Rapid assessment of genotype-by-environment interactions and heritability for growth rate in aquaculture species using in vitro fertilisation and DNA tagging

Commercial environments may receive only a fraction of expected genetic gains for growth rate as predicted from the selection environment. This fraction is result of undesirable genotype-by-environment interactions (GxE) and measured by the genetic correlation (rg) of growth between environments. Rapid estimates of genetic correlation achieved in one generation are notoriously difficult to estimate with precision. A new design is proposed where genetic correlations can be estimated by utilising artificial mating from cryopreserved semen and unfertilised eggs stripped from a single female. We compare a traditional phenotype analysis of growth to a threshold model where only the largest fish are genotyped for sire identification. The threshold model was robust to differences in family mortality differing up to 30%. The design is unique as it negates potential re-ranking of families caused by an interaction between common maternal environmental effects and growing environment. The design is suitable for rapid assessment of GxE over one generation with a true 0.70 genetic correlation yielding standard errors as low as 0.07. Different design scenarios were tested for bias and accuracy with a range of heritability values, number of half-sib families created, number of progeny within each full-sib family, number of fish genotyped, number of fish stocked, differing family survival rates and at various simulated genetic correlation levels.

Rapid microsatellite marker development for African mahogany ( Khaya senegalensis, Meliaceae) using next-generation sequencing and assessment of its intra-specific genetic diversity.

Khaya senegalensis (African mahogany or dry-zone mahogany) is a high-value hardwood timber species with great potential for forest plantations in northern Australia. The species is distributed across the sub-Saharan belt from Senegal to Sudan and Uganda. Because of heavy exploitation and constraints on natural regeneration and sustainable planting, it is now classified as a vulnerable species. Here, we describe the development of microsatellite markers for K. senegalensis using next-generation sequencing to assess its intra-specific diversity across its natural range, which is a key for successful breeding programs and effective conservation management of the species. Next-generation sequencing yielded 93943 sequences with an average read length of 234bp. The assembled sequences contained 1030 simple sequence repeats, with primers designed for 522 microsatellite loci. Twenty-one microsatellite loci were tested with 11 showing reliable amplification and polymorphism in K. senegalensis. The 11 novel microsatellites, together with one previously published, were used to assess 73 accessions belonging to the Australian K. senegalensis domestication program, sampled from across the natural range of the species. STRUCTURE analysis shows two major clusters, one comprising mainly accessions from west Africa (Senegal to Benin) and the second based in the far eastern limits of the range in Sudan and Uganda. Higher levels of genetic diversity were found in material from western Africa. This suggests that new seed collections from this region may yield more diverse genotypes than those originating from Sudan and Uganda in eastern Africa.

Preliminary assessment of a rapid leaf nitrogen test in mango

Nitrogen (N) is an essential nutrient in mango, influencing both productivity and fruit quality. In Australia, tree N is traditionally assessed once a year in the dormant pre-flowering stage by laboratory analysis of leaf N. This single assessment is insufficient to determine tree N status at all stages of the annual phenological cycle. Development of a field-based rapid N test would allow more frequent monitoring of tree N status and improved fertiliser management. This experiment examined the accuracy and useability of several devices used in other horticultural crops to rapidly assess mango leaf N in the field; the Konica Minolta 'SPAD-502 chlorophyll meter', Horiba 'Cardy Meter' and the Merck 'RQflex 10'. Regression and correlation analyses were used to determine the relationship between total leaf N and the measurements from the rapid test devices. The relationship between the chlorophyll index measured by the SPAD-502 meter and leaf N is highly significant at late fruit set (R 2=0.72, n=40) and post-harvest (R2=0.81, n=40) stages in the mango cultivar 'Kensington Pride' and significant (R2=0.51, n=40) at the flowering stage, indicating the device can be used to rapidly assess mango leaf N in the field. Correlation analysis indicated the relationship between petiole sap measured with the Cardy or Merck devices and leaf N is non-significant. © 2013 ISHS.

A new data source for fisheries resource assessment: genetic estimates of the effective number of spawners. Final Report to the Fisheries Research and Development Corporation.

The development of innovative methods of stock assessment is a priority for State and Commonwealth fisheries agencies. It is driven by the need to facilitate sustainable exploitation of naturally occurring fisheries resources for the current and future economic, social and environmental well being of Australia. This project was initiated in this context and took advantage of considerable recent achievements in genomics that are shaping our comprehension of the DNA of humans and animals. The basic idea behind this project was that genetic estimates of effective population size, which can be made from empirical measurements of genetic drift, were equivalent to estimates of the successful number of spawners that is an important parameter in process of fisheries stock assessment. The broad objectives of this study were to 1. Critically evaluate a variety of mathematical methods of calculating effective spawner numbers (Ne) by a. conducting comprehensive computer simulations, and by b. analysis of empirical data collected from the Moreton Bay population of tiger prawns (P. esculentus). 2. Lay the groundwork for the application of the technology in the northern prawn fishery (NPF). 3. Produce software for the calculation of Ne, and to make it widely available. The project pulled together a range of mathematical models for estimating current effective population size from diverse sources. Some of them had been recently implemented with the latest statistical methods (eg. Bayesian framework Berthier, Beaumont et al. 2002), while others had lower profiles (eg. Pudovkin, Zaykin et al. 1996; Rousset and Raymond 1995). Computer code and later software with a user-friendly interface (NeEstimator) was produced to implement the methods. This was used as a basis for simulation experiments to evaluate the performance of the methods with an individual-based model of a prawn population. Following the guidelines suggested by computer simulations, the tiger prawn population in Moreton Bay (south-east Queensland) was sampled for genetic analysis with eight microsatellite loci in three successive spring spawning seasons in 2001, 2002 and 2003. As predicted by the simulations, the estimates had non-infinite upper confidence limits, which is a major achievement for the application of the method to a naturally-occurring, short generation, highly fecund invertebrate species. The genetic estimate of the number of successful spawners was around 1000 individuals in two consecutive years. This contrasts with about 500,000 prawns participating in spawning. It is not possible to distinguish successful from non-successful spawners so we suggest a high level of protection for the entire spawning population. We interpret the difference in numbers between successful and non-successful spawners as a large variation in the number of offspring per family that survive – a large number of families have no surviving offspring, while a few have a large number. We explored various ways in which Ne can be useful in fisheries management. It can be a surrogate for spawning population size, assuming the ratio between Ne and spawning population size has been previously calculated for that species. Alternatively, it can be a surrogate for recruitment, again assuming that the ratio between Ne and recruitment has been previously determined. The number of species that can be analysed in this way, however, is likely to be small because of species-specific life history requirements that need to be satisfied for accuracy. The most universal approach would be to integrate Ne with spawning stock-recruitment models, so that these models are more accurate when applied to fisheries populations. A pathway to achieve this was established in this project, which we predict will significantly improve fisheries sustainability in the future. Regardless of the success of integrating Ne into spawning stock-recruitment models, Ne could be used as a fisheries monitoring tool. Declines in spawning stock size or increases in natural or harvest mortality would be reflected by a decline in Ne. This would be good for data-poor fisheries and provides fishery independent information, however, we suggest a species-by-species approach. Some species may be too numerous or experiencing too much migration for the method to work. During the project two important theoretical studies of the simultaneous estimation of effective population size and migration were published (Vitalis and Couvet 2001b; Wang and Whitlock 2003). These methods, combined with collection of preliminary genetic data from the tiger prawn population in southern Gulf of Carpentaria population and a computer simulation study that evaluated the effect of differing reproductive strategies on genetic estimates, suggest that this technology could make an important contribution to the stock assessment process in the northern prawn fishery (NPF). Advances in the genomics world are rapid and already a cheaper, more reliable substitute for microsatellite loci in this technology is available. Digital data from single nucleotide polymorphisms (SNPs) are likely to super cede ‘analogue’ microsatellite data, making it cheaper and easier to apply the method to species with large population sizes.

Production of microalgal concentrates by flocculation and their assessment as aquaculture feeds

A novel technique was developed for the flocculation of marine microalgae commonly used in aquaculture. The process entailed an adjustment of pH of culture to between 10 and 10.6 using NaOH, followed by addition of a non-ionic polymer Magnafloc LT-25 to a final concentration of 0.5 mg L-1. The ensuing flocculate was harvested, and neutralised giving a final concentration factor of between 200- and 800-fold. This process was successfully applied to harvest cells of Chaetoceros calcitrans, C. muelleri, Thalassiosira pseudonana, Attheya septentrionalis, Nitzschia closterium, Skeletonema sp., Tetraselmis suecica and Rhodomonas salina, with efficiencies >=80%. The process was rapid, simple and inexpensive, and relatively cost neutral with increasing volume (cf. concentration by centrifugation). Harvested material was readily disaggregated to single cell suspensions by dilution in seawater and mild agitation. Microscopic examination of the cells showed them to be indistinguishable from corresponding non-flocculated cells. Chlorophyll analysis of concentrates prepared from cultures of Concentrates of T. pseudonana prepared using pH-induced flocculation gave better growth of juvenile Pacific oysters (Crassostrea gigas) than concentrates prepared by ferric flocculation, or centrifuged concentrates using a cream separator or laboratory centrifuge. In follow up experiments, concentrates prepared from 1000 L Chaetoceros muelleri cultures were effective as supplementary diets to improve the growth of juvenile C. gigas and the scallop Pecten fumatus reared under commercial conditions, though not as effective as the corresponding live algae. The experiments demonstrated a proof-of-concept for a commercial application of concentrates prepared by flocculation, especially for use at a remote nursery without on-site mass-algal culture facilities.

Non-destructive assessment of Arundo donax (Poaceae) leaf quality

Leaf carbon (C) content, leaf nitrogen (N) content, and C:N ratio are especially useful for understanding plant-herbivore interactions and may be important in developing control methods for the invasive riparian plant Arundo donax L. We measured C content, N content, C:N ratio, and chlorophyll index (SPAD 502 reading) for 768 leaves from A. donax collected over a five year period at several locations in California, Nevada, and Texas. Leaf N was more variable than leaf C, and thus we developed a linear regression equation for estimating A. donax leaf N from the leaf chlorophyll index (SPAD reading). When applied to two independent data sets, the equation (leaf N content % = -0.63 + 0.08 x SPAD) produced realistic estimates that matched seasonal and spatial trends reported from a natural A. donax population. Used in conjunction with the handheld SPAD 502 meter, the equation provides a rapid, non-destructive method for estimating A. donax leaf quality.

Development of profitable milk production systems for northern Australia: a field assessment of the productivity of five potential farming systems using farmlets.

Farmlets, each of 20 cows, were established to field test five milk production systems and provide a learning platform for farmers and researchers in a subtropical environment. The systems were developed through desktop modelling and industry consultation in response to the need for substantial increases in farm milk production following deregulation of the industry. Four of the systems were based on grazing and the continued use of existing farmland resource bases, whereas the fifth comprised a feedlot and associated forage base developed as a greenfield site. The field evaluation was conducted over 4 years under more adverse environmental conditions than anticipated with below average rainfall and restrictions on irrigation. For the grazed systems, mean annual milk yield per cow ranged from 6330 kg/year (1.9 cows/ha) for a herd based on rain-grown tropical pastures to 7617 kg/year (3.0 cows/ha) where animals were based on temperate and tropical irrigated forages. For the feedlot herd, production of 9460 kg/cow.year (4.3 cows/ha of forage base) was achieved. For all herds, the level of production achieved required annual inputs of concentrates of similar to 3 t DM/animal and purchased conserved fodder from 0.3 to 1.5 t DM/animal. This level of supplementary feeding made a major contribution to total farm nutrient inputs, contributing 50% or more of the nitrogen, phosphorus and potassium entering the farming system, and presents challenges to the management of manure and urine that results from the higher stocking rates enabled. Mean annual milk production for the five systems ranged from 88 to 105% of that predicted by the desktop modelling. This level of agreement for the grazed systems was achieved with minimal overall change in predicted feed inputs; however, the feedlot system required a substantial increase in inputs over those predicted. Reproductive performance for all systems was poorer than anticipated, particularly over the summer mating period. We conclude that the desktop model, developed as a rapid response to assist farmers modify their current farming systems, provided a reasonable prediction of inputs required and milk production. Further model development would need to consider more closely climate variability, the limitations summer temperatures place on reproductive success and the feed requirements of feedlot herds.

Near infrared spectroscopy as a rapid non-invasive tool for agricultural and industrial process management with special reference to avocado and sandalwood industries

Near infrared spectroscopy (NIRS) can play a vital role as a cost effective, rapid, non-invasive, reproducible diagnostic tool for many environmental management, agricultural and industrial waste water monitoring applications. In this paper we highlight the ability of NIRS technology to be used as a diagnostic tool in agricultural and environmental applications through the successful assessment of Fourier Transform NIRS to predict α santalol in sandalwood chip samples, and maturity of ‘Hass’ avocado fruit based on dry matter content. Presented at the Third International Conference on Challenges in Environmental Science & Engineering, CESE-2010. 26 September – 1 October 2010, The Sebel, Cairns, Queensland, Australia.

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.

Rapid monitoring of nitrogen in mango trees

Nitrogen (N) is an essential nutrient in mango, influencing both productivity and fruit quality. In Australian mango orchards, tree N is traditionally assessed once a year at the dormant pre-flowering stage using laboratory analysis of leaf N. This single assessment is insufficient to determine tree N status at all stages of the annual phenological cycle. Development of a field-based rapid N test would allow more frequent monitoring of tree N status and improved fertiliser management. These experiments examined the accuracy and useability of several devices used in other horticultural crops to rapidly assess mango leaf N in the field; the Konica Minolta 'SPAD-502 chlorophyll meter', Horiba 'Cardy Meter' and the Merck 'RQflex 10.' Regression and correlation analyses were used to determine the relationship between total leaf N and the measurements from the rapid test devices. The relationship between the chlorophyll index measured by the SPAD-502 meter and leaf N was highly significant at late fruit set (R 2=0.72, n=40) and post-harvest (R 2=0.81, n=40) stages and significant at the flowering stage (R 2=0.51, n=40) in the cultivar 'Kensington Pride', indicating the device can be used to rapidly assess mango leaf N in the field. Correlation analysis indicated the relationship between petiole sap measured with the Cardy or Merck devices and leaf N was non-significant.

Rapid continuous chemical analysis of meat chicken shed emissions by SIFT–MS

Assessing and addressing odour impacts from poultry production is extremely difficult and subjective because the odorants involved and their dynamics over time and space are poorly understood. This knowledge gap is due, in part, to the lack of suitable analytical tools for measuring and monitoring odorants in the field. The emergence of Selected Ion Flow Tube – Mass Spectrometry (SIFT–MS) and similar instruments is changing that. These tools can rapidly quantify targeted odorants in ambient air in real time, even at very low concentrations. Such data is essential for developing better odour abatement strategies, assessment methods and odour dispersion models. This project trialled a SIFT–MS to determine its suitability for assessing the odorants in meat chicken shed emissions over time and space. This report details evaluations in New Zealand and Australia to determine the potential of SIFT–MS as a tool for the chicken meat industry, including odour measurement (as a proxy for dynamic olfactometry). The report is specifically targeted at those funding and conducting poultry odour research. It will be of interest to those involved with environmental odour monitoring and assessment in general. The high upfront cost of SIFT–MS will lead to potential users wanting compelling evidence that SIFT–MS will meet their needs before they invest in one.