Received date: 2009-09-11; Accepted date: 2010-01-20
Foundation items: National Natural Science Foundation of China (30370218); the program for New Century Excellent Talents in University
(NCET-07-0507); the Project of Science and Technology Development Plan in Shandong Province (2007GG2009011); Shandong Science Fund for Distinguished Young Scholars (2005BS02005).
收稿日期：2009-09-11；接受日期：2009-01-20
*
通讯作者 (Corresponding author)，E-mail ： 第一作者简介：钟华明(1984-), 男，硕士，E-mail:
动 物 学 研 究 2010，Apr. 31(2)：122−130 CN 53-1040/Q ISSN 0254-5853 Zoological Rearch DOI ：10.3724/SP.J.1141.2010.02122
Complete Mitochondrial Genome of the Red Fox (Vuples vuples ) and
Phylogenetic Analysis with Other Canid Species
ZHONG Hua-Ming 1, ZHANG Hong-Hai 1,*, SHA Wei-Lai 1, ZHANG Cheng-De 1, CHEN Yu-Cai 2
(1. College of Life Science, Qufu Normal University, Qufu 273165, China; 2. Ji’nan Paomaling World of Wildlife, Ji’nan 250000, China )
Abstract: The whole mitochondrial genome quence of red fox (Vuples vuples ) was determined. It had a total length of 16 723 bp. As in most mammal mitochondrial genome, it contained 13 protein coding genes, two ribosome RNA genes, 22 transfer RNA genes and one control region. The ba composition was 31.3% A, 26.1% C, 14.8% G and 27.8% T, respectively. The codon usage of red fox, arctic fox, gray wolf, domestic dog and coyote followed the same pattern except for an unusual ATT start codon, which initiates the NADH dehydrogena subunit 3 gene in the red fox. A long tandem repeat rich in AC was found between conrved quence block 1 and 2 in the control region. In order to confirm the phylogenetic relationships of red fox to other canids, phylogenetic trees were reconstructed by neighbor-joining and maximum parsimony methods using 12 concatenated heavy-strand protein-coding genes. The result indicated that arctic fox was the sister group of red fox and they both belong to the red fox-like clade in family Canidae, while gray wolf, domestic dog and coyote belong to wolf-like clade. The result was in accordance with existing phylogenetic results.
Key words: Red fox; Mitochondrial genome; Canidae; Phylogenetic analysis凯文杜兰特英文
赤狐线粒体全基因组及系统发育分析
钟华明1，张洪海1,*，沙未来1，张承德1，陈玉才2
（1. 曲阜师范大学 生命科学学院，山东 曲阜273165；2. 济南跑马岭野生动物世界，山东 济南 250000）
摘要：测定了赤狐的线粒体基因组全序列，总长度为16 723 bp ，碱基组成为：31.3% A 、26.1% C 、14.8% G 、27.8% T 。和大多数哺乳动物一样，赤狐的线粒体全基因组包含13个蛋白质编码基因、2个核糖体RNA 基因、22个转运RNA 基因和1个控制区。除ND3基因起始密码子为不常见的ATT 外，赤狐与北极狐、狼、家犬、郊狼的线粒体蛋白质编码遵循相同模式。在控制区的保守序列区段1和2之间发现一段较长的富含AC 的随机重复序列。为了验证赤狐与其他犬科动物的系统发育关系，利用12个重链蛋白质编码基因，分别通过邻接法和最大简约法构建了系统发育树。结果表明：赤狐与北极狐是姐妹群，它们在犬科中都属于赤狐型分支，而灰狼、家犬和郊狼则属于狼型分支，与现有的系统进化研究结果一致。
关键词：赤狐；线粒体全基因组；犬科；系统发育分析
中图分类号：Q344.13;Q786;Q959.838.09 文献标志码：A 文章编号：0254-5853-(2010)02-0122-09
Mammalian mitochondrial genomes typically have a t of 13 protein-coding genes, two ribosome RNA genes (12S RNA and 16S RNA) and 22 transfer RNA genes. The gene order is highly conrved among most vertebrates (Boore, 1999). The two strands that make up the genome are commonly known as the heavy strand (H-strand) and the light strand (L-strand). Twelve of the
13 protein-coding genes locate on the H-strand and only ND6 gene are located on the L-strand. Vertebrates usually contain two non-coding regions, the major of which contains mitochondrial replication and transcription promoters. Conquently, it is known as the control region (CR) (Wolstenholme, 1992; Boore, 1999). The small non-coding region is the origin of replication
boundedNo. 2 ZHONG Hua-Ming et al: Complete Mitochondrial Genome of the Red Fox (Vuples vuples) and Phylogenetic Analysis 123
of the L-strand (OL), thought to have a functional role in replication (Shadel & Clayton, 1997).
Belonging to Canivora, Canidae, Vuples, red fox (Vuples vuples) is one of the worldwide distributed
mammals. Comparing complete animal mitochondrial genome quences is now common for phylogenetic reconstruction and as a model for genome evolution (Wei et al, 2008). Mitochondrial DNA quences were also ud to clarify phylogenetic relationships within Canidae (Geffen et al, 1992; Wayne et al, 1997). Sequence data from three mitochondrial genes (Wayne et al, 1997) suggest that Canidae falls to four monophyletic groups: (1) the wolf- and jackal-like canids; (2) the red fox-like canids; (3) the South American foxes; and (4) the maned wolf (Chysocyon brachyurus) and bush dog (Speothos venaticus). Analysis of various morphological and mitochondrial DNA data (Zrzavy & Ricankova, 2004) as well as phylogenetic analysis using six nuclear loci combined with mitochondrial data (Bardeleben, 2005) both agree with the three clades, including the red fox-like canids, the South American foxes, and the wolf-like canids.
This paper reports a complete mitochondrial genome of red fox, which was also compared with that of other canids to discuss the red fox mitochondrial genome structure and evolution. A phylogenetic tree was reconstructed to confirm the evolution position of red fox in Canidae.
1 Materials and Methods
1.1 Samples and DNA extracting
A blood sample of 200 µL from a female red fox was obtained from Beijing Zoo. Total genomic DNA was extracted following the method of Sambrook & Rusll (2001) and the obtained DNA solution was prerved under −20 ℃ for next u.
1.2 PCR and quencing
Five pairs of primers were designed bad on the quences of wolf and coyote, which were available online with the accession number of NC008092 and NC008093, respectively. Primer quences are shown in Tab. 1.
Tab. 1 Five primer pairs for the first PCRs
Primer pair Primer Sequence(5'→3')
1 1-F TCCCTCTAGAGGAGCCTGTTC
1-R GGGTATGGGCCCGATAGCTT
2 2-F GGCGGATAAAAGAGTTACTTTGATAGAG
2-R GCGAATTTAACTTTGACAAAGTCATGT
3 3-F GAAGAAAGGAAGGAATCGAACC
3-R GCGTAGGGATGATAATTTTTAGCATT
4 4-F GTATTTGCTGCCTGCGAAGC
4-R TAGTGGTGGGATTGGTTGTGC
5 5-F GGGTATTGCTCAGTAGCCATAGC
5-R GGTTTGCTGAAGATGGCGGTATAT
PCR was implemented in a 50 µL system including 1 µL DNA, 5 µL 10 × Long-PCR Buffer, 4 µL dNTP mixtures (2.5 mmol/L each NTP), 15 mmol MgCl2; pfu polymera 1 µL (5 U/µL); 2 µL primers (10 µmol/L). After degeneration of 95℃for 5 min, 35 cycles of 95℃degeneration for 30 s, 57℃annealing for 30 s, and 72℃extension for 4.5 min were ran. Final extension was ran at 72℃for 10 min. PCR products were purified from a 1% agaro gel and were then ud as DNA templates in condary PCR. Secondary PCR was conducted with 10 pairs of primers (Tab. 2). PCR conditions were: initial degeneration of 95℃for 5 min, then 35 cycles of 95℃degeneration for 30 s, 57℃ annealing for 30 s, 72℃extension for 2.5 min and a final extension step of 72℃for 4.5 min. Reacting
system and purifying methods are the same as the first PCR. The purified PCR products were then nt out for quencing.
1.3 Sequence asmbly, annotation and analysis
Three times of quencing were performed to make sure correct target quences were amplified. The DNAMAN 6.0 program (Lynnon Biosoft, Quebec, Canada) was ud for editing and asmbling the quence. The positions of protein coding genes, rRNA genes and non-coding regions were located through alignment with that of wolf and coyote using BLAST (bi.i). Start and stop codons were identified using the vertebrate mitochon- drial code except some potential incomplete stop codons.
124 Zoological Rearch V ol. 31
Tab. 2 Ten primer pairs ud for condary PCRs
马其顿方阵Primer pair Primer Sequence(5'→3')
1 1-F
TCCCTCTAGAGGAGCCTGTTC
1-R TCCGAGGTCACCCCAACC
2 2-F GACGAGAAGACCCTATGGAGC
2-R GGGTATGGGCCCGATAGCTT
3 3-F GGCGGATAAAAGAGTTACTTTGATAGAG
3-R GCCTACTATACCGGCTCATGC
4 4-F GCTCAGCCATTTTACCTATGTTC
4-R GCGAATTTAACTTTGACAAAGTCATGT
5 5-F GAAGAAAGGAAGGAATCGAACC
5-R GCGAAGAGTTGTAGTGAAATCATAT
6 6-F GCTACCTAATGACCCACCAAAC
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6-R GCGTAGGGATGATAATTTTTAGCATT
7 7-F GTATTTGCTGCCTGCGAAGC
7-R CGCTTATCTGGAGTTGCACC
8 8-F CCGCAAGAACTGCTAATTCATG
8-R TAGTGGTGGGATTGGTTGTGC
9 9-F GGGTATTGCTCAGTAGCCATAGC
9-R GCAGAATTTCAGCTTTGGGTG
10 10-F CGCGATGAAGAGTCTTTGTAGTAT
10-R GGTTTGCTGAAGATGGCGGTATAT
21 tRNAs genes were identified by tRNAscan-SE Search Server v.1.21 (lowelab.ucsc.edu/tRNAscan-SE/). The tRNASer (AGC) gene not found by tRNAscan-SE was determined by comparison with the two canids mentioned above. RNA structure 4.6 (Lowe & Eddy, 1997) was ud to speculate the cond structure of the 22 tRNAs. MEGA v.4.0 (Tamura et al, 2007) was ud to compute ba composition, substitution and codon usage.
The mitochondrial genome quence of the red fox was submitted into GenBank with the accession number of GQ374180.
1.4 Phylogenetic analysis
Four complete protein quences available for Canidae were downloaded from GenBank, and tiger (Panthera tigris) was ud as an outgroup for phylogenetic analys (Tab. 3). All protein coding genes except the ND6 gene were chon to reconstruct phylogenetic tree. Multiple alignments were performed by ClustalX 1.83 (Thompson et al, 1997) with the default parameters. The results of alignments were also carefully checked and edited by eye. Phylogenetic analys were conducted using neighbor-joining (NJ) and maximum parsimony (MP). The NJ analysis was performed in MEGA 4.0 using Kimura 2-parameter as the nucleotide substitution model. Statistical confidence was assd by bootstrap analysis with 1000 replications. The MP analysis was performed in PAUP*4.0b10 (Swofford, 2003) using a
Tab. 3 The taxa and quence’s accession number of the six species ud for phylogenetic analys Species Common name Accession No. and references
Vuples vuples Red fox GQ374180,This paper
Alopex lagopus Arctic fox AH014073, Delisle & Strobeck, 2005
Canis lupus Gray wolf NC_008092, Bjornerfeldt et al, 2006
Canis lupus familiaris Dog NC_002008, Kim et al, 1998
Canis latrans Coyote NC_008093, Bjornerfeldt et al, 2006
Panthera tigris Tiger EF551003,
unpublished
No. 2 ZHONG Hua-Ming et al: Complete Mitochondrial Genome of the Red Fox (Vuples vuples) and Phylogenetic Analysis 125
heuristic arch. The robustness of the tree was tested with 100 bootstrap replications.
2 Results
2.1 Mitochondrial genome structure
The total length of red fox mitochondrial genome is 16723 bp, a little shorter than that of gray wolf(1 6729 bp) and coyote (16 724 bp). The red fox mitochondrial genome shares high similarity with tho of three other canids: gray wolf (84.93%), domestic dog (84.86%) and coyote (84.87%). Similar to other animals (Boore, 1999), the red fox mitochondrial genome has 13 protein-coding genes, 22 tRNA genes, two rRNA genes (12S rRNA, 16S rRNA) and one control region (CR). Except for the protein coding gene ND6 and 8 tRNA genes [tRNAGln, tRNAAla, tRNAAsn, tRNACys, tRNATyr, tRNASer (UCN), tRNAGlu, tRNAPro] encoded at the L-strand, the rest genes are encoded on the H-strand. Overall organization of the genome is comparable to the situation of other vertebrates(Tab. 4). As shown in Fig. 1, the genes are so compactly arranged that few gaps were found between them and some genes even overlap each other. The longest gap was found between COII and tRNALys by 16 nucleotides, while ATPa8 and ATPa6 overlap each other by 33 nucleotides, which is the longest one.
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The overall ba composition of the red fox mitochondrial genome is 31.3% A, 26.1% C, 14.8% G and 27.8% T, respectively (Tab. 5).The conquent order would be A>T>C>G, which was in coincidence with the order of other mammals. The A+T content is 59.1%, which indicated an A−T rich in the red fox mitochondrial genome.
2.2 Protein coding genes
Except ATA for ND2, ND5 and ATT for ND3, other protein coding genes u the start codon ATG. When it comes to stop codons, ND1, COI, COII, ATPa8, ATPa6, ND4L, ND5 and ND6 terminate with TAA, while ND2 and Cytb terminated with TAG and AGA respectively. Besides, three incomplete stop codons were detected: T (COIII), TA (ND3) and T (ND4). Codon usage analysis (Tab. 6) indicates obvious codon bias and CUA (Leu), AUA (Met), AUU (Ile) are the most ud three codons. Codon degenerate was also found by codon usage analysis. Both four-fold degenerate codons (e.g. codons specify Ala and Pro) and two-fold degenerate codons (e.g. codons specify Asn and Tyr) were found. Leu and Ser are found to be specified by both four-fold degenerate codons and two-fold degenerate codons.
The total length of 13 protein coding genes is 11411 bp, accounting for 68.24% of the complete quence of the red fox mitochondrial genome. As shown in Fig.1, three pairs of protein coding genes, ATP8/ATP6, ND4L/ND4 and ND5/ND6 overlap each other by 33 bp, 7 bp and 17 bp respectively. For the 13 protein-coding genes of the red fox mitochondrial DNA, the nucleotide distribution at three codon positions differ significantly (P<0.01).A strong bias against G (only 9.2%) at the third codon position was obrved. The frequency of A+T in all protein-coding genes ranges fr
om 53.6% to 61.6%.
2.3 Ribosomal RNA and transfer RNA genes
The 12S rRNA gene of the red fox mitochondrial genome was located between tRNA Phe gene and tRNA Val gene with a length of 957 bp, longer than that of gray wolf (954 bp) and coyote (955 bp) due to different numbers of inrtions. The ba composition is 35.6% A, 22.2% C, 18.6% G and 23.6% T respectively. The 16S rRNA gene was located between tRNA Val gene and tRNA Leu(UUR) gene. The length is 1579 bp, which was identical to coyote but shorter than gray wolf (1580 bp). The ba composition is 36.8% A, 20.6% C, 17.6% G and 25.0% T respectively. The computed A+T content of the 12S rRNA and 16S rRNA gene are 59.2% and 61.8%, which also indicates an A−
T rich.farmington
Fig. 1 Organizations of red fox mitochondrial
保鲜膜英文genome. Transfer RNA genes are depicted
by their one-letter amino acid codes
Numbers indicate non-coding nucleotides between genes or gene overlap (negative values). Arrows indicate orientation on (+) strand (clockwi) or (−) strand (counterclockwi).
daughter126 Zoological Rearch V ol. 31
Tab. 4 Organizations and characteristics of red fox mitochondrial genome
Position number Codon
Name of gene
Start
Stop
Size(bp)
Intervals
Start Stop Anti-codon Strand
tRNA Phe 1 69 69 0 GAA H 12SrRNA 70 1026 957 0
H tRNA Val 10271093 67 0 TAC H 16S rRNA 10942672 1579 0 H tRNA Leu(UUR) 26732747 75 2 TAA H ND1 27503706 957 0 ATG TAA
H tRNA Ile 37063774 69 -3 GAT H tRNA Gln 37723845 74 1 TTG L tRNA Met 38473916 70 0 CAT H ND2 39174960 1044 -2 ATA TAG
H tRNA Trp 49595026 68 12 TCA H tRNA Ala 50395107 69 1 TGC L tRNA Asn 51095179 71 0 GTT L OL 51805217 38 -4 L tRNA Cys 52145281 68 0 GCA L tRNA Tyr 52825349 68 1 GTA L COI 53516895 1545 -3 ATG TAA
H tRNA Ser(UCN) 68936961 69 6 TGA L tRNA Asp 69687035 68 0 GTC H COII 70367719 684 16 ATG TAA H tRNA Lys 77377803 67 1
TTT H ATPa8 78058008 204 -33 ATG TAA
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H ATPa6 79668646 681 -1 ATG TAA H COIII 86469429 0 ATG T
H tRNA Gly 94309497 68 0 TCC H ND3 94989844 347 0 ATT TA H tRNA Arg 98459913 69 0 TCG H ND4L 9914
10210 297 -7
ATG TAA
H ND4 10204 11582 1379 -1 ATG T H tRNA His 11582 11650 69 1 GTG H tRNA Ser(AGY) 11652 11710 59 0 H tRNA Leu(CUN) 11711 11780
70
0 TAG H ND5 11781 13601 1821 -17 ATA TAA H ND6 13585 14112 528 0 ATG TAA L tRNA Glu 14113 14181 69 4 TTC L Cytb 14186 15325 1140
0 ATG AGA H tRNA Thr 15326 15395 70 0 TGT H tRNA Pro 15395 15460
66
0 TGG L D-loop
15461 16723 1263
The length of the red fox mitochondrial tRNA genes
range from 59 bp to 75 bp. Among the adjacent tRNA genes, only tRNAIle and tRNAGln overlap each other by 3 bp. Except for tRNASer(AGY) lack the dihydrouridine
stem and loop (DHU Stem and loop), other tRNAs can fold into typical cloverleaf condary structure (Fig.2). The average A+T content in all tRNA genes is 63.6%, higher than that of protein-coding and rRNA genes (Tab.sound是什么意思
