Molecular phylogenetic analyses have suggested that extant sauropsids (reptiles and birds) are divided into two major groups, the lineage of Testudines (turtles) and Archosauria (crocodilians and birds) and the lineage of Lepidosauria (tuatara, lizards, worm lizards and snakes) although phylogenetic position of Testudines is still debatable [1–7]. The divergence time between the two lineages has been estimated at around 275 million years [3, 7–9]. Most sauropsidan species have karyotypes consisting of macrochromosomes and microchromosomes, as for birds [10–17], except for crocodilian species, whose karyotypes contain no microchromosomes [18, 19].
Whole genome sequencing of chicken revealed that the overall GC-content of chromosomes increases as chromosomal size decreases, that is, microchromosomes exhibit a higher GC-content than macrochromosomes [20, 21]. In a compositional map of GC-content constructed by 100-kb window analysis for the chicken whole genome sequence, most microchromosomes were occupied by GC-rich DNA segments, whereas GC-poor segments were more common in macrochromosomes . The differences of other features such as gene density, distribution in interphase nuclei and rate of nucleotide divergence were also identified between the two chromosomal groups of birds [23–27].
Reptiles are crucial taxon for tracking genome evolution in amniotes [21, 28, 29]. Intra-genomic GC heterogeneity has been found in reptiles by calculating GC-content in exonic third positions (GC3) [21, 30–33]. Although the use of GC3 as a proxy for genomic GC-content has been controversial , it is known that GC3 generally reflects the local GC-content of the introns and flanking regions of a gene [21, 35–37]. Chojnowski et al.  analyzed the GC3 of more than 6,000 ESTs in the American alligator (Alligator mississippiensis) and suggested that the alligator genome has a certain level of GC heterogeneity. They also examined the isochore structure of the red-eared slider turtle (
) and suggested that the isochore structure of the turtle is intermediate between that of a frog and the GC-rich isochore structures of archosaurs and mammals . However, the chromosomal distribution of the GC heterogeneity has not been fully investigated in reptiles.
We previously constructed a cytogenetic map with 90 cDNA clones for the Chinese soft-shelled turtle (Pelodiscus sinensis), which revealed that the chromosomes have been highly conserved between the turtle and chicken, with the six largest chromosomes being almost equivalent to each other . GC3 of the mapped genes showed a heterogeneous distribution, and orthologs exhibited similar GC3 levels between the turtle, chicken and human, suggesting that the intra-genome GC heterogeneity had already occurred in the last common ancestor of extant amniotes . Furthermore, our results suggested that the turtle microchromosomes tend to contain more GC-rich genes than GC-poor genes, as in chicken .
The green anole lizard (Anolis carolinensis) is the first reptilian species for which whole genomic sequence has been released . Anolis has a homogeneous genome composition compared with other amniotes [37, 39] and, unlike chicken, the GC-content is similar between macro- and microchromosomes . However, it remains unknown whether these genomic characteristics are common to other lepidosaurs or not. Snake karyotypes have been highly conserved within the group, and the usual diploid number is 2n = 36, consisting of eight pairs of macrochromosomes and 10 pairs of microchromosomes [10, 40, 41]. The chromosome number is largely different from the chicken karyotype (2n=78) because of the remarkable difference in the number of microchromosomes. The snake therefore provides an ideal system for exploring changes in GC-content between macro- and microchromosomes over the course of sauropsid evolution.
Previously we constructed a cytogenetic map with 109 cDNA clones for the Japanese four-striped rat snake, Elaphe quadrivirgata (Serpentes, Colubridae) [38, 42]. In this study, we have extended cDNA-based chromosome mapping of the snake genes and consequently constructed a cytogenetic map with a total of 183 genes. We compared GC3 of the mapped snake genes with GC3 of their orthologs of chicken, green anole lizard, Chinese soft-shelled turtle, human and Xenopus tropicalis. This highlighted the chromosome size-dependent GC heterogeneity in the snake genome and the shift of GC-content possibly caused by chromosome rearrangements during sauropsid evolution.