Structure and Evolution of the Mitochondrial Control Region of Leaf Beetles (Coleoptera: Chrysomelidae): A Hierarchical Analysis of Nucleotide Sequence Variation

To assess the levels of variation at different evolutionary scales in the mitochondrial (mt) control region of leaf beetles, we sequenced and compared the full mt control region in two genera ( Chrysomela and Gonioctena), in two species within a genus ( Gonioctena olivacea and G. pallida), in individuals from distant populations of these species in Europe, and in individuals from populations separated by moderate (10- to 100-km) to short (<5-km) distances. In all individuals, a highly repetitive section consisting of the tandem repetition of 12 to 17 imperfect copies of a 107- to 159-bp-long core sequence was observed. This repetitive fragment accounts for roughly 50% of the full control-region length. The sequence variability among repeated elements within the control region of a given individual depends on the species considered: the variability within any G. olivacea individual is much higher than that within G. pallida individuals. Comparisons of the repeated elements, in a phylogenetic framework, within and among individuals of G. olivacea and G. pallida suggests that the repetitive section of the control region experienced recurrent duplications/deletions, leading to some degree of concerted evolution. Comparisons between Chrysomela and Gonioctena control regions revealed virtually no significant sequence similarity, except for two long stretches of A's and several [T(T)A(A)] repeats, all found in the control region of other insect orders. Our analyses allowed us to identify portions of the control region with enough variation for population genetic or phylogeographic studies.     

Mitogenomic analysis of Montipora cactus and Anacropora matthai (cnidaria; scleractinia; acroporidae) indicates an unequal rate of mitochondrial evolution among Acroporidae corals

The complete nucleotide sequence of the mitochondrial (mt) genome was determined for specimens of the coral species Montipora cactus (Bernard 1897) and Anacropora matthai (Pillai 1973), representing two morphologically distinct genera of the family Acroporidae. These sequences were compared with the published mt genome sequence for the confamilial species, Acropora tenuis (Dana 1846). The size of the mt genome was 17,887 bp and 17,888 bp for M. cactus and A. matthai. Gene content and organization was found to be very similar among the three Acroporidae mt genomes with a group I intron occurring in the NADH dehyrogenase 5 (nad5) gene. The intergenic regions were also similar in length among the three corals. The control region located between the small ribosomal RNA (ms) and the cytochrome oxidase 3 (cox3) gene was significantly smaller in M. cactus and A. matthai (both 627 bp) than in A. tenuis (1086 bp). Only one set of repeated sequences was identified at the 3′-end of the control regions in M. cactus and A. matthai. A lack of the abundant repetitive elements which have been reported for A. tenuis, accounts for the relatively short control regions in M. cactus and A. matthai. Pairwise distances and relative rate analyses of 13 protein coding genes, the group I intron and the largest intergenic region, igr3, revealed significant differences in the rate of molecular evolution of the mt genome among the three species, with an extremely slow rate being seen between Montipora and Anacropora. It is concluded that rapid mt genome evolution is taking place in genus Acropora relative to the confamilial genera Montipora and Anacropora although all are within the relatively slow range thought to be typical of Anthozoa.     

A QTL for broad-spectrum resistance to cyst nematode species (Globodera spp.) maps to a resistance gene cluster in potato

 Broad-spectrum resistance in potato to the potato cyst nematode (PCN) species Globodera rostochiensis and G. pallida is commonly regarded as a polygenically inherited trait. Yet, by use of QTL analysis and a selected set of PCN populations, resistance to both PCN species could be ascribed to the action of locus Grp1. Grp1 confers major resistance to G. rostochiensis line Ro₅-22 and G. pallida population Pa₂-D383 and partial resistance to G. pallida population Pa₃-Rookmaker. Grp1 was mapped on chromosome 5 using previously characterized AFLP markers. Cleaved amplified polymorphic sequence (CAPS) markers available for RFLP loci GP21 and GP179 revealed that Grp1 maps on a genomic region harboring other resistance factors to viral, fungal and nematodal pathogens. The present data indicate that Grp1 is a compound locus which contains multiple genes involved in PCN resistance. Received: 10 September 1997 / Accepted: 6 October 1997     

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     

Observations on the distribution of potato cyst-nematodes, Globodera rostochiensis and G. pallida on two fenland potato farms

The distribution of potato cyst-nematodes, Globodera rostochiensis and G. pallida was surveyed in two widely separated fenland potato farms in Eastern England. Evidence was obtained of the suppression of G. rostochiensis and increase of G. pallida populations when Maris Piper and other potatoes with the same gene (H₁) for resistance to G. rostochiensis are grown. Numbers of G. rostochiensis and/or G. pallida in the soil could not be related directly to recent potato cropping histories of the fields. Identification of the two species of Globodera by the presence and colour of females on the roots of Désirée, Maris Piper, Cromwell (A27/20) and A25/11 potatoes were confirmed by agarose gel isoelectric focussing of general proteins. Cromwell exhibited little or no resistance to G. pallida at Woodwalton. A25/11 was more resistant to this species at Woodwalton but was susceptible to G. rostochiensis. The results of these surveys emphasise the need for carefully planned and integrated control programmes based on the use of resistant cultivars of potato, crop rotations and appropriate nematicide use to combat the growing problem of G. pallida, especially in organic soils.     

Single nucleotide polymorphism (SNP) genotyping as basis for developing a PCR-based marker highly diagnostic for potato varieties with high resistance to Globodera pallida pathotype Pa2/3

Globodera pallida is a parasitic root cyst nematode of potato, which causes reduction of crop yield and quality in infested fields. Field populations of G. pallida containing mixtures of pathotypes Pa2 and Pa3 (Pa2/3) are currently most relevant for potato cultivation in middle Europe. Genes for resistance to G. pallida have been introgressed into the cultivated potato gene pool from the wild, tuber bearing Solanum species S. spegazzinii and S. vernei. Selection of resistant genotypes in breeding programs is hampered by the fact that the phenotypic evaluation of resistance to G. pallida is time consuming, costly and often ambiguous. DNA-based markers diagnostic for resistance to G. pallida would facilitate the development of resistant varieties. A tetraploid F₁ hybrid family SR-Gpa segregating for quantitative resistance to G.␣pallida was developed and evaluated for resistance to G. pallida population ‘Chavornay’. Two subpopulations of 30 highly resistant and 30 susceptible individuals were selected and genotyped for 96 single nucleotide polymorphism (SNP) markers tagging 12 genomic regions on 10 potato chromosomes. Seven SNPs were found significantly linked to the nematode resistance, which were all located within a resistance ‘hotspot’ on potato chromosome V. A haplotype model for these seven SNPs was deduced from the SNP patterns observed in the SR-Gpa family. A PCR assay ‘HC’ was developed, which specifically detected the SNP haplotype c that was linked with high levels of nematode resistance. The HC marker was only found in accessions of S.␣vernei. Screening with the HC marker 34 potato varieties resistant to G. pallida pathotypes Pa2 and/or Pa3 and 22 susceptible varieties demonstrated that the HC marker was highly diagnostic for presence of high levels of resistance to G. pallida pathotype Pa2/Pa3.Amirali Sattarzadeh and Ute Achenbach contributed equally to the work     

Differentiation of Closely Related Globodera pallida (Stone) Populations by Means of DNA Restriction Fragment Length Polymorphisms (RFLPs)

A small genomic library from DNA of G. pallida population Delmsen (pathotype 3) was constructed. A number of clones were tested as hybridization probes with 21 G. pallida and 6 G. rostochiensis populations. The two species were easily distinguished by their hybridization patterns. A total of 32 RFLP markers were identified which permitted the differentiation of all G. pallida populations. By means of cluster analysis of the RFLP data the genetic distances between the 21,G. pallida populations were calculated and a dendrogram was constructed. The arrangement of the 21 G. pallida populations in the dendrogram suggests a partial correlation between genetic distance and virulence according to biological pathotyping.     

Emergence of juvenile potato cyst-nematodes Globodera rostochiensis and G. pallida and the control of G. pallida

Speed of emergence of juveniles from cysts in potato root diffusate (PRD) in vitro differed between Globodera rostochiensis and G. pallida and between populations within each species. Early emergence in vitro was slower in most populations of G. pallida than in most populations of G. rostochiensis. Fewer G. rostochiensis juveniles emerged from 4 or 6 month old than from 4 yr old cysts. More G. rostochiensis emerged in solutions of sodium metavanadate at concentrations of 10^-2 and 10^-3 M than in PRD and as many G. pallida emerged in the same solutions as in PRD. In plots of bare fallowed sandy loam, emergence of G. pallida was stimulated by 10^--³ M sodium metavanadate. The emergence of two populations of C. pallida in PRD was stimulated by the addition of benomyl at 0.1 ppm (3.4 × 10^--⁷ m). In microplots, cv. Cara potatoes grown for 8 wk decreased four populations of G. pallida by up to 93%. During a 4 wk period in PRD, more than 20 juveniles per gravid female emerged from five of 25 populations of G. pallida. In root observation boxes in which cv. Désirée was grown, oxamyl applied to the top 15 cm of a peaty loam soil greatly increased G. pallida in soil 1545 cm deep. In another peaty loam, but not in a sandy loam, the same treatment appeared to increase the nematode in soil 15–30 cm deep. Oxamyl incorporated in the uninfested top 15 cm of all three soils largely prevented nematode increase from juveniles migrating upwards from untreated heavily infested soil 15–30 cm deep. These experiments suggest that inadequate control of G. pallida increase on susceptible potatoes by an oximecarbamate nematicide of short persistence, such as oxamyl, is primarily due to the slow rate of juvenile emergence in most populations of G. pallida, with a second generation and the upward migration of juveniles from deeper untreated soil later in the growing season as potential contributory factors.     

Generation of VNTRs and heteroplasmy by sequence turnover in the mitochondrial control region of two elephant seal species

We describe an unusual repetitive DNA region located in the 3′ end of the light (L)-strand in the mitochondrial control region of two elephant seal species. The array of tandem repeats shows both VNTR (variable-number tandem repeat) and sequence variation and is absent from 12 compared mammalian species, except for the occurrence in the same location of a distinct repetitive region in rabbit mtDNA and a similar repeat in the harbor seal. The sequence composition and arrangement of the repeats differ considerably between the northern elephant seal (Mirounga angustirostris) and the southern species (M. leonina) despite an estimated divergence time of 1 MY (based on an mtDNA-RNA gene and the nonrepetitive control region). Analysis of repeat sequence relationships within and between species indicate that divergence in sequence and structure of repeats has involved both slippagelike and unequal crossingover processes of turnover, generating very high levels of divergence and heteroplasmy. Presented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Cloning and sequencing of cDNA encoding glutamine synthetase from the sea anemone Aiptasia pallida

Glutamine synthetase (GS) catalyzes the addition of ammonium to glutamic acid to form glutamine and plays a crucial role in the nitrogen assimilation of the sea anemone Aiptasia pallida and its endosymbiotic algae. We describe the cDNA cloning and sequence analysis of GS mRNA from A. pallida based on polymerase chain reaction (PCR) technology that employed a combination of degenerate and A. pallida-specific primers. The sequenced cDNA approximates 1620 nucleotides and is characterized by an open reading frame of 1107 nucleotides that encodes a protein of 369 amino acid residues. Comparisons of the deduced sea anemone GS protein to a wide range of species demonstrated greatest amino acid sequence identity to sea urchin GS (66%) and least identity to green algae GS (51%). The sequenced cDNA can be used in future research to study GS gene expression in A. pallida.     

Segregation and Recombination of a Multipartite Mitochondrial DNA in Populations of the Potato Cyst Nematode <Emphasis Type="Italic">Globodera pallida</Emphasis>

The discovery that the potato cyst nematode Globodera pallida has a multipartite mitochondrial DNA (mtDNA) composed, at least in part, of six small circular mtDNAs (scmtDNAs) raised a number of questions concerning the population-level processes that might act on such a complex genome. Here we report our observations on the distribution of some scmtDNAs among a sample of European and South American G. pallida populations. The occurrence of sequence variants of scmtDNA IV in population P4A from South America, and that particular sequence variants are common to the individuals within a single cyst, is described. Evidence for recombination of sequence variants of scmtDNA IV in P4A is also reported. The mosaic structure of P4A scmtDNA IV sequences was revealed using several detection methods and recombination breakpoints were independently detected by maximum likelihood and Bayesian MCMC methods. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. Rafael Zardoya]     

Morphological and Molecular Identification of Globodera pallida Associated with Potato in Idaho

The identity of a newly discovered population of pale potato cyst nematode Globodera pallida associated with potato in eastern Idaho was established by morphological and molecular methods. Morphometrics of cysts and second-stage juveniles were generally within the expected ranges for G. pallida with some variations noted. The Idaho population and paratype material from Epworth, Lincolnshire, England, both showed variations in tail shape, with bluntly rounded to finely pointed tail termini. Compared to literature values for the paratypes, second-stage juveniles of the Idaho population had a somewhat shorter mean body length, and cysts had a slightly higher mean distance from the anus to the nearest edge of the fenestra. PCR-RFLP of the rDNA ITS region, sequence-specific multiplex PCR and DNA sequence comparisons all confirmed the identity of the Idaho population as G. pallida. The ITS rDNA sequence of the Idaho isolate was identical to those from York, England, and the Netherlands. Species-specific primers that can positively identify the tobacco cyst nematode Globodera tabacum were also developed, providing a new assay for distinguishing this species from G. pallida and the golden potato cyst nematode Globodera rostochiensis.     

Estimation of genomic variation between British populations of the potato cyst nematode Globodera pallida using RAPD-PCR

Random amplified polymorphic DNA (RAPD) has been used to investigate the interrelationships of 20 populations of Globodera pallida collected originally from field soils around the UK. RAPD analysis revealed a high level of relative genomic diversity within British G. pallida but there was no general correlation of genomic similarity with geographic distribution. Two populations of pathotype Pa1 were clearly divergent from the bulk of G. pallida and might represent a distinct introduction. Two other populations, from Scotland and Wales, were also dissimilar from each other and from the rest of the G. pallida populations.     

Confirmation of natural hybrids between Gentiana straminea and G. siphonantha (Gentianaceae) based on molecular evidence

A few individuals with intermediate morphology always appeared in the sympatric distributions of Gentiana straminea and G. siphonantha. These intermediate individuals were hypothesized to be the hybrids of two species after a careful evaluation of their morphological characteristics. To test this hypothesis, sequence comparison of the internal transcribed spacer (ITS) regions of the nuclear ribosomal and trnS (GCU)-trnG (UCC) intergenic spacer region of the chloroplast DNA from Gentiana straminea, G. siphonantha and the putative hybrids was performed. The results suggest that most intermediate individuals were the natural hybrids between G. straminea and G. siphonantha. In addition, we examined the sequence variation among the individuals of both parent species and analyzed the possibility leading to the incongruent identification in some individuals based on morphologic and molecular evidences, respectively. The intraspecific diversification of DNA fragments within both parent species and their high variability in hybrid swarms probably resulted from chloroplast genome recombination and incomplete lineage sorting during the early stages of speciation origin of the parent species. __________ Translated from Acta Botanica Yunnanica, 2007, 29 (1): 91–97 [译自:云南植物研究]     

Changes in relative abundance of two potato cyst nematode species Globodera rostochiensis and G. pallida in one generation

Changes in relative abundance of the two potato cyst nematode species Globodera rostochiensis and Globodera pallida were studied during the 1983/84 season at two different population levels in small pots in the glasshouse and at a single population density on plants grown outdoors in 2 litre terylene cloth bags. In both environments G. rostochiensis was the more successful species. Although the ratio of the two species changed and G. pallida was at a lower level at the end of the experiment it was never eliminated. However, when the number of G. pallida in the mixture was small it did better than expected and demonstrated a frequency dependent response.     

Evidence of olive ridley mitochondrial genome introgression into loggerhead turtle rookeries of Sergipe, Brazil

The coastline of Sergipe state hosts the main Brazilian nesting sites of Lepidochelys olivacea (Eschscholtz, 1829). The second most abundant species of turtles in Sergipe is Caretta caretta (Linnaeus, 1758). Both sea turtle species, respectively known as olive ridley and loggerhead, are currently listed as endangered by the International Union for the Conservation of Nature and Natural Resources. The genetic diversity of the Sergipe loggerhead population (N = 51) was assayed by analyzing 627 bp from the control region of mitochondrial DNA in nesting females. Three haplotypes were identified: CC-A4, CC-A24 and CC × LO. The last one was recorded for specimens considered hybrids because they represent L. olivacea’s mtDNA, but had the external morphology of C. caretta or of a mixture of both species. Based on the two types of hybrids, it was hypothesized that at least two hybridization events had occurred: a more ancient hybridization event, accompanied by introgression (F2 or later backcrosses), and a recent one (F1), both of which involving the same L. olivacea haplotype. The incidence of L. olivacea mitochondrial genome introgression into the C. caretta rookeries was only observed in Sergipe, which could be related to the large numbers of L. olivacea in this region and an overlap of reproduction periods and distribution areas of both species. This may also be associated to global warming since it might alter the sex ratio of sea turtles, thus facilitating interspecific mating. Awareness of gene flow between these species will significantly influence the development and implementation of adequate management strategies.     

Unusual Origin of a Nuclear Pseudogene in the Italian Wall Lizard: Intergenomic and Interspecific Transfer of a Large Section of the Mitochondrial Genome in the Genus <Emphasis Type="Italic">Podarcis</Emphasis> (Lacertidae)

Two distinct cytochrome b-like sequences were discovered in the genome of Podarcis sicula. One of them represents a nuclear copy of a mitochondrial sequence (numt-sic) differing by 14.3% from the authentic mitochondrial (mt) sequence obtained from the same individual. This numt, however, differs by only 2.7% from the mt sequence found in one population of Podarcis muralis, a related species in which no corresponding numt was detected. The numt-sic sequence extends over at least 7637 bp and is homologous to a section of the mt genome spanning from the tRNA-Lys to the tRNA-Pro gene. Premature mt stop codons were detected in two of the nine protein coding genes of numt-sic. The distribution of substitutions among the three codon positions and the transition/transversion ratio of the numt-sic sequence resemble, with few exceptions, those of functional mt genes, indicating a rather recent transfer to the nucleus. Phylogenetic analyses performed on the data set including P. sicula numt-cytb sequences as well as mt-cytb sequences from the same individuals and mt sequences of various P. muralis populations suggest that numt-sic originated in P. muralis. In a geographic survey, P. sicula populations belonging to different mt lineages, covering most of the distribution area, were screened for the presence of numt-sic and for a 15-bp duplication polymorphism in the numt-nd5 sequence. Our results suggest that numt-sic has spread rapidly through the species range via sexual transmission, thereby being transferred to populations belonging to well-separated mt lineages that diverged 1–3 Mya. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Climate change is predicted to alter the current pest status of Globodera pallida and G. rostochiensis in the United Kingdom

The potato cyst nematodes Globodera pallida and G. rostochiensis are economically important plant pathogens causing losses to UK potato harvests estimated at £50 m/ year. Implications of climate change on their future pest status have not been fully considered. Here, we report growth of female G. pallida and G. rostochiensis over the range 15 to 25°C. Females per plant and their fecundity declined progressively with temperatures above 17.5°C for G. pallida, whilst females per plant were optimal between 17.5 and 22.5°C for G. rostochiensis. Relative reproductive success with temperature was confirmed on two potato cultivars infected with either species at 15, 22.5 and 25°C. The reduced reproductive success of G. pallida at 22.5°C relative to 15°C was also recorded for a further seven host cultivars studied. The differences in optimal temperatures for reproductive success may relate to known differences in the altitude of their regions of origin in the Andes. Exposure of G. pallida to a diurnal temperature stress for one week during female growth significantly suppressed subsequent growth for one week at 17.5°C but had no effect on G. rostochiensis. However, after two weeks of recovery, female size was not significantly different from that for the control treatment. Future soil temperatures were simulated for medium‐ and high‐emission scenarios and combined with nematode growth data to project future implications of climate change for the two species. Increased soil temperatures associated with climate change may reduce the pest status of G. pallida but benefit G. rostochiensis especially in the southern United Kingdom. We conclude that plant breeders may be able to exploit the thermal limits of G. pallida by developing potato cultivars able to grow under future warm summer conditions. Existing widely deployed resistance to G. rostochiensis is an important characteristic to retain for new potato cultivars.     

Control of potato pale cyst-nematode, Globodera pallida, with a granular nematicide and partially resistant potatoes

The effects of oxamyl applied to the seedbed and growing partially resistant potatoes in controlling potato pale cyst-nematode, Globodera pallida, were assessed in eleven field experiments on sandy, peaty and silty loam soils in England from 1986 to 1988. Standardised procedures allowed valid comparisons to be made between data from the three experiments in 1987 and from the seven experiments in 1988. In soil moderately or heavily infested with G. pallida, oxamyl frequently increased tuber yields of susceptible cv. Désirée and of partially resistant potatoes and lessened crude nematode increase (Pf/Pi) significantly at some sites but not at others. Although 2.8 kg oxamyl ha“¹ often increased tuber yields significantly, it was significantly less effective than 5.6 kg in controlling G. pallida at one site and at another site G. pallida increased more than in untreated plots. Potatoes partially resistant to G. pallida were more effective that oxamyl in controlling nematode increase at some sites but not at others. Combined use of oxamyl and partially resistant potatoes was generally more effective in controlling G. pallida than either measure alone. As measured by a ‘control coefficient’ (weight of tubers over 40mm diameter (t ha“¹) 4-crude nematode increase (Pf/Pi)), the most effective integrated control of G. pallida was obtained by growing cvs Glenna, Morag or Santé in soil treated with 5.6 kg oxamyl ha^-1.     

Potato cyst nematodes in England and Wales - occurrence and distribution

Potato cyst nematodes (PCN) have been known to occur in the UK for nearly a hundred years. They are the most problematic pests of potatoes and can cause severe yield losses. Previous work has shown the two species, Globodera rostochiensis and G pallida, to be distributed throughout the UK. This paper reports the results of the first structured and statistically unbiased survey undertaken to assess their occurrence and distribution in the potato growing land of England and Wales. The survey showed that PCN were present in 64% of sites sampled. Of the populations found, 67% were G pallida, 8% were G rostochiensis and 25% contained both species. The results show an increase in the incidence of PCN since previous studies were completed and confirm the perceived shift towards G pallida as the predominant species. Of the infestations found, 62% had a population density of less than 10 eggs g^?1 soil.