CN104087584A - Dasyatis zugei microsatellite sites, primers and application thereof - Google Patents

Dasyatis zugei microsatellite sites, primers and application thereof Download PDF

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CN104087584A
CN104087584A CN201410345130.9A CN201410345130A CN104087584A CN 104087584 A CN104087584 A CN 104087584A CN 201410345130 A CN201410345130 A CN 201410345130A CN 104087584 A CN104087584 A CN 104087584A
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dasyatis
zugei
primers
seq
microsatellite sites
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张洁
陈骁
张保卫
王慧
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Abstract

The invention discloses dasyatis zugei microsatellite sites, primers and application thereof. The dasyatis zugei microsatellite sites include 10 stable polymorphic microsatellite sites, wherein the sequences of the 10 stable polymorphic microsatellite sites are respectively as shown in SEQ ID NO:1-SEQ ID NO:10. The sequences of the primers of the dasyatis zugei microsatellite sites are respectively as shown in SEQ ID NO:11-SEQ ID NO:30. The application of the dasyatis zugei microsatellite sites or the primers comprises molecular ecology study, pedigree analysis, germplasm resource survey, assistant breeding and individual identification. The genotyping result of 10 pairs of primers in 15 dasyatis zugei individuals shows that the allelic gene number is 3-24, and the average allelic gene number is 13.6; the expected heterozygosity He is 0.5-0.984, and the observed heterozygosity Ho is 0.429-1, so that the 10 pairs of primers are indicated to have high polymorphism and meet the requirement for individual identification of dasyatis zugei.

Description

Dasyatis zugei microsatellite locus, primer and uses thereof
Technical field
The present invention relates to, in particular a kind of dasyatis zugei microsatellite locus sequence, primer and uses thereof.
Background technology
Dasyatis zugei (Dasyatis zugei) is subordinate to Myliobatiformes, ray section, is the selachian of one way of life at greater coasting area.Be subject to the impacts such as pollution, overfishing, fishery cultivating, population quantity reduces day by day at present, urgently protection.Need to understand the genetics informations such as its population structure and genetic background to its protection, at present the domestic molecule genetics research of still not carrying out selachian.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, microsatellite locus and the primer of dasyatis zugei is provided first, is also at present domestic first for the molecule genetics research result of chondrichthyes.
Technical scheme of the present invention is as follows:
Dasyatis zugei microsatellite locus, comprises 10 stable polymorphic micro-satellite sites, and its sequence is respectively as shown in SEQ ID NO:1-SEQ ID NO:10.
The primer of described dasyatis zugei microsatellite locus, its sequence is respectively as shown in SEQ ID NO:11-SEQ ID NO:30.
The purposes of described dasyatis zugei microsatellite locus or described primer, comprises molecular ecology research, pedigree analysis, Germplasm Resource Investigation, assistant breeding and individual recognition, described 10 stable polymorphic micro-satellite Sites Combination are used, or its corresponding combination of primers is used.
The gene type result demonstration of described 10 pairs of primers in 15 dasyatis zugei individualities, number of alleles is 3-24, average number of alleles is 13.6.Expect that heterozygosity He is 0.5-0.984, observation heterozygosity Ho is 0.429-1, shows that these 10 pairs of primer polymorphisms are high, can meet the individual recognition of dasyatis zugei.
Brief description of the drawings
Fig. 1 dasyatis zugei DNA 1% agarose-EB gel electrophoresis result; M:Trans2K DNA Marker; 1-4: dasyatis zugei complete genome DNA; Y: blank;
Fig. 2 AFLP amplified production 1% agarose-EB gel electrophoresis result; M:Trans2K DNA Marker; 1-8:AFLP amplified production; Y: blank;
Fig. 3 bacterium colony PCR 2% agarose-EB gel electrophoresis result; M:Trans2K DNA Marker (same to Fig. 2); 1-8: bacterium colony PCR result, wherein 2,6,7 and No. 8 is biobelt, is considered to positive recombinant; Y: blank;
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
1, DNA extracting
Utilize dasyatis zugei muscle tissue, extract genomic dna.Method for extracting adopts the cracking of SDS/ Proteinase K, phenol-chloroform extracting approach (Sambrook et al., 1999).DNA extraction rate was acquired is shown in Fig. 1 (exposition).
2, the structure of dasyatis zugei enriched microsatellite library
Get genomic dna 250ng left and right, use restriction enzyme MseI to interrupt, be connected with the AFLP acceptor joint (5'-TAC TCA GGA CTC AT-3 '/5 '-GAC GAT GAG TCC TGA G-3 ') of MseI simultaneously.After ten times of digestion product dilutions, use the primer (5'-GAT GAG TCC TGA GTA AN-3 ' is called for short MseI-N) of AFLP receptor-specific to carry out pcr amplification.Amplified production uses 1% agarose-EB gel detection, and result is the dispersion plating that is greater than 200bp, sees Fig. 2.
Use respectively the probe (AC) with vitamin H joint 12(AG) 12after hybridizing with above-mentioned pcr amplified fragment, use Streptavidin MagneSphere to carry out absorb-elute, to obtain the micro-satellite repetitive sequence of required strand.Taking the DNA that catches as template, carry out double-stranded DNA recovery taking MseI-N as primer.PCR product is through PCR cleaning agents box (Axygen) purifying, and by 1% agarose-EB gel detection, result is that the dispersion plating that is greater than 200bp is considered as successfully.
Two strands after purifying is recovered after product is connected with pEASY-T1 Simple Cloning Vector (TransGen Biotech) to transform in Trans5 α chemically competent cells (TransGen Biotech).The competent cell having transformed is coated and added 0.5mM IPTG and 0.5 μ g/mL X-Gal containing on the LB solid medium flat board of 0.06mg/ml ammonia benzyl (Amp), in constant incubator, cultivate 13h for 37 DEG C, obtain the micro-satellite library that comprises AC, two kinds of repetitions of AG.
3, the screening of positive colony, order-checking and design of primers
The full bacterium colony of picking white is placed in Amp+LB liquid nutrient medium enlarged culturing (37 DEG C, 5h).Adopt Tri-Primer-PCR (Zane et al, 2002) to detect the positive recombinant that contains micro-satellite fragment.In experiment, select the probe (AC) of the lifeless matter element mark that M13 carrier universal primer (M13-47) and clone are corresponding 12/ (AG) 12react.If there is two or more band in product in 2% agarose-EB gel electrophoresis result, think that this mono-clonal contains aim sequence, see Fig. 3 (exposition).Successfully check out 107 of the positive recombinants of aim sequence by three-primer method, use M13 universal primer to check order to the plasmid of these recons.
The preliminary screening of 4, sequential analysis, design of primers and microsatellite locus
Check sequencing result, remove after the carrier sequence of M13, use TRF software (Tandem Repeats Finder, Version3.2.1) to find the sequence (Benson, 1999) that wherein contains micro-satellite repeating unit.Use PRIMER5 software (Lalitha, 2000; Singh et al., 1998) in the flanking sequence of repeating unit sequence upstream and downstream, design primer, object fragment is set between 100-400bp.Select altogether 28 micro-satellites to repeat site as alternative mark.
5, reading and the acquisition of microsatellite locus of gene type data
One-sided primer 5 ' end to just select 28 microsatellite locus adds fluorophor mark (FAM/HEX/TAMRA), and the DNA that then uses 15 dasyatis zugei individualities is to the screening of increasing of above-mentioned microsatellite locus.First the PCR that treats bit selecting point carries out the optimization of amplification condition, and circulation is set to: 95 DEG C of denaturation 5 min; 95 DEG C of sex change 30 sec, select 8 thermogrades annealing 30sec in 48 DEG C to 60 DEG C, and 72 DEG C are extended 60sec, react 30 circulations; 72 DEG C are extended 10min.Amplified production uses 2% agarose-EB detected through gel electrophoresis, determines most suitable primer annealing temperature.Get rid of the alternative site that occurs following situation: in 8 thermogrades, all do not obtain single band clearly; Amplification is unstable, does not have repeatability in Different Individual.The amplified production of stablizing single band to having uses Tamara500 fluorescence molecule amount standard to use polyacrylamide gel to carry out gene type in ABI 3730DNA sequenator, and the result of genetic analysis is used Genescan 2.0 softwares to read.Gene type result by analysis after, remove and occur the alternative site of following situations: without the site of gene type result; In arbitrary individuality, number of alleles is in plural site; The site of allelotrope below two in 15 wild dasyatis zugeis.After screening, finally obtain 10 stable polymorphic micro-satellite sites, microsatellite sequence is as follows:
DzuI25(291bp)
ACATTGAATGACATTTAGAAATGGGAACAGAAGTGCTTGTCAGCCAACCATGAGTCTTCTTTAGATAACAGCAACAACAAACAGCAAAACAGCAAACCAGATCCTTTCCCACACACACACACACACACACACACACACACACACACACACACACACACCACACACACACACACTCACACACACACACTCACACACACACACATACACACACACACACACACACAGGCTATTATTTATACAAAATAGCATAGAAAGAAAGAGGCATTTATTCTCTTTTCACTCCTCACTCCT
DzuI45(520bp)
ATGGTGAAACTGAAGGAGCTGGACTTGGTGGGGATCACGCTATTGCCTGTGTTGGAGAGGCCTCGGTGTGCGAAGGTGGTGTGTAGTCCAACAGCGAGTGACTGTCTTAGTTGCTTGTCTTGTGATCACAAGACCCTGTTGGAAGTTGTTGGTGTGGAGCGCTGCAAGTCCAATTCCTGATTTATTGGCGGGGGGCGGGGGAGCTGCGTGGCCTCTGACCTGGCGAGGAGTGGGCCTCAAGCCGCAGTGTTGCCTGTTTACAGACACCCAGGAGAGAGGCATTGGAGTCGGAGGCGGCGTCAGAGCTGAAGCTGGGTGCTGCCATCTAGTGTTTGCCCAGCAGAATACAAGGTGCATGTTCGACTGCAACTGCTGCAGCATTAGCGGACTCACAGCCTTGGACTATTGTGTGTGTGTCTGTGTGTGTGTGTCTGTGTGTGTGTCTGTGTGTGATTGTCGGTACTGTGTCTTGGCCCCAGTACTAGCAACACTTTTGTTTGTCTGTAAACCTGGATATGGT
DzuI31(569bp)
ACTCCACCCTGCCACTTTCCCACTTGGCACAAGCTGCCATCACCCCTCCTCAGTCTGCCTATCATAGTTGCTGCATGTTTTACCTCTCCCGCTCTCCTCTTTACACTGGCCATCTTACCTCTCCAATCCTGGTCGTGACACAGGGTTTTGACCCAAAATGCCAGACCCCAGTTCCTCGATACACACAGATACTGCTTGATCACTGAGTTCATGCATCTGCATTCCGTCATATTGTAATGTGTGCGAGCAATATCTGATAACAGCATCTCCAGTGACATTGGGGGAGTGAGGGAAGTACTAACTTTCCAATGGGGCATAAATCAAGGGGATTTACAAATACAGTCTTGTGCAAAAGTCTTACACACACACACACACACACACACACACACACAGAGAGCTAGGGTTTGTAAGACTTTATCTGGCAGGAGCAAAGGATGTTGGGAATGGTGAGGGTGGAGGACTGTAGGACTGGTGGTAGAGAGGAGTGACAAGACTGGGAGGGTGGTGCAGGTGCAGACACACCCAGCCCTGAGACACCAGGCAAGGTCATTTGATTCTAAACAATTGGT
DzuI46(472bp)
CGTTATGTAGGCAGTCCTGGAAACAAAGTTATAGAGAAATATCAGACAGAGGCAAAATACTTAGATGAGTCATTCCTTCAATGTCATCATTTTGCTCTTACTTTGATCGCTGAAATCTCCGGAGAAGTTGGTGAGGTATATTTCATGTACCACAGTGGCAGAAATCGCTGCTTTCCTTCTCTTTGTGATACAGAATACATCCGTTTGATCCGTCAGAACCATGCAGGCCCCCGACGGAGGGATTACTTACTCTCCTCACTCTATTTTTTCTGTATCCCTGTAGTTCTGCCCTCTCTCTCTCTCACACACACTCTCTCTCTCTCTCTCACACACACACTCTCTCTCTCTCTCTCACACACACACATCCTACACACACACACACACACACACACACACACTCACACTCACACAGACACACACACACACACACACAAATATCCATGCCTCTTTGATTGTTTGCTGCTATGGAGAT
DzuI51(430bp)
CCCACCATGGGCAATAGAAAAGTCACTGTTGCAAACAGTGCAGCGAGCAACACTGTCATTATTTTTTACCCCTATTAGGCAGGGGTACACTTCGGTGTAGTCTGGGGTGAAGTATGTTTTATATTTTCATTTTTTGGAACACTTTGCCATGGTTCATGTTTTCTCTCTTACACACACACACACACACACACACACACACACACACACACACACACACACACCGCATATACTGTAGCAGAGGTCCCAATACAGACTGGTTACAGACCTCCAGCCAGAACCACTACCATTGACAGCTACTTTCTATCTTCTATGGTCTGAGTCCAACATCCAAGTCACTGCAGATTGCATGCATCCTTTTCTCTGCATGAGCCCTCCATGAGGGACCTGATCGAATAGGTTACTAAAATCCATGTAGACAACATCCTTACCT
DzuI57(720bp)
TGCCAAACAAAGTCACCCTCACTTTCCAAAACAAGAGAAAATCTGCAGATGCTCAAAATCCAAGCAACACACACACACACACACACACACACACACACACACAAAATGCTGCAGGAACTCTGCAGGCTAGGTAGCATCCATGGAAAATAATAAACAGTCGATGCTTCTGGCCAAGACTCCTTCGGCGCCCTCAGTTTCCCTCTGGATTTCACCCCAAGGGTTCAGGGAGTGATGATGGTTAGAACTGTTGGTCCTGAAACCCAAACCGTTTATACTGAGTAAATGCCTCGTCGCCATTGTCATTACTTTGCTAATGACTGAGGACAGACCAATCAGGTGCTAAATAGGTACATAATGGGTCTGTCATGTTTCTGTGATCAGAACTTATCTCAGAAGCTTTATGCATGGTTGGGCAAATGTCAGTGCTGTAACTACACTGGACAGCTTGGCTAGAGATGCGACTAATACTGGAATAGAAACCTTCACCACTGCAATGGTTTCCCTTTGCCCTGTCTTATAGCCTTTACTGCATCCTGCGCCTCTTGGCCGTTTCTTGGAGTGAAATGAGTGAGCTAAATCCTGGATACCAAGACAGCAGCTATCCCCAGGAGAAAGCATTTACTTTGAATGAAGATCGCTGTACATGGACTTATCAAAAGATGTTTGCAAATGCTGGAACAATACAAGTTTCTAACACTTAGATCAAAAGATTTTTTTTGT
DzuI71(811bp)
ACTAACTAATGCCACATAAATACTGCATGATTATTTATCCCCTCGTGTGGCCAGTAGTCATCCGCCATCATCTACAAATGGCCATTGAGATTCTTTTGACATAGGGAGGCCATTACCTCAAGGATTTGGCAGAGCAAGATCTTGGCAACAAGATCCAAAATGGTCAAACTGGCTCGGCTACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACACAAATTCCACCTAACTGCCCCCCATCTGTCAGACACTACCCTCTCAACTTCCTCCCCTTTCACTTGACCCCTTCCTTTCTCCCTCTTTCTTCTTCCGAATCCGGCCTCCTCCTCTCCATTTGCCAGGAGCCTGCTCCCTCTGATAATCACAGGTGTTCAGTTTATTAGTGTCTGCTTCCCAACTGCACGATACTTCCCCATTACAACTGGTCCCCAAAGTTACACCACCGCAGAATTTCTGAAATTTCCCCAGAAAACCACAGGATGTTCTGCAAGTGACTACCCAAAGCCGCTCAACGTTGGCAGATGGTGGTTTCTTGGTGCTCTGTTGGTGGAGGACATGTGGGTTTTGAGGGAAAGGGTAGTGTGTGGAAGGCAGAGTTTAAGATCATTGTATATGCTGAGCGATCCGATGATGAGCAATGAGGGACCCTCACTCCTGCAGAATGTAATCTCCTTCACAGTTAGGACTTACCAAAACCTTCACAGAGAGCTGAAAGAATGTTTGGTTTTGT
DzuI82(352bp)
CAAATAAAGGCGCCCTATACACACAGACAAGCTAAATCACACACACACACACACAATCTATAAATATTCTCTCTCCCTCACTCACTCACTCACACACATACACTACAGACATAGGCAGACTATCTCTCTTTTTCACCCACCTATCCCACCCCCTCTATACACACACACACACACACACACACACACACACACACACACACACACACAGTTGGAAGGAGACACAAATGAAATATAACGGGAAGTAACTCAATACAACTCTATCTTCGTTTATCACAGACACAATGGGACGAACCGTCTCCTCTGTGTGCGTCGGATTTCAAATATACAGAGATGCGTACACGATAAACCAGAT
DzuII15(410bp)
GGATAGTTCACGATAATACGGTATATATCCTGGAATGTAGAACGCAGCGGTACAACAGGTGGTGCTGCTGCATGACCTCCAGTGTCTAGCATTTGACCCTGACTTTGGATGCAAACTGTCTCCCTGTAGCTGCAAGGGTTTCTGCCAGATGCACTGCATTCCTGCCTTATCCCAAAGTTGTGCTGGCAGGTTAGCTGGCTGTTGTAAATTGCCGAGAGAGAGAGAGAGAGAGAGAGAGAGAGTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGTGTGTGTGTGTGTTGGCACGTGGCCTAGTGGGCAAGGCATCAGACTAGTAACCTGAAGGTCACAGGTTCAAGCCTCAGCTGAGGCAGCGAGTGTGTGTCCTTGAGCAAGGCAGT
DzuII64(649bp)
CTATTTATGCCTGCCAGCATCTTTTATATCTACTTCTATACATGTAACTGTTCAGTATTTTTCCCAGTGGTCGCAGGATACATATATAAATGTTCTGCATTTCTCCTACTGATTACATTTGTATCATTCTGGAAAGCTCTGGAAGTTTCTCCGGTGTTGCATTATTTGTATAGGGGCTATCTGATTGGTTGACTCTTATCTACAGATTTGAATGAAATGTTCCTCATCTATGGGGGTTCAAAAAGCTCCTTGCTTCTCTCTCTCTCTCTCTCCCTCTCTCTCTCTCCCTCTCTCTCTCTCTCTCTCTCTCCCTCTGCCCCTCTTACTGCCGGATTTCAATTTTCTTTCTTTCATTAGTGCTCCATAAAATGACAGGGAGGCAAGAAGTTTTGTGCCTCTTCCTGACTTCCGAAAGAATCTTGGATAAAATATATCAGAGTCCACGTGACACTGTCTGAGCGGAGCTTGTGCGTCCCCCCCAGGACCACGACGGTTCCCTCTGGGTTCTCTGGATCGCCCCTACTTCCGAAGACATACGGATTCAAGTTGGTCCGGTGGCATTGTGAACATTCTTTGCTGTGATTCGCCCTCCGAACAGTCTGTGTCAGTGGTGACTCACCGAGTGCCTCCTCCATTCATCCCTGTCGGT
The primer sequence in 10 sites is as shown in table 1 below:
The primer sequence in table 110 site
The 10 pairs of primers gene type result in 15 dasyatis zugei individualities is as shown in table 2 below:
Table 210 is the gene type result in 15 dasyatis zugei individualities to primer
Sample ID DzuI25 DzuI45 DzuI31 DzuI46 DzuI51 DzuI57 DzuI71 DzuI82 DzuII15 DzuII64
DZR01 198 201 218 375 386 250 236 174 270 304
? 198 213 272 381 386 252 258 184 288 306
DZR02 216 233 238 365 386 230 244 184 178 234
? 220 237 244 379 386 230 246 194 226 234
DZR03 198 233 206 381 386 230 242 144 182 256
? 216 245 282 385 386 256 244 148 226 256
DZR04 218 219 206 375 386 230 238 138 182 256
? 218 243 266 391 386 250 244 148 276 256
2010G1 184 211 208 369 386 230 240 170 184 276
? 192 261 264 387 388 246 246 178 192 272
2010G2 184 233 206 373 388 240 250 136 188 272
? 188 243 208 397 396 242 258 144 260 276
2010G3 142 211 238 403 386 230 244 174 194 274
? 166 223 274 485 396 230 246 178 218 280
2010G4 142 211 218 373 386 230 236 160 184 ?
? 166 261 220 373 396 236 244 168 218 ?
2010G5 144 187 206 379 386 236 236 144 196 280
? 148 211 274 373 388 244 244 162 268 274
2010B3 ? 211 ? 373 ? 242 242 160 182 248
? ? 243 ? 379 ? 246 246 166 202 268
2010B4 178 201 214 383 386 228 240 130 258 248
? 178 201 244 391 386 228 250 146 262 268
2010B5 196 211 220 391 386 230 242 200 190 344
? 196 243 274 395 396 248 248 202 206 356
2010B6 164 211 242 383 396 230 246 178 180 280
? 164 243 242 387 396 248 248 180 192 308
2010B7 174 187 230 373 386 230 238 178 214 344
? 184 223 250 401 386 254 242 204 306 356
2010B8 202 187 222 389 386 246 250 154 256 250
? 230 221 256 389 386 252 258 182 278 280
Somatotype data by analysis after, number of alleles is 3-24, average number of alleles is 13.6.Expect that heterozygosity He is 0.5-0.984, observation heterozygosity Ho is 0.429-1, and this shows that these 10 pairs of primer polymorphisms are high, can meet the individual recognition of dasyatis zugei.Concrete outcome is as shown in table 3 below:
Table 3
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Claims (3)

1. a dasyatis zugei microsatellite locus, comprises 10 stable polymorphic micro-satellite sites, and its sequence is respectively as shown in SEQ ID NO:1-SEQ ID NO:10.
2. the primer of dasyatis zugei microsatellite locus according to claim 1, its sequence is respectively as shown in SEQ ID NO:11-SEQID NO:30.
3. the purposes of dasyatis zugei microsatellite locus claimed in claim 1 or primer claimed in claim 2, comprise molecular ecology research, pedigree analysis, Germplasm Resource Investigation, assistant breeding and individual recognition, described 10 stable polymorphic micro-satellite Sites Combination are used, or its corresponding combination of primers is used.
CN201410345130.9A 2014-07-21 2014-07-21 Dasyatis zugei microsatellite sites, primers and application thereof Pending CN104087584A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104328118A (en) * 2014-11-14 2015-02-04 张保卫 Hyla tsinlingensis microsatellite sites, primers for microsatellite sites and application of microsatellite sites and primers
CN104328117A (en) * 2014-11-14 2015-02-04 智颖飙 Tugarinovia mongolica microsatellite sites, primers for microsatellite sites and application of microsatellite sites and primers

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
匡刚桥: "鳜鱼微卫星标记的开发及遗传多样性研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 *
李宁: "玉筋鱼和赤魟的分子***地理学研究", 《中国博士学位论文全文数据库农业科技辑》 *
王伟 等: "鱼类微卫星标记的研究进展", 《海洋科学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104328118A (en) * 2014-11-14 2015-02-04 张保卫 Hyla tsinlingensis microsatellite sites, primers for microsatellite sites and application of microsatellite sites and primers
CN104328117A (en) * 2014-11-14 2015-02-04 智颖飙 Tugarinovia mongolica microsatellite sites, primers for microsatellite sites and application of microsatellite sites and primers

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Application publication date: 20141008