HRM-facilitated rapid identification and genotyping of mutations induced by CRISPR/Cas9 mutagenesis in rice

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Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK) is encoded by six genes in rice (OsITPK1-6). A previous study had shown that nucleotide substitutions of OsITPK6 could significantly lower the phytic acid content in rice grains. In the present study, the possibility of establishing a genome editing-based method for breeding low-phytic acid cultivars in rice was explored, in conjunction with the functional determination of OsITPK6. Four OsITPK6 mutant lines were generated by targeted mutagenesis of the gene’s first exon using the CRISPR/Cas9 method, one (ositpk6_1) with a 6-bp in-frame deletion, and other three with frameshift mutations (ositpk6_2, _3, and _4). The frameshift mutations severely impaired plant growth and reproduction, while the effect of ositpk6_1 was relatively limited. The mutant lines ositpk6_1 and _2 had significantly lower levels (−10.1% and −32.1%) of phytic acid and higher levels (4.12- and 5.18-fold) of inorganic phosphorus compared with the wild-type (WT) line. The line ositpk6_1 also showed less tolerance to osmotic stresses. Our research demonstrates that mutations of OsITPK6, while effectively reducing phytic acid biosynthesis in rice grain, could significantly impair plant growth and reproduction.

Section: Methodsmentioning

confidence: 99%

Mutation of Inositol 1,3,4-trisphosphate 5/6-kinase6 Impairs Plant Growth and Phytic Acid Synthesis in Rice

Jiang

Yang

Liu

et al. 2019

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“…HRM analysis requires a simple set-up and allows rapid, high-throughput mutation screening, but requires a dedicated software. This particular technology has been found to work across different plant species [ 225 , 226 , 227 , 228 , 229 ].…”

Section: Techniques To Estimate and Quantify The Mutation Ratementioning

confidence: 99%

Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience

Sattar

Iqbal

Al-Khayri

et al. 2021

Fruit trees provide essential nutrients to humans by contributing to major agricultural outputs and economic growth globally. However, major constraints to sustainable agricultural productivity are the uncontrolled proliferation of the population, and biotic and abiotic stresses. Tree mutation breeding has been substantially improved using different physical and chemical mutagens. Nonetheless, tree plant breeding has certain crucial bottlenecks including a long life cycle, ploidy level, occurrence of sequence polymorphisms, nature of parthenocarpic fruit development and linkage. Genetic engineering of trees has focused on boosting quality traits such as productivity, wood quality, and resistance to biotic and abiotic stresses. Recent technological advances in genome editing provide a unique opportunity for the genetic improvement of woody plants. This review examines application of the CRISPR-Cas system to reduce disease susceptibility, alter plant architecture, enhance fruit quality, and improve yields. Examples are discussed of the contemporary CRISPR-Cas system to engineer easily scorable PDS genes, modify lignin, and to alter the flowering onset, fertility, tree architecture and certain biotic stresses.

“…The site-specific mutation was identified by amplification of PCR using primers (P1-F and P1-R, Table S1) encompassing the target sites in the gene of OsIPK1 ( Figure 1A). The analysis of high resolution melt (HRM) was carried out according to Li et al [54] to verify for the mutations. The fragments of a selected target with diverse colored lines on analysis of HRM were sequenced by Tsingke (Hangzhou, China) and the DSDecode (http://skl.scau.edu.cn/dsdecode/) program was used to decode the mutation sequences [55].…”

Section: Growth and Identification Of Mutantsmentioning

confidence: 99%

An Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase 1 Mutant with a 33-nt Deletion Showed Enhanced Tolerance to Salt and Drought Stress in Rice

Jiang

Liu

et al. 2020

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OsIPK1 encodes inositol 1,3,4,5,6-pentakisphosphate 2-kinase, which catalyzes the conversion of myo-inositol-1,3,4,5,6-pentakisphosphate to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in rice. By clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas9)-mediated mutagenesis in the 3rd exon of the gene, three OsIPK1 mutations, i.e., osipk1_1 (a 33-nt deletion), osipk1_2 (a 1-nt deletion), and osipk1_3 (a 2-nt deletion) were identified in T0 plants of the rice line Xidao #1 (wild type, WT). A transfer DNA free line with the homozygous osipk1_1 mutation was developed; however, no homozygous mutant lines could be developed for the other two mutations. The comparative assay showed that the osipk1_1 mutant line had a significantly lower level of phytic acid (PA, IP6; −19.5%) in rice grain and agronomic traits comparable to the WT. However, the osipk1_1 mutant was more tolerant to salt and drought stresses than the WT, with significantly lower levels of inositol triphosphate (IP3), reactive oxygen species (ROS) and induced IP6, and higher activities of antioxidant enzymes in seedlings subjected to these stresses. Further analyses showed that the transcription of stress response genes was significantly upregulated in the osipk1_1 mutant under stress. Thus, the low phytic acid mutant osipk1_1 should have potential applications in rice breeding and production.