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.

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 V. acanthurus 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.  

Text Editor

Rstudio (Packages required: dartR, orthofinder, genespace)

Genieous Prime

Circos