Why are there so few genetic markers available for coral population analyses?

Coral reefs are in serious decline, and research in support of reef management objectives is urgently needed. Reef connectivity analyses have been highlighted as one of the major future research avenues necessary for implementing effective management initiatives for coral reefs. Despite the number of new molecular genetic tools and the wealth of information that is now available for population-level processes in many marine disciplines, scleractinian coral population genetic information remains surprisingly limited. Here we examine the technical problems and approaches used, address the reasons contributing to this delay in understanding, and discuss the future of coral population marker development. Considerable resources are needed to target the immediate development of an array of relevant genetic markers coupled with the rapid production of management focused data in order to help conserve our globally threatened coral reef resources.

The influence of antioxidant supplementation on markers of inflammation and the relationship to oxidative stress after exercise

Interest in the relationship between inflammation and oxidative stress has increased dramatically in recent years, not only within the clinical setting but also in the fields of exercise biochemistry and immunology. Inflammation and oxidative stress share a common role in the etiology of a variety Of Chronic diseases. During exercise, inflammation and oxidative stress are linked via muscle metabolism and muscle damage. Because oxidative stress and inflammation have traditionally been associated with fatigue and impaired recovery from exercise, research has focused on nutritional strategies aimed at reducing these effects. In this review, we have evaluated the findings of studies involving antioxidant supplementation on alterations in markers of inflammation (e.g., cytokines, C-reactive protein and cortisol). This review focuses predominantly on the role of reactive oxygen and nitrogen species generated from muscle metabolism and muscle damage during exercise and on the modulatory effects of antioxidant supplements. Furthermore, we have analyzed the influence of factors such as the dose, timing, supplementation period and bioavailability of antioxidant nutrients. (C) 2007 Elsevier Inc. All rights reserved.

Characterisation of Aeromonas strains and species by pulsed field gel electrophoresis and principal components analysis

Aeromonas genomes were investigated by restriction digesting chromosomal DNA with the endonuclease XbaI, separation of restriction fragments by pulsed field gel electrophoresis (PFGE) and principal components analysis (PCA) of resulting separation patterns. A. salmonicida salmonicida were unique amongst the isolates investigated. Separation profiles of these isolates were similar and all characterised by a distinct absence of bands in the 250kb region. Principal components analysis represented these strains as a clearly defined homogeneous group separated by insignificant Euclidian distances. However, A. salmonicida achromogenes isolates in common with those of A. hydrophila and A. sobria were shown by principal components analysis to be more heterogeneous in nature. Fragments from these isolates were more uniform in size distribution but as demonstrated by the Euclidian distances attained through PCA potentially characteristic of each strain. Furthermore passaging of Aeromonas isolates through an appropriate host did not greatly modify fragment separation profiles, indicative of the genomic stability of test aeromonads and the potential of restriction digesting/PFGE/PCA in Aeromonas typing.

Biochemical markers and contact lens wear

The project objective was to develop a reliable selection procedure to match contact lens materials with individual wearers by the identification of a biochemical marker for assessment of in-eye performance of contact lenses. There is a need for such a procedure as one of the main reasons for contact lens wearers ceasing wearing contact lenses is poor end of day comfort i.e. the lenses become intolerable to the wearer as the day progresses. The selection of an optimal material for individual wearers has the potential benefit to reduce drop Qut, hence increasing the overall contact lens population, and to improve contact lens comfort for established wearers. Using novel analytical methods and statistical techniques, we were able to investigate the interactions between the composition of the tear film and of the biofilm deposited on the contact lenses and contact lens performance. The investigations were limited to studying the lipid components of the tear film; the lipid layer, which plays a key role in preventing evaporation and stabilising the tear film, has been reported to be significantly thinner and of different mixing characteristics during contact lens wear. Different lipid families were found to influence symptomatology, in vivo tear film structure and stability as well as ocular integrity. Whereas the symptomatology was affected by both the tear film lipid composition and the nature of the lipid deposition, the structure of the tear film and its stability were mainly influenced by the tear film lipid composition. The ocular integrity also appeared to be influenced by the nature of the lipid deposition. Potential markers within the lipid species have been identified and could be applied as follows: When required in order to identify a problematic wearer or to match the contact lens material to the contact lens wearer, tear samples collected by the clinician could be dispatched to an analytical laboratory where lipid analysis could be carried out by HPLC. A colorimetric kit based on the lipid markers could also be developed and used by clinician directly in the practice; such a kit would involve tear sampling and classification according to the colour into "Problem", "Border line" and "Good" contact lens wearers groups. A test kit would also have wider scope for marketing in other areas such as general dry-eye pathology.

Gram-positive infections in patients with end-stage renal disease

Gram-positive microorganisms, specifically coagulase-negative staphylococci are the most common species recovered from clinical culture specimens of patients with end-stage renal disease. The propensity of coagulase-negative staphylococci (CNS) to cause infection in this patient group has been widely debated. However, it is still unclear how this usually avirulent commensal microorganism produces infection that contributes to high rates of morbidity and mortality in patients with end-stage renal disease. The aim of this thesis was to investigate the rate, geographical distribution, molecular and phenotypic mechanisms of Gram-positive microorganisms associated with infection in renal dialysis patients. In addition, it sought to assess the value of early serological diagnosis of dialysis catheter-associated infection and the effect of antimicrobial treatment regimens on the faecal carriage of enteric microorganisms. In this study, the incidence of haemodialysis catheter-associated infection was established with the Meditrend audit tool. This tool was used to assess the infection outcomes of catheter insertion and management procedures until the catheter was explanted. Introduction of a catheter management protocol decreased the incidence of catheter-related infection. Staphylococcal species recovered from episodes of haemodialysis catheter-associated infection and continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis were genotyped by determination of macrorestriction profiles with pulsed-field gel electrophoresis. This highlighted horizontal transfer of microorganisms between different patients and the environment. The phenotypic characteristics of these strains were also investigated to determine characteristics that could be used as markers for dialysis catheter-associated infection. The expression of elastase, lipase and esterase by CNS was significantly associated with infection. A rapid enzyme-linked immunosorbent assay incorporating a novel staphylococcal antigen (lipid S) was used to evaluate the early detection of anti-staphylococcal immunoglobulin gamma in patient sera. The comparison of culture positive and culture negative patients demonstrated a steady state of immune activation in both groups. However anti-lipid S serum antibody titres > 1000 were found to be a predictor of infection. The effect on faecal carriage of vancomycin resistant enterococci (VRE) and Clostridium difficile toxins in patients treated with CAPD when empiric cephalosporin therapy was substituted for piperacillin/tazobactam was investigated. The introduction of piperacillin/tazobactam demonstrated a decrease in the faecal carriage of VRE.

Repetitive DNA fragments as taxonomic markers for {\it Penaeus\/} sibling taxa (Decapoda: Dendrobranchiata: Penaeidae) from the southern terminus of the Florida peninsula, U.S.A.

Sympatric populations of P. brasiliensis and P. duorarum from Biscayne Bay, Florida, revealed species-specific satellite DNA organizational patterns with the restriction endonuclease EcoRI. The species-specific satellite DNA patterns can be explained as resulting from differential amplification/deletion events having altered monomer arrays after the divergence of these two species. Two discontinuous populations of P. duorarum (Biscayne Bay and Dry Tortugas) were found to exhibit distinct EcoRI satellite fragment patterns; BamHI repetitive fragments specific to the Dry Tortugas P. duorarum population were also detected. In addition, the evolutionary conservation of the Penaeus (Farfantepenaeus) satellites was investigated. The putative conservation of sequences related to one cloned P. duorarum satellite monomer unit suggests that the FTR satellite DNA family may not only be of use as a genome tag to distinguish between sibling and cryptic Penaeus species but may also serve as a probe to better understand decapod crustacean genome organization and evolution. ^

The isolation, characterization, and application of WRKY genes as useful molecular markers in tropical trees

The improvement of tropical tree crops using conventional breeding methods faces challenges due to the length of time involved. Thus, like most crops, there is an effort to utilize molecular genetic markers in breeding programs to select for desirable agronomic traits. Known as marker assisted breeding or marker assisted selection, genetic markers associated with a phenotype of interest are used to screen and select material reducing the time necessary to evaluate candidates. As the focus of this research was improving disease resistance in tropical trees, the usefulness of the WRKY gene superfamily was investigated as candidates for generating useful molecular genetic markers. WRKY genes encode plant-specific transcriptional factors associated with regulating plants' responses to both biotic and abiotic stress. ^ One pair of degenerate primers amplified 48 WRKY gene fragments from three taxonomically distinct, economically important, tropical tree crop species: 18 from Theobroma cacao L., 21 from Cocos nucifera L. and 9 from Persea americana Mill. Several loci from each species were polymorphic because of single nucleotide substitutions present within a putative non-coding region of the loci. Capillary array electrophoresis-single strand conformational polymorphism (CAE-SSCP) mapped four WRKY loci onto a genetic linkage map of a T. cacao F2 population segregating for resistance to witches' broom disease. Additionally, PCR primers specific for four T. cacao loci successfully amplified WRKY loci from 15 members of the Byttneriae tribe. A method was devised to allow the reliable discrimination of alleles by CAE-SSCP using only the mobility assigned to the sample peaks. Once this method was validated, the diversity of three WRKY loci was evaluated in a germplasm collection of T. cacao . One locus displayed high diversity in the collection, with at least 18 alleles detected from mobility differences of the product peaks. The number of WRKY loci available within the genome, ease of isolation by degenerate PCR, codominant segregation demonstrated in the F2 population, and usefulness for screening germplasm collections and closely related wild species demonstrates that the WRKY superfamily of genes are excellent candidates for developing a number of genetic molecular markers for breeding purposes in tropical trees. ^

Investigating the origin of PM2.5 in Baltimore using highly time-resolved organic molecular markers measured during the Baltimore PM-supersite

The detailed organic composition of atmospheric fine particles with an aerodynamic diameter smaller than or equal to 2.5 micrometers (PM2.5) is an integral part of the knowledge needed in order to fully characterize its sources and transformation in the environment. For the study presented here, samples were collected at 3-hour intervals. This high time resolution allows gaining unique insights on the influence of short- and long-range transport phenomena, and dynamic atmospheric processes. A specially designed sequential sampler was deployed at the 2002-2003 Baltimore PM-Supersite to collect PM2.5 samples at a 3-hourly resolution for extended periods of consecutive days, during both summer and winter seasons. Established solvent-extraction and GC-MS techniques were used to extract and analyze the organic compounds in 119 samples from each season. Over 100 individual compounds were quantified in each sample. For primary organics, averaging the diurnal ambient concentrations over the sampled periods revealed ambient patterns that relate to diurnal emission patterns of major source classes. Several short-term releases of pollutants from local sources were detected, and local meteorological data was used to pinpoint possible source regions. Biogenic secondary organic compounds were detected as well, and possible mechanisms of formation were evaluated. The relationships between the observed continuous variations of the concentrations of selected organic markers and both the on-site meteorological measurements conducted parallel to the PM2.5 sampling, and the synoptic patterns of weather and wind conditions were also examined. Several one-to-two days episodes were identified from the sequential variation of the concentration observed for specific marker compounds and markers ratios. The influence of the meteorological events on the concentrations of the organic compounds during selected episodes was discussed. It was observed that during the summer, under conditions of pervasive influence of air masses originated from the west/northwest, some organic species displayed characteristics consistent with the measured PM2.5 being strongly influenced by the aged nature of these long-traveling background parcels. During the winter, intrusions from more regional air masses originating from the south and the southwest were more important.

Investigating the origin of PM2.5 in Baltimore using highly time-resolved organic molecular markers measured during the Baltimore PM-supersite

The detailed organic composition of atmospheric fine particles with an aerodynamic diameter smaller than or equal to 2.5 micrometers (PM 2.5) is an integral part of the knowledge needed in order to fully characterize its sources and transformation in the environment. For the study presented here, samples were collected at 3-hour intervals. This high time resolution allows gaining unique insights on the influence of short- and long-range transport phenomena, and dynamic atmospheric processes. A specially designed sequential sampler was deployed at the 2002-2003 Baltimore PM Supersite to collect PM2.5 samples at a 3-hourly resolution for extended periods of consecutive days, during both summer and winter seasons. Established solvent-extraction and GC-MS techniques were used to extract and analyze the organic compounds in 119 samples from each season. Over 100 individual compounds were quantified in each sample. For primary organics, averaging the diurnal ambient concentrations over the sampled periods revealed ambient patterns that relate to diurnal emission patterns of major source classes. Several short-term releases of pollutants from local sources were detected, and local meteorological data was used to pinpoint possible source regions. Biogenic secondary organic compounds were detected as well, and possible mechanisms of formation were evaluated. The relationships between the observed continuous variations of the concentrations of selected organic markers and both the on-site meteorological measurements conducted parallel to the PM2.5 sampling, and the synoptic patterns of weather and wind conditions were also examined. Several one-to-two days episodes were identified from the sequential variation of the concentration observed for specific marker compounds and markers ratios. The influence of the meteorological events on the concentrations of the organic compounds during selected episodes was discussed. It was observed that during the summer, under conditions of pervasive influence of air masses originated from the west/northwest, some organic species displayed characteristics consistent with the measured PM2.5 being strongly influenced by the aged nature of these long-traveling background parcels. During the winter, intrusions from more regional air masses originating from the south and the southwest were more important.

(Table 2) Collection sites and number of specimens of Usnea subgenus Neuropogon species

Usnea species of the Neuropogon group are amongst the most widespread and abundant macrolichens in Antarctic regions. Four principal species, U. antarctica, U. aurantiaco-atra, U. sphacelata and U. subantarctica, have been described on morphological grounds. However, identification to species level is often difficult and atypical morphologies frequently arise. Over 400 specimens were collected on the Antarctic Peninsula and Falkland Islands. Both morphological and molecular characters (ITS and RPB1) were used to compare samples to clarify taxonomic relationships. Morphological characteristics used included presence of apothecia, apothecial rays, soredia, papillae, fibrils, pigmentation and the diameter of the central axis as a proportion of branch diameter. Results revealed a very close relationship between U. antarctica and U. aurantiaco-atra, suggesting that they might constitute a species pair or be conspecific. Usnea sphacelata was comprised of at least two genetically distinct groups with no clear differences in morphology. One group included the first reported fertile specimen of this species. Usnea subantarctica was phylogenetically distinct from the other main Antarctic Usnea species, but clustered with U. trachycarpa. Genetic variation was evident within all species although there was no clear correlation between geographic origin and genetic relatedness. Phylogenetic analyses indicated that species circumscription in the Neuropogon group needs revision, with the principal species being non-monophyletic. None of the morphological characters, or groups of characters, used in this study proved to be completely unambiguous markers for a single species. However, axis thickness was supported as being informative for the identification of monophyletic lineages within the group.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

A timescale for diatom evolution based on four molecular markers: reassessment of ghost lineages and major steps defining diatom evolution.

ABSTRACT. – Phylogenies and molecular clocks of the diatoms have largely been inferred from SSU rDNA sequences. A new phylogeny of diatoms was estimated using four gene markers SSU and LSU rDNA rbcL and psbA (total 4352 bp) with 42 diatom species. The four gene trees analysed with a maximum likelihood (ML) and Baysian (BI) analysis recovered a monophyletic origin of the new diatom classes with high bootstrap support, which has been controversial with single gene markers using single outgroups and alignments that do not take secondary structure of the SSU gene into account. The divergence time of the classes were calculated from a ML tree in the MultliDiv Time program using a Bayesian estimation allowing for simultaneous constraints from the fossil record and varying rates of molecular evolution of different branches in the phylogenetic tree. These divergence times are generally in agreement with those proposed by other clocks using single genes with the exception that the pennates appear much earlier and suggest a longer Cretaceous fossil record that has yet to be sampled. Ghost lineages (i.e. the discrepancy between first appearance (FA) and molecular clock age of origin from an extant taxon) were revealed in the pennate lineage, whereas those ghost lineages in the centric lineages previously reported by others are reviewed and referred to earlier literature.

Development of a simple and fast “DNA extraction kit” for sea food identification and marine species

Seafood products fraud, the misrepresentation of them, have been discovered all around the world in different forms as false labeling, species substitution, short-weighting or over glazing in order to hide the correct identity, origin or weight of the seafood products. Due to the value of seafood products such as canned tuna, swordfish or grouper, these species are the subject of the commercial fraud is mainly there placement of valuable species with other little or no value species. A similar situation occurs with the shelled shrimp or shellfish that are reduced into pieces for the commercialization. Food fraud by species substitution is an emerging risk given the increasingly global food supply chain and the potential food safety issues. Economic food fraud is committed when food is deliberately placed on the market, for financial gain deceiving consumers (Woolfe, M. & Primrose, S. 2004). As a result of the increased demand and the globalization of the seafood supply, more fish species are encountered in the market. In this scenary, it becomes essential to unequivocally identify the species. The traditional taxonomy, based primarily on identification keys of species, has shown a number of limitations in the use of the distinctive features in many animal taxa, amplified when fish, crustacean or shellfish are commercially transformed. Many fish species show a similar texture, thus the certification of fish products is particularly important when fishes have undergone procedures which affect the overall anatomical structure, such as heading, slicing or filleting (Marko et al., 2004). The absence of morphological traits, a main characteristic usually used to identify animal species, represents a challenge and molecular identification methods are required. Among them, DNA-based methods are more frequently employed for food authentication (Lockley & Bardsley, 2000). In addition to food authentication and traceability, studies of taxonomy, population and conservation genetics as well as analysis of dietary habits and prey selection, also rely on genetic analyses including the DNA barcoding technology (Arroyave & Stiassny, 2014; Galimberti et al., 2013; Mafra, Ferreira, & Oliveira, 2008; Nicolé et al., 2012; Rasmussen & Morrissey, 2008), consisting in PCR amplification and sequencing of a COI mitochondrial gene specific region. The system proposed by P. Hebert et al. (2003) locates inside the mitochondrial COI gene (cytochrome oxidase subunit I) the bioidentification system useful in taxonomic identification of species (Lo Brutto et al., 2007). The COI region, used for genetic identification - DNA barcode - is short enough to allow, with the current technology, to decode sequence (the pairs of nucleotide bases) in a single step. Despite, this region only represents a tiny fraction of the mitochondrial DNA content in each cell, the COI region has sufficient variability to distinguish the majority of species among them (Biondo et al. 2016). This technique has been already employed to address the demand of assessing the actual identity and/or provenance of marketed products, as well as to unmask mislabelling and fraudulent substitutions, difficult to detect especially in manufactured seafood (Barbuto et al., 2010; Galimberti et al., 2013; Filonzi, Chiesa, Vaghi, & Nonnis Marzano, 2010). Nowadays,the research concerns the use of genetic markers to identify not only the species and/or varieties of fish, but also to identify molecular characters able to trace the origin and to provide an effective control tool forproducers and consumers as a supply chain in agreementwith local regulations.

Development of single nucleotide polymorphism (SNP) markers from the mango (Mangifera indica) transcriptome for mapping and estimation of genetic diversity

The development of molecular markers for genomic studies in Mangifera indica (mango) will allow marker-assisted selection and identification of genetically diverse germplasm, greatly aiding mango breeding programs. We report here our identification of thousands of unambiguous molecular markers that can be easily assayed across genotypes of the species. With origin centered in Southeast Asia, mangos are grown throughout the tropics and subtropics as a nutritious fruit that exhibits remarkable intraspecific phenotypic diversity. With the goal of building a high density genetic map, we have undertaken discovery of sequence variation in expressed genes across a broad range of mango cultivars. A transcriptome sequence reference was built de novo from extensive sequencing and assembly of RNA from cultivar 'Tommy Atkins'. Single nucleotide polymorphisms (SNPs) in protein coding transcripts were determined from alignment of RNA reads from 24 mango cultivars of diverse origins: 'Amin Abrahimpur' (India), 'Aroemanis' (Indonesia), 'Burma' (Burma), 'CAC' (Hawaii), 'Duncan' (Florida), 'Edward' (Florida), 'Everbearing' (Florida), 'Gary' (Florida), 'Hodson' (Florida), 'Itamaraca' (Brazil), 'Jakarata' (Florida), 'Long' (Jamaica), 'M. Casturi Purple' (Borneo), 'Malindi' (Kenya), 'Mulgoba' (India), 'Neelum' (India), 'Peach' (unknown), 'Prieto' (Cuba), 'Sandersha' (India), 'Tete Nene' (Puerto Rico), 'Thai Everbearing' (Thailand), 'Toledo' (Cuba), 'Tommy Atkins' (Florida) and 'Turpentine' (West Indies). SNPs in a selected subset of protein coding transcripts are currently being converted into Fluidigm assays for genotyping of mapping populations and germplasm collections. Using an alternate approach, SNPs (144) discovered by sequencing of candidate genes in 'Kensington Pride' have already been converted and used for genotyping.