- 著者
- Hiroaki Kusano Ami Takeuchi Hiroaki Shimada
- 出版者
- Japanese Society for Plant Biotechnology
- 雑誌
- Plant Biotechnology (ISSN:13424580)
- 巻号頁・発行日
- pp.23.0611a, (Released:2023-09-07)
- 参考文献数
- 29
- 被引用文献数
- 3
Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.
- 著者
- Ami Takeuchi Yuna Akatsu Takahiro Asahi Yukino Okubo Mariko Ohnuma Hiroshi Teramura Koji Tamura Hiroaki Shimada
- 出版者
- Japanese Society for Plant Biotechnology
- 雑誌
- Plant Biotechnology (ISSN:13424580)
- 巻号頁・発行日
- vol.39, no.2, pp.195-197, 2022-06-25 (Released:2022-06-25)
- 参考文献数
- 6
- 被引用文献数
- 3
Potato, Solanum tuberosum L. is an important crop. However, it is difficult to breed potato cultivars by applying conventional crossing methods because potato has a tetraploid genome and is vegetatively propagated. Flower formation and tuber development occur simultaneously. Many potato cultivars hardly produce any fruits after crossing and fail to produce seeds. We report an improved procedure for obtaining progeny seeds by grafting potatoes onto tomatoes. The rate of fruit formation was more than 19% when the grafted potatoes were used for the crossing experiments, whereas crossing using the ungrafted plants showed a rate of 1.1%. This result suggests that our procedure results in the easy acquisition of null-segregant progenies by crossing mutant lines. It is also expected to improve conventional potato breeding.
- 著者
- Mariko Ohnuma Kosuke Ito Karin Hamada Ami Takeuchi Kenji Asano Takahiro Noda Akira Watanabe Akiko Hokura Hiroshi Teramura Fuminori Takahashi Hiromi Mutsuro-Aoki Koji Tamura Hiroaki Shimada
- 出版者
- Japanese Society for Plant Biotechnology
- 雑誌
- Plant Biotechnology (ISSN:13424580)
- 巻号頁・発行日
- vol.40, no.3, pp.219-227, 2023-09-25 (Released:2023-09-25)
- 参考文献数
- 34
- 被引用文献数
- 2
Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze–thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.
- 著者
- Hiroaki Kusano Ami Takeuchi Hiroaki Shimada
- 出版者
- Japanese Society for Plant Biotechnology
- 雑誌
- Plant Biotechnology (ISSN:13424580)
- 巻号頁・発行日
- vol.40, no.3, pp.201-209, 2023-09-25 (Released:2023-09-25)
- 参考文献数
- 29
- 被引用文献数
- 3
Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.
- 著者
- Ami Takeuchi Mariko Ohnuma Hiroshi Teramura Kenji Asano Takahiro Noda Hiroaki Kusano Koji Tamura Hiroaki Shimada
- 出版者
- Japanese Society for Plant Biotechnology
- 雑誌
- Plant Biotechnology (ISSN:13424580)
- 巻号頁・発行日
- vol.38, no.3, pp.345-353, 2021-09-25 (Released:2021-09-25)
- 参考文献数
- 31
- 被引用文献数
- 16
The potato tuber starch trait is changed depending on the composition of amylose and amylopectin. The amount of amylopectin is determined by the activity of the starch branching enzymes SBE1, SBE2, and SBE3 in potato. SBE3, a homolog of rice BEI, is a major gene that is abundant in tubers. In this study, we created mutants of the potato SBE3 gene using CRISPR/Cas9 attached to the translation enhancer dMac3. Potato has a tetraploid genome, and a four-allele mutant of the SBE3 gene is desired. Mutations in the SBE3 gene were found in 89 of 126 transformants of potato plants. Among these mutants, 10 lines contained four mutant SBE3 genes, indicating that 8% efficiency of target mutagenesis was achieved. These mutants grew normally, similar to the wild-type plant, and yielded sufficient amounts of tubers. The potato starch in these tubers was similar to that of the rice BEI mutant. Western blot analysis revealed the defective production of SBE3 in the mutant tubers, suggesting that these transformants were loss-of-function mutants of SBE3.