The First Cytogenetic Map of the Tuatara, <i>Sphenodon punctatus</i>

Abstract

Tuatara, Sphenodon punctatus, is the last survivor of the distinctive reptilian order Rhynchocephalia and is a species of extraordinary zoological interest, yet only recently have genomic analyses been undertaken. The karyotype consists of 28 macrochromosomes and 8 microchromosomes. A Bacterial Artificial Chromosome (BAC) library constructed for this species has allowed the first characterization of the tuatara genome. Sequence analysis of 11 fully sequenced BAC clones (∼0.03% coverage) increased the estimate of genome wide GC composition to 47.8%, the highest reported for any vertebrate. Our physical mapping data demonstrate discrete accumulation of repetitive elements in large blocks on some chromosomes, particularly the microchromosomes. We suggest that the large size of the genome (5.0 pg/haploid) is due to the accumulation of repetitive sequences. The microchromosomes of tuatara are rich in repetitive sequences, and the observation of one animal that lacked a microchromosome pair suggests that at least this microchromosome is unnecessary for survival. We used BACs bearing orthologues of known genes to construct a low-coverage cytogenetic map containing 21 markers. We identified a region on chromosome 4 of tuatara that shares homology with 7 Mb of chicken chromosome 2, and therefore the orthologous region of the snake Z chromosome. We identified a region on tuatara chromosome 3 that is orthologous to the chicken Z, and a region on chromosome 9 orthologous to the mammalian X. Since the tuatara determines sex by temperature and has no sex chromosomes, this implies that different tuatara autosome regions are homologous with the sex chromosomes of mammals, birds and snakes. We have identified anchor BAC clones that can be used to reliably mark chromosomes 3–7, 10 and 13, some of which are difficult to distinguish based on morphology alone. Fluorescence in situ hybridization mapping of 18S rDNA confirms the presence of a single NOR located on the long arm of chromosome 7, as previously identified by silver staining. Further work to construct a dense physical map will lead to a better understanding of the dynamics of genome evolution and organization in this isolated species.

Conservation of chromosomes syntenic with avian autosomes in squamate reptiles revealed by comparative chromosome painting

“…Tuataras (Rhynchocephalia), the sister lineage to Squamata, can also be important for reconstruction of the ancestral state of Squamata. In tuatara only preliminary information on gene localization is currently available (O'Meally et al 2009). The few genes mapped so far in the tuatara karyotype do not give any evidence for the fusion of the avian ancestral chromosomes 3, 5 and 7 in this lineage.…”

Molecular cytogenetic map of the central bearded dragon, Pogona vitticeps (Squamata: Agamidae)

“…We also confirmed the locations of 22 P. vitticeps clones previously mapped and sequenced by Ezaz et al (2009b), Patel et al 2010 and Ezaz et al (in preparation). A tammar wallaby (Macropus eugenii) BAC clone (AGI 329J14) that contains the 18S rDNA locus (O'Meally et al 2009) was used to map the nucleolus organizer region (NOR). BAC DNA was extracted using the Promega Wizard Plus SV Minipreps DNA Purification System following the manufacturer's protocol, with volumes scaled up for 15-ml cultures.…”

“…Fluorescence in situ hybridization (FISH) was performed following the protocol described by O'Meally et al (2009). Approximately 200-500 ng of BAC DNA was directly labelled by nick translation incorporating either Spectrum Orange-dUTP or Spectrum Green-dUTP (Abbott Molecular, Inc., Des Plaines, IL).…”

“…Telomere peptide nucleic acid probe Telomeric repeats were mapped using a Cy3-(CCCTAA) 3 peptide nucleic acid (PNA) probe (Bio-Synthesis Inc., Lewisville, TX) as described previously (O'Meally et al 2009). A BAC clone (237P23) that identifies the Z and W microchromosomes, and which also distinguishes between them through differential hybridisation, was mapped to the same slides after hybridization and washing of the PNA probe as described above.…”

Genetic and Epigenetic Changes Involving (Retro)transposons in Animal Hybrids and Polyploids

“…Over-306 all, non-LTR retrotransposons are prevalent in amniote genomes, with LTR retrotransposons taking the back stage, and it has been argued that CR1 retrotransposons dominated the genomic landscape in the ancestral amniote [Shedlock, 2006;Shedlock et al, 2007]. It remains to be seen what kinds of repetitive sequences are associated with formation of microchromosomes in certain reptiles, such as the early-branching tuatara (Sphenodon punctatus) O'Meally et al, 2009;Miller et al, 2012].…”