Protocol for reconstitution of myTags in situ hybridization products

Cytogenomic resources have accelerated synteny and chromosome evolution studies in plant species, including legumes. Here, we established the first cytogenetic map of V. angularis (Va, subgenus Ceratotropis) and compared this new map with those of V. unguiculata (Vu, subgenus Vigna) and P. vulgaris (Pv) by BAC-FISH and oligopainting approaches. We mapped 19 Vu BACs and 35S rDNA probes to the 11 chromosome pairs of Va, Vu, and Pv. Vigna angularis shared a high degree of macrosynteny with Vu and Pv, with five conserved syntenic chromosomes. Additionally, we developed two oligo probes (Pv2 and Pv3) used to paint Vigna orthologous chromosomes. We confirmed two reciprocal translocations (chromosomes 2 and 3 and 1 and 8) that have occurred after the Vigna and Phaseolus divergence (~9.7 Mya). Besides, two inversions (2 and 4) and one translocation (1 and 5) have occurred after Vigna and Ceratotropis subgenera separation (~3.6 Mya). We also observed distinct oligopainting patterns for chromosomes 2 and 3 of Vigna species. Both Vigna species shared similar major rearrangements compared to Pv: one translocation (2 and 3) and one inversion (chromosome 3). The sequence synteny identified additional inversions and/or intrachromosomal translocations involving pericentromeric regions of both orthologous chromosomes. We propose chromosomes 2 and 3 as hotspots for chromosomal rearrangements and de novo centromere formation within and between Vigna and Phaseolus. Our BAC- and oligo-FISH mapping contributed to physically trace the chromosome evolution of Vigna and Phaseolus and its application in further studies of both genera.

Sex determination directs development as male or female in sexually reproducing organisms. Evolutionary transitions in sex determination have occurred frequently, suggesting simple mechanisms behind the transitions, yet their detail remains elusive. Here we explore the links between mechanisms of transitions in sex determination and sex chromosome evolution at both recent and deeper temporal scales (<1 Myr; ~79 Myr). We studied a rare example of a species with intraspecific variation in sex determination, Carinascincus ocellatus, and a relative, Liopholis whitii, using c-banding and mapping of repeat motifs and a custom Y chromosome probe set to identify the sex chromosomes. We identified both unique and conserved regions of the Y chromosome among C. ocellatus populations differing in sex determination. There was no evidence for homology of sex chromosomes between C. ocellatus and L. whitii, suggesting independent evolutionary origins. We discuss sex chromosome homology between members of the subfamily Lygosominae and propose links between sex chromosome evolution, sex determination transitions, and karyotype evolution.

Duckweeds represent a small, free-floating aquatic family (Lemnaceae) of the monocot order Alismatales with the fastest growth rate among flowering plants. They comprise five genera (Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia) varying in genome size and chromosome number. Spirodela polyrhiza had the first sequenced duckweed genome. Cytogenetic maps are available for both species of the genus Spirodela (S. polyrhiza and S. intermedia). However, elucidation of chromosome homeology and evolutionary chromosome rearrangements by cross-FISH using Spirodela BAC probes to species of other duckweed genera has not been successful so far. We investigated the potential of chromosome-specific oligo-FISH probes to address these topics. We designed oligo-FISH probes specific for one S. intermedia and one S. polyrhiza chromosome (Fig. 1a). Our results show that these oligo-probes cross-hybridize with the homeologous regions of the other congeneric species, but are not suitable to uncover chromosomal homeology across duckweeds genera. This is most likely due to too low sequence similarity between the investigated genera and/or too low probe density on the target genomes. Finally, we suggest genus-specific design of oligo-probes to elucidate chromosome evolution across duckweed genera.

Sugarcane (Saccharum spp.) is probably the crop with the most complex genome. Modern cultivars (2n=100-120) are derived from interspecific hybridization between the noble cane S. officinarum (2n=80) and the wild cane S. spontaneum (2n=40-128). We investigated the genome organization of important sugarcane cultivars and their parental species using chromosomespecific probes combined with genomic in situ hybridization (GISH). This allowed the genomic and genetic characterisation of Australian sugarcane cultivars and one of the major contributing parental clones, Mandalay. The S. spontaneum clone Mandalay follows the classical organization of S. spontaneum clones with x=8 with a major discrepancy related to an extra six chromosomes compared to the previously reported 2n=96 for Mandalay’s clone. Our previous results reported the rearrangements between the S. officinarum (x=10) and S. spontaneum (x=8) chromosomes, with a most likely scenario of a two-step process leading to x= 9 and then x=8, where each step involved three chromosomes that were rearranged into two. Further polyploidization led to the wide geographical dispersion of S. spontaneum clones with x= 8. In modern cultivars, the 13-20% of the S. spontaneum contribution originated from cytotypes with x=8. Modern cultivars have mainly 12 copies of each of the first four basic chromosomes and a more variable number for those basic chromosomes whose structure differs between the two parental species. These new insights and cytogenetic tools substantially improve our understanding of the extreme level of complexity of modern sugarcane cultivar genomes and could lead to guiding breeding strategies in the development of new improved varieties for the Australian industry.

Crop brassicas include three diploid [ Brassica rapa (AA; 2 n = 2 x = 16), B. nigra (BB; 2 n = 2 x = 18), and B. oleracea (CC; 2 n = 2 x = 20)] and three derived allotetraploid species. It is difficult to distinguish Brassica chromosomes as they are small and morphologically similar. We aimed to develop a genome-sequence based cytogenetic toolkit for reproducible identification of Brassica chromosomes and their structural variations. A bioinformatic pipeline was used to extract repeat-free sequences from the whole genome assembly of B. rapa . Identified sequences were subsequently used to develop four c. 47-mer oligonucleotide libraries comprising 27,100, 11,084, 9,291, and 16,312 oligonucleotides. We selected these oligonucleotides after removing repeats from 18 identified sites (500–1,000 kb) with 1,997–5,420 oligonucleotides localized at each site in B. rapa . For one set of probes, a new method for amplification or immortalization of the library is described. oligonucleotide probes produced specific and reproducible in situ hybridization patterns for all chromosomes belonging to A, B, C, and R ( Raphanus sativu s) genomes. The probes were able to identify structural changes between the genomes, including translocations, fusions, and deletions. Furthermore, the probes were able to identify a structural translocation between a pak choi and turnip cultivar of B. rapa. Overall, the comparative chromosomal mapping helps understand the role of chromosome structural changes during genome evolution and speciation in the family Brassicaceae. The probes can also be used to identify chromosomes in aneuploids such as addition lines used for gene mapping, and to track transfer of chromosomes in hybridization and breeding programs.

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.

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.

Protocol for using myTags probes to detect RNA in adherent cultured cells