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Background  Variations in genome size within and between species have been observed since the 1950 s in diverse taxonomic groups. Serving as model organisms, smooth pufferfish possess the smallest vertebrate genomes. Interestingly, spiny pufferfish from its sister family have genome twice as large as smooth pufferfish. Therefore, comparative genomic analysis between smooth pufferfish and spiny pufferfish is useful for our understanding of genome size evolution in pufferfish.    Results  Ten BAC clones of a spiny pufferfish  Diodon holocanthus  were randomly selected and shotgun sequenced. In total, 776 kb of non-redundant sequences without gap representing 0.1% of the  D. holocanthus  genome were identified, and 77 distinct genes were predicted. In the sequenced  D. holocanthus  genome, 364 kb is homologous with 265 kb of the  Takifugu rubripes  genome, and 223 kb is homologous with 148 kb of the  Tetraodon nigroviridis  genome. The repetitive DNA accounts for 8% of the sequenced  D. holocanthus  genome, which is higher than that in the  T. rubripes  genome (6.89%) and that in the  Te. nigroviridis  genome (4.66%). In the repetitive DNA, 76% is retroelements which account for 6% of the sequenced  D. holocanthus  genome and belong to known families of transposable elements. More than half of retroelements were distributed within genes. In the non-homologous regions, repeat element proportion in  D. holocanthus  genome increased to 10.6% compared with  T. rubripes  and increased to 9.19% compared with  Te. nigroviridis . A comparison of 10 well-defined orthologous genes showed that the average intron size (566 bp) in  D. holocanthus  genome is significantly longer than that in the smooth pufferfish genome (435 bp).    Conclusion  Compared with the smooth pufferfish,  D. holocanthus  has a low gene density and repeat elements rich genome. Genome size variation between  D. holocanthus  and the smooth pufferfish exhibits as length variation between homologous region and different accumulation of non-homologous sequences. The length difference of intron is consistent with the genome size variation between  D. holocanthus  and the smooth pufferfish. Different transposable element accumulation is responsible for genome size variation between  D. holocanthus  and the smooth pufferfish.

Genome size evolution in pufferfish: an insight from BAC clone-based Diodon holocanthus genome sequencing