Nuclear Mitochondrial DNA Research Articles

Systematics of Cyst Nematodes (Nematoda: Heteroderinae)

Systematics of Cyst Nematodes (Nematoda: Heteroderinae) . Nematology Monographs and Perspectives. Volume 8A, 8B . S.A. Subbotin, M. Mundo-Ocampo, J.B. Baldwin . ( David Hunt, Roland N. Perry ( eds )). 351 pp. 512 pp . Leiden, The Netherlands : Koninklijke Brill NV . 2010 . €139, €149. ISBN 978-90-04-16225 9, ISBN 978-90-04-16434 5 (hardback ). Cyst-forming nematodes are amongst the most economically important plant-parasitic nematodes of cultivated crops in the world, in both temperate and tropical regions. Many cyst nematode species, particularly those belonging to the genera Heterodera and Globodera, are pathogens on several important vegetable and fruit crops (carrot, chickpea, clover, fig, maize, olive, pea, potato, rice, soybean, sugar beet, tobacco, wheat, etc.), causing extensive damage and significant yield loss. Consequently, the accurate and timely identification of cyst nematodes infecting crops is a prerequisite for designing effective management strategies for these pathogens. This book is a useful publication that provides a comprehensive overview and an in-depth summary and review of the systematics and identification of cyst-forming nematodes, and includes research on taxonomy, biology, and methods of biochemical and molecular diagnostics. This book is Volume 8 in the Nematology Monographs & Perspectives series, and comprises two parts, A and B. Throughout its 11 chapters, part A presents summarized and specialized information on various key aspects of different exciting areas of morphology, taxonomy, biology, evolution and systematics. Chapters 1–4 describe the most important diagnostic characters of cyst nematodes, as well as summarized aspects on life cycles, symptoms on parasitized plants, their pathogenic variability (pathotypes), biogeography, methods for applying electron microscopy and biochemical and molecular approaches for diagnostics of cyst nematodes. A DNA-based diagnostic section includes a comprehensive review of genes used for molecular diagnostics, including nuclear RNA and mitochondrial DNA genes, as well as methods such as restriction fragment length polymorphism (RFLP), use of specific primers, and real-time PCR. Chapter 5 comprises general systematics of Heteroderinae, including a useful and practical identification key to genera, and a discussion on species delimitation. Chapters 6–11 deal with the systematics, key to species based on morphological features, molecular characterization, and species description in the genera Globodera, Punctodera, Cactodera, Dolichodera, Betulodera and Paradolichodera. For each species, the complete description of female, cyst, male and second-stage juveniles is provided, together with the type host and other host plants, as well as the distribution, biology, pathogenicity and biochemical and molecular characterization, if available. In addition, this volume is complemented with four useful and practical appendices dealing with methods for soil extraction of cyst nematodes, protocols for preparation of vulval cones, separation of G. pallida and G. rostochiensis by immuno-electro-focusing (IEF), and PCR-ITS-RFLP. Part B is completely dedicated to the genus Heterodera which comprises 80 species, including well known pathogens such as H. schachtii in sugar beet, H. avenae, H. filipjevi and H. hordecalis in cereals, H. ciceri in chickpea, H. cruciferae in crucifers, H. glycines in soybean, H. goettingiana in legumes, H. oryzae and H. oryzicola in rice, H. trifolii in clover and H. zeae in maize. As in part A, a key to species based on morphological features, molecular characterization, and species description, biology, distribution, host, pathogenicity, and biochemical and molecular characterization is provided for each species. The text is well written in both parts, and in their approach and choice of illustrations the authors make this a valuable tool for those involved in the fields of nematology and plant pathology. In summary, the authors are to be congratulated for their initiative and achievement in the production of such an enjoyable book that is the most comprehensive treatise currently available on cyst-forming nematodes and, as such, constitutes a unique and most valuable source of information, and a precious guide to anyone (scientists, teachers, students, extension officers) who is interested in Nematology at large, and in cyst nematodes in particular.

Evolutionary and biogeographic history of weasel-like carnivorans (Musteloidea)

We analyzed a concatenated (8492bp) nuclear–mitochondrial DNA data set from 44 musteloids (including the first genetic data for Lyncodon patagonicus) with parsimony, maximum likelihood, and Bayesian methods of phylogenetic and biogeographic inference and two Bayesian methods of chronological inference. Here we show that Musteloidea emerged approximately 32.4–30.9 million years ago (MYA) in Asia, shortly after the greenhouse–icehouse global climate shift at the Eocene–Oligocene transition. During their Oligocene radiation, which proceeded wholly or mostly in Asia, musteloids diversified into four primary divisions: the Mephitidae lineage separated first, succeeded by Ailuridae and the divergence of the Procyonidae and Mustelidae lineages. Mustelidae arose approximately 16.1 MYA within the Mid-Miocene Climatic Optimum, and extensively diversified in the Miocene, mostly in Asia. The early offshoots of this radiation largely evolved into badger and marten ecological niches (Taxidiinae, Melinae, Mellivorinae, Guloninae, and Helictidinae), whereas the later divergences have adapted to other niches including those of weasels, polecats, minks, and otters (Mustelinae, Ictonychinae, and Lutrinae). Notably, and contrary to traditional beliefs, the morphological adaptations of badgers, martens, weasels, polecats, and minks each evolved independently more than once within Mustelidae. Ictonychinae (which is most closely related to Lutrinae) arose approximately 9.5–8.9 MYA, most likely in Asia, where it diverged into the Old World Ictonychini (Vormela, Poecilictis, Ictonyx, and Poecilogale) and New World Lyncodontini (Lyncodon and Galictis) lineages. Ictonychini presumably entered Africa during the Messinian Salinity Crisis (at the Miocene–Pliocene transition), which interposed the origins of this clade (approximately 6.5–6.0 MYA) and its African Poecilictis–Ictonyx–Poecilogale subclade (approximately 4.8–4.5 MYA). Lyncodontini originated approximately 2.9–2.6 MYA at the Pliocene–Pleistocene transition in South America, slightly after the emergence of the Panamanian land bridge that provided for the Great American Biotic Interchange. As the genera Martes and Ictonyx (as currently circumscribed) are paraphyletic with respect to the genera Gulo and Poecilogale, respectively, we propose that Pekaniaand Poecilictis be treated as valid genera and that “Martes”pennanti and “Ictonyx”libyca, respectively, be assigned to these genera.

Comparative population genetics of seven notothenioid fish species reveals high levels of gene flow along ocean currents in the southern Scotia Arc, Antarctica

The Antarctic fish fauna is characterized by high endemism and low species diversity with one perciform suborder, the Notothenioidei, dominating the whole species assemblage on the shelves and slopes. Notothenioids diversified in situ through adaptive radiation and show a variety of life history strategies as adults ranging from benthic to pelagic modes. Their larval development is unusually long, lasting from a few months to more than a year, and generally includes a pelagic larval stage. Therefore, the advection of eggs and larvae with ocean currents is a key factor modulating population connectivity. Here, we compare the genetic population structures and gene flow of seven ecologically distinct notothenioid species of the southern Scotia Arc based on nuclear microsatellites and mitochondrial DNA sequences (D-loop/cytochrome b). The seven species belong to the families Nototheniidae (Gobionotothen gibberifrons, Lepidonotothen squamifrons, Trematomus eulepidotus, T. newnesi) and Channichthyidae (Chaenocephalus aceratus, Champsocephalus gunnari, Chionodraco rastrospinosus). Our results show low-population differentiation and high gene flow for all investigated species independent of their adult life history strategies. In addition, gene flow is primarily in congruence with the prevailing ocean current system, highlighting the role of larval dispersal in population structuring of notothenioids.

Mitochondrial-Nuclear DNA Interactions Contribute to the Regulation of Nuclear Transcript Levels as Part of the Inter-Organelle Communication System

Nuclear and mitochondrial organelles must maintain a communication system. Loci on the mitochondrial genome were recently reported to interact with nuclear loci. To determine whether this is part of a DNA based communication system we used genome conformation capture to map the global network of DNA-DNA interactions between the mitochondrial and nuclear genomes (Mito-nDNA) in Saccharomyces cerevisiae cells grown under three different metabolic conditions. The interactions that form between mitochondrial and nuclear loci are dependent on the metabolic state of the yeast. Moreover, the frequency of specific mitochondrial - nuclear interactions (i.e. COX1-MSY1 and Q0182-RSM7) showed significant reductions in the absence of mitochondrial encoded reverse transcriptase machinery. Furthermore, these reductions correlated with increases in the transcript levels of the nuclear loci (MSY1 and RSM7). We propose that these interactions represent an inter-organelle DNA mediated communication system and that reverse transcription of mitochondrial RNA plays a role in this process.

Molecular systematics of Batrachoseps (Caudata, Plethodontidae) in southern California and Baja California: Mitochondrial-nuclear DNA discordance and the evolutionary history of B. major

Inferences about species boundaries and evolutionary history are often complicated by discordance between datasets. In recent times, considerable effort has been devoted to understanding the causes of discordance between the patterns of genetic variation and structure shown by different unlinked molecular markers. The genus Batrachoseps (Caudata, Plethodontidae), the most diverse group of salamanders in western North America, is characterized by limited morphological variation and discordance between molecular datasets, making it a challenging group for taxonomists but also a good model to test newly developed analytical methods to sort out possible sources of discordance. In this study, we present a comprehensive assessment of the evolutionary history of B. major, one of the most widespread species in the genus, based on extensive sampling and phylogenetic and coalescent analyses of data from mitochondrial and nuclear markers. We found non-monophyly of mtDNA in B. major, with two lineages (northern and southern) that are more closely related to other species in the genus than to each other, but this division was not apparent in nuclear DNA. Despite non-monophyly in gene trees, species tree analyses recovered a sister group relationship between the two lineages of B. major, and coalescent simulations suggested that there is no need to invoke gene flow to account for the discordance across gene trees. The possibility that these two lineages represent sister, cryptic taxa, is discussed in the context of Bayesian methods of species/lineage delineation. Contrary to prior expectations, B. major has experienced extensive diversification on the Baja California Peninsula, where four endemic lineages have persisted for at least 4 million years.

Nuclear-mitochondrial cross-talk in global myocardial ischemia. A time-course analysis

Myocardial ischemia results in early and progressive damage to mitochondrial structure and function, but the molecular events leading to these changes have not been clearly established. We hypothesized that mitochondrial dysfunction and a coordinated expression of nuclear and mitochondrial genes occur in a time-dependent manner by relating the time courses of changes in parameters of mitochondrial bioenergetics after ischemia-reperfusion. Using a Langendorff rat heart model, mitochondrial bioenergetics and protein levels were assessed at different times of ischemia and ischemia/reperfusion. Mitochondrial and nuclear gene expression (super array analysis) and mitochondrial DNA levels were evaluated after late ischemia. Ischemia induced progressive and marked decreases in complex I, III, and V activities. Reperfusion (15, 30, and 60 min) after 45 min of ischemia had little further effect on enzyme activities or respiration. Super array analysis after 45 min ischemia revealed increased levels of the proteins with more pronounced increases in the corresponding mRNAs. Expression of mitochondrial and nuclear genes involved in oxidative phosphorylation increased after 45 min of ischemia but not after reperfusion. Myocardial ischemia induces mitochondrial dysfunction and differential but coordinated expression of nuclear and mitochondrial genes in a time-dependent manner. Our observations are pertinent to the search for molecular stimuli that generate mitochondrial defects and alter mitochondrial and nuclear transcriptional responses that may impact ischemic preconditioning and cardioprotection.

Hybridization between an invasive and a native species of the crayfish genus Orconectes in north-central Ohio

Hybridization between native and invasive species is an important but little-studied factor in crayfish invasions, with few documented natural cases. Here, we report genetic evidence of hybridization between invasive O. rusticus (Girard, 1852) and native O. sanbornii (Girard, 1852) in the Huron River in north-central Ohio based on a combination of molecular markers: nuclear DNA, mitochondrial DNA, and allozymes. Although we found no fixed differences between the species at nuclear DNA loci, fixed differences in mtDNA and allozyme loci confirmed the presence of individuals of hybrid ancestry. We also found preliminary evidence of possible mitochondrial recombination and biparental inheritance (though the existence of rare haplotypes cannot be excluded at the present time). We examined morphological features of both species in sympatry and allopatry and confirmed species-diagnostic morphological features including gonopod traits and shape of the annulus ventralis. This study is the second to detect hybridization between O. rusticus and a congener, which suggests that this may be an important mechanism of invasion by O. rusticus when closely related species are present. Further study of this system, including more extensive sampling and additional molecular markers, is necessary to understand the extent and implications of hybridization for the native species.

Roles of Mitochondrial DNA Changes on Cancer Initiation and Progression

Roles of Mitochondrial DNA Changes on Cancer Initiation and Progression Mitochondria are essential organelles that generate ATP through oxidative phosphorylation. This process is accomplished by a series of protein complexes and mitochondrial respiratory chains (MRC) encoded by nuclear DNA and mitochondrial DNA (mtDNA).

An Improved Method of DNA Extraction from the Shell of the Pacific Oyster, Crassostrea gigas

We earlier developed a method to extract DNA from in vivo-sampled mantle tissue of the bivalve Pacific oyster (Crassostrea gigas) for molecular marker-assisted selection (MAS) in breeding. However, mortality was too high when using this method. In the current study, a more efficient and safer method of extracting DNA from the oyster shell was developed, based on the improved phenol-chloroform DNA extraction method. Both nuclear DNA and mitochondrial DNA were successfully extracted from the oyster shell, and mortality was 0%. These results illustrate the safety of extracting DNA from the bivalve shell. In addition, because DNA is a cellular component, the results provide molecular evidence that cells are likely involved in shell formation.

Molecular Analysis of Aspermic <i>Fasciola</i> Flukes from Korea on the Basis of the Nuclear ITS1 Region and Mitochondrial DNA Markers and Comparison with Japanese Aspermic <i>Fasciola</i> Flukes

It has been speculated that populations of aspermic Fasciola flukes in Korea and Japan have a close phylogenetic relationship. To evaluate this, we analyzed 33 Korean aspermic Fasciola flukes on the basis of nuclear ribosomal internal transcribed spacer 1 (ITS1) and mitochondrial NADH dehydrogenase subunit 1 (nad1) and cytochrome c oxidase 1 (cox1) sequences. Fh, Fg, and Fh/Fg types were detected in the ITS1 region and displayed the fragment patterns of F. hepatica, F. gigantica, and both species, respectively by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Additionally, three concatenated haplotypes of nad1 and cox1(nad1/cox1) were detected, and 2 of these, Kor1/Kor1 (Fsp1/Fsp1) haplotype and Kor2a/Kor2 (Fsp2/Fsp2) haplotype, were shared by Korean and Japanese aspermic flukes. The Fst value (0.019), calculated using the concatenated sequences, indicated that Korean and Japanese aspermic Fasciola populations were genetically undifferentiated. Interestingly, a combination of the Fh/Fg type and Kor1/Kor1 haplotype was found at the highest frequency in Korean aspermic flukes, whereas the Fg type and Fsp2/Fsp2 haplotype combination was found at a conspicuously high frequency in Japanese aspermic flukes. This indicates that a founder effect caused by the introduction of infected hosts may have played a key role in the introduction of aspermic Fasciola flukes from Korea into Japan.

Genetic diversity of a newly established population of golden eagles on the Channel Islands, California

Gene flow can have profound effects on the genetic diversity of a founding population depending on the number and relationship among colonizers and the duration of the colonization event. Here we used data from nuclear microsatellite and mitochondrial DNA control region loci to assess genetic diversity in golden eagles of the recently colonized Channel Islands, California. Genetic diversity in the Channel Island population was low, similar to signatures observed for other recent colonizing island populations. Differences in levels of genetic diversity and structure observed between mainland California and the islands suggests that few individuals were involved in the initial founding event, and may have comprised a family group. The spatial genetic structure observed between Channel Island and mainland California golden eagle populations across marker types, and genetic signature of population decline observed for the Channel Island population, suggest a single or relatively quick colonization event. Polarity in gene flow estimates based on mtDNA confirm an initial colonization of the Channel Islands by mainland golden eagles, but estimates from microsatellite data suggest that golden eagles on the islands were dispersing more recently to the mainland, possibly after reaching the carrying capacity of the island system. These results illustrate the strength of founding events on the genetic diversity of a population, and confirm that changes to genetic diversity can occur within just a few generations.

Strategies of reducing input sample volume for extracting circulating cell-free nuclear DNA and mitochondrial DNA in plasma

Circulating cell-free (ccf) DNA in blood has been suggested as a potential biomarker in many conditions regarding early diagnosis and prognosis. However, misdiagnosis can result due to the limited DNA resources in Biobank's plasma samples or insufficient DNA targets from a predominant DNA background in genetic tests. This study explored several strategies for an efficient DNA extraction to increase DNA amount from limited plasma input. Ccf plasma DNA was extracted with three different methods, a phenol-chloroform-isoamylalcohol (PCI) method, a High Pure PCR Template Preparation Kit method and a method used for single cell PCR in this group. Subsequently, the total DNA was measured by Nanodrop and the genome equivalents (GE) of the GAPDH housekeeping gene and MTATP 8 gene were measured using a multiplex real-time quantitative PCR for the quantitative assessment of nDNA and mtDNA. Instead of 400-800 μL (routine input in the laboratory), 50 μLof plasma input enabled the extraction of ccf DNA sufficient for quantitative analysis. Using the PCI method and the kit method, both nDNA and mtDNA could be successfully detected in plasma samples, but nDNA extracted using protocol for single cell PCR was not detectable in 25% of plasma samples. In comparison to the other two methods, the PCI method showed lower DNA purity, but higher concentrations and more GE of nDNA and mtDNA. The PCI method was more efficient than the other two methods in the extraction of ccf DNA in plasma. Limited plasma is available for ccf DNA analysis.

Genetic population structure of the paper waspPolistes olivaceus(Hymenoptera: Vespidae) in Bangladesh

AbstractDispersal triggers gene flow, which in turn strongly affects the ensuing genetic population structure of a species. Using nuclear microsatellite loci and mitochondrial DNA (mtDNA), we estimated the genetic population structure of the waspPolistes olivaceusthroughout Bangladesh. The level of population differentiation using nuclear markers (FST) appeared to be much lower than that estimated using mtDNA haplotype sequences (ФST), even after correcting for effective population size differences between the two markers. These results suggest a philopatric tendency, in which gynes disperse less than males. We observed no isolation by distance among the study populations at either the nuclear or mtDNA level, suggesting nonequilibrium between gene flow and drift as a result of very frequent interpopulation movement. For the nuclear markers, an individual assignment test showed no genetically and geographically distinct groups. Instead, phylogenetic analyses as well as a minimum spanning network using mtDNA haplotypes consistently revealed two distinct lineages. The distribution of haplotypes indicated western populations with a single lineage and offered clear evidence for restricted gene flow across the Jamuna–Padma–Upper Meghna river system. Mismatch distributions exhibited a unimodal distribution, which along with a starlike haplotype network, suggested a population expansion in lineage I but not in lineage II. Overall, these results suggest that gene flow among populations ofP. olivaceuswas affected by both female philopatry and a major river system across Bangladesh.

Vertical and horizontal transmission drive bacterial invasion

A huge variety of Arthropod species is infected with endosymbiotic Wolbachia bacteria that manipulate their host's reproduction to invade populations. In addition to vertical transmission from mother to offspring through the egg cytoplasm, it has been demonstrated through phylogenetic analyses and natural transfer experiments that horizontal transmission of Wolbachia (i.e. contagion) can occur between Arthropod hosts. More recently, factors influencing horizontal transfer have also been explored. While it is clear that horizontal transmission between species plays a major role in the evolutionary history of Wolbachia infections among insects, its role in the spread of a new infection through a host population, notably through within-species transfers, remained unknown. In this issue of Molecular Ecology, Kraaijeveld etal. (2011) present the first evidence that horizontal transmission played a key role in the early spread of parthenogenesis-inducing Wolbachia through the parasitoid wasp Leptopilina clavipes. To support their finding, the authors studied genetic variation in three types of markers, including host nuclear DNA, mitochondrial DNA and Wolbachia DNA. Specifically, they examined potential associations between their diversity patterns. No diversity was detected in Wolbachia genes, indicating that a single Wolbachia strain must have infected and spread through L.clavipes. In addition, a correlation between substantial variation in mitochondrial and nuclear genotypes suggested that horizontal transmission played an important role in the current clonal genetic variation in this wasp. Such horizontal transmission could be facilitated by a specific host ecology (e.g. parasitoid wasps sharing the same host resource) and potentially impact co-evolution between host and symbiont.

Weak large‐scale population genetic structure in a philopatric seabird, the European Shag Phalacrocorax aristotelis

Quantifying population genetic structure is fundamental to testing hypotheses regarding gene flow, population divergence and dynamics across large spatial scales. In species with highly mobile life‐history stages, where it is unclear whether such movements translate into effective dispersal among discrete philopatric breeding populations, this approach can be particularly effective. We used seven nuclear microsatellite loci and mitochondrial DNA (ND2) markers to quantify population genetic structure and variation across 20 populations (447 individuals) of one such species, the European Shag, spanning a large geographical range. Despite high breeding philopatry, rare cross‐sea movements and recognized subspecies, population genetic structure was weak across both microsatellites and mitochondrial markers. Furthermore, although isolation‐by‐distance was detected, microsatellite variation provided no evidence that open sea formed a complete barrier to effective dispersal. These data suggest that occasional long‐distance, cross‐sea movements translate into gene flow across a large spatial scale. Historical factors may also have shaped contemporary genetic structure: cluster analyses of microsatellite data identified three groups, comprising colonies at southern, mid‐ and northern latitudes, and similar structure was observed at mitochondrial loci. Only one private mitochondrial haplotype was found among subspecies, suggesting that this current taxonomic subdivision may not be mirrored by genetic isolation.

Cryptic Diversity within the Major Trypanosomiasis Vector Glossina fuscipes Revealed by Molecular Markers

BackgroundThe tsetse fly Glossina fuscipes s.l. is responsible for the transmission of approximately 90% of cases of human African trypanosomiasis (HAT) or sleeping sickness. Three G. fuscipes subspecies have been described, primarily based upon subtle differences in the morphology of their genitalia. Here we describe a study conducted across the range of this important vector to determine whether molecular evidence generated from nuclear DNA (microsatellites and gene sequence information), mitochondrial DNA and symbiont DNA support the existence of these taxa as discrete taxonomic units.Principal FindingsThe nuclear ribosomal Internal transcribed spacer 1 (ITS1) provided support for the three subspecies. However nuclear and mitochondrial sequence data did not support the monophyly of the morphological subspecies G. f. fuscipes or G. f. quanzensis. Instead, the most strongly supported monophyletic group was comprised of flies sampled from Ethiopia. Maternally inherited loci (mtDNA and symbiont) also suggested monophyly of a group from Lake Victoria basin and Tanzania, but this group was not supported by nuclear loci, suggesting different histories of these markers. Microsatellite data confirmed strong structuring across the range of G. fuscipes s.l., and was useful for deriving the interrelationship of closely related populations.Conclusion/SignificanceWe propose that the morphological classification alone is not used to classify populations of G. fuscipes for control purposes. The Ethiopian population, which is scheduled to be the target of a sterile insect release (SIT) programme, was notably discrete. From a programmatic perspective this may be both positive, given that it may reflect limited migration into the area or negative if the high levels of differentiation are also reflected in reproductive isolation between this population and the flies to be used in the release programme.

Molecular basis for identification of species/isolates of gastrointestinal nematode parasites

Gastrointestinal (GI) parasitism is the most serious constraint throughout the world in small ruminants which causes significant production loss in animals. GI parasites are major contributor to reduce productivity in terms of meat, milk and wool in animals. Control of GI parasite is done primarily by anthelmintic treatment where choice and schedule of treatment is done after identification and quantitation of individual parasite. Identification of GI parasites is done through microscopic method by identifying specific morphological characteristics of egg and larva (L 3). Since most of parasite eggs are having similar morphological characteristics, identification up to species level through microscopy is not possible in most of cases. To address this issue, molecular techniques are the viable alternative for identification of species as well as molecular level differences within a species (isolates) of parasites. Different DNA based molecular techniques viz. PCR, AFLP, RAPD, RFLP, PCR-SSCP, real time PCR, DNA microarray etc. have been used for identification and to assess the genetic diversity among parasite population. For identification of species, the characteristic sequence of genomic DNA of different species should differ to allow the delineation of species, but at the same time, no/minor variation within the species should exist. In contrast, for purpose of identifying population variants (strains/isolates), a considerable degree of variation in the sequence should exist within a species. Various target regions, including nuclear ribosomal DNA (rDNA), mitochondrial DNA (mtDNA) or repetitive DNA elements (microsatellite loci), which show considerable variation in the number of repeats within individuals have been employed to achieve the identification of parasites species or strain.

Recent insertion of a 52-kb mitochondrial DNA segment in the wheat lineage

The assembly of a 1.3-Mb size region of the wheat genome has provided the opportunity to study a recent nuclear mitochondrial DNA insertion (NUMT). In the present study, we have studied two bacterial artificial chromosomes (BACs) and characterized a 52-kb NUMT segment from the tetraploid and hexaploid wheat BAC libraries. The conserved orthologous NUMT regions from tetraploid and hexaploid wheat Langdon and Chinese Spring shared identical gene haplotypes even though mutations (insertions, deletions, and substitutions) had occurred. The 52-kb NUMT was present in hexaploid variety Chinese Spring, but absent in variety Hope, by sequence comparison of their corresponding region. Amplifying the NUMT junctions using a set of the wheat materials including diploid, tetraploid, and hexaploid lines showed that none of the diploid wheat carried the region and only some tetraploid and hexaploid wheat were positive for the NUMT. Age estimation of the NUMT displayed the mean ages of Langdon NUMT and Chinese Spring NUMT to be 378,000 and 416,000years ago, respectively. Reverse transcription PCR and sequencing of the nad7 gene showed 28 C → U RNA editing sites and four partial editing sites, as expected for mitochondrial DNA expression. Specific SNPs discriminated between cDNA from the nucleus and the mitochondria and suggested that the nuclear copy was not expressed. The mitochondrial DNA studied was inserted into the genome quite recently within the wheat lineage and gave rise to the non-coding nuclear nad7 gene. The NUMT segment could be lost and acquired frequently during the wheat evolution.

Tracking origins of invasive herbivores through herbaria and archival DNA: the case of the horse‐chestnut leaf miner

Determining the native geographic range or origin of alien invasive species is crucial to developing invasive species management strategies. However, the necessary historical dimension is often lacking. The origin of the highly invasive horse‐chestnut leaf‐mining moth Cameraria ohridella has been controversial since the insect was first described in 1986 in Europe. Here, we reveal that herbarium collections across Europe indicate a Balkan origin for C ohridella. We successfully amplified nuclear DNA and mitochondrial DNA barcode fragments from larvae pressed within leaves of herbarium samples collected as early as 1879. These archival sequences confirm an identity of C ohridella and set back its history in Europe by more than a century. The herbarium samples uncovered previously unknown mitochondrial haplotypes and locally undocumented alleles, showing local outbreaks of C ohridella back to at least 1961 and dynamic frequency changes that may be associated with road development. This case history demonstrates that herbaria are greatly underutilized in studies of insect–plant interactions, herbivore biodiversity, and invasive species' origins.

Intracellular Glycation of Nuclear DNA, Mitochondrial DNA, and Cytosolic Proteins During Senescence-like Growth Arrest

To investigate the accumulation of intracellular advanced glycation end products (AGEs), a method was established for the simultaneous analysis of glycation products of cytosolic proteins, nuclear DNA, and mitochondrial DNA (mtDNA). Nuclear DNA, mtDNA, and cytosolic proteins were simultaneously isolated from one cell lysate by differential centrifugation and combined mechanical and chemical cell disruption methods. The major DNA-AGE N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) was quantified in nuclear DNA and mtDNA by ELISA, whereas the protein-AGEs N(ɛ)-(carboxymethyl)lysine (CML) and N(ɛ)-(carboxyethyl)lysine (CEL) were determined by western blot. The method was used to analyze NIH3T3 fibroblasts. In untreated cells, CEdG levels of mtDNA (14.84 ± 3.07 pg CEdG/μg mtDNA) were significantly higher compared with nuclear DNA (4.40 ± 0.64 pg CEdG/μg DNA; p < 0.001). Then, fibroblasts were analyzed after 7 days of senescence-like growth arrest. In senescent fibroblasts, the CEdG content of nuclear DNA significantly increased by 25%. However, the CEdG level of mtDNA significantly decreased to 52%; in parallel, an increase in mitochondrial mass and mtDNA was observed. Senescence did not lead to general accumulation of protein-AGEs, but two protein bands at 32 and 34 kDa showed a significant increase in the CML/CEL modification rate (208%, p < 0.001; 196%, p = 0.0016) in senescent fibroblasts compared with control cells.