The application of DArTseq technology to pineapple

DArTseq technology is potentially the most appropriate system to discover hundreds of polymorphic genomic loci, scoring thousands of unique genomic-wide DNA fragments in one single experiment, without requiring existing DNA sequence information. The DArT complexity reduction approach in combination with Illumina short read sequencing (Hiseq2000) was applied. To test the application of DArTseq technology in pineapple, a reference population of 13 Ananas genotypes from primitive wild accessions to modern cultivars was used. In a comparison of 3 systems, the combination of restriction enzymes PstI and MseI performed the best producing 18,900 DArT markers and close to 20,000 SNPs. Based on these markers genetic relationships between the samples were identified and a dendrogram was generated. The topography of the tree corresponds with our understanding of the genetic relationships between the genotypes. Importantly, the replicated samples of all genotypes have a dissimilarity of close to 0.0 and occupy the same positions on the tree, confirming high reproducibility of the markers detected. Eventually it is planned that molecular markers will be identified that are associated with resistance to Phytophthora cinnamomi (Pc), the most economically important pathogen of pineapple in Australia, as genetic resistance is known to exist within the Ananas. Marker assisted selection can then be utilized in a pineapple breeding program to develop cultivars resistant to Pc.

CRC50116 - Resistance Monitoring

National Monitoring for resistance to phosphine and grain protectants.

Methicillin Resistance In Staphylococci : Horizontal Transfer of Mobile Genetic Element (SCCmec) Between Staphylococcal Species

Regardless of the existence of antibiotics, infectious diseases are the leading causes of death in the world. Staphylococci cause many infections of varying severity, although they can also exist peacefully in many parts of the human body. Most often Staphylococcus aureus colonises the nose, and that colonisation is considered to be a risk factor for spread of this bacterium. S. aureus is considered to be the most important Staphylococcus species. It poses a challenge to the field of medicine, and one of the most problematic aspects is the drastic increase of the methicillin-resistant S. aureus (MRSA) strains in hospitals and community world-wide, including Finland. In addition, most of the clinical coagulase-negative staphylococcus (CNS) isolates express resistance to methicillin. Methicillin-resistance in S. aureus is caused by the mecA gene that encodes an extra penicillin-binding protein (PBP) 2a. The mecA gene is found in a mobile genomic island called staphylococcal chromosome cassette mec (SCCmec). The SCCmec consists of the mec gene and cassette chromosome recombinase (ccr)gene complexes. The areas of the SCCmec element outside the ccr and mec complex are known as the junkyard J regions. So far, eight types of SCCmec(SCCmec I- SCCmec VIII) and a number of variants have been described. The SCCmec island is an acquired element in S. aureus. Lately, it appears that CNS might be the storage place of the SCCmec that aid the S. aureus by providing it with the resistant elements. The SCCmec is known to exist only in the staphylococci. The aim of the present study was to investigate the horizontal transfer of SCCmec between the S. aureus and CNS. One specific aim was to study whether or not some methicillin-sensitive S. aureus (MSSA) strains are more inclined to receive the SCCmec than others. This was done by comparing the genetic background of clinical MSSA isolates in the health care facilities of the Helsinki and Uusimaa Hospital District in 2001 to the representatives of the epidemic MRSA (EMRSA) genotypes, which have been encountered in Finland during 1992-2004. Majority of the clinical MSSA strains were related to the EMRSA strains. This finding suggests that horizontal transfer of SCCmec from unknown donor(s) to several MSSA background genotypes has occurred in Finland. The molecular characteristics of representative clinical methicillin-resistant S. epidermidis (MRSE) isolates recovered in Finnish hospitals between 1990 and 1998 were also studied, examining their genetic relation to each other and to the internationally recognised MRSE clones as well, so as to ascertain the common traits between the SCCmec elements in MRSE and MRSA. The clinical MRSE strains were genetically related to each other; eleven PFGE types were associated with sequence type ST2 that has been identified world-wide. A single MRSE strain may possess two SCCmec types III and IV, which were recognised among the MRSA strains. Moreover, six months after the onset of an outbreak of MRSA possessing a SCCmec type V in a long-term care facility in Northern Finland (LTCF) in 2003, the SCCmec element of nasally carried methicillin-resistant staphylococci was studied. Among the residents of a LTCF, nasal carriage of MR-CNS was common with extreme diversity of SCCmec types. MRSE was the most prevalent CNS species. Horizontal transfer of SCCmec elements is speculated to be based on the sharing of SCCmec type V between MRSA and MRSE in the same person. Additionally, the SCCmec element of the clinical human S. sciuri isolates was studied. Some of the SCCmec regions were present in S. sciuri and the pls gene was common in it. This finding supports the hypothesis of genetic exchange happening between staphylococcal species. Evaluation of the epidemiology of methicillin-resistant staphylococcal colonisation is necessary in order to understand the apparent emergence of these strains and to develop appropriate control strategies. SCCmec typing is essential for understanding the emergence of MRSA strains from CNS, considering that the MR-CNS may represent the gene pool for the continuous creation of new SCCmec types from which MRSA might originate.

Enhancing supplies of high quality barley to meet pork industry demands

Producing barley varieties that have incrased grain yield and consistent or higher energy for pigs, as well as resistance to the major diseases of barley.

Flat grain beetle fumigation protocol

Flat grain beetle (FGB) is a major emergency plant pest (EPP) of stored grain in Australia. Populations of FGB have recently developed high level resistance to phosphine (the only viable fumigant available for non-quarantine use) resulting in control failures with current dosage regimes. As there is no practical alternative to phosphine, failure to control FGB with phosphine places at risk market access for Australian grain worth up to $7 billion in annual trade. Therefore there is an urgent need to develop appropriate phosphine fumigation protocols to eradicate outbreaks of strongly resistant FGB. Research outcomes: - Characterisation of high resistance to phosphine in flat grain beetles (FGB) for the first time internationally. - Establishment of fumigation protocols and an eradication strategy that will enable industry to eradicate infestations of phosphine-resistant flat grain beetle and prevent or delay further selection for resistance to phosphine. - Development of a rapid test to detect highly resistant FGB. -Facilitate continued market access of Australian grain.

Rain Damage to Strawberries Grown in Southeast Queensland: Evaluation and Genetic Control.

In Queensland, Australia, strawberries (Fragaria xananassa Duchesne) are grown in open fields and rainfall events can damage fruit. Cultivars that are resistant to rain damage may reduce losses and lower risk for the growers. However, little is known about the genetic control of resistance and in a subtropical climate, unpredictable rainfall events hamper evaluation. Rain damage was evaluated on seedling and clonal trials of one breeding population comprising 645 seedling genotypes and 94 clones and on a second clonal population comprising 46 clones from an earlier crossing to make preliminary estimates of heritability. The incidence of field damage from rainfall and damage after laboratory soaking was evaluated to determine if this soaking method could be used to evaluate resistance to rain damage. Narrow-sense heritability of resistance to rain damage calculated for seedlings was low (0.21 +/- 0.15) and not significantly different from zero; however, broad-sense heritability estimates were moderate in both seedlings (0.49 +/- 0.16) and clones (0.45 +/- 0.08) from the first population and similar in clones (0.56 +/- 0.21) from the second population. Immersion of fruit in deionized water produced symptoms consistent with rain damage in the field. Lengthening the duration of soaking of 'Festival' fruit in deionized water exponentially increased the proportion of damage to fruit ranging in ripeness from immature to ripe during the first 6-h period of soaking. When eight genotypes were evaluated, the proportion of sound fruit after soaking in deionized water in the laboratory for up to 5 h was linearly related (r(2) = 0.90) to the proportion of sound fruit in the field after 89 mm of rain. The proportion of sound fruit of the breeding genotype '2008-208' and 'Festival' under soaking (0.67, 0.60) and field (0.52, 0.43) evaluations, respectively, is about the same and these genotypes may be useful sources of resistance to rain damage.

Influence of plant traits on production costs and profitability of strawberry in southeast Queensland.

In Queensland the subtropical strawberry ( Fragaria * ananassa) breeding program aims to combine traits into novel genotypes that increase production efficiency. The contribution of individual plant traits to cost and income under subtropical Queensland conditions was investigated, with the overall goal of improving the profitability of the industry through the release of new strawberry cultivars. The study involved specifying the production and marketing system using three cultivars of strawberry that are currently widely grown annually in southeast Queensland, developing methods to assess the economic impact of changes to the system, and identifying plant traits that influence outcomes from the system. From May through September P (price; $ punnet -1), V (monthly mass; tonne of fruit on the market) and M (calendar month; i.e. May=5) were found to be related ( r2=0.92) by the function (SE) P=4.741(0.469)-0.001630(0.0005) V-0.226(0.102) M using data from 2006 to 2010 for the Brisbane central market. Both income and cost elements in the gross margin were subject to sensitivity analysis. 'Harvesting' and 'Handling/Packing' 'Groups' of 'Activities' were the major contributors to variable costs (each >20%) in the gross margin analysis. Within the 'Harvesting Group', the 'Picking Activity' contributed most (>80%) with the trait 'display of fruit' having the greatest (33%) influence on the cost of the 'Picking Activity'. Within the 'Handling/Packing Group', the 'Packing Activity' contributed 50% of costs with the traits 'fruit shape', 'fruit size variation' and 'resistance to bruising' having the greatest (12-62%) influence on the cost of the 'Packing Activity'. Non-plant items (e.g. carton purchases) made up the other 50% of the costs within the 'Handling/Packing Group'. When any of the individual traits in the 'Harvesting' and 'Handling/Packing' groups were changed by one unit (on a 1-9 scale) the gross margin changed by up to 1%. Increasing yield increased the gross margin to a maximum (15% above present) at 1320 g plant -1 (94% above present). A 10% redistribution of total yield from September to May increased the gross margin by 23%. Increasing fruit size increased gross margin: a 75% increase in fruit size (to ~30 g) produced a 22% increase in the gross margin. The modified gross margin analysis developed in this study allowed simultaneous estimation of the gross margin for the producer and gross value of the industry. These parameters sometimes move in opposite directions.

Bacterial extracellular polysaccharides

Extracellular polysaccharides are as structurally and functionally diverse as the bacteria that synthesise them. They can be present in many forms, including cell-bound capsular polysaccharides, unbound "slime", and as O-antigen component of lipopolysaccharide, with an equally wide range of biological functions. These include resistance to desiccation, protection against nonspecific and specific host immunity, and adherence. Unsurprisingly then, much effort has been made to catalogue the enormous structural complexity of the extracellular polysaccharides made possible by the wide assortment of available monosaccharide combinations, non-carbohydrate residues, and linkage types, and to elucidate their biosynthesis and export. In addition, the work is driven by the commercial potential of these microbial substances in food, pharmaceutics and biomedical industries. Most recently, bacteria-mediated environmental restoration and bioleaching have been attracting much attention owing to their potential to remediate environmental effluents produced by the mining and metallurgy industries. In spite of technological advances in chemistry, molecular biology and imaging techniques that allowed for considerable expansion of knowledge pertaining to the bacterial surface polysaccharides, current understanding of the mechanisms of synthesis and regulation of extracellular polysaccharides is yet to fully explain their structural intricacy and functional variability.

Spot Form of Net Blotch Resistance in a Diverse Set of Barley Lines in Australia and Canada.

The responses of 95 barley lines and cultivars to spot form of net blotch (SFNB) caused by Pyrenophora teres f. maculata were analyzed as seedlings and adults in Australia and Canada. Cluster analyses revealed complex reaction responses. Only 2 lines (Esperance Orge 289 and TR3189) were resistant to all isolates at the seedling stage, whereas 15 lines and cultivars (81-82/033, Arimont, BYDV-018, CBSS97M00855T-B2-M1-Y1-M2-Y-1M-0Y, C19776, Keel, Sloop, Torrens, TR326, VB0111, Yarra, VB0229, WI-2477, WI2553, and Wisconsin Pedigree) were resistant toward the two Canadian isolates and mixture of Australian isolates at the adult stages. In Australian field experiments, the effectiveness of SFNB resistance in three barley cultivars (Barque. Cowabbie, and Schooner) and one breeding line (VB9104) with a different source of resistance was tested. Barque, which possessed a resistance gene that provided complete resistance to SFNB, was the most effective and showed no effect on grain yield or quality in the presence of inoculum. Generally, cultivars with seedling or adult resistance had less disease and better grain quality than the susceptible control. Dash, but they were not as effective as Barque. A preliminary differential set of 19 barley lines and cultivars for P teres I. maculata is proposed.

Interactions between transgenic trees and mycorrhizal and pathogenic fungi

The development of biotechnology techniques in plant breeding and the new commercial applications have raised public and scientific concerns about the safety of genetically modified (GM) crops and trees. To find out the feasibility of these new technologies in the breeding of commercially important Finnish hardwood species and to estimate the ecological risks of the produced transgenic plants, the experiments of this study have been conducted as a part of a larger project focusing on the risk assessment of GM-trees. Transgenic Betula pendula and Populus trees were produced via Agrobacterium mediated transformation. Stilbene synthase (STS) gene from pine (Pinus sylvestris) and chitinase gene from sugar beet (Beta vulgaris) were transferred to (hybrid) aspen and birch, respectively, to improve disease resistance against fungal pathogens. To modify lignin biosynthesis, a 4-coumarate:coenzyme A ligase (4CL) gene fragment in antisense orientation was introduced into two birch clones. In in vitro test, one transgenic aspen line expressing pine STS gene showed increased resistance to decay fungus Phellinus tremulae. In the field, chitinase transgenic birch lines were more susceptible to leaf spot (Pyrenopeziza betulicola) than the non-transgenic control clone while the resistance against birch rust (Melampsoridium betulinum) was improved. No changes in the content or composition of lignin were detected in the 4CL antisense birch lines. In order to evaluate the ecological effects of the produced GM trees on non-target organisms, an in vitro mycorrhiza experiment with Paxillus involutus and a decomposition experiment in the field were performed. The expression of a transgenic chitinase did not disturb the establishment of mycorrhizal symbiosis between birch and P. involutus in vitro. 4CL antisense transformed birch lines showed retarded root growth but were able to form normal ectomycorrhizal associations with the mycorrhizal fungus in vitro. 4CL lines also showed normal litter decomposition. Unexpected growth reductions resulting from the gene transformation were observed in chitinase transgenic and 4CL antisense birch lines. These results indicate that genetic engineering can provide a tool in increasing disease resistance in Finnish tree species. More extensive data with several ectomycorrhizal species is needed to evaluate the consequences of transgene expression on beneficial plant-fungus symbioses. The potential pleiotropic effects of the transgene should also be taken into account when considering the safety of transgenic trees.

Current and emerging screening methods to identify post-head-emergence frost adaptation in wheat and barley

Cereal crops can suffer substantial damage if frosts occur at heading. Identification of post-head-emergence frost (PHEF) resistance in cereals poses a number of unique and difficult challenges. Many decades of research have failed to identify genotypes with PHEF resistance that could offer economically significant benefit to growers. Research and breeding gains have been limited by the available screening systems. Using traditional frost screening systems, genotypes that escape frost injury in trials due to spatial temperature differences and/or small differences in phenology can be misidentified as resistant. We believe that by improving techniques to minimize frost escapes, such ofalse-positive' results can be confidently identified and eliminated. Artificial freezing chambers or manipulated natural frost treatments offer many potential advantages but are not yet at the stage where they can be reliably used for frost screening in breeding programmes. Here we describe the development of a novel photoperiod gradient method (PGM) that facilitates screening of genotypes of different phenology under natural field frosts at matched developmental stages. By identifying frost escapes and increasing the efficiency of field screening, the PGM ensures that research effort can be focused on finding genotypes with improved PHEF resistance. To maximize the likelihood of identifying PHEF resistance, we propose that the PGM form part of an integrated strategy to (i) source germplasm;(ii) facilitate high throughput screening; and (iii) permit detailed validation. PGM may also be useful in other studies where either a range of developmental stages and/or synchronized development are desired.

Inheritance and relative dominance, expressed as toxicity response and delayed development, of phosphine resistance in immature stages of Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae)

Fumigation of stored grain with phosphine (PH 3) is used widely to control the lesser grain borer Rhyzopertha dominica. However, development of high level resistance to phosphine in this species threatens control. Effective resistance management relies on knowledge of the expression of resistance in relation to dosage at all life stages. Therefore, we determined the mode of inheritance of phosphine resistance and strength of the resistance phenotype at each developmental stage. We achieved this by comparing mortality and developmental delay between a strongly resistant strain (R-strain), a susceptible strain (S-strain) and their F 1 progenies. Resistance was a maternally inherited, semi-dominant trait in the egg stage but was inherited as an autosomal, incompletely recessive trait in larvae and pupae. The rank order of developmental tolerance in both the sensitive and resistant strains was eggs > pupae > larvae. Comparison of published values for the response of adult R. dominica relative to our results from immature stages reveals that the adult stage of the S-strain is more sensitive to phosphine than are larvae. This situation is reversed in the R-strain as the adult stage is much more resistant to phosphine than even the most tolerant immature stage. Phosphine resistance factors at LC 50 were eggs 400×, larvae 87× and pupae 181× with respect to reference susceptible strain (S-strain) adults indicating that tolerance conferred by a particular immature stage neither strongly nor reliably interacts with the genetic resistance element. Developmental delay relative to unfumigated control insects was observed in 93% of resistant pupae, 86% of resistant larvae and 41% of resistant eggs. Increased delay in development and the toxicity response to phosphine exposure were both incompletely recessive. We show that resistance to phosphine has pleiotropic effects and that the expression of these effects varies with genotype and throughout the life history of the insect. © 2012.

Individual-based modelling of the efficacy of fumigation tactics to control lesser grain borer (Rhyzopertha dominica) in stored grain

Increasing resistance to phosphine (PH 3) in insect pests, including lesser grain borer (Rhyzopertha dominica) has become a critical issue, and development of effective and sustainable strategies to manage resistance is crucial. In practice, the same grain store may be fumigated multiple times, but usually for the same exposure period and concentration. Simulating a single fumigation allows us to look more closely at the effects of this standard treatment.We used an individual-based, two-locus model to investigate three key questions about the use of phosphine fumigant in relation to the development of PH 3 resistance. First, which is more effective for insect control; long exposure time with a low concentration or short exposure period with a high concentration? Our results showed that extending exposure duration is a much more efficient control tactic than increasing the phosphine concentration. Second, how long should the fumigation period be extended to deal with higher frequencies of resistant insects in the grain? Our results indicated that if the original frequency of resistant insects is increased n times, then the fumigation needs to be extended, at most, n days to achieve the same level of insect control. The third question is how does the presence of varying numbers of insects inside grain storages impact the effectiveness of phosphine fumigation? We found that, for a given fumigation, as the initial population number was increased, the final survival of resistant insects increased proportionally. To control initial populations of insects that were n times larger, it was necessary to increase the fumigation time by about n days. Our results indicate that, in a 2-gene mediated resistance where dilution of resistance gene frequencies through immigration of susceptibles has greater effect, extending fumigation times to reduce survival of homozygous resistant insects will have a significant impact on delaying the development of resistance. © 2012 Elsevier Ltd.

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

Phosphine Resistance in the Rust Red Flour Beetle, Tribolium castaneum (Coleoptera: Tenebrionidae): Inheritance, Gene Interactions and Fitness Costs

The recent emergence of heritable high level resistance to phosphine in stored grain pests is a serious concern among major grain growing countries around the world. Here we describe the genetics of phosphine resistance in the rust red flour beetle Tribolium castaneum (Herbst), a pest of stored grain as well as a genetic model organism. We investigated three field collected strains of T. castaneum viz., susceptible (QTC4), weakly resistant (QTC1012) and strongly resistant (QTC931) to phosphine. The dose-mortality responses of their test- and inter-cross progeny revealed that most resistance was conferred by a single major resistance gene in the weakly (3.2x) resistant strain. This gene was also found in the strongly resistant (431x) strain, together with a second major resistance gene and additional minor factors. The second major gene by itself confers only 12-206x resistance, suggesting that a strong synergistic epistatic interaction between the genes is responsible for the high level of resistance (431x) observed in the strongly resistant strain. Phosphine resistance is not sex linked and is inherited as an incompletely recessive, autosomal trait. The analysis of the phenotypic fitness response of a population derived from a single pair inter-strain cross between the susceptible and strongly resistant strains indicated the changes in the level of response in the strong resistance phenotype; however this effect was not consistent and apparently masked by the genetic background of the weakly resistant strain. The results from this work will inform phosphine resistance management strategies and provide a basis for the identification of the resistance genes.