Physical Mapping and Refinement of the Painted Turtle Genome (Chrysemys picta) Inform Amniote Genome Evolution and Challenge Turtle-Bird Chromosomal Conservation

Abstract

Comparative genomics continues illuminating amniote genome evolution, but for many lineages our understanding remains incomplete. Here, we refine the assembly (CPI 3.0.3 NCBI AHGY00000000.2) and develop a cytogenetic map of the painted turtle (Chrysemys picta—CPI) genome, the first in turtles and in vertebrates with temperature-dependent sex determination. A comparison of turtle genomes with those of chicken, selected nonavian reptiles, and human revealed shared and novel genomic features, such as numerous chromosomal rearrangements. The largest conserved syntenic blocks between birds and turtles exist in four macrochromosomes, whereas rearrangements were evident in these and other chromosomes, disproving that turtles and birds retain fully conserved macrochromosomes for greater than 300 Myr. C-banding revealed large heterochromatic blocks in the centromeric region of only few chromosomes. The nucleolar-organizing region (NOR) mapped to a single CPI microchromosome, whereas in some turtles and lizards the NOR maps to nonhomologous sex-chromosomes, thus revealing independent translocations of the NOR in various reptilian lineages. There was no evidence for recent chromosomal fusions as interstitial telomeric-DNA was absent. Some repeat elements (CR1-like, Gypsy) were enriched in the centromeres of five chromosomes, whereas others were widespread in the CPI genome. Bacterial artificial chromosome (BAC) clones were hybridized to 18 of the 25 CPI chromosomes and anchored to a G-banded ideogram. Several CPI sex-determining genes mapped to five chromosomes, and homology was detected between yet other CPI autosomes and the globally nonhomologous sex chromosomes of chicken, other turtles, and squamates, underscoring the independent evolution of vertebrate sex-determining mechanisms.

Chromosomal Rearrangements during Turtle Evolution Altered the Synteny of Genes Involved in Vertebrate Sex Determination

“…c From Badenhorst et al [2015]. The chromosome location of Gata4 was determined in the present study.…”

“…Metaphase chromosome preparations were obtained from cell cultures following standard procedures as described previously [Badenhorst et al, 2015;Montiel et al, 2016Montiel et al, , 2017 for 12 turtle species with either TSD or GSD ranging in diploid number from 2n = 28 to 2n = 66 ( Fig. 1 A): Chrysemys picta (CPI), Trachemys scripta (TSC), Chelydra serpentina (CSE), Glyptemys insculpta (GIN), Staurotypus triporcatus (STR), Sternotherus odoratus (SOD), Apalone spinifera (ASP), Pelodiscus sinensis (PSI), Podocnemis unifilis (PUN), Pelomedusa subrufa (PSU), Chelodina oblonga (COB), and Emydura macquarii (EMA).…”

“…b Chrysemys assembly from NCBI Chrysemys_picta_bellii-3.0.3 [Badenhorst et al, 2015]. c From Badenhorst et al [2015].…”

“…j BAC originally mapped in Chrysemys [Badenhorst et al, 2015] but enriched in repeats which precluded clean hybridization in some more distant turtles.…”

“…These target genes were selected because turtle bacterial artificial chromosome (BAC) clones containing them were available from a BAC library of painted turtle ( Chrysemys picta ) (library VMRC CHY3 produced by the Joint Genome Institute), and they had been previously mapped in the chromosomes of painted turtle ( Table 2 ) [Badenhorst et al, 2015]. BAC clones contain DNA fragments of genomic sequences, 100-200 kb in size, which can be used as probes for in situ hybridization onto chromosomes to detect their physical locations [Janes et al, 2011].…”