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

Sam W Henderson,Steven T Henderson,Marc Goetz,Anna M G Koltunow

doi:10.3390/genes11091064

Sam W Henderson, Steven T Henderson + Show 2 more

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https://doi.org/10.3390/genes11091064

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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.

Highlights

Increasing seed yields in major crops requires new insights into the function of genes that regulate plant reproduction
The CRISPR/Cas9 editing constructs designed to disrupt endogenous Hieracium PDS (HPDS) gene function, contained three sgRNAs targeting a 252 bp region within exon 10 (Figure S1A,B), which is similar to the region that was used to target PHYTOENE DESATURASE (PDS) in cassava [30]
The completed HPDS-targeting CRISPR/Cas9 construct was introduced to cells of D36 leaf explants by Agrobacterium-mediated transformation

Summary

Introduction

Increasing seed yields in major crops requires new insights into the function of genes that regulate plant reproduction. Plants typically form seeds via a sexual pathway requiring meiosis to form male and female gametes and fertilization to initiate seed formation (Figure 1A). A number of non-agronomic plants can form seed asexually via apomixis. Most apomicts are facultative, meaning the sexual pathway remains intact in some ovules. Genetically identical seeds of a maternal genotype form because meiosis is avoided during female gametophyte or embryo sac formation, and fertilization is not required for embryo formation [1,2,3]. Harnessing apomixis in plant breeding would have significant benefits for

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