ABSTRACT: BACKGROUND: Mitochondrial DNA sequences are extensively used as genetic markers not only for studies of population or ecological genetics, but also for phylogenetic and evolutionary analyses. Complete mt-sequences can reveal information about gene order and its variation, as well as gene and genome evolution when sequences from multiple phyla are compared. Mitochondrial gene order is highly variable among mollusks, with bivalves exhibiting the most variability. Of the 41 complete mt genomes sequenced so far, 12 are from bivalves. We determined, in the current study, the complete mitochondrial DNA sequence of Crassostrea hongkongensis. We present here an analysis of features of its gene content and genome organization in comparison with two other Crassostrea species to assess the variation within bivalves and among main groups of mollusks. RESULTS: The complete mitochondrial genome of C. hongkongensis was determined using long PCR and a primer walking sequencing strategy with genus-specific primers. The genome is 16,475 bp in length and contains 12 protein-coding genes (the atp8 gene is missing, as in most bivalves), 22 transfer tRNA genes (including a suppressor tRNA gene), and 2 ribosomal RNA genes, all of which appear to be transcribed from the same strand. A striking finding of this study is that a DNA segment containing four tRNA genes (trnk1, trnC, trnQ1 and trnN) and two duplicated or split rRNA gene (rrnL5' and rrnS) are absent from the genome, when compared with that of two other extant Crassostrea species, which is very likely a consequence of loss of a single genomic region present in ancestor of C. hongkongensis. It indicates this region seem to be a "hot spot" of genomic rearrangements over the Crassostrea mt-genomes. The arrangement of protein-coding genes in C. hongkongensis is identical to that of Crassostrea gigas and Crassostrea virginica, but higher amino acid sequence identities are shared between C. hongkongensis and C. gigas than between other pairs. There exists significant codon bias, favoring codons ending in A or T and against those ending with C. Pair analysis of genome rearrangements showed that the rearrangement distance is great between C. gigas-C. hongkongensis and C. virginica, indicating a high degree of rearrangements within Crassostrea. The determination of complete mt-genome of C. hongkongensis has yielded useful insight into features of gene order, variation, and evolution of Crassostrea and bivalve mt-genomes. CONCLUSION: The mt-genome of C. hongkongensis shares some similarity with, and interesting differences to, other Crassostrea species and bivalves. The absence of trnC and trnN genes and duplicated or split rRNA genes from the C. hongkongensis genome is a completely novel feature not previously reported in Crassostrea species. The phenomenon is likely due to the loss of a segment that is present in other Crassostrea species and was present in ancestor of C. hongkongensis, thus a case of "tandem duplication-random loss (TDRL)". The mt-genome and new feature presented here reveal and underline the high level variation of gene order and gene content in Crassostrea and bivalves, inspiring more research to gain understanding to mechanisms underlying gene and genome evolution in bivalves and mollusks.
