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
TL;DR: In this article, the authors evaluated the CRIS-based approach for accelerating the breeding of many fruit crops and found that it may be useful to modify specific genomic loci to accelerate the breeding process.
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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TL;DR: In this paper, the relevance of model parametrizations, such as diploid or polyploid methods, and inclusion of dominance effects was discussed. And the importance of sequence depth of coverage and genotype dosage calling steps was assessed.
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.
16 citations
TL;DR: A review article on these Vaccinium berry crops on topics that span taxonomy to genetics and genomics to breeding is presented in this paper , highlighting the accomplishments made thus far for each of these crops, along their journey from the wild, and propose research areas and questions that will require investments by the community over the coming decades to guide future crop improvement efforts.
Abstract: Abstract The genus Vaccinium L. (Ericaceae) contains a wide diversity of culturally and economically important berry crop species. Consumer demand and scientific research in blueberry (Vaccinium spp.) and cranberry (Vaccinium macrocarpon) have increased worldwide over the crops’ relatively short domestication history (~100 years). Other species, including bilberry (Vaccinium myrtillus), lingonberry (Vaccinium vitis-idaea), and ohelo berry (Vaccinium reticulatum) are largely still harvested from the wild but with crop improvement efforts underway. Here, we present a review article on these Vaccinium berry crops on topics that span taxonomy to genetics and genomics to breeding. We highlight the accomplishments made thus far for each of these crops, along their journey from the wild, and propose research areas and questions that will require investments by the community over the coming decades to guide future crop improvement efforts. New tools and resources are needed to underpin the development of superior cultivars that are not only more resilient to various environmental stresses and higher yielding, but also produce fruit that continue to meet a variety of consumer preferences, including fruit quality and health related traits.
10 citations
TL;DR: Several published papers attest to the high efficiency of CRISPR-based systems in clonal crops and trees as discussed by the authors, however, nearly all studies in trees and clonally propagated crops retained all of the gene editing machinery in the genome.
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.
9 citations
TL;DR: A second round of regeneration enriched editing events and enhanced the production of edited blueberry shoots and the new protocol described facilitates high-precision breeding of blueberries using CRISPR Cas technologies.
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.
6 citations
TL;DR: In this paper , a protocol for in vitro polyploid induction of highbush blueberry (Vaccinium corymbosum L.) leaf tissues was studied by using different concentrations of colchicine and oryzalin.
Abstract: Polyploid induction is of utmost importance in horticultural plants for the development of new varieties with desirable morphological and physiological traits. Polyploidy may occur naturally due to the formation of unreduced gametes or can be artificially induced by doubling the number of chromosomes in somatic cells. In this experiment, a protocol for in vitro polyploid induction of highbush blueberry (Vaccinium corymbosum L.) leaf tissues was studied by using different concentrations of colchicine and oryzalin. Oryzalin was found to be highly toxic to this species, while the adventitious shoot organogenesis media enriched with 25 and 250 µM colchicine was able to induce polyploidization, with significant differences among the treatments used. Higher concentrations of both antimitotic agents led to the browning and death of the leaf tissues. The polyploids obtained showed several morphological differences when compared with the diploid shoots. Flow cytometry analysis was used to confirm the ploidy level of the regenerated shoots, demonstrating that a total of 15 tetraploids and 34 mixoploids were obtained. The stomatal sizes (length and width) of the tetraploids were larger than those of the diploids, but a reduced stomatal density was observed as compared to the controls. These shoots will be acclimatized and grown until they reach the reproductive phase in order to test their potential appeal as new varieties or their use for breeding and genetic improvement.
6 citations
References
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TL;DR: An online tool for the design of highly active sgRNAs for any gene of interest is provided, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9.
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.
1,377 citations
TL;DR: Different cultivars of four Vaccinium species were analyzed for total phenolics, total anthocyanins, and antioxidant capacity (oxygen radical absorbance capacity, ORAC).
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,...
1,341 citations
TL;DR: The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (Cas9) system is described, a recently developed tool for the introduction of site-specific double-stranded DNA breaks and the strengths and weaknesses are highlighted.
948 citations
TL;DR: The Antirrhinum gene CENTRORADIALIS and the Arabidopsis gene TERMINAL FLOWER 1 (TFL1) were shown to be homologous, which suggests that a common mechanism underlies indeterminacy in these plants.
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.
794 citations
TL;DR: 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.
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.
592 citations