Anthony Koutoulis

Heat shock, smoke and darkness: partner cues in promoting seed germination in Epacris tasmanica (Epacridaceae)

The Epacridaceae is one of the families that dominate Australian heathlands, environments prone to disturbance events such as fire and seasonal drought. To investigate the role of fire-related cues in breaking seed-dormancy mechanisms in the Epacridaceae, the influences of heat shock, darkness, direct smoke and varying concentrations of aqueous smoke solutions (5, 10 and 100%) on seed germination of the Tasmanian endemic Epacris tasmanica were examined. A small fraction (5.3%) of non-dormant E. tasmanica seed could germinate in the absence of fire-related cues. The most effective treatment for promoting seed germination was direct smoke (74.67%); however, germination with direct smoke was delayed by about two weeks when compared to other significant treatments, suggesting an initial inhibitory effect. Significant interactions were recorded between all classes of treatments (heat shock, darkness and smoke solutions), with treatments acting sequentially and additively to promote germination. The most effective combinatory treatment tested was 5% smoked water (5%S) in conjunction with darkness (D) and heat-shock (H) treatments (5%SDH), which raised germination levels to 49%. In the absence of heat shock, darkness and various concentrations of smoked water had no significant effect on seed germination. The 5%SDH treatment promoted seed germination significantly also in two wet-heathland (E. lanuginosa (42.7%) and E. obtusifolia (64.7%)) and two dry-heathland Epacris species (the Tasmanian endemic E. apsleyensis (72.7%) and the rare mainland Australian E. purpurascens (75%)). The results of this study indicate that fire-related dormancy-breaking cues act synergistically in promoting seed germination in E. tasmanica and suggesting that their level of influence may reflect the ecology of Epacris species.

Spine-scale reorientation in Apedinella radians (Pedinellales, Chrysophyceae) : the microarchitecture and immunocytochemistry of the associated cytoskeleton

SummaryMotile unicells ofApedinella radians have the extraordinary ability to instantaneously reorient six elongate spine-scales located on the cell surface. Extracellular striated fibrous connectors (termed microligaments) attach spine-scales to discrete regions of the plasma membrane underlain by intricate cytoplasmic plaques. A complex cytoskeleton is associated with the plaques and appears responsible for spine-scale movement. Three cytoskeletal proteins have thus far been identified by immunofluorescence using anti-tubulin, anti-actin, and anti-centrin. The three-dimensional configuration of the cytoskeleton has been established and consists of filamentous bundles of actin and centrin which form stellate systems interconnecting the plaques. Additionally, there is a network of microtubular triads which originate on the surface of the nuclear envelope and subtend the plasma membrane and also support several tentacular protrusions. It is proposed that contraction of the actin and/or centrin filamentous bundles is responsible for the reorientation of the spine-scales.

High-throughput genotyping of hop ( Humulus lupulus L.) utilising diversity arrays technology (DArT)

Implementation of molecular methods in hop (Humulus lupulus L.) breeding is dependent on the availability of sizeable numbers of polymorphic markers and a comprehensive understanding of genetic variation. However, use of molecular marker technology is limited due to expense, time inefficiency, laborious methodology and dependence on DNA sequence information. Diversity arrays technology (DArT) is a high-throughput cost-effective method for the discovery of large numbers of quality polymorphic markers without reliance on DNA sequence information. This study is the first to utilise DArT for hop genotyping, identifying 730 polymorphic markers from 92 hop accessions. The marker quality was high and similar to the quality of DArT markers previously generated for other species; although percentage polymorphism and polymorphism information content (PIC) were lower than in previous studies deploying other marker systems in hop. Genetic relationships in hop illustrated by DArT in this study coincide with knowledge generated using alternate methods. Several statistical analyses separated the hop accessions into genetically differentiated North American and European groupings, with hybrids between the two groups clearly distinguishable. Levels of genetic diversity were similar in the North American and European groups, but higher in the hybrid group. The markers produced from this time and cost-efficient genotyping tool will be a valuable resource for numerous applications in hop breeding and genetics studies, such as mapping, marker-assisted selection, genetic identity testing, guidance in the maintenance of genetic diversity and the directed breeding of superior cultivars.

Odd-chain polyunsaturated fatty acids in thraustochytrids

A series of unusual odd-chain fatty acids (OC-FA) were identified in two thraustochytrid strains, TC 01 and TC 04, isolated from waters off the south east coast of Tasmania, Australia. FA compositions were determined by capillary GC and GC-MS, with confirmation of polyunsaturated fatty acids (PUFA) structure performed by analysis of 4,4-dimethyloxazoline derivatives. PUFA constituted 68-74% of the total FA, with the essential PUFA; eicosapentaenoic acid (20:5ω3, EPA), arachidonic acid (20:4ω6, AA) and docosahexaenoic acid (22:6ω3, DHA), accounting for 42-44% of the total FA. High proportions of the saturated OC-FA 15:0 (7.1% in TC 01) and 17:0 (6.2% in TC 04) were detected. The OC-FA 17:1ω8 was present at 2.8% in TC 01. Of particular interest, the C₂₁ PUFA 21:5ω5 and 21:4ω7 were detected at 3.5% and 4.1%, respectively, in TC 04. A proposed biosynthesis pathway for these OC-PUFA is presented. It is possible that the unsaturated OC-PUFA found previously in a number of marine animals were derived from dietary thraustochytrids and they could be useful biomarkers in environmental and food web studies.

S-adenosylmethionine synthetase genes from eleven marine dinoflagellates

L.D. Harlow, A. Koutoulis and G.M. Hallegraeff. 2007. S-adenosylmethionine synthetase genes from eleven marine dinoflagellates. Phycologia 46: 46–53. DOI: 10.2216/06-28.1 Paralytic shellfish poisoning toxins (PSTs) such as saxitoxin are believed to be synthesised from precursor molecules including arginine, S-adenosylmethionine (SAM) and acetate. The enzyme SAM synthetase is a good candidate as an early acting enzyme in the PST biosynthetic pathway. We have used degenerate PCR to identify the gene encoding SAM synthetase, Sam, in dinoflagellates. Several different PCR products were cloned and sequenced from PST-producing strains of the dinoflagellates Alexandrium minutum, A. catenella and Gymnodinium catenatum and primers specific to dinoflagellate Sam used for further sequence analysis in 11 species of toxic and nontoxic dinoflagellate genera, including: Alexandrium, Gymnodinium, Karenia, Karlodinium, Noctiluca, Prorocentrum and Takayama. At the nucleotide level, Sam clones were unique to dinoflagellates and the most commonly identified Sam was highly conserved between dinoflagellate species. Dinoflagellate Sam G + C content was both typical (60.8%) or lower (40.7%) compared to A. tamarense. The derived protein sequences showed a low and equal level of similarity to Sam from other species representing a range of Kingdoms. DNA sequence information for dinoflagellate Sam provides important insights for dinoflagellate codon usage, which will facilitate primer design to target novel dinoflagellate genes.

Quantitative trait loci in hop (Humulus lupulus L.) reveal complex genetic architecture underlying variation in sex, yield and cone chemistry

BackgroundHop (Humulus lupulus L.) is cultivated for its cones, the secondary metabolites of which contribute bitterness, flavour and aroma to beer. Molecular breeding methods, such as marker assisted selection (MAS), have great potential for improving the efficiency of hop breeding. The success of MAS is reliant on the identification of reliable marker-trait associations. This study used quantitative trait loci (QTL) analysis to identify marker-trait associations for hop, focusing on traits related to expediting plant sex identification, increasing yield capacity and improving bittering, flavour and aroma chemistry.ResultsQTL analysis was performed on two new linkage maps incorporating transferable Diversity Arrays Technology (DArT) markers. Sixty-three QTL were identified, influencing 36 of the 50 traits examined. A putative sex-linked marker was validated in a different pedigree, confirming the potential of this marker as a screening tool in hop breeding programs. An ontogenetically stable QTL was identified for the yield trait dry cone weight; and a QTL was identified for essential oil content, which verified the genetic basis for variation in secondary metabolite accumulation in hop cones. A total of 60 QTL were identified for 33 secondary metabolite traits. Of these, 51 were pleiotropic/linked, affecting a substantial number of secondary metabolites; nine were specific to individual secondary metabolites.ConclusionsPleiotropy and linkage, found for the first time to influence multiple hop secondary metabolites, have important implications for molecular selection methods. The selection of particular secondary metabolite profiles using pleiotropic/linked QTL will be challenging because of the difficulty of selecting for specific traits without adversely changing others. QTL specific to individual secondary metabolites, however, offer unequalled value to selection programs. In addition to their potential for selection, the QTL identified in this study advance our understanding of the genetic control of traits of current economic and breeding significance in hop and demonstrate the complex genetic architecture underlying variation in these traits. The linkage information obtained in this study, based on transferable markers, can be used to facilitate the validation of QTL, crucial to the success of MAS.

Genetic Diversity of Cultured, Naturalized, and Native Pacific Oysters, Crassostrea Gigas, Determined from Multiplexed Microsatellite Markers

ABSTRACT Ten polymorphic microsatellite loci were multiplexed to analyze a total of 343 Pacific oysters (Crassostrea gigas) sampled from native (Japan and Korea), naturalized (France and Australia), and cultured (3 Australian programs) populations. Genetic diversity was high within the native and naturalized populations (average allelic richness, 18.7; expected heterozygosity, 0.89), but lower within samples from hatchery populations (allelic richness, 12.3; expected heterozygosity, 0.84). A significant decrease in diversity was found within Australian cultured populations. However, diversity was shown to be similar in samples from a well-managed, family-based selective breeding population and commercial hatchery mass spawning populations. The Bayesian analysis of population structure found no difference between native and naturalized samples, which, together with other results, indicate that the naturalized populations have not changed genetically since their introduction. This suggests that naturalized populations can provide a good source of genetic diversity for breeding programs.

Environmental adaptation in stomatal size independent of the effects of genome size

Cell sizes are linked across multiple tissues, including stomata, and this variation is closely correlated with genome size. These associations raise the question of whether generic changes in cell size cause suboptimal changes in stomata, requiring subsequent evolution under selection for stomatal size. We tested the relationships among guard cell length, genome size and vegetation type using phylogenetically independent analyses on 67 species of the ecologically and structurally diverse family, Proteaceae. We also compared how genome and stomatal sizes varied at ancient (among genera) and more recent (within genus) levels. The observed 60-fold range in genome size in Proteaceae largely reflected the mean chromosome size. Compared with variation among genera, genome size varied much less within genera (< 6% of total variance) than stomatal size, implying evolution in stomatal size subsequent to changes in genome size. Open vegetation and closed forest had significantly different relationships between stomatal and genome sizes. Ancient changes in genome size clearly influenced stomatal size in Proteaceae, but adaptation to habitat strongly modified the genome–stomatal size relationship. Direct adaptation to the environment in stomatal size argues that new proxies for past concentrations of atmospheric CO2 that incorporate stomatal size are superior to older models based solely on stomatal frequency.

Varietal characterization of hop (Humulus lupulus L.) by GC-MS analysis of hop cone extracts

An approach is described for use in the varietal characterization of hop (Humulus lupulus L.) varieties. The study focuses on commercial hop varieties and was timed to coincide with the 2008 commercial hop harvest in Tasmania, Australia. Analysis of hop extracts was performed using GC-MS. A 60 m capillary column was employed to increase efficiency to permit the use of a quadrupole mass spectrometer in place of a time of flight mass spectrometer that is more commonly used for this type of analysis. A set of characterization functions were derived from discriminant analysis which were highly suitable for varietal characterization of the eight commercial varieties included in the study, namely Willamette, Victoria, Pride of Ringwood, Cascade, Southern Hallertau, Millennium, Southern Saaz, and Super Pride.

Red to far-red ratio correction in plant growth chambers - growth responses and influence of thermal load on garden pea

Plant growth chambers are commonly used to minimize environmental variation but the light sources used vary considerably from natural light and from each other. Incandescent globes are often used to add more far-red light, with the aim of producing a more natural red to far-red ratio (R:FR), but also add to thermal load. High-intensity discharge lamps are often used to produce higher irradiances, more akin to natural light, but the thermal implications are rarely considered because air temperature is controlled. This paper examines the spectral properties and thermal implications of growth chamber light sources and takes a whole-plant physiology approach, by examining growth responses of a photoperiodic pea line (Pisum sativum L. cv. Torsdag) in the same growth chamber type under different light sources - in essence using plants to study the controlled environments rather than vice-versa. High R:FR delayed flowering and inhibited internode extension in pea. However, the addition of far-red-rich incandescent globes in the proportions provided in the growth chambers (400-500 W) did little to reduce R:FR, did not induce earlier flowering and actually further inhibited internode length. Leaflet size and yield were significantly reduced. While air temperature was maintained at 20 degrees C in all experiments, radiant temperature was significantly higher under high irradiance and/or with incandescent added, and soil temperatures were elevated. Growth responses under these lights were similar to the effect caused by elevating the air temperature. An alternative method of controlling R:FR, without thermal load implications, using light-emitting diodes is described.