Development of an immunomagnetic capture-reverse transcriptase-PCR assay for three pineapple ampeloviruses

A semi-automated, immunomagneticcapture-reverse transcription PCR(IMC-RT-PCR) assay for the detection of three pineapple-infecting ampeloviruses, Pineapple mealybug wilt-associated virus-1, -2 and -3, is described. The assay was equivalent in sensitivity but more rapid than conventional immunocapture RT-PCR. The assay can be used either as a one- or two-step RT-PCR and allows detection of the viruses separately or together in a triplex assay from fresh, frozen or freeze-dried pineapple leaf tissue. This IMC-RT-PCR assay could be used for high throughput screening of pineapple planting propagules and could easily be modified for the detection of other RNA viruses in a range of plant species, provided suitable antibodies are available.

How accurate are the marker orders in crop linkage maps generated from large marker datasets?

Marker ordering during linkage map construction is a critical component of QTL mapping research. In recent years, high-throughput genotyping methods have become widely used, and these methods may generate hundreds of markers for a single mapping population. This poses problems for linkage analysis software because the number of possible marker orders increases exponentially as the number of markers increases. In this paper, we tested the accuracy of linkage analyses on simulated recombinant inbred line data using the commonly used Map Manager QTX (Manly et al. 2001: Mammalian Genome 12, 930-932) software and RECORD (Van Os et al. 2005: Theoretical and Applied Genetics 112, 30-40). Accuracy was measured by calculating two scores: % correct marker positions, and a novel, weighted rank-based score derived from the sum of absolute values of true minus observed marker ranks divided by the total number of markers. The accuracy of maps generated using Map Manager QTX was considerably lower than those generated using RECORD. Differences in linkage maps were often observed when marker ordering was performed several times using the identical dataset. In order to test the effect of reducing marker numbers on the stability of marker order, we pruned marker datasets focusing on regions consisting of tightly linked clusters of markers, which included redundant markers. Marker pruning improved the accuracy and stability of linkage maps because a single unambiguous marker order was produced that was consistent across replications of analysis. Marker pruning was also applied to a real barley mapping population and QTL analysis was performed using different map versions produced by the different programs. While some QTLs were identified with both map versions, there were large differences in QTL mapping results. Differences included maximum LOD and R-2 values at QTL peaks and map positions, thus highlighting the importance of marker order for QTL mapping

Improving the efficiency of pig feed manufacturing and application of additives

This project aims to reduce production costs for high-quality pork through understanding how commercial processing conditions affect mill throughput, processing energy efficiency, product durability and the nutritional value of pig feed.

Differentiation of Solenopsis invicta social forms using high resolution melt PCR.

Solenopsis invicta Buren (red imported fire ant) are invasive pests that have the capability of major destructive impacts on lifestyle, ecology and economy. Control of this species is dependent, in part, upon ability to estimate the potential spread from newly discovered nests. The potential for spread and the spread characteristics differ between monogyne and polygyne social forms. Prior to this study, differentiation of the two social forms in laboratory test samples commonly used a method involving restriction endonuclease digestion of an amplified Gp-9 fragment. Success of this assay is limited by the quality of DNA, which in the field-collected insects may be affected by temporary storage in unfavourable conditions. Here, we describe an alternative and highly objective assay based upon a high resolution melt technique following preamplification of a significantly shorter Gp-9 fragment than that required for restriction endonuclease digestion. We demonstrate the application of this assay to a S. invicta incursion in Queensland, Australia, using field samples from which DNA may be partially degraded. The reductions in hands-on requirements and overall duration of the assay underpin its suitability for high-throughput testing.

Genetic control of nodal root angle in sorghum and its implications on water extraction

Genotypic variability in root system architecture has been associated with root angle of seedlings and water extraction patterns of mature plants in a range of crops. The potential inclusion of root angle as a selection criterion in a sorghum breeding program requires (1) availability of an efficient screening method, (2) presence of genotypic variation with high heritability, and (3) an association with water extraction pattern. The aim of this study was to determine the feasibility for inclusion of nodal root angle as a selection criterion in sorghum breeding programs. A high-throughput phenotypic screen for nodal root angle in young sorghum plants has recently been developed and has been used successfully to identify significant variation in nodal root angle across a diverse range of inbred lines and a mapping population. In both cases, heritabilities for nodal root angle were high. No association between nodal root angle and plant size was detected. This implies that parental inbred lines could potentially be used to asses nodal root angle of their hybrids, although such predictability is compromised by significant interactions. To study effects of nodal root angle on water extraction patterns of mature plants, four inbred lines with contrasting nodal root angle at seedling stage were grown until at least anthesis in large rhizotrons. A consistent trend was observed that nodal root angle may affect the spatial distribution of root mass of mature plants and hence their ability to extract soil water, although genotypic differences were not significant. The potential implications of this for specific adaptation to drought stress are discussed. Results suggest that nodal root angle of young plants can be a useful selection criterion for specific drought adaptation, and could potentially be used in molecular breeding programs if QTLs for root angle can be identified. (C) 2012 Elsevier B.V. All rights reserved.

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.

Light thresholds derived from seagrass loss in the coastal zone of the northern Great Barrier Reef, Australia

There is a world-wide trend for deteriorating water quality and light levels in the coastal zone, and this has been linked to declines in seagrass abundance. Localized management of seagrass meadow health requires that water quality guidelines for meeting seagrass growth requirements are available. Tropical seagrass meadows are diverse and can be highly dynamic and we have used this dynamism to identify light thresholds in multi-specific meadows dominated by Halodule uninervis in the northern Great Barrier Reef, Australia. Seagrass cover was measured at similar to 3 month intervals from 2008 to 2011 at three sites: Magnetic Island (MI) Dunk Island (DI) and Green Island (GI). Photosynthetically active radiation was continuously measured within the seagrass canopy, and three light metrics were derived. Complete seagrass loss occurred at MI and DI and at these sites changes in seagrass cover were correlated with the three light metrics. Mean daily irradiance (I-d) above 5 and 8.4 mol m(-2) d(-1) was associated with gains in seagrass at MI and DI, however a significant correlation (R = 0.649, p < 0.05) only occurred at MI. The second metric, percent of days below 3 mol m(-2) d(-1), correlated the most strongly (MI, R = -0.714, p < 0.01 and DI, R = -0.859, p = <0.001) with change in seagrass cover with 16-18% of days below 3 mol m(-2) d(-1) being associated with more than 50% seagrass loss. The third metric, the number of hours of light saturated irradiance (H-sat) was calculated using literature-derived data on saturating irradiance (E-k). H-sat correlated well (R = 0.686, p <0.01; and DI, R = 0.704, p < 0.05) with change in seagrass abundance, and was very consistent between the two sites as 4 H-sat was associated with increases in seagrass abundance at both sites, and less than 4 H-sat with more than 50% loss. At the third site (GI), small seasonal losses of seagrass quickly recovered during the growth season and the light metrics did not correlate (p > 0.05) with change in percent cover, except for I-d which was always high, but correlated with change in seagrass cover. Although distinct light thresholds were observed, the departure from threshold values was also important. For example, light levels that are well below the thresholds resulted in more severe loss of seagrass than those just below the threshold. Environmental managers aiming to achieve optimal seagrass growth conditions can use these threshold light metrics as guidelines; however, other environmental conditions, including seasonally varying temperature and nutrient availability, will influence seagrass responses above and below these thresholds. (C) 2012 Published by Elsevier Ltd.

Rapid phenotyping for adult-plant resistance to stripe rust in wheat

Stripe or yellow rust (YR) is a significant problem in wheat crops worldwide. The deployment of adult-plant resistance (APR) genes in wheat cultivars is considered a sustainable management strategy, as these genes confer partial resistance that is usually non-race specific. Screening for APR typically involves assessment of adult plants in the field, where expression may be influenced by environmental factors. We report a high-throughput screening method for YR APR that can be used to assess fixed lines or segregating populations grown under controlled environmental conditions (CEC). Inoculation of 3-week-old wheat plants from lines with known APR responses to YR, when grown under constant light and temperature, provided disease responses typical of adult plants. Two F-2 populations ('H45' x 'ST93' and 'Wyalkatchem' x 'ST93') segregating for APR were assessed under both CEC and field conditions. These populations showed similar variation in disease response and lines assessed in both environments attained similar rankings. Phenotypic screening using CEC and continuous light provides an opportunity to accelerate the development of new wheat cultivars with durable resistance.

A simple, one-tube assay for the simultaneous detection and diagnosis of ten Australian poultry Eimeria

Coccidiosis is a costly worldwide enteric disease of chickens caused by parasites of the genus Eimeria. At present, there are seven described species that occur globally and a further three undescribed, operational taxonomic units (OTUs X, Y, and Z) that are known to infect chickens from Australia. Species of Eimeria have both overlapping morphology and pathology and frequently occur as mixed-species infections. This makes definitive diagnosis with currently available tests difficult and, to date, there is no test for the detection of the three OTUs. This paper describes the development of a PCR-based assay that is capable of detecting all ten species of Eimeria, including OTUs X, Y, and Z in field samples. The assay is based on a single set of generic primers that amplifies a single diagnostic fragment from the mitochondrial genome of each species. This one-tube assay is simple, low-cost, and has the capacity to be high throughput. It will therefore be of great benefit to the poultry industry for Eimeria detection and control, and the confirmation of identity and purity of vaccine strains.

Bentonite can decrease ammonia volatilisation losses from poultry litter: laboratory studies

Ammonia volatilisation from manure materials within poultry sheds can adversely affect production, and also represents a loss of fertiliser value from the spent litter. This study sought to compare the ability of alum and bentonite to decrease volatilisation losses of ammonia from spent poultry litter. An in-vessel volatilisation trial with air flushing, ammonia collection, and ammonia analysis was conducted over 64 days to evaluate the mitigation potential of these two materials. Water-saturated spent litter was incubated at 25°C in untreated condition (control) or with three treatments: an industry-accepted rate of alum [4% Al2(SO4)3·18H2O by dry mass of litter dry mass; ALUM], air-dry bentonite (127% by dry mass; BENT), or water-saturated bentonite (once again at 127% by dry mass; SATBENT). A high proportion of the nitrogen contained in the untreated spent litter was volatilised (62%). Bentonite additions were superior to alum additions at retaining spent litter ammonia (nitrogen losses: 15%, SATBENT; 34%, BENT; 54%, ALUM). Where production considerations favour comparable high rates of bentonite addition (e.g. where the litter is to be re-formulated as a fertiliser), this clay has potential to decrease ammonia volatilisation either in-shed or in spent litter stockpiles or formulated products, without the associated detrimental effect of alum on phosphorus availability.

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.