Efficient CRISPR/Cas9-Mediated Knockout of an Endogenous PHYTOENE DESATURASE Gene in T1 Progeny of Apomictic Hieracium Enables New Strategies for Apomixis Gene Identification

Abstract

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.

Knockout of phytoene desaturase gene using CRISPR/Cas9 in highbush blueberry

“…In our experiments, a complete albino phenotype was observed even with low editing efficiency (23.8%) ( Figure 4A ), as well chimeric mutants showing green islands ( Figure 4B ) or anthocyanins ( Figure 4C ), that often appears with the albinism, as also observed in other species ( Henderson et al., 2020 ). Anyway, the CRISPR/Cas9 in polyploids is more challenging, due to the presence of multiple homoeologous copies (or alleles) of each gene ( Schaart et al., 2021 ), and low editing efficiency could be ascribed to different reasons.…”

CRISPR/Cas9-mediated editing of PHYTOENE DESATURASE gene in onion (Allium cepa L.)

“…Of these 21 shoots, two were complete albinos, four were chimeric, and 15 were pale green. Similar phenotypes have been reported in PDS knockout experiments in various plant species ( Pan et al., 2016 ; Naim et al., 2018 ; Hooghvorst et al., 2019 ; Henderson et al., 2020 ; Wang et al., 2020 ; Lu and Tian, 2022 ).…”

“…Rasthali ( Musa acuminata cv. Rasthali) ( Kaur et al., 2018 ), cavendish banana ( Musa acuminata subgroup Cavendish) ( Naim et al., 2018 ), melon ( Cucumis melo ) ( Hooghvorst et al., 2019 ), strawberry ( Fragaria ananassa ) ( Wilson et al., 2019 ), lettuce ( Lactuca sativa ) ( Henderson et al., 2020 ), poplar ( Populus tomentosa Carr ) ( Fan et al., 2015 ; Wang et al., 2020 ), and yam ( Dioscorea rotunda ) ( Syombua et al., 2021 ). Thus, we targeted the AcPDS gene to evaluate the editing efficiency induced by the CRISPR/Cas9 system.…”

The Genetic Components of a Natural Color Palette: A Comprehensive List of Carotenoid Pathway Mutations in Plants

“…MaPDS (Ma08_g16510) (Hu et al, 2017;Kaur et al, 2018;Naim et al, 2018;Ntui et al, 2020;Wu et al, 2020) Citrus (Jia and Nian, 2014;Jia and Wang, 2014;Jia et al, 2017Jia et al, , 2019Zhang et al, 2017;Zhu et al, 2019;Dutt et al, 2020) Grape (Nakajima et al, 2017;Ren et al, 2019aRen et al, ,b, 2021 Hop HlPDS (NCBI accession number: MT083893) (Awasthi et al, 2021) yellowish-white flower (EMS; Brassica napus) (Zhao et al, 2021) vp5 (maize) (Hable et al, 1998) (Nishitani et al, 2016;Osakabe et al, 2018;Charrier et al, 2019) Cassava (Manes.05G193700) (Odipio et al, 2017) Melon CmPDS (MELO3C017772.2) (Tian et al, 2017;Hooghvorst et al, 2019) Strawberry FvPDS (LG4-gene12690) (Wilson et al, 2019) Hieracium (Henderson et al, 2020) Populus (Fan et al, 2015;Liu et al, 2015;Wang et al, 2020a) Chicory CiPDS (GenBank accession MK455771) (Bernard et al, 2019) Yam (Syombua et al, 2021) Carrot (Genbank accession no. XM_017396654.1) (Xu et al, 2019) Rehmannia (Li et al, 2021) Witloof (De Bruyn et al, 2020) Sorghum (Liu et al, 2019) Soybean GmPDS11 (Glyma.11G253000), GmPDS18…”