The T0 plants generated in Montpellier are individually identified by a code consisting of 4 letters et 2 numbers (AAA A01). This code corresponds to the DNA isolation plate number followed by the coordinates of the well in 96-well extraction plate and is printed as a barcode which allow a reliable tracking of the information and seed stocks related to an individual line.� All the information related to the generation and phenotypic characterization of the primary transformant �including observations on T1 seeds borne by panicles of the primary transformant – are gathered in the Oryza Tag Line phenotype database.
Whether seeds of specific lines are increased under greenhouse conditions, panicles are individually harvested and are identified by numbers resuming from 101 (AAAA01/101) to 199 (AAAA01/199) for individual harvest or AAAA01/100 for a bulk harvest.
For a second user the numbering will be from 301 to 399 (individual harvest) or 300 (bulk harvest).
Most Hieracium subgenus Pilosella species are self-incompatible. Some undergo facultative apomixis where most seeds form asexually with a maternal genotype. Most embryo sacs develop by mitosis, without meiosis and seeds form without fertilization. Apomixis is controlled by dominant loci where recombination is suppressed. Loci deletion by γ-irradiation results in reversion to sexual reproduction. Targeted mutagenesis of genes at identified loci would facilitate causal gene identification. In this study, the efficacy of CRISPR/Cas9 editing was examined in apomictic Hieracium by targeting mutations in the endogenous PHYTOENE DESATURASE (PDS) gene using Agrobacterium-mediated leaf disk transformation. In three experiments, the expected albino dwarf-lethal phenotype, characteristic of PDS knockout, was evident in 11% of T0 plants, 31.4% were sectorial albino chimeras, and the remainder were green. The chimeric plants flowered. Germinated T1 seeds derived from apomictic reproduction in two chimeric plants were phenotyped and sequenced to identify PDS gene edits. Up to 86% of seeds produced albino seedlings with complete PDS knockout. This was attributed to continuing Cas9-mediated editing in chimeric plants during apomictic seed formation preventing Cas9 segregation from the PDS target. This successful demonstration of efficient CRISPR/Cas9 gene editing in apomictic Hieracium, enabled development of the discussed strategies for future identification of causal apomixis genes.
Conflict of interest statement
Keywords: CRISPR/Cas9; Hieracium piloselloides; PHYTOENE DESATURASE (PDS); amplicon sequencing; apomixis; genome editing; tissue culture.
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript or in the decision to publish the results.