Wold-McGimsey, F. | Search bioRxiv

bioRxiv2024

Disruption of recombination machinery alters the mutational landscape in plant organellar genomes

Land plant organellar genomes have extremely low rates of point mutation yet also experience high rates of recombination and genome instability. Characterizing the molecular machinery responsible for these patterns is critical for understanding the evolution of these genomes. While much progress has been made towards understanding recombination activity in land plant organellar genomes, the relationship between recombination pathways and point mutation rates remains uncertain. The organellar targeted mutS homolog MSH1 has previously been shown to suppress point mutations as well as non-allelic recombination between short repeats in Arabidopsis thaliana. We therefore implemented high-fidelity Duplex Sequencing to test if other genes that function in recombination and maintenance of genome stability also affect point mutation rates. We found small to moderate increases in the frequency of single nucleotide variants (SNVs) and indels in mitochondrial and/or plastid genomes of A. thaliana mutant lines lacking radA, recA1, or recA3. In contrast, osb2 and why2 mutants did not exhibit an increase in point mutations compared to wild type (WT) controls. In addition, we analyzed the distribution of SNVs in previously generated Duplex Sequencing data from A. thaliana organellar genomes and found unexpected strand asymmetries and large effects of flanking nucleotides on mutation rates in WT plants and msh1 mutants. Finally, using long- read Oxford Nanopore sequencing, we characterized structural variants in organellar genomes of the mutant lines and show that different short repeat sequences become recombinationally active in different mutant backgrounds. Together, these complementary sequencing approaches shed light on how recombination may impact the extraordinarily low point mutation rates in plant organellar genomes.

genetics↗

bioRxiv2023

Multi-target genome editing reduces polyphenol oxidase activity in wheat (Triticum aestivum L.) grains

Polyphenol oxidases (PPO) are dual activity metalloenzymes that catalyse the production of quinones. In plants, PPO activity may contribute to biotic stress resistance and secondary metabolism but is undesirable for food producers because it causes the discolouration and changes in flavour profiles of products during post-harvest processing. In wheat (Triticum aestivum L.), PPO released from the aleurone layer of the grain during milling results in the discolouration of flour, dough, and end-use products, reducing their value. Loss-of-function mutations in the PPO1 and PPO2 paralogous genes on homoeologous group 2 chromosomes confer reduced PPO activity in the wheat grain but limited natural variation and small intergenic distances between these genes complicates the selection of extremely low-PPO wheat varieties by recombination. In the current study, a CRISPR/Cas9 construct with one single guide RNA (sgRNA) targeting a conserved copper binding domain was used to edit all seven PPO1 and PPO2 genes in the spring wheat cultivar Fielder. Five of the seven edited T1 lines exhibited significant reductions in PPO activity, and T2 lines had PPO activity up to 86.7% lower than wild-type controls. In the elite winter wheat cultivars Guardian and Steamboat, which have five PPO1 and PPO2 genes, PPO activity was reduced by >90% in both T1 and T2 lines. This study demonstrates that multi-target editing at late stages of variety development could complement selection for beneficial alleles in crop breeding programmes by inducing novel variation in loci inaccessible to recombination.

plant biology↗