CN108753990B - Whole-genome microsatellite marker of Charybdis feriatus, screening method and application - Google Patents

Abstract

The invention relates to a whole-genome microsatellite marker of Charybdis feriatus as well as a screening method and application thereof, wherein the screening method mainly comprises the following steps: extracting genomic DNA of Charybdis feriatus; constructing a genome library and carrying out high-throughput sequencing; quality control, assembly and evaluation of sequencing data; analyzing and screening microsatellite loci; designing a microsatellite locus primer; carrying out PCR amplification on the genomic DNAs of different individuals of the Charybdis feriatus by using primers; detecting the PCR amplification product by using non-denaturing polyacrylamide gel electrophoresis; determining the genotype of each individual according to different migration distances of the amplification products, thereby obtaining the polymorphism map of the genetic variation of the Charybdis feriatus. The sequence number of the microsatellite loci obtained by screening reaches 24101990; the total number of microsatellite loci reaches 3779209. The method has the advantages of simple operation, rapidness, accuracy, sensitivity, time and labor conservation, low economic investment and the like, greatly improves the screening efficiency, and can be applied to genetic variation analysis and population genetic diversity research of the Charybdis feriatus.

Description

Whole-genome microsatellite marker of Charybdis feriatus, screening method and application

Technical Field

The invention belongs to the field of microsatellite molecular markers of Charybdis feriatus; in particular to a whole genome microsatellite marker of Charybdis feriatus, a screening method and application thereof.

Background

Microsatellites are ubiquitous in eukaryotic genomes, are random repetitive DNA sequences consisting of 1-6 nucleotides, and are a relatively mature molecular marker technology at present. The microsatellite has the advantages of large quantity, random distribution, high polymorphism, strong repeatability, co-dominant inheritance and the like, and is widely applied to the fields of population genetic diversity analysis, germplasm resource protection and management, genetic linkage map construction, QTL positioning and the like.

The first step in microsatellite analysis is to obtain high information content microsatellite loci by various experimental methods. There are many traditional experimental methods for obtaining microsatellite loci, among which the more classical method is to construct a genomic library, then use a probe for hybridization, clone sequencing, and finally select the microsatellite DNA of positive clones, but the method is relatively complicated. Another more traditional method for screening microsatellite loci, namely an enrichment method, comprises a magnetic bead enrichment method, a nylon membrane enrichment method and the like, and the principle of the method is that a section of mononucleotide chain with a special structure is fixed on a nylon membrane or small magnetic beads, then the mononucleotide chain is combined with a target fragment by a DNA hybridization method, and finally positive clones are screened. In addition, based on the principle of random amplified fragment polymorphism (RAPD), an improved method combining a magnetic bead enrichment method with an RAPD technology, namely a fast isolation by AFLP sequences associating enrichment method (FIASCO), has been developed.

The Charybdis feriatus belongs to Arthropoda (Arthropoda), Crustacea (Crustacea), decapetada (Decapoda), Discaceae (Portunidae), Charybdis (Charrybdis), commonly called Charybdis floribunda, Charybus floribunda, Scylla variegatus, Charybus zebrinus, Cross crab and Scylla serratus, is distributed in shallow sea areas of east and south China sea, inhabits on sand-mud rocky areas, rocky reefs or coral reef areas with water depth of 10-60 m, and is one of important marine fishery resources in China. In order to excavate widely distributed Charybdis feriatus resources in China, research on population genetic structure and genetic diversity is necessary. Microsatellite markers are ideal markers for carrying out the research, but the currently available microsatellite markers of Charybdis feriatus are very few, and the situation limits the development of genetic research related to Charybdis feriatus. Moreover, PCR amplification can only be carried out aiming at a certain sequence, and cannot cover the whole genome; the random property of the selected clone is very high, and the repetition rate is also very high. Therefore, in the existing screening method of the microsatellite marker of the Charybdis feriatus, the PCR amplification is utilized to clone and sequence the amplification product, the efficiency is low, the number of the obtained microsatellite loci is very small, and the microsatellite loci cannot cover the whole genome. Therefore, it is very necessary to develop a whole-genome microsatellite molecular marker of Charybdis feriatus.

Disclosure of Invention

The invention aims to provide a method for screening whole-genome microsatellite markers of Charybdis feriatus, which makes up the defects in the prior art.

A whole-genome microsatellite marker of Charybdis feriatus comprises 14 pairs of polymorphic microsatellite markers, wherein the 14 pairs of microsatellite markers are as follows:

further, the extraction of the genomic DNA of Charybdis feriatus in the step (1) mainly comprises the following steps: taking muscle tissues of Charybdis feriatus, putting the muscle tissues into a centrifugal tube containing a tissue lysate, and homogenizing; then adding RNA enzyme, mixing evenly, and incubating at room temperature; adding protease K, mixing, digesting in 55 deg.C water bath to obtain clear solution; then adopting Tris-saturated phenol and chloroform to continuously extract for a plurality of times; sucking the supernatant into a new centrifugal tube, adding precooled absolute ethyl alcohol to precipitate DNA, centrifugally collecting the precipitate, and cleaning; finally, the DNA was dried at room temperature and dissolved in sterile double distilled water and stored at-20 ℃ low temperature.

Further, the construction and high-throughput sequencing of the genomic library in step (2) comprises the following steps: the method mainly comprises the following steps: firstly, breaking genome DNA to 280bp and 450bp respectively by using a Biorupt ultrasonic crusher to establish two small fragment DNA libraries; second, DNA end filling: firstly, 60ul of fragmented DNA (namely 1ug) is taken, 40ul of illumina Endrepair buffer is added, mixed evenly and centrifuged, and the mixture is placed for 30 minutes at the temperature of 30 ℃; adding 160ul AmpureBeads, mixing well, and standing for 15 minutes at room temperature; placing the magnetic frame again, standing at room temperature for 5 minutes, removing supernatant, and keeping beads; adding 200ul 80% ethanol, standing at room temperature for 30 s, removing supernatant, repeating for 1 time, and drying at room temperature for 15 min; finally, adding 20ul RB, taking down from the magnetic frame, fully mixing, and standing at room temperature for 5 min; thirdly, adding A-tailing with the total volume of 30ul (12.5 ul of A-tailing buffer, 17.5ul of DNA after filling the tail end), and standing for 30 minutes at 37 ℃; fourthly, adding a joint, wherein RB is 2.5ul, ligation Mix is 2.5ul, the joint is 2.5ul, and the mixture is placed for 10 minutes at the temperature of 30 ℃; fifthly, enriching the connecting fragments by using a PCR amplification method; sixth, the DNA library was quantified with Qubit; seventh, clusters were generated and high throughput sequencing was performed with Hiseq 3000.

And a small fragment DNA library is established, so that the sequencing result is more accurate, and a genome sequence with a wider accurate range is obtained. Only by filling in the DNA end, the base A can be added at the DNA end in the third step, which facilitates the subsequent sequencing and splicing.

Further, the quality control, assembly and evaluation of the sequencing data in the step (3) comprises the following steps: firstly, filtering and evaluating the obtained sequences by using Trim Galore and FastQC respectively to obtain clean reads suitable for assembly; secondly, splicing a plurality of Kmer parameters of all the obtained clean reads by using SOAPdenovo splicing software to obtain contigs and scaffolds; thirdly, the resulting contigs and scafffolds were evaluated using QUAST; fourthly, GCE software is used for deeply analyzing the characteristics of the Charybdis feriatus genome.

The present invention assembles all clean reads without rejecting short contigs.

Further, the analysis and screening of the microsatellite loci in the step (4) comprises the following steps: when MISA is used for analyzing and screening the microsatellite loci, the conditions are set to repeat 1 base for at least 10 times, repeat 2 bases for at least 6 times and repeat 3-6 bases for at least 5 times, and on the basis, two SSR loci are considered to form a mixed SSR locus as the distance between the two SSR loci in a sequence is less than 100 bp.

Further, the design of the microsatellite locus primer in the step (5) comprises the following steps: under a linux system, Primer Premier3version:2.3.6 is used for Primer design of the searched microsatellite locus, the setting conditions are that the product length range is 100-280bp, the annealing temperature is 57-60 ℃, and the Primer length is 20-23 bp.

Further, in the step (6), different Charybdis feriatus can be subjected to primer pairThe PCR amplification of genomic DNA from a subject comprises the steps of: the designed primer is subjected to PCR amplification by taking the genomic DNA of Charybdis feriatus as a template, the reaction system is 12.5 mu L, the genomic DNA template comprises 0.5 mu L, the final concentration of the primer is 0.4 mu M, and the primer concentration is 1 XPCR buffer (+ Mg) ²⁺ ) The final concentration of dNTP is 0.2mM, Taq DNA polymerase is 0.5U, and finally sterile double distilled water is supplemented until the total volume is 12.5 mu L; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at a specific annealing temperature of the primer for 50 sec, extension at 72 ℃ for 50 sec, and 30 cycles; finally extension was carried out at 72 ℃ for 7 minutes.

Further, the step (7) of detecting the PCR amplification product by using native polyacrylamide gel electrophoresis comprises the following steps: mixing the PCR product with 6 XPCR loading buffer, loading 1. mu.L of the mixture into 8% non-denaturing polyacrylamide gel for electrophoresis; and obtaining a PCR product electrophoresis image of the Charybdis feriatus after dyeing and developing.

The method is mainly applied to genetic variation analysis and population genetic diversity research of Charybdis feriatus.

The invention comprises the construction and sequencing of a Charybdis feriatus genome library, the screening of microsatellite loci and the design of primers, and finally 14 polymorphic markers with clear amplification bands are obtained, and the 14 polymorphic markers are used for the genetic analysis of different individuals of the Charybdis feriatus to obtain the polymorphic map of the genetic variation of the Charybdis feriatus.

Compared with the prior art, the Charybdis feriatus is taken as a research object, sequences containing microsatellite loci in the whole genome are obtained through genome Survey analysis and related microsatellite analysis software, and primer design is carried out on the sequences to screen out primer pairs with polymorphism. The sequence number of the microsatellite loci obtained by screening reaches 24101990; the total number of the microsatellite loci obtained by screening the invention reaches 3779209. The method has the advantages of simple operation, rapidness, accuracy, sensitivity, time and labor conservation, low economic investment and the like, greatly improves the acquisition of the microsatellite locus number and the efficiency of screening the microsatellite marker, and provides a data basis for genetic variation analysis and population genetic diversity research of the Charybdis feriatus. Can be applied to genetic variation analysis and population genetic diversity research of Charybdis feriatus.

Drawings

FIG. 1 is a detection map of 40 individuals of Charybdis feriatus by using a screened primer XBX-16, wherein M is a molecular weight standard, and 1-40 are individuals of Charybdis feriatus.

FIG. 2 is a detection map of the screened primer XBX-28 on 40 individuals of Charybdis feriatus, wherein M is a molecular weight standard, and 1-40 are individuals of Charybdis feriatus.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

Example 1

1. Extraction of Charybdis feriatus genome DNA

40 adult crabs of Charybdis feriatus are collected, and about 10g of muscle tissues are taken and stored by 95% alcohol. Taking about 100mg of muscle tissue, putting the muscle tissue into a centrifuge tube with 1.5mL of 300 mu L of tissue lysate, and homogenizing; sequentially adding 10 mu L of RNase (20mg/mL) into a centrifugal tube, uniformly mixing, and incubating at room temperature for 2 min; adding 5 μ l protease K (20mg/mL), mixing, digesting in 55 deg.C water bath to clarify, shaking for several times, and mixing to accelerate its dissolution; then adopting Tris-saturated phenol and chloroform to continuously extract for 2 times; absorbing about 300. mu.L of supernatant into a new centrifuge tube, adding 1ml of precooled absolute ethyl alcohol to precipitate DNA, centrifuging at 12000rpm to collect precipitate, and washing with precooled 70% ethyl alcohol once again; finally, the DNA (precipitate) was dried at room temperature and dissolved in 40. mu.L of sterile double distilled water and stored at-20 ℃ for further use.

2. Construction and high throughput sequencing of genomic libraries

The construction and high-throughput sequencing of the genome library of the Charybdis feriatus individuals mainly comprises the following steps: firstly, breaking genome DNA to 280bp and 450bp respectively by using a Biorupt ultrasonic crusher to establish two small fragment DNA libraries; second, DNA end-filling: firstly, 60ul of fragmented DNA (namely 1ug) is taken, 40ul of illimination Endrepair buffer is added, mixed evenly and centrifuged, and the mixture is placed for 30 minutes at 30 ℃; adding 160ul AmpureBeads, mixing well, and standing for 15 minutes at room temperature; placing the magnetic frame again, standing at room temperature for 5 minutes, removing supernatant, and keeping beads; adding 200ul 80% ethanol, standing at room temperature for 30 s, removing supernatant, repeating for 1 time, and drying at room temperature for 15 min; finally, adding 20ul RB, taking down from the magnetic frame, fully mixing, and standing at room temperature for 5 min; thirdly, adding A-tailing with the total volume of 30ul (12.5 ul of A-tailing buffer, 17.5ul of DNA after filling the tail end), and standing for 30 minutes at 37 ℃; fourthly, adding a joint, wherein RB is 2.5ul, ligation Mix is 2.5ul, the joint is 2.5ul, and the mixture is placed for 10 minutes at the temperature of 30 ℃; fifthly, enriching the connecting fragments by using a PCR amplification method; the PCR reaction system, 50ul, included: 20ul of DNA, 5ul of PPC and 25ul of PCRmix; the PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 30 seconds, denaturation at 98 ℃ for 10 seconds, annealing at 60 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, circulation for 10 times, extension at 72 ℃ for 5 minutes, and storage at 10 ℃; sixth, the DNA library is quantified by the Qubit; seventh, clusters were generated and high throughput sequencing was performed with Hiseq 3000.

3. Quality control, assembly and evaluation of sequencing data

Firstly, processing original reads by using a Trim Galore version 0.3.7 tool to obtain clean reads; second, QC evaluation of clean reads results was performed using the FastQCversion 0.11.5 tool; thirdly, splicing a plurality of Kmer parameters of the optimized sequence by using SOAPdenovo (http:// soap. genomics. org. cn /) splicing software to obtain an optimal assembly result; fourth, some results were evaluated using QUAST version 4.5(http:// square. sourceforce. net /) for contigs greater than or equal to 100bp in length, contigs greater than or equal to 500bp in length, and scafffolds greater than or equal to 5000bp in length; fifth, GCE software (ftp:// ftp. genomics. org. cn/pub/GCE) was used to perform in-depth analysis of the characteristics of Charybdis feriatus genome.

4. Screening of microsatellite loci and primer design

When MISA is used for analyzing and screening the microsatellite loci, the conditions are set to repeat 1 base for at least 10 times, repeat 2 bases for at least 6 times and repeat 3-6 bases for at least 5 times, and on the basis, two SSR loci are considered to form a mixed SSR locus as the distance between the two SSR loci in a sequence is less than 100 bp. The SSR site search results are shown in table 1.

Under a linux system, Primer Premier3version:2.3.6 is used for Primer design of the searched microsatellite locus, the setting conditions are that the product length range is 100-280bp, the annealing temperature is 57-60 ℃, and the Primer length is 20-23 bp.

5. PCR amplification of microsatellite markers

Selecting 60 pairs of primer pairs, performing PCR amplification by using 40 Charybdis feriatus genome DNA as a template, wherein the reaction system is 12.5 mu L, the reaction system comprises 0.5 mu L of genome DNA template, the final concentration of the primer is 0.4 mu M, and 1 XPCR buffer (+ Mg) ²⁺ ) The final concentration of dNTP is 0.2mM, Taq DNA polymerase is 0.5U, and finally sterile double distilled water is supplemented until the total volume is 12.5 mu L; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at a primer-specific annealing temperature for 50 sec, extension at 72 ℃ for 50 sec, and 30 cycles; finally extension was carried out at 72 ℃ for 7 minutes.

6. Electrophoretic detection of PCR products

Mixing the PCR product with 6 XPCR loading buffer, loading 1. mu.L of the mixture into 8% non-denaturing polyacrylamide gel for electrophoresis; the molecular weight standard is pBR322/MspI, the electrophoretic fluid is 1 xTBE, the constant voltage is 35-40V/cm, and the electrophoresis is about 1-1.5 hours.

Dyeing and developing after electrophoresis is finished, wherein the operation process comprises the following steps: firstly, fixing the mixture by 70 percent ethanol for 10 minutes, and washing the mixture by distilled water for 5 minutes; then dyeing for 10 minutes by 1.5 per thousand silver nitrate, and washing for 8 seconds by distilled water; finally, coloring with a coloring solution (2% NaOH + 4% formaldehyde) until the band type is clear, and washing the gel with distilled water to obtain the polymorphism map of the genetic variation of the Charybdis feriatus. The final success involved 14 pairs of polymorphic microsatellite marker primers as shown in Table 2. FIG. 1 is a detection map of 40 individuals of Charybdis feriatus by using a screened primer XBX-16, and FIG. 2 is a detection map of 40 individuals of Charybdis feriatus by using a screened primer XBX-28, wherein M is a molecular weight standard, and 1-40 are individuals of Charybdis feriatus.

TABLE 1 SSR site search results

TABLE 2 14 polymorphic microsatellite markers of Charybdis feriatus screened by the invention

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

SEQUENCE LISTING

<110> Shantou university

<120> screening method of whole-genome microsatellite markers of Charybdis feriatus

<130> 2018.3.27

<160> 14

<170> PatentIn version 3.3

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attctcttct ctgctccttg cttgtaaatg aagttgacag agtgccttgg tcgttgtaag 1500

gatgtttcgt aagtatacga aacatccgta tagacaggag caaacgtgac gtagcagact 1560

acaggagtgt gatacaggca ggctatgctt acagcttaca acaaggaggc taaaagacat 1620

gtcctttagc ctccttggct tacaagccat gtgtttgaaa agaaatccgc aaatgcagtg 1680

tagcgtttca gcaagttttt ttccttctgt ttctaaatca ttctacatac tgacacagct 1740

actgagtgcc gcagatgacg taataatgca aatcatagta tgaatattga aacaaatcag 1800

cgataacagt attacgagtt tcacggttgg ctggtgtcga ttttcaatat tttagagact 1860

gcaagtatga atcagtggca gaataagaaa ataaattaat caaatattta ataattcaaa 1920

caatagccgg atattatctg caatagaaga cacgtaatag attctgcatt tgaacgagat 1980

ggtacaagag tggtgtgtca aattgcccgc ctgcctttgt tggtcactgg tcagtgtact 2040

ttactataga ctaataatgg gaattgagat gtgcctgcgt ttctctgaac ttatcttcgc 2100

ggtgtgtcgc ctcaggatgc aacgtaagta cacctatgat actctgatgg tgctatgaat 2160

ctgcataaaa ctcgatttat caggtgattc atgttacatt gtgtgcaagg gtgttaagcc 2220

tagtacacac tactcgtatc cacgcggagt gacgagcctc cacaatccct cgtcacgcgg 2280

caggccagcg cggcggtcaa gaggggtgag cgccgacaca cctaacccac cacgctggtc 2340

ctctgcatag aaataggaga ggtgtcacca tgaaactggt cataggaaat gcacctcaag 2400

tactctaata tactgaggaa aactgagaaa tcattttccc gtgccgagaa tcgaacccgg 2460

aacctctcga atgagaggcg agtagttatc cggtgagtca aagggaggta tcccctcagc 2520

gctgtgagat aaacaagttt tgtccttctc atcacacgac ttcctcctct gggtgggata 2580

tatttaacga tttttcctca gtgaatatac attagggtag cgtgagtcga aacggtcaag 2640

gctgtggtta aatctcttca tgggtccaca tgagtgtgtc ctcaggccaa tgtagtacct 2700

atctttaaaa aaggagataa aatttaaacg gctaattata gatctgttag cctaacttca 2760

gtagtaggta aattaatgga gtcagttata gcgaggagca ttagggaaca tttagagagg 2820

cacaaactga taaacgattc acagcatggc ttcacgaaga ggaagtcttg cttgacaaat 2880

ttattgagtt tttacggtaa agtttatgaa gcggctgaca atggagatag atatgacatc 2940

ttatacctgg attttagcaa agcattcgat aaagtacccc atcagaggct attgagaaag 3000

gttagggcgc atggcataga tggtaaaata ctaggatgga ttaggtcatg gctaacagat 3060

aggaggcaga gagttgtaat aaacggctcc aaatccaatt ggggtcaggt gataagtgga 3120

gtgccacagg gatcagtatt aggaccattg ttgtttctaa tatatattaa cgacttggac 3180

agtggaatta gtagtgatgt tagtaaattt gcagacgaca cgaagatagg taaatttatt 3240

aggtcagact cggatgttat cgccttacag gaggatttgg ataggatgaa cgaatggtca 3300

aacaaatggc aaatgcaatt taattaatac tagtaaatgc aaaggactca gagtaggtag 3360

aggaaaacca cacaataggt acacattaaa ccgagaggaa ctagtatgct ctgagtacga 3420

gaaagattta ggagttatag tcaactcaga tctccgtcca agtaagcaag gtatagaggc 3480

cagaaataaa gcaattaggg taactggatt catttctaga agtgttaaaa gtaggtgtcc 3540

caaggtaata ttaaagttat atttggcgct ggtcagacct catcttgact atgcggtaca 3600

attttggtcc cctcattaca ggaaggatat aggtctactc gaagtagtac agaggagaat 3660

gacaaagagg atacagggga tgagggatat tccctacgaa aggagattga agatgttaaa 3720

tttgcattcc ttagagaggc gtaggttaag aggagacctg atagaagttt ttaagtggta 3780

taggggttac aataaggggg acgtaagcaa ggttcttcga gtcagtaacc aggatagaac 3840

aagaaacaac gggtttaagt tagaaaaggg taggtttaag aaagagatag gaaaaaaatg 3900

gttcgcaaat agaatagtgg acgaatagaa cggactcagc agtcaggttg ttggagcgaa 3960

gacaataaag agctttaaaa gaagactgga taaatatatg gatggggatg ataggtggaa 4020

ttaggtagga tgttttcata cagggactgc ctagtgtagg cctggtggct tcttgcagct 4080

tcccttattt tcttatgttc ttattctctg caaaggcgaa accttgtcgg aagtgaaata 4140

ctgtccaggg ccaccgggcg gaccaaggag gctaaaggac cagagagttt agtgttcgtt 4200

aaatcttgga ggaaattacc acactgccct ctcgtggctg ggcaggtaac tattataaaa 4260

ttactagaaa acgggtggtt ttatggtaga ctattacata tttcttctcg ctcatgggtg 4320

ggtgttctat atatcatata ttctatatat tatatatgtt gtgtatcatg ccgtctttca 4380

ttcatagggt agctttatta aacgatgaat gcttatagtt caattcccag catggtgact 4440

attagagctg tgttcaaact cctttctctc tctctctctc tctctctctc tctctctctc 4500

tctctctctc tcnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 4560

nnnnnnnntt tcaaaggcta cagacggtaa acttgatttg cttgagtgtt tttcgtgtta 4620

attgtgcaga aacattgcta atctgacgcg agtacactta caacaccctt gaaaacccgc 4680

atttcatcta gacctcctgg aatgtagtga agatgttgtg ctttgttaat gattgatcag 4740

aggtggttct ccagtagcag acgaaatccg ggcttcagca ctgccacctg gtggctatac 4800

aaggatctaa aattttcttc ctcctgctcc ggtgagctaa accctccttg gttgttttgg 4860

ctccttggcg cgaaccaaac aagtcaccgg ctcccgggat agcctccccc ttgctactta 4920

atacccaagc tttgagagga ccgagaaact cgttccacag ctgccttacc aataataaac 4980

tgaggaaaac ttaaatagat aaatattccc gtgccgataa tcgaacccaa gacctctcgc 5040

atgagagacg agtacttatc cggcgagcca aagagaggta ccctctcggc gccgcgagat 5100

gagtgaggtt tctccttctc agcacactag gaacaagggc gagactgaaa tattcttgtt 5160

gcttacaccc ttctaaatgg taggaaatcg aatactaatt ggaaagtgga gggattctta 5220

tgcaattcat acgtaccccc acccccattc agggcgtatt gagggtaggg tctaatggtt 5280

gagtaatatt tcataaaaat acgtacgtac actgatctcg taaccaataa aaatgttttt 5340

gaactacagt agtaacaagt tgtaataaac gtgaaacaaa accattttcg ttgaggtgca 5400

gcctcacata caaagattac tcagaaataa ccatctgtat tagtacgtac gtacgtacgt 5460

actgctaagc tagccttggc tggtaagtcg atccagctcc tccaagaagc taaggcccct 5520

ttcagacgta acgacttttc gccgtgcacc gtccaccgca cgacgccgta cattccagag 5580

agagcgccat tgtttggaag gccgtccaca caacggcaca accatgtgcc gtccttttcc 5640

tgccagttgc ctccattctt tcataatgaa cgtgccgtta ggtacgatta acgataatgt 5700

ccatgcgaaa tatgataatt tagttgcaga ggaggaggag ggggaggtat gcatgctaaa 5760

acagcattaa atcaaataaa tctaataata actgtactga catgtttgta ctgcaaacat 5820

tccacggttt tctgattcaa tacagtaatt cagaccaaag tacgataatt ttgaccaaag 5880

atgtacgata ttaccggcac tgacatctgg tcaccctgat ttgaagtagt gtcgcggctg 5940

tcgccgtcta gttgagacgg tgaaacgtcg tcatgtctga acatgctcat tgagtcctgt 6000

gaacatttca cgccgtccac ggcgcctcgc cgtgccgttt ataaatgggc gccgttatga 6060

caacggtggc cgtgaacggc gttcagccgt tcacgaccgt ggtcggtacg tgtgaaagcg 6120

gccatagaca tccgtggtta tttgagagca cgacgtcgtg ccgcggacgg cgcacggcga 6180

aaagtcgtta cgtctgaaag gggcctaagg tgttctctta gttagcaata acatagtgag 6240

tattatgtgt atcatgaaga ttgccaccaa aaggtaaata atattgattt tagttcaatt 6300

cttatttact tggtaagaat acggagtctt ggaatatata tatatatata tatatatata 6360

tatatatata tnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6420

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6480

nnnnnnntgt agtggcgtgg gcccttacgc cttgaacggc ggcccacagg tgtcttttgg 6540

ttgaggtgga tgcgtcccaa ccagcgctcc aggagaagct ggccagctgg ccgggacgga 6600

gcgctgtagt gcactgtata atcccacgat gatcacttat cttgtaaggt acgacacgga 6660

tttttacaaa atttttagta ttgaaattat actgtaccat atttgaagaa aaatattata 6720

ctgcaattcc gtactttact tttcaattaa attttgccgt ccaataccgt attgcagtga 6780

ttcccagctg gggttccgcg agaaacagta ttataataat gtgctattac aaataaagaa 6840

gacacccgtg tattagtgta ttatacaatg gagttccgtg gaacaggaga aattattaca 6900

ggggttcctt caaggtgaaa aggttgggaa tcatgtaccg aattccaccc ttgtcgtcac 6960

caggtactgc gtgggagaaa taagagttag gtctgaacaa aaccgttttc tttgatcatg 7020

cgagactgaa agaaaacgaa acaactgaaa atttagatca gtgaccacgg taaaaagttg 7080

attctgaagc tctgaacttt taataagcaa gcccgttcct tgaaatgatg atgatgatga 7140

tggtgacagt ggtggtggtg gtggtggtgg tcgtagttgt ctgattgatg tggatagatg 7200

tgtagtattt gacagccttg accatattaa agaagatata ttttatttgt ttttgtgggg 7260

atgggatggt gagcaaggcc gttctttata atgattataa tagtgatgat ggtgatgact 7320

gactgactga ctgactgatg gagatgatag atatgtggta tttgagcgtt gactataaat 7380

ctatgatagc caaactctta ttaatttaga ataatggtga cgatgacggt gactgactga 7440

ctgaaggtag aagtgcacca gtccttacca atcccctgtc cccctctgta tccgcgcctg 7500

gtaaccgtag tgatcgctgc ccttacgaaa ggagattgaa gatattaagt tggcaatctt 7560

tagacgcaag ttaagagagg gcctgacaga agttcttaag tggcgtaagg gctgtaacaa 7620

aggggacgta agaaaaagta taatcaggat tgaacgagaa ataacgggtt caacctagaa 7680

aagagtaggt ataagaaaaa ataggaaagc acttgttccc taataggatg aatgaaacgg 7740

actcagtaat caggttgtta gtggtaatat aatagagctt taaaaagaaa attagacaaa 7800

tatatggatg gggatgatag gtggaagcag ataggcatgc tttaatacag gaactgccac 7860

gggggcctga cggcttcttg cagcttccct tactttctta tgctcttatg ttcttgtggc 7920

gagcgtggac ggtttgagga cagacggagg cggagaggga agtaatcaga aattattatt 7980

attattattg ttgttgttgt tgttgttgtt gttgtcgttg ttgttattat tattattatt 8040

atcattatta ttattattat tattattatt attattatta nnnnnnnnnn nnnnnnnnnn 8100

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnta tcattattat tactattatc 8160

attattgtta aaaaacacgt acacttgtat ataactctta gcatgtgcgt gttcactgcg 8220

tctgtgcctc tggcgggctg ttgaggctgc tgcaagtgac gcttcagata cttgagtcca 8280

catacgatat cttccccttt cgaaatattc aggatatatt catattggtt aatatgatct 8340

ctctgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtnn 8400

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnntt tttgctgatc tttctgactt 8460

cccaaaccgc cctagtgtac cacccacgcc atcaggtgct gcggctccgc ccacgccatc 8520

tggtgcggca gccagcagcc ctgaacctga tgatggagat gttgaaagtt tgctctctag 8580

cctttctcaa gaaaacaaga ccctggtaaa aataatttga gtgataataa cagaacaatt 8640

caagaatgaa atcaaaacac tgaaagaaga aatgaacaga aaagaaacgg aaatgaagca 8700

actcaagagt gaagtgaagg acatgaaaaa taaagtggca acacttaaaa cgcaaattga 8760

cgaggcagaa caatacgaac gtcgagacac gatcatccta aatggtcctt ctgtgccagc 8820

tgaaagccaa agtgaaaata cattgagtgc tgcagtcgct gccatgaaag accacataaa 8880

aataaatgtg aaagaaaatt atgtcagtgt tgctcacaga ttgggtccta agcaaaacca 8940

aatcagaccc atcatagtaa aactagtgaa caagtccctc aaatacgatc aggttgaggc 9000

atgcataaag ctgaaagcct gaaaccatgg cacttaccta aatgaaagcc taacccccta 9060

aagactaaat ataatgaaga aagtcctggc tatcagagaa caacatcgac agaaattcca 9120

gcagtgttac accaaagatg gtaaaataat aataaaattg aaaaattcca ctgtaaagca 9180

gacaatagtt aatgagtcaa ccttgctgga ctttctagaa caatacccgg ccatgaagga 9240

cacacaccaa gacttgctgt catcagacta gaccagctac tggcgatata aaagtatgaa 9300

ttgtattgtt atgtaattgt atgattgtat gcatctttat gtgaatctac atgtatgtac 9360

tgtactagta cgtacactac tactaccacc accactacta ctactactac tactactact 9420

actactacta ctactactcc tactactact actactacta tagtgaaacc aatcgaaacc 9480

caaggtcggg gcggggtcaa cttgggaatg tactacacct cagcgttgcc gtgtttcctc 9540

tgctctgatt ggtgtatgat gggcctgaga ttaggcttca ccgcgcttag aactgacacg 9600

tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg ttctcgtgcg cccttttaaa cgccatttgc 9660

tagtaatttt atggtgagtt atgcactgaa tgcccgaata atccttcata gtgttttacc 9720

agcgtcaaca atgcaagtga gctgtggaaa cagaccacct gtctcaggtc acgcaggtga 9780

ggcatgaggc ttgggtgtgt aggcctaagt gttgccatcc gccagggttt taacatttat 9840

tttaattagg aaaataacac atattttact gatattcatt ctagttgtgc tgaaaacatt 9900

attgtttcac tatctggcca tcttagagct acagggaaag tctgcgaaag cattgatatt 9960

ttttcagcaa cactgtaaaa atatgtatgc cacgtttcac tgttgttgta caagtatttt 10020

acgtatatta agttaatttt tctttgatgt gctagaatat ctacttagaa ctgtagcgga 10080

agtgtaaaat tacctatctg caatttcttt tacacatatc acaacaatta cataccgctg 10140

atttaggaag tacggcaccg ccgtagcagc ggaataccct gctgatcccg ccgttgacct 10200

tggttttagg ttggttggac tatactacta tactactact actactacta ctaccactac 10260

tgctgttgct gctgctactg ctactactac taccacgact attaatacta ctattgctgt 10320

tccaaccact tcatcttgga aatattgcta ctcagtgata aaacccccaa gattctgtgt 10380

gtcagtgcgg ctaccaatga tttaattacg tgctattttt ttctttaact tcttttcttt 10440

cttaattttt tattgtaatt ttattaacac cactgtctca cagaatgacg cgtttgtacg 10500

aaactttatt ccgtattgag gaacaactca taattaatca aataattaat aattattcaa 10560

atgcatggac acactgattt taataacata tatacttcta tgatctctct tcatataaca 10620

tcttctcaaa aggagatgaa aacaacacta atattttgca tatgaattca agatcattaa 10680

ctaaaaatta cgataaaatt accgctttct tgaacagtct atctacacct ccagatataa 10740

tagccatgac agaaacttgg cttaccaaca caaacaaaca cctgcatcaa ctccctggtt 10800

atctttcctc tcatttaact agaaacacta gaccacatgg aggggtttct gcgtttgtat 10860

caaatgactt acagtctgaa caaatacatg ctctaataat aatacatgaa taccttgaaa 10920

tcaatgctgt aaagataaca accaattcca ccagtcattt catatgtgca gtatatggac 10980

cacatggtaa acatattaat gttgaggaat ttaacaatac attatattcg cttcttcagg 11040

gagataatat aaagaataaa agagttataa ttgttggaga tctaaacata aatttacttg 11100

aacaagcaac acattagcca acaaataacc ttatcgcttc actgcaaaca attaatttta 11160

taccacatat ttccaggcca acacggttcc cagatagtgc aaaccttgcg gcgccctcac 11220

tactagatca catttttaca aatcttaaca gtaattttac atctggtata atccatttct 11280

ctatatctga tcacctccca atttttctga atattaaaac cccaaaagaa acgaataaac 11340

ttattttgat ccagttgcga gaccttgatc aaataaataa agcaaatttt ccaacaaatt 11400

aagagatatt gactgtgatg accttatcac ttcacaagat gtaaactaaa attttacttg 11460

attcatggat aaagtccatc atctgtacaa tgaatgctcc ccagttaaga caaagttcat 11520

atccgaaaag agactcagca acccttggat cacacgagaa atattaaatt gtactaaaat 11580

gaagaataat ctttacaaag attggaaaat tggggtaatt acagaaaatc agtatcaaga 11640

acacagaagc aagttaaatc gtactataaa acatgccaaa caatcatatt atgcgtcagc 11700

tttcaccaat ttcaaaaaca atgcaacaaa aatacggaac acgattaaca agatgcatga 11760

aaacaataaa gacagcgctt cagaatagat ttactctaat ggtattaaaa taagtaaccc 11820

aacgtaaatt acagaaacct ttaatgaatt ttacacaaac agctcacaaa taagataatg 11880

aattaccacc gtccaacatt atcacatcta cttttctgaa aggaaattac ccaacatcta 11940

tggctgtccc accagtaaat acacacgatg ttatcagtgt cataaactca ttgaaaaaca 12000

caaaaggtaa cattcacgag ttaccagtat ctctactcaa aaccaataga gatcaactag 12060

ctgccccatt aactactctt ttcaaccatt cagttcgctg tggtaaattt cctaattgcc 12120

ttaaacatcc tacagttatt cccattcaca aaaaacacaa aaataaaaat aaaaaattac 12180

cgtcttaagt gttaattcaa agttatttga aaaattgatg aaaaaatttc ttatgaatta 12240

tctcgattcc aaatccatca tatatcnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12300

nnnnnnnnng tttggctttc gacaggggct gagtacatta gatgctttat ctaccttcag 12360

tgaaaagaca tacaccacaa tggactccca aaggtcttta ctcagcatct ttattgattt 12420

tacaaaagcg tttgacacag tgaagcacga tatcttactg caaaagctaa atcattatgg 12480

gatacgagga actattcatg attggtttca ggactactta actaatagaa tgcaaactat 12540

taggttcatt aaccacacat ccataccaca acagataaag aatggcattc ctcaaggtag 12600

tgtcttaggt ccaatacttt tcctcatata tataaatgat ctggcatgca tattcacaaa 12660

cataaaagcc attctttttg cagacgacgc aactctctat ataacaagta aagtccctac 12720

aaatataata gatatagcca ataatgaact aaaaatgttt tacaaatggt gtctaagcaa 12780

cagatagagt gtaaatctaa acaaaactta tttcatgttg ttttccaata aagaaacaga 12840

agcattacct cctctaaccc tgcaactcag tcaattaaac agaaccaatc aacacacatg 12900

tctaggtata atttatgatg atgcgatatt tttcaaaact cacataacaa accttatact 12960

aaagttatct agaattgtat ctttattata tctagctaaa gaattcatgc cattctatgt 13020

actaagactt atgtatgtgt aatgcacata ctttaccaca cctgcaatac tgtacacctg 13080

tctcgagcaa cacatacccg acacgccaaa cacacctagt gcctctattc agacttcaaa 13140

agaaaattat aaggattata agaaataggg tttttttttt acatactcag gctctattta 13200

aaagcacaaa tatattaaag ctattcgaca taaataaaat acatattggc atatacatgt 13260

acaaattatt gcacaacggt cacaacacac tacttcattc tcagcatgac cacccagcac 13320

gcacacgtga tcatctccgt acaccagcac acaaccttac tatattccaa cactcattag 13380

ctgtcaccgg tcctaaaata tggaattcaa tccacaatac cataaaaaaa cttgtaaatc 13440

actatgttcc ttcaaaaaaa cattacaaaa gacacaatat tacacaatat taacaaacgt 13500

ccatgcaaca ctgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 13560

gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13620

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngtagggttt ggttatcaaa 13680

aatgttcttg agtaaacccc tttttgccta aaagttattt ctatagcttg aaatgggctg 13740

cctacgatat tatatatcat attgtatatg taaatatttt aaccaggagc ataataaagt 13800

gcttcaaggg tttcaagtgt ttccttgaac gtgagctgct gaagaagacg agtaccaaga 13860

tatctccaga acttactaaa tagtcttttg cattgggatg tatccagccc tgtctttgag 13920

accagcacac acacacacac acacacactc tctctctctc tctctctctc tctctctctc 13980

tctctnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14040

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14100

nnnnnnnnna tccctcaaac gtaaacatag ttatataata tttacctgct ttatcatggg 14160

aaaagatttt tttatgtttc aattcattat ttaatatgtt cgtttatttt tatttatgag 14220

cggtaatata cataggcagg gaacttatac taaacaggaa ccttagtttt tttttttttt 14280

tttttttttt tgcgggtaca actcccttat cagatagaaa tgaatatcat acaatatcgc 14340

catcgacact gaagatcctg gactcagtga caatgtaagc gtatacctat ttgcagggca 14400

ggcctatata ggtgcattca atggtatggc acgggcgaga gtggagcggc atgcggggag 14460

gcaccatatc gattacctgt caaaagtgga aagttgagtg aaaaggtcgg gaacctctgc 14520

ggtctatttc agttggtcca tcgggatttg ccgccctcgt ttctgtcatt tcatttttac 14580

tctcttattt ttagcgattt ttttttaaac ttcgtgcatc acccaatttt tttcttttcc 14640

cccttttttt ttacatgtgt gcaaaacagc acaggtaaat accagtttta taaaaaaaaa 14700

ataagataag aagggaaaat tttatttcct tttccctccc caaaaatctg agagagagag 14760

agagagagag agagagagag agagagagag agagnnnnnn nnnnnnnnnn nnnnnnnnnn 14820

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14880

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14940

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15000

nnnnnnnnnn nnnnnnnnnn nnnnnnnnng ttgtgataat cttttaaagc aatacaggta 15060

aaataagtta gaggtaagaa gataaaaaaa aatgataaaa acattatcaa tcatagaatc 15120

ccaaacttta aaatggccac cacagtgaat aaaaaaaaaa aaagtaatct ctcttcttga 15180

acttggtcat cgcacaaatt caacgtgaac acacacacac acacacacac acacacacac 15240

acacacacac acacacacac acacnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15300

nnactgccac attgtttaga attaaatgat cggcttcaac gttccttaaa atgtattgac 15360

attgggcagg aggcgggtta cccttcgtag gtgggcgatg acgagggtga atcattggag 15420

actttgaaaa ttgacgagtt tctccttctc cctcccggcc tgtggggcga atgcgtacaa 15480

ggcgcctcgc atggccggac aactgctgtc cttaaaaata gtgtggtgac cgcggggacg 15540

tgagttactg aggggcgaag agcgagggga cactcagcca tgtggcactg acccttgttg 15600

acctctgaag ggttgagcac cagctggtat tcgcctcgta tgtgggtcta taagtgtgtg 15660

tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtn nnnnnnnnnn 15720

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15780

nnnnnnnnnn nnnnnnnnnn nnnnnnnncc acggcgctgc gtcatgagtc acggcactgt 15840

ggcaatcttt gtaaaacttt tgcttgtaaa cttttccccg cgtgccccgc ctggcctgtg 15900

tctcctcggg gtgtgctcag tcagcctgct cctcgctttc ccacctactt tcttgatctc 15960

atgccctgat cacctcagct catctctttt tctttgttca tttctttata agttccttgt 16020

gacattctaa tttttccttg gttttttttt tcatttacct atcatcgtgc accattacac 16080

ataccttcag ttgtcctcct ttctctcttt attcatgtct atttataatc acacacacac 16140

acacacacac acacactctc tctctctctc tctctctctc tctctctctc tnnnnnnnnn 16200

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnta tgtaagcata 16260

tagtacggta tagttttatg taagcacttg aaagagagag agagagagag agagagagag 16320

agagagagag agagagagag agagaga 16347

<210> 6

<211> 1108

<212> DNA

<213> unknown

<220>

<221> misc_feature

<222> (283)..(367)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (439)..(590)

<223> n is a, c, g, or t

<400> 6

tacacttcac acttgcctct cctccacaca cacggaccta cgcacgcacg cacacacaca 60

cacggaccta cacacggtca cacacacacg caccacctct ttcacaccac cgacaccgcg 120

agttatatag agacttgtgt gtaaattact agtcagtcat tcagtcaaca tttttgcggc 180

gtgcatgtta ttagaggtac acacgcacgc acgcacacgc acctatgcac acacacacac 240

acacacacac acacacacac acacacacac acacacacac acnnnnnnnn nnnnnnnnnn 300

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360

nnnnnnnatg tgaaactaca cacaaatgaa actagagatg cacaaaccac gctaaaatat 420

atgaaacaaa tctctctcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn aatgtgggaa 600

gatctttccg cgccgatgag aaagactgaa agaaaattta atgaataatg tgatttatct 660

acatacactc ggatgccaaa tgtcgccgcc aggtggacac ggggttgctc ctttacacat 720

catccaattt tctttatgtt ttttctcttc gtacatctta ttccagtttc gcatttagca 780

gtctccattc caggcactcg ttttgttctt tctcctactc ttcctccttc tcctcctccc 840

ttcttccttc cttccttcct tatttctttg aattcgtggt gtctttctac tctcctcatt 900

cctcactttt ctgcaatcct ttcctctctt gctctctctc ttctcttatt ttttttctgg 960

caacaccggc cccctcacca tctccccttt tcccttctat tcctcttcct ccgtgctctc 1020

ttcacactgt ccacgcccct tttgtgtgtg tcagacgccc ccatcccccc cttctctctc 1080

tctctctctc tctctctctc tctctctc 1108

<210> 7

<211> 9591

<212> DNA

<213> unknown

<220>

<221> misc_feature

<222> (3334)..(3334)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (6437)..(6472)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (6531)..(6562)

<223> n is a, c, g, or t

<400> 7

ccctcgtccc tgcaggccac actgatgcac tggtttgtgt gtggtgtgtg tgtgtgtgtg 60

tgtgtggctg tggctgtggg ctgtgactgt ctggtctgtg taaggggctg agggctgttc 120

atgctgtgtt gtgttctgca tctagtgagt gttggccggg tctttaacac agcagattca 180

cttttagatg tttttttagg tcctggtaat gtagtgtttg taggcatcca cagcccctcg 240

ctccgttcct tatagtcact ggttttaata ttttccttat ctgccttccc ttctttcttc 300

attgttcaac ctatttctaa ctctgtgcag cctgtttgcc ataaagcgtc gcagatcatc 360

tgttcgagga tttaccctga acgcagactg gcactagtct tgctaactct tgcacagctt 420

tggaacagga gagttgtttg ggagtgggaa gatttttgag tagctaatga aaaaggcatt 480

tcagaataaa actatggtat gtaatagtat ttgattattt gtacctgatt gcacattgtt 540

tcatttttac ctcatttaac tttttttttt gttttttttt tagattttag aaatgaatta 600

atattccaga tcacttgtct ggtgggggtg atatcactgg tggtgctgct cctggccctc 660

tcccgcctgc aggtctcctc ccccaaggag atcacggatg agatcaagag agaaataaaa 720

agcaaaggta tgacttccac cgtcactcgt gtgtgctgac tgtttactgt cgcagatcag 780

cagtgtgtta tgtctcagtt gaaagtgtct agtttcctgt agagtaacaa ttaatgactg 840

tgtggtgtgt tcagaatcta tatactcatg cattttgctt tctgtctcct ctagtcgaca 900

atgtcaagga cttcgtgggc gtggggacgc ggccgaggtt ccgcaagcgg gacaagatct 960

acttctacgg caggaagatg ttgcgcaagg taatggctgg cgtgggtgtg agagaagtgg 1020

ccgctgtaga acggtgggca gaatactgat ggcagagaga ggagaaagaa aatgggtaac 1080

gggaactgcc aggaatgggg ggggcggggt cagagtacat ttaactaaat tggtgtttca 1140

gctttgcgat aatttcttgc aatagaattc cttgcaaaga aaagtaacat gcatagagtt 1200

gttttattca cttttctctc ataacataca cacgaagtgc cgacacagct catgttgaca 1260

taattttcct gtgtgtgact caacatttat gccttgagag ttacaatgtt tggtctgcag 1320

gtcaaggcca acatccccac ccggcctgca gcatcacggc tcctggccaa gcgtctggcg 1380

cggcagctgc tggggcgctc ggaccgagat tccccgcagc tggaggtgat tgagcccccc 1440

atggagtaca tgcaggaaga cctcacacac ttggacccca acgtacccac agagtttgtg 1500

ttcatgctgc ggaacatcag gtgaggcccg ccgacgtttg tgtgtctgtc gcgtcacctt 1560

ggtggagcgc cacactcgtt gtctgatgca gtttaattag gtgttcatgt ttaaatggaa 1620

ggtcagagag atttgtggca agataagttg tgatgggtga ggtttgataa agagttgctt 1680

atctttagtg tgtttagtca tagctgttat ctgccattga gaaataatta atgatggaag 1740

tggccctttg atccccccac cactagtcct ttagtgtggc cattgagggc agtcaggata 1800

aaggtttaat tatcacaaca acaaaaatat cacatgtttt agcattgtga gcagccatgc 1860

cgtctttcat gtactattaa tgtttttgtc caagcattgt acttaccctt aatatttcag 1920

taagctttgg ttcacattcc ccttcactca gttgtggtgt tgacctgtat ggtagttagt 1980

tggtgaaggc atggacagtt tgtagcttag tgctatttca tactcagtgt gtagcttgtc 2040

atgacctttg ttgttagtgt aaacatgctc ttgggatgga ctacctgaaa cacattcaca 2100

ttattcctgt tgttgctgag tgttctttct gtcaatcagg gagaaataga cacaggagtt 2160

gcaataggaa gaagtagaat acatgtttca gttgatccat cagcaaaatt gagtgtttaa 2220

tccctccact tagtgttggt aatgtgagaa atagtatccc tgtagtgttt gtggtgcctc 2280

gtggactgct cacctcaagc caaaatgtaa catgagtttc tacttgcttt gctggtgcta 2340

agtgacatgg atctttcgta tagcttcagg taaatgtcac tcttaaaatc atggtaatgc 2400

gtcctggaat attttttaat acgtctcatt cttcactatt tcacactgtc cgctgggtta 2460

tggcgggtgt gaagagggtg gtacacacac tggggtctta ttggaagcct tactacaaac 2520

ctaacagtta ccaactctta ggaaactctt tcatagggac cgtcacctct tttgttaccc 2580

ttggccgggc cccctctctg cataaaaaaa taatgtgcct gagacttcct ggcaattact 2640

gggctgtttt atcatctgca ggctctagat ctgtgaggtg tttgtgggtc atgctatcca 2700

gccgccctga gatttatcta taaaaaaccg caccaactga gtgtgttcag atgcttagct 2760

gttcatcttg ttactctgcc atcatctcag tataccaggg tcactgtagt ctcacagtcc 2820

ttgcaaatta ttaagacatc ctacttccgt cttggctgaa gttcattgtc tgtgacccaa 2880

agtgtgttca gtgatgcgct acacattttt ctagtgacat ctatcattct gtctctatat 2940

aatctataat atttatgaag tttattatta tttttttttt ttttttatta ttattatttt 3000

ttattattat tattattatt attattatta ttattattat tattagcatt aaactcttca 3060

tactggaact gtactgtata tatacccaat ggagctaaga gattttgttt tgcaagttca 3120

tgtaacagaa tagcattata gctgtaggct ccttcagctt taccaattct actttcgcat 3180

tagagtcatg tggatgtggg aggaggtgaa agagaaacac agctgtatgg agctgagttt 3240

attcctgact agttttgctt ttgacttctt ccagttgacc cagagtgaaa ctggtcggta 3300

ataagctcaa ctcctgacag ctgtggtttc cttntgtatg gagttgagtt tattcctgac 3360

tagttttgct ttgacttctt ccagttgacc cagagtgaaa ctggtcggta ataagctcaa 3420

ctcctgacag ctgtggtttc ctttcacctc ctcccccaac ctgtctaaat gtgtatacag 3480

tcatgtgaat gtcagaaaaa ttatgtgtgt accatcctta atgcagcact gtactgactc 3540

tgatccttgt cacacccagg ccaactttca aggctaggta atgtattcct cactcacacc 3600

acaggctgaa ggttatgctg cctcacttca ttatcatgca gacagtggcc acattataag 3660

aactcatgtc ttccccttcc tgtgttttag taagatgttc cacgctcagg tgtgcagact 3720

tgatgcaatg ccttggcagg gtcactacat tgttgtgtca agtgtgcatg ccagcgtgtt 3780

caccaaatga ctaaatagca ggaaacgaga agaaatttat ctttcagtgg gtgactaaca 3840

atctggccac cctctgtgtg gactctgggg aaacagtcag cccacctttc acagtattgt 3900

tgatgcacag caagtattct ctttcattgt ttgcactgag cacatagcca aggcgatgca 3960

tggaatactt tatggattct ttgcttagtt ataagttgga atgaaggctg attcaaagct 4020

gatttatcct ccagttctgt aatgtgtgag ttgaggcaat gctacacaga atgatagtta 4080

tttagtaacg ttgtgcatca tcagtatagt agaagggggc cagtctctgt ttggccaggg 4140

tctgtatgcc agtcttgctt acatcagcac tgagaggcag agtttcctgg ccgctgcttg 4200

ctggagtctc cagtggagga caagagaaaa tattaacatt ttaatgtaga catttttctg 4260

tttatcttta gtgttaccaa taggaactga caactaatgg ctaacttttt tctgtggtat 4320

ttatttaggc ctatttagaa caaatatact ggagtaatga ttgcaatagt atttgtgtga 4380

tgtcactgct cagctatagc ttcagcagct ggtttagaaa aatacatcca ctgtgttctt 4440

tctgggtggc tttcaatgac tagaagggct ggagggaggg atagaaggca ctttctgtag 4500

tttttattac aatcccactt tcagagagat gtcagcctgg tatatgataa tcagcacata 4560

attgccataa taatgagttg cactcacagc caagttcaac ctcaccaaat ttgcatcaca 4620

caagaatgaa aactgctgtc aaacactgta ctagcagtgt gtggaaggtt gtgtctttgt 4680

aacgacacat ggcactgcat atgttggtgt ggcagcatgg ctgtactaga cagaaggagg 4740

ctatcaatgt agtgaagtgc ctgtggcaga cgacatgttc ttgcctcttg tctggtgcag 4800

gtgtgtctcc aggaactcct gccgtcagtc atctcttttt gtacactgtc cctcggggct 4860

ggctgccttc agcctgtcct tgcctgtgcc tctcagcatc acgttgcgtt ctttctgtca 4920

tggccacaaa atttatcttg gctttgcact gtgctgactg atgaatgttt tgaaaaagtt 4980

aatttggctt gacttagtct ggatcttgat gggatggagt gttcatgcag cagctgcctg 5040

gagattaata ggcaaggaag caaatgctgt gttcatgcaa cacaagctca aattttagca 5100

gactaaagga gtaaagggtg tttgtggctg taaggaaagg aagggattgg tcgtctctta 5160

gcgaacctca ttgctgcgcg tggctggcct gaacagcgtg tccagcgcta gccgtggaag 5220

gtcagatttg tgatcatgct gggaaaggtt tttacatatt taggcactgt tctgtactta 5280

tgatcttcat cttaccccca tcatcatcca ctttgtcatt gcctggtgtg tgtgtgtgtg 5340

agagagagag agagagagaa agagagaagt atgcatgcat ttccaccaca tggctgaggg 5400

tgcatctgtt acatgattta ttatacattt gtatttattt ttgtatgctt gacatattaa 5460

ggattttcat ggatgcagca gactggtggt gactggtgcc agcttgtcct cagctttcaa 5520

tattatgttt tgggataatt atttctgtct aatggaaata gacaaagttt tttactatgg 5580

agaaacaaaa ttttctacta atgttttttc ccttcatata aattgctgat gtgagctgat 5640

gtttgtttta aagctgacat aatttcacaa aagtgtttat agttgtaact gcaaggcaaa 5700

ctgacaaaac tgctcagaaa gtgtgtgtgt gtgatggagt gatgggtgtg gatgtggtgt 5760

tatcaacagg ccttggtctt tgcagagtgt ttggccactt tgatacaccg ctgttcctgg 5820

agctttgcaa gagcttccag acaattcacc ttttcaaggg acaaaaactc ttttctattg 5880

gtgagttatt catgtttaag accattgctg tttggattgt tgttaccatg accaactatt 5940

acagcaaagt atcccattgc atctttacct tcatatatcc tcttgataag agaatccaac 6000

tctggccacc gtctgcctca tcctgaccac cctccccacc acccactgtg ttctgcaggt 6060

gacaatgacg agaacatcta catcgtccag aagggaagga tcagcgtgta cgtctccgag 6120

cccgacggca gcatgttctc cctgaaggaa gtcctgccag gggagtccat catctccctg 6180

ctgtccttct gcgacatcct gacaggccac cctcagccct acaagaccat tggggcgcgt 6240

gccgaggaga agtccatcgt catgaagatc cccgtggagg cattcaggga cgtgttcaag 6300

aagtacccgg agatgtttgt gcgcgtggtg cagatcatca tggtgcgcct catgagggtc 6360

accttcaccg ccctgcacca gtacctcggc ctctcctcag aactcataga caaggtgatg 6420

gctgctgctg ctgctgnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnctggcatg 6480

ctgtggctga gggacattgt tactattatt attcatttat ttttcatgtc nnnnnnnnnn 6540

nnnnnnnnnn nnnnnnnnnn nnagagggtc actggtcaag ggtgatgaaa agagtgaaaa 6600

aacagccgct taggtgctaa tccccccaaa aaaaaggtaa tctcaagtta ggattatatt 6660

tctgcattct tcccaggaaa cagtactttt ctgcagttga gaatgccaaa gctaaccatt 6720

gatgtatgtc aggtgccacg cagggacagc ggaggcaacc tggtgccgtc cctcgcctca 6780

cccaacaaag tgaagcgaga cagcggctcc acaccagacg gggaggagga cggcggcggg 6840

ccaggcacgc ccgtcaggca gagcagcgca cgcaagatcg ttctggtgga ccctaaggag 6900

gagacagacg acggccactc cctcagggtg gccacggaga agttccaggc gctgctgcac 6960

ctcgactcgg acgagatcct gcaggactcg gtggaggtga gggacgtggc ctcgggctcc 7020

ttcctcatga agcaggactc catgcaggag actgccctgg tctacatcct ggtggggact 7080

cttacagtgt cccagcacga tcccgaggga aacgaggacg tcaagctgtt caccgcatgc 7140

cccggggacc tggtgggggg cctggccgtc ctctccggcg acccttcctt cttcactgtg 7200

aaggccaagc acggcgcacg catcgccacc atcaccaggg caaccttctt caagtgagtc 7260

acgggctggc tggctgtgtt gtctgttaat cctccttccc ttcgaatgtg tgtccatatg 7320

acccttttgt ggcattcaaa gcctttgact gtgataggaa acaattaaca gcagtggagg 7380

taatgcatga aatgttttgt aagcatctgc aggaaggaag gggattaaag tcatgggttt 7440

tgcaatttat agtaagtttg aaaattggaa gtggctgtag cacaaataaa gatatctaag 7500

aggagttaat aatgaaggaa agatgtacca gtgatggagc taaatcagtc agctagtgat 7560

ttatcttaac tgtggtattt acaacagagg gaattaaggg agactttgta aatgtaaaac 7620

ttgtaaaaga aagaattctt gagcattaca gaaatttggc atgtagagac tcaagtgtgt 7680

actggtataa agcttggatt aggagacagt tatgtagaga aaaagcaaca caacacaaag 7740

tcatagaaga aggatgtggt gttaactgag tgcatcattt gtcacatgaa ggataatcaa 7800

gaagaaccca gaggtggtgt tgcacgtcgc taacaccgtc attcgccgcc tgtctccctt 7860

cgtcaggcag atagactttg cactggactg ggagcacctc gaggccggca gggcgctcta 7920

caagtaagtc attgaatgct gtccctcttg gctgactttc tgtaaatcag gtagtagact 7980

agactctttc tttcagtctt tctttctttc attctttgat tttttctttc ttttattctt 8040

tctttctctc caggcaagga cagaccacgg attccacctt cattgtgctg agtggacgcc 8100

tgcggtcggt catcactcac aaggatggac ggagggaggt ggtcgctgaa tacggcaaag 8160

gagatcttgt gggaattgtg agttaccttt cctcttactg atggcgcaaa ggagatgtgt 8220

agttgtggtt ccaatattgc aggatggata gaggtcagtt agaatcagtg caaaggagaa 8280

tgggtcaaag aatgcttggg atcttgtggg cattgtgagt caccttcctc ctcactgata 8340

gaggcaaaag agatgtgttg ttgtggttcc agtattgcag gaaacatata tgtcaattag 8400

aatcaatgca aaaaatagtt aaaaggatgc ttggagtgaa gggtgtttac aagaggagat 8460

tgaagctttt aaattcacat tccttagaga acgtaacttt agagggtgcc tgatagagaa 8520

gtggtgcttg ggatataaca aaggtgacat tagctgaatt tccaggatcg gtattcacat 8580

taaagcttga acgaagttag acttaaaaag agaaagaaat tggtagcaga ataggagttg 8640

taaatgaatg gattggctca gtaaatcaca cagtacggag atttaaaaga ttagacgaat 8700

ttaaagatga ggataacatc aaacagattg gcatattgtc ataaaacaac tgcccaagta 8760

atttttcacc gatttctgga ttttttgcct atatcacgtc tccaagatat ggcggacgtg 8820

ctggtgtaat tgcctgtgtc atatgttgag agtaaatggt ttaaaattcc ttaaatattc 8880

tgcctatatc gtaagaaaga tagagaaata aagtgggttt tatttcccag aaagtagaag 8940

aaatgacttg ggctgtaatt tataaggcta atgatatttc atacaggagc tgccacatgt 9000

aggcctgatg actttgcagc tttcctcatg ttcttatgtt atctgagctt gacacgacgg 9060

gttgacacag gtggagctgc tgaccaagac tgaacgcagc acgacagtca tggctgtgag 9120

agactcggag ttggccaagc tgccggaggg tctcttcaac aacattaagc tgaagcactc 9180

ggttgtgatg accaggctta tcaaactcct aggtgaaaga ctgctaggtg agtccccccc 9240

cccttgtgtt ttattgtttt atttttcgtt tttatttatt taaccccttt aagagccacc 9300

caacttttct ccttcatatt tataagcttt tttttttttg tcacttatgc tcatttcccg 9360

taaaacattt cgccatcaca aaagtcagta gcgacctctt gttttctcga tattttctcc 9420

aacatctaca tccaagaaac ttatacggtg aagattagga aagagtgacc gtggctgttc 9480

aagggttaag ctttcatttt tcaatcttaa gcctctaatt gttttcttat gttctcaatt 9540

cacctttttt gttgttcttg ttaatctttc atttctcacc cttaagcctc t 9591

<210> 8

<211> 751

<212> DNA

<213> unknown

<220>

<221> misc_feature

<222> (139)..(345)

<223> n is a, c, g, or t

<400> 8

ttcacatcat aactatagtc gctagatttt tctgtgatag tacaccagcc tgccacttgc 60

atgtcggatg aaacacacac acacacacac atcattgaag ttggcagtac agtacagtgt 120

ctcccttaca gagtgatann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 240

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnngtgtg tagagtagca 360

tgtgtggaag cagtgttcat ctcaaagttt gatgtgcgag tttgcaacac tggcaaaaaa 420

ttcaatataa agacacagtt ttgttttgtc catgtagcca cagagcagct ccctgtgtgt 480

cctcacacca tgtgtttctt tcctctccgg cggatcttct tcatctcggt taatttggat 540

tcctcttcca ccacaggcca agtggatcaa gaggctcgtc gataaagacc cagccgagcg 600

accgtcggcg gaggagctac tcaattcgtc cctgttccag cagccggagc cgccacaatt 660

acctgctgtc gccgggcaca ctgctgccgg cagcctcccg gtggatgctg cggccgcctc 720

tattaagttt tctcgttcgc aatttacgag t 751

<210> 9

<211> 788

<212> DNA

<213> unknown

<220>

<221> misc_feature

<222> (200)..(247)

<223> n is a, c, g, or t

<400> 9

agcgcttcct actcctcaag tcagcctaca ggcgctatag gccagcacat aaaaaaaaaa 60

aaacacttga gattgcttta agaacaggtt ggatagattt atggatagct acgataggtg 120

gtaacttggg aggcactagg tgtttttagg ttcgcaggag ctacctcgag taggtcaact 180

ggtcttttgt agtcttcttn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 240

nnnnnnnaat tcttatgttc ttatgaaaca agtgtaattc actgtttgat ctgctgcagt 300

cactgccatc gtctgccagt gtgttcgcta cacacaaaca tacctatgtg gtaacaccga 360

gagatcactg tctttccgct cctctaatat cacttacatt gcaaccaaat ggttacagaa 420

tgatagcaat atgagctgtc tgtatcatgt gagtgaggga ggaacagtcc atcggctagc 480

tgtgtttgtt ttgatcggta ccgtaacatg ttgaatcttg gatgataact gggtaatatg 540

aagtggctaa atcattcaaa tttgtagatg aggcagaatg tgcagccagg cagtggcagc 600

agccttatag tatgttgggg atgcttggtt attaactaac ccgtacagtg ttgctttcag 660

ggagtgaaac acatcatgtg gggttggatc ttggcacaac aaatttatta tcttataaaa 720

ttgcaaaaaa acagatgatt tctatggtcc aaatgcgata tctgagtgtg gcctgcaagc 780

catgattt 788

<210> 10

<211> 2294

<212> DNA

<213> unknown

<220>

<221> misc_feature

<222> (308)..(358)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (554)..(554)

<223> n is a, c, g, or t

<400> 10

gcggagaaag ggcggcctgg aggttctgcg agagcgagag agagagagag agagagagag 60

agagagggct gtatcaactg aatttcagaa acaaggaact aaatttgctg atgtgaatgt 120

tagaaggatc tctggcaagg actggcaact gagatggctt tccttgtcac atcagactgt 180

ttgctggtct aggctgagtc tctctctctc tctctgtgtg ccaggaagtc ttagtagctt 240

taataatgtc atgttgttta ttgatattaa tgttagttat agctgtgtgg agaaggtgtt 300

aaaattannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnat 360

gtgaatgtta gaaggatctc tggcaaggac tggcaactga gatagctttc cttgtcacat 420

cagactgttt gctggtctag gctgagtctc tctctctctc tgtgtgccag gaagtcttag 480

tagctataat aatgtcatgt tgtttattga tattaatgtt agttatagct gtgtggagaa 540

ggtgttaaaa ttanatgtaa tagcttgata tgatgtgcta tttgtgaagt cttgtcaata 600

atctccttct actactacta ctactactac tactactact actactagct gtcatttcaa 660

cagttgtatc attatgtaat agcttgatat gatgtgctat ttgtgaagtc ttgtcaataa 720

tctccttcta ctactactac tactactact actactacta ctactactac tactactagt 780

tcctctgttt gttgttggtg ttgttctttt cttcttcttc ttcttcttct tcttcttctt 840

cttcttcttc ttcttcttct tctcctcctc ctcctcctcc tcatcatcat catccatctc 900

ctcctcttcc tccacctctt cctcttcctc ctcctccttc ttcttctcct catcttcctc 960

cacctcctcc tcttcctcct cctcctcttc ttcctccttc tcctcctcct cctcttcctc 1020

ctcctcctca tccatctcct ccacctcttc ctcctcctcg ttcttcttct ccttgtcttc 1080

ctccacctcc tcctcttcct cctcctcgtt cttcttctcc ttgtcttcct ccacctcctc 1140

ctcttcctcc tcctcgttct tcttctcctt gtcttcctcc acctcctcct cttcctcctc 1200

ctcttcctct tcctccacct ccacctcctc cttctcctcc tcctcttcct ccttcttctc 1260

ctcctcctcc tcttcctcca cctccaactc ttcctcctcc tccttcttct cctcatcttc 1320

ctccacctcc aactcttcct cctcctcctg ctccttctcc tcctcttcct cctccttggc 1380

cagcagcaga gcgcactaag cacaccttgg ttggtagcac tgggagacca aactgcctta 1440

cctcttctct aatttcactt gcactggagg caaatgaata cataacaata gtgatatgag 1500

acgtcagcat cgcgaaagag agtagatgaa tggaaatgct ttggtaatgg ggggacaatg 1560

agggggggga gggtgtgcct gtctgtgatg ggtgtcggtg gtgatgcctg cctgacatca 1620

cccagcacac acatgtacta cctctgcagt agttctgaaa ggtcaaaatg ctttacgtcg 1680

ttaagatccc ttgtcctcga ggaattctgg tggctatgaa gttatccaga atgtttcact 1740

attctctaac acatctcttg taatgatgct ggctaggtga ggtggtgtta gtcagacaaa 1800

ggtatgttgg agaagttgta atggagggag tctacctggc cacttacctt taattctata 1860

atgagactgg atgtaaaggc tcacttaaca caaacaatac tacaggtgac tcttgattta 1920

cgcgatagat gcattccgag agcgatcgcg taagccgaaa atcgcgtaaa acgagaaacg 1980

caggatttgg tgctttttgg agctttctgg cgtgtttttt gcaaattttg ttcggaaaaa 2040

tcgcgtaaat ttaatcaatt ctaatatttt ttttgatcgc gttatagcga aatcgcgtaa 2100

aatgaactcg cgtaaattga gagttacctg tactaaagtt accaagaatg aacagttatc 2160

cagaatgttc actattctcg aacacacctc ttgtaatgat ggtggctagc tgaggtggta 2220

ttagtcagac aaaggtacag gtaactctcg atttacgcga tagatgcgtt ccgagagcga 2280

tcgcgtaaac cgaa 2294

<210> 11

<211> 1546

<212> DNA

<213> unknown

<220>

<221> misc_feature

<222> (1226)..(1392)

<223> n is a, c, g, or t

<400> 11

atagttaggg aagtactaca aaaaatggac atctgaggcc ttcctgtcgc tgctgctgct 60

gctactactg ctactggtgg tactaatgat aataatggtt aaggaaggta aagtggcgag 120

taaatatgtg ggttgtcctt gttacgtacg aggtatttgt aatgttggtt ggttactcta 180

atggttggaa tggaatatta atacataatg ttgaatagta atgatagtaa tgtgtaacta 240

aatatctaca gctactatta tttgtgaaag tgatggagaa acgatgcaag gcgtattaat 300

gtaatttgtt ggattgttac aaatacatat taagcggata tggaaagagc tgatgacgag 360

ggatcataat ataatgacac tgatttacta atgatgataa tagttaggaa ggcaaggttg 420

tgagtaaacg tgtgaattgt ctttggttac gatggaagta tttatgaagg tagagagttt 480

ttttttgttt ttttttttgt ttttacgtgc tggcctatta cgcctgtagg tatacttgaa 540

gagtatctag gaagcgctgt tcagcttcca cccattagtg gcgcaggcaa ttttatttat 600

agtggtaccc atattaggcc ccatatcacc acccaagctc atcattggtg caaggcagat 660

acaccttcac ctagacaccc aggtattaat gtgatatgta ggtgacttta aaccactcga 720

caaatgacaa aagtttcaag acggcacgtg atgggattcg aacctacgca tgaacgtctg 780

cccgatccca tgctcgccac cttatccact tcgccatcgc ctccctgtaa tagttggaat 840

attaatatat aacgttaaat agtaatgata gtaatgcttc actcatgatc tacagctact 900

attatttgtg aaagtgatgg agaaacgatg caaagcgaaa attgttggat tgctacgaat 960

atatatcaag cggaatgtca aaagctgatt acgatggaaa gacgccttgg aggctcaaat 1020

aacgccacta cagcaatgct aatgaggatt atgttgtaaa attgatacgg gaaactgtgg 1080

caaagacaat gatgatgata atgatgataa taataaaaga cataataata ataacaacaa 1140

caacaataat aataagacat aacaacaaca acaacaacaa caacaacaac aagaacaaca 1200

ataataataa taataataat aataannnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1260

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1320

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380

nnnnnnnnnn nnataataat agcaataata ataataataa caataatagc aataataata 1440

ataataataa taataataat tataaataat aatagcaata ataataataa taaaaataat 1500

aataacaata acaatacaca attgcaacca ccaccaccac caccac 1546

<210> 12

<211> 802

<212> DNA

<213> unknown

<220>

<221> misc_feature

<222> (592)..(645)

<223> n is a, c, g, or t

<400> 12

gtgacccaag gaggcagagg caagcacctt ccctcacaca cacacacaca cacacacaca 60

cacacacctg cctctccgac attccaggta cgtgacaggg acttgacggt gaacaatgtg 120

gccgcgtcgg gttactacag gggcgatgac aggcagagag gagacgagga taacagagta 180

tgggaaagaa agacaggaac agaaactcaa aaaaataaag ctacctatct acgagcgaag 240

aaaatacgac caatagatac acagatagga agaagaacag gtaaagatat ttgaagttag 300

taaatatagg acagtagata acagtgtata gggaaaaaaa agacaggaac aagaactcaa 360

agaaataaat aaaactacct atctactcgg gaagaaaata tgagaaatag atacacagag 420

aggaagaaga ataggtaaag atattagaag ttagtaaaca taggacagta aataacagag 480

tatggggaaa aaaagacagg aacaagaact caaaataaaa tatatataaa actagcaatc 540

tacaatctac tcacgaaaaa aaaaatgaga aatagatgca cagaaaatta annnnnnnnn 600

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnntgaaa aatgagttag 660

taaatatagg acagtagata acatagtata gggaaagcaa gacaggaaca gaacatcccc 720

cctgaaaaaa aataaattaa actaggaatc tactaactaa caaaatacga agaatagata 780

tacaagtaga aataataacc gg 802

<210> 13

<211> 1904

<212> DNA

<213> unknown

<220>

<221> misc_feature

<222> (289)..(371)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (825)..(911)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1414)..(1414)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (1600)..(1703)

<223> n is a, c, g, or t

<400> 13

ttcatataat tattttcctt ttcagctttt tcaaacattg catttcggcc tttcccttcc 60

aaccttctcc tcctcctcct cctcctcctc cacatcccca agaggccgct atacaggaat 120

aagtcgcgtc tcaatctcct tcacgcacac tcgaatgcaa atggcgtctc cagcgaatgt 180

taaagaaaaa tattttaatt ttgtggcgct gagatgcgtt atctatctgc gaggaggagg 240

aggaggagga ggaggaggag gaggaggagg aggaggagga ggaggaggnn nnnnnnnnnn 300

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360

nnnnnnnnnn nttaagggag aagaatgcag aggaagaagg gatcgggagg aagaaaagga 420

agataaggag gttaagaatg aaggaagggg aatatacaga taggaataca taggaaagac 480

gattgacagg aggccttgcg acttatggcg aggtcactcg ttgactacac tacatctgaa 540

attctacagt agtaggtaag gaaagaacag atgaaggctc ctccccaccc accactccct 600

ccagcgttat ccggcaggaa aaaagaacaa agaaatacct cgtttgttga aagataaacg 660

aggaaatctt atataggaaa gatggaaaac tagaagacaa atctataaga ctagttctta 720

ataactatct acatgaaaaa gtgaataatg aaccgcgtgt caaaattaaa aaccattttc 780

ttagaaatat ttgtctaatc gatctttaaa tgcagcaatt gtgtnnnnnn nnnnnnnnnn 840

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 900

nnnnnnnnnn nttagcttgg actactgatg ctggcagctt attccaaaaa tcaattattc 960

tgatgttgaa gtagctcttt ctcaaatcgg tgttgaagcg ttgtccttta agcttcaagc 1020

cgttatttcg cggtagcagt tgtggcgaga gctcaaaaaa gttatcataa tttaagtcat 1080

ctatcctctc tagtattttg aaggtttcaa tcagatcgct cgtctttctc gctcgtcttt 1140

gtacgctgaa agggaagagg gaggaaggtt gaagaaggtg taggaaggaa tgggaaggag 1200

aaggagcaga agattgtaag caggtaggaa gaagagaagg atggatacag agtaagggga 1260

gaaaaggaag tgtaggagca ggaagaggag gaggtaaaga aaaaaggaaa attaatgatt 1320

atgaaaaaaa gtaaaaaaaa aaaagtcaag aaacaacaac aacaacaaca gcaacaacaa 1380

caacaacaac aacaacaaca acaacaacaa caanggaaga ggaggaaggg tgaagaaggt 1440

gtaggaagga atgggaagga gaaggagcag gagaagattg taagcaggta ggaagaagag 1500

aaggatggat acagagtaag gggagaaaag gaagtgtagg aggaggaaga ggaggaggta 1560

aagaaaaaag gaaaattaat gattatgaaa aaaaagtaan nnnnnnnnnn nnnnnnnnnn 1620

nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680

nnnnnnnnnn nnnnnnnnnn nnnttaaaaa aaactgacac atttattgct acactctaca 1740

tacttacgta catacataca tacatacata catacataca tacatacata tatacataga 1800

aacatacgta cgtacataca taagaaataa ctcatcttct cctcttcttc ttcttcttct 1860

tcttcttctt cttcttcttc ttcttcttct tcttctcctt ctcc 1904

<210> 14

<211> 308

<212> DNA

<213> unknown

<400> 14

ataactctcc ccttccacca gaacacaaaa gaatgacagt aataacaata acaatcatca 60

tcatcatcat caccatcatc atcatcatca tcatcatcat catcatcatt ctcacctgtt 120

gtttgattca ccattatttc tcatcttcac tggcagcggc ggcggcggtg gtggcggtgg 180

cggtggaagt ggaggtggag gaggaggcgg gtggtggtgc tggaggagtg aagaaggtga 240

ggtgtccggc agtgcaggtg gaggaggtgg agatgacggt ggaggagaat acgatgagtg 300

tggagatg 308

Claims (10)

1. A primer group of whole-genome microsatellite markers of Charybdis feriatus is characterized by comprising 14 pairs of primers of polymorphic microsatellite markers, wherein the microsatellite markers and the primers are as follows:

。

2. the primer group of the whole-genome microsatellite marker of Charybdis feriatus according to claim 1, which is characterized by being screened by the following method, and mainly comprising the following steps:

(1) extracting genomic DNA of Charybdis feriatus;

(2) constructing a genome library and carrying out high-throughput sequencing;

(3) quality control, assembly and evaluation of sequencing data;

(4) analyzing and screening microsatellite loci;

(5) designing a microsatellite locus primer;

(6) carrying out PCR amplification on the genomic DNAs of different individuals of the Charybdis feriatus by using primers;

(7) detecting the PCR amplification product by using non-denaturing polyacrylamide gel electrophoresis;

(8) determining the genotype of each individual according to different migration distances of the amplification products, thereby obtaining the polymorphism map of the genetic variation of the Charybdis feriatus.

3. The primer group of the whole-genome microsatellite marker of Charybdis feriatus according to claim 2, which is characterized in that,

the extraction of the genomic DNA of the Charybdis feriatus in the step (1) mainly comprises the following steps: taking muscle tissues of Charybdis feriatus, putting the muscle tissues into a centrifugal tube containing a tissue lysate, and homogenizing; then adding RNA enzyme, mixing evenly, and incubating at room temperature; adding protease K, mixing, digesting in 55 deg.C water bath to obtain clear solution; then adopting Tris-saturated phenol and chloroform to continuously extract for a plurality of times; sucking the supernatant into a new centrifugal tube, adding precooled absolute ethyl alcohol to precipitate DNA, centrifugally collecting the precipitate, and cleaning; finally, the DNA was dried at room temperature and dissolved in sterile double distilled water and stored at-20 ℃ low temperature.

4. The primer group of the whole-genome microsatellite marker of Charybdis feriatus according to claim 2,

constructing the genomic library and performing high-throughput sequencing, wherein the construction and high-throughput sequencing method comprises the following steps: firstly, breaking genome DNA by ultrasonic wave to 280bp and 450bp respectively, and establishing two small fragment DNA libraries; secondly, filling in the ends of the DNA; thirdly, adding A-tailing, and standing for 30 minutes at 37 ℃; fourthly, adding a joint, and standing for 10 minutes at the temperature of 30 ℃; fifthly, enriching the connecting fragments by using a PCR amplification method; sixth, the DNA library is quantified by the Qubit; seventh, clusters were generated and high throughput sequencing was performed with Hiseq 3000.

5. The primer set of whole-genome microsatellite markers of Charybdis feriatus as claimed in claim 2, wherein the quality control, assembly and evaluation of the sequencing data in step (3) comprises the following steps: firstly, filtering and evaluating the obtained sequences by using Trim Galore and FastQC respectively to obtain clean reads suitable for assembly; secondly, splicing a plurality of Kmer parameters of all the obtained clean reads by using SOAPdenovo splicing software to obtain contigs and scaffolds; thirdly, the resulting contigs and scafffolds were evaluated using QUAST; fourthly, the GCE software is used for deeply analyzing the characteristics of the Charybdis feriatus genome.

6. The primer set of whole-genome microsatellite markers of Charybdis feriatus according to claim 2, wherein the analysis and screening of the microsatellite loci in the step (4) comprises the following steps: when MISA is used for analyzing and screening the microsatellite loci, the conditions are set to repeat 1 base for at least 10 times, repeat 2 bases for at least 6 times and repeat 3-6 bases for at least 5 times, and on the basis, two SSR loci are considered to form a mixed SSR locus as the distance between the two SSR loci in a sequence is less than 100 bp.

7. The primer group of the whole-genome microsatellite markers of the Charybdis feriatus according to claim 2, wherein the design of the microsatellite locus primers in the step (5) comprises the following steps: under a linux system, Primer Premier3version:2.3.6 is used for Primer design of the searched microsatellite locus, the setting conditions are that the product length range is 100-280bp, the annealing temperature is 57-60 ℃, and the Primer length is 20-23 bp.

8. The primer group of whole-genome microsatellite markers of Charybdis feriatus as claimed in claim 2, wherein the primer in the step (6) is used for carrying out PCR amplification on the genomic DNA of different individuals of Charybdis feriatus, and the primer group comprises the following steps: for the designed primerPCR amplification is carried out by taking genomic DNA of Charybdis feriatus as a template, a reaction system is 12.5 mu L, the reaction system comprises 0.5 mu L of genomic DNA template and 0.4 mu M, Mg of final concentration of primers ²⁺ 1 XPCR buffer, dNTP final concentration is 0.2mM, Taq DNA polymerase 0.5U, and finally sterile double distilled water is supplemented until the total volume is 12.5 mu L; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30 sec, annealing at a primer-specific annealing temperature for 50 sec, extension at 72 ℃ for 50 sec, and 30 cycles; finally extension was carried out at 72 ℃ for 7 minutes.

9. The primer group of whole-genome microsatellite markers of Charybdis feriatus according to claim 2, wherein the step (7) of detecting PCR amplification products by using non-denaturing polyacrylamide gel electrophoresis comprises the following steps: mixing the PCR product with 6 XPCR loading buffer, loading 1. mu.L of the mixture into 8% non-denaturing polyacrylamide gel for electrophoresis; and obtaining a PCR product electrophoresis image of the Charybdis feriatus after dyeing and color development.

10. The application of the primer group of the whole-genome microsatellite marker of the Charybdis feriatus as claimed in claim 1, wherein the primer group is applied to genetic variation analysis and population genetic diversity research of the Charybdis feriatus.

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