Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3

Authors: YOON, SANGWOONG - Former ARS Employee; Bragg, Jennifer; AUCAR-YAMATO, SHEYLA - Former ARS Employee; Chanbusarakum, Lisa; DLUGE, KURTIS - Former ARS Employee; CHENG, PRISCA - Former ARS Employee; BLUMWALD, EDUARDO - University Of California, Davis; Gu, Yong; Tobias, Christian

Submitted to: The Plant Genome
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2022
Publication Date: 4/25/2022
Citation: Yoon, S., Bragg, J.N., Aucar-Yamato, S., Chanbusarakum, L.J., Dluge, K., Cheng, P., Blumwald, E., Gu, Y.Q., Tobias, C.M. 2022. Haploidy and aneuploidy in switchgrass mediated by misexpression of CENH3. The Plant Genome. Article e20209. https://doi.org/10.1002/tpg2.20209.
DOI: https://doi.org/10.1002/tpg2.20209

Interpretive Summary: The ability to easily breed crops is facilitated by inbred lines where the specific genetic makeup is fixed. This allows individual beneficial genes and gene combinations to be manipulated in a predicable manner to ensure that traits such as disease resistance, or hybrid vigor are expressed in each individual from a cross. Inbred lines also enable the “unmasking” of undesirable genes so that these can easily be eliminated from breeding stock. Many species such as switchgrass are not inbreeding by nature and they might benefit by the ability to experimentally produce these inbreds in a rapid manner. This work describes strategies that were used to generate switchgrass seedlings with half the genetic compliment of wild-type switchgrass. These seedlings can be induced to undergo genome doubling and thus produce in two steps what would otherwise be extremely difficult if not impossible otherwise. To accomplish this feat, modifications were made to two genes encoding proteins controlling how chromosomes are transmitted during cell division. It experimentally mimics how in nature genome halfing or reduction sometimes occurs in the offspring of related but sexually compatible species.

Technical Abstract: Cross bred species such as switchgrass may benefit from advantageous breeding strategies requiring inbred lines. Doubled haploid (DH) production methods offer several ways that these lines can be produced which 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 switchgrass lines coexpressed Cas9, a poly-cistronic tRNA-gRNA (PTG) 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 biallelic and monoallelic frameshift mutations and deletions. Transgenic lines showed reduced fertility and pollen viability, and flow cytometry of T1 seedlings identified two T0 lines that produced five haploid individuals representing a rate of ~0.2%. Eight different T0 lines produced aneuploids at rates ranging from 1.6-7.3%. A sample of aneuploid lines were sequenced at low coverage and aligned to the reference genome, revealing missing chromosomes and chromosome arms. Haploid individuals grew poorly, an indication of genetic load.