Meiosis of newly formed allopolyploids frequently encounter perturbations induced by the merging of divergent and hybridizable genomes. However, to date, the meiotic properties of allopolyploids with dysploid parental karyotypes have not been studied in detail. The allotetraploid Cucumis ×hytivus (HHCC, 2n = 38) was obtained from interspecific hybridization between C. sativus (CC, 2n = 14) and C. hystrix (HH, 2n = 24) followed by chromosome doubling. The results of this study thus offer an excellent opportunity to explore the meiotic properties of allopolyploids with dysploid parental karyotypes.

Fluorescence in situ hybridization (FISH) was developed more than 30 years ago and has been the most paradigm-changing technique in cytogenetic research. FISH has been used to answer questions related to structure, mutation, and evolution of not only individual chromosomes but also entire genomes. FISH has served as an important tool for chromosome identification in many plant species. This review intends to summarize and discuss key technical development and applications of FISH in plants since 2006. The most significant recent advance of FISH is the development and application of probes based on synthetic oligonucleotides (oligos). Oligos specific to a repetitive DNA sequence, to a specific chromosomal region, or to an entire chromosome can be computationally identified, synthesized in parallel, and fluorescently labeled. Oligo probes designed from conserved DNA sequences from one species can be used among genetically related species, allowing comparative cytogenetic mapping of these species. The advances with synthetic oligo probes will significantly expand the applications of FISH especially in non-model plant species. Recent achievements and future applications of FISH and oligo-FISH are discussed.

The genomes of multicellular organisms are extensively folded into 3D chromosome territories within the nucleus1. Advanced 3D genome-mapping methods that combine proximity ligation and high-throughput sequencing (such as chromosome conformation capture, Hi-C)2, and chromatin immunoprecipitation techniques (such as chromatin interaction analysis by paired-end tag sequencing, ChIA-PET)3, have revealed topologically associating domains4 with frequent chromatin contacts, and have identified chromatin loops mediated by specific protein factors for insulation and regulation of transcription5–7. However, these methods rely on pairwise proximity ligation and reflect population-level views, and thus cannot reveal the detailed nature of chromatin interactions. Although single-cell Hi-C8 potentially overcomes this issue, this method may be limited by the sparsity of data that is inherent to current single-cell assays. Recent advances in microfluidics have opened opportunities for droplet-based genomic analysis9 but this approach has not yet been adapted for chromatin interaction analysis. Here we describe a strategy for multiplex chromatin-interaction analysis via droplet-based and barcode-linked sequencing, which we name ChIA-Drop. We demonstrate the robustness of ChIA-Drop in capturing complex chromatin interactions with single-molecule precision, which has not been possible using methods based on population-level pairwise contacts. By applying ChIA-Drop to Drosophila cells, we show that chromatin topological structures predominantly consist of multiplex chromatin interactions with high heterogeneity; ChIA-Drop also reveals promoter-centred multivalent interactions, which provide topological insights into transcription.

Background: The chromosome-specific probe is a fundamental tool of chromosome painting and has been commonly applied in mammalian species. The technology, however, has not been widely applied in plants due to a lack of methodologies for probe development. Identification and labeling of a large number of oligonucleotides (oligos) specific to a single chromosome offers us an opportunity to establish chromosome-specific probes in plants. However, never before has whole chromosome painting been performed in rice. Results: We developed a pooled chromosome 9-specific probe in rice, which contains 25,000 oligos based on the genome sequence of a japonica rice (Oryza sativa L., AA, 2n = 2× = 24). Chromosome 9 was easily identified in both japonica and indica rice using this chromosome 9-painting probe. The probe was also successfully used to identify and characterize chromosome 9 in additional lines of O. sativa, a translocation line, two new aneuploids associated with chromosome 9 and a wild rice (Oryza eichingeri A. Peter, CC, 2n = 2× = 24). Conclusion: The study reveals that a pool of oligos specific to a chromosome is a useful tool for chromosome painting in rice.

BackgroundArachis contains 80 species that carry many beneficial genes that can be utilized in the genetic improvement of peanut (Arachis hypogaea L. 2n = 4x = 40, genome AABB). Chromosome engineering is a powerful technique by which these genes can be transferred and utilized in cultivated peanut. However, their small chromosomes and insufficient cytological markers have made chromosome identification and studies relating to genome evolution quite difficult. The development of efficient cytological markers or probes is very necessary for both chromosome engineering and genome discrimination in cultivated peanut.ResultsA simple and efficient oligonucleotide multiplex probe to distinguish genomes, chromosomes, and chromosomal aberrations of peanut was developed based on eight single-stranded oligonucleotides (SSONs) derived from repetitive sequences. High-resolution karyotypes of 16 Arachis species, two interspecific F1 hybrids, and one radiation-induced M1 plant were then developed by fluorescence in situ hybridization (FISH) using oligonucleotide multiplex, 45S and 5S rDNAs, and genomic in situ hybridization (GISH) using total genomic DNA of A. duranensis (2n = 2x = 20, AA) and A. ipaënsis (2n = 2x = 20, BB) as probes. Genomes, chromosomes, and aberrations were clearly identifiable in the established karyotypes. All eight cultivars had similar karyotypes, whereas the eight wild species exhibited various chromosomal variations. In addition, a chromosome-specific SSON library was developed based on the single-copy sequence of chromosome 6A of A. duranensis. In combination with repetitive SSONs and rDNA FISH, the single-copy SSON library was applied to identify the corresponding A3 chromosome in the A. duranensis karyotype.ConclusionsThe development of repetitive and single-copy SSON probes for FISH and GISH provides useful tools for the differentiation of chromosomes and identification of structural chromosomal rearrangement. It facilitates the development of high-resolution karyotypes and detection of chromosomal variations in Arachis species. To our knowledge, the methodology presented in this study demonstrates for the first time the correlation between a sequenced chromosome region and a cytologically identified chromosome in peanut.