Effective label of XL/XR and Neo-X chromosomes of Drosophila miranda using oligopaints probes
Authors: Henry Bonilla 1; Isabela Pimentel de Almeida 1; Mara Lisboa Santana Pinheiros 1; Maria Vibranovski 1,2
Affiliations: 1) Bioscience Institute, University of São Paulo; 2) New college for Interdisciplinary Arts and Sciences, Arizona State University
Keywords: t. bioinformatic and genome tools; u. other (cytogenetics)
Experiments of fluorescent in-situ hybridization (FISH) are a valuable resource for investigating chromosome evolution, chromosome behavior in individual cells and, ultimately, for assessing the quality of a sequenced genome. Drosophila miranda is a model species to study the evolution of sex chromosomes due to their X chromosomes of different ages. The outcome of independent autosome-sex chromosome fusions produced the so-called XR and Neo-X that emerged about 15 MYA and 1.5 MYA ago, respectively.
Oligopaint represented a new generation of probes (Beliveau et al 2012) that are custom-synthesized oligonucleotides, short (∼26–46 bases), versatile and very specific for targeting single-copy chromosome regions that can be extended to any organism whose genome has been sequenced. Here, we developed oligopaints to label the entire set of X (XL/XR and Neo-X) and the fourth autosomal chromosomes. We used OligoMiner pipeline (Beliveau et al., 2018) and the latest D. miranda genome sequence (Manhajan et al., 2018). The specificity of oligopaints was assessed by FISH experiments on mitotic chromosomes. Shortly, we designed oligopaints under the stringent condition defined in OligoMiner in addition to Kmer and secondary structure filters to guarantee specificity. A Python script was developed to diminish the number of oligos but maintain an appropriate distribution along entire target regions. Finally, flanking primer regions were added to produce oligos from a custom complex oligo-pool library.
We obtained 25553 oligos covering the entire Neo-X chromosome with a density of 1.01 oligos/Kb. The XL/XR chromosome has been divided in four regions according to their different evolutionary history and the genome assembly. By separating the XL arm in pericentromeric and distal regions (XL-1 and XL-2), we obtained 11180 oligos (0.79 oligos/kb) and 27282 (1.08 oligos/kb), respectively. The XR arm was split in XR-A and XR-D. XR-A comes from the ancient X chromosome and it’s located in the pericentromeric region whereas XR-D in the distal portion of the XR arm. We obtained 12717 oligos (1.02) and 25382 oligos (0.99) in the XR-A and XR-D respectively. Only 10336 oligos (0.32 oligos/kb) were designed for the fourth chromosome.
FISH experiments on mitotic chromosomes revealed that all of our oligos are target region specific. A complete label on Neo-X and on the fourth chromosome was observed, despite the low oligo density of the latter. This finding is valuable, as there are not many studies on the minimum density of oligos necessary for visible marking along entire chromosomes. Finally, the XL/XR chromosome showed almost complete labeling. There is only a small region between XL-1 and XL-2 that has not been labeled because it could not be assembled into the genome. Our results represent the first oligopants designed for D. miranda which will allow further research on sex chromosomal behavior in different cell types.