Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing
The identification of full knockout plants is frequently the rate-limiting step of the research and commercial projects searching the complete loss of the function of target genes via the CRISPR/Cas9 system. This problem is particularly acute in polyploid crops such as alfalfa, where the common scre...
| Main Authors: | , , , , , , , , , |
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| Format: | Artículo |
| Language: | Inglés |
| Published: |
Springer
2022
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| Online Access: | http://hdl.handle.net/20.500.12123/12515 https://link.springer.com/article/10.1007/s11240-022-02358-6 https://doi.org/10.1007/s11240-022-02358-6 |
| _version_ | 1855484988983607296 |
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| author | Stritzler, Margarita Pascuan, Cecilia Gabriela Bottero, Ana Emilia Gomez, Maria Cristina Frare, Romina Alejandra Puebla, Andrea Fabiana Tajima, Hiromi Ayub, Nicolás Daniel Blumwald, Eduardo Soto, Gabriela Cynthia |
| author_browse | Ayub, Nicolás Daniel Blumwald, Eduardo Bottero, Ana Emilia Frare, Romina Alejandra Gomez, Maria Cristina Pascuan, Cecilia Gabriela Puebla, Andrea Fabiana Soto, Gabriela Cynthia Stritzler, Margarita Tajima, Hiromi |
| author_facet | Stritzler, Margarita Pascuan, Cecilia Gabriela Bottero, Ana Emilia Gomez, Maria Cristina Frare, Romina Alejandra Puebla, Andrea Fabiana Tajima, Hiromi Ayub, Nicolás Daniel Blumwald, Eduardo Soto, Gabriela Cynthia |
| author_sort | Stritzler, Margarita |
| collection | INTA Digital |
| description | The identification of full knockout plants is frequently the rate-limiting step of the research and commercial projects searching the complete loss of the function of target genes via the CRISPR/Cas9 system. This problem is particularly acute in polyploid crops such as alfalfa, where the common screening needs cloning and Sanger sequencing. Here, we produced and analyzed fifteen edited events containing different doses of edited alleles for three unrelated target genes. This analysis showed that next-generation sequencing (NGS) technology largely increases the efficiency of the determination of the exact genotype of an individual plant and that this increased performance is related to the high output of sequences produced by this alternative genotyping approach rather than to a higher balanced amplification of the alleles. Finally, we discuss the possible applications of NGS technology in other polyploid crops with complex genetics. |
| format | Artículo |
| id | INTA12515 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2022 |
| publishDateRange | 2022 |
| publishDateSort | 2022 |
| publisher | Springer |
| publisherStr | Springer |
| record_format | dspace |
| spelling | INTA125152022-08-08T10:28:26Z Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing Stritzler, Margarita Pascuan, Cecilia Gabriela Bottero, Ana Emilia Gomez, Maria Cristina Frare, Romina Alejandra Puebla, Andrea Fabiana Tajima, Hiromi Ayub, Nicolás Daniel Blumwald, Eduardo Soto, Gabriela Cynthia Medicago sativa Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Alelos Secuenciación de Alto Rendimiento Genotipado Edición de Genes CRISPR Alleles High-throughput Sequencing Genotyping Gene Editing Alfalfa Lucerne The identification of full knockout plants is frequently the rate-limiting step of the research and commercial projects searching the complete loss of the function of target genes via the CRISPR/Cas9 system. This problem is particularly acute in polyploid crops such as alfalfa, where the common screening needs cloning and Sanger sequencing. Here, we produced and analyzed fifteen edited events containing different doses of edited alleles for three unrelated target genes. This analysis showed that next-generation sequencing (NGS) technology largely increases the efficiency of the determination of the exact genotype of an individual plant and that this increased performance is related to the high output of sequences produced by this alternative genotyping approach rather than to a higher balanced amplification of the alleles. Finally, we discuss the possible applications of NGS technology in other polyploid crops with complex genetics. Instituto de Biotecnología Fil: Stritzler, Margarita. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Stritzler, Margarita. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética Ewald A. Favret; Argentina Fil: Stritzler, Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pascuan, Cecilia Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Pascuan, Cecilia Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética Ewald A. Favret; Argentina Fil: Pascuan, Cecilia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Bottero, Ana Emilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Bottero, Ana Emilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética Ewald A. Favret; Argentina Fil: Bottero, Ana Emilia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gomez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Gomez, Maria Cristina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética Ewald A. Favret; Argentina Fil: Gomez, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Frare, Romina Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Frare, Romina Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética Ewald A. Favret; Argentina Fil: Frare, Romina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Puebla, Andrea Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Puebla, Andrea Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Tajima, Hiromi. University of California. Department of Plant Sciences; Estados Unidos Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Ayub, Nicolás Daniel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética Ewald A. Favret; Argentina Fil: Ayub, Nicolás Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Blumwald, Eduardo. University of California. Department of Plant Sciences; Estados Unidos Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Soto, Gabriela Cynthia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Genética Ewald A. Favret; Argentina Fil: Soto, Gabriela Cynthia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina 2022-08-08T10:21:56Z 2022-08-08T10:21:56Z 2022-07 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://hdl.handle.net/20.500.12123/12515 https://link.springer.com/article/10.1007/s11240-022-02358-6 1573-5044 https://doi.org/10.1007/s11240-022-02358-6 eng info:eu-repograntAgreement/INTA/2019-PE-E6-I115-001/2019-PE-E6-I115-001/AR./Edición génica, transgénesis y mutagénesis como generadores de nueva variabilidad en especies de interés agropecuario info:eu-repograntAgreement/INTA/2019-PD-E6-I116-001/2019-PD-E6-I116-001/AR./Identificación y análisis funcional de genes o redes génicas de interés biotecnológico con fin agropecuario, forestal, agroalimentario y/o agroindustrial. info:eu-repo/semantics/restrictedAccess application/pdf Springer Plant Cell, Tissue and Organ Culture (Published: 27 July 2022) |
| spellingShingle | Medicago sativa Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Alelos Secuenciación de Alto Rendimiento Genotipado Edición de Genes CRISPR Alleles High-throughput Sequencing Genotyping Gene Editing Alfalfa Lucerne Stritzler, Margarita Pascuan, Cecilia Gabriela Bottero, Ana Emilia Gomez, Maria Cristina Frare, Romina Alejandra Puebla, Andrea Fabiana Tajima, Hiromi Ayub, Nicolás Daniel Blumwald, Eduardo Soto, Gabriela Cynthia Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing |
| title | Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing |
| title_full | Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing |
| title_fullStr | Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing |
| title_full_unstemmed | Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing |
| title_short | Rapid and cloning‑free screening of edited alfalfa via next‑generation sequencing |
| title_sort | rapid and cloning free screening of edited alfalfa via next generation sequencing |
| topic | Medicago sativa Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Interespaciadas Alelos Secuenciación de Alto Rendimiento Genotipado Edición de Genes CRISPR Alleles High-throughput Sequencing Genotyping Gene Editing Alfalfa Lucerne |
| url | http://hdl.handle.net/20.500.12123/12515 https://link.springer.com/article/10.1007/s11240-022-02358-6 https://doi.org/10.1007/s11240-022-02358-6 |
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