Genome engineering and plant breeding : impact on trait discovery and development

Key message: New tools for the precise modification of crops genes are now available for the engineering of new ideotypes. A future challenge in this emerging field of genome engineering is to develop efficient methods for allele mining. Abstract: Genome engineering tools are now available in plants, including major crops, to modify in a predictable manner a given gene. These new techniques have a tremendous potential for a spectacular acceleration of the plant breeding process. Here, we discuss how genetic diversity has always been the raw material for breeders and how they have always taken advantage of the best available science to use, and when possible, increase, this genetic diversity. We will present why the advent of these new techniques gives to the breeders extremely powerful tools for crop breeding, but also why this will require the breeders and researchers to characterize the genes underlying this genetic diversity more precisely. Tackling these challenges should permit the engineering of optimized alleles assortments in an unprecedented and controlled way

Development and evaluation of robust molecular markers linked to disease resistance in tomato for distinctness, uniformity and stability testing

Molecular markers linked to phenotypically important traits are of great interest especially when traits are difficult and/or costly to be observed. In tomato where a strong focus on resistance breeding has led to the introgression of several resistance genes, resistance traits have become important characteristics in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications. Evaluation of disease traits in biological assays is not always straightforward because assays are often influenced by environmental factors, and difficulties in scoring exist. In this study, we describe the development and/or evaluation of molecular marker assays for the Verticillium genes Ve1 and Ve2, the tomato mosaic virusTm1 (linked marker), the tomato mosaic virus Tm2 and Tm22 genes, the Meloidogyne incognita Mi1-2 gene, the Fusarium I (linked marker) and I2 loci, which are obligatory traits in PBR testing. The marker assays were evaluated for their robustness in a ring test and then evaluated in a set of varieties. Although in general, results between biological assays and marker assays gave highly correlated results, marker assays showed an advantage over biological tests in that the results were clearer, i.e., homozygote/heterozygote presence of the resistance gene can be detected and heterogeneity in seed lots can be identified readily. Within the UPOV framework for granting of PBR, the markers have the potential to fulfil the requirements needed for implementation in DUS testing of candidate varieties and could complement or may be an alternative to the pathogenesis tests that are carried out at present

Organismes génétiquement modifiés

chap. 11National audienc

Towards mastering CRISPR-induced gene knock-in in plants: Survey of key features and focus on the model Physcomitrella patens

Beyond its predominant role in human and animal therapy, the CRISPR-Cas9 system has also become an essential tool for plant research and plant breeding. Agronomic applications rely on the mastery of gene inactivation and gene modification. However, if the knock-out of genes by non-homologous end-joining (NHEJ)-mediated repair of the targeted double-strand breaks (DSBs) induced by the CRISPR-Cas9 system is rather well mastered, the knock-in of genes by homology-driven repair or end-joining remains difficult to perform efficiently in higher plants. In this review, we describe the different approaches that can be tested to improve the efficiency of CRISPR–induced gene modification in plants, which include the use of optimal transformation and regeneration protocols, the design of appropriate guide RNAs and donor templates and the choice of nucleases and means of delivery. We also present what can be done to orient DNA repair pathways in the target cells, and we show how the moss Physcomitrella patens can be used as a model plant to better understand what DNA repair mechanisms are involved, and how this knowledge could eventually be used to define more performant strategies of CRISPR-induced gene knock-in

Deciphering the DSB repair mechanisms involved in CRISPR-induced mutagenesis and gene targeting in the model plant, Physcomitrella patens.

Site-directed nucleases make it very easy now to mutate (knock-out) any genomic sequence and to direct the insertion of a template DNA at a specific target (knock-in). The DNA repair mechanisms that are presumed to be mainly involved in these two types of events are respectively canonical non-homologous end-joining (c-NHEJ) and, if the template shares homology to the target, homologous recombination (HR). By using, the model plant Physcomitrella patens, where efficient gene editing (knock-out or knock-in) via SDN can be obtained, we demonstrated that it may not be as simple. By applying the CRISPR/Cas9 system to a series of mutants impacted in different DNA repair pathways, we progressed in the understanding of the different mechanisms that could be involved in CRISPR-induced mutagenesis and gene targeting in plants. Without template DNA, we showed that the mutation efficiency was not decreased in absence of key factors of c-NHEJ, which could indicate that CRISPR-induced mutations may not only be due to c-NHEJ but also to alternative end-joining like micro-homology mediated end joining (MMEJ) and other types of non-canonical end joining. Targeted insertion of a circular template DNA presenting homology to the target appeared to be mainly dependent on the RAD51-dependent HR pathway but not entirely, and other pathways, including single-strand annealing (SSA), could potentially be involved

Genome engineering and plant breeding : impact on trait discovery and development

Key message: New tools for the precise modification of crops genes are now available for the engineering of new ideotypes. A future challenge in this emerging field of genome engineering is to develop efficient methods for allele mining. Abstract: Genome engineering tools are now available in plants, including major crops, to modify in a predictable manner a given gene. These new techniques have a tremendous potential for a spectacular acceleration of the plant breeding process. Here, we discuss how genetic diversity has always been the raw material for breeders and how they have always taken advantage of the best available science to use, and when possible, increase, this genetic diversity. We will present why the advent of these new techniques gives to the breeders extremely powerful tools for crop breeding, but also why this will require the breeders and researchers to characterize the genes underlying this genetic diversity more precisely. Tackling these challenges should permit the engineering of optimized alleles assortments in an unprecedented and controlled way

Evaluation of somatic hybrids of potato with Solanum stenotomum after a long-term in vitro conservation

International audienc

CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella patens

International audienceThe ability to address the CRISPR-Cas9 nuclease complex to any target DNA using customizable single-guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single-guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2-fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end-joining (alt-EJ)-driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology-driven repair (HDR) at the target locus but also that Cas9-induced double-strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR-mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR-induced HDR is only partially mediated by the classical homologous recombination pathway