MassARRAY and SABER Analyses of SNPs in Embryo DNA Reveal the Abscission of Self-Fertilised Progeny during Fruit Development of Macadamia (Macadamia integrifolia Maiden & Betche).

Yield in many crops is affected by abscission during the early stages of fruitlet development. The reasons for fruitlet abscission are often unclear but they may include genetic factors because, in some crops, self-pollinated fruitlets are more likely to abscise than cross-pollinated fruitlets. Pollen parentage can also affect final fruit size and fruit quality. Here, we aimed to understand the effects of pollen parentage on fruitlet retention and nut quality in orchards of macadamia (Macadamia integrifolia Maiden & Betche). We identified the pollen parent of macadamia 'cultivar '816' embryos by analysing single nucleotide polymorphisms (SNPs) in their DNA using customised MassARRAY and Single Allele Base Extension Reaction (SABER) methods. This allowed us to determine the proportions of self-fertilised and cross-fertilised progeny during premature fruit drop at 6 weeks and 10 weeks after peak anthesis, as well as at nut maturity. We determined how pollen parentage affected nut-in-shell (NIS) mass, kernel mass, kernel recovery, and oil concentration. Macadamia trees retained cross-fertilised fruitlets rather than self-fertilised fruitlets. The percentage of progeny that were cross-fertilised increased from 6% at 6 weeks after peak anthesis to 97% at nut maturity, with each tree producing on average 22 self-fertilised nuts and 881 cross-fertilised nuts. Three of the four cross-pollen parents provided fruit with significantly higher NIS mass, kernel mass, or kernel recovery than the few remaining self-fertilised fruit. Fruit that were cross-fertilised by '842', 'A4', or 'A203' had 16-29% higher NIS mass and 24-44% higher kernel mass than self-fertilised fruit. Nuts that were cross-fertilised by 'A4' or 'A203' also had 5% or 6% higher kernel recovery, worth approximately $US460-540 more per ton for growers than self-fertilised nuts. The highly selective abscission of self-fertilised fruitlets and the lower nut quality of self-fertilised fruit highlight the critical importance of cross-pollination for macadamia productivity.

Pollen limitation and xenia effects in a cultivated mass-flowering tree, Macadamia integrifolia (Proteaceae).

BACKGROUND AND AIMS: Pollen limitation is most prevalent among bee-pollinated plants, self-incompatible plants and tropical plants. However, we have very little understanding of the extent to which pollen limitation affects fruit set in mass-flowering trees despite tree crops accounting for at least 600 million tons of the 9200 million tons of annual global food production. METHODS: We determined the extent of pollen limitation in a bee-pollinated, partially self-incompatible, subtropical tree by hand cross-pollinating the majority of flowers on mass-flowering macadamia (Macadamia integrifolia) trees that produce about 200 000-400 000 flowers. We measured tree yield and kernel quality and estimated final fruit set. We genotyped individual kernels by MassARRAY to determine levels of outcrossing in orchards and assess paternity effects on nut quality. KEY RESULTS: Macadamia trees were pollen-limited. Supplementary cross-pollination increased nut-in-shell yield, kernel yield and fruit set by as much as 97, 109 and 92 %, respectively. The extent of pollen limitation depended upon the proximity of experimental trees to trees of another cultivar because macadamia trees were highly outcrossing. Between 84 and 100 % of fruit arose from cross-pollination, even at 200 m (25 rows) from orchard blocks of another cultivar. Large variations in nut-in-shell mass, kernel mass, kernel recovery and kernel oil concentration were related to differences in fruit paternity, including between self-pollinated and cross-pollinated fruit, thus demonstrating pollen-parent effects on fruit quality (i.e. xenia). CONCLUSIONS: This study is the first to demonstrate pollen limitation in a mass-flowering tree. Improved pollination led to increased kernel yield of 0.31-0.59 tons ha-1, which equates currently to higher farm-gate income of approximately $US3720-$US7080 ha-1. The heavy reliance of macadamia flowers on cross-pollination and the strong xenia effects on kernel mass demonstrate the high value that pollination services can provide to food production.

Hyperspectral Imaging of Adaxial and Abaxial Leaf Surfaces as a Predictor of Macadamia Crop Nutrition.

Tree crop yield is highly dependent on fertiliser inputs, which are often guided by the assessment of foliar nutrient levels. Traditional methods for nutrient analysis are time-consuming but hyperspectral imaging has potential for rapid nutrient assessment. Hyperspectral imaging has generally been performed using the adaxial surface of leaves although the predictive performance of spectral data has rarely been compared between adaxial and abaxial surfaces of tree leaves. We aimed to evaluate the capacity of laboratory-based hyperspectral imaging (400-1000 nm wavelengths) to predict the nutrient concentrations in macadamia leaves. We also aimed to compare the prediction accuracy from adaxial and abaxial leaf surfaces. We sampled leaves from 30 macadamia trees at 0, 6, 10 and 26 weeks after flowering and captured hyperspectral images of their adaxial and abaxial surfaces. Partial least squares regression (PLSR) models were developed to predict foliar nutrient concentrations. Coefficients of determination (R2P) and ratios of prediction to deviation (RPDs) were used to evaluate prediction accuracy. The models reliably predicted foliar nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), copper (Cu), manganese (Mn), sulphur (S) and zinc (Zn) concentrations. The best-fit models generally predicted nutrient concentrations from spectral data of the adaxial surface (e.g., N: R2P = 0.55, RPD = 1.52; P: R2P = 0.77, RPD = 2.11; K: R2P = 0.77, RPD = 2.12; Ca: R2P = 0.75, RPD = 2.04). Hyperspectral imaging showed great potential for predicting nutrient status. Rapid nutrient assessment through hyperspectral imaging could aid growers to increase orchard productivity by managing fertiliser inputs in a more-timely fashion.

Relationships between Nut Size, Kernel Quality, Nutritional Composition and Levels of Outcrossing in Three Macadamia Cultivars.

Tree nuts play an important role in healthy diets, but their economic value and nutritional quality may be affected by their size and paternity. We assessed relationships between nut size and kernel recovery, the incidence of whole kernels, fatty acid composition and mineral nutrient concentrations in three macadamia cultivars, "Daddow", "816" and "A4". We determined to what extent differences in nut size and quality were the result of different levels of cross- or self-paternity. Small nuts of all cultivars had lower kernel recovery than large nuts, and small nuts provided lower incidence of whole kernels in "Daddow" and "A4". Small kernels had a lower relative abundance of the saturated fatty acid, palmitic acid, in all cultivars and higher relative abundance of the unsaturated fatty acid, oleic acid, in "Daddow" and "A4". Small kernels had higher concentrations of many essential nutrients such as nitrogen and calcium, although potassium concentrations were lower in small kernels. Most nuts arose from cross-pollination. Therefore, nut size and kernel quality were not related to different levels of cross- and self-paternity. Identified cross-paternity was 88%, 78% and 90%, and identified self-paternity was 3%, 2% and 0%, for "Daddow", "816" and "A4", respectively. Small macadamia kernels are at least as nutritious as large macadamia kernels. High levels of cross-paternity confirmed that many macadamia cultivars are predominantly outcrossing. Macadamia growers may need to closely inter-plant cultivars and manage beehives to maximise cross-pollination.

In silico detection of polymorphic microsatellites in the endangered Isis tamarind, Alectryon ramiflorus (Sapindaceae).

PREMISE OF THE STUDY: Alectryon ramiflorus (Sapindaceae) is an endangered rainforest tree known from only two populations. In this study, we identified polymorphic microsatellites, in silico, improving the effectiveness and efficiency of microsatellite development of nonmodel species. The development of genetic markers will support future conservation management of the species. METHODS AND RESULTS: We used next-generation sequencing and bioinformatics to detect polymorphic microsatellites, in silico, reducing both the time and cost of marker development. A panel of 15 microsatellites, 12 of which were polymorphic, were subsequently characterized in 64 adult trees representing the entire species range. Mean observed heterozygosity and expected heterozygosity were 0.471 and 0.425, respectively. The polymorphism information content across loci ranged from 0.152 to 0.875. CONCLUSIONS: The microsatellite markers developed in this study will be useful in gaining an understanding of A. ramiflorus' genetic diversity, level of inbreeding, and population structure and for guiding future restoration and management efforts.

Tissue-specific modulation of glucocorticoid receptor expression in response to salinity acclimation in rainbow trout.

While studies clearly point to a role for cortisol signaling in seawater adaptation, very little is known about salinity impact on glucocorticoid receptor (GR) expression in fish. To this end, we investigated the temporal GR expression in the gill and liver of rainbow trout (Oncorhynchus mykiss) to salinity exposure. Trout were subjected to gradual salinity increases (11 ppt for 1 d, 17 ppt for 2 d and 23 ppt for 2 d) over a five day period. Gill Na(+), K(+)-ATPase alpha-subunit mRNA showed a transient elevation with salinity exposure, while gill cystic fibrosis transmembrane conductance regulator mRNA was not significantly affected by salinity. Liver PEPCK transcript levels showed a transient increase at day 1, but not at day 3 or day 5 of salinity exposure, while the activity of this enzyme was significantly depressed at all time points. Liver glycogen content was also significantly reduced by salinity exposure compared to the freshwater group. Gill GR transcript levels were 3-fold greater upon salinity exposure and this level was maintained over the 5 day period, while gill GR protein content remained unchanged except for a significant drop at day 1 of salinity exposure. Liver GR transcript levels showed no significant change with salinity exposure, while GR protein content was transiently elevated at day 3, but not at day 1 or day 5 of salinity exposure. The tissue-specific GR transcript response in the gill leads us to hypothesize a role for osmosensory signal transduction pathway in the regulation of GR expression in fish. Collectively, salinity exposure modulates GR expression and glucocorticoid signaling in rainbow trout.

Influence of salinity and organic matter on silver accumulation in Gulf toadfish (Opsanus beta).

To help extend the freshwater based biotic ligand model for silver (Ag) into brackish and saltwater conditions, 50g Gulf toadfish (Opsanus beta) were acclimated to 2.5%, 5%, 10%, 20%, 40%, 80%, or 100% salt water and exposed for 6d to 1.0microM AgNO(3), with or without 10mg C/L organic matter. Suwannee River natural organic matter collected by reverse osmosis was used. Silver accumulation in toadfish gills and plasma decreased as salinity increased, indicating low bioavailability of AgCl complexes. Complexation of Ag by organic matter, normally important in freshwater conditions, was less important as salinity increased. Although relatively little intestinal Ag uptake was observed, both liver and bile accumulated Ag from water imbibed past the isosmotic salinity point ( approximately 1/3 salt water). Toadfish also produced intestinal carbonate pellets, minerals which did not influence Ag accumulation. Our results further stress the importance of Ag speciation, physiological mechanisms, and intestinal Ag uptake when modelling Ag uptake and toxicity beyond freshwater conditions.

Influence of water chemistry and natural organic matter on active and passive uptake of inorganic mercury by gills of rainbow trout (Oncorhynchus mykiss).

To distinguish physiologically regulated uptake from passive uptake of inorganic Hg in fish, rainbow trout (Oncorhynchus mykiss) were exposed to inorganic Hg (0.5, 1, or 2 microM total Hg) in ion-poor water with various treatments. Addition of ions to the water (mM concentrations of Ca, K, Cl) did not consistently alter Hg accumulation by trout gills, although there was a trend to higher Hg accumulation at higher ion concentrations. The apical Ca channel blockers Verapamil and lanthanum also did not consistently affect Hg accumulation by trout gills. Pre-treatment of trout with the Na channel blocker Phenamil decreased Hg uptake by about half. These results suggest a combination of physiologically regulated and passive uptake of Hg by trout gills. Strong complexing agents of Hg (EDTA, NTA, ethylenediamine, cysteine) decreased Hg-binding by trout gills in a dose-dependent manner. From these data, a conditional equilibrium binding constant for Hg to the gills was estimated as logK(Hg-gill) = 18.0, representing very strong binding of Hg to the gills. This value is a first step in creating a biotic ligand model (BLM) for inorganic Hg and fish. Natural organic matter (2-10 mg C/L) also decreased Hg-binding by trout gills, although mM concentrations of Na, K, and Cl interfered with this effect. At low concentrations of these ions, natural organic matter samples isolated from various sources bound Hg to similar degrees, as judged by Hg accumulation by trout gills. A conditional binding constant to natural organic matter (NOM) was estimated as logK(Hg-NOM) = 18.0 with about 0.5 micromol binding sites per mg C, representing strong binding of Hg to NOM.

An in vitro biotic ligand model (BLM) for silver binding to cultured gill epithelia of freshwater rainbow trout (Oncorhynchus mykiss).

"Reconstructed" gill epithelia on filter supports were grown in primary culture from dispersed gill cells of freshwater rainbow trout (Oncorhynchus mykiss). This preparation contains both pavement cells and chloride cells, and after 7-9 days in culture, permits exposure of the apical surface to true freshwater while maintaining blood-like culture media on the basolateral surface, and exhibits a stable transepithelial resistance (TER) and transepithelial potential (TEP) under these conditions. These epithelia were used to develop a possible in vitro version of the biotic ligand model (BLM) for silver; the in vivo BLM uses short-term gill binding of the metal to predict acute silver toxicity as a function of freshwater chemistry. Radio-labeled silver ((110m)Ag as AgNO(3)) was placed on the apical side (freshwater), and the appearance of (110m)Ag in the epithelia (binding) and in the basolateral media (flux) over 3 h were monitored. Silver binding (greater than the approximate range 0-100 mug l(-1)) and silver flux were concentration-dependent with a 50% saturation point (apparent K(d)) value of about 10 mug l(-1) or 10(-7) M, very close to the 96-h LC50 in vivo in the same water chemistry. There were no adverse effects of silver on TER, TEP, or Na(+), K(+)-ATPase activity, though the latter declined over longer exposures, as in vivo. Silver flux over 3 h was small (<20%) relative to binding, and was insensitive to water chemistry. However, silver binding was decreased by elevations in freshwater Na(+) and dissolved organic carbon (humic acid) concentrations, increased by elevations in freshwater Cl(-) and reductions in pH, and insensitive to elevations in Ca(2+). With the exception of the pH response, these effects were qualitatively and quantitatively similar to in vivo BLM responses. The results suggest that an in vitro BLM approach may provide a simple and cost-effective way for evaluating the protective effects of site-specific waters.

Salinity acclimation affects the somatotropic axis in rainbow trout.

In this study, we set out to examine the role of the somatotropic axis in the ion-regulation process in rainbow trout. Specifically, our objective was to examine whether plasma insulin-like growth factor-binding proteins (IGFBPs) are modulated by gradual salinity exposure. To this end, freshwater (FW)-adapted rainbow trout were subjected to gradual salinity increases, up to 66% seawater, over a period of 5 days. During this acclimation process, minimal elevations in plasma Ca2+ and Cl- were seen in the salinity-acclimated groups compared with FW controls. There were no changes in plasma Na+ levels, and only a minor transient change in plasma cortisol levels was seen with salinity exposure. The salinity challenged animals responded with elevations in plasma growth hormone (GH) and IGF-I levels and gill Na+-K+-ATPase activity. We identified IGFBPs of 21, 32, 42, and 50 kDa in size in the plasma of these animals, and they were consistently higher with salinity. Despite the overall increase in IGFBPs with salinity, transient changes in individual BPs over the 5-day period were noted in the FW and salinity-exposed fish. Specifically, the transient changes in plasma levels of the 21-, 42-, and 50-kDa IGFBPs were different between the FW and salinity groups, while the 32-kDa IGFBP showed a similar trend (increases with sampling time) in both groups. Considered together, the elevated plasma IGFBPs suggest a key role for these binding proteins in the regulation of IGF-I during salinity acclimation in salmonids.