTTAACAGGTTTAATTATCTTTATTTTATTATTTTTAGAT CCTATCTTAATTTCTCCTAATTCTTCATCCCTTTCAACATCATCATTAAAATATTTAGTCTCGACCCCATTCATTG TTAATTGAGTTTATAATACCCCATCTATAATATTTACTATTTTTATTGTCTCCTATCTACTACTTATATTAATTGT TGTTGTTAAAATTATTAATTTGTTTAAAGGCCCACTTCGTCTTATATTATAATGACAACACCTATACGCAAATCCC ACCCTTTATTTAAAATTGCCAATGGAGCATTAGTAGATATACCTTTACCAAGTAACATTTCCACATTTTGAAACTT TGGTTCCTTGCTAGGCCTTTGTTTAGTTATCCAAATCGCGACAGGGTTATTCTTAGCTATACACTACTCTGCTCAC GTAGATCTAGCTTTTTCTAGAGTTGCCCACATTTGTCGTGACGTAAATTACGGTTGAATACTACGAACCCTTCACG CTAATGGGGCCTCTTTCTTCTTTATTTGCCTCTACCTTCATATTGGACGAGGCATCTACTTTAGATCTTATATAAA CCTTCATGCTTGAATAGTAGGGGTAGTAATTTTATTTATAATTATAGCGACTGCATTTTTAGGCTATGTTCTACCA TGAGGACAAATATCTTTTTGAGGCGCTACAGTTATTACAAATTTATTTTCCGCTATCCCATTTATCGGTACCGACC TAGTACAATGAATCTGAGGAGGGTTTTCTGTTGATAATGCCACTTTAACTCGATTTTTTTCATTCCATTTTATTTT ACCTTTAGTAGCATCAGCATTTTCTATAATTCACATTCTATTTTTACACCAAACAGGAGCTAATAACCCCTTAGGT ATTTCAAGACAAACTGATAAAGTACCATTTCATCCTTACTTTACATTTAAAGATATTGTTGGATTTGTAGTTATAT TAACAGCCCTAATTTTATTAACTCTTCTAGCCCCATATTTTCTAGGAGATCCAGATAATTTTATTCCTGCAAACCC TTTAGTAACCCCTCCTCATATCCAACCAGAATGGTATTTTCTTTTTGCTTACGCTATTTTACGATCAATTCCTAAT AAATTAGGAGGAGTTGTAGCATTAGCCGCTTCAGTGGCCATTCTTATAATTTTACCATTTACACACACTTCAAAAT TTCAAAGATTAGCATTTTACCCCATTAATCAAATGTTATTCTGAACCTTTGTAGTCATTGTAGTACTATTGACCTG AATTGGAGCACGCCCGGTTGAAGACCCTTATGTCATTACTGGACAGATTTTAACCGTATCCTATTTCTTGTTCTAT ATTATTAATCCTCTTATTCTTATTTGATGAGATAACATACTTAAATGACTATTTAGTTTAAAAAAAACAAATGTTT TGAAAACATTAAATAAGAATAAATTCTTAATAGTCGAGTGTTTCTTAAATAACTATTTACATTTATAAAATTTTAA TATCTTCCTATAAACTTAATTTTTACTCCCATTACTTCTAATATAGTGCCTGATAAAAGGATAGTTTTGATAGAAC TAATTATGTAATTTCCAGTTACCTATGTTATTAAAATCTTTATTAAAAAGATAAGCTAAATAAGCTAATGGGCTCA TACCCCGTAAATGAAAGTTTAATCTCTCTCTTTTTAATCGCTTTTCCTACCTCTTATTTCTTTTTCCTAATAACAC TAATCTCAGGGTCAATTATCTCTATTTCCTCAACTTCCTGATTTGGAGCTTGAATTGGATTAGAACTAAATCTCAT ATCATTTATTCCTCTTATTGCTTTTAAAATAAACCCACTTTTTTCTGAAGCAGCTTTAAAATATTTTCTTATTCAG GCATTAGGATCTGTGCTATTTATTTCTTCAAGATTCCTTTTAATGTCTTTTTATTCTTTTAGACTTTTATCTATTT TTTTAGCCCTCTTATTAAAACTAGGAGGAGCCCCTTTCCACTTCTGATTTCCTCAAGTTATAGAAGGTCTAAAATG ACCACAAATCTTCCTTCTATCTACAATTCAAAAACTAGCCCCCTTAACCTTAATTTCTTACCTAATAAACAACGAA ATCTTAATTAAAATTACAATTTTTTCAGCTATTTTATCAGCTTTAATCGGAAGACTTGGTGGTTTAAACTTAACCC TATTACGAAAAATTATTGCCTTTTCTTCAATTAATCACTTATCTTGAATATTAATAGCAATCTCAATTAGAGACTC CTCATGACTTTTTTATTTTTTTATTTATTCCCTTATTGTACTTTCTATTACCAGAGTATTTCATAAATTACAAACC TTCTCCCTTTCTATACTAATTCAATCTGATCAAAATAGAACACTTCACGCTACTATTATTTCACTTAATTTTTTAT CCTTGAGTGGTCTTCCACCTATAACAGGATTTATTCCCAAATGAATTGTTATTCAAGTTATATTAAATTTAAATAT ATTTATCCCCTTATTTTTTCTTCTTGTTTCTGCTTTGATTACTTTATATTTTTATTTGCGAATCATCATTACCATA ATCTTAATATTCAACCCTATTTTAAATTTTAATATGAAATATAAATCTCTAACAGATTCCCCATCTTCTCTATTTT TTAAATCCTCCTTCAACTTTTTTGGTCTTCTACTACCAGTATATTTCACCTTTATCTAGAATTTTAAGTTATTTAA ACTAAAAGCCTTCAAAGCTTTCTAAAAAAGTATTAACCTTTTAAATTCTATATTTTAATTATTATGCAACGATGAC TTTTTTCTACAAATCATAAAGATATTGGAACATTATATTTTATTTTCGGTGCTTGGGCCGGAATAGTAGGAACATC ATTAAGATTAATTATTCGAGCTGAATTAAGACAACCAGGCAGACTAATTGGTAACGATCAAATTTATAATGTAGTT GTAACAGCTCACGCTTTTGTAATAATTTTTTTTATAGTAATGCCCATTATAATTGGTGGATTTGGCAACTGACTTG TCCCTCTTATACTAGGGGCCCCAGATATAGCTTTCCCACGAATAAACAATATAAGATTCTGACTTTTACCCCCTTC TCTCTCTCTTTTACTTACAAGAAGAATAGTAGAAAGAGGAGTTGGTACTGGGTGGACTGTCTACCCACCTTTAGCA GCAGCCATCGCTCATGCTGGAGCATCAGTTGATCTTGGTATCTTCTCTCTACATCTTGCCGGAGTTTCCTCAATTT TAGGAGCTGTTAATTTCATAACAACAGTTATTAACATACGATCATATGGAATAACAATGGATCAAATACCTCTTTT TGTTTGAGCTGTATTTATTACTGCTATTTTACTTCTCTTATCCCTTCCAGTTTTAGCTGGAGCTATTACCATACTG CTTACAGATCGTAACCTAAATACGTCATTTTTCGACCCAGCAGGGGGAGGTGACCCAGTTCTATACCAGCACTTAT TCTGATTTTTCGGTCACCCAGAAGTTTATATTCTAATTTTGCCTGCCTTCGGTATAATCTCTCATATTGTAAGTCA AGAATCAGGTAAAAAGGAATCTTTTGGAACTTTAGGTATAATTTACGCTATACTAGCTATTGGAATTTTGGGCTTT GTAGTATGAGCTCATCATATATTTACAGTAGGTATAGACGTAGATACCCGAGCTTACTTTACCTCAGCCACTATAA TTATTGCTATCCCCACCGGAATTAAAATCTTTAGATGACTAAGGACCTTACATGGAACTCAAATAAATTATTCACC ATCTCTTCTATGAGCTTTAGGGTTTATCTTTTTATTTACTATTGGAGGTCTGACAGGAGTAGTTTTAGCTAATTCC TCAATTGATATTATTCTCCATGATACTTATTATGTTGTTGCCCACTTTCATTATGTACTCTCTATGGGAGCAGTTT TCGGAATCTTTGCTGGAGTTGCTCACTGATTTTCTCTAATAACAGGTTTATCTTTCAACCCTAAATGACTAAAAAT TCACTTTTTAGTAACTTTCGTCGGAGTCAATTTAACATTTTTTCCCCAACACTTTTTAGGATTAAACGGCATACCA CGACGATACTCTGATTATCCTGACGCCTATGCAACTTGAAATATTGTATCATCTATAGGTTCTATAATTTCTTTTG TAGCCGCATTAGGATTCTTATTAATCGTTTGAGAAGCATTTGTTTCTAATCGCCCAGTAATCTTTTCTCCTTTTCT GCCCTCTTCTATTGAATGGAAACATGCTTATCCTCCAGCCGACCATTCATATATGGAAATCCCACTAATTACTAAC TTCTAAAATGGCAGACAGATGTGTAGGATTTAAGCTCCTATAAGGAAGAATAATTTCTTCTTTTAGAATAACTTAT GGCAACATGAGCGTATTTAAATTTTCAAGACAGAGCTTCTCCTCTTATAGAACAATTAATTTTTTTTCACGATCAT ATTATATTAGTGATTGTGTTAATTGTAACATTTGTTGGTTATATAATAGCAACATTGTTTTTCAACTCCCTCATTA ACCGCTATATATTAGAAAATCAACCAATCGAAGTAATTTGAACATCAGTTCCAGCTTTTATTTTAATCTTTATTGC TTTACCATCCCTTCGCTTACTTTATCTTTTAGATGAAGTAAACAATCCTTCTATAACTCTTAAAACAATTGGCCAC CAATGATATTGAAGGTATGAATATTCAGATTTCCTTCAAGTAGAATTTGACTCATATATAGTACCATCTAATGAGC TGGAAGTATCAGGGTTCCGGCTACTAGACGTAGACAATCGTACCGTTCTTCCAATAAATACTCAAATCCGAGTTCT AATTACAGCTGCAGATGTCATCCATTCCTGGACAGTCCCATCCTTAGGTATTAAAGCCGATGCAATTCCCGGGCGG CTAAATCAATCAAGGTTTTTAATCAACCGACCTGGACTATTTTACGGTCAATGTTCAGAAATTTGCGGGGCAAACC ACAGATTTATGCCCATTGTTATTGAAAGAACTTCTATTAACTCATTTCTAAATTGAATTTCTCTATCAATTGAAGC GTCACCCTAACTCATCCTTAGTTTTACTAATTTTTGGGTGAAACAATTCCTTCTGTGAATTACTATTTATGCCACA AATAGCACCTATTTATTGACTATTTATATTTTTCTTTTTTTTAGCCAGATTCCTATTATTTTTCGCTCTTAATTAC TTCATTAAACCTTTTTATAAAGTTAATTTTCTTCAAGATAATAAAAAACCTTTAATTTATAAACATTGAAAATTAT AACAAATTTATTCTCAATTTTTGAACCTTCTTCAAGAATCTTTAATTTATCGATAAATTGAATCTCAACTATCCTA GGATTGATATTCTTACCTTATATATACTGAGCATCCCCTTCTCGTTGATCTTTACTTTGGGGAAAAATTATTTCAA CCTTGCATAAAGAATTTAAAGCTCTTTTACAGCCTTCACATAATGGAGCTTCTATAGTATTCATTGCTTTATTTAG GTTAATTGTATTTAATAATTTTCTAGGCTTATTACCTTATGTATTTACTAGCTCTAGTCATATGGTAATAACATTA TCTTTATCTTTGCCCTTGTGAATTACTTTAATAACATTTGGGTGAATTAAACACACTAAACATATATTTGCCCATT TAGTTCCCCAAGGGACGCCTTTCGCTTTAATGCCATTTATAGTGTTAATTGAAACCATTAGAAATGTAATTCGGCC TGGTACCTTAGCAATTCGGCTAGCCGCTAATATAATTGCCGGTCATTTACTTTTAACCTTATTAGGAAATACTGGC CCTTCATTATCAACCTCCATCCTATTCATTTTATTGTCAGCTCAAATACTCCTACTAATCTTAGAATCTGCCGTAG CTATTATTCAATCTTATGTTTTCGCTGTTTTAAGAACTCTCTACACTAGAGAAATTAACTAATGACATCATCACAC GGCTTTCATCCTTATCATTTAGTAGATATTAGACCCTGACCAATTACTGGGTCTATTAGAGCTATAATACTAACCA CTGGTTTAGTAAAATGATTTCACCAACACAATATAAACCTTTTAATTTTAAGATTTATTGCAACTTTGTTAACTAT AATTCAATGATGGCGAGATATTACCCGTGAAGGTACTTACCAAGGATCACACACTTTAACAGTAATAAAGGGGCTA CGCTGAGGTATAATTTTATTTATTGTATCAGAAGTTTTATTTTTCTTTTCTTTTTTTTGAGCTTTTTTCCATAGAA GACTATCTCCTGCTGTAGAAATTGGGATATATTGGCCGCCATTAGGTATTCAACCTTTTAACCCTTTCCAAATCCC CCTTCTAAACACTACGATTCTTCTATCGTCAGGTGCTACAGTTACTTGGGCTCATCACGCAATTCTAGAGGGTAAC TACTCTCAAGCTGTACAAAGACTAGGTTTAACTATTATCCTAGGTATTTATTTTACAATGCTCCAAGCATTTGAAT ATATTGAAGCTTCTTTCACTATTGCCGACTCAGTCTTTGGTTCAACATTTTTTGTAGCAACTGGGTTCCATGGATT ACACGTTATCATTGGAACTTCATTTTTATTTGTTTGTTTTATTCGACTTTATAAATGTCACTTTTCTTCTTCTCAT CATTTAGGATTTGAGGCAGCTGCTTGATACTGACACTTTGTTGATGTTGTTTGATTATTCTTATATATTTTCATTT ATTGATGAGGCGGATAATTTTTTTAATATAATAAGTATATCTGATTTCCAATCAGAAAGTCTAACTTTTAGAAAAA ATAATCTTTACAATATTGACAATTTCAGTTATTACTCTCATGGTTTCATATATCGTTATAACTTTAGCGACCCTCT TATCTAAAAAGACAATTATAGATCGAGAAAAAAATTCTCCGTATGAATGTGGATTCGATCCCAAAGGATCCGCCCG TCTACCTTTTTCTCTACGCTTTTTTTTAATTGCCGTAATTTTCCTAATCTTTGATGTAGAAATTACACTTCTGCTG CCTATTGCTTCTACATTAAACCTAACCAATATGTTTTCTTGATTTTTTACAAGTTTAATTTTTTTATTAATTTTGT TATTTGGTCTTTATTATGAATGATCACAAAGTGCTCTAGATTGATCTTAGTAAGACCATAGTCAATATATGACGTA TGAGTTGCATTCATAAAGAGTTTTCTAAACTGGTTTTAAAAATTAGAAAGCGAATAATTGCATTTAGTTTCGACCT AAATTTTAGACTTTAGTCTTCTAATTTTGGTGGAAACCAAAAAGAGGTATATCACTGTTAATGATAAAATTGAGTT CAAACTCCTACCAAGAAGGGATATTATGCTAAAGGCAGAAAGCTTCTAACTTTTTACTAAGCGGTTAAATTCCGTT TATATCCCTTTGTTATATATATGTACATATCTATCAACCTAGCCCTAGCACTTTGTTTTTATAGTTTAAACCAACC CAAAACATTGGTCTTGTAAACCAATAATGAATTATTTTCTAAAAACTTTCAGAAAAAAAGAGTAACTCTTTATCTT TAATTCCCAAAATTAATATTTTTTAAACTATTTCCTGAATTTATTACTCTAAATTTTATGCTGCTAGACAAAAACA AAACATTTTAAATCTAACAAAAAAAATCAAATAATTAATAGAAACAGGAAGATAACTTTTTCATGCTAAATATATC AATTTATCATACCGCAGTCGAGGTAAAGTACCTCGGACTCATACAAACCCAAAAGCTACTATTACACTCTTAAAAT AAAACATTACTCTGAACGGCCCCCTACCCAAAAAAAAAATAACAAATAAAACCCTCATGAATAGAATACTTGCGTA TTCAGCTATAAAAATTAATGCAAACCCGCCAGCCCCGTACTCTGTATTAAAGCCAGAGACTAGCTCAGATTCACCT TCAGAAAAATCAAATGGAGTTCGATTAGTCTCAGCTAAACATGACCTAAATCACACCATTCTTAAAGGAAATCTAA TTACAACAAATCAAAAATAATTTTGATATTTATTAAACAACTCTAAATTAAATCCACCAATTAATATTACAAAAGA TAATAAAATTAAAGCTAACCTAACTTCATAAGAAATAGTTTGAGCTACCGCTCGCAAAGAACCTAATAAAGAATAT TTACAATTTGATGATCATCCCGCCACTATAGTTGAATACACCCCTACACTTAAACAACACAAAAAAAACAGAATTC TCAGATTAAACCTTATTAATCCAGTTTCATAAGGTAAAACTGATCATACTAATAAAGAAATAAACAAACTAAATAC AGGACATAAATAGTAAACTAAAAAGTTTGATATAGTAGGAATAATTTGTTCCTTAGTGAAAAGCTTTACTGCATCA GAAAAAGGCTGTAAAATTCCTATATAGCCTACTTTATTTGGGCCTTTACGAATTTGAATATAACCTAAAATTTTTC GTTCAAGTAAAGTTACAAAAGCCACGCCAATTAAAACACACACTATTAATACCAAAAAATTAACTATCTCTACAAT TATTAAAGTCACAAAACAATTAGATTACTAATAATTTAACTATTTATAGAATTGCTCTATTTTATAGGATCCTAAA TCCAACACATATTATCTGCCAAAATAGTAATCATTATATAAAATAATTTTAATTATTTAATCCTTTCGTACTAAAA TAATTTTTCTTTATTAAAAGATAGAAACCAACCTGGCTTACGCCGGTTTGAACTCAAATCATGTAAAATTTTAAAG GTCGAACAGACCTACTTATATAACTGCTACAATTATAAAGATATTTTAATTCAACATCGAGGTCGCAAACTTTTCT TTCGATAAGTACTCTTAAGAAAAATAACGCTGTTATCCCTAAAGTAACTTAAACTTTTAATCTTTAATTAGGATCA TTTAAAATTTTAATCACATATAAGTGTACTTAATGTTAAGCAGTTAATAATCATTTTACTCTTATCGCCCCAATAA AATAAATTCAATTTACAAAATCTTTATAACCTAAAATTTGACTATATAAAATTTAATATTAAGCTTTATAGGGTCT TATCGTCCCTTTAATACATTTAAGCCTTTTCACTTAAAAGTAAAATTCAATAAACAATATAAAGACAGATCTTCTT TTGTCCAACCATTCATACAAGATTCCAATTAAAAAACTAACGATTATGCTACCTTTGCACGGTCAAAATACCGCGG CCATTTAATACTATCAATCAGTGGGCAGGTTAAACTTTTTATAACTTCAAACAGACATGTTTTTGATAAACAGGCA AAGAAGTTATTTTTGCCGAGTTCCTTTATTCCGCCCTATCAATTTTAAACAATATTTATTATTTAAATGTTTTGCT TTAATAAAAAACTTTTACTAATAAAATCATTATAACTTAACTCTTTATATTTAAATTAATTTTTTAATACTCCAAA AAAGTTTATATAAATATTATCCAATTTTAATTTAGCTAGAACACTTTATAAATATAGCTACTTTTAAGCCTAATTC ACCAATTAAACTATTTTAAACTATTTATCATTTTAATTATAAGAAGCTAATCCCTCCTACACTTAAATTTTACATC TTATCAACGTAAAAATTAAATTAAATTTACTTTTTAAAAAACTAGATATAATAAAACTCGACTAGCGTTTCACCTT TAATTTTATATTAAAAATTTATTTTCTACAAAAAATTTTTTATAAAATTAGCTATATTTTATTTCGAGATATTTAA TTTTTATTAACTTATATAAATTTTCCCTGATACACAAGGTACAATATTTAAAATTTTTTACTTCAAATTTTCTTGC TTTCCTTTACAGTACTCTTAACTATAGTTTATAAATTGTGTTTATTAAAAATTACTAGCATGAACAAATTACAAAT TATTTTTCATAAATATAAAATTTTCAAATTTATTTAACAAGTCATTAATAAATAGATGGGTAATTATTAATTTATA AAGAAAATATACATATCCTCATCTTTACTAGATACACTTTCCAGTACATCTACTATGTTACGACTTATTTCACTTA TAAATGAAAGCGACGGGCGATATGTACACAATTTAGAACTTATATCTTGTTAGCTTATTAAACTAAAAATACAATC AAATCCATCTTTATAATAATATTATCTTATTAATCCGCAGTAAAGTAATTTACTGTAACCCATTTTACCTTGCTTA TTGGCTGCACCATGATCTAATTTTACTAAACAACTAAATTTAATAATTTTTCCTTTAAGAATTATCTGATAATGAT GGTATACAAACTGTTAGAATTACTAAAGCAAGTAAGATTTTTCGTGGTTTATCGATTCTAAAACAGGTTCCTCTGA ATAGATTAAATTGCCGCCAAATTTTTTAAATTTTAAGTATTTATCTTTTACTAATTCAGTTTTATATTAAATTTAT TTTAGAATAATAGGGTATCTAATCCTAGTTTATTTCTAAACTTTTTTAGCTTCAATTATAATTTTACTCCATACAT AATAATATCCTAATTTTACCTTTTATTACCTGTATGAGATTTTTAATTTAATCATCTAACTATAACCTTAGAATTT TTACTCAATCTTAAAGTATAACCGCGAATGCTGGCACAAATATATATCAGATTTTAATATTATTACTATATCACAC CTTCATGTATAATTTTATAATATTTTATGCTGAGACAATAAAGGTTTATAATTTTATTTCACTCGAATACCAATTT AATATTTAATTAAATTGTTTAAATCACACTTTAATTTTCATACAATTTTAATAATAATGTAAAAACTTAAGCCAAA ATCAAACATTTGTCGTGATTGCAAATAATAACTTTAAAAAACTTAAATATACTATATATAACATAAAAATGAGAAT AAGCTTATACTTAAAGTATAACTTATACATCTTAAGCGCCACAAGCTAACGTTTTAATATTTTAAACTATTTAAGT TTTGTTATAACTAATATTGCTACAAATATATTTATTATCTACACAAAAACAAACAATATAATCACCAACCCAACCA CAAAAATTTTTATAAACACTTTAATATTATTAAGTTGTAACTCATTTAACAACCTAAAATATTTCTTAAACAAAAA ATACATCCCCTGGCCACCAAAATATTCAAATCACCCTTTGTCTCCTAATTTTTCAACACCTACCCCACTAATGAGA CTAGACCCTCTAATTTTTTTTGTCCTAAGAAAAGGTATAAATCATATAGACCCCCTTATTACTACTATCTTATAAT TATTAATAGATTTTAAATTGTAACTTACATTTAATAAATTAAATATATAACCTATAACTCCCCCTGCTAATCTTAC CCCCAACACTATATTTTTCATTATTACATTCAAACAAATTATATAAGGCTCTGGGAAAATAACCCAAGATAGAACA GCTCCTATAAAAATAGCTCTTACACCTAGTAAAATCATAGAATTTCTTATAATTTTATCCTCATCCCTAATATTTA TTATAACTCTTAAATTTGAATTACCTCTTAACCTATAAAAAATTAGACGTATAGTATACATAACTGTTAACCCTGT AGCTAAAATATATAACAGTAAAGAAATAAAATTAATCCATCTCATAAAAGCCACCTCTAAAATTATATCTTTAGAA TAAAACCCTGCTAAAAAAGGGAACCCACATAAGGCTAAATTTGAAACTCTTATAAAAGACACTCTTAACGGCATAA ATTTAATTAAACCACCTATACATCGAATATCCTGATAATTGCCAACCCTATGGATAATAATACCTGCGCATATAAA TAACAATGCTTTAAATAATGCATGACTCAGCAGATGAAAAAAAGAAAGATCAGGGTATCCTAAAGCTAAAATACTT AACATAACCCCCAATTGACTTAACGTTGATAAGGCAATAATTTTTTTTAAATCATATTCAAAATTGGCCCCTAATC CCGCCATAAATATAGTTAAGCAAGAAATAATTAATAATATTCCTTGGATTTTAGAACCTTCTAACGCACCACTAAA TCGAATTATTAAATACACCCCAGCAGTTACTAAAGTAGAAGAATGTACTAAAGCAGAAACAGGAGTAGGAGCTGCT ATAGCGGCAGGAAGCCATGCTGAGAAAGGAATTTGAGCACTTTTAGTCATAGCTCTTAACATTAACAAAAACTTAA ACAAAATTCACTCAGTATCTATAATATAGTAAGAATAAATAACAAAATTTCATCTACCTAACCTGGCCATTAGTCC AATACTAAGTAAAATAGCAACATCACCTACTCGATTAGATAAGATAGTTAACATCCCAGCATTAGCAGACTTTTCA TTTTGATAAAAAATTACTAAAGCATAAGAAACTAAACCTAATCCATCCCATCCTAAAAGAATACTTACCAAGTTAG GTCTTAAAACTATTATAGCCATAGATAAAACAAACATTAAAACTAAATATATAAATCGATTAAAGTTTTTATCCTC CTTCATATATCTACCTCTATAATACATCACACTTCCAGAAATAAGCAATACAAAACACAAAAAAAGTATCGAAATT CAATCAAAAATTAAAACAAACACCACTGATAATGAATTTAATCTTGCTAAATCCCATTCAATTACACTAGTAACAC CACTAAATAATTTTATTAAAGAAACTGTTCCACAAACTAAAGACCCCGCTCATAGCCCAAGTATTCTAATCTTATG TATATAAATTATTCAAACCATTATTTAAATATTAAATTATTAATTTAATACAAAATACAGTAACATTTCTACCAAT GGTACAAAACAATTAGTAGTAATTAAATCTAAATAAATCAAAGCAAATCGATTTGCACGATAAATCTTTTCAACGT AACTGAAATGCTGTTCTATTTTAGCTCTGCTTTGACACTATCATATTATATTTCCAATCTACTTTACATTTAAATA ATTTATTAAAAAAAGCTACTAAAAATATTAAGAAATGATAATACTATTATATTATACAATTTTATTTTTCTTTATT TATACGGAGGTATATAAGGTATATATAAATATATCACATTAGATATAAGTAAATAATATAAACAAATCAAATATAT AAAAATTAAATATGTATATTCGCCTCCACAATTATACCCCATCCAATATAATACAGGATAGATAAAACATTAAAAT TATAAAAGGGTAAAATTATAAATAAATAGAAGTATATAATGTCTTAGTTTATATGGAACATTGAAACATTTTATAT AAAAGTAAAAATTATATGTAACTTCATTATATAAATATATGATTTATACTATTTTTATTTATTCTATGGGTTGAAA AAAAGTAGTAGTCTCAACCCTAGACCCTAATTCCTCTAGAGGGGAAACGGGTCTAGACCGTTGAGACTACTACTAT AATAGGGGCAAAGAACATTATATTATATTTAGGTAAATATAAATTAAATATTTAATTATACTAACAAATAATTTAA CATTAATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATG TATATATGTATATGTATATATGTATATGTATATATGTATATGTATATATGTATATGTATATATGTATATGCTTTGT ACTCTACAATATATAATATCTACTATATAC(SEQ ID NO.58)。

The upstream primer sequence of primer 1 is as shown in SEQ ID NO:1:

CATGCAACGGAATTGCAC(SEQ ID NO:1)。

The downstream primer sequence of primer 1 is as shown in SEQ ID NO:2:

GGGTATGGTTTGATTCGG(SEQ ID NO:2)。

The upstream primer sequence of primer 2 is as shown in SEQ ID NO:3:

GGACTTTATATCTACCTGGCG(SEQ ID NO:3)。

The downstream primer sequence of primer 2 is as shown in SEQ ID NO:4:

GTATGTGAAGGAGCTCTTGG(SEQ ID NO:4)。

The upstream primer sequence of primer 3 is as shown in SEQ ID NO:5:

GAGCTGACTTTCAGTCATCC(SEQ ID NO:5)。

The downstream primer sequence of primer 3 is as shown in SEQ ID NO:6:

GCTAAGAATAACCCTGTCGC(SEQ ID NO:6)。

The upstream primer sequence of primer 4 is as shown in SEQ ID NO:7:

TCTCGACCCCATTCATTG(SEQ ID NO:7)。

The downstream primer sequence of primer 4 is as shown in SEQ ID NO:8:

GTGCTCCAATTCAGGTCAA(SEQ ID NO:8)。

The upstream primer sequence of primer 5 is as shown in SEQ ID NO:9:

CCCCATATTTTCTAGGAGAC(T)C(SEQ ID NO:9)。

The downstream primer sequence of primer 5 is as shown in SEQ ID NO:10:

AGTACAGATCCTAATGCCTG(SEQ ID NO:10)。

The upstream primer sequence of primer 6 is as shown in SEQ ID NO:11:

AAGCTAATGGGCTCATACCC(SEQ ID NO:11)。

The downstream primer sequence of primer 6 is as shown in SEQ ID NO:12:

TAGGTGGAAGACCACTCAAGG(SEQ ID NO:12)。

The upstream primer sequence of primer 7 is as shown in SEQ ID NO:13:

CCTTGAGTGGTCTTCCACCTA(SEQ ID NO:13)。

The downstream primer sequence of primer 7 is as shown in SEQ ID NO:14:

CCAAGATCAACTGATGCTCC(SEQ ID NO:14)。

The upstream primer sequence of primer 8 is as shown in SEQ ID NO:15:

TATTTTCGGTGCTTGGGC(SEQ ID NO:15)。

The downstream primer sequence of primer 8 is as shown in SEQ ID NO:16:

AGATTATACCGAAGGCAGGC(SEQ ID NO:16)。

The upstream primer sequence of primer 9 is as shown in SEQ ID NO:17:

CTACATCTTGCCGGAGTTTC(SEQ ID NO:17)。

The downstream primer sequence of primer 9 is as shown in SEQ ID NO:18:

ACGCTCAGGTTGCCATAAA(G)(SEQ ID NO:18)。

The upstream primer sequence of primer 10 is as shown in SEQ ID NO:19:

TGGCAGACAGATT(G)TGTAGGA(SEQ ID NO:19)。

The downstream primer sequence of primer 10 is as shown in SEQ ID NO:20:

AAGATCAACGAGAAGGGGAG(T)(SEQ ID NO:20)。

The upstream primer sequence of primer 11 is as shown in SEQ ID NO:21:

C(A)TCCCCTTCTCGTTGATCTT(SEQ ID NO:21)。

The downstream primer sequence of primer 11 is as shown in SEQ ID NO:22:

TAACTGTAGCACCG(T)GACGAT(SEQ ID NO:22)。

The upstream primer sequence of primer 12 is as shown in SEQ ID NO:23:

TTGA(G)CCGCCATTAGGTATTC(SEQ ID NO:23)。

The downstream primer sequence of primer 12 is as shown in SEQ ID NO:24:

GACCAATGTTTTGGGTTGG(SEQ ID NO:24)。

The upstream primer sequence of primer 13 is as shown in SEQ ID NO:25:

CAAACTCCTACCAAGAAGGG(SEQ ID NO:25)。

The downstream primer sequence of primer 13 is as shown in SEQ ID NO:26:

GTGTGTTTTAATTGGT(C)GTGGC(SEQ ID NO:26)。

The upstream primer sequence of primer 14 is as shown in SEQ ID NO:27:

CTGCATCAGAAAAAGGCTG(SEQ ID NO:27)。

The downstream primer sequence of primer 14 is as shown in SEQ ID NO:28:

ACCTGCCCACTGATTGATAG(SEQ ID NO:28)。

The upstream primer sequence of primer 15 is as shown in SEQ ID NO:29:

GAAACCAACCTGGCTTACG(SEQ ID NO:29)。

The downstream primer sequence of primer 15 is as shown in SEQ ID NO:30:

TTGTGTACATATCGCCCGTC(SEQ ID NO:30)。

The upstream primer sequence of primer 16 is as shown in SEQ ID NO:31:

CTCGACTAGCGTTTCACCTT(SEQ ID NO:31)。

The downstream primer sequence of primer 16 is as shown in SEQ ID NO:32:

CCAGCATTCGCGGTTATA(SEQ ID NO:32)。

The upstream primer sequence of primer 17 is as shown in SEQ ID NO:33:

TATAACCGCGAATGCTGG(SEQ ID NO:33)。

The downstream primer sequence of primer 17 is as shown in SEQ ID NO:34:

TAGCCTTATGTGGGTTCCCT(SEQ ID NO:34)。

The upstream primer sequence of primer 18 is as shown in SEQ ID NO:35:

AGGGAACCCACATAAGGCT(SEQ ID NO:35)。

The downstream primer sequence of primer 18 is as shown in SEQ ID NO:36:

CGGGGTCTTTAGTTTGTGGA(SEQ ID NO:36)。

The upstream primer sequence of primer 19 is as shown in SEQ ID NO:37:

CATGCCTGAATAGTAGGAGT(SEQ ID NO:37)。

The downstream primer sequence of primer 19 is as shown in SEQ ID NO:38:

GTGCTCCAATTCAGGTCAAT(SEQ ID NO:38)。

Present invention will be further explained below with reference to specific examples.Specific embodiment is to be with technical solution of the present invention Under the premise of implemented, give detailed embodiment and operating process.It should be understood that these embodiments are merely to illustrate this It invents rather than limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal Rule condition carries out.Unless otherwise indicated, percentage and number be by weight.

Embodiment 1

Eriocheir sinensis sample used in the present embodiment picks up from the Yangtze river basin between in December, 2017 in October, 2017, Specified place: industry, longitude and latitude: 119.27 ° of E, 32.11 ° of N.Sample acquisition mode is that cage is arrested or manually Fishing.Musculature is taken out, its hetero-organization is avoided to pollute, and is stored in -40 DEG C of refrigerator stand-by.

The extraction of 1.1DNA: this experiment takes classical phenol chlorine method to carry out DNA extraction, specific step is as follows (following reagent It is purchased from Sinopharm Chemical Reagent Co., Ltd.):

1. taking sample musculature 50mg or so, alcohol is dried, shreds processing, and is placed in the centrifuge tube of 1.5ml；

2. 500 μ L buffer (100mmol/LNaCl are added to centrifuge tube；10mmol/L Tris-Cl, pH 8.0； 1mmol/L EDTA, pH 8.0), the Proteinase K of 50 μ L 10%SDS solution and 10 μ L 20mg/ μ L, then at once using grinding The broken bulk musculature of rod milling, 56 DEG C of water-baths digest (or so general 3 hours), until solution is limpid in centrifuge tube；

3. (volume ratio is for saturation phenol and chloroform/isoamyl alcohol mixed liquor of 250 μ L that 250 μ L are added into centrifuge tube 24:1), then jog 10 minutes, room temperature 13000r/min are centrifuged 8 minutes；

4. Aspirate supernatant is into new centrifuge tube, and it is 3. primary to repeat step；

5. chloroform/isoamyl alcohol mixed liquor (body of 500 μ L into new centrifuge tube, is added in Aspirate supernatant into centrifuge tube Product is than being 24:1), then jog 10 minutes, room temperature 13000r/min are centrifuged 8 minutes；

6. Aspirate supernatant is into new centrifuge tube, and it is 5. primary to repeat step；

7. Aspirate supernatant into new centrifuge tube, is added the ice dehydrated alcohol of 50 μ L 3mol/L NaAc solution and 1mL, puts It is placed in -20 DEG C of refrigerators 1 hour, takes out centrifugation 13000r/min and be centrifuged 8 minutes, obtain nucleic acid precipitating；

8. the ice ethanol washing using 75% precipitates twice；

9. precipitating finally, being washed using ice dehydrated alcohol, after natural drying to be precipitated, the TE buffer that 50 μ L are added is molten Solution, and the ribonuclease A (concentration 0.05mg/mL) that 0.2 μ L is added removes RNA.

The quality testing of 1.2 DNA:

DNA is extracted from muscle according to 1.1 method, takes 2 μ l DNA samples in the (U.S. ultraviolet specrophotometer Q5000 Quawell company) on carry out concentration mensuration.DNA concentration requirement OD260/OD280 ratio is measured between 1.8~2.0, For OD260/OD230 ratio between 1.8~2.2, two above ratio may determine that the superiority and inferiority of extracted DNA purity.If super Above range out needs to extract again or repurity then it is believed that extract DNA purity undesirable.By quality testing qualification DNA sample according to the concentration of measurement, be diluted further to concentration 50ng/ μ l with DNA dissolution buffer, it is spare.

1.3 construction of gene library, high-flux sequence:

After sample gene group DNA detection is qualified, DNA is interrupted with physical method (ultrasonic wave), then fight each other the DNA having no progeny Purifying building sequencing library is carried out, step is successively are as follows: the end DNA is repaired, 3 ' ends plus A, connects sequence measuring joints, Ago-Gel electricity Swimming method recycles target fragment, carries out PCR amplification to target fragment, finally builds up sequencing library.To the library built before sequencing Quality inspection is carried out, is sequenced after quality inspection is qualified using Illumina HiSeq TM platform.

1.4 raw sequencing data quality evaluation:

Using biological information statistical method, the base distribution and quality fluctuation of all sequencing reads are counted, from It macroscopically can intuitively reflect sequencing sample sequencing quality.Fig. 1 is the base distribution figure of machine under initial data, this is reflected The distribution situation of 4 kinds of bases of A, T, G, C of high-flux sequence, it is good that base more uniformly represents sequencing result.Fig. 2 is initial data Base quality distribution diagram, in general, mass value are more than that the base ratio of 30 (sequencing error rate is less than one thousandth) is more, high The quality of data of flux sequencing is better.

The splicing of 1.5 mitochondrial genomes and annotation

Based on reference species chondriogene group sequence (eriocheir japonicus (Eriocheir japonica) (FJ455505.1), the closely square crab (Hemigrapsus sanguineus) (KX456205.1) of meat ball, Helice wuana (Helicana Wuana) (KX344898.1), Eriocheir sinensis mitochondrial genomes sequence (Eriocheir sinensis) (KP126617.1)), the survey read of high quality is filtered, except stoning sequence dna fragment；Utilize NOVOPlasty (https: //github.com/ndierckx/NOVOPlasty) splicing software carries out the splicing of more Kmer parameters, chooses optimal Assembling result；It is predicted using encoding egg white gene of the blast software to the mitochondrial genomes of each individual, using special Barrnap0.4.2 the and tRNAscan-SE v1.3.1 software of industry is respectively to rRNA and tRNA within the scope of mitochondrial genomes It is checked and accepted and is predicted.

1.6 Eriocheir sinensis mitochondrial genomes specific primer designs:

Referring to nearly edge species chondriogene group sequence (eriocheir japonicus (Eriocheir japonica) (FJ455505.1), the closely square crab (Hemigrapsus sanguineus) (KX456205.1) of meat ball, Helice wuana (Helicana Wuana) (KX344898.1), Eriocheir sinensis mitochondrial genomes sequence (Eriocheir sinensis) (KP126617.1)) it, using Primer premier version 6, is designed in 19 pairs of coverings in conjunction with inventor's experience The primer (referring to table 1) of magnificent Eriocheir mitochondria complete genome DNA sequence, primer sequence length range is within 1.2kb, phase The certain overlapping region of adjacent segment.

Table 1.PCR expands the primer:

Note: the above primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd.

1.7 carry out PCR amplification using the above primer:

PCR amplification uses Eppendorf thermal cycler, and it is 50 μ L that PCR, which reacts total volume, each including forward and reverse primer 2 μ L, 2 μ L of DNA profiling (concentration is 50ng/ μ L), 19 μ L, Taq PCR MasterMix of DNase-Free deionized water, 25 μ l (purchase is in Novoprotein Biotech company).The response procedures of PCR amplification are as follows: 94 DEG C initial denaturation 5 minutes；94 DEG C of denaturation 30 seconds, 55 DEG C were annealed 30 seconds, and 72 DEG C extend 1 minute, and totally 35 recycle；After circulation terminates, extending 10 minutes for 72 DEG C, 10 DEG C of ends Only react.The sequence actually obtained is sequenced by Sangon Biotech (Shanghai) Co., Ltd..Each primer PCR expands institute Sequence is obtained referring to table 2.

The pretreatment of 1.8 PCR product sequences:

For the reliability for ensuring sequence, when sequence assembly, sequencing quality need to be assessed, removal sequencing result both ends Low quality part, sequence direction should be consistent with PCR amplification forward primer direction.

The mitochondrial genomes of 1.9 pairs of splicings are corrected

After comparing with the Eriocheir sinensis mitochondrial genomes sequence (KP126617.1) announced, second generation sequencing is obtained Eriocheir sinensis mitochondrial genomes in often will appear overlap, gap and faulty sequence, at this time pass through the first generation survey Correlated series can be accurately positioned in sequence, and carry out artificial correction using 5 software of MEGA, finally can get complete Eriocheir sinensis Mitochondrial gene sequence (SEQ ID NO:58).

Other sequences of mitochondrial genomes other than the gene order of mitochondria control region are sequenced in the second generation that the present invention uses The error rate of column is 0.0173% (gene order of control zone, the present invention use first generation sequencing repairing), and the second generation is sequenced After original series and the reference sequences comparison of acquisition, it is sequenced with product of the first generation sequencing technologies to PCR amplification, and with Subject to the sequence, artificial correction has been carried out to the exception bits point of second generation sequencing sequence, specifically: extra 1- is cut off first 369 and 16808-17028 segment, obtains the sequence of 16438bp, then carries out artificial correction to the sequence spliced, wherein the " G " in 373 sites, manual amendment are " A ", and " A " in the 6442nd site is revised as " G "；" T " modification in the 9181st site For " C ", revised sequence is referring specifically to SEQ ID NO:58.

Resulting Eriocheir sinensis mitochondrial genomes DNA sequence dna is sequenced according to the present invention to give birth to the Eriocheir sinensis Object credit class show that the Eriocheir sinensis is under the jurisdiction of Arthropoda (Arthropoda), Crustachia (Crustacea), full Mesh (Decapoda), Grapsidae (Grapsidae), Eriocheir (Eriocheir).

Method of the invention applies also for the use of research and production unit, can fast and accurately measure Eriocheir sinensis category The mitochondria whole genome sequence of species improves scientific research and the genetic marker exploitation of production unit, germ plasm resource assessment and people The efficiency of work nursery etc., has great significance to scientific research and industry.

The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its Equivalent thereof.

Sequence table

<110>Shanghai Ocean University

<120>method based on high-flux sequence measurement Eriocheir sinensis mitochondria full-length genome

<130> 2018

<160> 58

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213>artificial sequence ()

<400> 1

catgcaacgg aattgcac 18

<210> 2

<211> 18

<212> DNA

<213>artificial sequence ()

<400> 2

gggtatggtt tgattcgg 18

<210> 3

<211> 21

<212> DNA

<213>artificial sequence ()

<400> 3

ggactttata tctacctggc g 21

<210> 4

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 4

gtatgtgaag gagctcttgg 20

<210> 5

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 5

gagctgactt tcagtcatcc 20

<210> 6

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 6

gctaagaata accctgtcgc 20

<210> 7

<211> 18

<212> DNA

<213>artificial sequence ()

<400> 7

tctcgacccc attcattg 18

<210> 8

<211> 19

<212> DNA

<213>artificial sequence ()

<400> 8

gtgctccaat tcaggtcaa 19

<210> 9

<211> 21

<212> DNA

<213>artificial sequence ()

<220>

<221> misc_feature

<222> (20)..(20)

<223>n=c or t

<400> 9

ccccatattt tctaggagan c 21

<210> 10

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 10

agtacagatc ctaatgcctg 20

<210> 11

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 11

aagctaatgg gctcataccc 20

<210> 12

<211> 21

<212> DNA

<213>artificial sequence ()

<400> 12

taggtggaag accactcaag g 21

<210> 13

<211> 21

<212> DNA

<213>artificial sequence ()

<400> 13

ccttgagtgg tcttccacct a 21

<210> 14

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 14

ccaagatcaa ctgatgctcc 20

<210> 15

<211> 18

<212> DNA

<213>artificial sequence ()

<400> 15

tattttcggt gcttgggc 18

<210> 16

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 16

agattatacc gaaggcaggc 20

<210> 17

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 17

ctacatcttg ccggagtttc 20

<210> 18

<211> 19

<212> DNA

<213>artificial sequence ()

<220>

<221> misc_feature

<222> (19)..(19)

<223>n=a or g

<400> 18

acgctcaggt tgccataan 19

<210> 19

<211> 20

<212> DNA

<213>artificial sequence ()

<220>

<221> misc_feature

<222> (13)..(13)

<223>n=t or g

<400> 19

tggcagacag atntgtagga 20

<210> 20

<211> 20

<212> DNA

<213>artificial sequence ()

<220>

<221> misc_feature

<222> (20)..(20)

<223>n=g or t

<400> 20

aagatcaacg agaaggggan 20

<210> 21

<211> 20

<212> DNA

<213>artificial sequence ()

<220>

<221> misc_feature

<222> (1)..(1)

<223>n=c or a

<400> 21

ntccccttct cgttgatctt 20

<210> 22

<211> 20

<212> DNA

<213>artificial sequence ()

<220>

<221> misc_feature

<222> (14)..(14)

<223>n=g or t

<400> 22

taactgtagc accngacgat 20

<210> 23

<211> 20

<212> DNA

<213>artificial sequence ()

<220>

<221> misc_feature

<222> (4)..(4)

<223>n=a or g

<400> 23

ttgnccgcca ttaggtattc 20

<210> 24

<211> 19

<212> DNA

<213>artificial sequence ()

<400> 24

gaccaatgtt ttgggttgg 19

<210> 25

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 25

caaactccta ccaagaaggg 20

<210> 26

<211> 21

<212> DNA

<213>artificial sequence ()

<220>

<221> misc_feature

<222> (16)..(16)

<223>n=t or c

<400> 26

gtgtgtttta attggngtgg c 21

<210> 27

<211> 19

<212> DNA

<213>artificial sequence ()

<400> 27

ctgcatcaga aaaaggctg 19

<210> 28

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 28

acctgcccac tgattgatag 20

<210> 29

<211> 19

<212> DNA

<213>artificial sequence ()

<400> 29

gaaaccaacc tggcttacg 19

<210> 30

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 30

ttgtgtacat atcgcccgtc 20

<210> 31

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 31

ctcgactagc gtttcacctt 20

<210> 32

<211> 18

<212> DNA

<213>artificial sequence ()

<400> 32

ccagcattcg cggttata 18

<210> 33

<211> 18

<212> DNA

<213>artificial sequence ()

<400> 33

tataaccgcg aatgctgg 18

<210> 34

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 34

tagccttatg tgggttccct 20

<210> 35

<211> 19

<212> DNA

<213>artificial sequence ()

<400> 35

agggaaccca cataaggct 19

<210> 36

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 36

cggggtcttt agtttgtgga 20

<210> 37

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 37

catgcctgaa tagtaggagt 20

<210> 38

<211> 20

<212> DNA

<213>artificial sequence ()

<400> 38

gtgctccaat tcaggtcaat 20

<210> 39

<211> 1194

<212> DNA

<213>artificial sequence ()

<400> 39

catgcaacgg aattgcaccg tttcttaaaa gatcaaaact tcttatgctc tttacatcag 60

catataaatt attttcaaac cctagtgatt tacacatctt taaacttgca atttaatatt 120

atttttatac tatagggcct aataaaggaa tctcattcgt ctacagattt acaatctgcc 180

gcttacaact cagccacttt attaattttt aaataaatag tatactcgat gactgaaagg 240

taagtataga tcttttaaat ctaatatagt aatttaacat ttacttcgag taaggaatta 300

gttaaaatat aataacattt gcttgtcatg caaaagtcat ctttaagata ctccttaaat 360

ttcagtttta tttgttttat ggtgtagata acacattgta ttttcgttac aaaaccgaaa 420

ctaatgtttc tagaacatta taatactttg cccacagata atatttatct cttttgcagc 480

ttcaatgcaa tattctatat aaattatata agctacttat attaatctag ctttacgtta 540

tacgccatta actaaattta aatttaaaat taacatattt aaaggaaatc aatgtaaaaa 600

tagaactaaa tattctcgaa cttttcctga cctacaagaa tataaactac tgacgaaaat 660

cccatgctga cttaaactat atatgtataa agtataagct gctctgaaaa aagaaagaaa 720

tataattccc attactgtaa atgtccttca ccttattaaa ctaataatca acctaatctc 780

cccgaataga tttaaagtag ggggagcagc catattaccc actctaaata aaaatcatca 840

caaacctatc ctaggtataa aatttaataa acccttacta actaaaagac tacgcctacc 900

aactcgctcg tatactatat tagccaaaca aaataatcca gaagaacata acccgtgtcc 960

gactattact acaacagctc ctattagtcc tcatcatcta aaaatcatta gaccacatag 1020

tactattctc atatgaacaa ccgaagaata agcaatcaag gactttatat ctacctggcg 1080

tagacacaaa agactcacta ctactccccc tactaaacta attctaaccc ataacgacga 1140

aaaattcaag ctaattttct gaaatatcaa taacacccga atcaaaccat accc 1194

<210> 40

<211> 905

<212> DNA

<213>artificial sequence ()

<400> 40

ggactttata tctacctggc gtagacacaa aagactcact actactcccc ctactaaact 60

aattctaacc cataacgacg aaaaattcaa gctaattttc tgaaatatca ataacacccg 120

aatcaaacca tacccgccta actttaataa aactcctgct aaaattatag aaccagcaac 180

cggggcttct acgtgagctt ttggcaacca taaatgaaat atatatatcg gtaatttaac 240

taaaaaagcc atcacccttc taaaatatca aatactgttt gcccagctaa cttctaaaat 300

aagatttctt aatattataa ctaacctata ttcttttata taaatataca ataaagaccc 360

caataaaggt aaagatagtg acaaagtata aaaaagtata taaactccag cttgaattcg 420

ttcaggttga tatccccatc caagaatcaa aattaaagtg ggaattaacg atatttcaaa 480

tataatataa aacaacaaat aatttaatga agaaaatgtt actaaaagcc taaataaaag 540

tactaaattt accaagataa acatacccct aaaattattt attattttta ctcgctccct 600

cgaaataaca ataagaaata taattcaaac acttaaataa actataatat aactgacata 660

atctattcct aaattaaatc ccaaactata tatatatata ttatgaaaac atcttaaacc 720

aactaaaaat cttaataatc ctaatcctat ctgagctgac tttcagtcat cctttaattt 780

tatcaataga actacaggta acaaaaactt taacattcca gaagattaaa tcttttaaaa 840

taatcatttc catgcctacg aacaataaga actaataaag ataaaccaag agctccttca 900

catac 905

<210> 41

<211> 1123

<212> DNA

<213>artificial sequence ()

<400> 41

gagctgactt tcagtcatcc tttaatttta tcaatagaac tacaggtaac aaaaacttta 60

acattccaga agattaaatc ttttaaaata atcatttcca tgcctacgaa caataagaac 120

taataaagat aaaccaagag ctccttcaca tacagctaaa gttaaaaaaa ataaagaaat 180

gtaaatttcc ctaccaacta tagacaactg taaccttaat aatcaaaaaa ctccaagcat 240

tataaattct aatcttaaca acgaatttaa taaatgttta ttataagaaa taaatctcca 300

taaaccacaa aaaattataa ataccgcccc aaacactctc aaaaatctaa aatttatcat 360

tagttttaat agttaattta aaaaactaaa acattggtgt taaaatccta aacatgaaat 420

tttttcttaa aactcaagag aattattatc tctattattt ttatattaac aatcccctta 480

ttaattgttt cttccttctt ttttatacaa ctctctcacc ctttggcaat aggccttata 540

ttatttattc aaacagtctt aatttcaatt acaattggag tttccaactt ttctttttga 600

ttttcttata ttctgttttt agtgttttta ggaggaatac tagttttatt catttatatt 660

gcttctttag cttcaaatga atcattttct ttttctttaa ttactttttt ctttgctatt 720

attttaacag gtttaattat ctttatttta ttatttttag atcctatctt aatttctcct 780

aattcttcat ccctttcaac atcatcatta aaatatttag tctcgacccc attcattgtt 840

aattgagttt ataatacccc atctataata tttactattt ttattgtctc ctatctacta 900

cttatattaa ttgttgttgt taaaattatt aatttgttta aaggcccact tcgtcttata 960

ttataatgac aacacctata cgcaaatccc accctttatt taaaattgcc aatggagcat 1020

tagtagatat acctttacca agtaacattt ccacattttg aaactttggt tccttgctag 1080

gcctttgttt agttatccaa atcgcgacag ggttattctt agc 1123

<210> 42

<211> 1169

<212> DNA

<213>artificial sequence ()

<400> 42

tctcgacccc attcattgtt aattgagttt ataatacccc atctataata tttactattt 60

ttattgtctc ctatctacta cttatattaa ttgttgttgt taaaattatt aatttgttta 120

aaggcccact tcgtcttata ttataatgac aacacctata cgcaaatccc accctttatt 180

taaaattgcc aatggagcat tagtagatat acctttacca agtaacattt ccacattttg 240

aaactttggt tccttgctag gcctttgttt agttatccaa atcgcgacag ggttattctt 300

agctatacac tactctgctc acgtagatct agctttttct agagttgccc acatttgtcg 360

tgacgtaaat tacggttgaa tactacgaac ccttcacgct aatggggcct ctttcttctt 420

tatttgcctc taccttcata ttggacgagg catctacttt agatcttata taaaccttca 480

tgcttgaata gtaggggtag taattttatt tataattata gcgactgcat ttttaggcta 540

tgttctacca tgaggacaaa tatctttttg aggcgctaca gttattacaa atttattttc 600

cgctatccca tttatcggta ccgacctagt acaatgaatc tgaggagggt tttctgttga 660

taatgccact ttaactcgat ttttttcatt ccattttatt ttacctttag tagcatcagc 720

attttctata attcacattc tatttttaca ccaaacagga gctaataacc ccttaggtat 780

ttcaagacaa actgataaag taccatttca tccttacttt acatttaaag atattgttgg 840

atttgtagtt atattaacag ccctaatttt attaactctt ctagccccat attttctagg 900

agatccagat aattttattc ctgcaaaccc tttagtaacc cctcctcata tccaaccaga 960

atggtatttt ctttttgctt acgctatttt acgatcaatt cctaataaat taggaggagt 1020

tgtagcatta gccgcttcag tggccattct tataatttta ccatttacac acacttcaaa 1080

atttcaaaga ttagcatttt accccattaa tcaaatgtta ttctgaacct ttgtagtcat 1140

tgtagtacta ttgacctgaa ttggagcac 1169

<210> 43

<211> 899

<212> DNA

<213>artificial sequence ()

<400> 43

ccccatattt tctaggagat ccagataatt ttattcctgc aaacccttta gtaacccctc 60

ctcatatcca accagaatgg tattttcttt ttgcttacgc tattttacga tcaattccta 120

ataaattagg aggagttgta gcattagccg cttcagtggc cattcttata attttaccat 180

ttacacacac ttcaaaattt caaagattag cattttaccc cattaatcaa atgttattct 240

gaacctttgt agtcattgta gtactattga cctgaattgg agcacgcccg gttgaagacc 300

cttatgtcat tactggacag attttaaccg tatcctattt cttgttctat attattaatc 360

ctcttattct tatttgatga gataacatac ttaaatgact atttagttta aaaaaaacaa 420

atgttttgaa aacattaaat aagaataaat tcttaatagt cgagtgtttc ttaaataact 480

atttacattt ataaaatttt aatatcttcc tataaactta atttttactc ccattacttc 540

taatatagtg cctgataaaa ggatagtttt gatagaacta attatgtaat ttccagttac 600

ctatgttatt aaaatcttta ttaaaaagat aagctaaata agctaatggg ctcatacccc 660

gtaaatgaaa gtttaatctc tctcttttta atcgcttttc ctacctctta tttctttttc 720

ctaataacac taatctcagg gtcaattatc tctatttcct caacttcctg atttggagct 780

tgaattggat tagaactaaa tctcatatca tttattcctc ttattgcttt taaaataaac 840

ccactttttt ctgaagcagc tttaaaatat tttcttattc aggcattagg atctgtgct 899

<210> 44

<211> 796

<212> DNA

<213>artificial sequence ()

<400> 44

aagctaatgg gctcataccc cgtaaatgaa agtttaatct ctctcttttt aatcgctttt 60

cctacctctt atttcttttt cctaataaca ctaatctcag ggtcaattat ctctatttcc 120

tcaacttcct gatttggagc ttgaattgga ttagaactaa atctcatatc atttattcct 180

cttattgctt ttaaaataaa cccacttttt tctgaagcag ctttaaaata ttttcttatt 240

caggcattag gatctgtgct atttatttct tcaagattcc ttttaatgtc tttttattct 300

tttagacttt tatctatttt tttagccctc ttattaaaac taggaggagc ccctttccac 360

ttctgatttc ctcaagttat agaaggtcta aaatgaccac aaatcttcct tctatctaca 420

attcaaaaac tagccccctt aaccttaatt tcttacctaa taaacaacga aatcttaatt 480

aaaattacaa ttttttcagc tattttatca gctttaatcg gaagacttgg tggtttaaac 540

ttaaccctat tacgaaaaat tattgccttt tcttcaatta atcacttatc ttgaatatta 600

atagcaatct caattagaga ctcctcatga cttttttatt tttttattta ttcccttatt 660

gtactttcta ttaccagagt atttcataaa ttacaaacct tctccctttc tatactaatt 720

caatctgatc aaaatagaac acttcacgct actattattt cacttaattt tttatccttg 780

agtggtcttc caccta 796

<210> 45

<211> 798

<212> DNA

<213>artificial sequence ()

<400> 45

ccttgagtgg tcttccacct ataacaggat ttattcccaa atgaattgtt attcaagtta 60

tattaaattt aaatatattt atccccttat tttttcttct tgtttctgct ttgattactt 120

tatattttta tttgcgaatc atcattacca taatcttaat attcaaccct attttaaatt 180

ttaatatgaa atataaatct ctaacagatt ccccatcttc tctatttttt aaatcctcct 240

tcaacttttt tggtcttcta ctaccagtat atttcacctt tatctagaat tttaagttat 300

ttaaactaaa agccttcaaa gctttctaaa aaagtattaa ccttttaaat tctatatttt 360

aattattatg caacgatgac ttttttctac aaatcataaa gatattggaa cattatattt 420

tattttcggt gcttgggccg gaatagtagg aacatcatta agattaatta ttcgagctga 480

attaagacaa ccaggcagac taattggtaa cgatcaaatt tataatgtag ttgtaacagc 540

tcacgctttt gtaataattt tttttatagt aatgcccatt ataattggtg gatttggcaa 600

ctgacttgtc cctcttatac taggggcccc agatatagct ttcccacgaa taaacaatat 660

aagattctga cttttacccc cttctctctc tcttttactt acaagaagaa tagtagaaag 720

aggagttggt actgggtgga ctgtctaccc acctttagca gcagccatcg ctcatgctgg 780

agcatcagtt gatcttgg 798

<210> 46

<211> 704

<212> DNA

<213>artificial sequence ()

<400> 46

tattttcggt gcttgggccg gaatagtagg aacatcatta agattaatta ttcgagctga 60

attaagacaa ccaggcagac taattggtaa cgatcaaatt tataatgtag ttgtaacagc 120

tcacgctttt gtaataattt tttttatagt aatgcccatt ataattggtg gatttggcaa 180

ctgacttgtc cctcttatac taggggcccc agatatagct ttcccacgaa taaacaatat 240

aagattctga cttttacccc cttctctctc tcttttactt acaagaagaa tagtagaaag 300

aggagttggt actgggtgga ctgtctaccc acctttagca gcagccatcg ctcatgctgg 360

agcatcagtt gatcttggta tcttctctct acatcttgcc ggagtttcct caattttagg 420

agctgttaat ttcataacaa cagttattaa catacgatca tatggaataa caatggatca 480

aatacctctt tttgtttgag ctgtatttat tactgctatt ttacttctct tatcccttcc 540

agttttagct ggagctatta ccatactgct tacagatcgt aacctaaata cgtcattttt 600

cgacccagca gggggaggtg acccagttct ataccagcac ttattctgat ttttcggtca 660

cccagaagtt tatattctaa ttttgcctgc cttcggtata atct 704

<210> 47

<211> 1182

<212> DNA

<213>artificial sequence ()

<400> 47

ctacatcttg ccggagtttc ctcaatttta ggagctgtta atttcataac aacagttatt 60

aacatacgat catatggaat aacaatggat caaatacctc tttttgtttg agctgtattt 120

attactgcta ttttacttct cttatccctt ccagttttag ctggagctat taccatactg 180

cttacagatc gtaacctaaa tacgtcattt ttcgacccag cagggggagg tgacccagtt 240

ctataccagc acttattctg atttttcggt cacccagaag tttatattct aattttgcct 300

gccttcggta taatctctca tattgtaagt caagaatcag gtaaaaagga atcttttgga 360

actttaggta taatttacgc tatactagct attggaattt tgggctttgt agtatgagct 420

catcatatat ttacagtagg tatagacgta gatacccgag cttactttac ctcagccact 480

ataattattg ctatccccac cggaattaaa atctttagat gactaaggac cttacatgga 540

actcaaataa attattcacc atctcttcta tgagctttag ggtttatctt tttatttact 600

attggaggtc tgacaggagt agttttagct aattcctcaa ttgatattat tctccatgat 660

acttattatg ttgttgccca ctttcattat gtactctcta tgggagcagt tttcggaatc 720

tttgctggag ttgctcactg attttctcta ataacaggtt tatctttcaa ccctaaatga 780

ctaaaaattc actttttagt aactttcgtc ggagtcaatt taacattttt tccccaacac 840

tttttaggat taaacggcat accacgacga tactctgatt atcctgacgc ctatgcaact 900

tgaaatattg tatcatctat aggttctata atttcttttg tagccgcatt aggattctta 960

ttaatcgttt gagaagcatt tgtttctaat cgcccagtaa tcttttctcc ttttctgccc 1020

tcttctattg aatggaaaca tgcttatcct ccagccgacc attcatatat ggaaatccca 1080

ctaattacta acttctaaaa tggcagacag atgtgtagga tttaagctcc tataaggaag 1140

aataatttct tcttttagaa taacttatgg caacatgagc gt 1182

<210> 48

<211> 1108

<212> DNA

<213>artificial sequence ()

<400> 48

tggcagacag atgtgtagga tttaagctcc tataaggaag aataatttct tcttttagaa 60

taacttatgg caacatgagc gtatttaaat tttcaagaca gagcttctcc tcttatagaa 120

caattaattt tttttcacga tcatattata ttagtgattg tgttaattgt aacatttgtt 180

ggttatataa tagcaacatt gtttttcaac tccctcatta accgctatat attagaaaat 240

caaccaatcg aagtaatttg aacatcagtt ccagctttta ttttaatctt tattgcttta 300

ccatcccttc gcttacttta tcttttagat gaagtaaaca atccttctat aactcttaaa 360

acaattggcc accaatgata ttgaaggtat gaatattcag atttccttca agtagaattt 420

gactcatata tagtaccatc taatgagctg gaagtatcag ggttccggct actagacgta 480

gacaatcgta ccgttcttcc aataaatact caaatccgag ttctaattac agctgcagat 540

gtcatccatt cctggacagt cccatcctta ggtattaaag ccgatgcaat tcccgggcgg 600

ctaaatcaat caaggttttt aatcaaccga cctggactat tttacggtca atgttcagaa 660

atttgcgggg caaaccacag atttatgccc attgttattg aaagaacttc tattaactca 720

tttctaaatt gaatttctct atcaattgaa gcgtcaccct aactcatcct tagttttact 780

aatttttggg tgaaacaatt ccttctgtga attactattt atgccacaaa tagcacctat 840

ttattgacta tttatatttt tctttttttt agccagattc ctattatttt tcgctcttaa 900

ttacttcatt aaaccttttt ataaagttaa ttttcttcaa gataataaaa aacctttaat 960

ttataaacat tgaaaattat aacaaattta ttctcaattt ttgaaccttc ttcaagaatc 1020

tttaatttat cgataaattg aatctcaact atcctaggat tgatattctt accttatata 1080

tactgagcat ccccttctcg ttgatctt 1108

<210> 49

<211> 1000

<212> DNA

<213>artificial sequence ()

<400> 49

atccccttct cgttgatctt tactttgggg aaaaattatt tcaaccttgc ataaagaatt 60

taaagctctt ttacagcctt cacataatgg agcttctata gtattcattg ctttatttag 120

gttaattgta tttaataatt ttctaggctt attaccttat gtatttacta gctctagtca 180

tatggtaata acattatctt tatctttgcc cttgtgaatt actttaataa catttgggtg 240

aattaaacac actaaacata tatttgccca tttagttccc caagggacgc ctttcgcttt 300

aatgccattt atagtgttaa ttgaaaccat tagaaatgta attcggcctg gtaccttagc 360

aattcggcta gccgctaata taattgccgg tcatttactt ttaaccttat taggaaatac 420

tggcccttca ttatcaacct ccatcctatt cattttattg tcagctcaaa tactcctact 480

aatcttagaa tctgccgtag ctattattca atcttatgtt ttcgctgttt taagaactct 540

ctacactaga gaaattaact aatgacatca tcacacggct ttcatcctta tcatttagta 600

gatattagac cctgaccaat tactgggtct attagagcta taatactaac cactggttta 660

gtaaaatgat ttcaccaaca caatataaac cttttaattt taagatttat tgcaactttg 720

ttaactataa ttcaatgatg gcgagatatt acccgtgaag gtacttacca aggatcacac 780

actttaacag taataaaggg gctacgctga ggtataattt tatttattgt atcagaagtt 840

ttatttttct tttctttttt ttgagctttt ttccatagaa gactatctcc tgctgtagaa 900

attgggatat attggccgcc attaggtatt caacctttta accctttcca aatccccctt 960

ctaaacacta cgattcttct atcgtcaggt gctacagtta 1000

<210> 50

<211> 1198

<212> DNA

<213>artificial sequence ()

<400> 50

ttggccgcca ttaggtattc aaccttttaa ccctttccaa atcccccttc taaacactac 60

gattcttcta tcgtcaggtg ctacagttac ttgggctcat cacgcaattc tagagggtaa 120

ctactctcaa gctgtacaaa gactaggttt aactattatc ctaggtattt attttacaat 180

gctccaagca tttgaatata ttgaagcttc tttcactatt gccgactcag tctttggttc 240

aacatttttt gtagcaactg ggttccatgg attacacgtt atcattggaa cttcattttt 300

atttgtttgt tttattcgac tttataaatg tcacttttct tcttctcatc atttaggatt 360

tgaggcagct gcttgatact gacactttgt tgatgttgtt tgattattct tatatatttt 420

catttattga tgaggcggat aattttttta atataataag tatatctgat ttccaatcag 480

aaagtctaac ttttagaaaa aataatcttt acaatattga caatttcagt tattactctc 540

atggtttcat atatcgttat aactttagcg accctcttat ctaaaaagac aattatagat 600

cgagaaaaaa attctccgta tgaatgtgga ttcgatccca aaggatccgc ccgtctacct 660

ttttctctac gctttttttt aattgccgta attttcctaa tctttgatgt agaaattaca 720

cttctgctgc ctattgcttc tacattaaac ctaaccaata tgttttcttg attttttaca 780

agtttaattt ttttattaat tttgttattt ggtctttatt atgaatgatc acaaagtgct 840

ctagattgat cttagtaaga ccatagtcaa tatatgacgt atgagttgca ttcataaaga 900

gttttctaaa ctggttttaa aaattagaaa gcgaataatt gcatttagtt tcgacctaaa 960

ttttagactt tagtcttcta attttggtgg aaaccaaaaa gaggtatatc actgttaatg 1020

ataaaattga gttcaaactc ctaccaagaa gggatattat gctaaaggca gaaagcttct 1080

aactttttac taagcggtta aattccgttt atatcccttt gttatatata tgtacatatc 1140

tatcaaccta gccctagcac tttgttttta tagtttaaac caacccaaaa cattggtc 1198

<210> 51

<211> 1181

<212> DNA

<213>artificial sequence ()

<400> 51

caaactccta ccaagaaggg atattatgct aaaggcagaa agcttctaac tttttactaa 60

gcggttaaat tccgtttata tccctttgtt atatatatgt acatatctat caacctagcc 120

ctagcacttt gtttttatag tttaaaccaa cccaaaacat tggtcttgta aaccaataat 180

gaattatttt ctaaaaactt tcagaaaaaa agagtaactc tttatcttta attcccaaaa 240

ttaatatttt ttaaactatt tcctgaattt attactctaa attttatgct gctagacaaa 300

aacaaaacat tttaaatcta acaaaaaaaa tcaaataatt aatagaaaca ggaagataac 360

tttttcatgc taaatatatc aatttatcat accgcagtcg aggtaaagta cctcggactc 420

atacaaaccc aaaagctact attacactct taaaataaaa cattactctg aacggccccc 480

tacccaaaaa aaaaataaca aataaaaccc tcatgaatag aatacttgcg tattcagcta 540

taaaaattaa tgcaaacccg ccagccccgt actctgtatt aaagccagag actagctcag 600

attcaccttc agaaaaatca aatggagttc gattagtctc agctaaacat gacctaaatc 660

acaccattct taaaggaaat ctaattacaa caaatcaaaa ataattttga tatttattaa 720

acaactctaa attaaatcca ccaattaata ttacaaaaga taataaaatt aaagctaacc 780

taacttcata agaaatagtt tgagctaccg ctcgcaaaga acctaataaa gaatatttac 840

aatttgatga tcatcccgcc actatagttg aatacacccc tacacttaaa caacacaaaa 900

aaaacagaat tctcagatta aaccttatta atccagtttc ataaggtaaa actgatcata 960

ctaataaaga aataaacaaa ctaaatacag gacataaata gtaaactaaa aagtttgata 1020

tagtaggaat aatttgttcc ttagtgaaaa gctttactgc atcagaaaaa ggctgtaaaa 1080

ttcctatata gcctacttta tttgggcctt tacgaatttg aatataacct aaaatttttc 1140

gttcaagtaa agttacaaaa gccacgccaa ttaaaacaca c 1181

<210> 52

<211> 874

<212> DNA

<213>artificial sequence ()

<400> 52

ctgcatcaga aaaaggctgt aaaattccta tatagcctac tttatttggg cctttacgaa 60

tttgaatata acctaaaatt tttcgttcaa gtaaagttac aaaagccacg ccaattaaaa 120

cacacactat taataccaaa aaattaacta tctctacaat tattaaagtc acaaaacaat 180

tagattacta ataatttaac tatttataga attgctctat tttataggat cctaaatcca 240

acacatatta tctgccaaaa tagtaatcat tatataaaat aattttaatt atttaatcct 300

ttcgtactaa aataattttt ctttattaaa agatagaaac caacctggct tacgccggtt 360

tgaactcaaa tcatgtaaaa ttttaaaggt cgaacagacc tacttatata actgctacaa 420

ttataaagat attttaattc aacatcgagg tcgcaaactt ttctttcgat aagtactctt 480

aagaaaaata acgctgttat ccctaaagta acttaaactt ttaatcttta attaggatca 540

tttaaaattt taatcacata taagtgtact taatgttaag cagttaataa tcattttact 600

cttatcgccc caataaaata aattcaattt acaaaatctt tataacctaa aatttgacta 660

tataaaattt aatattaagc tttatagggt cttatcgtcc ctttaataca tttaagcctt 720

ttcacttaaa agtaaaattc aataaacaat ataaagacag atcttctttt gtccaaccat 780

tcatacaaga ttccaattaa aaaactaacg attatgctac ctttgcacgg tcaaaatacc 840

gcggccattt aatactatca atcagtgggc aggt 874

<210> 53

<211> 1376

<212> DNA

<213>artificial sequence ()

<400> 53

gaaaccaacc tggcttacgc cggtttgaac tcaaatcatg taaaatttta aaggtcgaac 60

agacctactt atataactgc tacaattata aagatatttt aattcaacat cgaggtcgca 120

aacttttctt tcgataagta ctcttaagaa aaataacgct gttatcccta aagtaactta 180

aacttttaat ctttaattag gatcatttaa aattttaatc acatataagt gtacttaatg 240

ttaagcagtt aataatcatt ttactcttat cgccccaata aaataaattc aatttacaaa 300

atctttataa cctaaaattt gactatataa aatttaatat taagctttat agggtcttat 360

cgtcccttta atacatttaa gccttttcac ttaaaagtaa aattcaataa acaatataaa 420

gacagatctt cttttgtcca accattcata caagattcca attaaaaaac taacgattat 480

gctacctttg cacggtcaaa ataccgcggc catttaatac tatcaatcag tgggcaggtt 540

aaacttttta taacttcaaa cagacatgtt tttgataaac aggcaaagaa gttatttttg 600

ccgagttcct ttattccgcc ctatcaattt taaacaatat ttattattta aatgttttgc 660

tttaataaaa aacttttact aataaaatca ttataactta actctttata tttaaattaa 720

ttttttaata ctccaaaaaa gtttatataa atattatcca attttaattt agctagaaca 780

ctttataaat atagctactt ttaagcctaa ttcaccaatt aaactatttt aaactattta 840

tcattttaat tataagaagc taatccctcc tacacttaaa ttttacatct tatcaacgta 900

aaaattaaat taaatttact ttttaaaaaa ctagatataa taaaactcga ctagcgtttc 960

acctttaatt ttatattaaa aatttatttt ctacaaaaaa ttttttataa aattagctat 1020

attttatttc gagatattta atttttatta acttatataa attttccctg atacacaagg 1080

tacaatattt aaaatttttt acttcaaatt ttcttgcttt cctttacagt actcttaact 1140

atagtttata aattgtgttt attaaaaatt actagcatga acaaattaca aattattttt 1200

cataaatata aaattttcaa atttatttaa caagtcatta ataaatagat gggtaattat 1260

taatttataa agaaaatata catatcctca tctttactag atacactttc cagtacatct 1320

actatgttac gacttatttc acttataaat gaaagcgacg ggcgatatgt acacaa 1376

<210> 54

<211> 966

<212> DNA

<213>artificial sequence ()

<400> 54

ctcgactagc gtttcacctt taattttata ttaaaaattt attttctaca aaaaattttt 60

tataaaatta gctatatttt atttcgagat atttaatttt tattaactta tataaatttt 120

ccctgataca caaggtacaa tatttaaaat tttttacttc aaattttctt gctttccttt 180

acagtactct taactatagt ttataaattg tgtttattaa aaattactag catgaacaaa 240

ttacaaatta tttttcataa atataaaatt ttcaaattta tttaacaagt cattaataaa 300

tagatgggta attattaatt tataaagaaa atatacatat cctcatcttt actagataca 360

ctttccagta catctactat gttacgactt atttcactta taaatgaaag cgacgggcga 420

tatgtacaca atttagaact tatatcttgt tagcttatta aactaaaaat acaatcaaat 480

ccatctttat aataatatta tcttattaat ccgcagtaaa gtaatttact gtaacccatt 540

ttaccttgct tattggctgc accatgatct aattttacta aacaactaaa tttaataatt 600

tttcctttaa gaattatctg ataatgatgg tatacaaact gttagaatta ctaaagcaag 660

taagattttt cgtggtttat cgattctaaa acaggttcct ctgaatagat taaattgccg 720

ccaaattttt taaattttaa gtatttatct tttactaatt cagttttata ttaaatttat 780

tttagaataa tagggtatct aatcctagtt tatttctaaa cttttttagc ttcaattata 840

attttactcc atacataata atatcctaat tttacctttt attacctgta tgagattttt 900

aatttaatca tctaactata accttagaat ttttactcaa tcttaaagta taaccgcgaa 960

tgctgg 966

<210> 55

<211> 1085

<212> DNA

<213>artificial sequence ()

<400> 55

tataaccgcg aatgctggca caaatatata tcagatttta atattattac tatatcacac 60

cttcatgtat aattttataa tattttatgc tgagacaata aaggtttata attttatttc 120

actcgaatac caatttaata tttaattaaa ttgtttaaat cacactttaa ttttcataca 180

attttaataa taatgtaaaa acttaagcca aaatcaaaca tttgtcgtga ttgcaaataa 240

taactttaaa aaacttaaat atactatata taacataaaa atgagaataa gcttatactt 300

aaagtataac ttatacatct taagcgccac aagctaacgt tttaatattt taaactattt 360

aagttttgtt ataactaata ttgctacaaa tatatttatt atctacacaa aaacaaacaa 420

tataatcacc aacccaacca caaaaatttt tataaacact ttaatattat taagttgtaa 480

ctcatttaac aacctaaaat atttcttaaa caaaaaatac atcccctggc caccaaaata 540

ttcaaatcac cctttgtctc ctaatttttc aacacctacc ccactaatga gactagaccc 600

tctaattttt tttgtcctaa gaaaaggtat aaatcatata gaccccctta ttactactat 660

cttataatta ttaatagatt ttaaattgta acttacattt aataaattaa atatataacc 720

tataactccc cctgctaatc ttacccccaa cactatattt ttcattatta cattcaaaca 780

aattatataa ggctctggga aaataaccca agatagaaca gctcctataa aaatagctct 840

tacacctagt aaaatcatag aatttcttat aattttatcc tcatccctaa tatttattat 900

aactcttaaa tttgaattac ctcttaacct ataaaaaatt agacgtatag tatacataac 960

tgttaaccct gtagctaaaa tatataacag taaagaaata aaattaatcc atctcataaa 1020

agccacctct aaaattatat ctttagaata aaaccctgct aaaaaaggga acccacataa 1080

ggcta 1085

<210> 56

<211> 1019

<212> DNA

<213>artificial sequence ()

<400> 56

agggaaccca cataaggcta aatttgaaac tcttataaaa gacactctta acggcataaa 60

tttaattaaa ccacctatac atcgaatatc ctgataattg ccaaccctat ggataataat 120

acctgcgcat ataaataaca atgctttaaa taatgcatga ctcagcagat gaaaaaaaga 180

aagatcaggg tatcctaaag ctaaaatact taacataacc cccaattgac ttaacgttga 240

taaggcaata atttttttta aatcatattc aaaattggcc cctaatcccg ccataaatat 300

agttaagcaa gaaataatta ataatattcc ttggatttta gaaccttcta acgcaccact 360

aaatcgaatt attaaataca ccccagcagt tactaaagta gaagaatgta ctaaagcaga 420

aacaggagta ggagctgcta tagcggcagg aagccatgct gagaaaggaa tttgagcact 480

tttagtcata gctcttaaca ttaacaaaaa cttaaacaaa attcactcag tatctataat 540

atagtaagaa taaataacaa aatttcatct acctaacctg gccattagtc caatactaag 600

taaaatagca acatcaccta ctcgattaga taagatagtt aacatcccag cattagcaga 660

cttttcattt tgataaaaaa ttactaaagc ataagaaact aaacctaatc catcccatcc 720

taaaagaata cttaccaagt taggtcttaa aactattata gccatagata aaacaaacat 780

taaaactaaa tatataaatc gattaaagtt tttatcctcc ttcatatatc tacctctata 840

atacatcaca cttccagaaa taagcaatac aaaacacaaa aaaagtatcg aaattcaatc 900

aaaaattaaa acaaacacca ctgataatga atttaatctt gctaaatccc attcaattac 960

actagtaaca ccactaaata attttattaa agaaactgtt ccacaaacta aagaccccg 1019

<210> 57

<211> 875

<212> DNA

<213>artificial sequence ()

<400> 57

cctctataat acatcacact tccagaaata agcaatacaa aacacaaaaa aagtatcgaa 60

attcaatcaa aaattaaaac aaacaccact gataatgaat ttaatcttgc taaatcccat 120

tcaattacac tagtaacacc actaaataat tttattaaag aaactgttcc acaaactaaa 180

gaccccgctc atagcccaag tattctaatc ttatgtatat aaattattca aaccattatt 240

taaatattaa attattaatt taatacaaaa tacagtaaca tttctaccaa tggtacaaaa 300

caattagtag taattaaatc taaataaatc aaagcaaatc gatttgcacg ataaatcttt 360

tcaacgtaac tgaaatgctg ttctatttta gctctgcttt gacactatca tattatattt 420

ccaatctact ttacatttaa ataatttatt aaaaaaagct actaaaaata ttaagaaatg 480

ataatactat tatattatac aattttattt ttctttattt atacggaggt atataaggta 540

tatataaata tatcacatta gatataagta aataatataa acaaatcaaa tatataaaaa 600

ttaaatatgt atattcgcct ccacaattat accccatcca atataataca ggatagataa 660

aacattaaaa ttataaaagg gtaaaattat aaataaatag aagtatataa tgtcttagtt 720

tatatggaac attgaaacat tttatataaa agtaaaaatt atatgtaact tcattatata 780

aatatatgat ttatactatt tttatttatt ctatgggttg aaaaaaagta gtagtctcaa 840

ccctagaccc taattcctct agaggggaaa cgggt 875

<210> 58

<211> 16438

<212> DNA

<213>artificial sequence ()

<400> 58

ttacatgcaa cggaattgca ccgtttctta aaagatcaaa acttcttatg ctctttacat 60

cagcatataa attattttca aaccctagtg atttacacat ctttaaactt gcaatttaat 120

attattttta tactataggg cctaataaag gaatctcatt cgtctacaga tttacaatct 180

gccgcttaca actcagccac tttattaatt tttaaataaa tagtatactc gatgactgaa 240

aggtaagtat agatctttta aatctaatat agtaatttaa catttacttc gagtaaggaa 300

ttagttaaaa tataataaca tttgcttgtc atgcaaaagt catctttaag atactcctta 360

aatttcagtt ttatttgttt tatggtgtag ataacacatt gtattttcgt tacaaaaccg 420

aaactaatgt ttctagaaca ttataatact ttgcccacag ataatattta tctcttttgc 480

agcttcaatg caatattcta tataaattat ataagctact tatattaatc tagctttacg 540

ttatacgcca ttaactaaat ttaaatttaa aattaacata tttaaaggaa atcaatgtaa 600

aaatagaact aaatattctc gaacttttcc tgacctacaa gaatataaac tactgacgaa 660

aatcccatgc tgacttaaac tatatatgta taaagtataa gctgctctga aaaaagaaag 720

aaatataatt cccattactg taaatgtcct tcaccttatt aaactaataa tcaacctaat 780

ctccccgaat agatttaaag tagggggagc agccatatta cccactctaa ataaaaatca 840

tcacaaacct atcctaggta taaaatttaa taaaccctta ctaactaaaa gactacgcct 900

accaactcgc tcgtatacta tattagccaa acaaaataat ccagaagaac ataacccgtg 960

tccgactatt actacaacag ctcctattag tcctcatcat ctaaaaatca ttagaccaca 1020

tagtactatt ctcatatgaa caaccgaaga ataagcaatc aaggacttta tatctacctg 1080

gcgtagacac aaaagactca ctactactcc ccctactaaa ctaattctaa cccataacga 1140

cgaaaaattc aagctaattt tctgaaatat caataacacc cgaatcaaac catacccgcc 1200

taactttaat aaaactcctg ctaaaattat agaaccagca accggggctt ctacgtgagc 1260

ttttggcaac cataaatgaa atatatatat cggtaattta actaaaaaag ccatcaccct 1320

tctaaaatat caaatactgt ttgcccagct aacttctaaa ataagatttc ttaatattat 1380

aactaaccta tattctttta tataaatata caataaagac cccaataaag gtaaagatag 1440

tgacaaagta taaaaaagta tataaactcc agcttgaatt cgttcaggtt gatatcccca 1500

tccaagaatc aaaattaaag tgggaattaa cgatatttca aatataatat aaaacaacaa 1560

ataatttaat gaagaaaatg ttactaaaag cctaaataaa agtactaaat ttaccaagat 1620

aaacataccc ctaaaattat ttattatttt tactcgctcc ctcgaaataa caataagaaa 1680

tataattcaa acacttaaat aaactataat ataactgaca taatctattc ctaaattaaa 1740

tcccaaacta tatatatata tattatgaaa acatcttaaa ccaactaaaa atcttaataa 1800

tcctaatcct atctgagctg actttcagtc atcctttaat tttatcaata gaactacagg 1860

taacaaaaac tttaacattc cagaagatta aatcttttaa aataatcatt tccatgccta 1920

cgaacaataa gaactaataa agataaacca agagctcctt cacatacagc taaagttaaa 1980

aaaaataaag aaatgtaaat ttccctacca actatagaca actgtaacct taataatcaa 2040

aaaactccaa gcattataaa ttctaatctt aacaacgaat ttaataaatg tttattataa 2100

gaaataaatc tccataaacc acaaaaaatt ataaataccg ccccaaacac tctcaaaaat 2160

ctaaaattta tcattagttt taatagttaa tttaaaaaac taaaacattg gtgttaaaat 2220

cctaaacatg aaattttttc ttaaaactca agagaattat tatctctatt atttttatat 2280

taacaatccc cttattaatt gtttcttcct tcttttttat acaactctct caccctttgg 2340

caataggcct tatattattt attcaaacag tcttaatttc aattacaatt ggagtttcca 2400

acttttcttt ttgattttct tatattctgt ttttagtgtt tttaggagga atactagttt 2460

tattcattta tattgcttct ttagcttcaa atgaatcatt ttctttttct ttaattactt 2520

ttttctttgc tattatttta acaggtttaa ttatctttat tttattattt ttagatccta 2580

tcttaatttc tcctaattct tcatcccttt caacatcatc attaaaatat ttagtctcga 2640

ccccattcat tgttaattga gtttataata ccccatctat aatatttact atttttattg 2700

tctcctatct actacttata ttaattgttg ttgttaaaat tattaatttg tttaaaggcc 2760

cacttcgtct tatattataa tgacaacacc tatacgcaaa tcccaccctt tatttaaaat 2820

tgccaatgga gcattagtag atataccttt accaagtaac atttccacat tttgaaactt 2880

tggttccttg ctaggccttt gtttagttat ccaaatcgcg acagggttat tcttagctat 2940

acactactct gctcacgtag atctagcttt ttctagagtt gcccacattt gtcgtgacgt 3000

aaattacggt tgaatactac gaacccttca cgctaatggg gcctctttct tctttatttg 3060

cctctacctt catattggac gaggcatcta ctttagatct tatataaacc ttcatgcttg 3120

aatagtaggg gtagtaattt tatttataat tatagcgact gcatttttag gctatgttct 3180

accatgagga caaatatctt tttgaggcgc tacagttatt acaaatttat tttccgctat 3240

cccatttatc ggtaccgacc tagtacaatg aatctgagga gggttttctg ttgataatgc 3300

cactttaact cgattttttt cattccattt tattttacct ttagtagcat cagcattttc 3360

tataattcac attctatttt tacaccaaac aggagctaat aaccccttag gtatttcaag 3420

acaaactgat aaagtaccat ttcatcctta ctttacattt aaagatattg ttggatttgt 3480

agttatatta acagccctaa ttttattaac tcttctagcc ccatattttc taggagatcc 3540

agataatttt attcctgcaa accctttagt aacccctcct catatccaac cagaatggta 3600

ttttcttttt gcttacgcta ttttacgatc aattcctaat aaattaggag gagttgtagc 3660

attagccgct tcagtggcca ttcttataat tttaccattt acacacactt caaaatttca 3720

aagattagca ttttacccca ttaatcaaat gttattctga acctttgtag tcattgtagt 3780

actattgacc tgaattggag cacgcccggt tgaagaccct tatgtcatta ctggacagat 3840

tttaaccgta tcctatttct tgttctatat tattaatcct cttattctta tttgatgaga 3900

taacatactt aaatgactat ttagtttaaa aaaaacaaat gttttgaaaa cattaaataa 3960

gaataaattc ttaatagtcg agtgtttctt aaataactat ttacatttat aaaattttaa 4020

tatcttccta taaacttaat ttttactccc attacttcta atatagtgcc tgataaaagg 4080

atagttttga tagaactaat tatgtaattt ccagttacct atgttattaa aatctttatt 4140

aaaaagataa gctaaataag ctaatgggct cataccccgt aaatgaaagt ttaatctctc 4200

tctttttaat cgcttttcct acctcttatt tctttttcct aataacacta atctcagggt 4260

caattatctc tatttcctca acttcctgat ttggagcttg aattggatta gaactaaatc 4320

tcatatcatt tattcctctt attgctttta aaataaaccc acttttttct gaagcagctt 4380

taaaatattt tcttattcag gcattaggat ctgtgctatt tatttcttca agattccttt 4440

taatgtcttt ttattctttt agacttttat ctattttttt agccctctta ttaaaactag 4500

gaggagcccc tttccacttc tgatttcctc aagttataga aggtctaaaa tgaccacaaa 4560

tcttccttct atctacaatt caaaaactag cccccttaac cttaatttct tacctaataa 4620

acaacgaaat cttaattaaa attacaattt tttcagctat tttatcagct ttaatcggaa 4680

gacttggtgg tttaaactta accctattac gaaaaattat tgccttttct tcaattaatc 4740

acttatcttg aatattaata gcaatctcaa ttagagactc ctcatgactt ttttattttt 4800

ttatttattc ccttattgta ctttctatta ccagagtatt tcataaatta caaaccttct 4860

ccctttctat actaattcaa tctgatcaaa atagaacact tcacgctact attatttcac 4920

ttaatttttt atccttgagt ggtcttccac ctataacagg atttattccc aaatgaattg 4980

ttattcaagt tatattaaat ttaaatatat ttatcccctt attttttctt cttgtttctg 5040

ctttgattac tttatatttt tatttgcgaa tcatcattac cataatctta atattcaacc 5100

ctattttaaa ttttaatatg aaatataaat ctctaacaga ttccccatct tctctatttt 5160

ttaaatcctc cttcaacttt tttggtcttc tactaccagt atatttcacc tttatctaga 5220

attttaagtt atttaaacta aaagccttca aagctttcta aaaaagtatt aaccttttaa 5280

attctatatt ttaattatta tgcaacgatg acttttttct acaaatcata aagatattgg 5340

aacattatat tttattttcg gtgcttgggc cggaatagta ggaacatcat taagattaat 5400

tattcgagct gaattaagac aaccaggcag actaattggt aacgatcaaa tttataatgt 5460

agttgtaaca gctcacgctt ttgtaataat tttttttata gtaatgccca ttataattgg 5520

tggatttggc aactgacttg tccctcttat actaggggcc ccagatatag ctttcccacg 5580

aataaacaat ataagattct gacttttacc cccttctctc tctcttttac ttacaagaag 5640

aatagtagaa agaggagttg gtactgggtg gactgtctac ccacctttag cagcagccat 5700

cgctcatgct ggagcatcag ttgatcttgg tatcttctct ctacatcttg ccggagtttc 5760

ctcaatttta ggagctgtta atttcataac aacagttatt aacatacgat catatggaat 5820

aacaatggat caaatacctc tttttgtttg agctgtattt attactgcta ttttacttct 5880

cttatccctt ccagttttag ctggagctat taccatactg cttacagatc gtaacctaaa 5940

tacgtcattt ttcgacccag cagggggagg tgacccagtt ctataccagc acttattctg 6000

atttttcggt cacccagaag tttatattct aattttgcct gccttcggta taatctctca 6060

tattgtaagt caagaatcag gtaaaaagga atcttttgga actttaggta taatttacgc 6120

tatactagct attggaattt tgggctttgt agtatgagct catcatatat ttacagtagg 6180

tatagacgta gatacccgag cttactttac ctcagccact ataattattg ctatccccac 6240

cggaattaaa atctttagat gactaaggac cttacatgga actcaaataa attattcacc 6300

atctcttcta tgagctttag ggtttatctt tttatttact attggaggtc tgacaggagt 6360

agttttagct aattcctcaa ttgatattat tctccatgat acttattatg ttgttgccca 6420

ctttcattat gtactctcta tgggagcagt tttcggaatc tttgctggag ttgctcactg 6480

attttctcta ataacaggtt tatctttcaa ccctaaatga ctaaaaattc actttttagt 6540

aactttcgtc ggagtcaatt taacattttt tccccaacac tttttaggat taaacggcat 6600

accacgacga tactctgatt atcctgacgc ctatgcaact tgaaatattg tatcatctat 6660

aggttctata atttcttttg tagccgcatt aggattctta ttaatcgttt gagaagcatt 6720

tgtttctaat cgcccagtaa tcttttctcc ttttctgccc tcttctattg aatggaaaca 6780

tgcttatcct ccagccgacc attcatatat ggaaatccca ctaattacta acttctaaaa 6840

tggcagacag atgtgtagga tttaagctcc tataaggaag aataatttct tcttttagaa 6900

taacttatgg caacatgagc gtatttaaat tttcaagaca gagcttctcc tcttatagaa 6960

caattaattt tttttcacga tcatattata ttagtgattg tgttaattgt aacatttgtt 7020

ggttatataa tagcaacatt gtttttcaac tccctcatta accgctatat attagaaaat 7080

caaccaatcg aagtaatttg aacatcagtt ccagctttta ttttaatctt tattgcttta 7140

ccatcccttc gcttacttta tcttttagat gaagtaaaca atccttctat aactcttaaa 7200

acaattggcc accaatgata ttgaaggtat gaatattcag atttccttca agtagaattt 7260

gactcatata tagtaccatc taatgagctg gaagtatcag ggttccggct actagacgta 7320

gacaatcgta ccgttcttcc aataaatact caaatccgag ttctaattac agctgcagat 7380

gtcatccatt cctggacagt cccatcctta ggtattaaag ccgatgcaat tcccgggcgg 7440

ctaaatcaat caaggttttt aatcaaccga cctggactat tttacggtca atgttcagaa 7500

atttgcgggg caaaccacag atttatgccc attgttattg aaagaacttc tattaactca 7560

tttctaaatt gaatttctct atcaattgaa gcgtcaccct aactcatcct tagttttact 7620

aatttttggg tgaaacaatt ccttctgtga attactattt atgccacaaa tagcacctat 7680

ttattgacta tttatatttt tctttttttt agccagattc ctattatttt tcgctcttaa 7740

ttacttcatt aaaccttttt ataaagttaa ttttcttcaa gataataaaa aacctttaat 7800

ttataaacat tgaaaattat aacaaattta ttctcaattt ttgaaccttc ttcaagaatc 7860

tttaatttat cgataaattg aatctcaact atcctaggat tgatattctt accttatata 7920

tactgagcat ccccttctcg ttgatcttta ctttggggaa aaattatttc aaccttgcat 7980

aaagaattta aagctctttt acagccttca cataatggag cttctatagt attcattgct 8040

ttatttaggt taattgtatt taataatttt ctaggcttat taccttatgt atttactagc 8100

tctagtcata tggtaataac attatcttta tctttgccct tgtgaattac tttaataaca 8160

tttgggtgaa ttaaacacac taaacatata tttgcccatt tagttcccca agggacgcct 8220

ttcgctttaa tgccatttat agtgttaatt gaaaccatta gaaatgtaat tcggcctggt 8280

accttagcaa ttcggctagc cgctaatata attgccggtc atttactttt aaccttatta 8340

ggaaatactg gcccttcatt atcaacctcc atcctattca ttttattgtc agctcaaata 8400

ctcctactaa tcttagaatc tgccgtagct attattcaat cttatgtttt cgctgtttta 8460

agaactctct acactagaga aattaactaa tgacatcatc acacggcttt catccttatc 8520

atttagtaga tattagaccc tgaccaatta ctgggtctat tagagctata atactaacca 8580

ctggtttagt aaaatgattt caccaacaca atataaacct tttaatttta agatttattg 8640

caactttgtt aactataatt caatgatggc gagatattac ccgtgaaggt acttaccaag 8700

gatcacacac tttaacagta ataaaggggc tacgctgagg tataatttta tttattgtat 8760

cagaagtttt atttttcttt tctttttttt gagctttttt ccatagaaga ctatctcctg 8820

ctgtagaaat tgggatatat tggccgccat taggtattca accttttaac cctttccaaa 8880

tcccccttct aaacactacg attcttctat cgtcaggtgc tacagttact tgggctcatc 8940

acgcaattct agagggtaac tactctcaag ctgtacaaag actaggttta actattatcc 9000

taggtattta ttttacaatg ctccaagcat ttgaatatat tgaagcttct ttcactattg 9060

ccgactcagt ctttggttca acattttttg tagcaactgg gttccatgga ttacacgtta 9120

tcattggaac ttcattttta tttgtttgtt ttattcgact ttataaatgt cacttttctt 9180

cttctcatca tttaggattt gaggcagctg cttgatactg acactttgtt gatgttgttt 9240

gattattctt atatattttc atttattgat gaggcggata atttttttaa tataataagt 9300

atatctgatt tccaatcaga aagtctaact tttagaaaaa ataatcttta caatattgac 9360

aatttcagtt attactctca tggtttcata tatcgttata actttagcga ccctcttatc 9420

taaaaagaca attatagatc gagaaaaaaa ttctccgtat gaatgtggat tcgatcccaa 9480

aggatccgcc cgtctacctt tttctctacg ctttttttta attgccgtaa ttttcctaat 9540

ctttgatgta gaaattacac ttctgctgcc tattgcttct acattaaacc taaccaatat 9600

gttttcttga ttttttacaa gtttaatttt tttattaatt ttgttatttg gtctttatta 9660

tgaatgatca caaagtgctc tagattgatc ttagtaagac catagtcaat atatgacgta 9720

tgagttgcat tcataaagag ttttctaaac tggttttaaa aattagaaag cgaataattg 9780

catttagttt cgacctaaat tttagacttt agtcttctaa ttttggtgga aaccaaaaag 9840

aggtatatca ctgttaatga taaaattgag ttcaaactcc taccaagaag ggatattatg 9900

ctaaaggcag aaagcttcta actttttact aagcggttaa attccgttta tatccctttg 9960

ttatatatat gtacatatct atcaacctag ccctagcact ttgtttttat agtttaaacc 10020

aacccaaaac attggtcttg taaaccaata atgaattatt ttctaaaaac tttcagaaaa 10080

aaagagtaac tctttatctt taattcccaa aattaatatt ttttaaacta tttcctgaat 10140

ttattactct aaattttatg ctgctagaca aaaacaaaac attttaaatc taacaaaaaa 10200

aatcaaataa ttaatagaaa caggaagata actttttcat gctaaatata tcaatttatc 10260

ataccgcagt cgaggtaaag tacctcggac tcatacaaac ccaaaagcta ctattacact 10320

cttaaaataa aacattactc tgaacggccc cctacccaaa aaaaaaataa caaataaaac 10380

cctcatgaat agaatacttg cgtattcagc tataaaaatt aatgcaaacc cgccagcccc 10440

gtactctgta ttaaagccag agactagctc agattcacct tcagaaaaat caaatggagt 10500

tcgattagtc tcagctaaac atgacctaaa tcacaccatt cttaaaggaa atctaattac 10560

aacaaatcaa aaataatttt gatatttatt aaacaactct aaattaaatc caccaattaa 10620

tattacaaaa gataataaaa ttaaagctaa cctaacttca taagaaatag tttgagctac 10680

cgctcgcaaa gaacctaata aagaatattt acaatttgat gatcatcccg ccactatagt 10740

tgaatacacc cctacactta aacaacacaa aaaaaacaga attctcagat taaaccttat 10800

taatccagtt tcataaggta aaactgatca tactaataaa gaaataaaca aactaaatac 10860

aggacataaa tagtaaacta aaaagtttga tatagtagga ataatttgtt ccttagtgaa 10920

aagctttact gcatcagaaa aaggctgtaa aattcctata tagcctactt tatttgggcc 10980

tttacgaatt tgaatataac ctaaaatttt tcgttcaagt aaagttacaa aagccacgcc 11040

aattaaaaca cacactatta ataccaaaaa attaactatc tctacaatta ttaaagtcac 11100

aaaacaatta gattactaat aatttaacta tttatagaat tgctctattt tataggatcc 11160

taaatccaac acatattatc tgccaaaata gtaatcatta tataaaataa ttttaattat 11220

ttaatccttt cgtactaaaa taatttttct ttattaaaag atagaaacca acctggctta 11280

cgccggtttg aactcaaatc atgtaaaatt ttaaaggtcg aacagaccta cttatataac 11340

tgctacaatt ataaagatat tttaattcaa catcgaggtc gcaaactttt ctttcgataa 11400

gtactcttaa gaaaaataac gctgttatcc ctaaagtaac ttaaactttt aatctttaat 11460

taggatcatt taaaatttta atcacatata agtgtactta atgttaagca gttaataatc 11520

attttactct tatcgcccca ataaaataaa ttcaatttac aaaatcttta taacctaaaa 11580

tttgactata taaaatttaa tattaagctt tatagggtct tatcgtccct ttaatacatt 11640

taagcctttt cacttaaaag taaaattcaa taaacaatat aaagacagat cttcttttgt 11700

ccaaccattc atacaagatt ccaattaaaa aactaacgat tatgctacct ttgcacggtc 11760

aaaataccgc ggccatttaa tactatcaat cagtgggcag gttaaacttt ttataacttc 11820

aaacagacat gtttttgata aacaggcaaa gaagttattt ttgccgagtt cctttattcc 11880

gccctatcaa ttttaaacaa tatttattat ttaaatgttt tgctttaata aaaaactttt 11940

actaataaaa tcattataac ttaactcttt atatttaaat taatttttta atactccaaa 12000

aaagtttata taaatattat ccaattttaa tttagctaga acactttata aatatagcta 12060

cttttaagcc taattcacca attaaactat tttaaactat ttatcatttt aattataaga 12120

agctaatccc tcctacactt aaattttaca tcttatcaac gtaaaaatta aattaaattt 12180

actttttaaa aaactagata taataaaact cgactagcgt ttcaccttta attttatatt 12240

aaaaatttat tttctacaaa aaatttttta taaaattagc tatattttat ttcgagatat 12300

ttaattttta ttaacttata taaattttcc ctgatacaca aggtacaata tttaaaattt 12360

tttacttcaa attttcttgc tttcctttac agtactctta actatagttt ataaattgtg 12420

tttattaaaa attactagca tgaacaaatt acaaattatt tttcataaat ataaaatttt 12480

caaatttatt taacaagtca ttaataaata gatgggtaat tattaattta taaagaaaat 12540

atacatatcc tcatctttac tagatacact ttccagtaca tctactatgt tacgacttat 12600

ttcacttata aatgaaagcg acgggcgata tgtacacaat ttagaactta tatcttgtta 12660

gcttattaaa ctaaaaatac aatcaaatcc atctttataa taatattatc ttattaatcc 12720

gcagtaaagt aatttactgt aacccatttt accttgctta ttggctgcac catgatctaa 12780

ttttactaaa caactaaatt taataatttt tcctttaaga attatctgat aatgatggta 12840

tacaaactgt tagaattact aaagcaagta agatttttcg tggtttatcg attctaaaac 12900

aggttcctct gaatagatta aattgccgcc aaatttttta aattttaagt atttatcttt 12960

tactaattca gttttatatt aaatttattt tagaataata gggtatctaa tcctagttta 13020

tttctaaact tttttagctt caattataat tttactccat acataataat atcctaattt 13080

taccttttat tacctgtatg agatttttaa tttaatcatc taactataac cttagaattt 13140

ttactcaatc ttaaagtata accgcgaatg ctggcacaaa tatatatcag attttaatat 13200

tattactata tcacaccttc atgtataatt ttataatatt ttatgctgag acaataaagg 13260

tttataattt tatttcactc gaataccaat ttaatattta attaaattgt ttaaatcaca 13320

ctttaatttt catacaattt taataataat gtaaaaactt aagccaaaat caaacatttg 13380

tcgtgattgc aaataataac tttaaaaaac ttaaatatac tatatataac ataaaaatga 13440

gaataagctt atacttaaag tataacttat acatcttaag cgccacaagc taacgtttta 13500

atattttaaa ctatttaagt tttgttataa ctaatattgc tacaaatata tttattatct 13560

acacaaaaac aaacaatata atcaccaacc caaccacaaa aatttttata aacactttaa 13620

tattattaag ttgtaactca tttaacaacc taaaatattt cttaaacaaa aaatacatcc 13680

cctggccacc aaaatattca aatcaccctt tgtctcctaa tttttcaaca cctaccccac 13740

taatgagact agaccctcta attttttttg tcctaagaaa aggtataaat catatagacc 13800

cccttattac tactatctta taattattaa tagattttaa attgtaactt acatttaata 13860

aattaaatat ataacctata actccccctg ctaatcttac ccccaacact atatttttca 13920

ttattacatt caaacaaatt atataaggct ctgggaaaat aacccaagat agaacagctc 13980

ctataaaaat agctcttaca cctagtaaaa tcatagaatt tcttataatt ttatcctcat 14040

ccctaatatt tattataact cttaaatttg aattacctct taacctataa aaaattagac 14100

gtatagtata cataactgtt aaccctgtag ctaaaatata taacagtaaa gaaataaaat 14160

taatccatct cataaaagcc acctctaaaa ttatatcttt agaataaaac cctgctaaaa 14220

aagggaaccc acataaggct aaatttgaaa ctcttataaa agacactctt aacggcataa 14280

atttaattaa accacctata catcgaatat cctgataatt gccaacccta tggataataa 14340

tacctgcgca tataaataac aatgctttaa ataatgcatg actcagcaga tgaaaaaaag 14400

aaagatcagg gtatcctaaa gctaaaatac ttaacataac ccccaattga cttaacgttg 14460

ataaggcaat aatttttttt aaatcatatt caaaattggc ccctaatccc gccataaata 14520

tagttaagca agaaataatt aataatattc cttggatttt agaaccttct aacgcaccac 14580

taaatcgaat tattaaatac accccagcag ttactaaagt agaagaatgt actaaagcag 14640

aaacaggagt aggagctgct atagcggcag gaagccatgc tgagaaagga atttgagcac 14700

ttttagtcat agctcttaac attaacaaaa acttaaacaa aattcactca gtatctataa 14760

tatagtaaga ataaataaca aaatttcatc tacctaacct ggccattagt ccaatactaa 14820

gtaaaatagc aacatcacct actcgattag ataagatagt taacatccca gcattagcag 14880

acttttcatt ttgataaaaa attactaaag cataagaaac taaacctaat ccatcccatc 14940

ctaaaagaat acttaccaag ttaggtctta aaactattat agccatagat aaaacaaaca 15000

ttaaaactaa atatataaat cgattaaagt ttttatcctc cttcatatat ctacctctat 15060

aatacatcac acttccagaa ataagcaata caaaacacaa aaaaagtatc gaaattcaat 15120

caaaaattaa aacaaacacc actgataatg aatttaatct tgctaaatcc cattcaatta 15180

cactagtaac accactaaat aattttatta aagaaactgt tccacaaact aaagaccccg 15240

ctcatagccc aagtattcta atcttatgta tataaattat tcaaaccatt atttaaatat 15300

taaattatta atttaataca aaatacagta acatttctac caatggtaca aaacaattag 15360

tagtaattaa atctaaataa atcaaagcaa atcgatttgc acgataaatc ttttcaacgt 15420

aactgaaatg ctgttctatt ttagctctgc tttgacacta tcatattata tttccaatct 15480

actttacatt taaataattt attaaaaaaa gctactaaaa atattaagaa atgataatac 15540

tattatatta tacaatttta tttttcttta tttatacgga ggtatataag gtatatataa 15600

atatatcaca ttagatataa gtaaataata taaacaaatc aaatatataa aaattaaata 15660

tgtatattcg cctccacaat tataccccat ccaatataat acaggataga taaaacatta 15720

aaattataaa agggtaaaat tataaataaa tagaagtata taatgtctta gtttatatgg 15780

aacattgaaa cattttatat aaaagtaaaa attatatgta acttcattat ataaatatat 15840

gatttatact atttttattt attctatggg ttgaaaaaaa gtagtagtct caaccctaga 15900

ccctaattcc tctagagggg aaacgggtct agaccgttga gactactact ataatagggg 15960

caaagaacat tatattatat ttaggtaaat ataaattaaa tatttaatta tactaacaaa 16020

taatttaaca ttaatatata tatatatata tatatatata tatatatata tatatatata 16080

tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 16140

tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 16200

tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 16260

tatatatata tatatatata tatatatata tatatatata tatatatata tatatatata 16320

tatatatata tgtatatatg tatatgtata tatgtatatg tatatatgta tatgtatata 16380

tgtatatgta tatatgtata tgctttgtac tctacaatat ataatatcta ctatatac 16438

Claims (10)

1. a kind of method precise determination Eriocheir sinensis mitochondria of precise determination Eriocheir sinensis mitochondria whole genome sequence The method of whole genome sequence, which is characterized in that this method is based on high-flux sequence, comprising the following steps:

(1) complete genome DNA in the Eriocheir sinensis musculature is extracted, gene library is constructed, is sequenced using the second generation Technology is sequenced；

(2) it is screened by reference to sequencing read of the nearly edge species mitochondria whole genome sequence to high quality, except stoning DNA Then segment carries out the splicing of mitochondrial genomes, chooses optimal splicing result, then to the mitochondrial genomes spliced Encoding egg white gene, rRNA and tRNA carry out prediction annotation；

(3) by assembled good mitochondrial genomes sequence and nearly edge species mitochondrial genomes sequence alignment, according to conserved region, then With reference to the mitochondria whole genome sequence design primer of the nearly edge species, PCR expansion is carried out to corresponding second generation sequencing sample Increase sequencing, PCR product is carried out using first generation sequencing technologies, is finally corrected to the mitochondrial genomes sequence of splicing, i.e., It can get Eriocheir sinensis mitochondria whole genome sequence.

2. in the method precise determination of precise determination Eriocheir sinensis mitochondria whole genome sequence according to claim 1 The method of magnificent Eriocheir mitochondria whole genome sequence, which is characterized in that the Eriocheir sinensis muscle groups are extracted in step (1) Complete genome DNA in knitting uses phenol/chloroform method.

3. in the method precise determination of precise determination Eriocheir sinensis mitochondria whole genome sequence according to claim 1 The method of magnificent Eriocheir mitochondria whole genome sequence, which is characterized in that nearly edge species are eriocheir japonicus described in step (2) The closely square crab (Hemigrapsus sanguineus) of (Eriocheir japonica), meat ball, Helice wuana (Helicana ) and Eriocheir sinensis (Eriocheir sinensis) wuana.

4. the method for precise determination Eriocheir sinensis mitochondria whole genome sequence according to claim 1, feature exist In the splicing of step (2) Mitochondria genome is to carry out the splicing of more Kmer parameters using NOVOPlasty splicing software.

5. the method for precise determination Eriocheir sinensis mitochondria whole genome sequence according to claim 1, feature exist In, in step (2) to the encoding egg white gene, rRNA and tRNA of mitochondrial genomes carry out prediction annotation be utilized respectively blast Software, Barrnap 0.4.2 and tRNAscan-SE v1.3.1 software.

6. the method for precise determination Eriocheir sinensis mitochondria whole genome sequence according to claim 1, feature exist In, which is characterized in that PCR amplification the primer described in step (3) includes following 19 pairs of primers:

1st pair of primer: F:SEQ ID NO.1 and R:SEQ ID NO.2,

2nd pair of primer: F:SEQ ID NO.3 and R:SEQ ID NO.4,

3rd pair of primer: F:SEQ ID NO.5 and R:SEQ ID NO.6,

4th pair of primer: F:SEQ ID NO.7 and R:SEQ ID NO.8,

5th pair of primer: F:SEQ ID NO.9 and R:SEQ ID NO.10,

6th pair of primer: F:SEQ ID NO.11 and R:SEQ ID NO.12,

7th pair of primer: F:SEQ ID NO.13 and R:SEQ ID NO.14,

8th pair of primer: F:SEQ ID NO.15 and R:SEQ ID NO.16,

9th pair of primer: F:SEQ ID NO.17 and R:SEQ ID NO.18,

10th pair of primer: F:SEQ ID NO.19 and R:SEQ ID NO.20,

11st pair of primer: F:SEQ ID NO.21 and R:SEQ ID NO.22,

12nd pair of primer: F:SEQ ID NO.23 and R:SEQ ID NO.24,

13rd pair of primer: F:SEQ ID NO.25 and R:SEQ ID NO.26,

14th pair of primer: F:SEQ ID NO.27 and R:SEQ ID NO.28,

15th pair of primer: F:SEQ ID NO.29 and R:SEQ ID NO.30,

16th pair of primer: F:SEQ ID NO.31 and R:SEQ ID NO.32,

17th pair of primer: F:SEQ ID NO.33 and R:SEQ ID NO.34,

18th pair of primer: F:SEQ ID NO.35 and R:SEQ ID NO.36,

19th pair of primer: F:SEQ ID NO.37 and R:SEQ ID NO.38.

7. the method for precise determination Eriocheir sinensis mitochondria whole genome sequence according to claim 1, feature exist In the system total volume of PCR amplification described in step (3) is 50 μ L, including each 2 μ L of forward and reverse primer, 2 μ L of DNA profiling (concentration is 50ng/ μ L), 19 μ L, Taq PCR MasterMix of DNase-Free deionized water, 25 μ L.

8. the method for precise determination Eriocheir sinensis mitochondria whole genome sequence according to claim 1, feature exist In the response procedures of PCR amplification described in step (3) are as follows: 94 DEG C initial denaturation 5 minutes；94 DEG C are denaturalized 30 seconds, 55 DEG C of annealing 30 Second, 72 DEG C extend 1 minute, and totally 35 recycle；After circulation terminates, extending 10 minutes for 72 DEG C, 10 DEG C of termination reactions.

9. a kind of Eriocheir sinensis mitochondria complete genome DNA sequence, which is characterized in that the DNA sequence dna such as SEQ ID NO.58 It is shown.

10. a kind of for expanding the primer of Eriocheir sinensis mitochondria complete genome DNA, which is characterized in that the primer include with Lower 19 pairs of primers:

1st pair of primer: F:SEQ ID NO.1 and R:SEQ ID NO.2,

2nd pair of primer: F:SEQ ID NO.3 and R:SEQ ID NO.4,

3rd pair of primer: F:SEQ ID NO.5 and R:SEQ ID NO.6,

4th pair of primer: F:SEQ ID NO.7 and R:SEQ ID NO.8,

5th pair of primer: F:SEQ ID NO.9 and R:SEQ ID NO.10,

6th pair of primer: F:SEQ ID NO.11 and R:SEQ ID NO.12,

7th pair of primer: F:SEQ ID NO.13 and R:SEQ ID NO.14,

8th pair of primer: F:SEQ ID NO.15 and R:SEQ ID NO.16,

9th pair of primer: F:SEQ ID NO.17 and R:SEQ ID NO.18,

10th pair of primer: F:SEQ ID NO.19 and R:SEQ ID NO.20,

11st pair of primer: F:SEQ ID NO.21 and R:SEQ ID NO.22,

12nd pair of primer: F:SEQ ID NO.23 and R:SEQ ID NO.24,

13rd pair of primer: F:SEQ ID NO.25 and R:SEQ ID NO.26,

14th pair of primer: F:SEQ ID NO.27 and R:SEQ ID NO.28,

15th pair of primer: F:SEQ ID NO.29 and R:SEQ ID NO.30,

16th pair of primer: F:SEQ ID NO.31 and R:SEQ ID NO.32,

17th pair of primer: F:SEQ ID NO.33 and R:SEQ ID NO.34,

18th pair of primer: F:SEQ ID NO.35 and R:SEQ ID NO.36,

19th pair of primer: F:SEQ ID NO.37 and R:SEQ ID NO.38.