Thriving or Withering? Plant Molecular Cytogenetics in the First Quarter of the 21st Century

Abstract

Nearly four decades have passed since fluorescence in situ hybridisation was first applied in plants to support molecular cytogenetic analyses across a wide range of species. Subsequent advances in DNA sequencing, bioinformatic analysis, and microscopy, together with the immunolocalisation of various nuclear components, have provided unprecedented insights into the cytomolecular organisation of the nuclear genome in both model and non-model plants, with crop species being perhaps the most significant. The ready availability of sequenced genomes is now facilitating the application of state-of-the-art cytomolecular techniques across diverse plant species. However, these same advances in genomics also pose a challenge to the future of plant molecular cytogenetics, as DNA sequence analysis is increasingly perceived as offering comparable insights into genome organisation. This perception persists despite the continued relevance of FISH-based approaches for the physical anchoring of genome assemblies to chromosomes. Furthermore, cytogenetic approaches cannot currently rival purely genomic methods in terms of throughput, standardisation, and automation. This review highlights the latest key topics in plant cytomolecular research, with particular emphasis on chromosome identification and karyotype evolution, chromatin and interphase nuclear organisation, chromosome structure, hybridisation and polyploidy, and cytogenetics-assisted crop improvement. In doing so, it underscores the distinctive contributions that cytogenetic techniques continue to offer in genomic research. Additionally, we critically assess future directions and emerging opportunities in the field, including those related to CRISPR/Cas-based live-cell imaging and chromosome engineering, as well as AI-assisted image analysis and karyotyping.

Keywords: FISH; chromosome; chromosome markers; cytogenetics; cytomolecular analysis; fluorescence in situ hybridisation; interphase nucleus; karyotype; plant molecular cytogenetics; plant nuclear genome.

Figure 1

Milestones in the development and application of DNA–DNA in situ hybridisation, with a specific focus on plant research. The six milestones correspond to the following publications: [3,4,6,19,24,30]. Figure created using BioRender (https://BioRender.com, accessed on 15 July 2025).

Figure 2

Current major areas in plant cytomolecular research (I–V). (A) Karyogram of Cicer arietinum CDC Frontiers (kabuli type) chromosomes, discriminated using FISH on mitotic metaphase chromosomes with a combination of painting probes: CAF-OP1 (green), CAF-OP2 (red), and 5S rDNA (yellow) (adapted from [58]). (B) Ideogram showing the chromosome-level structure of the Catalobus pendulus genome, based on CCP analysis, indicating the positions of 44 genomic blocks on its 15 chromosomes (Cp1–Cp15). The photomicrograph shows an example of CCP with mitotic and pachytene chromosomes Cp9 and Cp10 painted using arabidopsis BAC contigs representing ancestral genomic blocks V, W, and X, respectively (adapted from [59]). (C) Exemplary chromosome translocations identified by oligo-FISH on mitotic metaphase chromosomes of Musa acuminata ITC0660 ‘Khae (Phrae)’, using probes specific for the long arm of chromosome 10 and the short and long arms of chromosome 7 (adapted from [60]). (D,E) FISH with centromeric, telomeric, and chromosome 2-specific probes applied to ultra-thin root sections of Oryza sativa, prepared using a cryomicrotome (adapted from [61]). The images show evidence of Rabl configuration in xylem (D) and cortex (E) cells. (F) 3D-SIM maximum intensity projections of FISH with the pAL centromeric repeat on structurally preserved, acrylamide-embedded 4C leaf nuclei of arabidopsis WT and cap-d3 mutants. The cap-d3 mutation affects centromere association but does not alter their overall spatial arrangement within the nuclei (adapted from [62]). (G) Changes in the localisation of the arabidopsis nuclear lamina protein CRWN1 under salt stress (adapted from [63]). (H) Multicolour FISH labelling of the Lathyrus sativus homoeologue of pea chromosome 6 using PS6 painting probes, along with probes for the satellite repeats FabTR-54, which fills the gap in the PS6-C signal, and FabTR-2, which is associated with CENH3 chromatin in L. sativus (adapted from [64]). (I) Interaction of the centromeric protein CENH3 (red) with α-tubulin (green) in metaphase chromosomes of Prionium serratum, visualised using spatial SIM (adapted from [65]). (J) Overview of the image analysis workflow for examining the ultrastructure of the Hordeum vulgare 5H metaphase chromosome after FISH using centromeric, 35S rDNA-targeting, telomeric, subtelomeric, and 5HL-specific oligo probes (adapted from [66]). (K,L) Meiotic chromosome behaviour in the autodecaploid Saccharum spontaneum clone Yunnan 82–106 revealed using CP oligo probes (adapted from [67]). (K) Pairing configurations at pachytene showing ten copies of chromosome 8 forming two bivalents and one hexavalent. (L) Five bivalents of chromosome 8 with their detailed structure visualised using dual-colour barcoded painting FISH. (M) GISH using gDNA of Agropyron cristatum (red) reveals the presence and stable meiotic behaviour of the translocated A. cristatum 1P chromosome segment in the wheat background of the T1AS.1PL translocation line (adapted from [68]). (N) Visualisation of Aegilops geniculata chromosomes (indicated by arrows) at mitotic metaphase in a Triticum aestivum–A. geniculata substitution line, using GISH with A. geniculata genomic DNA (green) and FISH with the pTa535 D-genome-specific probe (red) (adapted from [69]). Figure created using BioRender (https://BioRender.com, accessed on 15 July 2025). All materials presented in this figure, except for the photomicrograph in panel (M), were published under the terms of the Creative Commons Attribution (CC-BY 4.0) licence (https://creativecommons.org/licenses/by/4.0/). The image in panel (M) has been published under an exclusive licence to Springer-Verlag GmbH Germany, with the authors of the original publication retaining copyright.