The mitochondrial genome of the soybean cyst nematode, Heterodera glycines

We sequenced the entire coding region of the mitochondrial genome of Heterodera glycines. The sequence obtained comprised 14.9 kb, with PCR evidence indicating that the entire genome comprised a single, circular molecule of approximately 21-22 kb. The genome is the most T-rich nematode mitochondrial genome reported to date, with T representing over half of all nucleotides on the coding strand. The genome also contains the highest number of poly(T) tracts so far reported (to our knowledge), with 60 poly(T) tracts ≥ 12 Ts. All genes are transcribed from the same mitochondrial strand. The organization of the mitochondrial genome of H. glycines shows a number of similarities compared with Radopholus similis, but fewer similarities when compared with Meloidogyne javanica. Very few gene boundaries are shared with Globodera pallida or Globodera rostochiensis. Partial mitochondrial genome sequences were also obtained for Heterodera cardiolata (5.3 kb) and Punctodera chalcoensis (6.8 kb), and these had identical organizations compared with H. glycines. We found PCR evidence of a minicircular mitochondrial genome in P. chalcoensis, but at low levels and lacking a noncoding region. Such circularised genome fragments may be present at low levels in a range of nematodes, with multipartite mitochondrial genomes representing a shift to a condition in which these subgenomic circles predominate.     

Sequence and Characterization of Six Mitochondrial Subgenomes from <Emphasis Type="Italic">Globodera rostochiensis</Emphasis>: Multipartite Structure Is Conserved Among Close Nematode Relatives

Recently, a multipartite mitochondrial genome was characterized in the potato cyst nematode, Globodera pallida. Six subgenomic circles were detectable by PCR, while full-length genomes were not. We investigate here whether this subgenomic organization occurs in a close relative of G. pallida. We amplified and sequenced one entire mitochondrial subgenome from the cyst-forming nematode, Globodera rostochiensis. Comparison of the noncoding region of this subgenome with those reported previously for G. pallida facilitated the design of amplification primers for a range of subgenomes from G. rostochiensis. We then randomly sequenced five subgenomic fragments, each representative of a unique subgenome. This study indicates that the multipartite structure reported for G. pallida is conserved in G. rostochiensis. A comparison of subgenomic organization between these two Globodera species indicates a considerable degree of overlap between them. Indeed, we identify two subgenomes with an organization identical with that reported for G. pallida. However, other subgenomes are unique to G. rostochiensis, although some of these have blocks of genes comparable to those in G. pallida. Dot-plot comparisons of pairs of subgenomes from G. rostochiensis indicate that the different subgenomes share fragments with high sequence identity. We interpret this as evidence that recombination is operating in the mitochondria of G. rostochiensis. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Rafael Zardoya     

A multipartite mitochondrial genome in the potato cyst nematode Globodera pallida

The mitochondrial genome (mtDNA) of the plant parasitic nematode Globodera pallida exists as a population of small, circular DNAs that, taken individually, are of insufficient length to encode the typical metazoan mitochondrial gene complement. As far as we are aware, this unusual structural organization is unique among higher metazoans, although interesting comparisons can be made with the multipartite mitochondrial genome organizations of plants and fungi. The variation in frequency between populations displayed by some components of the mtDNA is likely to have major implications for the way in which mtDNA can be used in population and evolutionary genetic studies of G. pallida.     

A low rate of replacement substitutions in two major Ovis aries mitochondrial genomes

Two mitochondrial DNA molecules which represent major Ovis aries mtDNA haplogroups were cloned and comparatively sequenced to assess the degree of intraspecific variation. A total of 9623 bp that correspond to 58% of both mitochondrial genomes were determined. The control region, the Cyt b , ND2, ND3, ND4L, COIII and 12 tRNA genes, including the origin of L-strand replication, were completely characterized. Partial sequence information was obtained from the 12S and 16S rRNA and an additional six protein coding and six tRNA genes. The control regions of the two mtDNAs showed a nucleotide divergence of 4·34% while coding regions differed by 0·44%. The number of sheep coding region substitutions was similar to values observed in intraspecific comparisons of mitochondrial DNAs that represent remote points in genealogical trees of mice and humans. However, replacement substitutions were only observed at ∼30% of the rate in mice and ∼20% of the rate in humans. Nucleotide substitutions with a potential for phenotypic effects were found in the 12S and 16S rRNA and in the ND1 and COIII genes.     

The broad-spectrum potato cyst nematode resistance gene (Hero) from tomato is the only member of a large gene family of NBS-LRR genes with an unusual amino acid repeat in the LRR region

The Hero gene of tomato is a broad spectrum resistance gene that confers a high level of resistance to all pathotypes of the potato cyst nematodes Globodera rostochiensis and partial resistance to G. pallida. The gene was identified by map-based cloning, sequencing and complementation analysis of two susceptible tomato lines with an array of 13 overlapping cosmids spanning a total distance of 135 kb. Hero encodes a protein with a nucleotide-binding site (NBS) and a leucine-rich-repeat (LRR) domain and is a member of a gene family of 14 highly homologous genes, which are clustered within a continuous 118-kb region. The isolated Hero gene displayed resistance to various G. rostochiensis pathotypes and partial resistance to G. pallida pathotype Pa2/3 in transgenic tomato lines. None of the Hero homologues conferred resistance to G. rostochiensis pathotypes. Hero can be distinguished from its homologues by the length of a compound hexanucleotide microsatellite, which codes for a charged and repetitive amino acid domain within the LRR. We propose that the expansion of this microsatellite may be involved in the evolution of the Hero resistance gene.     

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea starPisaster ochraceus

Summary We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea starPisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA^glu and tRNA^thr are 3 to the 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.     

Multiplication of English isolates of potato pale cyst- nematode, Globodera pallida, on potatoes of differing nematode resistance and the identity of potato cyst- nematode populations

In pots, 25 populations of potato pale cyst-nematode, Globodera pallida Stone, differed significantly in their ability to multiply on potato clones P55/7 and ZC83/ 6, both fully resistant to G. pallida pathotype Pal. Neither clone was fully resistant to any of the populations. For 21 populations common to this and an earlier experiment, increase on the more resistant potatoes (P55/7, ZC83/6, cvs Sante, Paladin and Glenna) was correlated with their increase on less resistant potatoes (cvs Morag, Fingal and Valiant). Variation in virulence on these partially resistant potatoes was not matched by differences in the electrophoretic patterns of the nematodes' proteins. The identification of populations of G. rostochiensis (Woll.) Skarbilovich used in these experiments was confirmed by electrophoresis. All populations of G. pallida Stone, appeared to contain very small numbers of G. rostochiensis after subculture on susceptible potatoes (cv. Désirée).     

Microsatellite loci in the phytoparasitic nematode Globodera.

A Globodera pallida genomic library, population Guiclan (Pa2/3), was screened for TG and TC microsatellite motifs. Screening of 50,000 clones revealed 48 positive matches. After sequencing, primers were designed to amplify 14 microsatellite loci. The specificity of the loci was tested with DNA templates of other populations of G. pallida, and also on other species of Globodera. Appearance of amplification products on several of these DNA templates showed that the microsatellite flanking regions are relatively conserved between G. pallida populations as well as between Globodera species. Evidence for allele polymorphism between individuals was demonstrated by using nine loci primers, in G. pallida population Guiclan and from a population of a closely related species G. "mexicana". Some alleles appeared to be species specific.     

Complete mitochondrial genome of the Amur stickleback Pungitius sinensis (Gasterosteiformes, Gasterosteidae)

The complete mitochondrial genome was sequenced from the Amur stickleback Pungitius sinensis. The genome sequence was 16,581 bp in size, and the gene order and contents were identical with those of previously reported fish mitochondrial genomes. Of 13 protein-coding genes (PCGs), four genes (ND2, CO2, ND4, Cytb) had incomplete stop codons. The base composition of P. sinensis showed anti-G bias (9.53%) on the third position of PCGs.     

Mitochondrial DNA Genomes Organization and Phylogenetic Relationships Analysis of Eight Anemonefishes (Pomacentridae: Amphiprioninae)

Anemonefishes (Pomacentridae Amphiprioninae) are a group of 30 valid coral reef fish species with their phylogenetic relationships still under debate. The eight available mitogenomes of anemonefishes were used to reconstruct the molecular phylogenetic tree; six were obtained from this study (Amphiprion clarkii, A. frenatus, A. percula, A. perideraion, A. polymnus and Premnas biaculeatus) and two from GenBank (A. bicinctus and A. ocellaris). The seven Amphiprion species represent all four subgenera and P. biaculeatus is the only species from Premnas. The eight mitogenomes of anemonefishes encoded 13 protein-coding genes, two rRNA genes, 22 tRNA genes and two main non-coding regions, with the gene arrangement and translation direction basically identical to other typical vertebrate mitogenomes. Among the 13 protein-coding genes, A. ocellaris (AP006017) and A. percula (KJ174497) had the same length in ND5 with 1,866 bp, which were three nucleotides less than the other six anemonefishes. Both structures of ND5, however, could translate to amino acid successfully. Only four mitogenomes had the tandem repeats in D-loop; the tandem repeats were located in downstream after Conserved Sequence Block rather than the upstream and repeated in a simply way. The phylogenetic utility was tested with Bayesian and Maximum Likelihood methods using all 13 protein-coding genes. The results strongly supported that the subfamily Amphiprioninae was monophyletic and P. biaculeatus should be assigned to the genus Amphiprion. Premnas biaculeatus with the percula complex were revealed to be the ancient anemonefish species. The tree forms of ND1, COIII, ND4, Cytb, Cytb+12S rRNA, Cytb+COI and Cytb+COI+12S rRNA were similar to that 13 protein-coding genes, therefore, we suggested that the suitable single mitochondrial gene for phylogenetic analysis of anemonefishes maybe Cytb. Additional mitogenomes of anemonefishes with a combination of nuclear markers will be useful to substantiate these conclusions in future studies.     

Complete mitochondrial genome of <Emphasis Type="Italic">Ampittia dioscorides</Emphasis> (Lepidoptera: Hesperiidae) and its phylogenetic analysis

The complete mitochondrial genome of Ampittia dioscorides (Lepidoptera: Hesperiidae) was determined. The sequenced genome is a circular molecule of 15313 bp, containing 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an A + T-rich region. The gene arrangements and transcribing directions are identical to those in most of the reported lepidopteran mitogenomes. The base composition of the whole genome and genes or regions are also similar to those in other lepidopteran species. All the PCGs are initiated by typical ATN codons; the exception being COI, which begins with a CGA codon. Eight genes (ND2, ATPase8, ATPase6, COIII, ND5, ND4L, ND6, and Cytb) end with a TAA stop codon, and two genes (ND1 and ND3) end with TAG. The remaining three genes (COI and COII, which end with TA-, and ND4, which ends with T-) have incomplete stop codons. All tRNAs have the typical clover-leaf structure of mitochondrial tRNAs, with the exception of tRNA^Ser(AGY). On the basis of the concatenated nucleotide and amino acid sequences of the 13 PCGs and wingless gene of 22 butterfly species, maximum parsimony (MP) and Bayesian inference (BI) trees were constructed, respectively. Both MP and BI trees had the same topological structure: ((((Nymphalidae + Danaidae) + Lycaenidae) + Pieridae) + Papilionidae) + Hesperiidae). The results provide support for Hesperiidae as a superfamily-level taxon.     

Complete mitochondrial genome of the marine medaka Oryzias melastigma (Beloniformes, Adrianichthyidae)

The complete mitochondrial genome was obtained from the assembled genome data sequenced by next-generation sequencer from the marine medaka Oryzias melastigma. The mitochondrial genome sequence was 16,864 bp in size, and the gene order and contents were identical with those of previously reported fish mitochondrial genomes. Of 13 protein-coding genes (PCGs), 4 genes (CO3, ND3, ND4, and Cytb) had incomplete stop codons. The base composition of O. melastigma mitogenome showed high A+T (59.65%) and anti-G bias (8.73%) on the 3rd position of PCGs.     

Rapid identification of cyst (Heterodera spp., Globodera spp.) and root-knot (Meloidogyne spp.) nematodes on the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) in cultures and field samples

Cyst and root-knot nematodes show high levels of gross morphological similarity. This presents difficulties for the study of their ecology in natural ecosystems. In this study, cyst and root-knot nematode species, as well as some ectoparasitic nematode species, were identified using the second internal transcribed spacer (ITS2) sequence variation detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP). The ITS2 region was sufficiently variable within the taxa investigated to allow species to be separated on the basis of minor sequence variation. The PCR primers used in this study were effective for 12 species with three genera within the Heteroderinae (Globodera pallida, G. rostochiensis, Heterodera arenaria/avenae, H. ciceri, H. daverti, H. hordecalis, H. mani, H. schachtii, H. trifolii, Meloidogyne ardenensis, M. duytsi and M. maritima). However, pathotypes of Globodera pallida and G. rostochiensis could not be distinguished. The method was tested at two coastal dune locations in The Netherlands (one in the lime-poor dunes of the north and one in calcareous dunes of the south) to determine the population structure of cyst nematodes. At each site, cyst nematodes were associated with three plant species: two plant species on the foredune (Elymus farctus and Ammophila arenaria) and one plant species occurring further inland (Calamagrostis epigejos). Two species of cyst nematodes, H. arenaria and H. hordecalis, were found. H. arenaria associated with vigorous A. arenaria and H. hordecalis in association with degenerating A. arenaria and C. epigejos. The field survey demonstrated that in coastal dunes abiotic factors may be the important affecting the distribution of cyst nematodes.     

黑尾地鸦线粒体基因组序列测定与分析

采用长PCR扩增的线粒体DNA和引物步移法, 测定并注释了中国特有鸟类-黑尾地鸦(Podoces hendersoni)的线粒体基因组全序列。黑尾地鸦的mtDNA序列全长16 867 bp, GenBank登录号GU592504。基因含量和排列次序与原鸡的一致, 包含13个蛋白编码基因、22个tRNA、2个rRNA和1个控制区(D-loop)。除COI基因以GTG作为起始密码子外, 其余12个蛋白质编码基因均以典型ATG密码子起始。11个蛋白编码基因以完全终止密码子TAA、AGG或AGA终止, COIII和ND4基因终止密码子为不完整的T。tRNA^Ser(AGY)的DHU臂缺失, tRNA^Leu(CUN)的反密码子环由9个碱基构成, 而不是标准的7个碱基。其余的20个tRNA基因的二级结构均属典型的三叶草结构。预测了rRNA的二级结构, 其中, 12S rRNA二级结构包含4个结构域, 43个茎环结构; 16S rRNA的二级结构包含6个结构域, 55个茎环结构。此外, 在其他鸟类控制区中所发现的F-box、D-box、C-box、B-box、Bird similarity-box和CSB1-box也同样存在于黑尾地鸦中。     

Comparison of British populations of potato cyst nematodes with populations from continental Europe and South America using RAPDs.

Genetic variation between populations of Globodera pallida, primarily from Britain but including populations from continental Europe and South America and two Globodera rostochiensis populations, was examined using random amplified polymorphic DNA (RAPD). Fourteen primers were used and 250 amplification products observed. A comparison was made of the similarities between the species and, within G. pallida, between populations from Britain, The Netherlands, Germany, and Switzerland, of the pathotypes Pa2 and Pa3. In addition, one Pa1 population and two others from South America were included. On the basis of the RAPD analysis, all the Pa2-Pa3 populations, except one from Scotland (Luffness), constituted a single group with no clear distinction based on pathotype designation. The Luffness population is known to be distinct in its virulence. The data indicated that the main Pa2-Pa3 group could be subdivided based on geographic origin, but this is not well supported by bootstrap analysis. The Pa1 population and the two populations from South America all formed distinct groups.     

Phylogenetic inference of calyptrates,with the first mitogenomes for Gasterophilinae (Diptera: Oestridae) and Paramacronychiinae (Diptera: Sarcophagidae)

The complete mitogenome of the horse stomach bot fly Gasterophilus pecorum (Fabricius) and a near-complete mitogenome of Wohlfahrt''s wound myiasis fly Wohlfahrtia magnifica (Schiner) were sequenced. The mitogenomes contain the typical 37 mitogenes found in metazoans, organized in the same order and orientation as in other cyclorrhaphan Diptera. Phylogenetic analyses of mitogenomes from 38 calyptrate taxa with and without two non-calyptrate outgroups were performed using Bayesian Inference and Maximum Likelihood. Three sub-analyses were performed on the concatenated data: (1) not partitioned; (2) partitioned by gene; (3) 3rd codon positions of protein-coding genes omitted. We estimated the contribution of each of the mitochondrial genes for phylogenetic analysis, as well as the effect of some popular methodologies on calyptrate phylogeny reconstruction. In the favoured trees, the Oestroidea are nested within the muscoid grade. Relationships at the family level within Oestroidea are (remaining Calliphoridae (Sarcophagidae (Oestridae, Pollenia + Tachinidae))). Our mito-phylogenetic reconstruction of the Calyptratae presents the most extensive taxon coverage so far, and the risk of long-branch attraction is reduced by an appropriate selection of outgroups. We find that in the Calyptratae the ND2, ND5, ND1, COIII, and COI genes are more phylogenetically informative compared with other mitochondrial protein-coding genes. Our study provides evidence that data partitioning and the inclusion of conserved tRNA genes have little influence on calyptrate phylogeny reconstruction, and that the 3rd codon positions of protein-coding genes are not saturated and therefore should be included.     

Complete mitochondrial genome of the Amur stickleback Pungitius kaibarae (Gasterosteiformes, Gasterosteidae)

The complete mitochondrial genome was sequenced from the Amur stickleback Pungitius kaibarae. The genome sequence was 16,505 bp in size, and the gene order and contents were identical with the same genera Pungitius sinensis and other previously reported fish mitochondrial genomes. Of 13 protein-coding genes (PCGs), 3 genes (CO2, ND4, and Cytb) had incomplete stop codons. The base composition of P. kaibarae showed anti-G bias (8.86%) on the third position of PCGs.     

大卫绢蛱蝶线粒体基因组全序列测定和分析

目前有关蝶类线粒体基因组全序列及其分子进化的研究报道还不多见。本文利用long PCR和引物步移法得到大卫绢蛱蝶Calinaga davidis的线粒体基因组全序列, 同时就其基因组成和结构特点作了初步分析。结果显示： 其基因组全长为15 267 bp (GenBank登录号为HQ658143), 包括13个蛋白质编码基因(ATP6, ATP8, COI-III, ND1-6, ND4L, Cytb)、22个tRNA基因、2个rRNA基因(16S和12S)以及非编码的控制区。与其他鳞翅目昆虫相一致, 其基因组未出现基因重排现象。基因组共包含11个基因间隔区,总长度为130 bp, 间隔长度1~46 bp, 最大间隔在tRNA^Gln与ND2基因之间; 基因间共存在13处重叠, 总长度为66 bp, 重叠碱基数1~35 bp, 最长的重叠区位于COII与tRNA^Lys基因。lrRNA和srRNA基因长度分别为1 337 bp和773 bp; 除tRNA^Ser(AGN)缺少二氢尿嘧啶臂(DHU stem), 在相应的位置上只形成一个简单环外, 其余的tRNA基因都能形成典型的三叶草结构。13个蛋白编码基因总长度为11 247 bp, 共有3 737个密码子, 它们的碱基组成和密码子的使用具有明显的偏倚性; 除COI外(起始密码子TTG), 其余的12个蛋白质编码基因都以标准的ATN作为起始密码子; COI基因终止密码子为不完全T, ND4基因终止密码子为不完全TA, 其余基因都以TAA为终止密码子。A+T丰富区全长为389 bp, A+T含量高达92.0%, 其中存在2段类似微卫星的重复序列(TA)₆和(AAT)₄。本文的研究结果为探讨绢蛱蝶亚科在蛱蝶科中的系统学地位及其与其他亚科间的系统发生关系等问题提供了重要的分子生物学数据。     

The complete mitochondrial genome of the damsel bug Alloeorhynchus bakeri (Hemiptera: Nabidae)

The complete sequence of the mitochondrial DNA (mtDNA) of the damsel bug, Alloeorhynchus bakeri, has been completed and annotated in this study. It represents the first sequenced mitochondrial genome of heteropteran family Nabidae. The circular genome is 15, 851 bp in length with an A+T content of 73.5%, contains the typical 37 genes that are arranged in the same order as that of the putative ancestor of hexapods. Nucleotide composition and codon usage are similar to other known heteropteran mitochondrial genomes. All protein-coding genes (PCGs) use standard initiation codons (methionine and isoleucine), except COI, which started with TTG. Canonical TAA and TAG termination codons are found in eight protein-coding genes, the remaining five (COI, COII, COIII, ND5, ND1) have incomplete termination codons (T or TA). PCGs of two strands present opposite CG skew which is also reflected by the nucleotide composition and codon usage. All tRNAs have the typical clover-leaf structure, except the dihydrouridine (DHU) arm of tRNA(Ser (AGN))which forms a simple loop as known in many other metazoa. Secondary structure models of the ribosomal RNA genes of A. bakeri are presented, similar to those proposed for other insect orders. There are six domains and 45 helices and three domains and 27 helices in the secondary structures of rrnL and rrnS, respectively. The major non-coding region (also called control region) between the small ribosomal subunit and the tRNA(Ile )gene includes two special regions. The first region includes four 133 bp tandem repeat units plus a partial copy of the repeat (28 bp of the beginning), and the second region at the end of control region contains 4 potential stem-loop structures. Finally, PCGs sequences were used to perform a phylogenetic study. Both maximum likelihood and Bayesian inference analyses highly support Nabidae as the sister group to Anthocoridae and Miridae.     

Characterization of the red knot (Calidris canutus) mitochondrial control region.

We sequenced the complete mitochondrial control regions of 11 red knots (Calidris canutus). The control region is 1168 bp in length and is flanked by tRNA glutamate (glu) and the gene ND6 at its 5' end and tRNA phenylalanine (phe) and the gene 12S on its 3' end. The sequence possesses conserved sequence blocks F, E, D, C, CSB-1, and the bird similarity box (BSB), as expected for a mitochondrial copy. Flanking tRNA regions show correct secondary structure, and a relative rate test indicated no significant difference between substitution rates in the sequence we obtained versus the known mitochondrial sequence of turnstones (Charadriiformes: Scolopacidae). These characteristics indicate that the sequence is mitochondrial in origin. To confirm this, we sequenced the control region of a single individual using both purified mitochondrial DNA and genomic DNA. The sequences were identical using both methods. The sequence and methods presented in this paper may now serve as a reference for future studies using knot and other avian control regions. Furthermore, the discovery of five variable sites in 11 knots towards the 3' end of the control region, and the variability of this region in contrast to the more conserved central domain in the alignment between knots and other Charadriiformes, highlights the importance of this area as a source of variation for future studies in knots and other birds.