Association mapping and genetic dissection of drought-induced canopy temperature differences in rice.

Drought-stressed plants display reduced stomatal conductance, which results in increased leaf temperature by limiting transpiration. In this study, thermal imaging was used to quantify the differences in canopy temperature under drought in a rice diversity panel consisting of 293 indica accessions. The population was grown under paddy field conditions and drought stress was imposed for 2 weeks at flowering. The canopy temperature of the accessions during stress negatively correlated with grain yield (r= -0.48) and positively with plant height (r=0.56). Temperature values were used to perform a genome-wide association (GWA) analysis using a 45K single nucleotide polynmorphism (SNP) map. A quantitative trait locus (QTL) for canopy temperature under drought was detected on chromosome 3 and fine-mapped using a high-density imputed SNP map. The candidate genes underlying the QTL point towards differences in the regulation of guard cell solute intake for stomatal opening as the possible source of temperature variation. Genetic variation for the significant markers of the QTL was present only within the tall, low-yielding landraces adapted to drought-prone environments. The absence of variation in the shorter genotypes, which showed lower leaf temperature and higher grain yield, suggests that breeding for high grain yield in rice under paddy conditions has reduced genetic variation for stomatal response under drought.

Varietal variation and chromosome behaviour during meiosis in Solanum tuberosum.

Naturally occurring autopolyploid species, such as the autotetraploid potato Solanum tuberosum, face a variety of challenges during meiosis. These include proper pairing, recombination and correct segregation of multiple homologous chromosomes, which can form complex multivalent configurations at metaphase I, and in turn alter allelic segregation ratios through double reduction. Here, we present a reference map of meiotic stages in diploid and tetraploid S. tuberosum using fluorescence in situ hybridisation (FISH) to differentiate individual meiotic chromosomes 1 and 2. A diploid-like behaviour at metaphase I involving bivalent configurations was predominant in all three tetraploid varieties. The crossover frequency per bivalent was significantly reduced in the tetraploids compared with a diploid variety, which likely indicates meiotic adaptation to the autotetraploid state. Nevertheless, bivalents were accompanied by a substantial frequency of multivalents, which varied by variety and by chromosome (7-48%). We identified possible sites of synaptic partner switching, leading to multivalent formation, and found potential defects in the polymerisation and/or maintenance of the synaptonemal complex in tetraploids. These findings demonstrate the rise of S. tuberosum as a model for autotetraploid meiotic recombination research and highlight constraints on meiotic chromosome configurations and chiasma frequencies as an important feature of an evolved autotetraploid meiosis.

Construction of a dense SNP map of a highly heterozygous diploid potato population and QTL analysis of tuber shape and eye depth.

KEY MESSAGE: Generation of a dense SNP-based linkage map of a diploid potato population and identification of major QTLs for tuber shape and eye depth on chromosomes 2 and 10. This paper reports the construction of a genetic map of a highly heterozygous full-sib diploid potato population (06H1) based on the use of a set of 8,303 single nucleotide polymorphism (SNP) markers. The map contains 1,355 distinct loci and 2,157 SNPs, 802 of which co-segregate with other markers. We find high levels of collinearity between the 12 chromosomal maps with a recently improved version of the potato genome assembly, with the expected genetic clustering in centromeric regions. The linkage maps are used in combination with highly detailed phenotypic assessments conducted over two growing seasons to perform quantitative trait loci analysis of two important potato traits, tuber shape and eye depth. The major loci segregating for tuber shape in 06H1 map to loci on chromosomes 2 and 10, with smaller effects mapping to three other chromosomes. A major locus for tuber eye depth co-locates with the tuber shape locus on chromosome 10. To assess when tuber shape is established in the developing tuber, we have performed staged observations of tuber formation. Our observations suggest that tuber shape is determined very early in tuber development.

Optical Imaging Resources for Crop Phenotyping and Stress Detection.

With a rapidly increasing population, diminishing resource availability, and variation in environment, there is a need to change agricultural production to deliver long-term food security. To deliver such change, we need crops that are productive and tolerant to different stress factors. The traditional methods of obtaining data for phenotyping under field conditions, e.g., for morphological traits such as canopy structure or physiological traits such as plant stress-related traits, are laborious and time-consuming. A variety of imaging tools in the visible, spectral, and thermal infrared ranges allow data collection for quantitative studies of complex traits and crop monitoring. These tools can be used on crop phenotyping and monitoring platforms for high-throughput assessment of traits in order to better understand plant stress responses and the physiological pathways underlying yield. The applications and brief review of these imaging techniques are described and discussed in this chapter.

Transcriptome profiling shows a rapid variety-specific response in two Andigenum potato varieties under drought stress.

Potato is a drought-sensitive crop whose global sustainable production is threatened by alterations in water availability. Whilst ancestral Solanum tuberosum Andigenum landraces retain wild drought tolerance mechanisms, their molecular bases remain poorly understood. In this study, an aeroponic growth system was established to investigate stress responses in leaf and root of two Andigenum varieties with contrasting drought tolerance. Comparative transcriptome analysis revealed widespread differences in the response of the two varieties at early and late time points of exposure to drought stress and in the recovery after rewatering. Major differences in the response of the two varieties occurred at the early time point, suggesting the speed of response is crucial. In the leaves and roots of the tolerant variety, we observed rapid upregulation of ABA-related genes, which did not occur until later in the susceptible variety and indicated not only more effective ABA synthesis and mobilization, but more effective feedback regulation to limit detrimental effects of too much ABA. Roots of both varieties showed differential expression of genes involved in cell wall reinforcement and remodeling to maintain cell wall strength, hydration and growth under drought stress, including genes involved in lignification and wall expansion, though the response was stronger in the tolerant variety. Such changes in leaf and root may help to limit water losses in the tolerant variety, while limiting the reduction in photosynthetic rate. These findings provide insights into molecular bases of drought tolerance mechanisms and pave the way for their reintroduction into modern cultivars with improved resistance to drought stress and yield stability under drought conditions.

Development and Validation of Methodology for Estimating Potato Canopy Structure for Field Crop Phenotyping and Improved Breeding.

Traditional phenotyping techniques have long been a bottleneck in breeding programs and genotype- phenotype association studies in potato, as these methods are labor-intensive and time consuming. In addition, depending on the trait measured and metric adopted, they suffer from varying degrees of user bias and inaccuracy, and hence these challenges have effectively prevented the execution of large-scale population-based field studies. This is true not only for commercial traits (e.g., yield, tuber size, and shape), but also for traits strongly associated with plant performance (e.g., canopy development, canopy architecture, and growth rates). This study demonstrates how the use of point cloud data obtained from low-cost UAV imaging can be used to create 3D surface models of the plant canopy, from which detailed and accurate data on plant height and its distribution, canopy ground cover and canopy volume can be obtained over the growing season. Comparison of the canopy datasets at different temporal points enabled the identification of distinct patterns of canopy development, including different patterns of growth, plant lodging, maturity and senescence. Three varieties are presented as exemplars. Variety Nadine presented the growth pattern of an early maturing variety, showing rapid initial growth followed by rapid onset of senescence and plant death. Varieties Bonnie and Bounty presented the pattern of intermediate to late maturing varieties, with Bonnie also showing early canopy lodging. The methodological approach used in this study may alleviate one of the current bottlenecks in the study of plant development, paving the way for an expansion in the scale of future genotype-phenotype association studies.

Ex vivo magnetic resonance imaging of crystalline lens dimensions in chicken.

PURPOSE: A reduction in the power of the crystalline lens during childhood is thought to be important in the emmetropization of the maturing eye. However, in humans and model organisms, little is known about the factors that determine the dimensions of the crystalline lens and in particular whether these different parameters (axial thickness, surface curvatures, equatorial diameter, and volume) are under a common source of control or regulated independently of other aspects of eye size and shape. METHODS: Using chickens from a broiler-layer experimental cross as a model system, three-dimensional magnetic resonance imaging (MRI) scans were obtained at 115-microm isotropic resolution for one eye of 501 individuals aged 3-weeks old. After fixation with paraformaldehyde, the excised eyes were scanned overnight (16 h) in groups of 16 arranged in a 2x2x4 array. Lens dimensions were calculated from each image by fitting a three-dimensional mesh model to the lens, using the semi-automated analysis program mri3dX. The lens dimensions were compared to measures of eye and body size obtained in vivo using techniques that included keratometry and A-scan ultrasonography. RESULTS: A striking finding was that axial lens thickness measured using ex vivo MRI was only weakly correlated with lens thickness measured in vivo by ultrasonography (r=0.19, p<0.001). In addition, the MRI lens thickness estimates had a lower mean value and much higher variance. Indeed, about one-third of crystalline lenses showed a kidney-shaped appearance instead of the typical biconvex shape. Since repeat MRI scans of the same eye showed a high degree of reproducibility for the scanning and mri3dX analysis steps (the correlation in repeat lens thickness measurements was r=0.95, p<0.001) and a recent report has shown that paraformaldehyde fixation induces a loss of water from the human crystalline lens, it is likely that the tissue fixation step caused a variable degree of shrinkage and a change in shape to the lenses examined here. Despite this serious source of imprecision, we found significant correlations between lens volume and eye/body size (p<0.001) and between lens equatorial diameter and eye/body size (p<0.001) in these chickens. CONCLUSIONS: Our results suggest that certain aspects of lens size (specifically, lens volume and equatorial diameter) are controlled by factors that also regulate the size of the eye and body (presumably, predominantly genetic factors). However, since it has been shown previously that axial lens thickness is regulated almost independently of eye and body size, these results suggest that different systems might operate to control lens volume/diameter and lens thickness in normal chickens.

Season of birth, daylight hours at birth, and high myopia.

PURPOSE: Mandel et al recently reported that season of birth and daylight hours (photoperiod) at birth were associated with moderate and high levels of myopia in Israeli conscripts. We sought to investigate whether these associations were evident in subjects from the United Kingdom (UK). DESIGN: Retrospective cross-sectional study. PARTICIPANTS: The study population comprised 74,459 subjects aged 18 to 100 years attending UK optometry practices for an eye examination. METHODS: Data comprising non-cycloplegic spectacle prescription, sex, date of birth, and date of eye examination were collected from UK optometry practices. The average refractive error in fellow eyes was used to classify the degree of myopia in diopters (D) for each subject as follows: absent (>-0.75 D), low (-0.75 to -2.99 D), moderate (-3.00 to -5.99 D), or high (<-6.00 D). The average monthly hours of daylight for London, UK, were classified into 1 of 4 "photoperiod categories," following Mandel et al. The odds ratio (OR) for each level of severity of myopia was calculated using multivariate logistic regression with age, sex, and either season of birth or photoperiod category as risk factors. MAIN OUTCOME MEASURES: The OR for season of birth and photoperiod category as potential risk factors for myopia. RESULTS: Season of birth was significantly associated with the presence of high myopia: Subjects born in summer or autumn were more likely to be highly myopic compared with those born in winter (summer OR=1.17; 95% confidence interval [CI], 1.05-1.30; P=0.006; autumn OR=1.16; 95% CI, 1.04-1.30; P=0.007). However, season of birth was not a significant risk factor for low or moderate myopia. Photoperiod category was weakly associated with low myopia (OR=0.94; 95% CI, 0.89-0.99; P=0.019), but with a direction of effect opposite to that observed by Mandel et al. CONCLUSIONS: As in Israel, a disproportionate number of UK high myopes were born in summer or autumn rather than in winter. However, unlike the situation in Israel, this association does not seem to be related to daylight hours during the postnatal period, implicating alternative physiologic influences that vary with season, such as birth weight.

Common determinants of body size and eye size in chickens from an advanced intercross line.

Myopia development is characterised by an increased axial eye length. Therefore, identifying factors that influence eye size may provide new insights into the aetiology of myopia. In humans, axial length is positively correlated to height and weight, and in mice, eye weight is positively correlated with body weight. The purpose of this study was to examine the relationship between eye size and body size in chickens from a genetic cross in which alleles with major effects on eye and body size were segregating. Chickens from a cross between a layer line (small body size and eye size) and a broiler line (large body and eye size) were interbred for 10 generations so that alleles for eye and body size would have the chance to segregate independently. At 3 weeks of age, 510 chicks were assessed using in vivo high resolution A-scan ultrasonography and keratometry. Equatorial eye diameter and eye weight were measured after enucleation. The variations in eye size parameters that could be explained by body weight (BW), body length (BL), head width (HW) and sex were examined using multiple linear regression. It was found that BW, BL and HW and sex together predicted 51-56% of the variation in eye weight, axial length, corneal radius, and equatorial eye diameter. By contrast, the same variables predicted only 22% of the variation in lens thickness. After adjusting for sex, the three body size parameters predicted 45-49% of the variation in eye weight, axial length, corneal radius, and eye diameter, but only 0.4% of the variation in lens thickness. In conclusion, about half of the variation in eye size in the chickens of this broiler-layer advanced intercross line is likely to be determined by pleiotropic genes that also influence body size. Thus, mapping the quantitative trait loci (QTL) that determine body size may be useful in understanding the genetic determination of eye size (a logical inference of this result is that the 20 or more genetic variants that have recently been shown to influence human height may also be found to influence axial eye length). Furthermore, adjusting for body size will be essential in mapping pure eye size QTL in this chicken population, and may also have value in mapping eye size QTL in humans.

3-Dimensional modelling of chick embryo eye development and growth using high resolution magnetic resonance imaging.

Magnetic resonance imaging (MRI) is a powerful tool for generating 3-dimensional structural and functional image data. MRI has already proven valuable in creating atlases of mouse and quail development. Here, we have exploited high resolution MRI to determine the parameters necessary to acquire images of the chick embryo eye. Using a 9.4 Tesla (400 MHz) high field ultra-shielded and refrigerated magnet (Bruker), MRI was carried out on paraformaldehyde-fixed chick embryos or heads at E4, E6, E8, and E10. Image data were processed using established and custom packages (MRICro, ImageJ, ParaVision, Bruker and mri3dX). Voxel dimensions ranged from 62.5 microm to 117.2 microm. We subsequently used the images obtained from the MRI data in order to make precise measurements of chick embryo eye surface area, volume and axial length from E4 to E10. MRI was validated for accurate sizing of ocular tissue features by direct comparison with previously published literature. Furthermore, we demonstrate the utility of high resolution MRI for making accurate measurements of morphological changes due to experimental manipulation of chick eye development, thereby facilitating a better understanding of the effects on chick embryo eye development and growth of such manipulations. Chondroitin sulphate or heparin were microinjected into the vitreous cavity of the right eyes of each of 3 embryos at E5. At E10, embryos were fixed and various eye parameters (volume, surface area, axial length and equatorial diameter) were determined using MRI and normalised with respect to the un-injected left eyes. Statistically significant alterations in eye volume (p < 0.05; increases with chondroitin sulphate and decreases with heparin) and changes in vitreous homogeneity were observed in embryos following microinjection of glycosaminoglycans. Furthermore, in the heparin-injected eyes, significant disturbances at the vitreo-retinal boundary were observed as well as retinal folding and detachment confirming histological observations. These data reveal the utility and superiority of MRI for producing images enabling quantification of experimentally induced changes in eye volume and structure. The results indicate that MRI is an important tool that could become a routine approach for rapid and sensitive phenotypic analysis of normal chick ocular development and morphology as well as potentially the effects of surgical or genetic manipulations of chick embryo eyes in live embryos in ovo.

A method for automatic segmentation and splitting of hyperspectral images of raspberry plants collected in field conditions.

Hyperspectral imaging is a technology that can be used to monitor plant responses to stress. Hyperspectral images have a full spectrum for each pixel in the image, 400-2500 nm in this case, giving detailed information about the spectral reflectance of the plant. Although this technology has been used in laboratory-based controlled lighting conditions for early detection of plant disease, the transfer of such technology to imaging plants in field conditions presents a number of challenges. These include problems caused by varying light levels and difficulties of separating the target plant from its background. Here we present an automated method that has been developed to segment raspberry plants from the background using a selected spectral ratio combined with edge detection. Graph theory was used to minimise a cost function to detect the continuous boundary between uninteresting plants and the area of interest. The method includes automatic detection of a known reflectance tile which was kept constantly within the field of view for all image scans. A method to split images containing rows of multiple raspberry plants into individual plants was also developed. Validation was carried out by comparison of plant height and density measurements with manually scored values. A reasonable correlation was found between these manual scores and measurements taken from the images (r2 = 0.75 for plant height). These preliminary steps are an essential requirement before detailed spectral analysis of the plants can be achieved.

Assessing Drought Responses Using Thermal Infrared Imaging.

Canopy temperature, a surrogate for stomatal conductance, is shown to be a good indicator of plant water status and a potential tool for phenotyping and irrigation scheduling. Measurement of stomatal conductance and leaf temperature has traditionally been done by using porometers or gas exchange analyzers and fine-wire thermocouples attached to the leaves, which are labor intensive and point measurements. The advent of remote or proximal thermal sensing technologies has provided the potential for scaling up to leaves, plants, and canopies. Thermal cameras with a temperature resolution of <0.1 K now allow one to study the temperature variation within and between plants. This chapter discusses some applications of infrared thermography for assessing drought and other abiotic and biotic stress and outlines some of the main factors that need to be considered when applying this to the study of leaf or canopy temperature whether in controlled environments or in the field.

Coping with drought: stress and adaptive responses in potato and perspectives for improvement.

Potato (Solanum tuberosum L.) is often considered as a drought sensitive crop and its sustainable production is threatened due to frequent drought episodes. There has been much research aiming to understand the physiological, biochemical, and genetic basis of drought tolerance in potato as a basis for improving production under drought conditions. The complex phenotypic response of potato plants to drought is conditioned by the interactive effects of the plant's genotypic potential, developmental stage, and environment. Effective crop improvement for drought tolerance will require the pyramiding of many disparate characters, with different combinations being appropriate for different growing environments. An understanding of the interaction between below ground water uptake by the roots and above ground water loss from the shoot system is essential. The development of high throughput precision phenotyping platforms is providing an exciting new tool for precision screening, which, with the incorporation of innovative screening strategies, can aid the selection and pyramiding of drought-related genes appropriate for specific environments. Outcomes from genomics, proteomics, metabolomics, and bioengineering advances will undoubtedly compliment conventional breeding strategies and presents an alternative route toward development of drought tolerant potatoes. This review presents an overview of past research activity, highlighting recent advances with examples from other crops and suggesting future research directions.

Infra-red thermography for high throughput field phenotyping in Solanum tuberosum.

The rapid development of genomic technology has made high throughput genotyping widely accessible but the associated high throughput phenotyping is now the major limiting factor in genetic analysis of traits. This paper evaluates the use of thermal imaging for the high throughput field phenotyping of Solanum tuberosum for differences in stomatal behaviour. A large multi-replicated trial of a potato mapping population was used to investigate the consistency in genotypic rankings across different trials and across measurements made at different times of day and on different days. The results confirmed a high degree of consistency between the genotypic rankings based on relative canopy temperature on different occasions. Genotype discrimination was enhanced both through normalising data by expressing genotype temperatures as differences from image means and through the enhanced replication obtained by using overlapping images. A Monte Carlo simulation approach was used to confirm the magnitude of genotypic differences that it is possible to discriminate. The results showed a clear negative association between canopy temperature and final tuber yield for this population, when grown under ample moisture supply. We have therefore established infrared thermography as an easy, rapid and non-destructive screening method for evaluating large population trials for genetic analysis. We also envisage this approach as having great potential for evaluating plant response to stress under field conditions.

Heritability of ocular component dimensions in chickens: genetic variants controlling susceptibility to experimentally induced myopia and pretreatment eye size are distinct.

Purpose. To investigate the extent to which shared genetic variants control (1) multiple ocular component dimensions and (2) both normal eye length and susceptibility to visually induced myopic eye growth. Methods. Two laboratory-reared populations of chicks were examined. The first was a three-generation pedigree of White Leghorn (WL) birds used in a selective breeding experiment testing susceptibility to monocular deprivation of sharp vision (DSV). The chicks were assessed before (age, 4 days) and after 4 days of treatment with diffusers. The second was the 10th generation of an advanced intercross line (AIL) derived from a broiler-layer cross (age, 3 weeks). Variance components analysis was used to estimate heritability and to assess the evidence for shared genetic determination. Results. All measured ocular components were moderately or highly heritable (range, 0.36-0.61; all P < 0.001) in both chick populations, and there were strong genetic correlations across the traits, corneal curvature, vitreous chamber depth, and axial length. The genetic correlations between eye size and myopia susceptibility traits were not significantly different from 0. Conclusions. The genetic variants controlling ocular component dimensions in chicks are shared across some ocular traits (corneal curvature, vitreous chamber depth, and axial length) but distinct for others (lens thickness and corneal thickness). The genetic variants controlling susceptibility to visually induced myopia in chicks are different from those controlling normal eye size.

Selective breeding for susceptibility to myopia reveals a gene-environment interaction.

Purpose. To test whether the interanimal variability in susceptibility to visually induced myopia is genetically determined. Methods. Monocular deprivation of sharp vision (DSV) was induced in outbred White Leghorn chicks aged 4 days. After 4 days' DSV, myopia susceptibility was quantified by the relative changes in axial length and refraction. Chicks in the extreme tails of the distribution of susceptibility to DSV were kept and paired for breeding (high- and low-susceptibility lines). A second round of selection was then performed. The third generation of chicks, derived from the selected parents, was assessed after either monocular DSV (4 or 10 days) or lens wear. Results. After two rounds of selective breeding, the chicks from the high-susceptibility line developed approximately twice as much myopia in response to 4 days' DSV as did those from the low-susceptibility line (P < 0.001). All ocular component dimensions differed significantly (P < 0.001) between the two selected lines, both before treatment and in the responses of the treated eye. When DSV was conducted for 10 days, the relative changes in axial length and refractive error were still significantly different between the high and low lines (P < 0.001). The chicks bred for high or low susceptibility to DSV also showed significantly different responses to minus lens wear, but not to plus lens wear. Additive genetic effects explained ∼50% of the interanimal variability in response to DSV. Conclusions. Genes and environment interact to shape refractive development in chicks.

Sex, eye size, and the rate of myopic eye growth due to form deprivation in outbred white leghorn chickens.

PURPOSE: There is considerable variation in the degree of form-deprivation myopia (FDM) induced in chickens by a uniform treatment regimen. Sex and pretreatment eye size have been found to be predictive of the rate of FD-induced eye growth. Therefore, this study was undertaken to test whether the greater rate of myopic eye growth in males is a consequence of their larger eyes or of some other aspect of their sex. METHODS: Monocular FDM was induced in 4-day-old White Leghorn chicks for 4 days. Changes in ocular component dimensions and refractive error were assessed by A-scan ultrasonography and retinoscopy, respectively. Sex identification of chicks was performed by DNA test. Relationships between traits were assessed by multiple regression. RESULTS: FD produced (mean +/- SD) 13.47 +/- 3.12 D of myopia and 0.47 +/- 0.14 mm of vitreous chamber elongation. The level of induced myopia was not significantly different between the sexes, but the males had larger eyes initially and showed greater myopic eye growth than did the females. In multiple linear regression analysis, the partial correlation between sex and the degree of induced eye growth remained significant (P = 0.008) after adjustment for eye size, whereas the partial correlation between initial eye size and the degree of induced eye growth was no longer significant after adjustment for sex (P = 0.11). After adjustment for other factors, the chicks' sex accounted for 6.4% of the variation in FD-induced vitreous chamber elongation. CONCLUSIONS: The sex of the chick influences the rate of experimentally induced myopic eye growth, independent of its effects on eye size.

Axes of astigmatism in fellow eyes show mirror rather than direct symmetry.

PURPOSE: Most astigmats have a similar level of astigmatism in each eye. However, there is controversy over whether the astigmatic axes in fellow eyes typically show direct or mirror symmetry. We carried out a statistical analysis designed to address this issue. METHODS: The median absolute difference in the astigmatic axes of fellow eyes was calculated for a sample of 50 995 astigmats (subjects with at least 0.25 D of astigmatism in each eye). This was done, firstly, for a 'direct symmetry model' in which the difference in axis was calculated as |AxisR - AxisL| and secondly, for a 'mirror symmetry model' in which the difference in axis was calculated as |AxisR - (180 - AxisL)|. RESULTS: Under the direct symmetry model, the median absolute difference in the axis of astigmatism between fellow eyes was 20 degrees. Under the mirror symmetry model, the median absolute difference in the axis of astigmatism between fellow eyes was significantly lower, at 10 degrees (p < 10e-100). Comparable results were found when the analysis was restricted to subjects with: lower levels of astigmatism (< or =1.00 D), higher levels of astigmatism (>1.00 D), against-the-rule astigmatism, with-the-rule astigmatism or oblique astigmatism (all p < 10e-100). CONCLUSION: Our results show that mirror, rather than direct, symmetry is the norm.