William D. Bovill

Analysis of anther transcriptomes to identify genes contributing to meiosis and male gametophyte development in rice

BackgroundIn flowering plants, the anther is the site of male gametophyte development. Two major events in the development of the male germline are meiosis and the asymmetric division in the male gametophyte that gives rise to the vegetative and generative cells, and the following mitotic division in the generative cell that produces two sperm cells. Anther transcriptomes have been analyzed in many plant species at progressive stages of development by using microarray and sequence-by synthesis-technologies to identify genes that regulate anther development. Here we report a comprehensive analysis of rice anther transcriptomes at four distinct stages, focusing on identifying regulatory components that contribute to male meiosis and germline development. Further, these transcriptomes have been compared with the transcriptomes of 10 stages of rice vegetative and seed development to identify genes that express specifically during anther development.ResultsTranscriptome profiling of four stages of anther development in rice including pre-meiotic (PMA), meiotic (MA), anthers at single-celled (SCP) and tri-nucleate pollen (TPA) revealed about 22,000 genes expressing in at least one of the anther developmental stages, with the highest number in MA (18,090) and the lowest (15,465) in TPA. Comparison of these transcriptome profiles to an in-house generated microarray-based transcriptomics database comprising of 10 stages/tissues of vegetative as well as reproductive development in rice resulted in the identification of 1,000 genes specifically expressed in anther stages. From this sub-set, 453 genes were specific to TPA, while 78 and 184 genes were expressed specifically in MA and SCP, respectively. The expression pattern of selected genes has been validated using real time PCR and in situ hybridizations. Gene ontology and pathway analysis of stage-specific genes revealed that those encoding transcription factors and components of protein folding, sorting and degradation pathway genes dominated in MA, whereas in TPA, those coding for cell structure and signal transduction components were in abundance. Interestingly, about 50% of the genes with anther-specific expression have not been annotated so far.ConclusionsNot only have we provided the transcriptome constituents of four landmark stages of anther development in rice but we have also identified genes that express exclusively in these stages. It is likely that many of these candidates may therefore contribute to specific aspects of anther and/or male gametophyte development in rice. In addition, the gene sets that have been produced will assist the plant reproductive community in building a deeper understanding of underlying regulatory networks and in selecting gene candidates for functional validation.

Can citrate efflux from roots improve phosphorus uptake by plants? Testing the hypothesis with near‐isogenic lines of wheat

Phosphorus (P) deficiency in some plant species triggers the release of organic anions such as citrate and malate from roots. These anions are widely suggested to enhance the availability of phosphate for plant uptake by mobilizing sparingly-soluble forms in the soil. Carazinho is an old wheat (Triticum aestivum) cultivar from Brazil, which secretes citrate constitutively from its root apices, and here we show that it also produces relatively more biomass on soils with low P availability than two recent Australian cultivars that lack citrate efflux. To test whether citrate efflux explains this phenotype, we generated two sets of near-isogenic lines that differ in citrate efflux and compared their biomass production in different soil types and with different P treatments in glasshouse experiments and field trials. Citrate efflux improved relative biomass production in two of six glasshouse trials but only at the lowest P treatments where growth was most severely limited by P availability. Furthermore, citrate efflux provided no consistent advantage for biomass production or yield in multiple field trials. Theoretical modeling indicates that the effectiveness of citrate efflux in mobilizing soil P is greater as the volume of soil into which it diffuses increases. As efflux from these wheat plants is restricted to the root apices, the potential for citrate to mobilize sufficient P to increase shoot biomass may be limited. We conclude that Carazinho has other attributes that contribute to its comparatively good performance in low-P soils.

High-throughput phenotyping technologies allow accurate selection of stay-green

Improved genotypic performance in water-limited environments relies on traits, like ‘stay-green’, that are robust and repeatable, correlate well across a broader range of target environments and are genetically more tractable than assessment of yield per se. Christopher et al. (see pages 5159–5172) used multi-temporal, Normalised Difference Vegetative Index (NDVI) measurements with crop simulation modelling to demonstrate the value of various stay-green phenotype parameters for improving grain yield across different environment types.

Genetic approaches to enhancing phosphorus-use efficiency (PUE) in crops: challenges and directions

Abstract. Many soils have intrinsically low concentrations of available phosphorus (P), which is a major limitation to crop and pasture growth. Regular applications of P have underpinned agricultural productivity internationally, and fertiliser use now constitutes one of the largest variable input costs to farming. Globally, high-quality reserves of P are being depleted and price increases are likely in the future. In addition, the effects of P pollution on water quality are attracting legislative regulation. Hence, there is a need to improve P-use efficiency (PUE) in farming systems. Progress in improving PUE has been limited for several reasons, including: inconsistent definitions of PUE, inappropriate phenotyping, incomplete understanding of the controls of P uptake, lack of field validation, and little consideration of genotype × environment interactions that affect the expression of PUE. With greater consideration of these limitations, the powerful array of molecular and genomic tools currently available promises considerable advances in developing more P-efficient crops. Stronger interaction between molecular science and the traditional disciplines of plant breeding, crop physiology, soil science, and agronomy will allow new opportunities to study genetic differences in PUE, bringing P-efficient crops closer to reality.

Methodology for High-Throughput Field Phenotyping of Canopy Temperature Using Airborne Thermography

Lower canopy temperature (CT), resulting from increased stomatal conductance, has been associated with increased yield in wheat. Historically, CT has been measured with hand-held infrared thermometers. Using the hand-held CT method on large field trials is problematic, mostly because measurements are confounded by temporal weather changes during the time required to measure all plots. The hand-held CT method is laborious and yet the resulting heritability low, thereby reducing confidence in selection in large scale breeding endeavors. We have developed a reliable and scalable crop phenotyping method for assessing CT in large field experiments. The method involves airborne thermography from a manned helicopter using a radiometrically-calibrated thermal camera. Thermal image data is acquired from large experiments in the order of seconds, thereby enabling simultaneous measurement of CT on potentially 1000s of plots. Effects of temporal weather variation when phenotyping large experiments using hand-held infrared thermometers are therefore reduced. The method is designed for cost-effective and large-scale use by the non-technical user and includes custom-developed software for data processing to obtain CT data on a single-plot basis for analysis. Broad-sense heritability was routinely >0.50, and as high as 0.79, for airborne thermography CT measured near anthesis on a wheat experiment comprising 768 plots of size 2 × 6 m. Image analysis based on the frequency distribution of temperature pixels to remove the possible influence of background soil did not improve broad-sense heritability. Total image acquisition and processing time was ca. 25 min and required only one person (excluding the helicopter pilot). The results indicate the potential to phenotype CT on large populations in genetics studies or for selection within a plant breeding program.

Whole genome approaches to identify early meiotic gene candidates in cereals.

Early events during meiotic prophase I underpin not only viability but the variation of a species from generation to generation. Understanding and manipulating processes such as chromosome pairing and recombination are integral for improving plant breeding. This study uses comparative genetics, quantitative trait locus (QTL) analysis and a transcriptomics-based approach to identify genes that might have a role in genome-wide recombination control. Comparative genetics and the analysis of the yeast and Arabidopsis sequenced genomes has allowed the identification of early meiotic candidates that are conserved in wheat, rice and barley. Secondly, scoring recombination frequency as a phenotype for QTL analysis across wheat, rice and barley mapping populations has enabled us to identify genomic regions and candidate genes that could be involved in genome-wide recombination. Transcriptome data for candidate genes indicate that they are expressed in meiotic tissues. Candidates identified included a non-annotated expressed protein, a DNA topoisomerase 2-like candidate, RecG, RuvB and RAD54 homologues.

Responses to phosphorus among wheat genotypes

Abstract. Phosphorus (P) recovery and P-use efficiency (PUE) by wheat are low, and genetic improvement in PUE is a potential means of improving the effectiveness of P in farming systems. We examined variation in response to P in wheat to identify genotypes that showed consistent responsiveness to P fertiliser in the field and which may be the target of future studies, and examined differences in P uptake and partitioning. The response to P was studied among a diverse set of bread wheat germplasm at three sites in South Australia between 2009 and 2012. Up to 53 varieties and breeding lines were grown at two rates of P, 0 kg/ha and 30 kg/ha. Grain yield at 0 kg P/ha and response to P varied independently among genotypes. There were large effects of site and season on the response to P, but some genotypes showed consistently low and others high response to P. Analysis of a subset of lines revealed large responses in vegetative growth to P but the response diminished as crops matured, and variation in early vegetative growth was unrelated to the responses in biomass at maturity or grain yield. Genotypic variation in grain yield was more strongly related to variation in P utilisation efficiency than to variation in P uptake among wheat genotypes, which was associated with differences in P harvest index (PHI). Although breeding has improved yield, there has been no significant genetic gain in total P uptake; rather, improvements in PUE have been associated with an increase in P utilisation efficiency and PHI.

Genetic Mapping in the Triticeae

Genetic maps are the fundamental tools to identify features of phenotypes that are linked to specific genetic loci and eventually DNA sequences or genes. The major use of genetic linkage maps has, therefore, been to identify quantitative trait loci (QTL). Genetic maps are also essential for marker-assisted selection, comparative mapping, high-resolution mapping and map-based cloning. To date, over 40 maps with at least 300 markers have been published for different Triticeae populations. The quality of genetic maps can be affected by a number of factors and map curation ensures that map quality issues are identified and, where possible, resolved. We report on the issues involved in the production of quality genetic linkage maps by inspection of marker genotype data after map construction.

High Throughput Determination of Plant Height, Ground Cover, and Above-Ground Biomass in Wheat with LiDAR

Crop improvement efforts are targeting increased above-ground biomass and radiation-use efficiency as drivers for greater yield. Early ground cover and canopy height contribute to biomass production, but manual measurements of these traits, and in particular above-ground biomass, are slow and labor-intensive, more so when made at multiple developmental stages. These constraints limit the ability to capture these data in a temporal fashion, hampering insights that could be gained from multi-dimensional data. Here we demonstrate the capacity of Light Detection and Ranging (LiDAR), mounted on a lightweight, mobile, ground-based platform, for rapid multi-temporal and non-destructive estimation of canopy height, ground cover and above-ground biomass. Field validation of LiDAR measurements is presented. For canopy height, strong relationships with LiDAR (r2 of 0.99 and root mean square error of 0.017 m) were obtained. Ground cover was estimated from LiDAR using two methodologies: red reflectance image and canopy height. In contrast to NDVI, LiDAR was not affected by saturation at high ground cover, and the comparison of both LiDAR methodologies showed strong association (r2 = 0.92 and slope = 1.02) at ground cover above 0.8. For above-ground biomass, a dedicated field experiment was performed with destructive biomass sampled eight times across different developmental stages. Two methodologies are presented for the estimation of biomass from LiDAR: 3D voxel index (3DVI) and 3D profile index (3DPI). The parameters involved in the calculation of 3DVI and 3DPI were optimized for each sample event from tillering to maturity, as well as generalized for any developmental stage. Individual sample point predictions were strong while predictions across all eight sample events, provided the strongest association with biomass (r2 = 0.93 and r2 = 0.92) for 3DPI and 3DVI, respectively. Given these results, we believe that application of this system will provide new opportunities to deliver improved genotypes and agronomic interventions via more efficient and reliable phenotyping of these important traits in large experiments.

Responses to phosphorus among barley genotypes

Abstract. Genetic improvement in phosphorus (P) use efficiency (i.e. the ratio of biomass or yield at nil P to that at a given rate of application) is an important goal to improve P recovery and P efficiency of farming systems. Experiments were conducted at three sites in South Australia between 2009 and 2011 to characterise genetic variation in yield with no applied P and in the response to P fertiliser among a diverse range of barley (Hordeum vulgare L.) genotypes. In each experiment, 39–54 genotypes were grown at 0 or 30 kg P/ha. Responses to P were measured near the beginning of stem elongation by using normalised difference vegetation index (NDVI) and by harvesting the grain. Rhizosheath size was also measured on seedlings. Consistent differences in growth and yield at 0 kg P/ha were measured among the genotypes. By contrast, there were large environmental effects on responses to P, but some genotypes showed consistent responses. Measurements of growth, yield and P uptake on a subset of genotypes showed that most of the variation in biomass and yield could be attributed to variation in P-uptake efficiency (net total P uptake per unit available P) rather than to P-utilisation efficiency (biomass or yield per unit total P uptake). The size of the rhizosheath made a small contribution to variation in NDVI but not grain yield, suggesting that rhizosheath size may be of some benefit to early growth but that this does not persist through to yield. Genetic correlations between NDVI and yield were often weak but were generally positive at 0 kg P/ha. Correlations between responses in NDVI and responses in grain yield were low and often negative. The study identified several barley genotypes that showed consistent differences in yield at low P and responses to P; however, selection for P efficiency based solely on responses in vegetative growth may not be appropriate. Variation in P uptake appeared to be more important than P-utilisation efficiency for P efficiency in barley.