Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9
Abstract: Kiwifruit belong to the genus Actinidia with 54 species apparently all functionally dioecious. The sex-determinants of the type XX/XY, with male heterogametic, operate independently of the ploidy level. Recently, the SyGI protein has been described as the suppressor of female development....
- Autores:
-
De Mori, Gloria
Zaina, Giusi
Franco Orozco, Bárbara
Testolin, Raffaele
De Paoli, Emanuele
Cipriani, Guido
- Tipo de recurso:
- Article of investigation
- Fecha de publicación:
- 2020
- Institución:
- Tecnológico de Antioquia
- Repositorio:
- Repositorio Tdea
- Idioma:
- eng
- OAI Identifier:
- oai:dspace.tdea.edu.co:tdea/2791
- Acceso en línea:
- https://dspace.tdea.edu.co/handle/tdea/2791
- Palabra clave:
- Hermaphroditism
Genome Editing
Gene Editing
Edición Génica
Edição de Genes
Actinidia spp
Sex-determinant
Plant transformation
New breeding technologies
- Rights
- openAccess
- License
- https://creativecommons.org/licenses/by/4.0/
| id |
RepoTdea2_ac2bc46ad40b35206b5dd4d74cfbfe10 |
|---|---|
| oai_identifier_str |
oai:dspace.tdea.edu.co:tdea/2791 |
| network_acronym_str |
RepoTdea2 |
| network_name_str |
Repositorio Tdea |
| repository_id_str |
|
| dc.title.none.fl_str_mv |
Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 |
| title |
Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 |
| spellingShingle |
Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 Hermaphroditism Genome Editing Gene Editing Edición Génica Edição de Genes Actinidia spp Sex-determinant Plant transformation New breeding technologies |
| title_short |
Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 |
| title_full |
Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 |
| title_fullStr |
Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 |
| title_full_unstemmed |
Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 |
| title_sort |
Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 |
| dc.creator.fl_str_mv |
De Mori, Gloria Zaina, Giusi Franco Orozco, Bárbara Testolin, Raffaele De Paoli, Emanuele Cipriani, Guido |
| dc.contributor.author.none.fl_str_mv |
De Mori, Gloria Zaina, Giusi Franco Orozco, Bárbara Testolin, Raffaele De Paoli, Emanuele Cipriani, Guido |
| dc.subject.decs.none.fl_str_mv |
Hermaphroditism Genome Editing Gene Editing Edición Génica Edição de Genes |
| topic |
Hermaphroditism Genome Editing Gene Editing Edición Génica Edição de Genes Actinidia spp Sex-determinant Plant transformation New breeding technologies |
| dc.subject.proposal.none.fl_str_mv |
Actinidia spp Sex-determinant Plant transformation New breeding technologies |
| description |
Abstract: Kiwifruit belong to the genus Actinidia with 54 species apparently all functionally dioecious. The sex-determinants of the type XX/XY, with male heterogametic, operate independently of the ploidy level. Recently, the SyGI protein has been described as the suppressor of female development. In the present study, we exploited the CRISPR/Cas9 technology by targeting two different sites in the SyGI gene in order to induce a stable gene knock-out in two tetraploid male accessions of Actinidia chinensis var. chinensis. The two genotypes showed a regenerative efficiency of 58% and 73%, respectively. Despite not yet being able to verify the phenotypic effects on the flower structure, due to the long time required by tissue-cultured kiwifruit plants to flower, we obtained two regenerated lines showing near fixation of a unique modification in their genome, resulting in both cases in the onset of a premature stop codon, which induces the putative gene knock-out. Evaluation of gRNA1 locus for both regenerated plantlets resulted in co-amplification of a minor variant differing from the target region for a single nucleotide. A genomic duplication of the region in proximity of the Y genomic region could be postulated. Keywords: Actinidia spp.; sex-determinant; hermaphroditism; plant transformation; genome editing; new breeding technologies (NBTs) |
| publishDate |
2020 |
| dc.date.issued.none.fl_str_mv |
2020 |
| dc.date.accessioned.none.fl_str_mv |
2023-04-14T23:26:06Z |
| dc.date.available.none.fl_str_mv |
2023-04-14T23:26:06Z |
| dc.type.spa.fl_str_mv |
Artículo de revista |
| dc.type.coar.spa.fl_str_mv |
http://purl.org/coar/resource_type/c_2df8fbb1 |
| dc.type.content.spa.fl_str_mv |
Text |
| dc.type.driver.spa.fl_str_mv |
info:eu-repo/semantics/article |
| dc.type.redcol.spa.fl_str_mv |
http://purl.org/redcol/resource_type/ART |
| dc.type.version.spa.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
| dc.type.coarversion.spa.fl_str_mv |
http://purl.org/coar/version/c_970fb48d4fbd8a85 |
| format |
http://purl.org/coar/resource_type/c_2df8fbb1 |
| status_str |
publishedVersion |
| dc.identifier.uri.none.fl_str_mv |
https://dspace.tdea.edu.co/handle/tdea/2791 |
| dc.identifier.eissn.spa.fl_str_mv |
2223-7747 |
| url |
https://dspace.tdea.edu.co/handle/tdea/2791 |
| identifier_str_mv |
2223-7747 |
| dc.language.iso.spa.fl_str_mv |
eng |
| language |
eng |
| dc.relation.citationendpage.spa.fl_str_mv |
17 |
| dc.relation.citationissue.spa.fl_str_mv |
1 |
| dc.relation.citationstartpage.spa.fl_str_mv |
1 |
| dc.relation.citationvolume.spa.fl_str_mv |
10 |
| dc.relation.ispartofjournal.spa.fl_str_mv |
Plants |
| dc.relation.references.spa.fl_str_mv |
Liang, C.; Ferguson, A.R. Actinidia; Feng, K.-M., Ed.; Floria Reipublicae Pop. Sin; Science Press: Beijing, China, 1984; Volume 49/2, pp. 196–268, 309–324 Huang, H. Kiwifruit: The Genus ACTINIDIA; Elsevier Inc.: Amsterdam, The Netherlands, 2016; pp. 1–334. ISBN 9780128030660 McNeilage, M.A. Gender variation in Actinidia deliciosa, the kiwifruit. Sex. Plant Reprod. 1991, 4, 267–273. [CrossRef] Testolin, R.; Cipriani, G.; Costa, G. Sex segregation ratio and gender expression in the genus Actinidia. Sex. Plant Reprod. 1995, 8, 129–132. [CrossRef] Harvey, C.F.; Gill, G.P.; Fraser, L.G.; Mcneilage, M.A. Sex determination in Actinidia. 1. Sex-linked markers and progeny sex ratio in diploid A. chinensis. Sex. Plant Reprod. 1997, 10, 149–154. [CrossRef] Kawagoe, T.; Suzuki, N. Cryptic dioecy in Actinidia polygama: A test of the pollinator attraction hypothesis. Can. J. Bot. 2004, 82, 214–218. [CrossRef] McNeilage, M.A. Sex expression in fruiting male vines of kiwifruit. Sex. Plant Reprod. 1991, 8, 129–132. [CrossRef] Marsh, H.D.; Paterson, T.; Seal, A.G.; McNeilage, M.A. Heritability estimates in kiwifruit. Acta Hortic. 2003, 622, 221–229. [CrossRef] Testolin, R. Kiwifruit breeding: From the phenotypic analysis of parents to the genomic estimation of their breeding value (GEBV). Acta Hortic. 2011, 913, 123–130. [CrossRef] Van Nocker, S.; Gardiner, S.E. Breeding better cultivars, faster: Applications of new technologies for the rapid deployment of superior horticultural tree crops. Hortic. Res. 2014, 1, 22. [CrossRef] Mcneilage, M.A.; Steinhagen, S. Flower and fruit characters in a kiwifruit hermaphrodite. Euphytica 1998, 101, 69–72. [CrossRef] Testolin, R. Male density and arrangement in kiwifruit orchards. Sci. Hortic. 1991, 48, 41–50. [CrossRef] Goodwin, R.; McBrydie, H.; Taylor, M.Wind and honey bee pollination of kiwifruit (Actinidia chinensis‘HORT16A’). N. Z. J. Bot. 2013, 51, 229–240. [CrossRef] Stefani, E.; Giovanardi, D. Dissemination of Pseudomonas syringae pv. Actinidiae through pollen and its epiphytic life on leaves and fruits. Phytopathol. Mediterr. 2011, 50, 489–496. [CrossRef] Testolin, R.; Cipriani, G.; Messina, R. Sex control in Actinidia is monofactorial and remains so in polyploids. In Sex Determination in Plants; Ainsworth, C.C., Ed.; BIOS Scientific Publisher Limited: Milton Park, UK, 1999; pp. 173–181. Pilkington, S.M.; Tahir, J.; Hilario, E.; Gardiner, S.E.; Chagné, D.; Catanach, A.; McCallum, J.; Jesson, L.; Fraser, L.G.; Mcneilage, M.A.; et al. Genetic and cytological analyses reveal the recombination landscape of a partially differentiated plant sex chromosome in kiwifruit. BMC Plant Biol. 2019, 19, 172. [CrossRef] [PubMed] Akagi, T.; Henry, I.M.; Ohtani, H.; Morimoto, T.; Beppu, K.; Kataoka, I.; Tao, R. A Y-Encoded Suppressor of Feminization Arose via Lineage-Specific Duplication of a Cytokinin Response Regulator in Kiwifruit. Plant Cell 2018, 30, 780–795. [CrossRef] Akagi, T.; Pilkington, S.M.; Varkonyi-Gasic, E.; Henry, I.M.; Sugano, S.S.; Sonoda, M.; Firl, A.; Mcneilage, M.A.; Douglas, M.J.; Wang, T.; et al. Two Y-chromosome-encoded genes determine sex in kiwifruit. Nat. Plants 2019, 5, 801–809. [CrossRef] Gaj, T.; Gersbach, C.A.; Barbas, C.F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013, 31, 397–405. [CrossRef] Shan, Q.; Wang, Y.; Li, J.; Zhang, Y.; Chen, K.; Liang, Z.; Zhang, K.; Liu, J.; Xi, J.J.; Qiu, J.-L.; et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 2013, 31, 686–688. [CrossRef] Baltes, N.J.; Voytas, D.F. Enabling plant synthetic biology through genome engineering. Trends Biotechnol. 2015, 33, 120–131. [CrossRef] Wang, Z.;Wang, S.; Li, D.; Zhang, Q.; Li, L.; Zhong, C.; Liu, Y.; Huang, H. Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit. Plant Biotechnol. J. 2018, 16, 1424–1433. [CrossRef] Varkonyi-Gasic, E.; Wang, T.; Voogd, C.; Jeon, S.; Drummond, R.S.M.; Gleave, A.P.; Allan, A.C. Mutagenesis of kiwifruit CENTRORADIALIS -like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering. Plant Biotechnol. J. 2019, 17, 869–880. [CrossRef] Cˇ ermák, T.; Curtin, S.J.; Gil-Humanes, J.; Cˇ egan, R.; Kono, T.J.Y.; Konecˇná, E.; Belanto, J.J.; Starker, C.G.; Mathre, J.W.; Greenstein, R.L.; et al. A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants. Plant Cell 2017, 29, 1196–1217. [CrossRef] [PubMed] Fraser, L.G.; Tsang, G.K.; Datson, P.M.; De Silva, H.N.; Harvey, C.F.; Gill, G.P.; Crowhurst, R.N.; Mcneilage, M.A. A gene-rich linkage map in the dioecious species Actinidia chinensis (kiwifruit) reveals putative X/Y sex-determining chromosomes. BMC Genom. 2009, 10, 102. [CrossRef] [PubMed] Testolin, R. Kiwifruit Yield Efficiency, Plant Density, and Bud Number per Surface Unit. J. Am. Soc. Hortic. Sci. 1990, 115, 704–707. [CrossRef] Ferguson, A.R.; Huang, H. Genetic Resources of Kiwifruit: Domestication and Breeding. Hortic. Rev. 2007, 33, 1–122. Meyer, R.S.; Purugganan, M.D. Evolution of crop species: Genetics of domestication and diversification. Nat. Rev. Genet. 2013, 14, 840–852. [CrossRef] [PubMed] Schindele, A.; Dorn, A.; Puchta, H. CRISPR/Cas brings plant biology and breeding into the fast lane. Curr. Opin. Biotechnol. 2020, 61, 7–14. [CrossRef] Pinto, C.; Testolin, R.; Cipriani, G. Analysis of components of genetic variance and breeding value of kiwifruit (Actinidia chinensis Planch.) using best linear unbiased prediction (BLUP) and pedigree information. Acta Hortic. 2018, 1218, 73–80. [CrossRef] Alvarez, J.; Smyth, D.R. Carpel development genes in Arabidopsis. Flower. Newsl. 1997, 23, 12–17. Doench, J.G.; Hartenian, E.; Graham, D.B.; Tothova, Z.; Hegde, M.; Smith, I.; Sullender, M.; Ebert, B.L.; Xavier, R.J.; Root, D.E. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 2014, 32, 1262–1267. [CrossRef] Walton, E.F.; Fowke, P.J.;Weis, K.; McLeay, P.L. Shoot Axillary Bud Morphogenesis in Kiwifruit (Actinidia deliciosa). Ann. Bot. 1997, 80, 13–21. [CrossRef] Varkonyi-Gasic, E.; Moss, S.M.; Voogd, C.; Wu, R.; Lough, R.H.; Wang, Y.-Y.; Hellens, R.P. Identification and characterization of flowering genes in kiwifruit: Sequence conservation and role in kiwifruit flower development. BMC Plant Biol. 2011, 11, 72. [CrossRef] [PubMed] Gill, G.P.; Harvey, C.F.; Gardner, R.C.; Fraser, L.G. Development of sex-linked PCR markers for gender identification in Actinidia. Theor. Appl. Genet. 1998, 97, 439–445. [CrossRef] Fraser, L.G.; McNeilage, M.A. Reproductive Biology. In The kiwifruit genome; Springer: Cham, Switzerland, 2016; pp. 65–84. [CrossRef] Müller, N.A.; Kersten, B.; Leite Montalvão, A.P.; Mähler, N.; Bernhardsson, C.; Bräutigam, K.; Carracedo Lorenzo, Z.; Hoenicka, H.; Kumar, V.; Mader, M.; et al. A single gene underlies the dynamic evolution of poplar sex determination. Nat. Plants 2020, 6, 630–637. [CrossRef] [PubMed] Murashige, T.; Skoog, F. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiol. Plant. 1962, 15, 473–497. [CrossRef] Park, J.; Bae, S.; Kim, J.-S. Cas-Designer: A web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 2015, 31, 4014–4016. [CrossRef] Pilkington, S.M.; Crowhurst, R.; Hilario, E.; Nardozza, S.; Fraser, L.; Peng, Y.; Gunaseelan, K.; Simpson, R.; Tahir, J.; Deroles, S.C.; et al. A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants. BMC Genom. 2018, 19, 1–19. [CrossRef] Bae, S.; Park, J.; Kim, J.S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 2014, 30, 1473–1475. [CrossRef] Tsai, S.Q.; Wyvekens, N.; Khayter, C.; Foden, J.A.; Thapar, V.; Reyon, D.; Goodwin, M.J.; Aryee, M.J.; Joung, K.; Unit, P.; et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 2014, 32, 569–576. [CrossRef] Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 1983, 166, 557–580. [CrossRef] Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011, 17, 10. [CrossRef] Del Fabbro, C.; Scalabrin, S.; Morgante, M.; Giorgi, F.M. An Extensive Evaluation of Read Trimming Effects on Illumina NGS Data Analysis. PLoS ONE 2013, 8, e85024. [CrossRef] [PubMed] Warren, R.L.; Sutton, G.G.; Jones, S.J.M.; Holt, R.A.; Bateman, A. Assembling millions of short DNA sequences using SSAKE. Bioinformatics 2007, 23, 500–501. [CrossRef] [PubMed] Needleman, S.B.; Wunsch, C.D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 1970, 48, 443–453. [CrossRef] Wang, T.; Lin-Wang, K. High throughput transformation of Actinidia: A platform for kiwifruit functional genomics and molecular breeding. Transgenic Plant J. 2007, 1, 173–184. Wang, T.; Karunairetnam, S.;Wu, R.;Wang, Y.Y.; Gleave, A.P. High efficiency transformation platforms for kiwifruit (Actinidia spp.) functional genomics. Acta Hortic. 2012, 929, 143–148. [CrossRef] Gamborg, O.L.; Miller, R.A.; Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 1968, 50, 151–158. [CrossRef] Doyle, J.J.; Doyle, J. Isolation of Plant DNA from Fresh Tissue. Focus (Madison) 1990, 12, 13–15. Andrews, S. Fast QC: A Quality Control Tool for High Throughput Sequence Data. 2010. Available online: http://www. bioinformatics.babraham.ac.uk/projects/fastqc (accessed on 29 December 2020). Langmead, B.; Trapnell, C.; Pop, M.; Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009, 10, 25. [CrossRef] Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R.; Project, G.; et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [CrossRef] Magoˇc, T.; Magoˇc, M.; Salzberg, S.L. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011, 27, 2957–2963. [CrossRef] Pinello, L.; Canver, M.C.; Hoban, M.D.; Orkin, S.H.; Kohn, D.B.; Bauer, D.E.; Yuan, G.-C. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat. Biotechnol. 2016, 34, 695–697. [CrossRef] [PubMed] |
| dc.rights.uri.spa.fl_str_mv |
https://creativecommons.org/licenses/by/4.0/ |
| dc.rights.license.spa.fl_str_mv |
Atribución 4.0 Internacional (CC BY 4.0) |
| dc.rights.accessrights.spa.fl_str_mv |
info:eu-repo/semantics/openAccess |
| dc.rights.coar.spa.fl_str_mv |
http://purl.org/coar/access_right/c_abf2 |
| rights_invalid_str_mv |
https://creativecommons.org/licenses/by/4.0/ Atribución 4.0 Internacional (CC BY 4.0) http://purl.org/coar/access_right/c_abf2 |
| eu_rights_str_mv |
openAccess |
| dc.format.extent.spa.fl_str_mv |
17 páginas |
| dc.format.mimetype.spa.fl_str_mv |
application/pdf |
| dc.publisher.spa.fl_str_mv |
MDPI AG |
| dc.publisher.place.spa.fl_str_mv |
Suiza |
| dc.source.spa.fl_str_mv |
file:///C:/Users/user/Downloads/plants-10-00062.pdf |
| institution |
Tecnológico de Antioquia |
| bitstream.url.fl_str_mv |
https://dspace.tdea.edu.co/bitstream/tdea/2791/1/Targeted%20Mutagenesis%20of%20the%20Female-Suppressor%20SyGI%20Gene%20in%20Tetraploid%20Kiwifruit%20by%20CRISPR_CAS9.pdf https://dspace.tdea.edu.co/bitstream/tdea/2791/2/license.txt https://dspace.tdea.edu.co/bitstream/tdea/2791/3/Targeted%20Mutagenesis%20of%20the%20Female-Suppressor%20SyGI%20Gene%20in%20Tetraploid%20Kiwifruit%20by%20CRISPR_CAS9.pdf.txt https://dspace.tdea.edu.co/bitstream/tdea/2791/4/Targeted%20Mutagenesis%20of%20the%20Female-Suppressor%20SyGI%20Gene%20in%20Tetraploid%20Kiwifruit%20by%20CRISPR_CAS9.pdf.jpg |
| bitstream.checksum.fl_str_mv |
2a83d021b8b45e3d6ea6472d4af72761 2f9959eaf5b71fae44bbf9ec84150c7a ed385c1aa87b11a2bee0567eb5dff2fa e3f18c6f05f3ebe565e88aa16cb57a78 |
| bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 MD5 |
| repository.name.fl_str_mv |
Repositorio Institucional Tecnologico de Antioquia |
| repository.mail.fl_str_mv |
bdigital@metabiblioteca.com |
| _version_ |
1812189201505976320 |
| spelling |
De Mori, Gloriae5fe3985-c319-4f05-b9aa-3859144aa0d9Zaina, Giusib701f7a2-3803-4524-a2e5-6af5cd56cff3Franco Orozco, Bárbara49c7fb7f-9292-48f7-83e7-09cbac0fd8a2Testolin, Raffaele8da18219-2af5-4ba4-aedd-dd45f357fd54De Paoli, Emanuelee8a8e373-52ab-4e44-84ea-2fccfcba71d2Cipriani, Guido0ab67dc0-f8e6-42f8-8f14-09b18e82af7f2023-04-14T23:26:06Z2023-04-14T23:26:06Z2020https://dspace.tdea.edu.co/handle/tdea/27912223-7747Abstract: Kiwifruit belong to the genus Actinidia with 54 species apparently all functionally dioecious. The sex-determinants of the type XX/XY, with male heterogametic, operate independently of the ploidy level. Recently, the SyGI protein has been described as the suppressor of female development. In the present study, we exploited the CRISPR/Cas9 technology by targeting two different sites in the SyGI gene in order to induce a stable gene knock-out in two tetraploid male accessions of Actinidia chinensis var. chinensis. The two genotypes showed a regenerative efficiency of 58% and 73%, respectively. Despite not yet being able to verify the phenotypic effects on the flower structure, due to the long time required by tissue-cultured kiwifruit plants to flower, we obtained two regenerated lines showing near fixation of a unique modification in their genome, resulting in both cases in the onset of a premature stop codon, which induces the putative gene knock-out. Evaluation of gRNA1 locus for both regenerated plantlets resulted in co-amplification of a minor variant differing from the target region for a single nucleotide. A genomic duplication of the region in proximity of the Y genomic region could be postulated. Keywords: Actinidia spp.; sex-determinant; hermaphroditism; plant transformation; genome editing; new breeding technologies (NBTs)17 páginasapplication/pdfengMDPI AGSuizahttps://creativecommons.org/licenses/by/4.0/Atribución 4.0 Internacional (CC BY 4.0)info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2file:///C:/Users/user/Downloads/plants-10-00062.pdfTargeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9Artículo de revistahttp://purl.org/coar/resource_type/c_2df8fbb1Textinfo:eu-repo/semantics/articlehttp://purl.org/redcol/resource_type/ARTinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/version/c_970fb48d4fbd8a85171110PlantsLiang, C.; Ferguson, A.R. Actinidia; Feng, K.-M., Ed.; Floria Reipublicae Pop. Sin; Science Press: Beijing, China, 1984; Volume 49/2, pp. 196–268, 309–324Huang, H. Kiwifruit: The Genus ACTINIDIA; Elsevier Inc.: Amsterdam, The Netherlands, 2016; pp. 1–334. ISBN 9780128030660McNeilage, M.A. Gender variation in Actinidia deliciosa, the kiwifruit. Sex. Plant Reprod. 1991, 4, 267–273. [CrossRef]Testolin, R.; Cipriani, G.; Costa, G. Sex segregation ratio and gender expression in the genus Actinidia. Sex. Plant Reprod. 1995, 8, 129–132. [CrossRef]Harvey, C.F.; Gill, G.P.; Fraser, L.G.; Mcneilage, M.A. Sex determination in Actinidia. 1. Sex-linked markers and progeny sex ratio in diploid A. chinensis. Sex. Plant Reprod. 1997, 10, 149–154. [CrossRef]Kawagoe, T.; Suzuki, N. Cryptic dioecy in Actinidia polygama: A test of the pollinator attraction hypothesis. Can. J. Bot. 2004, 82, 214–218. [CrossRef]McNeilage, M.A. Sex expression in fruiting male vines of kiwifruit. Sex. Plant Reprod. 1991, 8, 129–132. [CrossRef]Marsh, H.D.; Paterson, T.; Seal, A.G.; McNeilage, M.A. Heritability estimates in kiwifruit. Acta Hortic. 2003, 622, 221–229. [CrossRef]Testolin, R. Kiwifruit breeding: From the phenotypic analysis of parents to the genomic estimation of their breeding value (GEBV). Acta Hortic. 2011, 913, 123–130. [CrossRef]Van Nocker, S.; Gardiner, S.E. Breeding better cultivars, faster: Applications of new technologies for the rapid deployment of superior horticultural tree crops. Hortic. Res. 2014, 1, 22. [CrossRef]Mcneilage, M.A.; Steinhagen, S. Flower and fruit characters in a kiwifruit hermaphrodite. Euphytica 1998, 101, 69–72. [CrossRef]Testolin, R. Male density and arrangement in kiwifruit orchards. Sci. Hortic. 1991, 48, 41–50. [CrossRef]Goodwin, R.; McBrydie, H.; Taylor, M.Wind and honey bee pollination of kiwifruit (Actinidia chinensis‘HORT16A’). N. Z. J. Bot. 2013, 51, 229–240. [CrossRef]Stefani, E.; Giovanardi, D. Dissemination of Pseudomonas syringae pv. Actinidiae through pollen and its epiphytic life on leaves and fruits. Phytopathol. Mediterr. 2011, 50, 489–496. [CrossRef]Testolin, R.; Cipriani, G.; Messina, R. Sex control in Actinidia is monofactorial and remains so in polyploids. In Sex Determination in Plants; Ainsworth, C.C., Ed.; BIOS Scientific Publisher Limited: Milton Park, UK, 1999; pp. 173–181.Pilkington, S.M.; Tahir, J.; Hilario, E.; Gardiner, S.E.; Chagné, D.; Catanach, A.; McCallum, J.; Jesson, L.; Fraser, L.G.; Mcneilage, M.A.; et al. Genetic and cytological analyses reveal the recombination landscape of a partially differentiated plant sex chromosome in kiwifruit. BMC Plant Biol. 2019, 19, 172. [CrossRef] [PubMed]Akagi, T.; Henry, I.M.; Ohtani, H.; Morimoto, T.; Beppu, K.; Kataoka, I.; Tao, R. A Y-Encoded Suppressor of Feminization Arose via Lineage-Specific Duplication of a Cytokinin Response Regulator in Kiwifruit. Plant Cell 2018, 30, 780–795. [CrossRef]Akagi, T.; Pilkington, S.M.; Varkonyi-Gasic, E.; Henry, I.M.; Sugano, S.S.; Sonoda, M.; Firl, A.; Mcneilage, M.A.; Douglas, M.J.; Wang, T.; et al. Two Y-chromosome-encoded genes determine sex in kiwifruit. Nat. Plants 2019, 5, 801–809. [CrossRef]Gaj, T.; Gersbach, C.A.; Barbas, C.F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 2013, 31, 397–405. [CrossRef]Shan, Q.; Wang, Y.; Li, J.; Zhang, Y.; Chen, K.; Liang, Z.; Zhang, K.; Liu, J.; Xi, J.J.; Qiu, J.-L.; et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nat. Biotechnol. 2013, 31, 686–688. [CrossRef]Baltes, N.J.; Voytas, D.F. Enabling plant synthetic biology through genome engineering. Trends Biotechnol. 2015, 33, 120–131. [CrossRef]Wang, Z.;Wang, S.; Li, D.; Zhang, Q.; Li, L.; Zhong, C.; Liu, Y.; Huang, H. Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit. Plant Biotechnol. J. 2018, 16, 1424–1433. [CrossRef]Varkonyi-Gasic, E.; Wang, T.; Voogd, C.; Jeon, S.; Drummond, R.S.M.; Gleave, A.P.; Allan, A.C. Mutagenesis of kiwifruit CENTRORADIALIS -like genes transforms a climbing woody perennial with long juvenility and axillary flowering into a compact plant with rapid terminal flowering. Plant Biotechnol. J. 2019, 17, 869–880. [CrossRef]Cˇ ermák, T.; Curtin, S.J.; Gil-Humanes, J.; Cˇ egan, R.; Kono, T.J.Y.; Konecˇná, E.; Belanto, J.J.; Starker, C.G.; Mathre, J.W.; Greenstein, R.L.; et al. A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants. Plant Cell 2017, 29, 1196–1217. [CrossRef] [PubMed]Fraser, L.G.; Tsang, G.K.; Datson, P.M.; De Silva, H.N.; Harvey, C.F.; Gill, G.P.; Crowhurst, R.N.; Mcneilage, M.A. A gene-rich linkage map in the dioecious species Actinidia chinensis (kiwifruit) reveals putative X/Y sex-determining chromosomes. BMC Genom. 2009, 10, 102. [CrossRef] [PubMed]Testolin, R. Kiwifruit Yield Efficiency, Plant Density, and Bud Number per Surface Unit. J. Am. Soc. Hortic. Sci. 1990, 115, 704–707. [CrossRef]Ferguson, A.R.; Huang, H. Genetic Resources of Kiwifruit: Domestication and Breeding. Hortic. Rev. 2007, 33, 1–122.Meyer, R.S.; Purugganan, M.D. Evolution of crop species: Genetics of domestication and diversification. Nat. Rev. Genet. 2013, 14, 840–852. [CrossRef] [PubMed]Schindele, A.; Dorn, A.; Puchta, H. CRISPR/Cas brings plant biology and breeding into the fast lane. Curr. Opin. Biotechnol. 2020, 61, 7–14. [CrossRef]Pinto, C.; Testolin, R.; Cipriani, G. Analysis of components of genetic variance and breeding value of kiwifruit (Actinidia chinensis Planch.) using best linear unbiased prediction (BLUP) and pedigree information. Acta Hortic. 2018, 1218, 73–80. [CrossRef]Alvarez, J.; Smyth, D.R. Carpel development genes in Arabidopsis. Flower. Newsl. 1997, 23, 12–17.Doench, J.G.; Hartenian, E.; Graham, D.B.; Tothova, Z.; Hegde, M.; Smith, I.; Sullender, M.; Ebert, B.L.; Xavier, R.J.; Root, D.E. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 2014, 32, 1262–1267. [CrossRef]Walton, E.F.; Fowke, P.J.;Weis, K.; McLeay, P.L. Shoot Axillary Bud Morphogenesis in Kiwifruit (Actinidia deliciosa). Ann. Bot. 1997, 80, 13–21. [CrossRef]Varkonyi-Gasic, E.; Moss, S.M.; Voogd, C.; Wu, R.; Lough, R.H.; Wang, Y.-Y.; Hellens, R.P. Identification and characterization of flowering genes in kiwifruit: Sequence conservation and role in kiwifruit flower development. BMC Plant Biol. 2011, 11, 72. [CrossRef] [PubMed]Gill, G.P.; Harvey, C.F.; Gardner, R.C.; Fraser, L.G. Development of sex-linked PCR markers for gender identification in Actinidia. Theor. Appl. Genet. 1998, 97, 439–445. [CrossRef]Fraser, L.G.; McNeilage, M.A. Reproductive Biology. In The kiwifruit genome; Springer: Cham, Switzerland, 2016; pp. 65–84. [CrossRef]Müller, N.A.; Kersten, B.; Leite Montalvão, A.P.; Mähler, N.; Bernhardsson, C.; Bräutigam, K.; Carracedo Lorenzo, Z.; Hoenicka, H.; Kumar, V.; Mader, M.; et al. A single gene underlies the dynamic evolution of poplar sex determination. Nat. Plants 2020, 6, 630–637. [CrossRef] [PubMed]Murashige, T.; Skoog, F. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiol. Plant. 1962, 15, 473–497. [CrossRef]Park, J.; Bae, S.; Kim, J.-S. Cas-Designer: A web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 2015, 31, 4014–4016. [CrossRef]Pilkington, S.M.; Crowhurst, R.; Hilario, E.; Nardozza, S.; Fraser, L.; Peng, Y.; Gunaseelan, K.; Simpson, R.; Tahir, J.; Deroles, S.C.; et al. A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants. BMC Genom. 2018, 19, 1–19. [CrossRef]Bae, S.; Park, J.; Kim, J.S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 2014, 30, 1473–1475. [CrossRef]Tsai, S.Q.; Wyvekens, N.; Khayter, C.; Foden, J.A.; Thapar, V.; Reyon, D.; Goodwin, M.J.; Aryee, M.J.; Joung, K.; Unit, P.; et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 2014, 32, 569–576. [CrossRef]Hanahan, D. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 1983, 166, 557–580. [CrossRef]Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011, 17, 10. [CrossRef]Del Fabbro, C.; Scalabrin, S.; Morgante, M.; Giorgi, F.M. An Extensive Evaluation of Read Trimming Effects on Illumina NGS Data Analysis. PLoS ONE 2013, 8, e85024. [CrossRef] [PubMed]Warren, R.L.; Sutton, G.G.; Jones, S.J.M.; Holt, R.A.; Bateman, A. Assembling millions of short DNA sequences using SSAKE. Bioinformatics 2007, 23, 500–501. [CrossRef] [PubMed]Needleman, S.B.; Wunsch, C.D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J. Mol. Biol. 1970, 48, 443–453. [CrossRef]Wang, T.; Lin-Wang, K. High throughput transformation of Actinidia: A platform for kiwifruit functional genomics and molecular breeding. Transgenic Plant J. 2007, 1, 173–184.Wang, T.; Karunairetnam, S.;Wu, R.;Wang, Y.Y.; Gleave, A.P. High efficiency transformation platforms for kiwifruit (Actinidia spp.) functional genomics. Acta Hortic. 2012, 929, 143–148. [CrossRef]Gamborg, O.L.; Miller, R.A.; Ojima, K. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 1968, 50, 151–158. [CrossRef]Doyle, J.J.; Doyle, J. Isolation of Plant DNA from Fresh Tissue. Focus (Madison) 1990, 12, 13–15.Andrews, S. Fast QC: A Quality Control Tool for High Throughput Sequence Data. 2010. Available online: http://www. bioinformatics.babraham.ac.uk/projects/fastqc (accessed on 29 December 2020).Langmead, B.; Trapnell, C.; Pop, M.; Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009, 10, 25. [CrossRef]Li, H.; Handsaker, B.; Wysoker, A.; Fennell, T.; Ruan, J.; Homer, N.; Marth, G.; Abecasis, G.; Durbin, R.; Project, G.; et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009, 25, 2078–2079. [CrossRef]Magoˇc, T.; Magoˇc, M.; Salzberg, S.L. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 2011, 27, 2957–2963. [CrossRef]Pinello, L.; Canver, M.C.; Hoban, M.D.; Orkin, S.H.; Kohn, D.B.; Bauer, D.E.; Yuan, G.-C. Analyzing CRISPR genome-editing experiments with CRISPResso. Nat. Biotechnol. 2016, 34, 695–697. [CrossRef] [PubMed]HermaphroditismGenome EditingGene EditingEdición GénicaEdição de GenesActinidia sppSex-determinantPlant transformationNew breeding technologiesORIGINALTargeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR_CAS9.pdfTargeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR_CAS9.pdfapplication/pdf2800483https://dspace.tdea.edu.co/bitstream/tdea/2791/1/Targeted%20Mutagenesis%20of%20the%20Female-Suppressor%20SyGI%20Gene%20in%20Tetraploid%20Kiwifruit%20by%20CRISPR_CAS9.pdf2a83d021b8b45e3d6ea6472d4af72761MD51open accessLICENSElicense.txtlicense.txttext/plain; charset=utf-814828https://dspace.tdea.edu.co/bitstream/tdea/2791/2/license.txt2f9959eaf5b71fae44bbf9ec84150c7aMD52open accessTEXTTargeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR_CAS9.pdf.txtTargeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR_CAS9.pdf.txtExtracted texttext/plain77591https://dspace.tdea.edu.co/bitstream/tdea/2791/3/Targeted%20Mutagenesis%20of%20the%20Female-Suppressor%20SyGI%20Gene%20in%20Tetraploid%20Kiwifruit%20by%20CRISPR_CAS9.pdf.txted385c1aa87b11a2bee0567eb5dff2faMD53open accessTHUMBNAILTargeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR_CAS9.pdf.jpgTargeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR_CAS9.pdf.jpgGenerated Thumbnailimage/jpeg16171https://dspace.tdea.edu.co/bitstream/tdea/2791/4/Targeted%20Mutagenesis%20of%20the%20Female-Suppressor%20SyGI%20Gene%20in%20Tetraploid%20Kiwifruit%20by%20CRISPR_CAS9.pdf.jpge3f18c6f05f3ebe565e88aa16cb57a78MD54open accesstdea/2791oai:dspace.tdea.edu.co:tdea/27912023-04-15 03:01:14.003An error occurred on the license name.|||https://creativecommons.org/licenses/by/4.0/open accessRepositorio Institucional Tecnologico de Antioquiabdigital@metabiblioteca.comTEEgT0JSQSAoVEFMIFkgQ09NTyBTRSBERUZJTkUgTcOBUyBBREVMQU5URSkgU0UgT1RPUkdBIEJBSk8gTE9TIFRFUk1JTk9TIERFIEVTVEEgTElDRU5DSUEgUMOaQkxJQ0EgREUgQ1JFQVRJVkUgQ09NTU9OUyAo4oCcTFBDQ+KAnSBPIOKAnExJQ0VOQ0lB4oCdKS4gTEEgT0JSQSBFU1TDgSBQUk9URUdJREEgUE9SIERFUkVDSE9TIERFIEFVVE9SIFkvVSBPVFJBUyBMRVlFUyBBUExJQ0FCTEVTLiBRVUVEQSBQUk9ISUJJRE8gQ1VBTFFVSUVSIFVTTyBRVUUgU0UgSEFHQSBERSBMQSBPQlJBIFFVRSBOTyBDVUVOVEUgQ09OIExBIEFVVE9SSVpBQ0nDk04gUEVSVElORU5URSBERSBDT05GT1JNSURBRCBDT04gTE9TIFTDiVJNSU5PUyBERSBFU1RBIExJQ0VOQ0lBIFkgREUgTEEgTEVZIERFIERFUkVDSE8gREUgQVVUT1IuCgpNRURJQU5URSBFTCBFSkVSQ0lDSU8gREUgQ1VBTFFVSUVSQSBERSBMT1MgREVSRUNIT1MgUVVFIFNFIE9UT1JHQU4gRU4gRVNUQSBMSUNFTkNJQSwgVVNURUQgQUNFUFRBIFkgQUNVRVJEQSBRVUVEQVIgT0JMSUdBRE8gRU4gTE9TIFRFUk1JTk9TIFFVRSBTRSBTRcORQUxBTiBFTiBFTExBLiBFTCBMSUNFTkNJQU5URSBDT05DRURFIEEgVVNURUQgTE9TIERFUkVDSE9TIENPTlRFTklET1MgRU4gRVNUQSBMSUNFTkNJQSBDT05ESUNJT05BRE9TIEEgTEEgQUNFUFRBQ0nDk04gREUgU1VTIFRFUk1JTk9TIFkgQ09ORElDSU9ORVMuCjEuIERlZmluaWNpb25lcwoKYS4JT2JyYSBDb2xlY3RpdmEgZXMgdW5hIG9icmEsIHRhbCBjb21vIHVuYSBwdWJsaWNhY2nDs24gcGVyacOzZGljYSwgdW5hIGFudG9sb2fDrWEsIG8gdW5hIGVuY2ljbG9wZWRpYSwgZW4gbGEgcXVlIGxhIG9icmEgZW4gc3UgdG90YWxpZGFkLCBzaW4gbW9kaWZpY2FjacOzbiBhbGd1bmEsIGp1bnRvIGNvbiB1biBncnVwbyBkZSBvdHJhcyBjb250cmlidWNpb25lcyBxdWUgY29uc3RpdHV5ZW4gb2JyYXMgc2VwYXJhZGFzIGUgaW5kZXBlbmRpZW50ZXMgZW4gc8OtIG1pc21hcywgc2UgaW50ZWdyYW4gZW4gdW4gdG9kbyBjb2xlY3Rpdm8uIFVuYSBPYnJhIHF1ZSBjb25zdGl0dXllIHVuYSBvYnJhIGNvbGVjdGl2YSBubyBzZSBjb25zaWRlcmFyw6EgdW5hIE9icmEgRGVyaXZhZGEgKGNvbW8gc2UgZGVmaW5lIGFiYWpvKSBwYXJhIGxvcyBwcm9ww7NzaXRvcyBkZSBlc3RhIGxpY2VuY2lhLiBhcXVlbGxhIHByb2R1Y2lkYSBwb3IgdW4gZ3J1cG8gZGUgYXV0b3JlcywgZW4gcXVlIGxhIE9icmEgc2UgZW5jdWVudHJhIHNpbiBtb2RpZmljYWNpb25lcywganVudG8gY29uIHVuYSBjaWVydGEgY2FudGlkYWQgZGUgb3RyYXMgY29udHJpYnVjaW9uZXMsIHF1ZSBjb25zdGl0dXllbiBlbiBzw60gbWlzbW9zIHRyYWJham9zIHNlcGFyYWRvcyBlIGluZGVwZW5kaWVudGVzLCBxdWUgc29uIGludGVncmFkb3MgYWwgdG9kbyBjb2xlY3Rpdm8sIHRhbGVzIGNvbW8gcHVibGljYWNpb25lcyBwZXJpw7NkaWNhcywgYW50b2xvZ8OtYXMgbyBlbmNpY2xvcGVkaWFzLgoKYi4JT2JyYSBEZXJpdmFkYSBzaWduaWZpY2EgdW5hIG9icmEgYmFzYWRhIGVuIGxhIG9icmEgb2JqZXRvIGRlIGVzdGEgbGljZW5jaWEgbyBlbiDDqXN0YSB5IG90cmFzIG9icmFzIHByZWV4aXN0ZW50ZXMsIHRhbGVzIGNvbW8gdHJhZHVjY2lvbmVzLCBhcnJlZ2xvcyBtdXNpY2FsZXMsIGRyYW1hdGl6YWNpb25lcywg4oCcZmljY2lvbmFsaXphY2lvbmVz4oCdLCB2ZXJzaW9uZXMgcGFyYSBjaW5lLCDigJxncmFiYWNpb25lcyBkZSBzb25pZG/igJ0sIHJlcHJvZHVjY2lvbmVzIGRlIGFydGUsIHJlc8O6bWVuZXMsIGNvbmRlbnNhY2lvbmVzLCBvIGN1YWxxdWllciBvdHJhIGVuIGxhIHF1ZSBsYSBvYnJhIHB1ZWRhIHNlciB0cmFuc2Zvcm1hZGEsIGNhbWJpYWRhIG8gYWRhcHRhZGEsIGV4Y2VwdG8gYXF1ZWxsYXMgcXVlIGNvbnN0aXR1eWFuIHVuYSBvYnJhIGNvbGVjdGl2YSwgbGFzIHF1ZSBubyBzZXLDoW4gY29uc2lkZXJhZGFzIHVuYSBvYnJhIGRlcml2YWRhIHBhcmEgZWZlY3RvcyBkZSBlc3RhIGxpY2VuY2lhLiAoUGFyYSBldml0YXIgZHVkYXMsIGVuIGVsIGNhc28gZGUgcXVlIGxhIE9icmEgc2VhIHVuYSBjb21wb3NpY2nDs24gbXVzaWNhbCBvIHVuYSBncmFiYWNpw7NuIHNvbm9yYSwgcGFyYSBsb3MgZWZlY3RvcyBkZSBlc3RhIExpY2VuY2lhIGxhIHNpbmNyb25pemFjacOzbiB0ZW1wb3JhbCBkZSBsYSBPYnJhIGNvbiB1bmEgaW1hZ2VuIGVuIG1vdmltaWVudG8gc2UgY29uc2lkZXJhcsOhIHVuYSBPYnJhIERlcml2YWRhIHBhcmEgbG9zIGZpbmVzIGRlIGVzdGEgbGljZW5jaWEpLgoKYy4JTGljZW5jaWFudGUsIGVzIGVsIGluZGl2aWR1byBvIGxhIGVudGlkYWQgdGl0dWxhciBkZSBsb3MgZGVyZWNob3MgZGUgYXV0b3IgcXVlIG9mcmVjZSBsYSBPYnJhIGVuIGNvbmZvcm1pZGFkIGNvbiBsYXMgY29uZGljaW9uZXMgZGUgZXN0YSBMaWNlbmNpYS4KCmQuCUF1dG9yIG9yaWdpbmFsLCBlcyBlbCBpbmRpdmlkdW8gcXVlIGNyZcOzIGxhIE9icmEuCgplLglPYnJhLCBlcyBhcXVlbGxhIG9icmEgc3VzY2VwdGlibGUgZGUgcHJvdGVjY2nDs24gcG9yIGVsIHLDqWdpbWVuIGRlIERlcmVjaG8gZGUgQXV0b3IgeSBxdWUgZXMgb2ZyZWNpZGEgZW4gbG9zIHTDqXJtaW5vcyBkZSBlc3RhIGxpY2VuY2lhCgpmLglVc3RlZCwgZXMgZWwgaW5kaXZpZHVvIG8gbGEgZW50aWRhZCBxdWUgZWplcmNpdGEgbG9zIGRlcmVjaG9zIG90b3JnYWRvcyBhbCBhbXBhcm8gZGUgZXN0YSBMaWNlbmNpYSB5IHF1ZSBjb24gYW50ZXJpb3JpZGFkIG5vIGhhIHZpb2xhZG8gbGFzIGNvbmRpY2lvbmVzIGRlIGxhIG1pc21hIHJlc3BlY3RvIGEgbGEgT2JyYSwgbyBxdWUgaGF5YSBvYnRlbmlkbyBhdXRvcml6YWNpw7NuIGV4cHJlc2EgcG9yIHBhcnRlIGRlbCBMaWNlbmNpYW50ZSBwYXJhIGVqZXJjZXIgbG9zIGRlcmVjaG9zIGFsIGFtcGFybyBkZSBlc3RhIExpY2VuY2lhIHBlc2UgYSB1bmEgdmlvbGFjacOzbiBhbnRlcmlvci4KCjIuIERlcmVjaG9zIGRlIFVzb3MgSG9ucmFkb3MgeSBleGNlcGNpb25lcyBMZWdhbGVzLgpOYWRhIGVuIGVzdGEgTGljZW5jaWEgcG9kcsOhIHNlciBpbnRlcnByZXRhZG8gY29tbyB1bmEgZGlzbWludWNpw7NuLCBsaW1pdGFjacOzbiBvIHJlc3RyaWNjacOzbiBkZSBsb3MgZGVyZWNob3MgZGVyaXZhZG9zIGRlbCB1c28gaG9ucmFkbyB5IG90cmFzIGxpbWl0YWNpb25lcyBvIGV4Y2VwY2lvbmVzIGEgbG9zIGRlcmVjaG9zIGRlbCBhdXRvciBiYWpvIGVsIHLDqWdpbWVuIGxlZ2FsIHZpZ2VudGUgbyBkZXJpdmFkbyBkZSBjdWFscXVpZXIgb3RyYSBub3JtYSBxdWUgc2UgbGUgYXBsaXF1ZS4KCjMuIENvbmNlc2nDs24gZGUgbGEgTGljZW5jaWEuCkJham8gbG9zIHTDqXJtaW5vcyB5IGNvbmRpY2lvbmVzIGRlIGVzdGEgTGljZW5jaWEsIGVsIExpY2VuY2lhbnRlIG90b3JnYSBhIFVzdGVkIHVuYSBsaWNlbmNpYSBtdW5kaWFsLCBsaWJyZSBkZSByZWdhbMOtYXMsIG5vIGV4Y2x1c2l2YSB5IHBlcnBldHVhIChkdXJhbnRlIHRvZG8gZWwgcGVyw61vZG8gZGUgdmlnZW5jaWEgZGUgbG9zIGRlcmVjaG9zIGRlIGF1dG9yKSBwYXJhIGVqZXJjZXIgZXN0b3MgZGVyZWNob3Mgc29icmUgbGEgT2JyYSB0YWwgeSBjb21vIHNlIGluZGljYSBhIGNvbnRpbnVhY2nDs246CgphLglSZXByb2R1Y2lyIGxhIE9icmEsIGluY29ycG9yYXIgbGEgT2JyYSBlbiB1bmEgbyBtw6FzIE9icmFzIENvbGVjdGl2YXMsIHkgcmVwcm9kdWNpciBsYSBPYnJhIGluY29ycG9yYWRhIGVuIGxhcyBPYnJhcyBDb2xlY3RpdmFzLgoKYi4JRGlzdHJpYnVpciBjb3BpYXMgbyBmb25vZ3JhbWFzIGRlIGxhcyBPYnJhcywgZXhoaWJpcmxhcyBww7pibGljYW1lbnRlLCBlamVjdXRhcmxhcyBww7pibGljYW1lbnRlIHkvbyBwb25lcmxhcyBhIGRpc3Bvc2ljacOzbiBww7pibGljYSwgaW5jbHV5w6luZG9sYXMgY29tbyBpbmNvcnBvcmFkYXMgZW4gT2JyYXMgQ29sZWN0aXZhcywgc2Vnw7puIGNvcnJlc3BvbmRhLgoKYy4JRGlzdHJpYnVpciBjb3BpYXMgZGUgbGFzIE9icmFzIERlcml2YWRhcyBxdWUgc2UgZ2VuZXJlbiwgZXhoaWJpcmxhcyBww7pibGljYW1lbnRlLCBlamVjdXRhcmxhcyBww7pibGljYW1lbnRlIHkvbyBwb25lcmxhcyBhIGRpc3Bvc2ljacOzbiBww7pibGljYS4KTG9zIGRlcmVjaG9zIG1lbmNpb25hZG9zIGFudGVyaW9ybWVudGUgcHVlZGVuIHNlciBlamVyY2lkb3MgZW4gdG9kb3MgbG9zIG1lZGlvcyB5IGZvcm1hdG9zLCBhY3R1YWxtZW50ZSBjb25vY2lkb3MgbyBxdWUgc2UgaW52ZW50ZW4gZW4gZWwgZnV0dXJvLiBMb3MgZGVyZWNob3MgYW50ZXMgbWVuY2lvbmFkb3MgaW5jbHV5ZW4gZWwgZGVyZWNobyBhIHJlYWxpemFyIGRpY2hhcyBtb2RpZmljYWNpb25lcyBlbiBsYSBtZWRpZGEgcXVlIHNlYW4gdMOpY25pY2FtZW50ZSBuZWNlc2FyaWFzIHBhcmEgZWplcmNlciBsb3MgZGVyZWNob3MgZW4gb3RybyBtZWRpbyBvIGZvcm1hdG9zLCBwZXJvIGRlIG90cmEgbWFuZXJhIHVzdGVkIG5vIGVzdMOhIGF1dG9yaXphZG8gcGFyYSByZWFsaXphciBvYnJhcyBkZXJpdmFkYXMuIFRvZG9zIGxvcyBkZXJlY2hvcyBubyBvdG9yZ2Fkb3MgZXhwcmVzYW1lbnRlIHBvciBlbCBMaWNlbmNpYW50ZSBxdWVkYW4gcG9yIGVzdGUgbWVkaW8gcmVzZXJ2YWRvcywgaW5jbHV5ZW5kbyBwZXJvIHNpbiBsaW1pdGFyc2UgYSBhcXVlbGxvcyBxdWUgc2UgbWVuY2lvbmFuIGVuIGxhcyBzZWNjaW9uZXMgNChkKSB5IDQoZSkuCgo0LiBSZXN0cmljY2lvbmVzLgpMYSBsaWNlbmNpYSBvdG9yZ2FkYSBlbiBsYSBhbnRlcmlvciBTZWNjacOzbiAzIGVzdMOhIGV4cHJlc2FtZW50ZSBzdWpldGEgeSBsaW1pdGFkYSBwb3IgbGFzIHNpZ3VpZW50ZXMgcmVzdHJpY2Npb25lczoKCmEuCVVzdGVkIHB1ZWRlIGRpc3RyaWJ1aXIsIGV4aGliaXIgcMO6YmxpY2FtZW50ZSwgZWplY3V0YXIgcMO6YmxpY2FtZW50ZSwgbyBwb25lciBhIGRpc3Bvc2ljacOzbiBww7pibGljYSBsYSBPYnJhIHPDs2xvIGJham8gbGFzIGNvbmRpY2lvbmVzIGRlIGVzdGEgTGljZW5jaWEsIHkgVXN0ZWQgZGViZSBpbmNsdWlyIHVuYSBjb3BpYSBkZSBlc3RhIGxpY2VuY2lhIG8gZGVsIElkZW50aWZpY2Fkb3IgVW5pdmVyc2FsIGRlIFJlY3Vyc29zIGRlIGxhIG1pc21hIGNvbiBjYWRhIGNvcGlhIGRlIGxhIE9icmEgcXVlIGRpc3RyaWJ1eWEsIGV4aGliYSBww7pibGljYW1lbnRlLCBlamVjdXRlIHDDumJsaWNhbWVudGUgbyBwb25nYSBhIGRpc3Bvc2ljacOzbiBww7pibGljYS4gTm8gZXMgcG9zaWJsZSBvZnJlY2VyIG8gaW1wb25lciBuaW5ndW5hIGNvbmRpY2nDs24gc29icmUgbGEgT2JyYSBxdWUgYWx0ZXJlIG8gbGltaXRlIGxhcyBjb25kaWNpb25lcyBkZSBlc3RhIExpY2VuY2lhIG8gZWwgZWplcmNpY2lvIGRlIGxvcyBkZXJlY2hvcyBkZSBsb3MgZGVzdGluYXRhcmlvcyBvdG9yZ2Fkb3MgZW4gZXN0ZSBkb2N1bWVudG8uIE5vIGVzIHBvc2libGUgc3VibGljZW5jaWFyIGxhIE9icmEuIFVzdGVkIGRlYmUgbWFudGVuZXIgaW50YWN0b3MgdG9kb3MgbG9zIGF2aXNvcyBxdWUgaGFnYW4gcmVmZXJlbmNpYSBhIGVzdGEgTGljZW5jaWEgeSBhIGxhIGNsw6F1c3VsYSBkZSBsaW1pdGFjacOzbiBkZSBnYXJhbnTDrWFzLiBVc3RlZCBubyBwdWVkZSBkaXN0cmlidWlyLCBleGhpYmlyIHDDumJsaWNhbWVudGUsIGVqZWN1dGFyIHDDumJsaWNhbWVudGUsIG8gcG9uZXIgYSBkaXNwb3NpY2nDs24gcMO6YmxpY2EgbGEgT2JyYSBjb24gYWxndW5hIG1lZGlkYSB0ZWNub2zDs2dpY2EgcXVlIGNvbnRyb2xlIGVsIGFjY2VzbyBvIGxhIHV0aWxpemFjacOzbiBkZSBlbGxhIGRlIHVuYSBmb3JtYSBxdWUgc2VhIGluY29uc2lzdGVudGUgY29uIGxhcyBjb25kaWNpb25lcyBkZSBlc3RhIExpY2VuY2lhLiBMbyBhbnRlcmlvciBzZSBhcGxpY2EgYSBsYSBPYnJhIGluY29ycG9yYWRhIGEgdW5hIE9icmEgQ29sZWN0aXZhLCBwZXJvIGVzdG8gbm8gZXhpZ2UgcXVlIGxhIE9icmEgQ29sZWN0aXZhIGFwYXJ0ZSBkZSBsYSBvYnJhIG1pc21hIHF1ZWRlIHN1amV0YSBhIGxhcyBjb25kaWNpb25lcyBkZSBlc3RhIExpY2VuY2lhLiBTaSBVc3RlZCBjcmVhIHVuYSBPYnJhIENvbGVjdGl2YSwgcHJldmlvIGF2aXNvIGRlIGN1YWxxdWllciBMaWNlbmNpYW50ZSBkZWJlLCBlbiBsYSBtZWRpZGEgZGUgbG8gcG9zaWJsZSwgZWxpbWluYXIgZGUgbGEgT2JyYSBDb2xlY3RpdmEgY3VhbHF1aWVyIHJlZmVyZW5jaWEgYSBkaWNobyBMaWNlbmNpYW50ZSBvIGFsIEF1dG9yIE9yaWdpbmFsLCBzZWfDum4gbG8gc29saWNpdGFkbyBwb3IgZWwgTGljZW5jaWFudGUgeSBjb25mb3JtZSBsbyBleGlnZSBsYSBjbMOhdXN1bGEgNChjKS4KCmIuCVVzdGVkIG5vIHB1ZWRlIGVqZXJjZXIgbmluZ3VubyBkZSBsb3MgZGVyZWNob3MgcXVlIGxlIGhhbiBzaWRvIG90b3JnYWRvcyBlbiBsYSBTZWNjacOzbiAzIHByZWNlZGVudGUgZGUgbW9kbyBxdWUgZXN0w6luIHByaW5jaXBhbG1lbnRlIGRlc3RpbmFkb3MgbyBkaXJlY3RhbWVudGUgZGlyaWdpZG9zIGEgY29uc2VndWlyIHVuIHByb3ZlY2hvIGNvbWVyY2lhbCBvIHVuYSBjb21wZW5zYWNpw7NuIG1vbmV0YXJpYSBwcml2YWRhLiBFbCBpbnRlcmNhbWJpbyBkZSBsYSBPYnJhIHBvciBvdHJhcyBvYnJhcyBwcm90ZWdpZGFzIHBvciBkZXJlY2hvcyBkZSBhdXRvciwgeWEgc2VhIGEgdHJhdsOpcyBkZSB1biBzaXN0ZW1hIHBhcmEgY29tcGFydGlyIGFyY2hpdm9zIGRpZ2l0YWxlcyAoZGlnaXRhbCBmaWxlLXNoYXJpbmcpIG8gZGUgY3VhbHF1aWVyIG90cmEgbWFuZXJhIG5vIHNlcsOhIGNvbnNpZGVyYWRvIGNvbW8gZXN0YXIgZGVzdGluYWRvIHByaW5jaXBhbG1lbnRlIG8gZGlyaWdpZG8gZGlyZWN0YW1lbnRlIGEgY29uc2VndWlyIHVuIHByb3ZlY2hvIGNvbWVyY2lhbCBvIHVuYSBjb21wZW5zYWNpw7NuIG1vbmV0YXJpYSBwcml2YWRhLCBzaWVtcHJlIHF1ZSBubyBzZSByZWFsaWNlIHVuIHBhZ28gbWVkaWFudGUgdW5hIGNvbXBlbnNhY2nDs24gbW9uZXRhcmlhIGVuIHJlbGFjacOzbiBjb24gZWwgaW50ZXJjYW1iaW8gZGUgb2JyYXMgcHJvdGVnaWRhcyBwb3IgZWwgZGVyZWNobyBkZSBhdXRvci4KCmMuCVNpIHVzdGVkIGRpc3RyaWJ1eWUsIGV4aGliZSBww7pibGljYW1lbnRlLCBlamVjdXRhIHDDumJsaWNhbWVudGUgbyBlamVjdXRhIHDDumJsaWNhbWVudGUgZW4gZm9ybWEgZGlnaXRhbCBsYSBPYnJhIG8gY3VhbHF1aWVyIE9icmEgRGVyaXZhZGEgdSBPYnJhIENvbGVjdGl2YSwgVXN0ZWQgZGViZSBtYW50ZW5lciBpbnRhY3RhIHRvZGEgbGEgaW5mb3JtYWNpw7NuIGRlIGRlcmVjaG8gZGUgYXV0b3IgZGUgbGEgT2JyYSB5IHByb3BvcmNpb25hciwgZGUgZm9ybWEgcmF6b25hYmxlIHNlZ8O6biBlbCBtZWRpbyBvIG1hbmVyYSBxdWUgVXN0ZWQgZXN0w6kgdXRpbGl6YW5kbzogKGkpIGVsIG5vbWJyZSBkZWwgQXV0b3IgT3JpZ2luYWwgc2kgZXN0w6EgcHJvdmlzdG8gKG8gc2V1ZMOzbmltbywgc2kgZnVlcmUgYXBsaWNhYmxlKSwgeS9vIChpaSkgZWwgbm9tYnJlIGRlIGxhIHBhcnRlIG8gbGFzIHBhcnRlcyBxdWUgZWwgQXV0b3IgT3JpZ2luYWwgeS9vIGVsIExpY2VuY2lhbnRlIGh1YmllcmVuIGRlc2lnbmFkbyBwYXJhIGxhIGF0cmlidWNpw7NuICh2LmcuLCB1biBpbnN0aXR1dG8gcGF0cm9jaW5hZG9yLCBlZGl0b3JpYWwsIHB1YmxpY2FjacOzbikgZW4gbGEgaW5mb3JtYWNpw7NuIGRlIGxvcyBkZXJlY2hvcyBkZSBhdXRvciBkZWwgTGljZW5jaWFudGUsIHTDqXJtaW5vcyBkZSBzZXJ2aWNpb3MgbyBkZSBvdHJhcyBmb3JtYXMgcmF6b25hYmxlczsgZWwgdMOtdHVsbyBkZSBsYSBPYnJhIHNpIGVzdMOhIHByb3Zpc3RvOyBlbiBsYSBtZWRpZGEgZGUgbG8gcmF6b25hYmxlbWVudGUgZmFjdGlibGUgeSwgc2kgZXN0w6EgcHJvdmlzdG8sIGVsIElkZW50aWZpY2Fkb3IgVW5pZm9ybWUgZGUgUmVjdXJzb3MgKFVuaWZvcm0gUmVzb3VyY2UgSWRlbnRpZmllcikgcXVlIGVsIExpY2VuY2lhbnRlIGVzcGVjaWZpY2EgcGFyYSBzZXIgYXNvY2lhZG8gY29uIGxhIE9icmEsIHNhbHZvIHF1ZSB0YWwgVVJJIG5vIHNlIHJlZmllcmEgYSBsYSBub3RhIHNvYnJlIGxvcyBkZXJlY2hvcyBkZSBhdXRvciBvIGEgbGEgaW5mb3JtYWNpw7NuIHNvYnJlIGVsIGxpY2VuY2lhbWllbnRvIGRlIGxhIE9icmE7IHkgZW4gZWwgY2FzbyBkZSB1bmEgT2JyYSBEZXJpdmFkYSwgYXRyaWJ1aXIgZWwgY3LDqWRpdG8gaWRlbnRpZmljYW5kbyBlbCB1c28gZGUgbGEgT2JyYSBlbiBsYSBPYnJhIERlcml2YWRhICh2LmcuLCAiVHJhZHVjY2nDs24gRnJhbmNlc2EgZGUgbGEgT2JyYSBkZWwgQXV0b3IgT3JpZ2luYWwsIiBvICJHdWnDs24gQ2luZW1hdG9ncsOhZmljbyBiYXNhZG8gZW4gbGEgT2JyYSBvcmlnaW5hbCBkZWwgQXV0b3IgT3JpZ2luYWwiKS4gVGFsIGNyw6lkaXRvIHB1ZWRlIHNlciBpbXBsZW1lbnRhZG8gZGUgY3VhbHF1aWVyIGZvcm1hIHJhem9uYWJsZTsgZW4gZWwgY2Fzbywgc2luIGVtYmFyZ28sIGRlIE9icmFzIERlcml2YWRhcyB1IE9icmFzIENvbGVjdGl2YXMsIHRhbCBjcsOpZGl0byBhcGFyZWNlcsOhLCBjb21vIG3DrW5pbW8sIGRvbmRlIGFwYXJlY2UgZWwgY3LDqWRpdG8gZGUgY3VhbHF1aWVyIG90cm8gYXV0b3IgY29tcGFyYWJsZSB5IGRlIHVuYSBtYW5lcmEsIGFsIG1lbm9zLCB0YW4gZGVzdGFjYWRhIGNvbW8gZWwgY3LDqWRpdG8gZGUgb3RybyBhdXRvciBjb21wYXJhYmxlLgoKZC4JUGFyYSBldml0YXIgdG9kYSBjb25mdXNpw7NuLCBlbCBMaWNlbmNpYW50ZSBhY2xhcmEgcXVlLCBjdWFuZG8gbGEgb2JyYSBlcyB1bmEgY29tcG9zaWNpw7NuIG11c2ljYWw6CgppLglSZWdhbMOtYXMgcG9yIGludGVycHJldGFjacOzbiB5IGVqZWN1Y2nDs24gYmFqbyBsaWNlbmNpYXMgZ2VuZXJhbGVzLiBFbCBMaWNlbmNpYW50ZSBzZSByZXNlcnZhIGVsIGRlcmVjaG8gZXhjbHVzaXZvIGRlIGF1dG9yaXphciBsYSBlamVjdWNpw7NuIHDDumJsaWNhIG8gbGEgZWplY3VjacOzbiBww7pibGljYSBkaWdpdGFsIGRlIGxhIG9icmEgeSBkZSByZWNvbGVjdGFyLCBzZWEgaW5kaXZpZHVhbG1lbnRlIG8gYSB0cmF2w6lzIGRlIHVuYSBzb2NpZWRhZCBkZSBnZXN0acOzbiBjb2xlY3RpdmEgZGUgZGVyZWNob3MgZGUgYXV0b3IgeSBkZXJlY2hvcyBjb25leG9zIChwb3IgZWplbXBsbywgU0FZQ08pLCBsYXMgcmVnYWzDrWFzIHBvciBsYSBlamVjdWNpw7NuIHDDumJsaWNhIG8gcG9yIGxhIGVqZWN1Y2nDs24gcMO6YmxpY2EgZGlnaXRhbCBkZSBsYSBvYnJhIChwb3IgZWplbXBsbyBXZWJjYXN0KSBsaWNlbmNpYWRhIGJham8gbGljZW5jaWFzIGdlbmVyYWxlcywgc2kgbGEgaW50ZXJwcmV0YWNpw7NuIG8gZWplY3VjacOzbiBkZSBsYSBvYnJhIGVzdMOhIHByaW1vcmRpYWxtZW50ZSBvcmllbnRhZGEgcG9yIG8gZGlyaWdpZGEgYSBsYSBvYnRlbmNpw7NuIGRlIHVuYSB2ZW50YWphIGNvbWVyY2lhbCBvIHVuYSBjb21wZW5zYWNpw7NuIG1vbmV0YXJpYSBwcml2YWRhLgoKaWkuCVJlZ2Fsw61hcyBwb3IgRm9ub2dyYW1hcy4gRWwgTGljZW5jaWFudGUgc2UgcmVzZXJ2YSBlbCBkZXJlY2hvIGV4Y2x1c2l2byBkZSByZWNvbGVjdGFyLCBpbmRpdmlkdWFsbWVudGUgbyBhIHRyYXbDqXMgZGUgdW5hIHNvY2llZGFkIGRlIGdlc3Rpw7NuIGNvbGVjdGl2YSBkZSBkZXJlY2hvcyBkZSBhdXRvciB5IGRlcmVjaG9zIGNvbmV4b3MgKHBvciBlamVtcGxvLCBsb3MgY29uc2FncmFkb3MgcG9yIGxhIFNBWUNPKSwgdW5hIGFnZW5jaWEgZGUgZGVyZWNob3MgbXVzaWNhbGVzIG8gYWxnw7puIGFnZW50ZSBkZXNpZ25hZG8sIGxhcyByZWdhbMOtYXMgcG9yIGN1YWxxdWllciBmb25vZ3JhbWEgcXVlIFVzdGVkIGNyZWUgYSBwYXJ0aXIgZGUgbGEgb2JyYSAo4oCcdmVyc2nDs24gY292ZXLigJ0pIHkgZGlzdHJpYnV5YSwgZW4gbG9zIHTDqXJtaW5vcyBkZWwgcsOpZ2ltZW4gZGUgZGVyZWNob3MgZGUgYXV0b3IsIHNpIGxhIGNyZWFjacOzbiBvIGRpc3RyaWJ1Y2nDs24gZGUgZXNhIHZlcnNpw7NuIGNvdmVyIGVzdMOhIHByaW1vcmRpYWxtZW50ZSBkZXN0aW5hZGEgbyBkaXJpZ2lkYSBhIG9idGVuZXIgdW5hIHZlbnRhamEgY29tZXJjaWFsIG8gdW5hIGNvbXBlbnNhY2nDs24gbW9uZXRhcmlhIHByaXZhZGEuCgplLglHZXN0acOzbiBkZSBEZXJlY2hvcyBkZSBBdXRvciBzb2JyZSBJbnRlcnByZXRhY2lvbmVzIHkgRWplY3VjaW9uZXMgRGlnaXRhbGVzIChXZWJDYXN0aW5nKS4gUGFyYSBldml0YXIgdG9kYSBjb25mdXNpw7NuLCBlbCBMaWNlbmNpYW50ZSBhY2xhcmEgcXVlLCBjdWFuZG8gbGEgb2JyYSBzZWEgdW4gZm9ub2dyYW1hLCBlbCBMaWNlbmNpYW50ZSBzZSByZXNlcnZhIGVsIGRlcmVjaG8gZXhjbHVzaXZvIGRlIGF1dG9yaXphciBsYSBlamVjdWNpw7NuIHDDumJsaWNhIGRpZ2l0YWwgZGUgbGEgb2JyYSAocG9yIGVqZW1wbG8sIHdlYmNhc3QpIHkgZGUgcmVjb2xlY3RhciwgaW5kaXZpZHVhbG1lbnRlIG8gYSB0cmF2w6lzIGRlIHVuYSBzb2NpZWRhZCBkZSBnZXN0acOzbiBjb2xlY3RpdmEgZGUgZGVyZWNob3MgZGUgYXV0b3IgeSBkZXJlY2hvcyBjb25leG9zIChwb3IgZWplbXBsbywgQUNJTlBSTyksIGxhcyByZWdhbMOtYXMgcG9yIGxhIGVqZWN1Y2nDs24gcMO6YmxpY2EgZGlnaXRhbCBkZSBsYSBvYnJhIChwb3IgZWplbXBsbywgd2ViY2FzdCksIHN1amV0YSBhIGxhcyBkaXNwb3NpY2lvbmVzIGFwbGljYWJsZXMgZGVsIHLDqWdpbWVuIGRlIERlcmVjaG8gZGUgQXV0b3IsIHNpIGVzdGEgZWplY3VjacOzbiBww7pibGljYSBkaWdpdGFsIGVzdMOhIHByaW1vcmRpYWxtZW50ZSBkaXJpZ2lkYSBhIG9idGVuZXIgdW5hIHZlbnRhamEgY29tZXJjaWFsIG8gdW5hIGNvbXBlbnNhY2nDs24gbW9uZXRhcmlhIHByaXZhZGEuCgo1LiBSZXByZXNlbnRhY2lvbmVzLCBHYXJhbnTDrWFzIHkgTGltaXRhY2lvbmVzIGRlIFJlc3BvbnNhYmlsaWRhZC4KQSBNRU5PUyBRVUUgTEFTIFBBUlRFUyBMTyBBQ09SREFSQU4gREUgT1RSQSBGT1JNQSBQT1IgRVNDUklUTywgRUwgTElDRU5DSUFOVEUgT0ZSRUNFIExBIE9CUkEgKEVOIEVMIEVTVEFETyBFTiBFTCBRVUUgU0UgRU5DVUVOVFJBKSDigJxUQUwgQ1VBTOKAnSwgU0lOIEJSSU5EQVIgR0FSQU5Uw41BUyBERSBDTEFTRSBBTEdVTkEgUkVTUEVDVE8gREUgTEEgT0JSQSwgWUEgU0VBIEVYUFJFU0EsIElNUEzDjUNJVEEsIExFR0FMIE8gQ1VBTFFVSUVSQSBPVFJBLCBJTkNMVVlFTkRPLCBTSU4gTElNSVRBUlNFIEEgRUxMQVMsIEdBUkFOVMONQVMgREUgVElUVUxBUklEQUQsIENPTUVSQ0lBQklMSURBRCwgQURBUFRBQklMSURBRCBPIEFERUNVQUNJw5NOIEEgUFJPUMOTU0lUTyBERVRFUk1JTkFETywgQVVTRU5DSUEgREUgSU5GUkFDQ0nDk04sIERFIEFVU0VOQ0lBIERFIERFRkVDVE9TIExBVEVOVEVTIE8gREUgT1RSTyBUSVBPLCBPIExBIFBSRVNFTkNJQSBPIEFVU0VOQ0lBIERFIEVSUk9SRVMsIFNFQU4gTyBOTyBERVNDVUJSSUJMRVMgKFBVRURBTiBPIE5PIFNFUiBFU1RPUyBERVNDVUJJRVJUT1MpLiBBTEdVTkFTIEpVUklTRElDQ0lPTkVTIE5PIFBFUk1JVEVOIExBIEVYQ0xVU0nDk04gREUgR0FSQU5Uw41BUyBJTVBMw41DSVRBUywgRU4gQ1VZTyBDQVNPIEVTVEEgRVhDTFVTScOTTiBQVUVERSBOTyBBUExJQ0FSU0UgQSBVU1RFRC4KCjYuIExpbWl0YWNpw7NuIGRlIHJlc3BvbnNhYmlsaWRhZC4KQSBNRU5PUyBRVUUgTE8gRVhJSkEgRVhQUkVTQU1FTlRFIExBIExFWSBBUExJQ0FCTEUsIEVMIExJQ0VOQ0lBTlRFIE5PIFNFUsOBIFJFU1BPTlNBQkxFIEFOVEUgVVNURUQgUE9SIERBw5FPIEFMR1VOTywgU0VBIFBPUiBSRVNQT05TQUJJTElEQUQgRVhUUkFDT05UUkFDVFVBTCwgUFJFQ09OVFJBQ1RVQUwgTyBDT05UUkFDVFVBTCwgT0JKRVRJVkEgTyBTVUJKRVRJVkEsIFNFIFRSQVRFIERFIERBw5FPUyBNT1JBTEVTIE8gUEFUUklNT05JQUxFUywgRElSRUNUT1MgTyBJTkRJUkVDVE9TLCBQUkVWSVNUT1MgTyBJTVBSRVZJU1RPUyBQUk9EVUNJRE9TIFBPUiBFTCBVU08gREUgRVNUQSBMSUNFTkNJQSBPIERFIExBIE9CUkEsIEFVTiBDVUFORE8gRUwgTElDRU5DSUFOVEUgSEFZQSBTSURPIEFEVkVSVElETyBERSBMQSBQT1NJQklMSURBRCBERSBESUNIT1MgREHDkU9TLiBBTEdVTkFTIExFWUVTIE5PIFBFUk1JVEVOIExBIEVYQ0xVU0nDk04gREUgQ0lFUlRBIFJFU1BPTlNBQklMSURBRCwgRU4gQ1VZTyBDQVNPIEVTVEEgRVhDTFVTScOTTiBQVUVERSBOTyBBUExJQ0FSU0UgQSBVU1RFRC4KCjcuIFTDqXJtaW5vLgoKYS4JRXN0YSBMaWNlbmNpYSB5IGxvcyBkZXJlY2hvcyBvdG9yZ2Fkb3MgZW4gdmlydHVkIGRlIGVsbGEgdGVybWluYXLDoW4gYXV0b23DoXRpY2FtZW50ZSBzaSBVc3RlZCBpbmZyaW5nZSBhbGd1bmEgY29uZGljacOzbiBlc3RhYmxlY2lkYSBlbiBlbGxhLiBTaW4gZW1iYXJnbywgbG9zIGluZGl2aWR1b3MgbyBlbnRpZGFkZXMgcXVlIGhhbiByZWNpYmlkbyBPYnJhcyBEZXJpdmFkYXMgbyBDb2xlY3RpdmFzIGRlIFVzdGVkIGRlIGNvbmZvcm1pZGFkIGNvbiBlc3RhIExpY2VuY2lhLCBubyB2ZXLDoW4gdGVybWluYWRhcyBzdXMgbGljZW5jaWFzLCBzaWVtcHJlIHF1ZSBlc3RvcyBpbmRpdmlkdW9zIG8gZW50aWRhZGVzIHNpZ2FuIGN1bXBsaWVuZG8gw61udGVncmFtZW50ZSBsYXMgY29uZGljaW9uZXMgZGUgZXN0YXMgbGljZW5jaWFzLiBMYXMgU2VjY2lvbmVzIDEsIDIsIDUsIDYsIDcsIHkgOCBzdWJzaXN0aXLDoW4gYSBjdWFscXVpZXIgdGVybWluYWNpw7NuIGRlIGVzdGEgTGljZW5jaWEuCgpiLglTdWpldGEgYSBsYXMgY29uZGljaW9uZXMgeSB0w6lybWlub3MgYW50ZXJpb3JlcywgbGEgbGljZW5jaWEgb3RvcmdhZGEgYXF1w60gZXMgcGVycGV0dWEgKGR1cmFudGUgZWwgcGVyw61vZG8gZGUgdmlnZW5jaWEgZGUgbG9zIGRlcmVjaG9zIGRlIGF1dG9yIGRlIGxhIG9icmEpLiBObyBvYnN0YW50ZSBsbyBhbnRlcmlvciwgZWwgTGljZW5jaWFudGUgc2UgcmVzZXJ2YSBlbCBkZXJlY2hvIGEgcHVibGljYXIgeS9vIGVzdHJlbmFyIGxhIE9icmEgYmFqbyBjb25kaWNpb25lcyBkZSBsaWNlbmNpYSBkaWZlcmVudGVzIG8gYSBkZWphciBkZSBkaXN0cmlidWlybGEgZW4gbG9zIHTDqXJtaW5vcyBkZSBlc3RhIExpY2VuY2lhIGVuIGN1YWxxdWllciBtb21lbnRvOyBlbiBlbCBlbnRlbmRpZG8sIHNpbiBlbWJhcmdvLCBxdWUgZXNhIGVsZWNjacOzbiBubyBzZXJ2aXLDoSBwYXJhIHJldm9jYXIgZXN0YSBsaWNlbmNpYSBvIHF1ZSBkZWJhIHNlciBvdG9yZ2FkYSAsIGJham8gbG9zIHTDqXJtaW5vcyBkZSBlc3RhIGxpY2VuY2lhKSwgeSBlc3RhIGxpY2VuY2lhIGNvbnRpbnVhcsOhIGVuIHBsZW5vIHZpZ29yIHkgZWZlY3RvIGEgbWVub3MgcXVlIHNlYSB0ZXJtaW5hZGEgY29tbyBzZSBleHByZXNhIGF0csOhcy4gTGEgTGljZW5jaWEgcmV2b2NhZGEgY29udGludWFyw6Egc2llbmRvIHBsZW5hbWVudGUgdmlnZW50ZSB5IGVmZWN0aXZhIHNpIG5vIHNlIGxlIGRhIHTDqXJtaW5vIGVuIGxhcyBjb25kaWNpb25lcyBpbmRpY2FkYXMgYW50ZXJpb3JtZW50ZS4KCjguIFZhcmlvcy4KCmEuCUNhZGEgdmV6IHF1ZSBVc3RlZCBkaXN0cmlidXlhIG8gcG9uZ2EgYSBkaXNwb3NpY2nDs24gcMO6YmxpY2EgbGEgT2JyYSBvIHVuYSBPYnJhIENvbGVjdGl2YSwgZWwgTGljZW5jaWFudGUgb2ZyZWNlcsOhIGFsIGRlc3RpbmF0YXJpbyB1bmEgbGljZW5jaWEgZW4gbG9zIG1pc21vcyB0w6lybWlub3MgeSBjb25kaWNpb25lcyBxdWUgbGEgbGljZW5jaWEgb3RvcmdhZGEgYSBVc3RlZCBiYWpvIGVzdGEgTGljZW5jaWEuCgpiLglTaSBhbGd1bmEgZGlzcG9zaWNpw7NuIGRlIGVzdGEgTGljZW5jaWEgcmVzdWx0YSBpbnZhbGlkYWRhIG8gbm8gZXhpZ2libGUsIHNlZ8O6biBsYSBsZWdpc2xhY2nDs24gdmlnZW50ZSwgZXN0byBubyBhZmVjdGFyw6EgbmkgbGEgdmFsaWRleiBuaSBsYSBhcGxpY2FiaWxpZGFkIGRlbCByZXN0byBkZSBjb25kaWNpb25lcyBkZSBlc3RhIExpY2VuY2lhIHksIHNpbiBhY2Npw7NuIGFkaWNpb25hbCBwb3IgcGFydGUgZGUgbG9zIHN1amV0b3MgZGUgZXN0ZSBhY3VlcmRvLCBhcXXDqWxsYSBzZSBlbnRlbmRlcsOhIHJlZm9ybWFkYSBsbyBtw61uaW1vIG5lY2VzYXJpbyBwYXJhIGhhY2VyIHF1ZSBkaWNoYSBkaXNwb3NpY2nDs24gc2VhIHbDoWxpZGEgeSBleGlnaWJsZS4KCmMuCU5pbmfDum4gdMOpcm1pbm8gbyBkaXNwb3NpY2nDs24gZGUgZXN0YSBMaWNlbmNpYSBzZSBlc3RpbWFyw6EgcmVudW5jaWFkYSB5IG5pbmd1bmEgdmlvbGFjacOzbiBkZSBlbGxhIHNlcsOhIGNvbnNlbnRpZGEgYSBtZW5vcyBxdWUgZXNhIHJlbnVuY2lhIG8gY29uc2VudGltaWVudG8gc2VhIG90b3JnYWRvIHBvciBlc2NyaXRvIHkgZmlybWFkbyBwb3IgbGEgcGFydGUgcXVlIHJlbnVuY2llIG8gY29uc2llbnRhLgoKZC4JRXN0YSBMaWNlbmNpYSByZWZsZWphIGVsIGFjdWVyZG8gcGxlbm8gZW50cmUgbGFzIHBhcnRlcyByZXNwZWN0byBhIGxhIE9icmEgYXF1w60gbGljZW5jaWFkYS4gTm8gaGF5IGFycmVnbG9zLCBhY3VlcmRvcyBvIGRlY2xhcmFjaW9uZXMgcmVzcGVjdG8gYSBsYSBPYnJhIHF1ZSBubyBlc3TDqW4gZXNwZWNpZmljYWRvcyBlbiBlc3RlIGRvY3VtZW50by4gRWwgTGljZW5jaWFudGUgbm8gc2UgdmVyw6EgbGltaXRhZG8gcG9yIG5pbmd1bmEgZGlzcG9zaWNpw7NuIGFkaWNpb25hbCBxdWUgcHVlZGEgc3VyZ2lyIGVuIGFsZ3VuYSBjb211bmljYWNpw7NuIGVtYW5hZGEgZGUgVXN0ZWQuIEVzdGEgTGljZW5jaWEgbm8gcHVlZGUgc2VyIG1vZGlmaWNhZGEgc2luIGVsIGNvbnNlbnRpbWllbnRvIG11dHVvIHBvciBlc2NyaXRvIGRlbCBMaWNlbmNpYW50ZSB5IFVzdGVkLgo= |