A unique tRNA gene family and a novel, highly expressed ORF in the mitochondrial genome of the silver-lip pearl oyster, Pinctada maxima (Bivalvia: Pteriidae)

Abstract

Characteristics of mitochondrial (mt) DNA such as gene content and arrangement, as well as mt tRNA secondary structure, are frequently used in comparative genomic analyses because they provide valuable phylogenetic information. However, most analyses do not characterize the relationship of tRNA genes from the same mt genome and, in some cases, analyses overlook possible novel open reading frames (ORFs) when the 13 expected protein-coding genes are already annotated. In this study, we describe the sequence and characterization of the complete mt genome of the silver-lip pearl oyster, Pinctada maxima. The 16,994-bp mt genome contains the same 13 protein-coding genes (PCGs) and two ribosomal RNA genes typical of metazoans. The gene arrangement, however, is completely distinct from that of all other available bivalve mt genomes, and a unique tRNA gene family is observed in this genome. The unique tRNA gene family includes two trnS^− AGY and trnQ genes, a trnM isomerism, but it lacks trnS^− CUN. We also report the first clear evidence of alloacceptor tRNA gene recruitment (trnP → trnS^− AGY) in mollusks. In addition, a novel ORF (orfUR1) expressed at high levels is present in the mt genome of this pearl oyster. This gene contains a conserved domain, “Oxidored_q1_N”, which is a member of Complex I and thus may play an important role in key biological functions. Because orfUR1 has a very similar nucleotide composition and codon bias to that of other genes in this genome, we hypothesize that this gene may have been moved to the mt genome via gene transfer from the nuclear genome at an early stage of speciation of P. maxima, or it may have evolved as a result of gene duplication, followed by rapid sequence divergence. Lastly, a 319-bp region was identified as the possible control region (CR) even though it does not correspond to the longest non-coding region in the genome. Unlike other studies of mt genomes, this study compares the evolutionary patterns of all available bivalve mt tRNA and atp8 genes.

Introduction

Though only 55 bivalve mitochondrial (mt) genomes are available in GenBank, they are considered to provide an extreme example of gene rearrangements that occur even among species from the same genus (Milbury and Gaffney, 2005, Xu et al., 2012). Unlike mt gene rearrangements in insects (e.g. Dowton and Austin, 1999), amphibians (e.g. Kurabayashi et al., 2005, Zhang et al., 2008), and even gastropods (e.g. Grande et al., 2008, Rawlings et al., 2010), the mechanism(s) by which the high level of genome rearrangement seen in the Bivalvia is currently unknown. It is very difficult to trace the evolutionary pattern of these rearrangements by comparative analyses using the limited number of representatives currently available (Boore et al., 2004). Therefore, it is not easy to use bivalve mt genomic architecture as characters for phylogenetic inference at high taxonomic levels (e.g., between different orders). Our recent comparative mt genomic analyses of four intrageneric clams (Paphia spp.) revealed that mt genome reorganization among congeneric species is not random but follows phylogenetic trends (Xu et al., 2012). Furthermore, there are many special features of bivalve mt genomes that do provide useful characters for studies of evolutionary genomics. Wu et al. (2009) reported a large number of tRNA gene lost in three scallops (Mimachlamys nobilis, Mizuhopecten yessoensis, Chlamys farreri), while Smith and Snyder (2007) found that the scallop, Placopecten magellanicus, contains 23 additional tRNA genes. Several bivalve lineages (e.g. some clams) lack the atp8 gene, while in Mytilus species and in freshwater mussels a fourteenth mt gene, thought to function in doubly uniparental inheritance (DUI), was identified (Breton et al., 2011a, Breton et al., 2011b).

Previous studies provide the basis for questions to which further genome annotation can be directed. For example, the loss of the atp8 gene in many bivalves raises the following questions: have these species completely lost the atp8 gene? Has the gene been transferred to the nucleus? Or do these bivalves possess a highly modified version of the gene that has been overlooked during genome annotation? Similarly, previous comparative mt genomic analyses were focused primarily on the differences in number and/or location of tRNA genes among different genomes but usually did not survey relationships among the copies of each tRNA gene from the same genome. Such investigation will not only provide direct evidence for tRNA gene recruitment (Lavrov and Lang, 2005, Saks et al., 1998, Wang and Lavrov, 2011) but also help us to understand the organization of tRNA gene duplication. Lastly, if all 13 expected protein-coding genes (PCG) were already annotated, the remaining mt regions were classified as intergenic and “non-coding” (Breton et al., 2011b). A novel mt open reading frame (ORF) would thus be overlooked if it lacked significant similarity to known proteins.

A total of sixteen mt genomes from two families of the order Pterioida were sequenced thus far; in this study, we report the complete mt genome of the silver-lip pearl oyster, Pinctada maxima. This mt genome sequence is the first representative from the third family, Pteriidae, in the order Pterioida. A unique tRNA gene set lacking trnS^− CUN but containing two trnS^− AGY genes and an additional trnQ gene was observed in this genome. We compare all available bivalve mt tRNA genes and discuss their evolutionary patterns. We also report the first clear evidence for alloacceptor tRNA gene recruitment in mollusks. In addition, we describe a novel, highly expressed gene and discuss its potential function and possible origin.