DOI: 10.5061/dryad.6djh9w14z
title: Fixed allele differences associated with the centromere reveal chromosome morphology and rearrangements in a reptile (Varanus acanthurus Boulenger)

description: Chromosome rearrangements are often implicated with genomic divergence and
 are proposed to be associated with species evolution. Rearrangements alter
 the genomic structure and interfere with homologous recombination by
 isolating a portion of the genome. Integration of multi-platform next
 generation DNA sequencing technologies has enabled putative identification
 of chromosome rearrangements in many taxa, however, integrating these data
 sets with cytogenetics is still uncommon beyond model genetic organisms.
 Therefore, to achieve the ultimate goal for the genomic classification of
 eukaryotic organisms, physical chromosome mapping remains critical. The
 ridge-tailed goannas (Varanus acanthurus BOULENGER) are a group
 of dwarf monitor lizards comprised of several species found throughout
 Northern Australia. These lizards exhibit extreme divergence at both the
 genic and chromosomal levels. The chromosome polymorphisms are widespread
 extending across much of their distribution, raising the question if these
 polymorphisms are homologous within the V.
 acanthurus complex. We used a combined genomic and cytogenetic
 approach to test for homology across divergent populations with
 morphologically similar chromosome rearrangements. We showed that more
 than one chromosome pair was involved with the widespread rearrangements.
 This finding provides evidence to support de
 novo chromosome rearrangements have occurred within populations.
 These chromosome rearrangements are characterised by fixed allele
 differences originating in the vicinity of the centromeric region. We then
 compared this region with several other assembled genomes of reptiles,
 chicken and the platypus. We demonstrated that the synteny of genes in
 chordates remains conserved despite centromere repositioning across these
 taxa.
description: Collection of specimens The geographic
 distinction between a fixed submetacentric race and a polymorphic race was
 along the Barkly tablelands (King et al. 1982), therefore collection
 efforts targeted populations in this region. Individuals were collected by
 hand when sheltering among rocks deposited by road graders along roadsides
 in the Northern Territory and Queensland (Figure 1). Upon collection, the
 animals were placed into muslin bags and then into an insulated box and
 freighted overnight to the Canberra Airport, and then transported to the
 University of Canberra and housed in terrariums as described by Retes and
 Bennett (2001). A laboratory colony was established with these individuals
 from four distinct geographical populations with known chromosome
 rearrangements (King et al. 1982, Dobry et al., 2022). From each
 individual, we collected blood for DNA extraction and tail tissue for
 chromosome preparation. Individuals were morphologically identified to
 species and then genetically profiled using SNP markers and compared with
 museum samples from a separate study that included several species in the
 ridge-tailed goanna species complex (Pavón-vázquez et al. 2022).
 Cell culture, chromosome preparations and karyotype
 analysis  Cell cultures were established from 34
 individuals as described in Dobry et al. (2022). Briefly, the animals were
 washed with chlorohexidine soap and any old scales were removed. A sterile
 scalpel was used to remove approximately 1 cm of the tail tip. The tail
 tissue was then soaked in 6% (v/v) hydrogen peroxide solution for five
 minutes, and then washed with betadine before macerating in Hanks Balanced Salt Solution (Sigma-Aldrich
 Corp. Milwaukee, Wis. USA) with 1x antibiotic-antimycotic (ThermoFisher
 Scientific Australia Pty Ltd., Scoresby,
 Victoria, Australia) as previously described in detail
 (Dobry et al., 2022). Metaphase chromosomes were prepared with standard
 methods as described elsewhere (Dobry et al., 2022, Matsubara et al. 2014;
 Ezaz et al. 2005; 2008). Slides were prepared with 10 microliters of cell
 suspension dropped onto each slide from a height of 20 cm and allowed to
 dry, and then washed with 100 % ethanol then stained using
 Vectashieldâ antifade mounting medium with DAPI (Vector Laboratories).
 Slides were viewed and photographed with a Zeiss Axio Scope A1
 epifluorescence microscope equipped with an AxioCam MRm Rev. 3 (Carl Zeiss
 Ltd., Cambridge, UK) camera,
 and Metasystems Isis FISH Imaging System V 5.5.10
 (Metasystems, Newton, MA, USA) software.  Genome
 sequencing, population genetics analysis and probe design
 A draft genome was sequenced and assembled as described in Zhu et
 al. (2022) and then aligned with flow-sorted chromosome pools for
 chromosomes 6/7 from the Komodo Dragon (Lind et al. 2019; Rovatsos et al.
 2019) using Nucmer (v4.0.0beta2) with parameters -b 500. We focused on the
 6/7 chromosome pools to reduce the complexity of the genome and target the
 predicted chromosome rearrangement which was hypothesized to be chromosome
 6 (King et al. 1982; Dobry et al. 2022). All individuals were sequenced
 using Diversity Arrays Technology (DArT, Bruce, ACT, Australia) for
 SNP analysis (Dobry et al. 2022). Diversity Arrays Technology used
 omicR (Talamantes-becerra et al. 2021) for the alignment of DArT data with
 the putative chromosome 6/7 scaffolds (this was provided by Diversity
 Arrays Technology as part of their service). DArT is a genome complexity
 reduction technology that utilizes restriction enzymes to fractionate the
 genome and incorporates Illumina sequencing to characterize the single
 nucleotide polymorphisms associated with the genome fractions (Kilian et
 al. 2012). We used the dartR package for further downstream analysis of
 the SNP data and chromosome scaffold alignments (Gruber et al. 2018;
 Mijangos et al. 2022). We sorted the SNP data to analyse the data aligning
 to the putative chromosome 6/7 scaffolds. To avoid population-specific
 differences confounding our analysis for chromosome homology between
 populations, we used the western population as a reference because it was
 the only population that had all three karyotype morphologies.
 To identify the specific rearranged region among the scaffolds
 comprising the chromosome 6/7 alignments, we used fixed allele analysis
 for each chromosome identity and observed a pattern of fixed differences
 between homozygous metacentric chromosomes compared to the homozygous
 acrocentric chromosomes (Figure 2). To isolate and identify those loci
 that showed fixed allele differences between karyotype morphologies and
 show the rearranged portion of the chromosome, we subset the SNPs based on
 the fixed differences. We then identified fixed allele differences between
 the two homozygous karyotypes and those that were heterozygous for the
 heterokaryomorphs (Table 2). We identified two scaffolds (178 and 185)
 with increased fixed differences from the other scaffolds. 
 We designed the probe set (myTags®, Arbor Biosciences, Ann Arbor,
 MI, USA) from scaf_178 which was comprised of a population of specific
 oligonucleotides ranging in size from 42 to 46 polynucleotides highly
 specific to single copy regions along the scaffold. First, the scaffold
 was blasted against the Komodo dragon genome sequences (Lind et al.
 2019) to identify regions of high homology. Those regions were then masked
 from the design. A hybrid genome assembly was generated with the Komodo
 dragon reference genome and the input scaffolds from the <em>V.
 acanthurus</em> genome (Zhu et al. 2022). Then the oligonucleotides
 were biotinylated for use in Fluorescence in situ hybridization
 (FISH).    Fluorescence in situ hybridization of
 Custom oligo probes To hybridize the probe, we
 followed the manufacturer's recommendations. In brief, we resuspended
 the probe to a concentration of 100 ng/mL and used 200 ng per slide
 diluted into 38 mL hybridization buffer (BioCare Medical, Pacheco CA), and
 then coverslips were sealed with rubber cement.  The slides were then
 denatured at 68°C for five minutes and then incubated at 37°C for 24-48
 hours. Following incubation, the coverslips were removed, and the slides
 were washed with 0.4x SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7, 0.3%
 (v/v) IGEPAL (Sigma-Aldrich) at 60°C for 2 minutes followed by a second
 wash at room temperature with 2x SSC: 3 M NaCl, 0.3% M sodium citrate, pH
 7, 0.1% (v/v) IGEPAL (Sigma-Aldrich) for 1 minute. The slides were then
 desiccated with an ethanol wash series of 70%, 90% and 100% (v/v) for one
 minute each and allowed to dry completely.  Once dry the slides were
 stained with Vectashield® antifade mounting medium with DAPI (Vector
 Laboratories) and viewed and photographed with a Leica Microsystems
 Thunder Imaging system. Karyotype images were constructed from metaphase
 chromosomes using Adobe Photoshop 2021. Reconstruction
 of the ancestral karyotype and direction of chromosome change 
 We extracted the trimmed SNP sequences for scaffold 178 and
 concatenated the sequence tags for phylogenetic analysis between
 karyotypes and populations using PAUP* 4.0a and Geneious Prime® 2022.2.1.
 The allele tags from DArT are unphased therefore we replaced heterozygous
 positions with standard ambiguity codes and used the concatenated SNPs to
 generate a single sequence across all loci for that scaffold generating a
 single sequence for each individual. We then aligned these sequences with
 Muscle 3.8.425 (Edgar 2004) and used PAUP* (Wilgenbusch and Swofford 2003)
 for inference of a phylogenetic tree.  Determination
 of synteny with other reptiles We sequenced the
 transcriptome as described in a previous study (Zhu et al. 2022). We then
 subset the genes identified from scaffold 178 and used orthofinder (Emms
 and Kelly 2015; Emms and Kelly 2017; Emms and Kelly 2019) and
 genespace  (Lovell et al. 2022) with default parameters for synteny
 visualization across species.  
description: Text Editor Rstudio (Packages required: dartR,
 orthofinder, genespace) Genieous Prime
 Circos