Characterisation of chicken Campylobacter jejuni isolates using resolution optimised single nucleotide polymorphisms and binary gene markers

The principal objective of this study was to determine if Campylobacter jejuni genotyping methods based upon resolution optimised sets of single nucleotide polymorphisms (SNPs) and binary genetic markers were capable of identifying epidemiologically linked clusters of chicken-derived isolates. Eighty-eight C. jejuni isolates of known flaA RFLP type were included in the study. They encompassed three groups of ten isolates that were obtained at the same time and place and possessed the same flaA type. These were regarded as being epidemiologically linked. Twenty-six unlinked C. jejuni flaA type I isolates were included to test the ability of SNP and binary typing to resolve isolates that were not resolved by flaA RFLP. The remaining isolates were of different flaA types. All isolates were typed by real-time PCR interrogation of the resolution optimised sets of SNPs and binary markers. According to each typing method, the three epidemiologically linked clusters were three different clones that were well resolved from the other isolates. The 26 unlinked C. jejuni flaA type I isolates were resolved into 14 SNP-binary types, indicating that flaA typing can be unreliable for revealing epidemiological linkage. Comparison of the data with data from a fully typed set of isolates associated with human infection revealed that abundant lineages in the chicken isolates that were also found in the human isolates belonged to clonal complex (CC) -21 and CC-353, with the usually rare C-353 member ST-524 being especially abundant in the chicken collection. The chicken isolates selected to be diverse according to flaA were also diverse according to SNP and binary typing. It was observed that CC-48 was absent in the chicken isolates, despite being very common in Australian human infection isolates, indicating that this may be a major cause of human disease that is not chicken associated.

Gene expression in the skin of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus

The cattle tick Rhipicephalus microplus (formerly Boophilus microplus) is responsible for severe production losses to the cattle industry worldwide. It has long been known that different breeds of cattle can resist tick infestation to varying degrees; however, the mechanisms by which resistant cattle prevent heavy infestation are largely unknown. The aim of this study was to determine whether gene expression varied significantly between skin sampling sites (neck, chest and tail region), and whether changes in gene expression could be detected in samples taken at tick attachment sites (tick attached to skin sample) compared with samples taken from non-attachment sites (no tick attachment). We present here the results of an experiment examining the expression of a panel of forty-four genes in skin sections taken from Bos indicus (Brahman) cattle of known high resistance, and Bos taurus (Holstein-Friesian) cattle of known low resistance to the cattle tick. The forty-four genes chosen for this study included genes known to be involved in several immune processes, some structural genes, and some genes previously suggested to be of importance in tick resistance by other researchers. The expression of fifteen gene transcripts increased significantly in Holstein-Friesian skin samples at tick attachment sites. The higher expression of many genes involved in innate inflammatory processes in the Holstein-Friesian animals at tick attachment sites suggests this breed is exhibiting a non-directed pathological response to infestation. Of the forty-four genes analysed, no transcripts were detected in higher abundance at tick attachment sites in the Brahman cattle compared with similar samples from the Holstein-Friesian group, nor difference between attachment site and non-attachment site samples within the Brahman group. The results presented here suggest that the means by which these two cattle breeds respond to tick infestation differ and warrant further investigation.

The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley

Using an established genetic map, a single gene conditioning covered smut resistance, Ruh.7H, was mapped to the telomere region of chromosome 7HS in an Alexis/Sloop doubled haploid barley population. The closest marker to Ruh.7H, abg704 was 7.5 cM away. Thirteen loci on the distal end of 7HS with potential to contain single nucleotide polymorphisms (SNPs) were identified by applying a comparative genomics approach using rice sequence data. Of these, one locus produced polymorphic co-dominant bands of different size while two further loci contained SNPs that were identified using the recently developed high resolution melting (HRM) technique. Two of these markers flanked Ruh.7H with the proximal marker located 3.8 cM and the distal marker 2.7 cM away. This is the first report on the application of the HRM technique to SNP detection and to rapid scoring of known cleaved amplified polymorphic sequence (CAPS) markers in plants. This simple, precise post-PCR technique should find widespread use in the fine-mapping of genetic regions of interest in complex cereal and other plant genomes.

Capsicum chlorosis virus infecting Capsicum annuum in the East Kimberley region of Western Australia

Capsicum chlorosis virus (CaCV) was detected in field grown Capsicum annuum from Kununurra in northeast Western Australia. Identification of the Kununurra isolate (WA-99) was confirmed using sap transmission to indicator hosts, positive reactions with tospovirus serogroup IV-specific antibodies and CaCV-specific primers, and amino acid sequence comparisons that showed >97% identity with published CaCV nucleocapsid gene sequences. The reactions of indicator hosts to infection with WA-99 often differed from those of the type isolate from Queensland. The virus multiplied best when test plants were grown at warm temperatures. CaCV was not detected in samples collected in a survey of C. annuum crops planted in the Perth Metropolitan area.

Identification of a mutation in the para-sodium channel gene of the cattle tick Rhipicephalus (Boophilus) microplus associated with resistance to synthetic pyrethroid acaricides

Resistance against synthetic pyrethroid (SP) products for the control of cattle ticks in Australia was detected in the field in 1984, within a very short time of commercial introduction. We have identified a mutation in the domain II S4-5 linker of the para-sodium channel that is associated with resistance to SPs in the cattle tick Rhipicephalus (Boophilus) microplus from Australia. The cytosine to adenine mutation at position 190 in the R. microplus sequence AF134216, results in an amino acid substitution from leucine in the susceptible strain to isoleucine in the resistant strain. A similar mutation has been shown to confer SP resistance in the whitefly, Bemisia tabaci, but has not been described previously in ticks. A diagnostic quantitative PCR assay has been developed using allele-specific Taqman® minor groove-binding (MGB) probes. Using the assay to screen field and laboratory populations of ticks showed that homozygote allelic frequencies correlated highly with the survival percentage at the discriminating concentration of cypermethrin.

Phylogenetic relationships of unclassified, satellitic Pasteurellaceae obtained from different species of birds as demonstrated by 16S rRNA gene sequence comparison

Avian haemophili demonstrating in vitro satellitic growth, also referred to as the V-factor or NAD requirement, have mainly been classified with Avibacterium paragallinarum (Haemophilus paragallinarum), Avibacterium avium (Pasteurella avium), Avibacterium volantium (Pasteurella volantium) and Avibacterium sp. A (Pasteurella species A). The aim of the present study was to assess the taxonomic position of 18 V-factor-requiring isolates of unclassified Haemophilus-like organisms isolated from galliforme, anseriforme, columbiforme and gruiforme birds as well as kestrels and psittacine birds including budgerigars by conventional phenotypic tests and 16S rRNA gene sequencing. All isolates shared phenotypical characteristics which allowed classification with Pasteurellaceae. Haemolysis of bovine red blood cells was negative. Haemin (X-factor) was not required for growth. Maximum-likelihood phylogenetic analysis including bootstrap analysis showed that six isolates were related to the avian 16S rRNA group and were classified as Avibacterium according to 16S rRNA sequence analysis. Surprisingly, the other 12 isolates were unrelated to Avibacterium. Two isolates were unrelated to any of the known 16S rRNA groups of Pasteurellaceae. Two isolates were related to Volucribacter of the avian 16S rRNA group. Seven isolates belonged to the Testudinis 16S rRNA group and out of these, two isolates were closely related to taxa 14 and 32 of Bisgaard, whereas four other isolates were found to form a genus-like group distantly related to taxon 40 and one isolated remained distantly related to other members of the Testudinis group. One isolate was closely related to taxon 26 (a member of Actinobacillus sensu stricto). The study documented major genetic diversity among V-factor-requiring avian isolates beyond the traditional interpretation that they only belong to Avibacterium, underlining the limited value of satellitic growth for identification of avian members of Pasteurellaceae. Our study also emphasized that these organisms will never be isolated without the use of special media satisfying the V-factor requirement.

Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1 B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

A new semi-dwarfing gene identified by molecular mapping of quantitative trait loci in barley.

Semi-dwarfing genes have been widely used in spring barley (Hordeum vulgare L.) breeding programs in many parts of the world, but the success in developing barley cultivars with semi-dwarfing genes has been limited in North America. Exploiting new semi-dwarfing genes may help in solving this dilemma. A recombinant inbred line population was developed by crossing ZAU 7, a semi-dwarf cultivar from China, to ND16092, a tall breeding line from North Dakota. To identify quantitative trait loci (QTL) controlling plant height, a linkage map comprised of 111 molecular markers was constructed. Simple interval mapping was performed for each of the eight environments. A consistent QTL for plant height was found on chromosome 7HL. This QTL is not associated with maturity and rachis internode length. We suggest the provisional name Qph-7H for this QTL. Qph-7H from ZAU 7 reduced plant height to about 3/4 of normal; thus, Qph-7H is considered a semi-dwarfing gene. Other QTLs for plant height were found, but their expression was variable across the eight environments tested.

Quantitative and molecular genetics of juvenile wood traits in radiata and slash/Caribbean pines.

The Juvenile Wood Initiative (JWI) project has been running successfully since July 2003 under a Research Agreement with FWPA and Letters of Association with the consortium partners STBA (Southern Tree Breeding Association), ArborGen and FPQ (Forestry Plantations Queensland). Over the last five and half years, JWI scientists in CSIRO, FPQ, and STBA have completed all 12 major milestones and 28 component milestones according to the project schedule. We have made benchmark progress in understanding the genetic control of wood formation and interrelationships among wood traits. The project has made 15 primary scientific findings and several results have been adopted by industry as summarized below. This progress was detailed in 10 technical reports to funding organizations and industry clients. Team scientists produced 16 scientific manuscripts (8 published, 1 in press, 2 submitted, and several others in the process of submission) and 15 conference papers or presentations. Primary Scientific Findings. The 15 major scientific findings related to wood science, inheritance and the genetic basis of juvenile wood traits are: 1. An optimal method to predict stiffness of standing trees in slash/Caribbean pine is to combine gravimetric basic density from 12 mm increment cores with a standing tree prediction of MoE using a time of flight acoustic tool. This was the most accurate and cheapest way to rank trees for breeding selection for slash/Caribbean hybrid pine. This method was also recommended for radiata pine. 2. Wood density breeding values were predicted for the first time in the STBA breeding population using a large sample of 7,078 trees (increment cores) and it was estimated that selection of the best 250 trees for deployment will produce wood density gains of 12.4%. 3. Large genetic variation for a suite of wood quality traits including density, MFA, spiral grain, shrinkage, acoustic and non-acoustic stiffness (MoE) for clear wood and standing trees were observed. Genetic gains of between 8 and 49% were predicted for these wood quality traits with selection intensity between 1 to 10% for radiata pine. 4. Site had a major effect on juvenile-mature wood transition age and the effect of selective breeding for a shorter juvenile wood formation phase was only moderate (about 10% genetic gain with 10% selection intensity, equivalent to about 2 years reduction of juvenile wood). 5. The study found no usable site by genotype interactions for the wood quality traits of density, MFA and MoE for both radiata and slash/Caribbean pines, suggesting that assessment of wood properties on one or two sites will provide reliable estimates of the genetic worth of individuals for use in future breeding. 6. There were significant and sizable genotype by environment interactions between the mainland and Tasmanian regions and within Tasmania for DBH and branch size. 7. Strong genetic correlations between rings for density, MFA and MoE for both radiata and slash/Caribbean pines were observed. This suggests that selection for improved wood properties in the innermost rings would also result in improvement of wood properties in the subsequent rings, as well as improved average performance of the entire core. 8. Strong genetic correlations between pure species and hybrid performance for each of the wood quality traits were observed in the hybrid pines. Parental performance can be used to identify the hybrid families which are most likely to have superior juvenile wood properties of the slash/Caribbean F1 hybrid in southeast Queensland. 9. Large unfavourable genetic correlations between growth and wood quality traits were a prominent feature in radiata pine, indicating that overcoming this unfavourable genetic correlation will be a major technical issue in progressing radiata pine breeding. 10. The project created the first radiata pine 18 k cDNA microarray and generated 5,952 radiata pine xylogenesis expressed sequence tags (ESTs) which assembled into 3,304 unigenes. 11. A total of 348 genes were identified as preferentially expressed genes in earlywood or latewood while a total of 168 genes were identified as preferentially expressed genes in either juvenile or mature wood. 12. Juvenile earlywood has a distinct transcriptome relative to other stages of wood development. 13. Discovered rapid decay of linkage disequilibrium (LD) in radiata pine with LD decaying to approximately 50% within 1,700 base pairs (within a typical gene). A total of 913 SNPS from sequencing 177,380 base pairs were identified for association genetic studies. 14. 149 SNPs from 44 genes and 255 SNPs from a further 51 genes (total 95 genes) were selected for association analysis with 62 wood traits, and 30 SNPs were shortlisted for their significant association with variation of wood quality traits (density, MFA and MoE) with individual significant SNPs accounting for between 1.9 and 9.7% of the total genetic variation in traits. 15. Index selection using breeding objectives was the most profitable selection method for radiata pine, but in the long term it may not be the most effective in dealing with negative genetic correlations between wood volume and quality traits. A combination of economic and biological approaches may be needed to deal with the strong adverse correlation.

Evidence for extensive population structure in the white-spotted eagle ray within the Indo-Pacific inferred from mitochondrial gene sequences

The white-spotted eagle ray Aetobatus narinari is a species complex that occurs circumglobally throughout warm-temperate waters. Aetobatus narinari is semi-pelagic and large (up to 300 cm disc width), suggesting high dispersal capabilities and gene flow on a wide spatial scale. Sequence data from two mitochondrial genes, cytochrome b (cytb) and NADH dehydrogenase subunit 4 (ND4), were used to determine the genetic variability within and among 18 sampling locations in the central Indo-Pacific biogeographical region. Populations in the Indo-Pacific were highly genetically structured with c. 70% of the total genetic variation found among three geographical regions (East China Sea, Southeast Asia and Australia). FST was 0.64 for cytb and 0.53 for ND4, with φST values being even larger, that is, 0.78 for cytb and 0.65 for ND4. This high-level genetic partitioning provides strong evidence against extensive gene flow in A. narinari. The degree of genetic population structuring in the Indo-Pacific was similar to that found on a global scale. Global FST was 0.63 for cytb and 0.57 for ND4, and global φST values were 0.94 for cytb and 0.82 for ND4. This suggests that the A. narinari complex may be more speciose than the two or three species proposed to date. Further sampling and genetic analyses are likely to uncover the ‘evolutionarily significant’ and ‘management’ units that are critical to determine the susceptibilities of individual populations to regional fishing pressures and to provide advice on management options. Network analyses showed a close genetic relationship between haplotypes from the central Indo-Pacific and South Africa, providing support for a proposed dispersal pathway from the possible centre of origin of the A. narinari species complex in the Indo-Pacific into the Atlantic Ocean.

Contagious dispersal of seeds of synchronously fruiting species beneath invasive and native fleshy-fruited trees

In subtropical Australia, many native and invasive plant species rely on a shared suite of frugivores, largely birds, for seed dispersal. Many native plants fruit during summer in this region, whereas most invasive plants fruit during winter, thus providing the opportunity for contagious dispersal of seeds beneath synchronously fruiting species. We sampled invasive and native seed rain beneath the canopy of a native summer-fruiting tree Guioa semiglauca and an invasive winter-fruiting tree Cinnamomum camphora, in three study sites over the course of a year. In July, during peak fruiting season for C. camphora and other invasive species, seed rain of invasive species was higher beneath C. camphora than G. semiglauca. This was partly due to the invasive tree Ligustrum lucidum, whose seed rain was three times higher beneath C. camphora than beneath the native tree. In February, seed rain of native species was more abundant beneath the canopy of G. semiglauca than beneath C. camphora, despite the fact that C. camphora was also fruiting at this time. This was probably due to the larger fruit crop produced by G. semiglauca at this time of year. Our study provides evidence that the presence of invasive bird-dispersed plants may facilitate contagious seed dispersal of other invaders, and likewise native species may facilitate seed spread of other native plants.

Coordinator's report: rules for nomenclature and gene symbolization in barley.

In this volume, the recommended rules for nomenclature and gene symbolization in barley are reprinted. The current lists of new and revised barley genetic stock descriptions are presented by BGS number order and by locus symbol in alphabetical order.

The spatiotemporal dynamics of Tribolium castaneum (Herbst): adult flight and gene flow.

Tribolium castaneum (Herbst) has been used as a model organism to develop and test important ecological and evolutionary concepts and is also a major pest of grain and grain products globally. This beetle species is assumed to be a good colonizer of grain storages through anthropogenic movement of grain, and active dispersal by flight is considered unlikely. Studies using T. castaneum have therefore used confined or walking insects. We combine an ecological study of dispersal with an analysis of gene flow using microsatellites to investigate the spatiotemporal dynamics and adult flight of T. castaneum in an ecological landscape in eastern Australia. Flying beetles were caught in traps at grain storages and in fields at least 1 km from the nearest stored grain at regular intervals for an entire year. Significantly more beetles were trapped at storages than in fields, and almost no beetles were caught in native vegetation reserves many kilometres from the nearest stored grain. Genetic differentiation between beetles caught at storages and in fields was low, indicating that although T. castaneum is predominantly aggregated around grain storages, active dispersal takes place to the extent that significant gene flow occurs between them, mitigating founder effects and genetic drift. By combining ecological and molecular techniques, we reveal much higher levels of active dispersal through adult flight in T. castaneum than previously thought. We conclude that the implications of adult flight to previous and future studies on this model organism warrant consideration.

Wood properties and processing outcomes for plantation grown African mahogany (Khaya senegalensis) trees from Clare, Queensland (18 and 20 year-old trees) and Katherine, Northern Territory (14 year-old trees).

Khaya senegalensis, African mahogany, a high-value hardwood, was introduced in the Northern Territory (NT) in the 1950s; included in various trials there and at Weipa, Q in the 1960s-1970s; planted on ex mine sites at Weipa (160 ha) until 1985; revived in farm plantings in Queensland and in trials in the NT in the 1990s; adopted for large-scale, annual planting in the Douglas-Daly region, NT from 2006 and is to have the planted area in the NT extended to at least 20,000 ha. The recent serious interest from plantation growers, including Forest Enterprises Australia Ltd (FEA), has seen the establishment of some large scale commercial plantations. FEA initiated the current study to process relatively young plantation stands from both Northern Territory and Queensland plantations to investigate the sawn wood and veneer recovery and quality from trees ranging from 14 years (NT – 36 trees) to 18-20 years (North Queensland – 31 trees). Field measures of tree size and straightness were complemented with log end splitting assessment and cross-sectional disc sample collection for laboratory wood properties measurements including colour and shrinkage. End-splitting scores assessed on sawn logs were relatively low compared to fast grown plantation eucalypts and did not impact processing negatively. Heartwood proportion in individual trees ranged from 50% up to 92 % of butt cross-sectional disc area for the visually-assessed dark coloured central heartwood and lighter coloured transition wood combined. Dark central heartwood proportion was positively related to tree size (R2 = 0.57). Chemical tests failed to assist in determining heartwood – sapwood boundary. Mean basic density of whole disc samples was 658 kg/m3 and ranged among trees from 603 to 712 kg/m3. When freshly sawn, the heartwood of African mahogany was orange-red to red. Transition wood appeared to be pinkish and the sapwood was a pale yellow colour. Once air dried the heartwood colour generally darkens to pinkish-brown or orange-brown and the effect of prolonged time and sun exposure is to darken and change the heartwood to a red-brown colour. A portable colour measurement spectrophotometer was used to objectively assess colour variation in CIE L*, a* and b* values over time with drying and exposure to sunlight. Capacity to predict standard colour values accurately after varying periods of direct sunlight exposure using results obtained on initial air-dried surfaces decreased with increasing time to sun exposure. The predictions are more accurate for L* values which represent brightness than for variation in the a* values (red spectrum). Selection of superior breeding trees for colour is likely to be based on dried samples exposed to sunlight to reliably highlight wood colour differences. A generally low ratio between tangential and radial shrinkages was found, which was reflected in a low incidence of board distortion (particularly cupping) during drying. A preliminary experiment was carried out to investigate the quality of NIR models to predict shrinkage and density. NIR spectra correlated reasonably well with radial shrinkage and air dried density. When calibration models were applied to their validation sets, radial shrinkage was predicted to an accuracy of 76% with Standard Error of Prediction of 0.21%. There was also a strong predictive power for wood density. These are encouraging results suggesting that NIR spectroscopy has good potential to be used as a non-destructive method to predict shrinkage and wood density using 12mm diameter increment core samples. Average green off saw recovery was 49.5% (range 40 to 69%) for Burdekin Agricultural College (BAC) logs and 41.9% (range 20 to 61%) for Katherine (NT) logs. These figures are about 10% higher than compared to 30-year-old Khaya study by Armstrong et al. (2007) however they are inflated as the green boards were not docked to remove wane prior to being tallied. Of the recovered sawn, dried and dressed volume from the BAC logs, based on the cambial face of boards, 27% could potentially be used for select grade, 40% for medium feature grade and 26% for high feature grades. The heart faces had a slightly higher recovery of select (30%) and medium feature (43%) grade boards with a reduction in the volume of high feature (22%) and reject (6%) grade boards. Distribution of board grades for the NT site aged 14 years followed very similar trends to those of the BAC site boards with an average (between facial and cambial face) 27% could potentially be used for select grade, 42% for medium feature grade, 26% for high feature grade and 5% reject. Relatively to some other subtropical eucalypts, there was a low incidence of borer attack. The major grade limiting defects for both medium and high feature grade boards recovered from the BAC site were knots and wane. The presence of large knots may reflect both management practices and the nature of the genetic material at the site. This stand was not managed for timber production with a very late pruning implemented at about age 12 years. The large amount of wane affected boards is indicative of logs with a large taper and the presence of significant sweep. Wane, knots and skip were the major grade limiting defects for the NT site reflecting considerable amounts of sweep with large taper as might be expected in younger trees. The green veneer recovered from billets of seven Khaya trees rotary peeled on a spindleless lathe produced a recovery of 83% of green billet volume. Dried veneer recovery ranged from 40 to 74 % per billet with an average of 64%. All of the recovered grades were suitable for use in structural ply in accordance to AS/NZ 2269: 2008. The majority of veneer sheets recovered from all billets was C grade (27%) with 20% making D grade and 13% B grade. Total dry sliced veneer recovery from the logs of the two largest logs from each location was estimated to be 41.1%. Very positive results have been recorded in this small scale study. The amount of colour development observed and the very reasonable recoveries of both sawn and veneer products, with a good representation of higher grades in the product distribution, is encouraging. The prospects for significant improvement in these results from well managed and productive stands grown for high quality timber should be high. Additionally, the study has shown the utility of non-destructive evaluation techniques for use in tree improvement programs to improve the quality of future plantations. A few trees combined several of the traits desired of individuals for a first breeding population. Fortunately, the two most promising trees (32, 19) had already been selected for breeding on external traits, and grafts of them are established in the seed orchard.

Forest health guide: symptoms of insect and fungal damage on trees

The Forest health guide: symptoms of insect and fungal damage on trees is intended to help forestry and quarantine staff undertake tree health assessments, in both forest and urban environments. The guide is designed to be used as a quick reference to common symptoms of damage, not as an identification guide to particular insect pests and pathogens.