Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3

Abstract

Cross bred species such as switchgrass may benefit from advantageous breeding strategies requiring inbred lines. Doubled haploid production methods offer several ways that these lines can be produced that often involve uniparental genome elimination as the rate limiting step. We have used a centromere‐mediated genome elimination strategy in which modified CENH3 is expressed to induce the process. Transgenic tetraploid switchgrass lines coexpressed Cas9, a poly‐cistronic tRNA‐gRNA tandem array containing eight guide RNAs that target two CENH3 genes, and different chimeric versions of CENH3 with alterations to the N‐terminal tail region. Genotyping of CENH3 genes in transgenics identified edits including frameshift mutations and deletions in one or both copies of the two CENH3 genes. Flow cytometry of T1 seedlings identified two T0 lines that produced five haploid individuals representing an induction rate of 0.5% and 1.4%. Eight different T0 lines produced aneuploids at rates ranging from 2.1 to 14.6%. A sample of aneuploid lines were sequenced at low coverage and aligned to the reference genome, revealing missing chromosomes and chromosome arms.

Accelerated Breeding for Helianthus annuus (Sunflower) through Doubled Haploidy: An Insight on Past and Future Prospects in the Era of Genome Editing

“…CRISPR/Cas9 technology has been used for the majority of CenH3 gene modification experiments [ 38 , 50 , 51 , 52 , 73 , 74 , 75 ]. This technology is more advantageous than other genome editing technologies as it does not require complicated designs and the assembly of DNA binding proteins, but it only requires a single Cas9 nuclease that can be programmed by engineering the guide RNA (gRNA) to direct target-specific cleavage [ 76 ].…”

Genome editing and chromosome engineering in plants

“…In this special focus issue, using the optimized CRISPR-Cas9 approach, Biswas et al (2022) and Nguyen et al (2021) characterized multiple novel genes involved in resistant starch in rice and N fixation and nodule development in soybean, respectively. In addition, Yoon et al (2022) applied CRISPR-Cas9 to develop double haploid technology in switchgrass that could significantly speed up its plant breeding processes. By targeting four functional domains of the CENH3 homolog genes, a number of aneuploid and haploid lines have been developed first time in this species.…”

“…In addition, Yoon et al. (2022) applied CRISPR‐Cas9 to develop double haploid technology in switchgrass that could significantly speed up its plant breeding processes. By targeting four functional domains of the CENH3 homolog genes, a number of aneuploid and haploid lines have been developed first time in this species.…”

Plant chromosome engineering – past, present and future

“…A haploid induction rate of up to 8% was obtained in wheat when a comparable haploid induction strategy was used (Lv et al, 2020). According to Yoon et al (2022), haploid induction rates between 0.5% and 1.4% were produced in switchgrass as a result of CENH3 misexpression. To develop a haploid inducer with a higher maternal haploid induction frequency in maize, different overexpressing maize CENH3 constructs were introduced into a Stock 6-derived haploid inducer (Meng et al, 2022).…”