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The complete mitochondrial genome of Macrobrachium nipponense
Gene 487 (2011) 160–165
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Gene j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e n e
Methods paper
The complete mitochondrial genome of Macrobrachium nipponense Keyi Ma, Jianbin Feng, Jingyun Lin, Jiale Li ⁎ Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai 201306, China
a r t i c l e
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Article history: Accepted 9 July 2011 Available online 30 July 2011 Received by A.J. van Wijnen Keywords: mtDNA Long PCR Sequence analysis Phylogeny
a b s t r a c t The complete mitochondrial (mt) genome sequence plays an important role in the accurate determination of phylogenetic relationships among metazoans. Herein, we determined the complete mt genome sequence, structure and organization of Macrobrachium nipponense (M. nipponense) (GenBank ID: NC_015073.1) and compared it to that of Macrobrachium lanchesteri (M. lanchesteri) and Macrobrachium rosenbergii (M. rosenbergii). The 15,806 base pair (bp) M. nipponense mt genome, which is comprised of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs), is slightly larger than that of M. lanchesteri (15,694 bp, GenBank ID: NC_012217.1) and M. rosenbergii (15,772 bp, GenBank ID: NC_006880.1). The M. nipponense genome contains a high AT content (66.0%), which is a common feature among metazoan mt genomes. Compared with M. lanchesteri and M. rosenbergii, we found a peculiar noncoding region of 950 bp with a microsatellite-like (TA)6 element and many hairpin structures. The 13 PCGs are comprised of a total of 3707 codons, excluding incomplete termination codons, and the most frequently used amino acid is Leu (16.0%). The predicted start codons in the M. nipponense mt genome include ATG, ATC and ATA. Seven PCGs use TAA as a stop codon, whereas two use TAG, three use T and only one uses TA. Twentythree of the genes are encoded on the L strand, and ND1, ND4, ND5, ND4L, 12S rRNA, 16S rRNA, tRNA His, tRNA Pro, tRNA Phe, tRNA Val, tRNA Gln, tRNACys, tRNA Tyr and a tRNA Leu are encoded on the H strand. The two rRNAs of M. nipponense and M. rosenbergii are encoded on the H strand, whereas the M. lanchesteri rRNAs are encoded on the L stand. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Mitochondria are eukaryotic organelles that are responsible for the majority of cellular ATP production (Dimijian, 2000; Hedges et al., 2001). Characteristics such as a relatively rapid mutation rate, a maternal inheritance pattern and a presumed lack of intermolecular recombination have led to the extensive use of mitochondrial DNA
Abbreviations: mt, mitochondrial; bp, base pair; tRNAs, transfer RNAs; ATP, adenosine triphosphate; nt, nucleotides; MP, Maximum-Parsimony; L strand, Light strand; Val(V), valine; Leu(L), leucine; Met(M), methionine; Pro(P), proline; Ser(S), serine; Gln(Q), glutamine; Trp(W), tryptophane; Ile(I), isoleucine; Thr(T), threonine; Tyr(Y), tyrosine; COX1, cytochrome c oxidase subunit 1; COX3, cytochrome c oxidase subunit 3; ATP8, ATP synthase F0 subunit 8; ND2, NADH dehydrogenase subunit 2; ND4, NADH dehydrogenase subunit 4; ND5, NADH dehydrogenase subunit 5; M., Macrobrachium; PCGs, protein-coding genes; rRNAs, ribosomal RNAs; NC, non-coding; ML, Maximum-Likelihood; CR, control region; H strand, High strand; Ala, alanine; Arg(R), arginine; Asn(N), asparagine; Asp(D), aspartic; Cys(C), cysteine; Glu(E), glutamic; His(H), histidine; Lys(K), lysine; Gly(G), glycine; CYTB, cytochrome b; COX2, cytochrome c oxidase subunit 2; ATP6, ATP synthase F0 subunit 6; ND1, NADH dehydrogenase subunit 1; ND3, NADH dehydrogenase subunit 3; ND4L, NADH dehydrogenase subunit 4L; ND6, NADH dehydrogenase subunit 6. ⁎ Corresponding author at: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China. Tel./fax: + 86 21 61900401. E-mail addresses: [email protected] (K. Ma), [email protected] (J. Li). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.07.017
(mtDNA) sequence information in investigations of population structure and phylogenetic relationships at different taxonomic levels (Avise, 1994; Avise, 2000). In general, the genomes of nearly all metazoan animals are small, extrachromosomal, circular DNA molecules that range in size from 14 to 48 kb (Boore, 1999; Wolstenholme, 1992). The typical mitochondrium contains 13 proteins that comprise the electron transport chain (COX1-3, CYTB, ND1-6, ND4L, ATP6 and ATP8), two ribosomal RNAs (16S rRNA and 12S rRNA) and 22 transfer RNAs (tRNAs). In addition, a large non-coding (NC) region contains sequences essential for the initiation of transcription and gene replication (Ki et al., 2010). Analyses of entire mitochondrial (mt) genomes have contributed to the resolution of many phylogenetic problems, because mt genes are present in a single copy, have a maternal mode of inheritance and exhibit a higher rate of base substitutions than most nuclear genes (Peng et al., 2007). Notably, complete mtDNA sequences can be informative at deep phylogenetic levels (Curole and Kocher, 1999), and their phylogenetic utility has been demonstrated in several animal taxa, including invertebrates (Boore and Brown, 2000; Grande et al., 2008). Crustaceans are one of the most diversified groups of aquatic invertebrates in freshwater and marine systems. Based on the recently revised taxonomic system, this phylum comprises 849 extant families in 42 orders and 6 classes (Martin and Davis, 2001). While a large number of crustacean species have been described morphologically, relatively few complete crustacean mt genomes have been sequenced
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to date: approximately 50 mt genomes from seven classes of Crustacea have been deposited in public databases (NCBI). This is primarily due to technical difficulties associated with the amplification of complete mt genomes using the long polymerase chain reaction (PCR) (Jung et al., 2006; Ki et al., 2009a, b; Machida et al., 2002). Failed amplification may be due to several factors, including gene rearrangements, AT-rich regions, poly-A (T) stretches or hairpin structures. The decapods are an extremely diverse group of crustaceans with many species of commercial importance. The oriental river prawn Macrobrachium nipponense (M. nipponense) is widely distributed in freshwaters of low salinity regions of the estuary in China, and they can also be found in most regions of Japan, Korea, Vietnam and Myanmar (Yu and Miyake, 1972). M. nipponense is the most important commercial freshwater prawn in both China and Vietnam (Feng et al., 2008). Although M. nipponense has been targeted commercially in China, genetic information essential to the sustainable management of this valuable resource, such as knowledge of its population structure, is lacking. In order to overcome these limitations, information from rapidly evolving gene regions is required. In the current study, we report the complete nucleotide sequence and gene arrangement of the M. nipponense mt genome, and we determine its genomic structure, gene order, codon usage and base composition. The results of the current study will contribute significantly to future investigations of crustacean genomics and phylogenetics. 2. Materials and methods 2.1. Tissue samples and mitochondrial DNA extraction Somatic tissues from a single female M. nipponense were collected from Shanghai, China, and were excised and stored at −70 °C. Mitochondrial DNA was isolated and purified from the somatic tissues (Tamura and Aptsuka, 1988).
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2.3. Gene identification and determination of tRNA structures ClustalW sequence alignments were performed using Lasergene 6 SeqMan, MegAlign and EditSeq 7 (DNASTAR). The boundaries of the protein-coding genes (PCGs) and rRNA genes were inferred by comparisons with the amino acid or nucleotide sequences of other metazoans (Zheng et al., 2010). The 5′ and 3′ ends of the 16S rRNA and 12S rRNA genes were assumed to be adjacent to the flanking tRNA genes or protein genes. Transfer RNA genes were identified using the tRNAscan-SE program (version 1.21, http://lowelab.ucsc.edu/tRNAscan-SE/Lowe and Eddy, 1997) with the invertebrate mt codon sequences. This specialized tRNA search server scans sequences for cloverleaf secondary structure, anticodon sequences and thermodynamic stability.
2.4. Phylogenetic analysis To illustrate the phylogenetic relationship of Decapoda, the other 34 available complete mt genomes were obtained from GenBank. Phobaeticus serratipes from Insecta served as outgroup. Amino acid sequences of 13 concatenated protein-coding genes (PCGs) were used in phylogenetic analysis. The alignment of the amino acid sequences of each 13 PCGs was aligned with Clustal X (Thompson et al., 1997) using default settings. Then the 13 separate amino acid sequence alignments were concatenated to a single multiple sequence alignment. We used two different inference methods: Maximum-Likelihood (ML) and Maximum-Parsimony (MP) to reconstruct phylogenetic relationships of Decapoda. The ML analysis was conducted with PHYML 3.0 program (Guindon and Gascuel, 2003) and 1000 bootstraps were used to estimate the node reliability. MP was performed using MEGA 4.0 (Tamura et al., 2007) at the default setting based on 100 replications of random addition and 500 bootstrap tests of inferred phylogeny.
2.2. Long-polymerase chain reaction (long PCR) PCR amplification of a portion of COX1 was performed using LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) primers (Folmer et al., 1994) and the following PCR cycle conditions: 94 °C for 3 min; a hot start by adding LA Taq polymerase (TaKaRa) at 72 °C; 37 cycles of 94 °C for 30 s, 43 °C for 30 s and 72 °C for 1 min and incubation at 72 °C for 5 min. In addition, PCR amplification of a portion of the 16S rRNA gene was performed using 16SA (5′-CGC CTG TTT AAC AAA AAC AT-3′) and 16SB (5′-CCG GTT GAA CTC AGA TCA-3′) primers (Ui et al., 2001) and the following PCR cycle conditions: 94 °C for 3 min; a hot start by adding LA Taq polymerase (TaKaRa) at 72 °C; 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min and incubation at 72 °C for 10 min. The two M. nipponense genes were partially sequenced, including 577 bp of COX1 and 317 bp of 16S rRNA. Based on the partial M. nipponense mt gene sequences, we designed and synthesized two pairs of primers (CS and SC) whose sequences were as follows: CS-F (5′-ATT GAA GGC TTG AAT GAA AGG TTG-3′), CS-R (5′-TAG AAA GAG GAG TAG GCA CAG GAT-3′), SC-F (5′CCT GTG CCT ACT CCT CTT TCT A-3′) and SC-R (5′-CTA CTG ACT ATG CTA CCT TCG C-3′). The CS fragment of approximately 4 kb and the SC fragment of approximately 12 kb were obtained using long PCR amplification (TaKaRa). The PCR reaction volume was 50 μl, and PCR conditions with the CS/SC primer sets were as follows: 94 °C for 1 min; 35 cycles of 98 °C for 10 s, 58 °C/53 °C for 30 s, and 68 °C for 6/15 min and an incubation at 72 °C for 10 min. With both primer sets, an aliquot of the PCR reaction yielded a single band on a 1% agarose gel stained with ethidium bromide. PCR products were sequenced using Shot Gun Sequencing by Map Biotechnology Co., Ltd. (Shanghai).
Fig. 1. Genetic map of the M. nipponense mt genome. COX1-3 indicates cytochrome oxidase subunits 1–3; ATP6/8, ATPase subunits 6–8; ND1-6/4L, NADH dehydrogenase subunits 1–6 and 4L and CYTB, cytochrome b. Transfer RNA genes are designated as tRNA with the corresponding three-letter amino acid code.
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(67.1%). Notably, the AT content of M. nipponense mtDNA is higher than that of many invertebrate mt genomes.
Table 1 Characteristics of the mt genomes of M. nipponense and two related species. Species
13 protein genes 22 tRNA genes 16S rRNA genes 12S rRNA genes Control region Noncoding regions mtDNA
M. nipponense
M. rosenbergii
M. lanchesteri
A + T (%)
A + T (%)
A + T (%)
64.3 65.7 68.8 69.0 79.9 73.6 66.0
60.1 64.7 66.0 66.0 75.7 60.3 62.3
65.2 66.4 71.6 70.3 82.7 72.4 67.1
3.2. Codon usage and sequence features of protein-coding genes
3. Results and discussion 3.1. Mitochondrial genome organization The complete mt genome sequence of M. nipponense is 15,806 bp in length, which is similar to M. lanchesteri (15,694 bp) and M. rosenbergii (15,772 bp), and its structural organization is depicted in Fig. 1. It consists of 13 PCGs, 2 rRNAs, 22 tRNAs and 17 NC regions (including a 950 bp control region [CR]). Genes including COX1 to tRNAGlu and seven additional genes (tRNAThr, ND6, CYTB, tRNASer, tRNAIle, tRNAMet and ND2) are encoded on the L strand, while the remaining genes (4 PCGs, 2 rRNAs, and 8 tRNAs) are encoded on the H strand. Overall, four overlaps between PCGs (7 bp between ATP8 and ATP6, 1 bp between ATP6 and COX3, 7 bp between ND4 and ND4L and 1 bp between ND6 and CYTB) were detected in the M. nipponense mt genome. In addition, three overlaps between tRNAs were identified. The overall AT content of M. nipponense mtDNA is 66.0%, which reflects the typical AT-rich pattern associated with invertebrate mt genomes. This value is higher than that of M. rosenbergii (62.3%) (Table 1) and lower than that of M. lanchesteri
Table 2 Codon usage for 13 mt PCGs of M. nipponense. Amino acid
Codon
N
%
Amino acid
Codon
N
%
Ala (A) Ala (A) Ala (A) Ala (A) Arg (R) Arg (R) Arg (R) Arg (R) Asn (N) Asn (N) Asp (D) Asp (D) Cys (C) Cys (C) Gln (Q) Gln (Q) Glu (E) Glu (E) Trp (W) Trp (W) His (H) His (H) Ile (I) Ile (I) Lys (K) Lys (K) Phe (F) Phe (F) Tyr (Y) Tyr (Y) Stop Stop
GCA GCC GCG GCU CGA CGC CGG CGU AAC AAU GAC GAU UGC UGU CAA CAG GAA GAG UGA UGG CAC CAU AUC AUU AAA AAG UUC UUU UAC UAU UAA UAG
72 55 13 116 43 1 6 13 49 71 39 33 9 41 62 18 53 25 80 17 50 27 70 224 60 24 85 200 35 93 7 2
1.95 1.49 0.35 3.14 1.16 0.03 0.16 0.35 1.33 1.92 1.05 0.89 0.24 1.11 1.68 0.49 1.43 0.68 2.16 0.46 1.35 0.73 1.89 6.06 1.62 0.65 2.30 5.41 0.95 2.51 0.19 0.05
Leu (L) Leu (L) Leu (L) Leu (L) Leu (L) Leu (L) Met (M) Met (M) Pro (P) Pro (P) Pro (P) Pro (P) Ser (S) Ser (S) Ser (S) Ser (S) Ser (S) Ser (S) Ser (S) Ser (S) Thr (T) Thr (T) Thr (T) Thr (T) Gly (G) Gly (G) Gly (G) Gly (G) Val (V) Val (V) Val (V) Val (V)
CUA CUC CUG CUU UUA UUG AUA AUG CCA CCC CCG CCU AGA AGC AGG AGU UCA UCC UCG UCU ACA ACC ACG ACU GGA GGC GGG GGU GUA GUC GUG GUU
113 48 21 98 247 65 135 47 51 33 5 58 61 4 33 37 79 28 8 109 98 44 8 78 108 24 47 70 104 24 29 100
3.06 1.30 0.57 2.65 6.68 1.76 3.65 1.27 1.38 0.89 0.14 1.57 1.65 0.11 0.89 1.00 2.14 0.76 0.22 2.95 2.65 1.19 0.22 2.11 2.92 0.65 1.27 1.89 2.81 0.65 0.78 2.70
N: total number of amino acids; %: percentage of total codon usage. Abbreviated stop codons were excluded.
The codon usage of M. nipponense PCGs is shown in Table 2. There is a clear preference for the use of AT-rich codons and for the presence of an A or T in the third codon position, reflecting the high AT content of the M. nipponense mt genome. The overall AT composition of PCGs in M. nipponense is 64.3%, which is higher than that of M. rosenbergii (60.1%) and similar to that of M. lanchesteri (65.2%). There is a total of 3707 codons comprising 13 PCGs, excluding termination codons. Analysis of the codon usage of the 13 PCGs revealed that three codons are used frequently. The UAA codon is used most frequently (247), followed by AUU (224) and UUU (200), while the CGC, AGC and CCG codons are used only one, four and five times each, respectively. The most frequently encoded amino acids include Leu (16.02%), Ser (9.72%), Ile (7.95%), Phe (7.71%) and Ala (6.93%). Our study showed that the entire mtDNA of M. nipponense excessively favor A and T. A common feature among metazoan mt genomes is a bias towards a higher representation of the nucleotides A and T, which leads to a subsequent bias in the corresponding encoded amino acids. This phenomenon is considered related to the ‘mutational bias-translational selection’ paradigm (Romero et al., 2000), meaning that both mutation and selection have an effect on the bias in codon usage. Ten protein-coding genes (ATP6, ATP8, COX2, COX3, CYTB, ND1, ND3, ND4, ND5 and ND4L) start with ATG, COX1 starts with ACG, ND6 starts with ATC and ND2 starts with ATA. Overall, the initiation codon inferred for 12 of the 13 genes is ATN, which is typical of metazoan mt genomes (Wolstenholme, 1992). Nine genes use complete stop codons, including seven genes with TAA (ATP8, ATP6, COX3, ND3, ND5, ND6 and ND4L) and two genes with TAG (ND1 and ND4), whereas four genes appear to end in incomplete stop codons, including three genes that end with T (COX2, CYTB and ND2) and one gene (COX1) that ends with TA, which are presumably completed as TAA by post-transcriptional polyadenylation (Anderson et al., 1981; Boore, 1999), as interpreted in the case of human mt DNA (Ojala et al., 1981). The boundaries between thirteen PCGs of M. nipponense were identified by sequence comparison with M. rosenbergii and M. lanchesteri, and determined by translation initiation and termination codons. The total length of M. nipponense PCGs is 11,211 bp. The longest PCG is the ND5 gene (1,707 bp), whereas the shortest PCG is the ATP8 gene (159 bp). ATP8 and ATP6 share a 7 bp overlap. Typically, ATP8 immediately precedes ATP6 or even overlaps with ATP6 in a different reading frame (Wolstenholme, 1992). In addition, ATP8 often begins with an MPQL motif and terminates shortly after the WXW sequence
Table 3 Comparisons of 13 mt PCGs in M. nipponense and two related species. Coding genes
No. of amino acids
Similarity (%)
M. nipponense
M. rosenbergii
M. lanchesteri
nr
nl
COX1 COX2 ATP8 ATP6 COX3 ND3 ND5 ND4 ND4L ND6 CYTB ND1 ND2
511 228 52 224 262 117 568 444 99 171 378 313 331
511 229 52 224 264 117 568 444 99 171 377 312 331
511 229 52 224 262 117 568 444 99 171 378 312 331
98 92 79 92 92 86 92 91 97 76 96 95 86
98 95 79 92 96 89 90 90 94 76 98 94 85
nr: similarity between M. nipponense and M. rosenbergii; nl: similarity between M. nipponense and M. lanchesteri.
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(Hoffmann et al., 1992); however, in M. nipponense, M. rosenbergii and M. lanchesteri, ATP8 begins with an MPQM motif. ATP6 shares 1 bp with COX3, ND6 shares 1 bp with CYTB and ND4L overlaps with ND4 by 7 bp. The similarity of amino acids encoded by PCGs between M. nipponense, M. rosenbergii and M. lanchesteri is somewhat different; amino acids encoded by COX1 have the highest similarity, followed by CYTB, whereas amino acids encoded by ND6 are the least similar (Table 3). 3.3. Transfer RNA and rRNA genes The 22 tRNAs encoded by the mt genome of the M. nipponense, which are identified on the basis of their respective anticodons and secondary structures (data not shown), vary in length from 60 (tRNA Leu) to 69 (tRNA Glu, tRNA Ser and tRNA Trp) nucleotides, and include two tRNA Leu and two tRNA Ser. These tRNAs correspond to the standard set found in other metazoan mtDNAs. Gene lengths and anticodon sequences are congruent with those described for other crustaceans. The anticodon sequences are identical to those reported for M. rosenbergii (Miller et al., 2005). The acceptor stem has seven nucleotides (nt) in M. nipponense, although there is one or two mismatched base pairs. The dihydrouridine arm consists of a stem of three or four paired nt and a loop of 4–9 unpaired nt, and the stems also have some mismatched termini (Tyr, Ala, Gln, etc.). Meanwhile, the TψC arm has a stem of 3–5 paired nt with one mismatched terminus (only found in Pro and Phe) and a loop of 3–7 unpaired nt. The anticodon stem consists of four or five nt pairs, while some tRNAs have one or two mismatched base pairs. The tRNALys gene of M. nipponense, like that of M. rosenbergii, contains UUU anticodons. Invertebrate tRNA anticodon sequences are generally conserved, although variation at the third (i.e., wobble) position (e.g. G–U) of the tRNALys gene is not uncommon (Beard et al., 1993; Boore and Brown, 1994; Yamauchi et al., 2002). The AT nucleotide frequency of the pooled tRNAs is similar to that of the PCGs and two rRNAs (65.7%). The lengths of the M. nipponense mtDNA-encoded 16S rRNA and 12S rRNA genes are 1305 and 852 bp, respectively, and the AT contents are 68.8% and 69.0%, respectively, which are within the range observed in other crustaceans. The 16S rRNA gene lies between the tRNALeu (CUA) and tRNAVal genes, while the 12S rRNA gene lies between the tRNAVal gene and the CR; both rRNA genes are encoded on the H strand.
Fig. 2. The largest potential hairpin structures found within CR located between 12S rRNA and tRNAIle genes of M. nipponense.
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3.4. NC regions There are as many as 17 NC regions found throughout the M. nipponense mt genome, ranging in size from 1 to 950 bp. Unlike vertebrates, the mtDNA control regions of invertebrates are not well characterized, due to lacking discrete conserved sequence blocks (Serb and Lydeard, 2003). In order to confirm the CR of M. nipponense, we searched for some peculiar patterns (e.g. AT-rich, hairpin structures), which were generally found in CR (Kumazawa et al., 1998; Saito et al., 2005). Finally, in all 17 NC regions, one large NC region of 950 bp found between the 12S rRNA and tRNA Ile genes, with all the patterns mentioned above, was identified as a putative CR. In M. nipponense, the AT content of the CR (79.9%) is higher than that of the PCG, tRNA and rRNA genes. Besides this, we searched a microsatellite-like (TA)6 element which was not find in the same region of M. rosenbergii and M. lanchesteri. There are 20 sections of nucleotide sequence existed in the putative CR, all of which can form a typical hairpin-like secondary structures (Fig. 2). The CR of M. nipponense has three ORFs that begin with an invertebrate mt initiation codon (ATA and ATG) and end with two termination codons (TAA and TAG). This region serves as a recognition signal for replication, transcription and RNA processing. When comparing the NC regions of M. nipponense with those of M. rosenbergii and M. lanchesteri, the nucleotide sequences vary to a greater extent, which is likely due to decreased stress associated with the evolution of the NC regions and the relatively low pressure of natural selection. Compared to the CR, the NC regions have a higher level of polymorphisms. Furthermore, the variation in lengths of the mtDNAs is mainly due to the different lengths of the NC regions (Harrison, 1989). 3.5. Phylogenetic analysis Phylogenetic analyses based on complete mt genome sequence data, containing comparatively enough information, have proved to enhance resolution, and statistical confidence of inferred phylogenetic trees when compared with analyses based only on partial mt genes (Mueller, 2006; Russo et al., 1996; Zardoya and Meyer, 1996). Therewithal, ML and MP trees based on amino acid sequence of 13 concatenated PCGs are performed to reconstruct phylogenetic relationships within Decapoda (Fig. 3). The topology of the phylogenetic tree with bootstrap values was obtained in ML and MP analyses with Phobaeticus serratipes (Insecta) as an outgroup. All the 34 species belonging to 16 families cluster together. Our study shows that Palaemonidae, Alpheidae and Atyidae have closer relationships compared to those sister groups of Penaeidae and the other Decapoda (except Squillidae). Squillidae was more far-related than other families of Decapoda. For the three Macrobrachium species, M. rosenbergii and M. lanchesteri are more closely related to each other than to M. nipponense. This finding is in accordance with the distribution of the three. M. rosenbergii and M. lanchesteri are found extensively in the tropical and subtropical waters, while M. nipponense extratropical. DNA sequences from different regions within the metazoan mt genome have proven to be powerful genetic markers for resolving population structure and evolution. DNA sequence data from the ATP8, ND2 and ND6 PCGs may have the potential to provide valuable information for the elucidation of stock structure within these species, given that these genes demonstrate high nucleotide substitution rates in crustacean mt genomes (Machida et al., 2002). The sequence data generated from the current study will specifically facilitate population-level research through the development of PCR primers for the survey of mtDNA sequence variation. Compared to MP bootstrap values, ML bootstrap values among some genera (e.g. Marsupenaeus japonicus and Fenneropenaeus chinensis, et al.; Charybdis japonica and Scylla olivacea, et al.) were poorly established (Fig. 3). Perhaps a larger
164 K. Ma et al. / Gene 487 (2011) 160–165 Fig. 3. Phylogenetic tree of Decapoda relationships derived from amino acids of the 13 PCGs using Maximum-Likelihood (ML) and Maximum-Parsimony (MP) analyses. Numbers in each branch indicate ML/MP bootstrap values. Alternative topologies (ML) were correspondingly shown on the right side.
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study of the evolutionary relationships among Decapoda using mtDNA is warranted. We expect that as the number of complete arthropod mtDNA sequences grows, so will our confidence in mt phylogenies for this group, and more broadly, our understanding of mt genome evolution. 3.6. Conclusions The M. nipponense mt genome consists of 37 genes, including 13 PCGs, 22 tRNAs and 2 rRNAs, of which 23 are encoded on the L strand. There are as many as 17 NC regions found throughout the M. nipponense mt genome. Similar to other metazoan mt genomes, the M. nipponense mt genome contains a high AT content. The 13 PCGs are comprised of 3707 codons, not including incomplete termination codons, and Leu was the most commonly encoded amino acid. Predicted start sites in the PCGs include ATG, ATC and ATA, and predicted stop codons include TAA, TAG, T, and TA. Phylogenetic tree analyses show that M. nipponense is more closely related to M. rosenbergii and M. lanchesteri than other Decapoda. The results of the current study should be useful for future investigations of arthropod mt genome evolution. Acknowledgments This research was supported by the Natural Science Foundation of China (31001111), Shanghai Leading Academic Discipline Project (Y1101), Key Torch-plan Projects of Science and Technology Commission of Shanghai (073205111), Selection and Training of Scientific Research for Outstanding Young Teachers of Shanghai Universities Special Foundation (SSC08004) and Technology Development Program of Northern Jiangsu Province (BN2009036). References Anderson, S., et al., 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465. Avise, J.C., 1994. Molecular Markers, Natural History and Evolution. Chapman and Hall, New York, NY. Avise, J.C., 2000. Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, Mass. Beard, C.B., Hamm, D.M., Collins, F.H., 1993. The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects. Insect Mol. Biol. 2, 103–124. Boore, J.L., 1999. Animal mitochondrial genomes. Nucleic Acids Res. 27, 1767–1780. Boore, J.L., Brown, W.M., 1994. Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicate. Genetics 138, 423–443. Boore, J.L., Brown, W.M., 2000. Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: sequence and gene arrangement comparisons indicate that Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa. Mol. Biol. Evol. 17, 87–106. Curole, A.P., Kocher, T.D., 1999. Mitogenomics: digging deeper with complete mitochondrial genomes. Trends Ecol. Evol. 14, 394–398. Dimijian, G.G., 2000. Evolving together: the biology of symbiosis, part 2. Proc. (Bayl UnivMed Cent) 13, 381–390. Feng, J.B., Li, J.L., Cheng, X., 2008. Research progress on germplasm resources exploitation and protection of Macrobrachium nipponense. J. Shanghai Fish. Univ. 15, 371–376. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome coxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Grande, C., Templado, J., Zardoya, R., 2008. Evolution of gastropod mitochondrial genome arrangements. BMC Evol. Biol. 8, 61. Guindon, S., Gascuel, O., 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704.
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Gene j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / g e n e
Methods paper
The complete mitochondrial genome of Macrobrachium nipponense Keyi Ma, Jianbin Feng, Jingyun Lin, Jiale Li ⁎ Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Shanghai Ocean University, Ministry of Education, Shanghai 201306, China
a r t i c l e
i n f o
Article history: Accepted 9 July 2011 Available online 30 July 2011 Received by A.J. van Wijnen Keywords: mtDNA Long PCR Sequence analysis Phylogeny
a b s t r a c t The complete mitochondrial (mt) genome sequence plays an important role in the accurate determination of phylogenetic relationships among metazoans. Herein, we determined the complete mt genome sequence, structure and organization of Macrobrachium nipponense (M. nipponense) (GenBank ID: NC_015073.1) and compared it to that of Macrobrachium lanchesteri (M. lanchesteri) and Macrobrachium rosenbergii (M. rosenbergii). The 15,806 base pair (bp) M. nipponense mt genome, which is comprised of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs (rRNAs), is slightly larger than that of M. lanchesteri (15,694 bp, GenBank ID: NC_012217.1) and M. rosenbergii (15,772 bp, GenBank ID: NC_006880.1). The M. nipponense genome contains a high AT content (66.0%), which is a common feature among metazoan mt genomes. Compared with M. lanchesteri and M. rosenbergii, we found a peculiar noncoding region of 950 bp with a microsatellite-like (TA)6 element and many hairpin structures. The 13 PCGs are comprised of a total of 3707 codons, excluding incomplete termination codons, and the most frequently used amino acid is Leu (16.0%). The predicted start codons in the M. nipponense mt genome include ATG, ATC and ATA. Seven PCGs use TAA as a stop codon, whereas two use TAG, three use T and only one uses TA. Twentythree of the genes are encoded on the L strand, and ND1, ND4, ND5, ND4L, 12S rRNA, 16S rRNA, tRNA His, tRNA Pro, tRNA Phe, tRNA Val, tRNA Gln, tRNACys, tRNA Tyr and a tRNA Leu are encoded on the H strand. The two rRNAs of M. nipponense and M. rosenbergii are encoded on the H strand, whereas the M. lanchesteri rRNAs are encoded on the L stand. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Mitochondria are eukaryotic organelles that are responsible for the majority of cellular ATP production (Dimijian, 2000; Hedges et al., 2001). Characteristics such as a relatively rapid mutation rate, a maternal inheritance pattern and a presumed lack of intermolecular recombination have led to the extensive use of mitochondrial DNA
Abbreviations: mt, mitochondrial; bp, base pair; tRNAs, transfer RNAs; ATP, adenosine triphosphate; nt, nucleotides; MP, Maximum-Parsimony; L strand, Light strand; Val(V), valine; Leu(L), leucine; Met(M), methionine; Pro(P), proline; Ser(S), serine; Gln(Q), glutamine; Trp(W), tryptophane; Ile(I), isoleucine; Thr(T), threonine; Tyr(Y), tyrosine; COX1, cytochrome c oxidase subunit 1; COX3, cytochrome c oxidase subunit 3; ATP8, ATP synthase F0 subunit 8; ND2, NADH dehydrogenase subunit 2; ND4, NADH dehydrogenase subunit 4; ND5, NADH dehydrogenase subunit 5; M., Macrobrachium; PCGs, protein-coding genes; rRNAs, ribosomal RNAs; NC, non-coding; ML, Maximum-Likelihood; CR, control region; H strand, High strand; Ala, alanine; Arg(R), arginine; Asn(N), asparagine; Asp(D), aspartic; Cys(C), cysteine; Glu(E), glutamic; His(H), histidine; Lys(K), lysine; Gly(G), glycine; CYTB, cytochrome b; COX2, cytochrome c oxidase subunit 2; ATP6, ATP synthase F0 subunit 6; ND1, NADH dehydrogenase subunit 1; ND3, NADH dehydrogenase subunit 3; ND4L, NADH dehydrogenase subunit 4L; ND6, NADH dehydrogenase subunit 6. ⁎ Corresponding author at: College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China. Tel./fax: + 86 21 61900401. E-mail addresses: [email protected] (K. Ma), [email protected] (J. Li). 0378-1119/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2011.07.017
(mtDNA) sequence information in investigations of population structure and phylogenetic relationships at different taxonomic levels (Avise, 1994; Avise, 2000). In general, the genomes of nearly all metazoan animals are small, extrachromosomal, circular DNA molecules that range in size from 14 to 48 kb (Boore, 1999; Wolstenholme, 1992). The typical mitochondrium contains 13 proteins that comprise the electron transport chain (COX1-3, CYTB, ND1-6, ND4L, ATP6 and ATP8), two ribosomal RNAs (16S rRNA and 12S rRNA) and 22 transfer RNAs (tRNAs). In addition, a large non-coding (NC) region contains sequences essential for the initiation of transcription and gene replication (Ki et al., 2010). Analyses of entire mitochondrial (mt) genomes have contributed to the resolution of many phylogenetic problems, because mt genes are present in a single copy, have a maternal mode of inheritance and exhibit a higher rate of base substitutions than most nuclear genes (Peng et al., 2007). Notably, complete mtDNA sequences can be informative at deep phylogenetic levels (Curole and Kocher, 1999), and their phylogenetic utility has been demonstrated in several animal taxa, including invertebrates (Boore and Brown, 2000; Grande et al., 2008). Crustaceans are one of the most diversified groups of aquatic invertebrates in freshwater and marine systems. Based on the recently revised taxonomic system, this phylum comprises 849 extant families in 42 orders and 6 classes (Martin and Davis, 2001). While a large number of crustacean species have been described morphologically, relatively few complete crustacean mt genomes have been sequenced
K. Ma et al. / Gene 487 (2011) 160–165
to date: approximately 50 mt genomes from seven classes of Crustacea have been deposited in public databases (NCBI). This is primarily due to technical difficulties associated with the amplification of complete mt genomes using the long polymerase chain reaction (PCR) (Jung et al., 2006; Ki et al., 2009a, b; Machida et al., 2002). Failed amplification may be due to several factors, including gene rearrangements, AT-rich regions, poly-A (T) stretches or hairpin structures. The decapods are an extremely diverse group of crustaceans with many species of commercial importance. The oriental river prawn Macrobrachium nipponense (M. nipponense) is widely distributed in freshwaters of low salinity regions of the estuary in China, and they can also be found in most regions of Japan, Korea, Vietnam and Myanmar (Yu and Miyake, 1972). M. nipponense is the most important commercial freshwater prawn in both China and Vietnam (Feng et al., 2008). Although M. nipponense has been targeted commercially in China, genetic information essential to the sustainable management of this valuable resource, such as knowledge of its population structure, is lacking. In order to overcome these limitations, information from rapidly evolving gene regions is required. In the current study, we report the complete nucleotide sequence and gene arrangement of the M. nipponense mt genome, and we determine its genomic structure, gene order, codon usage and base composition. The results of the current study will contribute significantly to future investigations of crustacean genomics and phylogenetics. 2. Materials and methods 2.1. Tissue samples and mitochondrial DNA extraction Somatic tissues from a single female M. nipponense were collected from Shanghai, China, and were excised and stored at −70 °C. Mitochondrial DNA was isolated and purified from the somatic tissues (Tamura and Aptsuka, 1988).
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2.3. Gene identification and determination of tRNA structures ClustalW sequence alignments were performed using Lasergene 6 SeqMan, MegAlign and EditSeq 7 (DNASTAR). The boundaries of the protein-coding genes (PCGs) and rRNA genes were inferred by comparisons with the amino acid or nucleotide sequences of other metazoans (Zheng et al., 2010). The 5′ and 3′ ends of the 16S rRNA and 12S rRNA genes were assumed to be adjacent to the flanking tRNA genes or protein genes. Transfer RNA genes were identified using the tRNAscan-SE program (version 1.21, http://lowelab.ucsc.edu/tRNAscan-SE/Lowe and Eddy, 1997) with the invertebrate mt codon sequences. This specialized tRNA search server scans sequences for cloverleaf secondary structure, anticodon sequences and thermodynamic stability.
2.4. Phylogenetic analysis To illustrate the phylogenetic relationship of Decapoda, the other 34 available complete mt genomes were obtained from GenBank. Phobaeticus serratipes from Insecta served as outgroup. Amino acid sequences of 13 concatenated protein-coding genes (PCGs) were used in phylogenetic analysis. The alignment of the amino acid sequences of each 13 PCGs was aligned with Clustal X (Thompson et al., 1997) using default settings. Then the 13 separate amino acid sequence alignments were concatenated to a single multiple sequence alignment. We used two different inference methods: Maximum-Likelihood (ML) and Maximum-Parsimony (MP) to reconstruct phylogenetic relationships of Decapoda. The ML analysis was conducted with PHYML 3.0 program (Guindon and Gascuel, 2003) and 1000 bootstraps were used to estimate the node reliability. MP was performed using MEGA 4.0 (Tamura et al., 2007) at the default setting based on 100 replications of random addition and 500 bootstrap tests of inferred phylogeny.
2.2. Long-polymerase chain reaction (long PCR) PCR amplification of a portion of COX1 was performed using LCO1490 (5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′) and HCO2198 (5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′) primers (Folmer et al., 1994) and the following PCR cycle conditions: 94 °C for 3 min; a hot start by adding LA Taq polymerase (TaKaRa) at 72 °C; 37 cycles of 94 °C for 30 s, 43 °C for 30 s and 72 °C for 1 min and incubation at 72 °C for 5 min. In addition, PCR amplification of a portion of the 16S rRNA gene was performed using 16SA (5′-CGC CTG TTT AAC AAA AAC AT-3′) and 16SB (5′-CCG GTT GAA CTC AGA TCA-3′) primers (Ui et al., 2001) and the following PCR cycle conditions: 94 °C for 3 min; a hot start by adding LA Taq polymerase (TaKaRa) at 72 °C; 30 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 1 min and incubation at 72 °C for 10 min. The two M. nipponense genes were partially sequenced, including 577 bp of COX1 and 317 bp of 16S rRNA. Based on the partial M. nipponense mt gene sequences, we designed and synthesized two pairs of primers (CS and SC) whose sequences were as follows: CS-F (5′-ATT GAA GGC TTG AAT GAA AGG TTG-3′), CS-R (5′-TAG AAA GAG GAG TAG GCA CAG GAT-3′), SC-F (5′CCT GTG CCT ACT CCT CTT TCT A-3′) and SC-R (5′-CTA CTG ACT ATG CTA CCT TCG C-3′). The CS fragment of approximately 4 kb and the SC fragment of approximately 12 kb were obtained using long PCR amplification (TaKaRa). The PCR reaction volume was 50 μl, and PCR conditions with the CS/SC primer sets were as follows: 94 °C for 1 min; 35 cycles of 98 °C for 10 s, 58 °C/53 °C for 30 s, and 68 °C for 6/15 min and an incubation at 72 °C for 10 min. With both primer sets, an aliquot of the PCR reaction yielded a single band on a 1% agarose gel stained with ethidium bromide. PCR products were sequenced using Shot Gun Sequencing by Map Biotechnology Co., Ltd. (Shanghai).
Fig. 1. Genetic map of the M. nipponense mt genome. COX1-3 indicates cytochrome oxidase subunits 1–3; ATP6/8, ATPase subunits 6–8; ND1-6/4L, NADH dehydrogenase subunits 1–6 and 4L and CYTB, cytochrome b. Transfer RNA genes are designated as tRNA with the corresponding three-letter amino acid code.
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(67.1%). Notably, the AT content of M. nipponense mtDNA is higher than that of many invertebrate mt genomes.
Table 1 Characteristics of the mt genomes of M. nipponense and two related species. Species
13 protein genes 22 tRNA genes 16S rRNA genes 12S rRNA genes Control region Noncoding regions mtDNA
M. nipponense
M. rosenbergii
M. lanchesteri
A + T (%)
A + T (%)
A + T (%)
64.3 65.7 68.8 69.0 79.9 73.6 66.0
60.1 64.7 66.0 66.0 75.7 60.3 62.3
65.2 66.4 71.6 70.3 82.7 72.4 67.1
3.2. Codon usage and sequence features of protein-coding genes
3. Results and discussion 3.1. Mitochondrial genome organization The complete mt genome sequence of M. nipponense is 15,806 bp in length, which is similar to M. lanchesteri (15,694 bp) and M. rosenbergii (15,772 bp), and its structural organization is depicted in Fig. 1. It consists of 13 PCGs, 2 rRNAs, 22 tRNAs and 17 NC regions (including a 950 bp control region [CR]). Genes including COX1 to tRNAGlu and seven additional genes (tRNAThr, ND6, CYTB, tRNASer, tRNAIle, tRNAMet and ND2) are encoded on the L strand, while the remaining genes (4 PCGs, 2 rRNAs, and 8 tRNAs) are encoded on the H strand. Overall, four overlaps between PCGs (7 bp between ATP8 and ATP6, 1 bp between ATP6 and COX3, 7 bp between ND4 and ND4L and 1 bp between ND6 and CYTB) were detected in the M. nipponense mt genome. In addition, three overlaps between tRNAs were identified. The overall AT content of M. nipponense mtDNA is 66.0%, which reflects the typical AT-rich pattern associated with invertebrate mt genomes. This value is higher than that of M. rosenbergii (62.3%) (Table 1) and lower than that of M. lanchesteri
Table 2 Codon usage for 13 mt PCGs of M. nipponense. Amino acid
Codon
N
%
Amino acid
Codon
N
%
Ala (A) Ala (A) Ala (A) Ala (A) Arg (R) Arg (R) Arg (R) Arg (R) Asn (N) Asn (N) Asp (D) Asp (D) Cys (C) Cys (C) Gln (Q) Gln (Q) Glu (E) Glu (E) Trp (W) Trp (W) His (H) His (H) Ile (I) Ile (I) Lys (K) Lys (K) Phe (F) Phe (F) Tyr (Y) Tyr (Y) Stop Stop
GCA GCC GCG GCU CGA CGC CGG CGU AAC AAU GAC GAU UGC UGU CAA CAG GAA GAG UGA UGG CAC CAU AUC AUU AAA AAG UUC UUU UAC UAU UAA UAG
72 55 13 116 43 1 6 13 49 71 39 33 9 41 62 18 53 25 80 17 50 27 70 224 60 24 85 200 35 93 7 2
1.95 1.49 0.35 3.14 1.16 0.03 0.16 0.35 1.33 1.92 1.05 0.89 0.24 1.11 1.68 0.49 1.43 0.68 2.16 0.46 1.35 0.73 1.89 6.06 1.62 0.65 2.30 5.41 0.95 2.51 0.19 0.05
Leu (L) Leu (L) Leu (L) Leu (L) Leu (L) Leu (L) Met (M) Met (M) Pro (P) Pro (P) Pro (P) Pro (P) Ser (S) Ser (S) Ser (S) Ser (S) Ser (S) Ser (S) Ser (S) Ser (S) Thr (T) Thr (T) Thr (T) Thr (T) Gly (G) Gly (G) Gly (G) Gly (G) Val (V) Val (V) Val (V) Val (V)
CUA CUC CUG CUU UUA UUG AUA AUG CCA CCC CCG CCU AGA AGC AGG AGU UCA UCC UCG UCU ACA ACC ACG ACU GGA GGC GGG GGU GUA GUC GUG GUU
113 48 21 98 247 65 135 47 51 33 5 58 61 4 33 37 79 28 8 109 98 44 8 78 108 24 47 70 104 24 29 100
3.06 1.30 0.57 2.65 6.68 1.76 3.65 1.27 1.38 0.89 0.14 1.57 1.65 0.11 0.89 1.00 2.14 0.76 0.22 2.95 2.65 1.19 0.22 2.11 2.92 0.65 1.27 1.89 2.81 0.65 0.78 2.70
N: total number of amino acids; %: percentage of total codon usage. Abbreviated stop codons were excluded.
The codon usage of M. nipponense PCGs is shown in Table 2. There is a clear preference for the use of AT-rich codons and for the presence of an A or T in the third codon position, reflecting the high AT content of the M. nipponense mt genome. The overall AT composition of PCGs in M. nipponense is 64.3%, which is higher than that of M. rosenbergii (60.1%) and similar to that of M. lanchesteri (65.2%). There is a total of 3707 codons comprising 13 PCGs, excluding termination codons. Analysis of the codon usage of the 13 PCGs revealed that three codons are used frequently. The UAA codon is used most frequently (247), followed by AUU (224) and UUU (200), while the CGC, AGC and CCG codons are used only one, four and five times each, respectively. The most frequently encoded amino acids include Leu (16.02%), Ser (9.72%), Ile (7.95%), Phe (7.71%) and Ala (6.93%). Our study showed that the entire mtDNA of M. nipponense excessively favor A and T. A common feature among metazoan mt genomes is a bias towards a higher representation of the nucleotides A and T, which leads to a subsequent bias in the corresponding encoded amino acids. This phenomenon is considered related to the ‘mutational bias-translational selection’ paradigm (Romero et al., 2000), meaning that both mutation and selection have an effect on the bias in codon usage. Ten protein-coding genes (ATP6, ATP8, COX2, COX3, CYTB, ND1, ND3, ND4, ND5 and ND4L) start with ATG, COX1 starts with ACG, ND6 starts with ATC and ND2 starts with ATA. Overall, the initiation codon inferred for 12 of the 13 genes is ATN, which is typical of metazoan mt genomes (Wolstenholme, 1992). Nine genes use complete stop codons, including seven genes with TAA (ATP8, ATP6, COX3, ND3, ND5, ND6 and ND4L) and two genes with TAG (ND1 and ND4), whereas four genes appear to end in incomplete stop codons, including three genes that end with T (COX2, CYTB and ND2) and one gene (COX1) that ends with TA, which are presumably completed as TAA by post-transcriptional polyadenylation (Anderson et al., 1981; Boore, 1999), as interpreted in the case of human mt DNA (Ojala et al., 1981). The boundaries between thirteen PCGs of M. nipponense were identified by sequence comparison with M. rosenbergii and M. lanchesteri, and determined by translation initiation and termination codons. The total length of M. nipponense PCGs is 11,211 bp. The longest PCG is the ND5 gene (1,707 bp), whereas the shortest PCG is the ATP8 gene (159 bp). ATP8 and ATP6 share a 7 bp overlap. Typically, ATP8 immediately precedes ATP6 or even overlaps with ATP6 in a different reading frame (Wolstenholme, 1992). In addition, ATP8 often begins with an MPQL motif and terminates shortly after the WXW sequence
Table 3 Comparisons of 13 mt PCGs in M. nipponense and two related species. Coding genes
No. of amino acids
Similarity (%)
M. nipponense
M. rosenbergii
M. lanchesteri
nr
nl
COX1 COX2 ATP8 ATP6 COX3 ND3 ND5 ND4 ND4L ND6 CYTB ND1 ND2
511 228 52 224 262 117 568 444 99 171 378 313 331
511 229 52 224 264 117 568 444 99 171 377 312 331
511 229 52 224 262 117 568 444 99 171 378 312 331
98 92 79 92 92 86 92 91 97 76 96 95 86
98 95 79 92 96 89 90 90 94 76 98 94 85
nr: similarity between M. nipponense and M. rosenbergii; nl: similarity between M. nipponense and M. lanchesteri.
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(Hoffmann et al., 1992); however, in M. nipponense, M. rosenbergii and M. lanchesteri, ATP8 begins with an MPQM motif. ATP6 shares 1 bp with COX3, ND6 shares 1 bp with CYTB and ND4L overlaps with ND4 by 7 bp. The similarity of amino acids encoded by PCGs between M. nipponense, M. rosenbergii and M. lanchesteri is somewhat different; amino acids encoded by COX1 have the highest similarity, followed by CYTB, whereas amino acids encoded by ND6 are the least similar (Table 3). 3.3. Transfer RNA and rRNA genes The 22 tRNAs encoded by the mt genome of the M. nipponense, which are identified on the basis of their respective anticodons and secondary structures (data not shown), vary in length from 60 (tRNA Leu) to 69 (tRNA Glu, tRNA Ser and tRNA Trp) nucleotides, and include two tRNA Leu and two tRNA Ser. These tRNAs correspond to the standard set found in other metazoan mtDNAs. Gene lengths and anticodon sequences are congruent with those described for other crustaceans. The anticodon sequences are identical to those reported for M. rosenbergii (Miller et al., 2005). The acceptor stem has seven nucleotides (nt) in M. nipponense, although there is one or two mismatched base pairs. The dihydrouridine arm consists of a stem of three or four paired nt and a loop of 4–9 unpaired nt, and the stems also have some mismatched termini (Tyr, Ala, Gln, etc.). Meanwhile, the TψC arm has a stem of 3–5 paired nt with one mismatched terminus (only found in Pro and Phe) and a loop of 3–7 unpaired nt. The anticodon stem consists of four or five nt pairs, while some tRNAs have one or two mismatched base pairs. The tRNALys gene of M. nipponense, like that of M. rosenbergii, contains UUU anticodons. Invertebrate tRNA anticodon sequences are generally conserved, although variation at the third (i.e., wobble) position (e.g. G–U) of the tRNALys gene is not uncommon (Beard et al., 1993; Boore and Brown, 1994; Yamauchi et al., 2002). The AT nucleotide frequency of the pooled tRNAs is similar to that of the PCGs and two rRNAs (65.7%). The lengths of the M. nipponense mtDNA-encoded 16S rRNA and 12S rRNA genes are 1305 and 852 bp, respectively, and the AT contents are 68.8% and 69.0%, respectively, which are within the range observed in other crustaceans. The 16S rRNA gene lies between the tRNALeu (CUA) and tRNAVal genes, while the 12S rRNA gene lies between the tRNAVal gene and the CR; both rRNA genes are encoded on the H strand.
Fig. 2. The largest potential hairpin structures found within CR located between 12S rRNA and tRNAIle genes of M. nipponense.
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3.4. NC regions There are as many as 17 NC regions found throughout the M. nipponense mt genome, ranging in size from 1 to 950 bp. Unlike vertebrates, the mtDNA control regions of invertebrates are not well characterized, due to lacking discrete conserved sequence blocks (Serb and Lydeard, 2003). In order to confirm the CR of M. nipponense, we searched for some peculiar patterns (e.g. AT-rich, hairpin structures), which were generally found in CR (Kumazawa et al., 1998; Saito et al., 2005). Finally, in all 17 NC regions, one large NC region of 950 bp found between the 12S rRNA and tRNA Ile genes, with all the patterns mentioned above, was identified as a putative CR. In M. nipponense, the AT content of the CR (79.9%) is higher than that of the PCG, tRNA and rRNA genes. Besides this, we searched a microsatellite-like (TA)6 element which was not find in the same region of M. rosenbergii and M. lanchesteri. There are 20 sections of nucleotide sequence existed in the putative CR, all of which can form a typical hairpin-like secondary structures (Fig. 2). The CR of M. nipponense has three ORFs that begin with an invertebrate mt initiation codon (ATA and ATG) and end with two termination codons (TAA and TAG). This region serves as a recognition signal for replication, transcription and RNA processing. When comparing the NC regions of M. nipponense with those of M. rosenbergii and M. lanchesteri, the nucleotide sequences vary to a greater extent, which is likely due to decreased stress associated with the evolution of the NC regions and the relatively low pressure of natural selection. Compared to the CR, the NC regions have a higher level of polymorphisms. Furthermore, the variation in lengths of the mtDNAs is mainly due to the different lengths of the NC regions (Harrison, 1989). 3.5. Phylogenetic analysis Phylogenetic analyses based on complete mt genome sequence data, containing comparatively enough information, have proved to enhance resolution, and statistical confidence of inferred phylogenetic trees when compared with analyses based only on partial mt genes (Mueller, 2006; Russo et al., 1996; Zardoya and Meyer, 1996). Therewithal, ML and MP trees based on amino acid sequence of 13 concatenated PCGs are performed to reconstruct phylogenetic relationships within Decapoda (Fig. 3). The topology of the phylogenetic tree with bootstrap values was obtained in ML and MP analyses with Phobaeticus serratipes (Insecta) as an outgroup. All the 34 species belonging to 16 families cluster together. Our study shows that Palaemonidae, Alpheidae and Atyidae have closer relationships compared to those sister groups of Penaeidae and the other Decapoda (except Squillidae). Squillidae was more far-related than other families of Decapoda. For the three Macrobrachium species, M. rosenbergii and M. lanchesteri are more closely related to each other than to M. nipponense. This finding is in accordance with the distribution of the three. M. rosenbergii and M. lanchesteri are found extensively in the tropical and subtropical waters, while M. nipponense extratropical. DNA sequences from different regions within the metazoan mt genome have proven to be powerful genetic markers for resolving population structure and evolution. DNA sequence data from the ATP8, ND2 and ND6 PCGs may have the potential to provide valuable information for the elucidation of stock structure within these species, given that these genes demonstrate high nucleotide substitution rates in crustacean mt genomes (Machida et al., 2002). The sequence data generated from the current study will specifically facilitate population-level research through the development of PCR primers for the survey of mtDNA sequence variation. Compared to MP bootstrap values, ML bootstrap values among some genera (e.g. Marsupenaeus japonicus and Fenneropenaeus chinensis, et al.; Charybdis japonica and Scylla olivacea, et al.) were poorly established (Fig. 3). Perhaps a larger
164 K. Ma et al. / Gene 487 (2011) 160–165 Fig. 3. Phylogenetic tree of Decapoda relationships derived from amino acids of the 13 PCGs using Maximum-Likelihood (ML) and Maximum-Parsimony (MP) analyses. Numbers in each branch indicate ML/MP bootstrap values. Alternative topologies (ML) were correspondingly shown on the right side.
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study of the evolutionary relationships among Decapoda using mtDNA is warranted. We expect that as the number of complete arthropod mtDNA sequences grows, so will our confidence in mt phylogenies for this group, and more broadly, our understanding of mt genome evolution. 3.6. Conclusions The M. nipponense mt genome consists of 37 genes, including 13 PCGs, 22 tRNAs and 2 rRNAs, of which 23 are encoded on the L strand. There are as many as 17 NC regions found throughout the M. nipponense mt genome. Similar to other metazoan mt genomes, the M. nipponense mt genome contains a high AT content. The 13 PCGs are comprised of 3707 codons, not including incomplete termination codons, and Leu was the most commonly encoded amino acid. Predicted start sites in the PCGs include ATG, ATC and ATA, and predicted stop codons include TAA, TAG, T, and TA. Phylogenetic tree analyses show that M. nipponense is more closely related to M. rosenbergii and M. lanchesteri than other Decapoda. The results of the current study should be useful for future investigations of arthropod mt genome evolution. Acknowledgments This research was supported by the Natural Science Foundation of China (31001111), Shanghai Leading Academic Discipline Project (Y1101), Key Torch-plan Projects of Science and Technology Commission of Shanghai (073205111), Selection and Training of Scientific Research for Outstanding Young Teachers of Shanghai Universities Special Foundation (SSC08004) and Technology Development Program of Northern Jiangsu Province (BN2009036). References Anderson, S., et al., 1981. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465. Avise, J.C., 1994. Molecular Markers, Natural History and Evolution. Chapman and Hall, New York, NY. Avise, J.C., 2000. Phylogeography: The History and Formation of Species. Harvard University Press, Cambridge, Mass. Beard, C.B., Hamm, D.M., Collins, F.H., 1993. The mitochondrial genome of the mosquito Anopheles gambiae: DNA sequence, genome organization, and comparisons with mitochondrial sequences of other insects. Insect Mol. Biol. 2, 103–124. Boore, J.L., 1999. Animal mitochondrial genomes. Nucleic Acids Res. 27, 1767–1780. Boore, J.L., Brown, W.M., 1994. Complete DNA sequence of the mitochondrial genome of the black chiton, Katharina tunicate. Genetics 138, 423–443. Boore, J.L., Brown, W.M., 2000. Mitochondrial genomes of Galathealinum, Helobdella, and Platynereis: sequence and gene arrangement comparisons indicate that Pogonophora is not a phylum and Annelida and Arthropoda are not sister taxa. Mol. Biol. Evol. 17, 87–106. Curole, A.P., Kocher, T.D., 1999. Mitogenomics: digging deeper with complete mitochondrial genomes. Trends Ecol. Evol. 14, 394–398. Dimijian, G.G., 2000. Evolving together: the biology of symbiosis, part 2. Proc. (Bayl UnivMed Cent) 13, 381–390. Feng, J.B., Li, J.L., Cheng, X., 2008. Research progress on germplasm resources exploitation and protection of Macrobrachium nipponense. J. Shanghai Fish. Univ. 15, 371–376. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome coxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3, 294–299. Grande, C., Templado, J., Zardoya, R., 2008. Evolution of gastropod mitochondrial genome arrangements. BMC Evol. Biol. 8, 61. Guindon, S., Gascuel, O., 2003. A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704.
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