Manipulation of Meiotic Recombination to Hasten Crop Improvement.

Reciprocal (cross-overs = COs) and non-reciprocal (gene conversion) DNA exchanges between the parental chromosomes (the homologs) during meiotic recombination are, together with mutation, the drivers for the evolution and adaptation of species. In plant breeding, recombination combines alleles from genetically diverse accessions to generate new haplotypes on which selection can act. In recent years, a spectacular progress has been accomplished in the understanding of the mechanisms underlying meiotic recombination in both model and crop plants as well as in the modulation of meiotic recombination using different strategies. The latter includes the stimulation and redistribution of COs by either modifying environmental conditions (e.g., T°), harnessing particular genomic situations (e.g., triploidy in Brassicaceae), or inactivating/over-expressing meiotic genes, notably some involved in the DNA double-strand break (DSB) repair pathways. These tools could be particularly useful for shuffling diversity in pre-breeding generations. Furthermore, thanks to the site-specific properties of genome editing technologies the targeting of meiotic recombination at specific chromosomal regions nowadays appears an attainable goal. Directing COs at desired chromosomal positions would allow breaking linkage situations existing between favorable and unfavorable alleles, the so-called linkage drag, and accelerate genetic gain. This review surveys the recent achievements in the manipulation of meiotic recombination in plants that could be integrated into breeding schemes to meet the challenges of deploying crops that are more resilient to climate instability, resistant to pathogens and pests, and sparing in their input requirements.

Efficient Genome Editing in Rice Protoplasts Using CRISPR/CAS9 Construct.

Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks' time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9-11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.

Beyond Seek and Destroy: how to Generate Allelic Series Using Genome Editing Tools.

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECAS9 is a viable and efficient alternative, although it has a lower efficiency than SpCAS9 and LbCPF1 for creating KO mutations.

Polycomb Repressive Complex 2 attenuates the very high expression of the Arabidopsis gene NRT2.1.

PRC2 is a major regulator of gene expression in eukaryotes. It catalyzes the repressive chromatin mark H3K27me3, which leads to very low expression of target genes. NRT2.1, which encodes a key root nitrate transporter in Arabidopsis, is targeted by H3K27me3, but the function of PRC2 on NRT2.1 remains unclear. Here, we demonstrate that PRC2 directly targets and down-regulates NRT2.1, but in a context of very high transcription, in nutritional conditions where this gene is one of the most highly expressed genes in the transcriptome. Indeed, the mutation of CLF, which encodes a PRC2 subunit, leads to a loss of H3K27me3 at NRT2.1 and results, exclusively under permissive conditions for NRT2.1, in a further increase in NRT2.1 expression, and specifically in tissues where NRT2.1 is normally expressed. Therefore, our data indicates that PRC2 tempers the hyperactivity of NRT2.1 in a context of very strong transcription. This reveals an original function of PRC2 in the control of the expression of a highly expressed gene in Arabidopsis.