A PCR assay for gender assignment in dugong (Dugong dugon) and West Indian manatee (Trichechus manatus)

Gender assignment for some aquatic mammals in the field is difficult. Molecular sexing from tissue biopsies is possible as males are heterogametic. Here we describe a multiplex PCR assay that amplifies the male specific SRY gene and differentiates ZFX and ZFY gametologues in two sirenian species, dugong (Dugong dugon) and West Indian manatee (Trichechus manatus). The assay was validated with animals of known gender and proved accurate and robust to experimental failure.

Seed persistence in the field may be predicted by laboratory-controlled aging

Weed management is complicated by the presence of soil seed banks. The complexity of soil-seed interactions means that seed persistence in the field is often difficult to measure, let alone predict. Field trials, although accurate in their context, are time-consuming and expensive to conduct for individual species. Some ex situ techniques for estimating seed life expectancy have been proposed, but these fail to simulate the environmental complexity of the field. Also, it has been questioned whether techniques such as the controlled aging test (CAT) are useful indicators of field persistence. This study aimed to test the validity of the standard CAT (seed aging at 45 C and 60% relative humidity) in use at the Royal Botanic Gardens, Kew, U.K., for predicting field seed-persistence. Comparison of seed persistence and CAT data for 27 northwest European species suggested a significant positive correlation of 0.31. Subsequently, 13 species of emerging and common weeds of Queensland were assessed for their seed longevity using the CAT. The seed longevity data of these species in the CAT were linked with field seed-persistence data according to three broad seed-persistence categories: <1 yr, 1 to 3 yr, and >3 yr. We discuss the scope for using the CAT as a tool for rapid assignment of species to these categories. There is a need for further studies that compare predictions of seed persistence based on the CAT with seed persistence in the field for a larger range of species and environments.

Refined Global Analysis of Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae) Mitochondrial Cytochrome Oxidase 1 to Identify Species Level Genetic Boundaries.

Identifying species boundaries within morphologically indistinguishable cryptic species complexes is often contentious. For the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae), the lack of a clear understanding about the genetic limits of the numerous genetic groups and biotypes so far identified has resulted in a lack of consistency in the application of the terms, the approaches use to apply them and in our understanding of what genetic structure within B. tabaci means. Our response has been to use mitochondrial gene cytochrome oxidase one to consider how to clearly and consistently define genetic separation. Using Bayesian phylogenetic analysis and analysis of sequence pairwise divergence we found a considerably higher to number of genetic groups than had been previously determined with two breaks in the distribution, one at 11% and another at 3.5%. At >11% divergence, 11 distinct groups were resolved, whereas at >3.5% divergence 24 groups were identified. Consensus sequences for each of these groups were determined and were shown to be useful in the correct assignment of sequences of unknown origin. The 3.5% divergence bound is consistent with species level separations in other insect taxa and Suggests that B. tabaci is it cryptic species composed of at least 24 distinct species. We further show that the placement of Bemesia atriplex (Froggatt) within the B. tabaci in, group adds further weight to the argument for species level separation within B. tabaci. This new analysis, which constructs consensus sequences and uses these its a standard against which unknown sequences call be compared, provides for the first time it consistent means of identifying the genetic hounds of each species with it high degree of certainty.

Deviant near-infrared spectra identifies Corymbia hybrids

The study examined the potential of Near Infrared Reflectance (NIR) spectroscopy for field diagnosis of hybrids between Corymbia (formerly Eucalyptus) species. NIR profiles were generated by scanning foliage from a total of 383 hybrid and 533 parental seedlings grown in a common garden and partial least squares discriminant analysis was used to test three-way model power to assign individuals to their appropriate taxon; either a parental or F1 hybrid class. Using the optimised conditions, fresh foliage from eight-month-old seedlings and a handheld NIR instrument (950–1800 nm), the mean assignment rates for the three hybrid groups ranged from 76% to 90%. Hybrid-parent contrast of NIR spectra deviated more so than parent–parent contrast. The F1 taxon assignment rates were usually higher than those for parents at 100% and 72%, respectively. Hybrid resolution was even greater for 2nd generation backcross hybrids. Similar to studies of morphology, taxon assignments tended to be more accurate for hybrid groups in which the parental taxa were more divergent. The practical application of this technique for hybrid diagnosis of seedlings in the nursery will require careful attention to control environmental factors because seedling age and storage effects influenced the ability of NIR to identify hybrids. The technique may also necessitate the generation of comparable reference populations, although exclusions approaches to analysis may circumvent the need for reference populations. The application of NIR in field diagnosis will be further complicated by the need to generate global models across environments but such models have been obtained for reliable prediction of chemistries in other situations.

Identification of intersectional Corymbia hybrids based on seedling morphology improves with parental divergence

Differences in morphology have provided a basis for detecting natural interspecific hybridisation in forest trees for decades but have come to prominence again more recently as a means for directly measuring gene flow from planted forests. Here we examined the utility of seedling morphology for hybrid discrimination in three hybrid groups relevant to the monitoring of gene flow from plantings of Corymbia (L.D. Pryor & L.A.S. Johnson ex Brooker) taxa in subtropical Australia. Thirty leaf and stem characters were assessed on 907 8-month old seedlings from four parental and six hybrid taxa grown in a common garden. Outbred F1 hybrids between spotted gums (Corymbia citriodora subspecies variegata, C. citriodora subspecies citriodora and Corymbia henryi) tended to more closely resemble their maternal Corymbia torelliana parent and the most discriminating characters were the ratio of blade length to maximum perpendicular width, the presence or absence of a lignotuber, and specific leaf weight. Assignment of individuals into genealogical classes based on a multivariate model limited to a set of the more discriminating and independent characters was highest in the hybrid group, where parental taxa were genetically most divergent. Overall power to resolve among outbred F1 hybrids from both parental taxa was low to moderate, but this may not be a limitation to its likely major application of identifying hybrids in seedlots from native spotted gum stands. Advanced generation hybrids (outbred F2 and outbred backcrosses) were more difficult to resolve reliably due to the higher variances of hybrid taxa and the tendency of backcrosses to resemble their recurrent parents. Visual assessments of seedling morphology may provide a filter allowing screening of the large numbers needed to monitor gene flow, but will need to be combined with other hybrid detection methods to ensure hybrids are detected.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically. Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

Stock assessment of the Queensland east coast common coral trout (Plectropomus leopardus) fishery

Common coral trout Plectropomus leopardus is an iconic fish of the Great Barrier Reef (GBR) and is the most important fish for the commercial fishery there. Most of the catch is exported live to Asia. This stock assessment was undertaken in response to falls in catch sizes and catch rates in recent years, in order to gauge the status of the stock. It is the first stock assessment ever conducted of coral trout on the GBR, and brings together a multitude of different data sources for the first time. The GBR is very large and was divided into a regional structure based on the Bioregions defined by expert committees appointed by the Great Barrier Reef Marine Park Authority (GBRMPA) as part of the 2004 rezoning of the GBR. The regional structure consists of six Regions, from the Far Northern Region in the north to the Swains and Capricorn–Bunker Regions in the south. Regions also closely follow the boundaries between Bioregions. Two of the northern Regions are split into Subregions on the basis of potential changes in fishing intensity between the Subregions; there are nine Subregions altogether, which include four Regions that are not split. Bioregions are split into Subbioregions along the Subregion boundaries. Finally, each Subbioregion is split into a “blue” population which is open to fishing and a “green” population which is closed to fishing. The fishery is unusual in that catch rates as an indicator of abundance of coral trout are heavily influenced by tropical cyclones. After a major cyclone, catch rates fall for two to three years, and rebound after that. This effect is well correlated with the times of occurrence of cyclones, and usually occurs in the same month that the cyclone strikes. However, statistical analyses correlating catch rates with cyclone wind energy did not provide significantly different catch rate trends. Alternative indicators of cyclone strength may explain more of the catch rate decline, and future work should investigate this. Another feature of catch rates is the phenomenon of social learning in coral trout populations, whereby when a population of coral trout is fished, individuals quickly learn not to take bait. Then the catch rate falls sharply even when the population size is still high. The social learning may take place by fish directly observing their fellows being hooked, or perhaps heeding a chemo-sensory cue emitted by fish that are hooked. As part of the assessment, analysis of data from replenishment closures of Boult Reef in the Capricorn–Bunker Region (closed 1983–86) and Bramble Reef in the Townsville Subregion (closed 1992–95) estimated a strong social learning effect. A major data source for the stock assessment was the large collection of underwater visual survey (UVS) data collected by divers who counted the coral trout that they sighted. This allowed estimation of the density of coral trout in the different Bioregions (expressed as a number of fish per hectare). Combined with mapping data of all the 3000 or so reefs making up the GBR, the UVS results provided direct estimates of the population size in each Subbioregion. A regional population dynamic model was developed to account for the intricacies of coral trout population dynamics and catch rates. Because the statistical analysis of catch rates did not attribute much of the decline to tropical cyclones, (and thereby implied “real” declines in biomass), and because in contrast the UVS data indicate relatively stable population sizes, model outputs were unduly influenced by the unlikely hypothesis that falling catch rates are real. The alternative hypothesis that UVS data are closer to the mark and declining catch rates are an artefact of spurious (e.g., cyclone impact) effects is much more probable. Judging by the population size estimates provided by the UVS data, there is no biological problem with the status of coral trout stocks. The estimate of the total number of Plectropomus leopardus on blue zones on the GBR in the mid-1980s (the time of the major UVS series) was 5.34 million legal-sized fish, or about 8400 t exploitable biomass, with an 2 additional 3350 t in green zones (using the current zoning which was introduced on 1 July 2004). For the offshore regions favoured by commercial fishers, the figure was about 4.90 million legal-sized fish in blue zones, or about 7700 t exploitable biomass. There is, however, an economic problem, as indicated by relatively low catch rates and anecdotal information provided by commercial fishers. The costs of fishing the GBR by hook and line (the only method compatible with the GBR’s high conservation status) are high, and commercial fishers are unable to operate profitably when catch rates are depressed (e.g., from a tropical cyclone). The economic problem is compounded by the effect of social learning in coral trout, whereby catch rates fall rapidly if fishers keep returning to the same fishing locations. In response, commercial fishers tend to spread out over the GBR, including the Far Northern and Swains Regions which are far from port and incur higher travel costs. The economic problem provides some logic to a reduction in the TACC. Such a reduction during good times, such as when the fishery is rebounding after a major tropical cyclone, could provide a net benefit to the fishery, as it would provide a margin of stock safety and make the fishery more economically robust by providing higher catch rates during subsequent periods of depressed catches. During hard times when catch rates are low (e.g., shortly after a major tropical cyclone), a change to the TACC would have little effect as even a reduced TACC would not come close to being filled. Quota adjustments based on catch rates should take account of long-term trends in order to mitigate variability and cyclone effects in data.

Understanding the barriers to the implementation of precision agriculture in the central region

There is an increasing requirement for more astute land resource management through efficiencies in agricultural inputs in a sugar cane production system. A precision agriculture (PA) approach can provide a pathway for a sustainable sugarcane production system. One of the impediments to the adoption of PA practices is access to paddock-scale mapping layers displaying variability in soil properties, crop growth and surface drainage. Variable rate application (VRA) of nutrients is an important component of PA. However, agronomic expertise within PA systems has fallen well behind significant advances in PA technologies. Generally, advisers in the sugar industry have a poor comprehension of the complex interaction of variables that contribute to within-paddock variations in crop growth. This is regarded as a significant impediment to the progression of PA in sugarcane and is one of the reasons for the poor adoption of VRA of nutrients in a PA approach to improved sugar cane production. This project therefore has established a number of key objectives which will contribute to the adoption of PA and the staged progression of VRA supported by relevant and practical agronomic expertise. These objectives include provision of base soils attribute mapping that can be determined using Veris 3100 Electrical Conductivity (EC) and digital elevation datasets using GPS mapping technology for a large sector of the central cane growing region using analysis of archived satellite imagery to determine the location and stability of yield patterns over time and in varying seasonal conditions on selected project study sites. They also include the stablishment of experiments to determine appropriate VRA nitrogen rates on various soil types subjected to extended anaerobic conditions, and the establishment of trials to determine nitrogen rates applicable to a declining yield potential associated with the aging of ratoons in the crop cycle. Preliminary analysis of archived yield estimation data indicates that yield patterns remain relatively stable overtime. Results also indicate the where there is considerable variability in EC values there is also significant variation in yield.

Genomic regions influencing seminal root traits in barley

Water availability is a major limiting factor for crop production, making drought adaptation and its many component traits a desirable attribute of plant cultivars. Previous studies in cereal crops indicate that root traits expressed at early plant developmental stages, such as seminal root angle and root number, are associated with water extraction at different depths. Here, we conducted the first study to map seminal root traits in barley (Hordeum vulgare L.). Using a recently developed high-throughput phenotyping method, a panel of 30 barley genotypes and a doubled-haploid (DH) population (ND24260 × 'Flagship') comprising 330 lines genotyped with diversity array technology (DArT) markers were evaluated for seminal root angle (deviation from vertical) and root number under controlled environmental conditions. A high degree of phenotypic variation was observed in the panel of 30 genotypes: 13.5 to 82.2 and 3.6 to 6.9° for root angle and root number, respectively. A similar range was observed in the DH population: 16.4 to 70.5 and 3.6 to 6.5° for root angle and number, respectively. Seven quantitative trait loci (QTL) for seminal root traits (root angle, two QTL; root number, five QTL) were detected in the DH population. A major QTL influencing both root angle and root number (RAQ2/RNQ4) was positioned on chromosome 5HL. Across-species analysis identified 10 common genes underlying root trait QTL in barley, wheat (Triticum aestivum L.), and sorghum [Sorghum bicolor (L.) Moench]. Here, we provide insight into seminal root phenotypes and provide a first look at the genetics controlling these traits in barley.