With the advent of fluorescence in situ hybridization (FISH) methods since the 1980s-1990s, we have rapidly gained insights into the structure, organization and evolution of plant genomes [1,2]. The potential of molecular cytogenetics was also recognized in the plant breeding sector, often with a focus on hybridization, introgressions and chromosomal additions. Now, with the advances in long-read sequencing and with the new genome-editing techniques in place, the plant cytogenetics field is repositioning itself at the interface between microscopy and genomics. FISH methods are more relevant than ever, for example to better resolve the inaccessible, repetitive genome fraction [3], the genomes’ three-dimensional organization [4,5], but also to identify chromosomes [6], assess haplotypes, predict recombination [7], and to determine genomic variation among individuals [8]. On top, FISH may also assist the synthetic genomics field seeking to steer chromosome mutations and to build artificial chromosomes [9,10]. Nevertheless, the application of FISH to plant chromosomes remains challenging.
First, plant genomes can contain over 80% of repetitive DNA [11] and up to 149 gigabasepairs [12]. Although these numbers reside at the extreme ends of the corresponding scales, they serve well to illustrate that plant genomes are often large and repetitive. Therefore, they remain difficult targets for sequence-based methods.
Second, only limited genome and haplotype information has been available for many plants, leading to less flexibility in experimental design. This gap is now closing at high speed, with many new crop and wild plant genome builds being released every year (see https://www.plabipd.de/timeline_view.ep). Similarly, many karyotyping tools that were originally designed for human chromosomes are now flexible in organism choice and can be used for plant chromosomes as well.
Finally, the preparation of plant chromosomes – a prerequisite for FISH – is not straight-forward. Plants contain a high amount of fibers and their cells are enclosed by a cell wall with a variable chemical composition, depending on species, tissue, and developmental stage. Finding the balance between cell wall degradation and removal of cytoplasm on the one hand, and preserving chromosome integrity on the other hand, is one of the key challenges of plant cytogenetics, a process which needs to be adapted or even established for each new plant species and tissue coming to the lab.