Bulked Oligo-FISH for Chromosome Painting and Chromosome Barcoding.

Recently developed bulked oligo-FISH is a highly versatile method, which is applicable in any plant species with an assembled genome sequence. This technique allows in situ identification of individual chromosomes, large chromosomal rearrangements, comparative karyotype analysis, or even the reconstruction of the three-dimensional organization of the genome. The method is based on the identification of thousands of short oligonucleotides, unique to specific genome regions, which are synthesized in parallel, fluorescently labeled and used as probes for FISH. In this chapter, we propose a detailed protocol for amplification and labeling of single-stranded oligo-based painting probes from so-called MYtags immortal libraries, the preparation of mitotic metaphase and meiotic pachytene chromosome spreads, and a protocol for the fluorescence in situ hybridization procedure using the synthetic oligo probes. The proposed protocols are demonstrated for banana (Musa spp.).

Genetic Mapping, Candidate Gene Identification and Marker Validation for Host Plant Resistance to the Race 4 of Fusarium oxysporum f. sp. cubense Using Musa acuminata ssp. malaccensis.

Fusarium wilt of banana is a devastating disease that has decimated banana production worldwide. Host resistance to Fusarium oxysporum f. sp. Cubense (Foc), the causal agent of this disease, is genetically dissected in this study using two Musa acuminata ssp. Malaccensis segregating populations, segregating for Foc Tropical (TR4) and Subtropical (STR4) race 4 resistance. Marker loci and trait association using 11 SNP-based PCR markers allowed the candidate region to be delimited to a 12.9 cM genetic interval corresponding to a 959 kb region on chromosome 3 of 'DH-Pahang' reference assembly v4. Within this region, there was a cluster of pattern recognition receptors, namely leucine-rich repeat ectodomain containing receptor-like protein kinases, cysteine-rich cell-wall-associated protein kinases, and leaf rust 10 disease-resistance locus receptor-like proteins, positioned in an interspersed arrangement. Their transcript levels were rapidly upregulated in the resistant progenies but not in the susceptible F2 progenies at the onset of infection. This suggests that one or several of these genes may control resistance at this locus. To confirm the segregation of single-gene resistance, we generated an inter-cross between the resistant parent 'Ma850' and a susceptible line 'Ma848', to show that the STR4 resistance co-segregated with marker '28820' at this locus. Finally, an informative SNP marker 29730 allowed the locus-specific resistance to be assessed in a collection of diploid and polyploid banana plants. Of the 60 lines screened, 22 lines were predicted to carry resistance at this locus, including lines known to be TR4-resistant, such as 'Pahang', 'SH-3362', 'SH-3217', 'Ma-ITC0250', and 'DH-Pahang/CIRAD 930'. Additional screening in the International Institute for Tropical Agriculture's collection suggests that the dominant allele is common among the elite 'Matooke' NARITA hybrids, as well as in other triploid or tetraploid hybrids derived from East African highland bananas. Fine mapping and candidate gene identification will allow characterization of molecular mechanisms underlying the TR4 resistance. The markers developed in this study can now aid the marker-assisted selection of TR4 resistance in breeding programs around the world.

Identification of a Major QTL-Controlling Resistance to the Subtropical Race 4 of Fusarium oxysporum f. sp. cubense in Musa acuminata ssp. malaccensis.

Vascular wilt caused by the ascomycete fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) is a major constraint of banana production around the world. The virulent race, namely Tropical Race 4, can infect all Cavendish-type banana plants and is now widespread across the globe, causing devastating losses to global banana production. In this study, we characterized Foc Subtropical Race 4 (STR4) resistance in a wild banana relative which, through estimated genome size and ancestry analysis, was confirmed to be Musa acuminata ssp. malaccensis. Using a self-derived F2 population segregating for STR4 resistance, quantitative trait loci sequencing (QTL-seq) was performed on bulks consisting of resistant and susceptible individuals. Changes in SNP index between the bulks revealed a major QTL located on the distal end of the long arm of chromosome 3. Multiple resistance genes are present in this region. Identification of chromosome regions conferring resistance to Foc can facilitate marker assisted selection in breeding programs and paves the way towards identifying genes underpinning resistance.

Migration of repetitive DNAs during evolution of the permanent translocation heterozygosity in the oyster plant (Tradescantia section Rhoeo).

Due to translocation heterozygosity for all chromosomes in the cell complement, the oyster plant (Tradescantia spathacea) forms a complete meiotic ring. It also shows Rabl-arrangement at interphase, featured by polar centromere clustering. We demonstrate that the pericentromeric regions of the oyster plant are homogenized in concert by three subtelomeric sequences: 45S rDNA, (TTTAGGG)n motif, and TSrepI repeat. The Rabl-based clustering of pericentromeric regions may have been an excellent device to combine the subtelomere-pericentromere sequence migration (via inversions) with the pericentromere-pericentromere DNA movement (via whole arm translocations) that altogether led to the concerted homogenization of all the pericentromeric domains by the subtelomeric sequences. We also show that the repetitive sequence landscape of interstitial chromosome regions contains many loci consisting of Arabidopsis-type telomeric sequence or of TSrepI repeat, and it is extensively heterozygous. However, the sequence arrangement on some chromosomal arms suggest segmental inversions that are fully or partially homozygous, a fact that could be explained if the inversions started to create linkages already in a bivalent-forming ancestor. Remarkably, the subterminal TSrepI loci reside exclusively on the longer arms that could be due to sharing sequences between similarly-sized chromosomal arms in the interphase nucleus. Altogether, our study spotlights the supergene system of the oyster plant as an excellent model to link complex chromosome rearrangements, evolution of repetitive sequences, and nuclear architecture.

Chromosome evolution and the genetic basis of agronomically important traits in greater yam.

The nutrient-rich tubers of the greater yam, Dioscorea alata L., provide food and income security for millions of people around the world. Despite its global importance, however, greater yam remains an orphan crop. Here, we address this resource gap by presenting a highly contiguous chromosome-scale genome assembly of D. alata combined with a dense genetic map derived from African breeding populations. The genome sequence reveals an ancient allotetraploidization in the Dioscorea lineage, followed by extensive genome-wide reorganization. Using the genomic tools, we find quantitative trait loci for resistance to anthracnose, a damaging fungal pathogen of yam, and several tuber quality traits. Genomic analysis of breeding lines reveals both extensive inbreeding as well as regions of extensive heterozygosity that may represent interspecific introgression during domestication. These tools and insights will enable yam breeders to unlock the potential of this staple crop and take full advantage of its adaptability to varied environments.

Draft Sequencing Crested Wheatgrass Chromosomes Identified Evolutionary Structural Changes and Genes and Facilitated the Development of SSR Markers.

Crested wheatgrass (Agropyron cristatum), a wild relative of wheat, is an attractive source of genes and alleles for their improvement. Its wider use is hampered by limited knowledge of its complex genome. In this work, individual chromosomes were purified by flow sorting, and DNA shotgun sequencing was performed. The annotation of chromosome-specific sequences characterized the DNA-repeat content and led to the identification of genic sequences. Among them, genic sequences homologous to genes conferring plant disease resistance and involved in plant tolerance to biotic and abiotic stress were identified. Genes belonging to the important groups for breeders involved in different functional categories were found. The analysis of the DNA-repeat content identified a new LTR element, Agrocen, which is enriched in centromeric regions. The colocalization of the element with the centromeric histone H3 variant CENH3 suggested its functional role in the grass centromere. Finally, 159 polymorphic simple-sequence-repeat (SSR) markers were identified, with 72 of them being chromosome- or chromosome-arm-specific, 16 mapping to more than one chromosome, and 71 mapping to all the Agropyron chromosomes. The markers were used to characterize orthologous relationships between A. cristatum and common wheat that will facilitate the introgression breeding of wheat using A. cristatum.

Advances in the Molecular Cytogenetics of Bananas, Family Musaceae.

The banana is a staple food crop and represents an important trade commodity for millions of people living in tropical and subtropical countries. The most important edible banana clones originated from natural crosses between diploid Musa balbisiana and various subspecies of M. acuminata. It is worth mentioning that evolution and speciation in the Musaceae family were accompanied by large-scale chromosome structural changes, indicating possible reasons for lower fertility or complete sterility of these vegetatively propagated clones. Chromosomal changes, often accompanied by changes in genome size, are one of the driving forces underlying speciation in plants. They can clarify the genomic constitution of edible bananas and shed light on their origin and on diversification processes in members of the Musaceae family. This article reviews the development of molecular cytogenetic approaches, ranging from classical fluorescence in situ hybridization (FISH) using common cytogenetic markers to oligo painting FISH. We discuss differences in genome size and chromosome number across the Musaceae family in addition to the development of new chromosome-specific cytogenetic probes and their use in genome structure and comparative karyotype analysis. The impact of these methodological advances on our knowledge of Musa genome evolution at the chromosomal level is demonstrated. In addition to citing published results, we include our own new unpublished results and outline future applications of molecular cytogenetics in banana research.

Fine structure and transcription dynamics of bread wheat ribosomal DNA loci deciphered by a multi-omics approach.

Three out of four RNA components of ribosomes are encoded by 45S ribosomal DNA (rDNA) loci, which are organized as long head-to-tail tandem arrays of nearly identical units, spanning several megabases of sequence. Due to this structure, the rDNA loci are the major sources of gaps in genome assemblies, and gene copy number, sequence composition, and expression status of particular arrays remain elusive, especially in complex genomes harboring multiple loci. Here we conducted a multi-omics study to decipher the 45S rDNA loci in hexaploid bread wheat. Coupling chromosomal genomics with optical mapping, we reconstructed individual rDNA arrays, enabling locus-specific analyses of transcription activity and methylation status from RNA- and bisulfite-sequencing data. We estimated a total of 6,650 rDNA units in the bread wheat genome, with approximately 2,321, 3,910, 253, and 50 gene copies located in short arms of chromosomes 1B, 6B, 5D, and 1A, respectively. Only 1B and 6B loci contributed substantially to rRNA transcription at a roughly 2:1 ratio. The ratio varied among five tissues analyzed (embryo, coleoptile, root tip, primary leaf, mature leaf), being the highest (2.64:1) in mature leaf and lowest (1.72:1) in coleoptile. Cytosine methylation was considerably higher in CHG context in the silenced 5D locus as compared with the active 1B and 6B loci. In conclusion, a fine genomic organization and tissue-specific expression of rDNA loci were deciphered, for the first time, in a complex polyploid species. The results are discussed in the context of wheat evolution and transcription regulation.

Telomere-to-telomere gapless chromosomes of banana using nanopore sequencing.

Long-read technologies hold the promise to obtain more complete genome assemblies and to make them easier. Coupled with long-range technologies, they can reveal the architecture of complex regions, like centromeres or rDNA clusters. These technologies also make it possible to know the complete organization of chromosomes, which remained complicated before even when using genetic maps. However, generating a gapless and telomere-to-telomere assembly is still not trivial, and requires a combination of several technologies and the choice of suitable software. Here, we report a chromosome-scale assembly of a banana genome (Musa acuminata) generated using Oxford Nanopore long-reads. We generated a genome coverage of 177X from a single PromethION flowcell with near 17X with reads longer than 75 kbp. From the 11 chromosomes, 5 were entirely reconstructed in a single contig from telomere to telomere, revealing for the first time the content of complex regions like centromeres or clusters of paralogous genes.

B Chromosomes in Genus Sorghum (Poaceae).

B chromosomes (Bs) are supernumerary dispensable genomic elements that have been reported in several thousand eukaryotic species. Since their discovery, Bs have been subjected to countless studies aiming at the clarification of their origin, composition, and influence on the carriers. Despite these efforts, we still have very limited knowledge of the processes that led to the emergence of Bs, the mechanisms of their transmission, and the effects of Bs on the hosts. In the last decade, sophisticated molecular methods, including next-generation sequencing, have provided powerful tool to help answer some of these questions, but not many species have received much attention yet. In this review, we summarize the currently available information about Bs in the genus Sorghum, which has so far been on the periphery of scientific interest. We present an overview of the occurrence and characteristics of Bs in various Sorghum species, discuss the possible mechanisms involved in their maintenance and elimination, and outline hypotheses of the origin of Bs in this genus.

Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes.

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

Karyotype Differentiation in Cultivated Chickpea Revealed by Oligopainting Fluorescence in situ Hybridization.

Chickpea (Cicer arietinum L.) is one of the main sources of plant proteins in the Indian subcontinent and West Asia, where two different morphotypes, desi and kabuli, are grown. Despite the progress in genome mapping and sequencing, the knowledge of the chickpea genome at the chromosomal level, including the long-range molecular chromosome organization, is limited. Earlier cytogenetic studies in chickpea suffered from a limited number of cytogenetic landmarks and did not permit to identify individual chromosomes in the metaphase spreads or to anchor pseudomolecules to chromosomes in situ. In this study, we developed a system for fast molecular karyotyping for both morphotypes of cultivated chickpea. We demonstrate that even draft genome sequences are adequate to develop oligo-fluorescence in situ hybridization (FISH) barcodes for the identification of chromosomes and comparative analysis among closely related chickpea genotypes. Our results show the potential of oligo-FISH barcoding for the identification of structural changes in chromosomes, which accompanied genome diversification among chickpea cultivars. Moreover, oligo-FISH barcoding in chickpea pointed out some problematic, most probably wrongly assembled regions of the pseudomolecules of both kabuli and desi reference genomes. Thus, oligo-FISH appears as a powerful tool not only for comparative karyotyping but also for the validation of genome assemblies.

The Puzzling Fate of a Lupin Chromosome Revealed by Reciprocal Oligo-FISH and BAC-FISH Mapping.

Old World lupins constitute an interesting model for evolutionary research due to diversity in genome size and chromosome number, indicating evolutionary genome reorganization. It has been hypothesized that the polyploidization event which occurred in the common ancestor of the Fabaceae family was followed by a lineage-specific whole genome triplication (WGT) in the lupin clade, driving chromosome rearrangements. In this study, chromosome-specific markers were used as probes for heterologous fluorescence in situ hybridization (FISH) to identify and characterize structural chromosome changes among the smooth-seeded (Lupinus angustifolius L., Lupinus cryptanthus Shuttlew., Lupinus micranthus Guss.) and rough-seeded (Lupinus cosentinii Guss. and Lupinus pilosus Murr.) lupin species. Comparative cytogenetic mapping was done using FISH with oligonucleotide probes and previously published chromosome-specific bacterial artificial chromosome (BAC) clones. Oligonucleotide probes were designed to cover both arms of chromosome Lang06 of the L. angustifolius reference genome separately. The chromosome was chosen for the in-depth study due to observed structural variability among wild lupin species revealed by BAC-FISH and supplemented by in silico mapping of recently released lupin genome assemblies. The results highlighted changes in synteny within the Lang06 region between the lupin species, including putative translocations, inversions, and/or non-allelic homologous recombination, which would have accompanied the evolution and speciation.

Chromosome Painting in Cultivated Bananas and Their Wild Relatives (Musa spp.) Reveals Differences in Chromosome Structure.

Edible banana cultivars are diploid, triploid, or tetraploid hybrids, which originated by natural cross hybridization between subspecies of diploid Musa acuminata, or between M. acuminata and diploid Musa balbisiana. The participation of two other wild diploid species Musa schizocarpa and Musa textilis was also indicated by molecular studies. The fusion of gametes with structurally different chromosome sets may give rise to progenies with structural chromosome heterozygosity and reduced fertility due to aberrant chromosome pairing and unbalanced chromosome segregation. Only a few translocations have been classified on the genomic level so far, and a comprehensive molecular cytogenetic characterization of cultivars and species of the family Musaceae is still lacking. Fluorescence in situ hybridization (FISH) with chromosome-arm-specific oligo painting probes was used for comparative karyotype analysis in a set of wild Musa species and edible banana clones. The results revealed large differences in chromosome structure, discriminating individual accessions. These results permitted the identification of putative progenitors of cultivated clones and clarified the genomic constitution and evolution of aneuploid banana clones, which seem to be common among the polyploid banana accessions. New insights into the chromosome organization and structural chromosome changes will be a valuable asset in breeding programs, particularly in the selection of appropriate parents for cross hybridization.

Fonio millet genome unlocks African orphan crop diversity for agriculture in a changing climate.

Sustainable food production in the context of climate change necessitates diversification of agriculture and a more efficient utilization of plant genetic resources. Fonio millet (Digitaria exilis) is an orphan African cereal crop with a great potential for dryland agriculture. Here, we establish high-quality genomic resources to facilitate fonio improvement through molecular breeding. These include a chromosome-scale reference assembly and deep re-sequencing of 183 cultivated and wild Digitaria accessions, enabling insights into genetic diversity, population structure, and domestication. Fonio diversity is shaped by climatic, geographic, and ethnolinguistic factors. Two genes associated with seed size and shattering showed signatures of selection. Most known domestication genes from other cereal models however have not experienced strong selection in fonio, providing direct targets to rapidly improve this crop for agriculture in hot and dry environments.

DNA replication and chromosome positioning throughout the interphase in three-dimensional space of plant nuclei.

Despite much recent progress, our understanding of the principles of plant genome organization and its dynamics in three-dimensional space of interphase nuclei remains surprisingly limited. Notably, it is not clear how these processes could be affected by the size of a plant's nuclear genome. In this study, DNA replication timing and interphase chromosome positioning were analyzed in seven Poaceae species that differ in their genome size. To provide a comprehensive picture, a suite of advanced, complementary methods was used: labeling of newly replicated DNA by ethynyl-2'-deoxyuridine, isolation of nuclei at particular cell cycle phases by flow cytometric sorting, three-dimensional immunofluorescence in situ hybridization, and confocal microscopy. Our results revealed conserved dynamics of DNA replication in all species, and a similar replication timing order for telomeres and centromeres, as well as for euchromatin and heterochromatin regions, irrespective of genome size. Moreover, stable chromosome positioning was observed while transitioning through different stages of interphase. These findings expand upon earlier studies in suggesting that a more complex interplay exists between genome size, organization of repetitive DNA sequences along chromosomes, and higher order chromatin structure and its maintenance in interphase, albeit controlled by currently unknown factors.

Comparative analyses of DNA repeats and identification of a novel Fesreba centromeric element in fescues and ryegrasses.

BACKGROUND: Cultivated grasses are an important source of food for domestic animals worldwide. Increased knowledge of their genomes can speed up the development of new cultivars with better quality and greater resistance to biotic and abiotic stresses. The most widely grown grasses are tetraploid ryegrass species (Lolium) and diploid and hexaploid fescue species (Festuca). In this work, we characterized repetitive DNA sequences and their contribution to genome size in five fescue and two ryegrass species as well as one fescue and two ryegrass cultivars. RESULTS: Partial genome sequences produced by Illumina sequencing technology were used for genome-wide comparative analyses with the RepeatExplorer pipeline. Retrotransposons were the most abundant repeat type in all seven grass species. The Athila element of the Ty3/gypsy family showed the most striking differences in copy number between fescues and ryegrasses. The sequence data enabled the assembly of the long terminal repeat (LTR) element Fesreba, which is highly enriched in centromeric and (peri)centromeric regions in all species. A combination of fluorescence in situ hybridization (FISH) with a probe specific to the Fesreba element and immunostaining with centromeric histone H3 (CENH3) antibody showed their co-localization and indicated a possible role of Fesreba in centromere function. CONCLUSIONS: Comparative repeatome analyses in a set of fescues and ryegrasses provided new insights into their genome organization and divergence, including the assembly of the LTR element Fesreba. A new LTR element Fesreba was identified and found in abundance in centromeric regions of the fescues and ryegrasses. It may play a role in the function of their centromeres.

The Formation of Sex Chromosomes in Silene latifolia and S. dioica Was Accompanied by Multiple Chromosomal Rearrangements.

The genus Silene includes a plethora of dioecious and gynodioecious species. Two species, Silene latifolia (white campion) and Silene dioica (red campion), are dioecious plants, having heteromorphic sex chromosomes with an XX/XY sex determination system. The X and Y chromosomes differ mainly in size, DNA content and posttranslational histone modifications. Although it is generally assumed that the sex chromosomes evolved from a single pair of autosomes, it is difficult to distinguish the ancestral pair of chromosomes in related gynodioecious and hermaphroditic plants. We designed an oligo painting probe enriched for X-linked scaffolds from currently available genomic data and used this probe on metaphase chromosomes of S. latifolia (2n = 24, XY), S. dioica (2n = 24, XY), and two gynodioecious species, S. vulgaris (2n = 24) and S. maritima (2n = 24). The X chromosome-specific oligo probe produces a signal specifically on the X and Y chromosomes in S. latifolia and S. dioica, mainly in the subtelomeric regions. Surprisingly, in S. vulgaris and S. maritima, the probe hybridized to three pairs of autosomes labeling their p-arms. This distribution suggests that sex chromosome evolution was accompanied by extensive chromosomal rearrangements in studied dioecious plants.

Chromosome Painting Facilitates Anchoring Reference Genome Sequence to Chromosomes In Situ and Integrated Karyotyping in Banana (Musa Spp.).

Oligo painting FISH was established to identify all chromosomes in banana (Musa spp.) and to anchor pseudomolecules of reference genome sequence of Musa acuminata spp. malaccensis "DH Pahang" to individual chromosomes in situ. A total of 19 chromosome/chromosome-arm specific oligo painting probes were developed and were shown to be suitable for molecular cytogenetic studies in genus Musa. For the first time, molecular karyotypes of diploid M. acuminata spp. malaccensis (A genome), M. balbisiana (B genome), and M. schizocarpa (S genome) from the Eumusa section of Musa, which contributed to the evolution of edible banana cultivars, were established. This was achieved after a combined use of oligo painting probes and a set of previously developed banana cytogenetic markers. The density of oligo painting probes was sufficient to study chromosomal rearrangements on mitotic as well as on meiotic pachytene chromosomes. This advance will enable comparative FISH mapping and identification of chromosomal translocations which accompanied genome evolution and speciation in the family Musaceae.

CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication.

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.