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Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9

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 pre...

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Autores principales: De Mori, Gloria, Zaina, Giusi, Franco-Orozco, Barbara, Testolin, Raffaele, De Paoli, Emanuele, Cipriani, Guido
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823651/
https://www.ncbi.nlm.nih.gov/pubmed/33396671
http://dx.doi.org/10.3390/plants10010062
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author De Mori, Gloria
Zaina, Giusi
Franco-Orozco, Barbara
Testolin, Raffaele
De Paoli, Emanuele
Cipriani, Guido
author_facet De Mori, Gloria
Zaina, Giusi
Franco-Orozco, Barbara
Testolin, Raffaele
De Paoli, Emanuele
Cipriani, Guido
author_sort De Mori, Gloria
collection PubMed
description 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.
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spelling pubmed-78236512021-01-24 Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9 De Mori, Gloria Zaina, Giusi Franco-Orozco, Barbara Testolin, Raffaele De Paoli, Emanuele Cipriani, Guido Plants (Basel) Article 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. MDPI 2020-12-30 /pmc/articles/PMC7823651/ /pubmed/33396671 http://dx.doi.org/10.3390/plants10010062 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
De Mori, Gloria
Zaina, Giusi
Franco-Orozco, Barbara
Testolin, Raffaele
De Paoli, Emanuele
Cipriani, Guido
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
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_short 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823651/
https://www.ncbi.nlm.nih.gov/pubmed/33396671
http://dx.doi.org/10.3390/plants10010062
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