CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid

Abstract

Summary Crossover formation during meiosis in plants is required for proper chromosome segregation and is essential for crop breeding as it allows an (optimal) combination of traits by mixing parental alleles on each chromosome. Crossover formation commences with the production of a large number of DNA double‐strand breaks, of which only a few result in crossovers. A small number of genes, which drive the resolution of DNA crossover intermediate structures towards non‐crossovers, have been identified in Arabidopisis thaliana. In order to explore the potential of modification of these genes in interspecific hybrids between crops and their wild relatives towards increased production of crossovers, we have used CRISPR/Cas9‐mutagenesis in an interspecific tomato hybrid to knockout RecQ4. A biallelic recq4 mutant was obtained in the F1 hybrid of Solanum lycopersicum and S. pimpinellifolium. Compared with the wild‐type F1 hybrid, the F1 recq4 mutant was shown to have a significant increase in crossovers: a 1.53‐fold increase when directly observing ring bivalents in male meiocytes microscopically and a 1.8‐fold extension of the genetic map when measured by analysing SNP markers in the progeny (F2) plants. This is one of the first demonstrations of increasing crossover frequency in interspecific hybrids by manipulating genes in crossover intermediate resolution pathways and the first to do so by directed mutagenesis. Significance statement Increasing crossover frequency during meiosis can speed up or simplify crop breeding that relies on meiotic crossovers to introduce favourable alleles controlling important traits from wild relatives into crops. Here we show for the first time that knocking out an inhibitor of crossovers in an interspecific hybrid between tomato and its relative wild species using CRISPR/Cas9‐mutagenesis results in increased recombination between the two genomes.

Counting on Crossovers: Controlled Recombination for Plant Breeding

“…Silencing in this study was achieved by RNA interference (RNAi), but could equally well be achieved today using fusions of nucleasedeactivated Cas9 (dCas9) to transcriptional repressors, also known as CRISPR interference (CRISPRi) [12,50]. In a recent study, active CRISPR/Cas was successfully used to promote homoeologous COs in an interspecific hybrid between wild and cultivated tomato by knockout of the anti-CO gene RECQ4 [23]. A related approach was effective in allopolyploid wheat (Triticum aestivum L.).…”

The Role of Structural Maintenance of Chromosomes Complexes in Meiosis and Genome Maintenance: Translating Biomedical and Model Plant Research Into Crop Breeding Opportunities

“…Biomedical research in yeast and mammalian cohesin and condensin complexes and their respective regulators has contributed significantly to our understanding of the processes they affect, namely genome maintenance, meiotic recombination, CO formation, and regulation of high-order chromatin structure in chromosomes. Research in the model plant A. thaliana and crops has also pushed the boundaries of our knowledge and facilitated charting the way to applied plant breeding by providing clear mutant phenotypes and solid genetic evidence ( de Maagd et al, 2020 ). Translation of basic research into crop breeding may speed the screening and retrieval of valuable alleles for tolerance to DNA damage by UVB and aluminum exposure (for a summary see Figure 6 ).…”

“…During meiosis, the reciprocal exchange of DNA between homologous chromosomes enables the reshuffling of parental genetic information and the transfer of the recombined material to the progeny (de Maagd et al, 2020). The formation of DSBs by the topoisomerase SPORULATION-DEFICIENT 11 (SPO11) is crucial for the initiation of synapsis between homologous chromosomes and CO formation (Esposito and Esposito, 1969;Ning et al, 2020).…”

“…The subsequent simultaneous assembly of the multi-layered chromosome axis and synaptonemal complex by the α-kleisin REC8/SYN1, the coiled-coil proteins ASY3 and ASY4, the HORMA-domain protein ASY1 and transverse filament protein ZYP1 catalyze the successful synapsis of homologous chromosomes and the formation of COs ( Ning et al, 2020 ). Thus, meiotic CO formation is an important target in crop breeding ( de Maagd et al, 2020 ), especially because in modern crops, successive rounds of selection have reduced their genetic variation, leaving them with less allelic diversity than their wild relatives, called the “domestication bottleneck” ( Dempewolf et al, 2017 ).…”

“…Meiosis starts with the formation of many DNA DSBs, most of which are repaired in non-CO events and do not result in recombinant chromosomes. In plants, only one to three DSBs per chromosome are processed into actual COs ( de Maagd et al, 2020 ), and most of these COs exhibit interference, the phenomenon that prevents multiple COs from occurring in close proximity along each chromosome pair ( Lambing et al, 2020b ). In Arabidopsis , type I COs are sensitive to interference and are associated with the activity of the ZMM class of proteins, including mutS homolog 4 (MSH4), a meiotic-specific reciprocal-recombination factor.…”