To interrogate shape and size variation over populations large enough to reveal associations with the underlying genetics requires measurement techniques that are scalable. Imaging techniques are readily scalable and have been widely used to measure growth rate , leaf number and size , leaf hyponasty and hypocotyl angle , and can be used to estimate morphological parameters. Image analysis can quantify size and shape variation due to defined genetic lesions in rosette plants and used to identify QTL for variation in rosette area, revealing a number of candidate genes for growth and size traits [3, 4, 62, 63, 65–67].
More recently, a multi-scale approach was used with the purpose of linking genes to plant shape at several scales, from the whole plant to cells and tissues attending to quantitative measurements extracted from digital images and models . It can be argued that research on plant morphology and gene mapping would benefit advanced automatic phenotyping methods scalable to large populations . For that reason, it is important to find and describe measurements that ensure an accurate capture of morphological variability with biological meaning. Principal component analysis generated an uncorrelated morphospace for variation in rosette morphology.
The first two principal components explained 75% of shape variation (51% PC1 and 24% PC2) in the RIL population. PC1 was a combination mainly of size components related to rosette age and RND and ECC components. The second PC represents just shape components indicating that compactness is independent of size and correlating with RCH and RND, while these two did not correlate among them. These 2 PCs indicated that younger rosettes were quite eccentric and when they grew older they either got rounder or elongated in one direction. PC 1 is composed of PRA, PL, RCH and SOL as positive loadings and RMS and ECC as negative ones.
PC2 is positively influenced by Compactness, RND, ISO and RCH, capturing the circularity of the rosette. PC3 (12% variance) is negatively influenced by Compactness, ECC and RMS and SOL, which are related with the asymmetry and the presence of inter-leaf gaps. PC5 (4% of variance), positively weighted by SOL, RCH and RMS and negatively by PRA, accounts for small leaves spread out in a circular fashion. PC6 (2% of variance) is dominated positively by RMS and less by PRA and negatively by SOL, phenotyping large rosettes with gaps between leaves but leaf blade overlapping. PC7 (1% of variance) oppose Compactness and RND accounting for dense filled-in rosettes.
PC8 and PC9 account for less that 1% of variance but PC9 identifies large rosettes with small perimeter, a sign of round shape. This study focuses on the global shape of an organ assemblage, the A. Rosette leaf shape varies along the ontogenetic development according to their local environment , e.g., in a single plant some leaves are longer than others, usually towards light sources, with crenate, undulate or entire borders.
Yet, rosette appearance is distinguishable among ecotypes and similar within individuals of the same ecotype, especially when grown in homogenous conditions . Thus, rosette shape variation is visible and genetically controlled within some range as predicted by the 'continuum and process morphology' [140–142]. Our results on shape descriptors heritability are consistent with these observations and build up on the concept that morphological traits act as functional ones . The QTLs found here agree with the theory that finely tuned genetic regulatory networks, linking and integrating environmental clues during ontogenetic development, are among the major contributions to plant local adaptations [144–147].
In this sense, our study introduces the automated plant phenomics as a relevant tool for the so called eco-evo-devo with particular emphasis on morphology at a subspecies taxa level . From an applied biology perspective, the QTLs reported may be useful for further research either in their role on phenotypic regulation or the type of genetic variants they bear. Here, we report machine-assisted acquisition of time-stamped images from MAGIC RILs during their rosette development and we extract a range of morphology descriptors for rosette size and shape variation.
Calculate The Area Of A Circle Whose Circumference Is 44Cm We continue the work of and extend their approach to quantify and explore the underlying genetic basis of size and shape variation by a QTL mapping approach. Combined phenomics and genomics analyses identify 116 loci linked to the shape in the early developmental stages of Arabidopsis rosette growth. Advanced mapping populations, like Multiparent Advanced Generation Inter-Cross , trade the advantages and disadvantages of classical mapping populations in terms of resolution and efficiency .
These are crosses of up to 20 genetically and phenotypically diverse parental types and several generations of selfing. The strategy reduces population structure and generates small haplotypic mosaics, providing finer resolution mapping than bi-parental with less false positives than natural populations. MAGIC allows haplotype reconstruction for markers alleles as founder-of-origin [89–91] tracking genetic variation back to parental sequence level . Tailored bayesian models improves parameter estimation and permutation-based procedures reduce False Discovery Rates . Rosettes display limited internode extension and generally are comprised of a spiral of leaves, overlapping to a greater or lesser extent .
This leaf assemblage has recurrently appeared along angiosperm phylogeny , yet its adaptive significance remains unclear. Rosettes probably occupy the space for photosynthesis, excluding nearby plants by establishing ground cover while remaining cryptic to larger herbivores . Leaf distribution and size may respond to environmental factors such as light interception , herbivore grazing , abiotic stress .
Plant development occurs throughout the individual lifetime [108–110] and the production of new organs, i.e. stems, roots, leafs or flowers, is constantly influenced by the environment [111–113]. Ecotypes adapted to local environments often differ in many traits, particularly in the arrangement, size and shape of such organs. Research on phenotypic variation often refers to "morphology", "form" and "shape" by implicit and informal definitions. As a consequence, the concept of shape can be reduced to adjectives like long-short, round-elongated, sparse-dense or into categories without explicit parameterization. Computer vision based shape descriptors are precisely defined, their measurement is objective, repeatable and interpretable as compared to visual human experience .
Understanding the role of environmental conditions on plant development is of increasing importance to counterbalance climate change effects in agricultural species . Natural variation found in nature can reveal features that are subject to selective pressure, including that exerted by the local environment [2–4]. For example, shoot architecture integrates the interaction between genetic determinants, developmental history, the environment and adaptation to particular lifestyles .
The rosette forms during the vegetative phase of many species including several major crops in the Brassicaceae family. The rosette is an attractive model to understand the genetic architecture of variation in plant form thanks to the quantity and quality of genetic and natural resources available in the model species, Arabidopsis . The MAGIC population has been previously characterised for flowering time, height and fitness with a specific advanced statistical method for QTL mapping . This method takes advantage of multiple parents to assign genetic variants as multi-allelic markers rather than bi-allelic.
In summary, MAGIC populations represent the best of both worlds, in the sense of high variation, low structure, high resolution and precision in the statistical methods. As a potential disadvantage the set of 485 RILs, with three replicates per RIL, became 1455 plants to keep under strict environmental control and daily phenotyping. To overcome this difficulty, a mechanised phenomics approach was necessary. Each shape descriptor and principal component were split by DAS and used as input for QTL mapping with the happy.hbrem R package.
This software was specifically designed for multi-parental population analysis and previously used for Arabidopsis MAGIC population . RILs genomes are reconstructed as parental haplotype mosaic working out the Identity By Descent using a Hidden Markov Model . For each phenotype, a genomic scan fits a Hierarchical Bayes random effects model with 19 random factors, corresponding to founder alleles, weighed by the IBD probabilities. A permutation test randomizing phenotypes 500 times, established a genome-wide threshold for statistically significant QTL and corrects for multiple testing according to .
Finally, a QTL location is defined as the peak marker with largest logP-value within an interval where other SNPs pass the genome-wide P-value . A boxplot with allelic phenotypic effect for each founder ecotype was calculated using the hbrem procedure. Principal components analysis (PCA, function prcomp from the package stats ) was calculated to generate an uncorrelated shape space, i.e. to eliminate remaining size-effects and correlations among descriptors. PCA was built with the correlation matrix and all 9 Principal Components were retained so that RILs distances remain constant, given overall mean and variance scaling.
Therefore, this PCA does not reduce dimensionality but constructs an uncorrelated morphospace with common aspects of rosettes grouped in each principal component. PCA was performed on RIL- averaged values rather than individual values. Under the county system certain densely populated municipalities, such as Copenhagen Municipality and Frederiksberg, had been given a status equivalent to that of counties, making them first-level administrative divisions.
These sui generis municipalities were incorporated into the new regions under the 2007 reforms. Cross comparisons across the RILs showed large variation in rosette morphology for the MAGIC RIL population. Fig 2 illustrates an example of six RILs and their values of Projected Rosette Area and Compactness.
S1 Fig includes the whole population and all traits and S1 Data contains averaged values per RIL for all nine shape descriptors and DAS. This relatively new situation has stirred some internal critique, but the Danish population has generally been very supportive, in particular of the War in Afghanistan. The Danish Defence has around 1,400 staff in international missions, not including standing contributions to NATO SNMCMG1.
Danish forces were heavily engaged in the former Yugoslavia in the UN Protection Force , with IFOR, and now SFOR. Between 2003 and 2007, there were approximately 450 Danish soldiers in Iraq. Denmark also strongly supported American operations in Afghanistan and has contributed both monetarily and materially to the ISAF. These initiatives are often described by the authorities as part of a new "active foreign policy" of Denmark. As a member of Development Assistance Committee , Denmark has for a long time been among the countries of the world contributing the largest percentage of gross national income to development aid. In 2015, Denmark contributed 0.85% of its gross national income to foreign aid and was one of only six countries meeting the longstanding UN target of 0.7% of GNI.
The country participates in both bilateral and multilateral aid, with the aid usually administered by the Ministry of Foreign Affairs. The organisational name of Danish International Development Agency is often used, in particular when operating bilateral aid. Three replicates per RIL were phenotyped for the 9 shape descriptors at Table 1 and their RIL group means were recorded for further analysis. Principal Components extracted from the correlation matrix–all DAS and RILs pooled–where calculated from RILs averaged phenotypic values. Colour code shows the trajectory of traits along time where every variable has been code in green for low values, yellow for intermediate and red for large. The color code is calculated for all DAS and RILs and recalculated for every feature.
Top view images were processed using an internal automatic workflow provided by the manufacturer. It performed the tasks of image processing, which calculated size and shape descriptors and stored them on the platform database. A detailed description of all descriptors can be found in and Table 1.
Rosette size was described by Projected Rosette Area and Perimeter Length . Rosette ground coverage comprised Compactness and Rotational Mass Symmetry descriptors. Rosette deviation from a circle was measured with Slenderness of Leaves , Roundness , Convex Hull Roundness , Isotropy and Eccentricity . Shape is a ubiquitous concept in biological research with context dependant morphometrical methodologies . Outline shape descriptors [18–21] define specific and quantitative aspects of form, e.g. roundness .
They can be combined with computer vision for high-throughput phenotyping of plants organs, assemblages, full plants and field plots. These are global descriptors with some degree of overlapping [29–31] and dependency . For example, a cogwheel, a starfish-like shape or Arabidopsis rosettes are visually different but with similar roundness scores and different circularity ones, due to their different definition .
Significant investment has been made in building road and rail links between regions in Denmark, most notably the Great Belt Fixed Link, which connects Zealand and Funen. It is now possible to drive from Frederikshavn in northern Jutland to Copenhagen on eastern Zealand without leaving the motorway. The main railway operator is DSB for passenger services and DB Cargo for freight trains. The North Sea and the Baltic Sea are intertwined by various, international ferry links. Construction of the Fehmarn Belt Fixed Link, connecting Denmark and Germany with a second link, will start in 2015. Copenhagen has a rapid transit system, the Copenhagen Metro, and an extensive electrified suburban railway network, the S-train.
In the four largest cities – Copenhagen, Aarhus, Odense, Aalborg – light rail systems are planned to be in operation around 2020. Land and water pollution are two of Denmark's most significant environmental issues, although much of the country's household and industrial waste is now increasingly filtered and sometimes recycled. The country has historically taken a progressive stance on environmental preservation; in 1971 Denmark established a Ministry of Environment and was the first country in the world to implement an environmental law in 1973. To mitigate environmental degradation and global warming the Danish Government has signed the Climate Change-Kyoto Protocol. However, the national ecological footprint is 8.26 global hectares per person, which is very high compared to a world average of 1.7 in 2010. In December 2014, the Climate Change Performance Index for 2015 placed Denmark at the top of the table, explaining that although emissions are still quite high, the country was able to implement effective climate protection policies.
In 2021 Denmark with Costa Rica launched the "Beyond Oil and Gas alliance" for stopping use fossil fuels. Located in Northern Europe, Denmark consists of the peninsula of Jutland and 443 named islands (1,419 islands above 100 square metres in total). Of these, 74 are inhabited , with the largest being Zealand, the North Jutlandic Island, and Funen. The island of Bornholm is located east of the rest of the country, in the Baltic Sea. Many of the larger islands are connected by bridges; the Øresund Bridge connects Zealand with Sweden; the Great Belt Bridge connects Funen with Zealand; and the Little Belt Bridge connects Jutland with Funen. The four cities with populations over 100,000 are the capital Copenhagen on Zealand; Aarhus and Aalborg in Jutland; and Odense on Funen.
Shape descriptors were correlated, suggesting ecologically related trait syndromes . PRA, PL, and SOL form a cluster capturing information on size and length. ECC and RMS form another cluster describing rosette elongation. RND and ISO describe the pattern of leaf arrangement as in a circle or a star-like shape.
A singleton comprising only RCH captures accurately the closeness of rosettes to a perfect cycle, regardless of the gaps between leaves. Compactness would be another singleton describing rosette coverage. Images were automatically segmented, rosettes were extracted, and analysed by device built-in pipelines. Shape descriptors and other metadata are recorded into a database. Shape descriptors and their PCA-derived morphospace were used for QTL mapping with happy.hbrem followed by gene search at ARAPORT11. High dimensional vectors of descriptors need multivariate statistics to discriminate shapes numerically.
Particularly, Principal Component Analysis build latent variables (or latent shapes ) generating a "morphospace" [40–42], with specific mathematical properties e.g. relative warps and synthetic shape axis [45–47]. PCA-based morphospace cleaves univariate morphological features that can be analysed as individual morphological phenotypes in quantitative trait loci mapping experiments [48–50]. Also, PCA is used to separate size from shape , study allometric growth or when sample size is much smaller than the number of variables measured. Painting has continued to be a prominent form of artistic expression in Danish culture, inspired by and also influencing major international trends in this area. These include impressionism and the modernist styles of expressionism, abstract painting and surrealism. Most Danish painters of modern times have also been very active with other forms of artistic expressions, such as sculpting, ceramics, art installations, activism, film and experimental architecture.
