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Journal ArticleDOI: 10.1080/14620316.2020.1822760

Targeted mutagenesis of CENTRORADIALIS using CRISPR/Cas9 system through the improvement of genetic transformation efficiency of tetraploid highbush blueberry

04 Mar 2021-Journal of Horticultural Science & Biotechnology (Informa UK Limited)-Vol. 96, Iss: 2, pp 153-161
Abstract: Genome editing technology, which enables researchers to modify specific genomic loci, may be useful for accelerating the breeding of many fruit crops. The aim of this study was to evaluate the CRIS...

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5 results found


Open accessJournal ArticleDOI: 10.3389/FPLS.2021.676326
Abstract: Blueberry (Vaccinium corymbosum and hybrids) is a specialty crop with expanding production and consumption worldwide. The blueberry breeding program at the University of Florida (UF) has greatly contributed to expanding production areas by developing low-chilling cultivars better adapted to subtropical and Mediterranean climates of the globe. The breeding program has historically focused on recurrent phenotypic selection. As an autopolyploid, outcrossing, perennial, long juvenile phase crop, blueberry breeding cycles are costly and time consuming, which results in low genetic gains per unit of time. Motivated by applying molecular markers for a more accurate selection in the early stages of breeding, we performed pioneering genomic selection studies and optimization for its implementation in the blueberry breeding program. We have also addressed some complexities of sequence-based genotyping and model parametrization for an autopolyploid crop, providing empirical contributions that can be extended to other polyploid species. We herein revisited some of our previous genomic selection studies and showed for the first time its application in an independent validation set. In this paper, our contribution is three-fold: (i) summarize previous results on the relevance of model parametrizations, such as diploid or polyploid methods, and inclusion of dominance effects; (ii) assess the importance of sequence depth of coverage and genotype dosage calling steps; (iii) demonstrate the real impact of genomic selection on leveraging breeding decisions by using an independent validation set. Altogether, we propose a strategy for using genomic selection in blueberry, with the potential to be applied to other polyploid species of a similar background.

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4 Citations


Journal ArticleDOI: 10.1007/S11240-021-02177-1
Xiaoyan Han1, Xiaoyan Han2, Yingzhen Yang3, Xue Han1  +5 moreInstitutions (4)
Abstract: To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T0) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies. A second round of regeneration enriched editing events and enhanced the production of edited blueberry shoots. The new protocol described facilitates high-precision breeding of blueberries using CRISPR Cas technologies.

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2 Citations


Open accessJournal ArticleDOI: 10.1007/S11627-021-10197-X
Abstract: Because of the limitations inherent in conventional breeding of trees and clonally propagated crops, gene editing is of great interest. Dozens of published papers attest to the high efficiency of CRISPR-based systems in clonal crops and trees. The opportunity for “clean” edits is expected to avoid or reduce regulatory burdens in many countries and may improve market acceptance. To date, however, nearly all studies in trees and clonal crops retained all of the gene editing machinery in the genome. Despite high gene editing efficiency, technical and regulatory obstacles are likely to greatly limit progress toward commercial use. Technical obstacles include difficult and slow transformation and regeneration, delayed onset of flowering or clonal systems that make sexual segregation of CRISPR-associated genes difficult, inefficient excision systems to enable removal of functional (protein- or RNA-encoding) transgenic DNA, and narrow host range or limited gene-payload viral systems for efficient transient editing. Regulatory obstacles include those such as in the EU where gene-edited plants are regulated like GMO crops, and the many forms of method-based systems that regulate stringently based on the method vs. product novelty and thus are largely applied to each insertion event. Other major obstacles include the provisions of the Cartagena Protocol with respect to international trade and the need for compliance with the National Environmental Policy Act in the USA. The USDA SECURE act has taken a major step toward a more science- and risk-based—vs. method and insertion event based—system, but much further regulatory and legal innovation is needed in the USA and beyond.

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Book ChapterDOI: 10.1007/978-3-030-74682-7_10
01 Jan 2021-
Abstract: Genome editing with artificially engineered nucleases is an advanced molecular technology for pursuing precise and effective genetic engineering. In this technology, engineered nucleases induce DNA double-strand breaks at targeted sites in a genome, stimulating the DNA repair system in cells, thus enabling site-directed mutagenesis. Genome editing using CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein9 (Cas9), originating from a defence system of prokaryotes, is a powerful technology that is now being widely utilized in molecular research studies, as well as in breeding programmes of various plant species, including fruit trees, to impart either novel or enhanced traits to established commercial cultivars or to new cultivars/genotypes. Recently, several reports have demonstrated successful apple genome editing and the introduction of important traits, such as those for early flowering and reduced fire blight susceptibility, to popular commercial cultivars, such as ‘Gala’ and ‘Golden Delicious’. It is important to point out that these reports reveal that such genome-edited/mutant apple plants or cell lines do not carry foreign genes. Nevertheless, during the process of precise genome editing, the coexistence of various types of mutations referred to as “mosaic mutations” and off-target effects are major concerns. Therefore, to minimize such effects, selection of target sequences and estimation of off-target effects for CRISPR/Cas9 has been developed for many organisms, and these have also been employed for apple by using in silico analysis based on genome information. On the other hand, apple genome heterozygosity has led to difficulties in genome editing, as the complex genome of apple precludes the use of some of these basic techniques for genome editing. Therefore, further studies focused on genome information and culture techniques tailored for apple are needed. It will be highly critical for each apple cultivar in developing precise and efficient genome editing for apple. This chapter will provide an overview of current studies of genome editing in apple and will discern and explore how this strategy will provide insights into molecular breeding technologies for genetic improvement of the apple.

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Topics: Genome editing (67%), Genome (59%), Cas9 (56%) ... read more

Open accessPosted ContentDOI: 10.1101/2021.03.05.434007
07 Mar 2021-bioRxiv
Abstract: Blueberry (Vaccinium corymbosum and hybrids) is a specialty crop, with expanding production and consumption worldwide. The blueberry breeding program at the University of Florida (UF) has greatly contributed to the expansion of production areas by developing low-chilling cultivars better adapted to subtropical and Mediterranean climates of the globe. The breeding program has historically focused on phenotypic recurrent selection. As an autopolyploid, outcrossing, perennial, long juvenile phase crop, blueberry’s breeding cycles are costly and time-consuming, which results in low genetic gains per unit of time. Motivated by the application of molecular markers for a more accurate selection in early stages of breeding, we performed pioneering genomic prediction studies and optimization for implementation in the blueberry breeding program. We have also addressed some complexities of sequence-based geno- typing and model parametrization for an autopolyploid crop, providing empirical contributions that can be extended to other polyploid species. We herein revisited some of our previous genomic prediction studies and described the current achievements in the crop. In this paper, our contribution for genomic prediction in an autotetraploid crop is three-fold: i) summarize previous results on the relevance of model parametrizations, such as diploid or polyploid methods, and inclusion of dominance effects; ii) assess the importance of sequence depth of coverage and genotype dosage calling steps; iii) demonstrate the real impact of genomic selection on leveraging breeding decisions by using an independent validation set. Altogether, we propose a strategy for the use of genomic selection in blueberry, with potential to be applied to other polyploid species of a similar background.

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References
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39 results found


Journal ArticleDOI: 10.1021/JF980145D
Ronald L. Prior1, Guohua Cao1, A. Martin1, Emin Sofic1  +7 moreInstitutions (1)
Abstract: Different cultivars of four Vaccinium species [Vaccinium corymbosum L (Highbush), Vaccinium ashei Reade (Rabbiteye), Vaccinium angustifolium (Lowbush), and Vaccinium myrtillus L (Bilberry)] were analyzed for total phenolics, total anthocyanins, and antioxidant capacity (oxygen radical absorbance capacity, ORAC). The total antioxidant capacity of different berries studied ranged from a low of 13.9 to 45.9 μmol Trolox equivalents (TE)/g of fresh berry (63.2−282.3 μmol TE/g of dry matter) in different species and cultivars of Vaccinium. Brightwell and Tifblue cultivars of rabbiteye blueberries were harvested at 2 times, 49 days apart. Increased maturity at harvest increased the ORAC, the anthocyanin, and the total phenolic content. The growing location (Oregon vs Michigan vs New Jersey) did not affect ORAC, anthocyanin or total phenolic content of the cv. Jersey of highbush blueberries. A linear relationship existed between ORAC and anthocyanin (rxy = 0.77) or total phenolic (rxy = 0.92) content. In general,...

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Topics: Vaccinium myrtillus (59%), Vaccinium (59%), Bilberry (59%) ... read more

1,284 Citations


Open accessJournal ArticleDOI: 10.1038/NBT.3026
John G. Doench1, Ella Hartenian1, Daniel B. Graham1, Zuzana Tothova2  +6 moreInstitutions (3)
Abstract: Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.

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Topics: CRISPR (56%), Cas9 (56%), Gene targeting (52%) ... read more

1,202 Citations


Open accessJournal ArticleDOI: 10.1016/J.BIOTECHADV.2014.12.006
Luisa Bortesi1, Rainer Fischer1Institutions (1)
Abstract: Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently developed tool for the introduction of site-specific double-stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments.

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786 Citations


Journal ArticleDOI: 10.1126/SCIENCE.275.5296.80
03 Jan 1997-Science
Abstract: Flowering plants exhibit one of two types of inflorescence architecture: indeterminate, in which the inflorescence grows indefinitely, or determinate, in which a terminal flower is produced. The indeterminate condition is thought to have evolved from the determinate many times, independently. In two mutants in distantly related species, terminal flower 1 in Arabidopsis and centroradialis in Antirrhinum, inflorescences that are normally indeterminate are converted to a determinate architecture. The Antirrhinum gene CENTRORADIALIS (CEN) and the Arabidopsis gene TERMINAL FLOWER 1 (TFL1) were shown to be homologous, which suggests that a common mechanism underlies indeterminacy in these plants. However, unlike CEN, TFL1 is also expressed during the vegetative phase, where it delays the commitment to inflorescence development and thus affects the timing of the formation of the inflorescence meristem as well as its identity.

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Topics: Inflorescence (57%), Meristem determinacy (56%), Antirrhinum (52%) ... read more

728 Citations


Open accessJournal ArticleDOI: 10.1186/1746-4811-9-39
11 Oct 2013-Plant Methods
Abstract: Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

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Topics: Transcription activator-like effector nuclease (68%), Genome editing (66%), Cas9 (65%) ... read more

537 Citations


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