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26.  From genetic improvement to commercial-scale mass culture of a Chilean strain of the green microalga Haematococcus pluvialis with enhanced productivity of the red ketocarotenoid astaxanthin 
AoB Plants  2013;5:plt026.
There is a great breach between research made at universities and applications of these “academic results” to commercial purposes. This research is a successful example of this interaction. We show that random mutagenesis/selection is an effective strategy for genetically improving strains of the astaxanthin-producing microalga, H. pluvialis and that improved carotenogenic capacity attained is maintained when the volume of the cultures is scaled up to a commercial size. This research allowed to the company dispose of an improved strain accumulating 30% more astaxanthin that the wild type strain (per dry weight basis) and a 72% more (per culture volume basis).
Astaxanthin is a red ketocarotenoid, widely used as a natural red colourant in marine fish aquaculture and poultry and, recently, as an antioxidant supplement for humans and animals. The green microalga Haematococcus pluvialis is one of the richest natural sources of this pigment. However, its slow growth rate and complex life cycle make mass culture difficult for commercial purposes. The aims of this research were (i) to standardize and apply a genetic improvement programme to a Chilean strain of H. pluvialis in order to improve its carotenogenic capacity and (ii) to evaluate the performance of a selected mutant strain in commercial-sized (125 000 L) open ponds in the north of Chile. Haematococcus pluvialis strain 114 was mutated by ethyl methanesulfonate. The level of mutagen dose (exposure time and concentration) was one that induced at least 90 % mortality. Surviving colonies were screened for resistance to the carotenoid biosynthesis inhibitor diphenylamine (25 µM). Resistant mutants were grown in a 30-mL volume for 30 days, after which the total carotenoid content was determined by spectrophotometry. Tens of mutants with improved carotenogenic capacity compared with the wild-type strain were isolated by the application of these standardized protocols. Some mutants exhibited curious morphological features such as spontaneous release of astaxanthin and loss of flagella. One of the mutants was grown outdoors in commercial-sized open ponds of 125 000 L in the north of Chile. Grown under similar conditions, the mutant strain accumulated 30 % more astaxanthin than the wild-type strain on a per dry weight basis and 72 % more on a per culture volume basis. We show that random mutagenesis/selection is an effective strategy for genetically improving strains of H. pluvialis and that improved carotenogenic capacity is maintained when the volume of the cultures is scaled up to a commercial size.
doi:10.1093/aobpla/plt026
PMCID: PMC3687207  PMID: 23789055
Astaxanthin; commercial-sized open ponds; Haematococcus pluvialis mutant; North Chile; random mutagenesis.
27.  In situ morphometric characterization of Aframomum melegueta accessions in Ghana 
AoB Plants  2013;5:plt027.
Misidentification of Aframomum melegueta, an important medicinal plant has huge adverse health implications. Thus, the scientific description and characterization of the accessions in Ghana was imperative. The study found the plant to bear creepy stolons which bear terminal bud for production of tillers instead of rhizomes which had earlier been reported by other investigators. All the accessions had a distinctive non storage bulbous structure at the base of the pseudostem. The UPGMA dendrogram clustered the accession into Ashanti and Eastern accessions based on ecological location. The Eastern accessions were sub-clustered in two groups based on fruit colour.
In spite of the huge economic importance of Aframomum melegueta in the herbal and pharmaceutical industries, its production is limited by lack of planting materials (propagules). The plant also lacks scientific descriptors, which has often led to misidentification with adverse health implications. We therefore aimed at developing a descriptor list to facilitate the identification of A. melegueta using 34 morphometric traits comprising 18 quantitative and 16 qualitative characters. The morphological traits showed that A. melegueta has a characteristic stolon that produces tillers instead of rhizomes. The unweighted pair group method with arithmetic mean using both the nearest-neighbour and complete-linkage methods based on the 34 morphometric traits clustered the eight accessions into two main groups based on ecological location. The accessions from the Eastern and Ashanti regions were separated at similarity coefficients of 0.822 and 0.644, respectively, with a highly significant discriminant function. The Eastern accessions were further clustered into red or yellow fruits at similarity indexes of 0.936 and 0.865 using the nearest-neighbour and complete-linkage methods, respectively. The present study has shown that morphometric traits of A. melegueta are greatly influenced by its ecological habitat. It is envisaged that the descriptor list developed coupled with a morphometric description would enhance its identification and utilization.
doi:10.1093/aobpla/plt027
PMCID: PMC3689184  PMID: 23799183
Characterization; cluster analysis; descriptors; morphometric traits; phenetic cluster; rhizome; stolon.
28.  Conserved versatile master regulators in signalling pathways in response to stress in plants 
AoB Plants  2013;5:plt033.
Environmental conditions have forced plants to develop elaborated molecular strategies to surpass natural obstacles to growth and proliferation. Elements in multiple signaling cascades allow plants to sense multiple and simultaneous ambient cues, and establish an opportune defensive response. A group of versatile master regulators of gene expression are decisive to control plant responses to stressing conditions. For crop breeding purposes, the task is to determine how to activate these key regulators to enable accurate and optimal responses to stressing conditions. In this review, we discuss how and which master regulators are implied in the responses to biotic and stresses, their evolution in the life kingdoms, and the interaction with other molecular factors that lead to a proper and efficient plant response.
From the first land plants to the complex gymnosperms and angiosperms of today, environmental conditions have forced plants to develop molecular strategies to surpass natural obstacles to growth and proliferation, and these genetic gains have been transmitted to the following generations. In this long natural process, novel and elaborate mechanisms have evolved to enable plants to cope with environmental limitations. Elements in many signalling cascades enable plants to sense different, multiple and simultaneous ambient cues. A group of versatile master regulators of gene expression control plant responses to stressing conditions. For crop breeding purposes, the task is to determine how to activate these key regulators to enable accurate and optimal reactions to common stresses. In this review, we discuss how plants sense biotic and abiotic stresses, how and which master regulators are implied in the responses to these stresses, their evolution in the life kingdoms, and the domains in these proteins that interact with other factors to lead to a proper and efficient plant response.
doi:10.1093/aobpla/plt033
PMCID: PMC3800984  PMID: 24147216
Biotic/abiotic stress; co-activators; gene expression regulation; integrators; key regulators; plant stress response.
29.  1-Aminocyclopropane-1-carboxylic acid oxidase reaction mechanism and putative post-translational activities of the ACCO protein 
AoB Plants  2013;5:plt031.
ACC oxidase (Malus. domestica ACCO1) catalyzes the final step in the biosynthesis of the plant hormone ethylene. ACCO converts 1-aminocyclopropane-1-carboxylic acid(ACC) to ethylene, cyanide, carbon dioxide and water in the presence of ferrous ion, oxygen, ascorbic acid and bicarbonate. Cyanide, a product of the reaction, activates ACCO. Site-directed mutagenesis investigations revealed binding sites for ACC, bicarbonate and ascorbic acid to include; Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction through post-translational modifications. ACCO is subject to auto-phosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner.
1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyses the final step in ethylene biosynthesis converting ACC to ethylene, cyanide, CO2, dehydroascorbate and water with inputs of Fe(II), ascorbate, bicarbonate (as activators) and oxygen. Cyanide activates ACCO. A ‘nest’ comprising several positively charged amino acid residues from the C-terminal α-helix 11 along with Lys158 and Arg299 are proposed as binding sites for ascorbate and bicarbonate to coordinately activate the ACCO reaction. The binding sites for ACC, bicarbonate and ascorbic acid for Malus domestica ACCO1 include Arg175, Arg244, Ser246, Lys158, Lys292, Arg299 and Phe300. Glutamate 297, Phe300 and Glu301 in α-helix 11 are also important for the ACCO reaction. Our proposed reaction pathway incorporates cyanide as an ACCO/Fe(II) ligand after reaction turnover. The cyanide ligand is likely displaced upon binding of ACC and ascorbate to provide a binding site for oxygen. We propose that ACCO may be involved in the ethylene signal transduction pathway not directly linked to the ACCO reaction. ACC oxidase has significant homology with Lycopersicon esculentum cysteine protease LeCp, which functions as a protease and as a regulator of 1-aminocyclopropane-1-carboxylic acid synthase (Acs2) gene expression. ACC oxidase may play a similar role in signal transduction after post-translational processing. ACC oxidase becomes inactivated by fragmentation and apparently has intrinsic protease and transpeptidase activity. ACC oxidase contains several amino acid sequence motifs for putative protein–protein interactions, phosphokinases and cysteine protease. ACC oxidase is subject to autophosphorylaton in vitro and promotes phosphorylation of some apple fruit proteins in a ripening-dependent manner.
doi:10.1093/aobpla/plt031
PMCID: PMC3828642  PMID: 24244837
ACC oxidase; ascorbate free radical; ascorbic acid; bicarbonate; cyanide; cysteine protease; phosphorylation; post-translational activities; reaction mechanism; site-directed mutagenesis.
30.  Phenotypic characters of rice landraces reveal independent lineages of short-grain aromatic indica rice 
AoB Plants  2013;5:plt032.
Crop domestication is a remarkable example of evolution of wildly growing plants into cultivable forms through human selection. Following the domestication of rice almost 10,000 years ago, ancient farmers selected many rice lineages for diverse agronomic and cultural traits, like grain size, shape and colour; awn length; pest resistance; and aroma etc. In this study, examining phenotypic traits of a large collection of Indian rice landraces (all accessed from Vrihi, rice seed bank, www.cintdis.org/vrihi) we characterize the huge phenotypic diversity, and find that a few grain, panicle and leaf traits are major drivers of this diversity. We also demonstrate the existence of short grain aromatic landraces perhaps with independently evolved aroma trait; unlike introgression from japonica into indica group, as evidenced in Basmati-type long grain varieties. The independent origin of aroma in indica rice is fascinating as it explores lesser known aspects of indica rice domestication and diversification.
Rice landraces are lineages developed by farmers through artificial selection during the long-term domestication process. Despite huge potential for crop improvement, they are largely understudied in India. Here, we analyse a suite of phenotypic characters from large numbers of Indian landraces comprised of both aromatic and non-aromatic varieties. Our primary aim was to investigate the major determinants of diversity, the strength of segregation among aromatic and non-aromatic landraces as well as that within aromatic landraces. Using principal component analysis, we found that grain length, width and weight, panicle weight and leaf length have the most substantial contribution. Discriminant analysis can effectively distinguish the majority of aromatic from non-aromatic landraces. More interestingly, within aromatic landraces long-grain traditional Basmati and short-grain non-Basmati aromatics remain morphologically well differentiated. The present research emphasizes the general patterns of phenotypic diversity and finds out the most important characters. It also confirms the existence of very unique short-grain aromatic landraces, perhaps carrying signatures of independent origin of an additional aroma quantitative trait locus in the indica group, unlike introgression of specific alleles of the BADH2 gene from the japonica group as in Basmati. We presume that this parallel origin and evolution of aroma in short-grain indica landraces are linked to the long history of rice domestication that involved inheritance of several traits from Oryza nivara, in addition to O. rufipogon. We conclude with a note that the insights from the phenotypic analysis essentially comprise the first part, which will likely be validated with subsequent molecular analysis.
doi:10.1093/aobpla/plt032
PMCID: PMC3828656  PMID: 24244838
Aromatic; Basmati; domestication; grain length; landraces; phenotypic diversity; rice
31.  Vegetative growth and cluster development in Shiraz grapevines subjected to partial root-zone cooling 
AoB Plants  2013;5:plt036.
Grapevine roots can be exposed to a range of temperatures at any particular moment because the root system can explore large volumes of soil over great depths and distances. A split-pot experiment was designed to assess how vegetative and reproductive development respond to partial and whole root-zone warming following winter dormancy. Simultaneous cooling and warming of parts of the root system slowed shoot elongation, leaf expansion and berry development compared to plants with a fully warmed root-zone, but not to the same extent as those with a fully cooled root-zone.
Heterogeneity in root-zone temperature both vertically and horizontally may contribute to the uneven vegetative and reproductive growth often observed across vineyards. An experiment was designed to assess whether the warmed half of a grapevine root zone could compensate for the cooled half in terms of vegetative growth and reproductive development. We divided the root system of potted Shiraz grapevines bilaterally and applied either a cool or a warm treatment to each half from budburst to fruit set. Shoot growth and inflorescence development were monitored over the season. Simultaneous cooling and warming of parts of the root system decreased shoot elongation, leaf emergence and leaf expansion below that of plants with a fully warmed root zone, but not to the same extent as those with a fully cooled root zone. Inflorescence rachis length, flower number and berry number after fertilization were smaller only in those vines exposed to fully cooled root zones. After terminating the treatments, berry enlargement and the onset of veraison were slowed in those vines that had been exposed to complete or partial root-zone cooling. Grapevines exposed to partial root-zone cooling were thus delayed in vegetative and reproductive development, but the inhibition was greater in those plants whose entire root system had been cooled.
doi:10.1093/aobpla/plt036
PMCID: PMC3828663  PMID: 24244839
Berry composition; grapevine; Shiraz; shoot growth; soil temperature; split pot; Vitis vinifera.
32.  Mapping pachytene chromosomes of coffee using a modified protocol for fluorescence in situ hybridization 
AoB Plants  2013;5:plt040.
FISH-mapping to meiotic chromosomes at pachytene is an important tool in plant cytogenetic research as it provides good resolution measurements of physical distances. This publication brings a new and more efficient protocol for the application of FISH technique for the first time in meiotic pachytene chromosomes of coffee.This new protocol involves some procedures for obtain suitable pachytene chromosomes that allows the making of a higher-resolution cytogenetic mapping on coffee chromosomes than that mapping on mitotic chromosomes. The use of this method expands the possibilities for high definition physical mapping of coffee chromosomes.
Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes.
doi:10.1093/aobpla/plt040
PMCID: PMC3828664  PMID: 24244840
Coffea; FISH; meiotic chromosomes; molecular cytogenetics; physical mapping; rDNA.
33.  Drought-induced trans-generational tradeoff between stress tolerance and defence: consequences for range limits? 
AoB Plants  2013;5:plt038.
In the study of geographic range boundary development, the focus has been on leading rather than on trailing edge dynamics. This is an important caveat as trailing edge dynamics will be critical for an understanding population level persistence. Our study begins to fill this knowledge gap and extends the conceptual framework of the field by focusing on trans-generational environmental effects. We found that while these effects may overcome some constraints on stress tolerance evolution and range expansion, other constraints may be created to limit range.
Areas just across species range boundaries are often stressful, but even with ample genetic variation within and among range-margin populations, adaptation towards stress tolerance across range boundaries often does not occur. Adaptive trans-generational plasticity should allow organisms to circumvent these problems for temporary range expansion; however, range boundaries often persist. To investigate this dilemma, we drought stressed a parent generation of Boechera stricta (A.Gray) A. Löve & D. Löve, a perennial wild relative of Arabidopsis, representing genetic variation within and among several low-elevation range margin populations. Boechera stricta is restricted to higher, moister elevations in temperate regions where generalist herbivores are often less common. Previous reports indicate a negative genetic correlation (genetic tradeoff) between chemical defence allocation and abiotic stress tolerance that may prevent the simultaneous evolution of defence and drought tolerance that would be needed for range expansion. In growth chamber experiments, the genetic tradeoff became undetectable among offspring sib-families whose parents had been drought treated, suggesting that the stress-induced trans-generational plasticity may circumvent the genetic tradeoff and thus enable range expansion. However, the trans-generational effects also included a conflict between plastic responses (environmental tradeoff); offspring whose parents were drought treated were more drought tolerant, but had lower levels of glucosinolate toxins that function in defence against generalist herbivores. We suggest that either the genetic or environmental tradeoff between defence allocation and stress tolerance has the potential to contribute to range limit development in upland mustards.
doi:10.1093/aobpla/plt038
PMCID: PMC3849778  PMID: 24307931
Boechera stricta; chemical defence; drought tolerance; epigenetic; glucosinolate; range limit; tradeoff
34.  Contrasting physiological responses to excess heat and irradiance in two tropical savanna sedges 
AoB Plants  2013;5:plt051.
Trinidad's Aripo Savanna is a rare example of an intact tropical grassland. It is a living laboratory in which to explore the mechanisms used by plants to survive the stress of life in the full glare of the equatorial sun. We found that the dominant species, Lagenocarpus rigidus, avoids overheating not through higher transpiration or more reflective leaf surfaces (as expected), but by altering the size and shape of its leaves to suit each location. This plasticity in leaf morphology is combined with plasticity in cell membrane properties, which allows the leaves to tolerate periods of extreme heat. In the absence of these traits a closely related species Lagenocarpus guianensis, finds its range restricted to the shaded savanna edges where heat and light are less overbearing
Tropical hyperseasonal savannas provide a rare example of a tropical climax community dominated by graminoid species. Species living in such savannas are frequently exposed to excess heat and light, in addition to drought and waterlogging, and must possess traits to avoid or tolerate these stress factors. Here we examine the contrasting heat and light stress adaptations of two dominant savanna sedges: Lagenocarpus guianensis, which is restricted to the sheltered forest edge, and Lagenocarpus rigidus, which extends from the forest edge to the open savanna. An ecotone extending from the forest edge to the open savanna was used to assess differences in a range of physiological traits (efficiency of photosystem II, cell membrane thermostability, stomatal conductance, leaf surface reflectance and canopy temperature depression) and a range of leaf functional traits (length : width ratio, specific leaf area and degree of folding). Lagenocarpus guianensis showed significantly less canopy temperature depression than L. rigidus, which may explain why this species was restricted to the forest edge. The range of leaf temperatures measured was within the thermal tolerance of L. guianensis and allowed photosystem II to function normally, at least within the cool forest edge. The ability of L. rigidus to extend into the open savanna was associated with an ability to decouple leaf temperature from ambient temperature combined with enhanced cell membrane thermostability. The high degree of canopy temperature depression seen in L. rigidus was not explained by enhanced stomatal conductance or leaf reflectance, but was consistent with a capacity to increase specific leaf area and reduce leaf length: width ratio in the open savanna. Plasticity in leaf functional traits and in cell membrane thermostability are key factors in the ability of this savanna sedge to survive abiotic stress.
doi:10.1093/aobpla/plt051
PMCID: PMC3875122  PMID: 24379971
Canopy temperature depression; cell membrane thermostability; environmental gradient; heat stress; leaf functional traits; leaf reflectance; light stress; tropical savanna.
35.  An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast India 
AoB Plants  2011;2012:plr032.
Submergence inhibits photosynthesis by terrestrial wetland plants, but less so in species that possess leaf gas films when submerged. Floodwaters are often supersaturated with dissolved CO2 enabling photosynthesis by submerged terrestrial plants, although rates remain well-below those in air. This important adaptation that enhances survival in submerged conditions is reviewed.
Background and aims
Dendrobium hookerianum is a rare and threatened epiphytic orchid of northeast India. Prospects for conservation would be strengthened by developing an in vitro method for mass propagation. Seeds are minute and difficult to use directly in the field for this purpose, being non-endospermous with a low nutrient content and dependent on a specific fungus for germination and early seedling development. Although produced in large numbers (2–3 million per capsule), <5 % germinate naturally in the wild. Our objective was to develop a rapid and successful method for in vitro propagation based on an initial in vitro asymbiotic seed germination step that achieved high percentages.
Methodology
Effects of four different media, i.e. (i) Murashige and Skoog (MS), (ii) Mitra et al., (iii) Knudson (KC) and (iv) Gamborg et al. (B5), were evaluated for large-scale multiplication by asymbiotic seed germination. Seedling leaf number, shoot number, shoot length, root number and root length were scored. After 7–8 months, large numbers of well-rooted plantlets were transferred to a glasshouse in thermocol pots containing compost. Six different composts based on broken brick and charcoal were compared for their ability to support further development over 90 days of hardening.
Principal results
The fastest and highest percentage seed germination was achieved using MS medium. Seeds on MS medium germinated in 3–4 weeks compared with 7–8 weeks on B5 medium. Seedling development was also superior on MS medium. The inclusion of plant growth regulators was unnecessary. Compost comprising broken brick and charcoal with an upper layer of moss was found to be the most suitable for the survival of transferred plantlets. Ninety per cent survival of plantlets was achieved 90 days after transfer to a glasshouse.
Conclusions
The use of MS culture medium is well suited for the mass multiplication of D. hookerianum plants intended for re-introducing this threatened orchid into the wild.
doi:10.1093/aobpla/plr032
PMCID: PMC3260561
36.  Identification of Stylosanthes guianensis varieties using molecular genetic analysis 
AoB Plants  2012;2012:pls001.
Molecular genetic diversity and population structure analysis were used to clarify the controversial botanical classification of Stylosanthes guianensis. The accessions were clustered in nine groups, each of which was mainly composed of only one of the four botanical varieties.
Background and aims
The botanical classification of Stylosanthes guianensis is controversial, and few studies have used molecular markers to analyse this species. We used microsatellite markers to study the genetic diversity and population structure of S. guianensis and compare our results with the current infraspecific botanical classification.
Methodology
A representative sample from the S. guianensis Brazilian germplasm collection (150 accessions) was analysed using 20 microsatellite loci. A model-based Bayesian approach implemented in the software STRUCTURE was used to assign accessions into clusters. A dendrogram was constructed based on Roger's genetic distances.
Principal results
The number of alleles per locus varied from 2 to 11, with an average of 4.7. The observed (HO) and expected (HE) heterozygosity values varied from 0 to 0.58 (mean of 0.18) and from 0.04 to 0.83 (mean of 0.55), respectively. Nine groups were assembled in STRUCTURE, and these groups were consistent with clusters inferred from the genetic distances and taxonomic varieties described for S. guianensis. The GST among the nine groups was 0.46.
Conclusions
The low HO and the GST values observed are in agreement with the outcrossing rate (26 %) estimated for this species. The data indicate a high genetic diversity among and within the botanical varieties and suggest that microsatellite-based information can be combined with classical taxonomy to elucidate infraspecific levels.
doi:10.1093/aobpla/pls001
PMCID: PMC3292737  PMID: 22479672
37.  Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don 
AoB Plants  2012;2012:pls002.
The genome size and organization of the important medicinal plant Catharanthus roseus is shown to correspond to 1C = 0.76 pg (~738 Mbps) and 2n = 16 chromosomes. The data provide a sound basis for future studies including cytogenetic mapping, genomics and breeding.
Background and aims
Catharanthus roseus is a highly valuable medicinal plant producing several terpenoid indole alkaloids (TIAs) with pharmaceutical applications, including the anticancer agents vinblastine and vincristine. Due to the interest in its TIAs, C. roseus is one of the most extensively studied medicinal plants and has become a model species for the study of plant secondary metabolism. However, very little is known about the cytogenetics and genome size of this species, in spite of their importance for breeding programmes, TIA genetics and emerging genomic research. Therefore, the present paper provides a karyotype description and fluorescence in situ hybridization (FISH) data for C. roseus, as well as a rigorous characterization of its genome size.
Methodology
The organization of C. roseus chromosomes was characterized using several DNA/chromatin staining techniques and FISH of rDNA. Genome size was investigated by flow cytometry using an optimized methodology.
Principal results
The C. roseus full chromosome complement of 2n = 16 includes two metacentric, four subtelocentric and two telocentric chromosome pairs, with the presence of a single nucleolus organizer region in chromosome 6. An easy and reliable flow cytometry protocol for nuclear genome analysis of C. roseus was optimized, and the C-value of this species was estimated to be 1C = 0.76 pg, corresponding to 738 Mbp.
Conclusions
The organization and size of the C. roseus genome were characterized, providing an important basis for future studies of this important medicinal species, including further cytogenetic mapping, genomics, TIA genetics and breeding programmes.
doi:10.1093/aobpla/pls002
PMCID: PMC3292738  PMID: 22479673
38.  Non-symbiotic haemoglobins—What's happening beyond nitric oxide scavenging? 
AoB Plants  2012;2012:pls004.
The ating evidence suggests non-symbiotic hemoglobins affect hormone responses by scavenging NO. Auxin, jasmonic acid, salicylic acid, ethylene and abscisic acid have altered responses when hemoglobins are expressed. Non-symbiotic hemoglobin is a factor during plant development, biotic and abiotic stress.
Background and aims
Non-symbiotic haemoglobins have been an active research topic for over 30 years, during which time a considerable portfolio of knowledge has accumulated relative to their chemical and molecular properties, and their presence and mode of induction in plants. While progress has been made towards understanding their physiological role, there remain a number of unanswered questions with respect to their biological function. This review attempts to update recent progress in this area and to introduce a hypothesis as to how non-symbiotic haemoglobins might participate in regulating hormone signal transduction.
Principal results
Advances have been made towards understanding the structural nuances that explain some of the differences in ligand association characteristics of class 1 and class 2 non-symbiotic haemoglobins. Non-symbiotic haemoglobins have been found to function in seed development and germination, flowering, root development and differentiation, abiotic stress responses, pathogen invasion and symbiotic bacterial associations. Microarray analyses under various stress conditions yield uneven results relative to non-symbiotic haemoglobin expression. Increasing evidence of the role of nitric oxide (NO) in hormone responses and the known involvement of non-symbiotic haemoglobins in scavenging NO provide opportunities for fruitful research, particularly at the cellular level.
Conclusions
Circumstantial evidence suggests that non-symbiotic haemoglobins may have a critical function in the signal transduction pathways of auxin, ethylene, jasmonic acid, salicylic acid, cytokinin and abscisic acid. There is a strong need for research on haemoglobin gene expression at the cellular level relative to hormone signal transduction.
doi:10.1093/aobpla/pls004
PMCID: PMC3292739  PMID: 22479675
39.  An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein 
AoB Plants  2012;2012:pls003.
Filter cubes made with machine-vision dichroic filters and illuminated with a royal blue light emitting diode can be used to produce an epifluorescent digital camera attachment that improves whole organism green fluorescent protein (GFP) photography. Mean pixel intensity responds linearly to purified GFP titration.
Background and aims
Studies have shown that levels of green fluorescent protein (GFP) leaf surface fluorescence are directly proportional to GFP soluble protein concentration in transgenic plants. However, instruments that measure GFP surface fluorescence are expensive. The goal of this investigation was to develop techniques with consumer digital cameras to analyse GFP surface fluorescence in transgenic plants.
Methodology
Inexpensive filter cubes containing machine vision dichroic filters and illuminated with blue light-emitting diodes (LED) were designed to attach to digital single-lens reflex (SLR) camera macro lenses. The apparatus was tested on purified enhanced GFP, and on wild-type and GFP-expressing arabidopsis grown autotrophically and heterotrophically.
Principal findings
Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ∼450 and ∼500 nm, and detects fluorescence between ∼510 and ∼595 nm. Epifluorescent photographs taken with SLR digital cameras were able to detect red-shifted GFP fluorescence in Arabidopsis thaliana leaves and cotyledons of pot-grown plants, as well as roots, hypocotyls and cotyledons of etiolated and light-grown plants grown heterotrophically. Green fluorescent protein fluorescence was detected primarily in the green channel of the raw image files. Studies with purified GFP produced linear responses to both protein surface density and exposure time (H0: β (slope) = 0 mean counts per pixel (ng s mm−2)−1, r2 > 0.994, n = 31, P < 1.75 × 10−29).
Conclusions
Epifluorescent digital photographs taken with complementary metal-oxide-semiconductor and charge-coupled device SLR cameras can be used to analyse red-shifted GFP surface fluorescence using visible blue light. This detection device can be constructed with inexpensive commercially available materials, thus increasing the accessibility of whole-organism GFP expression analysis to research laboratories and teaching institutions with small budgets.
doi:10.1093/aobpla/pls003
PMCID: PMC3296078  PMID: 22479674
40.  Estimates of nuclear DNA content in red algal lineages 
AoB Plants  2012;2012:pls005.
This paper compiles and discusses all currently available nuclear genome size data for red algae in relation to their most recent taxonomic classification.
Background and aims
The red algae are an evolutionarily ancient group of predominantly marine organisms with an estimated 6000 species. Consensus higher-level molecular phylogenies support a basal split between the unicellular Cyanidiophytina and morphologically diverse Rhodophytina, the later subphylum containing most red algal species. The Rhodophytina is divided into six classes, of which five represent early diverging lineages of generally uninucleate species, whose evolutionary relationships are poorly resolved. The remaining species compose the large (27 currently recognized orders), morphologically diverse and typically multinucleate Florideophyceae. Nuclear DNA content estimates have been published for <1 % of the described red algae. The present investigation summarizes the state of our knowledge and expands our coverage of DNA content information from 196 isolates of red algae.
Methodology
The DNA-localizing fluorochrome DAPI (4′,6-diamidino-2-phenylindole) and RBC (chicken erythrocytes) standards were used to estimate 2C values with static microspectrophotometry.
Principal results
Nuclear DNA contents are reported for 196 isolates of red algae, almost doubling the number of estimates available for these organisms. Present results also confirm the reported DNA content range of 0.1–2.8 pg, with species of Ceramiales, Nemaliales and Palmariales containing apparently polyploid genomes with 2C = 2.8, 2.3 and 2.8 pg, respectively.
Conclusions
Early diverging red algal lineages are characterized by relatively small 2C DNA contents while a wide range of 2C values is found within the derived Florideophyceae. An overall correlation between phylogenetic placement and 2C DNA content is not apparent; however, genome size data are available for only a small portion of red algae. Current data do support polyploidy and aneuploidy as pervasive features of red algal genome evolution.
doi:10.1093/aobpla/pls005
PMCID: PMC3303098  PMID: 22479676
41.  Genome size in Anthurium evaluated in the context of karyotypes and phenotypes 
AoB Plants  2012;2012:pls006.
Little is known about the genome of Anthurium other than chromosome observations, which frequently indicate supernumerary (“B”) chromosomes. New genome size estimates for 34 species and nine cultivars presented here provide insights into genome organization and evolution in this very large genus.
Background and aims
Anthurium is an important horticultural crop from the family Araceae, order Alismatales, a lineage considered to have diverged from other monocots prior to the cereals. Genome size and its distribution in Anthurium were investigated to gain a basic understanding of genome organization in this large genus and to forge a firm foundation for advancement of molecular approaches for the study of Anthurium. Currently, genome size estimates have been reported for only two Anthurium samples.
Methodology
Bulk nuclear DNA content estimates were obtained by flow cell cytometry using leaf tissue collected from Anthurium species of different subgeneric groups and from commercial cultivars. The most current and well-supported topology of subgeneric, sectional relationships was applied to present genome size estimates in the context of reported chromosome counts, karyotypes, putative phylogenetic relationships, observed phenotypes and pedigree.
Principal results
Genome size estimates based on bulk nuclear DNA content for 77 accessions representing 34 species and 9 cultivars were obtained, including initial estimates for 33 Anthurium species, and both the smallest (Anthurium obtusum; Tetraspermium) and largest (Anthurium roseospadix; Calomystrium) Anthurium genome sizes reported to date. Genome size did not distinguish any subgeneric section, but ranged 5-fold (4.42–20.83 pg/2 C) despite consistent 2N= 30 chromosome counts. Intraspecies genome size variation >20 % is reported for Anthurium ravenii, A. watermaliense and A. gracile.
Conclusions
Genome size estimates for Anthurium species spanning 13 recognized subgeneric sections indicate that genome size does not generally correlate with chromosome count or phylogenetic relationships. Mechanisms of genome expansion and contraction, including amplification and reduction of repetitive elements, polyploidy, chromosome reorganization/loss, may be involved in genome evolution in Anthurium as in other species. The new information on Anthurium genome sizes provides a platform for molecular studies supporting further research on genome evolution as well as cultivar development.
doi:10.1093/aobpla/pls006
PMCID: PMC3326669  PMID: 22509462
42.  Seed germination and seedling development ecology in world-wide populations of a circumboreal Tertiary relict 
AoB Plants  2012;2012:pls007.
Ecological traits of the circumboreal plant Viburnum opulus were examined to improve understanding of the variation of populations occurring in the same biome but on different continents. Seedling development/emergence is shown to be highly similar but some degree of variation was present in other traits, among populations.
Background and aims
Temperate forests are disjunct in the Northern Hemisphere, having become fragmented from the earlier widespread (Tertiary) boreotropical forest. We asked ‘What are the contemporary patterns of population variation in ecological traits of a Tertiary relict in a macroecological context?’. This issue underpins our understanding of variation in populations occurring in the same biome but on different continents.
Methodology
We examined characters associated with root and shoot emergences among populations of Viburnum opulus in temperate forests of Asia, North America and Europe. This species has complex seedling emergence extending over several years and requiring various temperature cues.
Principal results
Populations varied in germination responses and clustered into groups that were only partly related to varietal status. Whereas roots (at warm temperatures) and shoots (following a cold period) simultaneously emerged from seeds of all populations when simulated dispersal occurred in winter, they were delayed in some populations when dispersal occurred in summer.
Conclusions
Viburnum opulus populations, some separated by 10 300 km, showed high similarity in seedling development and in germination phenology, and we suggest that stabilizing selection has played a key role in maintaining similar dormancy mechanisms. Nevertheless, there was some degree of variation in other germination characters, suggesting local adaptation.
doi:10.1093/aobpla/pls007
PMCID: PMC3328982  PMID: 22514787
43.  Modelling leaf photosynthetic and transpiration temperature-dependent responses in Vitis vinifera cv. Semillon grapevines growing in hot, irrigated vineyard conditions 
AoB Plants  2012;2012:pls009.
Grapevines growing in Australia suffer from high temperatures which have major effects on photosynthesis and transpiration. To learn more, gas exchange was measured over several seasons and then modelled across temperatures from 20 to 45°C and validated with independent data.
Background and aims
Grapevines growing in Australia are often exposed to very high temperatures and the question of how the gas exchange processes adjust to these conditions is not well understood. The aim was to develop a model of photosynthesis and transpiration in relation to temperature to quantify the impact of the growing conditions on vine performance.
Methodology
Leaf gas exchange was measured along the grapevine shoots in accordance with their growth and development over several growing seasons. Using a general linear statistical modelling approach, photosynthesis and transpiration were modelled against leaf temperature separated into bands and the model parameters and coefficients applied to independent datasets to validate the model.
Principal results
Photosynthesis, transpiration and stomatal conductance varied along the shoot, with early emerging leaves having the highest rates, but these declined as later emerging leaves increased their gas exchange capacities in accordance with development. The general linear modelling approach applied to these data revealed that photosynthesis at each temperature was additively dependent on stomatal conductance, internal CO2 concentration and photon flux density. The temperature-dependent coefficients for these parameters applied to other datasets gave a predicted rate of photosynthesis that was linearly related to the measured rates, with a 1 : 1 slope. Temperature-dependent transpiration was multiplicatively related to stomatal conductance and the leaf to air vapour pressure deficit and applying the coefficients also showed a highly linear relationship, with a 1 : 1 slope between measured and modelled rates, when applied to independent datasets.
Conclusions
The models developed for the grapevines were relatively simple but accounted for much of the seasonal variation in photosynthesis and transpiration. The goodness of fit in each case demonstrated that explicitly selecting leaf temperature as a model parameter, rather than including temperature intrinsically as is usually done in more complex models, was warranted.
doi:10.1093/aobpla/pls009
PMCID: PMC3345123  PMID: 22567220
44.  The evolution of pollen germination timing in flowering plants: Austrobaileya scandens (Austrobaileyaceae) 
AoB Plants  2012;2012:pls010.
Austrobaileya has long served as a model for ancient angiosperm pollen structure. Its pollen germination is relatively rapid and requires < 10 % of the progamic phase. Extensive evidence suggests pollen germination underwent acceleration early in angiosperm history.
Background and aims
The pollination to fertilization process (progamic phase) is thought to have become greatly abbreviated with the origin of flowering plants. In order to understand what developmental mechanisms enabled the speeding of fertilization, comparative data are needed from across the group, especially from early-divergent lineages. I studied the pollen germination process of Austrobaileya scandens, a perennial vine endemic to the Wet Tropics area of northeastern Queensland, Australia, and a member of the ancient angiosperm lineage, Austrobaileyales.
Methodology
I used in vivo and in vitro hand pollinations and timed collections to study development from late pollen maturation to just after germination. Then I compared the contribution of pollen germination timing to progamic phase duration in 131 angiosperm species (65 families).
Principal findings
Mature pollen of Austrobaileya was bicellular, starchless and moderately dehydrated—water content was 31.5 % by weight and volume increased by 57.9 % upon hydration. A callose layer in the inner intine appeared only after pollination. In vivo pollen germination followed a logarithmic curve, rising from 28 % at 1 hour after pollination (hap) to 97 % at 12 hap (R2 = 0.98). Sufficient pollen germination to fertilize all ovules was predicted to have occurred within 62 min. Across angiosperms, pollen germination ranged from 1 min to >60 h long and required 8.3 ± 9.8 % of the total duration of the progamic phase.
Significance
Pollen of Austrobaileya has many plesiomorphic features that are thought to prolong germination. Yet its germination is quite fast for species with desiccation-tolerant pollen (range: <1 to 60 h). Austrobaileya and other early-divergent angiosperms have relatively rapid pollen germination and short progamic phases, comparable to those of many insect-pollinated monocots and eudicots. These results suggest that both the pollen germination and pollen tube growth periods were marked by acceleration of developmental processes early in angiosperm history.
doi:10.1093/aobpla/pls010
PMCID: PMC3345124  PMID: 22567221
45.  Vacuolar status and water relations in embryonic axes of recalcitrant Aesculus hippocastanum seeds during stratification and early germination 
AoB Plants  2012;2012:pls008.
Cells of embryonic axes in recalcitrant horse chestnut seeds are associated with a high water content and functionally preserved vacuoles with active aquaporins and invertase. These vacuoles function to the support rapid cell elongation of the hypocotyl that results in radicle emergence and thus the start of visible germination.
Backgrounds and aims
In tropical recalcitrant seeds, their rapid transition from shedding to germination at high hydration level is of physiological interest but difficult to study because of the time constraint. In recalcitrant horse chestnut seeds produced in central Russia, this transition is much longer and extends through dormancy and dormancy release. This extended time period permits studies of the water relations in embryonic axes during the long recalcitrant period in terms of vacuolar status and water transport.
Methodology
Horse chestnut (Aesculus hippocastanum) seeds sampled in Moscow were stratified in cold wet sand for 4 months. Vacuole presence and development in embryonic axes were examined by vital staining, light and electron microscopy. Aquaporins and vacuolar H+-ATPase were identified immunochemically. Water channel operation was tested by water inflow rate. Vacuolar acid invertase was estimated in terms of activity and electrophoretic properties.
Principal results
Throughout the long recalcitrant period after seed shedding, cells of embryonic axes maintained active vacuoles and a high water content. Preservation of enzyme machinery in vacuoles was evident from retention of invertase activity, substrate specificity, molecular mass and subunit composition. Plasmalemma and tonoplast aquaporins and the E subunit of vacuolar H+-ATPase were also present. In non-dormant seeds prior to growth initiation, vacuoles enlarged at first in hypocotyls, and then in radicles, with their biogenesis being similar. Vacuolation was accompanied by increasing invertase activity, leading to sugar accumulation and active osmotic functioning. After growth initiation, vacuole enlargement was favoured by enhanced water inflow through water channels formed by aquaporins.
Conclusions
Maintenance of high water content and desiccation sensitivity, as well as preservation of active vacuoles in embryonic axes after shedding, can be considered a specific feature of recalcitrant seeds, overlooked when studying tropical recalcitrants due to the short duration. The retained physiological activity of vacuoles allows them to function rapidly as dormancy is lost and when external conditions permit. Cell vacuolation precedes cell elongation in both hypocotyl and radicle, and provides impetus for rapid germination.
doi:10.1093/aobpla/pls008
PMCID: PMC3353218  PMID: 22593822
46.  Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica 
AoB Plants  2012;2012:pls011.
The paper describes the functional analysis of a class C heat shock transcription factor from rice (Oryza sativa). OsHsfC1b is shown to play a role in ABA-mediated salt stress tolerance and is required for plant growth under non-stress conditions.
Background and aims
Salt stress leads to attenuated growth and productivity in rice. Transcription factors like heat shock factors (HSFs) represent central regulators of stress adaptation. Heat shock factors of the classes A and B are well established as regulators of thermal and non-thermal stress responses in plants; however, the role of class C HSFs is unknown. Here we characterized the function of the OsHsfC1b (Os01g53220) transcription factor from rice.
Methodology
We analysed the expression of OsHsfC1b in the rice japonica cultivars Dongjin and Nipponbare exposed to salt stress as well as after mannitol, abscisic acid (ABA) and H2O2 treatment. For functional characterization of OsHsfC1b, we analysed the physiological response of a T-DNA insertion line (hsfc1b) and two artificial micro-RNA (amiRNA) knock-down lines to salt, mannitol and ABA treatment. In addition, we quantified the expression of small Heat Shock Protein (sHSP) genes and those related to signalling and ion homeostasis by quantitative real-time polymerase chain reaction in roots exposed to salt. The subcellular localization of OsHsfC1b protein fused to green fluorescent protein (GFP) was determined in Arabidopsis mesophyll cell protoplasts.
Principal results
Expression of OsHsfC1b was induced by salt, mannitol and ABA, but not by H2O2. Impaired function of OsHsfC1b in the hsfc1b mutant and the amiRNA lines led to decreased salt and osmotic stress tolerance, increased sensitivity to ABA, and temporal misregulation of salt-responsive genes involved in signalling and ion homeostasis. Furthermore, sHSP genes showed enhanced expression in knock-down plants under salt stress. We observed retarded growth of hsfc1b and knock-down lines in comparison with control plants under non-stress conditions. Transient expression of OsHsfC1b fused to GFP in protoplasts revealed nuclear localization of the transcription factor.
Conclusions
OsHsfC1b plays a role in ABA-mediated salt stress tolerance in rice. Furthermore, OsHsfC1b is involved in the response to osmotic stress and is required for plant growth under non-stress conditions.
doi:10.1093/aobpla/pls011
PMCID: PMC3357053  PMID: 22616023
47.  Long-term population demography of Trillium recurvatum on loess bluffs in western Tennessee, USA 
AoB Plants  2012;2012:pls015.
The paper uses modified population viability models and spatial structure via block analysis to assess population demography of Trillium recurvatum a clonal understory plant. The population is expanding, a likely outcome of the relatively high proportion of juvenile and non-flowering adult ramets and fast-replicating non-flowering adults. Further work is needed to elucidate the relative contributions of clonal vs seed recruitment to genetic structure and demography.
Background and aims
Understanding the demography of long-lived clonal herbs, with their extreme modularity, requires knowledge of both their short- and long-term survival and ramet growth patterns. The primary objective of this study was to understand the dynamics of a clonal forest herb, Trillium recurvatum, by examining temporal and small-scale demographic patterns. We hypothesized: (i) there would be more variability in the juvenile age class compared with non-flowering adult and flowering adult classes due to year-to-year fluctuations in recruitment; (ii) rates of population growth (λ) and increase (r) would be highest in non-flowering ramets due to a combination of transitions from the juvenile stage and reversions from flowering adults; and (iii) inter-ramet distances would be most variable between flowering and juvenile ramets due to a combination of clonal growth, seed dispersal by ants and ramet death over time.
Methodology
Census data were collected on the total number of stems in the population from 1990 to 2007, and placed within one of three life stages (juvenile, three-leaf non-flowering and three-leaf flowering). Modified population viability equations were used to assess temporal population viability, and spatial structure was assessed using block krigging. Correlations were performed using current and prior season weather to current population demography.
Principal results
The first hypothesis was rejected. The second hypothesis was supported: population increase (r) and growth rate (λ) were highest in non-flowering ramets. Finally, the third hypothesis was rejected: there was no apparent density dependence within this population of Trillium and no apparent spatial structure among life stages.
Conclusions
Overall population density fluctuated over time, possibly due to storms that move soil, and prior year's temperature and precipitation. However, density remained at some dynamic stable level. The juvenile age class had greater variability for the duration of this study and population growth rate was greatest for non-flowering ramets.
doi:10.1093/aobpla/pls015
PMCID: PMC3357055  PMID: 22616024
48.  Assessment of hybridization among wild and cultivated Vigna unguiculata subspecies revealed by arbitrarily primed polymerase chain reaction analysis 
AoB Plants  2012;2012:pls012.
This paper show that inter-subspecies hybridization among certain Vigna unguiculata subspecies, occurred during the course of evolution. This has affected several regions of the genome and is interfering with the dependable assessment of sub-species relationships using single (rRNA regions) or multilocus markers.
Background and aims
Intra-species hybridization and incompletely homogenized ribosomal RNA repeat units have earlier been reported in 21 accessions of Vigna unguiculata from six subspecies using internal transcribed spacer (ITS) and 5S intergenic spacer (IGS) analyses. However, the relationships among these accessions were not clear from these analyses. We therefore assessed intra-species hybridization in the same set of accessions.
Methodology
Arbitrarily primed polymerase chain reaction (AP-PCR) analysis was carried out using 12 primers. The PCR products were resolved on agarose gels and the DNA fragments were scored manually. Genetic relationships were inferred by TREECON software using unweighted paired group method with arithmetic averages (UPGMA) cluster analysis evaluated by bootstrapping and compared with previous analyses based on ITS and 5S IGS.
Principal results
A total of 202 (86 %) fragments were found to be polymorphic and used for generating a genetic distance matrix. Twenty-one V. unguiculata accessions were grouped into three main clusters. The cultivated subspecies (var. unguiculata) and most of its wild progenitors (var. spontanea) were placed in cluster I along with ssp. pubescens and ssp. stenophylla. Whereas var. spontanea were grouped with ssp. alba and ssp. tenuis accessions in cluster II, ssp. alba and ssp. baoulensis were included in cluster III. Close affinities of ssp. unguiculata, ssp. alba and ssp. tenuis suggested inter-subspecies hybridization.
Conclusions
Multi-locus AP-PCR analysis reveals that intra-species hybridization is prevalent among V. unguiculata subspecies and suggests that grouping of accessions from two different subspecies is not solely due to the similarity in the ITS and 5S IGS regions but also due to other regions of the genome.
doi:10.1093/aobpla/pls012
PMCID: PMC3357975  PMID: 22619698
49.  Hydrogen peroxide—a central hub for information flow in plant cells 
AoB Plants  2012;2012:pls014.
The review discusses recent advances in H2O2 plant biology, focusing on its signalling capabilities, the transduction pathways involved and its potential for interfering with other information transfer mechanisms in plant cells.
Background
Hydrogen peroxide (H2O2) was initially recognized as a toxic reactive oxygen species, able to cause damage to a variety of cellular structures. However, it became clear in the last decade that H2O2 can also act as a potent signalling molecule, involved in a plethora of physiological functions.
Scope
In the present review, we offer a brief summary of H2O2 signalling events and focus on the mechanisms of its perception and signal transduction, the factors that act downstream, as well as H2O2 interference with other information transfer mechanisms.
Conclusion
The significant scientific effort in the last 10 years to determine the position of H2O2 in signal transduction networks in plants demonstrated that it is essential for both the communication with external biotic and abiotic stimuli and the control of developmentally regulated processes. In addition, H2O2 complements, synergizes or antagonizes many cellular regulatory circuits by active interaction with other signals and plant hormones during growth, development and stress responses. Therefore, further understanding of H2O2 signal transduction is not only of fundamental, but also of practical importance, since this knowledge may contribute to improve agricultural practices and reduce stress-induced damage to crops.
doi:10.1093/aobpla/pls014
PMCID: PMC3366437  PMID: 22708052
50.  Morphological versus molecular markers to describe variability in Juniperus excelsa subsp. excelsa (Cupressaceae) 
AoB Plants  2012;2012:pls013.
This is a large scale investigation of morphological diversity in Juniperus excelsa excelsa. It offers complementary results to those obtained for the same populations using molecular markers. These two approaches are complementary and should be considered together in order to obtain a comprehensive view of the variability of J. excelsa excelsa.
Background and aims
Juniperus excelsa M.-Bieb. is a major forest element in the mountains of the eastern part of Mediterranean and sub-Mediterranean regions. This study comprises the first morphological investigation covering a large part of the geographical range of J. excelsa and aims to verify the congruency between the morphological results and molecular results of a previous study.
Methodology
We studied 14 populations sampled from Greece, Cyprus, Ukraine, Turkey and Lebanon, 11 of which have previously been investigated using molecular markers. Three hundred and ninety-four individuals of J. excelsa were examined using nine biometric features characterizing cones, seeds and shoots, and eight derived ratios. Statistical analyses were conducted in order to evaluate the intra- and inter-population morphological variability.
Principal results
The level of intra-population variability observed did not show any geographical trends. The total variation mostly depended on the ratios of cone diameter/seed width and seed width/seed length. The discrimination analysis, the Ward agglomeration method and barrier analysis results showed a separation of the sampled populations into three main clusters. These results confirmed, in part, the geographical differentiation revealed by molecular markers with a lower level of differentiation and a less clear geographical pattern. The most differentiated populations using both markers corresponded to old, isolated populations in the high altitudes of Lebanon (>2000 m). Moreover, a separation of the northern Turkish population from the southern Turkish populations was observed using both markers.
Conclusions
Morphological variation together with genetic and biogeographic studies make an effective tool for detecting relict plant populations and also populations subjected to more intensive selection.
doi:10.1093/aobpla/pls013
PMCID: PMC3357054  PMID: 22822421

Results 26-50 (132)