Comparative Biochemistry and Physiology, Part D 5 (2010) 256–264 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part D journal homepage: www.elsevier.com/locate/cbpd Complete mtDNA of Meretrix lusoria (Bivalvia: Veneridae) reveals the presence of an atp8 gene, length variation and heteroplasmy in the control region Hongxia Wang a, Suping Zhang a, Yang Li a,b, Baozhong Liu a,⁎ a Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China b Graduate School of the Chinese Academy of Sciences, Beijing 100039, China article info abstract Article history: The complete nucleotide sequence of the mitochondrial genome of the clam Meretrix lusoria (Bivalvia: Received 12 April 2010 Veneridae) was determined. It comprises 20,268 base pairs (bp) and contains 13 protein-coding genes, Received in revised form 16 July 2010 including ATPase subunit 8 (atp8), two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. Accepted 19 July 2010 The atp8 encodes a protein of 39 amino acids. All genes are encoded on the same strand. A putative control Available online 3 August 2010 region (CR or D-loop) was identified in the major non-coding region (NCR) between the tRNAGly and tRNAGln. A 1087 bp tandem repeat fragment was identified that comprises nearly 11 copies of a 101 bp motif and Keywords: Meretrix lusoria accounts for approximately 41% of the NCR. The 101 bp tandem repeat motif of the NCR can be folded into a mtDNA stem–loop secondary structure. Samples of eight individuals from Hainan and Fujian provinces were atp8 collected and their NCR regions were successfully amplified and sequenced. The data revealed a highly D-loop polymorphic VNTR (variable number of tandem repeats) associated with high levels of heteroplasmy in the VNTR D-loop region. The size of the CR ranged from 1942 to 3354 bp depending upon the copy number of the Heteroplasmy repeat sequence. © 2010 Elsevier Inc. All rights reserved. 1. Introduction due to its compact nature, maternal inheritance, and fast evolutionary rate compared to nuclear DNA (Moore, 1995). Recently, the number of The genus Meretrix contains few species, most of them have available complete mitogenome sequences has increased consider- commercial importance. The genus is widely distributed along the ably. To date, the whole bivalve mtDNA sequences in NCBI (http:// coastal and estuarine areas of the western Indian Ocean and Western www.ncbi.nlm.nih.gov) represent six orders and nine families within Pacific, including China, North Korea, Japan, and Southeast Asia the class. For the Meretrix genus, complete mitochondrial (mt) (Zhuang, 2001). Though research on the aquaculture and fishery of genome M. petechialis (Ren et al., 2009a)isavailable.More Meretrix species has received much attention, their classification is mitochondrial genomic information from various Meretrix species is ambiguous due to their low morphological variability and similar soft- required to effectively advance this research, because genome level tissue anatomies. Until now, the taxonomy of Meretrix species has characteristics are much more informative than single genes, such as been controversial (Jukes-Browne, 1914; Habe, 1977; Zhuang, 2001). the COI and lrRNA. Compared to other metazoans, the bivalve Among them, the clam Meretrix lusoria is the major commercial mitogenome displays an extraordinary amount of mtDNA variation, marine bivalve species currently being cultured in the benthic for example, the additional copy of the tRNA gene and the loss of tRNA environment of the South China area. Due to its commercial value, (Hoffmann et al., 1992; Milbury and Gaffney, 2005), the duplicated accurate species identification is needed for resource management rrnS and rrnL (Milbury and Gaffney, 2005), and the lack of an atp8 and conservation. (Zbawickaa et al., 2007). Furthermore, bivalve mitogenomes reported The various Meretrix species show morphological parallelism, so far have a high amount of mitochondrial gene rearrangement. systematic descriptions of Meretrix species are often confusing, and Among the currently available taxa, there are very few shared gene the specific name M. meretrix has apparently been used for various blocks. An additional complication in the Bivalvia (seven families: species (Yoosukh and Matsukuma, 2001). Therefore, DNA sequence Mytilidae, Unionidae, Margaritiferidae, Hyriidae, Donacidae, Soleni- information will be optional and hopeful for their identification. dae, and Veneridae) is the unusual mode of inheritance for mtDNA, MtDNA has been used for studying population structure, phylogeo- termed doubly uniparental inheritance (DUI) (Zouros et al., 1994; graphy, and phylogenetic relationships at various taxonomic levels Mizi et al., 2005; Theologidis et al., 2008, and references therein). This biparental transmission system challenged the paradigm of strict maternal inheritance (SMI), and it provides an opportunity for ⁎ Corresponding author. Tel.: +86 532 82898696; fax: +86 532 82898578. studying nuclear-cytoplasmic genome interactions and the evolu- E-mail address: [email protected] (B. Liu). tionary significance of different modes of mitochondrial inheritance 1744-117X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbd.2010.07.003 H. Wang et al. / Comparative Biochemistry and Physiology, Part D 5 (2010) 256–264 257 (Breton et al., 2007). Theologidis et al. (2008) discussed that the ation at 94 °C for 2 min followed by denaturation at 94 °C for 20 s, sporadic detection DUI phenomenon in bivalvia might be due to the annealing at 56 °C for 40 s, and extension at 72 °C for 7 min, for 34 difficulty of detection, as an alternative to the genuine lack of DUI. So it cycles. The amplified lengths of the three PCR products were suggested that DUI might be far more common than is currently approximately 7.5 kb, 4.6 kb, and 7.8 kb, respectively. thought. So far, whether the DUI phenomenon is present in genus Meretrix or not is a puzzle. 2.3. Cloning and DNA sequencing In addition, the mitochondrial DNA length variation and hetero- plasmy is a common phenomenon among animal taxa, including fish The three long PCR products were sub-cloned and sequenced using species (Cesaroni et al., 1997), mammals (Mignotte et al., 1990; primer walking. The PCR products were sheared into 1–2kb Wilkinson and Chapman, 1991), lizards (Moritz and Brown, 1987), fragments using a JY92-II ultrasonic cell crusher (Ningbo Scientz crickets (Rand and Harrison, 1989), nematodes (Powers et al., 1986), Biotechnology co., Ltd.). Fragments of approximately 1.5 kb were etc. In bivalves, the sea scallop Placopecten magellanicus (La Roche et recovered by QIAquick gel extraction kit (QIAGEN). Subsequently, the al., 1990), M. petechialis (Ren et al., 2009a) and genus Mytilus (Cao et 5′ ends of the purified DNA fragment were phosphorylated and the 3′ al., 2009) also have been reported about the existence of variable copy ends were blunted using the TaKaRa BKL Kit (Takara), followed by number of a repeated sequence and heteroplasmy in the mtDNA. A ligation into the pUC118-Hinc II vector, according to the manufac- comparison of the characteristics of the repeated arrays in animal turer's instructions. The ligation products were transferred into mtDNA might help to explain the generation and maintenance of this competent Escherichia coli DH10α cells using the heat shock method. source of genetic diversity. Three sub-clone plasmid libraries were constructed corresponding to In this paper, we report the complete nucleotide sequence of the the three long PCR products. The recombinant plasmids were screen mitochondrial genome from the clam M. lusoria, which exhibits via the white-blue plaque plate method (contain X-gal, IPTG, Amp LB), mtDNA size variation and heteroplasmy. We also detected the atp8 and the positive white colonies which contained inserts of about gene encoding a protein of 39 amino acids with complete start and 1.5 kb were screened by PCR using the vector universal primers, M13F stop codons in the genus Meretrix species. Furthermore, the boundary and M13R. Subsequently, the plasmids were extracted and sequenced annotations of rrnS and rrnL in some species of Veneroida were on an ABI 3730x1 DNA Analyzer. The complete mtDNA sequence was revised. The present study provides data that contributes to a better mostly obtained by clone sequencing and, to a lesser degree, by the understanding of the phylogenic relationships of genus Meretrix, and primer walking strategy for gap filling and correction. supply useful M. lusoria specific markers for identification. Also, this work may facilitate studies on population structure, genetic diversity, 2.4. VNTR amplification and sequence and broodstock management of the hard clam M. lusoria. Characterization of the M. lusoria VNTRs (variable number of 2. Materials and methods tandem repeats) was done through PCR amplification of the repeat region from two populations Sanya and Xiamen. A pair of species- 2.1. Sample collection and DNA isolation specific primers NCR1218 F (5′-AGT TGG GCG TTA ATC ATA GGG-3′) and trn-Gln R (5′-CAA AAA CCA AAC AAC TAC AC-3′) was designed to M. lusoria specimens were obtained from Sanya, Hainan, China (E amplify the tandem repeat region (Fig. 1A). For each individual 109°32′ N 18°13′) and Xiamen, Fujian, China (E 118°06′ N 24°27′). sample, about 20 ng of total genomic DNA was used to amplify the The adductor muscle was slit and the whole adult clam was VNTR of the NCR region, the DNA of the eight individuals come from immediately preserved in 95% ethanol. Total genomic DNA was 2.1. PCR conditions were as follows: initial denaturation at 94 °C for extracted from the foot muscle tissues of eight individuals using a 2 min followed by denaturation at 94 °C for 20 s, annealing at 55 °C for DNeasy tissue DNA extraction kit (Promega) following the manufac- 40 s, and extension at 72 °C for 2 min, for 34 cycles. The PCR product turer's instructions and stored at −20 °C.